free for authors, free for readers, free from publisher, free free neuropathology 1:2 (2020) opinion piece the “neuroepithelial tumor”: exchanging our trash can for an industrial size dumpster? arie perry division of neuropathology, department of pathology, university of california, san francisco, usa corresponding author: arie perry, department of pathology, university of california, 505 parnassus avenue, san francisco, ca 94117, usa arie.perry@ucsf.edu submitted: 30 november 2019 accepted: 07 december 2019 published: 01 january 2020    https://doi.org/10.17879/fnp-2020-2611   if “a rose by any other name would smell as sweet”, then does garbage by any other name smell as foul? before i argue the affirmative, i must first fully disclose that like every other neuropathologist, i’ve previously used the term “neuroepithelial tumor” in my own reports and manuscripts. nevertheless, i’ve been increasingly concerned that its usage is now pursuing an alarming crescendo with an inversely decreasing specificity. in the 2015 world health organization (who) consensus meeting in heidelberg, a decision was made to abandon the term, “primitive neuroectodermal tumor” or pnet from the subsequent 2016 scheme1 and this was generally hailed as a major breakthrough, with the promise of enhancing our diagnostic accuracy for central nervous system (cns) tumor classification. nonetheless, it was recognized that even with improved definitions, one still encounters occasional “pnet-like” cases that do not conform to currently known entities. as such, it was decided to introduce the term, cns embryonal tumor, nos for such cases. of course, everyone recognized that this was essentially trading in one trash can for another, but with the notion that the new trash can was smaller and with the hope that as additional entities are elucidated over time, eventually this category would disappear altogether. also, given that in 2013, the soft tissue and bone blue book similarly ditched “peripheral pnet” in favor of ewing sarcoma2, this new approach essentially eliminated the diagnosis of “pnet” altogether. unfortunately, since the who 2016 publication1, i feel that our trash can is yet again expanding, given that the term “neuroepithelial tumor” (net) is gaining momentum, both within the literature and in clinical practice. for instance, whereas we previously had only two known net entities, dysembryoplastic neuroepithelial tumor (dnet) (don’t even get me started on “dysembryoplastic”) and cribriform neuroepithelial tumor (crinet), we’ve since added: 1) high-grade neuroepithelial tumor (hgnet) with mn1 alteration (hgnet-mn1), 2) hgnet with bcor exon 15 internal tandem duplication (hgnet-bcor), 3) neuroepithelial tumor with h3 g34 mutation (net-h3-g34), and 4) polymorphous low-grade neuroepithelial tumor of the young (plnty)3-9. additionally, a fifth hgnet or hgnet, not elsewhere classified (hgnet-nec) is now being used in some clinical reports for malignant cns neoplasms that don’t fit neatly into a well-delineated tumor type, although some of these descriptive diagnoses are eventually replaced by a more specific one with further molecular testing. the oxford dictionary definition of neuroepithelium is: “1. a type of epithelium containing sensory nerve endings and found in certain sense organs (e.g. the retina, the inner ear, the nasal membranes, and the taste buds)” or more pertinent to net, “2. (in embryology) ectoderm that develops into nerve tissue.” https://www.lexico.com/en/definition/neuroepithelium). other definitions similarly focus on brain development. for instance, according to wikipedia, “neuroepithelial cells, or neuroectodermal cells, form the wall of the closed neural tube in early embryonic development. the neuroepithelial cells span the thickness of the tube's wall, connecting with the pial surface and with the ventricular or lumenal surface. they are joined at the lumen of the tube by junctional complexes, where they form a pseudostratified layer of epithelium called neuroepithelium. neuroepithelial cells are the stem cells of the central nervous system, known as neural stem cells, and generate the intermediate progenitor cells known as radial glial cells, that differentiate into neurons and glia in the process of neurogenesis.” (https://en.wikipedia.org/wiki/neuroepithelial_cell). this explains the intended use of neuroepithelial tumor in the original 1988 description of dnet10, wherein the authors emphasized their view that dnet is likely related to a developmental disorder or malformation, given the frequent histologic findings resembling focal cortical dysplasia in adjacent cortex. in other circumstances, net is utilized in a broader fashion to state a belief that a neoplasm is derived from cns precursor cells. unfortunately, net is now often utilized in an even less specific manner, essentially meaning: “i think this is probably a cns tumor because it’s located there, but i wouldn’t swear to it under oath in a court of law”. as long as the entire oncology team knows that this diagnosis represents our mea culpa of ignorance, then there’s no harm in using this term as a placeholder until we know more. however, busy people (including oncologists) often generalize and may assume that given the similar terminology, hgnet, nec is equivalent to hgnet-mn1 (replacing mostly what was previously diagnosed as astroblastoma, mainly behaving as who grade ii) or to hgnet-bcor and net-h3-g34 (both behaving predominantly as who grade iv tumors). in other words, one could falsely assume that all hgnets are biologically related and should therefore be treated in a similar fashion clinically. another major source of confusion comes from very different uses of “net” by various experts. as already mentioned, in the past, it was an abbreviation for neuroectodermal tumor within both central and peripheral forms of pnet. in neuropathology, it is now being used for neuroepithelial tumor as already discussed, but outside the cns, net is currently utilized far more commonly as an abbreviation for neuroendocrine tumor11. this newly sanctioned who term represents the lower grade or well differentiated subtype of “neuroendocrine neoplasm”. in other words, this is the more favorable tumor type, but nevertheless one that occasionally behaves more aggressively; in turn, net needs to be distinguished from neuroendocrine carcinoma, which is the overtly malignant and high-grade form of disease. within neuropathology, the most common manifestation of this newly proposed nomenclature is the pituitary neuroendocrine tumor or pitnet, in place of pituitary adenoma12,13. nonetheless, with so many different versions now entering the medical lexicon, no-one should be surprised if one net subtype is confused for another. in conclusion, by discarding pnet (i.e., who grade iv small blue cell tumor with neuronal features) in favor of net or hgnet, have we essentially exchanged our trash can for an industrial size dumpster? i occasionally wake up in a sweat from dreaming of a dystopic future wherein the who scheme is simply composed of a long list of entities all entitled “neuroepithelial tumor with ___ molecular alteration”. wouldn’t it be preferable to go as far as we can with what we know? in other words, if a tumor shows compelling astrocytic features, why not invoke astrocytoma or astrocytic neoplasm in the name? if the tumor has glioneuronal features, why not say so? if indeed, neuroepithelial tumor is the best we can do, then at least, let’s make a concerted effort to replace the name once we know more. of course, this is just one man’s opinion and an opinion is only worth the price one pays for it! references 1.       louis dn, ohgaki h, wiestler od, et al. who classification of tumours of the central nervous system (revised 4th edition). lyon, france: iarc; 2016. 2.       fletcher cdm, bridge ja, hogendoorn pcw, mertens f, eds. who classification of tumours of soft tissue and bone. 4th ed. lyon, france: iarc; 2013. 3.       sturm d, orr ba, toprak uh, et al. new brain tumor entities emerge from molecular classification of cns-pnets. cell. 2016;164(5):1060-1072. 4.       korshunov a, capper d, reuss d, et al. histologically distinct neuroepithelial tumors with histone 3 g34 mutation are molecularly similar and comprise a single nosologic entity. acta neuropathol. 2016;131(1):137-146. 5.       paret c, theruvath j, russo a, et al. activation of the basal cell carcinoma pathway in a patient with cns hgnet-bcor diagnosis: consequences for personalized targeted therapy. oncotarget. 2016;7(50):83378-83391. 6.       yoshida y, nobusawa s, nakata s, et al. cns high-grade neuroepithelial tumor with bcor internal tandem duplication: a comparison with its counterparts in the kidney and soft tissue. brain pathol. 2018;28(5):710-720. 7.       huse jt, snuderl m, jones dt, et al. polymorphous low-grade neuroepithelial tumor of the young (plnty): an epileptogenic neoplasm with oligodendroglioma-like components, aberrant cd34 expression, and genetic alterations involving the map kinase pathway. acta neuropathol. 2017;133(3):417-429. 8.       ferris sp, velazquez vega j, aboian m, et al. high-grade neuroepithelial tumor with bcor exon 15 internal tandem duplication a comprehensive clinical, radiographic, pathologic, and genomic analysis. brain pathol (in press). 9.       andreiuolo f, lisner t, zlocha j, et al. h3f3a-g34r mutant high grade neuroepithelial neoplasms with glial and dysplastic ganglion cell components. acta neuropathol commun. 2019;7(1):78. 10.    daumas-duport c, scheithauer bw, chodkiewicz jp, laws er, jr., vedrenne c. dysembryoplastic neuroepithelial tumor: a surgically curable tumor of young patients with intractable partial seizures. report of thirty-nine cases. neurosurgery. 1988;23(5):545-556. 11.    rindi g, klimstra ds, abedi-ardekani b, et al. a common classification framework for neuroendocrine neoplasms: an international agency for research on cancer (iarc) and world health organization (who) expert consensus proposal. mod pathol. 2018;31(12):1770-1786. 12.    asa sl, casar-borota o, chanson p, et al. from pituitary adenoma to pituitary neuroendocrine tumor (pitnet): an international pituitary pathology club proposal. endocr relat cancer. 2017;24(4):c5-c8. 13.    villa c, vasiljevic a, jaffrain-rea ml, et al. a standardised diagnostic approach to pituitary neuroendocrine tumours (pitnets): a european pituitary pathology group (eppg) proposal. virchows arch. 2019;475(6):687-692.   detailed neuropathologic report of covid-19 complicated by large intracerebral hemorrhage and periventricular lesions with macrophagic infiltrates feel free to add comments by clicking these icons on the sidebar free neuropathology 2:7 (2021) case report detailed neuropathologic report of covid-19 complicated by large intracerebral hemorrhage and periventricular lesions with macrophagic infiltrates adrian levine1, carol lee2, craig fava3, frankie tsang4, kelly macneil5, stephen t. yip1,5, veronica hirsch-reinshagen1 1 department of pathology and laboratory medicine, university of british columbia, vancouver, bc, canada 2 division of forensic pathology, royal columbian hospital, vancouver, bc, canada 3 division of critical care, royal columbian hospital, bc, canada 4 british columbia center for disease control, vancouver, bc, canada 5 cancer genetics & genomics laboratory, bc cancer, vancouver, bc, canada corresponding author: veronica hirsch-reinshagen · anatomical pathology · vancouver general hospital · 899 west 12th av · jpn rm 1401 · vancouver, bc · canada · v5z 1m9 veronica.hirsch@vch.ca submitted: 15 february 2021 accepted: 16 march 2021 copyedited by: deanna fang published: 25 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3266 keywords: covid-19, cerebral hemorrhage, brain, postmortem, histology, neuropathology abstract infection with the sars-cov-2 virus affects a wide range of systems. significant involvement of the central nervous system has been described, including ischemic and hemorrhagic strokes. thus far, neuropathologic reports of patients who passed away from covid-19 have generally described non-specific findings, such as variable reactive gliosis and meningeal chronic inflammatory infiltrates, as well as the consequences of the infection’s systemic complications on the brain, including ischemic infarcts and hypoxic/ischemic encephalopathy. the neuropathological changes in patients with covid-19 and large hemorrhagic strokes have not been described in detail. we report the case of an elderly male who had a long course of covid-19 and ultimately passed away from a large intracerebral hemorrhage. in addition to acute hemorrhage, neuropathologic examination demonstrated non-specific reactive changes and chronic periventricular lesions with macrophagic and perivascular lymphocytic infiltrates without evidence of demyelination or presence of sars-cov-2 by pcr test. this manuscript expands the spectrum of reported neuropathological changes in patients with covid-19. introduction intracerebral hemorrhage (ich) is an uncommon complication of infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus, affecting 0.5-7.9% of patients hospitalized with covid-191–3. therapeutic anticoagulation has been identified as the most common etiology1–3 and an important risk factor for ich in covid-194. in addition, other factors such as microand macrovascular thrombosis, endothelial dysfunction and endotheliitis may play important pathogenic roles in this setting. despite this plethora of hypothesized factors, only a few neuropathological reports have included detailed histopathological brain evaluations of covid-19 patients with large fatal ich. post-mortem neuropathological evaluation of patients with covid-19 have shown a range of histological changes and have been reviewed elsewhere5,6. the vast majority of cases show relatively mild and non-specific findings including variable and diffuse microand astrogliosis, and mild parenchymal and leptomeningeal infiltration by chronic inflammatory infiltrates, which could represent the histological features of an encephalopathy associated with severe systemic inflammation rather than specific covid-19-related changes7. in addition, many cases show variable hypoxic-ischemic encephalopathy, infarcts associated with large vessel thromboembolism and variable hemorrhagic lesions from microhemorrhages to fatal ich5,6. only three detailed neuropathological reports of patients with large ich have been published to date and include a case of cerebellar hemorrhage thought to be most likely secondary to hypertensive vasculopathy8, one of hemorrhagic transformation of a large middle cerebral artery stroke9 and two cases reported by the authors as ich in the context of lymphocytic panencephalitis, meningitis and diffuse petechial hemorrhages10. in the latter, no details are provided regarding the histological changes associated with, or the etiopathogenesis of, the ich or petechial hemorrhages. in this report, we expand on the neuropathological literature of covid-19 cases by detailing the neuropathological findings in an elderly male with covid-19 and fatal ich. some of the findings reported in this case were also seen in a younger individual with covid-19 and fatal ich for which we were unable to acquire consent for publication of clinical details. the histological similarities and differences between these two cases will be highlighted in the text. case presentation this male patient, in his early 70s, presented to hospital with bilateral pneumonia secondary to covid-19 infection that was confirmed by nasopharyngeal swab testing. parallel testing for influenza a, b and respiratory syncytial virus were negative. his prior medical history included hypertension and significant hip osteoarthritis. he was diagnosed with septic shock and transferred to the intensive care unit upon admission as he required vasopressors and mechanical ventilation. his mean arterial pressure was targeted to more than 65 mmhg while his systolic blood pressure (sbp) was kept below a ceiling of 180 to 200 mmhg to avoid ischemic brain injury. this was especially relevant in the context of his shock syndrome and prior history of hypertension. only two sbp episodes over 180 mmhg, both of less than 30 min in duration, were recorded 4 and 19 days prior to his terminal ich. during his first week of admission, he developed renal failure, which was thought clinically secondary to acute tubular necrosis in the context of multisystem organ failure. he was placed on continuous renal replacement therapy (crrt) one week after admission. this required local anticoagulation with unfractionated heparin titrated to obtain an activated partial thromboplastin time (aptt) post-crrt filter of 60 to 90 seconds. his international normalized ratio (inr) hovered around the upper normal limit of 1.2 during his entire hospitalization. other relevant laboratory findings included an elevated d-dimer, c reactive protein and procalcitonin. he did not have evidence of pulmonary embolism during his hospitalization. figure 1. (a) ct scan showed a large left frontal ich with intraventricular extension. (b) coronal sections confirmed this ich and left-to-right subfalcine herniation. (c) leptomeningeal chronic inflammatory infiltrates were most prominent in the brainstem. (d) immunohistochemistry for hla-dr, a major histocompatibility class ii cell surface receptor, highlighted the leptomeningeal chronic inflammatory infiltrates (arrow) and the mild-to-moderately increased brainstem microglial activation. nearly three weeks into his admission and two weeks after initiation of crrt, the patient showed evidence of decreased level of consciousness. a computerized tomography scan demonstrated a left frontal intracerebral hemorrhage with intraventricular extension and subfalcine herniation (fig. 1a). he died the next day after withdrawal of life-sustaining therapies. a brain-restricted autopsy was performed 7 days after death to aid in the determination of the etiology of the brain hemorrhage. gross examination of the brain revealed a weight of 1280 grams, an enlarged left hemisphere, subfalcine left-to-right herniation of the left anterior frontal lobe and bilateral hippocampal uncal herniations. coronal sections confirmed a large ich centered in the left frontal lobe in close proximity to the lateral ventricle with extension throughout the ventricular system (fig. 1b). away from the hemorrhage, no additional gross pathological changes were identified. microscopic examination revealed mild diffuse reactive changes (fig. 1c and d) and focal periventricular lesions at the angles of the ventricles (fig. 2). the diffuse reactive changes included rather mild leptomeningeal chronic inflammatory infiltrates, parenchymal reactive gliosis and very mildly increased perivascular cd3 t-lymphocytes. the leptomeningeal inflammatory infiltrates were composed predominantly of macrophages and cd3 t-cell lymphocytes found throughout the meninges, but were accentuated in the brainstem (fig. 1c). reactive gliosis, highlighted by mild-to-moderately increased immunoreactivity for human leukocyte antigen – dr isotype (hla-dr) and glial fibrillary acidic protein (gfap), was present predominantly in the brainstem and olfactory bulb. similar changes were also seen in the younger individual. no microglial nodules were identified on hematoxylin and eosin (he) stains. two possible microglial nodules were identified on hla-dr immunohistochemistry (ihc) in the medulla only. figure 2. (a) multifocal periventricular lesions showed loss of ependyma and significant infiltration by macrophages with scant accompanying lymphocytes. (b) cd68 highlighted the significant macrophagic infiltrates. (c) luxol-fast blue showed mild loss of myelin, while phosphorylated neurofilament (pnf) ihc (d) showed associated axonal pathology with significant numbers of axonal spheroids. atypical periventricular lesions (fig. 2) were seen at multiple locations including both temporal horns, left occipital horn, fourth ventricle and in close proximity to the anterior left frontal lateral ventricle at the edge of acutely hemorrhagic brain parenchyma. these lesions consisted of loss of ependymal lining, relatively well delimited macrophagic and lymphocytic infiltrates (fig. 2a and b) and reactive vessels with plump endothelial cells. axonal pathology, in the form of relatively frequent axonal spheroids without appreciable demyelination, was also present (fig. 2c and d). iron stains performed in several of these lesions were negative. the abundance of phagocytic macrophages and the absence of hemosiderin suggest that these lesions were older than the intracerebral hemorrhage and therefore not likely to be a reactive change to intraventricular blood. this type of lesion was not seen in the younger individual, who instead exhibited multifocal petechial hemorrhages and microscopic ischemic infarcts thought to be most compatible with embolic lesions. no definite evidence of thrombi or megakaryocytes was identified in either of the cases. the neocortex, hippocampi, deep grey nuclei and cerebellum were unremarkable aside from some mild age-related neurodegenerative pathology. pontine white matter tracts only showed several microscopic foci of amyloid precursor protein (app) immunoreactive axonal pathology associated with microand astrogliosis. cd68 revealed absence of mature phagocytic macrophages in these lesions. these were interpreted as foci of axonal damage secondary to traction due to the left frontal and intraventricular ich. no cerebral amyloid angiopathy, arteriolosclerosis, lacunar infarcts or other vascular changes were identified. polymerase chain reaction (pcr) test for the presence of sars-cov-2 rna in multiple sections (medulla, hippocampus and olfactory bulbs) of formalin-fixed paraffin-embedded (ffpe) brain tissue was negative, including representation of the periventricular lesions. similar results were obtained for the younger patient. discussion our cases showed three main pathological changes: (1) acute and fatal ich, (2) diffuse reactive changes and (3) additional parenchymal lesions, either as multifocal chronic periventricular lesions in the elderly individual or as multifocal microhemorrhages and infarcts in the younger patient. the histopathological features of the periventricular lesions make them most compatible with localized tissue necrosis of unclear etiology. they were negative for sars-cov-2 rna. to our knowledge, this type of lesion has not been studied extensively. a single report comparing the neuropathological substrate of periventricular white matter abnormalities in patients with major depression and in controls describes lesions with similar characteristics to those found in this case. these lesions were interpreted as corresponding most likely to ischemic insults and were found both in a patient with major depression and a control11. the pathogenesis of these lesions awaits further evaluation, but the fact that lesions similar to those seen in our older individual have been described in non-covid cases raises the possibility that they are either nonspecific in etiology (e.g. associated with an episode of severe systemic illness) and/or can be brought about by different injury mechanisms. furthermore, in our case, it is unclear whether these lesions predated the sars-cov-2 infection or developed as a consequence of it. the close relationship of the acute left frontal ich to changes suggestive of an underlying periventricular lesion raise the possibility that local loss of tissue integrity may play a predisposing role to ich in covid-19. a similar situation was observed in the younger individual, in whom multifocal petechial hemorrhages and microscopic ischemic infarcts were identified. these were interpreted as most likely due to microemboli in the context of severe covid-19 and therapeutic anticoagulation for extracorporeal membrane oxygenation (ecmo). both patients were locally or systemically anticoagulated for therapeutic reasons and the risk of ich in the setting of anticoagulation appears to be increased in patients with covid-19. a study of 10 patients with covid-19 supported on ecmo for acute respiratory distress syndrome (ards) showed a markedly increased incidence of hemorrhagic strokes compared to non-covid patients on ecmo12. the diffuse reactive changes identified in both covid cases are similar to those reported previously in the literature and are at least partially explained as a manifestation of severe illness-related encephalopathy5–7. several tissue blocks of both cases were tested for sars-cov-2 by pcr and were negative. neither patient received antiviral therapies that could have decreased sars-cov-2 tissue levels. formalin-fixed control lung tissue from an unrelated covid-19 autopsy case with a shorter postmortem interval served as a positive control. although the long post-mortem interval and formalin-fixation may have interfered with the detection of sars-cov-2 in our samples, these results are in line with previously published reports that have shown inconsistent and variable detection of sars-cov-2 in brain parenchyma by pcr and immunohistochemical methods7,8. overall, this would suggest that the brain is not a site consistently affected by high viral loads of sars-cov-2 and raises the possibility that direct infection of the cns tissue may not be the main pathogenic mechanism of covid-19 neurologic manifestations. in summary, the fatal ich in two cases of covid-19 were most likely due to a combination of anticoagulation and additional factors affecting the integrity of the cns parenchyma. additional chronic inflammatory infiltrates and glial reactive changes are at least partially explained by severe illness-related encephalopathy. references 1. kvernland, a. et al. anticoagulation use and hemorrhagic stroke in sars-cov-2 patients treated at a new york healthcare system [published online ahead of print, 2020 aug 24]. neurocrit care. (2020) 1-12. https://doi.org/10.1007/s12028-020-01077-0 2. dogra, s. et al. hemorrhagic stroke and anticoagulation in covid-19. j stroke cerebrovasc dis. (2020) 29(8):104984. https://doi.org/10.1016/j.jstrokecerebrovasdis.2020.104984 3. benger, m. v intracerebral haemorrhage and covid-19: clinical characteristics from a case series. brain behav immun. (2020) 88:940-944. https://doi.org/10.1016/j.bbi.2020.06.005 4. melmed, kr. et al. risk factors for intracerebral hemorrhage in patients with covid-19 [published online ahead of print, 2020 sep 24]. j thromb thrombolysis. (2020) 1-8. https://doi.org/10.1007/s11239-020-02288-0 5. lou, jj. et al. neuropathology of covid-19 (neuro-covid): clinicopathological update. free neuropathol. (2021) 2:2. https://doi.org/10.17879/freeneuropathology-2021-2993 6. mukerji, ss & solomon, ih. what can we learn from brain autopsies in covid-19?. neurosci lett. (2021) 742:135528. https://doi.org/10.1016/j.neulet.2020.135528 7. deigendesch, n. et al. correlates of critical illness-related encephalopathy predominate postmortem covid-19 neuropathology. acta neuropathol. (2020) 140(4):583-586. https://doi.org/10.1007/s00401-020-02213-y 8. al-dalahmah, o. et al. neuronophagia and microglial nodules in a sars-cov-2 patient with cerebellar hemorrhage. acta neuropathol commun. (2020) 8(1):147. published 2020 aug 26. https://doi.org/10.1186/s40478-020-01024-2 9. hanley, b. et al. histopathological findings and viral tropism in uk patients with severe fatal covid-19: a post-mortem study. lancet microbe. (2020) 1(6):e245-e253. https://doi.org/10.1016/s2666-5247(20)30115-4 10. von weyhern, ch. et al. early evidence of pronounced brain involvement in fatal covid-19 outcomes. lancet. (2020) 395(10241):e109. https://doi.org/10.1016/s0140-6736(20)31282-4 11. thomas, aj. et al. a neuropathological study of periventricular white matter hyperintensities in major depression. j affect disord. (2003) 76(1-3):49-54. https://doi.org/10.1016/s0165-0327(02)00064-2 12. usman, aa. et al. a case series of devastating intracranial hemorrhage during venovenous extracorporeal membrane oxygenation for covid-19. j cardiothorac vasc anesth. (2020) 34(11):3006-3012. https://doi.org/10.1053/j.jvca.2020.07.063 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples feel free to add comments by clicking these icons on the sidebar free neuropathology 1:14 (2020) letter optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples thibauld oullier 1*, alice prigent 1*, guillaume chapelet 1,2, michel neunlist 1, franck letournel 3, pascal derkinderen 1,4 1 université de nantes, inserm, tens, the enteric nervous system in gut and brain diseases, imad, nantes, france 2 chu nantes, clinical gerontology department, nantes, france 3 chu angers, neurobiology and neuropathology laboratory, angers, france 4 chu nantes, department of neurology, nantes, france * these authors contributed equally to this work. corresponding author: pascal derkinderen · inserm u1235 nantes · 1 rue gaston veil · 44035 nantes · france · tel: +33(0)240165202 · fax: +33(0)240165203 pascal.derkinderen@chu-nantes.fr ; derkinderenp@yahoo.fr submitted: 04 april 2020 accepted: 26 april 2020 copyedited by: christian thomas published: 28 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2749 additional resources and electronic supplementary material: supplementary material keywords: alpha-synuclein, parkinson’s disease, filter trap, dot blot, aggregates over the past two decades, the progress in molecular genetics and neuropathology enabled a better understanding of the pathogenesis of neurodegenerative disorders. the observation that abnormal protein accumulation is characteristic of a particular disease has led to a neuropathological classification according to the composition of the abnormal protein aggregates [1]. several methods have been developed for the detection of these insoluble protein aggregates including, for instance, dye binding assays and immunohistochemistry with proteinase k pretreatment [1]. filter retardation assay is a simple and rapid method which detects protein aggregates formed either in vivo or in vitro, including amyloid-beta/tau aggregates from alzheimer’s disease (ad) [2–4] and polyglutamine expansion from huntington’s disease [5,6]. in this assay, ‘lysis buffer’-resistant protein aggregates, but not soluble monomeric species, are retained by a cellulose acetate membrane and subsequently detected by immunoblotting. as loading control, a ‘classical’ dot blot with either polyvinylidene fluoride (pvdf) or nitrocellulose membranes is run in parallel with the same lysates. in contrast to polyglutamine expansion, tau and beta-amyloid, there are only few studies on using filter retardation assays for the detection of alpha-synuclein aggregates, which are characteristic of parkinson’s disease (pd) and dementia with lewy bodies (dlb) [7]. in addition, a full characterization of any filter-based assay for alpha-synuclein, including the effect of membrane composition, lysis procedure and antibodies has not yet been reported. we therefore set out the current study in which we characterize a vacuum-based 96-well format filter assay for the detection of aggregated alpha-synuclein in human brain specimens. frozen brain samples from neuropathologically confirmed cases of dlb, progressive supranuclear palsy (psp) and ad were obtained from the neuropathology department of angers (dr franck letournel). brain samples were lysed with the “precellys 24™” tissue homogenizer (bertin technologies, cat# p000669-pr240-a, montigny-le-bretonneux, france) followed by a brief sonication (one minute, 5 seconds on, 5 seconds off, amplitude 70%; vcx130 ultrasonic processor with a 6 mm probe, sonics and materials, newton, ct, usa) either in (i) radioimmunoprecipitation assay (ripa) buffer (merck millipore, cat# 20-188, molsheim, france) containing 2 mm orthovanadate (sigma, cat# s6505, saint quentin fallavier, france), 1% (v/v) phosphatase inhibitor cocktail iii (sigma, cat# p0044) and a protease inhibitors cocktail (roche, cat# 14424700, meylan, france) at 4°c; (ii) ‘sodium dodecyl sulfate (sds) buffer’ containing 1% (w/v) sds, 2 mm orthovanadate (sigma, cat# s6505) and 1% (v/v) phosphatase inhibitor cocktail iii (sigma, cat# p0044); (iii) ‘urea buffer’ containing 7 m urea (sigma, cat# t7875), 2 m thiourea (sigma, cat# l3771), 4% (v/v) chaps (sigma, cat# c3023), 2 mm orthovanadate (sigma, cat# s6505), 1% (v/v) phosphatase inhibitor cocktail iii (sigma, cat# p0044) and a protease inhibitors cocktail (roche, cat# 14424700). lysates were centrifuged at 10,000 x g for 5 minutes; supernatants were recovered and protein concentration determined using the pierce bca protein assay kit (thermofisher scientific, cat# 23227, saint herblain, france) along with the varioskan™ multimode microplate reader (thermofisher scientific, cat# vl0l00d0); samples lysed in urea buffer were diluted 5 times for compatibility with the bca assay (https://assets.thermofisher.com/tfs-assets/lsg/application-notes/tr0068-protein-assay-compatibility.pdf). twenty µg of these supernatants were subjected to vacuum filtration through a 96-well dot blot apparatus (bio-rad, cat# 1706545, marnes-la-coquette, france) with 0.45 µm pore size cellulose acetate (sterlitech, cat# ca0453001, kent, usa), nitrocellulose (thermofisher scientific, cat# 77010) or pvdf membranes (thermofisher scientific, cat# 88518). the resultant membranes were blocked for one hour at room temperature in tris-buffered saline (tbs, sigma, cat# t5912) with 0.1% (v/v) tween-20 (sigma, cat# p1379) and 5% (w/v) non-fat dry milk, then incubated overnight at 4°c with the primary antibodies listed in table 1. bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit (life technologies cat# 31460, diluted 1:5,000) or anti-mouse antibodies (sigma, cat# a9044, diluted 1:5,000) and visualized by enhanced chemiluminescent detection (supersignal™ west pico plus chemiluminescent substrate, thermofisher scientific, cat# 34580). oligomeric aggregates of alpha-synuclein, which were obtained by incubating monomeric recombinant alpha-synuclein (sigma, cat# 575001) in phosphate-buffered saline under agitation at a concentration of 10 mg/ml at 37°c for one week, served as a control [8]. table 1. alpha-synuclein and phospho-alpha-synuclein antibodies used in the current study. when cellulose acetate membranes were used, recombinant oligomeric alpha-synuclein and dlb lysates were barely or not detected by two of the total alpha-synuclein antibodies (syn211 and syn-1), regardless of the lysis procedure (figure 1a). in contrast, mjfr1 antibody efficiently detected both oligomeric alpha-synuclein and aggregated alpha-synuclein from ripa-lysed dlb samples (figure 1a). no specific signal was observed with this antibody when dlb samples were lysed with sds or urea buffers (figure 1a). two of the phosphospecific antibodies (ep1536y and to a lesser extent psyn#64) detected aggregated alpha-synuclein in ripa-dlb lysates. no signal was observed when psp and ad lysates were filtered and analyzed with either mjfr1 or ep1536y antibody. these findings show that sample lysis with ripa sample lysis together with mjfr1 and/or ep1536y immunoblotting is the most specific and reproducible approach. we therefore used these two antibodies and ripa-lysed samples to perform additional dot blots with pvdf or nitrocellulose membranes, that could serve as a loading control. the results show that pvdf achieved better reproducibility and specificity for the detection of total and phosphorylated alpha-synuclein than nitrocellulose membranes (figure 1b). figure 1. results from the filter trap and dot blot assays. (a) recombinant oligomeric alpha-synuclein (oligo) and samples from dlb, psp and ad brains lysed in ripa, sds and urea buffers were subjected to vacuum filtration through a 96-well dot blot apparatus with an acetate cellulose membrane; immunoblotting (ib) was then performed with 3 different antibodies to total alpha-synuclein (mjfr1, syn-1 and syn211) and 3 antibodies specific for the phosphorylated form of the protein (ep1536y, psyn#64 and phospho s129). (b) recombinant oligomeric alpha-synuclein (oligo) and ripa-lysed samples from dlb, psp and ad brains were subjected to vacuum filtration through a 96-well dot blot apparatus with a pvdf or a nitrocellulose membrane ; immunoblotting (ib) was then performed with mjfr1 or ep1536y antibodies. results shown are representative of 3 to 5 independent experiments. the results presented here, although preliminary, provide the basis for reproducible and specific detection of pathological alpha-synuclein in diseased brains using a filter trap assay. we show that the selection of primary antibody and lysis buffer has important effects on the performance of the assay. as an optimized protocol, we suggest that brain homogenates should be lysed in ripa buffer and that immunoblotting should be performed with mjfr1 for the detection of total alpha-synuclein and ep1536y for the detection of the phosphorylated form of the protein (a step by step standard operating procedure is provided as supplementary material). owing to the high-throughput capability of the filter trap assay, such an approach is not only potentially interesting for detecting aggregated alpha-synuclein in post mortem brain samples but also in the peripheral nervous system of living pd patients in order to develop original biomarkers of the disease [9]. references 1. kovacs gg. molecular pathological classification of neurodegenerative diseases: turning towards precision medicine. int j mol sci. 2016;17:189. 2. bieschke j, cohen e, murray a, dillin a, kelly jw. a kinetic assessment of the c. elegans amyloid disaggregation activity enables uncoupling of disassembly and proteolysis. protein sci. 2009;18:2231–41. 3. boyé-harnasch m, cullin c. a novel in vitro filter trap assay identifies tannic acid as an amyloid aggregation inducer for het-s. j biotechnol. 2006;125:222–30. 4. chang e, kuret j. detection and quantification of tau aggregation using a membrane filter assay. anal biochem. 2008;373:330–6. 5. novoselova tv, margulis ba, novoselov ss, sapozhnikov am, van der spuy j, cheetham me, et al. treatment with extracellular hsp70/hsc70 protein can reduce polyglutamine toxicity and aggregation. j neurochem. 2005;94:597–606. 6. van waarde-verhagen mawh, kampinga hh. measurement of chaperone-mediated effects on polyglutamine protein aggregation by the filter trap assay. methods mol biol. 2018;1709:59–74. 7. recasens a, dehay b, bové j, carballo-carbajal i, dovero s, pérez-villalba a, et al. lewy body extracts from parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. ann neurol. 2014;75:351–62. 8. corbillé a-g, neunlist m, derkinderen p. cross-linking for the analysis of α-synuclein in the enteric nervous system. j neurochem. 2016;139:839–47. 9. lebouvier t, tasselli m, paillusson s, pouclet h, neunlist m, derkinderen p. biopsable neural tissues: toward new biomarkers for parkinson’s disease? front psychiatry. 2010;1:128. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. free for authors, free for readers, free from publisher, free free neuropathology 1:1 (2020) editorial free for authors, free for readers, free from publisher, free formatting and free opinion: this is free neuropathology werner paulus institute of neuropathology, university hospital muenster, muenster, germany corresponding author: werner paulus, institute of neuropathology, pottkamp 2, 48149 muenster, germany werner.paulus@uni-muenster.de submitted: 05 december 2019 published: 01 january 2020    https://doi.org/10.17879/fnp-2020-2610   as reader, author, referee and editor i have often discussed and reflected upon the nature of the "perfect" journal what are the qualities that make up a great scientific journal? the following ingredients came to my mind: •    excellent science •    interesting and relevant content •    high visibility •    a high impact factor •    short turnaround times (from submission to first decision, from accept to online publication) •    fair reviews •    no unnecessary major revisions just to satisfy referees •    a responsive and supportive editorial office •    insightful editors •    no subscription rates for readers (open access) •    low (ideally no) article processing fees for authors •    a convenient and easy electronic submission system •    high technical standard (figures, copyediting, layout) •    a clear and informative website •    electronic tools for enabling interaction between authors and readers i am not aware of a journal that fulfills all of these requirements. since the great majority of established journals are published by commercial publishers, some of these features are out of control of the editor, such as quality of copyediting, layout design, time until online publication, charges for authors and/or readers, promotion, and appearance and content of the website. however, there is no need for these latter features to fall into the responsibility of the publisher. should scientists be willing to perform these activities themselves, they are in principle well capable to found, shape and run a wonderful journal that fulfills the expectations of the scientific community. it was precisely this insight that sparked the development of free neuropathology. why is the new journal called free neuropathology? the word "free" bears various meanings. first, it means no cost, like in "free beer". accordingly, the journal is free of cost for everyone. when discussing the name, a few colleagues expressed concern that "free" may have a connotation of "poor quality", because something without price might be considered as having no value. i am not convinced of this argument, because some of our highest ethical values cannot be bought for money (and thus are for free), and because some  universities with the word "free" in their name are among the most distinguished academic institutions, such as frije universiteit amsterdam and freie universität berlin. second, "free" means having the freedom to do something your own way. in fact, at free neuropathology we are able to decide on every aspect of the journal without having to obey non-scientific paper-shufflers, and we put much emphasis on freedom from unnecessary formalities and bureaucracy that increasingly abound in the publishing business. third, we encourage authors to submit not only original papers, letters and reviews, but also opinion pieces, critiques and annotations, because we feel that frank views, open-minded discussion and critical analysis of prevailing approaches and trendy hypotheses are integral parts of science. in the end, the name of the journal reflects a mix of freebie, freedom and liberty. free for authors and free for readers most scientists, scientific organizations and politicians believe that publishing is expensive and that someone has to pay for the publication process, either the reader through subscription models or the author through open-access models. i believe that there is a third option if the activities of the publisher are rendered by scientists working in the field of the journal, i.e. by authors, referees and editors who have put the most time, energy and enthusiasm into the journal anyway. it is true that some technical infrastructure and manpower is necessary to keep a journal running and that this is not without cost, but compared to the time that scientists spend as voluntary referees and editors (let alone the work of authors), the additional time and expenses for taking over the publishers´ classical activities are moderate and they can easily be covered by scientific institutions. furthermore, while in past years handling of manuscripts, layout and printing of journals required considerable time, staff, technical skills and money, digital publishing has made these activities easier, increasingly automated and cheaper. at free neuropathology we take advantage of the open journal systems platform for the management of peer-reviewed academic journals. many thanks to the developers of this open-source software and to my university library! green open access means self-archiving of the accepted version of the manuscript in the authors´ format. in the gold open access model, articles are made immediately and freely available, while authors must pay article processing fees. diamond open access (also referred to as platinum open access), as will be employed at free neuropathology, means that the whole process, including submission, handling by the editorial team, peer review, copyediting, layout and retrieving full text content, is completely free. these tasks are taken over by colleagues who provide high quality editorial, peer reviewing and publishing services. free neuropathology´s diamond open access model is based on the enthusiasm of volunteers who love to be engaged in the scientific publication process, thereby serving science and society. we also believe that volunteers will enjoy career benefits and recognition from institutions for doing the work. our young members of the layout/copyediting board feel that this is a great opportunity to learn about publishing and a great way to expand personal networks. free from publishers virtually everyone in science criticizes the high profit margins and pricing policies of commercial publishers. it has been estimated that the major fifty-seven academic publishers generate a combined revenue of 60 billion € per year, with profits in the range of 20 to 50%. increases in subscription fees by 60% per year for individual journals are not unusual – the more prestigious the journal, the more impertinent the increase. while from an economical point of view this is understandable because shareholders and private equity must be satisfied; however, the excessive cash outflow endangers science. this scenario is even more absurd, because scientists working voluntarily as authors, referees and members of editorial boards do the bulk of the work, thereby serving as useful dupes for publishing houses and maximizing the profit of people who have zero interest in science per se. the authors, who are largely funded by governments, even offer their work to publishers for free, who then sell it back to government-funded institutions at astronomical prices. publishers are inventive in obfuscating their business models. the cash flow is often organized in a way so that the individual scientist does not see the problem, because subscription fees or article processing charges are covered by universities or funding agencies. needless to say that, in the end, all scientists have to pay the bill because the money transferred to publishers needs to be detracted from personnel, infrastructure and funding of scientific institutions. furthermore, publishers have invented a variety of new services that nobody needs, in order to justify their prices. finally, publishers sell not only their few top journals but also bunches of hundreds of low-impact or irrelevant journals to large scientific organizations. nobody is in need of these journals, but all scientists have to pay for them. established publishers tend to condemn so-called predatory publishers because they rip off scientists, but do they mean themselves? let me relate to you a parable (it´s a bit lengthy and needs pondering, so if you have little time feel free to skip to the next paragraph): an artist has created a painting after spending years of work, money, creativity, care and enthusiasm. because this is the way it has always been, the artist does not try to sell the opus, but prefers to donate it to a company called jumpartize, which is owned by private equity that operates amusement parks and museums. the businesspeople of jumpartize do not understand art nor do they appreciate it, but they are very good at making the most money out of it. artists must pay art processing charges (apc) for the art they donate, and they find this ok because jumpartize builds and maintains museums, frames and dusts the paintings, employs staff (custodians, cleaners, clerks), and counts how often each piece of art is mentioned in newspaper articles and social media, resulting in the artificialfactor®. alternatively, artists can commit to lifelong work for one week per year in one of jumpartize´s amusements parks as clowns or as animate figures in haunted houses (open joy program). many artists love to become famous and they try to endow their artwork to the most prestigious museums boasting the highest artificialfactor®. jumpartize asks several art historians to evaluate the offered paintings and to write up art critiques within two weeks for free, which they happily accept because they consider the invitation to be an honor. admission fees for the museum are high at about 120 € and they rise by 30 % every year, but the public accepts this because art is considered to be high value and because prices for dusters have increased recently. the artists themselves must also pay admission fees to see their own works, but they are allowed to hang a low-quality poster of their paintings in their private rooms. some museums of jumpartize offer free entrance for everyone if artists are willing to defray the costs of running the museum and serve as building workers for jumpartize´s new 20-story headquarters building. politicians and several national academies of fine arts are very proud of having successfully negotiated with jumpartize that artists are allowed to terminate work in haunted houses by the age of 80 (plan artistique or in short plan a). meanwhile jumpartize, including all fun parks, museums and artwork has been sold to another private equity investor for double the original price. if you find this scenario absurd or crazy, what does it mean for the behavior of scientists? scientists, librarians and politicians complain about costs of journals but at the same time they continue to support commercial publishers. science politicians have been very proud of negotiations or declarations such as plan s or deal, whereby article processing charges are limited or somewhat reduced, but in the end they have surrendered. financially supporting journals by national or international funding organizations (such as gates open research) is also not helpful, because funding is usually restricted to a limited period of time, so that permanent structures which are mandatory for scientific journals are endangered. at first glance, journals that are owned by scientific societies and published by commercial publishers may be in a more comfortable position, because societies can replace the publisher in case of disagreement or disservice. however, the problem is that scientific societies usually develop dollar signs in their eyes as soon as the publisher offers sharing part of the profit. this corrupts science. i am deeply convinced that the purpose of scientific journals is publishing science, ideally the best science, but definitely not making money, for whomsoever. there are other good reasons for proceeding without publishers. we, the scientists and developers of a journal, will be in a position to decide on website, layout, copyediting and promotion, and we no longer depend on publishers´ decisions which are led by financial considerations. for example, publishers tend to decline suggestions of modifying individual journal websites because, for economical or branding reasons, they want the websites of their hundreds or thousands of journals to appear identical. publishers promote journals at commercial exhibitions at scientific meetings (often largely unnoticed by scientists), while scientists know their field, their colleagues and the appropriate communication channels better than publishers do. scientists therefore could perform more efficiently and more cost-effectively in undertaking these classical publishers´ activities. it is true that publishers have much more experience because at least the handful of oligopolistic publishers have published thousands of journals before. but we will learn and we will learn fast. and yes, we may be somewhat naïve, we will make mistakes and some technical issues will not work perfectly right from the start, but in my experience scientists work more efficiently and creatively than publishers´ staff, and in the end our approach will be successful. publishers like to talk about ethics. they request from authors disclosures of potential conflicts of interest such as shareholding of family members in relevant companies, they ask for reference numbers of ethical committees, they request statements of author contributions, and they analyze papers using software for detecting plagiarism and manipulation of figures. while these activities may be considered to be measures for increasing quality and transparency (as well as subscription rates and article processing fees), publishers play the role of science police, thereby undermining the general credibility of scientists and considering them as potential cheaters and criminals. to be clear, i find that ethical standards and guidelines are important, but they should be developed and released by scientific societies and not by individual publishers who just try to increase sales and the prestige of their products. publishers stress the highest ethical standards, but in the end they are interested in nothing more than profit and they harness scientists in their dull business. commercial publishers have had a detrimental effect on science. publishing scientific work together with commercial publishers should be discontinued for reasons of economy, quality and ethics. free formatting many scientists are concerned and annoyed by the increasing number of formal requirements when submitting papers, such as rigid regulations on references, tables, figures, organization of manuscript, font type/size, abbreviations and nomenclature. we believe that most of these regulations are unnecessary and they detract from the gist of the purpose, i.e. prompt publication of good science. at free neuropathology authors can format their paper as they like, as long as formatting is consistent within the paper and the paper has been well written and carefully prepared. free opinion controversial discussion and deviating views represent an integral part of science, which is often somewhat neglected for technical, psychological and political reasons. at free neuropathology we have implemented features so that authors and readers can take part in scientific discussion. we encourage the submission of “opinion pieces” which are in a separate category of papers in this journal. this is a channel for expressing personal but scientifically founded views on hypotheses, terminology, key papers, opinion making, politics or anything else related to neuropathology. in addition, and in order to stimulate discussion on papers published in free neuropathology, we use the open-source software hypothesis which basically introduces an annotation layer over any paper. after signing-in and clicking the arrow on the right side of the browser window, the hypothesis side-bar will appear and you will be able to provide sentence-level comments, add critique, share information and engage in discussion. finally, even the editor can express frank views in editorials or social media channels -this cannot be taken for granted under other circumstances. feel free to join if you support our concept and share our spirit: feel free to join the movement. when you have interesting data in the fields of human or experimental neuropathology: submit an original paper to free neuropathology. when you have concisely written up something as a letter: consider free neuropathology for prompt publication. when you have strong views about a controversial issue or if you disagree with anything in the field: send us an opinion piece. if you like grassroots movements and soft revolutions: support and recommend free neuropathology. if you would like to become actively involved in our editorial activities, if you are willing to share your technical skills, or if you have comments, questions, critique, ideas or stimulating suggestions: send me a note. 62nd meeting of the french society of neuropathology meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 1:34 (2020) meeting abstracts 62nd meeting of the french society of neuropathology meeting abstracts december 4th, 2020 the french society of neuropathology was created in 1989, succeeding the french club of neuropathology set up in 1965. the society organizes two scientific meetings per year.   submitted: 11 december 2020 accepted: 14 december 2020 published: 15 december 2020 https://doi.org/10.17879/freeneuropathology-2020-3134 keywords: french society of neuropathology, sfnp, meeting abstracts, 62nd meeting dec. 2020 short communications:     plu i, evrard b, duchesne m, regnault b, pérot p, chrétien d, eloit m, seilhean d (2020) etiological diagnosis of a plasma cell encephalitis by next generation sequencing. free neuropathol 1, 34: 2 etiological diagnosis of a plasma cell encephalitis by next generation sequencing isabelle plu1, bruno evrard2, mathilde duchesne3, béatrice regnault4, philippe pérot4, delphine chrétien4, marc eloit4, danielle seilhean1 1 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 2 intensive care department, university hospital limoges, france 3 department of pathology, university hospital limoges, france 4 pathogen discovery unit, department of virology, pasteur institute, paris a 59-year-old man, working as a forester, developed a fever and temporal headache associated with cranial nerves deficits. etiological investigations including multiplex pcr and the search for autoantibodies remained negative. his condition quickly worsened leading to coma and death after 2 months.   neuropathology revealed lymphoplasmacytic infiltrates in the leptomeninges, cranial nerve roots and around the vessels in cerebral gray and white matter. perivascular cuffs were associated with astrocytic gliosis and microglial activation. immunochemistry against known viruses was negative. the new generation dna sequencing (ngs) without a priori has led to the identification of a type 1 lyssavirus of the european bat (eblv1), the reservoir of which is an insectivorous bat. only two cases of transmission to human have been reported to date. this case of zoonosis illustrates the interspecies transmission of an unusual virus leading to a potential emerging disease. ngs coupled with neuropathology provide a valuable tool for identifying new causes of encephalitis.     teyssou e, muratet f, del mar amador m, gyorgy b, guegan j, marie y, meininger v, salachas f, millecamps s, seilhean d (2020) a novel mutation in sod1 causing unusual neuropathological findings. free neuropathol 1, 34: 3 a novel mutation in sod1 causing unusual neuropathological findings elisa teyssou1*, françois muratet1*, maria del mar amador1,2, beata gyorgy1, justine guegan1, yannick marie1, vincent meininger2,3, françois salachas1,2, stéphanie millecamps1, danielle seilhean1,4 1 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 2 department of neurology, reference center for als ile de france, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 3 peupliers hospital, ramsay générale de santé, paris, france 4 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france * equal contribution a 63-year-old man with no personal or family history developed amyotrophic lateral sclerosis (als) at age 63, fatal in 17 months. in the anterior horns of the spinal cord, neuronal loss was moderate. cystatin c immunohistochemistry (ihc) showed numerous bunina bodies. ihc of ubiquitin, p62 or tdp43, did not show skein-like inclusions. swollen neurons in clusters, were labelled with antiubiquitin, sod-1 and phosphorylated neurofilaments. they were different from inclusions usually observed in sod-1 mutations.   whole exome sequencing analysis identified a novel sod1 mutation c.164c>t, p.thr55ileu, confirmed by sanger sequencing. no other rare variant was identified in any other als-related genes. association of neuropathology and whole exome sequencing can provide a useful tool for the identification of unknown forms of the disease, better understanding of the physiopathology and lead to new therapeutic targets.     siegert e, dittmayer c, schneider u, preuße c, goebel hh, stenzel w (2020) myositis in scleroderma – capillary pathology is fundamental. free neuropathol 1, 34: 4 myositis in scleroderma – capillary pathology is fundamental elise siegert, carsten dittmayer, udo schneider, corinna preuße, hans-hilmar goebel, werner stenzel department of neuropathology, charité universitätsmedizin, corporate member of freie universität berlin, humboldt-universität zu berlin, and berlin institute of health (bih), berlin, germany systemic sclerosis is a chronic disease of connective tissues characterized by fibosis, vasculopathy and autoimmunity. affected patients show signs of the skin, internal organs and sometimes overlap myositis. the vasculopathy is considered obliterative but the pathogenic basis is not known to date.   we are presenting light-microscopic and ultrastructural as well as clinical data of 18 patients suffering form scleroderma and myositis. we have applied a new electron microscopical technique which we call large scale electron microscopy allowing a ‘pan and zoom’ approach similar to ‘google earth’ viewing.   this analysis allows to study >1000 capillaries of patients and controls, highlighting reduplications of basement.   we show that this type of ultrastructural changes is membranes, endothelial activation and proliferation of pericytes specific for a subtype of scleromyositis and discus possible pathogenic mechanisms.     boluda s, wallon d, rovelet-lecrux a, campion d, nicolas g, duyckaerts c (2020) neuropathological variability of four tauopathy cases with mapt microduplication. free neuropathol 1, 34: 5 neuropathological variability of four tauopathy cases with mapt microduplication susana boluda1,2, david wallon3,4,5, anne rovelet-lecrux3,4,6, dominique campion3,4,6, gaël nicolas3,4,6, charles duyckaerts1,2 1 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 2 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 3 inserm u1245, university of normandy, unirouen, rouen, france 4 cnr-maj, normandy center for genomic and personalized medicine, rouen, france 5 department of neurology, university hospital, rouen, france 6 department of genetics, university hospital, rouen, france we report the neuropathology of four mapt duplication carriers, a rare chromosomal rearrangement involving the 17q21.31 chromosomal region that causes an early onset dementia (eod) clinically mimicking alzheimer disease or an atypical extrapyramidal syndrome, as recently described. they were three males and one female with ages ranging between 37 and 57 years. they were all tauopathies with a variability in the morphology and distribution of the aggregates. the cases either mimicked pick disease with neuronal globular aggregates similar to pick bodies and pathology involving predominantly the cortical and limbic regions and subcortical nuclei or they resembled progressive supranuclear palsy with tufted astrocytes with major involvement of the brain stem and subcortical nuclei. the tau isoform expression also varied from either only 3r, only 4r or a mixed 3r/4r expression.   in conclusion, mapt duplication may lead to a tauopathy spectrum with a range of 3r and 4r expression.     seilhean d, mokhtari k, plu i, boluda-casas s, mathon b, cao a, hervé d, mégarbane b, bielle f, levavasseur e, malet i, marot s, el hachimi h, marty s, prigent a, duyckaerts c, potier mc, haïk s, delatour b, marcelin ag (2020) multiple cerebral angiopathy in sars-cov-2 infection. free neuropathol 1, 34: 6 multiple cerebral angiopathy in sars-cov-2 infection danielle seilhean1,2, karima mokhtari1, isabelle plu1, susana boluda-casas1,2, bertrand mathon3, albert cao4, dominique hervé5, bruno mégarbane6, franck bielle1,2, etienne levavasseur2, isabelle malet7, stéphane marot7, hamid el hachimi2, serge marty2, annick prigent2, charles duyckaerts1,2, marie-claude potier2, stéphane haïk1,2, benoît delatour2, anne-geneviève marcelin7 1 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 2 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 3 department of neurosurgery, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 4 department of neurology, neuro-icu, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 5 department of neurology, lariboisière hospital, ap-hp nordparis university, paris, france 6 department of intensive care, lariboisière hospital, ap-hp nordparis university, paris, france 7 department of virology, inserm umrs 1136, pitie salpetriere hospital, ap-hp sorbonne university, paris, france sars-cov-2 is the cause of a pandemic characterized by its severity in elderly subjects or those presenting metabolic or vascular risk factors. brain damage, although relatively rare, is often fatal. from four cases (one autopsy and three brain biopsies) we analyzed the involvement of small cerebral arteries associated with white matter lesions, without signs of vasculitis or encephalitis. viral rna has not been detected in the brain. sars-cov2 spike protein (s) has been detected in the golgi apparatus of endothelial cells, colocalized with a host protease. our observations suggest the possibility of hematogenous neuroinvasion. the interaction of a small amount of protein s with endogenous proteases is thought to be able to disrupt the permeability of brain endothelial cells causing vascular damage. this result could provide therapeutic avenues to prevent or cure severe brain forms in patients at risk.     bourhis a, peyre m, bielle f (2020) a rare tumor of the peripheral nerve mimicking a schwannoma. free neuropathol 1, 34: 7 a rare tumor of the peripheral nerve mimicking a schwannoma amélie bourhis1, matthieu peyre2,3, franck bielle2,4 1 department of pathology, university hospital, brest, france 2 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 3 department of neurosurgery, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 4 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france a 38 year-old man presented with pain of the left forearm for five years. he had a history of old hiv infection and several past opportunistic infections. the pain was paroxysmal, favored by pronation and was increasing for two years. mri evidenced a tumor nodule on the path of the posterior interosseous nerve mimicking a schwannoma. intracapsular tumor resection stopped the pain. microscopic examination showed a tumor proliferation of fascicled eosinophilic fusiform cells. immunostaining showed the expression of the muscular markers smooth muscle actin and desmin, and the absence of expression of sox10. in situ hybridization for eber rna of ebv was positive in tumor cells. we diagnosed an ebv-associated leiomyoma of the immunocompromised, which was located in the peripheral nerve, a localization not previously reported. we discuss the tumorigenesis of this rare neoplasm.   conferences:     eloit m (2020) virus spillover from animal reservoirs and vectors to human detection using agnostic tools. free neuropathol 1, 34: 8 virus spillover from animal reservoirs and vectors to human detection using agnostic tools marc eloit head of pathogen discovery laboratory, pasteur institute, paris, france oie collaborating centre for detection and identification in humans of emerging animal pathogens, pasteur institute, paris, france professor of virology, veterinary school of maisons alfort, france viruses from wildlife including arboviruses vectored by mosquitoes have caused dramatic outbreaks over the last 25 years. on the one hand, new sequencing capacities have deeply affected the means by which new viruses are discovered, leading to the identification of ever more viruses in animal reservoirs and arthropods. on the other hand, it is worth noting that the continually growing knowledge regarding these viruses does not per se serve to identify potential human or animal threats. our laboratory aims to fill this gap by identifying, in selected ecosystems, whether as yet unknown, unexpected or neglected mosquito or wildlife viruses are responsible for frequent but sub-clinical or mild infection in humans highly exposed to wild life/arthropods, and thus represent good candidates for global spreading. it combines high throughput screening of viruses in animal/arthropods linked to antibody screening in healthy exposed human populations and virus search in patients presenting with severe diseases of unknown etiology. i will show our methodology and some results.     ludes b (2020) migration of siberian populations of the past: contribution of a multi-genetic markers approach. free neuropathol 1, 34: 9 migration of siberian populations of the past: contribution of a multi-genetic markers approach bertrand ludes university of paris-cnrs, fre 2029 head of the forensic institute of paris (institut médico-légal de paris) molecular markers of nuclear dna (autosomes and y chromosome) and mitochondrial dna from human samples help clarifying the migrations of the first peoples of the eurasian steppes. we analyzed samples dating from the middle of the 2nd millennium bc to 4th century ad and originating from the krasnoyarsk region (south siberia). we confirmed that, during the bronze and iron ages, southern siberia was a region dominated by european peoples, suggesting an eastward migration of the kurgan peoples across the russo-kazak steppe. the results further showed that at that time, the inhabitants of southern siberia must have had fair eyes, skin and hair.     copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. taylor’s focal cortical dysplasia revisited: history, original specimens and impact feel free to add comments by clicking these icons on the sidebar free neuropathology 2:11 (2021) flashback taylor’s focal cortical dysplasia revisited: history, original specimens and impact burkhard s. kasper epilepsy center, department of neurology, friedrich-alexander university erlangen-nuremberg, germany corresponding author: burkhard s. kasper · epilepsy center · department of neurology · friedrich-alexander university erlangen-nuremberg · schwabachanlage 6 · 91054 erlangen · germany burkhard.kasper@uk-erlangen.de submitted: 01 april 2021 accepted: 18 april 2021 copyedited by: jerry lou published: 23 april 2021 https://doi.org/10.17879/freeneuropathology-2021-3324 additional resources and electronic supplementary material: supplementary material keywords: dysplasia, epilepsy, history, taylor, fcd original paper:   focal dysplasia of the cerebral cortex in epilepsy. dc taylor, ma falconer, cj bruton, j.a.n. corsellis, journal of neurology, neurosurgery and psychiatry 1971; 34: 369-387   free full text link at jnnp: https://dx.doi.org/10.1136/jnnp.34.4.369 abstract 50 years ago back in 1971, david c. taylor and colleagues from england reported on a small series of surgical epilepsy cases proposing a new type of tissue lesion as a cause of difficult-to-treat focal epilepsy: a localized malformation of cerebral cortex. the lesion is now known as focal cortical dysplasia (fcd) type ii or taylor’s cortical dysplasia. fcd ii is not rare, and today is a frequent finding in neurosurgical epilepsy specimens. medical progress has been achieved in that the majority of fcd ii is diagnosed non-invasively by magnetic resonance imaging today. detailed studies on fcd revealed that the lesion belongs to a spectrum of mtor-o-pathies, thereby confirming the authors´ initial hypothesis of a relationship to tuberous sclerosis. here, selected original materials from taylor´s series are presented as virtual slides, supplemented by original clinical records, in order to give a first-hand impression of this milestone finding in neuropathology of epilepsy. what exactly did the authors report in this study? tissue findings from surgical brain specimens from 10 epilepsy patients were presented in detail [1], illustrating intriguing but formerly un-appreciated abnormalities of neocortical architecture. as core findings, neurons of bizarre size and shape were described throughout cortical layers ii-vi (today known as dysmorphic neurons) causing laminar disruption, in most cases accompanied by another pathological cell type, named grotesque cells of probable glial origin (today known as balloon cells). disorganization of cortical architecture and these cellular components were defined as pathognomonic features and suggested a new type of malformative cns lesion, i.e. focal dysplasia, today known as focal cortical dysplasia (fcd) type ii or taylor’s dysplasia [2]. why is this a milestone paper? the authors’ synopsis was groundbreaking since it defined a new category of circumscript lesion responsible for intractable focal epilepsy, adding to the spectrum of better-known epileptogenic lesions such as hippocampal sclerosis and benign neoplasms. epilepsy surgery was a rather young discipline back then. most causal epileptogenic lesions could be detected ex-post by histopathology only, since other supporting diagnostic modalities were unavailable at the time. dc taylor, a psychiatrist (!), recognized the similarities between these few cases out of several hundreds of cases just by reading through the reports, un-aided by any data such as imaging techniques [3]. these lesions were largely invisible to naked eye macroscopic examination in the operating theater or during tissue preparation [1]. by now, fcd has become a classic cause of epilepsy, ranking among the top 3 structural causes in epilepsy surgical series and in the majority is detected pre-surgically by magnetic resonance imaging (mri) [2, 4] what had been known before this seminal contribution? the authors in 1971 stated that “no reports of closely similar observations had been traced thus far” [1]. findings with some similarity to focal dysplasia had been sporadically reported under different names, such as “abortive tuberous sclerosis” [5], “infantile cerebral gliosis with giant nerve cells” [6] or “localized cerebral gliosis with giant neurons” [7]. maybe the earliest reference to a dysplasia-like pathology from epilepsy brains goes back to 1896 [8], but reports were overall scarce before the era of epilepsy surgery. which of the authors central hypotheses or conclusions turned out right or wrong? the histological reminiscence to tuberous sclerosis (ts) was noted and pointed out [1]. corsellis extensively searched for hints towards ts in both the patients and their families (see supplements 2-6). in a preceding conference presentation, the term “cryptic tuber” had already been used [9]. however, absence of any other signs of ts or suspicious familial traits was emphasized [1]. meanwhile, fcd ii has been identified as closely relating to genetic alterations within the mtor-signalling-cascade and belonging to the spectrum of “mtor-o-pathies” together with tuberous sclerosis and other malformations [10, 11]. fcd was correctly proposed to be a solitary lesion in most cases, its removal likely predicting seizure improvement or freedom. the authors proposed that dysplasia of their type would directly underlie the electrical and clinical manifestations of certain epilepsies. this has been strikingly verified later, since fcd turned out as intrinsically epileptogenic, i.e. the dysmorphic neurons themselves show the abnormal firing behavior, as proven by many techniques today such as in-vivo invasive recordings, electrocorticography, non-invasive measurements and in-vitro cellular recordings in fcd (for reference, see [4]). while taylor and the group did not comment on the presumed frequency of fcd, it is remarkable, that they found their 10 cases in a cohort of 300 epilepsy surgeries corresponding to 3%, while nowadays fcd ranks among the top pathologies in surgical series [2], see also below. at what stage of the authors´ career was the paper published? three out of four authors were well-known authorities in their fields, since murray falconer had established his fine epilepsy surgical series at the maudsley cooperating with the experienced neuropathology service at runwell, provided by j.a.n corsellis and clive joseph bruton. david taylor had been a research fellow at the beginning of his thirties and had started researching the psychiatric aspects of epilepsy surgery at the maudsley, giving rise to several publications from 1967 on (see also a letter by him in suppl. 7). after recognizing the commonalities in the tissue reports, he and the co-authors meticulously collected all available data leading to this seminal paper. how was the paper received over the time? more than 800 citations have cumulated since publication up to 2020 with about 20 citations per decade in the seventies and eighties and >200 citations per decade since then, 75% citations from clinical neurology [12]. the paper was translated early, e.g. to german, french and russian abstracts [13] and was widely recognized. “taylor’s dysplasia” as a term was coined by montreal epileptologist fred andermann (personal communication, see [3]). it was discussed later that the microscopic features were not strictly unique to taylor’s dysplasia, but may be seen in cases of hemimegalencephaly, for example [14]. dysplasia still was estimated to account for a minority of 2-5% of epilepsy surgery specimens as late as 1992 [14], while numbers have raised in modern series up to 30% [2], which relates to largely improved imaging and recording techniques. what happened to the original histological slides until today? starting early after 1950 j.a.n. corsellis built a large collection of brain specimen recruited from his neuropathology service including both surgical and autopsy tissues [15]. this collection came to be known as the corsellis collection later on [3]. it was the first of its kind and located in runwell hospital/wickford essex for years [15,16], and later moved to london (held by the west london mental health nhs trust), after runwell hospital closed down. of note, corsellis in addition to meticulously performing macroand microinvestigations documented in great detail the clinical patient findings and obtained many clinical notes (see supplemental material). his activities were funded by the medical research council as early as 1953 [16], he became a reference pathologist consulted by international colleagues [16], and after his death in 1994 his activities were continued by clive bruton. the original slides of focal dysplasia remained undiscovered as part of the corsellis collection brain bank in london for decades. an incomplete but significant part of slides, paraffin blocks and accompanying material including microphotographs and patient notes were found there and saved during research on the life and work of j.a.n. corsellis [3] and given to bs kasper by the corsellis collection to act as official custodian of this material when it became clear that the collection would be dismantled. did the re-discovery of this material elicit any surprising insights? vast material accompanying the tissue specimen impressively illustrates the working strategy at the institution led by corsellis. clinical notes and findings not only were systematically searched for, collected and saved (see suppl. 1-6), but also valued and integrated into the reports and discussions. there was a close exchange between the pathology group and the clinicians involved, not only the active neurosurgeons, but also the hospital and external neurologists, doctors and caregivers of the patients, as well as possible. this included postoperative years also. corsellis insisted on postsurgical cognitive tests, for example, and with taylor even visited the patient at home. no challenge seemed too difficult to overcome for the sake of obtaining further important information, as exemplified by correspondence between corsellis and neuropathologist professor h. orthner from göttingen/germany (see suppl. 4) which showed the length to which corsellis labored to obtain details about this patients mother who had died and had autopsy (see suppl. 6) as well as about the patients son, in order to check for any manifestations of tuberous sclerosis. what can be seen on the original slides? we present case 2 from the taylor paper for this re-appreciation. original slides in various stains were found preserved (he, van gieson, bielschowsky, ptah, nissl, oil-red, marchi). these slides correspond to figure 6 from the 1971 paper (for orientation see original figure and compare to macro-photographs and drawings in suppl. 1). exploring the original slides in h&e and nissl stains as well as new stainings obtained from the original paraffin blocks (see figure) nicely illustrates the presence of dysmorphic giant neurons, especially found at the bottom part of a sulcus. you are invited to explore the interactive, virtual microscopy slides in detail, just as taylor and corsellis did back then. this lesion nowadays would be classified as focal cortical dysplasia type iia, since balloon cells (then described as grotesque cells of probable glial origin) were not found, as confirmed by review. however, the majority of specimens in taylor´s series did contain balloon cells, i.e. focal cortical dysplasia type iib, representing the most common correlate of “fcd” or “taylor type dysplasia” as defined by imaging today. remember that in 1971 no magnetic resonance imaging (mri) was available. figure 1. original (historical) slides from taylors series (nissl, he) and new slides cut from the original paraffin blocks (evg, gfap, vimentin, nestin). clicking into the respective picture will lead you to the full virtual slide. links to the slides: nissl: https://omero-imaging.uni-muenster.de/webclient/img_detail/34739 he: https://omero-imaging.uni-muenster.de/webclient/img_detail/34169 evg: https://omero-imaging.uni-muenster.de/webclient/img_detail/34358 gfap: https://omero-imaging.uni-muenster.de/webclient/img_detail/34360 vimentin: https://omero-imaging.uni-muenster.de/webclient/img_detail/34363 nestin: https://omero-imaging.uni-muenster.de/webclient/img_detail/35885 supplementary material supplements 1-6 as well as their content appear in chronological order. to follow, click on the links. free full text taylor 1971: https://dx.doi.org/10.1136/jnnp.34.4.369 suppl. 1: tissue workup incl. notes, macroand microphotographs by j.a.n. corsellis suppl. 2: clinical notes on the patient’s surgery: presurgical investigations, intraoperative electrocorticography, early postsurgery eeg and summary suppl. 3: documentation relating to corsellis’ inquiries for clinical data and early follow up suppl. 4: correspondence with prof. orthner, göttingen, germany suppl. 5: documents illustrating the patient’s long term follow up suppl. 6: medical history of the patient´s mother suppl. 7: a personal letter from david c. taylor the notes and records presented here accompanied the preserved patient’s material, as collected, stored and delivered by the corsellis collection. the full original material (photos & notes) were digitized in high quality to jpg-format at erlangen university. information enabling for identification of the patient or her mother was eliminated by image editing using acdsee pro7 software (bsk). acknowledgement this contribution, 50 years after the original publication in jnnp, is dedicated to the memory of professor david c. taylor (1933-2021) [19] and professor j.a.n. corsellis (1915-1994) [17,18,3], two great men of neuroscience, who were the right people at the right place in the right time back then. thanks to dr. thomas zobel from the wwu imaging network for support and hosting the omero image server. thanks to christian thomas, münster, for producing the virtual slides. thanks to frank bittner, erlangen, for excellent digitizing service and to thomas sebille (fotomas) for help with image editing. references 1. taylor dc, falconer ma, bruton cj, corsellis jan. focal dysplasia of the cerebral cortex in epilepsy. journal of neurology, neurosurgery and psychiatry 1971; 34: 369-387. 2. blümcke i, thom m, aronica e, armstrong dd, vinters hv, palmini a, jacques ts, avanzini g, barkovich aj, battaglia g, becker a, cepeda c, cendes f, colombo n, crino p, cross jh, delalande o, dubeau f, duncan j, guerrini r, kahane p, mathern g, najm i, ozkara c, raybaud c, represa a, roper sn, salamon n, schulze-bonhage a, tassi l, vezzani a, spreafico r. the clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc task force of the ilae diagnostic methods commission. epilepsia 2011; 52: 158-174. 3. kasper bs, taylor dc, janz d, kasper em, maier m, williams mr, crow tj. neuropathology of epilepsy and psychosis: the contributions of j.a.n. corsellis. brain. 2010;133: 3795-3805. 4. kasper bs, rössler k, hamer hm, dörfler a, blümcke i, coras r, roesch j, mennecke a, wellmer j, sommer b, lorber b, lang jd, graf w, stefan h, schwab s, buchfelder m, rampp s. coregistrating magnetic source and magnetic resonance imaging for epilepsy surgery in focal cortical dysplasia. neuroimage clin. 2018;19: 487-496. 5. yakovlev pi. congenital morphologic abnormalities of the brain in a case of abortive tuberous sclerosis. arch neurol psychiat (chic) 1939; 41: 119-139. 6. crome l. infantile cerebral gliosis with giant nerve cells. journal of neurology, neurosurgery and psychiatry 1957; 20: 117-124. 7. cravioto h, feigin i. localized vertebral gliosis with giant neurons histologically resembling tuberous sclerosis. j neuropathol exp neurol 1960; 19: 572-579. 8. roncoroni l. la fine morfologia del cervello degli epilettici e dei delinquenti. arch psich scienze penali antropol criminale 1896; 17: 92-116. 9. corsellis jan. “cryptic tubers” as a cause of focal epilepsy. proceedings of the society of british neurosurgeons (abstract) journal of neurology, neurosurgery and psychiatry 1971; 34: 104-105. 10. d'gama am, woodworth mb, hossain aa, bizzotto s, hatem ne, lacoursiere cm, najm i, ying z, yang e, barkovich aj, kwiatkowski dj, vinters hv, madsen jr, mathern gw, blümcke i, poduri a, walsh ca.somatic mutations activating the mtor pathway in dorsal telencephalic progenitors cause a continuum of cortical dysplasias. cell rep. 2017;21: 3754-3766. 11. baldassari s, ribierre t, marsan e, adle-biassette h, ferrand-sorbets s, bulteau c, dorison n, fohlen m, polivka m, weckhuysen s, dorfmüller g, chipaux m, baulac s. dissecting the genetic basis of focal cortical dysplasia: a large cohort study. acta neuropathol. 2019;138(6):885-900. 12. https://apps.webofknowledge.com (searched aug 13 2020). 13. corsellis jan, bruton cj, taylor dc, falconer ma. lokalisierte dysplasie der hirnrinde auf grund von gewebeuntersuchungen, die durch neurochirurgische eingriffe bei epileptikern gewonnen wurden. psychiatr. neurl. med. psychol (leipz) 1973; 17-18: 43-53. 14. janota i, polkey ce. cortical dysplasia in epilepsy a study on material from surgical resections for intractable epilepsy. recent advances in epilepsy 1992; 5: 37-49. 15. schoefert, anna kathryn. research on the human brain post mortem in britain, c.1950-1980: constituting the corsellis collection. m phil thesis, cambridge university, 2011. 16. metters js. isaacs report. tso, may 2003; page 235-239 see: http://image.guardian.co.uk/sys-files/society/documents/2003/05/12/isaacs_report.pdf (last looked up 16.9.2020). 17. janota i. obituary j.a.n corsellis (1915-1994). neuropathology appl neurobiol 1995; 21: 168. 18. crow tj. obituary j.a.n corsellis (1915-1994). psychiatric bulletin 1996; 20: 508-509. 19. kasper bs. in memoriam: david c. taylor. epilepsy behav 2021 apr 15; 118:107960 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. from neurology to neuropathology and back feel free to add comments by clicking these icons on the sidebar free neuropathology 2:10 (2021) reflections from neurology to neuropathology and back arnulf h. koeppen research service (151), va medical center, albany, ny, usa corresponding author: arnulf h. koeppen · research service (151) · va medical center · 113 holland avenue · albany, ny 12208 · usa arnulf.koeppen@va.gov submitted: 13 april 2021 accepted: 17 april 2021 copyedited by: deanna c. fang published: 21 april 2021 https://doi.org/10.17879/freeneuropathology-2021-3333 keywords: neuropathology, neurology, reflections, autobiography chemistry or medicine 1958-1963? in the fifties, high school students in germany had to select their career goals without much guidance or college education. i was a student at a gymnasium of mathematics and natural sciences in osterode, a provincial town near the harz mountains in lower saxony of germany. my father, a general practitioner in a small town in the harz, had different ideas. he wanted me to be a medical student and ultimately take over his practice. the university town, göttingen, was located 50 km from our home. göttingen was then a small town, but medical education was excellent and diverse. i stayed in a room near the max planck institute where otto hahn, the nobel laureate in nuclear fission, was still active. my inaugural dissertation involved measurements of nuclear sizes in rat liver, adrenals, and endometrium after the animals were treated with drugs affecting the autonomic nervous system (koeppen, 1963). my interest in the neurosciences began during my last year of medical school. the inaugural dissertation received the annual faculty award in 1963; and the sum of 800 deutschmarks was sufficient to defray my cost of a trip by ocean liner to the united states where a non-profit organization, the ventnor foundation, had offered me a position as a medical assistant (intern) at a new jersey hospital. after an internship of 18 months, i joined the neurology training program at montefiore hospital and medical center in new york city in 1965. neurology education was disappointing, but i met the late drs. harry zimmerman, robert terry, and asao hirano who stimulated my interest in neuropathology and, above all, clinicoanatomic correlation in neurology and neurosurgery. after one year at montefiore, i was recruited to the department of neurology and psychiatry of northwestern university school of medicine in chicago. my first assignment was to the veterans affairs (va) medical center in hines, illinois, where the late dr. kevin d. barron was the chief neurologist. dr. barron had been trained by drs. zimmerman and terry and continued to practice neuropathology at northwestern and the va medical center. dr. barron was a master of clinicoanatomic correlation and stressed that neurologists should have training in neuropathology. his training had a major impact on my further career as a neurologist and neuropathologist. while opposition to neurologists and psychiatrists practicing neuropathology was evolving, we were reminded of non-pathologists who had made major contributions to the practice of neuropathology. we thought of alois alzheimer and his 1906 illustrations of neurofibrillary tangles, and of king engel who gave us modified trichrome in the diagnosis of mitochondrial myopathy. my training in neuropathology from 1969 to 1971 was enough to gain acceptance to the neuropathology board examination in 1980, and, since that time, i was convinced that i was a better neurologist because i was also a neuropathologist. during my time in chicago, dr. barron insisted that i should learn neurochemistry as well, and i was assigned to the research laboratory of the late dr. joseph bernsohn. over a one-year period, i learnt standard and advanced laboratory techniques, and my initial interest in chemistry came to belated fruition. since joining the department of neurology at albany medical college in 1969, i have maintained a neurochemical and neuropathological laboratory at the albany va medical center (figure 1) while also running the clinical neurology service. among my valued neurochemical discoveries was a sensitive assay of brain malonyl-coenzyme a, a critical molecule in the biosynthesis of fatty acids (mitzen and koeppen, 1984). the autopsy practice generated access to specimens from unusual cases, such as pelizaeus-merzbacher disease (pmd). the neurochemical laboratory helped me identify the cause of the disease, namely, a deficiency of proteolipid protein (plp) (koeppen, et al., 1987). the mutation in this patient with pmd was a point mutation (hudson, et al., 1989) though later, the mutation in most cases was identified as a duplication of the plp gene. the plp protein is unusual because it is soluble in a mixture of chloroform and methanol (folch and lees, 1951). through the work on pmd, i met the co-discoverers of plp, the late dr. marjorie b. lees (figure 2), and the late prof. franz seitelberger who had advanced the existence of a “connatal” form of pmd. prof. seitelberger invited me to the neurological institute in vienna where i gave a lecture on plp and pmd. profs. kurt jellinger and herbert budka were in audience. i also discovered that the late neuropathologist, dr. wolfgang zeman (figure 3), had written to dr. lees in 1963, proposing that plp was a likely candidate in the genetic mutation underlying the disease. pelizaeus-merzbacher disease is an x-linked disorder, and pelizaeus, a german balneologist, understood the mechanism of x-linked transmission before the existence of an x-chromosome was known. pelizaeus (1885) stated succinctly that the “disease goes through the mother but does not do anything to her”. it is peculiar that at the time of my work on pmd, a spontaneous x-linked central nervous system myelin deficiency in rats was discovered in albany, ny. the animal model was called "myelin-deficient (md) rat", and the neuropathologic and genetic similarity to pelizaeus-merzbacher disease was remarkable (koeppen et al, 1988). pmd patients also show a lack of other myelin proteins, and the injection of a white matter homogenate into guinea pigs (type 13) did not cause experimental allergic encephalomyelitis (eae). antibodies to plp were not commercially available, and i isolated the protein from frozen normal human white matter. injection into rabbits caused encephalitis that closely resembled eae. one rabbit with advanced encephalitis generated a high-titer anti-plp antibody that worked very well on paraffin sections of human central nervous tissue. the antibody became very popular, and i forwarded samples to several foreign and domestic myelin investigators. figure 1. laboratory staff in albany, ny. from left to right: alyssa sossei, research assistant; sarah bjork, medical student; r. liane ramirez, research assistant; the author; pamela kruger, post-doctoral scientist. a new goal: the hereditary ataxias in 1972, a patient with autosomal dominant ataxia presented with an unusual disturbance of ocular motility that became known as supranuclear pseudoophthalmoplegia. many years later, the mutation was identified as a cytosine-adenine-guanine (cag) trinucleotide repeat expansion on chromosome 12, and the disease was named spinocerebellar ataxia type 2 (sca-2). the mutation was like the cag trinucleotide expansions in sca-1 and sca-3 (machado-joseph disease). sca-6, sca-7, and sca-17 were later also identified as the consequence of cag trinucleotide repeat expansions. the expansions are expressed by changing the length of polyglutamine in the cognate proteins, hence, became known as polyglutamine (or polyq) diseases. antibodies raised against the polyq stretch of the normal tata-binding protein, designated “1c2”, became useful in the visualization of intranuclear inclusion bodies in the ataxic polyq diseases and huntington's chorea. stimulated by the experience with a single patient with supranuclear pseudoophthalmoplegia, i traveled to scotland, his ancestral home, to search for other members of this family. over a period of 40 days, i could not find any but discovered unrelated patients with an identical disturbance of eye movements. at that time, commercial genetic testing for sca was not yet fully available, and the lack of saccades in some patients with ataxia became a "genetic marker" of the disease. the discovery of supranuclear pseudoophthalmoplegia remains important but due to its variability can no longer be called a genetic marker. the scottish experience (koeppen, et al, 1977) prompted me to apply to the alexander-von-humboldt foundation for a research fellowship. the foundation agreed, and i moved to the university of hamburg, germany, where i found a warm welcome in the department of human genetics and the neuropathological institute of the eppendorf university hospital. the plan was to backtrack from autopsy records to surviving family members. the director of eppendorf neuropathology was prof. hans-joachim colmant, a psychiatrist. he made available a huge archive of neuropathological specimens in the basement of the hospital that had survived the bombings of world war ii. the chief psychiatrist during the war, prof. hans bürger-prinz, decided that the collection of slides, reaching back to 1891, should be protected against the allied bombings and moved the collection to a secure location. beyond specimens of ataxia, the collection also contained invaluable other cases, including jakob's original 1920 specimens of prion disease. it was an exciting experience to study these slides and be allowed to make photographs. german neuropathologists favored cresyl violet as their routine stain, and it was apparent that this stain does not properly show the sponginess that is readily apparent on hematoxylin-and-eosin. my stay in hamburg was intermittent, but i benefitted from the collection at the eppendorf hospital over an aggregate period of 2 years. on return to albany, ny, i began to collect autopsy specimens of hereditary ataxia in a tissue repository and, to this day, make available fixed and frozen samples to other ataxia investigators. figure 2. marjorie b. lees another change in direction: from dominant to recessive ataxia, 1996-2021 after dr. massimo pandolfo and his group discovered the mutation in friedreich ataxia (fa) in 1996, my interest changed, and my research now focuses on this autosomal recessive disease. though fa is a monogenic disease, the pathology offers a fascinating array of lesions in multiple organs: brain, spinal cord, dorsal root ganglia, sensory peripheral nerves, eyes, heart, and the insulin-producing beta cells of the pancreas. many clinicians and fa researchers consider fa a "neurodegenerative" disease, implying the existence of onset, progression, and death. the term neurodegenerative also means atrophy, and clinical onset is thought to be related to a degree of atrophy that translates to nervous system dysfunction. neuropathologists, however, will agree that fa is also a hypoplastic disease as far as the spinal cord and dorsal root ganglia are concerned. sectioning of the spinal cord longitudinally to represent dorsal root entry zones as obersteiner and redlich did in 1894 in vienna invariably showed loss of demarcation between the peripheral nervous tissue of dorsal roots and the central nervous tissue of the spinal cord (koeppen, et al, 2017). a surprising observation in fa was the protrusion of glial tissue from the spinal cord into dorsal roots, in support of dysfunction of a local barrier called the boundary cap. this work received the 2018 stout award from the united states and canadian academy of pathology for “resolving a scientific medical problem by studying its anatomic features”. this award is my most prized recognition. figure 3. wolfgang zeman the mystery of brain iron in 1967, i participated in the examination of a brain that was incrusted by hemosiderin. the diagnosis, superficial siderosis, was not difficult, but the disorder was thought to be rare, and magnetic resonance imaging with its extraordinary ability to detect brain iron, was still to come. dr. kevin d. barron and i studied the specimen in detail, and i entered a 10-year period of research in normal and pathological brain iron (koeppen et al, 1992-2002). this iron work correlated well with my later studies in fa because other researchers thought that this disease was due to iron accumulation in mitochondria of all affected organs. america, 1964-2021 my fifty-seven years in the united states constitute two thirds of my life. my early youth was troubled by the experience of world war ii, america has been good to me. i waved goodbye to europe when the maasdam, a passenger liner of the holland-america line put out to sea in late january 1964, and i greeted the statue of liberty 10 days later. i was never homesick, but occasionally think of my time as a refugee at the end of world war ii. in january 1945, my mother packed some essentials and took me, two brothers, and a sister by bus to the west, leaving the then german province of silesia. at that time, our father was a prisoner of war in an american camp near naples, italy, and did not return to germany until 1947. things did not stabilize until 1949 when the family walked across the border between the newly established communist german democratic republic and the american zone of west germany. food, drink, and clothing were scarce, and we spent one year in a displaced-persons camp. the privations ended in 1950 when the german economy improved with the outstanding help of the marshall plan. after i became more established in the united states, i raised a family and met many good people. i want to thank my teachers of neurology and neuropathology, among whom my friend and colleague for 47 years, the late dr. kevin d. barron, was the most important (figure 4). the united states continues to fascinate me, and, during my chicago years, the sky of the midwest seemed higher and bluer. i did not forget my european origin or my years of medical education in germany. i am still trilingual (english, german, and spanish) and enjoy the occasional opportunity to practice three languages. i also became an amateur radio operator (a “ham” in american lingo), and my last conversation with my late father was by ham radio. while on sabbatical in hamburg, i received the call sign of dj0rf wherein the “dj0” was reserved for foreign nationals who had an amateur radio license in their home countries. ham radio will be my main pursuit after i finally retire from research or the practice of neurology and neuropathology. figure 4. kevin d. barron and former trainees. from left to right; suhandsu chokroverty, mbbs; the late kevin d. barron, md, the late cesar mayo, md; ahk; and the late artemio ordinario, md. the group photo was taken during the combined 1967 meeting of the american association of neuropathologist and the american neurological association in atlantic city, new jersey. a final word: do we teach enough clinicoanatomic correlation? we hear a lot from proponents of “evidence-based” medicine but rarely about the art of medicine. clinicoanatomic correlation benefits from both, and thinking of brain structure while considering a differential diagnosis is a rewarding exercise. senior physicians will agree that anatomy and pathology are the basis of medicine. telehealth has arrived and is perhaps not in line with the art of medicine. its supporters may argue that it is more artful than face-to-face and hands-on medicine. whatever we prefer as clinicians, we should strive to emphasize clinicoanatomic correlation in our teaching and convey the joy of a successful diagnosis based on traditional knowledge of anatomy. such thought should, of course, precede the ordering of advanced imaging. in a recent survey of neurology training programs in the united states, 86% of program directors considered a rotation through neuropathology essential (rayi, et al, 2020). let me add my voice to this goal. references folch j, lees m. proteolipides, a new type of tissue lipoproteins. their isolation from brain. j biol chem 1951; 191 807-17 hudson l.d., puckett c., berndt .j, chan j., gencic s. mutation of the proteolipid protein (plp) gene in human x-linked myelin disorder. proc natl acad sci 1989; 86: 8128-31 koeppen a.h.: (transl.) the influence of pharmacological agents of the autonomic nervous system on the cellular nuclei of the liver, uterus, and adrenal cortex measured by karyometric methods). inaugural dissertation of university of göttingen medical school, 1963 koeppen a.h., hans m.b., shepherd d.i., best p.v.: adult-onset hereditary ataxia in scotland. arch neurol 1977; 34: 611-18 koeppen a.h., ronca n.a., greenfield e.a., hans m.b.: defective biosynthesis of proteolipid protein in pelizaeus-merzbacher disease. ann neurol 1987; 21: 159-70 koeppen a.h., barron k.d., csiza c.k., greenfield e.a.: comparative immunocytochemistry of pelizaeus-merzbacher disease, the jimpy mouse, and the myelin-deficient rat. j neurol sci 1988; 84: 315-27 koeppen a.h., borke r.c.: experimental superficial siderosis of the central nervous system. i. morphological observations, j neuropathol exp neurol.1991; 50: 579-9 koeppen a.h., hurwitz c.g., dearborn r.e., dickson a.c., borke r.c., chu r.c.: experimental superficial siderosis of the central nervous system: biochemical correlates. j neurol sci 1992; 112: 38-45 koeppen a.h., dickson a.c., chu r.c., thach r.e.: the pathogenesis of superficial siderosis of the central nervous system. ann neurol1993; 34: 646-53 koeppen a.h.: the history of iron in the brain. j neurol sci 1995; 134 (suppl.): 1-9 koeppen a.h.: a brief history of brain iron research. j neurol sci 2002; 207: 95-7 koeppen a.h., becker a.b., qian j., gelman b.b., mazurkiewicz j.e.: friedreich ataxia: developmental failure of the dorsal root entry zone. j neuropathol exp neurol 2017; 76 (11): 969-77 mitzen e.j., koeppen a.h.: malonate, malonyl-coenzyme a and acetyl-coenzyme a in developing rat brain. j neurochem 1984; 43: 499-506 obersteiner h., redlich e. (transl): on the nature and pathogenesis of the tabetic dorsal column degeneration. arbeit neurol inst wien univ 1894; 1-3: 158-72 pelizaeus f.: (transl) on a peculiar form of spastic paralysis with brain symptoms on hereditary basis (multiple sclerosis). arch psychiat nervenkrankh 1885; 16: 698-710 rayi a. et al.: how are residents trained in neuropathology? a survey of neurology program directors in the united states. j neuropathol exp neureol 2020; 79:1218-22 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuronal intermediate filament inclusion disease may be incorrectly classified as a subtype of ftld-fus feel free to add comments by clicking these icons on the sidebar free neuropathology 1:9 (2020) original paper neuronal intermediate filament inclusion disease may be incorrectly classified as a subtype of ftld-fus kevin f. bieniek1, keith a. josephs2, wen-lang lin3, dennis w. dickson3 1 department of pathology & laboratory medicine, university of texas health science center, san antonio, tx, usa 2 department of neurology (behavioral neurology & movement disorders) mayo clinic, rochester, mn, usa 3 department of neuroscience (neuropathology), mayo clinic, jacksonville, fl, usa corresponding author: keith a josephs, md, mst, msc · mayo clinic · 200 1st street s.w. · rochester, mn, 55902 · usa · tel: +1-507-538-1038 · fax: +1-507-538-6012 josephs.keith@mayo.edu submitted: 02 february 2020 accepted: 05 march 2020 copyedited by: aivi t. nguyen published: 11 march 2020 https://doi.org/10.17879/freeneuropathology-2020-2639 keywords: fus, tdp-43, atypical frontotemporal lobar degeneration, nifid, electron microscope abstract background: the majority of cases of frontotemporal lobar degeneration (ftld) are characterized by focal cortical atrophy with an underlying tau or tdp-43 proteinopathy. a subset of ftld cases, however, lack tau and tdp-43 immunoreactivity, but have neuronal inclusions positive for ubiquitin, referred to as atypical ftld (aftld-u). studies have demonstrated that ubiquitin-positive inclusions in aftld-u are immunoreactive for fused in sarcoma (fus). as such, the current nosology for this entity is ftld-fus, which is thought to include not only aftld-u but also neuronal intermediate filament inclusion disease (nifid) and basophilic inclusion body disease. objective: to compare pathological features of cases of aftld-u and nifid. methods: we reviewed the neuropathology of 15 patients (10 males and 5 females; average age at death 54 years (range 41-69 years)) with an antemortem clinical diagnosis of a frontotemporal dementia and pathological diagnosis of aftld-u (n=8) or nifid (n=7). sections were processed for immunohistochemistry and immunoelectron microscopy with fus, tdp-43, and α-internexin (αinx) antibodies. results: eight cases had pathologic features consistent with ftld-fus, with severe striatal atrophy (7/8 cases), as well as fus-positive neuronal cytoplasmic and vermiform intranuclear inclusions, but no αinx immunoreactivity. five cases had features consistent with nifid, with neuronal inclusions positive for both fus and αinx. striatal atrophy was present in only two of the nifid cases. two cases had αinx-positive neuronal inclusions consistent with nifid, but both lacked striatal atrophy and fus immunoreactivity. surprisingly, one of these two nifid cases had lesions immunoreactive for tdp-43. discussion: while fus pathology remains a prominent feature of aftld-u, there is pathologic heterogeneity, including rare cases of nifid with tdp-43rather than fus-positive inclusions. introduction the pathological term frontotemporal lobar degeneration (ftld) assumes the presence of focal frontal and anterior temporal lobar atrophy(1). histologically, the majority of ftlds are pathologically classified into two broad categories of tau-positive ftld (ftld-tau) and tdp-43-positive ftld (ftld-tdp) based on the presence of tau and tdp-43 immunoreactive inclusions, respectively(2, 3). a subset of ftld cases however, lack tau and tdp-43 immunoreactivity, and instead have neuronal inclusions that are immunoreactive to ubiquitin(4). such cases have been referred to in the literature as atypical ftld with ubiquitin inclusion (aftld-u)(5). in the past decade, immunohistochemical studies have revealed that the ubiquitinated protein in cases of aftld-u is the fused in sarcoma (fus) protein(6). interestingly, there are two other relatively rare ftld pathological variants that also have neuronal inclusions that are immunoreactive to fus(7, 8). these include the entity neuronal intermediate filament inclusion disease (nifid)(9), previously known as neurofilament inclusion body disease(10), and basophilic inclusion body disease(11) that have also been referred to as the generalized variant of pick’s disease(12). as a result, aftld-u, nifid and basophilic inclusion body disease are all currently classified as subtypes of ftld-fus(3, 13). in keeping with pathologically lumping these three entities as ftld-fus, is the fact that all three subtypes of ftld-fus, particularly aftld-u and nifid, are strongly associated with a clinical presentation of the behavioral variant of frontotemporal dementia (bvftd)(13-15). basophilic inclusion body disease is more strongly associated with the juvenile form of amyotrophic lateral sclerosis(16). in addition, aftld-u and nifid have both been found to be associated with striatal atrophy on mri(17, 18). hence, aftld-u and nifid have a lot of features in common. one study has directly compared aftld-u and nifid(19). hence, little is known about which clinical and pathologically differences between the two variants may further help to distinguish them, and whether all cases of aftld-u and nifid do indeed show fus immunoreactivity. in this study, we set out to address these two unknowns in a cohort of 15 ftld cases that including aftld (n=8) and nifid (n=7). materials and methods subject selection the neuropathological databases at the mayo clinic, jacksonville, florida were queried to identify all cases of ftld that had been given a pathological diagnosis of nifid or aftld-u. a total of 15 cases were identified. all 15 cases were evaluated by a single expert neuropathologist (dwd). clinical data the medical records of all 15 cases were reviewed by one clinician with expertise in neurodegenerative diseases (kaj) to abstract demographic and clinical information. data abstracted included sex, age at onset, prominent symptoms during the disease course, family history of any neurodegenerative diseases and final clinical diagnosis prior to death. pathological methods all 15 cases underwent histologic and ultrastructural evaluation. tissue sections were stained with hematoxylin and eosin, luxol fast blue-periodic acid schiff (lfb-pas) and bielschowsky silver stains. immunohistochemical staining was performed using standard methods. the deparaffinized and rehydrated sections were steamed in distilled water for 30 min and immunostained in batches to assure consistency with a dako autostainer (dako, carpinteria, ca) using 3, 3’diaminobenzidine as the chromogen. after immunostaining, the sections were lightly counterstained with hematoxylin. the following antibodies were used: phosphorylated neurofilament (smi-31, 1:20,000; covance, berkeley, ca); ubiquitin (mouse monoclonal ubi-1, 1:40,000; encor biotechnology, alachua, fl; rabbit polyclonal ubq(20), 1:500 and rabbit polyclonal uh-19(21), 1:2,500); phospho-tau (cp13, 1:100; peter davies, albert einstein college of medicine, bronx, ny); alpha-synuclein (nacp(22), 1:3,000), alpha-internexin (1:100; encor biotechnology, alachua, fl); tdp-43 (mc2085, dr. petrucelli, 1:1500), ptdp-43 (s409/410, cosmo bio co., 1:5000) and rabbit polyclonal anti-fus (1:500; hpa008784; sigma, st. louis. mo and bethyl lab; a300-302a; montgomery, tx). the sigma antibody gave consistent and better staining and was used throughout the study. the presence or absence of motor neuron disease was assessed and defined as previously described, including stains for activated microglia(23). electron microscopy small pieces of formalin-fixed brains were immersed in 2.5% glutaraldehyde-0.1 m cacodylate buffer overnight at 4°c. after washing in buffer, they were post-fixed in aqueous 2% osmium tetroxide for 1 hr, washed and fixed in 1% uranyl acetate-50% ethanol for 30 min, followed by dehydration in 70%, 80%, 95%, 100% ethanols and propylene oxide. they were infiltrated and embedded in epon 812. thin sections were stained with uranyl acetate and lead citrate and examined in a philips 208s electron microscope fitted with a bottom-mount ccd camera (orius 831, gatan, pleasanton, ca). immunoelectron microscopy small pieces of formalin-fixed brains were dehydrated in serial washes of 30%, 50%, 70%, 90% ethanol for 10 min each, infiltrated and embedded in lr white resin. they were polymerized in a vacuum oven at 50°c for 2 days. thin sections were collected on formvar-coated nickel grids. grids were floated with section-sides down on citrate buffer, ph 6.0, in a 100°c oven for 10 min, cooled to room temperature for 15 min followed by immunogold labeling. the sigma anti-fus was used at 1:20 in pbs. results demographics and clinical data for all 15 cases are shown in table 1. there were 10 males and 5 females with median age at death of 54 years (range 41-69 years). the median disease duration was 5 years (range 3-13 years). the most common final clinical diagnosis in this series was behavioral variant of frontotemporal dementia (bvftd)(4, 24), rendered in 10 (67%) cases. for the other 5 cases, the final clinical diagnoses were corticobasal syndrome in two cases(25), and one each diagnosed with progressive supranuclear palsy(26), primary lateral sclerosis(27) and multiple system atrophy-parkinsonian type (msa-p)(28). table 1: demographic and clinical features of all 15 cases als = amyotrophic lateral sclerosis; bvftd = behavioral variant frontotemporal dementia; cbs = corticobasal syndrome ocb = obsessive compulsive behavior; psp = progressive supranuclear palsy, snd = striatonigral degeneration † previously published (josephs et al. acta neuropathol 2008; 116: 159-167) ‡ previously published (josephs et al. brain 2003; 126: 2291-2303 * previously published (josephs et al. acta neuropathol 2005; 109: 427-32) the median age of onset of the nifid cases was 52 years old (range: 41-61 years) while for aftld-u it was 54 years old (range: 42-69 years). disease duration in nifid was only 3.5 years (range: 2.0-5.0 years) and was much shorter than the median disease duration of the aftld-u group which was 10 years (range: 4-13 years) (p<0.05). the clinical diagnoses were heterogeneous in the nifid cases, with three cases (43%) diagnosed as bvftd. on the other hand, of the aftld-u cases all but one (88%) had been diagnosed with bvftd. the nifid cases were more likely to have had pyramidal tract signs and motor dysfunction compared to the aftld-u cases; myoclonic jerks and excessive startle were also observed in nifid but not aftld-u. pathological findings gross examination all 15 cases had evidence of frontal and temporal lobe atrophy, and hence all met criteria for ftld. striatal atrophy was observed in nine of the 15 cases (table 2, figure 1). table 2: nci immunohistochemical profile in 15 aftld-u and nifid cases β diffuse cytoplasmic fus staining with mini-pick body-like nci na = not able to find any neuronal intranuclear inclusions (nii) figure 1: striatal atrophy on gross examination striatal atrophy was not seen in many nifid cases (a; case 5) and was only observed in two cases (b; case 6). conversely, only one aftld-u case lacked marked striatal atrophy (c; case 14) while the rest of the aftld-u cases demonstrated severe atrophy (d; case 13) [bar: 1 cm]. light microscopy results of the light microscopic examination are shown in table 2 and figure 2. none of the 15 cases had evidence of tau deposition, and none met criteria for alzheimer’s or lewy body disease. all 15 cases were immune-reactive to ubiquitin. thirteen of the 15 cases had neuronal inclusions that were immune-reactive to fus. seven of the 15 cases had eosinophilic inclusions that were seen on hematoxylin and eosin and were immunostained for α-internexin in keeping with their pathological diagnosis of nifid. none of the remaining eight cases that had been diagnosed as aftld-u showed α-internexin immunoreactivity. seven of the eight aftld-u cases (88%) showed severe striatal atrophy with fus-positive neuronal cytoplasmic and vermiform intranuclear inclusions. of the seven nifid cases, five cases had neuronal inclusions that were immunoreactive to fus. unlike in aftld-u, striatal atrophy was only present in only 2/7 (29%) of the nifid cases, both of which were fus positive. hence, neither of the two fus negative nifid cases had striatal atrophy. therefore, 9/13 cases with fus immunoreactive inclusions showed striatal atrophy while 0/2 without fus showed striatal atrophy. surprisingly, one of the two nifid cases without fus immunoreactivity had lesions that were immunoreactive for both α-internexin and tdp-43 (figure 3). figure 2: alpha-internexin and fus pathology hematoxylin and eosin (a/e/i/m), α-internexin (b/f/j/n) and fus (c/g/k/o) in the frontal cortex and fus (d/h/l/p) in the hippocampus of cases 4 (a-d), 5 (e-h), 7 (i-l), and 15 (m-p) [bar:100 μm] figure 3: fus negative tdp-43 positive nifid case neuronal eosinophilic (a/b) inclusions in the hippocampus (a/c/e/g) and frontal cortex (b/d/f/h) of case 3 are negative for fus (c/d) but positive for tdp-43 (e/f/h), as well as αinx (g/h). α-internexin (brown) and tdp-43 (blue) colocalizes on double-labeling immunohistochemistry (h)[bar:100 μm] electron and immunoelectron microscopy results of the electron microscopic examination are shown in figure 4. in the fus-positive nifid cases, we found that fus was localized to loose granulofilaments that were in close proximity to intermediate filament inclusions that contained tightly packed uncoated filaments unlabeled by fus antibody. the two types of filaments did not mix. the compact intermediate filament inclusions were similar to those we previously reported(29). in the tdp-positive nifid case (#3), the tdp-positive ncis were composed of granulofilaments in tightly packed bundles or loose orientations. cytoplasmic organelles, e.g. mitochondria, were occasionally encompassed by these inclusions. figure 4: electron microscopy variable α-internexin (a/d/g), fus (b/e/h), and tdp-43 (c/f/i) immunoreactivity on electron microscopy in the neuronal inclusions of cases 3 (a-c), 2 (d-f), and 9 (g-i)[bar: 2 μm main; 0.3 μm inset] these granulofilaments were not labeled with fus. the characteristic compact intermediate filament inclusions were not as widespread as the tdp-positive inclusions in this case. importantly, they were located in separate neurons. in addition to their ultrastructural difference from the tdp-positive inclusions, these intermediate filament inclusions were not labeled by tdp-43. immunohistochemistry showed that the intermediate filament inclusions were immuno-negative for fus. the tdp-negative, fus-negative nifid case (#4) had compact intermediate filament inclusions similar to those described above. these compact intermediate filament inclusions were immuno-negative to fus and tdp-43. neuronal cytoplasmic inclusions in all the aftld-u cases consisted of granulofilaments inclusions in loose arrangement and all were immuno-positive for fus. discussion in this study we found pathological evidence for aftld-u to be a homogeneous entity that is strongly associated with a clinical diagnosis of bvftd, and pathologically by fus immunoreactivity and striatal atrophy. on the other hand, we found nifid to be more heterogeneous with more variable clinical presentations. furthermore, nifid does not always appear to be associated with fus immunoreactivity and is typically not associated with striatal atrophy. interestingly, we found evidence of overlap between a case that would meet criteria for ftld-tdp as well as nifid. in this study, aftld-u was a very homogeneous entity and the evidence supports aftld-u being classified as ftld-fus. from a clinical standpoint aftld-u is strongly associated with clinical features of bvftd as previously reported(14, 30) and hence should be considered in patients presenting with bvftd especially in the presence of striatal atrophy. indeed striatal atrophy has been reported in aftld-u on antemortem mri imaging(17). nifid, on the other hand, as currently defined does not appear to be as distinct an entity clinically and pathologically as aftld-u. supportive of this statement is the fact that of the seven nifid cases in this study, patients were given five different clinical diagnoses at the last evaluation prior to death. secondly, striatal atrophy, although present in two nifid case was absent in the rest. interestingly, both nifid cases with striatal atrophy showed fus immunoreactivity, one with a clinical diagnosis of bvftd. it therefore appears that striatal atrophy is predictive of fus, but fus is not necessarily predictive of striatal atrophy. regardless, nifid should also be considered in the differential diagnosis of bvftd with striatal atrophy and should suggest the presence of ftld-fus. the one feature that may be helpful in predicting ftld-fus nifid from ftld-fus aftld-u in patients with bvftd and striatal atrophy may be the rapidity of progression, with faster progression being more suggestive of ftld-fus nifid. to understand what may be happening with nifid it is worth further discussion. neuronal intermediate filament inclusion body disease is a type of ftld with previously reported clinical presenting features of bvftd, corticobasal syndrome, and motor neuron disease, especially the primary lateral sclerosis variant(10, 31, 32). typically, patients with nifid have a relatively rapidly progressive course, becoming mute and unable to ambulate, dying around 3 ½ years after onset(10). these features were observed in this cohort of seven nifid cases. from a pathological standpoint, in nifid, neuronal cytoplasmic inclusions (ncis) are easily visible on hematoxylin and eosin, show variable staining to ubiquitin and silver stains but are strikingly immunoreactive to type iv intermediate filaments, including neurofilament and α-internexin(9, 10, 33, 34). these nci, however, are not morphologically homogeneous and it has been known from the original description of nifid(10) that nci appeared to separate into two types: rounded inclusions that are similar to pick bodies, hence called ‘pick-body like (pbl) inclusions,’ and smaller more compact inclusions with a glass like appearance called ‘compact hyaline inclusions’(7, 10, 19, 29). could this nci inclusion type difference be playing any role in the heterogeneity we observed in this study? the ultrastructual analysis in our seven nifid cases shows that these two different types of nci also have different ultrastructual appearances. the pbl inclusions were ultrastructually granulofilamentous while the compact hyaline inclusions had a more tightly compact appearance. with immunoelectron microscopy we found that the granulofilamentous inclusions were immunoreactive to fus while the compact hyaline inclusions were immunoreactive to intermediate filament. interestingly all seven nifid cases had compact hyaline inclusions, and all seven showed α-internexin immunoreactivity. however, unlike in previous reports(35, 36), two of our seven nifid cases did not show fus immunoreactivity and one of these two nifid cases showed predominantly, almost exclusively, compact hyaline inclusions (case #4). this nifid case was immuno-negative to fus, suggesting that the absence of the granulofilamentous inclusions may be the explanation for the absence of fus immunoreactivity. the other fus negative case (case #3) is also unique. in this case, both granulofilamentous inclusions and compact hyaline inclusions were present. as expected, the presence of the compact hyaline inclusions was associated with α-internexin immunoreactivity. surprisingly though, the granulofilamentous inclusions in this case (case #3) showed immunoreactivity to tdp-43 but not fus. it is therefore possible that this is a case of ftld-tdp in which compact hyaline inclusions happen to also be present and hence accounts for the α-internexin immunoreactivity observed. this would not be counterintuitive since granulofilamentous morphology is the typical appearance of the ncis in ftld-tdp(37). on the other hand, we cannot exclude the possibility that this is a case of nifid with tdp-43 immunoreactivity given that tdp-43 immunoreactivity has been described in many different diseases(38). compared to our nifid cases, all eight aftld-u cases showed a homogeneous pattern of fus immunoreactivity similar to what has been previously reported(6, 8, 19, 36). in our eight aftld-u cases, ultrastructural analysis demonstrated granulofilamentous inclusions as we previously reported(4), and immunoelectron microscopy revealed fus immunoreactivity. of note is the fact that compact hyaline inclusions were absent in all eight aftld-u cases. this finding would also support our hypothesis that fus immunoreactivity in nifid and aftld-u is associated with the presence of the granulofilamentous inclusions while α-internexin immunoreactivity is associated with the presence of the compact hyaline inclusions. it remains unclear however; why some granulofilamentous inclusions show exclusive fus immunoreactivity (e.g. aftld-u) and others show exclusive tdp-43 immunoreactivity (e.g. ftld-tdp). only one study to date has reported both fus and tdp-43 immunoreactivity in the same ncis(39). overall, the data from this study support the importance of fus in the pathogenesis of aftld-u and the classification of aftld-u as an ftld-fus. however, the role of fus in nifid is less clear, with evidence supporting nifid being somewhat different from aftld-u with a concern that nifid, at least not all cases, may be incorrectly classified as a subtype of ftld-fus. supporting this statement is the fact that another fus negative case with α-internexin positive inclusions has been described(40). in this other case, however, a sod1 mutation was identified and some inclusions were immunoreactive to sod1. on a different note, the presence of intermediate filament inclusions and tdp-43 immunoreactive inclusions in the same case makes one contemplate the current sub-classification of ftld into strict categories. the relationship between fus and intermediate filament is reminiscent of the relationship between alzheimer’s disease pathology and lewy body disease. alzheimer’s disease pathology can occur in isolation, as can lewy body disease, but there are instances in which both alzheimer’s disease and lewy body disease co-occur. it would be naïve to argue that when two pathologies co-occur, that one pathology is more important or more relevant than the other. in summary, aftld-u and nifid share some but not all features. striatal atrophy, while appearing to be a characteristic feature of aftld-u, does not appear to be a feature of nifid although it can occur in some cases. further analyses are needed to better understand the relationship of aftld-u to nifid and of nifid to fus. such analysis should include the assessment of other proteins that have been reported to be associated with aftld-u and nifid including taf15 and ews(41), as well as transportin1(42). references 1. cairns nj, bigio eh, mackenzie ir, neumann m, lee vm, hatanpaa kj, et al. neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the consortium for frontotemporal lobar degeneration. acta neuropathol. 2007;114(1):5-22. 2. mackenzie ir, neumann m, bigio eh, cairns nj, alafuzoff i, kril j, et al. nomenclature for neuropathologic subtypes of frontotemporal lobar degeneration: consensus recommendations. acta neuropathol. 2009;117(1):15-8. 3. mackenzie ir, neumann m, bigio eh, cairns nj, alafuzoff i, kril j, et al. nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an update. acta neuropathol. 2010;119(1):1-4. 4. josephs ka, lin wl, ahmed z, stroh da, graff-radford nr, dickson dw. frontotemporal lobar degeneration with ubiquitin-positive, but tdp-43-negative inclusions. acta neuropathol. 2008;116(2):159-67. 5. mackenzie ir, foti d, woulfe j, hurwitz ta. atypical frontotemporal lobar degeneration with ubiquitin-positive, tdp-43-negative neuronal inclusions. brain. 2008;131(pt 5):1282-93. 6. neumann m, rademakers r, roeber s, baker m, kretzschmar ha, mackenzie ir. a new subtype of frontotemporal lobar degeneration with fus pathology. brain. 2009;132(pt 11):2922-31. 7. neumann m, roeber s, kretzschmar ha, rademakers r, baker m, mackenzie ir. abundant fus-immunoreactive pathology in neuronal intermediate filament inclusion disease. acta neuropathol. 2009;118(5):605-16. 8. munoz dg, neumann m, kusaka h, yokota o, ishihara k, terada s, et al. fus pathology in basophilic inclusion body disease. acta neuropathol. 2009;118(5):617-27. 9. cairns nj, zhukareva v, uryu k, zhang b, bigio e, mackenzie ir, et al. alpha-internexin is present in the pathological inclusions of neuronal intermediate filament inclusion disease. am j pathol. 2004;164(6):2153-61. 10. josephs ka, holton jl, rossor mn, braendgaard h, ozawa t, fox nc, et al. neurofilament inclusion body disease: a new proteinopathy? brain. 2003;126(pt 10):2291-303. 11. yokota o, tsuchiya k, terada s, ishizu h, uchikado h, ikeda m, et al. basophilic inclusion body disease and neuronal intermediate filament inclusion disease: a comparative clinicopathological study. acta neuropathol. 2008;115(5):561-75. 12. munoz-garcia d, ludwin sk. classic and generalized variants of pick's disease: a clinicopathological, ultrastructural, and immunocytochemical comparative study. ann neurol. 1984;16(4):467-80. 13. josephs ka, hodges jr, snowden js, mackenzie ir, neumann m, mann dm, et al. neuropathological background of phenotypical variability in frontotemporal dementia. acta neuropathol. 2011;122(2):137-53. 14. urwin h, josephs ka, rohrer jd, mackenzie ir, neumann m, authier a, et al. fus pathology defines the majority of tauand tdp-43-negative frontotemporal lobar degeneration. acta neuropathol. 2010;120(1):33-41. 15. snowden js, hu q, rollinson s, halliwell n, robinson a, davidson ys, et al. the most common type of ftld-fus (aftld-u) is associated with a distinct clinical form of frontotemporal dementia but is not related to mutations in the fus gene. acta neuropathol. 2011;122(1):99-110. 16. matsumoto s, kusaka h, murakami n, hashizume y, okazaki h, hirano a. basophilic inclusions in sporadic juvenile amyotrophic lateral sclerosis: an immunocytochemical and ultrastructural study. acta neuropathol. 1992;83(6):579-83. 17. josephs ka, whitwell jl, parisi je, petersen rc, boeve bf, jack cr, jr., et al. caudate atrophy on mri is a characteristic feature of ftld-fus. eur j neurol. 2010;17(7):969-75. 18. seelaar h, klijnsma ky, de koning i, van der lugt a, chiu wz, azmani a, et al. frequency of ubiquitin and fus-positive, tdp-43-negative frontotemporal lobar degeneration. j neurol. 2010;257(5):747-53. 19. lashley t, rohrer jd, bandopadhyay r, fry c, ahmed z, isaacs am, et al. a comparative clinical, pathological, biochemical and genetic study of fused in sarcoma proteinopathies. brain. 2011;134(pt 9):2548-64. 20. dickson dw, wertkin a, mattiace la, fier e, kress y, davies p, et al. ubiquitin immunoelectron microscopy of dystrophic neurites in cerebellar senile plaques of alzheimer's disease. acta neuropathol. 1990;79(5):486-93. 21. lee s, park yd, yen sh, ksiezak-reding h, goldman je, dickson dw. a study of infantile motor neuron disease with neurofilament and ubiquitin immunocytochemistry. neuropediatrics. 1989;20(2):107-11. 22. gwinn-hardy k, mehta nd, farrer m, maraganore d, muenter m, yen sh, et al. distinctive neuropathology revealed by alpha-synuclein antibodies in hereditary parkinsonism and dementia linked to chromosome 4p. acta neuropathol. 2000;99(6):663-72. 23. josephs ka, parisi je, knopman ds, boeve bf, petersen rc, dickson dw. clinically undetected motor neuron disease in pathologically proven frontotemporal lobar degeneration with motor neuron disease. arch neurol. 2006;63(4):506-12. 24. neary d, snowden js, gustafson l, passant u, stuss d, black s, et al. frontotemporal lobar degeneration: a consensus on clinical diagnostic criteria. neurology. 1998;51(6):1546-54. 25. boeve bf, lang ae, litvan i. corticobasal degeneration and its relationship to progressive supranuclear palsy and frontotemporal dementia. ann neurol. 2003;54 suppl 5:s15-9. 26. litvan i, agid y, calne d, campbell g, dubois b, duvoisin rc, et al. clinical research criteria for the diagnosis of progressive supranuclear palsy (steele-richardson-olszewski syndrome): report of the ninds-spsp international workshop. neurology. 1996;47(1):1-9. 27. zhai p, pagan f, statland j, butman ja, floeter mk. primary lateral sclerosis: a heterogeneous disorder composed of different subtypes? neurology. 2003;60(8):1258-65. 28. papp mi, kahn je, lantos pl. glial cytoplasmic inclusions in the cns of patients with multiple system atrophy (striatonigral degeneration, olivopontocerebellar atrophy and shy-drager syndrome). j neurol sci. 1989;94(1-3):79-100. 29. uchikado h, li a, lin wl, dickson dw. heterogeneous inclusions in neurofilament inclusion disease. neuropathology. 2006;26(5):417-21. 30. rohrer jd, lashley t, schott jm, warren je, mead s, isaacs am, et al. clinical and neuroanatomical signatures of tissue pathology in frontotemporal lobar degeneration. brain. 2011;134(pt 9):2565-81. 31. bigio eh, lipton am, white cl, 3rd, dickson dw, hirano a. frontotemporal and motor neurone degeneration with neurofilament inclusion bodies: additional evidence for overlap between ftd and als. neuropathol appl neurobiol. 2003;29(3):239-53. 32. cairns nj, grossman m, arnold se, burn dj, jaros e, perry rh, et al. clinical and neuropathologic variation in neuronal intermediate filament inclusion disease. neurology. 2004;63(8):1376-84. 33. josephs ka, uchikado h, mccomb rd, bashir r, wszolek z, swanson j, et al. extending the clinicopathological spectrum of neurofilament inclusion disease. acta neuropathol. 2005;109(4):427-32. 34. uchikado h, shaw g, wang ds, dickson dw. screening for neurofilament inclusion disease using alpha-internexin immunohistochemistry. neurology. 2005;64(9):1658-9. 35. armstrong ra, gearing m, bigio eh, cruz-sanchez ff, duyckaerts c, mackenzie ir, et al. the spectrum and severity of fus-immunoreactive inclusions in the frontal and temporal lobes of ten cases of neuronal intermediate filament inclusion disease. acta neuropathol. 2011;121(2):219-28. 36. mackenzie ir, munoz dg, kusaka h, yokota o, ishihara k, roeber s, et al. distinct pathological subtypes of ftld-fus. acta neuropathol. 2011;121(2):207-18. 37. lin wl, dickson dw. ultrastructural localization of tdp-43 in filamentous neuronal inclusions in various neurodegenerative diseases. acta neuropathol. 2008;116(2):205-13. 38. josephs ka, mackenzie i, frosch mp, bigio eh, neumann m, arai t, et al. late to the part-y. brain. 2019;142(9):e47. 39. deng hx, zhai h, bigio eh, yan j, fecto f, ajroud k, et al. fus-immunoreactive inclusions are a common feature in sporadic and non-sod1 familial amyotrophic lateral sclerosis. ann neurol. 2010;67(6):739-48. 40. nakamura m, bieniek kf, lin wl, graff-radford nr, murray me, castanedes-casey m, et al. a truncating sod1 mutation, p.gly141x, is associated with clinical and pathologic heterogeneity, including frontotemporal lobar degeneration. acta neuropathol. 2015;130(1):145-57. 41. neumann m, bentmann e, dormann d, jawaid a, dejesus-hernandez m, ansorge o, et al. fet proteins taf15 and ews are selective markers that distinguish ftld with fus pathology from amyotrophic lateral sclerosis with fus mutations. brain. 2011;134(pt 9):2595-609. 42. brelstaff j, lashley t, holton jl, lees aj, rossor mn, bandopadhyay r, et al. transportin1: a marker of ftld-fus. acta neuropathol. 2011;122(5):591-600. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. enteric synucleinopathy: from trendy concept to real entity. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:26 (2020) opinion piece enteric synucleinopathy: from trendy concept to real entity. adrien de guilhem de lataillade 1,2, thibaud lebouvier 3, wendy noble 4, laurène leclair-visonneau 1, pascal derkinderen 1,2 1 université de nantes, inserm, tens, the enteric nervous system in gut and brain diseases, imad, nantes, france 2 chu nantes, department of neurology, nantes, f-44093, france 3 univ. lille, inserm urm_s1172, chu lille, distalz, licend, f-59000 lille, france 4 king’s college london, institute of psychiatry, psychology and neuroscience, department of basic and clinical neuroscience, maurice wohl clinical neuroscience institute, 5 cutcombe road, camberwell, london. se5 9rx, uk corresponding author: pascal derkinderen · inserm u1235 nantes · 1 rue gaston veil · 44035 nantes · france · tel: +33(0)240165202 · fax: +33(0)240165203 pascal.derkinderen@chu-nantes.fr submitted: 29 july 2020 accepted: 23 august 2020 copyedited by: aivi t. nguyen published: 28 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2920 keywords: synucleinopathies, tauopathies, enteric nervous system, aggregates, alpha-synuclein, parkinson’s disease abstract an accumulating body of literature has emerged in the past 25 years to show that parkinson’s disease (pd) is not only a disorder of the brain but also of the gastrointestinal tract and more generally of the gut-brain axis. gastrointestinal symptoms occur in almost every pd patient at some point and in nearly every case examined pathologically autopsy studies find alpha-synuclein deposits, the pathological hallmarks of pd, in the enteric nervous system. this concept of ‘enteric synucleinopathy’ led to the hypothesis that the enteric nervous system might play a pivotal role in the initiation and spreading of pd. although this hypothesis opens up interesting perspectives on the pathogenesis of neurodegenerative disorders, some important questions are still pending. the present opinion paper describes and compares the physiological and pathophysiological properties of alpha-synuclein in the brain and the enteric nervous system. we conclude that the existing data supports the existence of pathological alpha-synuclein species in the gut in pd. we also discuss if gut-brain interactions are important in other neurodegenerative disorders. abbreviations ad alzheimer’s disease, ens enteric nervous system, gi gastrointestinal, lb lewy bodies, ln lewy neurites, pd parkinson’s disease, pmca protein misfolding cyclic amplification, psp progressive supranuclear palsy the enteric nervous system (ens) is an intricate neural network embedded within the gastrointestinal (gi) tract and distributed from the lower oesophagus to the rectum. compared to other components of the peripheral nervous system, the ens shows some unique features that closely resemble the cns and therefore it is sometimes referred to as ‘the brain-in-the-gut’ or the ‘second brain’ [1] (figure 1). this close homology between the cns and ens suggests that a disease process affecting the cns could also involve its enteric counterpart. parkinson’s disease (pd) is the best example of this assumption. pd is the most common synucleinopathy (or synuclein proteinopathy), a group of neurodegenerative disorders characterized by a common pathological lesion composed of aggregates of alpha-synuclein in selectively vulnerable neuron populations in the cns [2]. although pd has traditionally been considered a disease of dopaminergic neurons in the substantia nigra, analyses of gi samples from pd patients have consistently found neural pathology, with the presence of alpha-synuclein deposits being detected in the ens in nearly every pd patient examined [3–11]. this has led to the emerging concept of enteric synucleinopathy. although these observations open up interesting perspectives on the pathogenesis of neurodegenerative disorders, some important questions are still pending, among which are: what are the biochemical and pathological characteristics of alpha-synuclein deposits in the ens? are they similar to those observed in the cns? is there any neuronal loss in the ens in pd? besides pd, are gut-brain interactions important in other neurodegenerative disorders? here, we first provide a brief overview of the normal expression profiles of alpha-synuclein in the ens, before discussing these questions and the arguments for and against the existence of enteric synucleinopathies. figure 1. anatomical organisation and local reflexes of the enteric nervous system. the ens is a neuronal network embedded within the gi tract and distributed from the lower oesophagus to the rectum. it is organized in two major ganglionated plexuses, the myenteric (mp or auerbach’s plexus) mainly involved in the control of smooth muscle activity, and the submucosal (smp or meissner’s plexus), which regulates secretion (sn, secretory neurons) and microvasculature. compared to other sections of the peripheral nervous system, the ens shows unique features that closely resemble some of the cns: it contains a variety of functionally distinct enteric neurons along with a vast repertoire of neurotransmitters and intercellular messengers which are the basis for enteric neurotransmission. it also harbours a prominent component of glial cells (egc for enteric glial cells) which, like astrocytes in the cns, contribute to support, protection and maintenance of the neural networks. local distention of the intestinal wall and chemical contents in the gut lumen activate intrinsic primary afferent neurons (ipan) located in both the smp and mp. the ipan projects both in oral and anal directions to synapse with interneurons (in) and motor neurons (mn). cholinergic (ach) mn in red are excitatory while nitrergic (no) mn in green are inhibitory. the peristaltic reflex includes an ascending excitatory reflex mediated by cholinergic mn and elicits contraction of the circular or longitudinal smooth muscles located orally to the site of stimulation. the descending inhibitory reflex involves inhibitory nitrergic mn that elicit relaxation of the circular muscles and longitudinal muscles located anally to the site of stimulation. alpha-synuclein is physiologically expressed by enteric neurons alpha-synuclein was first isolated in 1988 from the electric organ of the pacific electric ray torpedo californica. in addition to its strong presynaptic localisation, maroteaux et al., also identified alpha-synuclein in the nucleus, thus accounting for the name ‘synuclein’ (synapse + nucleus) [12]. several studies have documented the physiological function of alpha-synuclein in modulating synaptic vesicle release [13]. human alpha-synuclein, 140 amino acids in length, is mainly expressed by cns neurons and erythrocytes and is composed of 3 different functional regions (figure 2a) [13]. there is still open debate about the physiological structure of brain alpha–synuclein as some research groups concluded that it occurs as a helically folded tetramer [14], while others counterclaim that it exists primarily as a disordered monomer [15]. alpha-synuclein has a natural propensity to aggregate as amyloid structures in a nucleation-dependent process in which monomers assemble via oligomers into fibrils [16] (figure 2b). figure 2. alpha-synuclein structure and molecular mechanism of its oligomerisation and fibrillogenesis. (a) schematic representation of alpha-synuclein, which is composed of 3 distinct regions : (i) an n-terminal domain (amino acids 1–60) that binds lipids and contains the lysines that are ubiquitinated (ii) a central domain known as the non-amyloid component (nac) (amino acids 61–95) which is involved in aggregation, and (iii) a c-terminal acidic tail (amino acids 96–140) accountable for most interactions with other proteins and small molecules and that contains most of the phosphorylation sites including serine 129. some of the truncated c terminal forms of alpha-synuclein are also shown. (b) illustration of the molecular steps involved in alpha-synuclein oligomerisation and fibrillogenesis leading to lewy body formation. soluble alpha-synuclein is natively unstructured and monomeric. under pathological conditions, soluble α-synuclein forms β-sheet-like oligomers (protofibrils), which convert into amyloid fibrils and eventually deposit in lewy bodies. alpha-synuclein is also expressed by enteric neurons. in rodents, guinea-pigs and human alpha-synuclein positive neurons are present in the two plexuses of the ens and along the entire digestive tract [5, 17, 18] (figure 3). using amine-reactive cross-linking, we showed that, unlike brain neurons, alpha-synuclein exists primarily as a monomer in enteric neurons [19]. as far as we know, the expression of alpha-synuclein by enteric glial cells, the enteric counterpart of cns astrocytes, has not been evaluated. detailed immunohistochemical characterisation showed that alpha-synuclein immunoreactive neurons are mostly cholinergic [17, 18] (figure 1) and that alpha-synuclein is closely associated with the vesicular apparatus [20]. although the role of alpha-synuclein in the ens is still mostly unknown, a recent study suggested that it is involved in the development and electrophysiological properties of enteric cholinergic neurons [21]. figure 3. physiological expression of alpha-synuclein and phospho-alpha-synuclein histopathology in the ens. left: anti-alpha-synuclein antibody syn-1 was used to detect alpha-synuclein in the myenteric ganglia (colon) in a control subject devoid of neurodegenerative disorders; βiii tubulin antibody was used to label the neuronal network. scale bar is 50 µm. right: the colon from a pd patient was microdissected into mucosa, whole mount of submucosa and myenteric plexus. each part was stained with an antibody specific for the phosphorylated form of alpha-synuclein. phospho-alpha-synuclein histopathology with a ln-like pattern is observed in the mucosa (arrow, the asterisk is for the crypt) and submucosa, while a lb-like structure is found in the myenteric plexus. scale bar is 40 µm. does the ens contain pathological aggregated forms of alpha-synuclein? lewy bodies (lb) and lewy neurites (ln) are the defining neuropathological characteristics of pd. lb typically appear in neuronal somata as eosinophilic, rounded inclusions while ln are strand-like structures observed in axons. in 1997, it was demonstrated that lb and ln isolated from pd brain were highly immunoreactive for alpha-synuclein, thereby suggesting that this protein was one of the main components of lewy pathology [22]. subsequent work showed that alpha-synuclein in lb and ln was hyperphosphorylated at serine residue 129 [23] (figure 2). because alpha and phospho-alpha-synuclein immunohistochemical staining has a much greater sensitivity than hematoxylin and eosin staining for the detection of lb [24], this method quickly became the method of choice for the neuropathological diagnosis of pd [25]. using this approach, several neuropathology laboratories demonstrated that lb and ln-like structures were observed in the ens (in both the myenteric and submucosal plexus) in the vast majority of pd patients [3–7] (figure 3). in some of these studies, proteinase k [3] or alkaline protease [7] pre-treatments were used primarily to unmask antigens and enhance immunolabeling, but such treatments might also allow protease-resistant misfolded, aggregated and hyperphosphorylated alpha-synuclein to be distinguished from soluble forms of the protein. on the whole these findings suggest that pathological aggregated alpha-synuclein is present within the gi tract of pd subjects. two key issues should however be borne in mind. first, the mere detection of alpha-synuclein phosphorylated at serine 129 is not synonymous with aggregation since soluble alpha-synuclein is also physiologically phosphorylated at this residue [26]. secondly, immunohistochemical approaches are not sufficient to show that alpha-synuclein is misfolded/aggregated; biochemical confirmation of altered solubility is required. indeed, in the cns, a comprehensive biochemical characterisation of alpha-synuclein forms in lb has already been carried out. using one and two-dimensional immunoblot analysis with modification-specific synuclein antibodies and mass spectroscopy, anderson et al. confirmed that the predominant modification of alpha-synuclein in lb was phosphorylation at serine 129. they also found a set of additional characteristic modifications including ubiquitination at amino-terminal lysine residues and specific carboxy-terminal truncations [27] (figure 2). an additional property of alpha-synuclein aggregates found in diseased brains is their seeding ability: the amyloid fibrils formed by alpha-synuclein aggregates can act as templates for the conversion of physiological alpha-synuclein, resulting in the growth of the fibrils and spread of alpha-synuclein pathology [28]. how about the biochemical characterisation of alpha-synuclein in diseased ens? there are some existing data but it is definitely less complete and robust than those available for the cns. using one and two-dimensional analysis of colonic biopsies, we were unable to detect any differences in the expression levels, phosphorylation or aggregation status of alpha-synuclein between controls and pd specimens [29]. these negative findings might however be explained by the relative sparsity of neuronal structures and/or alpha-synuclein inclusions in the gi samples that were used; due to a shortage of samples, only 2 routine colonic biopsies per subject were pooled and analysed. however, when we had the opportunity to analyse 4 pooled biopsies per subject, we were able to detect acidic and high molecular weight alpha-synuclein species in the gi tract of pd patients but not controls, which likely represent hyperphosphorylated and aggregated forms of the protein, respectively (lebouvier-derkinderen unpublished results, figure 4). such a pattern is reminiscent of that observed in pd brain [27] (figure 4). the limitations of the two-dimensional immunoblotting technique prompted us to use more sensitive approaches, based on the seeding efficiency of alpha-synuclein. using an assay inspired by the protein misfolding cyclic amplification (pmca) assay, we showed that gi biopsies from pd patients (2 to 4 biopsies per subject taken from the upper or lower gi tract) seeded alpha-synuclein aggregation in 10 out of 18 cases [30]. more recently, viviane labrie and patrik brundin’s group focused on the vermiform appendix, a structure which is particularly enriched in alpha-synuclein [31]. using mass spectrometry, they identified full-length alpha-synuclein together with a set of 10 truncated forms in the healthy human appendix [32]. additional experiments performed with an in vitro shaking assay (also inspired by the pmca approach), showed that appendix lysates from either control or pd subjects seeded aggregation and truncation of alpha-synuclein. these intriguing and provocative findings suggest that aggregated and truncated alpha-synuclein are consistently found in the human healthy appendix, thereby supporting the assumption that the appendix may act as a reservoir for pathogenic forms of alpha-synuclein [31, 32]. figure 4. high molecular weight and post-translationally modified alpha-synuclein species in the gi tract of pd patients. immunoblots of total mesencephalon lysates (a and b) from a control (a) and a pd patient autopsy sample (b), and immunoblots of colonic biopsy lysates (c and d) from a control (c) and pd patient (d) were resolved by two-dimensional page using ph 3–10 ief gradients. immunoblots were probed with the alpha-synuclein antibody syn-1. white asterisks mark full-length synuclein. boxes highlight differences between pd patients and controls: acidic modifications of full-length monomer (solid boxes), and high molecular weight species (dotted boxes) are present in pd patients and absent in controls, in both substantia nigra and colonic biopsies (n=2); pi values and positions of molecular weight standards are indicated. an equivalent amount of protein was loaded in a-b and c-d, respectively. it has also been shown that pathological alpha-synuclein obtained from pd brain has the ability to trigger alpha-synuclein pathology in the cns of rodents and non-human primates [33, 34]. for example, the intranigral inoculation of alpha-synuclein-containing lb extracts obtained from pd brains leads to widespread alpha-synuclein pathology together with dopaminergic neurodegeneration in mice and monkeys [35]. for the sake of comparisons between brain and enteric alpha-synuclein, it is important to know if pathological alpha-synuclein obtained from the gi tract shares the same pathogenic capacity. although the precise answer to this question remains unknown, a recent study evaluated the effects of alpha-synuclein aggregates from post-mortem pd stellate ganglia (pooled from 3 patients) injected into mouse brain [36]. stellate ganglion is a paravertebral ganglion, which like the gut, exhibits marked lewy pathology in almost all pd subjects [4, 37]. in contrast to the findings obtained with substantia nigra-derived alpha-synuclein, no pathological effects were observed when peripheral aggregates from stellate ganglion were injected into the brain of wild-type mice, at least up to 6 months following injection [36]. this intriguing observation, which casts doubt on the pathogenicity of peripheral alpha-synuclein, requires replication using extracts from other peripheral organs affected by lewy pathology, such as the gi tract or the salivary glands [3, 38]. is there any enteric neuronal loss in pd? in the substantia nigra of pd subjects, the presence of alpha-synuclein aggregates is accompanied by severe neuronal loss and clinicopathological findings strongly suggest that the classical pd motor symptoms, including bradykinesia and hypertonia, are driven primarily by neuronal loss rather than only the aggregation of alpha-synuclein [39]. as such, the quantitative evaluation of enteric neuron populations is important to determine the pathological underpinnings of gi symptoms, which are so frequently observed in pd [40]. with that said, and despite the publication of proposed guidelines and consensus techniques, the quantification of submucosal and myenteric neuron number remains challenging [41], mainly because of the fishnet-like architecture of the ens plexus. in the most comprehensive post-mortem study to date, annerino et al. used formalin-fixed paraffin embedded sections to compare myenteric neuron density along the length of the gi tract in 6 patients with pd and 12 controls. there were no differences in total myenteric neuron density between controls or patients in any segment examined [42]. similarly, when whole mounts of submucosa obtained from colonic biopsies were analysed by immunohistochemistry, no major decrease in neuronal density was observed in pd relative to controls in two independent studies that included a total of 58 pd and 30 control cases [8, 43]. on the whole, these data strongly suggest that enteric neuron loss is not a feature of pd. besides alpha-synuclein, are other aggregated proteins found in the ens? the observation that alpha-synuclein deposits are a feature of pd gut logically leads to speculation that such a phenomenon might also occur in other neurodegenerative disorders such as tauopathies. like alpha-synuclein, tau is physiologically expressed by enteric neurons. however, in contrast to the cns neurons that express all six tau isoforms, adult human ens primarily express only two tau isoforms [44]. tau aggregates found in tauopathies generally contain tau in an elevated state of phosphorylation that is often aberrantly cleaved [45]. in a preliminary report, we analysed by western blot colonic biopsies from 5 patients with probable progressive supranuclear palsy (psp) using 2 different phospho-tau antibodies and one antibody specific for caspase-cleaved tau [44]. the phosphorylation and truncation patterns of tau in psp were indistinguishable from those in controls [44]. in a subsequent study, brittany dugger et al. performed a comprehensive immunohistochemical analysis of the ens in tauopathies. using formalin fixed paraffin embedded sections, they examined the sigmoid colon in 26 psp, 21 ad and 19 controls using one antibody for total tau and two phospho-tau antibodies [46]. no differences in the staining pattern were observed between colonic specimen from tauopathy patients and controls [46]. so, enteric synucleinopathy: myth or reality? even if they are still preliminary, recent findings and in particular the ones obtained with ultrasensitive amplification techniques such as pmca, convincingly showed that alpha-synuclein aggregates with seeding capacity are found in the ens [30, 32]. it therefore seems justified to use the word ‘enteric synucleinopathy’. that said, some outstanding questions remain: (i) existing findings suggest that enteric neuron loss is not a feature of pd but there could be more subtle morphologic changes [47] that could be evaluated in the future using computer-assisted analysis of the enteric neurons [48] (ii) a comprehensive inventory of alpha-synuclein forms present the ens from patients with pd still need to be carried out, as has already been performed for the cns [27] (iii) the results obtained with pmca need to be independently replicated and confirmed by others amplification techniques such as rt-quic (real-time quaking-induced conversion) [49]. in addition, it still remains to be determined if pathological alpha-synuclein purified from the ens is capable of promoting alpha-synuclein pathology when intracerebrally inoculated, as already demonstrated for cns-derived alpha-synuclein aggregates [50]. this is a critical issue with regard to pd pathogenesis as it has been speculated that enteric alpha-synuclein aggregates may spread to the cns via the vagal connections. however, performing such experiments, which require purification of lb-containing fractions from the gut, can be challenging due to the low frequency and density of aggregated alpha-synuclein in the ens [3, 51]. on the whole, current knowledge strongly supports the existence of enteric synucleinopathy and these data encourage future studies aimed at expanding our knowledge of peripheral pathology in neurodegenerative disorders to gain clues that enable further understanding of the differential pathogenesis of these disorders. references 1. schemann m, frieling t, enck p (2020) to learn, to remember, to forget-how smart is the gut? acta physiol (oxf) 228:e13296. https://doi.org/10.1111/apha.13296 2. goedert m, spillantini mg (1998) lewy body diseases and multiple system atrophy as alpha-synucleinopathies. mol psychiatry 3:462–465. https://doi.org/10.1038/sj.mp.4000458 3. beach tg, adler ch, sue li, et al (2010) multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with lewy body disorders. acta neuropathol 119:689–702. https://doi.org/10.1007/s00401-010-0664-3 4. gelpi e, navarro-otano j, tolosa e, et al (2014) multiple organ involvement by alpha-synuclein pathology in lewy body disorders. mov disord 29:1010–1018. https://doi.org/10.1002/mds.25776 5. punsoni m, friedman jh, resnick m, et al (2019) enteric pathologic manifestations of alpha-synucleinopathies. appl immunohistochem mol morphol 27:543–548. https://doi.org/10.1097/pai.0000000000000613 6. aldecoa i, navarro-otano j, stefanova n, et al (2015) alpha-synuclein immunoreactivity patterns in the enteric nervous system. neurosci lett 602:145–149. https://doi.org/10.1016/j.neulet.2015.07.005 7. stokholm mg, danielsen eh, hamilton-dutoit sj, borghammer p (2016) pathological α-synuclein in gastrointestinal tissues from prodromal parkinson disease patients. ann neurol 79:940–949. https://doi.org/10.1002/ana.24648 8. lebouvier t, neunlist m, bruley des varannes s, et al (2010) colonic biopsies to assess the neuropathology of parkinson’s disease and its relationship with symptoms. plos one 5:e12728. https://doi.org/10.1371/journal.pone.0012728 9. braak h, de vos rai, bohl j, del tredici k (2006) gastric alpha-synuclein immunoreactive inclusions in meissner’s and auerbach’s plexuses in cases staged for parkinson’s disease-related brain pathology. neurosci lett 396:67–72. https://doi.org/10.1016/j.neulet.2005.11.012 10. qualman sj, haupt hm, yang p, hamilton sr (1984) esophageal lewy bodies associated with ganglion cell loss in achalasia. similarity to parkinson’s disease. gastroenterology 87:848–856. https://doi.org/10.1016/0016-5085(84)90079-9 11. wakabayashi k, takahashi h, takeda s, et al (1988) parkinson’s disease: the presence of lewy bodies in auerbach’s and meissner’s plexuses. acta neuropathol 76:217–221. https://doi.org/10.1007/bf00687767 12. maroteaux l, campanelli jt, scheller rh (1988) synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. j neurosci 8:2804–2815. https://doi.org/10.1523/jneurosci.08-08-02804.1988 13. burré j, sharma m, südhof tc (2018) cell biology and pathophysiology of α-synuclein. cold spring harb perspect med 8(3):a024091. https://doi.org/10.1101/cshperspect.a024091 14. bartels t, choi jg, selkoe dj (2011) α-synuclein occurs physiologically as a helically folded tetramer that resists aggregation. nature 477:107–110. ttps://doi.org/10.1038/nature10324 15. fauvet b, mbefo mk, fares m-b, et al (2012) α-synuclein in central nervous system and from erythrocytes, mammalian cells, and escherichia coli exists predominantly as disordered monomer. j biol chem 287:15345–15364. https://doi.org/10.1074/jbc.m111.318949 16. conway ka, harper jd, lansbury pt (2000) fibrils formed in vitro from alpha-synuclein and two mutant forms linked to parkinson’s disease are typical amyloid. biochemistry 39:2552–2563. https://doi.org/10.1021/bi991447r 17. sharrad df, de vries e, brookes sjh (2013) selective expression of α-synuclein-immunoreactivity in vesicular acetylcholine transporter-immunoreactive axons in the guinea pig rectum and human colon. j comp neurol 521:657–676. https://doi.org/10.1002/cne.23198 18. phillips rj, walter gc, wilder sl, et al (2008) alpha-synuclein-immunopositive myenteric neurons and vagal preganglionic terminals: autonomic pathway implicated in parkinson’s disease? neuroscience 153:733–750. https://doi.org/10.1016/j.neuroscience.2008.02.074 19. corbillé a-g, neunlist m, derkinderen p (2016) cross-linking for the analysis of α-synuclein in the enteric nervous system. j neurochem 139:839–847. https://doi.org/10.1111/jnc.13845 20. böttner m, fricke t, müller m, et al (2015) alpha-synuclein is associated with the synaptic vesicle apparatus in the human and rat enteric nervous system. brain res 1614:51–59. https://doi.org/10.1016/j.brainres.2015.04.015 21. swaminathan m, fung c, finkelstein di, et al (2019) α-synuclein regulates development and function of cholinergic enteric neurons in the mouse colon. neuroscience 423:76–85. https://doi.org/10.1016/j.neuroscience.2019.10.029 22. spillantini mg, schmidt ml, lee vm, et al (1997) alpha-synuclein in lewy bodies. nature 388:839–840. https://doi.org/10.1038/42166 23. fujiwara h, hasegawa m, dohmae n, et al (2002) alpha-synuclein is phosphorylated in synucleinopathy lesions. nat cell biol 4:160–164. https://doi.org/10.1038/ncb748 24. kuusisto e, parkkinen l, alafuzoff i (2003) morphogenesis of lewy bodies: dissimilar incorporation of alpha-synuclein, ubiquitin, and p62. j neuropathol exp neurol 62:1241–1253. https://doi.org/10.1093/jnen/62.12.1241 25. beach tg, white cl, hamilton rl, et al (2008) evaluation of alpha-synuclein immunohistochemical methods used by invited experts. acta neuropathol 116:277–288. https://doi.org/10.1007/s00401-008-0409-8 26. muntané g, ferrer i, martinez-vicente m (2012) α-synuclein phosphorylation and truncation are normal events in the adult human brain. neuroscience 200:106–119. https://doi.org/10.1016/j.neuroscience.2011.10.042 27. anderson jp, walker de, goldstein jm, et al (2006) phosphorylation of ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic lewy body disease. j biol chem 281:29739–29752. https://doi.org/10.1074/jbc.m600933200 28. jung bc, lim y-j, bae e-j, et al (2017) amplification of distinct α-synuclein fibril conformers through protein misfolding cyclic amplification. exp mol med 49:e314. https://doi.org/10.1038/emm.2017.1 29. corbillé a-g, preterre c, rolli-derkinderen m, et al (2017) biochemical analysis of α-synuclein extracted from control and parkinson’s disease colonic biopsies. neurosci lett 641:81–86. https://doi.org/10.1016/j.neulet.2017.01.050 30. fenyi a, leclair-visonneau l, clairembault t, et al (2019) detection of alpha-synuclein aggregates in gastrointestinal biopsies by protein misfolding cyclic amplification. neurobiol dis 129:38–43. https://doi.org/10.1016/j.nbd.2019.05.002 31. gray mt, munoz dg, gray da, et al (2014) alpha-synuclein in the appendiceal mucosa of neurologically intact subjects. mov disord 29:991–998. https://doi.org/10.1002/mds.25779 32. killinger ba, madaj z, sikora jw, et al (2018) the vermiform appendix impacts the risk of developing parkinson’s disease. sci transl med 10(465):earr5280. https://doi.org/10.1126/scitranslmed.aar5280 33. recasens a, ulusoy a, kahle pj, et al (2018) in vivo models of alpha-synuclein transmission and propagation. cell tissue res 373:183–193. https://doi.org/10.1007/s00441-017-2730-9 34. dehay b, vila m, bezard e, et al (2016) alpha-synuclein propagation: new insights from animal models. mov disord 31:161–168. https://doi.org/10.1002/mds.26370 35. recasens a, dehay b, bové j, et al (2014) lewy body extracts from parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. ann neurol 75:351–362. https://doi.org/10.1002/ana.24066 36. recasens a, carballo-carbajal i, parent a, et al (2018) lack of pathogenic potential of peripheral α-synuclein aggregates from parkinson’s disease patients. acta neuropathol commun 6:8. https://doi.org/10.1186/s40478-018-0509-1 37. forno ls, norville rl (1976) ultrastructure of lewy bodies in the stellate ganglion. acta neuropathol 34:183–197. https://doi. org/10.1007/bf00688674 38. del tredici k, hawkes ch, ghebremedhin e, braak h (2010) lewy pathology in the submandibular gland of individuals with incidental lewy body disease and sporadic parkinson’s disease. acta neuropathol 119:703–713. https://doi.org/10.1007/s00401-010-0665-2 39. greffard s, verny m, bonnet a-m, et al (2006) motor score of the unified parkinson disease rating scale as a good predictor of lewy body-associated neuronal loss in the substantia nigra. arch neurol 63:584–588. https://doi.org/10.1001/archneur. 63.4.584 40. edwards ll, quigley em, pfeiffer rf (1992) gastrointestinal dysfunction in parkinson’s disease: frequency and pathophysiology. neurology 42:726–732. https://doi.org/10.1212/wnl.42.4.726 41. knowles ch, de giorgio r, kapur rp, et al (2009) gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the gastro 2009 international working group. acta neuropathol 118:271–301. https://doi.org/10.1007/s00401-009-0527-y 42. annerino dm, arshad s, taylor gm, et al (2012) parkinson’s disease is not associated with gastrointestinal myenteric ganglion neuron loss. acta neuropathol 124:665–680. https://doi.org/10.1007/s00401-012-1040-2 43. giancola f, torresan f, repossi r, et al (2017) downregulation of neuronal vasoactive intestinal polypeptide in parkinson’s disease and chronic constipation. neurogastroenterol motil 29(5):10.1111. https://doi.org/10.1111/nmo.12995 44. lionnet a, wade ma, corbillé a-g, et al (2018) characterisation of tau in the human and rodent enteric nervous system under physiological conditions and in tauopathy. acta neuropathol commun 6:65. https://doi.org/10.1186/s40478-018-0568-3 45. guo t, noble w, hanger dp (2017) roles of tau protein in health and disease. acta neuropathol 133:665–704. https://doi.org/10.1007/s00401-017-1707-9 46. dugger bn, hoffman br, scroggins a, et al (2019) tau immunoreactivity in peripheral tissues of human aging and select tauopathies. neurosci lett 696:132–139. https://doi.org/10.1016/j.neulet.2018.12.031 47. ohlsson b, englund e (2019) atrophic myenteric and submucosal neurons are observed in parkinson’s disease. parkinsons dis 2019:7935820. https://doi.org/10.1155/2019/7935820 48. ozawa t, shimizu h, matsui h, et al (2019) shrinkage of the myenteric neurons of the small intestine in patients with multiple system atrophy. auton neurosci 221:102583. https://doi.org/10.1016/j.autneu.2019.102583 49. rossi m, candelise n, baiardi s, et al (2020) ultrasensitive rt-quic assay with high sensitivity and specificity for lewy body-associated synucleinopathies. acta neuropathol 140:49–62. https://doi.org/10.1007/s00401-020-02160-8 50. arotcarena m-l, dovero s, prigent a, et al (2020) bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates. brain 143:1462–1475. https://doi.org/10.1093/brain/awaa096 51. beach tg, corbillé a-g, letournel f, et al (2016) multicenter assessment of immunohistochemical methods for pathological alpha-synuclein in sigmoid colon of autopsied parkinson’s disease and control subjects. j parkinsons dis 6:761–770. https://doi.org/10.3233/jpd-160888 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurodegeneration free neuropathology 1:12 (2020) review top ten discoveries of the year: neurodegeneration john f. crary neuropathology brain bank & research core, department of pathology, nash family department of neuroscience, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary, md-phd · friedman brain institute · ronald m. loeb center for alzheimer’s disease · icahn school of medicine at mount sinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 26 january 2020 accepted: 02 april 2020 copyedited by: nima sharifai published: 08 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2634 keywords: neurodegeneration, neuropathology, alzheimer’s disease, limbic-predominant age-related tdp-43 encephalopathy, amyotrophic lateral sclerosis, parkinson’s disease, huntington’s disease abstract as we embark on a new year of scientific inquiry in neurodegenerative disease research, it is helpful to take a look back and consider the contributions in the field with the potential to be the most impactful. the purpose of this review is to highlight recent advances in 2019 which have the potential to be transformative in the field of neurodegenerative neuropathology. substantive scientific progress rarely occurs as a “eureka moment”, and when possible, we opted to highlight collaborative efforts and research trends. we also included groundbreaking methodologies and tools. the generous increases in federal funding in the united states and elsewhere have massively expanded the total number of active programs researching alzheimer’s disease. this exacerbates an imbalance, and an effort was made to highlight innovations across disease categories, and not to permit dementia to crowd out movement disorders, motor neuron disease, ataxias, etc. thus, our overall goal was to highlight some of the most important discoveries, tools or methods that we feel will most likely directly enhance our ability to understand and diagnose neurodegenerative brain diseases. given space limitations and the targeted readership of this journal, we selected ten topics most relevant to neuropathologists and clinical neuroscientists: 1. a new neurodegenerative disease category, 2. a new approach to probing gene expression on the single cell level, 3. a new approach merging histology and gene expression profiling, 4. a new computational approach using deep machine learning and computer vision, 5. a neuropathological substrate for sleep disturbance in alzheimer’s disease, 6. a candidate pathogenic agent for alzheimer’s disease, 7. a comprehensive approach to morphometric analysis of cerebellar neurodegeneration, 8. the strongest evidence yet linking neurodegeneration to contact sports, 9. mounting evidence for gut to central nervous system transmission in parkinson’s disease, and 10. a spotlight on glia in huntington’s disease. 1. late, a new old neurodegenerative disease as the population ages, researchers are gleaning fundamental new insights from the oldest-old population (i.e., those over the age of 80 years) whose vulnerability to neuropathology diverges from the younger-old. in a paper published in brain, a large group of neuropathologists led by dr. peter nelson at the university of kentucky introduced a new diagnostic category, limbic-predominant age-related tdp-43 encephalopathy (late), describing a type of neurodegeneration that mostly strikes elderly individuals over 80 years old (nelson et al., 2019). this work was the culmination of many years of study and a consensus group report, the goal of which was to address how to deal with the surprisingly large number of patients that variably develop an amnestic dementia that is very similar to that seen in alzheimer’s disease (ad), but with abnormalities in the transactive response dna binding protein of 43 kda (tdp-43). historically, the first study to recognize that there was something different occurring in these patients did so by identifying 13 autopsied subjects with dementia who had marked neuronal loss with reactive gliosis in the medial temporal lobe, but had no alzheimer’s neuropathological changes; this was termed “hippocampal sclerosis” (dickson et al., 1994). this neuropathological signature is not specific, and it was not until tdp-43 was discovered in amyotrophic lateral sclerosis (als) and frontotemporal lobar degeneration (ftld) patients that investigators thought to also look in hippocampal sclerosis patients and ultimately link the two. it was also recognized that subjects with tdp-43 proteinopathy only variably present with a pattern histologically considered hippocampal sclerosis on hematoxylin and eosin (h&e) stain. neuropathologically, subjects with late have the tell-tale abnormal translocation of tdp-43 from the nucleus to the cytoplasm, where it becomes phosphorylated (figure 1). in late, tdp-43 pathology can be detected in numerous brain regions, some of which overlap with ftld, but the amygdala is most severely affected, followed by the hippocampal formation. nelson et al. provided diagnostic criteria and a proposed hierarchical staging system for assessing patients for late neuropathologic change: tdp-43 immunohistochemistry is essential (hippocampal sclerosis pathology only is not sufficient) and should be performed in the amygdala (stage 1), mid-level hippocampus (stage 2) and middle frontal gyrus (stage 3).   figure 1. limbic-predominant age-related tdp-43 encephalopathy (late). (a) late neuropathological change in an 83-year-old woman with dementia and the clinical diagnosis of ad. marked atrophy and neuronal loss in the hippocampal formation, but with minimal ad neuropathological change. (b) tdp-43 proteinopathy is shown in the dentate gyrus. image courtesy of dr. peter nelson (nih grant p30 ag028383). there is a great deal that we have to learn by studying late. while a few genes have been implicated as risk factors (i.e., apoe, grn, tmem106b, abcc9, kcnmb2), larger genetic studies have the potential to better delineate mechanisms and causal pathways that could serve as biomarkers and therapeutic targets. further, we have very little understanding of the extent to which late affects non-caucasian populations; studying more diverse populations has the potential to expand on risk factors, genetic and otherwise. biochemical and molecular studies of tdp-43, including secondary modifications such as phosphorylation, have the potential to lead to biomarkers that could be used pre-mortem. finally, co-morbidities are nearly universal in the oldest-old population, and the extent to which they interact with late pathology will be important for understanding its clinical significance. 2. single-cell transcriptomics nothing has plagued molecular profiling of post-mortem neurodegenerative disease brain tissue more than the problem of normalization in the context of variable and often massive neuronal loss with commensurate reactive gliosis. numerous approaches have been developed to address this problem (e.g., deconvolution of bulk rna sequencing or expression profiling of pooled cells isolated by laser capture microdissection), but they are laborious and results often fall short. recent advances in high-throughput single cell sorting using microfluidics coupled with next-generation sequencing have enabled a paradigm shift in how gene expression profiles can be investigated. the first papers deploying single-cell rna sequencing (scrna-seq) in human post-mortem neurodegenerative disease tissues have emerged in 2019 and we expect this to transform transcriptomic studies (del-aguila et al., 2019; grubman et al., 2019; mathys et al., 2019). one of the major barriers that investigators encountered in applying single cell technology to human post-mortem brain is that it is not possible to isolate single cells from frozen tissue given the membrane breakdown. thus, scrna-seq is currently best done from cells isolated from fresh brain tissue, and precludes using archival frozen banked brains. previous work in animal models, however, has shown that single nuclei can withstand the freezing process and yield nuclear rna profiles that can serve as a proxy for those generated from cytoplasmic rna. because of this, groups are focusing on this variant approach termed single nuclei rna-seq (snrna-seq, or just nuc-seq). three studies were published in 2019 using nuc-seq in post-mortem human brain tissue from patients with neurodegenerative disease. in the first, published online in august by del-aguila et al, investigators at washington university in saint louis used nuc-seq to profile a patient with the psen1 p.a79v mutation and compared it to two related family members with sporadic ad (non-mutational), and proposed a collection of best practices (del-aguila et al., 2019). all of these studies have made the data available online (figure 2). in november, grubman et al., from monash university (australia) and duke-national university of singapore medical school, examined the entorhinal cortex from 6 sporadic patients and 6 controls. their group found that ad is associated with downregulation of apoe in oligodendrocyte precursors and astrocytes, and with upregulation of apoe in a subpopulation of microglia. they also explored transcription factor regulatory modules (grubman et al., 2019). finally, in the largest study published in june by mathys et al., investigators at the broad institute of mit/harvard profiled the prefrontal cortex from 48 individuals from the rush rosmap cohort and identified sex differences in disease-associated subpopulations of cells, and found interesting recurrent differences in myelination that might play a role in ad (mathys et al., 2019). together, these results are the first step towards providing a highly detailed cell-type specific molecular atlas of neurodegenerative diseases.   figure 2. single-nuclei rna-seq (snrna-seq) brain expression browser. cell type specific expression profiles can be shown using a publicly available interactive web-based resource (http://ngi.pub/snuclrna-seq). 3. spatial transcriptomics essentially all approaches to measuring gene expression are limited by the fact that isolation of nucleic acids from tissue by necessity disrupts the spatial relationship of the cells of interest. losing the cellular architecture is a major barrier to understanding the context of genome-wide changes in rna expression. this year we saw the first use of spatial transcriptomics, a method first introduced in 2016 that bridges rna-sequencing and histopathology by allowing researchers to generate thousands of gene expression profiles from a single histological section using a slide printed with an array of barcoded specialized mrna-capturing probes (ståhl et al., 2016). in a study led by hemali phatnani at the new york genome center and published in science (maniatis et al., 2019), spatial transcriptomics was applied to post-mortem human amyotrophic lateral sclerosis tissue from seven patients with either lumbaror bulbar-onset sporadic als (figure 3). they also profiled sod1-g93a transgenic mice. together, they quantitated the spatial distribution of 11,138 mouse and 9624 human genes in spinal cord sections and confirmed the alteration in expression of some known als genes. they were also able to identify an abnormal microglial signature that preceded astroglial dysfunction. they chose to follow up on a mechanism involving trem2 and its partner tyrobp, revealing a spatiotemporal ordering with tyrobp expression upregulated pre-symptomatically before trem2. changes in other genes that are part of this mechanism (e.g., apoe, cx3cr1, lpl, b2m and cx3cr1) occurred later. these results demonstrate how this technology can lead to novel insights by being deployed as part of an interdisciplinary approach to neurodegenerative disease.   figure 3. identification of expression programs using spatially resolved transcriptomics. (a) example data for a single gene from spatial transcriptomics profiling of postmortem lumbar and cervical spinal cord sections from a sporadic als patient presenting with bulbar symptom onset. (b) data from many spatial transcriptomics arrays were aligned to a common coordinate system, and expression levels were hex binned spatially. this procedure enables reconstruction of larger regions than can be profiled with a single array and reveals expected spatial expression patterns for genes expressed by cell types present in specific tissue domains. (c) spatially resolved coexpression analysis reveals gene sets with tissue domain specific expression patterns, a subset of which also exhibit differences with respect to proximity to the site of symptom onset. figure courtesy of drs. hemali phatnani and silas maniatis. 4. artificial intelligence and computer vision since alois alzheimer first described the disease that bears his name in 1906, revealing striking morphological changes using ammoniacal silver stains, there has been very little progress in the way these cellular changes are interpreted. visual microscopic examination remains state-of-the-art, but it is slow, imprecise, marginally reproducible and requires the experience of a highly trained neuropathologist, the numbers of which are dwindling. the task is highly repetitive and fatigue inducing, which increases the likelihood of error. the whole process would be best suited to automation, but the concept of a machine that could substitute for a neuropathologist seemed implausible until recent developments in the field of artificial intelligence. increases in computational power combined with improved algorithms for training artificial neural networks have placed us at the precipice of a transformation moment in neuropathology. the most common machine learning approach to analyzing visual imagery is the convolutional neural network (cnn). these are a type of deep learning, a class of artificial neural networks with multiple layers that progressively extract more complex features from the input. cnns are based on the architecture of the organization of the animal visual cortex, encompassing simulations of overlapping receptive fields what were described by hubel and wiesel in the cat and monkey in the 1950s and 1960s. this year we saw the first two papers deploying deep learning to analyze digital whole slide images from patients with neurodegenerative diseases to identify both amyloid plaques and neurofibrillary tangles. one paper, published in nature communications and led by dr. brittany dugger (university of california davis), reported a deep learning pipeline that identifies amyloid plaques and cerebral amyloid angiopathy in immunohistochemically stained slides (tang et al., 2019). to build this, they annotated over 70,000 plaques that served as ground truth to train and evaluate the cnn achieving 0.993 receiver operating characteristic (roc) curve and 0.743 precision recall curve. in another paper, investigators from the center for computational and systems pathology at the icahn school of medicine at mount sinai, led by dr. john f. crary (author of this review) and published in laboratory investigation, focused on neurofibrillary tangle pathology (signaevsky et al., 2019). this study used 3177 images derived from patients with primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy and alzheimer’s disease to train and test the network, achieving recall, precision and f1 scores of 0.91, 0.80 and 0.85 respectively. we expect that this approach will be transformative in the near future with the development of sophisticated neural networks capable of rapidly and reproducibly analyzing slides alongside, or perhaps instead of, the neuropathologist. in the long term, this will certainly transform the skillset of the neuropathologist, who will perhaps be less adept at picking up subtle morphological features by eye, but hopefully more able to deploy computational and other modern tools. 5. a neuropathological substrate for sleep-wake disruption in alzheimer’s disease sleep-wake disruption is common in aging and associated with cognitive decline and decreased quality of life. sleep disturbance is seen in a number of neurodegenerative diseases and is amongst the earliest symptoms of ad. this includes reduced total, slow wave, and rapid eye movement (rem) sleep time. there are arousal deficiencies, including daytime sleepiness, that can precede cognitive impairment. competition between subcortical populations of both wake promoting neurons (wpns) and sleep promoting neurons (spns) provide a switch that coordinates sleep behaviors. among these populations, wpns in the locus coeruleus, orexin/hypocretin-producing neurons in the lateral hypothalamic area, and histaminergic neurons in the tuberomammillary nucleus (tmn) play critical roles in stimulating the cerebral cortex and contributing to arousal. the neuropathological substrate for sleep disturbances in ad remains unclear, but attention has been focused on amyloid-beta (aβ) as sleep promotes its clearance. accumulation of abnormal hyperphosphorylated tau (p-tau), however, is a stronger correlate of neurodegeneration and functional decline, and accumulation of p-tau in wpns is among the first ad neuropathological changes to appear in the brain. in a powerful study published in alzheimer’s & dementia, a team led by dr. lea grinberg (ucsf) directly addressed the question of p-tau pathology in brainstem wpns in ad by performing rigorous quantitative study, using stereological measures of wake-promoting cells in the locus coeruleus, lateral hypothalamic area and tuberomammillary nucleus in ad, compared to two amyloid-independent primary tauopathies (progressive supranuclear palsy and corticobasal degeneration) and healthy controls (oh et al., 2019). they found that while all three of these diseases displayed the presence of p-tau in wpns, only the ad patients exhibited clinically relevant cell loss (figure 4). the implications are that loss of these cells are an important component of sleep disturbance in ad, but also that amyloid-independent tauopathies may benefit from alternative clinical approaches (e.g., suppression of the arousal system).   figure 4. tau pathology in the locus coeruleus in alzheimer’s disease compared to progressive supranuclear palsy, corticobasal degeneration and controls. double-label immunohistochemistry showing colocalization of tau pathology (brown) and norepinephrine (red). figure courtesy of dr. lea grinberg. 6. periodontal disease as a risk factor for alzheimer’s disease neuroinflammation is a well-recognized feature of ad, with activation of microglia, inflammasomes, and complement. over the years, a number of infectious agents have been postulated to trigger this inflammation and participate in disease pathogenesis. the aβ peptide itself has even been demonstrated to have antimicrobial properties, supporting this hypothesis. one candidate pathogenic agent that has emerged is porphyromonas gingivalis, a pathogen that plays a critical role as a keystone pathogen in chronic periodontitis (cp). previously, among other supportive data, p. gingivalis was shown to be associated with cognitive decline in ad patients compared to controls without cp. furthermore, oral infection of apoe-/transgenic mice resulted in brain infection and complement activation. in a paper published in science advances, researchers led by drs. jan potempa (university of louisville) and stephen s. dominy (cortexyme) have provided the strongest evidence yet that infiltration of the brain with p. gingivalis might be causal and that their toxicity is through their virulence factors termed gingipains (dominy et al., 2019). gingipains are secreted cysteine proteases that play critical roles related to host colonization, inactivation of host defenses, iron/nutrient acquisition, and destruction of tissue. these researchers demonstrated the presence of gingipains in post-mortem ad brain tissue alongside the presence of p. gingivalis dna. remarkably, treatment with a small molecule gingipain inhibitor blocks gingipain-induced neurodegeneration in an animal model with an associated decrease in host aβ response to p. gingivalis. this orally available gingipain inhibitor is currently being tested in human clinical studies. more work needs to be done, and it is anticipated that this work might trigger a new research trend around periodontal disease in ad. 7. deconstructing cerebellar degeneration using “patholog-omics” essential tremor (et) is a common condition that can be associated with significant morbidity. for many years, the neuropathological features underlying et had been a complete mystery, with autopsy studies being limited and qualitative. recently, investigators have been building large cohorts of et patients and a series of et-related neuropathological changes have been reported, but many had been observed in other neurodegenerative diseases affecting the cerebellum, raising questions of specificity. how these features and the other cerebellar degenerations are related had never been formally or systematically studied. in what could be considered a tour de force, investigators led by drs. elan louis (yale university) and phyllis faust (columbia university) published as landmark paper in acta neuropathologica applying what they termed a “patholog-omics” approach using 37 quantitative morphological metrics (louis et al., 2019). they compared 156 brains from healthy controls and patients with a spectrum of cerebellar diseases, including essential tremor, spinocerebellar ataxia, multiple system atrophy, parkinson’s disease, and dystonia, mapping unique cellular patterns of neurodegeneration. measures included purkinje cell loss, axonal changes (e.g., torpedoes), basket cell hypertrophy, climbing fiber synaptic changes and others. they then used principal component analysis to derive distinctive and overlapping signatures marking each of these disorders. this study, which is the first of its kind to attempt to apply “omics” approaches to histomorphology, paves the way towards applying the approach to other neurodegenerative diseases. 8. chronic traumatic encephalopathy and american football: a dose-response relationship while the fact that repetitive mild traumatic brain injury leads to devastating long-term sequelae in professional boxers has been recognized for close to a century without controversy, chronic traumatic encephalopathy (cte) only recently began to achieve substantial scientific scrutiny and public interest when it was identified in american football players. while there is strong evidence that repetitive head injuries are causal for cte, skeptics remain. among the criticisms is that while clinicopathological studies are powerful, they are association analyses that ostensibly cannot prove a causal relationship on their own. this is not the whole story. in 1965, sir austin bradford hill, an english statistician, proposed nine criteria that, when met, provide epidemiologic evidence of causality. in terms of cte, eight of these criteria had been met, including strength (effect size), reproducibility, specificity, temporality, plausibility, coherence, experimentation and analogy. the missing criterion, until now, was evidence of a biological gradient: the presence of a dose-response relationship showing that increased exposure leads to a greater incidence/magnitude of the effect. in a report published online in november 2019 in the annals of neurology, researchers led by drs. jesse mez and ann c. mckee (boston university) measured exposure to contact sports in the largest autopsy series of american football players ever assembled and calculated cte risk. they found that the odds of cte doubled with every 2.6 years of american football played (mez et al., 2020). this paper firmly establishes that there is a dose-response relationship between exposure to american football and cte, which the strongest indicator of causality obtainable. this study also provides hard data for athletes and families trying to determine how much exposure might be considered safe. 9. parkinson’s disease: the gut-brain connection the pathological spread of abnormal protein aggregates through “prion-like” templated misfolding is strongly implicated in a host of neurodegenerative diseases. parkinson’s disease is no exception, with rigorous neuroanatomical studies by heiko braak and colleagues describing a hierarchical spread of α-synuclein pathology caudally to rostrally in the central nervous system (braak et al., 2003). parkinson’s disease is notable in that it is not restricted to the central nervous system, with α-synuclein aggregates present throughout the body including the gastrointestinal tract. this, together with the fact that the dorsal motor nucleus of the vagus nerve, which heavily innervates the gut, is among the first regions to show α-synuclein pathology, has fueled investigation around whether parkinson’s disease may be initiated in the gut. this hypothesis is plausible given that a number of cohort studies of human subjects have a reduction in parkinson’s disease risk following truncal vagotomy. further, injection of various forms of human or recombinant α-synuclein fibrils into the gut at various sites leads to central pathology, but this pathology was minimal and transient (holmqvist et al., 2014; manfredsson et al., 2018; uemura et al., 2018). investigators at the johns hopkins university school of medicine published a manuscript in neuron showing results using a new experimental paradigm, which demonstrated severe and widespread α-synuclein pathology in the brains of transgenic mice following injection of preformed fibrils in the gut (kim et al., 2019). they injected α-synuclein fibrils directly into the muscularis layer of the duodenum and pylorus and tracked the progression of pathological aggregate generation to the dorsal motor nucleus, caudal hindbrain, and onward to the substantia nigra with accompanying cell loss and functional impairment. truncal vagotomy blocked this spread, confirming the direct spread through this nerve, confirming what was seen previously. further, knockout of α-synuclein prevented the spread as well, indicating that the propagation is dependent on the molecule. this new animal model provides a robust experimental system, and together with the previous studies adds to the growing trend focusing on gut to brain transmission in parkinson’s disease. 10. huntington’s disease: a spotlight on astrocytes astrocytes play a critical role in a broad spectrum of nervous system functions, through direct interactions with neurons, blood vessels and other glial cell populations. thus, it is not surprising that they have been implicated in a number of neurodegenerative disorders. glial pathology is well-documented in tauopathies (e.g., tufted astrocytes of psp) and synucleinopathies (e.g., papp-lantos bodies in oligodendrocytes in msa). huntington’s disease (hd) principally affects the striatum and cortico-striatal-thalamic circuitry with medium spiny neurons being most vulnerable. intriguingly, the huntingtin gene (htt) gene is expressed in all cell types, but abnormalities in astrocytes have been demonstrated in post-mortem hd brains tissue. astrocytes express mutant htt (mhtt) with increased gfap expression and reactive changes. reactive astrocytosis is a constant feature of neurodegeneration that accompanies neuronal loss, but how these changes contribute to disease and the extent to which they are secondary changes or contribute causally is unclear. emerging evidence from transcriptomic studies has begun to paint a picture suggesting that astrocytes may not be passive bystanders, but may play an active role in some diseases, including hd. these studies have revealed two broad categories of astrocytes, termed a1 and a2, with a1 being neurotoxic (liddelow et al., 2017; zamanian et al., 2012). these and other studies prompted a group of investigators at ucla, led by dr. baljit s. khakh, to take a closer look at astrocytes in hd. their study, published in october 2019 in science translational medicine, used transcriptomic approaches in post-mortem human brain tissue from 36 huntington’s disease patients, vonsattel stage 0-3, alongside two transgenic mouse models to probe astrocyte pathology in hd (diaz-castro et al., 2019). in their transgenic animals, they deployed a powerful approach, termed ribotag, which allowed them to specifically profile rna expression in astrocytes. the result of this analysis was the elucidation of a novel core disease-associated astrocyte signature consisting of 62 genes seen in both human post-mortem brain and the transgenic animals. remarkably, alterations in this profile could be ameliorated in their models using transcriptional repression of the mhtt specifically in astrocytes using zinc finger protein (zfp) transcriptional repressors. these findings are certain to accelerate the intensification of interest in gliobiology in hd and other neurodegenerative disorders. references braak, h., del tredici, k., rüb, u., de vos, r. a. i., jansen steur, e. n. h., & braak, e. (2003). staging of brain pathology related to sporadic parkinson’s disease. neurobiology of aging, 24(2), 197–211. https://doi.org/10.1016/s0197-4580(02)00065-9 del-aguila, j. l., li, z., dube, u., mihindukulasuriya, k. a., budde, j. p., fernandez, m. v., ibanez, l., bradley, j., wang, f., bergmann, k., davenport, r., morris, j. c., holtzman, d. m., perrin, r. j., benitez, b. a., dougherty, j., cruchaga, c., & harari, o. (2019). a single-nuclei rna sequencing study of mendelian and sporadic ad in the human brain. alzheimer’s research & therapy, 11(1), 71. https://doi.org/10.1186/s13195-019-0524-x diaz-castro, b., gangwani, m. r., yu, x., coppola, g., & khakh, b. s. (2019). astrocyte molecular signatures in huntington’s disease. science translational medicine, 11(514), eaaw8546. https://doi.org/10.1126/scitranslmed.aaw8546 dickson, d.w., davies, p., bevona, c., van hoeven, k.h., factor, s.m., grober, e., aronson, m.k., crystal, h.a. (1994) hippocampal sclerosis: a common pathological feature of dementia in very old (> or = 80 years of age) humans. acta neuropathol 88(3), 212–221. https://doi.org/10.1007/bf00293396 dominy, s. s., lynch, c., ermini, f., benedyk, m., marczyk, a., konradi, a., nguyen, m., haditsch, u., raha, d., griffin, c., holsinger, l. j., arastu-kapur, s., kaba, s., lee, a., ryder, m. i., potempa, b., mydel, p., hellvard, a., adamowicz, k., … potempa, j. (2019). porphyromonas gingivalis in alzheimer’s disease brains: evidence for disease causation and treatment with small-molecule inhibitors. science advances, 5(1), eaau3333. https://doi.org/10.1126/sciadv.aau3333 grubman, a., chew, g., ouyang, j. f., sun, g., choo, x. y., mclean, c., simmons, r. k., buckberry, s., vargas-landin, d. b., poppe, d., pflueger, j., lister, r., rackham, o. j. l., petretto, e., & polo, j. m. (2019). a single-cell atlas of entorhinal cortex from individuals with alzheimer’s disease reveals cell-type-specific gene expression regulation. nature neuroscience, 22(12), 2087–2097. https://doi.org/10.1038/s41593-019-0539-4 holmqvist, s., chutna, o., bousset, l., aldrin-kirk, p., li, w., björklund, t., wang, z.-y., roybon, l., melki, r., & li, j.-y. (2014). direct evidence of parkinson pathology spread from the gastrointestinal tract to the brain in rats. acta neuropathologica, 128(6), 805–820. https://doi.org/10.1007/s00401-014-1343-6 kim, s., kwon, s.-h., kam, t.-i., panicker, n., karuppagounder, s. s., lee, s., lee, j. h., kim, w. r., kook, m., foss, c. a., shen, c., lee, h., kulkarni, s., pasricha, p. j., lee, g., pomper, m. g., dawson, v. l., dawson, t. m., & ko, h. s. (2019). transneuronal propagation of pathologic α-synuclein from the gut to the brain models parkinson’s disease. neuron, 103(4), 627-641.e7. https://doi.org/10.1016/j.neuron.2019.05.035 liddelow, s. a., guttenplan, k. a., clarke, l. e., bennett, f. c., bohlen, c. j., schirmer, l., bennett, m. l., münch, a. e., chung, w.-s., peterson, t. c., wilton, d. k., frouin, a., napier, b. a., panicker, n., kumar, m., buckwalter, m. s., rowitch, d. h., dawson, v. l., dawson, t. m., … barres, b. a. (2017). neurotoxic reactive astrocytes are induced by activated microglia. nature, 541(7638), 481–487. https://doi.org/10.1038/nature21029 louis, e. d., kerridge, c. a., chatterjee, d., martuscello, r. t., diaz, d. t., koeppen, a. h., kuo, s.-h., vonsattel, j.-p. g., sims, p. a., & faust, p. l. (2019). contextualizing the pathology in the essential tremor cerebellar cortex: a patholog-omics approach. acta neuropathologica, 138(5), 859–876. https://doi.org/10.1007/s00401-019-02043-7 manfredsson, f. p., luk, k. c., benskey, m. j., gezer, a., garcia, j., kuhn, n. c., sandoval, i. m., patterson, j. r., o’mara, a., yonkers, r., & kordower, j. h. (2018). induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient cns pathology. neurobiology of disease, 112, 106–118. https://doi.org/10.1016/j.nbd.2018.01.008 maniatis, s., äijö, t., vickovic, s., braine, c., kang, k., mollbrink, a., fagegaltier, d., andrusivová, ž., saarenpää, s., saiz-castro, g., cuevas, m., watters, a., lundeberg, j., bonneau, r., & phatnani, h. (2019). spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis. science, 364(6435), 89–93. https://doi.org/10.1126/science.aav9776 mathys, h., davila-velderrain, j., peng, z., gao, f., mohammadi, s., young, j. z., menon, m., he, l., abdurrob, f., jiang, x., martorell, a. j., ransohoff, r. m., hafler, b. p., bennett, d. a., kellis, m., & tsai, l.-h. (2019). single-cell transcriptomic analysis of alzheimer’s disease. nature, 570(7761), 332–337. https://doi.org/10.1038/s41586-019-1195-2 mez, j., daneshvar, d. h., abdolmohammadi, b., chua, a. s., alosco, m. l., kiernan, p. t., evers, l., marshall, l., martin, b. m., palmisano, j. n., nowinski, c. j., mahar, i., cherry, j. d., alvarez, v. e., dwyer, b., huber, b. r., stein, t. d., goldstein, l. e., katz, d. i., … mckee, a. c. (2020). duration of american football play and chronic traumatic encephalopathy. annals of neurology, 87(1), 116–131. https://doi.org/10.1002/ana.25611 nelson, p. t., dickson, d. w., trojanowski, j. q., jack, c. r., boyle, p. a., arfanakis, k., rademakers, r., alafuzoff, i., attems, j., brayne, c., coyle-gilchrist, i. t. s., chui, h. c., fardo, d. w., flanagan, m. e., halliday, g., hokkanen, s. r. k., hunter, s., jicha, g. a., katsumata, y., … schneider, j. a. (2019). limbic-predominant age-related tdp-43 encephalopathy (late): consensus working group report. brain: a journal of neurology, 142(6), 1503–1527. https://doi.org/10.1093/brain/awz099 oh, j., eser, r. a., ehrenberg, a. j., morales, d., petersen, c., kudlacek, j., dunlop, s. r., theofilas, p., resende, e. d. p. f., cosme, c., alho, e. j. l., spina, s., walsh, c. m., miller, b. l., seeley, w. w., bittencourt, j. c., neylan, t. c., heinsen, h., &grinberg, l. t. (2019). profound degeneration of wake-promoting neurons in alzheimer’s disease. alzheimer’s & dementia, 15(10), 1253–1263. https://doi.org/10.1016/j.jalz.2019.06.3916 signaevsky, m., prastawa, m., farrell, k., tabish, n., baldwin, e., han, n., iida, m. a., koll, j., bryce, c., purohit, d., haroutunian, v., mckee, a. c., stein, t. d., white, c. l., walker, j., richardson, t. e., hanson, r., donovan, m. j., cordon-cardo, c., … crary, j. f. (2019). artificial intelligence in neuropathology: deep learning-based assessment of tauopathy. laboratory investigation; a journal of technical methods and pathology, 99(7), 1019–1029. https://doi.org/10.1038/s41374-019-0202-4 ståhl, p. l., salmén, f., vickovic, s., lundmark, a., navarro, j. f., magnusson, j., giacomello, s., asp, m., westholm, j. o., huss, m., mollbrink, a., linnarsson, s., codeluppi, s., borg, å., pontén, f., costea, p. i., sahlén, p., mulder, j., bergmann, o., … frisén, j. (2016). visualization and analysis of gene expression in tissue sections by spatial transcriptomics. science, 353(6294), 78–82. https://doi.org/10.1126/science.aaf2403 tang, z., chuang, k. v., decarli, c., jin, l.-w., beckett, l., keiser, m. j., &dugger, b. n. (2019). interpretable classification of alzheimer’s disease pathologies with a convolutional neural network pipeline. nature communications, 10(1), 2173. https://doi.org/10.1038/s41467-019-10212-1 uemura, n., yagi, h., uemura, m. t., hatanaka, y., yamakado, h., & takahashi, r. (2018). inoculation of α-synuclein preformed fibrils into the mouse gastrointestinal tract induces lewy body-like aggregates in the brainstem via the vagus nerve. molecular neurodegeneration, 13(1), 21. https://doi.org/10.1186/s13024-018-0257-5 zamanian, j. l., xu, l., foo, l. c., nouri, n., zhou, l., giffard, r. g., & barres, b. a. (2012). genomic analysis of reactive astrogliosis. the journal of neuroscience: the official journal of the society for neuroscience, 32(18), 6391–6410. https://doi.org/10.1523/jneurosci.6221-11.2012 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathologists play a key role in establishing the extent of covid-19 in human patients feel free to add comments by clicking these icons on the sidebar free neuropathology 1:11 (2020) opinion piece neuropathologists play a key role in establishing the extent of covid-19 in human patients lokman cevik, michele joana alves, josé javier otero div. of neuropathology, dept. of pathology, the ohio state university school of medicine, columbus, oh, usa corresponding author: josé javier otero, md, phd · the ohio state university college of medicine · department of pathology · 4166 graves hall · 333 w 10th avenue · columbus, oh 43210 · usa jose.otero@osumc.edu submitted: 01 april 2020 accepted: 02 april 2020 published: 02 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2736 keywords: covid-19, sars-cov2 abstract sars-cov2 infection causes covid-19, and represents the most emergent health care crisis of our generation. ample evidence in the scientific literature suggests that sars-cov, mers-cov, and endemic human coronaviruses infect brain cells. we delineate a rationale for encouraging evaluation of the brain, and in particular the brainstem, in covid-19 so that potential neuropathological mechanisms can be delineated.   sars-cov2 infection causes covid-19, and represents the most emergent health care crisis of our generation. sars-cov2 is a member of the betacoronavirus family that includes the severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), both of which have caused fatal infections in the past two decades (huang et al., 2020). although most people recover from the disease, sars-cov2 can cause severe respiratory distress syndrome, particularly in older patients and patients with underlying comorbidities (mahase, 2020). most of the patients who require intensive care ultimately become unable to breathe spontaneously (wang et al., 2020). although sars-cov2 shows its effects predominantly on the respiratory system, the primary pathophysiology behind the respiratory dysfunction and mortality remains elusive. previous findings of sars-cov infection and other coronaviruses in the nervous system bring to mind the possibility that sars-cov2 infection can cause respiratory failure by disrupting the cardiorespiratory center in the brainstem and are briefly reviewed below. it is known that most coronaviruses share similar viral structures and infection pathways (baig et al., 2020). these structural and pathophysiological similarities between other coronaviruses and sars-cov2 likely indicate that pathophysiological insights from other coronavirus studies may be generalizable to sars-cov2. sars-cov and sars-cov2 have high protein homology (baig et al., 2020), and sars-cov2 uses the same receptor to enter the host cells with at least 10 times higher affinity relative to sars-cov (wrapp et al., 2020). although multiple candidate receptors have been proposed, cellular infection through interaction with the angiotensin (ang) converting enzyme (ace2), a transmembrane carboxypeptidates sharing homology to ace1’s extracellular domain but with unique transmembrane and intracellular domains (riordan, 2003), seems to be critical in the pathogenesis in sars-cov (baig et al., 2020). although ace2 is capable of modifying angiotensin i, it catalyzes angiotensin i to ang(1-9) rather than to ang ii, and plays diverse physiological roles (reviewed by (clarke and turner, 2012)). ace2 is expressed in airway epithelia, lung parenchyma, vascular endothelia, kidney cells, small intestine cells (li et al., 2020) and also in the brain, particularly in glial cells and neurons (baig et al., 2020). we also note that endemic human covs have neuroinvasive potential (desforges et al., 2014). with this in mind, several studies have explored the extent to which zoonotically transmitted covs affect human brain function. studies using different mouse lines transgenic for the expression of ace2 showed extensive virus replication in the brain, likely mediated through retrograde transport through the olfactory bulb. among the brain regions affected by the virus, thalamus, cerebrum and brainstem were severely impacted. while in the k18-hace2 transgenic line, the sars-cov infection induces neuronal death as a result directly from the neuronal and not pulmonary infection. an abundant neuronal loss was found along with increased inflammatory cytokines, and proliferation of microglia, but not of astrocytes (mccray et al., 2007; netland et al., 2008). additional studies using transgenic mice demonstrated enhanced levels of il-6, il-12p40, g-csf, cxcl1, mip-a and mcp-1 in brain homogenates 3 days after the infection resulting in an inflammatory cytokine reaction (tseng et al., 2007). such cytokine elevations and extensive neuronal pathologies in the brainstem were also noted in mers-cov infections (li et al., 2016). in this same study, the authors showed evidence of viral replication in primary and porcine astrocytes and human glioblastoma and neuroblastoma cell lines (li et al., 2016). it is not well established how sars-cov infection affects astrocytes in vivo and how it mediates the neuronal damage of this syndrome, despite the fact that ace2 is expressed in isolated astrocytes from brainstem, cerebellum and medulla (gallagher et al., 2006; gowrisankar and clark, 2016). on the other hand, expression of ace2 at mrna and protein levels in neurons is also documented. in vivo findings have shown ace2 expression in neurons of the paraventricular nucleus (pvn), area postrema (ap), dorsal motor nucleus of the vagus (dmnv), nucleus of tractus solitarii (nts), the rostroventrolateral medulla (rvlm), and the nucleus ambiguous (na), all brain structures related to cardiovascular and respiratory function (doobay et al., 2007). furthermore, we do not know the extent to which sars-cov affects brains of newborn babies. for instance, a timely study emerging from china performed on covid-19 outcomes on pediatric patients demonstrated that the severity of covid-19 in children less than 1-year-old was very high (53.8% showing critical course) relative to other age groups (dong et al., 2020). neurological manifestations of covid-19 have also been documented. in a preprint study from china, one of 3 patients suffering from sars-cov2 had neurological manifestations, including dizziness, headache, impaired consciousness, hypogeusia (reduced ability to taste) and hyposmia (reduced ability to smell) (mao et al., 2020), the latter symptoms suggesting neuronal involvement of areas in proximity to the olfactory bulb. another possibility of trans-synaptic transfer of sars-cov2 is the usage of neuroanatomic interconnections of the respiratory and gastrointestinal system to nuclei of the brainstem as has been noted by the avian influenza virus (li et al., 2020). finally, sars-cov2 could disseminate through the blood to the brain via crossing endothelial cells that express the ace2 receptor as another way of transfer (baig et al., 2020). in conclusion, there is growing evidence that the brain could be the main trigger in the severity of covid-19, but there is a paucity of evidence derived from human patients. according to the interim guidance of the american centers for disease control (cdc) in march 2020, cdc recommends collecting swab specimens, samples for postmortem microbiologic and infectious disease testing, formalin-fixed autopsy tissues from lung, upper airway, and other major organs, if an autopsy is performed for a confirmed covid-19 case (center of disease control and prevention, 2020, march 25). however, additional concerns have also been raised regarding the use of oscillating saws, a tool commonly used during brain procurement, as these saws have been shown to promote aerosolization. nevertheless, we believe that documenting the extent of cns involvement in severe manifestations, including documenting which brainstem nuclei may be affected in the autopsies is crucial to clarify the pathophysiology of covid-19. we also recognize that in areas ravaged by the scale of infection and covid-19 disease, investment in personal protective equipment for autopsy would represent an unwise decision when front-line workers remain unprotected. we therefore suggest that a coordinated effort between health systems work together to meet this goal. drawing inspiration from the neuropathological evaluations of brains of hiv infected patients (petito et al., 2003), we suggest that a study design composed of 20 brains procured from covid-19 infected decedents be performed, with extensive sampling of brainstem nuclei to include structures implicated in human control of respiration, including the locus coeruleus (nobuta et al., 2015), ventral medulla (rudzinski and kapur, 2010), and prebotzinger complex (schwarzacher et al., 2011). it would also be important to evaluate covid19 infected decedents without significant neurological manifestations as potential controls. in this way, highly impactful descriptive studies of covid-19 disease can be achieved and the burden of investing in personal protective equipment can be shared by various centers. references baig, a.m., khaleeq, a., ali, u., syeda, h., 2020. evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms. acs chem neurosci. center of disease control and prevention, 2020, march 25. collection and submission of postmortem specimens from deceased persons with known or suspected covid-19, march 2020 (interim guidance). clarke, n.e., turner, a.j., 2012. angiotensin-converting enzyme 2: the first decade. int j hypertens 2012, 307315. desforges, m., le coupanec, a., brison, e., meessen-pinard, m., talbot, p.j., 2014. neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans. adv exp med biol 807, 75-96. dong, y., mo, x., hu, y., qi, x., jiang, f., jiang, z., tong, s., 2020. epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china-10.1542/peds.2020-0702 pediatrics. doobay, m.f., talman, l.s., obr, t.d., tian, x., davisson, r.l., lazartigues, e., 2007. differential expression of neuronal ace2 in transgenic mice with overexpression of the brain renin-angiotensin system. am j physiol regul integr comp physiol 292, r373-381. gallagher, p.e., chappell, m.c., ferrario, c.m., tallant, e.a., 2006. distinct roles for ang ii and ang-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. am j physiol cell physiol 290, c420-426. gowrisankar, y.v., clark, m.a., 2016. angiotensin ii regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. j neurochem 138, 74-85. huang, c., wang, y., li, x., ren, l., zhao, j., hu, y., zhang, l., fan, g., xu, j., gu, x., cheng, z., yu, t., xia, j., wei, y., wu, w., xie, x., yin, w., li, h., liu, m., xiao, y., gao, h., guo, l., xie, j., wang, g., jiang, r., gao, z., jin, q., wang, j., cao, b., 2020. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet 395, 497-506. li, k., wohlford-lenane, c., perlman, s., zhao, j., jewell, a.k., reznikov, l.r., gibson-corley, k.n., meyerholz, d.k., mccray, p.b., jr., 2016. middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4. j infect dis 213, 712-722. li, y.c., bai, w.z., hashikawa, t., 2020. the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients. j med virol. mahase, e., 2020. coronavirus covid-19 has killed more people than sars and mers combined, despite lower case fatality rate. bmj 368, m641. mao, l., wang, m., chen, s., he, q., chang, j., hong, c., zhou, y., wang, d., li, y., jin, h., hu, b., 2020. neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study. medrxiv, 2020.2002.2022.20026500. mccray, p.b., jr., pewe, l., wohlford-lenane, c., hickey, m., manzel, l., shi, l., netland, j., jia, h.p., halabi, c., sigmund, c.d., meyerholz, d.k., kirby, p., look, d.c., perlman, s., 2007. lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus. j virol 81, 813-821. netland, j., meyerholz, d.k., moore, s., cassell, m., perlman, s., 2008. severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2. j virol 82, 7264-7275. nobuta, h., cilio, m.r., danhaive, o., tsai, h.h., tupal, s., chang, s.m., murnen, a., kreitzer, f., bravo, v., czeisler, c., gokozan, h.n., gygli, p., bush, s., weese-mayer, d.e., conklin, b., yee, s.p., huang, e.j., gray, p.a., rowitch, d., otero, j.j., 2015. dysregulation of locus coeruleus development in congenital central hypoventilation syndrome. acta neuropathologica. petito, c.k., adkins, b., mccarthy, m., roberts, b., khamis, i., 2003. cd4+ and cd8+ cells accumulate in the brains of acquired immunodeficiency syndrome patients with human immunodeficiency virus encephalitis. j neurovirol 9, 36-44. riordan, j.f., 2003. angiotensin-i-converting enzyme and its relatives. genome biol 4, 225. rudzinski, e., kapur, r.p., 2010. phox2b immunolocalization of the candidate human retrotrapezoid nucleus. pediatr dev pathol 13, 291-299. schwarzacher, s.w., rub, u., deller, t., 2011. neuroanatomical characteristics of the human pre-botzinger complex and its involvement in neurodegenerative brainstem diseases. brain 134, 24-35. tseng, c.t., huang, c., newman, p., wang, n., narayanan, k., watts, d.m., makino, s., packard, m.m., zaki, s.r., chan, t.s., peters, c.j., 2007. severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor. j virol 81, 1162-1173. wang, d., hu, b., hu, c., zhu, f., liu, x., zhang, j., wang, b., xiang, h., cheng, z., xiong, y., zhao, y., li, y., wang, x., peng, z., 2020. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. wrapp, d., wang, n., corbett, k.s., goldsmith, j.a., hsieh, c.l., abiona, o., graham, b.s., mclellan, j.s., 2020. cryo-em structure of the 2019-ncov spike in the prefusion conformation. science 367, 1260-1263. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. the definition and role of brain invasion in meningioma grading: still controversial after all these years feel free to add comments by clicking these icons on the sidebar free neuropathology 2:8 (2021) opinion piece the definition and role of brain invasion in meningioma grading: still controversial after all these years arie perry department of pathology, division of neuropathology, university of california san francisco (ucsf), san francisco, ca 94143, usa corresponding author: arie perry · department of pathology, division of neuropathology · university of california, san francisco (ucsf) · 505 parnassus avenue, m551, box 0102 · san francisco, ca 94143 · usa arie.perry@ucsf.edu submitted: 05 march 2021 accepted: 23 march 2021 copyedited by: nima sharifai published: 24 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3276 keywords: meningioma, brain invasion, grading in their landmark 1938 monograph on meningiomas, cushing and eisenhardt stated that brain invasion, defined similarly to today by tumoral penetration of the pia, had long been considered a sign of malignancy1. although this was accepted as undisputed gospel over the following decades, the precise role of brain invasion in meningioma grading and how to best define it remains unanswered nearly a century later. for instance, in 1993, mclean and colleagues questioned the role of brain invasion in grading, since they found no additional prognostic significance of this finding within their 28 otherwise atypical and anaplastic meningiomas; unfortunately, they did not address the controversial issue of brain invasive otherwise benign (biob) meningiomas2. similarly, the second edition of the world health organization (who) classification scheme in 1993 highlighted that “some participants maintained that gross brain invasion, not only metastasis, qualifies a tumor for the designation malignant; such lesions do not invariably exhibit histological malignancy and their immunocytochemical features may be similar to those of ordinary meningiomas”3. i suspect that the term “gross” in this phrase meant well-developed or extensive, rather than the surgeon’s gross impression. of course, the significance of brain invasion is a topic near and dear to my heart, as it was one of the first questions i tried to tackle in my neuropathology career, first in a 1997 mayo clinic clinicopathology study of meningioma grading criteria and then in a followup 1999 series addressing the issue of brain invasion and other pathologically defined forms of potential malignancy4,5. along with my mentor, bernd scheithauer, scott stafford (radiation oncology), and other collaborators, we proposed a grading scheme based on statistically significant associations between histopathologic variables and estimated patient survival times (recurrence-free and overall). a fact that is often forgotten today is that these associations were specifically limited to the cohort of patients treated with gross total resection, given that subtotally resected meningiomas frequently recurred regardless of microscopic features. to a large extent, our grading criteria were adopted in the 3rd edition of the who scheme in 20006 and have remained fairly intact ever since. based on our data that biob meningiomas had recurrence and mortality rates similar to atypical meningiomas without brain invasion, it was emphasized that such cases are prognostically equivalent to who grade ii meningiomas. however, it was not until the revised 4th edition of the who in 2016 that brain invasion was adopted as a definitive criterion for the diagnosis of atypical meningioma, who grade ii7. figure 1. examples of well-developed and extensive brain invasion that would likely be associated with high interobserver reproducibility. a, b. note the tongue-like or finger-like protrusions into adjacent brain with no obvious leptomeningeal layer between the two. c. a gfap stain shows entrapped slivers of gliotic cns parenchyma deep within the main tumor mass (upper portion). d. this brain invasive otherwise benign (biob) meningioma showed benign histologic features, including extensive progesterone receptor expression. images c and d are courtesy of dr. robert schmidt, washington university, st. louis, mo, usa. given that we found brain invasion in 4% of cases in our 1997 series, and just 23% of brain invasive meningiomas were otherwise benign in our 1999 expanded series, i expected that this classification change in 2016 would only have a minimal impact on clinical practice (with an expected increase in the incidence of who grade ii meningiomas of 1% at best). in other words, this would be the estimate if biob meningiomas only account for a fourth of the roughly 4% of brain invasive tumors, assuming that everyone was previously diagnosing such cases as who grade i rather than ii, which is clearly not true since some were already designating such cases as grade ii. to my surprise, however, i have personally noticed a mini-epidemic in biob meningiomas in my personal consults and especially in a clinical trial for which i am serving as the central pathology reviewer. of interest, i previously served in a similar role for a pre-2016 rtog/nrg clinical trial that included meningiomas of all three grades; in this series we found a strong concordance rate of 92.4% between the home pathologist’s interpretations of brain invasion and my own (kappa statistic 0.76)8. however, in this post-2016 followup trial focused specifically on atypical meningiomas, there have already been quite a few cases that i have rejected because they initially qualified as grade ii based on focal brain invasion alone, but i disagreed with that call during my central review. admittedly, this is merely an anecdotal impression on my part rather than hard data, given that the study is still accruing cases and no formal analyses have been performed. nonetheless, it might not be surprising if people have loosened their minimal criteria for calling brain invasion after the 2016 who, since it is only human nature (especially among pathologists) to not want to miss even the tiniest example of something if it has a potentially important association with outcome. however, i would argue the opposite approach in favor of stricter criteria so that one does not dilute the potential impact of this prognostic marker with questionable examples. in other words, if brain invasion is the only reason to upgrade a tumor, then i would rather it to be irrefutable before assigning that higher grade. figure 2. a, b. most meningiomas with attached cns parenchyma can be clearly interpreted as being non-invasive, given the broad, linear interface and clear leptomeningeal layer between the tumor and adjacent brain parenchyma. additionally, the modified who 2016 criteria sparked a renewed interest in this controversy and some of the subsequent clinicopathologic series have failed to find any prognostic differences between their biob and non-invasive who grade i meningiomas9-11. in contrast, a 2016 study of 206 who grade ii meningiomas found brain invasion to be an independent negative prognostic variable on multivariate analysis12. another study of who grade ii meningiomas found that brain invasion was prognostic only if specifically combined with the presence of necrosis13, even though a non-invasive meningioma with necrosis only would normally be considered as otherwise benign. for a more detailed summary of the strengths and limitations of previously published studies on this topic, the reader is referred to the review of brokinkel, hess, and mawrin14. suffice it to say that there are still no large, highly powered studies of strictly defined, gross totally resected biob meningiomas with long clinical followup times, given the overall rarity of such cases. as such, this question remains unanswered and the upcoming 5th edition of the who (in press) will include the following text on the question of brain invasion: “brain invasion by meningioma is characterized by irregular, tongue-like protrusions of tumour cells into underlying gfap-positive parenchyma, without intervening leptomeninges. extension along perivascular virchow-robin spaces does not constitute brain invasion because the pia is not breached. such perivascular spread and hyalinization is most commonly encountered in children and can mimic meningioangiomatosis, but does not constitute true brain invasion15,16. brain invasion occurs most often in meningiomas with additional high-grade features. nonetheless, the presence of brain invasion in gross totally resected, otherwise benign-appearing meningiomas remains controversial, as it has been associated with recurrence rates similar to other who grade 2 meningiomas in some, but not all studies5,10,11; larger series with longer follow-up times may be needed to resolve this issue.” given the issues raised above, i decided to write this opinion piece and offer my personal views on what does or does not constitute brain invasion, recognizing that this is at best an educated guess and there is no guarantee that i am any more likely to be correct than authors with opposing viewpoints. as a reminder, most cases with brain invasion will qualify for a higher histologic grade using other criteria anyway, and cases with absolutely no other worrisome features are generally rare. nevertheless, when encountering a biob meningioma on routine histopathology, it is perhaps best at this time to: 1) obtain gfap and other immunostains (ihc) to clarify the brain invasion in borderline cases, 2) order molecular studies to look for other prognostically relevant biomarkers (e.g., specific chromosomal losses, cdkn2a homozygous deletion, tert promoter mutation, etc.), 3) spell out the remaining uncertainties over the clinical significance of these rare cases in the pathology report, and/or 4) suggest close clinical followup just in case the tumor does behave in a more aggressive fashion. figure 3. challenging cases that are more likely to yield discordant interpretations. a. although the interface is irregular rather than linear, there is a discernible leptomeningeal layer between meningioma and brain; as such, i would not call this brain invasion. b. there is a focally irregular interface in this case and it is difficult to determine whether or not there is intervening leptomeninges; a gfap stain could help push me in one direction or the other in such a case. c, d. gfap stains in this case highlight a gently undulating tumor-cns interface; however, in contrast to figure 1c, there is minimal if any definite gfap-positive tissue within the main tumor mass. if this was the only worrisome feature in this meningioma, i would err on the side of caution and not accept this as definite brain invasion. in terms of minimal criteria for brain invasion, a common question is whether or not the surgeon’s intraoperative impression can serve as a surrogate. to my knowledge, all studies published to date have shown a much higher frequency of brain invasion on gross impression than that which is confirmed under the microscope, possibly due to cases with simple adhesion to leptomeninges being overcalled macroscopically. more importantly, with only one exception17, none of the published studies have found a statistical association with grossly diagnosed brain invasion and subsequent recurrence-free survival. as such, the gross intraoperative impression of brain invasion should not be considered a reliable surrogate. that said, of course, the detection of brain invasion is highly dependent on sampling issues and it therefore could be especially useful for the pathologist if the surgeon labels the tumor-brain interface or areas most suspicious for invasion intraoperatively. the importance of appropriate sampling has also been emphasized by other authors and, not surprisingly, the frequency of finding brain invasion increases with greater sampling or perhaps with additional guidance from the surgeon, more targeted sampling. that said, the reported frequency of brain invasion in 33% of otherwise benign meningiomas, by pizem and colleagues in 201418, seems remarkably excessive to me, even in meningiomas that are extensively sampled. in any case, i believe that the original definition of brain invasion, which requires a breach of the pial barrier, is still the best method. in my experience, the two scenarios that are most often mistaken for (or do not show sufficient evidence of) microscopic brain invasion are: 1) an irregular interface with adjacent brain that nevertheless shows an intervening layer of leptomeninges/collagen and 2) perivascular spread along virchow-robin spaces. examples of these and less controversial scenarios are illustrated. additionally, one should be cautious of overcalling brain invasion in highly cauterized or otherwise poorly preserved fragments of tissue. figure 4. a. there is extensive tumoral spread along perivascular virchow-robin spaces in this example, which could easily be misinterpreted as either brain invasion or associated meningioangiomatosis. however, careful examination shows the perivascular arrangement and since this is not considered a breach of the pial barrier, it does not qualify as brain invasion. this type of spread is often associated with radial collars of spindled tumor cells and hyalinization, similar to that seen in meningioangiomatosis. b. a gfap stain fails to show any convincingly entrapped glial tissue within the main tumor mass (upper portion). c. a somatostatin receptor 2a stain highlights the onion-like radial layers of perivascular tumor cells; note that the brain is also normally positive but displays a fibrillar staining pattern distinct from the spindled meningioma cells. d. the mostly centrally placed blood vessels within the virchow-robin spaces are highlighted on this smooth muscle actin immunostain. although this pattern of spread is more often encountered in pediatric patients, this one was from an adult. based on the above, my sincere plea is that we all adopt strict rather than loose criteria for brain invasion going forward, so that if this variable really does provide independent prognostic information, we will be in a better position to detect it. to quote the haarlem white paper from 2014, “diagnostic entities should be defined as narrowly as possible to optimize interobserver reproducibility”19 (or in this case a grading parameter rather than an entity). references 1. cushing h, eisenhardt l. meningiomas: their classification, regional behaviour, life history, and surgical end results. springfield, il: cc thomas; 1938. 2. mclean ca, jolley d, cukier e, giles g, gonzales mf. atypical and malignant meningiomas: importance of micronecrosis as a prognostic indicator. histopathology. 1993;23(4):349-353. 3. kleihues p, burger p, scheithauer b. histological typing of tumours of the central nervous system. in: sobin l, ed. world health organization. international histological classification of tumours. 2nd ed. berlin: springer; 1993. 4. perry a, stafford sl, scheithauer bw, suman vj, lohse cm. meningioma grading: an analysis of histologic parameters. am j surg pathol. 1997;21(12):1455-1465. 5. perry a, scheithauer bw, stafford sl, lohse cm, wollan pc. "malignancy" in meningiomas: a clinicopathologic study of 116 patients, with grading implications. cancer. 1999;85(9):2046-2056. 6. louis d, scheithauer b, budka h, von deimling a, kepes j. meningiomas. in: kleihues p, cavenee w, eds. world health organization classification of tumours. 3rd ed. lyon: iarc press; 2000:176–189. 7. perry a, louis dn, budka h, et al. chapter 10: meningioma. in: louis dn, ohgaki h, wiestler od, et al., eds. who classification of tumours of the central nervous system (revised 4th edition). lyon: iarc; 2016:232-245. 8. rogers cl, perry a, pugh s, et al. pathology concordance levels for meningioma classification and grading in nrg oncology rtog trial 0539. neuro oncol. 2016;18(4):565-574. 9. spille dc, hess k, sauerland c, et al. brain invasion in meningiomas: incidence and correlations with clinical variables and prognosis. world neurosurg. 2016;93:346-354. 10. baumgarten p, gessler f, schittenhelm j, et al. brain invasion in otherwise benign meningiomas does not predict tumor recurrence. acta neuropathol. 2016;132(3):479-481. 11. biczok a, jungk c, egensperger r, et al. microscopic brain invasion in meningiomas previously classified as who grade i is not associated with patient outcome. j neurooncol. 2019; 145(3):469-477. 12. champeaux c, dunn l. world health organization grade ii meningiomas. acta neurochir (wien). 2016. 13. garcia-segura me, erickson aw, jairath r, munoz dg, das s. necrosis and brain invasion predict radio-resistance and tumor recurrence in atypical meningioma: a retrospective cohort study. neurosurgery. 2021;88(1):e42-e48. 14. brokinkel b, hess k, mawrin c. brain invasion in meningiomas-clinical considerations and impact of neuropathological evaluation: a systematic review. neuro oncol. 2017;19(10):1298-1307. 15. giangaspero f, guiducci a, lenz fa, mastronardi l, burger pc. meningioma with meningioangiomatosis: a condition mimicking invasive meningiomas in children and young adults: report of two cases and review of the literature. am j surg pathol. 1999;23(8):872-875. 16. perry a, kurtkaya-yapicier o, scheithauer bw, et al. insights into meningioangiomatosis with and without meningioma: a clinicopathologic and genetic series of 24 cases with review of the literature. brain pathol. 2005;15(1):55-65. 17. behling f, fodi c, gepfner-tuma i, et al. cns invasion in meningioma-how the intraoperative assessment can improve the prognostic evaluation of tumor recurrence. cancers (basel). 2020;12(12). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurovascular disease feel free to add comments by clicking these icons on the sidebar free neuropathology 1:5 (2020) review top ten discoveries of the year: neurovascular disease anna m. planas department of brain ischemia and neurodegeneration, spanish national research council (csic), barcelona, spain corresponding author: anna m. planas · department of brain ischemia and neurodegeneration · institute for biomedical research of barcelona (iibb) · spanish national research council (csic) · institute for biomedical research august pi i sunyer (idibaps) · rosselló 161 · planta 6 · 08036-barcelona · spain anna.planas@iibb.csic.es submitted: 09 january 2020 accepted: 25 january 2020 published: 30 january 2020 https://doi.org/10.17879/freeneuropathology-2020-2615 abstract the aim of this review is to highlight novel findings in 2019 in the area of neurovascular disease. experimental studies have provided insight into disease development, molecular determinants of pathology, and putative novel therapeutic targets. studies in genetic experimental models as well as monogenic forms of human cerebrovascular diseases identified pathogenic molecules that may also be relevant to sporadic cases. there have been advances in understanding the development of cerebral cavernous angiomas and arteriovenous malformations, and putative curative treatments have been suggested from experimental models. key pathogenic pathways involved in vessel calcification and stiffness have also been identified. at the cellular level, studies showed that proper function of endothelial and mural cells, particularly pericytes, is crucial to ensure full endothelial differentiation and blood-brain barrier integrity. moreover, recent discoveries support the existence of a homeostatic crosstalk between vascular cells and other neural cells, including neurons. cerebrovascular diseases are strongly associated with inflammation. beyond pathogenic roles of specific components of the inflammatory response, new discoveries showed interesting interactions between inflammatory molecules and regulators of vascular function. clinical investigation on cerebrovascular diseases has progressed by combining advanced imaging and genome-wide association studies. finally, vascular cognitive impairment and dementia are receiving increasing attention. recent findings suggest that high-salt intake may cause cerebrovascular dysfunction and cognitive impairment independent of hypoperfusion and hypertension. these and other recent reports will surely inspire further research in the field of cerebrovascular disease that will hopefully contribute to improved prevention and treatment. abbreviations apoe, apolipoprotein e; avm, arteriovenous malformations; bbb, blood-brain barrier; caa, cerebral amyloid angiopathy; cadasil, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; carasil, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy; cbf, cerebral blood flow; ccm, cerebral cavernous angiomas; cdk5, cyclin-dependent kinase 5; dti, diffusion tensor imaging; endmt, endothelial-to-mesenchymal transition; gom, granular osmiophilic material; ipscs, induced pluripotent stem cells; pdgfb, platelet-derived growth factor subunit β; pfbc, primary familial brain calcification, fahr’s disease; sah, subarachnoid hemorrhage; smcs, smooth muscle cells; timp3, tissue inhibitor of metalloproteinase 3; tgfβ, transforming growth factor-β introduction in the next pages i will discuss a few topics in the field of cerebrovascular disease that i believe have made advances in 2019 supported by excellent studies covering genetic and sporadic forms of the diseases in humans and experimental animals. 1. cavernous angiomas originate from clonal expansion of mutated endothelial cells there have been recent advances in understanding the development of cerebral cavernous angiomas (ccm). new results suggest that small angiomas may derive from a clonal expansion of mutant endothelial cells that undergo endothelial-to-mesenchymal transition and express stem-cell like features (fig. 1). ccm are vascular lesions composed of clusters of capillary-venous blood vessels with thin walls and impaired endothelial tight junctions that usually contain stagnant blood. these abnormal vessels may rupture causing intracranial hemorrhage (fig. 1) or small subclinical bleeds, which may lead to seizures. most cases of ccm are spontaneous but 20% of cases may be attributed to autosomal dominant inheritance. the disease is caused by sporadic or inherited mutations in one of the following three genes: ccm1 (krit1), ccm2 (mgc4607), or ccm3 (pdcd10). in the affected vessels, endothelial cells suffer endothelial-to-mesenchymal transition (endmt), express stem cell markers, and undergo loss of typical endothelial cell properties, with increased cell migration, reduced adherence, and increased blood-brain barrier (bbb) permeability. in familial cases, the vascular lesions are discrete although the above genetic mutations affect all cells. furthermore, heterozygous mice do not develop the disease. the prevalent explanation is the two-hit hypothesis. endothelial cells carrying a germline mutation in one of the ccm genes need to suffer a second, likely a somatic, mutation that will trigger the pathology. malinverno et al. (2019) showed that only a few endothelial cells bearing a ccm3-/mutation, undergoing endmt, and expressing progenitor markers, are needed to generate cavernomas because the affected cells undergo clonal expansion. demonstration of clonal expansion was carried out using an elegant strategy by crossing the ‘r26r-confetti’ mouse, a multicolor reporter mouse model, first with cdh5(pac)-creert2 mice for an endothelial specific expression of cre, and then with ccm3fl/fl mice. endothelial deletion of ccm3 was accompanied by color-based clonal lineage tracing. the majority of small vascular lesions were composed of cells of the same color suggesting a clonal origin from proliferation of the affected endothelial cells. this study followed two previous studies (detter et al., 2018; manavski et al., 2018) where clonal expansion of mutant endothelial cell in ccm endmt was also demonstrated using a reporter confetti strategy. in contrast to the clonal origin of small lesions, large cavernomas are mosaics containing clonally dominant mutant cells and wild type endothelial cells suggesting recruitment of neighboring wild type cells to the cavernous lesion. those wild type endothelial cells also overexpressed endmt markers. transplantation of ccm3-/cells into the brain of wild type mice generated abnormal vessels and recruited endothelial cells from the host (malinverno et al., 2019). by means of several in vitro studies, these authors demonstrated that wild type endothelial cells acquire features of endmt after contacting ccm3-/cells. furthermore, by using genetic mouse models, malinverno et al. deleted ccm3 in endothelial progenitors. they hypothesized that the early steps of cavernoma formation are due to clonal expansion of resident endothelial progenitors. in this way they provided evidence supporting the view that endothelial progenitors suffering ccm3 mutation trigger cavernoma formation. hopefully these new discoveries will provide novel avenues to prevent or attenuate ccm. figure 1. cavernomas. schematic representation of cavernoma generation from vessels with mutated ccm genes (see text section 1). according to malinverno et al., (2019), the process involves endothelial-to-mesenchymal transition (endmt) and clonal expansion. susceptibility weighted imaging shows a cavernoma with hemorrhage in the right occipital lobe (arrow). the mri image was obtained in the comprehensive stroke center of hospital clinic of barcelona. 2. cerebral arteriovenous malformations: from origin to treatment failure to achieve full endothelial cell differentiation could underlie the pathology of arteriovenous malformations (avms) where recent studies identified β-adrenergic antagonists as putative curative treatments. avms involve the formation of arteriovenous shunts amongst other vascular abnormalities including vessel calcifications, and the presence of surrounding macrophages and gliosis. this condition impairs oxygen delivery to the parenchyma and predisposes to vessel rupture and hemorrhage. alterations in endothelial cell endmt are associated with this pathology. yao et al. (2019) recently investigated the presence of endmt markers in the endothelium of human avms. through complementary strategies they identified the expression of the stem cell marker sry-box 2 (sox2) and the mesenchymal marker n-cadherin in lesional endothelial cells, together with a reduction in the expression of typical endothelial markers. the authors also investigated a mouse model of avms, the matrix gla protein null (mgp-/-) mouse, which develops arterial calcifications, enlarged vessels, and arterial-venous shunts. cerebral endothelial cells of these mice also overexpressed sox2.they limited sox2 expression in endothelial cells by generating heterozygous cdh5-cre/sox2fl/wt mice with endothelial deletion of sox2 in heterozygosis. these mice were bred with mgp-/mice, which no longer overexpressed sox2 and, importantly, the pathological vascular features of avms were strongly attenuated. moreover, reduction of sox2 abolished the expression of endmt markers. chip-seq analysis showed alterations in histone methylation affecting the expression of several genes that participate in stem cell pathways and epithelial-mesenchymal signaling. the analysis identified overexpression of the gene jmjd5 in mgp-/cerebral endothelial cells as a downstream target of sox2. then, a human brain microvascular endothelial cell line depleted of mgp was generated using crisp/cas9 technology. this line showed higher expression of n-cadherin than wild type cells, suggesting endmt. in addition, these cells overexpressed sox2 and jmjd5. sox2 depletion reduced jmjd5 expression. further experiments suggested that sox2 also interacts with jmjd5 to regulate endmt. to identify drugs that could prevent avms by inhibiting sox2, a high-throughput robotic model was generated using a cell line that allowed screening of more than 3,000 products. the system identified a drug called pronethanol, which was shown to effectively reduce sox2 expression. furthermore, treatment of mgp-/mice with pronethanol for 14 days improved the cerebral vasculature in avm. interestingly, pronethanol is a non-selective beta-blocker with the drawback of causing neurological side effects in humans and carcinogenesis in mice. a number of ß-adrenergic drugs were subsequently tested. the results showed that ß-adrenergic antagonists decreased sox2 and endmt, and improved lumen formation. this study is a good example of the way mechanistic studies in human tissue, human cells, and animal models lead to the discovery of potential drugs to improve the disease. previous studies in mgp-/mice had shown the contribution of endmt and the emergence of multipotent cells to the process of ossification and vascular calcification (e.g. yao et al., 2013). like in the case of ccms, failure of brain endothelial cells to acquire or maintain their differentiation state generates aberrant vessels and vascular diseases. 3. arterial stiffness and calcifications: molecular insights and consequences vessel calcification and arterial stiffness are common in the aged population. arterial stiffness is caused by structural alterations reducing arterial wall distensibility and the arterial capacity to buffer pulsatile cardiac ejection. this condition is epidemiologically associated with hypertension and cognitive decline. arterial calcification is an important cause of arterial stiffness but the contribution of calcifications to cognitive decline over other associated features is unclear. muhire et al. (2019) showed in experimental animals that carotid calcification attenuated resting cerebral blood flow (cbf), impaired cerebral autoregulation, and induced cognitive deficits. the study used a mouse model of arterial stiffness induced by direct application of cacl2 on the carotid artery. the model is characterized by reduced arterial compliance and distensibility, increased thickness of the intima-media, and fragmentation of the internal elastic lamina, without increased systolic blood pressure. carotid stiffness increased bbb permeability in the hippocampus where the number of collagen iv+ vessels decreased, suggesting a pathogenic role of these calcifications. regarding the molecular mechanisms underlying vessel calcifications in brain arteries, a recent study pointed to the involvement of osteopontin and tgfβ signaling in the pathogenesis of calcifications (grand moursel et al., 2019). osteopontin is an extracellular glycoprotein with diverse features. it is cleaved by thrombin and cleaved forms can bind to several integrin receptors expressed by immune cells. given that vascular calcifications are detected in the cerebral cortex in severe forms of cerebral amyloid angiopathy (caa), the study investigated molecular modulators of arterial calcifications in post-mortem tissue of eight patients with hereditary cerebral hemorrhage with amyloidosis-dutch type (hchwa-d or d-caa). calcified vessel walls showed overexpression of medial and abluminal collagen type i. they also overexpressed osteopontin and tgfβ signaling factor phospho-smad2/3, and accumulation of these factors preceded overt vessel calcifications. the study found significant positive correlations between the level of osteopontin and phospho-smad2/3 and disease severity and suggested the involvement of these molecules in cerebral artery calcifications in caa. more severe brain vessel calcifications are found in certain rare genetic diseases. primary familial brain calcification (pfbc) is a heritable disease also termed fahr’s disease. it is a rare form of brain calcification affecting vessels of the basal ganglia and often manifests with neuropsychiatric symptoms. zarb et al. (2019) used a mouse model with mutations in platelet-derived growth factor subunit-β (pdgfb) gene causing reduced endothelial expression of this factor, resulting in vascular calcifications associated with pericyte loss and bbb alterations. interestingly, the mouse model recapitulated several behavioral alterations overlapping with pfbc, including sensorimotor deficits, hyperactivity, anxiety and impaired working memory. cells surrounding brain vessel calcifications expressed osteoblast, osteoclast, and osteocyte markers. the extracellular matrix contained bone matrix proteins. importantly, blood vessel ossification was also demonstrated in brain tissue from primary familial brain calcification patients. another consequence of vessel calcifications was activation of microglial and astroglial cells. given the reported astrogliosis in pfbc patients, astrocytes were investigated in the mouse model. notably, gfap+ astrocytes surrounding calcifications expressed oxidative stress and inflammatory neurotoxic markers, such as complement component 3 (c3) and lipocalin 2. 4. inflammation/innate immunity: role of complement activation inflammation is present in most brain diseases affecting the elderly, including cerebrovascular disease. recently, activation of the complement cascade was shown to play a pathogenic role in subarachnoid hemorrhage. moreover, interaction between initiator complement component c1q and apoe was reported. complement activation has been reported in cerebrovascular diseases and several lines of evidence strongly support the involvement of complement in secondary tissue injury following acute stroke. c3 is amongst key biomarkers associated with increased risk of major disability, mortality and vascular events following ischemic stroke (zhong et al., 2019). complement activation is also associated with poor functional outcome following aneurysmal subarachnoid hemorrhage (sah). a recent study showed that complement components c1q, c3/c3b/ic3b were more abundant in the brain of sah patients than controls (van dijk et al., 2019). a single nucleotide polymorphism in c5 correlated with poor outcome in sah patients, which showed increased levels of c5a in csf and plasma. in a mouse model of sah, blocking c5 activation, using mice deficient in c5a or after administration of c5a blocking antibodies, reduced microglial activation and neural cell death. interestingly, a recent study discovered molecular links between complement and apolipoprotein-e (apoe). apoe is a factor associated with alzheimer’s disease, cerebrovascular diseases, and atherosclerosis (yin et al., 2019). human apoe has four isoforms apoe, apoe2, apoe3, and apoe4. the apoe4 allele is an important risk factor for late onset alzheimer’s disease, whereas several studies indicate that altered apoe2 could be associated with white matter hyperintensities in small vessel diseases. yin et al. (2019) showed absence of apoe-mediated activation of the classical complement cascade in the choroid plexus. lipid deposits were noticed in choroid plexus of aged apoe-/mice regardless of whether they were fed a high fat diet or normal chow. knockin mice expressing the human form of apoe4 (apoe4-ki) also showed lipid deposits in the choroid plexus but only when fed a high fat diet. in contrast, apoe3-ki mice did not develop those lipid deposits irrespective of diet regimen. lipid accumulation was paralleled by leukocyte accumulation in the choroid plexus and csf. using laser-capture microdissection of choroid plexus followed by differential gene expression profiling, an interferon signature was detected in apoe4-ki mice. additionally, upregulation of complement genes was detected in apoe-/mice. accordingly, the choroid plexus of apoe-/mice showed accumulation of immunoglobulins, and complement proteins c1q, c3, c4, as well as c5 and anaphylatoxin c3a. through various in vitro strategies, the study demonstrated that apoe inhibits the classical pathway of complement activation through ca2+-dependent high affinity binding to the activated form of c1q, which was elicited by all apoe isoforms. notably, the formation of lipid droplets in the choroid plexus was found in human brains with various degrees of alzheimer’s disease pathology, and higher lipid droplet numbers were found in patients with dementia. lipid deposits were associated with c1q-apoe complex. furthermore, c1q and apoe colocalized in atherosclerotic plaques. overall, the study suggests that apoe may prevent complement activation. altered inhibitory effects of apoe on complement due to allelic alterations is proposed as a pathogenic mechanism in alzheimer’s disease and atherosclerosis. further studies will hopefully elucidate the role of this new molecular interaction between apoe and complement in cerebrovascular and neurodegenerative diseases. 5. cerebral amyloid angiopathy: microbleeds, microinfarcts and white matter hyperintensities progress has been made in understanding the neuropathology of caa as well as the neuropathological correlates of mri alterations in this condition. caa can lead to symptomatic lobar intracerebral hemorrhage. mri of caa patients may show microbleeds, microinfarcts, and white matter hyperintensities (reijmer et al., 2015). van veluw et al. (2019a) reported that microbleeds and microinfarcts represent different microvascular alterations in caa. the vessels showing microbleeds had accumulation of amyloid β (aβ) and fibrin/fibrinogen upstream and downstream of the microbleed, but not in the microbleed region devoid of smooth muscle cells (smcs). likewise, smcs were missing in the regions of microinfarcts, but in this case aβ was present in the injured core. the study suggested that removal of aβ in vessels that have lost smcs might increase the risk of bleeding, which, if confirmed, might potentially have future therapeutic implications. using high resolution mri in post-mortem brain tissue with correlative histopathological analyses, van veluw et al (2019b) provided relevant information on the neuropathological correlates of neuroimaging alterations. diffusion tensor imaging (dti) is the mri modality best suited to study white matter fiber track orientation and physical features that can assess microstructural integrity of the brain tissue. the post-mortem brain of caa patients was fixed with 10% formalin for at least 3 weeks. one brain hemisphere was scanned in a 3t mri scanner for 14 hours. image analysis included fiber tractography, and 3-mm thick regions of interest were highlighted in parts of the tracts later taken for histology. formalin fixation alters the mri diffusion properties, but fractional anisotropy measured in vivo was linearly related with fractional anisotropy acquired ex vivo in the same brains. the tissue was embedded in paraffin and 6-µm thick sections were obtained for histological analyses. the studies found reduced fractional anisotropy in caa patients compared to controls. in multivariate analysis, fractional anisotropy data was independently associated with tissue rarefaction as assessed with luxol fast blue & hematoxylin, lower myelin density as assessed with immunohistochemistry against myelin basic protein, and decreased axonal density determined by immunohistochemistry against neurofilament (nf200). another dti parameter, mean diffusivity, was increased in cca patients and was independently associated with myelin density (van veluw et al., 2019b). moreover, increased mean diffusivity in the frontal white matter was associated with caa severity in the frontal cortex. in contrast, the dti changes were not related to markers of gliosis or with oligodendrocyte counts. investigations on the neuropathological correlates of imaging alterations are important because mri is widely available and can be used to follow the progression of disease. 6. novel mechanisms underlying cadasil vasculopathy cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) is the most common hereditary monogenic form of cerebral small vessel disease. cadasil leads to early onset hemorrhagic or ischemic stroke and vascular dementia. recent findings in cadasil unraveled novel molecular targets by describing attenuated vascular dysfunction due to inhibitors of endoplasmic reticulum stress and modulators of the cytoskeleton, or due to vascular endothelial growth factor supplementation. cadasil pathology develops due to mutations in the notch3 gene which result in dysfunction of vascular smcs. notch3 is involved in vasculogenesis and is mainly expressed in smcs in adults. however, no specific therapies have been developed so far, possibly because there are many aspects of notch3 function that remain currently unknown. a recent study (neves et al. 2019) identified molecular targets involved in vascular dysfunction in cadasil. notably, the study replicates in a cadasil mouse model, the tgnotch3r169c mouse, the same findings as in human cadasil. samples were obtained from peripheral biopsies of cadasil patients. molecular alterations were identified in peripheral arteries despite the fact that cadasil pathology manifests principally in cerebral vessels. peripheral vessels of cadasil patients showed impaired vascular reactivity and altered vascular structure with features suggesting reduced stiffness. alterations in smc proliferation, apoptosis, and cytoskeletal disorganization was also noted. vascular smcs of cadasil patients showed increased notch3 signaling, notch3 ectodomain (notch3ecd) accumulation, and expression of notch3 target genes relative to control patients. the study also showed nox5 upregulation linked to an exaggerated endoplasmic reticulum stress response and aberrant cytoskeleton-associated protein phosphorylation in cadasil. moreover, the study reports that inhibitors of notch3, nox5, er stress, and rhoa/rho kinase attenuate vascular dysfunction. this finding identifies putative pharmacologic therapeutic targets. vascular smcs in cadasil are surrounded by deposits of granular osmiophilic material (gom). however, the mechanisms underlying gom formation and their contribution to disease progression are unknown. a recent study investigated the course of gom deposit evolution in humanized tgnotch3arg182cys mice (gravesteijn et al., 2019) and in human cadasil tissue. gom deposits are electron dense and are composed of notch3ecd and extracellular matrix proteins, notably tissue inhibitor of metalloproteinase 3 (timp3) and clusterin, that accumulate in the basement membrane of vascular smcs and pericytes. the study proposes a five-stage gom classification system based on size, morphology, and electron density of the gom deposits in order to generate a more systematic, uniform, and unbiased way of analyzing gom accumulations. it appears that new gom deposits are continuously generated. however, gom in tgnotch3arg182cys mice did not reach the end-stage of gom accumulation as in human disease. a limitation of the study is that unlike other cadasil mouse models, the tgnotch3arg182cys mouse lacks histologic vascular pathology and functional deficits. the authors suggest that this phenomenon could be attributed to absence of end-stage gom accumulation in these mice. beyond mouse models, the cellular cadasil pathology has been investigated in patient-specific vascular mural cells generated from induced pluripotent stem cells (ipscs) obtained from skin biopsies of cadasil patients (kelleher et al., 2019). this study showed alterations in endothelial capillary structures caused by the aberrant notch3 expression in mural cells, and corrective effects of vascular endothelial growth factor supplementation. these novel advances are steps toward the identification of novel therapeutic targets in this monogenic form of small vessel disease. 7. genetic variants in small vessel disease and molecular target discovery in addition to studies examining monogenic forms of small vessel disease, there are efforts to study genetic variation in large cohorts of patients to identify genes involved in sporadic small vessel disease. genome-wide association studies correlating variants with mri features of sporadic small vessel disease may lead to the identification of new putative pharmacologic targets. small vessel disease is often diagnosed on mri, with characteristic findings including white matter hyperintensities and signs of lacunar infarcts (fig. 2). some recent studies investigated the relationship between genetics and white matter hyperintensities in small vessel disease. traylor et al (2019) carried out a genome-wide association meta-analysis of white matter hyperintensity volumes in 11,226 subjects, including 2,797 stroke patients. the study identified a locus at genome-wide significance in an intron of pleckstrin homology and rhogef domain-containing family g member 1 gene (plekhg1) associated with white matter hyperintensities. the association was validated in an independent cohort where this polymorphism was related with ischemic strokes. the strongest association was found with small vessel stroke. plekhg1 plays a role in vascular endothelial cell reorientation in response to mechanical stress. therefore, the polymorphism in this gene could generate some form of vascular alteration leading to the development of white matter hyperintensities. this study also validated 2 previously identified genes efemp1 and trim47/trim65. according to current knowledge, efemp1 encodes the fibulin3 protein, which is an extracellular matrix glycoprotein that inhibits timp3. timp3 contributes to cerebrovascular dysfunction in cadasil through accumulation in the vascular extracellular matrix. therefore, there seems to be some concordance between genetic alterations in sporadic small vessel disease and the monogenic form of cadasil. based on the concept that polymorphisms in genes involved in monogenic forms of small vessel disease may have some contribution in the sporadic form of the disease, mishra et al. (2019) carried out the first whole exome sequencing study on mri white matter hyperintensities in small vessel disease. this work used a composite extreme phenotype study focused on candidate genes with mutations causing mendelian heritable forms of small vessel disease, i.e. notch3, htra1, col4a1, col4a2 and trex1. the study identified significant associations of gene variants in htra1 and notch3 with the development of white matter hyperintensities. overall the study demonstrates shared mechanisms between monogenic forms and multifactorial forms of small vessel disease. the study suggested that the risk htra1 allele reduced htra1 expression. mutations in htra1 are associated with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, carasil, a condition inherited in an autosomal recessive pattern. htra1 is a serine-protease involved in cleaving extracellular matrix proteins, including fibulin3, and regulating important signaling pathways such as the insulin growth factor and tgfß pathway, which suffers an inhibitory effect. notably, htra1 is differentially expressed in astrocytes (chen et al., 2018) suggesting that dysfunction of this pathway may alter astrocyte function leading to compromised integrity of the neurovascular unit and the bbb. hopefully additional studies using ipsc from patients and genetically modified animal models further investigating molecular targets identified in prior genetic studies will lead to the discovery of treatments to prevent and attenuate the progression of small vessel disease. figure 2. lacunar infarcts typical of small vessel disease. a) human brain tissue showing a lacunar infarct (arrow) in the centrum semiovale. image obtained by dr. iban aldecoa, neurological tissue bank of the biobank of hospital clínic idibaps university of barcelona. b) mri showing a thalamic lacunar infarct (arrow) and c) corresponding ct-perfusion time-to-drain (ttd) image obtained in the acute phase showing a delay in blood flow in the lacunar infarction (arrow). images in b and c were obtained in the comprehensive stroke center of hospital clinic of barcelona. 8. cerebral hypoperfusion and vascular cognitive impairment: risks of high-salt diet chronic cerebral hypoperfusion has been associated with diffuse white matter damage and cognitive impairment of vascular origin. vascular pathology is also frequently observed in neurodegenerative dementias. hase et al. (2019) investigated the histopathologic features of white matter capillaries by studying approximately 0.7 million capillary profiles from the frontal lobe white matter of 153 subjects with different dementia types including alzheimer’s disease, dementia with lewy bodies, dementia in parkinson’s disease, vascular dementia, mixed dementia, post-stroke dementia and post-stroke no dementia. control samples were examined from old and young individuals devoid of brain pathology. all dementias showed higher vascular pathology scores than corresponding age-matched controls. in relation to these controls, white matter lesion scores were also higher in all dementias, with the highest values corresponding to vascular dementia and post-stroke dementia. the two latter conditions showed the highest abundance of string vessels, which were observed to a lesser extent in the other dementia types. the density of col4+ and glut1+ capillaries was lower in the white matter than in the cortex and was also lower in dementia patients versus aged controls, with the exception of individuals that had suffered a stroke. the capillary width in the white matter was 31% greater than in the cortex. notably, white matter capillaries were wider in all dementias compared to all controls irrespective of age. moreover, there was a moderate positive correlation between capillary width and white matter lesion scores. these results indicate adaptive compensatory mechanisms for chronic hypoperfusion in aging-related dementias. an emerging risk factor recently recognized as a driver of cerebral vascular dysfunction and hypoperfusion is high-salt intake (faraco et al., 2018). interestingly, the underlying mechanism is not dependent on salt-induced hypertension but on a complex innate immune response mediated by alterations in the gut immune system (faraco et al., 2018). last year, a study by the same team reported that chronic dietary high-salt intake in mice promotes cognitive impairment by inducing phosphorylation of the microtubule-associated protein tau rather than by insufficient cerebral perfusion (faraco et al., 2019). the presence of hyperphosphorylated tau is one of the hallmarks of alzheimer’s disease that has also been reported in patients with vascular cognitive impairment. mice exposed to high-salt diet for several months developed signs of cognitive impairment that correlated with the phosphorylation and aggregation of tau in the brain tissue. through different experimental strategies, the study demonstrated that high-salt diet impaired the production of endothelial nitric oxide following denitrosylation of calpain. deficient nitric oxide generation activated cyclin-dependent kinase 5 (cdk5) which caused tau phosphorylation. although these important findings were obtained in mice with unrealistically high levels of salt intake, the study identifies a causal relation between high-salt intake, vascular dysfunction, tau pathology, and cognitive impairment independent of cerebral hypoperfusion. 9. critical role of pericytes in bbb function and more pericytes are mural cells surrounding capillaries that play essential functions in development and maintenance of bbb integrity. pericytes facilitate molecular pathways critical in preventing vascular malformations, and it appears that these cells can generate factors that directly support neuronal viability. recent studies have provided valuable information regarding the biology of pericytes as well as their role in vascular function. using pdgfrb-creert2 mice with cre expression induced in mural cells by tamoxifen administration, diéguez-hurtado et al. (2019) deleted the expression of the rbpj gene encoding a transcriptional regulator of the notch signaling pathway. ablation of rbpj at early postnatal stages caused brain hemorrhages, avms at the pial surface, endothelial cell hyperplasia and apoptosis, enlargement of the subendothelial basement membrane, and reduced blood flow. these changes were accompanied by astroglial and microglia reactions, inflammation, edema, and some neuronal loss around severely affected blood vessels. rbpj deletion upregulated genes of the tgfß pathway. the study provides evidence showing that mice with rbpj gene deficiency in mural cells developed signs of ccms. interestingly, when rbpj was deleted in mural cells of adult mice, the above phenotype was no longer observed. however, induction of permanent brain ischemia in these mice caused increased edema, larger brain lesion volumes, and exacerbated inflammation and vascular abnormalities. beyond the critical role of pericytes in brain vascular function, nikolakopoulou et al. (2019) elucidated the role of pericytes in neuronal survival under conditions of vascular dysfunction. a mouse pericyte inducible cre-line was generated using a double gene promoter strategy including both pdgfrb and cspg4 promoters to increase the specificity of cre expression in pericytes over other cells. this line was crossed with mice expressing inducible human diphtheria toxin receptor under control of an upstream loxp-flanked stop sequence. injection of tamoxifen induced dtr expression in pericytes which then became susceptible to diphtheria toxin. ablation of pericytes in adult mice reduced tight junction proteins zonula occludens and occludin, reduced adherens junction ve-cadherin on cortical and hippocampal capillaries, decreased cbf, and increased bbb breakdown. pericyte loss was followed by vasogenic edema, confirming the involvement of pericytes in maintaining adult bbb function. furthermore, neuron loss in the cortex and hippocampus as well as behavioral deficits were detected several days after pericyte ablation. a brain-specific pericyte-secreted growth factor termed pleiotropin was hypothesized to prevent neuronal loss. in vitro, pleiotropin was able to protect neurons under hypoxic conditions. in vivo, silencing pleiotropin expression was not sufficient to cause neuronal loss. however, ischemia or excitotoxic lesions in mice with silenced pleiotropin expression caused larger infarctions and more neuronal degeneration. the study concluded that pericyte-dependent pleiotropin-mediated neurotrophic support promotes neuronal survival under circulatory stress. the expedient neuronal loss found in this model of pericyte ablation is intriguing because it was not found in previous pericyte-deficient models. the findings could be relevant to other neurodegenerative conditions if the dependence of neuronal survival on neurotrophic factors provided by pericytes under vascular stress is confirmed. 10. molecules derived from the vascular endothelium impact neuronal function in addition to pericytes, endothelial cells seem to signal to neurons. tan et al. (2019) showed that the endothelium secretes semaphorin 3g (sema3g) which regulates synaptic structure and plasticity in hippocampal neurons through actions on the neuronal neuropilin-2/plexina4 holoreceptor. this study suggests a direct link between endothelial-derived factors and neuronal function beyond the supply of oxygen and nutrients through blood flow and proposes a molecular mechanism by which endothelial dysfunction can alter neuronal circuits and cognition. the study shows that sema3g increases excitatory synapse density via neuropilin-2/plexina4 signaling and activation of rac1. to determine whether sema3g was derived from endothelial cells, mice bearing a sema3g deletion selectively in endothelial cells were obtained by crossing cdh5-cre mice with floxed sema3g mice. these mice showed less dendritic spine density in the ca1 and impaired memory. nonetheless, sema3g deletion with this strategy would cause sema3g deficiency during developmental stages which may cause indirect alterations and compensatory changes. generation of tamoxifen inducible cdh5-creert2/sema3gfl/fl mice allowed deleting sema3g in the endothelium of adult mice. this inducible deletion significantly impaired hippocampal long-term potentiation frequency and altered miniature excitatory post-synaptic currents in ca1 pyramidal neurons. furthermore, sema3g overexpression improved spine density loss and cognitive defects in cdh5-creert2/sema3gfl/fl mice. the results suggest that endothelium-derived sema3g is required for normal hippocampal synaptic plasticity in adulthood. this study raises many questions regarding the mode of transit of sema3g from the vascular endothelium to the neurons through the vascular and astrocytic basal laminae and astrocytic end-feet, and whether the source of sema3g is the capillary endothelium. the discovery of molecular interactions between endothelial cells and neurons suggest that endothelial cells influence neuronal function. therefore, alterations in the vascular endothelium might exert a direct effect on dendritic spine function and contribute to cognitive decline and memory loss. other recent studies further support the view that molecules generated by the cerebrovascular endothelium have a direct impact on neuronal function. liu et al. (2020) showed that mice with a selective deletion of cdk5 gene show a higher endothelial chemokine cxcl1 expression. therefore, dysregulation of cdk5 expression and/or function seems to be critical in different forms of cerebrovascular alterations. endothelial cdk5 knockout mice showed astrogliosis in the hippocampus and weakened astrocytic glutamate current mediated by the astroglial glutamate transporter glt1. the effects on astroglia were mediated by endothelial-derived cxcl1 acting on the astrocytic receptor cxcr2. these alterations had consequences for neurons since they reduced glutamate uptake and increased the excitability of hippocampal pyramidal neurons causing seizures in mice with endothelial-deficient cdk5. although current evidence is still scarce and based on experimental animal studies, the possibility that vascular endothelial cells generate molecules that signal to neurons and astrocytes and govern neuronal function warrants future studies. conclusion the above studies discovered contributing factors to neurovascular pathology. adequate cbf supply and bbb integrity require homeostasis in the cellular and extracellular environment around blood vessels. moreo-ver, the concept is emerging that pericytes and endothe-lial cells can signal to neurons by releasing certain specif-ic molecular cues. ideally, the novel knowledge of genet-ic risk factors, molecular mechanisms, cell and system interactions, as well as pathologic and imaging hallmarks in cerebrovascular diseases should translate into curative treatments that are currently unavailable. acknowledgements supported by the spanish ministry of science, innovation and universities (saf2017-87459-r). note i apologize to the authors that have provided highly valuable contributions in the field in 2019 but could not be cited here due to limitations in the number of selected references. references chen j, van gulden s, mcguire tl, fleming ac, oka c, kessler ja, peng cy. bmp-responsive protease htra1 is differentially expressed in astrocytes and regulates astrocytic development and injury response. j neurosci. 2018; 38:3840-3857. doi: 10.1523/jneurosci.2031-17.2018. detter mr, snellings da, marchuk da. cerebral cavernous malformations develop through clonal expansion of mutant endothelial cells. circ res. 2018; 123(10):1143-1151. doi: 10.1161/circresaha.118.313970. diéguez-hurtado r, kato k, giaimo bd, nieminen-kelhä m, arf h, ferrante f, bartkuhn m, zimmermann t, bixel mg, eilken hm, adams s, borggrefe t, vajkoczy p, adams rh. loss of the transcription factor rbpj induces disease-promoting properties in brain pericytes. nat commun. 2019; 10(1):2817. doi: 10.1038/s41467-019-10643-w. faraco g, brea d, garcia-bonilla l, wang g, racchumi g, chang h, buendia i, santisteban mm, segarra sg, koizumi k, sugiyama y, murphy m, voss h, anrather j, iadecola c. dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated th17 response. nat neurosci. 2018; 21(2):240-249. doi: 10.1038/s41593-017-0059-z. faraco g, hochrainer k, segarra sg, schaeffer s, santisteban mm, menon a, jiang h, holtzman dm, anrather j, iadecola c. dietary salt promotes cognitive impairment through tau phosphorylation. nature. 2019 oct;574(7780):686-690. doi: 10.1038/s41586-019-1688-z. grand moursel l, van der graaf lm, bulk m, van roon-mom wmc, van der weerd l. osteopontin and phospho-smad2/3 are associated with calcification of vessels in d-caa, an hereditary cerebral amyloid angiopathy. brain pathol. 2019; 29(6):793-802. doi: 10.1111/bpa.12721. gravesteijn g, munting lp, overzier m, mulder aa, hegeman i, derieppe m, koster aj, van duinen sg, meijer oc, aartsma-rus a, van der weerd l, jost cr, van den maagdenberg amjm, rutten jw, lesnik oberstein saj. progression and classification of granular osmiophilic material (gom) deposits in functionally characterized human notch3 transgenic mice. transl stroke res. 2019 oct 30. doi: 10.1007/s12975-019-00742-7. hase y, ding r, harrison g, hawthorne e, king a, gettings s, platten c, stevenson w, craggs ljl, kalaria rn. white matter capillaries in vascular and neurodegenerative dementias. acta neuropathol commun. 2019; 7(1):16. doi: 10.1186/s40478-019-0666-x. kelleher j, dickinson a, cain s, hu y, bates n, harvey a, ren j, zhang w, moreton fc, muir kw, ward c, touyz rm, sharma p, xu q, kimber sj, wang t. patient-specific ipsc model of a genetic vascular dementia syndrome reveals failure of mural cells to stabilize capillary structures. stem cell reports. 2019; 13(5):817-831. doi: 10.1016/j.stemcr.2019.10.004. liu xx, yang l, shao lx, he y, wu g, bao yh, lu nn, gong dm, lu yp, cui tt, sun nh, chen dy, shi wx, fukunaga k, chen hs, chen z, han f, lu ym. endothelial cdk5 deficit leads to the development of spontaneous epilepsy through cxcl1/cxcr2-mediated reactive astrogliosis. j exp med. 2020; 217(1). pii: e20180992. doi: 10.1084/jem.20180992. malinverno m, maderna c, abu taha a, corada m, orsenigo f, valentino m, pisati f, fusco c, graziano p, giannotta m, yu qc, zeng ya, lampugnani mg, magnusson pu, dejana e. endothelial cell clonal expansion in the development of cerebral cavernous malformations. nat commun. 2019; 10(1):2761. doi: 10.1038/s41467-019-10707-x. manavski y, lucas t, glaser sf, dorsheimer l, günther s, braun t, rieger ma, zeiher am, boon ra, dimmeler s. clonal expansion of endothelial cells contributes to ischemia-induced neovascularization. circ res. 2018; 122(5):670-677. doi: 10.1161/circresaha.117.312310. mishra a, chauhan g, violleau mh, vojinovic d, jian x, bis jc, li s, saba y, grenier-boley b, yang q, bartz tm, hofer e, soumaré a, peng f, duperron mg, foglio m, mosley th, schmidt r, psaty bm, launer lj, boerwinkle e, zhu y, mazoyer b, lathrop m, bellenguez c, van duijn cm, ikram ma, schmidt h, longstreth wt, fornage m, seshadri s, joutel a, tzourio c, debette s. association of variants in htra1 and notch3 with mri-defined extremes of cerebral small vessel disease in older subjects. brain. 2019; 142(4):1009-1023. doi: 10.1093/brain/awz024. muhire g, iulita mf, vallerand d, youwakim j, gratuze m, petry fr, planel e, ferland g, girouard h. arterial stiffness due to carotid calcification disrupts cerebral blood flow regulation and leads to cognitive deficits. j am heart assoc. 2019; 8(9):e011630. doi: 10.1161/jaha.118.011630. neves kb, harvey ap, moreton f, montezano ac, rios fj, alves-lopes r, nguyen dinh cat a, rocchicciolli p, delles c, joutel a, muir k, touyz rm. er stress and rho kinase activation underlie the vasculopathy of cadasil. jci insight. 2019; 4(23). pii: 131344. doi: 10.1172/jci.insight.131344. nikolakopoulou am, montagne a, kisler k, dai z, wang y, huuskonen mt, sagare ap, lazic d, sweeney md, kong p, wang m, owens nc, lawson ej, xie x, zhao z, zlokovic bv. pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss. nat neurosci. 2019; 22(7):1089-1098. doi: 10.1038/s41593-019-0434-z. reijmer yd, fotiadis p, martinez-ramirez s, salat dh, schultz a, shoamanesh a, ayres am, vashkevich a, rosas d, schwab k, leemans a, biessels gj, rosand j, johnson ka, viswanathan a, gurol me, greenberg sm. structural network alterations and neurological dysfunction in cerebral amyloid angiopathy. brain. 2015;138(pt 1):179-88. doi: 10.1093/brain/awu316. tan c, lu nn, wang ck, chen dy, sun nh, lyu h, körbelin j, shi wx, fukunaga k, lu ym, han f. endothelium-derived semaphorin 3g regulates hippocampal synaptic structure and plasticity via neuropilin-2/plexina4. neuron. 2019; 101(5):920-937.e13. doi: 10.1016/j.neuron.2018.12.036. traylor m, tozer dj, croall id, lisiecka-ford dm, olorunda ao, boncoraglio g, dichgans m, lemmens r, rosand j, rost ns, rothwell pm, sudlow clm, thijs v, rutten-jacobs l, markus hs; international stroke genetics consortium. genetic variation in plekhg1 is associated with white matter hyperintensities (n = 11,226). neurology. 2019; 92(8):e749-e757. doi: 10.1212/wnl.0000000000006952. van dijk bj, meijers jcm, kloek at, knaup vl, rinkel gje, morgan bp, van der kamp mj, osuka k, aronica e, ruigrok ym, van de beek d, brouwer m, pekna m, hol em, vergouwen mdi. complement c5 contributes to brain injury after subarachnoid hemorrhage. transl stroke res. 2019 dec 6. doi: 10.1007/s12975-019-00757-0. van veluw sj, reijmer yd, van der kouwe aj, charidimou a, riley ga, leemans a, bacskai bj, frosch mp, viswanathan a, greenberg sm. histopathology of diffusion imaging abnormalities in cerebral amyloid angiopathy. neurology. 2019b; 92(9):e933-e943. doi: 10.1212/wnl.0000000000007005. van veluw sj, scherlek aa, freeze wm, ter telgte a, van der kouwe aj, bacskai bj, frosch mp, greenberg sm. different microvascular alterations underlie microbleeds and microinfarcts. ann neurol. 2019a; 86(2):279-292. doi: 10.1002/ana.25512. yao j, wu x, zhang d, wang l, zhang l, reynolds ex, hernandez c, boström ki, yao y. elevated endothelial sox2 causes lumen disruption and cerebral arteriovenous malformations. j clin invest. 2019; 129(8):3121-3133. doi: 10.1172/jci125965. yao y, jumabay m, ly a, radparvar m, cubberly mr, boström ki. a role for the endothelium in vascular calcification. circ res. 2013; 113(5):495-504. doi: 10.1161/circresaha.113.301792. yin c, ackermann s, ma z, mohanta sk, zhang c, li y, nietzsche s, westermann m, peng l, hu d, bontha sv, srikakulapu p, beer m, megens rta, steffens s, hildner m, halder ld, eckstein hh, pelisek j, herms j, roeber s, arzberger t, borodovsky a, habenicht l, binder cj, weber c, zipfel pf, skerka c, habenicht ajr. apoe attenuates unresolvable inflammation by complex formation with activated c1q. nat med. 2019;25(3):496-506. doi: 10.1038/s41591-018-0336-8. zarb y, weber-stadlbauer u, kirschenbaum d, kindler dr, richetto j, keller d, rademakers r, dickson dw, pasch a, byzova t, nahar k, voigt ff, helmchen f, boss a, aguzzi a, klohs j, keller a. ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response. brain. 2019; 142(4):885-902. doi: 10.1093/brain/awz032. zhong c, zhu z, wang a, xu t, bu x, peng h, yang j, han l, chen j, xu t, peng y, wang j, li q, ju z, geng d, he j, zhang y. multiple biomarkers covering distinct pathways for predicting outcomes after ischemic stroke. neurology. 2019; 92(4):e295-e304. doi: 10.1212/wnl.0000000000006717. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. charcot identifies and illustrates amyotrophic lateral sclerosis feel free to add comments by clicking these icons on the sidebar free neuropathology 2:12 (2021) flashback charcot identifies and illustrates amyotrophic lateral sclerosis charles duyckaerts1,2, thierry maisonobe1,3, jean-jacques hauw4, danielle seilhean1,5 1 raymond escourolle neuropathology department, la salpêtrière hospital, assistance publique des hôpitaux de paris, sorbonne université, paris, france 2 alzheimer-prions team, paris brain institute icm (inserm u1127, cnrs umr7225), sorbonne université-umrs1127), paris, france 3 clinical neurophysiology department, la salpêtrière hospital, assistance publique des hôpitaux de paris, sorbonne université, paris, france 5 académie nationale de médecine, paris, france 6 als: causes and mechanisms of motor neuron degeneration, paris brain institute icm (inserm u1127, cnrs umr7225, sorbonne université-umrs1127), paris, france corresponding author: charles duyckaerts · raymond escourolle neuropathology department · la salpêtrière hospital · assistance publique des hôpitaux de paris · sorbonne université · paris · france charles.duyckaerts@gmail.com submitted: 31 march 2021 accepted: 08 may 2021 copyedited by: lauren walker and henry robbert published: 18 may 2021 https://doi.org/10.17879/freeneuropathology-2021-3323 keywords: amyotrophic lateral sclerosis, charcot, history of medicine, pyramidal tract abstract jean-martin charcot described what he called amyotrophic lateral sclerosis in his 12th and 13th lessons published in 1873 by bourneville. he distinguished the symptoms that were related to the lesion of the anterior horn of the spinal cord and those that were due to the degeneration (that he named “sclerosis”) of its lateral column. he thought that “inflammation” progressed from the lateral column to the anterior horn (but the term inflammation is not to be taken in the current meaning): the lesion of the anterior horn was thus “deuteropathic”. an album containing drawings made by charcot is kept in la salpêtrière neuropathology department. four drawings are pasted on one of its pages, showing the degeneration of the pyramidal tract. they constitute the original of the engravings illustrating charcot’s 12th lesson. the illustration of the fascicular atrophy of the adductor pollicis presented in the album does not appear in the lessons, even though this alteration is widely discussed and linked to the lesion of the anterior horn, which was supposed to ensure the “nutrition” of the muscle. the technique used by charcot and his interpretation of the microscopic pictures, as exposed in his lessons, are discussed. introduction jean-martin charcot identified amyotrophic lateral sclerosis (als) on clinical-pathological grounds. the data, which he collected, and his conception are remarkably summarized in the second volume of his lessons on the diseases of the nervous system, edited by bourneville (charcot, 1873) and in his lessons on cerebral localization, edited by bourneville et brissaud (charcot, 1876). the neuropathology laboratory of la salpêtrière hospital keeps an album containing original drawings by charcot. one of its pages concerns an als case. this paper compares the two documents: the drawings and the description of the disease, both by charcot. the album the album bears the title “charcot museum, pathological anatomy”, handwritten in ink on the black hardcover (fig. 1). it contains 100 light-blue pages of a format known as “couronne” in france (37 x 47 cm), commonly used by the artists of the time. pages 15 to 100 are blank. they were numbered with stencils. “foxing” (i.e. brown spots and browning of the edges) suggests they are original. the first fourteen pages have been restored, as well as the binding, some twenty years ago, and were renumbered in pencil. the drawings, usually on white papers, are pasted on those pages. there are also a few engravings, some taken from charcot’s articles. all the drawings have the same style and, although not signed, are reputed to be by charcot himself. charcot had hesitated between medicine and the fine arts. he left numerous drawings of pathological anatomy and sketches of patients (see for instance, the case bachère, charcot, 1892, fig. 64, p 337, https://gallica.bnf.fr/ark:/12148/bpt6k55646784/f343.item.texteimage; or a case of parkinson’s disease ibidem, fig. 63, p 336; the illustration of “a labioglossal spasm”, charcot, 1892, fig. 19 and 20, p 211, https://gallica.bnf.fr/ark:/12148/bpt6k55646784/f217.image.r=fig; or the picture of the face in a case of parkinson’s disease, charcot, 1892, fig. 60, p 334, https://gallica.bnf.fr/ark:/12148/bpt6k55646784/f340.image.r=fig). fig. 1. hardcover of the album in which charcot’s drawings were collected. these drawings demonstrate his talent for illustrating the cases that he examined. the style of the drawings found in the album is identical to that of the drawings found in the notes that he prepared for his “lessons” (still visible at charcot library, now located at paris brain institute-icm, https://institutducerveau-icm.org/en/actualite/charcot-library): there is no reason to question their authorship. the drawings, initially made on loose sheets, were secondarily collected and pasted in the album that was passed down from charcot’s own laboratory to the current department of neuropathology. each page generally concerns a particular topic (microaneurysm, multiple sclerosis, myelitis, lateral funiculus in a foetus, syringomyelia, pott’s disease, vertebral metastases, anatomy of the posterior funiculi, lead paralysis, tumor of the spinal cord, als, cysticercosis). the album has never been the subject of a specific study, although some of its pictures have been published occasionally (the drawing of the central nervous system of the als case under investigation here has been published in seilhean, 2020). cases and samples three original drawings of the als case, p 12 of the album (fig. 2), made on white papers, are pasted on a grey (faded green) cardboard and concern a case, identified as “trouillet’s case” at its bottom (fig. 2 and fig. 4). they include sections of the medulla oblongata and spinal cord, shown in the superior part of the card (fig. 3) and labeled a, b, c, d, and two views of the adductor pollicis muscle, which occupy the inferior part and are labeled a and b (fig. 4). a microscopic view of “altered slender columns” of the spinal cord (fig. 5), possibly from another case, has been directly pasted on the right side of the page as shown in fig. 2. fig. 2. general view of page 12 of the album dealing with amyotrophic lateral sclerosis. three drawings are pasted on a grey (faded green) cardboard (arrows). they show sections of the central nervous system (at the top) and of the adductor pollicis (at the bottom). in the lower right corner of the card, “cas de trouillet” (trouillet’s case) can be read. an additional drawing on the right illustrates two microscopic aspects of an “altered slender column [of the spinal cord]”. the views of “trouillet’s case” are not dated but, as we found a great similarity between the drawings of the album and the engravings of the 12th lesson (charcot, 1873, pp 221, fig. 16-19), we are inclined to believe that they are anterior to 1873. we could not elucidate the identity of that trouillet, probably the doctor who referred the patient to charcot. charcot gave the names of the two als cases that he published previously, as was usual at the time. the first case, catherine aubel, was published in a paper entitled “two cases of progressive muscular atrophy with lesions of the grey matter and of the anterolateral fascicles of the white matter” (charcot and joffroy, 1869, first case, p 354 reproduced in charcot, 1873, p 402). the disease of the second case of that paper (named a.c.) is not reported in the lessons and is not identified as als by charcot. the second case identified as als, elisabeth p., was published with only gombault as its author (gombault, 1871). it is reproduced in the lessons (charcot, 1873, p 416) with charcot’s commentary “with the collaboration of gombault” as if he, charcot, known for his authoritarianism, was the first coauthor. trouillet is not mentioned in those two articles. as charcot indicated that his lesson was based on the study of five clinical-pathological cases (charcot, 1873, p 228, footnote 1) and as three cases are identified (catherine aubel, elisabeth p. and trouillet’s case), two remain unidentified and, as far as we know, unpublished. fig. 3. upper part: page 12 of the album. trouillet’s case (see fig. 2). four sections of the central nervous system. the section are labeled a, b, c, d by charcot himself. the legend reads: a: bulbe b: moelle cervicale c: moelle dorsale d: moelle lombaire which means: a: medulla oblongata; b: spinal cord at cervical level; c: at thoracic level, d: at lumbar level. lower part: below the original drawings of charcot found in the album, the engravings of lesson 12 (charcot, 1873, pp 221, fig. 16-19). the legend in french reads: fig. 19. cross section of the medulla through the middle part of the olive. a, a, sclerotic anterior pyramids fig. 16. – transverse section of the spinal cord through the middle part of the cervical enlargement. fig. 17. – transverse section through the middle of the dorsal region. fig. 18. – transverse section through the middle of the lumbar enlargement. preparation of microscopic sections by charcot (et bouchard) the technique that has been used to obtain the illustrated sections is not reported. we can rely on two articles in which charcot indicated how he proceeded (charcot and joffroy, 1869 reproduced in charcot, 1873, pp 402-416; charcot, 1865). the central nervous system was fixed for “nearly one month” with a “much diluted solution of chromic acid” (the fixation properties of formaldehyde were described by f. blum two decades later – blum, 1893). the microtome, as we know it today, was probably not in use in paris at the time. its invention by purkinje in 1841 (chvátal, 2017) or his in 1866 (dupont, 2018) is controversial but it is clear that it was commonly used much later. the manual of histological techniques by mathias duval (1878), for instance, does not mention microtomes (in the current meaning) and concludes: “a large number of instruments have been invented [to cut fixed pieces]. one comes always back to the simple razor which is more efficient than all complicated ‘discotomes’ [we put in quotes] if the habit required to use it skillfully has been acquired.” the fixed sample was usually stuck in elderberry pith and cut, freehand, with a razor blade. the sections were not dehydrated and mounted in balsam or resin, as they are today. the manual of mathias duval (1878) indicates that one common practice was to cover the section with a coverslip (much thicker than they are today) and to introduce between the slide and the coverslip, on one side, a drop of glycerol that would diffuse and push the water out on the other side. the coverslip could then be sealed with wax or bitumen of judea (duval, 1878). fig. 4. two drawings, labeled a and b, are visible in the lower part of the page. a: one aspect of the adductor pollicis. a tendon appears as an unstained mass in the center of the section. numerous atrophic fibers are visible. b: another aspect of the adductor pollicis. fascicular atrophy of the muscle fibers are clearly seen around an artery in the center of the section. the staining was obtained with a few drops of “a concentrated solution of ammoniacal carmine”. “[…] the sick parts take a violet color, darker when the alteration is more severe. the stain of the healthy part remains unchanged.” (charcot, 1865, p 31). “this procedure, to make more sensitive to the eye the alterations of sclerosis, which has been erased by the maceration in chromic acid, belongs to m. bouchard”, then an intern in charcot’s department and later professor of general pathology at paris medicine faculty. what did ammoniacal carmine stain in sections fixed by chromic acid? we have no definite answer to this question. astrogliosis is a good candidate. several methods were developed later to stain “fibrous nevroglia” with crystal violet or other phenyl methane dyes (such as weigert’s or holzer’s methods), but the intimate mechanism of the staining remained unknown (proescher, 1934). the lower square view (at the highest magnification) shows “altered slender columns [literally cords]”, most probably the lateral column, after treatment by acetic acid (as mentioned: “après acide acétique”), a practice that was usual at the time. mathias duval (1878) writes in his manual (p 211-212) “the most important [of the “insulating reagents”] and the most used is acetic acid: its special action is to swell and make the connective or laminous fibers disappear, and as there are few tissues where these fibers are not in a certain abundance and do not veil the other elements, there are few preparations in which acetic acid is not used.” charcot mentions that “we have taken for comparison some very nice preparations of healthy spinal cord which we owe to the kindness of mr. lockhart clarke” (charcot and joffroy, 1869, p 366). it is interesting to note that the technique used by lockhart clarke was different: the fixation of the tissue was obtained with spirit of wine; the section was then treated with one part of acetic acid and three parts of spirit of wine before being cleared in turpentine oil and observed under a coverslip: in other words, the fixation was alcoholic and there was no staining. lockhart clarke’s pictures of the spinal cord were of an exceptional precision and beauty (lockhart clarke, 1851, 1858). a case of lockhart clarke is cited by charcot as possible cases of als (turner et al., 2010; radcliffe and lockhart clarke, 1862). the method used to prepare the muscle samples that were drawn in trouillet’s case is also not indicated. charcot and joffroy examined the muscle directly and immediately (“in the fresh state”), dissociating the fibers with needles in the case of catherine aubel (charcot and joffroy, 1869, p 362). vulpian (1869), at the same time, mentions that he examined the muscle fibers after fixation in a dilute solution of chromic acid. in the case of trouillet, this fixation also must have been applied, as charcot’s careful drawings necessarily took a certain amount of time that an unmounted and unfixed preparation would not have allowed. fig. 5. microscopic view of an “altered slender column” of the spinal cord. at the top, one reads “350 to 400 d[iameters]” (the magnification) and below the lower view “after acetic acid” (“après acide acétique” – see text for explanation). the drawings the drawing of trouillet’s case shows in red the pyramids of the medulla oblongata and the lateral column of the spinal cord, over-colored by ammionacal carmine. charcot insisted upon the sparing of a fascicle of white matter, outside the pyramidal tract and just below the surface of the spinal cord: “on transverse sections, at the level of the cervical enlargement, the symmetrical alteration is greater in width than anywhere else. the area invaded by the sclerosis reaches anteriorly, and even exceeds the external angle of the anterior horn. posteriorly, it almost reaches the posterior grey matter. laterally, however, it is constantly separated from the cortical layer of the spinal cord by a spared bundle of white matter” (charcot, 1873, pp 220-221). the microscopic view shown on the right of the page (fig. 5) may have belonged to another case and illustrate “altered slender columns” [of the spinal cord] at a magnification of “350 to 400 d[iameters]”. it shows oligodendroglial nuclei and scanty myelin sheaths. the loss of fibers that it probably intended to show is impossible to ascertain without comparison with a normal case. two views of the adductor pollicis muscle are shown (fig. 4, a and b). the fascicular atrophy is evident on panel b. charcot reported “infiltration by leukocytes, predominant near the tendon” (fig. 4, a). the tendon is the unstained mass in the center. it is difficult to ascertain the presence of leucocytes. it could as well be atrophic muscle fibers. charcot’s interpretation charcot describes the effects of the lesions of the anterior horn of the spinal cord in his 11th lesson, and of the lateral column of the spinal cord in the 12th and 13th lessons, the association of both lesions affecting anterior horn and lateral column, defining als. the lesion of the anterior horn and muscle atrophy: according to charcot’s description, the “large cells” of the anterior horn appear too few and atrophic, and the “neuroglia” is “sclerotic” (charcot, 1873, p 223). the anterior roots and the nerves are atrophic. charcot had understood that the muscle atrophy, visible at clinical examination, was due to the lesion of the anterior horn and was related to atrophy of the muscle fibres, visible at microscopic examination, contrarily to what is observed in muscle atrophy due to inactivity. “motor inertia caused in the lower limbs by the suppression of cerebral action may be complete, absolute. muscles, however, in such cases, do not lack nutrition or only in the long run because of protracted inaction” (charcot, 1873, p 199). the anterior horn provides the “nutrition” for the muscle – “nutrition” is a term charcot uses repeatedly in the study of the lesions of the anterior horn. the atrophy of denervated muscles had been previously described among others by vulpian (1869), a colleague and friend of charcot. charcot thought that the lesion of the anterior horn was responsible for the fasciculations that he named “fibrillar twitching”. (he added a remark that experienced neurologists could still make today: “i could add that [such fibrillar twitching] is not only seen in progressive muscular atrophy, but also in healthy subjects. it may then constitute one of the symptoms of a peculiar form of hypochondria, rather frequent, incidentally, in medical students.”) he considered that the involvement of the anterior horn spared the “faradic contractility” (contrarily to the primary involvement of the muscle). in his view, the involvement of the anterior horn was “secondary” to the lesion of the lateral column (hence, the term “deuteropathic” applied to the “chronic spinal amyotrophy” observed in als). “the progression of the inflammatory lesion from the fascicles of the white matter to the grey matter very likely takes place […] through the nervous tubes that physiologically connect more or less directly the two regions”. what was the meaning of the word “inflammation” at that time? certainly not the meaning of “neuroinflammation” as we understand it today. cohnheim (1873) was just publishing his observation on the course of inflammation as observed in thin, transparent tissue. with this experimental model, he could observe diapedesis. metchnikoff had not yet discovered phagocytosis (1893, metchnikoff, 1968). it is also worth mentioning here that the microglia have been described much later by del rio hortega (1919). the use of the term inflammation by charcot does not correspond, from today’s perspective, to his neuropathological description of the lesions: he did not describe leucocyte infiltration of the white or grey matter of the spinal cord. how could an “inflammatory lesion” progress “through the nervous tubes”? in this context, was the term “inflammation” used as a synonym for “lesion”? the lesion of the “lateral fascicle”, named later “pyramidal tract”: although the plantar reflex had not yet been described by babinski (1896), charcot believed that the motor deficit was initially due to the lesion of the lateral fascicles, rather than to the lesion of the anterior horns. muscle stiffness was a sign of the involvement of the same fascicle. the autonomy of the “system of the lateral fascicles” or simply of the “lateral fascicle” was discussed at length. he uses embryology to support his reasoning (charcot, 1873, 12th lesson, pp 215-218), showing that both the lateral fascicle and “türck’s bundle”, localized in the anterior column and altered in some cases of als, developed late in the spinal cord, in an “autonomous” manner. the description of the anatomy is both more precise and functional in his lessons of 1876 (charcot, 1876; translated in english in 1883 charcot, 1883), probably in relation with his recent acquaintance with flechsig’s work, abundantly referred to but without quotation. flechsig, head of the histology department and later professor of psychiatry in leipzig, used the chronology of myelination during development to recognize the spinal cord fascicles. they are correctly identified in charcot’s figure 46 “after flechsig” (charcot, 1876, p 179): the term “system of the lateral fasciculus” (charcot, 1873, p 220 with figures on p 221) is replaced by that of the crossed “pyramidal tract” (charcot, 1876, p 179), due to türck, who, as a professor of neurology in vienna, developed the method of secondary degeneration to follow the spinal cord bundles (türck, 1851). charcot shows pictures of the spinal cord stained by osmic acid: “thus, on a plate of flechsig that i am passing before your eyes and relating to the spinal cord of a newborn, you can see the following particularities: all the parts tinted in black are the developed parts: the cylinder-axis is surrounded by myelin sheath…” the pyramidal tracts, still unmyelinated, remain white (charcot, 1876, fig. 48 and 49, p 188-189). charcot had understood that the “autonomous development of the pyramidal tracts” was related to a late myelination. the spared bundle of white matter, outside of the pyramidal tract, precisely illustrated in the album (fig. 3) is in his 1875 lessons (charcot, 1876) correctly identified as the dorsal spinocerebellar tract (flechsig’s bundle or direct spinocerebellar tract): “as for the base of the triangle [made by the pyramidal tract], directed outwards, it is separated from the pia-mater by a band of nervous substance forming a sort of mantle for it and consisting of the direct cerebellar fascicles. but this arrangement occupies only the upper half of the medullary cord; below the dorsal region, the cerebellar bundles end and, in the lumbar region, where there is no trace of them left, the crossed pyramidal bundles touch the pia-mater” (charcot, 1876, pp 191-192). charcot’s description of the motor symptoms, based on his clinical-pathological method (goetz, 2000) is now fully achieved and underlines the systematic aspect of the atrophy, and its functional significance. charcot underlined the absence of sensory deficit in als. he knew that the lesion of the posterior columns were linked with “locomotor ataxia” but there is a stark contrast, in the lessons dealing with “localization”, between the knowledge that charcot had of the pyramidal tract and the difficulties that he still met in understanding the somatosensory pathways. he could state, for example, that for sensitivity, there is “indifferent conduction through this or that element of the spinal cord” (charcot, 1876, p 282). in charcot’s view the typical progression of als started in the upper limbs on one side, affecting secondarily the lower limbs, sparing the nerves innervating the vesical and rectal muscles, and ending with “labio-glosso-pharyngeal” paralysis, the nerves located above the viith cranial nerve being unaffected. the various clinical aspects of als remained, at the time, to be described. a note on the historical context charcot used to quote rather extensively the literature, at least according to the standards of the time. his library, still visible today, testifies of the extent of his interests. he cited several cases of the literature from france, germany and the united kingdom in support of his analysis. the second volume of the lessons, however, was published in 1873, three years only after the siege of paris by the prussian troops. people caught in paris during the siege were hungry and ate dogs, horses, rats and even elephants and antelopes from the zoo (as told by victor hugo in his book “choses vues” – hugo, 1900 passim and pp 299, 308)! charcot had sent his wife and his three children to normandy, outside paris where he, personally, continued to work at the hospital (bonduelle et al. 1996, p. 296). if charcot used to remain on purely scientific grounds, it is not impossible that the comments on a paper by nikolaus friedreich (professor of pathological anatomy in würzburg, later professor of pathology and therapy in heidelberg) were tinged with a certain resentment: “such a neglect in matters of nosographic distinctions, especially in a question in itself rather obscure, is at least regrettable and can only maintain the confusion” (charcot, 1873, footnote 1, p 208). such a critical remark is counterbalanced by a good knowledge of the german literature. it is charcot who names the direct corticospinal tract türck’s bundle (referring to türck, probably türck, 1851) and underlines the importance of flechsig (unfortunately without quotation) in the functional understanding of the disease that he was identifying (charcot, 1876, p 191). flechsig published an early and impressive synthesis of his work in 1876 (flechsig, 1876); he was only 29. conclusion it may finally be considered that the wish that charcot expressed in his lesson has been fulfilled: “i must first declare that the reports on which my description is based, are still few, twenty at the most. but one must notice that it was also the case, some time ago, concerning progressive locomotor ataxia. the clinical picture drawn by duchenne (de boulogne) with the help of a small number of facts, nearly 20 years ago, has not aged. [...] may my description of amyotrophic lateral sclerosis experience the same fate!” (charcot, 1873, p 228) acknowledgement the help of véronique leroux-hugon, previous curator of charcot library, and of florian horrein, the current curator, is greatly acknowledged. charcot’s album belongs to the “musée de l’assistance publique-hôpitaux de paris”, which we thank, and kept in the neuropathology department of la salpêtrière hospital. references babinski j. (1896) sur le réflexe cutané plantaire dans certaines affections du système nerveux central. c r seances soc biol. 3: 207-208. blum f. (1893) der formaldehyd als hartungsmittel. z wiss mikrosk. 10: 314-315. bonduelle m., gelfand t., goetz c.g. (1996) charcot, un grand médecin dans son siècle. michalon publisher, paris. charcot j.-m. (1865) sclérose des cordons latéraux de la moelle épinière, chez une femme hystérique, atteint de contracture permanente des quatre membres. bull mém soc méd hôp paris. 2: 24-35. [the document may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k5440456t/f36.item ] charcot j.-m. (1873) leçons sur les maladies du système nerveux faites à la salpêtrière par j.m. charcot, recueillies et éditées par bourneville. iie série. adrien delahaye publisher, paris. [the book may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k6149090r/f12.item ] charcot j.-m. (1876) leçons sur les localisations dans les maladies du cerveau et de la moëlle épinière, faites à la faculté de médecine de paris, recueillies et publiées par bourneville et brissaud (1875). a. delahaye & lecrosnier publisher, paris. [the book may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k773614/f177.item ] charcot j.-m. (1883) lectures on the localization of cerebral and spinal diseases delivered at the faculty of medicine of paris translated and edited by walter baugh madden. the new sydenham society. volume cil. [the book may be accessed on the internet: https://archive.org/details/lecturesondiseas02char ] charcot j.-m. (1892) leçons du mardi à la salpêtrière. policlinique 1887-1888. notes de cours de mm. blin, charcot et h. colin, élèves du service. vve babé publisher, paris. [the book may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k55646784/f4.item.r=fig ] charcot j.-m., joffroy a. (1869) deux cas d’atrophie musculaire progressive avec lésions de la substance grise et des faisceaux antérolatéraux de la moelle épinière. arch physiol norm pathol. 2 : 354-367, 629-649, 744-760 (note that the paper was published in three parts, in different issues of the journal.) [the paper may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k432734s/f354.item ] chvátal a. (2017) jan evangelista purkyně (1787-1869) and his instruments for microscopic research in the field of neuroscience. j hist neurosci. 26: 238-256. cohnheim j. (1873) neue untersuchungen über die entzündung. august hirschwald, publisher, berlin. dupont j.c. (2018) historical perspective on neuroembryology: wilhelm his and his contemporaries. genesis. 56: 6-7. duval m. (1878) précis de technique microscopique et histologique ou introduction à l’anatomie générale. j.-b. baillière et fils publisher, paris. [the book may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k9618339x.texteimage ] flechsig p. (1876) die leitungsbahnen im gehirn und rückenmark des menschen auf grund entwickelungsgeschichtlichter untersuchungen. wilhelm engelmann publisher, leipzig. goetz c.g. (2000) amyotrophic lateral sclerosis: early contributions of jean-martin charcot. muscle nerve. 23: 336-43. hugo v. (1900) choses vues. nouvelle série. calmann-lévy publisher, paris. [the book may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k141436z.r=victor%20hugo%20choses%20vues?rk=21459 ] gombault m. (1871) sclérose symétrique des cordons latéraux de la moelle et des pyramides antérieures dans le bulbe. – atrophie des cellules des cornes antérieures de la moelle.—atrophie musculaire progressive. – paralysie glosso-laryngée, arch physiol norm pathol. 4: 509-518 [the paper may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k432736j/f517.item ] lockhart clarke j. (1851) researches into the structure of the spinal chord. 141: 607-621 [the paper may be accessed on the internet: https://royalsocietypublishing.org/doi/pdf/10.1098/rstl.1851.0029 ] lockhart clarke j. (1858) researches into the intimate structure of the brain, human and comparative. first series. on the structure of medulla oblongata. philos trans r soc lond. 148: 231-259 [the paper may be accessed on the internet: https://wellcomelibrary.org/item/b22297169 ] metchnikoff e. (1968) lectures on the comparative pathology of inflammation, delivered at the pasteur institute in 1891 dover publications publisher, mineola. proescher f. (1934) contribution to the staining of neuroglia. stain technology. 9: 33-38 [the paper may be accessed on the internet: https://doi.org/10.3109/10520293409116130 ] radcliffe c.b., lockhart clarke j. (1862) an important case of paralysis and muscular atrophy with disease of the nervous centres. brit & foreign medico-chirurgical rev. 30: 215–225 río-hortega p. (1919) el "tercer elemento” de los centros nerviosos. i. la microglía en estado normal, boletín de la sociedad española de biología. 8: 67-82 seilhean d. (2020) neuropathology in pitié-salpêtrière hospital: past, present and prospect. neuropathology. 40: 3–13. doi:10.1111/neup.12616. [the paper may be accessed on the internet: https://onlinelibrary.wiley.com/doi/full/10.1111/neup.12616 ] türck l. (1852) über secundäre erkrankung einzelner rückenmarkstränge und ihrer fortsetzung zum gehirne. akad wissensch wien math naturwiss class sitzungber. 8: 511-534. turner m.r., swash m., ebers g.c. (2010) lockhart clarke’s contribution to the description of amyotrophic lateral sclerosis. brain. 133: 3470-3479. [the paper may be accessed on the internet: https://academic.oup.com/brain/article-pdf/133/11/3470/882976/awq097.pdf ] vulpian a. (1869) sur les modifications que subissent les muscles sous l’influence de la section de leurs nerfs. arch physiol norm pathol. 2: 558-578. [the paper may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k432734s/f558.item ] copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. deposits of disease-associated alpha-synuclein may be present in the dura mater in lewy body disorders: implications for potential inadvertent transmission by surgery feel free to add comments by clicking these icons on the sidebar free neuropathology 1:6 (2020) original paper deposits of disease-associated alpha-synuclein may be present in the dura mater in lewy body disorders: implications for potential inadvertent transmission by surgery ellen gelpi md1,2, naomi p. visanji phd3, selma hönigschnabl md4, angelika reiner md4, peter fischer md5, anthony e. lang md3, herbert budka md1, gabor g. kovacs md3,6,7 1 division of neuropathology and neurochemistry, department of neurology, medical university of vienna, vienna, austria 2 neurological tissue bank of the biobanc-hospital clinic-idibaps, barcelona, spain 3 edmond j. safra program in parkinson’s disease and the morton and gloria shulman movement disorders clinic, toronto western hospital, toronto, ontario, canada 4 institute of pathology, danube hospital of vienna, austria 5 department of psychiatry, medical research society vienna d.c., danube hospital, vienna, austria 6 laboratory medicine program & krembil brain institute, university health network, toronto, ontario, canada; 7 department of laboratory medicine and pathobiology and tanz centre for research in neurodegenerative disease, university of toronto corresponding author: professor gabor g kovacs md phd frcpc · laboratory medicine program · toronto general hospital · university health network · 200 elizabeth street · toronto on · canada · m5g 2c4 gabor.kovacs@uhnresearch.ca submitted: 09 january 2020 accepted: 06 february 2020 published: 12 february 2020 https://doi.org/10.17879/freeneuropathology-2020-2616 keywords: alpha-synuclein, dura mater, lymphatic drainage, prion, propagation, transmission abstract deposition of alpha-synuclein in the brain is a hallmark of lewy body disorders. alpha-synuclein has been considered to show prion-like properties. prion diseases can be transmitted by the transplantation of cadaveric dura mater causing iatrogenic creutzfeldt-jakob disease. recent observations of amyloid-β deposition in dural grafts support the seeding properties of amyloid-β. here we assessed the presence of alpha-synuclein in dura mater samples as a potential transmissible seed source. we immunostained 32 postmortem dura mater samples; 16 cases with lewy-body disorder (lbd) showing different pathology stages and 16 non-lbd cases for phosphorylated (ser129) and disease-associated (5g4) alpha-synuclein. disease-associated alpha-synuclein aggregates were identified in intradural nerve fibres and associated with a vessel in a single lbd-braak stage 4 case. we conclude that alpha-synuclein is detectable, although rarely, in dura mater samples in patients with lbd. the risk of potential transmissibility of dural alpha-synuclein deserves assessment by complementary experimental studies. introduction neurodegenerative diseases are considered protein-misfolding disorders. as described in prion diseases, the host physiological protein adopts an abnormal conformation (seed), capable of inducing misfolding and aggregation of the neighboring molecules (e.g. for prion protein: the physiological prpc converting to disease-associated prpsc, and prpsc recruiting more prpc). in prion disease, strong clinical, epidemiological and experimental evidence has demonstrated human and interspecies transmissibility of prpsc(1). for several other proteins associated with neurodegenerative conditions, such as amyloid-β (aβ), tau, alpha-synuclein, and tar dna binding protein (tdp-43), properties similar to those of prp have been shown predominantly in an experimental setting (2). the potential of aβ peptides to be transmitted between humans has been a topic of great interest for public health. young adults with iatrogenic creutzfeldt-jakob-disease (cjd) following cadaveric pituitary-derived growth hormone in childhood were found to have aβ deposits in the brain parenchyma and blood vessels (3, 5, 6), as well as in iatrogenic cjd after dura grafting (4). two studies have reported the presence of aβ deposits in dura mater samples of postmortem brains of patients with alzheimer’s disease (ad) and other neurodegenerative pathologies (7, 8). additionally, neurosurgical procedures and dural grafting during childhood or teenage years have been related to increased cerebral amyloid angiopathy (caa) decades later (9, 10). these findings indicate that dura mater grafts could be also a source of aβ seeds capable of inducing the aggregation of aβ in the cns of the graft recipients. lewy-body disorders (lbd) and multiple system atrophy (msa) are characterized by the accumulation of alpha-synuclein. a growing body of experimental evidence supports prion-like propagation in alpha-synucleinopathies (11-13). observations in a small number of subjects with parkinson`s disease (pd) treated with fetal mesencephalic dopaminergic grafts in the striatum, who years later were found to have alpha-synuclein positive lbs in grafted neurons, first suggested the potential of alpha-synuclein propagation from host to graft cells (14, 15). while experimental studies have also successfully demonstrated the transmission potential of msa alpha-synuclein in genetically modified mouse models (16), observations in humans argue against a human-to-human iatrogenic transmission of msa and pd (17). given the potential implications for public health, we posit that thorough assessment of the potential transmissibility of all neurodegenerative proteinopathies is essential (18). therefore, in this study, we assessed human postmortem dura mater samples, some of which have been previously shown to harbor aβ deposits (7), for the presence of alpha-synuclein aggregates, which may represent seeds with the potential of propagating further synucleinopathy. material and methods formalin-fixed and paraffin-embedded postmortem tissue samples of the dura mater from the left temporal and posterior region as well as cross-sections from the area of the superior sagittal sinus and confluence of sinuses (7), were cut into 5-micrometer thick sections. this study was approved by the ethics committee of the medical university of vienna. brains were previously evaluated neuropathologically and a final primary diagnosis and concomitant pathologies were recorded. fixation time of dura mater samples ranged from 3 to 10 years. immunohistochemistry of the dura mater samples was performed on an automated immunostainer (dako autostainer link 48, glostrup, denmark) using two different anti-alpha-synuclein antibodies: clone 5g4 (1:4000), roboscreen, leipzig, germany; specific for disease-associated forms and the phospho-specific anti-alpha-synuclein phosphorylated at serine 129 (wako) (1:2000). in addition, we performed immunohistochemical staining to depict the nerve fibres within the connective tissue of the dura mater: anti-neurofilament protein (nfp), clone 2f11 (1:800), dako; pgp 9.5 (1:100), dako, rabbit polyclonal; anti-tyrosine hydroxylase, clone 1b5 (1:200), novocastra, newcastle, uk; and anti-phosphorylated neurofilaments, clone smi31 (1:5000), former covance research products, princeton, nj, usa, and for blood and lymphatic vascular endothelia (anti-cd34, clone qbend/10 (1:100), novocastra; and podoplanin (1:3000), rabbit polyclonal). dako flex system (peroxidase/dab) was used for visualization of antibody reactions. samples were assessed blind to the neuropathological diagnosis. the presence or absence of each specific immunoreactivity was recorded. in addition, double immunofluorescence combining pgp 9.5 and 5g4 antibodies and immunofluorescence staining with nfp and 5g4 on adjacent tissue sections was performed in one selected case. results we analyzed postmortem dura mater samples from 32 donors (50% females) aged between 84 and 87 years (mean 84.9) from the vienna transdanube aging (vita) study (19). table 1: demographic and neuropathological features of the study cohort abbreviations: ad: alzheimer’s disease; pd: parkinson’s disease; copd: chronic obstructive pulmonary disease; ad-np: alzheimer’s disease neuropathological changes; caa: cerebral amyloid angiopathy; agd: argyrophilic grain disease; hs: hippocampal sclerosis; part: primary age-related tauopathy; svd: small vessel disease; cbd: corticobasal degeneration. n.a.: not available; n.e.: not evaluable due to unsteady immunoreactivity these cases were analyzed in a previous study for the presence of aβ deposits neuropathological diagnoses included 16 cases with alpha-synuclein pathology (table 1; six cases with amygdala predominant lewy body pathology, one msa case, single cases of braak stage 1, stage 2, stage 3, stage 5 and stage 6, and four cases with braak stage 4 of pd-related pathology) (20); and 16 cases without lb pathology but other neuropathological conditions. nerve fibers were detected mainly by nfp immunohistochemistry in all cases in variable distribution: large nerves surrounding large vessels (fig. 1a, b) or smaller nerve branches in between both dura layers (fig. 1c, d). ). these patterns were also observed by immunofluorescence (fig. 2). no immunoreactivity for tyrosine-hydroxylase could be detected. cd34 and podoplanin also gave negative results. figure 1 a-d: histologic appearance of dura mater consisting of two lamellae of connective tissue. within the connective tissue there are large and small vessels, which are surrounded by nerve fibres (a-d, neurofilament immunohistochemistry). nerve fibres are also identified between the lamellae and within the connective tissue (arrows). e-i: immunohistochemistry for phosphorylated alpha-synuclein at serine 129 and for oligomeric forms with the 5g4 antibody did not show abnormal alpha-synuclein aggregates in most of the studied cases (fig. 1e, f). in one case (case #12 table 1), coarse (g) and punctate (h) aggregates within nerve fibres and perivascular (i) aggregates were identified with the 5g4 antibody. detailed immunohistochemical analysis of dura samples identified pathological alpha-synuclein aggregates with the 5g4 antibody in one case. these aggregates consisted of small punctate deposits along nerve fibres (fig. 1h, 2b, d), coarse aggregates (fig. 1g, 2f) and perivascular deposits (fig. 1i, 2a). we observed a variable staining intensity of axons among the different cases and also within the same case, which was not related to the presence/absence of alpha-synuclein. this variability might have been related to the fixation time or tissue quality, among other potential variables, so we could not reliably assess axonal preservation in the patient with alpha-synuclein aggregates. we found no tortuous axonal profiles or axonal swellings. this individual was an 85-year-old male with coronary disease with severe and generalized atherosclerosis, an old myocardial infarction, as well as pulmonary adenocarcinoma. no parkinsonism or dementia were clinically reported. neuropathological examination revealed a vascular encephalopathy and braak stage 4 lewy-body pathology, with only mild neurofibrillary pathology in the limbic system and mild neuritic plaque / aβ pathology (table 1, case 12). in the remaining cases included in our study, with or without cns lewy body pathology, there was a complete lack of alpha-synuclein immunoreactivity in dural samples applying both antibodies. figure 2: immunofluorescence images a, b: double immunofluorescence for pgp 9.5 depicts delicate perivascular fibres (red) which focally also accumulate alpha-synuclein (5g4, green). other regions show nerve fascicles with variable density of axonal profiles: some have abundant axons (b, pgp 9.5, red) and show only tiny alpha-synuclein accumulations (5g4, green, white arrow). c-f: in adjacent tissue sections some nerve fascicles have only few axons (c, nfp) while others show an intermediate density of axons (e, nfp red) with relatively abundant and coarse alpha-synuclein aggregates (f, 5g4). discussion in this study, we found alpha-synuclein aggregates in dura mater samples from one patient with lewy-body pathology (braak stage 4) (20). alpha-synuclein aggregates were not identified in the dura mater of 31 other cases with different stages of lbd or in other neurodegenerative conditions. these findings might have implications for public health as well as for the understanding of alpha-synuclein propagation. concerning public health, individuals with symptomatic or prodromal alpha-synucleinopathies may undergo neurosurgical interventions contacting the dura mater (for example, the widespread use of deep brain stimulation in pd). while there is still no epidemiological evidence of human-to-human transmission of alpha-synuclein (17), the application of appropriate prevention measures will be increasingly required if experimental studies strengthen evidence of the potential transmissibility of synucleinopathies. this further emphasizes the need for effective biomarkers for the early diagnosis of alpha-synuclein related diseases (21-24). furthermore, there is a clear need for the detailed mapping of tissues that may harbor potential seeds. indeed, with respect to the present findings, since dura mater transplants are in direct contact with brain tissues, they might provide an increased potential to propagate seeds to the recipient’s cns. concerning protein propagation, the dura mater has been a source of disease transmission in human prion disease (1) and is also a source for aβ seeds (9, 10). recent studies support the notion that these seeds are able to propagate pathological proteins, even without inducing the classical alzheimer´s phenotype. however, they can lead to unexpectedly increased frequency of cerebral amyloid angiopathy and associated brain hemorrhages (9, 10). propagation of human alpha-synuclein has been considered since the detection of alpha-synuclein aggregates in grafted fetal mesencephalic neurons in pd patients (14, 15). this has subsequently been demonstrated in experimental studies (13) including inoculation of human substantia nigra extracts containing lewy bodies into wild-type mice and macaque monkeys (26). in contrast, the inoculation of extracts of peripheral alpha-synuclein from the autonomic sympathetic stellate ganglion did not induce pathological conversion of endogenous alpha-synuclein, alpha-synuclein propagation, or neurodegeneration in mice (27). thus, central and peripheral nervous system-related alpha-synuclein might have different propagation potential and therefore different transmissibility. the dura mater is thoroughly innervated by afferent fibres arising from the ipsilateral trigeminal ganglion and nerve (including its ophthalmic, maxillary and mandibular divisions) and by sympathetic fibres arising from the ipsilateral superior cervical ganglion. the posterior cranial fossa dura mater receives sensory meningeal branches from the vagus and glossopharyngeal nerves. the sympathetic ganglion is upstream from the stellate ganglion, which has been shown to harbor prominent alpha-synuclein or lewy-type pathology in postmortem studies of patients with pd and dementia with lewy bodies (23, 24), along with the dorsal motor nucleus of the vagal nerve in the cns (20). therefore, it might be reasonable to consider that these sympathetic and parasympathetic fibres innervating the dura mater might also accumulate alpha-synuclein. although in that particular case where we found alpha-synuclein in the dura mater, we did not have the opportunity to stain the trigeminal ganglion or nerve nor the stellate ganglion, we have previously evaluated the trigeminus nerve using immunostaining for alpha-synuclein. in that study we found alpha-synuclein immunoreactivity in two out of four pd/lbd cases where a total of six trigeminal nerves (in two cases both sides available) were examined (28). alternatively, we have previously reported that the presence of lymphatic vessels in dura mater might be a drainage route for molecules from the brain parenchyma in neurodegenerative disorders, including alpha-synuclein in lbd (25). although we found discrete perivascular alpha-synuclein aggregates in dura mater samples, we were not able to confirm whether these represent lymphatic vessels, possibly due to the prolonged fixation time of this dura mater sample (<6 years), and therefore this question deserves further study. the present study has some limitations. first, as mentioned above, the dura mater samples were obtained after a prolonged fixation time, which may influence immunohistochemical results, not only for cd34, podoplanin and th, but for phospho-synuclein as well (29). therefore, the interpretation concerning the perivascular association of alpha-synuclein aggregates needs some caution. we could also not reliably assess axonal preservation in the case harboring alpha-synuclein aggregates. the prolonged fixation also does not allow complementary molecular/biochemical studies. while the identification of characteristic pathological aggregates of alpha-synuclein with only one antibody is limiting, the 5g4 antibody has shown to be a robust marker of early alpha-synuclein pathology, even in brains with prolonged fixation time (30). second, the level of pathological forms of alpha-synuclein detectable by conventional immunohistochemistry may be low. moreover, the analysis of the supratentorial, rather than the infratentorial dura could also have been a limiting factor due to differences in their innervation; however, the supratentorial dura mater also receives sympathetic and parasympathetic innervation, and thus could also be exposed to transport of misfolded proteins. the negative findings in the other lbd cases of the series might reflect a sampling limitation, as it was not feasible to examine the whole dura mater. alternatively, the lack of staining could also reflect the diverse innervation of the dura and different pathogenic capacities of central and peripheral alpha-synuclein, or it could also indicate that the dura mater is not a primary vulnerable region for alpha-synuclein aggregation and could be considered to be a low risk tissue for the accumulation of potentially transmissible seeds. it must be emphasized that this study raises a potentially important issue but is unable to provide a conclusion on the risk of transmission of alpha-synuclein via dura mater grafts or neurosurgical procedures. this observation would ideally be complemented by experimental studies using prospectively collected freshly harvested, short fixed and frozen dura samples, including a larger cohort of patients of different age groups. conclusion disease-associated alpha-synuclein is detectable by conventional immunohistochemistry in postmortem supratentorial dura mater, here in a single patient with lewy-body-type alpha-synucleinopathy in the cns. this suggests propagation of alpha-synuclein from the brain and may indicate that dura mater could be a potential risk tissue for inadvertent transmission of alpha-synuclein by dura mater grafts or surgery. further experimental and biochemical studies are needed to assess the risk of alpha-synuclein transmission via dura mater and its relationship with human disease. acknowledgements we are grateful to susi schmid, irene ebner, gerda ricken and sigrid klotz from the division of neuropathology and neurochemistry, department of neurology, medical university of vienna, for excellent technical support. we are indebted to brain donors and their family of the vienna transdanube study (vita) for generous brain and sample donation for research. this work has been supported by the bishop karl golser award to ggk. references 1. brown p, brandel jp, sato t, et al. iatrogenic creutzfeldt-jakob disease, final assessment. emerg infect dis. 2012; 18:901-907. 2. duyckaerts c, clavaguera f, potier mc. the prion-like propagation hypothesis in alzheimer's and parkinson's disease. curr opin neurol. 2019; 32:266-271. 3. jaunmuktane z, mead s, ellis m, et al. evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy. nature. 2015; 525:247-250. 4. frontzek k, lutz mi, aguzzi a, et al. amyloid-β pathology and cerebral amyloid angiopathy are frequent in iatrogenic creutzfeldt-jakob disease after dural grafting. swiss med wkly. 2016; 146:w14287. 5. silvia a. purro, mark a. farrow, jacqueline linehan, et al. transmission of amyloid-β protein pathology from cadaveric pituitary growth hormone. nature. 2018; 564:415-419. 6. burwinkel m, lutzenberger m, heppner fl, et al. intravenous injection of beta-amyloid seeds promotes cerebral amyloid angiopathy (caa). acta neuropathol commun. 2018; 6:23. 7. kovacs gg, lutz mi, ricken g, et al. dura mater is a potential source of aβ seeds. acta neuropathol. 2016; 131:911-923. 8. hamaguchi t, taniguchi y, sakai k, et al. significant association of cadaveric dura mater grafting with subpial aβ deposition and meningeal amyloid angiopathy. acta neuropathol. 2016; 132:313-315. 9. jaunmuktane z, quaegebeur a, taipa r, et al. evidence of amyloid-β cerebral amyloid angiopathy transmission through neurosurgery. acta neuropathol. 2018; 135:671-679. 10. banerjee g, adams me, jaunmuktane z, et al. early onset cerebral amyloid angiopathy following childhood exposure to cadaveric dura. ann neurol. 2019; 85:284-290. 11. recasens a, dehay b, bové j, et al. lewy body extracts from parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. ann neurol. 2014; 75:351-362. 12. karpowicz rj jr, trojanowski jq, lee vm. transmission of α-synuclein seeds in neurodegenerative disease: recent developments. lab invest. 2019; 99:971-981. 13. desplats p, lee hj, bae ej, et al. inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. proc natl acad sci u s a. 2009; 106:13010-13015. 14. kordower jh, chu y, hauser ra, et al. lewy body-like pathology in long-term embryonic nigral transplants in parkinson's disease. nat med. 2008; 14:504-506. 15. li jy, englund e, holton jl, et al. lewy bodies in grafted neurons in subjects with parkinson's disease suggest host-to-graft disease propagation. nat med. 2008; 14:501-503. 16. woerman al, oehler a, kazmi sa, et al. multiple system atrophy prions retain strain specificity after serial propagation in two different tg(snca*a53t) mouse lines. acta neuropathol. 2019; 137:437-454. 17. de pablo-fernandez e, cerdán santacruz d, warner tt, et al. no evidence of iatrogenic human transmission in autopsy confirmed multiple system atrophy. mov disord. 2018; 33:1183-1184. 18. de sousa pa, ritchie d, green a, et al. renewed assessment of the risk of emergent advanced cell therapies to transmit neuroproteinopathies acta neuropathol. 2018; 137:363-377. 19. fischer p, jungwirth s, krampla w, et al. vienna transdanube aging "vita": study design, recruitment strategies and level of participation. j neural transm suppl. 2002; 62:105-116. 20. braak h, del tredici k, rüb u, et al. staging of brain pathology related to sporadic parkinson's disease. neurobiol aging. 2003; 24:197-211. 21. vilas d, iranzo a, tolosa e, et al. assessment of α-synuclein in submandibular glands of patients with idiopathic rapid-eye-movement sleep behaviour disorder: a case-control study. lancet neurol. 2016; 15:708-718. 22. tolosa e, gaig c, santamaría j, et al. diagnosis and the premotor phase of parkinson disease. neurology. 2009; 72(7 suppl):s12-20. 23. gelpi e, navarro-otano j, tolosa e, et al. multiple organ involvement by alpha-synuclein pathology in lewy body disorders. mov disord. 2014; 29:1010-1018. 24. beach tg, adler ch, sue li, et al; arizona parkinson's disease consortium. multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with lewy body disorders. acta neuropathol. 2010; 119:689-702. 25. visanji np, lang ae, munoz dg. lymphatic vasculature in human dorsal superior sagittal sinus: implications for neurodegenerative proteinopathies. neurosci lett. 2018; 665:18-21. 26. recasens a, dehay b, bové j, et al. lewy body extracts from parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. ann neurol. 2014; 75:351-362. 27. recasens a, carballo-carbajal i, parent a, et al. lack of pathogenic potential of peripheral α-synuclein aggregates from parkinson's disease patients. acta neuropathol commun. 2018; 6:8. 28 kovacs gg, breydo l, green r, et al. intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread. neurobiol dis. 2014; 69:76-92. 29. pikkarainen m, martikainen p, alafuzoff i. the effect of prolonged fixation time on immunohistochemical staining of common neurodegenerative disease markers. j neuropathol exp neurol. 2010; 69:40-52. 30. kovacs gg, wagner u, dumont b, et al. an antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology. acta neuropathol. 2012; 124:37-50. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. between two worlds life in neuropathology and beyond feel free to add comments by clicking these icons on the sidebar free neuropathology 1:22 (2020) reflections between two worlds life in neuropathology and beyond peter lantos professor emeritus, king’s college london address for correspondence: peter lantos · www.peter-lantos.com · peter.lantos@btinternet.com submitted: 10 august 2020 accepted: 12 august 2020 published: 17 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2933 keywords: hungary; middlesex hospital, london; neuropathology; institute of psychiatry/maudsley, london; papp-lantos inclusions; bergen-belsen; memoir, fiction and plays introduction on a rainy october afternoon in 1968, as my plane landed at heathrow airport from budapest, i did not know that i would never return to the country of my birth and early youth. i arrived with a small suitcase and five pounds in my pocket. i was a fortunate and happy recipient of a wellcome research fellowship at £1,200 per annum, which had been awarded two years earlier. the hungarian authorities, however, for reasons known only to themselves, did not give me an exit permit. my visit was on a knife edge until the last minute, for on the day when i was going to the british consulate to apply for a visa, the hungarian state radio announced that the armies of the warsaw pact (including hungary’s) were giving “brotherly help” to the people of czechoslovakia. translating this orwellian newspeak, it meant invading a friendly, neighbouring country. arrival in london my first impression was compensation for all the preceding stress: my future boss, professor stanley holt, met me at the airport and brought one of his assistants, who happened to also be a hungarian and who left after the revolution of 1956. holt (fig 1), or “holtie” to his friends, was a cell biologist of international reputation. his department of histochemistry of the courtauld institute of biochemistry within the middlesex hospital medical school, london, w1, which despite its small size, was a beehive of activity with young scientists from all over the world. he originally excelled in histochemistry and later applied electron microscopy to the demonstration of enzymes – a methodology i learned from him. this expertise stood me in good stead one year later when i started to work on my phd. he had lexicographic knowledge of a wide range of subjects (including an in-depth personal expertise of scottish single malt whiskies), and was an excellent teacher and a most entertaining raconteur. fig 1 professor stanley holt in the late summer of 1969, as my fellowship was coming to an end, i realised that, whatever happens, i was not returning to hungary. the reason for this decision was complex, but at its core was the discovery of personal freedom with the liberating feeling that i was in charge of my own destiny. having experienced a new way of life, i felt that i could not conform to the rules and regulations of life under communism. “defection”, my trial and prison sentence in hungary when i told holtie of my decision, his response was rapid and efficient as he went to see the director of the institute and the secretary of the medical school. the next couple of months remain memorable for the kindness and help of people who allayed my way of getting permission to stay in the united kingdom, to find accommodation and, most importantly, a job. in the meantime i had to write an official letter of resignation to my workplace, the institute of morbid anatomy and histopathology, medical university in szeged, hungary. it was not an easy task. the response was immediate and brutal. the rector of the university ordered a disciplinary action on the basis that i refused to return to hungary. since i failed to appear personally to present my case, i was summarily dismissed from employment. but this was not the end. i was also indicted with a criminal charge of refusing to return to hungary. the trial took place, in my absence, on 9 february 1970 in the district court of szeged. i was sentenced to 16 months imprisonment as principal punishment and complete confiscation of all my belongings as secondary punishment. the latter i viewed with a sense of bitter irony, for it was for the third time in less than three decades that we lost all our material possessions: first, the fascists took away everything in 1944, then the communists closed down the family’s enterprise in 1949 and finally i lost everything in 1970, for my flat in szeged had been sealed and sold with its contents. for many weeks after my “defection”, i had a recurring nightmare. i was getting on the plane at budapest airport and, as i was fastening my seatbelt, a stewardess called my name and frogmarched me off the plane. i was terrified; i would never fly to london. the nightmare always ended the same way i awoke in a pool of sweat, but happily realising that i was in london after all. many years later in 2015, i learned that the hungarian state security (avo) had a file on me (fig 2). at my request its archives sent a copy which is now in my possession. it consists of 39 pages, with all the details of my trial and some other information that had been kept for decades. “big brother” had a long memory. fig 2 copy of my photograph in the file of the hungarian state security, taken circa 1966 the middlesex hospital medical school, london my first appointment was a research assistant in the bland-sutton institute of pathology of the middlesex hospital at an annual salary of £2,160. not knowing at the time, i was the first to be employed from the so-called “biscuit money.” in the 1960s, garfield weston, multi-millioner owner of fortnum and mason (the queen’s grocery shop) and many other outlets, donated a large sum of money to promote research into neurological diseases at the middlesex hospital. this donation was, by the way, the seed from which the reta lila weston institute of neurological studies at university college london later blossomed. professor george dick, director of the bland-sutton, and dr. helen (“wendy”) grant, a consultant neuropathologist (fig 3), were looking for a research worker who would examine ultrastructural abnormalities in progressive multifocal leukoencephalopathy (pml). this was an appointment clearly tailored for me, nevertheless i was formally interviewed. fig 3 dr. helen (wendy) grant in 1987 i started to investigate post-mortem brains with pml, but soon realised that the information we could get would not add a great deal to the existing knowledge. after an extensive literature search and discussions with professor dick and dr grant, i suggested that my project be in the field of experimental brain tumours. two british biochemists, peter magee and john barnes, had discovered the carcinogenic actions of nitroso compounds in 1956. subsequent extensive investigations revealed that two nitrosoureas, methyland ethyl-nitrosourea, have preferential carcinogenic actions in the nervous system. ethyl-nitrosourea (enu) when injected in a single dose to pregnant rats after the 15th day of gestation induces tumours at a rate of nearly 100% in the nervous systems of offspring. this is of paramount importance in carcinogenesis; because the precise time of interaction between the carcinogen and the cells is known, it became possible to study carcinogenesis and the evolution of tumours from their inception. phd on brain tumours it was at this time that i met a young german neuropathologist who expressed his ideas with enthusiasm and some force. we started to collaborate and became friends: his name is paul kleihues and he came to work with magee, a professor at the courtauld institute of biochemistry. i started my project on the development of enu-induced tumours of the nervous system in bd-ix rats in 1970. the tumour yield of the offspring approached 100% and i sequentially followed their growth from the earliest abnormal cell proliferations to fully developed malignant tumours. pleomorphic gliomas, resembling human glioblastoma multiforme, have originated from undifferentiated cells of the subependymal plate. my first collaborator who joined me was also funded from the “biscuit money”: geoffrey pilkington, 21 years old at the time at the time of our collaborations, recently retired at the end of 2019 as professor of cellular and molecular neuro-oncology. i submitted my phd thesis to the university of london with the title, the fine structure and enzyme cytochemistry of tumours induced by n-ethyl-n-nitrosourea and was awarded in 1973. it might have been the last word in the field, but leafing through it now, its two volumes are fossils of a bygone era. while working on the phd, i was also doing the occasional neurological post-mortems and looking at brain biopsies. at the advice of senior colleagues, i decided to prepare and sit for the mrcpath examination. to learn the “trade,” i visited many neuropathological departments in england and abroad. on the basis of my phd and previous experience in hungary, i was exempted from the primary and got successfully through the final examination in the winter of 1975. this i celebrated by treating myself to a round-the-world air ticket. in the summer of 1976, i was appointed senior lecturer in neuropathology at the middlesex hospital medical school and honorary consultant to the hospital. i already had my small research team working on experimental brain tumours and my first phd student, who later became a professor of neurology in athens, greece. the changing ethos of medicine the middlesex hospital exists no more. first it fused with university college hospital london (uchl) to become “unisex” for a couple of years while the new uchl was built. it was later demolished to give space to modern blocks of flats. only its beautiful chapel – an oasis of peace and a place for reflection – has survived as a protected building. while i worked there from 1968 to 1980, it was a most pleasant and stimulating environment; the competence of its consultant staff was matched by the excellence of its academic life. medicine by that time might not have been a vocation, but it was still a profession and not the business to which it has been degraded today. it was run by doctors for patients and not by managers for profit and quotas. when the uk went through turbulent economic and financial difficulties, we were encouraged to raise funds for research; yet the basic principle that it was the university which should provide funds for salaries and infrastructure still prevailed. this changing world was reflected even in the everyday attitude of employers. young doctors of today would not believe that, for many years, it was a daily tradition at the middlesex to lay out tea with cakes and sandwiches in the senior common room – free. this is a far cry from now, when staff are expected to pay for parking at their work place. the institute of psychiatry/maudsley in 1979, the chair of neuropathology at the institute of psychiatry (iop) was advertised and i applied. to my surprise (and perhaps to everybody’s), i was appointed in october of that year; exactly ten years after i had decided to remain in england. in the immediate euphoria and surprise, i did not realise that i arrived at the wrong place, at the wrong time. the chair at the iop was, at the time, one of the two established chairs of neuropathology in the uk, created to acknowledge the work of professor alfred meyer who escaped from nazi germany and built up the neuropathology laboratory at the maudsley hospital, london (fig 4). i was the fourth incumbent of the chair and did not realise that i was going to face an uphill struggle. in the late 1970s the uk was literally bankrupt and, after the “winter of discontent” when the country was paralysed by strikes, margaret thatcher had won the election for the conservative party in may 1979. the new government embarked on an austerity policy that did not spare universities. the vacant jobs of two senior lecturers and one lecturer, advertised with the chair, were “frozen” – the dreaded term i heard for the first, but unfortunately not the last time. the department was decimated; there was one full-time and one part-time consultant, one senior registrar and a group of three neurophysiologists. however, there was no shortage of advice, with the best coming from lucien rubinstein in his letter of congratulation. i will quote the penultimate sentences, for it is wise advice even for future generations: “the main thing is to plan carefully and deliberately, and recognize where action is possible and where it is futile. if you insist, however, long enough, often enough, and nicely enough, you will, we are sure, get your own way.” and this i tried to follow during the next 22 years, with more or less success. fig 4 professor alfred meyer i made a plan for the first five years and decided that i would resign if i did not succeed. first, with a major grant from the mrc and with geoff pilkington’s help, who came with me from the middlesex hospital, we redesigned and upgraded the electron microscope unit. this was followed by modernising the histopathology laboratory and creating laboratories for immunohistochemistry, molecular biology, genetics and tissue culture. with the late professor david marsden, we successfully applied to the mrc for a grant to establish an alzheimer’s and parkinson’s diseases brain bank. after he was appointed to the chair of neurology at the institute of neurology and neurosurgery in london, we had a friendly “divorce.” the parkinsonian component moved with him, whilst the larger share stayed at the iop to become a major resource of research. this brain bank is still flourishing under the directorship of professor safa al-sarraj, head of clinical neuropathology. in 1985, with the spreading of hiv, the mrc funded an aids brain bank which became one of the national centres of aids research. by this time, the department of neuropathology expanded to occupy two floors in the iop and had a foothold in the new clinical neuroscience building of king’s college hospital. research during the 1980s and 1990s thrived, chiefly for two reasons. first, the department was fortunate to attract talented young researchers, including trainees in neuropathology, other clinicians (including psychiatrists), and a large group of non-medical neuroscientists. there were several independent, but collaborating research groups in neurodegeneration, hiv/aids, prion diseases, brain tumours, neuro-immunology, schizophrenia and autism. second, although there was never enough money to cover everything we wanted to do, the department was successful in getting longand short-term grants. by this time the department had some 50 members, including four consultants and three secretaries. when the iop merged with king’s college london as part of the major re-structuring of medical schools and postgraduate institutes in london, i became the clinical director (lead clinician) of neuropathology services covering a catchment area of 3.5 million people. i also held honorary consultant contracts with the maudsley, king’s college, guy’s and st thomas’ hospitals. in 1992, i received the degree of dsc of the university of london, and in 2001 i was elected fellow of the academy of medical sciences. my main research interest was in neurodegeneration, particularly in familial and rarer dementias and prion diseases. this included successful collaboration with psychiatrists and neurologists of the iop/maudsley and elsewhere, both in england and abroad. i was also invited to participate in three editions of the who classification of brain tumours (1993,1997 and 2000), the clinico-pathological definition of pick’s disease, the european network of brain tissue bank (european union), hiv infection of the nervous system diseases and creutzfeldt-jakob disease (international society of neuropathology), and the definition and diagnostic criteria of corticobasal degeneration, supranuclear palsy and multiple system atrophy (national institutes of health of the usa). it is in multiple system atrophy in which we have made a significant contribution to the diagnosis and definition of disease. the discovery of glial cytoplasmic inclusions, also (unfortunately not) known as papp-lantos inclusions in 1987, mátyás papp, professor of neurology and neuropathology at the semmelweis medical university of budapest (who in the early 1980s spent one year in my department), sent me several cases of striatonigral degeneration, olivopontocerebellar atrophy and shy-drager’s syndrome. he observed, by using the gallyas’ technique, tangle-like inclusions which occurred predominantly in oligodendrocytes. in london, we further characterised these inclusions by electron microscopy and immunocytochemistry (a state-of-the-art technique available at the time) and established that they were present in all three clinical syndromes. later, it was found that all three processes also contain ɑ-synuclein. the fundamental importance of glial cytoplasmic inclusions are twofold. first, they are the hallmark cellular lesions – not in any way less important than lewy bodies – by which these elusive disorders can be confidently diagnosed. second, their presence in all three clinical syndromes demonstrated that they are a single nosological entity: multiple system atrophy. editing greenfield’s neuropathology when professor david graham asked me in 1992 whether i would co-edit the sixth edition of greenfield’s neuropathology, i had no hesitation to accept. whilst editing textbooks never appealed to me, two titles were tempting: “greenfield’s” and “russell and rubinstein”. earlier, i was approached by the publisher of the latter, but the transatlantic negotiations came to a halt and i felt better not to be involved. the first edition of greenfield’s was published in 1958 and the fifth edition of 1992 was not an unqualified success. we decided to have a different concept, content and design from the previous editions. with the support of georgina bentliff, this was the beginning of our harmonious collaboration of some ten years with the publishers: first edward arnold, then hodder and stoughton and finally hodder headline. the sixth edition, published in 1997, was well received, collected a couple of prizes and had to be reprinted. the seventh edition appeared on the market in 2002 and must have been regarded as a success, for the publisher gave a special, leather-bound copy to the two editors. the british neuropathological society british neuropathologists have two excellent forums dedicated to neuropathology: the british neuropathological society (bns) and the bimonthly scientific journal, neuropathology and applied neurobiology. in the past, the bns had two annual meetings: in the winter in london with rotating venues and in the summer in the country at clinical neuroscience centres. the meetings were open to everybody who paid the registration fee. they were attended by neuropathologists and neuroscientists from other countries – mainly from continental europe. at their best, the meetings lasted two days and were frequently preceded by a symposium with invited speakers on selected, topical subjects. these were occasions as informative and stimulating scientifically as they were enjoyable socially. i joined the bns in 1972, becoming its vice-president in 1993-1995 and president in 1995-1997. the bns first published neuropathology and applied neuropathology in 1975 under the editorship of professor john cavanagh, an outstanding neuroscientist with razor-sharp critical sense. he edited the journal until 1988 when, under professor roy weller’s editorship, the journal continued to flourish as it does to the present day. the problems of british neuropathology as head of a large department of neuropathology, i was aware of three chronic problems of our specialty. first, the complex and sometimes difficult relationship with histopathology, as reflected by the chronic “independence” fight with the royal college of pathologists for recognition as a separate specialty. this was less of a problem for departments which were part of a large clinical neuroscience centre, but more difficult for those which were de facto part of a histopathology department. the royal college of pathologists eventually recognised neuropathology as a subspecialty, organised a sub-committee of neuropathology of which i was chairman (1987-1990), and allowed the final examination to be taken in neuropathology, overseen by a panel of examiners which i also chaired (1985-1990). second, irrespective of location, the cost of neuropathology services were always a concern – increasingly so with the advent of managers and during the endless reorganisations. the argument that the post-mortem examination of brains and the diagnosis of biopsies from the nervous system may exceed the average cost was not always readily accepted. the third problem was recruitment. unfortunately, neuropathology is a small specialty and during my time the number of consultants did not exceed 50 in the uk. choosing neuropathology as a career in medicine had the potential difficulty of finding a consultant appointment after the completion of training – let alone getting one at a desired location. this clearly was a limiting factor, although both the mrc and the wellcome trust were most helpful by funding training positions in neuropathology. despite the rumours: there is life after retirement the publication of the seventh edition of greenfield’s in 2002, coupled with other reasons, seemed to be the ideal time for my retirement. i also remembered my fore-teacher at school who advised to leave the party when one enjoys it most. whilst i admire my colleagues who do not seem ever want to retire, i knew that i was not going to select this avenue, despite a couple of tempting offers for part-time work. i did love neuropathology and do not think, if i were starting again, i would choose another specialty within medicine. overall i enjoyed my work, both at the middlesex hospital and at the iop, but, apart from working pro bono for medical charities, retirement was a closure of my professional life. however, even after retirement, i followed the careers of those who worked with me. it is with some satisfaction to record that all my phd students (with the exception of one who has a leading position in the british pharmaceutical industry) became professors in their fields in this country, the usa, ireland, hungary and greece. one is now executive dean of the iop, which is still one of the leading centres of its type and now appropriately renamed the institute of psychiatry, psychology and neuroscience. some of my young colleagues and trainees also had stellar careers: two at the institute of ophthalmology and one as the director of the wellcome sanger institute, having been knighted a few years ago. immediately after retirement, i enrolled in a two-year theology course on judaism, french and hebrew classes and short cookery courses and demonstrations. however, these were only diversions, for i had a project to pursue. bergen-belsen in the summer of 1944, as a child of five, i was deported with my parents first to austria and then to bergen-belsen concentration camp in germany. i was prisoner 8431, my mother 8517 and my father 8432. my father died there of starvation; my mother and i survived (fig 5). before the british army reached the camp, we were put on a train with destination theresienstadt, another concentration camp outside prague. on the way our train was bombed by the allies for they thought that it was carrying german troops, but finally we were liberated by the 30th division of the 9th us army on 13 april 1945 outside a small village in eastern germany. after travelling across war-torn europe, we arrived home to an empty family house at the end of august 1945. my mother never talked about this experience – not until i was a teenager when i started to ask questions. fig 5 with my mother in 1946 life under the communists after the war, hungary was a democratic country with a multi-party political system, free elections and an increasing living standard. however, at the end of 1949 our normal life had abruptly changed. insidiously increasing its power with the assistance of the red army, which by then had changed from a liberating to an occupying force, the communist party came to power to inaugurate years’ of terror. the revolution of 1956 brought a few days’ of hope and freedom, only to be squashed by the red army and with the introduction of reinvigorated oppression. in 1958, after some difficulties and at the protestation of my mother, my social classification was upgraded from “class alien” (which meant social death) to “other” (a dubious category with which one could just get by). i was admitted to the medical university of szeged (now named after albert szent-györgyi, the nobel prize-winning scientist who worked there). despite oppression until 1960 – for example we knew there was at least one “student” who regularly reported on us to the police – i greatly enjoyed my university years. the main reason was the quality of teaching. there was an extensive and demanding curriculum, including a good balance between theory and practice in the form of lectures and practical classes. despite the “iron curtain,” medical and scientific journals were available from all over the world, and from the late 1950s senior staff could apply for fellowships in western countries. and this is how some years later i received a wellcome research fellowship in 1966, as a lecturer in the institute of morbid anatomy and histopathology. revisiting belsen i realised that as an adult, i knew very little of what had happened to us. after all, 21 members of my family perished in the holocaust. the aim of the project i embarked on was to fill this gap. however, i encountered a formidable obstacle at the very beginning in that most of the family documents had been lost during the war. those which survived had been left, and presumably destroyed, in my flat in szeged. to rekindle memories, during 2003 and 2004 i traced the footsteps my family made 60 years earlier in 1944 and 1945. it was an exciting and rewarding time – a sort of research i had not done before: diving into the archives of four different countries and meeting other survivors and witnesses. two witnesses, amongst the many, were unforgettable: the late george gross, the american tank commander who liberated our train and whom i visited in san diego, california (fig 6) and the late professor john hankinson, professor of neurosurgery in newcastle, who as a last-year medical student volunteered to help in belsen after liberation. fig 6 with george gross in san diego in 2003 for his work, the british army presented him with a beautiful silver pocket watch which he passed on to me when i visited him in newcastle. this is one of my most cherished possessions (fig7). fig 7 the silver pocket watch from bergen-belsen on the top of my bureau reinventing myself as a self-employed author i had not planned to write a book about my childhood. however, with a large amount of material from my travels, i started to make notes as a sort of aide memoire. the experience at the beginning was quite unsettling, for there was an element of emotional involvement, completely different from the impersonal, objective and sparse style of medical/scientific writing – something i had practised for four decades. however, in the end, i had a manuscript and one of my friends, an editor, found a small publisher. parallel lines – a journey from childhood to belsen was first published in 2006 by arcadia books london (fig 8a). it was well received both in this country and abroad; it was translated into hungarian (sínek és sorsok, scolar 2009), italian (tracce di memoria, giunti 2015) and german (von ungarn nach bergen-belsen und zurück, wallstein 2017). after attending medical/scientific meetings, symposia and workshops, it was a great change and exciting to be invited to literary festivals and book launches in this country, the usa, germany, hungary, australia and new zealand. after parallel lines i wrote a novel, closed horizon, also published by arcadia books in 2012 (fig 8b). it is a dystopic novel which takes place in the surveillance state of the near future and it is a clash between the individual and the darker power of the state. “the play’s the thing” (shakespeare) i never had any intention of writing plays and it was only the love of theatre which lead me to a short course, organised by the writing academy of the publisher faber and faber, on writing plays in 2012. the course was good fun with two excellent tutors: one being a well-known playwright and the other the artistic director of a central london theatre. thus, my playwriting “career” is entirely accidental, but i enjoyed the idea of trying to bring characters to life by creating inner tensions and external conflicts. the research, which was essential for the historical background and characters of the plays, was very similar to the excitement of discoveries of medical research. yet the world of theatres, actors, directors and agents is far removed from the medical/scientific environment. it was not any less revelatory and exhilarating, yet far more uncertain and changeable – as if someone attempted to enter a mirage. nothing exemplifies this more than the author relationships with publishers. whilst medical publishers came to ask, or even begged, authors to write for them, one has to find a publisher in the in the literary world where there are over 160,000 titles a year. the first three play were collected into a volume, stolen lives to form a trilogy (regent books london 2018; fig 8c). all three take place in hungary against historical turning points: the visitor during the second world war; distorting mirrors in the 1956 revolution and stolen years at the fall of communism in 1989. in all three, the source of drama is the same: human tragedy, in the form of an irredeemable conflict between vulnerable individuals and brutal authoritarian dictatorships relentlessly motivated by an obnoxious ideology, be it fascism or communism. i have completed two more plays. light and shadow is the extraordinary love affair which shook puritanical victorian london between the french painter james (jacques) tissot and kathleen newton, a catholic irish divorcée with an illegitimate child. my last play, bread from air: the strange case of dr haber has perhaps the most complex of characters: dr fritz haber the nobel laureate in chemistry (1918), who by producing ammonia from hydrogen and nitrogen, the basic substance of fertilisers, is credited with saving the lives of two out of the seven billion people alive today. yet, it was also he who produced (on a scientific basis) poisonous gases, one of which was first introduced in 1915 against french troops at ypres. his private life was as contradictory and colourful as his professional career. he lived long enough to be refused entry by the nazis into the institute he founded and that bore his name. fortunately, he did not survive to witness the death of some of his relatives in auschwitz, who were murdered by the same poisonous gas his team had experimented with in the 1920s. these two plays were published in a volume, love and obsession (regent books, 2019; fig 8d). fig 8 covers of my books all these plays had professional rehearsed readings with actors – mostly in the theatre and one in the tate gallery – but none has had a full production yet (fig 9). now, with the pandemic creating grave consequences for theatres, it is even less likely that this will occur in the near future. nevertheless, i am working on my sixth play which, through its extraordinary heroine, brings together my country of birth and my adopted country. fig 9 at the reading of one of my plays in 2015 epilogue or final curtain after 21 years of absence, i arrived at budapest airport on a sunny september day in 1989. the city was more beautiful than i ever remembered and was floating on a wave of expectation and hope. the still “socialist” government had just opened the border to austria and this was the “beginning of the end” of communism, for the berlin wall came down two months later in november. for me this journey was of great importance, for it built a bridge between the two worlds in which i have lived: hungary of my childhood and youth and england of my adult life. in the sunshine of euphoria and wellbeing, i felt that a new golden age was dawning over hungary, europe and the world. looking back now, i could not have been more wrong. yet we should hope that common sense and decency will still prevail over madness and outrage; liberal democracy over dictatorships and populism (fig 10). fig 10 peter lantos today copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurooncology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:8 (2020) review top ten discoveries of the year: neurooncology pieter wesseling1,2 1 department of pathology, amsterdam university medical centers, location vumc, brain tumor center amsterdam, de boelelaan 1117, 1081hv amsterdam, the netherlands 2 princess máxima center for pediatric oncology, heidelberglaan 25, 3584 cs utrecht, the netherlands corresponding author: pieter wesseling, md, phd · department of pathology · amsterdam university medical centers/vumc · de boelelaan 1117 · 1081 hv amsterdam · the netherlands · tel: +31-20-4444979 p.wesseling@amsterdamumc.nl submitted: 30 january 2020 accepted: 23 february 2020 published: 26 february 2020 https://doi.org/10.17879/freeneuropathology-2020-2671 keywords: brain tumor, neuropathology, molecular diagnostics, glioma, medulloblastoma, methylome analysis abstract this article briefly discusses 10 topics that were selected by the author as top 10 discoveries published in 2019 in the broader field of neuro-oncological pathology (so including neurosciences as well as clinical neuro-oncology but with implications for neuro-oncological pathology). some topics concern new information on immunohistochemical and molecular markers that enable improved diagnosis of particular tumors of the central nervous system (cns) and information on a refined classification of medulloblastomas. subsequently, several papers are discussed that further elucidate some pathobiological aspects of especially medulloblastomas (histogenesis, molecular evolution) and diffuse gliomas (mechanisms involved in cns infiltration, role of cancer stem(-like) cells, longitudinal molecular evolution). the remaining topics concern progress made in vaccination therapy for glioblastomas and in using cerebrospinal fluid for liquid biopsy diagnosis of gliomas. clearly, substantial, and sometimes even amazing progress has been made in increasing our understanding in several areas of neuro-oncological pathology. at the same time, almost every finding raises new questions, and translation of new insights in improving the outcome for patients suffering from cns tumors remains a huge challenge. introduction looking back from time to time is good. the request of the editor-in-chief of this journal to contribute a review on ‘top 10 discoveries’ in neuro-oncology published in 2019 was the reason for the author to look back in a somewhat more structured way in order to select which topics could/should qualify for such a label. from the start it was clear that there are multiple ways to shape such a selection process. to cut a long story short, keeping the readership of free neuropathology in mind and with some help of the editor-in-chief of this journal, the choice was made to select topics based on original papers published in 2019 from a broad range of journals, important criteria being that indeed relevant progress was made and that in one way or another the findings can be expected to have substantial implications for neuro-oncological pathology. furthermore, it was decided to not just focus on articles in the high(est)-impact journals and to aim for a somewhat broader blend of topics. some topics presented in this review were selected based on just one article, while the information of two or three papers was combined for the discussion of other topics. the 10 topics that emerged from this (quite subjective!) selection process are:   1. lack of h3k27me3 staining is a promising surrogate marker for oligodendrogliomas [1] 2. improved molecular diagnosis of rare cns tumor types [2, 3] 3. refined molecular classification of non-wnt/non-shh medulloblastomas [4] 4. histogenesis of posterior fossa tumors in children [5-7] 5. longitudinal molecular analysis of diffuse gliomas [8] 6. notch1-sox2 signaling controls glioma cell invasion in white matter tracts [9] 7. ‘synaptic cooption’ in glial and metastatic cns tumors [10-12] 8. the role of cancer stem(-like) cells in glioblastomas [13] 9. personalized (neo)antigen vaccination therapy for glioblastoma [14, 15] 10. liquid biopsy diagnosis of gliomas using cerebrospinal fluid (csf) [16, 17]   of note, the ranking in this list is not based on importance of the findings, but rather on an attempt to create a flow in this review (from information that may quite readily be implemented in daily diagnostic practice via more hard-core neurobiology/neuroscience findings to some future perspectives). also, there is a bias in the selection of the topics in this list towards diffuse gliomas and embryonal tumors/medulloblastomas. obviously, other colleagues might have presented a quite different list of ‘top 10 discoveries’, also because many more very good and interesting papers with implications for neuro-oncological pathology were published in 2019. for example, the study of paramasivam et al [18], providing novel information on mutational patterns and regulatory networks of subgroups of meningiomas would be a good alternative for a topic. having said that, it is hoped that this review not only provides easily digestible information on the top 10 discoveries in neuro-oncological pathology as selected by this author, but will also be used as a stepping stone to appreciate the much more detailed information in the original papers that were used as building blocks for this review. topic 1. lack of h3k27me3 staining is a promising surrogate marker for oligodendrogliomas [1] unequivocal histological recognition of (anaplastic) oligodrogliomas, idh-mutant & 1p/19q-codeleted (‘canonical oligodendrogliomas’) has been hindered by the lack of specific immunohistochemical markers for these tumors [19]. in a study analyzing an epigenetically well-defined cohort of diffuse gliomas in adults (idh-mutant & 1p/19q-codeleted oligodendrogliomas, n = 26; idh-mutant astrocytic tumors, n = 34; idh-wildtype glioblastomas, n = 101), filipski et al report that 25 out of the 26 oligodendrogliomas showed lack of nuclear h3k27me3 staining, in the remaining case h3k27me3 staining was interpreted as non-conclusive [1]. interestingly, in an otherwise ‘nucleonegative’ oligodendroglioma of a female patient only dot-like nuclear staining was seen. this phenomenon has previously been described to represent the inactivated x chromosome, with h3k27me3 functioning as a transcriptional silencing mechanism via chromatin remodeling [20] (fig. 1). the vast majority of idh-mutant and idh-wildtype astrocytic tumors showed clear nuclear h3k27me3 staining. based on these findings in combination with the results of immunohistochemistry for atrx, idh1r132h and h3k27m, filipski et al report that in their cohort of h3k27-wildtype diffuse gliomas the tumors showing lack of nuclear h3k27me3 staining, retention or non-conclusive nuclear atrx staining, and idh1r132h mutation are oligodendroglioma, idh-mutant & 1p/19q codeleted with a predicted probability of 0.9678. ideally, in case of h3k27me3and h3k27m-nucleonegative diffuse gliomas that in addition show retained or non-conclusive atrx staining but no idh1r132h-mutant protein staining, sequencing analysis is performed to demonstrate presence/absence of an idh1 or idh2 mutation. obviously, the results of immunohistochemistry should be interpreted with caution in especially biopsies that are small and/or only show low tumor cell percentage. the use of an antibody panel including h3k27me3, h3k27m mutant protein, atrx, and idh1r132h may thus greatly facilitate recognition (anaplastic) oligodendrogliomas, idh-mutant & 1p/19q-codeleted, especially so in a situation where molecular testing is not readily available/possible. the pathobiology underlying the global lack of h3k27me3 in tumor cell nuclei and 1p/19q codeletion needs further elucidation. of note, while in diffuse gliomas in adults lack of h3k27me3 staining may thus indicate canonical oligodendroglioma with a relatively favorable prognosis, in several other tumors of the (central) nervous system (e.g. posterior fossa ependymomas, malignant peripheral nerve sheath tumors, meningiomas) such a lack of nuclear staining is associated with worse prognosis [21-23]. fig. 1. loss of nuclear h3k27me3 immunohistochemical staining; a surrogate marker for oligodendroglioma, idh-mutant and 1p/19q-codeleted. in the past, the diagnosis oligodendroglioma was mainly based on histology in hematoxylin and eosin (h&e) stained sections, the ‘fried egg’ phenotype of the tumor cells being an important clue for this diagnosis (a); nowadays, demonstration of idh-mutant status and (complete) 1p/19q codeletion is required for the diagnosis of ‘canonical’ oligodendroglioma (b; result of methylome analysis showing (with almost perfect calibrated score of 0.99) suggested diagnosis of oligodendroglioma, idh-mutant and 1p/19q-codeleted, as well as copy number profile based on methylome analysis with loss of chromosome arms 1p and 19q, and hypermethylated status of mgmt promoter); filipski et al report that immunohistochemistry can be used to recognize ‘canonical’ oligodendrogliomas as well, the combination of lack of nuclear h3k27me3 staining, retention or non-conclusive nuclear atrx staining, and positive staining for idh1r132h mutant protein in h3k27-wildtype diffuse gliomas being highly suggestive for this diagnosis (c; while the nuclei of non-neoplastic cells in the microvessel wall show strong h3k27me3 staining (arrowheads), the tumor cell nuclei (arrows) are largely negative; in this female patient, the remaining positive dots in the tumor cell nuclei represent the inactivated x chromosome [20]). topic 2. improved molecular diagnosis of rare cns tumor types [2, 3] only recently, genome-wide dna methylation analysis has been introduced as a very helpful tool for improved diagnosis of cns tumor types and subtypes [24-29]. in 2019, two papers were published by the ‘heidelberg team’ + coauthors from other centers/countries demonstrating the power of methylome analysis. sievers et al [2] report the results of in-depth analysis of 30 tumors initially identified through methylome analysis as a separate group in a heidelberg cohort of > 25,000 tumors. most tumors in this group were histologically diagnosed as rosette-forming glioneuronal tumor (rgnt), and all revealed fgfr1 hotspot mutations, with in about two-thirds of the cases co-occurrence of a pik3ca mutation and in one third of the tumors an additional loss-of-function mutation in nf1. in contrast to most low-grade gliomas, rgnts displayed co-occurrence of two or even all three of these mutations. these highly recurrent combined genetic alterations affecting both mapk and pi3k signaling pathways may offer potential therapeutic targets for rgnts [2]. in a study of hou et al [3], 17 of the 28 tumors initially histologically diagnosed as papillary glioneuronal tumor (pgnt) showed methylation profiles typical for other tumor entities (mostly dysembryoplastic neuroepithelial tumor and pilocytic astrocytoma). the remaining 11 cases exhibited a unique profile and were considered as a distinct pgnt methylation class. three additional tumors in the heidelberg cohort clustered with this methylation class as well but were originally not diagnosed as pgnt. in all 12 cases of this methylation class of which material was available for further analysis, fusions involving prkca were identified (slc44a1–prkca, n = 11; notch1-prkca, n = 1), whereas such fusions were not found in the tumors belonging to other methylation classes. both the study of sievers et al and of hou et al thus provide information on molecular characteristics that can be used for improved recognition of such rare cns tumor types and again illustrate the power of methylome analysis in this context. topic 3. refined molecular classification of non-wnt/non-shh medulloblastomas [4] the 2016 who classification of cns tumors recognizes four molecular variants of medulloblastoma: wnt-activated, shh-activated and tp53-mutant, shh-activated and tp53-wildtype, and non-wnt/non-shh. the non-wnt/non-shh subgroup, accounting for about 65% of all medulloblastomas, encompasses the group 3 and group 4 molecular variants of medulloblastoma. however, these groups have heterogeneous clinical characteristics and survival outcomes, and their biology remained less clear. studies published in 2017 suggested the existence of 4, 6 or 8 subgroups within the overarching non-wnt/non-shh group [30-32]. in an attempt to provide clarity and a basis for further biological studies, sharma et al analyzed the number and nature of subtypes that could be identified in a cohort of 1501 genomically characterized non-wnt/non-shh medulloblastomas [4]. in this study, rather than advocating one single analytical approach or method, multiple class-definition approaches were used and equal weight was given to each analytical technique. in a lower complexity analysis group 3 and group 4 were identified again, but more complex analysis strongly supported the existence of eight robust group 3/group 4 subtypes (i-viii). these subtypes could generally be recognized based on their dna-methylation profiles and showed differences in enrichment for specific driver gene alterations, cytogenetic events, and ages of incidence with mostly unimodal age distribution. cytogenetic signatures, chromosomal copy-number aberrations and information on one gene or a set of genes in isolation were found to be insufficient for recognition of subtypes i-viii. the relevance of these subtypes is supported by differences in survival, with e.g. subtype iv being associated with low-risk clinical behavior, and with relatively frequent late relapses in patients with a subtype viii medulloblastoma. these findings can be expected to improve risk stratification, therapy and thereby the outcome for patients with non-wnt/non-shh medulloblastomas. of note, although the bimodal age distribution that was found for subtypes v and vii may indicate that some further refinement of the classification is still possible, the authors report that they did not find strong evidence for more than eight molecular subgroups in the non-wnt/non-shh category. subtyping of non-wnt/non-shh medulloblastomas into subtype i-viii is now available for the community by performing methylome analysis as described by capper et al [24, 25] and using an extension of the heidelberg brain tumor classifier (https://www.molecularneuropathology.org/mnp/classifier/7). topic 4. histogenesis of posterior fossa tumors in children [5-7] for quite some time it is clear that different subtypes of medulloblastomas have different developmental origin [33]. in fact, cerebellar tumors may be the result of a disorder of early cerebellar development. in an attempt to further elucidate cerebellar development at the single cell level, vladiou et al performed large-scale single-cell rna sequencing (scrna-seq) of more than 60,000 cells from the developing mouse cerebellum [5]. these analyses allowed them to reconstruct the cellular hierarchy in development at various points in time, with many of the normal mouse cerebellar cell populations only being present for a restricted time period during the fetal or very early postnatal period. subsequently, this transcriptome information was compared to that of pediatric cerebellar tumors: medulloblastomas, posterior fossa (pf) ependymomas, and pilocytic astrocytoma. the authors find that (different molecular subgroups of) these tumors indeed mirror the transcription of cells from distinct, temporally restricted cerebellar lineages: shh medulloblastomas, group 3 and group 4 medulloblastomas were found to transcriptionally resemble respectively the granule cell hierarchy, nestin+ stem cells, and unipolar brush cells, while pf type a/pf type b (pfa/pfb) ependymomas and cerebellar pilocytic astrocytomas resembled prenatal gliogenic progenitor cells. these findings indicate that each of these cerebellar tumor types arises from a particular cell type. however, across the medulloblastoma subgroups (shh, group 3, group 4), as well as pfa ependymoma and pilocytic astrocytomas, the scrnaseq data also demonstrated high levels of single-cell heterogeneity, with evidence of multiple lineages of differentiation and tumor cells matching different time points in the differentiation hierarchy [5]. in another study, hovestadt et al performed single-cell transcriptomics to investigate intraand intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. they find that wnt, shh and group 3 tumors comprise subgroup-specific undifferentiated and differentiated neuronal-like populations of malignant cells, whereas group 4 tumors consist of differentiated neuronal-like neoplastic cells. the tumor cells in shh medulloblastomas closely resembled granule neurons of varying differentiation states that correlated with patient age. using cross-species transcriptomic analysis, it appeared that distinct glutamatergic populations are the putative cells-of-origin for shh and group 4 medulloblastomas. furthermore, the tumor cells of group 3 and group 4 medulloblastomas exhibited characteristics ranging from primitive progenitor-like to more mature neuronal-like cells, with the relative proportions of these cells distinguishing these subgroups [6]. in a third study, based on single-cell transcriptome analysis of >65,000 cells of the embryonal pons and forebrain, jessa et al derived signatures for 191 distinct cell populations of these regions [7]. bulk transcriptome analysis of wnt-activated medulloblastomas revealed a match with the mossy fiber neuronal lineage of the rhombic lip. embryonal tumors with multilayered rosettes (etmrs) appeared to be derived from a neuronal lineage as well, but especially the tyr and myc subgroups of atypical teratoid/rhabdoid tumor (at/rt) seemed to originate from non-neuroectodermal cells. all in all, these studies provide strong evidence that pediatric cns tumors are the result of a disorder of early development, although the possibility that they arise from more mature cells undergoing de-differentiation at later time points or from trans-differentiation of other cell lineages cannot yet be completely ruled out. topic 5. longitudinal molecular analysis of diffuse gliomas [8] genomic characterization efforts such as the cancer genome atlas (tcga) have greatly increased our understanding of glioma biology [34-37]. based on these findings three major, clinically relevant subgroups of diffuse gliomas in adults were introduced in the who 2016 classification of cns tumors: (1) idh-mutant and chromosome 1p/19q codeleted (idh-mutant-codel); (2) idh-mutant without codeletion of chromosome 1p/19q (idh-mutant-noncodel); and (3) idh-wildtype. so far, however, such studies were generally limited to analysis of tumor tissue as obtained by first operation, and how the genetic landscape of these gliomas evolves over time and in response to therapy remained largely unknown. in december 2019, the glioma longitudinal analysis (glass) consortium published the results of analysis of 222 diffuse glioma patients (25 idh-mutant-codel, 63 idh-mutant-noncodel, and 134 idh-wildtype) with high-quality data on mutations and chromosomal copy numbers in samples of at least two time points [8]. the study revealed that the driver genes detected in the initial sample were retained in the recurrent tumor, and there was little evidence of recurrence-specific gene alterations. also, current standard of care therapies (chemotherapy, irradiation) generally did not seem to coerce the tumors down predictable paths. idh-mutant-noncodels were found to be most sensitive to developing a hypermutator phenotype after therapy with alkylating agents, a phenomenon that has been reported before [38, 39]. importantly, no differences were found in overall survival between hypermutators and non-hypermutators independent of age, subtype and mgmt promoter methylation status. in line with a recent study demonstrating that homozygous cdkn2a loss is a marker for high-grade malignancy in idh-mutant-noncodels [40], in the glass study recurrent idh-mutant-noncodels were enriched for homozygous cdkn2a deletions and this was associated with shorter survival compared to patients without these alterations. no differences in the levels of immunoediting were found between initial and recurrent gliomas. the glass study thus indicates that the strongest selective pressure in these three major subgroups of diffuse gliomas occurs during early glioma development and that current therapies shape this evolution in a largely stochastic manner. hopefully, such information on how diffuse gliomas evolve over time and in response to therapy will help to design more efficacious therapeutic strategies for these tumors. topic 6. notch1-sox2 signaling controls glioma cell invasion in white matter tracts [9] one of the main reasons that patients with diffuse gliomas so far cannot be cured is the diffuse infiltrative growth of the tumor cells along white matter tracts. one would hope that unraveling the mechanisms underlying this phenomenon may help to identify novel therapeutic targets. in an analysis of human glioma tissue samples, wang et al found that at the invasive front, cd133+ ‘glioma stem cells’ (gscs) were preferentially located along white matter tracts [41]. also, these tracts showed significant swelling and seemed to have discontinuous myelin, indicating that some gscs in the glioma-brain interface are preferentially located adjacent to unmyelinated white matter tracts. based on their findings the authors postulate that glioma invasion along these tracts occurs along axons inside myelin sheaths rather than along the outer surface of such sheaths, and that glioma-associated edema may play a role in local destruction of white matter fibers and thereby pave the way for direct interaction of glioma cells and axons. additionally, based on their findings in preclinical experiments that nearly all cd133+ gscs were also notch1+ and that nerve fibers at the invasive frontier all expressed jagged1, wang et al postulate that the interaction between jagged1 and notch1 may be an important determinant for the distribution of gscs. indeed, axonally expressed jagged1 was found to activate the notch signaling pathway in gscs and to subsequently promote the transcription of sox2 via sox9. conversely, sox2 upregulation decreased the methylation of the notch1 promoter to reinforce the high expression of notch1 in gscs and facilitate their white matter-tract tropism, while inhibition of notch signaling was found to attenuate this tropism. the findings of wang et al indicate that the notch1-sox2 positive-feedback loop is an important determinant of gsc invasion along white matter tracts, and that molecules in this loop may be exploited as therapeutic targets. however, the authors acknowledge that the mechanisms underlying glioma cell invasion along white matter tracts are probably more complex, and may also involve mechanisms as discussed in the next topic (topic 7). topic 7. ‘synaptic cooption’ in glial and metastatic cns tumors [10-12] after reporting earlier that neuroligin-3 secreted by active neurons promotes glioma growth [42, 43], in a recent study published in nature, venkatesh et al found that electrochemical communication exists between neurons and glioma cells through bona fide ampa (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor-dependent synapses between these cells [10]. also, neuronal activity was found to evoke non-synaptic activity-dependent potassium currents that were amplified by gap junction-mediated tumor interconnections and resulting in an electrically coupled network. depolarization of glioma membranes was found to promote proliferation, whereas blocking electrochemical signaling inhibited the growth of glioma xenografts in mice. intraoperative electrocorticography in glioma patients revealed increased cortical excitability in the glioma-infiltrated brain areas. based on these findings the authors suggest that glioma growth is promoted by synaptic and electrical integration into neural circuits, with glioma-induced increase in neuronal excitability and activity-regulated glioma growth as elements of a positive feedback loop. in the same issue of nature, venkaratamani et al also report that functional synapses between neurons and glioma cells exist [11]. previously, these authors already demonstrated that many tumor cells in astrocytic tumors including glioblastomas extend ultra-long membrane protrusions (microtubes) that are used for brain invasion and proliferation, and that interconnect over long distances [44] (fig. 2). in their recent study, venkaratamani et al show the presence of ‘neurogliomal synapses’ in human glioma samples and in different disease models. such synapses were found to be located on tumor microtubes and produce postsynaptic currents that are mediated by glutamate receptors of the ampa subtype. in their experiments, neuronal activity led to synchronized calcium transients in tumor-microtube-connected glioma networks. furthermore, perturbation of ampa receptors and use of an ampa receptor antagonist reduced calcium-related invasiveness of tumor cells and of glioma growth. intriguingly, in a third study published in that same issue of nature, zeng et al demonstrate the existence of ‘pseudo-tripartite synapses’ between breast cancer cells and glutamatergic neurons and show that such cancer cells in the brain can co-opt a neuronal signaling pathway involving activation by glutamate ligands of n-methyl-d-aspartate receptors (nmdars) [12]. these three studies thus suggest very peculiar brain tumor-microenvironment interactions in the form of direct, biologically relevant synaptic communication between neurons and tumor cells with potential therapeutic implications. fig. 2. human glioma cells forming a mycelium-like network in mouse brain. based on findings in especially two-dimensional histological slides, tumor cells in the periphery of diffuse gliomas have been considered as ‘guerilla warriors’ that move as single cells into the preexistent cns tissue [41]. more recent findings, however, show that such cells in diffuse astrocytic tumors form a network of microtubes. this figure shows such network formation by human glioblastoma cells 28 days after implantation into a mouse brain (yellow: glioblastoma cells labeled by human-specific anti-vimentin antibody; magenta: mouse astrocytes labeled by anti-gfap antibody; blue: cell nuclei labeled by dapi; figure kindly provided by pavel gritsenko and peter friedl and based on work recently published [61]). in addition to such a mycelium-like network formation, studies discussed under topic 7 now report that direct, biologically relevant synaptic communication exists between neurons and tumor cells as well. topic 8. cancer stem cells in glioblastomas; do they exist? [13] it is been proposed that in glioblastomas cancer stem cells (cscs) or stem-like cells reside at the top of a hierarchical organization, are able to (re)create intra-tumoral heterogeneity by generating more differentiated offspring, and are relatively resistant to therapy and thus contribute significantly to tumor recurrence [45]. expression of particular cell membrane antigens (cd133, cd15/ssea, cd44, and/or a2b5) has often been used for identification of the csc subset in glioblastomas. however, it is increasingly clear that no single marker allows for unequivocal identification of a csc population in glioblastomas. moreover, there is still controversy if a bona fide csc population exists at all (and if so, if it concerns a quiescent or proliferative subpopulation). in this context, dirkse et al addressed the question whether glioblastoma cells expressing csc-associated cell membrane markers are indeed a defined entity at the apex of a hierarchical organization. they found that, like in patient biopsies, csc markers were heterogeneously expressed in patient-derived glioblastoma xenografts and stem-like cell cultures. more precisely, all facs-sorted subpopulations of tumor cells of glioblastomas were able to self-renew over multiple passages without significant differences between each other. this suggests that phenotypically heterogeneous glioblastoma cells can adapt to a variety of environmental changes and acquire similar stem cell properties in vitro. dirkse et al also found that accelerated reconstitution of heterogeneity provided a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than csc multipotency. the study of dirkse et al thus provides strong evidence that cscs do not constitute a defined cellular entity, but rather a cellular state adapting to microenvironmental cues. the capacity of any given tumor cell to reconstitute heterogeneity in glioblastomas cautions against therapies targeting only a small subpopulation of tumor cells with stem cell(-like) characteristics. meanwhile, inherent cancer cell plasticity emerges as a novel relevant target for treatment [13]. topic 9. personalized (neo)antigen vaccination therapy for glioblastoma [14, 15] in patients with e.g. melanomas, high tumor mutational load (tml) is associated with increased frequency of neoantigens and improved response to checkpoint inhibition. administration of personalized neoantigen vaccines has been shown to successfully recruit t cells to the tumor and can lead to tumor regression. unfortunately, also after the introduction of immunotherapies, so far the very poor survival rates for patients with glioblastoma have not improved much. of note, glioblastomas often have a relatively low mutational burden and are considered as immunologically ‘cold’. recent studies suggest that for patients with glioblastoma, a personalized molecular approach is needed to improve the benefit of treatment with programmed cell death protein 1 (pd-1) inhibitors nivolumab or pembrolizumab, and that the neoadjuvant administration of pd-1 blockade may represent a more efficacious approach for these tumors [46, 47]. two recent studies published in nature explored the feasibility of vaccination therapy using tumor (neo)antigens for patients with glioblastoma. keskin et al performed a phase i/ib multi-epitope, personalized neoantigen vaccination study [14]. patients who did not receive dexamethasone generated circulating polyfunctional neoantigen-specific cd4+ and cd8+ t cell responses that were enriched in a memory phenotype. after vaccination, neoantigen-specific t cells were found to migrate from peripheral blood into the glioblastoma, suggesting that they may favorably alter the immune milieu of the tumor [14]. hilf et al studied vaccination using both unmutated tumor antigens and neoepitopes for more effective immunotherapy of glioblastomas, including those with a low mutational load [15]. highly individualized vaccinations with both types of tumor antigens were integrated into standard care for patients with newly diagnosed glioblastoma. fifteen patients were first treated with a vaccine with a previously constructed library of non-mutated antigens that are over-represented in glioblastomas (apvac1), followed by a second vaccine (apvac2) targeted against mutated neoantigens or non-mutated antigens that were not present in apvac1. the vaccines were personalized by analysis of mutations and of the transcriptomes and immunopeptidomes of the individual tumors. both vaccines showed favorable safety and elicited t cell responses against the proteins in the vaccine, with apvac1 inducing a sustained cd8+ t cell response, and apvac2 both cd4+ and cd8+ t cell responses [15]. topic 10. liquid biopsy diagnosis of gliomas using csf [16, 17] cancer cells release nucleic acids, vesicles, proteins, and other components into the blood stream and other body fluids. already for quite some time, circulating tumor dna (ctdna) in blood is considered as an easily accessible source of potentially very useful diagnostic, prognostic and/or predictive information that can be used to improve the management of cancer patients [48]. so far, however, liquid biopsy diagnosis for detection and monitoring of patients with gliomas has not yet entered the clinic. part of the problem may be that, compared to other cancers, gliomas release relatively limited amounts of ctdna into the bloodstream [49]. sequencing of ctdna from the cerebrospinal fluid (csf) may provide an alternative way to diagnose glial and other cns tumors. indeed, in 42 out of 85 adult patients with diffuse glioma (49.4%), miller et al detected ctdna in csf that was obtained by lumbar puncture [16]. presence of ctdna was associated with disease burden and adverse outcome. the glioma genomes detected in csf closely resembled those in matched tumor samples. alterations occurring early in gliomagenesis (idh1/idh2 mutation, 1p/19q codeletion) were shared in all matched ctdna-positive csf-tumor pairs, whereas growth factor receptor signaling pathways showed considerable evolution. most patients with ctdna-positive csf did not have detectable malignant cells in the csf, and no significant association was found between ctdna-positive csf and glioma grade, disease duration or prior therapy. of note, in this study of miller et al the csf samples were derived from patients relatively late in their disease course (median disease duration before csf collection for patients with idh-wildtype glioblastomas about a year, and for patients with idh-mutant lower grade gliomas over 5 years), and all patients already had treatment for their glial tumor. in contrast, pan et al explored the potential of ctdna analysis in csf obtained prior to surgical manipulation in a cohort of 57 patients with brainstem glioma [17]. over 90% of these csf samples were obtained intraoperatively. pan et al report that alterations were identified in the csf ctdna in 36/37 cases (97.3%) in which the primary tumors harbored at least one mutation, while in 31/37 of cases (83%) all primary tumor alterations were detected in the csf. in these patients, mutation detection using plasma ctdna was found to be much less sensitive than sequencing the csf ctdna. these studies thus suggest that indeed csf may be a much more promising biosource for liquid biopsy diagnostics of gliomas than blood. combination of such an approach with analyses going beyond dna sequence information (e.g. analysis of epigenetic and immune signatures in cell free dna) may further boost the exploitation of liquid biopsy diagnostics in patients with (glial) cns tumors [49]. discussion the papers discussed in this review as top 10 discoveries illustrate the substantial and sometimes even amazing progress that has been made in increasing our understanding of particular topics in neuro-oncological pathology. hopefully, this review not only provides easily digestible information on the topics selected by the author, but also works as a stepping stone to read and appreciate the original papers that were used as building blocks for the present manuscript. obviously, most of the findings presented here immediately elicit next questions. for example, are the findings presented by filipski et al already ‘mature’ enough to be used as surrogate markers for the diagnosis of (anaplastic) oligodendroglioma, idh-mutant and 1p/19q-codeleted in clinical practice? and while wang et al report that the notch1-sox2 positive-feedback loop is an important (and possibly targetable) determinant of invasion of glioma cells along white matter tracts, the authors acknowledge that other mechanisms as discussed in topic 7 may be involved as well. but then again, is synapse formation between neuronal and glioma cell processes especially relevant for the invasive front? or also for proliferation of tumor cells in the highly cellular center of glioblastomas where neurites can be expected to be (much) more scarce? what about the presence of ‘pseudo-tripartite synapses’ in the ‘bulky’ areas of metastatic breast cancer in the cns? and (see topic 8): do bona fide glioma stem cells exist? nowadays, a rapidly increasing number of diagnostic and prognostic molecular markers can be used for an improved, ‘histo-molecular’ diagnosis of cns tumors [50]. multiple assays have been developed for this purpose, ranging from single gene tests to high-throughput, integrated techniques enabling detection of multiple genetic aberrations in a single workflow [51]. immunohistochemistry is a helpful, relatively inexpensive alternative for further molecular characterization of particular cns tumors. within a few years after its introduction, genome-wide methylation profiling has already had a revolutionary impact on cns tumor classification [24, 25, 52]. the papers discussed in topic 2 (‘improved molecular diagnosis of rare cns tumor types’) provide examples of the strength of this platform. however, such methylome analysis is relatively expensive. obviously, a test providing clinically very valuable information at low costs will much more easily be accepted as a routine diagnostic tool compared to an expensive test with limited clinical benefits. unfortunately, a clear framework for assessment of cost-effectiveness of molecular testing of cns (and other) tumors is lacking [53]. it is important to realize though that molecular diagnostics of (cns) tumors is relatively inexpensive compared to e.g. neuro-imaging and chemotherapy, and that a diagnosis that is ‘on target’ will not only have substantial clinical benefit for the patients but may also help to avoid unnecessary health care costs [52, 54]. meanwhile, since the publication of the who classification of cns tumours in 2016, the consortium to improve molecular and practical approaches into cns tumor taxonomy (cimpact-now) has already published several updates with suggestions on how exactly particular molecular markers can be used for improved diagnosis in clinical practice (see for summary of round 1 updates [55]). the findings described in topic 3 (‘refined molecular classification of non-wnt/non-shh medulloblastomas‘) and topic 4 (‘histogenesis of posterior fossa tumors in children’) may soon be incorporated into a more refined next who classification of these tumors (5th edition expected to be published within a year from now!). when shaping a who classification, one may indeed follow such a more progressive approach (i.e. introduce the latest tools and findings and include e.g. the most refined subclassification of non-wnt/non-shh medulloblastomas), or be somewhat more conservative in an attempt to better preserve long-term correlations, to avoid major disruption of patient management, and/or because of limited availability of (often expensive) diagnostic tools. obviously, finding the right balance is key, as there are many ‘strings attached’ (fig. 3). fig. 3. ‘strings attached’ to designing a next who classification of (cns) tumors. finding the right balance between a conservative and progressive attitude with regard to incorporating the latest findings into a next who tumor classification is key but can be challenging, not only because there are many strings attached, but also because of different aspects of the underlying dynamics. figure based on input of dr. david n. louis. next to diagnostic and prognostic markers, clinical neuropathologists are increasingly asked to perform testing for predictive biomarkers, e.g. for assessment of the likelihood of response of gliomas to particular immunotherapeutic approaches. only a limited number of newly diagnosed gliomas is reported to have an (occasionally inherited) mismatch repair (mmr) defect and/or a ‘hypermutator’ phenotype. recurrent gliomas more often show such a phenotype, especially so after alkylating chemotherapy [8]. assuming that the hypermutator status leads to an increase in expression of neoantigens, these gliomas/glioblastomas with a hypermutator phenotype could be good candidates for immune checkpoint blockade. unequivocal scoring of immunohistochemical staining for pd1/pd-l1 (surface proteins on tumor-infiltrating lymphocytes and tumor cells, respectively, and involved in suppression of the immune system) can be challenging. alternatively, mmr deficiency, microsatellite instability (msi), dna polymerase epsilon (pole) mutations, and high tumor mutational load (tml) are being used in this context, but assessment of these biomarkers can be challenging as well. for example, mmr gene (mlh1, msh2, msh6, and/or pms2) mutations often, but not always lead to lack of immunostaining of tumor cell nuclei for the corresponding protein(s). also, the techniques and thresholds to be used for optimal assessment of high tml are not yet settled. while for non-small cell lung carcinoma ≥10 mutations per mb is considered as high tml, it is unclear if this threshold should be used for gliomas as well [56, 57]. of course, identification and optimization of biomarkers for response to immunotherapy only makes sense if at least some patients can be expected to significantly benefit from that particular therapy. furthermore, the studies reported under topic 9 (‘personalized (neo)antigen vaccination therapy for glioblastoma’) indicate that biomarker discovery in this context is a moving target. in conclusion, ‘the times they are a-changin’ and one can expect that there is much more change to come. liquid biopsy-based diagnoses may emerge soon as a clinically helpful source of information. furthermore, a very recently published study showed that the use of artificial intelligence outperformed radiologists in the mammogram-based prediction of breast cancer [58]. similarly, deep learning/machine learning approaches may increasingly be used for automated, histology-based brain tumor classification and/or for predicting molecular markers based on mri or other neuro-imaging modalities [59, 60]. such developments could have significant impact on the ‘core business’ of the (neuro)pathologist. however, to quote the editor-in-chief of free neuropathology: ‘as long as neuropathology continues to be creative and innovative at the forefront of classifying and diagnosing neurological disorders and revealing their molecular pathogenesis …, its future will be bright’ [52]. references 1. filipski, k., et al., lack of h3k27 trimethylation is associated with 1p/19q codeletion in diffuse gliomas. acta neuropathol, 2019. 138(2): p. 331-334. 2. sievers, p., et al., rosette-forming glioneuronal tumors share a distinct dna methylation profile and mutations in fgfr1, with recurrent co-mutation of pik3ca and nf1. acta neuropathol, 2019. 138(3): p. 497-504. 3. hou, y., et al., papillary glioneuronal tumor (pgnt) exhibits a characteristic methylation profile and fusions involving prkca. acta neuropathol, 2019. 137(5): p. 837-846. 4. sharma, t., et al., second-generation molecular subgrouping of medulloblastoma: an international meta-analysis of group 3 and group 4 subtypes. acta neuropathol, 2019. 138(2): p. 309-326. 5. vladoiu, m.c., et al., childhood cerebellar tumours mirror conserved fetal transcriptional programs. nature, 2019. 572(7767): p. 67-73. 6. hovestadt, v., et al., resolving medulloblastoma cellular architecture by single-cell genomics. nature, 2019. 572(7767): p. 74-79. 7. jessa, s., et al., stalled developmental programs at the root of pediatric brain tumors. nat genet, 2019. 51(12): p. 1702-1713. 8. barthel, f.p., et al., longitudinal molecular trajectories of diffuse glioma in adults. nature, 2019. 576(7785): p. 112-120. 9. wang, j., et al., invasion of white matter tracts by glioma stem cells is regulated by a notch1-sox2 positive-feedback loop. nat neurosci, 2019. 22(1): p. 91-105. 10. venkatesh, h.s., et al., electrical and synaptic integration of glioma into neural circuits. nature, 2019. 573(7775): p. 539-545. 11. venkataramani, v., et al., glutamatergic synaptic input to glioma cells drives brain tumour progression. nature, 2019. 573(7775): p. 532-538. 12. zeng, q., et al., synaptic proximity enables nmdar signalling to promote brain metastasis. nature, 2019. 573(7775): p. 526-531. 13. dirkse, a., et al., stem cell-associated heterogeneity in glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment. nat commun, 2019. 10(1): p. 1787. 14. keskin, d.b., et al., neoantigen vaccine generates intratumoral t cell responses in phase ib glioblastoma trial. nature, 2019. 565(7738): p. 234-239. 15. hilf, n., et al., actively personalized vaccination trial for newly diagnosed glioblastoma. nature, 2019. 565(7738): p. 240-245. 16. miller, a.m., et al., tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid. nature, 2019. 565(7741): p. 654-658. 17. pan, c., et al., molecular profiling of tumors of the brainstem by sequencing of csf-derived circulating tumor dna. acta neuropathol, 2019. 137(2): p. 297-306. 18. paramasivam, n., et al., mutational patterns and regulatory networks in epigenetic subgroups of meningioma. acta neuropathol, 2019. 138(2): p. 295-308. 19. wesseling, p., m. van den bent, and a. perry, oligodendroglioma: pathology, molecular mechanisms and markers. acta neuropathol, 2015. 129(6): p. 809-27. 20. schaefer, i.m., a. minkovsky, and j.l. hornick, h3k27me3 immunohistochemistry highlights the inactivated x chromosome (xi) and predicts sex in non-neoplastic tissues. histopathology, 2016. 69(4): p. 702-4. 21. panwalkar, p., et al., immunohistochemical analysis of h3k27me3 demonstrates global reduction in group-a childhood posterior fossa ependymoma and is a powerful predictor of outcome. acta neuropathol, 2017. 134(5): p. 705-714. 22. cleven, a.h., et al., loss of h3k27 tri-methylation is a diagnostic marker for malignant peripheral nerve sheath tumors and an indicator for an inferior survival. mod pathol, 2016. 29(6): p. 582-90. 23. katz, l.m., et al., loss of histone h3k27me3 identifies a subset of meningiomas with increased risk of recurrence. acta neuropathol, 2018. 135(6): p. 955-963. 24. capper, d., et al., dna methylation-based classification of central nervous system tumours. nature, 2018. 555(7697): p. 469-474. 25. capper, d., et al., practical implementation of dna methylation and copy-number-based cns tumor diagnostics: the heidelberg experience. acta neuropathol, 2018. 136(2): p. 181-210. 26. jaunmuktane, z., et al., methylation array profiling of adult brain tumours: diagnostic outcomes in a large, single centre. acta neuropathol commun, 2019. 7(1): p. 24. 27. pickles, j.c., et al., dna methylation-based profiling for paediatric cns tumour diagnosis and treatment: a population-based study. lancet child adolesc health, 2020. 4(2): p. 121-130. 28. perez, e. and d. capper, dna-methylation-based classification of paediatric brain tumours. neuropathol appl neurobiol, 2020. 29. priesterbach-ackley, l.p., et al., brain tumour diagnostics using a dna methylation-based classifier as a diagnostic support tool. neuropathol appl neurobiol, 2020. 30. schwalbe, e.c., et al., novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study. lancet oncol, 2017. 18(7): p. 958-971. 31. cavalli, f.m.g., et al., intertumoral heterogeneity within medulloblastoma subgroups. cancer cell, 2017. 31(6): p. 737-754 e6. 32. northcott, p.a., et al., the whole-genome landscape of medulloblastoma subtypes. nature, 2017. 547(7663): p. 311-317. 33. gibson, p., et al., subtypes of medulloblastoma have distinct developmental origins. nature, 2010. 468(7327): p. 1095-9. 34. cancer genome atlas research, n., comprehensive genomic characterization defines human glioblastoma genes and core pathways. nature, 2008. 455(7216): p. 1061-8. 35. brennan, c.w., et al., the somatic genomic landscape of glioblastoma. cell, 2013. 155(2): p. 462-77. 36. cancer genome atlas research, n., et al., comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. n engl j med, 2015. 372(26): p. 2481-98. 37. ceccarelli, m., et al., molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. cell, 2016. 164(3): p. 550-63. 38. johnson, b.e., et al., mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. science, 2014. 343(6167): p. 189-193. 39. van thuijl, h.f., et al., evolution of dna repair defects during malignant progression of low-grade gliomas after temozolomide treatment. acta neuropathol, 2015. 129(4): p. 597-607. 40. shirahata, m., et al., novel, improved grading system(s) for idh-mutant astrocytic gliomas. acta neuropathol, 2018. 136(1): p. 153-166. 41. claes, a., a.j. idema, and p. wesseling, diffuse glioma growth: a guerilla war. acta neuropathol, 2007. 114(5): p. 443-58. 42. venkatesh, h.s., et al., neuronal activity promotes glioma growth through neuroligin-3 secretion. cell, 2015. 161(4): p. 803-16. 43. venkatesh, h.s., et al., targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma. nature, 2017. 549(7673): p. 533-537. 44. osswald, m., et al., brain tumour cells interconnect to a functional and resistant network. nature, 2015. 528(7580): p. 93-8. 45. gimple, r.c., et al., glioblastoma stem cells: lessons from the tumor hierarchy in a lethal cancer. genes dev, 2019. 33(11-12): p. 591-609. 46. zhao, j., et al., immune and genomic correlates of response to anti-pd-1 immunotherapy in glioblastoma. nat med, 2019. 25(3): p. 462-469. 47. cloughesy, t.f., et al., neoadjuvant anti-pd-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma. nat med, 2019. 25(3): p. 477-486. 48. best, m.g., et al., liquid biopsies in patients with diffuse glioma. acta neuropathol, 2015. 129(6): p. 849-65. 49. van der pol, y. and f. mouliere, toward the early detection of cancer by decoding the epigenetic and environmental fingerprints of cell-free dna. cancer cell, 2019. 36(4): p. 350-368. 50. kristensen, b.w., et al., molecular pathology of tumors of the central nervous system. ann oncol, 2019. 30(8): p. 1265-1278. 51. priesterbach-ackley, l.p., et al., molecular tools for the pathologic diagnosis of central nervous system tumors. neurooncol pract, 2019. 6(1): p. 4-16. 52. paulus, w., heireca! the heidelberg revolution of cancer classification and what it means for neurooncology and neuropathology. acta neuropathol, 2018. 136(2): p. 177-179. 53. wesseling, p., the abcs of molecular diagnostic testing of cns tumors: acceptance, benefits, costs. neuro oncol, 2019. 21(5): p. 559-561. 54. karimi, s., et al., the central nervous system tumor methylation classifier changes neuro-oncology practice for challenging brain tumor diagnoses and directly impacts patient care. clin epigenetics, 2019. 11(1): p. 185. 55. louis, d.n., et al., cimpact-now: a practical summary of diagnostic points from round 1 updates. brain pathol, 2019. 29(4): p. 469-472. 56. hodges, t.r., et al., mutational burden, immune checkpoint expression, and mismatch repair in glioma: implications for immune checkpoint immunotherapy. neuro oncol, 2017. 19(8): p. 1047-1057. 57. tan, a.c., a.b. heimberger, and m. khasraw, immune checkpoint inhibitors in gliomas. curr oncol rep, 2017. 19(4): p. 23. 58. mckinney, s.m., et al., international evaluation of an ai system for breast cancer screening. nature, 2020. 577(7788): p. 89-94. 59. ker, j., et al., automated brain histology classification using machine learning. j clin neurosci, 2019. 66: p. 239-245. 60. korfiatis, p. and b. erickson, deep learning can see the unseeable: predicting molecular markers from mri of brain gliomas. clin radiol, 2019. 74(5): p. 367-373. 61. gritsenko, p.g., et al., p120-catenin-dependent collective brain infiltration by glioma cell networks. nat cell biol, 2020. 22(1): p. 97-107. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodegeneration: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:9 (2021) review neurodegeneration: 2021 update john f. crary neuropathology brain bank & research core, department of pathology, nash family department of neuroscience, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary , md-phd · friedman brain institute · ronald m. loeb center for alzheimer’s disease · icahn school of medicine at mount sinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 28 march 2021 accepted: 15 april 2021 copyedited by: jeffrey nirschl published: 21 april 2021 https://doi.org/10.17879/freeneuropathology-2021-3317 keywords: neurodegeneration, neuropathology, alzheimer disease, tauopathy, α-synucleinopathy, chronic traumatic encephalopathy, cryogenic electron microscopy, proteomics abstract this article reviews a collection of manuscripts in the field of neurodegenerative disease chosen from what are considered by the author to be among the 10 most important and potentially impactful topics or research trends of 2020 relevant to the field of experimental and diagnostic neuropathology. a deliberate effort was made to provide balance among disease categories covered. the result is a varied selection that includes not just individual papers but also research topics and trends. the association of covid-19 with longer-term neurological symptoms has launched a research trend fueled by speculation that the sars-cov-2 might trigger neurodegenerative changes. the onslaught of transcriptomic studies has begun to give way to proteomics, with three transformative studies published examining glial contributions to alzheimer disease, cerebral atherosclerosis in cognitive decline, and the complex sequence of post-translational modifications of the tau protein. plasma biomarkers for alzheimer disease have continued to make rapid advances, especially around highly sensitive assays capable of detecting different forms of abnormal hyperphosphorylated tau in peripheral blood. two studies using cryo-electron microscopy showed the power of the approach by continuing to elucidate the diversity of filamentous tau inclusions, and a third study gave the first glimpse of α-synuclein aggregates at near atomic resolution. another study continued to delineate how different α-synuclein conformers (“strains”) target specific brain regions and lead to neurodegeneration. in huntington’s disease, we saw compelling molecular data showing how cells adapt to endoplasmic reticulum stress through the unfolded protein response. finally, the role of astrocytes in chronic traumatic encephalopathy has emerged as a critical area of interest. introduction in a year like no other, experimental neuropathology research in neurodegeneration persevered. confronted with a devastating pandemic, we endured despite shuttered laboratories, working from home, supply shortages, the loss of loved ones and a myriad of other challenges. our laboratories demonstrated inspiring resilience, adapting to the new landscape and soldiering on. many rose to the challenge, finding purpose and inspiration in adversity. dr. rita levy montalcini once said, “i should thank mussolini for having declared me to be of an inferior race. this led me to the joy of working, not any more unfortunately in university institutes, but in a bedroom.” upon completing this review of just a few of the accomplishments of the past year, it was clear that this spirit is alive, the same that compelled levy-montalcini to work tirelessly at home to study chick embryos using scalpels fashioned from sewing needles. the spirit could be felt emanating from our colleagues sheltering at home, many also literally working in their bedrooms! so, it is with great pride and appreciation that we take this look back and celebrate the accomplishments of our colleagues and recognize just a few of the many transformative advances in 2020. the pandemic is a reminder that substantive scientific progress comes with great human effort and rarely a “eureka moment.” here, we also highlight collaborative efforts, research trends, groundbreaking methodologies, and tools. a concerted effort was made to seek out innovations across all neurodegenerative disease categories. given the readership and mission of this journal, we emphasized pathoanatomical studies most relevant to neuropathologists and clinical neuroscientists. 1. the long-term neuropathological sequelae of covid-19 over the past year, the sars-cov-2/covid-19 pandemic has permeated essentially every aspect of our lives, and research in neurodegenerative disease is no exception, launching a new, albeit speculative, research trend. early in the pandemic, neuropathologists began to wonder about the extent to which covid-19 might impact brain health. theoretically, this could be through direct neurotropism as some coronaviruses are known to cause encephalitis, or perhaps indirectly through complications related to critical illness and systemic disease (morgello, 2020). early reports documented encephalitis in the setting of covid-19 (reichard et al., 2020), but sars-cov-2 encephalitis was considered a rare event predominantly restricted to susceptible populations and of modest public health concern. larger autopsy series emerged, highlighting infarction and microthrombi in the brain in the acute setting (figure 1), more commonly in hospitalized patients, but this too was thought to be uncommon (bryce et al., 2020). nevertheless, researchers began to speculate that there may be long-term neurological sequelae that follow covid-19 as examples of post-viral tauopathy and neurodegeneration are well documented and now there is increasing alarm that there might be an incipient rise in neurological complications, including dementia and neurodegeneration, that might follow the pandemic (badrfam & zandifar, 2020). figure 1. post-mortem findings in a patient with sars-cov-2/covid-19. a. fresh gross image of a right hemibrain from a 68 male with covid-19 showing diffuse swelling and leptomeningeal hemorrhage. b. fixed right hemibrain from the same patient showing acute and subacute infarction in the distribution of the middle cerebral artery territory. c. luxol fast blue counterstained hematoxylin and eosin (lh& e) section showing subacute hemorrhagic infarction. d. lh& e-stained section from the neocortical white matter in a 29-year-old male covid-19 patient with acute disseminated encephalomyelitis (adem). neurologists and other clinicians have been describing persistent neurological symptomatology in covid-19 survivors, including inattentiveness, memory impairment and a constellation of other cognitive symptoms (i.e., “brain fog”). these are part of an entity now termed the post-acute covid-19 syndrome (pacs). the underlying neuropathological substrate for these findings remains unclear, but the effects of hypoxic-ischemic injury, chronic neuroinflammation, toxic encephalopathy from breakdown of the blood brain barrier are candidates. direct effects of the virus are not completely ruled out, and the potential for neurotropic strains to emerge remains a possibility. given the existence of encephalitis lethargica and measles related subacute sclerosing panencephalitis, both virus related and associated with neurofibrillary tangles, it is not unreasonable to speculate that a degenerative component of pacs might exist. critically, it is imperative to understand whether these pacs neurological symptoms are progressive and we expect a surge in translational studies in the coming years focused on the long-term changes that follow covid-19.  2. proteomic analysis: spotlight on microglial and astrocyte inflammation in alzheimer disease in the previous decades, transformative technologies have enabled large-scale genomic and transcriptomic studies of post-mortem brain tissues that have been directed towards neurodegenerative diseases, yielding a flood of data out of which has emerged a rich and complex picture of alzheimer disease (ad). but what about the proteins? owning to their relatively more complex chemical structures, measuring structural changes on the protein level in a high throughput manner is much more challenging. but now, proteomic technologies have advanced sufficiently, and we are beginning to see them take center stage. here and in the next two sections we highlight three such studies. the first study, published in nature medicine, led by nicholas seyfried and allan levey at emory university, profiled 2,000 brain samples, comparing more than 3,000 proteins from multiple cohorts, including controls, people with asymptomatic ad neuropathology and those with dementia due to ad (johnson et al., 2020). cases were derived from a total of eight collections, including the religious orders study and memory and aging project (ros-map), mayo clinic brain bank, baltimore longitudinal study of aging (blsa), baltimore coroner’s office, banner sun health research institute, mount sinai school of medicine brain bank, adult changes in thought study (act), and university of pennsylvania school of medicine brain bank. researchers identified 13 different gene sets (“modules”) based on co-expression patterns (figure 2). of these, six were correlated with ad neuropathological features (i.e., amyloid plaques, neurofibrillary tangles) or cognitive impairment. of the three that were most closely correlated with ad features, two were downregulated, including the m1 that was composed of proteins involved in synaptic function and the m3 that tracked with mitochondrial proteins. m4 had the strongest correlation with disease traits and contained astrocyte and microglial proteins involved in sugar metabolism. intriguingly, many of the proteins in the m4 module were encoded by genes that are ad risk loci. importantly, regardless of their precise function and role in disease, 27 of them were detected in csf, underscoring their potential utility as biomarkers. because this was funded by the accelerating medicine partnership ad (amp-ad) project, all of these data are publicly available for the research community and will surely catalyze continued proteomic studies. figure 2. protein network analysis in post-mortem alzheimer disease (ad) brain. a. brain tissue from the dorsolateral prefrontal cortex from ad patients and compared to cognitively normal subjects with age-related pathology and pathologically negative controls from four cohorts. b. a correlation network consisting of 13 modules using 3334 proteins was created and correlated with neuropathological assessments, cell type markers, and the associated biological processes identified using gene ontology (go) analysis. images courtesy of dr. erik c. johnson, allan i. levy, nicholas t. seyfried, reproduced with permission under the creative commons license. 3. proteomic analysis: the contribution of cerebral atherosclerosis to cognitive decline another proteomics paper published in nature neuroscience, also from emory university led by nicholas seyfried, allan levey, and thomas s. wingo, examined cerebral atherosclerosis as it relates to cognitive decline and ad (wingo et al., 2020). here, the investigators performed a proteome-wide association study (pwas), which is similar to a genome-wide association study (gwas), to look more broadly at relevant neuropathological changes, including amyloid-beta deposition, neurofibrillary tangles, infarcts (both macro and microinfarcts), cerebral amyloid angiopathy, tar dna-binding protein 43 (tdp-43, transactive response dna binding protein 43 kda), lewy body pathology, hippocampal sclerosis, and atherosclerosis. out of this analysis, 114 proteins emerged, independently of cerebrovascular risk factors (e.g., hypertension, diabetes, smoking; figure 3). strikingly, many were from oligodendrocytes involved in myelination. other genes were involved in mrna processing and splicing. they performed co-expression network analysis and 31 modules were identified, with five being linked independently to cerebral atherosclerosis. this analysis pointed to oligodendrocyte function alongside downregulation of mrna processing in neurons and astrocytes with impaired synaptic functioning in brains of individuals with cerebral atherosclerosis. this may be related to another finding of higher levels of neurofilament light (nfl) and medium (nfm) which were higher in cases with atherosclerosis, perhaps reflecting axonal injury. this study will drive continued research into the effects of cerebral atherosclerosis on the brain proteome and their contribution to ad. figure 3. co-expression network analysis of protein modules in post-mortem human brain. a, b. a total of 31 modules were identified and protein signatures mapped to cell type. c. these modules were then associated with neuropathological outcomes. d. differences in module eigen proteins were seen modules 3 and 9 for atherosclerosis and alzheimer disease-related dementia. images curtesy of drs. thomas wingo, aliza wingo, nicholas seyfried, allan i. levey (emory university school of medicine). 4. proteomic analysis: a high-resolution quantitative map of post-translational modifications of tau proteoforms neurofibrillary tangles exist in a spectrum of morphological forms that are immediately recognizable using immunohistochemistry with antisera targeting hyperphosphorylated tau (p-tau). the earliest detectable neurofibrillary change is the presence faint granular p-tau labeling in pre-tangles; this staining accumulates and coalesces into intracellular tangles that eventually become extracellular (“tombstone” or “ghost”) tangles following neuronal death. understanding the molecular events that correspond to this sequence would be extremely helpful for developing the next generation of diagnostics (see below) and therapeutics. the pathobiology of tau pathology is complex, with multiple tau isoforms undergoing innumerable secondary structural modifications in disease states beyond just hyperphosphorylation, including truncation, acetylation, ubiquitination, glycosylation and methylation. it has been challenging to temporally map the order of these events using traditional approaches (e.g., phospho-specific tau antisera-based methods) which lack the ability to synchronously and comprehensively detect these critical changes. in the last proteomics paper, published in cell in november of 2020, a powerful study, led by dr. judith steene at boston children’s hospital, detailed their focused analysis that was entirely directed towards the tau protein (wesseling et al., 2020). they deployed a previously published mass spectrometry-based assay, that they term “flexi-tau” (mair et al., 2016), which allowed them to generate a high-resolution quantitative proteomic map of 95 post-translational modifications on multiple tau isoforms from 91 human post-mortem brains. they then used unsupervised analyses to predict sequential addition of modifications. they found that while there is a great degree of heterogeneity, there appears to be a minimal set of modifications that develop in a processive fashion and are associated with tissue fractions associated with disease stage as well as tau seeding activity. the analysis also highlighted a specific alternatively spliced tau isoform with four microtubule binding domain repeats (4r), but lacking n-terminal domain inserts (0n). the 0n4r isoform is highly overrepresented in aggregates isolated from early disease stages alongside modifications associated with increased negative charge in the proline-rich region and decreased positive charge in the microtubule binding domain. these findings are promising because they give us the most comprehensive picture of tau secondary structure and provide a pathway towards continuing to advance the biomarkers that are rapidly evolving and identify the critical modifications that are responsible for the pathogenicity of tau paving the way towards new therapeutics. 5. plasma biomarkers for alzheimer disease obtaining a definitive diagnosis of a neurodegenerative disease continues to require an autopsy, the perennial gold standard, but continued advances in blood-based biomarkers are continuing to challenge this and offer a low-cost, convenient alternative. over the past decade, substantial progress has been made in our ability to assess molecular changes in the central nervous system using non-invasive amyloid positron emission tomography (pet) scans and cerebrospinal fluid (csf) markers. yet these modalities are expensive, inconvenient and come with a mild degree of risk. the long-sought development of a blood-based biomarker for alzheimer disease (ad), had been considered unrealistic because the levels of brain proteins in the blood had been thought to be simply too low to be reliably and reproducibly detected. further, there was concern that the extent to which blood levels of various factors reflect ongoing disease processes might be too disconnected or tangential to be clinically useful. so, it comes as no surprise that the research community has been stunned by the rapid progress in blood-based biomarkers in neurodegeneration. this year a milestone was achieved, with the “precivityad™” mass-spec amyloid-β assay, which was developed at washington university saint louis in the laboratory directed by dr. randall bateman, receiving approval under the clinical laboratory improvement amendments (clia) as well as an fda breakthrough designation. however, this is only half of the story. the other essential neuropathological hallmark of ad, the neurofibrillary tangle composed of abnormal tau, is also required for a diagnosis. remarkably, similar rapid progress is being made on this front with a series of groundbreaking studies that were published over that past year, continuing the momentum. in previous years, the first papers emerged describing highly sensitive immunoassays identifying p-tau phosphorylated at threonines 181 or 217 as early promising biomarkers. in 2020, phospho-threonine 231 (p-tau231) was reported to be an excellent early marker for tau pathology in csf (suárez-calvet et al., 2020). this was followed up with results from a study using an ultrasensitive single molecule array (simoa) for the quantification of p-tau231 in blood plasma (ashton et al., 2021). the study included a total of 588 subjects and successfully differentiated ad from amyloid-β negative cognitively normal individuals. plasma p-tau231 also differentiated ad patients from non-ad neurodegenerative disorders and amyloid-β negative mci patients. in samples taken from patients with post-mortem autopsy confirmation, plasma p-tau231 was extremely accurate in identifying ad neuropathology in comparison to non-ad neurodegenerative disorders (auc = 0.99). plasma p-tau231 was highly correlated with other ad biomarkers, i.e., csf p-tau231, tau pet ([18f]mk-6240) and amyloid-β pet ([18f]azd). the elevations in p-tau231 were a very early change, preceding amyloid-β pet and plasma p-tau181. plasma p-tau231 had the power to resolve even subtle differences, differentiating subjects across even early braak stages. together, we are witnessing impressive progress in blood-based ad biomarkers with the potential to detect the earliest disease stages, prior to significant irreversible brain destruction, and enable the next generation of clinical trials. 6. cryo-electron microscopy: ultrahigh resolution structure of tau in corticobasal degeneration in 2017, the nobel prize in chemistry was awarded to jacques dubochet, joachim frank and richard henderson for cryo-electron microscopy (cryo-em). the approach, which involves cooling samples to cryogenic temperatures and embedding them in an environment of vitreous water, has been in development since the 1970s, but recent technical advances in detectors and software algorithms have enabled solving molecular structures at near-atomic resolution. the power of cryo-em was first demonstrated that year to the neurodegenerative disease research community when the approach was applied to paired-helical filament tau fibrils in alzheimer disease, illuminating at near atomic resolution the c-shaped conformation of the core (fitzpatrick et al., 2017). this report was quickly followed by the solving of the structure for the j-shaped tau filament core in pick disease which contains additional residues making it slightly longer than in ad (falcon et al., 2018). next came chronic traumatic encephalopathy, which has a similar c-shape to that in alzheimer disease (falcon et al., 2019). also described was an additional non-proteinaceous hydrophobic density that has yet to be defined. these findings support the notion that there exists a spectrum of hitherto unrecognized tau structural features and conformations, often termed “strains” in a nod to the prion literature that has informed much of the research on protein misfolding, that may be distinct in different disease states. now, we are continuing to see additional insights with two papers describing the structure of the tau filament core in corticobasal degeneration as well as another which is the first cryo-em structure of α-synuclein (see next section). in a paper published in cell in february 2020, a team led by anthony fitzpatrick at columbia university detailed their study showing the cryo-em structure of filaments from corticobasal degeneration (arakhamia et al., 2020). in additional analyses, they further integrated these cryo-em structures and those from ad with mass spectrometry data to localize post-translational secondary modifications. a separate study, published in nature, led by sjors scheres and michel goedert at the mrc laboratory of molecular biology in cambridge, england, u.k. (zhang et al., 2020). this team also showed a similar structure in cbd (figure 4). together, in addition to providing a validated atomic level resolution c-shaped structure of the tau filament in cbd, these studies both revealed a density buried in the structure that represents a molecule of unknown identity, akin to what was identified in cte. the fibrils in cbd are composed entirely of tau with 4 microtubule binding domain repeats (4r), which differs from pick disease which is composed of 3r tau as well as ad and cte which are mixed 3r and 4r. additional future studies directed towards further delineation of the diversity of tau filament structures has the potential to pave the way towards improved diagnostics and advancing our understanding of the diversity of conformers in tauopathy that may be associated with different clinical symptomatology. figure 4. cryo-em of tau filaments in corticobasal degeneration. a. negative-stain electron micrographs of type i and type ii tau filaments extracted from the frontal cortex of cbd. b. cryo-em maps of type i and type ii tau filaments from the frontal cortex of cbd. c. top, the microtubule-binding repeats (r1–r4) of tau and the sequence after r4 that is present in the core of cbd filaments (all shown in different colors). bottom, atomic model of the cbd type ii tau filament. the extra density is shown in light blue, with k290, k294 and k370 indicated. images courtesy of drs. wenjuan zhang, sjors scheres and michel goedert mrc laboratory of molecular biology, cambridge). 7. cryo-electron microscopy: ultrahigh resolution structures of α-synuclein conformers this year we also saw reports of the first ultrahigh resolution cryo-em structures of α-synuclein fibrils published in nature also from sjors scheres and michel goedert at the mrc laboratory of molecular biology (schweighauser et al., 2020). the synucleinopathies are a group of neurodegenerative disorders that include parkinson’s disease (pd), pd dementia, diffuse lewy body disease (dlbd) and multiple system atrophy (msa). co-first authors schweighauser and shi et al., studied three cases of dlbd and five with msa. in msa, they found two types of filament, that they termed type 1 and type 2, each with filaments with different protofibrils that make up the core. the conformation of α-synuclein filaments derived from dlbd brains by cryo-em precluded 3d imaging, so 2d class averaging was used to show that there were differences in the structure from msa. overall, these structural differences are of great interest as accumulating evidence suggests that α-synuclein might behave in a prion-like manner, with templating and propagation of abnormal structures/conformers. this knowledge may be helpful in understanding the pathogenesis and natural history of the synucleinopathies, and especially in the development of specific α-synuclein pet tracers, which have been so far elusive. 8. characterizing α-synuclein strains the α-synucleinopathies, including parkinson disease, diffuse lewy body disease, and dementia with lewy bodies, while linked by aggregation of α-synuclein, have been hypothesized to diverge based on the prion-like properties unique disease conformational structural folding that templates replication of the abnormality. these strains are hypothesized to have different properties and may influence neuropathological features and symptomatology. this year, a study was published that took us one step closer to understanding the pathobiology and diversity of α-synuclein strains. in an elegant set of experiments, lau et al. recreated an array of α-synuclein fibrils with different confirmations in vitro using recombinant proteins by altering buffer conditions (lau et al., 2020). they generated a host of fibrils that varied in their biophysical properties and compared them with those derived from human post-mortem brain samples. then, the fibrils were inoculated into a transgenic mouse line that overexpresses human mutant α-synuclein and observed them. over time, the mice developed a range of traits that were strain specific, including aggregate morphology, incubation periods and behavioral changes, that could be propagated serially (figure 5). in the brains of these animals, distinct neuroanatomical vulnerability was observed that was dictated by strain type. these findings give us an additional model for testing questions related to how these different α-synuclein conformers target specific cells and brain regions leading to neurodegeneration. figure 5. distinct midbrain pathology in transgenic α-synuclein (m83) mice inoculated with salt (s) fibrils or no salt (ns) fibrils. a. the ns fibril-injected mice show the “lewy body-like” α-synuclein pathology. b. the s fibril-injected mice exhibit the “ring-like” α-synuclein pathology. both images show stains with the ep1536y antibody that recognizes synuclein phosphorylated at serine 129. images courtesy of dr. joel watts (university of toronto). 9. huntington’s disease and chorea abnormalities in proteostasis are a key feature of essentially all neurodegenerative disorders. this change is associated with endoplasmic reticulum (er) stress, which then triggers the unfolded protein response (upr). xbp1 is a key mediator of the er stress response thought to be a master regulator of the upr that drives the adaptation response to recover proteostasis through a number of mechanisms. in a series of compelling experiments, a team from the university of chile showed that treatment with igf2, a factor that they previously implicated as a downstream effector of xbp1 in animal models deficient in xbp1, may be an important contributor to this finding (garcía-huerta et al., 2020). treatment with igf1 led to a marked reduction in the burden of intracellular aggregates of mutant huntingtin in cellular models, including an induced pluripotent stem cell (ipsc)-derived model of medium spiny neurons from hd patients. when the autophagy and the ubiquitin proteasome were assessed, they surprisingly found that these pathways were not responsible. remarkably, they found igf2 signaling enhanced secretion of soluble mutant huntingtin into exosomes/microvesicles. these findings were not limited to cell culture but were recapitulated when igf2 was infused into the brain of hd mice. finally, these findings were validated in human tissue samples, with a reduction in igf2 in post-mortem hd brain and blood. these findings take us one step closer to understanding the role of the upr in hd and suggest a mechanism that could be targeted for therapeutics. 10. chronic traumatic encephalopathy: what about the astrocytes? slightly over ten years ago, chronic traumatic encephalopathy (cte) was propelled to the fore of neurodegenerative disease research where it has remained. since that time, broad consensus has emerged among neuropathologists that cte has a distinct neuropathological presentation that is distinguishable from other tauopathies, with a unique pathognomonic perivascular lesion that was codified in consensus criteria (mckee et al., 2016). in this report, the pathognomonic lesion was described as consisting of “p-tau aggregates in neurons, astrocytes, and cell processes around small vessels in an irregular pattern at the depths of the cortical sulci”. while this working definition was considered sufficiently precise, confusion emerged and persisted in the literature around the perivascular astrocytes in aging related tau astrogliopathy (artag) (kovacs et al., 2016) that can generally be differentiated based on the absence of neurons and localization in the white matter or subpial compartments. clarification was not sufficient to put the astrocyte question to rest (mckee et al., 2020). setting aside artag, there is a tremendous burden of p-tau positive gray matter astrocytes in cte. a study led by john trojanowski at the university of pennsylvania and william stewart at the university of glasgow published in brain communications sought to neuroanatomically map these astrocytes and found a preferential accumulation in the sulcal depths, the precise region that is biomechanically vulnerable in cte and where tau pathology first emerges (arena et al., 2020). this is in contrast to neuronal tau pathology which they found was more diffuse. this seems to suggest that astrocyte pathology is an early event. in another study published in brain pathology led by ann mckee and john crary (writer of this review), a spectrum of cases that also included very early cte lesions was able to pinpoint abnormal tau in early pathognomonic lesions to perivascular neurons (figure 6), but not astrocytes, which appeared to accumulate later in the disease progression (cherry et al., 2020). this tau pathology consisted predominantly of tau isoforms containing four microtubule binding domain repeats (4r tau), an isoform that has been proposed to be especially toxic given its increased propensity towards aggregation. together, these findings point to a complex picture of evolving tau pathology in neurons and astrocytes early in cte and we expect that research in the coming years around the role of astrocytes in early cte will intensify. figure 6. immunofluorescence microscopy in a sulcal region from an individual who passed away in their 20s with chronic traumatic encephalopathy (cte). a. low power image highlighting two perivascular lesions. b. high power image showing staining with markers for astroglia (gfap, blue), neurons (map2, green) and abnormal hyperphosphorylated p-tau (at8; green). white arrows mark colocalization. in these lesions, there is only map2 overlap with p-tau. no gfap/at8 colocalization was seen in this case. scale bar is 50 um. images courtesy of dr. jonathan cherry and ann mckee (boston university and boston va). acknowledgement dr. crary receives research funding from the nih (p30ag066514, r01ag054008, r01ns095252, r01ag062348, r01ns086736, u54ns115266, u54ns115322). we further acknowledge the rainwater charitable trust/tau consortium, david and elsie werber, alexander saint amand scholar award and karen strauss cook research scholar award. references arakhamia, t., lee, c. e., carlomagno, y., duong, d. m., kundinger, s. r., wang, k., williams, d., deture, m., dickson, d. w., cook, c. n., seyfried, n. t., petrucelli, l., & fitzpatrick, a. w. p. (2020). posttranslational modifications mediate the structural diversity of tauopathy strains. cell, 180(4), 633-644.e12. https://doi.org/10.1016/j.cell.2020.01.027 arena, j. d., johnson, v. e., lee, e. b., gibbons, g. s., smith, d. h., trojanowski, j. q., & stewart, w. (2020). astroglial tau pathology alone preferentially concentrates at sulcal depths in chronic traumatic encephalopathy neuropathologic change. brain communications, 2(2), fcaa210. https://doi.org/10.1093/braincomms/fcaa210 ashton, n. j., pascoal, t. a., karikari, t. k., benedet, a. l., lantero-rodriguez, j., brinkmalm, g., snellman, a., schöll, m., troakes, c., hye, a., gauthier, s., vanmechelen, e., zetterberg, h., rosa-neto, p., & blennow, k. (2021). plasma p-tau231: a new biomarker for incipient alzheimer’s disease pathology. acta neuropathologica. https://doi.org/10.1007/s00401-021-02275-6 badrfam, r., & zandifar, a. (2020). from encephalitis lethargica to covid-19: is there another epidemic ahead? clinical neurology and neurosurgery, 196, 106065. https://doi.org/10.1016/j.clineuro.2020.106065 bryce, c., grimes, z., pujadas, e., ahuja, s., beasley, m. b., albrecht, r., hernandez, t., stock, a., zhao, z., rasheed, m. a., chen, j., li, l., wang, d., corben, a., haines, k., westra, w., umphlett, m., gordon, r. e., reidy, j., … fowkes, m. (2020). pathophysiology of sars-cov-2: targeting of endothelial cells renders a complex disease with thrombotic microangiopathy and aberrant immune response. the mount sinai covid-19 autopsy experience. medrxiv, 2020.05.18.20099960. https://doi.org/10.1101/2020.05.18.20099960 cherry, j. d., kim, s. h., stein, t. d., pothast, m. j., nicks, r., meng, g., huber, b. r., mez, j., alosco, m. l., tripodis, y., farrell, k., alvarez, v. e., mckee, a. c., & crary, j. f. (2020). evolution of neuronal and glial tau isoforms in chronic traumatic encephalopathy. brain pathology (zurich, switzerland). https://doi.org/10.1111/bpa.12867 falcon, b., zhang, w., murzin, a. g., murshudov, g., garringer, h. j., vidal, r., crowther, r. a., ghetti, b., scheres, s. h. w., & goedert, m. (2018). structures of filaments from pick’s disease reveal a novel tau protein fold. nature, 561(7721), 137–140. https://doi.org/10.1038/s41586-018-0454-y falcon, b., zivanov, j., zhang, w., murzin, a. g., garringer, h. j., vidal, r., crowther, r. a., newell, k. l., ghetti, b., goedert, m., & scheres, s. h. w. (2019). novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules. nature, 568(7752), 420–423. https://doi.org/10.1038/s41586-019-1026-5 fitzpatrick, a. w. p., falcon, b., he, s., murzin, a. g., murshudov, g., garringer, h. j., crowther, r. a., ghetti, b., goedert, m., & scheres, s. h. w. (2017). cryo-em structures of tau filaments from alzheimer’s disease. nature, 547(7662), 185–190. https://doi.org/10.1038/nature23002 garcía-huerta, p., troncoso-escudero, p., wu, d., thiruvalluvan, a., cisternas-olmedo, m., henríquez, d. r., plate, l., chana-cuevas, p., saquel, c., thielen, p., longo, k. a., geddes, b. j., lederkremer, g. z., sharma, n., shenkman, m., naphade, s., sardi, s. p., spichiger, c., richter, h. g., … hetz, c. (2020). insulin-like growth factor 2 (igf2) protects against huntington’s disease through the extracellular disposal of protein aggregates. acta neuropathologica, 140(5), 737–764. https://doi.org/10.1007/s00401-020-02183-1 johnson, e. c. b., dammer, e. b., duong, d. m., ping, l., zhou, m., yin, l., higginbotham, l. a., guajardo, a., white, b., troncoso, j. c., thambisetty, m., montine, t. j., lee, e. b., trojanowski, j. q., beach, t. g., reiman, e. m., haroutunian, v., wang, m., schadt, e., … seyfried, n. t. (2020). large-scale proteomic analysis of alzheimer’s disease brain and cerebrospinal fluid reveals early changes in energy metabolism associated with microglia and astrocyte activation. nature medicine, 26(5), 769–780. https://doi.org/10.1038/s41591-020-0815-6 kovacs, g. g., ferrer, i., grinberg, l. t., alafuzoff, i., attems, j., budka, h., cairns, n. j., crary, j. f., duyckaerts, c., ghetti, b., halliday, g. m., ironside, j. w., love, s., mackenzie, i. r., munoz, d. g., murray, m. e., nelson, p. t., takahashi, h., trojanowski, j. q., … dickson, d. w. (2016). aging-related tau astrogliopathy (artag): harmonized evaluation strategy. acta neuropathologica, 131(1), 87–102. https://doi.org/10.1007/s00401-015-1509-x lau, a., so, r. w. l., lau, h. h. c., sang, j. c., ruiz-riquelme, a., fleck, s. c., stuart, e., menon, s., visanji, n. p., meisl, g., faidi, r., marano, m. m., schmitt-ulms, c., wang, z., fraser, p. e., tandon, a., hyman, b. t., wille, h., ingelsson, m., … watts, j. c. (2020). α-synuclein strains target distinct brain regions and cell types. nature neuroscience, 23(1), 21–31. https://doi.org/10.1038/s41593-019-0541-x mair, w., muntel, j., tepper, k., tang, s., biernat, j., seeley, w. w., kosik, k. s., mandelkow, e., steen, h., & steen, j. a. (2016). flexitau: quantifying post-translational modifications of tau protein in vitro and in human disease. analytical chemistry, 88(7), 3704–3714. https://doi.org/10.1021/acs.analchem.5b04509 mckee, a. c., cairns, n. j., dickson, d. w., folkerth, r. d., keene, c. d., litvan, i., perl, d. p., stein, t. d., vonsattel, j.-p., stewart, w., tripodis, y., crary, j. f., bieniek, k. f., dams-o’connor, k., alvarez, v. e., gordon, w. a., & tbi/cte group. (2016). the first ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. acta neuropathologica, 131(1), 75–86. https://doi.org/10.1007/s00401-015-1515-z mckee, a. c., stein, t. d., crary, j. f., bieniek, k. f., cantu, r. c., & kovacs, g. g. (2020). practical considerations in the diagnosis of mild chronic traumatic encephalopathy and distinction from age-related tau astrogliopathy. journal of neuropathology and experimental neurology, 79(8), 921–924. https://doi.org/10.1093/jnen/nlaa047 morgello, s. (2020, august). coronaviruses and the central nervous system. journal of neurovirology; j neurovirol. https://doi.org/10.1007/s13365-020-00868-7 reichard, r. r., kashani, k. b., boire, n. a., constantopoulos, e., guo, y., & lucchinetti, c. f. (2020). neuropathology of covid-19: a spectrum of vascular and acute disseminated encephalomyelitis (adem)-like pathology. acta neuropathologica, 140(1), 1–6. https://doi.org/10.1007/s00401-020-02166-2 schweighauser, m., shi, y., tarutani, a., kametani, f., murzin, a. g., ghetti, b., matsubara, t., tomita, t., ando, t., hasegawa, k., murayama, s., yoshida, m., hasegawa, m., scheres, s. h. w., & goedert, m. (2020). structures of α-synuclein filaments from multiple system atrophy. nature, 585(7825), 464–469. https://doi.org/10.1038/s41586-020-2317-6 suárez-calvet, m., karikari, t. k., ashton, n. j., lantero rodríguez, j., milà-alomà, m., gispert, j. d., salvadó, g., minguillon, c., fauria, k., shekari, m., grau-rivera, o., arenaza-urquijo, e. m., sala-vila, a., sánchez-benavides, g., gonzález-de-echávarri, j. m., kollmorgen, g., stoops, e., vanmechelen, e., zetterberg, h., … alfa study. (2020). novel tau biomarkers phosphorylated at t181, t217 or t231 rise in the initial stages of the preclinical alzheimer’s continuum when only subtle changes in aβ pathology are detected. embo molecular medicine, 12(12), e12921. https://doi.org/10.15252/emmm.202012921 wesseling, h., mair, w., kumar, m., schlaffner, c. n., tang, s., beerepoot, p., fatou, b., guise, a. j., cheng, l., takeda, s., muntel, j., rotunno, m. s., dujardin, s., davies, p., kosik, k. s., miller, b. l., berretta, s., hedreen, j. c., grinberg, l. t., … steen, j. a. (2020). tau ptm profiles identify patient heterogeneity and stages of alzheimer’s disease. cell, 183(6), 1699-1713.e13. https://doi.org/10.1016/j.cell.2020.10.029 wingo, a. p., fan, w., duong, d. m., gerasimov, e. s., dammer, e. b., liu, y., harerimana, n. v., white, b., thambisetty, m., troncoso, j. c., kim, n., schneider, j. a., hajjar, i. m., lah, j. j., bennett, d. a., seyfried, n. t., levey, a. i., & wingo, t. s. (2020). shared proteomic effects of cerebral atherosclerosis and alzheimer’s disease on the human brain. nature neuroscience, 23(6), 696–700. https://doi.org/10.1038/s41593-020-0635-5 zhang, w., tarutani, a., newell, k. l., murzin, a. g., matsubara, t., falcon, b., vidal, r., garringer, h. j., shi, y., ikeuchi, t., murayama, s., ghetti, b., hasegawa, m., goedert, m., & scheres, s. h. w. (2020). novel tau filament fold in corticobasal degeneration. nature, 580(7802), 283–287. https://doi.org/10.1038/s41586-020-2043-0 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. studies on inflammation and stroke provide clues to pathomechanism of central nervous system involvement in covid-19 feel free to add comments by clicking these icons on the sidebar free neuropathology 1:16 (2020) opinion piece studies on inflammation and stroke provide clues to pathomechanism of central nervous system involvement in covid-19 ádám dénes 1, stuart m. allan 2, tibor hortobágyi 3,4, craig j. smith 5 1 laboratory of neuroimmunology, institute of experimental medicine, szigony u. 43. 1083, budapest, hungary 2 division of neuroscience and experimental psychology, lydia becker institute of immunology and inflammation, the university of manchester, manchester academic health science centre, av hill building, manchester, m13 9pt, uk 3 department of pathology, faculty of medicine, university of szeged, szeged, hungary 4 mta-de cerebrovascular and neurodegenerative research group, department of neurology, university of debrecen, debrecen, hungary 5 division of cardiovascular sciences, lydia becker institute of immunology and inflammation, the university of manchester, manchester academic health science centre, manchester centre for clinical neurosciences, salford royal nhs foundation trust, salford, m6 8hd, uk corresponding author: ádám dénes · laboratory of neuroimmunology · institute of experimental medicine · szigony u. 43. 1083 · budapest · hungary denesa@koki.hu submitted: 26 may 2020 accepted: 28 may 2020 copyedited by: calixto-hope g lucas, jr. published: 05 june 2020 https://doi.org/10.17879/freeneuropathology-2020-2818 keywords: covid-19, sars-cov-2, neuro-covid, stroke, neuroinflammation, neuropathology recent data, including a number of controversial findings from clinical covid-19 studies, have initiated an intense debate regarding central nervous system (cns) involvement in sars-cov-2 infection-associated pathologies and overall outcomes. in our opinion, involvement of the brain may be an important contributor to the highly complex pathophysiology caused by sars-cov-2 and lessons from studies on stroke and systemic inflammation provide important clues. numerous cns symptoms, including loss of smell and taste, headache, dizziness, nausea, seizures and respiratory distress have been reported in covid-19. several neurological syndromes, such as stroke, encephalitis, epilepsy, and guillain-barre syndrome have been associated with covid-19. more recently, large-vessel occlusive stroke has been described in younger patients without typical covid-19 symptoms1-3. in addition, history of stroke is associated with increased severity of covid-194. these observations highlight the brain as an important target of this multiorgan disease. while neuroinvasion of sars-cov-2 has been associated with cerebral thrombosis, hemorrhagic infarction, demyelinating lesions and encephalopathy (termed as neuro-covid)5,6, it has also been suggested that some respiratory symptoms in patients with covid-19 could indicate neurological involvement7. post-mortem examination of a series of patients with positive polymerase chain reaction testing for covid-19 in pleural effusions revealed negative testing in all cerebrospinal fluid (csf) samples along with no signs of encephalitis or cns vasculitis8. this data may suggest that brain involvement in covid-19 does not play a major role in the disease pathogenesis. however, it appears difficult to draw firm conclusions from these observations. sars-cov-2 might not be detectable in the csf due to low viral load, increased clearance, or the sensitivity of detection5, while macroscopic analysis may not be sufficient to reveal the presence of infection in the brain tissue. nevertheless, other arguments also indicate that neurological symptoms reported to date may be nonspecific and not necessarily imply cns disease, while respiratory failure alone does not suggest cns invasion by sars-cov-29. however, in the absence of comprehensive neuropathological analysis, the extent and anatomical distribution of sars-cov-2 infection in the cns remain unanswered. the robust inflammatory and prothrombotic response directly and indirectly affecting the cns could explain some of the major neurological complications, which may be complemented by effects of possible sars-cov-2 infection in the brain. coronaviruses have long been recognised as potentially neurovirulent microorganisms10. neuroinvasiveness of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), has been previously reported with pronounced infection in brainstem nuclei involved in respiratory and cardiovascular control2,11. based on these data, sars-cov-2 might also reach the cns via multiple routes. in viraemia, sars-cov-2 binds to the angiotensin-converting enzyme-2 (ace2) receptor on the endothelium and, after crossing the blood-brain barrier (bbb), also binds to neurons expressing the receptor12. one of the currently available case reports with post-mortem neuropathology limited to electron microscopic examination in a frontal lobe sample demonstrates the virus in endothelial cells with features suggestive of transit of the virus towards the neuropil, and neuronal ‘viral-like’ particles in cytoplasmic vacuoles13. via the olfactory route, the virus infects the olfactory epithelium, enters the nervous system across the cribriform plate through axons of olfactory bulb neurons, and then infects the sustentacular cells that maintain the integrity of olfactory sensory neurons. sars-cov-2 may also enter and spread via the cerebral lymphatic (glymphatic) drainage system since the virus can infect the endothelial cells of the olfactory lymphatic system which connect to the brain12,14-16. in addition, similar to herpesviruses or the avian influenza virus, sars-cov-2 could reach the brain via peripheral nerves, possibly via retrograde transport and trans-synaptic spread17-19. deficient systemic immune response due to older age, chronic disease or immunosuppressive therapy, altered ace2 expression in diabetic or hypertensive patients, cerebrovascular disease (major risk factors for covid-19-associated mortality)1,4, as well as vascular inflammation or impaired blood-brain barrier (bbb) function in aged or comorbid patients could also increase the risk and severity of infection. if cns infection occurs, the outcome largely depends on the ability of the cns immune system to control the spread of the virus. studies on neurotropic virus infection suggest that microglia, the main inflammatory cells of the cns, are important in controlling both the spread of the virus and shaping the cerebral inflammatory response. this key role of microglia has been experimentally demonstrated in coronavirus, herpesvirus, theiler’s virus and vesicular stomatitis virus infection among others20-24. importantly, lack of normal microglial function not only increases viral spread in the brain, but is also associated with markedly worsened neurological symptoms (motor function deficits, neuronal injury, brain oedema, seizures, etc.) and increased mortality in animal models20-24. microglial phenotype is heavily influenced by age, comorbidities and systemic inflammation25. therefore, it is likely that compromised microglial function is an important contributor to poor outcome in neuro-covid, especially if brain areas involved in respiratory, cardiovascular and neuroendocrine control are affected by sars-cov-2. effective antiviral drug delivery through the bbb may therefore be important in the management of neurological complications. irrespective of whether cns sars-cov-2 infection occurs, the cerebral effects of systemic inflammation associated with “cytokine storm” and prothrombotic state have profound impact on outcome in severe covid-19 cases. high serum levels of inflammatory cytokines, such as il-6, predict poor outcome26 and may contribute to cardiacand respiratory arrest, coma and multiorgan failure through complex mechanisms that include microcirculatory deficits, hypotension, oedema and thrombosis. circulating inflammatory cytokines stimulate the autonomic nervous system and the hypothalamic-pituitary-adrenal (hpa) axis with major impact on blood pressure and flow, respiration and neuroendocrine function. the autonomic nervous system and hpa axis also play an important role in the regulation of immune cell responses, cytokine production, cell trafficking and cell death via both humoral and neural mechanisms. the sustained increase in serum adrenaline, noradrenaline and cortisol levels due to prolonged autonomic and hpa axis activation eventually lead to dysregulation of inflammatory responses. this, in line with excessive cytokine production, results in insufficient elimination of infectious agents. the lymphopenia due to apoptosis and impaired lymphopoiesis shifts immune cell balance towards excessive monocyte and granulocyte load, further increasing the production myeloid cell-derived proinflammatory cytokines. the excessive systemic inflammatory response also contributes to the neurological complications via direct and indirect actions. findings from clinical and experimental stroke studies with preceding or post-stroke infection may help to shed light on the mechanisms of covid-19-related neurological impairment. stroke in apparently healthy youngand middle-aged people with rapid formation of thrombi in the cerebral circulation and high mortality of older patients with chronic inflammatory disease collectively suggest a high impact of altered coagulation in patients with covid-1927,28. infections in general (e.g. seasonal flu) increase stroke incidence29. as known from studies of viral and bacterial sepsis and from comorbid models of stroke, increased systemic inflammatory burden promotes vascular inflammation, platelet activation and alters coagulation cascades, leading to a procoagulant state leading to thrombosis and disseminated intravascular coagulation. such coagulopathy, predictive of worse clinical outcome, has been reported in covid-1930,31. this promotes thrombus formation in both veins and arteries – a known feature of severe covid-19 with thromboembolic complications reported in around one third of infected patients32. the cerebrovascular endothelial cells have particularly high sensitivity to proinflammatory cytokines and sars-cov-2 infection could further boost the expression of adhesion molecules and increase vascular permeability. brain injury and mortality are generally far more severe in patients with additional stroke risk factors (e.g. in obese, diabetic, hypertensive patients or after infection), similarly to that seen in experimental stroke models. as an example, localized infection of the lungs by influenza virus or streptococcus pneumoniae leads to marked increases in circulating proinflammatory cytokines, endothelial activation (indicated by increased levels of adhesion molecules) and platelet aggregation. these infection-driven changes are associated with augmented brain inflammation and leukocyte recruitment, leading to increased neuronal injury and worse neurological outcome33-35. targeted blockade of proinflammatory cytokines (e.g. il-6, il-1 or tnf) or platelet-endothelial interactions attenuated infection-induced brain injury in experimental models33,34,36-38, while the potential efficacy of il-6 receptor antagonist tocilizumab to reduce mortality in severe covid-19 cases has been suggested39. recent reports have described patients with ischaemic stroke complicating covid-19 infection, often manifesting as large-vessel infarcts occurring in multiple territories and associated with features of prothrombotic coagulopathy3,40,41. however, these small case series may not be representative of wider clinical practice, and no causal relationship has yet been established between covid-19 infection and stroke. other mechanisms may also be relevant, such as destabilisation of atheromatous plaques resulting in thrombosis and cerebral atheroembolism, atrial fibrillation in critically ill patients causing thromboembolism of cardiac origin, or haemorrhage secondary to microangiopathy and cerebral vasculitis. importantly, patients may also acquire sars-cov-2 infection following stroke. suppression of both innate and adaptive immune response is well-documented after stroke, driven by autonomic nervous system failure and activation of the hpa axis, which could exacerbate subclinical infection, or increase susceptibility to nosocomial infection. an important issue to address is whether neurological manifestations (reported in up to one third of cases with different severity of infection1,42) result from systemic effects on the brain, direct cns infection by sars-cov-2, or both, and if the latter is a major contributor to covid-19-related severe illness and mortality. firm conclusions cannot be drawn at this stage due to limited data availability, but it is likely that the impact of cns-related effects on disease outcome is considerable. most neurological manifestations appear to occur early in the illness, preceding severe respiratory distress and the need for mechanical ventilation, while several patients are admitted to the hospital merely based on neurologic manifestation with no respiratory symptoms1. at this time, data are largely from single case reports. for example, occurrence of neurological symptoms such as fever, anosmia, dysgeusia, headache and possible seizure in line with respiratory distress and severe ventilator asynchronies were found in a patient where autopsy later confirmed the presence of sars-cov-2 infection in the brain. findings included widespread tissue damage involving the neurons, glia, nerve axons, and myelin sheath, progressively more severe from the olfactory nerve to the gyrus rectus and to the brainstem43. a recent report showed that after positive diagnosis for sars-cov-2, a patient developed complete anosmia and dysgeusia, with mri showing signs of bilateral olfactory bulb oedema, followed by normalization of both sensory symptoms and mri signal by day 1444. another report found that 44% of patients admitted to the intensive care unit with covid-19 and neurological symptoms showed cns abnormalities on mri, which included cortical (frontal, parietal, occipital, temporal, insular) and deep white matter flair signal abnormalities, in the absence of sars-cov-2 in the csf (50% of cases tested). thus, while focal neuropathologies appear to be frequent in severe cases, the extent of associated brain infection remains unclear presently. of note, the incidence of epileptiform discharges, seizure-like events and new onset encephalopathy is more than two-fold higher in acutely ill covid-19 patients with neurological symptoms compared to non-infected patients, but it is not known if this is through direct or indirect actions on the cns45. while sars-cov-2 infection shares many similarities with bacterial sepsis, the inflammatory response was considered more modest (e.g. lower il-6 levels), and progressive and profound suppression of adaptive immunity was noted in covid-19 relative to sepsis46. therefore, further studies are required to assess the nature of systemic inflammatory changes and their impact on neurological symptoms and disease outcome. in conclusion, the available evidence strongly indicates that the brain is an important target of sars-cov-2 and the impact of its brain-related pathophysiology on survival and outcome in covid-19 is substantial. while diagnostic efforts and research studies to investigate the presence of infection in the brain and to reveal the mechanisms of both central and systemic inflammation in covid-19 are necessary, lessons from previous work on infection, stroke and systemic inflammation should be considered. regarding the clinical management of covid-19 patients, immune modulatory therapies are attractive candidates. in this respect it will be intriguing to learn the outcomes of ongoing clinical trials of anti-cytokine therapies, including anakinra (il-1 receptor antagonist) and tocilizumab (il-6 receptor antibody). references 1. mao l, jin h, wang m, hu y, chen s, he q, chang j, hong c, zhou y, wang d, miao x, li y, hu b. neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china. jama neurology. 2020;e201127. doi:10.1001/jamaneurol.2020.1127 2. wu y, xu x, chen z, duan j, hashimoto k, yang l, liu c, yang c. nervous system involvement after infection with covid-19 and other coronaviruses. brain, behavior, and immunity. 2020; s0889-1591(20)30357-3. doi:10.1016/j.bbi.2020.03.031 3. oxley tj, mocco j, majidi s, kellner cp, shoirah h, singh ip, de leacy ra, shigematsu t, ladner tr, yaeger ka, skliut m, weinberger j, dangayach ns, bederson jb, tuhrim s, fifi jt. large-vessel stroke as a presenting feature of covid-19 in the young. the new england journal of medicine. 2020;382:e60. doi:10.1056/nejmc2009787 4. aggarwal g, lippi g, michael henry b. cerebrovascular disease is associated with an increased disease severity in patients with coronavirus disease 2019 (covid-19): a pooled analysis of published literature. international journal of stroke. 2020;1747493020921664. doi:10.1177/1747493020921664 5. panciani pp, saraceno g, zanin l, renisi g, signorini l, battaglia l, fontanella mm. sars-cov-2: "three-steps" infection model and csf diagnostic implication. brain, behavior, and immunity. 2020 may 5, doi: 10.1016/j.bbi.2020.05.002 6. zanin l, saraceno g, panciani pp, renisi g, signorini l, migliorati k, fontanella mm. sars-cov-2 can induce brain and spine demyelinating lesions. acta neurochirurgica. 2020 may 4;1-4. doi: 10.1007/s00701-020-04374-x 7. recasens bb, llorens jmm, sevilla jjr, rubio ma. lack of dyspnea in patients with covid-19: another neurological conundrum? european journal of neurology. 2020 apr 17, doi: 10.1111/ene.14265 8. schaller t, hirschbuhl k, burkhardt k, braun g, trepel m, markl b, claus r. postmortem examination of patients with covid-19. jama. may 21, 2020. doi:10.1001/jama.2020.8907 9. turtle l. respiratory failure alone does not suggest central nervous system invasion by sars-cov-2. journal of medical virology. 04, april 2020. https://doi.org/10.1002/jmv.25828 10. desforges m, le coupanec a, brison e, meessen-pinard m, talbot pj. neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans. advances in experimental medicine and biology. 2014;807:75-96 11. li yc, bai wz, hashikawa t. the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients. journal of medical virology. 2020;10.1002/jmv.25728. doi:10.1002/jmv.25728 12. natoli s, oliveira v, calabresi p, maia lf, pisani a. does sars-cov-2 invade the brain? translational lessons from animal models. european journal of neurology. 2020;10.1111/ene.14277. doi:10.1111/ene.14277 13. paniz-mondolfi a, bryce c, grimes z, gordon re, reidy j, lednicky j, sordillo em, fowkes m. central nervous system involvement by severe acute respiratory syndrome coronavirus -2 (sars-cov-2). journal of medical virology. 2020;10.1002/jmv.25915. doi:10.1002/jmv.25915 14. varga z, flammer aj, steiger p, haberecker m, andermatt r, zinkernagel as, mehra mr, schuepbach ra, ruschitzka f, moch h. endothelial cell infection and endotheliitis in covid-19. lancet. 2020;395:1417-1418 15. bostanciklioglu m. sars-cov-2 entry and spread in the lymphatic drainage system of the brain. brain, behavior, and immunity. 2020; doi: 10.1016/j.bbi.2020.04.080 16. leon fodoulian jt, daniel rossier, basile n. landis, alan carleton, ivan rodriguez. sars-cov-2 receptor and entry genes are expressed by sustentacular cells in the human olfactory neuroepithelium.biorxiv2020.03.31.013268;doi: https://doi.org/10.1101/2020.03.31.013268 17. desforges m, le coupanec a, dubeau p, bourgouin a, lajoie l, dube m, talbot pj. human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? viruses. 2019;12 18. matsuda k, shibata t, sakoda y, kida h, kimura t, ochiai k, umemura t. in vitro demonstration of neural transmission of avian influenza a virus. the journal of general virology. 2005;86:1131-1139 19. csonka t, szepesi r, bidiga l, peter m, klekner a, hutoczky g, csiba l, mehes g, hortobagyi t. [the diagnosis of herpes encephalitis--a case-based update]. ideggyogyaszati szemle. 2013;66:337-342 20. reinert ls, lopusna k, winther h, sun c, thomsen mk, nandakumar r, mogensen th, meyer m, vaegter c, nyengaard jr, fitzgerald ka, paludan sr. sensing of hsv-1 by the cgas-sting pathway in microglia orchestrates antiviral defence in the cns. nature communications. 2016;7:13348 21. fekete r, cserep c, lenart n, toth k, orsolits b, martinecz b, mehes e, szabo b, nemeth v, gonci b, sperlagh b, boldogkoi z, kittel a, baranyi m, ferenczi s, kovacs k, szalay g, rozsa b, webb c, kovacs gg, hortobagyi t, west bl, kornyei z, denes a. microglia control the spread of neurotropic virus infection via p2y12 signalling and recruit monocytes through p2y12-independent mechanisms. acta neuropathologica. 2018;136:461-482 22. drokhlyansky e, goz ayturk d, soh tk, chrenek r, o'loughlin e, madore c, butovsky o, cepko cl. the brain parenchyma has a type i interferon response that can limit virus spread. proceedings of the national academy of sciences of the united states of america. 2017;114:e95-e104 23. wheeler dl, sariol a, meyerholz dk, perlman s. microglia are required for protection against lethal coronavirus encephalitis in mice. the journal of clinical investigation. 2018;128:931-943 24. waltl i, kaufer c, gerhauser i, chhatbar c, ghita l, kalinke u, loscher w. microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage. brain, behavior, and immunity. 2018;74:186-204 25. dubbelaar ml, kracht l, eggen bjl, boddeke e. the kaleidoscope of microglial phenotypes. frontiers in immunology. 2018;9:1753 26. moore jb, june ch. cytokine release syndrome in severe covid-19. science. 2020;368:473-474 27. lippi g, wong j, henry bm. hypertension and its severity or mortality in coronavirus disease 2019 (covid-19): a pooled analysis. polish archives of internal medicine. 2020;130:304‐309. doi:10.20452/pamw.152722020 28. terpos e, ntanasis-stathopoulos i, elalamy i, kastritis e, sergentanis tn, politou m, psaltopoulou t, gerotziafas g, dimopoulos ma. hematological findings and complications of covid-19. american journal of hematology. 2020;10.1002/ajh.25829. doi:10.1002/ajh.25829 29. miller ec, elkind ms. infection and stroke: an update on recent progress. current neurology and neuroscience reports. 2016;16:2 30. zhou f, yu t, du r, fan g, liu y, liu z, xiang j, wang y, song b, gu x, guan l, wei y, li h, wu x, xu j, tu s, zhang y, chen h, cao b. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet. 2020;395:1054-1062 31. tang n, li d, wang x, sun z. abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia. journal of thrombosis and haemostasis. 2020;18:844-847 32. klok fa, kruip m, van der meer njm, arbous ms, gommers d, kant km, kaptein fhj, van paassen j, stals mam, huisman mv, endeman h. incidence of thrombotic complications in critically ill icu patients with covid-19. thrombosis research. 2020;s0049-3848(20)30120-1. doi:10.1016/j.thromres.2020.04.013 33. denes a, pradillo jm, drake c, sharp a, warn p, murray kn, rohit b, dockrell dh, chamberlain j, casbolt h, francis s, martinecz b, nieswandt b, rothwell nj, allan sm. streptococcus pneumoniae worsens cerebral ischemia via interleukin 1 and platelet glycoprotein ib-alpha. annals of neurology. 2014;75:670-683 34. denes a, humphreys n, lane te, grencis r, rothwell n. chronic systemic infection exacerbates ischemic brain damage via a ccl5 (regulated on activation, normal t-cell expressed and secreted)-mediated proinflammatory response in mice. the journal of neuroscience. 2010;30:10086-10095 35. muhammad s, haasbach e, kotchourko m, strigli a, krenz a, ridder da, vogel ab, marti hh, al-abed y, planz o, schwaninger m. influenza virus infection aggravates stroke outcome. stroke. 2011;42:783-791 36. mccann sk, cramond f, macleod mr, sena es. systematic review and meta-analysis of the efficacy of interleukin-1 receptor antagonist in animal models of stroke: an update. translational stroke research. 2016;7:395-406 37. bonetti nr, diaz-canestro c, liberale l, crucet m, akhmedov a, merlini m, reiner mf, gobbato s, stivala s, kollias g, ruschitzka f, luscher tf, beer jh, camici gg. tumour necrosis factor-alpha inhibition improves stroke outcome in a mouse model of rheumatoid arthritis. scientific reports. 2019;9:2173 38. lambertsen kl, finsen b, clausen bh. post-stroke inflammation-target or tool for therapy? acta neuropathologica. 2019;137:693-714 39. zhang c, wu z, li jw, zhao h, wang gq. the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality. international journal of antimicrobial agents. 2020:105954 40. zhang y, xiao m, zhang s, xia p, cao w, jiang w, chen h, ding x, zhao h, zhang h, wang c, zhao j, sun x, tian r, wu w, wu d, ma j, chen y, zhang d, xie j, yan x, zhou x, liu z, wang j, du b, qin y, gao p, qin x, xu y, zhang w, li t, zhang f, zhao y, li y. coagulopathy and antiphospholipid antibodies in patients with covid-19. the new england journal of medicine. 2020;382:e38 41. beyrouti r, adams me, benjamin l, cohen h, farmer sf, goh yy, humphries f, jager hr, losseff na, perry rj, shah s, simister rj, turner d, chandratheva a, werring dj. characteristics of ischaemic stroke associated with covid-19. journal of neurology, neurosurgery, and psychiatry. 2020;jnnp-2020-323586. doi:10.1136/jnnp-2020-323586 42. kandemirli sg, dogan l, sarikaya zt, kara s, akinci c, kaya d, kaya y, yildirim d, tuzuner f, yildirim ms, ozluk e, gucyetmez b, karaarslan e, koyluoglu i, demirel kaya hs, mammadov o, kisa ozdemir i, afsar n, citci yalcinkaya b, rasimoglu s, guduk de, kedir jima a, ilksoz a, ersoz v, yonca eren m, celtik n, arslan s, korkmazer b, dincer ss, gulek e, dikmen i, yazici m, unsal s, ljama t, demirel i, ayyildiz a, kesimci i, bolsoy deveci s, tutuncu m, kizilkilic o, telci l, zengin r, dincer a, akinci io, kocer n. brain mri findings in patients in the intensive care unit with covid-19 infection. radiology. 2020:201697 43. bulfamante g, chiumello d, canevini mp, priori a, mazzanti m, centanni s, felisati g. first ultrastructural autoptic findings of sars-cov-2 in olfactory pathways and brainstem. minerva anestesiologica. 2020;10.23736/s0375-9393.20.14772-2. 44. laurendon t, radulesco t, mugnier j, gerault m, chagnaud c, el ahmadi aa, varoquaux a. bilateral transient olfactory bulbs edema during covid-19-related anosmia. neurology. may 22, 2020; 10.1212/wnl.0000000000009850 45. galanopoulou as, ferastraoaru v, correa dj, cherian k, duberstein s, gursky j, hanumanthu r, hung c, molinero i, khodakivska o, legatt ad, patel p, rosengard j, rubens e, sugrue w, yozawitz e, mehler mf, ballaban‐gil k, haut sr, moshé sl, boro a. eeg findings in acutely ill patients investigated for sars-cov-2/covid-19: a small case series preliminary report. epilepsia open. 2020; https://doi.org/10.1002/epi4.12399 46. remy ke, brakenridge sc, francois b, daix t, deutschman cs, monneret g, jeannet r, laterre pf, hotchkiss rs, moldawer ll. immunotherapies for covid-19: lessons learned from sepsis. lancet respiratory medicine. 2020;s2213-2600(20)30217-4. doi:10.1016/s2213-2600(20)30217-4 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. sars-cov-2 vaccination induced cerebral venous sinus thrombosis: do megakaryocytes, platelets and lipid mediators make up the orchestra? feel free to add comments by clicking these icons on the sidebar free neuropathology 2:18 (2021) opinion piece sars-cov-2 vaccination induced cerebral venous sinus thrombosis: do megakaryocytes, platelets and lipid mediators make up the orchestra? kate chander chiang1, ravi raghavan2, ajay gupta1,3 1 kare biosciences, orange, ca 92869, usa 2 department of pathology, loma linda university medical center, loma linda, ca 92354, usa 3 department of medicine, university of california irvine (uci) school of medicine, orange, ca 92868, usa corresponding author: ajay gupta · division of nephrology, hypertension and kidney transplantation · university of california irvine (uci) school of medicine · orange, ca 92868 · usa ajayg1@hs.uci.edu submitted: 6 june 2021 accepted: 1 july 2021 copyedited by: biswarathan ramani published: 7 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3395 keywords: sars-cov-2, covid-19, vaccine, thrombosis, cerebral venous sinus thrombosis, aspirin, thromboxane, cox-2, adenoviral vector, megakaryocyte abstract the covid-19 vaccines comprised of adenoviral vectors encoding the spike (s) glycoprotein of sars-cov-2 are highly effective but associated with rare thrombotic complications. the adenovirus vector infects epithelial cells expressing the coxsackievirus and adenovirus receptor (car). the s glycoprotein expressed locally stimulates neutralizing antibody and cellular immune responses. these vaccines have been associated with thromboembolic events including cerebral venous sinus thrombosis (cvst). s glycoprotein stimulates the expression of cyclooxygenase-2 (cox-2) and leads to massive generation of thromboxane a2 in covid-19. megakaryocytes express car and we postulate that s glycoprotein stimulated generation of thromboxane a2 leads to megakaryocyte activation, biogenesis of activated platelets and thereby increased thrombogenicity. cerebral vein sinuses express podoplanin, a natural ligand for clec2 receptors on platelets. platelets traversing through the cerebral vein sinuses could be further activated by thromboxane a2-dependent podoplanin-clec2 signaling, leading to cvst. a prothrombotic hormonal milieu, and increased generation of thromboxane a2 and platelet activation in healthy females compared to males is consistent with increased risk for cvst observed in women. we propose that antiplatelet agents targeting thromboxane a2 receptor signaling such as low-dose aspirin merit consideration for chemoprophylaxis when administering the adenovirus based covid-19 vaccines to young adults at risk of thrombosis provided there are no contraindications. abbreviations txa2 thromboxane a2, dic disseminated intravascular coagulopathy, cox cyclooxygenase, ttp thrombotic thrombocytopenic purpura, cvst cerebral venous sinus thrombosis, clec c-type lectin-like receptor tlr, toll-like receptor, car coxsackievirus and adenovirus receptor, covid-19 coronavirus disease 2019, sars-cov-2 severe acute respiratory syndrome coronavirus 2, pf4 platelet factor 4 introduction covid-19 disease is caused by a novel positive-strand rna coronavirus (sars-cov-2), which belongs to the coronaviridae family, along with the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) coronaviruses.1 the genome of these viruses encodes several non-structural and structural proteins, including spike, envelope, membrane, and nucleocapsid proteins.2 the majority of the vaccines for covid-19 that employ administration of viral antigens or viral gene sequences aim to induce neutralizing antibodies against the viral spike protein (s), preventing uptake through the ace2 receptor, and thereby blocking infection.3 the janssen covid-19 vaccine (johnson & johnson) is comprised of a recombinant, replicationincompetent ad26 vector, encoding a stabilized variant of the sars-cov-2 spike protein. the chadox1 ncov-19 vaccine (azd1222, vaxzevria®) was developed at oxford university and consists of a replication-deficient chimpanzee adenoviral vector chadox1, encoding the s protein.4 in us phase iii trials, vaxzevria has been demonstrated to have 79% efficacy at preventing symptomatic covid-19, and 100% efficacy against severe or critical disease and hospitalization, with comparable efficacy across ethnicity, gender and age.5 however, vaxzevria has been associated with thrombotic and embolic events including disseminated intravascular coagulation (dic) and cerebral venous sinus thrombosis (cvst), occurring within 14 days after vaccination, mostly in people under 55 years of age, the majority of whom have been women.6 data from europe suggests that the event rate for thromboembolic events may be about 10 per million vaccinated. antibodies to platelet factor 4 (pf4)/heparin complexes have been recently reported in a few patients.7 however, the significance of this finding remains to be established. as of april 12, 2021, about 6.8 million doses of the janssen vaccine have been administered in the u.s.8 cdc and fda are reviewing data involving six reported u.s. cases of cvst in combination with thrombocytopenia.8 all six cases occurred among women between the ages of 18 and 48, and symptoms occurred 6 to 13 days after vaccination.8 sars-cov-2 is known to cause thromboinflammation leading to thrombotic microangiopathy, pulmonary thrombosis, pedal acro-ischemia (“covid-toes”), arterial clots, strokes, cardiomyopathy, coronary and systemic vasculitis, deep venous thrombosis, pulmonary embolism, and microvascular thrombosis in renal, cardiac and brain vascularture.9-14 cerebral venous sinus thrombosis (cvst) has also been reported in covid-19 patients.15 amongst 34,331 hospitalized covid-19 patients, cvst was diagnosed in 28.16 in a multicenter, multinational, cross sectional, retrospective study of 8 patients diagnosed with cvst and covid-19, seven were women with an average age of 63 years.17 in a combined series of 41 patients with covid-19 and cvst, the average age was about 50 years (sd, 16.5 years).17 the pathobiology of thrombotic events associated with the astrazeneca vaccine should be viewed in the context of mechanisms underlying thromboinflammation that complicates sars-cov-2 infection and covid-19 disease. a. role of cox-2 and thromboxane a2 in thromboinflammation complicating adenovirus-based covid-19 vaccine encoding the spike protein of sars-cov-2 thromboinflammation in covid-19 seems to be primarily caused by endothelial, platelet and neutrophil activation, platelet-neutrophil aggregates and release of neutrophil extracellular traps (nets).13,18 platelet activation in covid-19 is fueled by a lipid storm characterized by massive increases in thromboxane a2 (txa2) levels in the blood and bronchoalveolar lavage fluid.19,20 cyclooxygenase (cox) enzymes catalyze the first step in the biosynthesis of thromboxane a2 from arachidonic acid, and cox-2 expression is induced by the spike protein of coronaviruses.21 we postulate that an aberrant increase in thromboxane a2 generation induced by the spike protein expression from adenovirus vaccines leads to thromboinflammation and cvst. the support for the above proposed mechanism comes from the following observations. first, when mice of different age groups were infected with sars-cov virus, the generation of txa2 was markedly increased in younger mice compared to middle aged mice.22 furthermore, in children with asymptomatic or mildly symptomatic sars-cov-2 infection, microvascular thrombosis and thrombotic microangiopathy occur early in infection.19 these observations are consistent with the higher risk for thrombosis in adults under 60 years of age, compared with the older age group.6,7 second, platelets from female mice are much more reactive than from male mice.23 furthermore, txa2 generation, txa2-platelet interaction and platelet activation is increased in women compared to men.24,25 these observations are consistent with disproportionately increased risk of thrombosis in women following astrazeneca and janssen covid-19 vaccines. the adenoviral vector chadox1, containing the ncov-19 spike protein gene, infects host cells through the coxsackievirus and adenovirus receptor (car).26 car-dependent cell entry of the viral vector allows insertion of the sars-cov-2 spike protein gene and expression of spike protein by host cells (figure 1). car is primarily expressed on epithelial tight junctions including on enterocytes.27,28 car expression has also been reported in platelets,29 and, since platelets are anucleate cells, car expression by megakaryocytes can be inferred. therefore, astrazeneca and janssen vaccines would be expected to induce expression of spike protein in megakaryocytes, platelets (figure 1) and enterocytes. we postulate that the expression of car receptors by basolateral membrane on enterocytes allows uptake of adenovirus vector,28 leading to spike protein-induced cox-2 expression and generation of thromboxane a2. delivery of thromboxane a2 into the splanchnic circulation could activate platelets locally, leading to splanchnic vein thrombosis associated with adenovirus vaccines.30 figure 1. astrazeneca or janssen covid-19 vaccine-induced thromboinflammation and cerebral venous sinus thrombosis (cvst) proposed mechanisms: adenovirus carrier delivers sars-cov-2 dna encoding the spike (s) protein to the lung megakaryocytes via the coxsackie-adenovirus receptor (car). spike protein induces cox-2 expression in megakaryocytes leading to megakaryocyte activation, biogenesis of activated platelets that express cox-2 and generate thromboxane a2 (txa2). cerebral venous sinuses express podoplanin, a natural ligand for clec2 receptors on platelets. platelets traversing through the cerebral venous sinuses would be further activated by thromboxane a2-dependent podoplanin-clec2 signaling, leading to release of extracellular vesicles, thereby promoting clec5aand tlr2-mediated neutrophil activation, thromboinflammation and cvst. young age and female gender are associated with increased txa2 generation and platelet activation, respectively, and hence increased risk of thromboembolic complications following sars-cov-2 vaccination with adenovirus-based vaccines. spike protein of coronaviruses is known to induce cox-2 gene expression.21,31 cox-2 expression is induced during normal human megakaryopoiesis and characterizes newly formed platelets.32 while in healthy controls <10% of circulating platelets express cox-2, in patients with high platelet generation, up to 60% of platelets express cox-2.32 generation of txa2 by platelets is markedly suppressed by cox-2 inhibition in patients with increased megakaryopoiesis versus healthy subjects.32 therefore, we postulate that expression of spike protein induces cox-2 expression and generation of thromboxane a2 by megakaryocytes. txa2 promotes biogenesis of activated platelets expressing cox-2. platelet txa2 generation leads to platelet activation and aggregation, and thereby thromboinflammation (figure 1). extravascular spaces of the lungs comprise populations of mature and immature megakaryocytes that originate from the bone marrow, such that lungs are a major site of platelet biogenesis, accounting for approximately 50% of total platelet production or about 10 million platelets per hour.33 more than 1 million extravascular megakaryocytes have been observed in each lung of transplant mice.33 following intramuscular injection of the astrazeneca and janssen vaccines, the adenovirus vector will traverse the veins and lymphatics to be delivered to the pulmonary circulation, thereby exposing lung megakaryocytes in the first pass. interestingly, under thrombocytopenic conditions, haematopoietic progenitors migrate out of the lung to repopulate the bone marrow and completely reconstitute blood platelet count.33 since thrombocytopenia is a common feature of cvst associated with adenovirus sars-cov-2 vaccines, migration of megakaryocytes from the lungs into the systemic circulation including the cerebral circulation could further predispose to cvst. in this regard, it is relevant that megakaryocytes have been found in the cerebral circulation in patients deceased from severe covid-19.34 the prothrombotic mechanisms postulated above could be operative alone or in conjunction with other underlying factors that have been proposed including the presence of anti-platelet factor 4 (pf4) antibodies as reported by greinacher and others.7 notably, amongst 31 cases of cvst in close temporal relationship with a covid-19 vaccination, anti-pf4 antibodies were positive in 22 cases and negative in 9.35 therefore, cvst following covid-19 vaccination appears to be a multifactorial disease. further studies using cultured megakaryocytes and animal models are needed to define the underlying mechanisms. even though the risk of cvst is increased about 10-fold by the adenovirus based covid-19 vaccines,36 it remains unclear why cvst remains a rare event. to further define the importance of these causative factors, biomarkers such as plasma thromboxane b2, anti-pf4 antibodies and anti-phospholipid antibodies should be measured in the thrombosis-free vaccinated population. such studies may help define the role of anti-pf4 antibodies as causative versus an epiphenomenon. interestingly, almost all patients with thrombosis and thrombocytopenia were found to have anti-pf4 antibodies.35 on the other hand, majority of patients with thrombosis without thrombocytopenia lacked anti-pf4 antibodies.35 the postulated mechanisms may be especially relevant to thrombotic events occurring in the absence of thrombocytopenia or anti-pf4 antibodies. b. predilection of cerebral venous sinuses for thrombosis following vaccination recent studies have demonstrated that arterial, venous and sinusoidal endothelial cells in the brain uniquely express markers of the lymphatic endothelium including podoplanin.37 podoplanin (d2-40) serves as a ligand for clec2 receptors on platelets.38 thromboxane a2-dependent clec2 signaling leads to platelet activation (figure 1), while a txa2 receptor antagonist nearly abolished clec2 signaling and platelet activation.38 txa2-dependent clec2 signaling promotes release of exosomes and microvesicles from platelets, leading to activation of clec5a and tlr2 receptors respectively on neutrophils, neutrophil activation and release of neutrophil extracellular traps (nets) (figure 1).39 neutrophil activation, more than platelet activation, is associated with thrombotic complications in covid-19.13,18,40 as proposed above, the expression of podoplanin by cerebral venous sinuses may be responsible for the predilection of brain vascular bed to thromboinflammation and cvst as a complication of covid-19 vaccines. the valves at the lymphovenous junction and the podoplanin-clec2 lymphovenous hemostasis act as “fail-safe” mechanisms to prevent movement of blood into the lymphatic system, thereby preventing coagulation in the lymphatics despite expression of podoplanin in the lymphatic endothelium.41 c. chemoprophylaxis with antiplatelet agents in animal models of endotoxin-mediated endothelial injury and thromboinflammation, antagonism of thromboxane a2 signaling prevents ards, reduces myocardial damage and increases survival.42-44 considering the key role played by platelets in thromboinflammation, we propose consideration of anti-platelet agents, either aspirin or txa2 receptor antagonists, as chemoprophylactic agents when the astrazeneca vaccine is administered to adults between 18 and 60 years of age.45 high bleeding risk because of another medical condition or medication would be contraindications to use of antiplatelet agents.45 medical conditions that increase bleeding risk include previous gastrointestinal bleeding, peptic ulcer disease, blood clotting problems, and kidney disease.45 medications that increase bleeding risk include nonsteroidal anti-inflammatory drugs, steroids, and other anticoagulants or anti-platelet agents.45 aspirin appears to be safe in covid-19. in a retrospective observational study in hospitalized patients with covid-19, low-dose aspirin was found to be effective in reducing morbidity and mortality and was not associated with any safety issues including major bleeding.46 therefore, aspirin is likely to be safe as an adjunct to covid-19 vaccines even in the event of a subsequent infection with sars-cov-2 virus. can aspirin influence the host immune response to the covid-19 vaccines? this issue merits further investigation. when healthy adults > 65 years of age were given influenza vaccine and randomized to receive 300 mg aspirin or placebo on days 1, 2, 3, 5 and 7, the aspirin group showed 4-fold or greater rise in influenza specific antibodies.47 the risk-benefit analysis, based on above information, suggests that a one to three week course of low-dose aspirin merits consideration in order to prevent the thromboembolic events associated with the astrazeneca vaccine. summary thromboembolic disease, cvst and thrombocytopenia have been reported in association with astrazeneca and janssen covid-19 vaccines, especially in younger women. many countries have halted use of these vaccines for adults under 30 to 60 years of age. european and north american countries generally have access to mrna vaccines. however, in many asian and african countries the choices are limited to adenovirus-based covid-19 vaccines. the governments in such countries are forging ahead with vaccinating all adults, including those under 60 years of age, with vaxzevria, covishield (the version of vaxzevria manufactured by the serum institute of india) or the janssen vaccines. this has led to grave concern and anxiety amongst the citizens and medical professionals. considering the profound global public health implications of limiting the use of these vaccines, it is critical to understand the pathobiology of vaccination induced thrombotic events in order to guide strategies aimed at prevention. in this regard, studies are urgently needed to examine lipid mediators and the thromboxane a2 platelet axis following vaccination with these vaccines compared with mrna vaccines. the risk-benefit analysis based on information presented here suggests that chemoprophylaxis using a short course of low-dose aspirin in adults under 60 years of age and lacking any contraindications to aspirin may be justified in conjunction with adenovirus-based covid-19 vaccines in order to prevent thromboembolic events and enhance safety. acknowledgment we thank prof. andrzej breborowicz, md, phd, department of pathophysiology, poznan university of medical sciences, poland for a careful review and critique of this work. funding no funding was required. conflict of interest ag and kcc have filed a patent for use of ramatroban as an anti-thrombotic and immune modulator in sars-cov-2 infection. the patents have been licensed to kare biosciences. kcc is an employee of kare biosciences. author contributions ag and kcc conceptualized, created the framework and drafted the original version. all authors reviewed and edited the final version. references 1. ortiz-prado e, simbaña-rivera k, gómez-barreno l, et al. clinical, molecular, and epidemiological characterization of the sars-cov-2 virus and the coronavirus disease 2019 (covid-19), a comprehensive literature review. diagnostic microbiology and infectious disease. 2020;98(1):115094. 2. du l, he y, zhou y, liu s, zheng b-j, jiang s. the spike protein of sars-cov — a target for vaccine and therapeutic development. nature reviews microbiology. 2009;7(3):226-236. 3. kyriakidis nc, lópez-cortés a, gonzález ev, grimaldos ab, prado eo. sars-cov-2 vaccines strategies: a comprehensive review of phase 3 candidates. npj vaccines. 2021;6(1). 4. voysey m, clemens sac, madhi sa, et al. safety and efficacy of the chadox1 ncov-19 vaccine (azd1222) against sars-cov-2: an interim analysis of four randomised controlled trials in brazil, south africa, and the uk. the lancet. 2021;397(10269):99-111. 5. astrazeneca. azd1222 us phase iii trial met primary efficacy endpoint in preventing covid-19 at interim analysis. https://www.astrazeneca.com/media-centre/press-releases/2021/astrazeneca-us-vaccine-trial-met-primary-endpoint.html. published 2021. accessed april 5, 2021. 6. european medicines agency. covid-19 vaccine safety update vaxzevria. https://www.ema.europa.eu/en/documents/covid-19-vaccine-safety-update/covid-19-vaccine-safety-update-vaxzevria-previously-covid-19-vaccine-astrazeneca-29-march-2021_en.pdf. published 2021. accessed april 4, 2021. 7. greinacher a, thiele t, warkentin te, weisser k, kyrle pa, eichinger s. thrombotic thrombocytopenia after chadox1 ncov-19 vaccination. new england journal of medicine. 2021;384(22):2092-2101. 8. cdc. joint cdc and fda statement on johnson & johnson covid-19 vaccine. https://www.cdc.gov/media/releases/2021/s0413-jj-vaccine.html. published 2021. accessed april 13, 2021. 9. ackermann m, verleden se, kuehnel m, et al. pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19. new england journal of medicine. 2020;383(2):120-128. 10. goyal p, choi jj, pinheiro lc, et al. clinical characteristics of covid-19 in new york city. new england journal of medicine. 2020;382(24):2372-2374. 11. guan w-j, ni z-y, hu y, et al. clinical characteristics of coronavirus disease 2019 in china. new england journal of medicine. 2020;382(18):1708-1720. 12. hottz ed, azevedo-quintanilha ig, palhinha l, et al. platelet activation and platelet-monocyte aggregates formation trigger tissue factor expression in severe covid-19 patients. blood. 2020;136(11):1330-1341. 13. nicolai l, leunig a, brambs s, et al. immunothrombotic dysregulation in covid-19 pneumonia is associated with respiratory failure and coagulopathy. circulation. 2020;142(12):1176-1189. 14. song w-c, fitzgerald ga. covid-19, microangiopathy, hemostatic activation, and complement. journal of clinical investigation. 2020;130(8):3950-3953. 15. mowla a, shakibajahromi b, shahjouei s, et al. cerebral venous sinus thrombosis associated with sars-cov-2; a multinational case series. journal of neurological sciences. 2020;419:117183. 16. baldini t, asioli gm, romoli m, et al. cerebral venous thrombosis and severe acute respiratory syndrome coronavirus-2 infection: a systematic review and meta-analysis. european journal of neurology. 2021;10.1111/ene.14727. 17. abdalkader m, shaikh sp, siegler je, et al. cerebral venous sinus thrombosis in covid-19 patients: a multicenter study and review of literature. journal of stroke and cerebrovascular diseases. 2021;30(6):105733. 18. petito e, falcinelli e, paliani u, et al. association of neutrophil activation, more than platelet activation, with thrombotic complications in coronavirus disease 2019. the journal of infectious diseases. 2021;223(6):933-944. 19. diorio c, mcnerney ko, lambert m, et al. evidence of thrombotic microangiopathy in children with sars-cov-2 across the spectrum of clinical presentations. blood advances. 2020;4(23):6051-6063. 20. archambault as, zaid y, rakotoarivelo v, et al. high levels of eicosanoids and docosanoids in the lungs of intubated covid‐19 patients. the faseb journal. 2021;35(6). 21. liu m, gu c, wu j, zhu y. amino acids 1 to 422 of the spike protein of sars associated coronavirus are required for induction of cyclooxygenase-2. virus genes. 2006;33(3):309-317. 22. vijay r, hua x, meyerholz dk, et al. critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection. journal of experimental medicine. 2015;212(11):1851-1868. 23. leng x-h, hong sy, larrucea s, et al. platelets of female mice are intrinsically more sensitive to agonists than are platelets of males. arteriosclerosis, thrombosis, and vascular biology. 2004;24(2):376-381. 24. kim bs, auerbach da, sadhra h, et al. a sex-specific switch in platelet receptor signaling following myocardial infarction. in: cold spring harbor laboratory; 2019. 25. eikelboom jw, hirsh j, weitz ji, johnston m, yi q, yusuf s. aspirin-resistant thromboxane biosynthesis and the risk of myocardial infarction, stroke, or cardiovascular death in patients at high risk for cardiovascular events. circulation. 2002;105(14):1650-1655. 26. cohen cj, xiang zq, gao g-p, ertl hcj, wilson jm, bergelson jm. chimpanzee adenovirus cv-68 adapted as a gene delivery vector interacts with the coxsackievirus and adenovirus receptor. journal of general virology. 2002;83(1):151-155. 27. cohen cj, shieh jt, pickles rj, okegawa t, hsieh jt, bergelson jm. the coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction. proceedings of the national academy of sciences. 2001;98(26):15191-15196. 28. kesisoglou f, schmiedlin-ren p, fleisher d, zimmermann em. adenoviral transduction of enterocytes and m-cells using in vitro models based on caco-2 cells: the coxsackievirus and adenovirus receptor (car) mediates both apical and basolateral transduction. molecular pharmaceutics. 2010;7(3):619-629. 29. assinger a. platelets and infection an emerging role of platelets in viral infection. frontiers in immunology. 2014;5:649. 30. porres-aguilar m, lazo-langner a, panduro a, uribe m. covid-19 vaccine-induced immune thrombotic thrombocytopenia: an emerging cause of splanchnic vein thrombosis. annals of hepatology. 2021;23:100356. 31. yan x, hao q, mu y, et al. nucleocapsid protein of sars-cov activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein. the international journal of biochemistry & cell biology. 2006;38(8):1417-1428. 32. rocca b, secchiero p, ciabattoni g, et al. cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets. proceedings of the national academy of sciences. 2002;99(11):7634-7639. 33. lefrançais e, ortiz-muñoz g, caudrillier a, et al. the lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. nature. 2017;544(7648):105-109. 34. nauen dw, hooper je, stewart cm, solomon ih. assessing brain capillaries in coronavirus disease 2019. jama neurology. 2021;78(6):760-762 35. schultz nh, sørvoll ih, michelsen ae, et al. thrombosis and thrombocytopenia after chadox1 ncov-19 vaccination. new england journal of medicine. 2021;384(22):2124-2130. 36. torjesen i. covid-19: risk of cerebral blood clots from disease is 10 times that from vaccination, study finds. bmj. 2021;373:n1005. 37. mezey é, szalayova i, hogden ct, et al. an immunohistochemical study of lymphatic elements in the human brain. proceedings of the national academy of sciences. 2021;118(3):e2002574118. 38. badolia r, inamdar v, manne bk, dangelmaier c, eble ja, kunapuli sp. g(q) pathway regulates proximal c-type lectin-like receptor-2 (clec-2) signaling in platelets. journal of biological chemistry. 2017;292(35):14516-14531. 39. sung p-s, huang t-f, hsieh s-l. extracellular vesicles from clec2-activated platelets enhance dengue virus-induced lethality via clec5a/tlr2. nature communications. 2019;10(1). 40. ng h, havervall s, rosell a, et al. circulating markers of neutrophil extracellular traps are of prognostic value in patients with covid-19. arteriosclerosis, thrombosis, and vascular biology. 2021;41(2):988-994. 41. welsh jd, kahn ml, sweet dt. lymphovenous hemostasis and the role of platelets in regulating lymphatic flow and lymphatic vessel maturation. blood. 2016;128(9):1169-1173. 42. carey ma, bradbury ja, seubert jm, langenbach r, zeldin dc, germolec dr. contrasting effects of cyclooxygenase-1 (cox-1) and cox-2 deficiency on the host response to influenza a viral infection. the journal of immunology. 2005;175(10):6878-6884. 43. kuhl pg, bolds jm, loyd je, snapper jr, fitzgerald ga. thromboxane receptor-mediated bronchial and hemodynamic responses in ovine endotoxemia. american journal of physiology. 1988;254(2 pt 2):r310-319. 44. altavilla d, canale p, squadrito f, et al. protective effects of bay u 3405, a thromboxane a2 receptor antagonist, in endotoxin shock. pharmacological research. 1994;30(2):137-151. 45. peters at, mutharasan rk. aspirin for prevention of cardiovascular disease. jama. 2020;323(7):676. 46. chow jh, khanna ak, kethireddy s, et al. aspirin use is associated with decreased mechanical ventilation, intensive care unit admission, and in-hospital mortality in hospitalized patients with coronavirus disease 2019. anesthesia & analgesia. 2021;132(4). 47. saleh e, moody ma, walter eb. effect of antipyretic analgesics on immune responses to vaccination. human vaccines & immunotherapeutics. 2016;12(9):2391-2402. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neuroinflammation feel free to add comments by clicking these icons on the sidebar free neuropathology 1:3 (2020) review top ten discoveries of the year: neuroinflammation hans lassmann center for brain research, medical university of vienna, vienna, austria corresponding author: hans lassmann · center for brain research · medical university of vienna · spitalgasse 4 · a-1090 wien · austria hans.lassmann@meduniwien.ac.at submitted: 25 december 2019 accepted: 08 january 2020 published: 11 january 2020 https://doi.org/10.17879/fnp-2020-2612 keywords: neuroimmunology, brain inflammation, lymphocytes, microglia, autoantibodies, multiple sclerosis abstract ten neuropathological studies, published in 2019, are discussed, which address important aspects of neuroimmunology and inflammatory brain disease. they include topics related to new mechanisms of inflammation and immune mediated neurodegeneration, which are relevant for multiple sclerosis (publications 1 to 4) and discuss the role of specific autoantibodies against myelin oligodendrocyte glycoprotein or aquaporin 4 in neuromyelitis optica spectrum disorders (publications 5 and 6). other studies highlight the discovery of new virus induced diseases of the nervous system and their relevance for clinical neurology and diagnostic neuropathology (publications 7 and 8). finally, very interesting studies are discussed dealing with microglia and immune mechanisms in neurodegeneration (publication 9) and the neuropathological long-term outcome of aß vaccination in alzheimer’s disease (publication 10). all these studies highlight the central role of neuropathology in neurological disease research. introduction there is a common belief that it is modern technology in molecular neurobiology, immunology and genetics, which drives progress in the understanding, diagnosis and therapy of human diseases of the nervous system, while neuropathology is sometimes considered an old fashioned and descriptive discipline which has little to add. however, very important new insights for human disease, achieved during the last years, came from neuropathological studies. in the field of neuroim-munology this included the discovery of new disease entities, new insights into the mechanisms of immune surveillance of the brain, of brain inflammation and of inflammation induced tissue damage in the nervous system. although all these discoveries were based on a combined multidisciplinary approach, the interpretation of the structural changes in the brain tissue in human disease and experimental models, based on neuropathological competence and experience, was essential to draw the correct conclusions. here i will discuss some recent work published during the last months, which contributed to a shift in the understanding of disease mechanisms. 1. neuronal vulnerability and multilineage diversity in multiple sclerosis (schirmer et al 2019) this study is based on a potentially revolutionary technical development. up to now gene expression was performed either on homogenized tissue, which did not discriminate spatial, temporal or cellular changes during lesion development, or by in situ hybridization, which generally only provided information on the expression of single genes. recently new technologies of single nucleus rna sequencing together with multi-color or multi-channel-based methods of in situ hybridization and immunohistochemistry have been developed, which promise to overcome some of the above listed limitations. the study by schirmer et al (2019) is one of the first to apply such technology for the analysis of multiple sclerosis (ms) brain tissue and combined this investigation with detailed confirmation by in situ hybridization and immunohistochemistry. the study supports the importance of meningeal inflammation with high b-lymphocyte content for cortical lesions, the heterogeneous astrocyte and microglia activation in the lesions, the loss of oligodendrocytes, the phagocytosis of myelin debris in macrophages during lesion activity and the importance of oxidative injury as a mechanism of neuronal injury. the most innovative finding is that a distinct subpopulation of excitatory neurons is dominantly affected in subpial cortical lesions of ms patients. thus, this study impressively shows that this new technology can be applied on autopsy material of the human brain, and that the results reliably confirm what has been described with conventional technology before (mahad et al 2015, reich et al 2018). even the main finding of a selective loss of a subset of excitatory neurons in the cortex is not surprising, since these cells are dominantly located in the outer cortical layers, which have been described before to show the most severe neuronal loss in subpial lesions (magliozzi et al 2010). a similar approach has been used recently to determine oligodendrocyte heterogeneity in normal human controls and multiple sclerosis patients (jäkel et al 2019). this study, too, showed that this new technology is suitable for the analysis of post mortem tissue of human inflammatory brain diseases and it reveals a new dimension regarding the complexity of cell phenotypes already in the normal tissue, being even more complex in multiple sclerosis lesions. however, to draw firm conclusions related to disease pathogenesis such studies have to be performed on much larger samples of patients and lesions and on the basis of much more elaborate selection and characterization of different lesion types. thus, in future studies such technologies have to be applied on cases and lesions, which have been very carefully selected according to the specific research question. unfortunately, availability of such material is very limited when the technology requires the use of fresh frozen tissue. thus, major efforts are necessary to adapt these new technologies to formaldehyde fixed and paraffin embedded archival material. 2. epstein-barr virus specific cd8 t cells selectively infiltrate the brain in multiple sclerosis and interact locally with virus-infected cells: clue for a virus driven immunopathological mechanism (serafini et al 2019) even in the absence of an overt inflammatory disease of the central nervous system there is a small to moderate number of t-lymphocytes present in the brain and spinal cord, while infiltration of the tissue by t-cells and b-cells is massively increased in inflammatory brain diseases. from experimental data in normal mice and mouse models of neurodegenerative diseases or autoimmune encephalomyelitis it was suggested that these cells are mhc class ii restricted cd4+ t-cells, which enter the brain in the course of immune surveillance and, when directed against autoantigens of the cns, may exert both, disease-promoting as well as regulatory or protective functions. these concepts however, were difficult to harmonize with previous observations that the majority of t-cells in the normal human brain and in the cns of patients with neurodegenerative disease are cd8+ mhc class i restricted t-cells, displaying the phenotype of tissue resident memory cells (smolders et al 2018). similarly, in the ms brain cd8+ t-cells with a phenotype of tissue resident memory cells prevail, where they show focally restricted activation in particular in active lesion (van nierop et al 2017, machado santos et al 2018; figure 1c). so far, such tissue resident memory cells have been found, defined and characterized in solid tissues, including the brain, and in models of virus infection, where they form a very effective barrier against re-infection by the same agent (steinbach et al 2018). thus, a key question is, what target antigen is recognized by the cd8+ tissue resident t-cells in the ms brain. multiple sclerosis is regarded by immunologists as an autoimmune disease, driven by t-lymphocytes directed against myelin anti-gen(s). however, attempts to identify an ms-specific autoimmune response have so far failed (hohlfeld et al 2016). even when the investigation specifically focused on activated cd8+ t-cells in the ms lesions, no reactivity was found against the classical candidates of myelin directed autoimmunity, but a substantial number of isolated t-cells recognized epitopes from epstein barr virus (ebv; van nierop et al 2017, serafini et al 2019). furthermore, serafini et al (2019) showed that these ebv reactive t-cells form close contacts with b-lymphocytes expressing epitopes of ebv related proteins, and these t-cells display cd 107 on their surface, suggesting the release of the content of their cytotoxic granules. overall these data suggest that the chronic inflammatory response in the ms brains may in part be driven by a t-cell response against ebv (serafini et al 2019). however, the question whether there is productive ebv expression in the ms brain is still controversial. it is described by some groups, while other groups using similar techniques in material from comparable disease phenotypes and stages failed to confirm the data (van nierop et al 2017). the reason for these discrepancies is still unresolved (lassmann et al 2011). another recent study (konjevic sabolek et al 2019) describes the interaction of activated cd8+ cytotoxic cells with local macrophages, but not with b-cells, which are the potential target for ebv infection. thus, the specific antigenic target of t-cells in ms lesions is still unresolved, but recent data indicate that the concept of autoimmun-ity against myelin or other cns antigens may be wrong. figure 1: one well established and several new mechanisms triggering or propagating inflammation in the central nervous system (cns) a: the basic well-established mechanism of immune surveillance and inflammation of the central nervous system is that t-cells, activat-ed in the peripheral immune system, are able to pass the blood brain barrier and enter the cns in the process of immune surveillance. when they encounter their specific antigen, presented in the meninges, the perivascular space or the cns parenchyma they get re-activated, produce proinflammatory cytokines and attract additional immune cells into the lesion. red cells: activated t-cells, red ring cells: resting t-cells; star like cells: macrophages and microglia involved in antigen presentation; the blue circles indicate the endotheli-um of the blood brain barrier and the glia limitans. b: an alternative way to start inflammation in the cns (as described by publication 3) is the local presence of damaged tissue or foreign antigens from infectious agents (red dots); they interact with microglia and induce inflammasome activation and the production of pro-inflammatory cytokines (green triangles). these cytokines activate endothelial cells at the blood brain barrier and allow the recruitment of t-cells into the focal site of tissue injury. in case these t-cells (red circles) find their cognate antigen within the lesion, they become activated (red cells) and trigger the full-blown inflammatory lesion. c: in conditions of chronic brain inflammation, such as for instance in multiple sclerosis (as discussed in context with publication 2), high numbers of tissue resident memory t-cells (green circles) are present within the brain and lesions and they are mainly in an inactivated stage. when there is re-appearance of their cognate antigen (for instance a foreign antigen or a modified self-antigen) presented by perivascular b-cells (blue circles) or by macrophages / microglia (blue stars), they get re-activated and propagate the chronic inflammatory process. d: a new mechanism of brain inflammation is outlined in publication 6. when high titers of antibodies against aquaporin 4 are present in the circulation, the very low amount of antibodies which can pass the normal blood-brain barrier, is sufficient to induce astrocyte dysfunction and their pro-inflammatory activation (green cells). this leads to the production of pro-inflammatory cytokines (green triangles), which activate the endothelium and massively disturb the blood brain barrier. this allows more antibodies together with leukocytes (red circles) and complement to enter the perivascular space, to destroy the astrocytes at the glia limitans and to promote inflammation. 3. microglia nodules provide the environment for pathogenic t cells in human encephalitis (tröscher et al 2019) a key question in the pathogenesis of inflammatory brain diseases is, why and how t-cells enter the brain and initiate the lesions. one mechanism is that they are activated in the peripheral immune system and get access to the brain in the course of immune surveillance (wekerle et al 1986; figure 1a). the other possibility, which may be particularly important in conditions of virus infection or neurodegeneration, is that local cells in the brain, such as for instance microglia, sense focal tissue injury and provide signals which drives the initial recruitment of cd8+ t-cells into the cns (figure 1b). rasmussen’s encephalitis is a rare inflammatory seizure disorder mediated by cytotoxic t-cells which attack neurons and astrocytes (bauer et al 2012). in the study by tröscher et al (2019) it is shown that the initial stage of lesion formation is characterized by microglia activation and the formation of microglia nodules, which occurs before the first cd8+ t-cells have entered the brain. the activated microglia initially show inflammasome activation and up-regulation of endosomal toll-like receptors, which is followed by the production of chemokines, the expression of major histocompatibility complex class i antigen, the activation of interferon signaling pathways and the recruitment of cd8+ t-lymphocytes. these data suggest that the initial trigger of inflammatory lesions in rasmussen’s encephalitis is not the passage of activated t-cells through the blood brain barrier in the course of immune surveillance, but a local cell infection or tissue damage, which creates a focal pro-inflammatory environment through inflammasome activation. subsequently, a subset of these recruited t-cells has to recognize their cognate antigen within the cns to propagate the inflammatory reaction. such a scenario may play a major role in the induction of inflammation in viral diseases of the nervous system, but similar microglia activation and the formation of microglia nodules is also seen around active lesions in the ms brain (burm et al 2016). 4. post mortem multiple sclerosis lesion pathology is influenced by single nu-cleotide polymorphism (fransen et al 2020) irrespective of the specific antigen, recognized by the inflammatory cells in ms lesions, the incidence and severity of brain inflammation appears to be influenced by the genetic background of the patients. this is now well established from genome wide association studies (gwas), which define ms as a disease with a highly polygenic background (international multiple sclerosis genetics consortium 2019). more than 200 gene loci have been found associated with disease incidence, each of them with only very little individual impact. the global interpretation of these findings is that ms, as described before by pathology, is a chronic inflammatory disease of the nervous system, and its likelihood to affect a patient is influenced by multiple genes which in their interaction may enhance pro-inflammatory immune mechanisms. it is likely that such genetic polymorphisms also modify the pathological evolution of ms lesions, but considering their low impact on the disease in the general ms population, it was unexpected that such an effect may become apparent in autopsy cohorts. fransen et al (2020) addressed this question on a material of 179 ms brains, containing a very high number of very well staged and characterized individual ms lesions. on this basis they were able to show that certain variants of immune related genes, which were before shown to be associated with disease severity, were also associated with the proportion of active lesions. the respective genes were in part related to apoptosis (fas), t-cell activation (ctla4) or are playing a role both in inflammation and neurodegeneration (clec16a). this study provides a first hint on how the genetic background of ms patients may affect pathological outcome of the lesions. however, there are still controversial issues regarding the statistical design for such investigations. performing genotype/phenotype correlations with multiple potential risk associ-ated gene polymorphisms and multiple different pathological outcomes requires enormous stringency related to multiple testing, and there is some doubt that this can ever be reached with the limited autopsy material available. 5. characterization of human myelin oligodendrocyte glycoprotein antibody response in demyelination (tea et al 2019) pathological studies performed during the last decade suggest that immunological mechanisms responsible for the induc-tion of ms-like inflammatory demyelinating lesions are heterogeneous between patients, in particular at the early stages of the disease and in patients with aggressive disease course (lucchinetti et al 2000). one subtype of lesions was defined by immunoglobulin and complement deposition in active lesions with initial stages of demyelination. this was interpreted as evidence for the involvement of pathogenic autoantibodies, as it is reflected in models of experimental autoimmune encephalomyelitis, in which demyelination is triggered by antibodies against myelin oligodendrocyte glycoprotein (mog) on the background of a t-cell mediated inflammatory reaction in the brain and spinal cord. new technologies, using trans-fected cell lines, are now available to identify potential pathogenic autoantibodies and using these techniques it was re-cently possible to identify a subset of patients with inflammatory demyelinating disease with a systemic antibody response against mog (borisow et al 2018). identification of such demyelinating anti-mog antibodies is difficult, since they are directed against complex conformational epitopes, while antibodies directed against conventional linear mog epitopes are not pathogenic. the study by tea et al (2019) describes the results of a detailed epitope mapping, which provides new information on the relevant target epitopes, recognized by potentially pathogenic mog auto-antibodies. this is important for diagnosis of mog antibody associated inflammatory demyelinating disease and for further analysis of pathogenetic mechanisms involved in antibody mediated demyelination. the identification of pathogenic mog-antibodies is a major breakthrough in multiple sclerosis research, since current data suggest that the presence of anti-mog antibodies defines an inflammatory demyelinating disease distinct from ms. it presents with clinical features of acute or relapsing disseminated encephalomyelitis or neuromyelitis optica and shows a different response to current anti-inflammatory treatments (jarius et al 2016). these new findings further suggest that the experimental models of autoimmune encephalomyelitis more closely reflect mog antibody associated disease than multiple sclerosis itself. 6. circulating aqp4-specific auto-antibodies alone can induce neuromyelitis optica spectrum disorder in the rat (hillebrand et al 2019) another disease, which originally has been classified as a variant of multiple sclerosis, is neuromyelitis optica (nmo). however, using similar techniques of auto-antibody detection as described above, it became clear that the majority of patients with nmo have auto-antibodies against the astrocytic water channel aquaporin 4 (aqp4). further comprehensive clinical characterization of patients with aqp4 auto-antibodies enlarged the clinical phenotype, now designated as neuro-myelitis optica spectrum disorders (nmosd), which are pathologically characterized by a primary inflammatory astro-cytopathy with secondary demyelination (fujihara et al 2019). experimental studies showed that patient derived auto-antibodies induce tissue damage in vitro or after direct injection into the brain, or when systemically injected into animals with t-cell mediated autoimmune encephalomyelitis. the study by hillebrand et al (2019) shows for the first time that circulating aqp4 directed autoantibodies alone can induce nmosd like disease in rats, provided that the injection resulted in a very high titer of circulating antibodies with very high specificity and affinity. circulating antibodies reached the cns tissue not only through circumventricular organs but also by the physiological leakage through cerebral veins (figure 1d). thus, a very low degree of aqp4 antibody leakage through the intact blood brain barrier may target perivascular astrocytes at the perivascular glia limitans and may augment further blood-brain barrier damage in the course of antibody mediated damage of the astrocytic process. this property is unique for aqp4 auto-antibodies and not seen with other antibodies such as those directed against mog. the study further shows that brain damage by the leakage of the antibody in circumventricular organs is very limited and not associated with complement activation, possibly by the expression of complement inhibitory proteins. in contrast, when present in the perivenous space aqp4 antibodies induce profound blood-brain barrier injury, leakage of complement, recruitment of granulocytes and the activation of macrophages, and this gives rise to the full pathological spectrum of nmosd lesions. this study, thus, provides some explanation for the heterogeneous spectrum of pathology in different brain regions of nmosd patients. 7. the neuropathology of fatal encephalomyelitis in human borna virus infection (liesche et al 2019) accurate diagnosis of non-purulent lymphocytic encephalitis in humans is a challenge. when no positive identification of a specific virus infection is possible, a diagnosis of encephalitis of autoimmune or probable infectious cause is assigned (venkatesan and murphy 2018, seilhean 2019). the study by liesche et al (2019) defines a new entity of lymphocytic scle-rosing panencephalomyelitis, which is caused by infection with borna disease virus 1 (bodv-1). although the disease may follow generalized immunosuppression, in the majority of cases it developed spontaneously. the study provides a clear account of the pathological features of this new disease, consisting of mononuclear cell infiltrates, edema, microglia acti-vation and the presence of eosinophilic spherical inclusion bodies. virus rna was detected in neurons, ependymal cells, astrocytes and oligodendrocytes, but not in lymphocytes, macrophages or microglia. the distribution of the lesions in the brain and spinal cord was variable ranging from a dominant brain stem encephalitis, to prominent affection of the hippo-campus or a multifocal or diffuse pathology in other cases. in addition, infection may also involve the peripheral nervous system. this study adds borna virus infection to the list of potential causative agents driving an acute or subacute inflam-matory disease of the central and peripheral nervous system, which has to be included in the spectrum of clinical differen-tial diagnoses. how frequent this condition is in the global population of patients with encephalitis has to be determined in the future. 8. zika virus replicates in adult human brain tissue and impairs synapses and memory in mice (figueiredo et al 2019) another recently discovered disease of the nervous system is associated with zika virus infection. although originally be-lieved to disturb brain development in the course of intrauterine infection (counotte et al 2019), it became clear in the last years that it may also infect the adult human nervous system and give rise to inflammatory diseases of the central and peripheral nervous system (da silva et al 2017). the underlying mechanisms were up to now not defined. in this study the authors show that zika virus can infect neurons and replicate not only in the human, but also in the mouse brain tissue in vitro. this allowed to study in more detail the consequences of zika virus brain infection. in the mouse it mainly accumu-lates in memory associated brain regions, such as for instance the hippocampus, where it induces disturbances of long-term potentiation and impairs memory. these effects are associated and possibly mediated by microglia activation with the up-regulation of pro-inflammatory cytokines and the complement components c1q and c3. in line with this view it was also found that blockade of microglia activation or neutralization of tnf-α prevented synapse and memory dysfunction. these findings suggest that analysis of cognitive disturbances in zika virus infected patients should be performed in a more systematic manner. however, it has also to be noted that similar mechanisms of cytokine and microglia activation associated synapse and memory disturbance occur in many different inflammatory conditions of the central nervous system, including even experimental autoimmune encephalomyelitis (musella et al 2016). it is, thus, rather a general consequence of brain exposure to pro-inflammatory cytokines. whether the mechanisms inducing synaptic dysfunction are different and specific in zika virus infected brain has to be clarified in the future by direct comparison. figure 2: microglia activation in neurodegeneration and brain inflammation the normal brain is populated by so called resting or homeostatic microglia. when they sense neurodegeneration, they become activat-ed through the trem2/apoe pathway. in this activation state there are phagocytic cells responsible for the removal of tissue debris, which is essential to limit further injury and to allow tissue repair. when these activated microglia are further exposed to inflammatory cytokines, they differentiate into immunological effector cells, which are actively involved in tissue damage. thus, both, an impairment of the initial step of microglia activation as well as the proinflammatory activation are detrimental for the brain. 9. impact of trem2 risk variants on brain region-specific immune activation and plaque microenvironment in alzheimer’s disease patient brain samples (prokop et al 2019) for many years our views on the pathogenesis of alzheimer’s disease focused on protein misfolding and deposition of aß and tau in the extracellular space or in the neuronal cytoplasm. in recent years, however, evidence accumulated that ele-ments of the innate immune system may play a role in promoting the cognitive disturbance in patients and the neuropa-thological damage in the brain. in particular, recent genome wide association studies identified polymorphisms of genes involved in microglia activation to be associated with the disease (sims et al 2017). one of these genes is the trem2 re-ceptor (johnsson et al 2013), which when activated by neurodegeneration triggers initial microglia activation through the trem2 / apoe pathway (krasemann et al 2017). although the role of these molecules in microglia activation in general and in experimental models of neurodegeneration is well understood, it is important to determine the neuropathological effects of the respective gene variants in alzheimer’s disease patients. this question was addressed by the study of prokop et al (2019). the authors describe that carriers of the respective trem2 risk variants had lower numbers of plaque-associated microglia and this was associated with a higher degree of axonal injury and tau pathology in comparison to carefully matched alzheimer’s disease patients without these trem2 variants. in addition, microglia senescence was found to be enhanced and some global decrease in microglia activation in trem2 variant ad cases was apparent. the study clearly underlines the difficulty of such an investigation in human autopsy material, since such differences only became apparent when exactly matched lesion stages were compared in patients with similar disease severity. this is due to the fact that these gene polymorphisms do not enable or prevent ad pathology, but just modify its speed of development. overall, the data suggest that impairment of microglia activation not only enhances the progression of cognitive impairment, but also promotes axonal degeneration and tau pathology. whether this is due to a reduced neuroprotective function of microglia or a reduced clearance of misfolded proteins due to impaired phagocytosis remains to be determined in the future. 10. persistent neuropathological effects 14 years following amyloid-ß immunization in alzheimer’s disease (nicoll et al 2019) following promising results, documenting the removal of amyloid plaques in mice after active immunization with aß, a first clinical trial had been initiated almost 20 years ago, which was followed by several other trials using immunization strategies or the systemic application of specific aß-antibodies (wisniewski and goni 2014, 2015). the first trial was stopped due to the appearance of autoimmune inflammatory disease of the central nervous system as a complication of the vaccination strategy. a detailed analysis of the neuropathology of patients who died in the years after the trial suggested that this vaccination resulted in clearance of aß-deposits from the brain in association with the development of an aß antibody response, but that this had no significant effect of the progression of dementia (holmes et al 2008). the present study by nicoll et al (2019) in detail describes the neuropathology in relation to the clinical outcome seen in 22 patients of this trial, who died within 14 years after the immunization. this comprehensive analysis provides important insights related to the design and outcome of aß directed immunotherapies in alzheimer’s disease. only 77% of the patients included in this cohort had alzheimer’s disease, while dementia in the others was due to different diseases. in the ad patients with aß vaccination the vast majority provided evidence for plaque removal and the extent for amyloid deposition correlated inversely with the titer of antibodies directed against the n-terminal region of aß, documenting the sustained effect of aß vaccination reducing amyloid load. despite a significantly lower extent of neurofibrillary pathology in patients with low amyloid load, there was still profound neurofibrillary tangle pathology in all cases. in addition, severe cerebral amyloid angiopathy was noted in the majority of patients, irrespective of the effect on aß plaque removal. progression of dementia was seen in most patients, even in those with extensive plaque removal. in conclusion, this study impressively documents the value of systematic neuropathological studies to monitor clinical dementia trials. it shows that alzheimer’s disease patients can remain plaque-free even for 14 years after active immunization, but that this has little effect on tau pathology and the further development of dementia. conclusion in this review a number of recent neuropathological studies are presented, which contributed to the dynamic change in our understanding of the pathogenesis of neuroinflammatory diseases. they highlight and confirm that the field of neuro-pathology occupies a central position in human disease research. experimental models provide important insights into basic and molecular mechanisms, but their relevance for the disease in humans has to be determined by studying the disease in patients. correct interpretation of the changes occurring in damaged tissues requires profound neuropathological expertise. references bauer j, vezzani a, bien cg. epileptoic encephalitis: the role of the innate and adaptive immune system. brain pathology 2012; 22:412-21 borisow n, mori m. kuwabara s, scheel m, paul f. diagnosis and treatment of nmo spectrum disorder and mog encephalomyelitis. front neurol 2018; 9:888 burm sm, peferoen la, zuiderwijk-sick ea, et al. expression of il-1ß in rhesus eae and ms lesions is mainly induced in the cns itself. j neuroinflammation 2016; 13:138 counotte mj, meili kw, taghavi k, calvet g, low n. zika virus infection as a cause of coingenital abnormalities and guillain barre syndrome. a living systematic review. f1000res 2019; 8:1433 da silva irf, frontera ja, bispo de filippis am, nascimento ojmd. neurologic complications associated with the zika virus in brazilian adults. jama neurol 2017; 74:1190-1198 figueiredo cp, barris-aragao fgq, neris rls, frost ps. et al. zika virus replicates in adult human tissue and impairs synapses and memory in mice. nature commun 2019; 10:3890 fransen nl, crusius jba, smolders j, mizee mr et al. post-mortem multiple sclerosis lesion pathology is influenced by single nucleotide polymorphisms. brain pathol 2020; 30:106-119 fujihara k. neuromyelitis optica spectrum disorders: still evolving and broadening. curr opin neurol 2019; 32: 385-394 hillebrand s, schanda k, nigritinou m, tsymala i et al. circulating aqp4-specific auto-antibodies alone can induce neuromyelitis optica spectrum disorder in the rat. acta neuropathol 2019; 137:467-485 hohlfeld r, dornmair k, meinl e, wekerle h. the search for the target antigens in multiple sclerosis, part 2: cd8+ t-cells, b-cells and antibodies in the focus of reverse ´translational research. lancet neurol 2016; 15: 317-31 holmes c, boche d, wilkinson d, yadegarfar g, et al. long-term effects of abeta42 immunisation in alzheimer’s disease: follow-up of a randomized, placebo-controlled phase i trial. lancet 2008; 372: 216-23 international multiple sclerosis genetics consortium. a systems biology approach uncovers cell-specific gene regulatory effects of genetic associations in multiple sclerosis. nat commun 2019; 10:2236 jäkel s, agirre e, mendana falcao a, van bruggen d, et al. altered human oligodendrocyte heterogeneity in multiple sclerosis. nature 2019; 566: 543-547 jarius s, ruprecht k, kleiter i, borisow n et al. mog-igg in nmo and related disorders: a multicenter study of 50 patients. part 2: epidemiology, clinical presentation, radiological and laboratory features, treatment response and long-term outcome. j neuroinflammat 2016; 13:280 johnsson t, stefanson h, steinberg s, jonsdottir i, et al. variant of trem2 associated with risk of alzheimer’s disease. new engl j med 2013; 368: 107-116 konjevic sabolek m, held k, beltran e, et al. communication of cd8(+) t cells with mononuclear phagocytes in multiple sclerosis. ann clin transl neurol 2019; 6:1151-1164 krasemann s, madore c, cialic r, baufeld c, et al. the trem-apoe pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. immunity 2018; 47:566-581 lassmann h, niedobitek g, aloisi f, middeldorp jm; the neuropromise ebv working group. epstein-barr virus in the multiple sclerosis brain: a controversial issue--report on a focused workshop held in the centre for brain research of the medical university of vienna, austria brain 2011; 134: 2772-2786 liesche f, ruf v, zoubaa s, kaletka g et al. the neuropathology of fatal encephalomyelitis in human borna virus infection. acta neuropathol 2019; 138: 653-665 lucchinetti c, brück w, parisi j, scheithauer b, rodriguez m, lassmann h. heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination ann neurol 2000; 47: 707-717 machado-santos j, saji e, tröscher ar, et al. the compartmentalized inflammatory response in the multiple sclerosis brain is composed of tissue-resident cd8+ t lymphocytes and b cells. brain 2018; 141:2066-2082 magliozzi r, howell ow, reeves c, et al. a gradient of neuronal loss and meningeal inflammation in multiple sclerosis. ann neurol 2010; 68:477-493 mahad dh, trapp bd, lassmann h. pathological mechanisms in progressive multiple sclerosis. lancet neurol 2015; 14:183-193 musella a, mandolesi g, mori f, gentile a, centonze d. linking synaptopathy and gray matter damage in multiple sclerosis. mult scler 2016; 22: 149-149 nicoll jar, buckland gr, harrison ch, page a, et al. persistent neuropathological effects 14 years following amyloid ß-immunization in alzheimer’s disease. brain 2019; 142: 2113-2126 prokop s, miller kr, labra sr, pitkin rm, et al. impact of trem2 risk variants on brain region-specific immune activation and plaque microenvironment in alzheimer’s disease patient brain samples. acta neuropathol 2019; 138: 613-630 reich da, lucchinetti cf, calabresi ps. multiple sclerosis. new engl j med 2018; 378:169-180. schirmer l, velmeshev d, holmqvist s, et al. neuronal vulnerability and multilineage diversity in multiple sclerosis. nature 2019; 573:75-82 seilhean d. infections of the central nervous system: neuropathology. rev neurol (paris) 2019; 175:431-435 serafini b, rosicarelli b, veroni c, mazzola ga, aloisi f. epstein-barr virus-specific cd8 t cells selectively infiltrate the multiple sclerosis brain and interact locally with virus infected cells: clue for a virus-driven immunopathological mechanism. j virol 2019; 93(24). pii: e00980-19. doi: 10.1128/jvi.00980-19. sims r, van der lee sj, naj ac, bellenguez c, et al. rare coding variants in plc2, abi3 and trem2 implicate microglial-mediated innate immunity in alzheimer’s disease. nat genet 2017, 49: 1373-1384 smolders j, heutinck km, fransen nl, et al. tissue-resident memory t cells populate the human brain. nat commun 2018; 9:4593 steinbach k, vincenti i, merkler d. resident-memory t cells in tissue restricted immune responses: for better or worse. front immunol 2018; 9:2827 tea f, lopez ja, ramanathan s, merheb v. et al. characterization of human myelin oligodendrocyte glycoprotein antibody response in demyelination. acta neuropathol commun 2019; 7:145 tröscher a, wimmer i, quemada-garrido l, köck u, et al. (2019) microglial nodules provide the environment for pathogenic t cells in human encephalitis acta neuropathol 2019; 137:619-635 van nierop gp, van luijn mm, michels ss, et al. phenotypic and functional characterization of t cells in white matter lesions of multiple sclerosis patients. acta neuropathol 2017; 134:383-401 venkatesan a, murphy oc. viral encephalitis. neurol clin 2018; 36:705-724 wisniewski t, goni f. immunotherapeutic approaches for alzheimer’s disease. neuron 2015; 85: 1162-1176 wekerle h, linington c, lassmann h, meyermann r (1986) cellular immune reactivity within the cns. trends neurosci 1986; 9:271-277 wisniewski t, goni f. immunotherapy of alzheimer’s disease. biochem pharmacol 2014; 88: 499-507 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. association of acute disseminated encephalomyelitis (adem) and covid-19 in a pediatric patient feel free to add comments by clicking these icons on the sidebar free neuropathology 2:19 (2021) case report association of acute disseminated encephalomyelitis (adem) and covid-19 in a pediatric patient liam chen department of laboratory medicine and pathology, university of minnesota medical school, minneapolis, mn, usa corresponding author: liam chen · department of laboratory medicine and pathology · university of minnesota medical school · c515 mayo memorial building · 420 delaware street · minneapolis, mn 55455 · usa llchen@umn.edu submitted: 8 june 2021 accepted: 8 july 2021 copyedited by: deborah mcintyre published: 12 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3388 keywords: sars-cov-2, covid-19, acute disseminated encephalomyelitis (adem), acute hemorrhagic leukoencephalitis (ahle), acute necrotizing encephalopathy of childhood (anec), demyelinating abstract cases of acute disseminated encephalomyelitis (adem) and its hyperacute form, acute hemorrhagic leukoencephalitis (ahle), have been reported in coronavirus disease 2019 (covid-19) patients as rare, but most severe neurological complications. however, histopathologic evaluations of adem/ahle pathology in covid patients are extremely limited, so far having only been reported in a few adult autopsy cases. here we compare the findings with an adem-like pathology in a pediatric patient taken through a biopsy procedure. understanding the neuropathology may shed informative light on the autoimmune process affecting covid-19 patients and provide critical information to guide the clinical management. numerous evidence has confirmed that coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1], affects the nervous system [2]. most of the associated neurological dysfunctions are mild symptoms such as anosmia and dysgeusia [3] while severe debilitating neurological disorders such as stroke and meningoencephalitis are less frequent [4, 5]. these manifestations are likely caused by sars-cov-2 entering brain through direct infection of olfactory neuroepithelium [6], or transmitted by circulating lymphocytes and monocytes that are able to cross the blood-brain barrier [7]. within the brain and spinal cord, various cell types including endothelial cells, vascular pericytes and smooth muscle, neurons and glia all express angiotensin converting enzyme 2 (ace2) receptor [8, 9], the primary receptor for sars-cov-2 spike protein [10]. it is true that sars-cov-2 could be detected in olfactory epithelium [11], brain and cranial nerves by rt-pcr [12, 13], immunohistochemistry (ihc) [14], in situ hybridization (ish) [11] and electron microscopy [15]. in addition, neuropathological findings such as leptomeningeal and parenchymal lymphocytic inflammation, microglial nodules and neuronophagy are compatible with a diagnosis of viral meningoencephalitis [12, 16]. nevertheless, neither viral inclusions nor cytopathic changes have been observed on hematoxylin and eosin (h&e) stained slides. furthermore, in the few studies that have reported evidence of viral rna or protein in the cranial nerves or brainstem, the degree and distribution of neuropathologic changes have shown no correlation with the amount of virus in a given area of pathology, suggesting that the pathology is rather secondary to the systemic effects of viral infection. indeed, convincing evidence has elucidated that systemic hypercoagulability plays an important role in covid-19 related stroke process, as sars-cov-2 could induce cytokine storm and endotheliopathy [17, 18], which in turn lead to the histologic findings of microthrombi, hemorrhages and infarcts [19, 20]. in contrast, there is still considerable debate over whether parainfectious, autoimmune mediated process such as acute disseminated encephalomyelitis (adem), or its hyperacute form, acute hemorrhagic leukoencephalopathy (ahle), can truly be attributed to covid-19. one reason for this controversy is the lack of neuropathological descriptions of adem and ahle despite that a handful clinical and imaging case studies of covid-19 patients have suggested lesions characteristic of adem or ahle [21-31]. so far, there are only few case reports with neuropathological features of adem and ahle in adult patients who died from complications of covid-19 [32-34]. another reason for the controversy, as will be discussed further below, is whether the observed ademor ahle-like pathology is truly due to a primary demyelinating process or merely a secondary white matter injury of a comorbid vascular disease or a combination. r. ross reichard et al. reported the neuropathological findings of a 71-year-old patient who died from complications of covid-19 [32]. hemorrhagic white matter lesions were present throughout the cerebral hemispheres. luxol fast blue/ periodic acid–schiff (lfb/pas) identified loss of myelin, pas-positive macrophages, and fragmented axonal processes within these lesions. it has features of ahle, although necrotic blood vessels and perivascular inflammation were not identified within the hemorrhagic lesions. a second distinct pathology identified was characterized by small subcortical white matter pallor with a variable perivenular distribution, resembling an adem-like histological appearance. notably, prominent acute axonal injury was present in the regions of myelin loss, which is not characteristic of classic adem [32]. jamie m. walker et al. presented two cases of fatal covid-19 with severe neurologic sequalae [33]. one was a 51-year-old woman with an irregular pattern of demyelination centered primarily around veins and venules. both perivascular cd3+ t-lymphocytes as well as frequent perivascular and parenchymal cd68+ histiocytes and activated microglial cells were present, consistent with a diagnosis of adem. another autopsy examination of a 64-year-old demonstrated myelin pallor, perivascular cd3+ t-lymphocytes, axonal spheroids, as well as numerous ringand ball-hemorrhages throughout the white matter with central blood vessels showing fibrinoid necrosis, most consistent with a diagnosis of ahle. it should be noted that in all three aforementioned cases, global hypoxic-ischemic injury was present, in addition to microscopic cortical infarcts in the first case [32] and large areas of cerebral intraparenchymal hemorrhages in the third case [33]. thus, the neuropathological lesions should be interpreted with caution and the possibility of a cerebrovascular origin with secondary demyelinating pathology should be considered. adem and ahle usually affects children and young adults after an infection or vaccination [35]. however there are very few reported cases of adem in pediatric population with sars-cov-2 infection based on clinical features and mri findings [25, 36, 37]. this is consistent with the differing clinical presentations of covid-19 in children and adult patients. recently, we have examined a brain biopsy taken from an 8-year-old girl who was admitted with new-onset seizures. on hospital day 2, covid-19 spike igg antibody testing was positive. brain mri demonstrated extensive t2 hyperintensity centered at bilateral basal ganglia, extending to the frontal white matter, external and internal capsules, corpus callosum, thalami, insula, as well as the cerebellar hemispheres, brainstem and the spinal cord. interval mri imaging demonstrated persistent diffusion restriction in the affected white matter throughout much of the central white matter tracts, cerebral peduncles, corticospinal tracts and cerebellar white matter, suggesting ongoing demyelination, but no evidence to suggest hemorrhages. three weeks later, there was vacuolating necrosis in bilateral basal ganglia. ventriculoperitoneal shunt placement and stereotactic brain biopsy were performed on hospital day 48. neuropathological examination of the brain biopsy tissue revealed subcortical white matter pallor with perivascular lymphocytic infiltrates centered primarily around venules (figure 1a). immunohistochemical staining for sars-cov-2 spike protein was negative. no microglial nodules or evidence of neuronophagia was present. demyelination was confirmed by lfb stain (figure 1b) whereas axons were relatively preserved as shown by neurofilament immunostain (figure 1c). interestingly, app immunostain highlighted damaged, swollen axons in areas surrounding the vessels, a pattern seen in adult patient as well (figure 1d). the lymphocytes were composed predominantly of cd3-positive t cells (figure 1e) whereas cd68 stain highlighted numerous perivascular macrophages and the diffuse distribution of activated microglia (figure 1f). figure 1. neuropathological findings of a brain biopsy from a child post sars-cov-2 infection. a. h&e section of white matter pallor, reactive astrogliosis and perivascular lymphocytic infiltrates around small veins and venules. b. lfb stain demonstrates the perivascular myelin loss within the subcortical white matter lesion. c. neurofilament immunostain shows preservation of most axons in the region of myelin loss. d. app immunostain identifies axonal swellings adjacent to the perivascular areas. e. perivascular lymphocytes are predominantly cd3-positive t cell. f. cd68 immunostain confirms both perivascular and diffuse distributions of macrophages within the area of white matter pallor. scale bar, 100 μm in a-e. in this case, the absence of confounding concomitant cerebrovascular lesions makes the pathological interpretation relatively straightforward. these adem/ahle cases represent rare, but the most severe end of the covid-19 neuropathologic spectrum. consequently, adem/ahle should be a consideration during evaluation of patients, especially children with encephalopathy, seizures and/or focal neurologic deficits after recovering from covid-19. another intriguing development in this pediatric patient is the later mri findings of bilateral basal ganglia necrosis, suggestive an even rarer entity, acute necrotizing encephalopathy (ane), also referred as acute necrotizing encephalopathy of childhood. ane is characterized by multiple, symmetrical lesions in the thalami, striatum, cerebral white matter, and brain stem [38]. despite its association with viral infection, ane is not considered an inflammatory encephalitis in comparison to adem and ahle. in fact, it has been suggested that an intense surge of cytokines causes damage to the blood-brain barrier with necrosis as a secondary effect [39]. given sars-cov-2 is a cytokine storm trigger, it is not surprising to observe ane as a probable association of covid-19 [40, 41]. it would be important to understand whether these parainfectious demyelinating diseases reflect distinct pathological processes or a continuum of a single disease.  references 1. zhu, n., et al., a novel coronavirus from patients with pneumonia in china, 2019. n engl j med, 2020. 382(8): p. 727-733. 2. lou, j.j., et al., neuropathology of covid-19 (neuro-covid): clinicopathological update. free neuropathol, 2021. 2. 3. lechien, j.r., et al., olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study. eur arch otorhinolaryngol, 2020. 277(8): p. 2251-2261. 4. merkler, a.e., et al., risk of ischemic stroke in patients with coronavirus disease 2019 (covid-19) vs patients with influenza. jama neurol, 2020. 5. dogra, s., et al., hemorrhagic stroke and anticoagulation in covid-19. j stroke cerebrovasc dis, 2020. 29(8): p. 104984. 6. fodoulian, l., et al., sars-cov-2 receptors and entry genes are expressed in the human olfactory neuroepithelium and brain. iscience, 2020. 23(12): p. 101839. 7. salamanna, f., et al., body localization of ace-2: on the trail of the keyhole of sars-cov-2. front med (lausanne), 2020. 7: p. 594495. 8. brann, d.h., et al., non-neuronal expression of sars-cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-associated anosmia. sci adv, 2020. 6(31). 9. xia, h. and e. lazartigues, angiotensin-converting enzyme 2 in the brain: properties and future directions. j neurochem, 2008. 107(6): p. 1482-1494. 10. gheblawi, m., et al., angiotensin-converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2. circ res, 2020. 126(10): p. 1456-1474. 11. meinhardt, j., et al., olfactory transmucosal sars-cov-2 invasion as a port of central nervous system entry in individuals with covid-19. nat neurosci, 2021. 24(2): p. 168-175. 12. matschke, j., et al., neuropathology of patients with covid-19 in germany: a post-mortem case series. lancet neurol, 2020. 19(11): p. 919-929. 13. von weyhern, c.h., et al., early evidence of pronounced brain involvement in fatal covid-19 outcomes. lancet, 2020. 395(10241): p. e109. 14. solomon, i.h., et al., neuropathological features of covid-19. n engl j med, 2020. 383(10): p. 989-992. 15. morbini, p., et al., ultrastructural evidence of direct viral damage to the olfactory complex in patients testing positive for sars-cov-2. jama otolaryngol head neck surg, 2020. 146(10): p. 972-973. 16. al-dalahmah, o., et al., neuronophagia and microglial nodules in a sars-cov-2 patient with cerebellar hemorrhage. acta neuropathol commun, 2020. 8(147). 17. song, p., et al., cytokine storm induced by sars-cov-2. clin chim acta, 2020. 509: p. 280-287. 18. goshua, g., et al., endotheliopathy in covid-19-associated coagulopathy: evidence from a single-centre, cross-sectional study. lancet haematol, 2020. 7(8): p. e575-e582. 19. fabbri, v.p., et al., brain ischemic injury in covid-19-infected patients: a series of 10 post-mortem cases. brain pathol, 2021. 31(1): p. 205-210. 20. hernandez-fernandez, f., et al., cerebrovascular disease in patients with covid-19: neuroimaging, histological and clinical description. brain, 2020. 143(10): p. 3089-3103. 21. parsons, t., et al., covid-19-associated acute disseminated encephalomyelitis (adem). j neurol, 2020. 267(10): p. 2799-2802. 22. novi, g., et al., acute disseminated encephalomyelitis after sars-cov-2 infection. neurol neuroimmunol neuroinflamm, 2020. 7(5). 23. abdi, s., a. ghorbani, and f. fatehi, the association of sars-cov-2 infection and acute disseminated encephalomyelitis without prominent clinical pulmonary symptoms. j neurol sci, 2020. 416: p. 117001. 24. sawlani, v., et al., covid-19-related intracranial imaging findings: a large single-centre experience. clin radiol, 2021. 76(2): p. 108-116. 25. de miranda henriques-souza, a.m., et al., acute disseminated encephalomyelitis in a covid-19 pediatric patient. neuroradiology, 2021. 63(1): p. 141-145. 26. yong, m.h., et al., a rare case of acute hemorrhagic leukoencephalitis in a covid-19 patient. j neurol sci, 2020. 416: p. 117035. 27. umapathi, t., et al., encephalopathy in covid-19 patients; viral, parainfectious, or both? eneurologicalsci, 2020. 21: p. 100275. 28. mccuddy, m., et al., acute demyelinating encephalomyelitis (adem) in covid-19 infection: a case series. neurol india, 2020. 68(5): p. 1192-1195. 29. lopes, c.c.b., et al., acute disseminated encephalomyelitis in covid-19: presentation of two cases and review of the literature. arq neuropsiquiatr, 2020. 78(12): p. 805-810. 30. langley, l., et al., acute disseminated encephalomyelitis (adem) associated with covid-19. bmj case rep, 2020. 13(12). 31. handa, r., et al., covid-19-associated acute haemorrhagic leukoencephalomyelitis. neurol sci, 2020. 41(11): p. 3023-3026. 32. reichard, r.r., et al., neuropathology of covid-19: a spectrum of vascular and acute disseminated encephalomyelitis (adem)-like pathology. acta neuropathol, 2020. 140(1): p. 1-6. 33. walker, j.m., et al., covid-19 patients with cns complications and neuropathologic features of acute disseminated encephalomyelitis and acute hemorrhagic leukoencephalopathy. j neuropathol exp neurol, 2021. 34. bryce, c., et al., pathophysiology of sars-cov-2: the mount sinai covid-19 autopsy experience. mod pathol, 2021. 35. cole, j., et al., acute disseminated encephalomyelitis in children: an updated review based on current diagnostic criteria. pediatr neurol, 2019. 100: p. 26-34. 36. mclendon, l.a., et al., post-covid-19 acute disseminated encephalomyelitis in a 17-month-old. pediatrics, 2021. 147(6). 37. vraka, k., et al., two paediatric patients with encephalopathy and concurrent covid-19 infection: two sides of the same coin? case rep neurol med, 2021. 2021: p. 6658000. 38. wong, a.m., et al., acute necrotizing encephalopathy of childhood: correlation of mr findings and clinical outcome. ajnr am j neuroradiol, 2006. 27(9): p. 1919-23. 39. mizuguchi, m., acute necrotizing encephalopathy of childhood: a novel form of acute encephalopathy prevalent in japan and taiwan. brain dev, 1997. 19(2): p. 81-92. 40. lazarte-rantes, c., et al., acute necrotizing encephalopathy associated with sars-cov-2 exposure in a pediatric patient. cureus, 2021. 13(5): p. e15018. 41. kumar, n., et al., acute necrotizing encephalitis as a probable association of covid-19. indian j crit care med, 2020. 24(10): p. 991-994. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurotrauma: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:4 (2021) review neurotrauma: 2021 update daniel p. perl j. edward hebert school of medicine, uniformed services university, bethesda, md, usa corresponding author: daniel p. perl, md · j. edward hebert school of medicine · uniformed services university · bethesda, md 20814 · usa daniel.perl@usuhs.edu submitted: 12 february 2021 accepted: 09 march 2021 copyedited by: shino magaki published: 15 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3264 keywords: traumatic brain injury, chronic traumatic encephalopathy, biomarkers, neuroinflammation, microglia, tau abstract despite the interruptions and restrictions to the progress of science that the covid-19 pandemic has introduced, 2020 was marked by a number of important advances in the field of neurotrauma. here, i will highlight what i believe are among the most important contributions. this year there were notable advances towards providing clinically useful information on neurotrauma outcome through the use of fluid biomarkers. i also introduce fascinating approaches to studying the role of microglia in nervous system repair and neuroinflammatory mechanisms leading to dysfunction through the use of colony-stimulating factor 1 receptor inhibitors, especially plexxikon 5622 (plx5622). oral administration of this compound is able to deplete microglial elements and then, following withdrawal from the drug, a new population of microglia then repopulates the brain. use of this approach in traumatic brain injury experimental models has produced important insights into the pathogenetic role of microglia in responding to this process. important new data on the nature and distribution of tau involvement of neurons and astrocytes in cases of chronic traumatic encephalopathy (cte) also appeared suggesting differences and similarities to alzheimer’s disease. additionally, the use of tau-specific pet scan ligands in at-risk populations has suggested that this approach may be able to identify cases with cte. lastly, we note the death in the past year of a major contributor to the field of neurotrauma neuropathology, professor j. hume adams. introduction for virtually all of us, 2020 will mostly be remembered as the year of covid-19. with numerous calls for mask wearing, social distancing, partial-to-complete shutdowns of offices and laboratories (including our own) and other similar restrictions, this pandemic has definitely had a dampening effect on everyone’s scientific productivity. nevertheless, by viewing the traumatic brain injury (tbi) literature that appeared during 2020, it is hard to see any significant slowing of progress. i have the impression that with most scientists working within a telecommuting mode, rather than being active in the lab, many took the time to get to accumulated data waiting to be analyzed, write papers waiting to be written, etc., and thus scientific output did not appear to slow, and possibly even increased. it might be predicted that following the achievement of widespread vaccination allowing for a final full return to our lab benches, there will likely be a lag in output as new experiments need to be put in place and additional data collected. time will tell. nevertheless, in surveying the field of neurotrauma, many important contributions were reported in 2020 and here i would like to pick some of what i believe were among the more important advances that were published. this is an individual viewpoint, reflecting my own biases, and i will apologize, in advance, if i have omitted any of your favorites on this year’s list. so, among the more than 2,000 publications appearing in the literature on neurotrauma in 2020, here are those that i chose to highlight for you. contributions to the development of fluid biomarkers for tbi: finally some approaches that appear to be useful over the past few years, a large number of groups have engaged in efforts to identify useful blood-based biomarkers in the field of tbi that hopefully would be able to reflect outcome, especially with respect to predicting a transition to clinically persistent sequelae. by and large, the results of these efforts have been relatively inconsistent and have not achieved robust clinical validity. however, in 2020 a group reported [1] that increases in exosome and plasma levels of neurofilament light (nfl) chain protein in patients with repeated mild traumatic brain injuries (mtbis) were predictive of subsequent development of chronic post-concussion syndrome, post-traumatic stress disorder (ptsd) and depression. the study involved a cohort of 195 retired service members enrolled in the chronic effects of neurotrauma consortium longitudinal study. the cohort received detailed assessments to document and quantify episodes of prior mtbi and were grouped into repetitive mtbi (3 or more episodes), 1-2 mtbis and controls with no tbi history. these assessments were obtained through interviews with the subjects. these data have particular importance, because they were obtained from retired service members, and thus their tbi exposures mostly occurred years prior to obtaining the biomarker assay. the results reported suggest that their assays are reflecting a chronic, ongoing pathologic process that extended from the mtbi exposures to the time when the blood was drawn. the nature of that process remains unclear, although in their discussion the authors noted literature pointing towards the elevation of nfl levels in association with axonal injury. the use of isolated exosomes, brain-derived membrane-bound components that cross the blood-brain-barrier to enter the blood stream, is particularly noteworthy. since these exosomes can be linked back to a central nervous system origin, it suggests that this approach is capable of providing indicators of more specific brain-related pathology and thus more closely reflect relevant biologic processes. since the results were predictive for the development of depression and symptoms indicative of ptsd, this result represents another piece of evidence for a biologic substrate of the long-term persistent behavioral consequences of tbi. further advances in the blood biomarker field were reported by okonkwo et al. [2] in a study where the predictive accuracy of determining glial fibrillary acidic protein (gfap) levels was compared with s100 calcium-binding protein b (s100b) assays in the blood of tbi patients. the u.s. centers for disease control and prevention reports that each year about 4,800,000 evaluations for tbi occur in emergency departments in the united states. about 80-90% of these patients have mild forms of tbi (mtbi, as defined by a glasgow coma scale score of 13-15) and only 10% of the mtbi patients will subsequently demonstrate abnormalities on computed tomography (ct) studies of the head and thus need further evaluation and monitoring. accordingly, rapid reliably predictive studies are needed to lighten this huge clinical diagnostic load and better identify which patients need further ct scrutiny. in the united states, assays of serum gfap have been approved by the u.s. food and drug administration (fda) so that they can be used in a clinical setting to determine the need for a head ct within 12 hours of an episode of mtbi. alternatively, in europe, approval has been granted for s100b assays that are currently used to serve this clinical assessment function. importantly, in the study now reported, plasma gfap was determined using a newly developed point-of-care prototype assay that can be completed within 15 minutes. serum s100b was determined by a more standard laboratory-based analytic method that requires transfer of the specimen to the laboratory, adding considerably greater time to provide a report. the endpoint used for the 1,359 tbi patients in the study, who ranged from mild to more severe degrees of injury, was the ability to predict the presence of structural abnormalities on a head ct scan. using a predetermined cut-off value for serum gfap of 22 pg/ml, this study found that gfap levels were an excellent predictor of ct abnormalities and could do so even on samples taken up to 24 hours from the time of trauma. gfap assays substantially outperformed that of s100b as a ct abnormality predictor. further, the mean level of gfap correlated with increasing numbers of distinct lesions identified on head ct, suggesting a dose response. indeed, patients with severe to moderate tbi (gcs 3-12) had 10-fold greater levels than the levels assayed on patients with mtbi (gcs 13-15). overall, the gfap assay had a predictive sensitivity of 0.987. it should be pointed out that the point-of-care assay, with its rapid reporting time (15 minutes) is much faster than the currently fda approved lab-based methodology and, by its nature, can be performed in obscure locations without access to laboratory facilities. it is my understanding that this new and faster technology is in the process of being evaluated by the fda for approval as a clinical diagnostic tool. this promises to become an extremely valuable approach for clinicians faced with a staggering number of tbi patients to evaluate. a rapid point-of-care assay method will also be of great value for military medicine where many tbis are clinically evaluated downrange in isolated battlefield locations. assays on the playing field of contact sport events might also find this approach quite attractive for clinicians involved in such evaluations. the role of microglia in repair of tbi microglia are abundant cellular constituents of the brain that play important roles in brain development, maintenance and the pathogenesis of most disease states. we are taught to consider microglia as playing a central role in virtually all nervous system repair processes, including that of tbi. indeed, although the precise mechanism for their action remains unknown, microglia have been thought to mediate ongoing neuroinflammatory damage to the brain that is responsible for at least some of the long-term functional sequelae of tbi and even aspects of potential subsequent neurodegenerative phenomena. recently, a series of drugs have been identified that inhibit colony-stimulating factor 1 receptor (csf-1r), which microglia depend upon for their survival. these drugs penetrate the brain after oral administration and within two to three weeks are able to almost completely deplete the entire microglial cellular compartment of the brain in an exposed animal. following withdrawal of the drug, there will be a steady repopulation of the brain by microglia (figure 1). importantly, the repopulated microglia appear to be a new rejuvenated population of cells without a prior history of antigen exposure. the primary agent used in such microglial repopulation experiments is referred to as plexxikon (plx) 5622. figure 1. microglia are depicted in 8 weeks old cx3cr1-gfp mice (microglia – green; dapi – blue) a) control mouse, normal diet, b) 3 weeks following a diet containing 1200 mg/kg of plx5622 (note virtual complete absence of microglia) and c) after 3 weeks on the plx5622 diet then 2 weeks on the control diet (note the return of prominent green staining from the repopulated microglia). from ongoing studies of k. whiting and z. galdzicki of uniformed service university neurosciences program. the use of drugs such as plx5622 have begun to be used to explore the specific roles that microglia play in the repair processes and sequelae of experimental tbi, such as occurs with controlled cortical impact. henry and colleagues [3] administered plx5622 one month after controlled cortical impact in mice and then withdrew the drug to allow for microglial repopulation. by 3 months post-injury, they noticed that the plx5622 treated animals possessed a smaller cortical lesion, reduced hippocampal neuron cell death and decreased expression of nox2 and nlrp3 inflammasome-associated neuroinflammatory modulators, when compared to non-treated animals receiving equivalent controlled cortical impact injury. the plx5622 treatment animals also showed improved long-term motor and cognitive function. these intriguing experiments demonstrated that removal of microglia in the chronic phase of tbi repair reduced subsequent neuroinflammation as well as lessening subsequent motor and cognitive functional deficits. further, it showed that such inflammatory effects extended far longer than has been traditionally believed. willis and coworkers [4] also used plx5622 for microglial depletion and repopulation in a mouse model of tbi (they too used closed cortical impact), however these workers showed that removal of microglia had little effect on the clinical outcome of their tbi model. nevertheless, examination of the treated animals showed neuroprotective effects that appeared to aid in recovery. these beneficial effects were mostly modulated through interleukin-6 (il-6) signaling via the soluble il-6 receptor and its support of neurogenesis. these authors suggested that the presence of activated microglia associated with neurotrauma may not have a negative effect on outcomes and that it would appear that, as they state, “these cells lack an ability to support endogenous repair processes.” obviously, more needs to be done to further dissect out the role that microglia play in the brain’s response to tbi. the timing of when the microglial removal and repopulation takes place in these models appears to be critical to the results obtained. it may be predicted that the use of tools such as plx5622 will greatly help in unraveling such issues. in the discussion, the authors wonder if plx5622 might even represent a clinically feasible therapeutic approach to reducing some of the long-term complications of tbi. plx5622 has received limited fda approval for clinical use in patients with other non-tbi indications. for those who are interested in pursuing microglial depletion and repopulation, in a companion publication, this group has provided detailed protocols for such experiments in mice [5]. this would appear to be a valuable new tool for the investigation of the role of microglia, both positive and negative, in the pathogenesis of not only neurotrauma but many other disease processes [6, 7]. studies of the long-term effects of subconcussive blast exposure in breachers the effects of blast tbi on the brain have mostly been studied either experimentally in small animal models or in humans following a single significant blast event, typically related to exposure to an improvised explosive device (ied). it should be kept in mind that, in the combat setting, blunt impact tbi also commonly occurs in conjunction with these blast injuries. breaching represents a process where explosions are used to blast open doors and thus gain entry to buildings (figure 2). as such, breaching is associated with blast exposure in the absence of impact trauma. stone and colleagues [8] reported studies comparing a cohort of career breachers (typically breaching instructors) to a matched but minimally exposed control group. of the 20 experienced breachers studied, they reported having experienced an average of 4,628 breaching blast exposures over their careers, as opposed to the control group (n=14) who experienced an average of 3 exposures. keep in mind that these exposures are all considered to be sub-concussive in nature. figure 2. breaching is a procedure whereby an explosive charge, typically placed on a door, is used by combatants to enter a building and engage the enemy. service members participate in breaching in both training exercises and on the field of battle. repeated training exercises, such as portrayed here, expose participants to numerous sub-concussive blast wave exposures, in the absence of impact tbi. courtesy of sofia echelmeyer. using detailed neuroimaging approaches, they found, somewhat surprisingly, evidence of a significant degree of cerebral cortical thickening in the breacher group. this change was widespread, throughout the cerebral cortex. the nature of this cerebral cortical enlargement remains unclear, as no neuropathologic studies of deceased career breachers have yet to be reported. the authors also noted differences between the heavily exposed and control groups related to regional blood flow, various neuropsychological assessment results and serum biomarkers, however none of these differences survived bonferroni correction for multiple comparisons. this suggested that further studies using a larger cohort would be instructive. service members carrying out other duties, such as those who spend a career firing high caliber artillery ordinances or explosive ordinance disposal personnel, may also have an equivalent degree of blast exposure, and it would appear that similar studies of these groups are warranted. tbl-related neuroinflammatory markers neuroinflammatory response to tbi is a subject that has received considerable attention in both experimental models and clinical settings. clinically, studies of cytokine expression have mostly focused on moderate to severe tbi patients which have demonstrated slightly elevated plasma cytokine levels in the acute phase post-injury. post-mortem studies by johnson and colleagues [9] have demonstrated that activated microglia can persist for decades after the initial traumatic incident. a recent study by chaban and colleagues [10] published in 2020 looked at plasma levels of 12 different cytokines in a cohort including 207 patients with mtbi and 82 matched uninjured community controls. plasma samples were drawn at admission for evaluation of the tbi, as well as 2 weeks, 3 months and 12 months following the injury. brain magnetic resonance imaging (mri) was also performed on all the participants. comparing the mtbi group with the controls, they found significant elevations in plasma interferon gamma, il-8, macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, il-17a, il-9, tumor necrosis factor, and basic fibroblast growth factor at all time points, whereas a number of the other cytokines remained unchanged. the presence of persistent cytokine levels did not correlate with the mri findings, suggesting that the cytokine changes were not related to the extent of tissue damage. this is one of the few longitudinal studies of plasma neuroinflammatory markers of patients with mtbi that showed evidence of persistent systemic inflammation lasting up to a year. the nature of intracellular tau accumulations in cases of chronic traumatic encephalopathy (cte) tbi, especially repeated impact tbi, constitutes a risk factor for the development of chronic traumatic encephalopathy (cte), a tauopathy associated with neurofibrillary tangles (nfts) and tau accumulation in astrocytes, primarily in the form of what are referred to as thorn-shaped astrocytes (tsas). arena and colleagues [11] reported a detailed immunohistochemical study on the nature of tau accumulations in nfts and tsas in cases of cte and compared them to staining results obtained in several other forms of tau-related neurodegenerative disorders (alzheimer’s disease, frontotemporal dementia, pick’s disease) and other tau-related conditions seen in the elderly (age-related tau astrogliopathy, or artag, and primary age-related tauopathy, or part). this study showed that the nfts of the cte cases contained both 3r and 4r isoforms that are also classically seen in association with the tangles of alzheimer’s disease and part. the tsas of cte stained virtually entirely for 4r tau, similar to what is seen in cases of artag but distinct from what has been observed in alzheimer’s disease. the use of antibodies directed towards various post-translational modifications of tau (primarily related to the presence of specific phosphorylation residues) also showed consistent similarities between the tsas of cte and those of alzheimer’s disease and artag. further, the authors employed recently developed anti-tau antibodies that are configuration-dependent [12] (gt-7 and gt-38) which showed that the nfts in the depths of sulci in the cases of cte were strongly immunoreactive, which is consistent with the staining reaction that is seen in the nfts of alzheimer’s disease. however, in all but a few of the cte cases, the astrocytes failed to stain with these antibodies. in their discussion, the authors noted the similarity between the tau immunophenotype seen in the nfts of cte and that of alzheimer’s disease and part. in contrast, the tau-positive astrocytes of cte were indistinguishable from those of artag but distinctly different from the alzheimer’s disease results. this led to the suggestion that cte and artag may share pathogenetic mechanisms that separate these two diseases from that of alzheimer’s disease. clearly, these are concepts that need to be explored further and may be of importance in sorting out the complex and confusing nosology and diagnostic criteria for these various neuropathologic entities. obviously, additional work needs to be done and neuropathologists involved in this area will follow it with interest. animal models of cte and the use of pet ligand for tau for clinical diagnosis of cte further on tau and its association with repeated tbi, dickstein and colleagues [13] investigated various biomarkers in rats that were experimentally exposed to repeated low-level blast over-pressures as well as military service members who had been exposed to ieds on the battlefield and subsequently suffered persistent behavioral, cognitive and/or memory complaints. in the rats, six weeks following blast exposure, by western blot, abnormally phosphorylated tau (thr181, p-tau) was shown to be increased in the right anterior cortex and right hippocampus but not in contralateral locations. by ten months post-exposure, p-tau levels had further increased and were now more likely to be encountered bilaterally. using immunohistochemistry, involved animals primarily showed fine dendritic staining although some did show perikaryal accumulations, a most unique observation among repeated tbi experimental rodent models. the human military cases were studied by pet scanning using [18f]av45 (flortaucipir), a pet ligand that is reported to be selective for tau. half of the ten blast-exposed service members receiving pet scans showed excessive accumulation of the ligand at gray-white matter junctions in frontal, parietal and temporal regions, a pattern the authors interpreted to be “a typical localization of cte tauopathy.” in addition, levels of plasma nfl were elevated in the blast-exposed subjects who showed excess [18f]av45 retention on pet analysis. this study suggests the potential value of the rat as an experimental model of tau accumulation following repeated blast exposure. this stands in direct contrast to the mostly negative findings in mice exposed to repetitive impact tbi. the reported results also point to the potential for further use of tau pet ligands in the study of human subjects at risk for cte. of note, since in the human studies they engaged living patients, no opportunity for neuropathologic confirmation of the proposed diagnosis of cte could be made. follow-up from last year’s neurotrauma 2019 report in my contribution last year, i highlighted a paper suggesting that the use of tranexamic acid treatment showed a significant lowering of mortality in a very large randomized placebo-controlled clinical trial in patients with more severe forms of acute tbi [14]. i felt this was important in view of the dismal record, despite numerous attempts, for introducing therapeutic agents with evidence-based positive results in the treatment of tbi patients. at the time, i was encouraged by the size of the study and their positive results. since tranexamic acid appears to be quite safe and is a very inexpensive drug, i was further encouraged by this approach. i also used this publication to highlight the importance of vascular pathology and bleeding in tbi pathophysiology, morbidity and mortality. despite my optimism over the positive results of prehospital administration of tranexamic acid in tbi patients, in 2020, bossers et al. [15] reported the results of a multicenter cohort study of using the drug to treat 1,827 severe tbi patients. this new study showed that prehospital administration of tranexamic acid produced increased mortality in those patients receiving the drug. of course, one negative study does not settle matters here, but these results suggest that care must be taken before proceeding in this direction. it would appear that, once again, those who seek an effective therapy for tbi patients are left with few, if any, therapeutic approaches showing efficacy. the toolbox for clinicians dealing with the effects of tbi continues to remain virtually empty of drugs that carry evidence of improved clinical outcome. finally for those of us with an interest in the neuropathology of neurotrauma, there is one additional event that occurred in 2020 that i felt was important to note. on april 12, 2020, professor james hume adams died [16]. professor adams was a pioneering, major contributor to our understanding of the neuropathology of neurotrauma, spending virtually all of his career studying the disorder. he trained in neuropathology at the institute of psychiatry at the maudsley hospital in london, where he initially came in contact with several people with an interest in neurotrauma, namely sabina stritch, peter daniel and j.a.n. corsellis. it is clear that the seeds for his life-long interest in neurotrauma were planted at this time. he subsequently took a position in glasgow at the department of pathology, western infirmary, eventually moving to the new facility at the southern general hospital. while in glasgow, he developed and ran the glasgow database of human head injury, and its brain bank repository has represented a major resource for the study of the effects of impact tbi on the human brain. importantly, this unique facility represents one of the only available collections in which one would be able to characterize the long-term effects of impact tbi. over the years, this facility has been extensively used by the glasgow group, now under the direction of dr. willie stewart. professor adams was best known for his seminal contributions in describing and then further characterizing diffuse axonal injury (dai), both clinically and experimentally. much of this work was carried out in conjunction with colleagues in glasgow and at the university of pennsylvania. the critical concepts related to the effects of trauma on the brain that he first proposed continue to be understood and used to this day. although in more recent years he had been in retirement as an emeritus professor, the influence of this work continues and according to his obituary, the 60 papers he published with the university of pennsylvania group on neurotrauma have been cited more than 9,000 times. in 2020, we lost a towering figure who contributed greatly to the field of neurotrauma. acknowledgement the opinions expressed herein are those of the author and are not necessarily representative of those of the uniformed services university of the health sciences, the united states department of defense, the united states army, navy or air force. references 1. guedes, v. a. et al. exosomal neurofilament light: a prognostic biomarker for remote symptoms after mild traumatic brain injury? neurology, 2020. 94(23): p. e2412-e2423. 2. okonkwo, d. o. et al. point-of-care platform blood biomarker testing of glial fibrillary acidic protein versus s100 calcium-binding protein b for prediction of traumatic brain injuries: a transforming research and clinical knowledge in traumatic brain injury study. j neurotrauma, 2020. 37(23): p. 2460-2467. 3. henry, r. j. et al. microglial depletion with csf1r inhibitor during chronic phase of experimental traumatic brain injury reduces neurodegeneration and neurological deficits. j neurosci, 2020. 40(14): p. 2960-2974. 4. willis, e. f. et al. repopulating microglia promote brain repair in an il-6 dependent manner. cell, 2020. 180(5): p. 833-846. 5. willis, e. f. and vukovic, j. protocol for brain-wide or region-specific microglia depletion and repopulation in adult mice. star protoc, 2020. 1(3): p. 100211. 6. qu, w. et al. inhibition of colony-stimulating factor 1 receptor early in disease ameliorates motor deficits in sca1 mice. j neuroinflammation, 2017. 14(1): p. 107. 7. tahmasebi, f. et al. the effect of microglial ablation and mesenchymal stem cell transplantation on a cuprizone-induced demyelination model. j cell physiol, 2021. 236(5): p.3552-3564. 8. stone, j. r. et al. functional and structural neuroimaging correlates of repetitive low-level blast exposure in career breachers. j neurotrauma, 2020. 37(23): p. 2468-2481. 9. johnson, v. e. et al. inflammation and white matter degeneration persist for years after a single traumatic brain injury. brain, 2013. 136(pt 1): p. 28-42. 10. chaban, v. et al. systemic inflammation persists the first year after mild traumatic brain injury: results from the prospective trondheim mild traumatic brain injury study. j neurotrauma, 2020. 37(19): p. 2120-2130. 11. arena, j. d. et al. tau immunophenotypes in chronic traumatic encephalopathy recapitulate those of ageing and alzheimer's disease. brain, 2020. 143(5): p. 1572-1587. 12. gibbons, g. s. et al. detection of alzheimer disease (ad)-specific tau pathology in ad and nonad tauopathies by immunohistochemistry with novel conformation-selective tau antibodies. j neuropathol exp neurol, 2018. 77(3): p. 216-228. 13. dickstein, d. l. et al. brain and blood biomarkers of tauopathy and neuronal injury in humans and rats with neurobehavioral syndromes following blast exposure. mol psychiatry, 2020. 14. crash-3 trial collaborators. effects of tranexamic acid on death, disability, vascular occlusive events and other morbidities in patients with acute traumatic brain injury (crash-3): a randomised, placebo-controlled trial. lancet, 2019. 394(10210): p. 1713-1723. 15. bossers, s. m. et al. association between prehospital tranexamic acid administration and outcomes of severe traumatic brain injury. jama neurol, 2020. 16. graham, d. and smith, c. obituary of emeritus professor j hume adams. neuropathol appl neurobiol, 2020. 46(6): p. 618-620. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. molecular clarification of brainstem astroblastoma with ewsr1-bend2 fusion in a 38-year-old man feel free to add comments by clicking these icons on the sidebar free neuropathology 2:16 (2021) case report molecular clarification of brainstem astroblastoma with ewsr1-bend2 fusion in a 38-year-old man matthew a. smith-cohn,1,2 zied abdullaev,3 kenneth d. aldape,3 martha quezado,3 marc k. rosenblum,4 chad m. vanderbilt,4 fausto j. rodriguez,5 john laterra,**2 charles g. eberhart**5 1 neuro-oncology branch, national cancer institute, national institutes of health, bethesda, md, usa 2 department of neurology, the johns hopkins university school of medicine, baltimore, md, usa 3 laboratory of pathology, center for cancer research, national cancer institute, bethesda, md, usa 4 department of pathology, memorial sloan-kettering cancer center, new york, ny, usa 5 department of pathology, the johns hopkins university school of medicine, baltimore, md, usa   ** these authors contributed equally to this manuscript corresponding author: charles eberhart, md, ph.d. · department of pathology · the johns hopkins university school of medicine · 1800 orleans st. · sheikh zayed tower · baltimore, md 21287 · usa ceberha@jhmi.edu submitted: 21 april 2021 accepted: 17 june 2021 copyedited by: calixto-hope lucas published: 21 june 2021 https://doi.org/10.17879/freeneuropathology-2021-3334 keywords: astroblastoma, brainstem, fusion, methylation, neoplasms, neuro-oncology abstract the majority of astroblastoma occur in a cerebral location in children and young adults. here we describe the unusual case of a 38-year-old man found to have a rapidly growing cystic enhancing circumscribed brainstem tumor with high grade histopathology classified as astroblastoma, mn1-altered by methylome profiling. he was treated with chemoradiation and temozolomide followed by adjuvant temozolomide without progression to date over one year from treatment initiation. astroblastoma most frequently contain a mn1-bend2 fusion, while in this case a rare ewsr1-bend2 fusion was identified. only a few such fusions have been reported, mostly in the brainstem and spinal cord, and they suggest that bend2, rather than mn1, may have a more critical functional role, at least in these regions. this unusual clinical scenario exemplifies the utility of methylome profiling and assessment of gene fusions in tumors of the central nervous system. introduction astroblastomas are rare central nervous system (cns) neoplasms that most frequently occur in cerebral locations in children and young adults. here we describe the unusual case of a 38-year-old man with a brainstem tumor with an integrated diagnosis of malignant neoplasm consistent with astroblastoma with mn1 alteration following methylome profiling, and found to have a non-canonical ewsr1-bend2 fusion. this unusual clinical scenario exemplifies the utility of methylome profiling and assessment of gene fusions in tumors of the cns. case report a 38-year-old male with a past medical history of melanoma in situ of the trunk, status post excision, and no family history of cancer, presented with subacute onset of progressively worsening dysesthesia first involving his upper extremities and progressing to affect his right chest and leg over two weeks. mri of the brain and cervical spine demonstrated a well-localized cystic enhancing anterior medullary-cervical lesion measuring 1.3 x 1.1 x 1.7 cm, with t2 hyperintensity of the lesion and medullary pyramids (fig.1a, 1b, 1c). he was admitted to the neurology service for an expedited evaluation of infectious, rheumatologic, neoplastic, and inflammatory etiologies. mri of the spine and whole-body pet/ct revealed no other lesions. serum studies were unremarkable, and lumbar csf contained 1 wbc, 3 rbc, glucose 55, protein 57 (ref 15-45), no oligoclonal bands and negative infectious studies, flow cytometry, and cytopathology. he was treated with high dose iv methylprednisolone for five days and discharged home on a steroid taper. he had progression of symptoms, and a repeat mri two months later showed growth of the lesion to 3.3 x 1.7 x 1.5 cm (fig.1d, 1e, 1f). subsequently, he underwent a suboccipital c1 laminectomy and subtotal surgical resection of the mass. figure 1. radiographic findings. coronal (a, d), sagittal (b, e), axial (c, f) post-contrast t1 mri of the brain. the top row (a-c) shows mri imaging of the brain at presentation, and the bottom row (d-f) is two months later before tumor resection. pathology microscopic evaluation showed a cellular tumor with compact growth. large, pleomorphic to epithelioid cells predominated in some regions (fig.2a), while in other areas prominent perivascular growth was noted. this included scattered cells with a somewhat astroblastic phenotype, exhibiting stout processes extending to the surface of blood vessels (fig.2b). necrotic foci without pseudopalisading were present. cellular regions of tumor had 1 to 5 mitotic figures per high power field, and the ki67 proliferation index was moderate to high, up to 20-30% (fig.2c). s100, olig2 and ema were strongly positive on immunohistochemical analysis (fig.2d, e and data not shown), and gfap was focally positive, supporting glial differentiation. in contrast, markers of melanocytic (melana, sox10, hmb45), epithelial (cytokeratin ae1/ae3), and neuronal (synaptophysin) differentiation were all negative (fig.2f and data not shown). figure 2. histopathology. the tumor included more pleomorphic regions (a), as well as neoplastic cells with thick, short processes (arrow, b) arrayed around proliferating blood vessels (asterisks, b). the tumor was quite proliferative on ki67 immunostain (c), and diffusely positive for olig2 (d) and s100 (e), while blood vessels did not express these glial markers (asterisks, e). melana was not expressed in the tumor (f). (original magnifications: a-e 400x, f 200x). molecular diagnostics next-generation sequencing (ngs) found a tumor mutation rate of 0.88 mutations per megabase, no known pathologic variants, and several variants of unknown significance, including adgra2 (p.g407s), ar (p.a646d), blm (p.r643h), epha5 (p.l907v), and prkdc (p.i1013v), all with allele frequencies of 46% or higher. no alterations in tp53, atrx, braf, h3f3a or hist1h3b were detected on ngs. dna methylation profiling was consistent with a “high-grade neuroepithelial tumor with mn1 alteration”, with a calibrated score of 0.994 (fig.3a). copy number evaluation using data from the methylation array demonstrated alterations in chromosome 22 and x (fig.3b). subsequent gene fusion testing found a ewing sarcoma breakpoint region 1/ews rna binding protein 1 (ewsr1) ben domain containing 2 (bend2) fusion between loci on chromosomes 22 and x (fig.3c). the integrated diagnosis was malignant neoplasm consistent with astroblastoma with mn1 alteration. figure 3. methylation and copy number profiling of astroblastoma with an ewsr1-bend2 fusion. (a) the lesion (blue box) was plotted on an x–y coordinate graph (red dot to the lower left of the x–y intersection), where closer proximity to other dots indicates greater similarity of the index tumor’s genomic cpg methylation pattern to existing cases in the library. (b) copy number profiling demonstrates the location for ewsr1-bend2 fusion at chromosome 22 and x (blue arrow). (c) fusion between exon 7 of ewsr1 and exon 5 of bend2 genes with breakpoints at genomics positions chr22:29683123 and chrx:18234853 respectively. the red arrow represents the direction of the gene specific primary utilized by the archer assay to enrich the amplicons for ewsr1 fusion events. legend: mb,g3, medulloblastoma, subclass group 3; epn, pf a, ependymoma, posterior fossa group a; epn, pf b, ependymoma, posterior fossa group b; subepn, pf, subependymoma, posterior fossa; mb, shh chl ad, medulloblastoma, subclass shh a (children and adult); mb, shh inf, medulloblastoma, subclass shh b (infant); contr, cebm, control tissue, cerebellar hemisphere; lipn, cerebellar liponeurocytoma; cns nb, foxr2, cns neuroblastoma with foxr2 activation; sp-epn-mycn, mycn amplified spinal cord ependymoma; epn, yap, ependymoma, yap fusion; hgnet, mn1; high grade neuroepithelial tumor with mn1 alteration; gbm, g34, glioblastoma, idh wildtype, h3.3 g34 mutant; gbm, mes, glioblastoma, idh wildtype, subclass mesenchymal; gbm, mid, glioblastoma, idh wildtype, subclass midline; gbm, mycn, glioblastoma, idh wildtype, subclass mycn; gbm, rtk i, glioblastoma, idh wildtype, subclass rtk i; gbm, rtk ii, glioblastoma, idh wildtype, subclass rtk ii; gbm, rtk iii, glioblastoma, idh wildtype, subclass rtk iii; hgnet, bcor, cns high grade neuroepithelial tumor with bcor alteration; ptpr, a, papillary tumor of the pineal region group a; ptpr, b, papillary tumor of the pineal region group b; contr, pons, control tissue, pons; contr, wm, control tissue, white matter; contr, hypthal, control tissue, hypothalamus; contr, hemi, control tissue, hemispheric cortex; cn, central neurocytoma; subepn, spine, subependymoma, spinal; lgg, myb, low grade glioma, myb/mybl1; lgg, dnt, low grade glioma, dysembryoplastic neuroepithelial tumor; lgg, gg, low grade glioma, ganglioglioma; ihg, infantile hemispheric glioma; lgg, pa/gg st, low grade glioma, rosette forming glioneuronal tumor; lgg, rgnt, rosette forming glioneuronal tumor; contr, react, reactive tumor microenvironment; dlgnt, diffuse leptomeningeal glioneuronal tumor; ana pa, anaplastic pilocytic astrocytoma; pxa, (anaplastic) pleomorphic xanthoastrocytoma; lympho, lymphoma; dmg, k27, diffuse midline glioma h3 k27m mutant; lgg, pa pf, subclass posterior fossa pilocytic astrocytoma; lgg, pa mid, midline pilocytic astrocytoma; contr, inflam, control tissue, inflammatory tumor microenvironment; epn, rela, ependymoma, rela fusion; atrt, tyr, atypical teratoid/rhabdoid tumor, subclass tyr; chgl, chordoid glioma of the third ventricle; lgg, sega, subependymal giant cell astrocytoma. subsequent clinical course the patient was treated with fractionated radiation (5040 cgy in 28 fractions) with concurrent daily temozolomide (75 mg/m2) followed by adjuvant temozolomide (150-200 mg/m2, 5 days-on/23 days-off) with a partial response. at the time of writing, the patient has not progressed since initiation of treatment over one year ago. clinically the patient was able to return to work full time with continued central neuropathic pain of the left face and right-side extremities and trunk managed with gabapentin. discussion astroblastoma has historically been a controversial entity since its introduction in the 1924 classification of cns brain neoplasms by cushing and bailey.1 mn1-altered astroblastomas arise primarily in cerebral locations in pediatric patients and young adults.2 in addition to the presence of a ewsr1-bend2 fusion, our case is unusual compared to other described mn1-altered astroblastoma in that the patient is near his fourth decade of life and with tumor located in the brainstem and upper cervical spine, rather than the cerebrum. including the case presented, there are three other described cases of cns tumors with a ewsr1-bend2 fusion (tab. 1).3–5 aside from one case that did not provide additional information, these tumors occurred in males and were infratentorial, involving the spinal cord and sometimes the brainstem.3–5 although non-specific, mri of astroblastomas have been described to enhance, as well as appear well-demarcated, cystic, and lobulated, which is consistent with our case and similar to other cases of ewsr1-bend2 fused astroblastoma tumors (fig.1).3,4,6 comparable to conventional mn1-altered astroblastomas and other cases of ewsr1-bend2 fused tumors, our case showed perivascular growth and immunohistochemistry was positive for s100, olig2, ema and gfap.2–4 ultrastructural observations in astroblastomas suggest a relationship to tanycytes, an ependymal cell subtype, but it is unclear if this applies also to mn1-altered and ewsr1-bend2 fused astroblastomas.1,2 molecular profiling has had a huge impact on the diagnosis of “astroblastic” tumors and has helped clarify subtypes of astroblastoma. in 2016, sturm et al. identified a group of 41 tumors with a common methylation profile and mn1 fusions, designating them ‘cns high-grade neuroepithelial tumor with mn1 alteration’ (cns-hgnet-mn1).7 notably, the majority of these contained astroblastic or ependymal perivascular pseudorosettes, although a number of other histopathological appearances were also represented. subsequent studies of mn1-altered brain tumors have confirmed that many, but not all, have ependymal or astroblastic features.8 based on these and other studies, the consortium to inform molecular and practical approaches to cns tumor taxonomy (cimpact-now) has proposed designating these tumors “astroblastoma, mn1-altered”.2 early case series of astroblastoma reported prior to these newer molecular tools likely contained other tumor subtypes, as exemplified by a recent study in which molecular profiling of 14 adult tumors with histologic feature of astroblastoma found they were pleomorphic xanthoastrocytomas or high grade gliomas with alterations activating the mitogen-activated protein kinase pathway, and none had clear evidence of a mn1-bend2 fusion, or clustered with that group on methylation profiling analysis.9 the mn1 alteration is usually a fusion between mn1 and bend2.2 while half or more of microscopically defined astroblastomas harbor an mn1 alteration, a significant number do not.3,10 our patient is unique due to the presence of a rare non-canonical ewsr1-bend2 fusion between chromosomes 22 and x. interestingly, early molecular analyses of astroblastomas identified deletions in chromosomes 22q and x.1,2 including our case, there are at least four known cases of cns tumor with ewsr1-bend2 fusions in the literature (table 1).3–5 one case was a 3-month -old male with tumor spanning the lower medulla to the c4 spinal cord level,3 with a second in a 36-year-old male patient with a thoracic spinal “ependymoma” with ewsr1-bend2 fusion.4 a third case was found in a series of molecularly characterized pediatric tumors which did not provide clinical information.5 remarkably, in at least two of these cases, as in ours, methylation clustering led to the tumor falling into the category of astroblastoma/hgnet with mn1 alteration.3,4 these reports highlight the possibility of astroblastic or ependymal tumors with a methylation profile consistent with “astroblastoma, mn1-altered” being driven by fusions in genes other than mn1, and also suggest that it may be bend2, rather than mn1, which has a more critical functional role. supporting this notion, there is one reported case of an neuro-epithelial tumor with an mn1-patz1 fusion found with rna sequencing a supratentorial mass in a 1-year-old girl that suggest a similar pathogenic role as a chimeric protein.11 however, this case lacked astroblastomatous rosettes and did not have epigenetic similarities with cns hgnet-mn1, but did have similarities to patz1-sarcomas.11 table 1: known cases of astroblastoma with an ewsr1-bend2 fusion in the literature the grading and clinical behavior of astroblastomas is not well understood. as noted by the 6th cimpact-now update and other case series, a significant proportion of tumors in the methylation class astroblastoma/cns-hgnet-mn1 do not conform to astroblastoma histologically, and it is unclear if disparate histologic patterns have biologic relevance aside from high grade features.1,2 the 2016 who, completed before identification of the mn1-altered molecular group, does not assign grades, but observed that astroblastic neoplasms generally fall into two general categories: well-differentiated or anaplastic/malignant.1,12 case series that predated molecular evaluation of astroblastomas likely included other tumor subtypes, but found that in histologically defined astroblastomas, an elevated proliferation rate is associated with worse outcomes.1,12 a case series of 14 neuroepithelial tumors with mn1 alterations, including three spinal cases, two of which were the oldest (14.6 and 36 years old) and only males in the series, describes heterogeneous treatments consisting of focal or cranial spinal radiation without chemotherapy.8 the event-free survival ranged considerably from 6 to 100 months, with some cases resulting in metastasis in the cns, arguing that intermittent monitoring with completed neuroaxis imaging is warranted.8 a meta-analysis of 73 patients with mn1-altered neuroepithelial tumors (astroblastomas) found a 5and 10-year progression-free survival of 38% and 0%, and 5and 10-year overall survival of 89% and 55%, respectively.13 the natural history of astroblastoma with ewsr1-bend2 fusion has not been well described in the literature, and it is unclear if the clinical behavior is significantly different from astroblastomas with a mn1 alteration. the case we present showed anaplastic microscopic features, as well as aggressive clinical behavior with rapid growth over two months histologic features and clinical behavior was similar to other described cases of astroblastoma with a ewsr1-bend2 fusion.3,4 given the rarity of these tumors, there is no defined standard established treatment. based on studies of non-molecularly characterized astroblastoma, resection remains the cornerstone of therapy to improve outcomes, as supported by an analysis of 116 patients that found that gross total resection improved outcome with a 5-year progression-free survival of 83% versus 55% in those with subtotal resection.1 regarding radiation, a systemic review of 95 histologic astroblastoma patients did not show a survival benefit with radiation therapy; however, the series had a broad age range from 1 to over 61 years of age and did not compare survival differences in astroblastoma with and without anaplastic features.14 review of the literature found that a 20-year-old woman with a spinal cord mn1-altered astroblastoma, and a 36-year-old man with a spinal astroblastoma with ewsr1-bend2 both had a reduction in tumor size after radiation, temozolomide, and bevacizumab.4,15 in another case, a 3-month old boy with ewsr1-bend2 astroblastoma had progressive disease after five days of temozolomide and etoposide.3 given the significant growth of the presented patient's tumor in two months (fig.1) and dramatically elevated ki67 proliferation index, he was treated with concurrent radiation with temozolomide followed by adjuvant temozolomide based on the “stupp” protocol and this appears to be a viable option, as he has had a partial response control of his tumor over one year later at the time of writing.16 this case exemplifies the use of advanced molecular testing, including the evaluation of chromosome fusions and methylation profiling, to accurately diagnose rare neoplasms of the cns. through these technologies, improved diagnosis accuracy provides clinically impactful insights that enhance the understanding of tumor biology. funding this work was supported by the national institutes of health t32 research training grant. authors’ contributions m.a.s case report concept, design, and critical revision of content. z.a., k.d.a, m.q., m.k.r., f.j.r., c.g.e and l.j.l. provided critical revision of content. acknowledgments the authors thank rust turakulov at the nih for help with formatting the methylation plot for publication. the authors also thank the patient and his caregivers. references 1. brat, d. j., hirose, y., cohen, k. j., feuerstein, b. g. & burger, p. c. astroblastoma: clinicopathologic features and chromosomal abnormalities defined by comparative genomic hybridization. brain pathology 10, 342–352 (2006). 2. louis, d. n. et al. cimpact-now update 6: new entity and diagnostic principle recommendations of the cimpact-utrecht meeting on future cns tumor classification and grading. brain pathol bpa.12832 (2020) doi:10.1111/bpa.12832. 3. yamasaki, k. et al. spinal cord astroblastoma with an ewsr1-bend2 fusion classified as a high-grade neuroepithelial tumour with mn1 alteration. neuropathol appl neurobiol 46, 190–193 (2020). 4. tsutsui, t. et al. path-23. adult spinal cord astroblastoma with ewsr1-bend2 fusion. neuro-oncology 22, iii429–iii429 (2020). 5. ramkissoon, s. h. et al. clinical targeted exome-based sequencing in combination with genome-wide copy number profiling: precision medicine analysis of 203 pediatric brain tumors. neuro oncol 19, 986–996 (2017). 6. sener, r. n. astroblastoma: diffusion mri, and proton mr spectroscopy. comput med imaging graph 26, 187–191 (2002). 7. sturm, d. et al. new brain tumor entities emerge from molecular classification of cns-pnets. cell 164, 1060–1072 (2016). 8. baroni, l. v. et al. treatment response of cns high-grade neuroepithelial tumors with mn1 alteration. pediatr blood cancer 67, (2020). 9. boisseau, w. et al. molecular profiling reclassifies adult astroblastoma into known and clinically distinct tumor entities with frequent mitogen-activated protein kinase pathway alterations. oncologist 24, 1584–1592 (2019). 10. mhatre, r. et al. mn1 rearrangement in astroblastoma: study of eight cases and review of literature. brain tumor pathol 36, 112–120 (2019). 11. burel-vandenbos, f. et al. a polyphenotypic malignant paediatric brain tumour presenting a mn1-patz1 fusion, no epigenetic similarities with cns high-grade neuroepithelial tumour with mn1 alteration (cns hgnet-mn1) and related to patz1 -fused sarcomas. neuropathol appl neurobiol 46, 506–509 (2020). 12. bonnin, j. m. & rubinstein, l. j. astroblastomas: a pathological study of 23 tumors, with a postoperative follow-up in 13 patients. neurosurgery 25, 6–13 (1989). 13. chen, w. et al. central nervous system neuroepithelial tumors with mn1-alteration: an individual patient data meta-analysis of 73 cases. brain tumor pathol 37, 145–153 (2020). 14. sughrue, m. e. et al. clinical features and post-surgical outcome of patients with astroblastoma. j clin neurosci 18, 750–754 (2011). 15. yamada, s. m. et al. primary spinal cord astroblastoma: case report. journal of neurosurgery: spine 28, 642–646 (2018). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. the multifaceted appearance of supratentorial ependymoma with zfta-maml2 fusion feel free to add comments by clicking these icons on the sidebar free neuropathology 2:24 (2021) case report the multifaceted appearance of supratentorial ependymoma with zfta-maml2 fusion ming liang oon1, lutfi hendriansyah2, patricia diana pratiseyo3, eka j wahjoepramono2, jian yuan goh4, chik hong kuick4, kenneth te chang4,5, arie perry6, char loo tan1,4 1 department of pathology, national university health system, singapore, singapore 2 department of neurosurgery, siloam hospital lippo village karawaci, tangerang, indonesia 3 department of pathology, siloam hospital lippo village karawaci, tangerang, indonesia 4 department of pathology and laboratory medicine, kk women's and children's hospital, singapore, singapore 5 duke-nus medical school, singapore, singapore 6 departments of pathology and neurological surgery, university of california, san francisco, california, usa corresponding author: char loo tan · department of pathology · national university hospital · 5 lower kent ridge road · singapore 119074 char_loo_tan@nuhs.edu.sg submitted: 12 june 2021 accepted: 11 september 2021 copyedited by: jerry j. lou published: 20 september 2021 https://doi.org/10.17879/freeneuropathology-2021-3397 keywords: ependymoma, zfta, c11orf95, maml2, myogenic abstract ependymomas are glial neoplasms with a wide morphological spectrum. the majority of supratentorial ependymomas are known to harbor zfta fusions, most commonly to rela. we present an unusual case of a 9-year-old boy with a supratentorial ependymoma harboring a noncanonical zfta-maml2 fusion. this case had unusual histomorphological features lacking typical findings of ependymoma and bearing resemblance to a primitive neoplasm with focal, previously undescribed myogenic differentiation. we discuss the diagnostic pitfalls in this case and briefly review the histological features of ependymoma with noncanonical gene fusions. our report underscores the importance of molecular testing in such cases to arrive at the correct diagnosis. supratentorial ependymomas with noncanonical fusions are rare, and more studies are necessary for better risk stratification and identification of potential treatment targets. introduction ependymomas (epn) form a diverse group of morphologically and molecularly heterogeneous glial neoplasms [5, 14]. while the classical histological appearance of epn is well-described, the morphological spectrum is broad and may confound diagnosis. unusual features encountered in ependymomas include chondro-osseous elements [13, 17], neuropil-like islands [6], melanin-containing cells [4], tumor giant cells [8], neuronal differentiation [16], granular cell features [18], et cetera. recent advances in genomic and methylome profiling have identified distinct molecular subgroups characterized by recurrent genetic or epigenetic alterations that may better inform diagnosis, clinical outcomes and risk stratification of epn as compared to conventional light microscopic diagnosis and histological grading [5]. the majority of supratentorial (st) epns have gene fusions involving either zfta (zinc finger translocation associated; previously known as c11orf95) or yap1. these gene fusions characterize the two molecular subgroups of st-epn: st-epn-zfta and st-epn-yap1, respectively [1, 14]. here, we present an unusual case of a st-epn with zfta-maml2 fusion demonstrating diagnostic challenges due to primitive morphology, suggesting an embryonal neoplasm and divergent differentiation, including hitherto undescribed myogenic differentiation. our case underscores the importance of molecular testing in such cases to arrive at the correct diagnosis. clinical summary a 9-year-old boy presented with a visible, enlarging right forehead lump for 2 months. the child complained of an occasional headache but was otherwise well without any focal neurological deficits. brain magnetic resonance imaging (mri) revealed a well-demarcated, right frontal cystic tumor with a mural nodule, surrounding vasogenic edema and midline shift (fig. 1a-c). the tumor showed a high choline peak and high choline:n-acetylaspartate (naa) ratio. the child underwent craniotomy, revealing a well-demarcated bosselated tumor with a clear interface between tumor and brain (fig. 1d-e). gross total resection of the tumor was achieved (fig. 1f), and the postoperative course was uneventful. he received adjuvant radiation therapy followed by temozolomide. the child remained well at follow up 9 months post-resection, with no radiological evidence of recurrence. figure 1. mri findings of the tumor. a) axial, b) coronal and c) sagittal views showing a cystic tumor with an enhancing multilobulated mural nodule in the right frontal lobe with mass effect. d) intraoperative view after lifting of dural flap, prior to resection e) dissection of a well-demarcated multinodular cystic tumor. f) final intraoperative view after gross total resection of the solid tumor showing part of cyst lining with ventricular wall. material and methods the specimen was routinely processed for paraffin embedding and staining with hematoxylin and eosin (h&e). immunohistochemistry (ihc) with commercially available primary antibodies, including gfap, olig2, synaptophysin, desmin, myogenin, myod1, cam 5.2, ema, ini1, brg1, lin28a, bcor, hmb45 and ki67, was performed following locally validated technical protocols. gene fusion detection was performed using a next-generation sequencing-based anchored multiplex polymerase chain reaction (pcr) assay (archer® fusionplex, boulder, co, usa) that can detect and identify gene fusions involving any of 101 covered genes, including rela, yap1, maml2, ncoa1 and ncoa2. briefly, total ribonucleic acid (rna) was extracted from formalin-fixed paraffin-embedded tissue sections of tumor. 150 ng of rna was used for library preparation utilizing the archer® fusionplex kit, according to the manufacturer’s protocol (archerdx, boulder, co, usa). the prepared library was sequenced using an illumina miniseq sequencer. the data obtained was analyzed by the archer data analysis (version 6.2.3) portal. a confirmatory reverse transcription-pcr was designed flanking the breakpoint of the two genes and the pcr product was sanger-sequenced. dna methylation analysis was performed using the illumina infinium human methylation epic beadchip kit according to published protocols [2, 7]. the results of the methylation profiling and copy number variation analysis were obtained using an automated web-based deoxyribonucleic acid (dna) methylation profiling program (molecularneuropathology.org) [12]. the case was also integrated onto the t-distributed stochastic neighbor embedding (t-sne) analysis in the german cancer research center (dkfz) database. results light microscopy sections showed a discrete tumor composed of hypercellular sheets of small, round to ovoid cells with hyperchromatic nuclei and indistinct cell boundaries, imparting a primitive appearance to the tumor. in other areas, the tumor cells formed nodular aggregates, short fascicles and palisades/ trabeculae, separated by fibrous septa imparting a desmoplastic appearance (fig. 2a). a minor subset of tumor cells showed prominent intracytoplasmic fine eosinophilic granules, some with a more globular appearance (fig. 2b). a minor component of the tumor was composed of spindled cells with eosinophilic cytoplasm reminiscent of skeletal muscle fibers (fig. 2c). focally, aggregates of tumor cells with eccentric nuclei and mucinous cytoplasm were also identified (fig. 2d). no other heterologous differentiation was seen. there was a distinct lack of a fibrillary background in the tumor. while some tumor cells appeared to congregate around large thick-walled vessels, no distinctive perivascular pseudorosettes were present. geographic areas of necrosis accompanied by dystrophic calcification were present. the mitotic count reached up to 8 per 10 high power fields (3 per mm2). figure 2. light microscopy and immunohistochemical findings of the tumor. a) low-power appearance of the tumor consisting of nests of tumor cells separated by fibrous septa, imparting a desmoplastic appearance. the bulk of the tumor cells show primitive cytomorphology with minimal cytoplasm, imparting a nondescript “small blue cell” appearance. no perivascular pseudorosettes are seen, although tumor cells aggregate along blood vessels. b) a minor subset of tumor cells showed prominent intracytoplasmic fine eosinophilic granules (arrowheads). c) rare tumor cells showed spindled morphology, resembling skeletal muscle fibers. d) aggregates of tumor cells with eccentric nuclei and mucinous cytoplasm (arrowheads). the tumor cells were positive for e) gfap. very focal areas showed myogenic differentiation, being positive for f) desmin, g) myogenin and h) myod1 (f-h were images taken from same area of the tumor.) clicking the figure will lead you to the full virtual slide (h&e). the tumor cells showed patchy positivity for gfap (fig. 2e), and focal reactivity for synaptophysin, cam 5.2 and ema (cytoplasmic; no dot/ring-like pattern was found). focally, some cells expressed desmin (fig. 2f) and nuclear reactivity for myogenin (fig. 2g) and myod1 (fig. 2h). no neurofilament-positive axons were identified, in keeping with the non-infiltrative nature of this tumor. there was retained expression for ini1 and brg1. olig2, lin28a, bcor and hmb45 stains were negative. the ki67 labeling index reached up to 40%. gene fusion detection and confirmation the archer fusionplex assay detected a zfta (exon 5)maml2 (exon 2) fusion (fig. 3a). this gene fusion was confirmed by rt-pcr with sanger sequencing of the amplicon product (fig. 3b). figure 3. a) archer fusionplex assay detected a zfta (exon 5; previously known as c11orf95)-maml2 (exon 2) fusion, which was confirmed by b) sanger sequencing. methylation analysis dna-based methylation analysis of the tumor revealed a methylation class corresponding most closely to “ependymoma, rela fusion”, with a calibrated score of 0.65. concurrent copy number analysis revealed 1q gain and cdkn2a/b loss (fig. 4a). no chromothripsis of chromosome 11 was present. on the t-sne plot, our case localized to the methylation cluster of st-epn-rela, consisting of 444 cases including our case, out of a total of 3272 cases of ependymomas of different anatomical locations and molecular groups, lending further diagnostic confidence (fig. 4b). a final diagnosis of st-epn with zfta-maml2 fusion was rendered after synthesizing the pathological and molecular findings of this case. figure 4. a) copy number variation profile of the tumor, with 1q gain and cdkn2a/b losses. b) t-distributed stochastic neighbor embedding (t-sne) plot containing a total of 3272 cases of epns for cluster analysis. our case localized to methylation cluster of supratentorial epn with rela fusion (n= 444, including our case) (inset, case indicated in black). clicking the figure will lead you to a high-resolution version. discussion zfta, now recognized as a key player in the oncogenesis of st-epn, confers transforming capabilities upon oncogenic fusion proteins by increasing their translocation to the nucleus and permitting access to the transcriptional machinery [9]. most commonly, zfta is fused to rela [9, 14, 19]. being promiscuous, zfta may also rarely fuse with alternative partners such as maml2, maml3, ncoa1, ncoa2 and ctnna2 [14, 18, 19]. of note, the zfta-maml2 fusion has only been identified in 18 cases of st-epn in the literature thus far, including the current case [14, 18, 19]. tumors with such alternative fusion partners form satellite clusters around the previously known st-epn-rela cluster in the t-sne plot of methylation profiles and show distinct transcriptional profiles [19]. while most st-epn-zfta demonstrate classical features of epn such as perivascular pseudorosettes and fibrillary matrix, a recent paper by zheng et al found that epns with zfta fusions with alternative partners tend to demonstrate unusual histological features. these include sarcomatous features, areas mimicking high grade central nervous system (cns) neoplasms like diffuse high-grade glioma, cns embryonal tumors and other primitive tumors [19]. such atypical histological findings are in keeping with the case we present herein. similar to the case by tamai et al, our case also demonstrated granular cell features [18]. in their case, the granular cells showed eccentrically placed nuclei and cytoplasm filled with eosinophilic granules which were positive for periodic acid-schiff (pas) and alpha-1-antitrypsin stains. in addition, our case showed a small population of tumor cells with unequivocal expression for desmin, myogenin and myod1, consistent with myogenic differentiation, a finding which has not been previously described. initial diagnosis on the basis of light microscopy was difficult because of the atypical morphologic features of the tumor including the conspicuous absence of a fibrillary stroma, the absence of classical epn morphology, and the presence of primitive-appearing cells in a desmoplastic background. coupled with the polyphenotypic immunoprofile, which included desmin reactivity, the differential of desmoplastic small round cell tumor was initially entertained [10]. however, the subsequent molecular finding of the zfta-maml2 gene fusion and the absence of an ewsr1-wt1 fusion clinched the diagnosis of st-epn with zfta-maml2 fusion and led to revision of the histologic diagnosis. recurrent fusions involving zfta are characteristic of epn and fusions involving this gene have only been described previously in chondroid lipoma [15]. no primitive tumor has been described in the literature with this characteristic fusion to date. while lacking in histological elements of classical epn, the circumscribed, non-infiltrative nature of this tumor and the presence of the signature fusion and methylation profile were all in keeping with a diagnosis of st-epn. the novel finding of myogenic differentiation, likely attributable to metaplasia of the neoplastic glial component or the mesenchymal component of the tumor cells, broadens the spectrum of heterologous differentiation in epn [3]. the protean morphological manifestations of epn has the potential to obfuscate morphological diagnosis, particularly when classical epn features are not present. this underscores the limitations of morphological evaluation alone and the necessity of molecular testing. there has been longstanding controversy surrounding the utility of histological grading of epn. while st-epn-rela was found to be associated with adverse patient outcomes in one retrospective study [14], this was not reproduced in another trial-based study [11]. interestingly, mice with epn induced by zfta-maml2 fusion demonstrated a reduced survival compared to those with zfta-rela fusion [19]. out of the 18 cases of st-epn with zfta-maml2 fusion, follow up data was only available in two cases. both cases showed anaplastic features, corresponded to who grade 3. the case reported by tamai et al. showed no recurrence for 30 months without chemoradiotherapy, which may indicate a favorable prognosis [18]. our patient remained disease free at 9 months follow up. owing to the paucity of survival data, it remains to be seen if differences in survival exist in st-epn with alternative fusions. similarly, the utility of assignment of a who grade to molecularly defined epn remains insufficiently characterized [5]. additional studies are necessary to better delineate the behavior of these tumors, which may inform treatment decisions and risk stratification. in conclusion, we present an unusual case of st-epn with zfta-maml2 fusion exhibiting primitive morphology and a focal area of previously undescribed myogenic differentiation. awareness of the multifaceted appearance of epn is critical and an integrated clinico-pathological-molecular diagnosis is essential for the contemporary diagnosis of this entity. acknowledgements we would like to thank drs. felix hinz and felix sahm from the german cancer research center (dkfz) for their invaluable help in getting the t-sne data of our case. references andreiuolo f, varlet p, tauziède-espariat a, jünger st, dörner e, dreschmann v, et al (2019) childhood supratentorial ependymomas with yap1-mamld1 fusion: an entity with characteristic clinical, radiological, cytogenetic and histopathological features. brain pathol 29: 205-216 https://doi.org/10.1111/bpa.12659 capper d, jones dtw, sill m, hovestadt v, schrimpf d, sturm d, et al (2018) dna methylation-based classification of central nervous system tumours. nature 555: 469-474 https://doi.org/10.1038/nature26000 chakraborti s, govindan a, alapatt jp, radhakrishnan m, santosh v (2012) primary myxopapillary ependymoma of the fourth ventricle with cartilaginous metaplasia: a case report and review of the literature. brain tumor pathology 29: 25-30 https://doi.org/10.1007/s10014-011-0059-8 chan ac, ho lc, yip ww, cheung fc (2003) pigmented ependymoma with lipofuscin and neuromelanin production. archives of pathology & laboratory medicine 127: 872-875 https://doi.org/10.5858/2003-127-872-pewlan ellison dw, aldape kd, capper d, fouladi m, gilbert mr, gilbertson rj, et al (2020) cimpact-now update 7: advancing the molecular classification of ependymal tumors. brain pathol 30: 863-866 https://doi.org/10.1111/bpa.12866 gessi m, marani c, geddes j, arcella a, cenacchi g, giangaspero f (2005) ependymoma with neuropil-like islands: a case report with diagnostic and histogenetic implications. acta neuropathologica 109: 231-234 https://doi.org/10.1007/s00401-004-0927-y illumina (2019) infinium hd methylation assayreference guide. jeon yk, jung hw, park sh (2004) infratentorial giant cell ependymoma: a rare variant of ependymoma. pathology, research and practice 200: 717-725 https://doi.org/10.1016/j.prp.2004.08.003 kupp r, ruff l, terranova s, nathan e, ballereau s, stark r, et al (2021) zfta-translocations constitute ependymoma chromatin remodeling and transcription factors. cancer discovery: candisc.1052.2020 https://doi.org/10.1158/2159-8290.cd-20-1052 lee jc, villanueva-meyer je, ferris sp, cham em, zucker j, cooney t, et al (2020) clinicopathologic and molecular features of intracranial desmoplastic small round cell tumors. brain pathol 30: 213-225 https://doi.org/10.1111/bpa.12809 merchant te, bendel ae, sabin nd, burger pc, shaw dw, chang e, et al (2019) conformal radiation therapy for pediatric ependymoma, chemotherapy for incompletely resected ependymoma, and observation for completely resected, supratentorial ependymoma. j clin oncol 37: 974-983 https://doi.org/10.1200/jco.18.01765 molecularneuropathology.org (2020) mnp welcome https://www.molecularneuropathology.org/mnp. accessed 10 august 2021. mridha ar, sharma mc, sarkar c, garg a, singh mm, suri v (2007) anaplastic ependymoma with cartilaginous and osseous metaplasia: report of a rare case and review of literature. journal of neuro-oncology 82: 75-80 https://doi.org/10.1007/s11060-006-9239-5 pajtler kw, witt h, sill m, jones dt, hovestadt v, kratochwil f, et al (2015) molecular classification of ependymal tumors across all cns compartments, histopathological grades, and age groups. cancer cell 27: 728-743 https://doi.org/10.1016/j.ccell.2015.04.002 parker m, mohankumar km, punchihewa c, weinlich r, dalton jd, li y, et al (2014) c11orf95-rela fusions drive oncogenic nf-κb signalling in ependymoma. nature 506: 451-455 https://doi.org/10.1038/nature13109 rodriguez fj, scheithauer bw, robbins pd, burger pc, hessler rb, perry a, et al (2007) ependymomas with neuronal differentiation: a morphologic and immunohistochemical spectrum. acta neuropathologica 113: 313-324 https://doi.org/10.1007/s00401-006-0153-x salazar mf, tena-suck ml, ortiz-plata a, salinas-lara c, rembao-bojórquez d (2017) lipomatous/extensively vacuolated ependymoma with signet-ring cell-like appearance: analysis of a case with extensive literature review. case reports in pathology 2017: 8617050 https://doi.org/10.1155/2017/8617050 tamai s, nakano y, kinoshita m, sabit h, nobusawa s, arai y, et al (2021) ependymoma with c11orf95-maml2 fusion: presenting with granular cell and ganglion cell features. brain tumor pathol 38: 64-70 https://doi.org/10.1007/s10014-020-00388-6 zheng t, ghasemi dr, okonechnikov k, korshunov a, sill m, maass kk, et al (2021) cross-species genomics reveals oncogenic dependencies in zfta/c11orf95 fusion-positive supratentorial ependymomas. cancer discov: https://doi.org/10.1158/2159-8290.cd-20-0963 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. reflection on my 37 years of practice as a neuropathologist: home alone. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:30 (2020) reflections reflection on my 37 years of practice as a neuropathologist. home alone. mara popović institute of pathology, faculty of medicine, university of ljubljana corresponding author: mara popović · mara popović · institute of pathology · faculty of medicine · university of ljubljana · korytkova 2, 1000 ljubljana · slovenia mara.popovic@mf.uni-lj.si submitted: 20 october 2020 accepted: 27 october 2020 copyedited by: nima sharifai published: 2 november 2020 https://doi.org/10.17879/freeneuropathology-2020-3062 additional resources and electronic supplementary material: supplementary material keywords: reflections, neuropathology early life and education i was born 70 years ago in the small town of sinj, dalmatia, croatia, yugoslavia. when i was 5 months old, my father was sent to prison for two years on goli otok (bare island), an ill-famed prison for politically inappropriate people at that time. at 24 years old, he could not understand that stalin, who was an idol for young communists, had become an enemy overnight after the informbiro resolution, by which the yugoslav communist party was expelled from the international communist party led by stalin. it was a price for living free on the outer side of the iron curtain, between east and west, where yugoslavia remained until the end of 1990, when the balkan wars started. my father has stuck with communist ideas until today, almost 95 years old (figure 1). under his influence, i entered the communist party in high school, but very quickly realized that politics is not something i would like to practice. figure 1. my father and our late dog maša 15 years ago. after primary school, i attended classical gymnasium “natko nodilo” in split, croatia, where, in addition to other high school subjects, i learned latin and ancient greek. at that time, my life consisted of studying, taking care of my two younger brothers, and helping my mother in everyday domestic jobs. after gymnasium in 1969, i decided to study medicine, mainly because it was not available in split at that time. i wanted to distance myself from my primary family to escape my mother’s control. i wanted to be free. my mother supported me in my decision (figure 2). i finished faculty of medicine at the university of zagreb in january 1975. after one year of obligatory practice, my first employment was in the emergency clinic in karlovac, where i worked for three years alternating between twelve-hour day and night shifts, as i traveled from my residence in zagreb. it was a very stressful job for a young, inexperienced m.d., and i was terrified of the possibility of doing something wrong. i was looking for a residency in pathology in zagreb, but the salary for a beginner was so small that i could not pay rent for my small apartment. i therefore moved to a small town in slovenia, ravne na koroškem, which has a large iron factory and where i in cared for factory workers’ health. i received an apartment from the health centre, only having to pay a small rent. figure 2. my mother in her youth in the national costume of continental dalmatia, which she was wearing just for the photo. my family i met my husband srđan during my third year of medical faculty. he had just entered the faculty of mechanical engineering. he was a city boy from zagreb, full of energy, honest and kind. he introduced another dimension into my life – fun and entertainment. every weekend we spent together, we joined his friends and enjoyed life. three years later, i completed my study of medicine. we married in 1978, just before he was conscripted into the army for one year. figure 3. me and my husband long ago on the top of one of the slovene hills we had reached by bike. after military service of one year, my husband joined me in the small town in slovenia, but he was not very happy there. he missed the greater opportunities of city life. our daughter saša was born in 1980, and our son marko in 1983. in 1982 i obtained a residency in pathology in the nearby small town of slovenj gradec, and went to study pathology at the institute of pathology, faculty of medicine, university of ljubljana (ip fm ul). after two years of residency, the institute offered me a staff position, and i accepted. my husband was very happy. he obtained a good position at an engineering company in ljubljana and we have remained in ljubljana to this day. my husband has supported me throughout my career, taking care of our children, but asking that the weekends must be ours (at least sundays). so throughout our life together we spent our weekends practicing many sports: tennis, jogging, hiking, mountain climbing, cycling. during the summer we would wind-surf as well, and during the winter we would ski (and later cross country skiing). we became serious mountain bikers, practicing twice a week for years (figure 3). last year, for my 69th birthday, i switched to an e-mtb bike. i use only eco power, the smallest of five levels of support, but it helps me a lot and i no longer suffer. our daughter saša is an architect, single, loves cats and especially loves her dog cloe, a young beagle with a particularly stubborn character (figure 4). our son marko is a happily married father of two sons (figure 5). figure 4. our daughter saša and her dog cloe. figure 5. our son marko with family. my mother (91) and my father (almost 95) live in split, in their own home, with no sign of dementia and without any medication except vitamin b12 for my mother, who looks after the two of them (figure 6). my father goes to a nearby café every day to drink coffee and read the newspaper, and my mother goes every day to the market across the street to buy fresh vegetables, fish, fruit and other things. last year, they celebrated 70 years of marriage. figure 6. my parents, 94 and 90 years old, last year. neuropathology in the third year of my residency in pathology, in 1984, prof. danilo tavčar, the pathologist who established the department of neuropathology (np) at the ip fm ul in 1972, invited me to join the department and become his assistant. i was also invited by prof. nina gale, who was head of the department of head and neck pathology. i needed some days to decide which invitation to accept. prof tavčar was almost 70 years old, and i would very soon remain alone in a quite unknown field of pathology. nina gale was only a little older than me, a very successful and prominent head and neck pathologist. it would be much easier to accept her offer, but the unknown field of np attracted and challenged me more. so i accepted prof. tavčar’s offer, and i did not regret the decision, even though it was not easy. i worked with prof. tavčar for two years parallel to my residency in pathology, which i completed in 1986. in the meantime, i attended postgraduate study for neurology in zagreb, which lasted four months during the academic year 1984/1985. my working days were in zagreb, and i spent the weekends with my family in ljubljana. my husband took care of our 4-year-old daughter and our 1.5-year-old son spent the working days with my husband’s parents in zagreb, and travelled with me for weekends to ljubljana. prof. tavčar took me to the international congress of neuropathology in stockholm in 1986 where we met srečko pogačar (1929 to 2018) (figure 7), a slovene neurologist and the first slovenian neuropathologist to have gone to rhode island medical centre in the usa to study neuropathology, in 1965. srečko had been the first to establish a np lab at the clinic of neurology in ljubljana in the early sixties. when he came back from the usa to ljubljana, the np lab no longer existed. disappointed, srečko decided to leave slovenia and continue his career in the usa practicing psychiatry and neuropathology, especially brain cuttings. he was a lecturer on neuropathology and neurology at harvard and brown universities [1]. figure 7. international congress of neuropathology stockholm 1986. from the left: prof. danilo tavčar, myself, prof. tavčar’s wife maca, and srečko pogačar. prof. tavčar undertook further training in np in massachusetts general hospital, boston, under dr. e.p. richardson, for six months in 1974. he shared all his knowledge of np with me. in the first half of the 1980s, immunohistochemistry started to become a useful tool in pathology. it was a very attractive challenge for me. prof. tavčar left me to do that job. after we returned from stockholm, he experienced an epileptic attack in his left leg – jackson’s epilepsy for the first time. he knew what it meant. after a ct-scan, which showed a focal lesion in the right parietal lobe, on which he was operated, he did not want to see the biopsy. he worked until spring 1987, going to radiotherapy from his office, and did not want to take any of the alternative therapies that his wife had asked him to. he died in december 1987, twelve months after his first epileptic attack. i stayed in contact with srečko pogačar, who offered to organize a six-month fellowship for me at rhode island hospital (rih) in providence, where i could learn neuropathology under the mentorship of the now deceased dr. mary ambler, a neurologist and neuropathologist. it was a great opportunity that i had to seize. i reached an agreement with the current chief of ip mf ul, prof. dušan ferluga, and with my family. my daughter was 9-years-old and my son 6-years-old. my husband had a good job in ljubljana and could not drop everything to go with me. i was enthusiastic and happy for the opportunity to be more confident in np. i remember the early morning in zagreb when my husband drove me to the airport. my children stayed with their grandpa and grandma. i had a cramp in my epigastrium from the time i boarded the airplane in zagreb until i landed six months later at the same airport and hugged my children and my husband. at that moment the cramp disappeared. professionally and otherwise, i had a good time in the usa, but i missed my family a lot. no skype, no facebook, no sms, no whatsapp, no e-mail. only atlantic surface mail and expensive telephone calls once a week with my husband. figure 8. six months training in neuropathology in rhode island hospital. a. mentor mary ambler, me and rosemarie guglielmi. b. mary ambler organized a leaving party for me. dr. mary ambler was a great person and teacher, and her technician rosemarie guglielmi still remains my dear friend (figure 8). while in the usa, i spent one week in the department of pathology at cleveland university hospital, where i worked with another slovene, uroš roessmann, whom i met at icnp in stockholm in 1986 (figure 9). he gave me the opportunity to see more child brain tumours, which rih did not have. uroš became a good friend and, after he retired in 1991, he visited slovenia twice, spending three months each time at the ip mf ul as a visiting professor. it was another valuable opportunity to learn more from an experienced neuropathologist. figure 9. international congress of neuropathology stockholm 1986. from the left: maca and danilo tavčar, milči and uroš roesmann, and me. before going to the usa, i had joined the research group of janez sketelj (figure 10), pathophysiologist, eminent researcher and head of the institute of pathophysiology mf, ul. we had planned research on the regeneration of the rat sciatic nerve after various types of injury, which i continued to pursue after coming back to ljubljana. this research resulted in my msc in 1992, phd in 1996 and four articles [2-5]. figure 10. janez sketelj, my mentor for msc and phd. i was left without a mentor or colleague at the institute who could help me in cases of rare brain tumours and brain pathology. in september 1987, i attended the danube neurology symposium in innsbruck, where i met herbert budka, a well-known neuropathologist and head of the institute of neurology in vienna (figure 11). figure 11. herbert budka, who i met for the first time at the danube symposium of neurology in innsbruck in 1987. throughout almost my entire career in np, until his retirement in 2011, he and his staff were a great help to me and to slovene np. i visited them several times and spent three months with them in 2007. it was an excellent way of learning, through a daily microscopic session with herbert and all the others around a multiple head microscope. i remember asking herbert why they had to look at every meningioma. “you have to see a lot of ordinary meningiomas to recognize a peculiar one” herbert said, and he was right. in 1995, herbert budka invited slovenia to join a cjd surveillance project led by him and financed by the eu. i have very pleasant memories of the annual meetings in baden near vienna, with excellent scientific and social programs, where i learned a lot about prion diseases and had an enjoyable time with scandinavian neuropathologists (figure 12). as a result of that program, ip mf ul established a slovene cjd surveillance system together with the national institute of public health and slovenian neurologists [6, 7]. ip mf ul became the referral centre for cjd in slovenia. to date, we have confirmed 88 scjd cases, one genetic cjd, two cases of gerstmann-sträussler-scheinker syndrome (gss, a a mother in 2007 [8] and her son in 2020). a case of clinically obvious fatal insomnia with a new n181s prnp mutation, without family history, was recognized in 2015. despite regulations surrounding this project by slovenian law in 2001, some clinically suspected cjd cases and the case of fatal insomnia escaped autopsy. fifty-three clinically suspected cjd cases were disproved by autopsy. a prion laboratory with containment level 3 was established in 2000, supported by the ministry of health and stimulated by the work of vladka čurin šerbec and her team from the centre for transfusion medicine in ljubljana, who produced an anti-prp antibody named v5b2 with the same quality as other commercially available anti-prp antibodies. four phds and several valuable papers have been produced in that lab [9-12]. thanks to herbert and the cjd surveillance system, the annual incidence of scjd increased in slovenia from 0,5 per 1 mil (in the period 1985-2000) to 1,5 per 1 mil (in the period 2001-2020). in the last ten years, specifically, the incidence reached 2,5 per 1 mil [13, 14]. slovenia is still a member of the cjd surveillance project, which is now lead by the united kingdom. we have not identified any variant cjd (vcjd) cases in slovenia, however a case of scjd, mv2a molecular subtype, had clinical, radiological and pathological similarities with vcjd [15]. figure 12. scandinavian neuropathologists, from the left: sverre mork, me, peter stubbe teglbjaerg and henning laursen. excellent company at the annual cjd meetings in baden by vienna. my collaboration with the institute of neurology at akh in vienna was also fruitful in tumour pathology. herbert budka, christine haberler, johannes hainfellner and ellen gelpi were of great help in my rare and difficult brain tumour cases, which resulted in several papers [16-18]. ellen gelpi and gabor g kovacs recognized one of our cases of brainstem tauopathy [19], to probably be a new entity of anti-iglon5-related tauopathy [20]. figure 13. a. the late john kepes, an excellent neuropathologist and person. b. his letter announcing that he would no longer be available for consultation. another neuropathologist from the usa also provided me with great help, the late john kepes, who was a very nice person. not only would he send me his opinions on cases i had sent him for consult, but he also sent microphotographs to point out the main changes important for the case diagnosis. i was very touched when he sent me a letter with an apology that he was unable to help any more (figure 13). i developed my academic career at the ip mf ul and became full professor of pathology in 2011. even though it is my duty to research, i am not a researcher at heart. i don’t like statistics but i really enjoy studying every brain, especially neurodegenerative ones. during my career i have attended all european and some international congresses of np, presenting these cases (my cv in supplement). i would like to highlight some of which i am proud. i identified and published neuropathological changes in four family members with a p364s mapt mutation. the mutation was very new and had been described by italian researchers two years previously [21]. they described the clinical presentation of the patient, made recombinant tau with the mutation, and showed that under in vitro conditions “p364s tau revealed a remarkable increase in aggregation rate with respect to wt tau, displaying mostly protofibrils and some short fibrils after 24 hours, and a consistent presence of both straight and twisted long fibrils after 5 days. the propensity of p364s tau to aggregate was even higher than p301l tau used as well-known mutated control.” figure 14. composite neuronal tau inclusion (cnti) in a family with a p136s mutation in mapt. a. h&e, b. gallyas silver staining displays only the peripheral part of the inclusion, which is 4rtau positive, while the central part is only 3rtau positive in double immunohistochemistry on 3r and 4r tau (in c). i recognized that this mutant tau produced in all affected family members a new neuronal tau inclusion consisting of two components (figure 14). we designated it composite neuronal tau inclusion (cnti) [22]. additionally, i discovered that in one family member’s brain, in addition to cnti, all other neuronal tau inclusions known so far were present [23]. another interesting result of brain research was a case of a young male with paranoid schizophrenia, who developed bulbar symptoms and died in his sleep. a few months before death, he was diagnosed by mri as having multiple sclerosis. the brain cutting revealed that he had light chain deposition disease (lcdd) brain vasculopathy with multifocal hypoxic brain injury, especially prominent and fatal in the medulla (figure 15). when we submitted the manuscript to the journal human pathology, it was the first case of lcdd restricted to the brain [24]. by the time of publication, however, the first published case of lcdd restricted to the brain had been published by fischer et al. [25] figure 15. severe light chain deposition disease vasculopathy (lcddv) of medulla. h&e, original magnitude 40x. my most exciting and successful research in brain pathology was a case of zika virus-induced microcephaly, with the first confirmation of causality displaying the virus in the foetal brain by pcr, immunohistochemistry using maternal sera, and by electron microscopy [26]. several papers were produced on this case thanks to our young resident at that time, jernej mlakar, who performed a perfect brain autopsy (figure 16) [27-29]. jernej is now a general pathologist involved in np together with three other fields of pathology. i was lucky and privileged only having done np throughout my career as a pathologist. even though i was a solitary neuropathologist for most of my np career, several valuable people were included in np, without whom my work would not have been possible. two excellent np technicians, dori jazbec and marija zupančič; two administrators, marija blejc and mojca keber; immunohistochemical masters, daniel velkavrh, majda dimnik, miša omerzel and ajla hajrlanović; geneticists alenka matjašič, ema boštjančič and andrej zupan (the most invaluable people in degenerative and especially in tumour brain pathology today) [8, 15, 22, 23, 30-32], and brain cutting assistants, janez caserman, srđan čekić and damir novak. last but not least, i should mention the numerous students and residents of pathology, neurology and neurosurgery to whom i have tried to impart all my knowledge of np. i have also learned a lot from them. many of the residents prepared clinico-pathological conferences, which we had almost every fourth wednesday of the month before the coronavirus pandemic. figure 16. me (actual) and jernej mlakar, future head of department of neuropathology at the institute of pathology, faculty of medicine, university of ljubljana, slovenia. so i am approaching the end of my almost 37-year career in np. i have never regretted my choice, and even though i was the only neuropathologist in slovenia most of the time, i have always favoured mondays to fridays. i know that after the 1st july 2021, when my retirement will start, my life will not be the same. i will miss my beloved np a lot. acknowledgment i am deeply grateful for technical support to our it team, metod perme and miha juvan and our researcher, nina hauptman, who have always been available when my pc did not obey. new technology is great and very useful, but never makes mistakes, which is unbearable. references 1. pogacar, s. and m. popovic, dr. srecko pogacar: from a castle in slovenia to a clinic in ri. r i med j (2013), 2013. 96(10): p. 38-40. 2. popović, m., m. bresjanac, and j. sketelj, regenerating axons enhance differentiation of perineurial-like cells involved in minifascicle formation in the injured peripheral nerve. j neuropathol exp neurol, 1994. 53(6): p. 590-7. 3. sketelj, j., m. bresjanac, and m. popović, rapid growth of regenerating axons across the segments of sciatic nerve devoid of schwann cells. j neurosci res, 1989. 24(2): p. 153-62. 4. popović, m., m. bresjanac, and j. sketelj, role of axon-deprived schwann cells in perineurial regeneration in the rat sciatic nerve. neuropathol appl neurobiol, 2000. 26(3): p. 221-31. 5. popović, m., j. sketelj, and m. bresjanac, changes of schwann cell antigenic profile after peripheral nerve injury. pflugers arch, 1996. 431(6 suppl 2): p. r287-8. 6. popović, m., et al., creutzfeldt-jakob disease in slovenia from 1985 to 2003. wien klin wochenschr, 2004. 116(15-16): p. 524-9. 7. budka, h., concern about mad cow disease: end of the beginning, or beginning of the end? wien klin wochenschr, 2004. 116(15-16): p. 505-7. 8. kojović, m., et al., de novo p102l mutation in a patient with gerstmann-sträussler-scheinker disease. eur j neurol, 2011. 18(12): p. e152-3. 9. curin serbec, v., et al., monoclonal antibody against a peptide of human prion protein discriminates between creutzfeldt-jacob’s disease-affected and normal brain tissue. j biol chem, 2004. 279(5): p. 3694-8. 10. colja venturini, a., et al., anti-idiotypic antibodies: a new approach in prion research. bmc immunol, 2009. 10: p. 16. 11. lukan, a., et al., regional distribution of anchorless prion protein, prp226*, in the human brain. prion, 2014. 8(2): p. 203-9. 12. vranac, t., et al., a single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies. peptides, 2006. 27(11): p. 2695-705. 13. čakš jager, n., et al., analysis of 22 years of surveillance for prion diseases in slovenia, 1996 to 2017. zdr varst, 2018. 57(4): p. 227-33. 14. rus, t., et al., high incidence of sporadic creutzfeldt-jakob disease in slovenia in 2015: a case series. dement geriatr cogn dis extra, 2018. 8(1): p. 42-50. 15. bošnjak, m., et al., a case of mv2k subtype of sporadic creutzfeldt-jakob disease with florid-like plaques: similarities and differences to variant creutzfeldt-jakob disease. neuropathology, published online april 5, 2020. 16. gelpi, e., et al., pleomorphic xanthoastrocytoma with anaplastic features presenting without gfap immunoreactivity: implications for differential diagnosis. neuropathology, 2005. 25(3): p. 241-6. 17. filbin, m.g., et al., developmental and oncogenic programs in h3k27m gliomas dissected by single-cell rna-seq. science, 2018. 360(6386): p. 331-35. 18. clarke, m., et al., infant high-grade gliomas comprise multiple subgroups characterized by novel targetable gene fusions and favorable outcomes. cancer discov, 2020. 10(7): p. 942-63. 19. pretnar-oblak, j., et al., isolated bulbar paralysis in a patient with medullar tau pathology: a case report. j neurol neurosurg psychiatry, 2010. 81(8): p. 847-9. 20. gelpi, e., et al., neuropathological criteria of anti-iglon5-related tauopathy. acta neuropathol, 2016. 132(4): p. 531-43. 21. rossi, g., et al., new mutations in mapt gene causing frontotemporal lobar degeneration: biochemical and structural characterization. neurobiol aging, 2012. 33(4): p. 834.e1-6. 22. popović, m., et al., tau protein mutation p364s in two sisters: clinical course and neuropathology with emphasis on new, composite neuronal tau inclusions. acta neuropathol, 2014. 128(1): p. 155-7. 23. štrafela, p., et al., familial tauopathy with p364s mapt mutation: clinical course, neuropathology and ultrastructure of neuronal tau inclusions. neuropathol appl neurobiol, 2018. 44(6): p. 550-62. 24. popovic, m., et al., light chain deposition disease restricted to the brain: the first case report. hum pathol, 2007. 38(1): p. 179-84. 25. fischer, l., et al., a 19-year-old male with generalized seizures, unconsciousness and a deviation of gaze. brain pathol, 2006. 16(2): p. 185-6, 187. 26. mlakar, j., et al., zika virus associated with microcephaly. n engl j med, 2016. 374(10): p. 951-8. 27. onorati, m., et al., zika virus disrupts phospho-tbk1 localization and mitosis in human neuroepithelial stem cells and radial glia. cell rep, 2016. 16(10): p. 2576-592. 28. štrafela, p., et al., zika virus-associated micrencephaly: a thorough description of neuropathologic findings in the fetal central nervous system. arch pathol lab med, 2017. 141(1): p. 73-81. 29. vesnaver, t.v., et al., zika virus associated microcephaly/micrencephaly-fetal brain imaging in comparison with neuropathology. bjog, 2017. 124(3): p. 521-25. 30. matjašič, a., et al., a novel ptprz1-etv1 fusion in gliomas. brain pathol, 2020. 30(2): p. 226-34. 31. matjasic, a., et al., expression of loc285758, a potential long non-coding biomarker, is methylation-dependent and correlates with glioma malignancy grade. radiol oncol, 2017. 51(3): p. 331-41. 32. matos, b., et al., dynamic expression of 11 mirnas in 83 consecutive primary and corresponding recurrent glioblastoma: correlation to treatment, time to recurrence, overall survival and mgmt methylation status. radiol oncol, 2018. 52(4): p. 422-32. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neuromuscular disease feel free to add comments by clicking these icons on the sidebar >free neuropathology 1:4 (2020) review top ten discoveries of the year: neuromuscular disease marta margeta department of pathology, university of california, san francisco, ca, usa corresponding author: marta margeta · ucsf pathology, box 0511 · 513 parnassus ave., hsw-514 · san francisco, ca 94143 · usa marta.margeta@ucsf.edu submitted: 15 january 2020 accepted: 21 january 2020 published: 23 january 2020 https://doi.org/10.17879/freeneuropathology-2020-2627 keywords: anterior horn disease, congenital myopathy, inflammatory myopathy, muscular dystrophy, neuropathy, radiculopathy, rna-seq, gene therapy abstract this review highlights ten important advances in the neuromuscular disease field that either were first reported in 2019, or have reached a broad consensus during that year. the overarching topics include (i) new / emerging diseases; (ii) advances in understanding of disease etiology and pathogenesis; (iii) diagnostic advances; and (iv) therapeutic advances. within this broad framework, the individual disease entities that are discussed in more detail include myoglobinopathy, popdc3-mutated limb-girdle muscular dystrophy, neuromuscular adverse events associated with the immune checkpoint inhibition therapy, neuroglial stem cell-derived inflammatory pseudotumor of the spinal cord and spinal cord roots, acute flaccid myelitis, congenital myopathies, idiopathic inflammatory myopathies (with particular emphasis on immune-mediated necrotizing myopathies and sporadic inclusion body myositis), spinal muscular atrophy, and duchenne muscular dystrophy. in addition, the review highlights several diagnostic advances (such as diagnostic rna sequencing and development of digital diagnostic tools) that will likely have a significant impact on the overall neuromuscular disease field going forward. abbreviations aav, adeno-associated virus; afm, acute flaccid myelitis; ai, artificial intelligence; asm, antisynthetase syndrome-associated myositis; aso, antisense oligonucleotide; casa, chaperone-assisted selective autophagy; dm, dermatomyositis; dmd, duchenne muscular dystrophy; ema, european medicines agency; ev, enterovirus; fda, federal drug administration; hmgcr, 3-hydroxy-3-methylglutaryl-coa reductase; ifn, interferon; iim, idiopathic inflammatory myopathy; imnm, immune-mediated necrotizing myopathy; irae, immune-related adverse event secondary to immune checkpoint inhibition; irmg, myasthenia gravis secondary to immune checkpoint inhibition; irmyositis, inflammatory myopathy secondary to immune checkpoint inhibition; irneuropathy, immune-mediated neuropathy secondary to immune checkpoint inhibition; mhc, major histocompatibility complex; mngs, metagenomic next generation sequencing; msa, myositis-specific antibodies; mxa, myxovirus resistance protein a; nm, nemaline myopathy; nmd, neuromuscular disease; n-scipt, neuroglial stem cell-derived inflammatory pseudotumor; pm, polymyositis; rna-seq, transcriptome analysis (rna sequencing); sibm, sporadic inclusion body myositis; sma, spinal muscular atrophy; srp, signal recognition particle; temra cells, terminally differentiated effector memory t cells; wes, whole exome sequencing introduction 2019 was an exciting year for the neuromuscular disease (nmd) field, with important developments on multiple fronts and in many different disease categories. in this review paper, i will summarize ten 2019 nmd discoveries that i consider to be most important and/or interesting; they are grouped in four different “discovery clusters” and listed in no particular order. (like most classifications, this one is not perfect – a few discoveries included in the “etiology/pathogenesis cluster” have direct implications for diagnostics and/or treatment of nmds – but i hope it will improve the overall readability of the review.) newly defined / emerging neuromuscular diseases 1. newly defined genetic diseases even as our understanding of the nmd genetics inches closer to completion, new genetic nmds continue to be defined; in this review, i will summarize key features of two particularly interesting entities that were first characterized in 2019. myoglobinopathy is caused by the c.292c>t, p.his98tyr mutation in myoglobin, a small cytoplasmic hemoprotein that buffers intracellular o2 concentration and regulates the intracellular redox potential in cardiomyocytes and type 1 muscle fibers (olive et al., 2019). through an effort that involved two research teams on two different continents, this mutation was detected in 14 individuals from six different families, all of whom were initially identified based on the unique clinicopathologic features of their disease. clinically, myoglobinopathy presents with a weakness of the proximal lower limb and axial muscles; the symptoms start in the fourth to fifth decade and are slowly progressive, ultimately involving distal lower limb, upper limb, and respiratory muscles. creatine kinase levels are usually normal, while cardiac involvement is clinically apparent in approximately half of the affected individuals. pathologically, all muscle biopsies (as well as the hearts from two individuals who underwent autopsy) showed characteristic sarcoplasmic bodies that are brown on h&e stain, red on modified gomori trichrome stain, and highly electron dense on electron microscopy (fig. 1); interestingly, these sarcoplasmic inclusions rarely contain myoglobin and seem to be a product of increased lipid oxidation. (late stage biopsies also show chronic myopathic changes and autophagic impairment including rimmed vacuoles.) although additional work will be required to fully elucidate the molecular mechanisms underlying this newly defined disease, the initial biochemical experiments suggest that mutant myoglobin is more prone to the loss of the heme moiety and is associated with elevated intracellular superoxide levels, suggesting that impaired redox balance plays a role in the pathogenesis of this disease. figure 1. ultrastructural characterization of sarcoplasmic bodies, the morphological hallmark of myoglobinopathy: electron micrographs (a–d) show collections of highly electron-dense bodies with some less dense material at their periphery. the sarcoplasmic bodies are seen under the sarcolemma (a), often next to the nuclei. some sarcoplasmic bodies are surrounded by a membrane (b). sarcoplasmic bodies of different electron densities are seen near several vesicular structures (c) and were observed in the cardiac muscle obtained post mortem from one of the affected individuals (d). electron micrographs (e–g) and the corresponding nanoscale secondary ion mass spectrometry images (h–j). blue indicates sulfur (32s), red phosphorus (31p), and green iron (56fe), respectively. sarcoplasmic bodies are seen interspersed between the myofibrils (e), next to nuclei (f), or inside an autophagic vacuole (g). note the high-sulfur signal in the sarcoplasmic bodies (h–j), and the iron signal (green dots within the sarcoplasmic bodies in i, j). scale bar in a = 2 µm, b = 5 µm, c = 0.5 µm, d = 1 µm, h–j = 4 µm. [this figure and its legend were adopted from figure 5 in (olive et al., 2019); this use is permitted under the creative commons attribution 4.0 international license.] a different, purely genetic approach was used to define a new autosomal recessive form of limb-girdle muscular dystrophy that is caused by biallelic mutations in popdc3 gene: 5 affected individuals from 3 different families were identified via exome sequencing of a cohort of 1500 patients with presumed genetic limb-girdle weakness and elevated creatine kinase levels; their muscle biopsies showed dystrophic features of variable severity (vissing et al., 2019). popdc3 (popeye domain containing 3) gene encodes a membrane protein that regulates camp signaling, is highly expressed in skeletal and cardiac muscles, and has not yet been associated with any human disease. interestingly, patients with popdc3 mutations do not show a cardiac phenotype, while mutations in two other members of the same gene family (popdc1 and popdc2) cause a cardiac phenotype (generally a conduction disorder) accompanied by a variable skeletal muscle involvement (de ridder et al., 2019; vissing et al., 2019). the pathogenesis of this new disorder remains to be elucidated; however, initial studies suggest a loss-of-function mechanism that leads to altered camp modulation. 2. emerging therapy-related diseases introduction of new therapies has led to the emergence of unusual or completely novel nmd phenotypes; in 2019, two new nmd categories, previously described only in individual case reports or small case series, have been more thoroughly defined. immune-checkpoint inhibitor therapy has revolutionized treatment of patients with advanced cancers, but is often associated with immune-related adverse events (iraes) that most commonly affect the skin, gi tract, and endocrine glands, as well as the pns. neuromuscular iraes can be severe and include myasthenia gravis (irmg), myopathy (irmyositis), and peripheral neuropathy (irneuropathy; most commonly acute demyelinating polyradiculoneuropathy) (johansen et al., 2019; mohn et al., 2019; psimaras et al., 2019). while these neuromuscular iraes share some commonalties with their sporadic counterparts, several unique features have emerged. for example, up to 25% of the patients show more than one neuromuscular irae, with the combination of irmg and irmyositis encountered most often (johansen et al., 2019). furthermore, irmg is more likely to be negative for achr antibodies than idiopathic mg, while irmyositis is more likely to involve oculomotor and bulbar muscles than any of idiopathic inflammatory myopathies (iims); these features make clinical distinction between irmg and irmyositis difficult in many cases (johansen et al., 2019; mohn et al., 2019). in addition, irmyositis often involves axial muscles, while iims do not (touat et al., 2018). finally, a significant fraction of patients with irmyositis shows cardiac involvement, which is not typically seen in iim patients and has a poor prognosis (anquetil et al., 2018; johansen et al., 2019; mohn et al., 2019). interestingly, while the rates of irneuropathies (as well as the overall irae rates) are higher in patients treated with ipilimubab (the anti-ctla-4 antibody), irmg and irmyositis are particularly common in patients treated with nivolumab or pembrolizumab (the pd-1 blocking antibodies) (mohn et al., 2019). the pathogenesis of neuromuscular iraes is incompletely understood, but is thought to involve both type iv (t cell-mediated) and type ii (antibody-mediated) immune mechanisms (psimaras et al., 2019). on a tissue level, irmyositis can take multiple forms but the most commonly observed pattern [perimysial foci of cd8+ t cells and cd68+ macrophages that are associated with necrosis of adjacent muscle fibers; touat et al. (2018) and fig. 2] is distinct from the patterns seen in iims. interestingly, in some cases identical clonal cd8+ cells have been detected within the cancer tissue, skeletal muscle, and myocardium, suggesting that cross-reactivity between cancer cells and myocytes plays at least some role in the pathogenesis of irmyositis (psimaras et al., 2019). figure 2. myositis secondary to therapy with immune checkpoint inhibitors (irmyositis): a representative h&e-stained cryosection (a) shows patches of dense perimysial inflammation that focally extend into the endomysium and abut clusters of degenerating/regenerating muscle fibers; away from the inflammatory foci, muscle fibers have a relatively normal appearance. immunohistochemistry for cd8 (b) and histochemistry for acid phosphatase (c) show that the inflammatory infiltrate largely consists of cytotoxic t cells and macrophages, respectively. the patient was treated with pembrolizumab and presented with evidence of myocarditis as well as limb and bulbar weakness approximately 6 weeks following the last antibody dose. scale bars: a, 100 µm; b-c, 50 µm. in recent years, stem cell treatments have also risen in popularity, although they have no documented health benefits and are – at least for now – completely unregulated. in 2019, it was recognized that one relatively common form of stem cell treatment, intrathecal injection of allogeneic stem cells, can lead to development of distinctive lesions of the lumbosacral spinal cord and spinal cord nerve roots. these lesions, named neuroglial stem cell-derived inflammatory pseudotumors [n-scipts; (sloan et al., 2019)], can clinically mimic inflammatory and neoplastic disease processes and usually consist of a haphazardly organized neuroglial tissue that resembles a low-grade neuroglial tumor (fig. 3); typically, there is at least focal inflammatory infiltrate, which likely represents a host immune response to foreign antigens. while it seems likely that direct injection of allogeneic stem cells into other parts of the body could lead to development of stem-cell derived pseudotumors with non-neuroglial differentiation, only neuroglial scipts have been reported to date (sloan et al., 2019). from a diagnostic perspective, it is critical to be aware of the n-scipt existence, so that these lesions (which at the moment seem to be best handled through watchful waiting) are distinguished from the spinal cord tumors that would generally warrant a more aggressive treatment. advances in understanding of etiology and pathogenesis of neuromuscular diseases 3. acute flaccid myelitis: mounting evidence for causation by non-polio enteroviruses acute flaccid myelitis (afm) is a clinical syndrome characterized by flaccid limb weakness that develops acutely and has a lower motor neuron pattern; the current diagnostic criteria also include spinal cord imaging findings suggestive of anterior myelitis (messacar et al., 2018). the most common form of afm used to be poliomyelitis, which is caused by poliovirus [a member of the en terovirus (ev) genus]; as a result, afm became rare worldwide after introduction of the anti-polio vaccine in the 1950s. a small number of afm cases in the period from 1970-90 was attributed to infection with ev-a71 and a few other viruses (dyda et al., 2018; messacar et al., 2018). since 2012, however, afm has started to increase in incidence. the current form of this disease, first documented in california, is characterized by limb paralysis that occurs 5-7 days after a flu-like respiratory illness; it primarily affects young children (3-9 years of age) and peaks in incidence every other year during the summer/fall season. the largest number of confirmed cases (>500 to date) has been reported in the us, with sporadic cases confirmed throughout the world (schubert et al., 2019). in 2014, the peak of afm incidence has shown a temporal and geographic overlap with the outbreak of ev-d68 infection, raising the possibility that ev-d68 (which rarely caused human disease prior to 2008) might be the cause of recent afm outbreaks (dyda et al., 2018). indeed, ev-d68 infection meets many of the bradford-hill criteria (nine principles applied to examine causality) that are required to establish it as a cause of afm (dyda et al., 2018; messacar et al., 2018). in particular, afm-like disease can be recapitulated in a mouse model through infection with a 2014 strain (but not the originally isolated 1962 strain) of ev-d68, suggesting that neurotropism might have emerged only recently in ev-d68 evolution (hixon et al., 2017). however, 2016 afm cases have not shown a clear association with an ev-d68 outbreak, raising doubts about the causal association between ev-d68 and afm (dyda et al., 2018). moreover, ev has not been detected in the respiratory or gi specimens from more than half of afm children, and ev rna is very rarely detected in the csf of afm patients (schubert et al., 2019); thus, concerns have persisted that recent afm outbreaks might be caused by a still unidentified pathogen or an autoimmune mechanism. the 2019 study by schubert et al. has used a multi-pronged approach [metagenomic ultra-deep next generation sequencing (mngs) of the csf to detect the pathogen-derived nucleic acids, and antiviral antibody detection in the csf using virscan phage display library and ev elisa) to analyze 42 afm cases (covering all outbreaks from 2012-2018) and 58 neurologic disease controls. except for one already known ev-d68-positive case, mngs failed to discover any csf pathogens in either group. however, virscan and ev elisa revealed significantly higher levels of anti-ev antibodies in cases compared to controls, providing a strong support for evs as causative agents of all recent afm outbreaks. interestingly, csf from afm cases commonly contained antibodies targeting more than one ev; thus, viral detection in the spinal cord tissue will likely be required to definitively establish which ev species can cause afm. in addition, further work will be required to determine the sensitivity and specificity of csf ev serology in afm diagnosis (schubert et al., 2019). figure 3. neuroglial stem cell-derived inflammatory pseudotumor (n-scipt): a representative h&e-stained paraffin section (a) shows a spinal cord nerve root surrounded by haphazardly organized but otherwise mature-appearing neuroglial tissue. neurofilament immunohistochemistry (b) highlights intensely stained but disorganized nerve cell processes (arrowheads) that surround the centrally located nerve root (arrow); the tissue at the periphery of the specimen shows glial differentiation and is neurofilament-negative. chronic inflammatory infiltrate was apparent on other sections; for additional images and clinical history details, see the original publication (sloan et al., 2019). scale bar, 100 µm. 4. congenital myopathies: new pathogenetic mechanisms congenital myopathies are a group of genetically and clinically heterogeneous disorders that typically present early in life with weakness / hypotonia and delayed motor development, and are subsequently characterized by a stable or slowly progressive disease course. histopathologic findings are variable and include type 1 fiber predominance and atrophy, cores and/or minicores, mislocalization of intracellular organelles (including nuclei), and various sarcoplasmic inclusions (such as nemaline rods, caps, and cytoplasmic bodies). the well-established pathogenetic mechanisms include impairments of calcium homeostasis, excitation-contraction coupling, sarcomeric filament assembly, force generation, and membrane remodeling. excitingly, this mechanistic spectrum has been significantly widened by the work published in 2019. some of the newly discovered mechanisms include (i) cell cycle acceleration accompanied by reduced cell growth (villar-quiles et al., 2019), (ii) impaired redox balance (lornage et al., 2019), (iii) altered muscle kinetics due to impaired sarcomere relaxation (de winter et al., 2020), and (iv) nuclear dysfunction secondary to impaired contractility (ross et al., 2019); these last two studies are particularly groundbreaking and will therefore be described in a greater detail. nemaline myopathy (nm) can be caused by mutations in at least 13 genes, the great majority of which encode proteins that either form thin filaments or are associated with these filaments. a recent tour-de-force study by de winter et al. (2020; advanced electronic publication in 2019) has established that kbtbd13 (kelch repeat and btb domain containing 13), the mutations in which cause nem6 subtype of nm, is also an actin-binding protein; when mutated, kbtbd13 impairs sarcomere relaxation by altering the structure of thin filaments. specifically, thin filaments are more tightly wound and stiffer than normal when they are bound to mutant kbtbd13r408c; this change in the thin filament compliance disrupts the positive feedback loop that is required for thin-thick filament detachment, resulting in slowing of muscle relaxation that clinically characterizes nem6. given the 8:1 stoichiometry of kbtbd13-actin binding, which is close to the 7:1 stoichiometry between troponin / tropomyosin and actin, it is possible that kbtbd13 acts in concert with those well-known sarcomeric regulatory proteins to fine-tune muscle contraction kinetics. interestingly, deletion of kbtbd13 in mice does not lead to slower muscle relaxation, suggesting that kbtbd13r408c is a gain-of-function mutation. it remains to be shown whether other mutations in this gene lead to similar functional deficits and to elucidate how defects in muscle relaxation ultimately lead to production of nemaline rods (which are recapitulated in the mouse kbtbd13r408c knock-in model). in 2019, we have also learned that impaired contractility and abnormal cytoskeletal organization produce secondary nuclear deficits, which structurally – and possibly functionally – mimic nuclear abnormalities seen in patients with mutations in the nuclear envelope proteins, such as lamins, nesprins, and emerin (ross et al., 2019). abnormal spacing of myonuclei, nuclear envelope defects, and chromatin alterations were present not only in the skeletal muscle from patients with two different genetic forms of nm and nm model mice, but also in skeletal muscle from patients with acquired (immune-mediated) forms of nm, underscoring that these nuclear abnormalities are secondary to impaired contractility rather than a direct consequence of nm mutations. in addition, muscle fibers from both nm patients and nm model mice showed extensive cytoskeletal disruption (disorganized microtubules, mislocalized intermediate filaments composed of desmin, and altered cortical microfilaments composed of actin); given that treatment with microtubule-disrupting agents was shown to alter nuclear shape in vitro, these cytoskeletal alterations likely also contribute to nuclear deficits seen in the nm muscle. indeed, we have recently observed very similar nuclear defects in a patient with a desmin mutation (fig. 4); thus, secondary nuclear defects (and consequent gene expression changes) may be an important but overlooked cause of muscle dysfunction that is more widespread than currently recognized. figure 4. nuclear abnormalities in a case of myofibrillar myopathy caused by a desmin mutation: representative electron micrographs show marked nuclear abnormalities (a-e) including invaginations of the nuclear membrane (a and b, arrowheads), abnormally dense chromatin (the nucleus highlighted by the arrow in b, as well as nuclei in c and d), and nuclear fragmentation (c-e). granulofilamentous material (f), a clue to the correct diagnosis, was seen only focally. subsequent genetic testing revealed a heterozygous des gene mutation [c.1237_1239delgag (p.e413del)]. scale bars: a and e, 1 µm; b-d, 2 µm; f, 4 µm. 5. sporadic inclusion body myositis: the role of terminally differentiated cytotoxic t cells sporadic inclusion body myositis (sibm) is the most common skeletal myopathy affecting people over the age of 50; it typically presents with a weakness of the quadriceps and deep finger flexor muscles, has a slowly progressive course resulting in eventual loss of mobility, and is currently without effective therapy. pathologically, sibm shows both inflammatory and myodegenerative features (fig. 5a); together with a lack of response to immunosuppressive treatment, this “dual” nature of sibm pathology has led to a long-standing disagreement about the role of inflammation in sibm pathogenesis.three studies published in 2019 (dzangue-tchoupou et al., 2019; greenberg et al., 2019; knauss et al., 2019) will likely break this impasse by providing a strong support for the autoimmune nature of sibm: starting with different questions and using different experimental approaches, these studies have demonstrated that terminally differentiated effector memory t cells (so-called temra cells) are enriched in sibm muscle biopsies (greenberg et al., 2019; knauss et al., 2019) as well as peripheral blood of sibm patients (dzangue-tchoupou et al., 2019), and that the presence of these cells is fairly unique for sibm when compared to other iims [although similar cells were also seen in biopsies from two patients with severe, treatment-resistant irmyositis (knauss et al., 2019)]. temra cells are positive for cd8, t-bet (a transcription factor that drives differentiation of naïve t cells into cytotoxic t lymphocytes), cd57 (a marker of replicative senescence), and klrg1 (killer cell lectin-like receptor g1; a marker of t cell exhaustion). they contain high concentration of cytotoxic enzymes (granzymes and perforin) that likely directly cause muscle fiber damage, are known to increase in peripheral blood with age, and are resistant to corticosteroids and drugs that target actively dividing cells (such as methotrexate); the latter two characteristics likely provide at least partial explanation for the age dependence and resistance to immunosuppression that are characteristic of sibm. figure 5. distinct patterns of lc3 and p62 immunostaining in sporadic inclusion body myositis (sibm) and immune-mediated necrotizing myopathy (imnm): a typical sibm case (a-c) shows inflammatory and myodegenerative changes on h&e staining (a) and dense, largely subsarcolemmal protein aggregates and vacuoles on lc3 (b) and p62 (c) immunostaining. in contrast, an imnm case (d-f) shows randomly distributed degenerating/regenerating fibers and the absence of significant inflammation on h&e (d) and multiple fibers with diffuse, finely punctate staining on lc3 (e) and p62 (f) immunostaining. for both cases, h&e stain was performed on cryosections, while lc3 and p62 immunohistochemistries were performed on formalin-fixed, paraffin-embedded tissue sections. scale bars, 50 µm. in addition to providing insight into sibm pathogenesis, these studies have important implications for the sibm diagnosis and treatment. of particular interest, the study by greenberg at al. (2019) included 10 samples from patients diagnosed with polymyositis (pm), an iim that was historically thought to share the sibm inflammatory profile but lack its myodegenerative phenotype and its refractoriness to therapy. the existence of pm as a distinct iim subtype has recently been called into question, given that most patients diagnosed with pm based on the 1975 bohan & peter criteria can be reclassified as one of the other iims [early stage sibm, immune-mediated necrotizing myopathy (imnm), antisynthetase syndrome-associated myositis (asm), or dermatomyositis (dm)] using the current clinicoseropathologic criteria (allenbach et al., 2017; tanboon and nishino, 2019). however, greenberg and co-authors have demonstrated an important difference between their “pm” and sibm samples: while cd8+ lymphocytes invading muscle fibers in sibm biopsies were abundant and cd57-positive, the cd8+ t lymphocytes invading muscle fibers in “pm” biopsies were sparse and cd57-negative. additional work will be required to establish whether pathologically diagnosed pm is a distinct (albeit rare) disease entity or whether it corresponds to an early stage of sibm that lacks senescent, cd57+klrg1+ temra cells and thus remains responsive to immunosuppressive therapy. either way, these findings raise a possibility that the presence of cd8+cd57+klrg1+ t cells in an iim biopsy could serve as a prognostic marker of resistance to the currently available immunosuppressive therapies, a clinically important application that warrants future study. in addition, expression of klrg1 by sibm temra cells may have therapeutic implications: given that this protein is not expressed by central memory t cells and regulatory t cells, it could be used as a basis for development of a future sibm therapeutic (greenberg et al., 2019). 6. immune-mediated necrotizing myopathies: mechanisms of cell injury mmune-mediated necrotizing myopathies (imnms) are a unique class of iims for which the consensus clinicoseropathologic criteria were defined at a workshop held in 2016 (allenbach et al., 2018). imnms have been linked to autoantibodies against signal recognition particle (srp) and 3-hydroxy-3-methylglutaryl-coa reductase (hmgcr), but approximately one third of cases is seronegative and requires histopathologic diagnosis (bergua et al., 2019). regardless of the serologic subtype, pathologic features of imnm include randomly distributed necrotic and regenerating muscle fibers (fig. 5d), variable mhc-1 upregulation, sarcolemmal complement deposition, and a scant to absent lymphocytic infiltrate. previous work has shown that in vitro treatment with either anti-srp or anti-hmgcr antibodies leads to impaired myoblast fusion and muscle fiber atrophy (arouche-delaperche et al., 2017), suggesting that these autoantibodies play a causative role in imnm pathogenesis. a study from the same group published in 2019 has taken the field a step further by demonstrating the causative effect of these two antibodies in vivo: mice treated with either anti-srp+ or anti-hmgcr+ patient-derived plasma develop weakness and necrotizing myopathy that closely resembles human imnm (bergua et al., 2019). in a further agreement with human disease, muscle deficiency is worse following treatment with anti-srp than with anti-hmgcr antibodies, but both antisera require functional complement pathway to produce the disease phenotype. taken together, these studies provide a conclusive evidence for the key role of type ii (antibodyand complement-mediated) immune mechanisms in imnm pathogenesis. interestingly, target antigens of the two known imnm autoantibodies (the signal peptide-binding 54 kda subunit of the srp ribonucleoprotein complex and hgmcr, the rate-limiting enzyme in the cholesterol biosynthetic pathway) are both expressed in the er of all cell types (bergua et al., 2019). given the ubiquitous expression of these antigens, it remains unclear why anti-srp and anti-hmgcr antibodies selectively target skeletal muscle in both humans and mice. however, the er localization of both autoantibody targets may be significant for imnm pathogenesis: a 2019 study has shown that human imnm muscle biopsies of any serotype show increased er stress, activation of the unfolded protein response, and upregulation of the chaperone-assisted selective autophagy (casa) pathway (fischer et al., 2019). notably, activation of the casa pathway in imnms is associated with a unique pattern of immunostaining for autophagy-associated proteins lc3 and p62/sqstm1: instead of the coarse lc3and p62-positive vacuoles and protein aggregates seen in autophagic vacuolar myopathies and sibm [figs. 5b and 5c; also reviewed in (margeta, 2019)], imnm biopsies show diffusely punctate and often quite strong staining of a variable subset of non-necrotic muscle fibers (figs. 5e and 5f). while it remains to be shown how antibody-mediated cell injury leads to activation of the casa pathway [the impairment of which plays a role in the pathogenesis of myofibrillar myopathies; reviewed in (margeta, 2019)], this unique lc3and p62-staining pattern is likely to prove useful in diagnostically challenging imnm cases; however, additional work will be required to establish its sensitivity and specificity for imnms relative to other conditions in the histopathologic differential diagnosis (toxic necrotizing myopathies and other iims). advances in neuromuscular disease diagnostics 7. idiopathic inflammatory myopathies: myositis-specific antibodies and interferon activation signatures as already mentioned, iims are currently divided into four distinct classes dm, asm, imnm, and sibm based on the most up-to-date clinicoseropathologic criteria [first comprehensively outlined in a review by allenbach at al. (2017) and further updated in a recent review by tanboon and nishino (2019)]. this classification has been developed based on advances made over the last 5-10 years and is largely (although not entirely) based on the association of individual iim classes with different myositis-specific antibodies (msa); while these autoantibodies sometimes directly mediate muscle injury (as already discussed for imnms in discovery #6), more often they represent a diagnostically useful epiphenomenon / disease marker. the discovery of msa has revolutionized iim diagnostics because of their high specificity, but the sensitivity remains relatively low. for example, two retrospective studies of large european iim cohorts published in 2019 found that msa are negative in 40-60% of iim patients (betteridge et al., 2019; montagnese et al., 2019); in these msa-negative cases (as well as in rare cases with more than one msa present or in cases for which msa information is lacking), muscle biopsy remains the cornerstone of iim diagnosis. figure 6. dermatomyositis (dm) variants: (a) a “classic” dm case shows marked perifascicular fiber atrophy but no significant inflammation in the perimysium or endomysium; the patient’s antibody status is not known, but the overall clinicopathologic findings fit best with the anti-tif1γ-associated dm. (b) a typical case of anti-mi-2-associated dm also shows perifascicular atrophy, but could be confused with anti-synthetase syndrome-associated myositis given the presence of perimysial and focally endomysial inflammation, perimysial fragmentation, and patchy necrosis of perifascicular muscle fibers. (c) a case of anti-mda5-associated dm shows moderate processing artifacts but no significant pathologic changes; the only significant abnormality was mild upregulation of mhc-i complex in all muscle fibers (not shown). for all three cases, images show representative h&e-stained cryosections. scale bar, 100 µm. histopathologically, most cases of dm, imnm, asm, and sibm are easily distinguishable [for a review of up-to-date pathologic criteria, see tanboon and nishino (2019)]; however, a few entities can create diagnostic difficulties. for example, anti-mda5-associated subtype of dm generally lacks perifascicular atrophy (a key diagnostic feature of dm; fig. 6a) and can therefore mimic imnm or even normal muscle (fig. 6c), while anti-mi-2-associated dm shows prominent perimysial inflammation and perifascicular fiber necrosis that can mimic asm (fig. 6b). the interferon-induced gene expression signatures associated with different iim classes, which were defined in two separate studies published in 2019 (pinal-fernandez et al., 2019; rigolet et al., 2019), confirm the validity of the current iim classification and identify immunohistochemical markers of these interferon responses, which will likely prove diagnostically useful. in particular, both studies have shown that all subtypes of dm (including the histologically atypical cases associated with anti-mda5 and anti-mi-2 antibodies) show activation of the type i interferon (infα/β) pathway and prominent upregulation of ifn1-inducible genes such as mxa (myxovirus resistance protein a), which can be used as an immunohistochemical marker of the inf1 response. in dm, mxa generally shows sarcolemmal staining in perifascicular fibers; the exception is anti-mda5-associated dm, where mxa staining is scattered or diffuse but not perifascicular (tanboon and nishino, 2019). in contrast, both asm cases (regardless of the nature of the associated anti-synthetase antibody) and sibm cases show activation of the interferon type ii pathway (i.e. upregulation of infγ-inducible genes); mhc-ii, an immunohistochemical marker of infγ activation, can be used to monitor this pathway in muscle biopsies (rigolet et al., 2019) and could therefore be useful for distinguishing asm from anti-mi-2-dm in challenging cases. [of note, dm muscle biopsies do show a weak activation of infγ pathway (pinal-fernandez et al., 2019; rigolet et al., 2019); however, at least for mhc-ii proteins that activation is several orders of magnitude lower than in asm and sibm biopsies. thus, it should be possible to optimize mhc-ii staining to effectively differentiate between dm and asm cases.] finally, both studies found that imnm biopsies do not exhibit either type i or type ii interferon signature, further validating their separation into a separate iim category (pinal-fernandez et al., 2019; rigolet et al., 2019). thus, neuromuscular pathology laboratories that see a lot of iim cases should consider adding mxa and mhc-ii immunostains to their stain repertoire, so that these markers can be evaluated in diagnostically challenging iim cases. what are the sources of infα/β and infγ that drive these differential responses? that remains to be definitively established; however, the most likely candidate for the infα/β source are plasmacytoid dendritic cells, which are increased in dm muscle, while the most likely candidate for the infγ source are cd8+ t cells, which were observed in a close vicinity of mhc-ii-expressing fibers in both asm and sibm (rigolet et al., 2019). 8. emerging diagnostic techniques the reduced cost and widespread availability of advanced genetic testing techniques [such as disease gene panels and whole exome sequencing (wes)] have led to a dramatic diagnostic improvement for many inherited nmds. however, even with the aid of these advanced molecular techniques, 50-75% of presumed genetic nmd cases currently remains undiagnosed (cummings et al., 2017); the underlying reasons include challenges involved in interpretation of the variants of unknown significance and of variants in the genes previously not linked to human disease, the inability of these techniques to detect certain types of genetic alterations (such as structural re-arrangements and copy number variants), and the lack of coverage of intronic and regulatory regions (gonorazky et al., 2019). in such diagnostically challenging cases, transcriptome analysis (rna-seq) will likely prove beneficial. indeed, a recent study has shown that rna-seq resolved 36% of their paneland/or wes-negative nmd cases (gonorazky et al., 2019); an essentially identical diagnostic yield (35%) was reported in a 2017 study that used a different nmd patient cohort (cummings et al., 2017). importantly, gene expression is tissue-dependent; thus, the effectiveness of rna-seq for diagnostic testing hinges on the use of disease-relevant source material. in the context of genetic muscle disease, biopsied muscle represents the ideal specimen, while peripheral blood is inadequate; unmodified skin-derived fibroblasts can be useful for some diseases (such as congenital muscular dystrophies), but the diagnostic yield is substantially enhanced if fibroblasts are transdifferentiated into myotubes in vitro (gonorazky et al., 2019). importantly, expression of the muscle-specific transcripts was significantly lower in transdifferentiated myotubes than in the matched muscle biopsy samples for most genes; therefore, muscle biopsies will likely remain the gold standard for diagnostic transcriptomics. however, the transdifferentiation approach may prove useful when muscle biopsy cannot be performed or when accurate diagnosis requires correlation of gene expression patterns between the proband and other family members (gonorazky et al., 2019). another approach for improving muscle disease diagnostics is based on the use of automated image-analysis methods and artificial intelligence (ai) tools during muscle biopsy evaluations. to this end, a french group has recently developed a fully automated macro script for image j that performs muscle fiber measurements (such as total fiber number, type 1 and type 2 fiber proportion, and muscle fiber diameter) that are routinely assessed during muscle biopsy evaluation, but are typically estimated and reported in a semi-quantitative fashion (reyes-fernandez et al., 2019). in addition to validating the macro script (which analyzes images from immunofluorescence-stained sections and is available for download as the manuscript supplementary material), this study has used quantitative morphometry to definitively demonstrate that the size of type 2 fibers decreases with age but increases with obesity, an observation that has relevance for routine muscle biopsy interpretation (reyes-fernandez et al., 2019). notably, the authors are in the process of adopting their macro script for use on immunoperoxidase-stained sections, which will make it more routinely applicable to most pathology practices. more provocatively, a study presented at the 2019 world muscle society congress (kabeya et al., 2019) has shown that a deep neural network model (“ai-based muscle histopathologist”) can use images from h&e-stained slides to differentiate several major muscular dystrophies subtypes better than physicians (although the study abstract does not indicate whether those physicians were expert muscle pathologists). while intriguing, the validity of these results needs to be replicated by future studies. in particular, nmd diagnosis generally requires interpretation of many different stains and integration of clinical, laboratory, and histopathologic findings; thus, it is fairly unlikely that the diagnostic proficiency of an “ai-based muscle histopathologist” will generalize to a broader set of nmds. at the same time, it is likely that the best diagnostic yield will ultimately be achieved through the combined efforts of ai and human pathologists, as was previously demonstrated for the other fields of medicine that are based on image interpretation (such as radiology). advances in neuromuscular disease treatment development of new disease therapies takes decades of work, with clinical trials that span years and report their results thorough multiple papers published over time; as such, it is not entirely appropriate to designate any of the recently approved genetic treatments as a 2019 discovery. at the same time, these new treatments were among of the “hottest” nmd advances in 2019, and therefore had to be included in this review. 9. a success story: genetic therapies for spinal muscular atrophy spinal muscular atrophy (sma) is an autosomal recessive neurodegenerative disease that affects the alpha motor neurons in the anterior horn of the spinal cord, resulting in neurogenic muscle atrophy and weakness; the onset of disease varies from the first 6 months of age for the most severe infantile form (sma1) to the second or third decade for the mildest form (sma4). sma is caused by loss-of-function mutations in both copies of smn1 (survival motor neuron 1) gene, but clinical phenotype is modified by the presence of the paralog gene, smn2, which yields only a small number of full length transcripts (due to a mutation in the exon 7 that alters gene splicing) and is present in a variable number of copies in different individuals. the first genetic therapy for sma, an intrathecally delivered antisense oligonucleotide (aso) called nusinersen, was approved in the us in 2016 and in europe in 2017 based on the results of clinical trials done with sma1 and sma2 infants; additional clinical trials for other sma subgroups are still ongoing. [for a succinct but comprehensive review of all treatment approaches summarized in this section, see (vita et al., 2019).] nusinersen binds to the smn2 pre-mrna downstream of exon 7, resulting in an increase in the expression of smn protein and a significant improvement in motor function and patient survival. risdiplam, an orally available small molecule therapeutic that also acts as a splicing modifier of smn2 mrna, is still in clinical trials; however, based on the promising early results, it has been placed on an accelerated pathway for the approval by ema (european medicines agency). an alternate approach for sma treatment is to directly replace the defective smn1 gene; this approach has led to development of aav9-smn (also called avxs-101 or zolgensma), an aav9 (adeno-associated virus 9) vector carrying human smn transgene that was approved for sma treatment by fda (federal drug administration) in may 2019 (al-zaidy and mendell, 2019). the availability of multiple treatment options raises many interesting questions that will need to be addressed going forward. are some of these treatments superior to others? (an indirect comparison between nusinersen and avxs-101 has been done and suggests that avxs-101 is more effective (dabbous et al., 2019); however, that study had significant limitations. thus, a direct comparison of these two drugs is needed, but will be challenging to perform given the associated cost and ethical considerations.) if two treatments are based on different genetic strategies, would a combination therapy be more effective than either treatment alone? how will treatment efficacy differ depending on the sma subtype and the age at which the treatment is started? as expected for a neurodegenerative disease, the treatment is more effective if it is initiated before significant neuron loss has occurred (de vivo et al., 2019); depending on the sma subtype, this may require perinatal or even prenatal diagnosis. indeed, a large autopsy study published in 2019 has shown that smn protein levels in the human spinal cord decline rapidly during development, with a critical window that spans from 3 months before to 3 months after birth (ramos et al., 2019); this finding highlights the need for development of newborn sma screening programs, which are already being piloted in several different countries (dangouloff et al., 2019; vill et al., 2019). 10. on the horizon: genetic therapies for inherited myopathies aside from sma, the only currently approved genetic therapies for neuromuscular disease are three new drugs for duchenne muscular dystrophy (dmd), which is a severe progressive x-linked muscle disorder caused by the lack of membrane-associated dystrophin protein; however, based on their rather modest effects to date and on the conditional and inconsistent nature of the approvals that were rendered by fda and ema, these treatments have yet to prove their therapeutic utility and are best still considered as being “under development” (vita et al., 2019). ataluren is an orally bioavailable drug that binds ribosomal rna and interferes with recognition of premature stop codons present in 10-15% of dmd patients; it received a conditional approval from ema in 2014, but the evaluation of long-term outcomes is still ongoing. the other two conditionally approved treatments for dmd are based on the exon-skipping aso technology; the goal of this therapy is to “trick” the cellular machinery to restore the reading frame by skipping over the mutant exon, resulting in an internally truncated but functional protein. eteplirsen, which targets the splice-donor region of exon 51 (mutated in 13% of dmd patients), was conditionally approved by fda in 2016 but failed to receive an approval from ema (aartsma-rus and goemans, 2019); a phase 3 clinical trial to evaluate its efficacy is still ongoing. golodirsen (exon 53; 8% of dmd patients) has been approved by fda in december 2019 (https://www.drugs.com/newdrugs/fda-approves-vyondys-53-golodirsen-duchenne-muscular-dystrophy-dmd-patients-amenable-skipping-exon-5119.html; accessed on 12/26/2019), while clinical trials for casimirsen (exon 45; 8% of dmd patients) and suvodirsen (exon 51) are still ongoing (vita et al., 2019). based on the animal model studies, similar exon skipping treatments may also prove valuable for limb-girdle and congenital muscular dystrophies (hwang and yokota, 2019). excitingly, new treatments for other genetic myopathies are also under active development; several different therapeutic strategies are being evaluated through ongoing or planned clinical trials and include aav vector-based gene replacement therapies (for x-linked myotubular myopathy and pompe disease), aso-based therapy aimed at reduction of toxic rna production (for myotonic dystrophy type 1), chaperone therapy (for pompe disease), and the second generation enzyme-replacement therapy (also for pompe disease) (vita et al., 2019). initial results that are available to date are promising; thus, with some luck, at least some of these new treatments will prove beneficial enough to be covered in a future update. at the same time, all these new gene therapies are likely to be highly expensive; for example, the list price of nusinersen treatment in the us is $750,000 in the first year and $375,000 / year after that. thus, an increase in the availability of genetic therapies raises important socio-economic and ethical questions that will have to be addressed before their full benefits can be realized. disclosure statement the author is a consultant for audentes therapeutics; she serves as a member of the muscle biopsy review committee for the aspiro clinical trial (nct03199469), which is evaluating the safety and efficacy of gene transfer in x-linked myotubular myopathy. acknowledgements i am grateful to drs. michael lawlor, nigel g. laing, and benedikt schoser for helpful input during the conceptualization stage of this review, to dr. emily sloan for n-scipt images, and to ms. christine lin for assistance with figure preparation. this work was supported by the muscular dystrophy association grant mda514303. references aartsma-rus, a., and goemans, n. (2019). a sequel to the eteplirsen saga: eteplirsen is approved in the united states but was not approved in europe. nucleic acid ther 29, 13-15. allenbach, y., benveniste, o., goebel, h.h., and stenzel, w. (2017). integrated classification of inflammatory myopathies. neuropathol appl neurobiol 43, 62-81. allenbach, y., mammen, a.l., benveniste, o., stenzel, w., on behalf of the immune-mediated necrotizing myopathies working group (2018). 224th enmc international workshop: clinico-sero-pathological classification of immune-mediated necrotizing myopathies zandvoort, the netherlands, 14-16 october 2016. neuromuscul disord 28, 87-99. anquetil, c., salem, j.e., lebrun-vignes, b., johnson, d.b., mammen, a.l., stenzel, w., leonard-louis, s., benveniste, o., moslehi, j.j., and allenbach, y. (2018). immune checkpoint inhibitor-associated myositis. circulation 138, 743-745. al-zaidy, s.a., and mendell, j.r. (2019). from clinical trials to clinical practice: practical considerations for gene replacement therapy in sma type 1. pediatr neurol 100, 3-11. arouche-delaperche, l., allenbach, y., amelin, d., preusse, c., mouly, v., mauhin, w., tchoupou, g.d., drouot, l., boyer, o., stenzel, w., et al. (2017). pathogenic role of anti-signal recognition protein and anti-3-hydroxy-3-methylglutaryl-coa reductase antibodies in necrotizing myopathies: myofiber atrophy and impairment of muscle regeneration in necrotizing autoimmune myopathies. ann neurol 81, 538-548. bergua, c., chiavelli, h., allenbach, y., arouche-delaperche, l., arnoult, c., bourdenet, g., jean, l., zoubairi, r., guerout, n., mahler, m., et al. (2019). in vivo pathogenicity of igg from patients with anti-srp or anti-hmgcr autoantibodies in immune-mediated necrotising myopathy. ann rheum dis 78, 131-139. betteridge, z., tansley, s., shaddick, g., chinoy, h., cooper, r.g., new, r.p., lilleker, j.b., vencovsky, j., chazarain, l., danko, k., et al. (2019). frequency, mutual exclusivity and clinical associations of myositis autoantibodies in a combined european cohort of idiopathic inflammatory myopathy patients. j autoimmun 101, 48-55. cummings, b.b., marshall, j.l., tukiainen, t., lek, m., donkervoort, s., foley, a.r., bolduc, v., waddell, l.b., sandaradura, s.a., o'grady, g.l., et al. (2017). improving genetic diagnosis in mendelian disease with transcriptome sequencing. sci transl med 9. dabbous, o., maru, b., jansen, j.p., lorenzi, m., cloutier, m., guerin, a., pivneva, i., wu, e.q., arjunji, r., feltner, d., et al. (2019). survival, motor function, and motor milestones: comparison of avxs-101 relative to nusinersen for the treatment of infants with spinal muscular atrophy type 1. adv ther 36, 1164-1176. dangouloff, t., burghes, a., tizzano, e.f., servais, l., and group, n.s.s. (2019). 244th enmc international workshop: newborn screening in spinal muscular atrophy may 10-12, 2019, hoofdorp, the netherlands. neuromuscul disord, epub ahead of print. de ridder, w., nelson, i., asselbergh, b., de paepe, b., beuvin, m., ben yaou, r., masson, c., boland, a., deleuze, j.f., maisonobe, t., et al. (2019). muscular dystrophy with arrhythmia caused by loss-of-function mutations in bves. neurol genet 5, e321. de vivo, d.c., bertini, e., swoboda, k.j., hwu, w.l., crawford, t.o., finkel, r.s., kirschner, j., kuntz, n.l., parsons, j.a., ryan, m.m., et al. (2019). nusinersen initiated in infants during the presymptomatic stage of spinal muscular atrophy: interim efficacy and safety results from the phase 2 nurture study. neuromuscul disord 29, 842-856. de winter, j.m., molenaar, j.p., yuen, m., van der pijl, r., shen, s., conijn, s., van de locht, m., willigenburg, m., bogaards, s.j., van kleef, e.s., et al. (2020). kbtbd13 is an actin-binding protein that modulates muscle kinetics. j clin invest, epub ahead of print. dyda, a., stelzer-braid, s., adam, d., chughtai, a.a., and macintyre, c.r. (2018). the association between acute flaccid myelitis (afm) and enterovirus d68 (ev-d68) what is the evidence for causation? euro surveill 23. dzangue-tchoupou, g., mariampillai, k., bolko, l., amelin, d., mauhin, w., corneau, a., blanc, c., allenbach, y., and benveniste, o. (2019). cd8+t-bet+ cells as a predominant biomarker for inclusion body myositis. autoimmun rev 18, 325-333. fischer, n., preusse, c., radke, j., pehl, d., allenbach, y., schneider, u., feist, e., von casteleyn, v., hahn, k., ruck, t., et al. (2019). sequestosome-1 (p62) expression reveals chaperone-assisted selective autophagy in immune-mediated necrotizing myopathies. brain pathol, epub ahead of print. gonorazky, h.d., naumenko, s., ramani, a.k., nelakuditi, v., mashouri, p., wang, p., kao, d., ohri, k., viththiyapaskaran, s., tarnopolsky, m.a., et al. (2019). expanding the boundaries of rna sequencing as a diagnostic tool for rare mendelian disease. am j hum genet 104, 466-483. greenberg, s.a., pinkus, j.l., kong, s.w., baecher-allan, c., amato, a.a., and dorfman, d.m. (2019). highly differentiated cytotoxic t cells in inclusion body myositis. brain 142, 2590-2604. hixon, a.m., yu, g., leser, j.s., yagi, s., clarke, p., chiu, c.y., and tyler, k.l. (2017). a mouse model of paralytic myelitis caused by enterovirus d68. plos pathog 13, e1006199. hwang, j., and yokota, t. (2019). recent advancements in exon-skipping therapies using antisense oligonucleotides and genome editing for the treatment of various muscular dystrophies. expert rev mol med 21, e5. johansen, a., christensen, s.j., scheie, d., hojgaard, j.l.s., and kondziella, d. (2019). neuromuscular adverse events associated with anti-pd-1 monoclonal antibodies: systematic review. neurology 92, 663-674. kabeya, y., iwamori, t., yonezawa, s., takeuchi, y., nakano, h., nagisa, y., okubo, m., inoue, m., tokumasu, r., ozawa, i., et al. (2019). ai-based muscle histopathologist can differentiate major muscular dystrophies better than human. neuromusc disord 29, s126-127. (abstract) knauss, s., preusse, c., allenbach, y., leonard-louis, s., touat, m., fischer, n., radbruch, h., mothes, r., matyash, v., bohmerle, w., et al. (2019). pd1 pathway in immune-mediated myopathies: pathogenesis of dysfunctional t cells revisited. neurol neuroimmunol neuroinflamm 6, e558. lornage, x., schartner, v., balbueno, i., biancalana, v., willis, t., echaniz-laguna, a., scheidecker, s., quinlivan, r., fardeau, m., malfatti, e., et al. (2019). clinical, histological, and genetic characterization of pyroxd1-related myopathy. acta neuropathol commun 7, 138. margeta, m. (2019). autophagy defects in skeletal myopathies. annu rev pathol, epub ahead of print. messacar, k., asturias, e.j., hixon, a.m., van leer-buter, c., niesters, h.g.m., tyler, k.l., abzug, m.j., and dominguez, s.r. (2018). enterovirus d68 and acute flaccid myelitis-evaluating the evidence for causality. lancet infect dis 18, e239-e247. mohn, n., beutel, g., gutzmer, r., ivanyi, p., satzger, i., and skripuletz, t. (2019). neurological immune related adverse events associated with nivolumab, ipilimumab, and pembrolizumab therapy-review of the literature and future outlook. j clin med 8, epub ahead of print. montagnese, f., babacic, h., eichhorn, p., and schoser, b. (2019). evaluating the diagnostic utility of new line immunoassays for myositis antibodies in clinical practice: a retrospective study. j neurol 266, 1358-1366. olive, m., engvall, m., ravenscroft, g., cabrera-serrano, m., jiao, h., bortolotti, c.a., pignataro, m., lambrughi, m., jiang, h., forrest, a.r.r., et al. (2019). myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions. nat commun 10, 1396. pinal-fernandez, i., casal-dominguez, m., derfoul, a., pak, k., plotz, p., miller, f.w., milisenda, j.c., grau-junyent, j.m., selva-o'callaghan, a., paik, j., et al. (2019). identification of distinctive interferon gene signatures in different types of myositis. neurology 93, e1193-e1204. psimaras, d., velasco, r., birzu, c., tamburin, s., lustberg, m., bruna, j., and argyriou, a.a. (2019). immune checkpoint inhibitors-induced neuromuscular toxicity: from pathogenesis to treatment. j peripher nerv syst 24 suppl 2, s74-s85. ramos, d.m., d'ydewalle, c., gabbeta, v., dakka, a., klein, s.k., norris, d.a., matson, j., taylor, s.j., zaworski, p.g., prior, t.w., et al. (2019). age-dependent smn expression in disease-relevant tissue and implications for sma treatment. j clin invest 129, 4817-4831. reyes-fernandez, p.c., periou, b., decrouy, x., relaix, f., and authier, f.j. (2019). automated image-analysis method for the quantification of fiber morphometry and fiber type population in human skeletal muscle. skelet muscle 9, 15. rigolet, m., hou, c., baba amer, y., aouizerate, j., periou, b., gherardi, r.k., lafuste, p., and authier, f.j. (2019). distinct interferon signatures stratify inflammatory and dysimmune myopathies. rmd open 5, e000811. ross, j.a., levy, y., ripolone, m., kolb, j.s., turmaine, m., holt, m., lindqvist, j., claeys, k.g., weis, j., monforte, m., et al. (2019). impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy. acta neuropathol 138, 477-495. schubert, r.d., hawes, i.a., ramachandran, p.s., ramesh, a., crawford, e.d., pak, j.e., wu, w., cheung, c.k., o'donovan, b.d., tato, c.m., et al. (2019). pan-viral serology implicates enteroviruses in acute flaccid myelitis. nat med 25, 1748-1752. sloan, e.a., sampognaro, p.j., junn, j.c., chin, c., jacques, l., ramachandran, p.s., derisi, j.l., wilson, m.r., kriegstein, a.r., bollen, a.w., et al. (2019). neuroglial stem cell-derived inflammatory pseudotumor (n-scipt): clinicopathologic characterization of a novel lesion of the lumbosacral spinal cord and nerve roots following intrathecal allogeneic stem cell intervention. acta neuropathol 138, 1103-1106. tanboon, j., and nishino, i. (2019). classification of idiopathic inflammatory myopathies: pathology perspectives. curr opin neurol 32, 704-714. touat, m., maisonobe, t., knauss, s., ben hadj salem, o., hervier, b., aure, k., szwebel, t.a., kramkimel, n., lethrosne, c., bruch, j.f., et al. (2018). immune checkpoint inhibitor-related myositis and myocarditis in patients with cancer. neurology 91, e985-e994. vill, k., kolbel, h., schwartz, o., blaschek, a., olgemoller, b., harms, e., burggraf, s., roschinger, w., durner, j., glaser, d., et al. (2019). one year of newborn screening for sma results of a german pilot project. j neuromuscul dis 6, 503-515. villar-quiles, r.n., catervi, f., cabet, e., juntas-morales, r., genetti, c.a., gidaro, t., koparir, a., yuksel, a., coppens, s., deconinck, n., et al. (2019). asc-1 is a cell cycle regulator associated with severe and mild forms of myopathy. ann neurol, epub ahead of print. vissing, j., johnson, k., topf, a., nafissi, s., diaz-manera, j., french, v.m., schindler, r.f., sarathchandra, p., lokken, n., rinne, s., et al. (2019). popdc3 gene variants associate with a new form of limb girdle muscular dystrophy. ann neurol 86, 832-843. vita, g., vita, g.l., musumeci, o., rodolico, c., and messina, s. (2019). genetic neuromuscular disorders: living the era of a therapeutic revolution. part 2: diseases of motor neuron and skeletal muscle. neurol sci 40, 671-681. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. viral infection and dementia: a brief synthesis feel free to add comments by clicking these icons on the sidebar free neuropathology 2:15 (2021) opinion piece viral infection and dementia: a brief synthesis clayton a. wiley division of neuropathology, department of pathology, university of pittsburgh, pittsburgh, pa, usa corresponding author: clayton a. wiley · division of neuropathology · department of pathology · university of pittsburgh · 200 lothrop street · scaife hall s-701 · pittsburgh, pa 15213 · usa wileyca@upmc.edu submitted: 17 may 2021 accepted: 4 june 2021 copyedited by: shino magaki published: 8 june 2021 https://doi.org/10.17879/freeneuropathology-2021-3347 keywords: dementia, emergent infections, immunosenescence, neurodegeneration, syphilis, viral encephalitis abstract for the past 400 years, the most common cause of dementia was tertiary syphilis [1]. its prevalence declined dramatically with the advent of potent antibiotics in the 20th century, but these same antibiotics also helped increase our average lifespan, leading to dramatic increases in the prevalence of age-related dementias. abundant progress has been made connecting early onset dementias with mutations in neural genes. late onset dementias have been linked to a more enigmatic set of genes, some of which have been connected to neuroinflammation, begging the question: are age-related dementias linked to infection? numerous studies have reported an association between dementia and infections in general and viral infections in particular. while these associations have been subject to extensive reviews, the purpose of this synthesis is to examine the hypothesized link of viral infections and dementia from the opposite perspective: what do we know about acute and chronic encephalitides that could forge a link with dementias? there appears to be little support for the concept that viral infections are a major contributor to today’s common dementias. however, the emergence of new central nervous system (cns) viral infections, coupled with senescent immune and nervous systems in our aged population, create new opportunities for infections to contribute to dementia. introduction there may be as many clinical definitions for dementia as there are pathological definitions for neurodegeneration. a simple clinical definition of dementia might be “a chronic irreversible encephalopathy affecting multiple cognitive domains” (memory being the most prominently involved domain in the most common form of dementia, alzheimer’s disease [ad]). clinical dementias can be measured with a variety of clinical scales but are most commonly appreciated from the perspective of impaired capacity to perform activities of daily living. a simple pathological definition of neurodegeneration could encompass loss of neurons, their axonal connections or synapses, but given difficulties in quantifying these degenerative changes, current pathological diagnoses rely on distinctive histopathology (figure 1) [2, 3]. with recent refinements in our understanding of dementia and neurodegeneration, is there support for an infectious etiology in age-related dementias? figure 1. bielschowsky-stained section of neocortex from a patient with alzheimer’s disease. individual pyramidal neurons show intense staining of neurofibrillary tangles in cell bodies and proximal processes. neuropil plaques show staining of dystrophic neuritic processes. there are no infiltrating adaptive immune cells. syphilis: the most common form of dementia it has been hypothesized that syphilis was brought to europe with the return of various european conquistadors [1]. in the old-world, syphilis spread mercilessly infecting up to 20% of the population, 5% of whom eventually developed dementia [4]. so prevalent was tertiary syphilis that sir william osler held it as the paradigmatic neurologic disease saying, “know paretic neurosyphilis in all aspects and you know all of psychiatry.” [5] by the beginning of the 20th century tertiary syphilis was the most common form of dementia [4]. fortunately, the advent of antibiotics in the 20th century coupled with the exquisite sensitivity of treponema pallidum, led to rapid decline in prevalence and incidence of infection and commensurate decrease in syphilitic dementia. which is not to say syphilis is gone. today it infects 0.5% of the world’s population with 6 million new infections per year (~100,000 in north america). the agent treponema pallidum (figure 2) causes a protean complex of diseases of which its tertiary form, mediates dementia. without antibiotic therapy, syphilis can establish a lifelong infection that evades and exhausts the immune system. tertiary syphilis is the result of years of organism replication to the point that unfettered growth throughout the brain along with macrophage activation causes a global chronic encephalitis with neurodegeneration and dementia [5]. it is an unusual form of encephalitis, distinct from the acute viral infections described below, with little evidence of cell mediated immunity and instead a remarkable abundance of activated microglia in gray matter regions of abundant t. pallidum. as with most infections, perivascular infiltrates are prominent and with the spirochetal tropism for vascular walls can progress to an end arteritis infarctive syndrome. histopathologic changes are widespread with minimal evidence of a system specific distribution except for a frontal/temporal cortical predominance. there is extensive neuronal loss and reactive astrocytosis but no intraneuronal protein aggregates. figure 2a. warthin starry stain of rabbit testes infected with treponema pallidum. short thin corkscrew like organisms (black) are seen adjacent to a small blood vessel. figure 2b. immunostain (red) for t. pallidum shows a dense collection of microorganisms. age-related dementia perversely, the cure of the most common cause of infectious dementia was followed years later by an increasing prevalence of age-related dementias that plague us today. the same antibiotic development that fueled elimination of syphilis also protected people from common pathogens and thus (along with improvements in sanitation) indirectly increased our average life span. prior to the introduction of agriculture, the average life span of homo sapiens has been estimated to be less than 35 years [6]. with agriculture and the industrial revolution, by 1900 the average lifespan increased in the us to 47 years. add in modern sanitation and medicine and by 1950 the average lifespan was 66 for males and 71 for females. a mere 70 years later the average bumped another decade to 76 and 81 for males and females, respectively. unfortunately, one thing we know about dementia today with absolute certainty is, it is age dependent. the alzheimer’s association estimates that in the us, one in 10 people over the age of 65 has ad (one of the most common forms of dementia) (https://www.alz.org/alzheimers-dementia/facts-figures, accessed 12.15.20). despite many of those dying 3-5 years after diagnosis, by 85 years of age 30% of the population is demented. said another way, syphilitic dementia disappeared and was more than replaced by age-related dementia. is there evidence that this age-related dementia is related to an infectious agent? infection-related dementia there have been scattered but intense investigations to discover an infectious etiology for today’s common dementias. these studies have spanned simple attempts to morphologically identify an infectious agent within the brains of affected individuals, to sophisticated and sensitive metagenomics sequencing studies. numerous reports, some of which were replicated, have identified a gamut of bacterial or viral agents in the brains of deceased previously demented individuals [7]. however, given the prevalence of dementia and the abundance of opportunities to find an associated infectious agent, it is difficult to imagine that a prevalent direct etiologic agent has been missed. that conclusion should be stated with some diffidence given the famous example of helicobacter pylori linked to gastric and duodenal ulcers [8], but it is a fair summary to say, no infectious agent has been consistently associated with common forms of age-related dementia known today. but is presence of the infectious agent within the brain tissue the sine qua non linking an agent to dementia? it is theoretically possible that an infectious agent could initiate the neurodegenerative process or mediate it remotely without directly invading the brain. for example, direct infection of neurons perturbs cellular metabolism that could potentially disrupt normal protein synthesis or proteasome/autophagy degradation leading to accumulation of aggregated proteins and neuronal dysfunction. indirect effects of systemic infection on neurophysiology could include decreased perfusion secondary to general cytokine induced blood coagulation or altered neuronal gene expression induced by specific cytokines like interferons. this brief review will be limited to assessing potential direct viral etiologies of human dementias from the perspective of what we know about acute and chronic viral encephalitis that could link them to dementias. the reader is referred to a selection of reviews assessing potential of bacterial etiologies [7]. prions (proteinaceous infectious particles) which are infectious agents known to cause dementia will also not be discussed because they are exquisitely rare, part of the host genome rather than a viral agent and have been the subject of many comprehensive reviews [9]. why do we care about viruses? our biosphere has evolved from competition amongst replicating agents. uniand multicellular hosts have evolved numerous defenses to ward off invasion by infectious agents. this dynamic state persists with continuous evolution in host defense and agent offense. viruses are one class of infectious agents. perhaps most succinctly defined by peter medawar as a piece of bad news wrapped in a protein [10], viruses have evolved to co-opt the host cell energy and machinery to replicate the viral genome. the strategies for accomplishing this are legion and in rare cases involve infecting the host brain. how do viruses infect the brain? viral infection can occur through a variety of pathways [11]. to move from infected to naïve host, viruses must traverse the environment and access the surface of living cells which, in the case of droplet and aerosol transmission, implies contact with a mucous membrane, but in the case of arthropod born viruses, is the result of direct subcutaneous injection. at the cell surface, specific host membrane ligands bind viral proteins vastly facilitating viral entry. intracellular replication increases the number of infectious particles (virion) that can disseminate to sites for a secondary amplification (frequently in lymphoid structures) before greater dissemination throughout the host (e.g., the brain). viral entry into a host cell immediately triggers the host cell’s innate immunity, launching a race between the virus and host immune system that ends with either cessation of viral replication and clearance of the virus, or a variety of debilitating outcomes for the host. upon cell entry cytoplasmic and membrane bound host proteins of the innate immune system immediately detect viral proteins and nucleic acids initiating a cascade of antiviral machinery (e.g., interferon) to block viral replication in the infected and adjacent cells. unfortunately, evolution has selected for an array of viral mechanisms to evade the host innate immune response. the partial thwarting of explosive viral replication gives the host’s second line of defense, adaptive immunity, a chance to mount a complex humoral and cellular response. adaptive immunity takes precious days to develop, so if the virus gains entry to the cns during that window, it may gain a temporal advantage over the growing adaptive immune response resulting in a costly destructive battle inside the cns. the cns is protected from viral invasion by an elaborate system of physical, chemical and physiological barriers [12]. encased within the cranial vault, direct introduction of a viral agent is essentially impossible, so viruses take two other routes to enter the brain: hematogenous or transaxonal. both of these other routes require initially establishing an infection in the periphery. hematogenous dissemination requires that the virus reach a concentration (titer) in the blood that can broach the blood brain barrier (bbb) by passage through, or infection of, endothelial cells. an intriguing variation on this route is viral entry into white blood cells and transit through the bbb hidden within a monocyte trojan horse. a second route, transaxonal, has been perfected by many viruses and requires infection of peripheral nerves that naturally extend cellular processes into the brain, such that the virus can utilize intracellular transport mechanisms to rapidly enter the cns sanctuary. viral encephalitis viruses that successfully circumvent peripheral innate immunity and access the cns through pathways described above, have the opportunity to encounter cns cells [11]. if viral surface proteins bind to host neuroglial membrane receptors, the virus is taken into the cell where it can be detected by innate immune machinery similar to that in the periphery. neuroglial elements may not be able to unleash the same potent anti-viral innate immunity as peripheral cells. within a closed chamber the brain can tolerate only limited cytotoxic edema. additionally, given exquisite balance of rna processing within the cns, it may not tolerate potent interferon related arrest of rna metabolism. like other organs, the brain has histiocytes (microglia) that are specialized in detecting pathogens and attracting peripheral immune surveillance [13]. as adaptive immunity is brought to bear on infected cns cells, cellular destruction leads to neurological dysfunction which is accentuated by cytotoxic edema. simple brain edema associated with immune attack is enough to cause neurological dysfunction and through swelling, brain herniation and death. add to that the viral and cytotoxic effector arms of the immune system and it is not surprising that encephalopathy is the clinical outcome. viral-host duals display a spectrum of histopathologies. rapidly growing viruses with the capacity to lyse cells combined with robust immune responses can lead to frank necrosis, while less dramatic infectious battles result in non-necrotic inflammatory reactions [14]. most viral encephalitides are acute infections ending with either host death or viral clearance achieved in a matter of days. chronic viral encephalitis is a more nuanced outcome, characterized by persistent adaptive immune response in the context of continued viral replication. do acute viral encephalitides lead to dementia? viruses mediating acute encephalitis cover a wide range of families but are mostly rna viruses. because of their temporal time course and systemic symptoms, acute viral encephalitides are not likely to be confused clinically with a progressive dementia. as the host immune response brings viral replication under control, cerebral edema can mediate global neural dysfunction, clinically manifest as delirium. with clearance of virus and abatement of the immune response, a brain lesion resulting from the inflammatory process can manifest as a static neurologic deficit, but the clinical picture is not one of a progressive neurodegenerative disease. firstly, the time course of acute encephalitis is in days not months to years. secondly, distribution of infection within the cns, with rare exception, is global rather than system specific as seen with most dementias. thirdly, with the exception of immune compromised hosts, acute encephalitis pathology demonstrates a severe inflammatory response rather than subtle neurodegenerative changes with pathognomonic aggregated proteins (figure 3). figure 3a. immunostain for cd3 of neocortical tissue from the brain of a child who died of coxsackie viral encephalomyocarditis shows dense infiltration of lymphocytes (red). figure 3b. immunostain for coxsackie viral antigens shows staining of multiple neuroglial cells in both the nucleus and cytoplasmic processes. do chronic viral encephalitides lead to dementia? the spectrum of viruses mediating chronic encephalitis is more limited and has proportionally more representation from dna viruses. some chronic encephalitides (e.g., herpes simplex virus [hsv]) are the end product of millennia of co-evolution between host and virus. while hsv can cause an acute encephalitis in the immunologically compromised, in the immune intact host hsv has achieved a unique latent infection in the peripheral nervous system (pns) where viral synthesis is shut down and re-awakened in times of stress permitting active disease and transmission. regardless of organ, chronic infection results from a dynamic balance where the virus does not kill the host and the host does not develop an adequate immune response that can eradicate infection. in addition to outflanking the innate immune response, many viruses have developed the means to mediate some level of general or specific immunosuppression. if this down regulation occurs prior to viral eradication, it could result in viral persistence with or without some low level of immune reaction. alternatively, rather than causing immunosuppression themselves, viruses can take advantage of genetic or iatrogenic compromises of the host immune system. finally like all other organs (including the brain), the immune system undergoes a natural senescence [15]. with age, stem cell elements dissipate and immunological memory fades. loss of immunological surveillance can result in selective holes in adaptive immunity permitting chronic viral infections (e.g., progressive multifocal leukoencephalopathy, figure 4). figure 4. in situ hybridization (red) for jc viral nucleic acids in the brain of a patient succumbing to progressive multifocal leukoencephalopathy. individual oligodendroglial nuclei are filled with viral nucleic acid while surrounding parenchyma shows no t-cell infiltration. potential chronic viral infections associated with dementia an example of a rare chronic viral encephalitis seen in children and young adults can be caused by two different viruses: rubeola and rubella. childhood infection with these agents is usually followed by a self-limiting systemic disease marked by a distinctive skin rash. in rare children, for unknown reasons, but hypothesized to be related to an aberrant immune response, chronic viral infection persists in the cns [16]. unlike acute viral encephalitis, the immune system only dampens exponential viral growth, without destroying infected host cells to eradicate the virus. for years viral replication and a low-grade immune response compromise neuronal function and lead to atrophy and a global encephalopathy. pathologically these diseases show muted inflammatory changes. hsv: of perhaps all chronic viral infections potentially associated with neurodegeneration, herpesviruses may be the most studied. herpesviridae are ancient viruses that have co-evolved with many mammalian species. of the 8 known human herpesviruses, hsv is the most notorious for mediating neurological disease. in the fetus, newborn and immunocompromised, hsv mediates an acute and frequently necrotic pan-encephalitis. for unknown reasons, rare apparently immunologically intact individuals also develop an acute necrotizing encephalitis predominantly focused in the frontal and temporal lobes. while readily treatable, there are case reports where this acute infection transitions into a smoldering chronic infection, raising the prospects that it could be associated with a chronic neurodegenerative disease. the literature on this topic is immense and controversial with proponents on both sides of the debate for and against hsv involvement in neurodegeneration (reviews [17, 18]). while there is no consensus, the cumulative evidence would suggest that while low levels of hsv are detectable in cns and pns (latent infection), there is minimal to no evidence that active hsv replication is temporally or spatially associated with common neurodegenerative diseases. whether hsv infection could initiate a cascade of inflammatory events that subsequently drives a degenerative process awaits construction of a pathogenically logical and testable hypothesis. sars-cov-2: in 2021 it is almost impossible to write anything without mentioning covid-19. much heat and little light has been published on the effect of sars-cov-2 infection on the nervous system. as with many severe infections, a variety of neurological signs and symptoms have been observed in covid patients [19-21]. observations of olfactory neuroepithelial infection [22] have led to the conjecture that sars-cov-2 enters the cns by ascending axonal connections to the olfactory bulb. such a dissemination has been demonstrated in transgenic murine models expressing human angiotensin converting enzyme 2 (ace2; the human viral receptor) under a keratin promoter [23]. but this convenient animal system does not seem to actually model the human disease [24]. intensive attempts have been made to identify sars-cov-2 infection in human autopsy tissue to little avail. the preponderance of evidence would suggest a non-direct infection effect on the nervous system such as overwhelming systemic immune activation (cytokine storm) leading to dissolution of the bbb and compromised cns perfusion. in addition to these effects of acute infection, more chronic cns symptoms have been documented in a subset of patients recovering from covid (long-haulers). while early in our studies of covid pathology, clearly this disease is open to the same theories that have developed with hsv associated neurodegeneration. the challenge will be to convert these theories into testable hypotheses. is hiv the infectious dementia of our generation? before aids was even proven to be caused by a viral infection, it was clearly associated with severe neurologic disease. beyond the devastating opportunistic infections related to severe immunosuppression, late-stage aids patients also exhibited a unique dementia. in retrospect, the histopathology of aids dementia was exactly what should have been expected for a macrophage tropic virus in a patient with severe immunosuppression, minimal to no lymphocyte infiltration in the context of abundant virus [25] (figure 5). indeed, the concentration of virus in the cns exceeded that in lymphoid organs [26]. the big mystery that has yet to be solved is, how did infection of microglia in the absence of neuroglial infection lead to a clinical dementia [27]? it has been hypothesized that infected microglia either produced a neurotoxin (e.g., quinolinic acid [28]) or were unable to carry out critical physiological functions that non-infected microglia normally perform (e.g., synaptic stripping). hypotheses range from remote or local responses to immunologic stimulation to hypothesized abortive infection of astrocytes. before intensive investigation could elucidate the pathogenesis of aids dementia, highly effective anti-retroviral therapy eradicated hiv encephalitis. while hiv infected individuals still experience neurologic symptoms of unknown etiology, aids dementia, like syphilis dementia, disappeared. figure 5a. low power h&e-stained section of cortical tissue from a patient with hiv encephalitis. figure 5b. higher power h&e-stained section showing a multinucleated giant cell. what would make a dementia virus? with what we have learned from infections of the nervous system, what would be the characteristics of a host-viral interaction that would result in a neurodegenerative disorder of dementia? the abundance of rna viruses that infect the brain would suggest that something about their biology makes them particularly effective at replication in the cns. to effectively infect cns cells, the hypothetical dementia virus would need a viral coat protein capable of binding host cns cell membranes. through its intricate specialization and regionalization there are an abundance of potential cns region and system specific target proteins (e.g., neurotransmitter receptors). infection of cns cells would not necessarily lead to lysis of neuroglial elements but would incapacitate normal cell physiology leading to dysfunction or degeneration. competition between normal host cns rna metabolism, viral rna metabolism and innate immune responses could mimic many of the neurodegenerative mechanisms being investigated in neurodegenerative disorders like amyotrophic lateral sclerosis [29]. alternatively, as seen with syphilis and hiv, infection of microglia might indirectly mediate neuronal dysfunction [27, 30]. how would the hypothetical virus reach the cns and propagate between hosts? while many pathways could be used, aerosol transmission is the most effective means of spreading between high density host populations (figure 6). additionally, for entirely mysterious reasons, aerosol dissemination is highly effective at transferring virus to the brain. viruses that normally are limited to peripheral infections, when aerosolized, lead to severe pan-cns infection (e.g., rift valley fever virus, figure 7). figure 6a. in situ hybridization autoradiography (black grains) for influenza a in the brain of a ferret 8 days after aerosol exposure to h5n1. numerous foci throughout the brain demonstrate abundant flu infected cells. figure 6b. higher power image showing regions of intense neuroglial infection. but the most important feature of the hypothetical dementia virus infection is that it would occur in the context of immunosuppression. many viruses elicit general or specific immunosuppression, with hiv notable for a particularly severe cd4 t-cell suppression. however, it is also possible that rather than causing immunosuppression on its own, the hypothetical virus could take advantage of senescence of the human immune system accompanying the aging of our population. with greater than 20% of our population over the age of 65 by 2050, there is no shortage of potential hosts. the covid-19 pandemic has taught us the cost of not being prepared to combat emergent infections. the highly effective mrna vaccines are the result of decades of intensive investigation. we need to broaden and deepen our understanding of how vaccination can be employed to confer protection of the brain particularly from aerosol infection in the immunosenescent host. figure 7. when rift valley fever virus (rvfv) is transmitted by mosquito bite, infection does not cause an encephalitis. however, when delivered as an aerosol, rvfv causes a global encephalitis. in situ hybridization autoradiography (black grains) for rvfv in the brain of a mouse 7 days after aerosol exposure to rvfv. essentially all of the neurons are infected. conclusion historically infectious agents have been the primary cause of dementia. introduction of antibiotics eradicated syphilitic dementia while at the same time helping humans live longer allowing them to develop even more prevalent age-related dementias. the recent emergence of hiv has once again proved the potential for an infectious agent to mediate a dementing illness. fortunately, we discovered pharmaceuticals to arrest hiv infection and block development of immunosuppression and aids dementia. in the context of an increasingly aged population, living with senescent immune and nervous systems offers up new targets for emergent viral infections to wreak havoc on our cognitive capacities. however, for the most common age-related neurodegenerative diseases (e.g., ad, lewy body disease, frontotemporal dementia) there is little in the histopathology defining these disorders to connect them to a viral etiology. references [1] tampa m, sarbu i, matei c, benea v, georgescu sr. brief history of syphilis. j med life. 2014;7:4-10. [2] montine tj, phelps ch, beach tg, bigio eh, cairns nj, dickson dw, et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol. 2012;123:1-11. [3] hyman bt, phelps ch, beach tg, bigio eh, cairns nj, carrillo mc, et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease. alzheimers dement. 2012;8:1-13. [4] merritt hh, adams rd, solomon hc. neurosyphilis. new york: oxford university press; 1946. [5] ghanem kg. evaluation and management of syphilis in the hiv-infected patient. curr infect dis rep. 2010;12:140-6. [6] pahl kp. life expectancy in ancient and modern man. acta anthropogenet. 1981;5:119-28. [7] honjo k, van reekum r, verhoeff np. alzheimer's disease and infection: do infectious agents contribute to progression of alzheimer's disease? alzheimers dement. 2009;5:348-60. [8] de brito bb, da silva faf, soares as, pereira va, santos mlc, sampaio mm, et al. pathogenesis and clinical management of helicobacter pylori gastric infection. world j gastroenterol. 2019;25:5578-89. [9] walker lc. prion-like mechanisms in alzheimer disease. handb clin neurol. 2018;153:303-19. [10] medawar pb, medawar js. aristotle to zoos: a philosophical dictionary of biology. cambridge: harvard university press; 1985. [11] johnson rt. viral infections of the nervous system. 2nd ed. philadelphia: lippincott-raven; 1998. [12] johnson rt. neurovirology: evolution of a new discipline. j neurovirol. 1995;1:2-4. [13] kofler j, wiley ca. microglia: key innate immune cells of the brain. toxicol pathol. 2011;39:103-14. [14] love s, budka h, ironside jw, perry a eds. greenfield's neuropathology. 9th ed. boca raton: crc press; 2015. [15] fukushima y, minato n, hattori m. the impact of senescence-associated t cells on immunosenescence and age-related disorders. inflamm regen. 2018;38:24. [16] townsend jj, baringer jr, wolinsky js, malamud n, mednick jp, panitch hs, et al. progressive rubella panencephalitis. late onset after congenital rubella. n engl j med. 1975;292:990-3. [17] itzhaki rf. corroboration of a major role for herpes simplex virus type 1 in alzheimer's disease. front aging neurosci. 2018;10:324. [18] allnutt ma, johnson k, bennett da, connor sm, troncoso jc, pletnikova o, et al. human herpesvirus 6 detection in alzheimer's disease cases and controls across multiple cohorts. neuron. 2020;105:1027-35 e2. [19] paterson rw, brown rl, benjamin l, nortley r, wiethoff s, bharucha t, et al. the emerging spectrum of covid-19 neurology: clinical, radiological and laboratory findings. brain. 2020;143:3104-20. [20] zubair as, mcalpine ls, gardin t, farhadian s, kuruvilla de, spudich s. neuropathogenesis and neurologic manifestations of the coronaviruses in the age of coronavirus disease 2019: a review. jama neurol. 2020;77:1018-27. [21] berlit p, bosel j, gahn g, isenmann s, meuth sg, nolte ch, et al. "neurological manifestations of covid-19" guideline of the german society of neurology. neurol res pract. 2020;2:51. [22] meinhardt j, radke j, dittmayer c, franz j, thomas c, mothes r, et al. olfactory transmucosal sars-cov-2 invasion as a port of central nervous system entry in individuals with covid-19. nat neurosci. 2021;24:168-75. [23] song e, zhang c, israelow b, lu-culligan a, prado av, skriabine s, et al. neuroinvasion of sars-cov-2 in human and mouse brain. j exp med. 2021;218:e20202135. [24] matschke j, lutgehetmann m, hagel c, sperhake jp, schroder as, edler c, et al. neuropathology of patients with covid-19 in germany: a post-mortem case series. lancet neurol. 2020;19:919-29. [25] wiley ca, schrier rd, nelson ja, lampert pw, oldstone mb. cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients. proc natl acad sci u s a. 1986;83:7089-93. [26] pang s, koyanagi y, miles s, wiley c, vinters hv, chen is. high levels of unintegrated hiv-1 dna in brain tissue of aids dementia patients. nature. 1990;343:85-9. [27] wang t, rumbaugh ja, nath a. viruses and the brain: from inflammation to dementia. clin sci (lond). 2006;110:393-407. [28] valle m, price rw, nilsson a, heyes m, verotta d. csf quinolinic acid levels are determined by local hiv infection: cross-sectional analysis and modelling of dynamics following antiretroviral therapy. brain. 2004;127:1047-60. [29] butti z, patten sa. rna dysregulation in amyotrophic lateral sclerosis. front genet. 2018;9:712. [30] mcmanus rm, heneka mt. role of neuroinflammation in neurodegeneration: new insights. alzheimers res ther. 2017;9:14. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurotrauma feel free to add comments by clicking these icons on the sidebar free neuropathology 1:10 (2020) review top ten discoveries of the year: neurotrauma daniel p. perl j. edward hebert school of medicine, “america’s medical school”, uniformed services university, bethesda, md, usa corresponding author: daniel p. perl · j. edward hebert school of medicine · “america’s medical school” · uniformed services university · 4301 jones bridge road, room b-3138 · bethesda, md 20814 · usa daniel.perl@usuhs.edu submitted: 12 february 2020 accepted: 22 march 2020 copyedited by: nicole schwab published: 30 march 2020 https://doi.org/10.17879/freeneuropathology-2020-2662 keywords: neurotrauma, tbi, chronic traumatic encephalopathy, cte, tau, mri, pet, cryo-em abstract neurotrauma represents a major public health problem and is one of the leading causes of death and disability worldwide. despite its high prevalence, there are major gaps in our understanding of the underlying pathophysiology leading to the substantial morbidity and mortality associated with this problem. here, ten studies published in 2019 are reviewed that addressed issues related to the acute and long-term effects of neurotrauma. these studies can be broken down into three separate categories, namely, the importance of neurotrauma-based damage to the cerebrovascular unit, white matter damage following neurotrauma, and research related to the long-term neurodegenerative consequences of repeated head trauma, especially chronic traumatic encephalopathy. the advances highlighted here indicate that progress has been made. however, major gaps in knowledge remain which will require additional neuropathologic studies of clinical specimens, as well as the development and investigation of a wide range of relevant pre-clinical models. further efforts in this field are clearly needed if there are to emerge better clinical outcomes for the numerous patients that suffer neurotrauma each year as well as those currently suffering from its long-term effects. abbreviations chimera closed-head impact model of engineered rotational acceleration, cryo-em cryo electron microscopy, crash clinical randomisation of an antifibrinolytic in significant haemorrhage, ct computerized tomography, cte chronic traumatic encephalopathy, gcs glasgow coma scale, gfap glial fibrillary acidic protein, mri magnetic resonance imaging, mtbi mild traumatic brain injury, nfl national football league, pet positron emission tomography, tbi traumatic brain injury, wt wild type. introduction according to pubmed, in 2019 there appeared 2,508 publications which included traumatic brain injury (tbi) as a keyword. this compares to 886 papers with tbi as a keyword published in 2009. each year, over the past decade, there has been a steady increase in publications, addressing this topic (see figure 1). this almost three-fold increase in published reports over the past decade reflects, i believe, increasing awareness of the importance of the health issues related to neurotrauma, opportunities for neurotrauma research, as well as increased funding for research on the topic. however, as will be pointed out, despite this increased research attention, there is much more to be done. figure 1: no. of tbi papers published (source: www.ncbi.nlm.nih.gov/pubmed) according to the centers for disease control and prevention, in the united states, in 2014 there were 2.87 million tbi-related emergency room admissions, 288,000 hospital admissions and 56,800 deaths (155 per day) (see www.cdc.gov/traumaticbraininjury). that number of annual deaths is greater than occurs each year related to other, more widely recognized, health issues such as breast cancer (42,170 deaths, data from the american cancer society), colon and rectal cancer (53,200 deaths, data from the american cancer society), pancreatic cancer (47,050 deaths, data from the american cancer society) and influenza (51,376 deaths, data from 2014-15 from the cdc) (see www.cancer.org and www.cdc.gov/flu as sources of data on death rates). a further reason for increasing attention to neurotrauma has been concerns about the long-term effects of tbi on the brain and the possible link to the subsequent development of neurodegenerative disorders. there has also been increasing interest among the us department of defense and veterans administration in both the acute and the long-term effects of impact and blast tbi among the more than 2 million military service members who have been deployed to the middle east to fight the war against terrorism. finally, there has been particular concern about the long-term effects of participation in contact sports, such as american football, with the occurrence of repeated concussive and subconcussive impact tbis and a risk of developing chronic traumatic encephalopathy (cte). all of this has combined to promote increasing attention to neurotrauma research. obviously i was not able to review all 2,508 papers appearing in the past year in making my selections of the most important contributions to this field. i readily admit that there is a degree of personal bias to those i have chosen, however, the following is a list of what i consider to be the most influential papers to have appeared in the past year, plus comments on how i believe they have influenced both clinical and pre-clinical concepts. role of the vasculature in tbi pathophysiology in 2019, there have been several reports that highlighted the importance of the cerebrovascular unit in the pathophysiology of tbi. in 2019, this was emphasized by the positive results of a very large clinical trial (crash 2019), the use of magnetic resonance imaging (mri) and subsequent neuropathology correlation in tbi patients (griffin, turtzo et al. 2019) and a pre-clinical model of sub-concussive blast exposure in the rat (gama sosa, de gasperi et al. 2019). over the past several years, unfortunately, there have been a large number of failed clinical trials seeking to improve outcomes of acute victims of tbi. however, in 2019, a randomized placebo-controlled clinical trial on the effects of treatment by tranexamic acid on death and disability in patients with acute tbi (crash 2019) was successful. the rationale for this approach is that tranexamic acid reduces bleeding by inhibiting the enzymatic breakdown of fibrin blood clots. fibrinolysis, with increased concentrations of fibrinogen degradation products, is commonly seen in the brains of acute tbi patients and predicts the development of further intracranial hemorrhagic expansion (zhang, he et al. 2018). the report involved an extremely large trial, consisting of a total of 12,737 hospitalized patients with acute tbi. enrollment criteria included adults with tbi who were within 3 hours of injury, had a glasgow coma scale (gcs) of 12 or lower or showed evidence of intracranial bleeding on ct examination. the study clearly demonstrated that administration of tranexamic acid within 3 hours of head injury significantly reduced the risk of death. the authors stressed that early treatment was essential to elicit this effect and that patients with a gcs score of 3 or less or who had non-reactive pupils at baseline failed to show a reduction in death-rate. in the tranexamic acid-treated group, excluding patients with a gcs of 3 or with bilateral unreactive pupils at baseline, the risk of head-injury-related death was 12.5% versus 14.9% in the placebo group (485 vs 525 events; rr 0·89 [95% ci 0·80–1·00]). in addition, their study demonstrated that tranexamic acid administration in the tbi patients had a very good safety record. the risks of serious vascular occlusive events (deep vein thrombosis, pulmonary embolism, stroke or myocardial infarction) or of seizures were not increased in the tranexamic treated patients when compared to the placebo group. importantly, tranexamic acid is a widely available drug which is quite inexpensive. indeed, the total cost of this medication, as given in the trial, was less than $10/patient! tbi is not just a problem in rich industrialized countries, but is seen worldwide, especially in developing countries. tbi is a leading cause of death and disability worldwide, with an estimated 10,000,000 cases being hospitalized or dying each year (roberts, belli et al. 2018). the low cost and demonstrated safety of this treatment makes it ideal for use throughout the world and, according to the authors, promises a dramatic impact on the survival of large numbers of patients. this important paper reports what i believe is the first clinical drug trial to ever show significantly reduced mortality from tbi. it is promising for changing clinical practice and saving numerous lives, worldwide. for neuropathologists, this paper emphasizes the importance of bleeding and vascular ischemic lesions as important factors leading to fatal outcomes following head trauma. these aspects have been poorly documented and understood in the neuropathology literature and will require further investigation, both in a clinical setting and in pre-clinical models. an example of this approach is my next chosen publication which involves mri and correlative neuropathology in the evaluation of acute tbi patients (griffin, turtzo et al. 2019). full disclosure requires that i identify myself as a participant in, and an author of this study which reports on the identification and characterization of microbleeds in the work-up of acute patients with tbi (griffin, turtzo et al. 2019). nevertheless, the results reported are sufficiently important to include it on my list of important contributions to the field of neurotrauma. within the initial clinical workup of patients coming into an emergency department following tbi is the use of ct examination looking for evidence of major sites of intracranial bleeding that might require surgical intervention. some research groups have now begun to employ mri imaging as a means of providing the more detailed morphologic spatial resolution inherent to this technology to the information available for acute tbi clinical management. in doing so, it has been noted that the brains of many tbi patients being evaluated in this way show the presence of multiple small, often punctate or linear hypointensities on t2-weighted imaging (huang, kuo et al. 2015, chiara ricciardi, bokkers et al. 2017). mri lacks a cellular level of resolution leading to conjecture as to what the underlying pathology of these lesions might represent. some have interpreted them to be traumatic microbleeds, while others have suggested them to represent the sites of hemorrhagic axonal injuries. importantly, whether such mri findings may be used as a biomarker for predicting successful, partial, or poor recovery remains an unanswered question. the data in this study (griffin, turtzo et al. 2019) is from a large number of civilian tbi victims who were clinically evaluated in several urban trauma centers in the washington, dc area. eligible patients were entered into an mri screening protocol along with further longitudinal clinical follow-up. a total of 439 patients met inclusion criteria for the initial neuroradiologic analysis. most of those enrolled were determined to have mtbi (83%), with a mean gcs at the time of initial evaluation of 15. importantly, approximately one-third of these mild patients showed evidence of punctate and/or linear microbleeds on initial mri exam, indicating that such lesions are not uncommon following mild and mostly asymptomatic head trauma. the presence of traumatic microbleeds was shown to be an independent predictor of subsequent disability in the cohort (p >0.05; odds ratio = 2.5). one of the patients who had received mri within 48 hours of injury and showed the presence of multiple punctate and linear intraparenchymal hemorrhages, died 7 months following the accident and was subjected to post-mortem examination with brain donation for use in research. using the clinical mri exams, supplemented by ex vivo imaging of the formalin fixed brain specimen, a correlational neuroimaging/neuropathology investigation was performed on the specimen in an attempt to identify the histopathologic nature of the punctate/linear hemorrhages that had been identified. using co-registration of the clinical and ex vivo mri images with histopathologic stained sections, the post-traumatic microhemorrhages showed the presence of iron-laden macrophages, predominantly within perivascular spaces. in these colocalized histopathologic slides, evidence of axonal injury was not observed in conjunction with the microhemorrhages. this study highlights the importance of damage to the cerebral vasculature as a consequence of mtbi and its effect on clinical outcome. this result, combined with the findings of the previously discussed paper on the effectiveness of tranexamic acid, underscores the importance of studying the effects of tbi on the integrity of the cerebrovasculature unit. we are far from knowing how best to use such mri findings for a more rational therapeutic approach to patients. however, additional correlational neuroimaging / neuropathology studies of this type will clearly lead to important information that will, in the future, refine clinical diagnostic and treatment guidelines. in a further investigation of the role of the cerebrovascular unit in the pathophysiology of tbi, gama-sosa and colleagues reported findings in a pre-clinical rat model of repetitive mild blast tbi (gama sosa, de gasperi et al. 2019). using a blast tube facility, the dose of blast exposure to the rats was mild and determined to represent 74.5 kpa (equivalent to 10.8 psi) and animals were exposed to one blast per day, over three consecutive days. exposed rats were sacrificed at 6 weeks, 8 months and 10 months following blast exposure. this model was considered to be a low level of blast exposure, comparable to those endured by military service members in training activities and combat exercises (thus emphasizing the clinical relevance of this model), and exposed animals failed to show evidence of generalized neuronal pathology at either the light or electron microscopic level. however, upon detailed examination of the neurovascular unit, significant pathology was identified. using purified vascular fractions, decreased levels of vascular-associated glial fibrillary acidic protein (gfap) and that of several neuronal intermediate filament proteins (α-internexin and low, middle and high neurofilament proteins) were identified 6 weeks following the blast exposure. electron microscopy showed evidence of damage to astrocytic end-feet associated with blood vessels. at 8 months following blast exposure, there was evidence of alterations of the vascular smooth muscle layer. these findings indicate that despite the low dose of blast exposure, significant chronic vascular damage had been induced. these intriguing findings need to be further investigated and serve to add additional support to the importance of the above discussed papers. white matter involvement in tbi a consistent clinical finding in patients with tbi is slowing of information processing. damage to neuronal circuitry, with disruption of long axons, is thought to play a major role in this phenomenon. in an attempt to model this aspect of tbi, a group experimentally studied aspects of tbi damage using a direct impact concussion model in mice (marion, radomski et al. 2018) that has previously been shown to induce traumatic axonal injury to the corpus callosum (mierzwa, marion et al. 2015). animals were studied neurophysiologically and morphologically. following impact, compound action potential velocities along myelinated axons were slowed within 3 days of injury with partial recovery of function by 2 weeks. this suggested to the authors that partial demyelination, followed by remyelination was responsible for the changed action potential velocities. using electron microscopy, dispersed demyelinated axons and disorganized myelin attachments to axons at paranodes were noted within the corpus callosum of the injured mice. animals examined after longer periods following tbi exhibited an overall loss of axons at 6 weeks post-injury and observable corpus callosum thinning was seen by 8 weeks. white matter damage is a consistent finding in the brains of patients who survive tbi and have significant cognitive issues and conspicuous thinning of the corpus callosum is often noted in such patients (johnson, stewart et al. 2013). this paper provides a useful experimental model for this phenomenon and is promising for a better understanding of the pathophysiology of such white matter damage and a substrate to explore treatment options for patients with this important aspect of head trauma’s clinical effects. the year 2019 has, among other things, witnessed increasing attention to the metoo movement, recognizing the high prevalence and importance of sexual harassment and violence against women. i was intrigued to find a publication which identified the importance of intimate partner violence as a substrate for tbi, as evidenced by its effects on the integrity of white matter (valera, cao et al. 2019). the authors point out that it is estimated that in the united states, about 42,000,000 women over age 15 have experienced physical or sexual abuse and that 75% of women with a history of intimate-partner violence have sustained at least one partner-related tbi, while 50% have sustained repeated tbis. despite this staggering number of victims, there has been almost no study of the long-term functional and structural consequences of such violence. the valera, et al preliminary study attempted to address this gap in knowledge by recruiting 20 women who had sustained such intimate partner assaults, based on a detailed semi-structured interview. participants underwent detailed study, including 3t mri neuroimaging. the neuroimaging aspects of the study revealed evidence of abnormalities in fractional anisotropy in the posterior and superior corona radiata as well as the posterior thalamic radiation. however, this was a preliminary study with limited power and lacked a control population for comparison. nevertheless, this paper identified a potentially very large group of women who are at-risk for significant tbi and has been largely overlooked. their preliminary results indicate that the degree of neurotrauma in women who sustain tbi through intimate partner violence is sufficient to produce changes in white matter integrity visible with 3t mri neuroimaging. as the authors point out, the number of women that could be affected vastly overwhelms that of professional contact sport athletes or even those with non-professional participation in such sporting activities. the underlying neuropathologic nature of damage related to this form of neurotrauma remains unstudied and is an issue that clearly needs further investigation. advances in the investigation of the effects of repeated impact tbi on the development of neurodegeneration (chronic traumatic encephalopathy, cte) there has been considerable interest in the subsequent development of neurodegeneration following repeated impact tbi, particularly in the form of cte. in specific, this has involved concerns about how prevalent the disorder is among professional contact sport athletes and, more broadly, about possible risks to the general public following much lesser extents of tbi exposure. in 2019, these concerns have been addressed in a number of ways. currently, cte can only be diagnosed by neuropathologic examination of the brain at autopsy. clinically, there is a considerable need to identify an approach by which patients at-risk for this condition can be evaluated and confidently diagnosed during life. for the most part, this effort has involved the use of a variety of positron emission tomography (pet)-scan ligands directed against tau deposits in the brains of at-risk subjects (dickstein, pullman et al. 2016, marquie, normandin et al. 2017). the use of similar pet ligands directed at beta-amyloid has been employed with considerable success in the diagnostic work-up of patients thought to be suffering from alzheimer’s disease and this paper (stern, adler et al. 2019) represents the use of similar approaches for cte, a form of tauopathy. in a 2019 study (stern, adler et al. 2019), 26 former national football league (nfl) players and 31 controls were evaluated using both flortaucipir and florbetapir pet ligands, reagents directed against tau and beta-amyloid, respectively (wong, rosenberg et al. 2010, marquie, siao tick chong et al. 2017) . the former football players had a minimum of 2 years playing in the nfl (mean 9.1 years ± 3.0) and a minimum of 12 years total football playing experience (mean 18.8 years ± 4.6). all participants reported to be suffering from cognitive, mood, and behavioral symptomatology. the controls were free of a history of tbi or evidence of cognitive impairment. the former football player cohort showed significantly elevated uptake by the tau pet ligand in the three neuroanatomic regions that had been examined (bilateral superior frontal, bilateral medial temporal and left parietal areas) when compared to that of the control group. there was no difference between groups in the beta-amyloid directed pet results. as the authors point out, the differences noted were based on collective group findings and the results “do not inform the use of testing in individual patients to determine cte pathology during life.” at the time of publication, all of the participants in this study are presumably still alive and thus there was no opportunity to validate the findings with post-mortem assessment. this is an important first step but validation using a much large sample will be necessary to determine the specificity and sensitivity of this approach to the diagnosis of at-risk individual patients. neuropathologists will need to be actively involved in such studies, particularly in attempts to identify individuals with cte and distinguish them from patients with other forms of neurodegenerative disorders, especially other forms of tauopathies. most studies to date have depicted cte in patients who participated in boxing or american professional football. in 2019, an epidemiologic study appeared documenting evidence of an increased incidence of neurodegenerative disorders among individuals who had been former professional soccer players (mackay, russell et al. 2019). the authors used mortality data derived from the death certificates of 7,676 former scottish professional soccer players and compared them to 23,028 controls that were drawn from the general population. further, they compared prescription information on the use of medications prescribed for the treatment of dementia between the two groups. the results indicated that scottish professional soccer players had a lower all-cause mortality rate than the general population but showed a significantly increased tendency to die of all forms of neurodegenerative disease. indeed, significantly higher mortality rates from 1) dementia, not otherwise specified, 2) alzheimer’s disease, 3) non-alzheimer’s dementias, 4) motor neuron disease and 5) parkinson’s disease were all seen in the former soccer players when compared to general population controls. recognizing the weakness of possible errors in death certificates’ stated causes of death, the authors turned to the prescription data for drugs used to treat dementia, showing a higher rate of use among the former soccer players. obviously, there was no opportunity to verify by autopsy examination the indicated causes of death on the death certificates. however, errors in ascribing such deaths would be expected to be roughly equal among the athletes and the controls. this study expands the growing epidemiologic literature supporting the concept that repeated head trauma has a correlation with the subsequent development of neurodegenerative diseases and dementia. the nature of that association and, in particular, whether it speaks to the induction of cte and/or alzheimer’s disease, parkinson’s disease or amyotrophic lateral sclerosis, will require additional study and neuropathologic investigation of incident cases. this paper points to the importance of such studies in the future. as an additional point, the authors remind their readers that soccer is played in over 200 countries worldwide with more than a quarter billion participants. while this study could not address the risks of more casual amateur play, it does raise further concerns regarding the long-term consequences of repeated head trauma, a subject that is likely to attract increasing interest and research in the coming years. attempts to model chronic traumatic encephalopathy (cte) in rodents chronic traumatic encephalopathy (cte) is a progressive neurodegenerative disease that has been closely linked to repeated head trauma, primarily in the setting of participation in contact sport athletics. the neuropathologic features of cte include deposition of abnormally phosphorylated tau in neurons and astrocytes in a unique and distinguishable pattern (mckee, cairns et al. 2016) (see figure 2). despite considerable efforts, no laboratory has been able to produce a consistent and appropriate experimental model of cte. in 2019, two papers (gangolli, benetatos et al. 2019, mouzon, bachmeier et al. 2019) described efforts to do so using differing impact rodent models and both failed to induce intraneuronal tau deposits in their injured animals. figure 2. characteristic tau neuropathology features of chronic traumatic encephalopathy (cte), including: a. depth of sulcus involvement, b,c. perivascular foci with involvement of neurons and astrocytes, and d. neurofibrillary tangles in substantia nigra, pars compacta neurons (arrows). hyperphosphorylated tau immunohistochemistry (at-8) in a former collegiate american football player. first and foremost, one needs to recognize that rodent tau is different from human tau and there has been an almost universal failure, under any experimental condition, to induce tau aggregates comparable to human neurofibrillary tangles in wild-type mice. accordingly, both groups used a humanized mouse transgenic model with knock out of the expression of the native mouse tau, in the hopes that this maneuver would enhance their chances of inducing such changes. in the gangolli et al. (gangolli, benetatos et al. 2019) experiment, head trauma was produce using the closed-head impact model of engineered rotational acceleration (chimera) approach. chimera is a relatively newly developed technology that is capable of producing both concussive and subconcussive injury to experimental animals and also produces a significant rotational acceleration component (namjoshi, cheng et al. 2017). mice were exposed to 20 daily impacts for 20 consecutive days and the energy used for the chimera impact was controlled to yield animals with either a subconcussive or concussive level of injury. all animals underwent behavioral testing at 3 months and 12 months post-injury and were sacrificed for morphologic and biochemical studies at the later time period. behavioral abnormalities were seen in both the subconcussive and concussive-exposed animals (more prominent following the higher energy impact model) and this was accompanied by evidence of white matter disruption. importantly, there was no evidence of tau pathology in any of the injured animals. clearly, these experiments demonstrated that repeated exposure to head trauma through the chimera approach could induce white matter damage, important in and of itself, but this failed to induce a model that could be used in studying the underlying tau-related pathophysiology of cte. the mouzon, et al. paper (mouzon, bachmeier et al. 2019) reports exposing humanized transgenic mice to closed-head impact injury with either a single blow or 5 successive blows over a ten day interval (48 hours apart). the animals were then allowed to survive for either 24 hours or 12 months, post-injury. evaluations included behavioral testing and, on sacrifice, morphologic and biochemical assays. the behavioral testing showed persistent and progressively worsening deficits in visuospatial learning from 2 to 12 months and significant deficits in visuospatial memory consolidation at 12 months post-injury. neuropathologic evaluation at both 24 hours and 12 months revealed evidence of axonal injury, thinning of the corpus callosum and activation of microglia and astrogliosis in the white matter. tau immunohistochemistry and elisa studies showed an increase in phosphorylated tau in the cortex at the site below the impact point and in the ca1/ca3 region of the hippocampus of both the single and repeated injury mice. however, this response was transitory in the immediate post-injury (24 hours) phase and was not detected at the 12-month survival time period. no neuronal tau aggregates were detected in the injured animals. the authors point out that the “post-tbi neuropathology in htau and wt mice was otherwise comparable, with essentially similar levels of axonal injury and chronic neuroinflammation in both study groups.” they further suggested that the observation of transient tau hyperphosphorylation at acute time periods post-injury “may be a normal physiologic response of the brain to mtbi.” both articles failed to show that the use of humanized tau mice with repeated impact tbi was able to develop a rodent model of cte and thus their results could not be used to mimic the human disease. in my opinion, despite considerable effort, the neurotrauma field has yet to identify a consistent and valid animal model of cte. characterization of tau filaments in chronic traumatic encephalopathy cases by cryo-electron microscopy cryo-electron microscopy is a technology that is capable of imaging the three-dimensional structure of complex biomolecules at the single digit angstrom level of resolution. this newly developed approach is so powerful, that its developers, drs. jacques dubochet, joachim frank and richard henderson, were honored with the 2017 nobel prize in chemistry for their work. tau filaments isolated from the brains of patients with alzheimer’s disease (fitzpatrick, falcon et al. 2017) and pick’s disease (falcon, zhang et al. 2018) have been reported using this technique and have been shown to form differing configurations. in 2019, falcon, et al. (falcon, zivanov et al. 2019) reported a cryo-em study of tau filaments isolated from three cases of cte (a former american football player and two former boxers). the configuration of the filaments isolated from each of the cte cases were identical and were distinctly different than that isolated from the alzheimer’s disease and pick’s disease brains. in the cte-derived tau filaments, the β-helix region of the molecule displayed a distinct hydrophobic cavity that was not seen in tau filaments from alzheimer’s or pick’s disease specimens. this cavity contained an additional density whose identify has yet to be identified but was thought to be a non-proteinaceous co-factor. these exciting findings serve to define cte as representing a distinct and different neurodegenerative process from alzheimer’s disease. in addition, the identity and function of the associated co-factor represents a potentially exciting therapeutic target for patients with cte. conclusions in this review a number of studies are presented, some involving clinically relevant issues while others use preclinical models to investigate pathophysiologic mechanisms related to neurotrauma. the studies discussed here have relevance to neuropathologists with an interest in aspects of neurotrauma, whether they be involved in clinical diagnostic issues or preclinical models. despite the clinical importance of neurotrauma and its acute and long-term consequences, there exist major knowledge gaps throughout the field that require further investigation. i hope the studies discussed here will help to stimulate neuropathologists, especially those newly entering the field, to become active in this important and fertile area of investigation. disclosure statement the opinions expressed herein are those of the author and are not necessarily representative of those of the uniformed services university of the health sciences, the united states department of defense or of the united states army, navy or air force. references chiara ricciardi, m., r. p. bokkers, j. a. butman, d. a. hammoud, d. l. pham, s. warach and l. l. latour (2017). "trauma-specific brain abnormalities in suspected mild traumatic brain injury patients identified in the first 48 hours after injury: a blinded magnetic resonance imaging comparative study including suspected acute minor stroke patients." j neurotrauma 34(1): 23-30. crash, collaborators (2019). "effects of tranexamic acid on death, disability, vascular occlusive events and other morbidities in patients with acute traumatic brain injury (crash-3): a randomised, placebo-controlled trial." lancet 394(10210): 1713-1723. dickstein, d. l., m. y. pullman, c. fernandez, j. a. short, l. kostakoglu, k. knesaurek, l. soleimani, b. d. jordan, w. a. gordon, k. dams-o'connor, b. n. delman, e. wong, c. y. tang, s. t. dekosky, j. r. stone, r. c. cantu, m. sano, p. r. hof and s. gandy (2016). "cerebral [(18) f]t807/av1451 retention pattern in clinically probable cte resembles pathognomonic distribution of cte tauopathy." transl psychiatry 6(9): e900. falcon, b., w. zhang, a. g. murzin, g. murshudov, h. j. garringer, r. vidal, r. a. crowther, b. ghetti, s. h. w. scheres and m. goedert (2018). "structures of filaments from pick's disease reveal a novel tau protein fold." nature 561(7721): 137-140. falcon, b., j. zivanov, w. zhang, a. g. murzin, h. j. garringer, r. vidal, r. a. crowther, k. l. newell, b. ghetti, m. goedert and s. h. w. scheres (2019). "novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules." nature 568(7752): 420-423. fitzpatrick, a. w. p., b. falcon, s. he, a. g. murzin, g. murshudov, h. j. garringer, r. a. crowther, b. ghetti, m. goedert and s. h. w. scheres (2017). "cryo-em structures of tau filaments from alzheimer's disease." nature 547(7662): 185-190. gama sosa, m. a., r. de gasperi, g. s. perez garcia, g. m. perez, c. searcy, d. vargas, a. spencer, p. l. janssen, a. e. tschiffely, r. m. mccarron, b. ache, r. manoharan, w. g. janssen, s. j. tappan, r. w. hanson, s. gandy, p. r. hof, s. t. ahlers and g. a. elder (2019). "low-level blast exposure disrupts gliovascular and neurovascular connections and induces a chronic vascular pathology in rat brain." acta neuropathol commun 7(1): 6. gangolli, m., j. benetatos, t. j. esparza, e. m. fountain, s. seneviratne and d. l. brody (2019). "repetitive concussive and subconcussive injury in a human tau mouse model results in chronic cognitive dysfunction and disruption of white matter tracts, but not tau pathology." j neurotrauma 36(5): 735-755. griffin, a. d., l. c. turtzo, g. y. parikh, a. tolpygo, z. lodato, a. d. moses, g. nair, d. p. perl, n. a. edwards, b. j. dardzinski, r. c. armstrong, a. ray-chaudhury, p. p. mitra and l. l. latour (2019). "traumatic microbleeds suggest vascular injury and predict disability in traumatic brain injury." brain 142(11): 3550-3564. huang, y. l., y. s. kuo, y. c. tseng, d. y. chen, w. t. chiu and c. j. chen (2015). "susceptibility-weighted mri in mild traumatic brain injury." neurology 84(6): 580-585. johnson, v. e., j. e. stewart, f. d. begbie, j. q. trojanowski, d. h. smith and w. stewart (2013). "inflammation and white matter degeneration persist for years after a single traumatic brain injury." brain 136(pt 1): 28-42. mackay, d. f., e. r. russell, k. stewart, j. a. maclean, j. p. pell and w. stewart (2019). "neurodegenerative disease mortality among former professional soccer players." n engl j med 381(19): 1801-1808. marion, c. m., k. l. radomski, n. p. cramer, z. galdzicki and r. c. armstrong (2018). "experimental traumatic brain injury identifies distinct early and late phase axonal conduction deficits of white matter pathophysiology, and reveals intervening recovery." j neurosci 38(41): 8723-8736. marquie, m., m. d. normandin, a. c. meltzer, m. siao tick chong, n. v. andrea, a. anton-fernandez, w. e. klunk, c. a. mathis, m. d. ikonomovic, m. debnath, e. a. bien, c. r. vanderburg, i. costantino, s. makaretz, s. l. devos, d. h. oakley, s. n. gomperts, j. h. growdon, k. domoto-reilly, d. lucente, b. c. dickerson, m. p. frosch, b. t. hyman, k. a. johnson and t. gomez-isla (2017). "pathological correlations of [f-18]-av-1451 imaging in non-alzheimer tauopathies." ann neurol 81(1): 117-128. marquie, m., m. siao tick chong, a. anton-fernandez, e. e. verwer, n. saez-calveras, a. c. meltzer, p. ramanan, a. c. amaral, j. gonzalez, m. d. normandin, m. p. frosch and t. gomez-isla (2017). "[f-18]-av-1451 binding correlates with postmortem neurofibrillary tangle braak staging." acta neuropathol 134(4): 619-628. mckee, a. c., n. j. cairns, d. w. dickson, r. d. folkerth, c. d. keene, i. litvan, d. p. perl, t. d. stein, j. p. vonsattel, w. stewart, y. tripodis, j. f. crary, k. f. bieniek, k. dams-o'connor, v. e. alvarez, w. a. gordon and t. c. group (2016). "the first ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy." acta neuropathol 131(1): 75-86. mierzwa, a. j., c. m. marion, g. m. sullivan, d. p. mcdaniel and r. c. armstrong (2015). "components of myelin damage and repair in the progression of white matter pathology after mild traumatic brain injury." j neuropathol exp neurol 74(3): 218-232. mouzon, b., c. bachmeier, j. ojo, c. acker, s. ferguson, g. crynen, p. davies, m. mullan, w. stewart and f. crawford (2019). "chronic white matter degeneration, but no tau pathology at one-year post-repetitive mild traumatic brain injury in a tau transgenic model." j neurotrauma 36(4): 576-588. namjoshi, d. r., w. h. cheng, a. bashir, a. wilkinson, s. stukas, k. m. martens, t. whyte, z. a. abebe, k. a. mcinnes, p. a. cripton and c. l. wellington (2017). "defining the biomechanical and biological threshold of murine mild traumatic brain injury using chimera (closed head impact model of engineered rotational acceleration)." exp neurol 292: 80-91. roberts, i., a. belli, a. brenner, r. chaudhri, b. fawole, t. harris, r. jooma, a. mahmood, t. shokunbi, h. shakur and c.-t. collaborators (2018). "tranexamic acid for significant traumatic brain injury (the crash-3 trial): statistical analysis plan for an international, randomised, double-blind, placebo-controlled trial." wellcome open res 3: 86. stern, r. a., c. h. adler, k. chen, m. navitsky, j. luo, d. w. dodick, m. l. alosco, y. tripodis, d. d. goradia, b. martin, d. mastroeni, n. g. fritts, j. jarnagin, m. d. devous, sr., m. a. mintun, m. j. pontecorvo, m. e. shenton and e. m. reiman (2019). "tau positron-emission tomography in former national football league players." n engl j med 380(18): 1716-1725. valera, e. m., a. cao, o. pasternak, m. e. shenton, m. kubicki, n. makris and n. adra (2019). "white matter correlates of mild traumatic brain injuries in women subjected to intimate-partner violence: a preliminary study." j neurotrauma 36(5): 661-668. wong, d. f., p. b. rosenberg, y. zhou, a. kumar, v. raymont, h. t. ravert, r. f. dannals, a. nandi, j. r. brasic, w. ye, j. hilton, c. lyketsos, h. f. kung, a. d. joshi, d. m. skovronsky and m. j. pontecorvo (2010). "in vivo imaging of amyloid deposition in alzheimer disease using the radioligand 18f-av-45 (florbetapir [corrected] f 18)." j nucl med 51(6): 913-920. zhang, j., m. he, y. song and j. xu (2018). "prognostic role of d-dimer level upon admission in patients with traumatic brain injury." medicine (baltimore) 97(31): e11774 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. desmoplastic myxoid tumor of pineal region, smarcb1-mutant, in young adult feel free to add comments by clicking these icons on the sidebar free neuropathology 2:14 (2021) case report desmoplastic myxoid tumor of pineal region, smarcb1-mutant, in young adult branavan manoranjan1, yves p. starreveld1, robert a. nordal2, christopher dunham3, susanne bens4, christian thomas5, martin hasselblatt5, jeffrey t. joseph6 1 department of clinical neurosciences, division of neurosurgery, university of calgary, canada 2 department of radiation oncology, foothills medical centre, canada; department or diagnostic imaging, university of calgary, canada 3 department of pathology and laboratory medicine, university of british columbia, canada 4 institute of human genetics, ulm university & ulm university medical center, ulm, germany 5 institute of neuropathology, university hospital münster, germany 6 department of pathology and laboratory medicine, university of calgary, canada corresponding author: jeffrey t. joseph · department of pathology, mccaig tower 7539 · foothills medical centre and university of calgary · 1403 29 st nw, calgary, ab, t2n 2t9 · canada jtjoseph@ucalgary.ca submitted: 21 april 2021 accepted: 23 may 2021 copyedited by: bert m. verheijen published: 01 june 2021 https://doi.org/10.17879/freeneuropathology-2021-3340 additional resources and electronic supplementary material: supplementary material keywords: desmoplastic myxoid tumor, smarcb1-mutant, atypical teratoid/rhabdoid tumor, pineal, csf dissemination abstract we present a young adult woman who developed a myxoid tumor of the pineal region having a smarcb1 mutation, which was phenotypically similar to the recently described desmoplastic myxoid, smarcb1-mutant tumor of the pineal region (dmt-smarcb1). the 24-year-old woman presented with headaches, nausea, and emesis. neuroimaging identified a hypodense lesion in ct scans that was t1-hypointense, hyperintense in both t2-weighted and flair mri scans, and displayed gadolinium enhancement. the resected tumor had an abundant, alcian-blue positive myxoid matrix with interspersed, non-neoplastic neuropil-glial-vascular elements. it immunoreacted with cd34 and individual cells for ema. immunohistochemistry revealed loss of nuclear ini1 expression by the myxoid component but its retention in the vascular elements. molecular analyses identified a smarcb1 deletion and dna methylation studies showed that this tumor grouped together with the recently described dmt-smarcb1. a cerebrospinal fluid cytologic preparation had several cells morphologically similar to those in routine and electron microscopy. we briefly discuss the correlation of the pathology with the radiology and how this tumor compares with other smarcb1-mutant tumors of the nervous system. case presentation the patient is a 24-year-old woman who presented following 2-weeks of progressive headache, nausea, emesis, and diplopia. her medical history was significant for right sided hearing loss that began with tinnitus in 2014. neurological examination revealed bilateral grade i papilledema but no other focal motor or sensory deficits. preoperative non-contrast head computed tomography (ct) revealed a hypodense pineal region lesion extending into the posterior third ventricle and causing obstructive hydrocephalus (figure 1a). magnetic resonance imaging (mri) demonstrated a 3.0 x 3.4 x 2.6 cm mass in the pineal region (see below). beta-human chorionic gonadotropin (beta-hcg) and alpha-fetoprotein (afp) from serum and cerebrospinal fluid (csf) were within normal limits. a spinal mri did not identify visible leptomeningeal disease; however, csf cytology had atypical cells (see below). figure 1: preoperative neuroimaging. the tumor in the pineal region was hypodense on non-contrast ct head (a), hypointense in t1-weighted (b) and hyperintense in t2-weighted (c) mri sequences. it was strongly hyperintense in flair sequences (d), showed heterogeneous gadolinium enhancement (e), and did not demonstrate restricted diffusion (f). she underwent a successful endoscopic third ventriculostomy (etv) and biopsy of the pineal lesion on post-admit day 3. the initial biopsy did not identify a germ cell tumor or a lymphoma, so the patient underwent tumor resection via a suboccipital craniotomy. grossly, the tumor was opaque, grey-white in color, firm, and moderately vascular. the tumor was debulked, however it could not be completely resected where its superior edge was adherent to the vein of galen and the basal vein of rosenthal, and where it was adherent to the pulvinar. post-operative mri showed nodular enhancement along the right lateral margin of the surgical cavity. she was discharged home on postoperative day 8 but subsequently developed a pseudomeningocele that was treated with a programmable ventriculoperitoneal shunt. she remains well on follow-up at 5 months post-shunt insertion. because of the csf cytologic results (see below), she received craniospinal irradiation. curative intent craniospinal irradiation with a radiation boost to the primary tumor region was prescribed. 36 gy was delivered to the neuraxis in 1.8 gy fractions, and an additional 19.8 gy was given to the primary tumor volume. at last follow-up, five months after surgery, the patient remained well. neuroradiology preoperative non-contrast computed tomography (ct) revealed a hypodense pineal region tumor extending into the posterior third ventricle and causing obstructive hydrocephalus (figure 1a). magnetic resonance imaging (mri) showed that this mass was hypointense in t1-weighted images (figure 1b), hyperintense in t2-weighted (figure 1c) and flair images (figure 1d). the mass heterogeneously enhanced with gadolinium (figure 1e) and was in close proximity to the deep cerebral veins, including the vein of galen. it did not show diffusion restriction (figure 1f; adc map not shown). no leptomeningeal disease or drop metastases were demonstrable on spinal mri (data not shown). these indicated that the tumor was likely sparsely cellular, its matrix contained abundant but not free water, and it had vessels that lacked a blood-brain barrier. csf cytology the patient's initial csf cytology (figure 2), which was obtained from a lumbar puncture before surgical intervention, included several small clusters of atypical cells that had a high nuclear-to-cytoplasmic ratio, prominent nucleoli (white arrows in 2a), and variable amounts of cytoplasm. some cells had small zones of clear cytoplasm (black arrows in 2b) that were interpreted as vacuoles, while others lacked this feature (cell cluster in 2a). these cells were morphologically similar to those in the routine sections (figure 3f) and in the electron microscopy from the later tumor resection (see figure 6). although these cells had some features of chondrocytes (see arya, 2019; chen, 1990; takeda, 1981; bigner, 1981; and the chapter on csf cytology by cibas, 2014), they showed increased pleomorphism and displayed considerably less cytoplasm than previously illustrated chondrocytes in csf and lacked other debris that was illustrated in arya, 2019. figure 2: csf cytology. these cells from the preoperative csf, imaged under oil at 100x magnification, were stained with toluidine blue. they had large nuclei with prominent nucleoli (a, white arrows). some appeared multinucleated (a and b). their cytoplasm showed moderate staining cytoplasm (a), although several cells had partial cytoplasmic clearing or vacuoles (b, black arrows). tumor histology the tumor had two distinct features: a major myxoid component (figure 3) and a minor, intermixed, vascular component that was associated with neuropil and astrocytes (figure 5, see below). the former had sparsely (figure 3, black arrow in a and d) to moderately cellular regions (white arrow in 3a) containing occasional microcysts (3c, black arrow) that were delineated by a single layer of cells. although fragmented, the tumor had regions displaying a clear collagenous capsule (3b, black arrows). because this capsule was not adherent to other tissues, it likely indicated that the tumour grew into a fluid-filled space, either within the third ventricle or into a meningeal cistern. in more myxoid, less cellular areas, the tumor cells formed small clusters or chains of cells (3d, black arrow) that were embedded in the loose, myxoid stroma (3d, white arrow). fine eosinophilic strands meandered through the background stroma (3, d and e). although these regions were histologically reminiscent of chordoma, the tumor did not express brachyury (data not shown). tumor cells displayed a minor predilection for the perivascular region (3e, black arrow), although they did not form ependymal pseudorosettes or pilomyxoid rosettes. they often grew along barely visible eosinophilic strands (swirling pattern in 3e, chains of nuclei in 3d). at high magnification, the cells had small amounts of often eccentric, eosinophilic cytoplasm (3f, black arrow) and large, hyperchromatic nuclei. many cells appeared binucleated or multinucleated (3f, white arrow) these were morphologically similar to the few available cells in the csf cytology (compare with cells in figure 2). mitoses were present (3f, red arrow) but infrequent (less than one per ten high-power fields) and the ki67 proliferation index was 9% (see here). figure 3: routine histology. all panels stained with hematoxylin-eosin. the resection (a; 4x) had hypocellular (black arrow) and moderately cellular (white arrows) areas. in some fragments (b; 10x), the tumor was delimited by an outer collagenous layer (black arrows). more densely cellular regions (c; 10x) had microcysts (black arrow) lined by single cells. a loose, hypocellular area (d; 20x) displayed chain-like growth (black arrow), with cells set in a myxoid matrix having fine, eosinophilic strands. tumor cells showed a weak predilection for the perivascular region (e, white arrows; 20x) around thin-walled vessels (black arrow). at high magnification in f (oil immersion, 100x), the tumor cells had scant amount of often eccentric, eosinophilic cytoplasm (black arrow) and occurred in scattered small groups (white arrow). the red arrow highlights a mitotic figure. clicking the picture will lead you to the full virtual slide (h&e). the tumor's myxoid matrix and its scattered microcysts (figure 4a) diffusely and strongly stained with alcian blue (figure 4b), which indicates that the tumor elaborated a ground substance containing carbohydrate moieties. this myxoid matrix was pervasive throughout the tumor and was only absent in non-tumor vascular elements (see pink-staining vessels and islands in 4b). unlike similar tumors, this neoplasm did not display an overt desmoplasia in the surrounding parenchyma (see figure 3). however, the tumor did produce an extensive network of fine, eosinophilic strands (see figure 3d and 4c), which stained strongly blue in masson trichrome stains (figure 4d). this finding indicates that the tumor created an intrinsic desmoplastic collagenous stroma and likely explains why this myxoid tumor felt firm during surgery. figure 4: myxoid matrix and desmoplasia. matched panels a – b (10x) and c – d (20x) are from the separate histologic regions. the hematoxylin-eosin section in panel a includes several microcysts (black arrow), which stain with alcian blue (b, black arrow). in addition, the loose, slightly basophilic myxoid matrix in a and c also stains strongly for alcian blue (b). throughout the tumor, cells elaborated fine eosinophilic strands (c), which stained strongly blue in the masson trichrome (d). clicking the picture will lead you to the full virtual slide (elastica-van gieson). tumor immunophenotype immunohistochemistry was performed using the dako omnis. the overall results are presented in supplemental table 1. many areas of the tumor strongly expressed cd34 (figure 5a), which was positive in the perinuclear cytoplasm and to a lesser extent in the long processes extending from the cells. in contrast, only a minor population tumor cells expressed ema and only in a perinuclear location (panel 5b). however, this ema expression occurred throughout the tumor. gliovascular islands, composed of vessels encased within an astrocytic (glial fibrillary acidic protein, gfap; figure 5c) and synaptophysin-positive matrix (data not shown) were dispersed throughout the tumor. the myxoid component showed no expression of either of these markers, indicating it did not derive from primary brain parenchyma. the most informative stain was ini1, whose expression was lost in the tumor (figure 5d) but retained in the reactive gliovascular islands (black arrow in 5d). these findings indicated that the tumor had 1) a defect in the smarcb1 gene or its expression and 2) incorporated adjacent brain tissue. while individual tumor cells had cytological features reminiscent of rhabdoid cells, they did not express myogenin or desmin. see supplemental table 1. figure 5: immunohistochemistry. the tumor strongly immunoreacted for cd34 in many regions (panel a; 20x), in both perinuclear regions and cytoplasmic processes. although widespread, the tumor showed only perinuclear reactivity for ema, and only in a small subset of tumor cells (panel b; 40x). many regions of tumor had islands of neuropil and reactive astrocytes that stained for glial fibrillary acidic protein (gfap, panel c, black arrow; 20x). these islands retained strong reactivity for ini1 (panel d, from same region as panel c, black arrow; 20x), however, the encasing tumor had lost expression of this molecular marker. clicking the picture will lead you to the full virtual slide (smarcb1/ini1). electron microscopy toluidine-stained plastic 1-micron sections again showed a relatively hypocellular neoplasm have cells widely disbursed in the loose matrix (figure 6a). vessels and some surrounding cells represented portions of the gliovascular element (6a, red arrows). ultrastructurally, tumor cells had scant cytoplasm and large nuclei with prominent nucleoli (figure 6b, red arrowhead). although by both routine histology and in cytological preparation, the tumor had frequent cells that appeared to have several nuclei, in ultrastructure, at least some of these cells were separated by a plasmalemma (6c, red arrowheads). the ultrastructure of these tumor cells was similar to the cells in the csf cytology and resected tumor routine stains (compare figure 6 with figures 2 and 3f). the cytoplasm typically contained small vacuoles (6b, red arrows) and intermediate filaments (6d, red arrow). figure 6: electron microscopy. the toluidine-stained 1-micron plastic section (a, 40x) demonstrated a hypocellular tumor having cells embedded in a loose matrix, with embedded gliovascular elements (red arrows). in ultrastructural examination using a hitachi h-7650 electron microscope at 75 kv, the tumor cells have large nuclei with prominent nucleoli (b, arrowhead, and c), scant cytoplasm containing both vacuoles (b, red arrows) and intermediate filaments (d, red arrows). closely apposed cells were separated by plasmalemma (c, red arrowheads). molecular results on dna-methylation profiling (illumina methylation epic beadchip) using the heidelberg cns tumor classifier [capper, 2018], the neoplasm was not classifiable (calibrated score <0.3). copy number alterations derived from dna-methylation intensity values (figure 7a) revealed gains of chromosome 11q and 17q, losses of chromosomes 1q and 4p as well as heterozygous loss affecting the smarcb1 region on chromosome 22q11.23 (black arrow). to confirm the specific loss, smarcb1 fish (figure 7b) was performed as described previously [frühwald, 2020]. this demonstrated a heterozygous loss of smarcb1 locus. figure 7: copy number variation. copy number alterations (a) derived from dna-methylation intensity values revealed chromosomal gains (green values) and losses (red values), including losses affecting the smarcb1 region on chromosome 22q11.23 (arrow). fluorescence in situ hybridization (fish) analysis using a probe consisting of the two clones rp11-71g19 (spectrum orange) and rp11-911f12 (spectrum green) (b) showed one colocalized signal per nucleus (orange arrows) representing the intact smarcb1 locus and a heterozygous loss of the deleted smarcb1 allele (green arrows) containing the clone rp11-71g19 (loss of red signal) and parts of clone rp11-911f12 (diminished intensity of remaining single green signal). next, t-distributed stochastic neighbor embedding (t-sne) analysis was performed in comparison with dna methylation profiles of 2.801 previously published samples comprising 82 molecularly distinct cns tumor entities [capper, 2018] as well as dna methylation profiles of six previously published dmt cases [thomas, 2020]. here, the dna methylation profile of the present case clearly grouped with that of dmt (figure 8, right, gray oval). this grouping was close to but distinct from three different groupings of smarcb1-mutant atypical teratoid-rhabdoid tumors (atrt) (figure 8, right, blue ovals). figure 8: dna methylation studies and smarcb1 gene analysis. on t-distributed stochastic neighbor embedding (t-sne) analysis, the dna methylation profile of 82 distinct tumor entities (left), this patient's tumor grouped with that of dmt (right). discussion the tumour presented here is histologically and immunophenotypically similar to cases presented in thomas, 2020, matsumura, 2021, wang, 2021. as demonstrated in the dna methylation studies (figure 8), this tumour also falls into the same cluster as the desmoplastic myxoid tumour, smarcb1-mutant tumor described in thomas, 2020. available demographic data from all prior cases indicate no gender differences (f: 6; m: 4) and an average age of onset of 37 years (range 15-61 years old). of note, all prior cases have been in the pineal region. the smarcb1 gene, which is also known as ini1 (integrase interactor 1) and hsnf5 (snf5 homolog), encodes the swi/snf-related, matrix-associated actin-dependent regulator of chromatin, subfamily b, member 1 protein, which is a core component of the atp-dependent swi/snf chromatin-remodeling complex [phelan, 1999] and acts as a tumor suppressor [versteege, 2002]. biallelic inactivation of smarcb1 has been associated with the growth of benign and malignant tumors in children and adults [hollmann, 2011]. intracranial smarcb1-deficient tumors span a range of malignancies and include childhood atypical teratoid/rhabdoid tumor (atrt) [biegel, 1999], cribriform neuroepithelial tumor [hasselblatt, 2009], poorly differentiated chordoma [mobley, 2010], meningeal tumors having challenging histology [dadone, 2017], a young adult superficial (not pineal) rhabdoid tumour [bodi, 2018], poorly differentiated sinonasal carcinoma [agaimy, 2017], and the desmoplastic myxoid tumor, smarcb1-mutant, of the pineal region (dmt) that we present here [thomas, 2020; matsumura, 2021; wang, 2021]. two previous reports discussed dmt radiologic features. unlike the tumour described here, which was hypodense in ct and hypointense in t1-weighted mri (figure 1a and 1b), the tumor described in wang, 2021 was hyperdense on ct scans and slightly hyperintense on t1-weighted mri scans. it is likely that the more prominent desmoplasia illustrated in wang, 2021, compared to our tumor, correlated with their increased ct signal density. in our patient's tumor, the alcian blue myxoid component (see figure 5b) was predominant, which correlates with our increased t2 and flair signal (see figure 1c and 1d) and is dissimilar to the isointense t2-weighted signal described in matsumura, 2021. similar to the image illustrated in matsumura, 2021, gadolinium enhancement was heterogeneous in our patient's tumour. this tumor had several features not yet described in dmts. it had a pervasive myxoid matrix and had little of the overt and possibly reactive desmoplasia illustrated in thomas, 2020, wang, 2021, or matsumura, 2021. it did, however, diffusely elaborate fine, eosinophilic strands similar to those illustrated in thomas, 2020 and wang 2021 (see figure 4c and 3d), which stain like collagen (figure 4d). we feel that this represents intrinsic tumor desmoplasia, rather than the reactive desmoplasia illustrated in the other cases and common to many systemic cancers. the dmt also had non-neoplastic elements, including gliovascular islands (figure 5c) that strongly expressed gfap but retained ini1 expression (figure 5d). these islands likely originated from the underlying pineal gland or surrounding brain. ultrastructural studies demonstrated the tumour cells had large nuclei with prominent nucleoli. the cytoplasm was scant and had vacuoles and intermediate filaments (figure 6). unlike the prior reports, our patient had atypical cells in her cerebrospinal fluid (figure 2). because of the scant available material in the csf cytology, we have not been able to verify these cells as neoplastic; however, we feel their cytologic features were morphologically similar to those in light (figure 3f) and electron microscopy (figure 6) from the tumor resection and considered them neoplastic rather than chondrocytes. our tumor cells lacked desmin or myogenin expression. as in our tumor, most cases expressed cd34 (figure 5a). ema expression has been variable, with strong positivity in 5 of 6 cases in thomas, 2020, focally positive in matsumura, 2020, and absent in wang, 2021. in our patient, the ema expression was subtle but distinct (figure 5b). given these data, we feel the salient pathological features of this tumor are: regions having a pale or slightly basophilic, alcian-blue positive myxoid matrix areas demonstrating either frank desmoplasia or intrinsic, fine eosinophilic collagenous bands, which stain strongly blue in masson trichrome cytologic features include small to medium size cells having an increased nuclear-to-cytoplasmic ratio, a large nucleus having a prominent nucleolus, and variable amounts of eosinophilic cytoplasm, which can sometimes be scant or can elaborate fine eosinophilic filaments and small vacuoles variable expression of cd34 and ema; neoplastic cells lack expression of specific muscle, glial, or neuronal proteins loss of ini1 protein expression or demonstration of smarcb1 mutation the advent of integrated genomics has revolutionized our understanding of atrt through the identification of three molecular subgroups (atrt-shh, atrt-tyr, and atrt-myc), which differ in their demographics, location, prognosis, and smarcb1 mutational profile [johann, 2016; torchia, 2016]. comparative methylation profiling of dmt with other intracranial smarcb1-mutant tumors has shown dmt, smarcb1-mutant tumors most closely resemble atrt-myc and poorly differentiated chordomas [thomas, 2020]. by contrast, the superficial cd34+ smarcb1-deficient tumor described in bodi, 2018, clustered with the atrt-myc, using the heidelberg brain tumor classifier, and not with the dmt, smarcb1-mutant group [thomas, 2020]. the patient we present here also has a methylation profile that clusters together with the other described smarcb1-mutant myxoid pineal tumours (see figure 8). while it is interesting to speculate about the origins or common features of smarcb1-mutant tumours, and although it has some features that overlap with atrt, the neoplasm described here is histologically distinct from other smarcb1-mutant tumours. references agaimy a, hartmann a, antonescu cr, chiosea si, el-mofty sk, geddert h, iro h, lewis js jr, märkl b, mills se, riener mo, robertson t, sandison a, semrau s, simpson rh, stelow e, westra wh, bishop ja. smarcb1 (ini-1)-deficient sinonasal carcinoma: a series of 39 cases expanding the morphologic and clinicopathologic spectrum of a recently described entity. am j surg pathol. 2017 apr;41(4):458-471. arya a. cartilage cells a potential mimicker of malignant cells in cerebrospinal fluid and a diagnostic pitfall. j cytol. 2019 oct-dec;36(4):218-219. biegel ja, zhou jy, rorke lb, stenstrom c, wainwright lm, fogelgren b. germ-line and acquired mutations of ini1 in atypical teratoid and rhabdoid tumors. cancer res. 1999 jan;59(1):74-79. bigner sh, jonston ww. the cytopathology of cerebrospinal fluid. i. nonneoplastic conditions, lymphoma and leukemia. acta cytol. 1981 jul-aug;25(4):345-353. bodi i, giamouriadis a, sibtain n, laxton r, king a, vergani f. primary intracerebral ini1-deficient rhabdoid tumor with cd34 immunopositivity in a young adult. surg neurol int. 2018 feb;9(45). capper d, jones dtw, sill m, et al. dna methylation-based classification of central nervous system tumours. nature. 2018 mar;555(7697):469-474. chen kt, moseley d. cartilage cells in cerebrospinal fluid. arch pathol lab med. 1990 feb;114(2):212. cibas es. chapter 6. cerebrospinal fluid. in cibas es, ducatman bs, editors. cytology e-book: diagnostic principles and clinical correlates, fourth edition. 2014, elsevier, health sciences division, philadelphia. dadone b, fontaine d, mondot l, cristofari g, jouvet a, godfraind c, varlet p, ranchère-vince d, coindre jm, gastaud l, baudoin c, peyron ac, thyss a, coutts m, michiels jf, pedeutour f, burel-vandenbos f, renop. meningeal swi/snf related, matrix-associated, actin-dependent regulator of chromatin, subfamily b member 1 (smarcb1)-deficient tumours: an emerging group of meningeal tumours. neuropathol appl neurobiol. 2017 aug;43(5):433-449. frühwald mc, hasselblatt m, nemes k, bens s, steinbügl m, johann pd, kerl k, hauser p, quiroga e, solano-paez p, biassoni v, gil-da-costa mj, perek-polnik m, van de wetering m, sumerauer d, pears j, stabell n, holm s, hengartner h, gerber nu, grotzer m, boos j, ebinger m, tippelt s, paulus w, furtwängler r, hernáiz-driever p, reinhard h, rutkowski s, schlegel p-g, schmid i, kortmann r-d, timmermann b, warmuth-metz m, kordes u, gerss j, nysom k, schneppenheim r, siebert r, kool m, graf n. age and dna methylation subgroup as potential independent risk factors for treatment stratification in children with atypical teratoid/rhabdoid tumors. neuro oncol. 2020 jul;22(7):1006-1017. hasselblatt m, oyen f, gesk s, kordes u, wrede b, bergmann m, schmid h, frühwald mc, schneppenheim r, siebert r, paulus w. cribriform neuroepithelial tumor (crinet): a nonrhabdoid ventricular tumor with ini1 loss and relatively favorable prognosis. j neuropathol exp neurol. 2009 dec;68(12):1249-1255. hollmann tj, hornick jl. ini1-deficient tumors: diagnostic features and molecular genetics. am j surg pathol. 2011 oct;35(10):e47-e63. johann pd, erkek s, zapatka m, kerl k, buchhalter i, hovestadt v, jones dtw, sturm d, hermann c, segura wang m, korshunov a, rhyzova m, gröbner s, brabetz s, chavez l, bens s, gröschel s, kratochwil f, wittmann a, sieber l, geörg c, wolf s, beck k, oyen f, capper d, van sluis p, volckmann r, koster j, versteeg r, von deimling a, milde t, witt o, kulozik ae, ebinger m, shalaby t, grotzer m, sumerauer d, zamecnik j, mora j, jabado n, taylor md, huang a, aronica e, bertoni a, radlwimmer b, pietsch t, schüller u, schneppenheim r, northcott pa, korbel jo, siebert r, frühwald mc, lichter p, eils r, gajjar a, hasselblatt m, pfister sm, kool m. atypical teratoid/rhabdoid tumors are comprised of three epigenetic subgroups with distinct enhancer landscapes. cancer cell. 2016 mar;29(3):379-393. matsumura n, goda n, yashige k, kitagawa m, yamazaki t, nobusawa s, yokoo h. desmoplastic myxoid tumor, smarcb1-mutant: a new variant of smarcb1-deficient tumor of the central nervous system preferentially arising in the pineal region. virchows arch. 2021 jan. doi: 10.1007/s00428-020-02978-3. epub ahead of print. mobley bc, mckenney jk, bangs cd, callahan k, yeom kw, schneppenheim r, hayden mg, cherry am, gokden m, edwards ms, fisher pg, vogel h. loss of smarcb1/ini1 expression in poorly differentiated chordomas. acta neuropathol. 2010 dec;120(6):745-753. phelan ml , sif s, narlikar gj, kingston re. reconstitution of a core chromatin remodeling complex from swi/snf subunits. mol cell. 1999 feb;3(2):247-253 . takeda m, king de, choi hy, gomi k, lang wr. diagnostic pitfalls in cerebrospinal fluid cytology. acta cytol. 1981 may-jun;25(3):245-250. thomas c, wefers a, bens s, nemes k, agaimy a, oyen f, vogelgesang s, rodriguez fj, brett fm, mclendon r, bodi i, burel-vandenbos f, keyvani k, tippelt s, poulsen fr, lipp es, giannini c, reifenberger g, kuchelmeister k, pietsch t, kordes u, siebert r, frühwald mc, johann pd, sill m, kool m, von deimling a, paulus w, hasselblatt m. desmoplastic myxoid tumor, smarcb1-mutant: clinical, histopathological and molecular characterization of a pineal region tumor encountered in adolescents and adults. acta neuropathol. 2020 feb;139(2):277-286. torchia j, golbourn b, feng s, ho kc, sin-chan p, vasiljevic a, norman jd, guilhamon p, garzia l, agamez nr, lu m, chan ts, picard d, de antonellis p, khuong-quang da, planello ac, zeller c, barsyte-lovejoy d, lafay-cousin l, letourneau l, bourgey m, yu m, gendoo dma, dzamba m, barszczyk m, medina t, riemenschneider an, morrissy as, ra ys, ramaswamy v, remke m, dunham cp, yip s, ng hk, lu jq, mehta v, albrecht s, pimentel j, chan ja, somers gr, faria cc, roque l, fouladi m, hoffman lm, moore as, wang y, choi sa, hansford jr, catchpoole d, birks dk, foreman nk, strother d, klekner a, bognár l, garami m, hauser p, hortobágyi t, wilson b, hukin j, carret as, van meter te, hwang ei, gajjar a, chiou sh, nakamura h, toledano h, fried i, fults d, wataya t, fryer c, eisenstat dd, scheinemann k, fleming aj, johnston dl, michaud j, zelcer s, hammond r, afzal s, ramsay da, sirachainan n, hongeng s, larbcharoensub n, grundy rg, lulla rr, fangusaro jr, druker h, bartels u, grant r, malkin d, mcglade cj, nicolaides t, tihan t, phillips j, majewski j, montpetit a, bourque g, bader gd, reddy at, gillespie gy, warmuth-metz m, rutkowski s, tabori u, lupien m, brudno m, schüller u, pietsch t, judkins ar, hawkins ce, bouffet e, kim sk, dirks pb, taylor md, erdreich-epstein a, arrowsmith ch, de carvalho dd, rutka jt, jabado n, huang a. integrated (epi)-genomic analyses identify subgroup-specific therapeutic targets in cns rhabdoid tumors. cancer cell. 2016 dec;30(6):891-908. versteege i, medjkane s, rouillard d, delattre o. a key role of the hsnf5/ini1 tumour suppressor in the control of the g1-s transition of the cell cycle. oncogene. 2002 sep;21(42):6403-6412. wang ye, chen jj, wang w, zhang al, zhou w, wu hb. a case of desmoplastic myxoid tumor, smarcb1 mutant, in the pineal region. neuropathology. 2021 feb;41(1):37-41. wilson bg, roberts cw. swi/snf nucleosome remodellers and cancer. nat rev cancer. 2011 jun;11(7):481-492. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. loss of ramified microglia precedes axonal spheroid formation in adult-onset leukoencephalopathy with axonal spheroids. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:27 (2020) original paper loss of ramified microglia precedes axonal spheroid formation in adult-onset leukoencephalopathy with axonal spheroids. murad alturkustani 1,2,3, qi zhang 2,3, basma alyamany 2,3, lee-cyn ang 2,3 1 department of pathology, king abdulaziz university, jeddah, saudi arabia 2 london health sciences centre, london, ontario, canada 3 western university, london, ontario, canada corresponding author: murad alturkustani · pathology department · king abdulaziz university · abdullah sulayman, po box 80205 · jeddah · saudi arabia alturkustani.murad@gmail.com submitted: 22 august 2020 accepted: 9 september 2020 copyedited by: deanna c. fang and henry robbert published: 5 october 2020 https://doi.org/10.17879/freeneuropathology-2020-2971 additional resources and electronic supplementary material: supplementary material keywords: adult-onset leukoencephalopathy with axonal spheroids, leukodystrophy, microglia, axonal spheroids, white matter, hdls, alsp abstract two different pathological mechanisms have been suggested to underlie adult-onset leukoencephalopathy with axonal spheroids (alas). pathological studies have suggested that alas involves primary axonopathy with secondary demyelination. however, the identification of mutations in colony stimulating factor 1 receptor (csf1r), important for microglial survival, has suggested that alas is a microgliopathy. this study examines the correlation between microglial changes and axonopathy in alas. a total of 6 alas cases were studied. white matter lesions were classified into three evolving stages: 1) numerous axonal spheroids among well-myelinated fibers; 2) moderate loss of myelinated fibers with or without axonal spheroids; and 3) a leukodystrophy-like pattern of severe confluent axonal and myelin loss. axonal spheroids and ramified microglia were semi-quantified and the lesions were assigned a score of 0–3. we found a strong correlation between the preponderance of axonal spheroids and ramified microglial loss. all areas with a predominance of axonal spheroids showed a near-complete absence of ramified microglia, which was also apparent in small cortical and white matter lesions. in contrast, some areas with no ramified microglia showed no axonal pathology. our findings support the suggestion that ramified microglia loss precedes axonal spheroids formation. this observation will help to better understand the pathogenesis of alas and suggests a protective role of microglia. introduction two different pathological mechanisms have been suggested to underlie adult-onset leukoencephalopathy with axonal spheroids (alas). pathological studies have proposed a primary axonopathy with secondary demyelination (1, 2), while genetic/molecular studies have identified a mutation in a gene important for microglial function and, hence, implied that alas is a microgliopathy (3). moreover, a recent pathological study has identified a microglial morphological alteration (i.e. “dysplastic” microglia) as the pathological correlate for microgliopathy (4). alas is also known by other names in the literature with most common ones being hereditary diffuse leukoencephalopathy with spheroids (hdls) and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (alsp). recent advances in our understanding of the biology and functions of microglia have demonstrated both its beneficial and harmful effects on various diseases (5). investigating the relationship between microgliopathy and axonopathy in the pathogenesis of alas may provide better insight into the role of microglia in this disease. recent attempts to explain the pathogenesis yielded different conclusions: 1) generalized colony stimulating factor 1 receptor (csf1r) signaling impairment in cd68-immunopositive microglia is followed by multifocal axonal degeneration (6); 2) increase in ionizing calcium-binding molecule 1 (iba1) and cd68-immunopositive cell numbers precedes axonal pathology (7); 3) uneven distribution of iba1-immunopositive cells in the white matter precludes any firm conclusions regarding the role of these cells in the pathology (4); and 4) csf1r mutations lead to aberrant microglia density and distribution, and regional loss of microglia (8). in this study, we investigated the relationship between axonal pathology and microglial changes in alas using the pathological staging system of alas proposed by alturkustani et al. (1). this system focuses on the specific stages of the lesions rather than the overall pathological stage of the entire case at a point in time (7) and should provide a more precise correlation. materials and methods a formal autopsy consent was provided in all cases and the western university research ethics board approved the study. a total of 6 alas cases were studied. four cases were described before (cases 1–4) (1). two additional cases of alas were referred to the london health sciences center laboratory in 2013–2017. the brains were fixed and prepared as described before (1). representative samples were submitted for microscopic examination and staining, and immunostaining procedures were performed as described previously (1). these procedures include luxol fast blue (lfb) with hematoxylin and eosin (he) staining. for immunohistochemistry, the primary antibodies used were anti-amyloid precursor protein (app; chemicon, temecula, ca), anti-phosphorylated neurofilament (smi31; covance, berkeley, ca), and anti-glial fibrillary acidic protein (dako, carpinteria, ca). mouse monoclonal anti-hla-dr antibody (1:200; clone cr3/43; dako, carpinteria, ca) was used to label microglial cells. anti-ionized calcium binding adaptor molecule 1 (iba1) antibody (1:500, 019-19741, wako) was stained in dr. v. wee yong’s laboratory (calgary, canada) following their previously published protocol (9). the white matter lesions were classified into three evolving stages, as discussed in our previous work (1): 1) numerous axonal spheroids within well-myelinated fibers; 2) moderate loss of myelinated fibers with or without axonal spheroids; and 3) a leukodystrophy-like pattern with severe confluent axonal and myelin loss. digital images of lfb-he, app, hla-dr, iba1, and smi31 staining slides were obtained using the aperio system. particular areas of interest were demarcated on the computer screen of these digital images. at least five areas for each pathological stage from each case were examined. the pathological features, regardless of the size of the lesion, defined the pathological stages. any lesion can range from very small areas (usually in stage 1 pathology) to very large (common in stage 3 pathology). a single area in the white matter could contain more than one pathological stage. axonal spheroids and the hla-dr-expressing cells were semi-quantified (see supplemental figure 1) and lesions were assigned a score of 0–3. the scoring for axonal spheroids was as follows: 0 = absence of axonal spheroids; 1 = few axonal spheroids (1–2/high power field; hpf); 2 = moderate number of axonal spheroids (3–5/hpf); and 3 = frequent axonal spheroids (6 or more/hpf). for the hla-dr and/or iba1-expressing cells, we subdivided them into ramified-phenotype and amoeboid-phenotype. their numbers were scored in the following manner: 0 = no hla-dr staining; 1= few cells (1–9/hpf); 2 = moderate number of cells (10–19/hpf); and 3 = frequent cells (20 or more/hpf). several morphological shapes of microglia have been identified (10). however, for the sake of simplicity, in this study we divided microglia into two different types: 1) ramified microglia with a small cell body with branching processes radiating from the cell bodies, including a broad spectrum of reactive microglial shapes with varying lengths and thicknesses of processes and different cell body dimensions; and 2) amoeboid microglia with a macrophage-like or amoeboid phenotype characterized by a round cytoplasm with no processes. the white matter from the same autopsy tissue in the areas adjacent to the affected white matter and separate from it was used as control tissue to confirm the reliability of the immunostains (i.e. internal positive control) used in this study. as the autopsy brain tissue from relatively normal cases was not expected to show axonal spheroids, we did not use such controls in our study. results the clinical information for the first four cases has been discussed before (1) and is summarized in table 1. in general, clinical presentations were dependent on the location of the white matter areas affected, which were preferentially frontotemporal. case 5 was a 57-year-old female who had a rapid 2-year progression of neurobehavioral symptoms (self-neglect and apathy) and seizures until death. she had a sister with cognitive impairments, but slower progression. both patients had a previously unreported variant of csf1r (c.2563 c>t) mutation (11). their mother had a history of early-onset dementia. case 6 was a 38-year-old female with a rapid decline in cognition and shortand long-term memory impairment. she was placed in a nursing home as she required assistance for daily life activities and died two years after the initial presentation. her first-degree relatives had a positive history of dementia. she had another novel variant of csf1r (c.2377a>g) mutation. the csf1r gene mutational analysis for the first 4 cases was unsuccessful as the quality of the dna extracted from fixed tissue was inadequate, as discussed before (1). both mutations were analyzed via the varsome website (https://varsome.com/variant/hg19/nm_005211.3%3ac.2563c%3et and https://varsome.com/variant/hg19/nm_005211.3%3ac.2377a%3eg). various software tools including dann (score 0.9991 and 0.9989, respectively), mutationtaster (disease causing), fathmm (damaging), fathmm-mkl (damaging), metasvm (damaging), and metalr (damaging) predicted pathogenicity, which was supported by locations within the protein kinase functional domain. antibodies against hla-dr and iba1 are considered the most sensitive markers for microglia as both stain all the morphological forms of activated microglia, phagocytic, and dystrophic microglia (5). although iba1 may be considered to be a more sensitive marker for these microglia by some authors (5), we found that hla-dr is more sensitive for microglia staining in the white matter and comparable to iba1 staining in the gray matter. iba1 immunostaining also showed background staining, requiring careful examination to distinguish the immunopositive cells from the background (see supplemental figure 2). this resulted in an inferior quality of images for analysis. the following results were confirmed by both hla-dr and iba1 immunostains but are better demonstrated with the hla-dr images, which we will utilize in the rest of this article. hla-dr and iba1 immunostaining showed a wide variation of microglial morphology in both gray and white matter in all cases. microglial processes showed variations in number (single to multiple), thickness, and length. some of the processes showed discontinuous staining and some areas showed only fragmented processes, which could be considered dysplastic (4) or dystrophic (5). microglia with fragmented processes were observed along with those with intact processes in areas of normal-appearing white matter. this wide morphological variation made it difficult to correlate between microglial morphology and axonal spheroids. therefore, as indicated in materials and methods, we only considered microglia with the amoeboid phenotype (i.e. globular cytoplasm with no processes) as a separate form of microglia. all other morphological variations of microglia, including those with fragmented processes, were considered ramified/reactive microglia. using this simplified categorization of hla-dr and iba1-expressing cells (ramified vs. amoeboid phenotype), we consistently detected the following changes in all 6 cases (table 2): the white matter lesions classified as stage 1 pathology showed well-myelinated areas with moderate to frequent (score 2–3) axonal spheroids and a consistent loss of hla-dr-expressing cells with ramified morphology (score 0) in close proximity to the axonal spheroids (figure 1a–f). some areas had a few (score 1) hla-dr-expressing microglia with amoeboid phenotype, while others showed none (score 0). this may indicate that these areas had a slightly longer pathology duration than the areas without these cells. the white matter adjacent to the affected areas was unremarkable, with no axonal spheroids, no pigmented microglia, or other abnormality (figure 1g). however, hla-dr immunostaining showed denser forms of “ramified” microglia (figure 1h). the remarkable association of axonal spheroids and the complete loss of ramified microglia was also evident in the affected cortex (figure 2a–d). this association was apparent even in a very small area of cortical involvement, while the immediately adjacent area showed ramified microglia (figure 2e–h). in stage 2 pathology, white matter showed reduced myelin staining (figure 3a–b), a variable number of axonal spheroids ranging from none to frequent (score 0–3), moderate to frequent amoeboid microglia (score 2–3), and none to few ramified microglia. according to the distribution of axonal spheroids, these areas were subclassified into three components: 1) areas with axonal spheroids (score 1–3), showing no ramified microglia (score 0), but moderate to frequent amoeboid microglia (score 2–3). these areas may represent an “active/ongoing injury.” the white matter surrounding these areas showed widespread dense ramified microglia (score 3), while cells with amoeboid-phenotype were present in the central area (score 2–3), and the surrounding white matter (figure 3c–f), used as an internal control for hla-dr immunostaining. 2) areas with no axonal spheroids (score 0), few-moderate amoeboid microglia (score 1–2), and no ramified microglia (score 0; figure 3g–h). these can be considered “susceptible areas” and can have a different degree of myelinated axons loss. 3) areas with no axonal spheroids (score 0), few-moderate amoeboid microglia (score 1–2), and none to few ramified microglial cells (score 1). these may represent areas of secondary axonal degeneration. figure 1. microglia and axonal spheroids in stage 1 pathology. (a) normal-appearing white matter with frequent spheroids inside the rectangular inset (lfb-he; scale bar: 500 µm). (b) higher magnification of (a) shows many spheroids and no pigmented microglia (lfb-he; scale bar: 50 µm). (c) app immunostaining highlights frequent axonal spheroids inside the rectangular inset (app; scale bar: 500 µm). (d) higher magnification of (c) (app; scale bar: 100 µm). (e) the affected area in (c) with many axonal spheroids in the white matter and a small area of adjacent cortex that shows a nearly complete loss of hla-dr-expressing ramified microglia (inside the polygonal inset) and only a few scattered hla-dr-expressing amoeboid-phenotype cells. the adjacent normal-appearing areas show an increased expression of hla-dr-positive ramified microglia (hla-dr; scale bar: 1 mm). (f) higher magnification of (e) (hla-dr; scale bar: 500 µm). (g) normal-appearing white matter adjacent to the affected area shows no recognizable abnormalities (lfb-he; scale bar: 50 µm). (h): hla-dr immunostaining shows a frequent expression of hla-dr-immunopositive ramified microglia (hla-dr; scale bar: 50 µm). all images in this figure are representative of case 2. figure 2. cortical involvement in alas. (a) focal involvement of the upper cortex with frequent axonal spheroids (inside ellipse) compared to the cortex on the right side. the underlying u-fibers are also affected (lfb-he; scale bar: 500 µm). (b) higher magnification of (a) from the cortical area inside the ellipse (lfb-he; scale bar: 50 µm). (a) and (b) represent an inset (higher magnification of the upper rectangle) in figure 4a. (c) hla-dr immunostaining shows a complete focal loss of staining in the area with frequent axonal spheroids (inside ellipse) (hla-dr; scale bar: 500 µm). (d) higher magnification of (c) to highlight the presence of axonal spheroids (arrows) associated with a complete absence of hla-dr-expressing ramified microglia (hla-dr; scale bar: 50 µm). (e): a small cortical area (inside the rectangle) with frequent axonal spheroids (app; scale bar: 500 µm). (f) higher magnification of (e) (app; scale bar: 50 µm.). (g) hla-dr loss is limited to the very small affected area (inside the rectangle), while the surrounding cortical areas show hla-dr-expressing ramified microglia (hla-dr; scale bar: 500 µm). (h) higher magnification of (g) (hla-dr; scale bar: 200 µm). all images in this figure are representative of case 6. figure 3. microglia and axonal spheroids in stage 2 pathology. (a) decreased myelin staining in the deep subcortical white matter (mostly inside the inset) compared to the surrounding subcortical u-fibers (lfb-he, scale bar: 500 µm). (b) higher magnification of (a) (lfb-he;, scale bar: 200 µm). (c) the area showing decreased myelin staining in (a) shows a complete loss of hla-dr-expressing ramified microglia (mostly inside the inset) but moderate numbers of hla-dr-immunopositive cells with amoeboid phenotype. the adjacent white matter shows frequent hla-dr-expressing ramified microglia (hla-dr; scale bar: 500 µm). (d) higher magnification of (c) (scale bar: 200 µm). (e) app-immunopositive axonal spheroids are limited to the area (mostly inside the inset) showing an extensive loss of ramified microglia and presence of hla-dr expressing cells with amoeboid phenotype (app; scale bar: 500 µm). (f) higher magnification of (e) (app; scale bar: 200 µm). (g): another area with decreased myelin staining due to a prominent loss of myelinated fibers but without axonal spheroids (lfb-he; scale bar: 50 µm). (h) scattered hla-dr-immunopositive cells with amoeboid phenotype in the same area (hla-dr; scale bar: 100 µm). all images in this figure are representative of case 5. in stage 3 pathology, the white matter showed a marked loss of myelinated axons and a relative preservation of the subcortical u-fibers (figure 4a), which was classified as a “leukodystrophy-like” pattern. although the white matter in this area as a whole was considered stage 3 pathology, hla-dr immunostaining highlighted different patterns of staining (figure 4b), which allowed us to appreciate variations in the preservation of the white matter and the distribution of hla-dr-expressing cells in stage 3. we could observe areas of compact neuropil in the background, few small myelinated axons, a moderate number of oligodendrocytes, and mild to moderate gliosis (figure 4c–e) along with rarefied areas showing a marked loss of oligodendrocytes and moderate gliosis (figure 4g). hla-dr immunostaining highlighted dense (score 3) ramified microglia in the areas showing higher white matter preservation and in the relatively preserved myelinated subcortical u-fibers (figure 4b, d, f), while rarefied areas showed no ramified microglia (score 0) and a few to moderate (score 1–2) amoeboid microglia (figure 4h). axonal spheroids were absent (score 0) in most areas, yet scattered and rare (score 1) in others. unfortunately, the imaging characteristics are not available for the radiological-pathological correlation. in general, stage 3 pathology, which denotes long-standing lesions, are prominent in frontal and temporal lobes, less prominent in parietal lobes, and rare in occipital lobes. discussion axonal spheroids and ramified microglia we found a strong association between the presence of axonal spheroids and a complete loss of ramified microglia in all pathological stages of alas lesions. this association was also apparent in very small areas with frequent axonal spheroids and in focal cortical lesions, strengthening the reliability of the findings. although this was a semi-quantitative study, our findings demonstrate a strong correlation between the complete absence of ramified microglia (score 0) and the presence of axonal spheroids in affected areas. this finding could be considered “quantitative”, as score 0 denotes a complete absence of the ramified microglia. in stage 3, the presence of ramified microglia was associated with areas of better white matter preservation. these findings provide a link between pathological features observed (i.e. axonopathy) and the genetic abnormality identified (i.e. microgliopathy) in alas, supporting the beneficial effects of the reactive ramified microglia in maintaining axonal integrity. csf1r and alas identifying csf1r mutations as the underlying genetic abnormality in alas suggested microgliopathy as the underlying abnormality (3, 12) in alas. as it is the most common mutation in alas, konno et al. proposed the term “csf1r-related leukoencephalopathy” for this entity (13). as the pathological features are the gold standard for alas diagnosis and not all alas cases have csf1r alterations, this term would be specific to cases with confirmed csf1r mutations only and would not be suitable for all alas cases. aars2 mutations have been described in cases suspected to represent alas, including one case with biopsy findings supportive of this diagnosis (14). however, we concur with konno et al. that “further studies, including detailed analyses of autopsied brains, are required to characterize aars2 associated leukoencephalopathy” (13). csf1r is a tyrosine kinase receptor whose signaling is fundamental for microglial survival (15). two different mechanisms have been proposed for csf1r mutations to affect the function of microglia in patients with alas: 1) haploinsufficiency mechanism that results in a loss of function of csf1r (8, 16, 17), and 2) a dominant-negative model (18). it is still unclear what the lost functions are or what is constitutive of the dominant-negative effect on the microglia. oosterhof et al. concluded that the haploinsufficiency resulted in the maldistribution of microglia that leads to their local loss (8). their recent work showed that homozygous mutations in csf1r could result in the complete absence of microglia in pediatric-onset leukodystrophy (19). our results confirmed the regional loss of ramified microglia in alas and found that the axonal pathology is mostly limited to these areas, supporting the importance of microglia in alas’ pathogenesis and suggesting a protective role of ramified microglia. figure 4. microglia and axonal spheroids in stage 3 pathology. (a) marked loss of myelinated fibers in the deep white matter with relative preservation of the subcortical u-fibers (lfb-he; scale bar: 5 mm). (b) variable degree of hla-dr expression in the subcortical deep white matter with multifocal areas containing hla-dr-expressing ramified microglia. these areas are found mainly in the subcortical u-fibers and small foci of the adjacent white matter. the deeper white matter shows only scattered hla-dr-expressing ramified microglia (hla-dr; scale bar: 5 mm). (c) higher magnification of the area within the lower rectangle outlined in (a). the deep white matter shows a variable loss of myelinated fibers. areas of prominent loss show marked rarefaction, while areas with better preserved myelinated fibers in the center show a more compact parenchyma (lfb-he; scale bar: 500 µm). (d) a better-preserved central area in (c) shows a marked expression of hla-dr ramified microglia, while rarefied areas show only scattered hla-dr-expressing ramified microglia and moderate to frequent hla-dr-expressing cells with amoeboid phenotype (hla-dr; scale bar: 500 µm). (e) higher magnification of the better-preserved area in (c). although there is a marked loss of myelinated fibers, a few scattered myelinated fibers remain, neuropil appears more compacted, and the loss of oligodendrocytes is mild to moderate (lfb-he; scale bar: 50 µm). (f) higher magnification of the central area in (d) (hla-dr; scale bar: 50 µm). (g) the rarefied white matter areas show only occasional myelinated fibers, the neuropil is rarefied and mainly formed by astrocytic processes, and oligodendrocytes loss is moderate to severe (lfb-he; scale bar: 50 µm). (h) hla-dr immunostaining of the area in (g) shows mainly microglia with the amoeboid phenotype (hla-dr; scale bar: 100 µm). all images in this figure are representative of case 6. morphological correlation of microglia and axonal degeneration tada et al. described “dysplastic” microglia in brain samples from six patients with confirmed csf1r mutations (4). these dysplastic microglia were interpreted as the morphological manifestation of the csf1r mutation, but no association with axonal spheroids was established. in this study, we observed various reactive microglia morphologies, including some that can be considered “dystrophic”/“dysplastic”. however, the significance of these changes was hard to interpret and to correlate with the pathology. a different sequence of events in the pathogenesis of alas has been suggested. riku et al. concluded that broad microglial impairment and low csf1r expression on microglia preceded multifocal axonal degeneration (6). meanwhile, oyanagi et al. concluded that microglia proliferation preceded axonal swelling and loss. this conclusion suggests that microglial proliferation rather than loss leads to pathology (i.e. microglia is harmful and not beneficial; 7), which stands in contrast to our conclusions. in this study, we concluded that microglial loss preceded axonal degeneration based on the following observations: 1) there is a consistent complete loss of ramified microglia (by both hla-dr & iba1 immunostains) in all areas with axonal spheroids. as we did not observe any area with axonal spheroids to have reactive ramified microglia, it is unlikely that axonal spheroids precede microglia loss. 2) there is a presence of “susceptible areas” in stage 2 (i.e. areas with no ramified microglia and no axonal spheroids). ramified microglia loss in these areas are significant as reactive microglia should be present in areas with white matter pathology. the presence of microglia with amoeboid phenotypes in these areas confirm the reliability of microglia immunostaining in these sections. 3) there is a presence of morphologically abnormal microglia in normal-appearing white matter. tada et al., using ultrastructural examination, considered these dysplastic (4). methodological differences between the studies might explain the difference in conclusions between this study and previous studies. in this study we described the correlation between the axonal spheroids and ramified microglia in each lesion based on its stage rather than the overall brain pathology used by oyanagi et al. the latter system is useful for the correlation of clinical findings and the duration of pathology, while the former is more suitable to evaluate the mechanisms of a multifocal disease showing lesions with different pathological stages. similar limitations may also apply to the conclusion drawn by tada et al. on the microglial distribution in alas patients. they described spatial differences in microglia distribution with areas showing reduced numbers of microglia and others showing microgliosis in both the cortex and the white matter. they concluded that this differential distribution interferes with any meaningful quantitative statistical study of microglia depending on the area examined (4). this conclusion is valid if all lesions are studied together as one stage but not specifically for the stage of the individual lesion as analyzed in this study. we found that this spatial distribution did correlate with the axonal pathology in alas. the second significant difference between the previous studies and our study is that the other authors did not distinguish between ramified microglia and those with amoeboid phenotype. many morphological variations of microglia have been identified (10). although morphology is not a reliable predictor of function, microglia with amoeboid phenotype is an exception as it is consistently associated with phagocytosis (10). therefore, we suggest that at least the microglia with amoeboid phenotype should be distinguished from other phenotypes. microglia functions and pathology microglia are regarded as immune cells of the central nervous system (cns) and play a role in tackling viruses, phagocytosis, and as antigen-presenting cells (10, 20). recently, additional functions have been demonstrated, such as active surveillance of synaptic integrity, regulation of brain development and potentially in blood vessel formation (10, 20). microglia have been considered both pro-inflammatory and anti-inflammatory as well as neurotoxic and neuroprotective depending on their functional profiles (10, 20). the beneficial effects of microglia described include limiting the effects of cerebral ischemia in animal models since their absence through csf1r blockade results in increased infarct size (21–23), while microglial repopulation reverses this effect (23). microglial loss due to absence of csf1r affects brain development and results in olfactory deficits in mice (24). most harmful effects of microglia are associated with the concept of neuroinflammation, especially in multiple sclerosis (25), and after traumatic brain injury (26, 27). however, the term and the concept of neuroinflammation should be used with extreme caution, as suggested by graeber and streit (20). microglia and axonal pathology there are only few studies that deal with the association between the axonal pathology and microglial reaction. in the experimental micropig model for mild traumatic brain injury, lafrenaye et al. found that activated microglia contacted the proximal segments of injured axons (i.e. axonal spheroids) within 6 hours of the induced injury (28). lafrenaye et al. suggested that microglia activation is beneficial and may enhance axonal regeneration through this contact (29). in a human study, microglia activation and microglia nodules were associated with axonal swelling in the perilesional white matter. these changes were demonstrated in the periplaque white matter lesions in multiple sclerosis, in white matter areas adjacent to infarcts, and in traumatic brain injury (30). in this study, the preferred hypothesis was that microglial reaction/nodules resulted in the axonal damage (30). however, no evidence of the timing of the events was provided to support this conclusion. both studies (29, 30) showed the close association of reactive microglia to the axonal spheroids in affected white matter areas. the interpretation is quite different as the latter study suggested that neuroinflammation (defined as a microglial reaction) resulted in the axonal pathology (30), while the former study found that microglia reaction is secondary to axonal injury and suggested a neuroprotective function to these activated microglia. our study described a different association between the axonal pathology and the ramified microglia, which, to our knowledge, has not been described before, and in which axonal spheroids were associated with loss of ramified microglia. the remarkable restriction of axonal spheroids to these areas, best appreciated in the small early lesions (i.e. stage 1 pathology), suggests that the loss of microglia is protective in preventing this pathology. pathogenesis of alas the focus on the pathogenesis of alas has changed in the literature since the recognition of csf1r mutations in alas. previous pathological studies focused on the role of axonal pathology in the pathogenesis. the most relevant conclusions were: 1) the pathological features of the affected white matter suggest that the main pathological process is axonal degeneration with secondary demyelination (1); 2) the recurring or ongoing nature of the pathology is supported by the gradual progression of the white matter pathologic processes (31, 32); and 3) a possible role of hypoxic-ischemic injuries in the pathogenesis of alas (32, 33). studies after the discovery of csf1r mutations in alas focused on the effect of these mutations on microglia and the morphological changes of microglia in alas. the most relevant conclusions from these studies include: 1) csf1r mutations can affect microglia through loss of function (haploinsufficiency model; 17) or dominant-negative effect (18); 2) demonstration of dystrophic microglia as the possible pathological correlate of these mutations (4); 3) change of microglia density precedes the white matter pathology, but with contradicting results. oyanagi et al. proposed that microglia proliferation preceded axonal degeneration (7), while oosterhof et al. found that microglia loss preceded it (8); and 4) csf1r haploinsufficiency may lead to the maldistribution of microglia and result in the regional loss (8). there are many different ways to associate the previous observations and conclusions about the lesions in alas with meaningful pathogenesis. we propose the following pathogenesis: microglia loss precedes the axonal pathology and most likely represents an early stage of the pathology. however, it is still unclear what causes axonal injuries to develop in these restricted areas (and how), and whether this progression occurs with time only (e.g. aging process) or if an additional etiology (e.g. hypoperfusion) is required. axonal injuries that result in axonal spheroids formation have many causes, including hypoxic-ischemic, metabolic, toxic, and traumatic causes. in previous studies of alas, evidence of oxidative stress caused by hypoxic-ischemic etiology was presented (32, 33) and this may represent possible additional etiological factors in the pathogenesis of alas. the effects of hypoxic-ischemic injury depend on the severity and the duration of the hypoxic-ischemic events. as there are no morphological features of moderate to severe hypoxic-ischemic injury in alas, and the axonal injuries are limited to areas of microglia loss, we concluded that this should be viewed as a mild form of injury. one possible cause of mild hypoxic-ischemic events that occur with aging is age-related cerebral hypoperfusion (34), which could potentially explain the delayed presentation and progression of lesions in alas. we emphasize that this is speculative, and not a conclusion of this study, but it might be an explanation of the observed correlation between the axonal pathology and the microglial loss. however, this proposed pathogenesis indicates that progression of alas is potentially preventable through: 1) replacement of the lost ramified microglia to prevent further damage through their protective function, and 2) improving or preventing the reduced cerebral blood flow associated with aging. the first suggestion supports the promising results from allogeneic haematopoietic stem cell transplantation (to repopulate the microglial niche) in patients with alas (35, 36). the second suggestion is currently unpractical, even in the animal model, but this may change in the future. limitation of the study the major limitation of this study was the inability to test for csf1r mutation from the paraffin sections in four of the six cases. however, the histopathological diagnosis of alas is reliable and considered the current gold standard as new mutations in different genes are being discovered in alas. the pathological features of all cases were similar and the characteristic three pathological stages were present in all cases. there was no difference in the morphological features between cases with confirmed csf1r alterations (case 5 and 6), and cases with no genetic confirmation (cases 1–4). the association of microglia distribution to the axonal spheroids and the pathological stages were similar in all cases, as well (table 2). our results and conclusions in this study (i.e. the correlation between microglia loss and axonal spheroids) are based on the pathological findings only, regardless of the underlying genetic alterations. as such, this limitation should not depreciate our results and conclusions. however, it restricted our ability to study the correlation between csf1r mutation and the morphological changes. conclusions the main finding of this study is a multifocal loss of ramified microglia in alas. this loss was seen in the affected areas of all pathological stages and best correlated with the presence of axonal spheroids. the presence of dense ramified microglia was associated with better preservation of the white matter. we concluded that ramified microglia most likely have a protective function for the white matter and that their absence contributes to axonal pathology observed in alas. these findings will contribute to a better understanding of the pathogenesis of alas and the protective roles of microglia, thus providing possible areas of intervention to prevent the progression of the disease. acknowledgments we are grateful for the contributions of professor v. wee yong, university of calgary and hotchkiss brain institute, alberta, canada (performing iba1 immunohistochemistry), the neuropathology technologists at the london health sciences centre, ontario, canada (technical support), dr. ashraf dallol, king abdulaziz university, jeddah, saudi arabia (interpretation of genetic data) and the department of pathology, western university, ontario, canada (research funding for lee-cyn ang). part of this work was presented as a poster at the xixth international congress of neuropathology, tokyo, september 23–27, 2018. references 1. alturkustani m, keith j, hazrati ln, rademakers r, ang lc. pathologic staging of white matter lesions in adult-onset leukoencephalopathy/leukodystrophy with axonal spheroids. j neuropathol exp neurol 2015:74;233-40. 2. alturkustani m, sharma m, hammond r, ang lc. adult-onset leukodystrophy: review of 3 clinicopathologic phenotypes and a proposed classification. j neuropathol exp neurol 2013:72;1090-103. 3. rademakers r, baker m, nicholson am, rutherford nj, finch n, soto-ortolaza a, lash j, wider c, wojtas a, dejesus-hernandez m, adamson j, kouri n, sundal c, shuster ea, aasly j, mackenzie j, roeber s, kretzschmar ha, boeve bf, knopman ds, petersen rc, cairns nj, ghetti b, spina s, garbern j, tselis ac, uitti r, das p, van gerpen ja, meschia jf, levy s, broderick df, graff-radford n, ross oa, miller bb, swerdlow rh, dickson dw, wszolek zk. mutations in the colony stimulating factor 1 receptor (csf1r) gene cause hereditary diffuse leukoencephalopathy with spheroids. nat genet 2012:44;200-5. 4. tada m, konno t, tada m, tezuka t, miura t, mezaki n, okazaki k, arakawa m, itoh k, yamamoto t, yokoo h, yoshikura n, ishihara k, horie m, takebayashi h, toyoshima y, naito m, onodera o, nishizawa m, takahashi h, ikeuchi t, kakita a. characteristic microglial features in patients with hereditary diffuse leukoencephalopathy with spheroids. ann neurol 2016:80;554-65. 5. streit wj, xue qs, tischer j, bechmann i. microglial pathology. acta neuropathol commun 2014:2;142. 6. riku y, ando t, goto y, mano k, iwasaki y, sobue g, yoshida m. early pathologic changes in hereditary diffuse leukoencephalopathy with spheroids. j neuropathol exp neurol 2014:73;1183-90. 7. oyanagi k, kinoshita m, suzuki-kouyama e, inoue t, nakahara a, tokiwai m, arai n, satoh ji, aoki n, jinnai k, yazawa i, arai k, ishihara k, kawamura m, ishizawa k, hasegawa k, yagisita s, amano n, yoshida k, terada s, yoshida m, akiyama h, mitsuyama y, ikeda si. adult onset leukoencephalopathy with axonal spheroids and pigmented glia (alsp) and nasu-hakola disease: lesion staging and dynamic changes of axons and microglial subsets. brain pathol 2017:27;748-69. 8. oosterhof n, kuil le, van der linde hc, burm sm, berdowski w, van ijcken wfj, van swieten jc, hol em, verheijen mhg, van ham tj. colony-stimulating factor 1 receptor (csf1r) regulates microglia density and distribution, but not microglia differentiation in vivo. cell rep 2018:24;1203-17 e6. 9. sarkar s, doring a, zemp fj, silva c, lun x, wang x, kelly j, hader w, hamilton m, mercier p, dunn jf, kinniburgh d, van rooijen n, robbins s, forsyth p, cairncross g, weiss s, yong vw. therapeutic activation of macrophages and microglia to suppress brain tumor-initiating cells. nat neurosci 2014:17;46-55. 10. boche d, perry vh, nicoll ja. review: activation patterns of microglia and their identification in the human brain. neuropathol appl neurobiol 2013:39;3-18. 11. taylor r, alyamany b, pandey s, kertesz a, ang lc, finger e. two distinct clinical phenotypes in a family with alsp caused by a novel csf-1r mutation.(p2. 176). aan enterprises, 2018. 12. van der knaap ms, bugiani m. leukodystrophies: a proposed classification system based on pathological changes and pathogenetic mechanisms. acta neuropathol 2017:134;351-82. 13. konno t, kasanuki k, ikeuchi t, dickson dw, wszolek zk. csf1r-related leukoencephalopathy: a major player in primary microgliopathies. neurology 2018:91;1092-104. 14. lynch ds, zhang wj, lakshmanan r, kinsella ja, uzun ga, karbay m, tufekcioglu z, hanagasi h, burke g, foulds n, hammans sr, bhattacharjee a, wilson h, adams m, walker m, nicoll ja, chataway j, fox n, davagnanam i, phadke r, houlden h. analysis of mutations in aars2 in a series of csf1r-negative patients with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia. jama neurol 2016:73;1433-9. 15. elmore mr, najafi ar, koike ma, dagher nn, spangenberg ee, rice ra, kitazawa m, matusow b, nguyen h, west bl, green kn. colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. neuron 2014:82;380-97. 16. pridans c, sauter ka, baer k, kissel h, hume da. csf1r mutations in hereditary diffuse leukoencephalopathy with spheroids are loss of function. sci rep 2013:3;3013. 17. konno t, tada m, tada m, koyama a, nozaki h, harigaya y, nishimiya j, matsunaga a, yoshikura n, ishihara k, arakawa m, isami a, okazaki k, yokoo h, itoh k, yoneda m, kawamura m, inuzuka t, takahashi h, nishizawa m, onodera o, kakita a, ikeuchi t. haploinsufficiency of csf-1r and clinicopathologic characterization in patients with hdls. neurology 2014:82;139-48. 18. hume da, caruso m, ferrari-cestari m, summers km, pridans c, irvine km. phenotypic impacts of csf1r deficiencies in humans and model organisms. j leukoc biol 2019. 19. oosterhof n, chang ij, karimiani eg, kuil le, jensen dm, daza r, young e, astle l, van der linde hc, shivaram gm, demmers j, latimer cs, keene cd, loter e, maroofian r, van ham tj, hevner rf, bennett jt. homozygous mutations in csf1r cause a pediatric-onset leukoencephalopathy and can result in congenital absence of microglia. am j hum genet 2019:104;936-47. 20. graeber mb, streit wj. microglia: biology and pathology. acta neuropathol 2010:119;89-105. 21. berezovskaya o, maysinger d, fedoroff s. colony stimulating factor-1 potentiates neuronal survival in cerebral cortex ischemic lesion. acta neuropathol 1996:92;479-86. 22. fedoroff s, berezovskaya o, maysinger d. role of colony stimulating factor-1 in brain damage caused by ischemia. neurosci biobehav rev 1997:21;187-91. 23. szalay g, martinecz b, lenart n, kornyei z, orsolits b, judak l, csaszar e, fekete r, west bl, katona g, rozsa b, denes a. microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. nat commun 2016:7;11499. 24. erblich b, zhu l, etgen am, dobrenis k, pollard jw. absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits. plos one 2011:6;e26317. 25. lassmann h. mechanisms of white matter damage in multiple sclerosis. glia 2014:62;1816-30. 26. ramlackhansingh af, brooks dj, greenwood rj, bose sk, turkheimer fe, kinnunen km, gentleman s, heckemann ra, gunanayagam k, gelosa g, sharp dj. inflammation after trauma: microglial activation and traumatic brain injury. ann neurol 2011:70;374-83. 27. cherry jd, tripodis y, alvarez ve, huber b, kiernan pt, daneshvar dh, mez j, montenigro ph, solomon tm, alosco ml, stern ra, mckee ac, stein td. microglial neuroinflammation contributes to tau accumulation in chronic traumatic encephalopathy. acta neuropathol commun 2016:4;112. 28. lafrenaye ad, todani m, walker sa, povlishock jt. microglia processes associate with diffusely injured axons following mild traumatic brain injury in the micro pig. j neuroinflammation 2015:12;186. 29. lafrenaye ad. physical interactions between activated microglia and injured axons: do all contacts lead to phagocytosis? neural regen res 2016:11;538-40. 30. singh s, metz i, amor s, van der valk p, stadelmann c, bruck w. microglial nodules in early multiple sclerosis white matter are associated with degenerating axons. acta neuropathol 2013:125;595-608. 31. yamashita m, yamamoto t. neuroaxonal leukoencephalopathy with axonal spheroids. eur neurol 2002:48;20-5. 32. freeman sh, hyman bt, sims kb, hedley-whyte et, vossough a, frosch mp, schmahmann jd. adult onset leukodystrophy with neuroaxonal spheroids: clinical, neuroimaging and neuropathologic observations. brain pathol 2009:19;39-47. 33. ali zs, van der voorn jp, powers jm. a comparative morphologic analysis of adult onset leukodystrophy with neuroaxonal spheroids and pigmented glia--a role for oxidative damage. j neuropathol exp neurol 2007:66;660-72. 34. tarumi t, zhang r. cerebral blood flow in normal aging adults: cardiovascular determinants, clinical implications, and aerobic fitness. j neurochem 2018:144;595-608. 35. eichler fs, li j, guo y, caruso pa, bjonnes ac, pan j, booker jk, lane jm, tare a, vlasac i, hakonarson h, gusella jf, zhang j, keating bj, saxena r. csf1r mosaicism in a family with hereditary diffuse leukoencephalopathy with spheroids. brain 2016:139;1666-72. 36. gelfand jm, greenfield al, barkovich m, mendelsohn ba, van haren k, hess cp, mannis gn. allogeneic hsct for adult-onset leukoencephalopathy with spheroids and pigmented glia. brain 2020:143;503-11. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated hydrophilic polymer embolism identified in brain tumor specimens following wada testing: a report of 2 cases feel free to add comments by clicking these icons on the sidebar free neuropathology 2:23 (2021) case report hydrophilic polymer embolism identified in brain tumor specimens following wada testing: a report of 2 cases vanessa s. goodwill md*1, michael g. brandel md, mas*2, jeffrey a. steinberg md2, thomas l. beaumont md, phd2, lawrence a. hansen md1 1 department of pathology, university of california, san diego, usa 2 department of neurosurgery, university of california, san diego, usa corresponding author: vanessa goodwill, md · uc san diego health, department of pathology · east campus office bldg (ecob), ste. 1-200 · 9444 medical center drive · la jolla, ca 92037 · usa vgoodwill@health.ucsd.edu submitted: 28 july 2021 accepted: 28 august 2021 copyedited by: deborah mcintyre published: 2 september 2021 https://doi.org/10.17879/freeneuropathology-2021-3457 keywords: embolism, vascular access devices, brain infarction, glioma, vasculitis abstract hydrophilic polymers are commonly used as coatings on intravascular medical devices. as intravascular procedures continue to increase in frequency, the risk of embolization of this material throughout the body has become evident. these emboli may be discovered incidentally but can result in serious complications including death. here, we report the first two cases of hydrophilic polymer embolism (hpe) identified on brain tumor resection following wada testing. one patient experienced multifocal vascular complications and diffuse cerebral edema, while the other had an uneventful postoperative course. wada testing is frequently performed during preoperative planning prior to epilepsy surgery or the resection of tumors in eloquent brain regions. these cases demonstrate the need for increased recognition of this histologic finding to enable further correlation with clinical outcomes. introduction intravascular devices, such as catheters, guidewires, and stents are commonly coated with hydrophilic polymer material. this hydrophilic polymer material has significant procedural benefits, such as acting as a lubricant to decrease vascular trauma, and reducing vascular spasm.1 over the past several decades there has been a drastic increase in the prevalence of minimally invasive endovascular medical procedures. as a result, there has been growing recognition of the risk of embolization of hydrophilic polymer coating material throughout the body.2,3 wada tests (also known as intracarotid sodium amobarbital procedure, isap) are commonly performed before ablative procedures or resections for epilepsy or tumor to assess hemispheric dominance for language. there is a low risk of complications following wada testing including seizures, status-epilepticus, vascular spasm, and transient encephalopathy.4 hydrophilic polymer emboli, however, have not previously been reported following pre-operative wada testing. here, we describe two patients who underwent primary brain tumor resection following wada testing. in both resection specimens, intravascular hydrophilic polymer material with associated foreign body giant cell reaction was identified, and in one case there was evidence of associated acute ischemic change in surrounding neurons. to our knowledge, the current work represents the first report of this histologic finding after wada testing. case summaries case 1 clinical findings: a 55-year-old right-handed man presented with new-onset generalized tonic-clonic seizures. magnetic resonance imaging (mri) revealed a non-enhancing infiltrative and expansile t2-hyperintense mass centered within the right superior temporal gyrus (fig. 1a-b). the patient underwent bilateral wada testing. vascular access was obtained via the right femoral artery using the modified seldinger technique. a 5-french angled glide diagnostic catheter was introduced over a 0.035 terumo glidewire into the descending thoracic aorta and then the cervical internal carotid artery under direct fluoroscopic visualization. a codman prowler select plus infusion microcatheter with a synchro 2 microwire was used to catheterize the internal carotid artery just distal to the posterior communicating artery. an angiographic run was performed to confirm catheter position prior to brevital (methohexitol) infusion (fig. 1c). the wada test demonstrated clear left hemisphere language dominance and greater left hemisphere memory support. figure 1: case 1 imaging and pathology. (a) axial t2-flair and (b) t1 post-contrast sequences of a brain mri demonstrating a non-enhancing infiltrative and expansile t2-hyperintense mass centered within the right superior temporal gyrus. (c) a pa view of digital subtraction angiogram demonstrating a microcatheter within the right internal carotid artery. (d-e) the tumor resection demonstrates a moderately to highly cellular infiltrating glioma, with significant nuclear atypia and scattered mitoses (arrow). (f-g) parenchymal vessels within the resection display giant cell vasculitis and contain basophilic granular foreign material consistent with hydrophilic polymer. five weeks later, the patient underwent a right temporo-parieto-occipital craniotomy for tumor resection. his postoperative course was complicated by a seizure upon awakening with right frontal venous infarct and layering remote cerebellar hemorrhage. he experienced weakness in his left upper and lower extremities and was discharged to a rehabilitation facility with antiepileptic and steroid medications. at 5 months follow-up, the patient had recovered his motor function with no additional seizures, was able to walk over a mile per day without assistance and was tolerating adjuvant chemoradiation. subsequently, post-treatment mris demonstrated a heterogeneously enhancing, centrally necrotic mass in the right temporo-parietal tumor bed with no associated hyperperfusion, characteristic of evolving pseudoprogression. these findings were associated with marked hemispheric cerebral edema refractory to steroid treatment and requiring bevacizumab (fig. 2). figure 2: case 1 post-treatment imaging findings. an 8-month post-operative mri following chemo-radiation showed evidence of pseudoprogression with significant associated cerebral edema. axial t1 post-contrast (a) shows a heterogeneously enhancing, centrally necrotic mass, with restricted diffusion on dwi (b), and no associated hyperperfusion (c). pathology: evaluation of the primary resection specimen revealed a moderately to highly cellular diffusely infiltrating glioma. there was significant pleomorphism and focally frequent mitoses, but no microvascular proliferation or necrosis, meeting histologic criteria for anaplastic astrocytoma, who grade iii (fig. 1d-e). molecular analysis by next-generation sequencing (ngs) and microarray revealed features of primary idh-wildtype glioblastoma, including egfr amplification, and the tumor was upgraded to who grade iv.5 additionally noted on pathologic examination were scattered foci of giant cell vasculitis and intravascular foreign material. the foreign material was granular, blue-gray and non-polarizable, morphologically consistent with hydrophilic polymer material. no surrounding ischemic changes were identified (fig. 1f-g). case 2 clinical findings: a 24-year-old right-handed female with no significant past-medical history presented after suffering a generalized tonic-clonic seizure at work. mri showed a large, non-enhancing infiltrative t2 hyperintense mass centered in the right hippocampus and inferior temporal lobe with regional mass effect (fig. 3a-b). she underwent stereotactic biopsy at an outside facility, which demonstrated a who grade ii diffuse astrocytoma. figure 3: case 2 imaging and pathology. (a) axial t2-flair and (b) t1 post-contrast mri brain sequences demonstrate a non-enhancing infiltrative t2 hyperintense mass centered in the right hippocampus and inferior temporal lobe. (c) pa view of a digital subtraction angiogram demonstrates a microcatheter within the left internal carotid artery. (d) the tumor resection shows a moderately cellular glioma infiltrating white matter and (e) cortex with perineuronal satellitosis. (f) a hypereosinophilic ischemic neuron (arrow and inset) is seen next to a vessel occluded by hydrophilic polymer material with associated inflammation. (g) some vessels contain multinucleate foreign-body giant cells consuming basophilic polymer material. the patient underwent bilateral wada testing with the same microcatheterization technique described in case 1 (fig. 3c). injections of both sodium methohexital (brevital) and amobarbitol (amytal) were completed, and language representation was identified in the left hemisphere. three weeks later, the patient underwent right frontotemporal craniotomy for tumor resection. she had an uncomplicated postoperative course and was discharged home on postoperative day 3 at her baseline functional status, with no neurologic deficits. she remains neurologically intact and seizure-free 9 months postoperatively. pathology: histologic examination of the resection specimen showed a paucicellular infiltrating glioma composed of atypical astrocytes with enlarged, irregular, and hyperchromatic nuclei. no mitoses, necrosis, or microvascular proliferation were detected (fig. 3d-e). ngs was performed and an idh1-r132h mutation was identified, along with mutations in tp53, and atrx. the final diagnosis was diffuse astrocytoma, idh-mutant, who grade ii. also noted multifocally were a few small parenchymal vessels containing non-polarizable granular blue-gray foreign material consistent with hpe. surrounding these vessels was foreign-body giant cell reaction, and a few hyper-eosinophilic acutely ischemic neurons (fig. 3f-g). discussion hydrophilic polymer embolization in cerebral vessels was first reported in 1997 by barnwell et al. in four cases involving use of a microcatheter with hydrophilic coating for cerebral angiography (three autopsy specimens after endovascular thrombolysis for acute ischemic stroke, and one biopsy specimen of a previously-embolized meningioma).6 emboli were described as basophilic and granular, identical in histologic appearance to hydrophilic polymer material seen on sections of microcatheter submitted for histologic analysis. hpe have since been identified widely throughout the body, including the lungs, skin, heart, and central nervous system (cns).3,7–11 hpe may be discovered incidentally or in association with a wide range of complications. reported sequelae of hpe throughout the body include vasculitis, thrombosis, infarction, and even death.12 additionally, there is a significant range in the time-course of presentation and duration, spanning acute, subacute, and delayed symptomatology. one study described evidence of persistent inflammation over three years after the intravascular procedure.13 hpe has been reported following various neuro-interventional procedures such as aneurysm coil embolization, aneurysm flow diversion, intra-arterial thrombolysis, and diagnostic cerebral angiography, and may result in a variety of cns complications.12,14,15 mehta et al. reported a series of 32 cases of hpe within the cns, with findings including vasculitis, granuloma formation, chemical meningitis, ischemia, and hemorrhage.8 these findings were associated with a 38% incidence of stroke and 28% rate of death. the high rate of morbidity and mortality secondary to hpe in this study, however, may be an overestimate as patients were identified in part by their adverse clinical outcome, and types of catheterization also included central venous access and hemodialysis catheterization. here, we report two cases of hpe identified in brain tumor resection following pre-operative wada testing. to perform a wada test, sodium methohexital (brevital) or amobarbital (amytal) is injected into right or left internal carotid artery, which results in ipsilateral hemispheric anesthetization. at our institution, a neuropsychologist conducts a baseline evaluation prior to wada testing, and then repeats this assessment during injections of each hemisphere. in addition, an epileptologist interprets the continuous eeg and compares background activity in each hemisphere to activity during injection to confirm appropriate post-anesthetic changes. a variety of microcatheters are utilized at our institution, including the prowler select plus which is a braid/coil microcatheter with a radiopaque tip and hydrophilic coating. to our knowledge, no prior reports have described an association between this microcatheter and hpe. the rate of complications following cerebral angiography is between 0.3-1.3%, and only a fraction of these are neurologic complications with lasting effects.16,17 the complication rate for wada testing is likely around 2%, although some studies have reported up to 10%.4 of particular interest, our patient from case 1 suffered immediate post-craniotomy vascular complications possibly induced or exacerbated by hpe-associated vasculitis and thrombosis, although there were no immediate or delayed complications from the wada test on its own. although this patient had recovery of neurological function and was ambulatory and independent with activities of daily living, he had early radiologic evidence of pseudoprogression complicated by cerebral edema refractory to steroid treatment and requiring bevacizumab. though one can only speculate, as no additional tissue resection was performed following chemo-radiation, it is possible hpe associated vascular changes in this patient may have also contributed to his significant refractory cerebral edema and extent of pseudoprogression. the patient from case 2 has suffered no adverse clinical sequela to date despite histologic evidence of vasculitis and surrounding acute ischemia. hpe-induced vasculitis and thrombosis may contribute to surgical and post-operative vascular complications in patients undergoing craniotomy for tumor resection or during epilepsy surgery. as the clinical sequelae of hydrophilic polymer emboli can be quite severe based on the findings of several case series,8,12 clinicians and surgeons should be made aware of their presence when identified histologically. therefore, we recommend including the presence of hpe and any associated vascular or ischemic changes as a separate diagnostic line in pathology reports when identified in biopsy or resection specimens to allow for clinical correlation in the event of any unforeseen vascular complications (see example below, case 2). right temporal brain mass, resection: integrated diagnosis: astrocytoma, idh-mutant, who grade ii. histological diagnosis: diffuse astrocytoma. histological grade: who grade ii. molecular information: molecular alterations including: activating mutation of idh1 (r132h), and inactivating mutations of tp53 and atrx. hydrophilic polymer emboli with associated giant cell vasculitis and focal acute ischemic changes (see comment). comment: hydrophilic polymers are applied as surface coatings on vascular devices, and embolization of this material has been reported following various vascular procedures. hydrophilic polymer emboli may be an incidental finding, but can also result in a range of vascular complications. clinical correlation is suggested. furthermore, the incidence of hpe following neuro-interventional procedures is likely under-recognized. published studies are limited by selection bias; however, and neither the overall rate of hpe after cerebral angiography nor the true rate of clinical complications in the setting of cerebral hpe can be identified from the current literature. as such, it is important to recognize hpe histologically so that prospective clinical outcome studies of patients undergoing neuroendovascular procedures prior to cerebral tissue biopsy may be performed. disclosures the following authors have nothing to disclose: vanessa s. goodwill, michael g. brandel, jeffrey a. steinberg, thomas l. beaumont, lawrence a. hansen. references 1. koga s, ikeda s, futagawa k, et al. the use of a hydrophilic-coated catheter during transradial cardiac catheterization is associated with a low incidence of radial artery spasm. int j cardiol. 2004;96(2). 2. mehta ri, mehta ri. hydrophilic polymer embolism: an update for physicians. am j med. 2017;130(7). 3. mehta ri, mehta ri, choi jm, mukherjee a, castellani rj. hydrophilic polymer embolism and associated vasculopathy of the lung: prevalence in a retrospective autopsy study. hum pathol. 2015;46(2). 4. beimer nj, buchtel ha, glynn sm. one center’s experience with complications during the wada test. epilepsia. 2015;56(8). 5. brat dj, aldape k, colman h, et al. cimpact-now update 5: recommended grading criteria and terminologies for idh-mutant astrocytomas. acta neuropathol. 2020;139(3). 6. barnwell sl, d'agostino an, shapiro sl, nesbit gm, kellogg jx. foreign bodies in small arteries after use of an infusion microcatheter. ajnr am j neuroradiol. 1997 nov-dec;18(10):1886-9. 7. komoda s, ozawa h, yuasa t. hydrophilic polymer embolism in the lung as an adverse event: autopsy case of thromboembolisms in multiple organs associated with heparin-induced thrombocytopenia. circ j. 2018;82(2). 8. mehta ri, mehta ri. polymer-induced central nervous system complications following vascular procedures: spectrum of iatrogenic injuries and review of outcomes. hum pathol. 2016;53. 9. mehta ri, mehta ri. hydrophilic polymer embolism: implications for manufacturing, regulation, and postmarket surveillance of coated intravascular medical devices. j patient saf. 2018 mar 19:10.1097 10. french b, ranguelov r, johansen k, tan sl. ischemic toe ulceration due to foreign body embolus from hydrophilic polymer-coated intravascular device. vasc endovascular surg. 2019 oct;53(7):606-8. 11. rosen le, singh ri, mahon b. myocardial hydrophilic polymer emboli following cardiac catheterization: a case report and literature review. cardiovasc pathol. 2014;23(3). 12. mehta ri, mehta ri, solis oe, et al. hydrophilic polymer emboli: an under-recognized iatrogenic cause of ischemia and infarct. mod pathol. 2010;23(7). 13. sequeira a, parimoo n, wilson j, traylor j, bonsib s, abreo k. polymer embolization from minimally invasive interventions. am j kidney dis. 2013;61(6). 14. shapiro m, ollenschleger md, baccin c, et al. foreign body emboli following cerebrovascular interventions: clinical, radiographic, and histopathologic features. am j neuroradiol. 2015;36(11). 15. geisbush tr, marks mp, heit jj. cerebral foreign body reaction due to hydrophilic polymer embolization following aneurysm treatment by pipeline flow diversion device. interv neuroradiol. 2019;25(4). 16. willinsky ra, taylor sm, terbrugge k, farb ri, tomlinson g, montanera w. neurologic complications of cerebral angiography: prospective analysis of 2,899 procedures and review of the literature. radiology. 2003;227(2). 17. fifi jt, meyers pm, lavine sd, et al. complications of modern diagnostic cerebral angiography in an academic medical center. j vasc interv radiol. 2009;20(4). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathological findings in possible normal pressure hydrocephalus: a post-mortem study of 29 cases with lifelines feel free to add comments by clicking these icons on the sidebar free neuropathology 3:2 (2022) original paper neuropathological findings in possible normal pressure hydrocephalus: a post-mortem study of 29 cases with lifelines joni j. hänninen1,2, madoka nakajima3, aleksi vanninen1,2, santtu hytönen4, jaana rummukainen5, anne m. koivisto6,7,8, juha e. jääskeläinen2, hilkka soininen9, anna sutela10, ritva vanninen10, mikko hiltunen11, ville leinonen2, tuomas rauramaa1 1 the department of pathology, kuopio university hospital and the institute of clinical medicine – pathology, university of eastern finland, kuopio, finland 2 the department of neurosurgery, kuopio university hospital and the institute of clinical medicine – neurosurgery, university of eastern finland, kuopio, finland 3 the department of neurosurgery, juntendo university faculty of medicine, tokyo, japan 4 the national institute for health and welfare, forensic medicine, kuopio, finland 5 the department of pathology, kuopio university hospital, kuopio, finland 6 the department of neurology, kuopio university hospital and the institute of clinical medicine – neurology, university of eastern finland, kuopio, finland 7 the department of neurosciences, medical faculty, university of helsinki, helsinki, finland 8 the department of geriatrics/rehabilitation and internal medicine, helsinki university hospital, helsinki, finland 9 the department of neurology, kuopio university hospital, kuopio, finland 10 the department of radiology, kuopio university hospital and the institute of clinical medicine – radiology, university of eastern finland, kuopio, finland 11 the institute of biomedicine, university of eastern finland, kuopio, finland corresponding author: tuomas rauramaa · department of clinical pathology · kuopio university hospital · p.o. box 100 fin-70029 kys · kuopio · finland tuomas.rauramaa@kuh.fi submitted: 09 april 2021 accepted: 19 january 2022 copyedited by: elisabeth g. hain published: 26 january 2022 https://doi.org/10.17879/freeneuropathology-2022-3331 keywords: hydrocephalus, normal pressure hydrocephalus, post-mortem, neuropathology, neurodegeneration abstract aims: there are very few detailed post-mortem studies on idiopathic normal-pressure hydrocephalus (inph) and there is a lack of proper neuropathological criteria for inph. this study aims to update the knowledge on the neuropathology of inph and to develop the neuropathological diagnostic criteria of inph. methods: we evaluated the clinical lifelines and post-mortem findings of 29 patients with possible nph. pre-mortem cortical brain biopsies were taken from all patients during an intracranial pressure measurement or a cerebrospinal fluid (csf) shunt surgery. results: the mean age at the time of the biopsy was 70± 8 sd years and 74± 7 sd years at the time of death. at the time of death, 11/29 patients (38%) displayed normal cognition or mild cognitive impairment (mci), 9/29 (31%) moderate dementia and 9/29 (31%) severe dementia. two of the demented patients had only scarce neuropathological findings indicating a probable hydrocephalic origin for the dementia. amyloid-β (aβ) and hyperphosphorylated τ (hpτ) in the biopsies predicted the neurodegenerative diseases so that there were 4 aβ positive/low alzheimer’s disease neuropathological change (adnc) cases, 4 aβ positive/intermediate adnc cases, 1 aβ positive case with both low adnc and progressive supranuclear palsy (psp), 1 hpτ/psp and primary age-related tauopathy (part) case, 1 aβ/hpτ and low adnc/synucleinopathy case and 1 case with aβ/hpτ and high adnc. the most common cause of death was due to cardiovascular diseases (10/29, 34%), followed by cerebrovascular diseases or subdural hematoma (sdh) (8/29, 28%). three patients died of a postoperative intracerebral hematoma (ich). vascular lesions were common (19/29, 65%). conclusions: we update the suggested neuropathological diagnostic criteria of inph, which emphasize the rigorous exclusion of all other known possible neuropathological causes of dementia. despite the first 2 probable cases reported here, the issue of “hydrocephalic dementia” as an independent entity still requires further confirmation. extensive sampling (with fresh frozen tissue including meninges) with age-matched neurologically healthy controls is highly encouraged. introduction normal pressure hydrocephalus (nph) has a classic triad of clinical symptoms: impaired cognition, urinary incontinence and gait disturbance [1]. nph is considered idiopathic (inph) in the absence of known predisposing factors such as neurotrauma or hemorrhage [1, 2]. the surgical insertion of a shunt in order to bypass cerebrospinal fluid (csf) usually eases the symptoms [3], but unfortunately the improvements often decline over the years [4]. a considerable number of the patients with inph seem to develop cognitive impairment, eventually reaching dementia, commonly due to concomitant alzheimer’s disease (ad), frontotemporal lobar degeneration (ftld), vascular degeneration, or compellingly due to inph by yet unknown neurodegenerative mechanisms [5, 6]. in the norwegian population, the prevalence of inph is estimated to be approximately 22 cases per 100,000 people [7] and it is more common in the older age groups [8]. recent studies reporting familiar aggregation indicate that inph may be an independent neurodegenerative disorder that potentially has its own specific molecular biological basis and etiology [9]. in a study of 10 patients – 9 of whom are included in this study – it was shown that signs of other diseases such as ad, corticobasal degeneration (cbd) and vascular lesions can be identified in the post-mortem brains of the patients suspected to have nph [10]. the number of detailed post-mortem studies on inph is very small and there is a lack of defined and universally accepted neuropathological criteria for inph. the kuopio nph registry (http://www.uef.fi/nph) consists of data on the patients with presumed nph. they underwent intraventricular pressure (icp) monitoring together with right frontal cortical biopsies from 1991 up until 2010. from 2010 onwards, the shunt surgery has included the cortical brain biopsy. this study contains 29 patients with full post-mortem neuropathological examinations and with clinical follow-ups after the procedures with the biopsies. materials and methods kuopio nph registry the neurosurgical department of kuopio university hospital (kuh) alone provides full-time acute and elective neurosurgical services for the kuh catchment population in eastern finland. the medical evaluation of the kuh neurosurgery for possible nph has contained a clinical examination by a neurologist and a neurosurgeon, a computed tomography (ct) or a magnetic resonance imaging (mri) scan, and a 24-h icp monitoring together with a right frontal cortical biopsy up until 2010. in 2010, a 3-step prognostic test protocol was launched. first, csf tap tests are performed on all patients with suspected nph, where at least a 20% improvement in the gait speed in a 10-m walk is considered a positive result. in the second phase, those with negative tap tests undergo lumbar infusion tests, where a conductance of ≤ 10 is considered a positive result. in the third step, the participants with negative findings in both of the tests mentioned above undergo a 24-h monitoring of the icp or undergo a shunt surgery based on a clinical and radiological evaluation. brain biopsies are acquired from the patients who undergo the icp monitoring or the csf shunt surgery [11]. kuopio nph registry includes (i) the clinical baseline and the follow-up data and (ii) other hospital diagnoses, medications and the causes of death from the national registries, as well as the autopsy findings. in this study, nph is defined as a state with the typical clinical and imaging findings, and it is classified as inph when no predisposing factors are found. the shunt response may be present or absent. basic study cohort from 1991 up until 2017, 879 patients with possible nph had been studied with the diagnostic protocol described above including the right frontal cortical biopsies. final study cohort the construction of this study is outlined in figure 1. the patients in this study cohort were followed up until their deaths. all clinical data available from the hospitals in the kuh catchment area was collected. overall, 565 patients had died and 92 of them were autopsied, 29 of whom had had a full neuropathological examination. out of the 29 patients, 23 were shunted according to the diagnostic procedure. the development of the clinical symptoms was documented by neurologists, general practitioners, or both. the mini-mental state examination (mmse) was tested on 26 patients at the baseline. all available data was re-evaluated by 2 neurologists subspecialized in memory disorders (a. m. k. or h. s.), blinded for the cortical biopsy and the autopsy findings. dementia was diagnosed according to the diagnostic and statistical manual of mental disorders, fourth edition (dsm-iv) criteria [12]. the cognitive status was evaluated at the time of the biopsy and retrospectively at the time of death and classified as normal, mild cognitive impairment (mci), moderate or severe dementia (bedridden). figure 1. flow chart of 879 consecutive patients with possible nph. nph = normal pressure hydrocephalus. radiological evaluation all available ct and mri scans were re-evaluated separately by 2 experienced neuroradiologists (a. s. and r. v.), blinded for the biopsy and the neuropathological findings. in the incongruent evaluations, a consensus reading was performed. cortical and subcortical (lacunar) infarctions and cerebral white matter changes (wmcs) were especially looked for. the wmcs were categorized according to the modified fazekas scale: no wmc (grade 0), punctuate (grade 1), early confluent (grade 2) and confluent (grade 3) [13]. biopsy the biopsy procedure has been described previously [14]. briefly, 1 to 3 cylindrical right frontal cortical brain biopsies of 1-3 mm in diameter and 1-8 mm in length were obtained with biopsy forceps or a needle before inserting the intraventricular catheter for the 24-h icp monitoring or the shunt. the samples were fixed in buffered formalin overnight and then embedded in paraffin. apolipoprotein e genotyping dna was extracted and the apolipoprotein e (apoe) genotypes were determined as described previously [15]. autopsies and the neuropathological examination the main findings and the causes of death were assessed from the original autopsy reports. the brains were stored in 10% buffered formaldehyde for at least 1 week before being cut into 1-cm-thick coronal slices and assessed macroscopically by a neuropathologist. the brain specimens were taken from 16 standard regions and embedded in paraffin. the included brain regions were: the frontal cortex, the temporal cortex, the cingular gyrus at the level of the mammillary bodies, the parietal cortex, the motor cortex, the occipital cortex, the anterior and the posterior hippocampus with the entorhinal cortex, the basal forebrain including the amygdala, the striatum, the thalamus, the midbrain, the pons, the medulla, the cerebellar vermis and the cortex [16]. additional samples were taken if necessary for the diagnostics. histology and immunochemistry the 7-μ m sections were deparaffinized and rehydrated through the standard procedures. all sections were stained by using haematoxylin-eosin (h&e) and a selection of the sections also with immunohistochemical (ihc) methods. the antibodies used are described in table 1. shortly after the pretreatment, the sections were incubated with normal goat serum for 30 min at room temperature to block non-specific reactions. after unmasking the epitope, the antibodies were added to the dilution. the sections were then incubated overnight at 4 °c. during the next day, the sections were incubated with a biotinylated second antibody for 30 min, followed by streptavidin enzyme conjugate (labsa zymed laboratories, south san francisco, ca, usa) for 30 min at room temperature. the reaction products were visualized by using 3-amino-9-ethyl-carbazole (aec) or 3-3’-diaminobenzidin (dab). all immunostained sections were counterstained with harris’ haematoxylin, dehydrated, and mounted in depex (bdh chemicals, hull, uk). an amyloid-β (aβ) staining was performed on the biopsies and 5 neuroanatomical regions (the frontal cortex, the hippocampus and the basal forebrain including the amygdala, the substantia nigra and the cerebellar cortex). hyperphosphorylated tau (hpτ) was performed on the biopsies and 5 neuroanatomical regions (the temporal cortex, the occipital cortex, the anterior and the posterior hippocampus, the basal forebrain including the amygdala). an α -synuclein staining was performed on the biopsies and 7 neuroanatomical regions (the cingular gyrus, the parietal cortex, the posterior hippocampus, the substantia nigra, the pons, the medulla oblongata and the basal forebrain including the amygdala). transactive response dna-binding protein 43 (tdp43) was used on the biopsies and the posterior hippocampal sample. p62 was used on selected cases. all sections were evaluated with light microscopy by an experienced neuropathologist (t.r., j.r.). the pathology observed while applying the ihc techniques was assessed as described earlier [17–22]. in the biopsy samples, hpτ was classified as being present or absent and the type of the specific lesions was noted. the aβ pathology was classified as being present in mild, moderate or extensive extent, and the vessel wall positivity was noted, suggesting cerebral amyloid angiopathy (caa). alzheimer’s disease neuropathological change (adnc) was assessed according to the national institute on aging–alzheimer's association (nia-aa) criteria [23]. table 1. immunohistochemistry. hpτ = hyperphosphorylated τ, tdp43 = transactive response dna-binding protein 43, ac = autoclave, cb = citrate buffer, fa = formic acid, dab = 3-3’-diaminobenzidin, aec = 3-amino-9-ethylcarbazole (biocare medical). the statistical methods spss statistics (version 22.0 spss inc., chicago, il, usa) was used for all statistical analyses. spearman’s rank correlation coefficient was used for correlating aβ in the biopsies and the autopsies. ethical issues the study was approved by the ethics committee of the hospital district of northern savonia. all patients recruited after 2008 provided a written informed consent. patient treated in neurosurgery prior to that, were studied according to the permissions from the finnish national supervisory authority for welfare and health, and the finnish ministry of social affairs and health. results demographics: the patient demographics, the clinical findings including the brain imaging and the cortical biopsies, are described in table 2. more than half of the patients were female (17/29, 59%). the mean age at the time of the biopsy was 70± 8 sd (standard deviation) years and 74± 7 sd years at the time of death. the patients with clinically severe dementia died at the average age of 76± 5 sd years. the age at death did not differ between the cognitively normal/mci or the moderately demented patients. the follow-up time from the brain biopsy to the autopsy was 47± 44 sd months, ranging from less than 1 month to 165 months. table 2. patient characteristics at the time of the biopsies and the final clinical diagnoses. * no amyloid-β. mmse = mini-mental state examination, aβ = amyloid-β, hpτ = hyperphosphorylated τ, inph = idiopathic normal pressure hydrocephalus, ct = computerized tomography, mri = magnetic resonance imaging, wmc = white matter change (fazekas grade [13] at least 1), pv = periventricular, na = not available, ad = alzheimer’s disease, hta = arterial hypertension, chd = coronary heart disease, t2d = type 2 diabetes. clinical findings: at the time of the clinical work-up and the cortical biopsy, the leading symptom was gait difficulty in 75% of the subjects with normal cognition or mci (n=20). in the moderately demented group, half of the subjects (n=3/6) presented with cognitive impairment as the leading symptom, followed by gait difficulty (n=2) and tremors (n=1). in the severely demented group, 2 out of the 3 patients presented with cognitive impairment and 1 with gait difficulty as the leading symptom. in this study, inph is defined as a state in which the clinical findings, the imaging and the post-mortem findings are typically consistent with nph, the predisposing factors are excluded, and the shunting procedure has been performed with a positive shunt response. idiopathic nph was seen in 70% of the subjects with normal cognition or mci and 33% of the clinically demented subjects. secondary nph (snph) was seen in 3/20 (15%) of the subjects in the group with normal cognition or mci. the most common clinical diagnosis was hypertension (18/29, 62%), present in all groups. other common clinical diagnoses were coronary heart disease, type ii diabetes (t2d), ad, psychiatric disorders, cancer and asthma. two of the patients in the group with normal cognition or mci had clinically cognitive impairment related to alcohol, 1 patient presented with parkinsonism and 1 with epilepsy. regarding the psychiatric morbidities, 1 patient had a history of schizoaffective disorder, 2 patients had schizophrenia and 2 had a long history of psychiatric symptoms and 1 of maniac episodes. no specific neuropathological findings were seen in this group. shunting: one patient in the group with normal cognition or mci was not shunted and 15/19 (79%) of the shunted patients had positive responses. in the moderately demented group, 3/6 of the patients were shunted and 2 of them responded positively. one patient in the severely demented group (1/3) was shunted without a response. imaging: radiologically, cortical and lacunar infarctions were found in the normal or mci and the moderately demented groups. the findings are summarized in table 2. the infarctions were of grades 1 and 2. both periventricular and deep white matter changes were present in all groups. the changes ranged from grades 0 to 3. cortical biopsies: aβ aggregates were seen in 10 patients: 4/20 (20%) in the group with normal cognition or mci, 3/6 (50%) and 3/3 (100%) in the moderately and the severely demented groups, respectively. hpτ was not seen in the group with normal cognition or mci. one patient in the moderately demented group had hpτ immunopositive lesions (without aβ) and 2 out of the 3 patients in the severely demented group had hpτ positivity. prominent hpτ pathology was seen upon the neuropathological examinations in all the 3 cases with hpτ in the biopsies. the presence of aβ in the biopsies correlated with the extent of aβ (thal phase) [22] defined at the neuropathological examinations (spearman’s ρ 0.775, p = 0.01). table 3. causes of death. at the time of death, 11/29 patients (38%) displayed normal cognition or mci, 9/29 (31%) moderate dementia, and 9/29 (31%) severe dementia. the causes of death are described in table 3. the most common cause of death was due to cardiovascular diseases (10/29, 34%), followed by cerebrovascular diseases (n=5/29, 17%) or acute subdural hematoma (sdh) (3/29, 10%), pneumonia (5/29, 17%) and metastatic carcinoma (4/29, 14%). two patients died of postoperative intracerebral hematoma (ich) indicating the 0.2% (2/879) risk of fatal ich, related to the combined procedure of the brain biopsy and the insertion of the intraventricular catheter in the nph population. table 4. post-mortem neuropathological findings. caa = cerebral amyloid angiopathy, apoe = apolipoprotein e, na = not available, adnc = alzheimer’s disease neuropathological change (nia-aa [24]), part = primary age-related tauopathy, psp = progressive supranuclear palsy, cbd = corticobasal degeneration, aβ = amyloid-β deposition, ich = intracerebral hematoma, sah = subarachnoidal hematoma, sdh = subdural hematoma. neuropathology: the neuropathological findings are described in table 4. enlarged ventricles were seen to variable degrees in all cases. the neuropathological examination revealed that in the group with normal cognition or mci, almost half (5/11, 45%) of the patients had cerebrovascular atherosclerosis and more than a half (5/9, 55%) of the moderately demented patients had notable cerebrovascular atherosclerosis. all of the severely demented patients had cerebrovascular atherosclerosis to variable degrees. one patient in the least cognitively affected group had a ruptured basilar artery aneurysm resulting in subarachnoid hemorrhage (sah), 1 had sdh and 1 patient had a hippocampal infarct. in the moderately demented group, 4 out of the 9 patients had acute sdh and 75% of these were traumatic in origin, and 1 patient had traumatic sah. in the severely demented group, 1 patient had a multifocal metastatic (including meninges) carcinoma explaining the dementia, and 1 patient a temporal cavernoma with an ipsilateral hippocampal infarct. multifocal brain infarcts sufficient to be causative for the cognitive decline were seen in 3 patients, 2 demented (occurred after the shunting procedure) and 1 with mci (occurred during the year of death). in these patients, small lacunar infarctions were seen in the ct during the evaluation prior to the icp measurement or the shunting. gliosis in the subependymal region was seen in all cases to variable degrees and at least mild meningeal thickening was seen in 60% of the patients. the most common secondary findings are summarized in table 5, among the pooled findings from previous literature reviewed in 2012 [10]. table 5. neuropathological findings in nph. nph = normal pressure hydrocephalus, na = not available, ad = alzheimer’s disease. for adnc evaluation, nia-aa criteria were used [23]. four of the subjects with normal cognition or mci had adnc and 6 had primary age-related tauopathy (part) of braak stage iii at maximum. one patient with low adnc had also progressive supranuclear palsy (psp). in the moderately demented group, 3 had intermediate adnc and 4 had braak i part. in the severely demented group, 6 patients had adnc and 2 had part. in addition, single cases of psp, cbd, α -synucleinopathy and aβ depositions of thal stage 3 (without hpτ) were displayed. cerebral amyloid angiopathy (caa) was seen in 1 out of the 11 subjects with normal cognition or mci, 22% of the moderately demented patients and 44% of the severely demented patients. the apoe genotype was found in 15/29 patients. at the time of biopsy, in the group with normal cognition or mci, 8/10 (80%) had genotype ε 3/3, 1 ε 3/4 and 1 ε 2/3. among the moderately demented patients, 2/3 (67%) had the ε 3/4 and 1 had the ε 3/3 genotype. in the group of the severely demented patients, 1 had ε 3/3 and 1 ε 4/4 genotype. neuropathological wmcs in the form chronic vascular type were seen to a variable extent and did not correlate to the cognitive status. in figure 2, we describe a model for the neuropathological evaluation of the nph cases. figure 2. our suggested model for neuropathological evaluation of nph cases. (i/s)nph = (idiopathic/secondary) normal pressure hydrocephalus, ihc = immunohistochemistry, aβ = amyloid-β , hpτ = hyperphosphorylated τ, tdp43 = transactive response dna-binding protein 43, ad = alzheimer’s disease. figure 3. radiological and post-mortem sectional findings. a: coronal mri demonstrating ventricular enlargement pre-shunting, b: same case post-mortem section after shunting (time interval 2 years), c: coronal mri demonstrating ventricular enlargement pre-shunting, d: intracerebral hematoma after the shunting procedure (time interval 3 months). we present examples of radiological and post-mortem sectional findings in figure 3. table 6 is the case-by-case representation of the patients. two subjects with disproportionate neuropathological findings to the degree of cognitive impairment at death are highlighted, representing cases of probable “hydrocephalic dementia”. table 6. case-by-case representation of the patients. probable nph-dementia subjects are highlighted. + = yes, = no. mci = mild cognitive impairment, mmse = mini-mental state examination, aβ = amyloid-β, hpτ = hyperphosphorylated τ, n/a = not available, (i/s)nph = (idiopathic/secondary) normal pressure hydrocephalus, ct = computed tomography, mri = magnetic resonance imaging, wmc = white matter changes (fazekas grades [13]), ad = alzheimer’s disease, tia = transient ischemic attack, ich = intracerebral hematoma, t2d = type 2 diabetes, mcc = morbus cordis coronarius, af = atrial fibrillation, caa = cerebral amyloid angiopathy, apoe = apolipoprotein e, adnc = alzheimer’s disease neuropathological change, part = primary age-related tauopathy, psp = progressive supranuclear palsy, cbd = corticobasal degeneration, sah = subarachnoid hemorrhage, ich = intracerebral hemorrhage. click here for high resolution. discussion hakim and adams [24] described the clinical triad of nph, including the gait difficulty, the cognitive decline and the urinary incontinence, more than 50 years ago. due to its relative rarity, inph is still a rather poorly understood disease and the number of studies with full systematic neuropathological examinations is limited. in this study, we describe the post-mortem findings in 29 patients with presumed nph. idiopathic nph differs from most neurodegenerative diseases presenting with dementia, as the symptoms can be alleviated surgically [3, 25]. idiopathic nph mostly affects the elderly population. the median age of the patients studied here at the time of the diagnosis is comparable to previous studies [7, 26]. in this study, all patients were followed from the time of the shunting or the icp monitoring up until their deaths, and cortical biopsies were taken from all subjects during the operation. overall, in previous studies, the neuropathological findings have been reported to be highly variable and no specific findings of “hydrocephalic dementia” have been described up to date [10, 27, 28]. del bigio describes 5 patients with inph, 3 with snph and 20 patients in whom the ventriculomegaly could not be explained by the other disease processes identified. neuropathologically, these groups did not differ [2]. the classical hakim’s triad is only seen in full in 50% of the nph cases [1]. in our study, gait difficulty was the most common leading symptom in the group with normal cognition or mci. in the moderately demented group, half of the subjects presented clinically with cognitive impairment as the leading symptom and in the group of the severely demented patients, cognitive symptoms were more common than gait difficulty or incontinence. previous studies have shown that more than 20% of the initially shunt-responsive inph patients develop ad later in life [5, 10]. the outcome of the shunting is more favorable if the procedure is done at the earlier stages of the disease [29, 30]. therefore, the candidates for the shunting procedure need to be carefully selected as comorbid ad can make the shunting outcome less favorable [5, 31]. the correlation of aβ seen in the biopsies and the overall aβ load justify the use of the biopsies in the clinical evaluation, although many neurodegenerative diseases generally cannot be ruled out by examining a small cortical biopsy representing a small fraction of the brain tissue. however, hpτ in the biopsies predicted the neuropathological diagnosis of tauopathy or ad. clinically, it is especially important to rule out other types of dementia in patients with cognitive impairment as the leading symptom [33]. the shunting procedure itself may results in complications such as postoperative ich. in our study cohort, the neuropathological examination revealed that 2 patients had psp and 1 cbd. in previous studies, it has been shown that psp and parkinson’s disease can clinically mimic inph [28, 33]. in this study, the most common neuropathological finding in the demented group was adnc or early ad/part, followed by multifocal infarcts. in the study published by del bigio and coworkers [34], significant co-morbidity was frequent in nph. it is noteworthy that mixed diagnoses and co-pathologies are common especially in the elderly age groups [35]. mccarty et al. pointed out that a feature of nph, disproportionately enlarged subarachnoid-space hydrocephalus (desh), is often misdiagnosed as cortical atrophy [36]. in a recent study, it was shown that schizophrenia occurs 3 times more frequently among the inph patients when compared to the general population in finland [37]. studies using different imaging modalities have revealed that there are several progressive structural changes in the brains of the patients suffering from schizophrenia. the most common findings are the enlargements of the lateral ventricles and the reduction of the grey matter volume [38, 39] and the smaller hippocampi [40]. at the microanatomical level, various cytoarchitectural alterations such as a decreased number of neurons and increased neuronal densities in various neuronal populations have been reported [41]. in our study, we did not use a stereological methodology to quantify the cytoarchitectural alterations. overall, our cases with comorbid psychiatric diseases did not differ from the other nph cases. among our study subjects, acute sdh was a relatively common cause of death. one possible explanation for this is that gait disturbance is common in nph and this might lead to falls resulting in sdh. another explanation may be that the csf shunt can be a risk factor for sdh [42]. it should also be noted that atrophic brains may be more prone to develop sdh. three of the patients in this study died of postoperative ich, indicating the 0.2% (2/879) risk of fatal ich related to the combined procedure of the brain biopsy and the insertion of the intraventricular catheter in the nph population. previous studies have shown that inph patients often have cardiovascular and cerebrovascular diseases [10, 43–45]. in a recent study by israelsson and coworkers, it was shown that inph patients are more prone to vascular risk factors such as dyslipidemia and obesity [46]. in functional imaging studies, cerebral blood flow has been shown to be reduced in people with inph [47, 48]. in our study cohort, cerebrovascular atherosclerosis was a common finding to variable degrees and cardiovascular diseases were the most common cause of death. cerebral infarcts were seen in all groups, and some of these were multifocal, suggesting that dementia could be attributed to these lesions in some cases, i.e. representing “vascular dementia.” the etiology and the pathophysiology of inph are still poorly understood. the components of the nph clinical triad are likely to result from subcortical disconnections and disrupted axons. this differs from white matter damage in pediatric hydrocephalus where axonal stretch and local ischemia occur in a simple tissue environment. secondary changes occur in the neuronal cell bodies and at the synaptic level, but the neurons are viable. the csf shunting may reverse the dysfunction up to some degree, but the axonal damage cannot be restored [34, 49]. the subependymal gliosis and the fragmented ependymal lining seen in most of the cases in this study to variable extents may be reactive changes to the chronically increased csf pressure, rather than causative alterations. similarly, the white matter rarefaction may result from the csf leakage into the brain tissue and white matter changes are common in vascular pathologies in any case. another common finding was the meningeal thickening. this can be a secondary alteration to the edema caused by chronic hydrocephalus or the result of mild meningitis in the past, among other possible etiologies. the meningeal tissue assessed here represents only a fraction of the whole meninges and therefore, there is a risk of sampling bias. there has been a recent interest in the role of the disrupted glial-lymphatic, the “glymphatic circulation” in inph [50, 51]. aquaporin 4 (aqp4) may be one of the key molecules involved in the glymphatic system [52]. however, the concept of the cns glymphatic system is currently evolving and needs to be studied further [53]. a recent study by eide and hansson revealed that astrogliosis is common in the cortical inph biopsies and that the expression of aqp4 and dystrophin protein 71 is reduced in the astrocytic perivascular endfeet [54]. the expression of the perivascular aqp4 water channel has been shown to be altered in the inph patients [55]. other interesting structures possibly involved in inph are the ependymal cilia [56–58]. however, the number of studies assessing these structures is very limited. the csf is mostly produced by the choroid plexuses and flows through the arachnoid villi and the granulations into the blood. it has been suggested that the chronic increase in the icp causes the downregulation of the csf regulation [59]. in this study, we did not study these structures in detail, but hypothetically the function of these structures could be altered in inph patients and therefore should be further studied. our study has several strengths and limitations. the cases selected for this study presented with clinically presumed nph. first of all, we have studied only selected cases (only 29 out of the 565 deceased were available for the neuropathological examination) and therefore, there is a risk of selection bias. however, to the best of our knowledge, this is the most comprehensive study of its kind in the literature. the cognitive status of the patients at death was retrospectively assessed based on the clinical records and there is a chance that this could be biased. however, it is unlikely that cases with full-blown dementia were missed. another strength is the availability of the follow-up data with the cortical biopsies. in conclusion, in this study we report the neuropathological findings on nph in a post-mortem series, which is the largest to the best of our knowledge. the neuropathological diagnostic criteria of inph are still lacking, and despite the first probable cases reported here, the issue of “hydrocephalic dementia” as an independent entity still requires further confirmation. the neuropathological findings in our cohort of possible nph patients were highly heterogeneous, which is in line with previous literature. esiri and rosenberg have recommended that the principal goal in the neuropathological work-up in hydrocephalic dementia is the exclusion of the defined entities [60]. here, we described a model for the neuropathological evaluation of the nph cases. future studies may eventually reveal that inph belongs to the family of “proteinopathies” with the dysfunctional protein yet to be discovered. the use of more extensive sampling (with fresh frozen tissue including meninges) than in the protocol used here and the use of age-matched healthy controls is highly encouraged. special interest should be directed towards the demented patients with neuropathological findings disproportionate to the clinical presentation. conflict of interest the authors declare that they have no conflicts of interest. references relkin n, marmarou a, klinge p, bergsneider m, black pm. diagnosing idiopathic normal-pressure hydrocephalus. neurosurgery 2000; 57: s4-16 del bigio mr. neuropathology of human hydrocephalus. in: adult hydrocephalus. ed. d rigamonti. cambridge: cambridge university press. 2014: 14–27 kazui h, miyajima m, mori e, ishikawa m. lumboperitoneal shunt surgery for idiopathic normal pressure hydrocephalus (sinphoni-2): an open-label randomised trial. lancet neurol 2017; 14: 585-94 eide pk, sorteberg w. outcome of surgery for idiopathic normal pressure hydrocephalus: role of preoperative static and pulsatile intracranial pressure. world neurosurg 2016; 86: 186-93.e1 koivisto am, alafuzoff i, savolainen s, sutela a, rummukainen j, kurki m et al. poor cognitive outcome in shunt-responsive idiopathic normal pressure hydrocephalus. neurosurgery 2013; 72: 1-8 koivisto am, kurki mi, alafuzoff i, sutela a, rummukainen j, savolainen s et al. high risk of dementia in ventricular enlargement with normal pressure hydrocephalus related symptoms. j alzheimers dis 2016; 52: 497-507 brean a, eide pk. prevalence of probable idiopathic normal pressure hydrocephalus in a norwegian population. acta neurol scand 2008; 118: 48-53 jaraj d, rabiei k, marlow t, jensen c, skoog i, wikkelsø c. prevalence of idiopathic normal-pressure hydrocephalus. neurology 2014; 82: 1449-54 huovinen j, kastinen s, komulainen s, oinas m, avellan c, frantzen j et al. familial idiopathic normal pressure hydrocephalus. j neurol sci 2016; 368: 11-8 leinonen v, koivisto am, savolainen s, rummukainen j, sutela a, vanninen r et al. post-mortem findings in 10 patients with presumed normal-pressure hydrocephalus and review of the literature. neuropathol appl neurobiol 2012; 38: 72-86 junkkari a, luikku aj, danner n, jyrkkänen hk, rauramaa t, korhonen ve et al. the kuopio idiopathic normal pressure hydrocephalus protocol: initial outcome of 175 patients. fluids barriers cns 2019; 16: 21 frances a, pincus ha, first mb et al. diagnostic and statistical manual of mental disorders. in: psychiatric diagnosis. eds. je mezzich, mc kastrup, y honda, 4th edn. new york, ny: springer. 1994: 69-81 fazekas f, chawluk jb, alavi a, hurtig hi, zimmerman ra. mr signal abnormalities at 1.5 t in alzheimer’s dementia and normal aging. ajr am j roentgenol 1987; 149: 351–6 leinonen v, koivisto am, savolainen s, rummukainen j, tamminen jn, tillgren t et al. amyloid and tau proteins in cortical brain biopsy and alzheimer’s disease. ann neurol 2010; 68: 446-53 solomon a, turunen h, ngandu t, peltonen m, levälahti e, helisalmi s et al. effect of the apolipoprotein e genotype on cognitive change during a multidomain lifestyle intervention. jama neurol 2018; 75: 462-70 alafuzoff i, gelpi e, al-sarraj s, arzberger t, attems j, bodi i et al. the need to unify neuropathological assessments of vascular alterations in the ageing brain: multicentre survey by the brainnet europe consortium. exp gerontol 2012; 47: 825-33 alafuzoff i, arzberger t, al-sarraj s, bodi i, bogdanovic n, braak h et al. staging of neurofibrillary pathology in alzheimer’s disease: a study of the brainnet europe consortium. brain pathol 2018; 18: 484-96 alafuzoff i, pikkarainen m, arzberger t, thal dr, al-sarraj s, bell j et al. inter-laboratory comparison of neuropathological assessments of β-amyloid protein: a study of the brainnet europe consortium. acta neuropathol 2008; 115: 533-46 alafuzoff i, ince pg, arzberger t, al-sarraj s, bell j, bodi i et al. staging/typing of lewy body related α -synuclein pathology: a study of the brainnet europe consortium. acta neuropathol 2009; 117: 635-52 alafuzoff i, thal dr, arzberger t, bogdanovic n, al-sarraj s, bodi i et al. assessment of β-amyloid deposits in human brain: a study of the brainnet europe consortium. acta neuropathol 2009; 117: 309-20 josephs ka, murray me, whitwell jl, parisi je, petrucelli l, jack cr et al. staging tdp-43 pathology in alzheimer’s disease. acta neuropathol 2014; 127: 441-50 thal dr, rüb u, orantes m, braak h. phases of aβ-deposition in the human brain and its relevance for the development of ad. neurology 2002; 58: 1791-800 montine tj, phelps ch, beach tg, bigio eh, cairns nj, dickson dw et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol 2012; 123: 1-11 hakim s, adams rd. the special clinical problem of symptomatic hydrocephalus with normal cerebrospinal fluid pressure. observations on cerebrospinal fluid hydrodynamics. j neurol sci 1965; 2: 307-27 katzen h, ravdin ld, assuras s, heros r, kaplitt m, schwartz th et al. postshunt cognitive and functional improvement in idiopathic normal pressure hydrocephalus. neurosurgery 2011; 68: 416-9 vanneste jal, augustijn p, dirven c, tan wf, goedhart zd. shunting normal-pressure hydrocephalus: do the benefits outweigh the risks? a multicenter study and literature review. neurology 1992; 42: 54-9 cabral d, beach tg, vedders l, sue li, jacobson s, myers k et al. frequency of alzheimer’s disease pathology at autopsy in patients with clinical normal pressure hydrocephalus. alzheimers dement 2011; 7: 509-13 magdalinou nk, ling h, smith jds, schott jm, watkins ld, lees aj. normal pressure hydrocephalus or progressive supranuclear palsy? a clinicopathological case series. j neurol 2013; 260: 1009-13 petersen rc, mokri b, laws er. surgical treatment of idiopathic hydrocephalus in elderly patients. neurology 1985; 35: 307-11 andrén k, wikkelsø c, tisell m, hellström p. natural course of idiopathic normal pressure hydrocephalus. j neurol neurosurg psychiatry 2014; 85: 806-10 junkkari a, häyrinen a, rauramaa t, sintonen h, nerg o, koivisto am, roine rp, viinamäki h, soininen h, luikku a, jääskeläinen je, leinonen v. health-related quality-of-life outcome in patients with idiopathic normal-pressure hydrocephalus a 1-year follow-up study. eur j neurol 2017; 24: 58-66 golomb j, de leon mj, george ae, kluger a, convit a, rusinek h et al. hippocampal atrophy correlates with severe cognitive impairment in elderly patients with suspected normal pressure hydrocephalus. j neurol neurosurg psychiatry 1994; 57: 590-3 starr bw, hagen mc, espay aj. hydrocephalic parkinsonism: lessons from normal pressure hydrocephalus mimics. j clin mov disord 2014; 1: 2 del bigio mr, cardoso er, halliday wc. neuropathological changes in chronic adult hydrocephalus: cortical biopsies and autopsy findings. can j neurol sci 1997; 24: 121-6 kovacs gg, alafuzoff i, al-sarraj s, arzberger t, bogdanovic n, capellari s et al. mixed brain pathologies in dementia: the brainnet europe consortium experience. dement geriatr cogn disord 2008; 26: 343-50 mccarty am, jones dt, dickson dw, graff-radford nr. disproportionately enlarged subarachnoid-space hydrocephalus (desh) in normal pressure hydrocephalus misinterpreted as atrophy: autopsy and radiological evidence. neurocase 2019; 25: 151-5 vanhala v, junkkari a, korhonen ve, kurki mi, hiltunen m, rauramaa t et al. prevalence of schizophrenia in idiopathic normal pressure hydrocephalus. neurosurgery 2019; 84: 883–9 olabi b, ellison-wright i, mcintosh am, wood sj, bullmore e, lawrie sm. are there progressive brain changes in schizophrenia? a meta-analysis of structural magnetic resonance imaging studies. biol psychiatry 2011; 70: 88-96 harrison pj, freemantle n, geddes jr. meta-analysis of brain weight in schizophrenia. schizophr res 2003; 64: 25-34 arnold sjm, ivleva ei, gopal ta, reddy ap, jeon-slaughter h, sacco cb et al. hippocampal volume is reduced in schizophrenia and schizoaffective disorder but not in psychotic bipolar i disorder demonstrated by both manual tracing and automated parcellation (freesurfer). schizophr bull 2015; 41: 233-49 bakhshi k, chance sa. the neuropathology of schizophrenia: a selective review of past studies and emerging themes in brain structure and cytoarchitecture. neuroscience 2015; 303: 82-102 sundström n, lagebrant m, eklund a, koskinen l-od, malm j. subdural hematomas in 1846 patients with shunted idiopathic normal pressure hydrocephalus: treatment and long-term survival. j neurosurg 2018; 129: 797-804 krauss jk, regel jp, vach w, droste dw, borremans jj, mergner t. vascular risk factors and arteriosclerotic disease in idiopathic normal-pressure hydrocephalus of the elderly. stroke 1996; 27: 24-9 eide pk, pripp ah. increased prevalence of cardiovascular disease in idiopathic normal pressure hydrocephalus patients compared to a population-based cohort from the hunt3 survey. fluids barriers cns 2014; 11: 19 jaraj d, agerskov s, rabiei k, marlow t, jensen c, guo x et al. vascular factors in suspected normal pressure hydrocephalus: a population-based study. neurology 2016; 86: 592-9 israelsson h, carlberg b, wikkelsö c, laurell k, kahlon b, leijon g, et al. vascular risk factors in inph: a prospective case-control study (the inph-crash study). neurology 2017; 88: 577-85 momjian s, owler bk, czosnyka z, czosnyka m, pena a, pickard jd. pattern of white matter regional cerebral blood flow and autoregulation in normal pressure hydrocephalus. brain 2004; 127: 965-72 chang c-c, asada h, mimura t, suzuki s. a prospective study of cerebral blood flow and cerebrovascular reactivity to acetazolamide in 162 patients with idiopathic normal-pressure hydrocephalus. j neurosurg 2009; 111: 610-7 del bigio mr. neuropathology and structural changes in hydrocephalus. dev disabil res rev 2010; 16: 16-22 ringstad g, vatnehol sas, eide pk. glymphatic mri in idiopathic normal pressure hydrocephalus. brain 2017; 140: 2691-705 ringstad g, valnes lm, dale am, pripp ah, vatnehol s-as, emblem ke et al. brain-wide glymphatic enhancement and clearance in humans assessed with mri. jci insight 2018; 3: e121537 rasmussen mk, mestre h, nedergaard m. the glymphatic pathway in neurological disorders. lancet neurol 2018; 17: 1016-24 abbott nj, pizzo me, preston je, janigro d, thorne rg. the role of brain barriers in fluid movement in the cns: is there a ‘glymphatic’ system? acta neuropathol 2018; 135: 387-407 eide pk, hansson ha. astrogliosis and impaired aquaporin-4 and dystrophin systems in idiopathic normal pressure hydrocephalus. neuropathol appl neurobiol 2017; 44: 474-90 hasan-olive mm, enger r, hansson h-a, nagelhus ea, eide pk. loss of perivascular aquaporin-4 in idiopathic normal pressure hydrocephalus. glia 2019; 67: 91-100 yang hw, lee s, yang d, dai h, zhang y, han l et al. deletions in cwh43 cause idiopathic normal pressure hydrocephalus. embo mol med 2021; 13: e13249 yamada s, ishikawa m, nozaki k. exploring mechanisms of ventricular enlargement in idiopathic normal pressure hydrocephalus: a role of cerebrospinal fluid dynamics and motile cilia. fluids barriers cns 2021; 18: 20 morimoto y, yoshida s, kinoshita a, satoh c, mishima h, yamaguchi n et al. nonsense mutation in cfap43 causes normal-pressure hydrocephalus with ciliary abnormalities. neurology 2019; 92: e2364-74 silverberg gd, huhn s, jaffe r a, chang sd, saul t, heit g et al. downregulation of cerebrospinal fluid production in patients with chronic hydrocephalus. j neurosurg 2002; 97: 1271-5 esiri m, rosenberg g. hydrocephalus and dementia. in: the neuropathology of dementia. eds. m esiri, v lee, j trojanowski, 2nd edn. cambridge: cambridge university press. 2004: 442–56 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. quantitative proteomic profiling of white matter in cases of cerebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin feel free to add comments by clicking these icons on the sidebar free neuropathology 1:28 (2020) original paper quantitative proteomic profiling of white matter in cases of cerebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin antigoni manousopoulou1**, ho ming yuen2**, matthew macgregor sharp2, satoshi saito2, roxana aldea3, norman mazer3, spiros d. garbis4, roxana o. carare2* 1 beckman research institute, city of hope, duarte, ca, usa 2 faculty of medicine, university of southampton, southampton so16 6yd, uk 3 roche pharma research and early development, pharmaceutical sciences, roche innovation center basel, f. hoffmann-la roche ltd, basel, switzerland 4 proteome exploration laboratory, beckman institute, division of biology and biological engineering, california institute of technology, pasadena, ca, usa ** authors contributed equally corresponding author: prof roxana o carare · university of southampton · faculty of medicine · southampton general hospital · south academic block, mp806 · tremona road · southampton, hampshire · so16 6yd · united kingdom r.o.carare@soton.ac.uk submitted: 14 august 2020 accepted: 06 october 2020 copyedited by: biswa ramani published: 08 october 2020 https://doi.org/10.17879/freeneuropathology-2020-2955 additional resources and electronic supplementary material: supplementary material (zip) keywords: extracellular matrix, cell adhesion, clusterin, cerebral amyloid angiopathy, caa, white matter, proteomics abstract aims: cerebral amyloid angiopathy (caa) is the accumulation of amyloid beta (aβ) in the walls of cerebral arterioles, arteries and capillaries. changes in the white matter in caa are observed as hyperintensities and dilated perivascular spaces on mri suggesting impairment of fluid drainage but the pathophysiology behind these changes is poorly understood. we tested the hypothesis that proteins associated with clearance of aβ peptides are upregulated in the white matter in cases of caa. methods: in this study, we compare the quantitative proteomic profile of white matter from post-mortem brains of patients with caa and age-matched controls in order to gain insight into the cellular processes and key molecules involved in the pathophysiology of caa. results: our proteomic analysis resulted in the profiling of 3,734 proteins (peptide fdr p<0.05). of these, 189 were differentially expressed in caa vs. control. bioinformatics analysis of these proteins showed significant enrichment of proteins related to cell adhesion | cell-matrix interaction, mitochondrial dysfunction and hypoxia. upregulated proteins in caa included emilin2, col4a2, tln1, clu, hspg2. downregulated proteins included dsp, ide, hbg1. conclusions: the present study reports an in-depth quantitative proteomic profiling of white matter from patients with caa, highlighting extracellular matrix proteins and clusterin as key molecules in the pathophysiology of white matter changes in cases of caa. abbreviations caa cerebral amyloid angiopathy, wmh white matter hyperintensities, ipa ingenuity pathway analysis, col4a2 collagen iv alpha 2, hspg2 heparan sulfate proteoglycan 2, emilin2 emi domain endowed, tln1 talin1, clu clusterin, ide insulin degrading enzyme, dsp desmoplakin, ipad intramural periarterial drainage. introduction cerebral amyloid angiopathy (caa) is characterized by the deposition of amyloid proteins including amyloid beta (aβ), cystatin c, prion protein, abri/adan, transthyretin, gelsolin and immunoglobulin light chain amyloid (1) in the walls of leptomenigeal arteries and cortical arterioles (caa-type 2) (2) and in the walls of arteries or capillaries (caa-type 1), but rarely in the walls of venules (1-3). sporadic caa is a common feature of alzheimer’s disease (1-3) and reflects the failure of clearance of proteins and interstitial fluid from the ageing brain (4, 5). although caa is uncommon in the white matter, (6) there are radiological features in the white matter in cases of caa that include white matter hyperintensities (wmh) and dilated perivascular spaces (7, 8). the underlining pathophysiology is poorly understood, but the wmh are associated with small vessel disease (9). in addition to damage to nerve fibres particularly in the deep white matter, the wmh suggest that there is an increase in the volume of interstitial fluid (isf) in the affected white matter. pathologically, wmh are characterised by pallor of myelin staining in the affected white matter with morphological changes in astrocytes and axons and disruption of the distribution of aquaporin 4 in astrocyte processes (10). imaging studies in the early stages of wmh suggest that the lesions are due to altered interstitial fluid mobility and water content that may be reversible (11). interstitial fluid and aβ enter the basement membranes of capillaries and arteries to be cleared from the brain along the intramural periarterial drainage (ipad) pathways (12, 13). with increasing age, the structure of basement membranes changes and aβ accumulates in the walls of arteries as caa (14-16). our proteomic studies performed on leptomeningeal arteries from brains with caa demonstrated an upregulation of clusterin (apolipoprotein j) and tissue inhibitors of metalloproteinases 3 (timp3) (17). the aim of the present study was to compare the quantitative proteomic profile of white matter from patients with caa to the corresponding brain region of age-matched controls in order to gain insight in the pathophysiology of white matter changes in caa and to identify novel therapeutic targets. an overview of the present study workflow is presented in figure 1. figure 1: outline of experimental workflow. labelled peptides were analysed using two-dimensional liquid chromatography and tandem mass spectrometry. materials and methods tissue samples fresh frozen white matter from the occipital lobe of severe caa cases (n=6) and non-demented aged matched controls (n=4) was supplied by newcastle brain tissue resource (ethics rec 08/h0906/136) (table 1). all cases were diagnosed according to internationally-used criteria including neuritic braak stages (18), thal amyloid phases (19), and cerad scores (20) (table 1). table 1: header table 1: demographics of the cases used for this study. pm: post-mortem. the pm delay refers to the number of hours post-mortem until the tissue was dissected and frozen. tissue quantitative proteomics analysis samples of 1mg per case were dissolved in 0.5 m triethylammonium bicarbonate in 0.05% sodium dodecyl sulphate. these were then subjected to pulsed probe sonication (misonix, farmingdale, ny, usa) and lysates were centrifuged (16,000 g, 10 min, 4°c). supernatants were measured for protein content using the bicinchoninic acid assay (pierce bca protein assay kit, thermo fisher scientific, waltham, ma, us). 100 μg of protein was used per sample, adjusted to the highest volume. proteins were reduced with 4μl 50mm tris(2-carboxyethyl)phosphine hydrochloride at 60°c for 1 hour and alkylated using 2 μl 200mm methanethiosulfonate at room temperature for 10 min. proteins were enzymatically digested using trypsin overnight in dark at 37°c (sigma aldrich, st. louis, mo, usa). peptides from each sample were labelled using the 10plex tandem mass tag (tmt) reagent kit (thermo fisher scientific, waltham, ma, us). labelled peptides were mixed and analyzed using two-dimensional liquid chromatography and tandem mass spectrometry as reported previously (17). database searching unprocessed raw files were submitted to proteome discoverer 1.4 (thermo fisher scientific, waltham, ma, us) for target decoy search using sequest. the uniprotkb homo sapiens database which comprised 20,159 entries (release date january 2015) was utilized. the search allowed for up to two missed cleavages, a precursor mass tolerance of 10ppm, a minimum peptide length of six and a maximum of two variable (one equal) modifications of; oxidation (m), deamidation (n, q), or phosphorylation (s, t, y). methylthio (c) and tmt 6-plex (k, y and n-terminus) were set as fixed modifications. false discovery rate (fdr) corrected p value at the peptide level was set at <0.05. percent co-isolation excluding peptides from quantitation was set at 50. reporter ion ratios from unique peptides only were taken into consideration for the quantitation of the respective protein. quantitation per protein in each of the six caa patients was divided by each of the two matched same sex controls, which yielded 12 quantitative ratios per protein. these ratios, which represent the overall fold change in protein in the caa patients with respect to the controls, were median-normalized and log2 transformed. we then combined the two respective log2 ratios per caa patient when compared to the two controls by their mean which generated one observation per caa patient per protein (see supplementary table 1). due to small sample size, statistical analysis was performed per protein regardless of gender (n=6). based on a previous study (16), we set the cut point of log2 ratio higher than or equals to 0.6, or lower than or equals to -0.6 to identify differentially expressed proteins (deps) (equivalent to a 1.5-fold change), but this was considered too restrictive for complex structure such as the white matter (number of proteins included = 51). hence, we have narrowed the gap by halving the cut point to ±0.3 (equivalent to 1.2-fold change) instead in this study (number of proteins included = 189). proteins identified with at least two unique peptides were included in the analysis. one-sample t test was performed on these log2 ratios per protein, comparing to 0, to identify the proteins that were statistically significant and differentially expressed in caa patients comparing to controls (p <0.05). bioinformatics analysis ingenuity pathway analysis (ipa) (qiagen, hilden, germany) and metacore (clarivate analytics, philadelphia, pa, usa) were used to identify significantly enriched processes and pathways in the differentially expressed proteins of white matter from patients with caa vs. control. p values in the context of canonical pathway analysis reflect how significantly the pathway is over-represented in the dataset of deps in white matter from patients with caa vs. controls. significance was set at p < 0.05. results the global, untargeted proteomic analysis resulted in the profiling of 3,734 proteins (peptide fdr p<0.05) (supplementary tables 1, 2, 3). of these, 189 were differentially expressed in caa (supplementary table 4). bioinformatics analysis of the differentially expressed proteins (deps) using metacore showed significant enrichment of the cell adhesion | cell-matrix interaction process (figure 2). mapping on this process network, emilin-2 (emilin2), fibrillin-1 (fbn1), collagen alpha-2(iv) (col4a2) and the extracellular protein heparan sulfate proteoglycan (hspg2) were upregulated; inter-alpha trypsin inhibitor heavy chain h3 (itih3) was downregulated in caa vs. control. figure 3 is a bar graph showing the relative expression levels of key upregulated (emilin2, col4a2, talin-1 (tln1), clusterin (clu)) and downregulated (desmoplakin (dsp), insulin-degrading enzyme (ide), hemoglobin subunit gamma-1 (hbg1)) proteins in the white matter of patients with caa vs. controls (figure 3). figure 2: bioinformatics analysis using metacore showed significant enrichment of the cell-adhesion | cell-matrix interaction processes in the differentially expressed proteins of white matter from patients with caa vs. controls. upregulated proteins (emilin-2, fibrillin, collagen iv, perlecan) are highlighted with a red circle whereas downregulated proteins (itih3) with a blue circle. the p represents significance of enrichment (i.e. this pathway is significantly enriched in the list of differentially expressed proteins). figure 3: up-regulated (red) and downregulated (blue) proteins in white matter from patients with caa vs. controls. a positive log2ratio (ratio>1) reflects upwhereas a negative log2ratio (ratio<1) reflects downregulation of the respective protein in patients with caa vs. controls. metacore demonstrated dysregulation of the hypoxia-inducible factor 1alpha (hif1a) in caa with upregulation of diamine acetyltransferase 2 (sat2) and downregulation of heat shock 70kda protein 4 (hspa4), heat shock 70kda protein 1a/1b (hspa1a), 78kda glucose-related protein (hspa5), endoplasmin (hsp90b1), transitional endoplasmic reticulum atpase (vcp) and guanine nucleotide binding protein subunit beta-2-like-1 (gnb2l1), suggesting altered response to hypoxia in caa (figure 4). figure 4: negative regulation of hif1a was a significantly enriched pathway map in the differentially expressed proteins of white matter from patients with caa vs. controls. upregulated proteins are highlighted with a red circle whereas downregulated proteins with a blue circle. more specifically, sat2 was upregulated whereas hsp70, hspa4, hsp90, rack1 were downregulated in caa vs. control. the p represents significance of enrichment (i.e. this pathway is significantly enriched in the list of differentially expressed proteins). mitochondrial function also appeared altered in caa with upregulation of the mitochondrial fission 1 protein (fis1), a marker of mitochondrial and mitophagy (21) and reductions in proteins associated with the normal function of the mitochondria (mitochondrial succinate dehydrogenase flavoprotein subunit (sdha), mitochondrial aconitate hydratase (aco2), mitochondrial phospholipid hydroperoxide glutathione peroxidase (gpx4), mitochondrial atp synthase subunit beta (atp5f1b), mitochondrial thioredoxin reductase 2 (txnrd2), mitogen-activated protein kinase 9 (mapk9)). mitochondrial dysfunction was a significantly over-represented canonical pathway (figure 5). figure 5: mitochondrial dysfunction was a significantly enriched canonical pathway in the differentially expressed proteins of white matter from patients with caa vs. controls. upregulated proteins are denoted with red whereas downregulated proteins with blue. more specifically, fis1 was upregulated whereas sdha, aco2, gpx4, atp5f1b, txnrd2 and mapk9 were downregulated in caa vs. control. the p represents significance of enrichment (i.e. this pathway is significantly enriched in the list of differentially expressed proteins). discussion the present study reports a comprehensive global proteomic profile of post-mortem white matter samples from patients with caa compared to the corresponding brain region of age-matched controls. out of 189 proteins identified as differentially expressed with both metacore and ipa, several proteins associated with mitochondrial dysfunction, hypoxia, and clearance of aβ are significantly upor downregulated. the pathways reflecting hypoxia and mitochondrial dysfunction have been observed in previous analyses of the white matter in alzheimer’s disease (22). hypoxia-inducible factor-1 (hif1) regulates oxygen homeostasis and thus has a major role in the control of energy/metabolism and angiogenesis. diamine-acetyltransferase-2 (sat2), guanine nucleotide binding protein subunit beta-2-like 1 (gnb2l1) and heat shock proteins are involved in the degradation of hif1 (23, 24) and they were expressed at altered levels in the white matter of caa cases (figure 4). in the present study there was an increase in fis1, a marker of mitochondrial and mitophagy (21), and a reduction in proteins associated with the normal function of the mitochondria (sdha, aco2, gpx4, atp5f1b, txnrd2, mapk9), suggesting an overall dysregulation of the functions of the mitochondria in the white matter from cases of caa (figure 5). most of the novel proteins showing substantial changes in the present study of the white matter in caa were proteins associated with the extracellular matrix constituents and clearance of aβ. proteins with increased expression collagen iv alpha 2 chain (col4a2) is a highly conserved protein across species and is present in almost all vascular basement membranes (bm) (25). a common variation in the col4a2 gene is associated with patients with intracerebral haemorrhage (26), which is one of the most frequent caa-related cerebrovascular complications. consistent with the current proteomic results, previous studies have reported increased collagen iv expression in cerebral microvessels in patients with alzheimer’s disease (27) and increased collagen iv immunostaining in the leptomeningeal arteries in hereditary cystatin c amyloid angiopathy (28). a recent proteomic study of the grey matter in ad with caa type 1 (predominantly capillary caa) has identified collagen alpha-2(vi) (col6a2) as increased in the grey matter of caa type 1(29). perlecan is a heparan sulfate proteoglycan 2 (hspg2), also a component of bm and provides dynamic flexibility to the bm, while promoting the aggregation of aβ (30, 31). pathological studies have reported increased expression of hspg2 in alzheimer’s disease (32) with a significant association of hspg2 polymorphism in apolipoprotein ε4 allele carriers (33). the upregulation of col4a2 and hspg2 have been observed in other studies in the grey matter, but their increase in the white matter of caa cases in the present study demonstrate the widespread changes in the extracellular matrix in the brain, reflected in a failure of intramural periarterial drainage (ipad). emilin-2 was expressed at higher levels in the white matter from brains with caa vs. controls. emilin-2 is part of the eden (emi domain endowed) spectrum of proteins (34), with roles in angiogenesis, as well as cell adhesion/migration (34, 35). emilin-2 possesses the gc1q domain, which interacts with α4β1 integrin, which in turn binds to fibronectin, a major component of bm (36). recent studies demonstrated a reduction in fibronectin in post-mortem brains with white matter hyperintensities as well as on cerebrovascular smooth muscle cell cultures exposed to hypercapnia as a model of hypoperfusion (37). although the expression of emilin-2 in brain is relatively small compared to other organs and the physiological role of emilin-2 is unclear (38), its association with bm (39) suggests that the upregulation of emilin-2 reflects a compensatory upregulation of angiogenesis and ipad in the face of the hypoperfusion and altered extracellular matrix characteristics of the white matter in caa (15, 40). talin-1 (tln1) is a heavy protein that contains a c-terminal flexible rod domain which binds to vinculin and actin while the n-terminal head binds to integrin cytoplasmic tails (41-43). binding of the talin head to integrin is a key step required for integrin activation (44, 45). integrins are essential for cell adhesion and α5β1 is anti-apoptotic and a receptor for aβ (46). as talin also promotes axon growth (47), it is possible that it is upregulated as a compensatory effect of axonal and synapse destruction in white matter lesions (22). in the present study, the expression of clu (clusterin) was higher in the white matter of caa cases compared to controls. our own prior proteomic study of leptomeningeal arteries in caa revealed a high expression of clu, and a recent study on the proteomic expression of grey matter in caa also revealed an increased expression of clu (17). genome-wide association studies identified variations in clu expression associated with alzheimer’s disease (48, 49). clusterin may act as a chaperone molecule that facilitates intramural periarterial drainage of soluble aβ (50). increased expression of clu may be a compensatory mechanism to the cerebrovascular accumulation of aβ in these cases. proteins with decreased expression ide (insulin-degrading enzyme) was significantly decreased in our study. ide is a key enzyme that degrades aβ in the extracellular spaces (51). in diabetes mellitus there is inhibition of aβ degradation directly associated with a reduced activity of ide (52-54). overexpression of ide ameliorates aβ pathology (55). ide is expressed within the vessel walls and its activity is decreased in caa (56). the decreased expression of ide may reflect a failure of the proteolysis and consequent increased toxicity of aβ. the expression of hbg1, the γ(a)-globin subunit of fetal haemoglobin, was also decreased in our study. hbg1 is capable of strongly binding to aβ (57). an animal model of caa exhibited impaired aβ transcytosis from brain to blood, resulting in decreased level of aβ in the blood (58). decreased expression of hbg1 may result from reduced level of the aβ and hbg1 complex in the blood in cases of caa. desmoplakin (dsp) is a desmosome-associated transmembrane glycoprotein and maintains the integrity of the epidermis and myocardium (59). the physiological role of desmoplakin in the brain remains unknown. interestingly, it was reported that desmoplakin is a specific marker of lymphatic vessels in human tongue (60). since ipad represents the lymphatic drainage pathways of the brain parenchyma, (61) a decreased level of desmoplakin may simply reflect impaired ipad. the present study has some limitations: firstly, the sample size is low due to the difficulty in obtaining fresh frozen white matter. this has also prevented us from performing an analysis by sex or any other stratification. secondly, it was not possible to specifically select areas of wmh from the frozen samples of brain and to study them separately. nevertheless, our study has revealed generalised changes in the white matter in cases of caa. thirdly, we have not performed validation of the results by immunocytochemistry for the proteins involved. however, we have provided evidence from other studies that support our findings and provide a working hypothesis. while previous proteomic studies in the white matter in alzheimer’s disease have identified modifications in proteins that are involved in cell survival and cell adhesion (22), in our study, we highlight that in the white matter of severe cases of caa there is upregulation of the proteins that mark cell-matrix interaction, hypoxia, mitochondrial dysfunction and intramural periarterial drainage of soluble aβ. the study from castano et al in 2013 used ad cases with a very low caa score (2 out of a maximum of 12) and identified gfap, tropomyosins, calmodulin, annexin 1 and alpha-internexin as significantly upregulated, while ubiquitin carboxyl-terminal esterase and fascin-1 were down-regulated (22). the upregulated proteins are part of the cytoskeleton maintenance, calcium metabolism and cell survival pathways. we also have identified annexin and ubiquitin carboxyl-terminal esterase modified in the same pattern. the different results between the two studies are likely to be due to the very different caa status of the cases; the present study points to pathways that are involved in the clearance of aβ along the vessel walls. our results suggest a working hypothesis that there is upregulation of the intramural periarterial drainage (ipad) pathways in the white matter as a compensatory mechanism to the failure of drainage of interstitial fluid and solutes from the white matter and the grey matter that is a key pathogenetic factor in caa. acknowledgments the authors thank the newcastle brain tissue research facility, the patients and their families. ethical approval newcastle brain tissue resource (ethics rec 08/h0906/136). author contributions antigoni manousopoulou performed the proteomic experiments, analysed data, interpreted data and wrote manuscript; roxana o. carare and spiros d. garbis designed the study; ho ming yuen assisted with statistical analysis; matthew macgregor sharp, satoshi saito, norman mazer and roxana aldea had important intellectual contributions to the design of the study and analysis. references 1. jellinger ka, attems j. prevalence and pathogenic role of cerebrovascular lesions in alzheimer disease 1. j neurolsci. 2005;229-230:37-41. 2. banerjee g, carare r, cordonnier c, greenberg sm, schneider ja, smith ee, et al. the increasing impact of cerebral amyloid angiopathy: essential new insights for clinical practice. j neurol neurosurg psychiatry. . 2017;88(11):982-94. 3. schreiber s, wilisch-neumann a, schreiber f, assmann a, scheumann v, perosa v, et al. invited review: the spectrum of age-related small vessel diseases: potential overlap and interactions of amyloid and nonamyloid vasculopathies. neuropathol appl neurobiol. 2020;46(3):219-239. 4. carare ro, hawkes ca, jeffrey m, kalaria rn, weller ro. review: cerebral amyloid angiopathy, prion angiopathy, cadasil and the spectrum of protein elimination failure angiopathies (pefa) in neurodegenerative disease with a focus on therapy. neuropathol appl neurobio. 2013;39(6):593-611. 5. weller ro, hawkes ca, kalaria rn, werring dj, carare ro. white matter changes in dementia: role of impaired drainage of interstitial fluid. brain pathol. 2015;25(1):63-78. 6. revesz t, holton jl, lashley t, plant g, frangione b, rostagno a, et al. genetics and molecular pathogenesis of sporadic and hereditary cerebral amyloid angiopathies. acta neuropathol. 2009;118(1):115-30. 7. maniega sm, valdes hernandez mc, clayden jd, royle na, murray c, morris z, et al. white matter hyperintensities and normal-appearing white matter integrity in the aging brain. neurobiol aging. 2015;36(2):909-18. 8. wardlaw jm, benveniste h, nedergaard m, zlokovic bv, mestre h, lee h, et al. perivascular spaces in the brain: anatomy, physiology and pathology. nat rev neurol. 2020;16(3):137-53. 9. al-mashhadi s, simpson je, heath pr, dickman m, forster g, matthews fe, et al. oxidative glial cell damage associated with white matter lesions in the aging human brain. brain pathol. 2015;25(5):565-74. 10. chen a, akinyemi ro, hase y, firbank mj, ndung'u mn, foster v, et al. frontal white matter hyperintensities, clasmatodendrosis and gliovascular abnormalities in ageing and post-stroke dementia. brain. 2016;139(pt 1):242-58. 11. wardlaw jm, valdes hernandez mc, munoz-maniega s. what are white matter hyperintensities made of? relevance to vascular cognitive impairment. j am heart assoc. 2015;4(6). 12. morris aw, sharp mm, albargothy nj, fernandes r, hawkes ca, verma a, et al. vascular basement membranes as pathways for the passage of fluid into and out of the brain. acta neuropathol. 2016;131(5):725-36. 13. albargothy nj, johnston da, macgregor-sharp m, weller ro, verma a, hawkes ca, et al. convective influx/glymphatic system: tracers injected into the csf enter and leave the brain along separate periarterial basement membrane pathways. acta neuropathol. 2018;136(1):139-152. 14. hawkes ca, hartig w, kacza j, schliebs r, weller ro, nicoll ja, et al. perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy. acta neuropathol. 2011;121(4):431-43. 15. keable a, fenna k, yuen hm, johnston da, smyth nr, smith c, et al. deposition of amyloid beta in the walls of human leptomeningeal arteries in relation to perivascular drainage pathways in cerebral amyloid angiopathy. biochimica et biophysica acta. 2016;1862(5):1037-46. 16. hawkes ca, gatherer m, sharp mm, dorr a, yuen hm, kalaria r, et al. regional differences in the morphological and functional effects of aging on cerebral basement membranes and perivascular drainage of amyloid-beta from the mouse brain. aging cell. 2013;12(2):224-36. 17. manousopoulou a, gatherer m, smith c, nicoll ja, woelk ch, johnson m, et al. systems proteomic analysis reveals that clusterin and tissue inhibitor of metalloproteinases 3 increase in leptomeningeal arteries affected by cerebral amyloid angiopathy. neuropathol appl neurobiol. 2017 oct;43(6):492-504. 18. braak h, braak e. neuropathological stageing of alzheimer-related changes. acta neuropathol. 1991;82:239-59. 19. thal dr, rub u, schultz c, sassin i, ghebremedhin e, del tredici k, et al. sequence of abeta-protein deposition in the human medial temporal lobe. journal of neuropathology and experimental neurology. 2000;59(8):733-48. 20. morris jc, mohs rc, rogers h, fillenbaum g, heyman a. consortium to establish a registry for alzheimer's disease (cerad) clinical and neuropsychological assessment of alzheimer's disease. psychopharmacol bull. 1988;24(4):641-52. 21. gomes lc, scorrano l. high levels of fis1, a pro-fission mitochondrial protein, trigger autophagy. biochimica et biophysica acta. 2008;1777(7-8):860-6. 22. castano em, maarouf cl, wu t, leal mc, whiteside cm, lue lf, et al. alzheimer disease periventricular white matter lesions exhibit specific proteomic profile alterations. neurochemistry international. 2013;62(2):145-56. 23. baek jh, liu yv, mcdonald kr, wesley jb, hubbi me, byun h, et al. spermidine/spermine-n1-acetyltransferase 2 is an essential component of the ubiquitin ligase complex that regulates hypoxia-inducible factor 1alpha. j biol chem. 2007;282(32):23572-80. 24. liu yv, semenza gl. rack1 vs. hsp90: competition for hif-1 alpha degradation vs. stabilization. cell cycle. 2007;6(6):656-9. 25. khoshnoodi j, pedchenko v, hudson bg. mammalian collagen iv. microsc res tech. 2008;71(5):357-70. 26. rannikmae k, davies g, thomson pa, bevan s, devan wj, falcone gj, et al. common variation in col4a1/col4a2 is associated with sporadic cerebral small vessel disease. neurology. 2015;84(9):918-26. 27. kalaria rn, pax ab. increased collagen content of cerebral microvessels in alzheimer's disease. brain research. 1995;705(1-2):349-52. 28. snorradottir ao, isaksson hj, kaeser sa, skodras aa, olafsson e, palsdottir a, et al. deposition of collagen iv and aggrecan in leptomeningeal arteries of hereditary brain haemorrhage with amyloidosis. brain research. 2013;1535:106-14. 29. hondius dc, eigenhuis kn, morrema thj, van der schors rc, van nierop p, bugiani m, et al. proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in alzheimer's disease. acta neuropathol commun. 2018;6(1):46. 30. trout al, rutkai i, biose ij, bix gj. review of alterations in perlecan-associated vascular risk factors in dementia. int j mol sci. 2020;21(2). 31. castillo gm, ngo c, cummings j, wight tn, snow ad. perlecan binds to the beta-amyloid proteins (a beta) of alzheimer's disease, accelerates a beta fibril formation, and maintains a beta fibril stability. j neurochem. 1997;69(6):2452-65. 32. lepelletier fx, mann dm, robinson ac, pinteaux e, boutin h. early changes in extracellular matrix in alzheimer's disease. neuropathol appl neurobiol. 2017;43(2):167-82. 33. iivonen s, helisalmi s, mannermaa a, alafuzoff i, lehtovirta m, soininen h, et al. heparan sulfate proteoglycan 2 polymorphism in alzheimer's disease and correlation with neuropathology. neurosci lett. 2003;352(2):146-50. 34. colombatti a, spessotto p, doliana r, mongiat m, bressan gm, esposito g. the emilin/multimerin family. front immunol. 2012;2:93. 35. paulitti a, andreuzzi e, bizzotto d, pellicani r, tarticchio g, marastoni s, et al. the ablation of the matricellular protein emilin2 causes defective vascularization due to impaired egfr-dependent il-8 production affecting tumor growth. oncogene. 2018;37(25):3399-414. 36. danen eh, sonneveld p, brakebusch c, fassler r, sonnenberg a. the fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate rhoa-gtp loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis. j cell biol. 2002;159(6):1071-86. 37. macgregor sharp m, saito s, keable a, gatherer m, aldea r, agarwal n, et al. demonstrating a reduced capacity for removal of fluid from cerebral white matter and hypoxia in areas of white matter hyperintensity associated with age and dementia. acta neuropathol commun. 2020;8(1):131. 38. doliana r, bot s, mungiguerra g, canton a, cilli sp, colombatti a. isolation and characterization of emilin-2, a new component of the growing emilins family and a member of the emi domain-containing superfamily. j biol chem. 2001;276(15):12003-11. 39. amma ll, goodyear r, faris js, jones i, ng l, richardson g, et al. an emilin family extracellular matrix protein identified in the cochlear basilar membrane. mol cell neurosci. 2003;23(3):460-72. 40. okamoto y, yamamoto t, kalaria rn, senzaki h, maki t, hase y, et al. cerebral hypoperfusion accelerates cerebral amyloid angiopathy and promotes cortical microinfarcts. acta neuropathol. 2012;123(3):381-94. 41. kim c, ye f, ginsberg mh. regulation of integrin activation. annu rev cell dev biol. 2011;27:321-45. 42. ye f, lagarrigue f, ginsberg mh. snapshot: talin and the modular nature of the integrin adhesome. cell. 2014;156(6):1340e1. 43. calderwood da, campbell id, critchley dr. talins and kindlins: partners in integrin-mediated adhesion. nat rev mol cell biol. 2013;14(8):503-17.4. goult bt, xu xp, gingras ar, swift m, patel b, bate n, et al. structural studies on full-length talin1 reveal a compact auto-inhibited dimer: implications for talin activation. j struct biol. 2013;184(1):21-32. 45. tadokoro s, shattil sj, eto k, tai v, liddington rc, de pereda jm, et al. talin binding to integrin beta tails: a final common step in integrin activation. science. 2003;302(5642):103-6. 46. matter ml, zhang z, nordstedt c, ruoslahti e. the alpha5beta1 integrin mediates elimination of amyloid-beta peptide and protects against apoptosis. j cell bio. 1998;141(4):1019-30. 47. tan cl, kwok jc, patani r, ffrench-constant c, chandran s, fawcett jw. integrin activation promotes axon growth on inhibitory chondroitin sulfate proteoglycans by enhancing integrin signaling. j neurosci. 2011;31(17):6289-95. 48. lambert jc, heath s, even g, campion d, sleegers k, hiltunen m, et al. genome-wide association study identifies variants at clu and cr1 associated with alzheimer's disease. nat genet. 2009;41(10):1094-9. 49. harold d, abraham r, hollingworth p, sims r, gerrish a, hamshere ml, et al. genome-wide association study identifies variants at clu and picalm associated with alzheimer's disease. nat genet. 2009;41(10):1088-93. 50. wojtas am, kang ss, olley bm, gatherer m, shinohara m, lozano pa, et al. loss of clusterin shifts amyloid deposition to the cerebrovasculature via disruption of perivascular drainage pathways. proc natl acad sci u s a . 2017;114(33):e6962-e71. 51. saito s, ihara m. new therapeutic approaches for alzheimer's disease and cerebral amyloid angiopathy. front aging neurosci. 2014;6:290. 52. qiu wq, folstein mf. insulin, insulin-degrading enzyme and amyloid-beta peptide in alzheimer's disease: review and hypothesis. neurobiol aging. 2006;27(2):190-8. 53. craft s, watson gs. insulin and neurodegenerative disease: shared and specific mechanisms. lancet neurol. 2004;3(3):169-78. 54. farris w, mansourian s, chang y, lindsley l, eckman ea, frosch mp, et al. insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. proc natl acad sci u s a. 2003;100(7):4162-7. 55. leissring ma, farris w, chang ay, walsh dm, wu x, sun x, et al. enhanced proteolysis of beta-amyloid in app transgenic mice prevents plaque formation, secondary pathology, and premature death. neuron. 2003;40(6):1087-93. 56. morelli l, llovera re, mathov i, lue lf, frangione b, ghiso j, et al. insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced activity in cerebral amyloid angiopathy. j biol chem. 2004;279(53):56004-13. 57. perry rt, gearhart da, wiener hw, harrell le, barton jc, kutlar a, et al. hemoglobin binding to a beta and hbg2 snp association suggest a role in alzheimer's disease. neurobiol aging. 2008;29(2):185-93. 58. davis j, xu f, miao j, previti ml, romanov g, ziegler k, et al. deficient cerebral clearance of vasculotropic mutant dutch/iowa double aβ in human aβpp transgenic mice. neurobiol aging. 2006;27(7):946-54. 59. kam cy, dubash ad, magistrati e, polo s, satchell kjf, sheikh f, et al. desmoplakin maintains gap junctions by inhibiting ras/mapk and lysosomal degradation of connexin-43. j cell biol. 2018;217(9):3219-35. 60. ebata n, nodasaka y, sawa y, yamaoka y, makino s, totsuka y, et al. desmoplakin as a specific marker of lymphatic vessels. microvasc res. 2001;61(1):40-8. 61. engelhardt b, carare ro, bechmann i, flugel a, laman jd, weller ro. vascular, glial, and lymphatic immune gateways of the central nervous system. acta neuropathol. 2016;132(3):317-38. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurooncology: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:5 (2021) review neurooncology: 2021 update pieter wesseling1,2 1 department of pathology, amsterdam university medical centers, location vumc, brain tumor center am-sterdam, amsterdam, the netherlands 2 laboratory for childhood cancer pathology, princess máxima center for pediatric oncology, utrecht, the netherlands corresponding author: pieter wesseling, md, phd · department of pathology · amsterdam university medical centers/vumc · de boelelaan 1117 · 1081 hv amsterdam · the netherlands p.wesseling@amsterdamumc.nl submitted: 26 february 2021 accepted: 09 march 2021 copyedited by: deanna fang published: 17 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3271 keywords: brain tumor, molecular diagnostics, glioma, medulloblastoma, tumor microenvironment, covid-19 abstract this article briefly presents 10 topics that were selected by the author as ‘top 10 discoveries’ published in 2020 in the broader field of neurooncological pathology including neurosciences as well as clinical neurooncology of interest for neurooncological pathology. the selected topics concern new information on the molecular characteristics of gliomas (infratentorial idh-mutant diffuse astrocytomas, pediatric low-grade gliomas, infant-type high-grade gliomas, hypermutation in gliomas), the immunological aspects of the brain tumor microenvironment (tme), the impact of the tme on preclinical glioma models, and the importance of lymphatic drainage on brain tumor surveillance. furthermore, important papers were published on two ‘new’ genetic syndromes predisposing to medulloblastoma, on liquid biopsy-based diagnosis of central nervous system (cns) tumors, and on the ‘microbiome’ in glioblastomas (and other cancers). in the last part of this review, a dozen of papers are given as examples of papers that did not make it to the top 10 list of the author, underscoring the subjective component in the selection process. acknowledging that 2020 will be remembered as the year in which the world changed because of the covid-19 pandemic, some of the consequences of this pandemic for neurooncological pathology are briefly discussed as well. hopefully, this review forms an incentive to appreciate the wealth of information provided by the papers that were used as building blocks for the present manuscript. introduction even though, in 2020, the covid-19 pandemic had a quite dramatic, worldwide impact on the way we interacted (e.g. wearing of face masks, social distancing, travel restrictions, or even complete lockdowns), the publication of articles in international, peer-reviewed scientific journals seemed to continue more or less as usual. the author of this review was asked to contribute a review on the ‘top 10 discoveries’ in neurooncology published in 2020. as a search for candidate papers in pubmed using ‘brain & tumor & 2020’ or ‘spinal cord & tumor & 2020’ as key words resulted in over 10,000 hits, it took some time before the selection process for the ‘top ten discoveries’ was finished. here follows the list of the discoveries that emerged because they can be expected to have substantial implications for neurooncological pathology: infratentorial idh-mutant astrocytomas are different [1] molecular landscape of pediatric low-grade gliomas [2] gene fusions in infant-type high-grade gliomas [3] hypermutation in gliomas [4] immunological aspects of the brain tumor microenvironment [5-7] lymphatic drainage enables brain tumor surveillance [8, 9] impact of tumor microenvironment on preclinical glioblastoma models [10] newly recognized medulloblastoma predisposition syndromes [11, 12] liquid biopsy diagnosis of cns tumors [13] the glioblastoma microbiome [14] the order in this list is not based on any ranked importance of the findings but reflects an attempt to create some flow in this review. furthermore, acknowledging that authors did their best to optimally summarize the essence of their findings in (especially the abstracts of) the papers, in this review several phrases were copied and pasted from the original papers, always accompanied by adding the reference. of course, there is quite a subjective component in the selection of papers presented in this review. for example, the selection was not per se solely based on articles in the high(est)-impact journals but also aimed for a somewhat broader blend of topics. ergo, other colleagues would probably have selected other papers, but, as far as i am concerned, that is all in the game (‘de gustibus non est disputandum’). to make up for this situation to some degree, a few papers that just did not make it to my top 10 list will be briefly mentioned in the discussion. because of the extraordinary impact of the covid-19 pandemic in 2020 on our (professional) lives, some covid-19 aspects related to neurooncological pathology are very briefly discussed as well. topic 1. infratentorial idh-mutant astrocytomas are different [1] in the world health organization (who) 2016 classification, three main groups of adult-type diffuse gliomas are listed: astrocytomas, idh-mutant; oligodendrogliomas, idh-mutant and 1p/19q-codeleted; and astrocytomas, idh-wildtype (with glioblastoma idh-wildtype as by far the most frequent representative in this last group) [15]. in the upcoming 5th edition of the who classification (to be published in 2021), some changes can be expected in the definitions and nomenclature of idh-mutant astrocytomas. this is in line with what has been proposed by different updates of the consortium to improve molecular and practical approaches to cns tumor taxonomy (cimpact-now). cimpact-now update 5 suggested to grade the idh-mutant astrocytomas as who grade iv if molecular analysis reveals the presence of homozygous cdkn2a/b deletion and not only if the tumor histologically shows features of the highest malignancy grade [16]. furthermore, cimpact-now update 6 proposed to group the who grade ii-iv diffuse idh-mutant astrocytic tumors under a single name (“astrocytoma, idh mutant”) with arabic numerals assigned for the grade. the most malignant form in this group would then be ‘astrocytoma, idh-mutant, grade 4’ (rather than ‘glioblastoma, idh-mutant’), facilitating discrimination from the much more frequent and more aggressive ‘idh–wildtype glioblastoma’ [17]. in these considerations, the location of the idh-mutant astrocytic tumors is not taken into account. indeed, in the vast majority of patients presenting with a diffuse idh-mutant astrocytoma, the tumor is located in a cerebral hemisphere. banan r et al (with hartmann c and reuss d as corresponding authors) published in acta neuropathologica the clinical and pathological characteristics of a series of 42 primary infratentorial idh-mutant astrocytic tumors [1]. the mean age of the patients in this series was 37 years (similar to the patients with supratentorial tumors), the male:female ratio was 1.8:1, and approximately 30%, 40% and 30% were considered to be who grade ii, iii and iv, respectively. interestingly, only 1 out of 4 of these infratentorial idh-mutant tumors had the idh1 r132h mutation (in supratentorial tumors this is > 80%), with idh1 r132c/g and idh2 r172s/g as the most frequent ‘other’ idh mutations. also, atrx loss and mgmt promoter methylation were significantly less frequently found in the infratentorial compartment. of note, gene panel sequencing revealed two samples with idh1 r132c/h3f3a k27m co-mutations in this series. overall, the clinical outcomes of patients with infratentorial idh-mutant astrocytomas were significantly better than for patients with diffuse midline gliomas, h3k27m-mutant but were significantly worse than for patients with supratentorial idh-mutant astrocytomas when the two cases with idh1/h3k27m co-mutation were included. after exclusion of these two cases, only a trend toward worse outcome for infratentorial idh-mutant astrocytomas was seen. this study shows that diffuse gliomas in the brainstem and cerebellum are easily missed as being idh-mutant in cases where only immunohistochemistry for idh1 r132h mutant protein is performed and that h3k27m mutations co-occur in a small subset of these tumors and signify worse prognosis. topic 2. molecular landscape of pediatric low-grade gliomas [2] ryall s et al (with tabori u and hawkins c as co-senior authors) published a paper in cancer cell reporting the results of combined clinical, morphological and molecular profiling of > 1000 well-annotated, pediatric low-grade gliomas (plggs) with extensive clinical follow-up. the vast majority of the cases that could be adequately analyzed at the molecular level were found to harbor a driver alteration leading to activation of the ras/mapk pathway, while those without an identified alteration often showed upregulation of this pathway as well. based on the type of molecular alteration, plggs could be broadly classified in different classes: rearrangement-driven/fusion-positive tumors versus tumors with a single-nucleotide variant (snv). tumors in the former category were diagnosed at a younger age, enriched for who grade i histology, progressed infrequently, and rarely resulted in death as compared to tumors in the latter category. correlation of type of molecular alteration with outcome (incidence of disease progression or death) allowed for stratification of plggs into three risk categories: low risk (braf fusions, nf1 alterations, fgfr2 fusions, myb/mybl1 rearrangements, fgfr1 tyrosine kinase duplications, fgfr1-tacc1 fusion); intermediate risk (braf p.v600e, idh1 p.r132h, fgfr1 snv, met snv); and high risk (braf p.v600e + cdkn2a deletion, h3.3 p.k27m). additionally, a small, remaining group of ‘unknown risk’ was found with fusions of or snvs in other genes. importantly, while most plggs in children with the genetic pre-disposition disorder nf1 occurred as optic pathway glioma and had a good prognosis, nf1 plggs arising outside the optic pathway had significantly worse overall and progression-free survival. also, the most common kiaa1549-braf fusion, i.e. 16:09 (involving exon 16 of kiaa1549 and exon 9 of braf), was found to be associated with cerebellar location and good prognosis. in contrast, the kiaa1549-braf 15:09 fusion was the only fusion type seen in hemispheric tumors, the primary fusion found in tumors with ‘extensive dissemination’, and associated with a worse progression-free survival. these findings indeed provide very helpful guidance for the prognostication and clinical management of patients with plggs (e.g. ‘watch & wait’ for low-risk tumors versus a more aggressive approach for intermediate and high-risk tumors). obviously, for individual patients, such information should be evaluated in the context of the clinical situation, other molecular findings, and the exact who diagnosis (figure 1). fig. 1. kiaa-braf1549 duplication (and fusion) as seen in the copy number variation (cnv)-profile of a pilocytic astrocytoma (a) and a diffuse leptomeningeal glioneuronal tumor (dlgnt) (b). a tandem duplication of chromosome 7q34, indicative of the kiaa1549-braf fusion, is a frequent event in pilocytic astrocytomas and dlgnts. such a duplication is generally clearly visible as a relatively high position for this region (indicated by arrows in a and b) compared to the rest of chromosome 7 in the detailed cnv profiles that can be obtained using e.g. methylome profiling [55]. ryall s et al propose disease stratification of pediatric low-grade gliomas (plggs) based on the type of molecular alteration(s) in the tumor, with most kiaa1549-braf fusion-positive tumors belonging to the low-risk category [2]. it is important to put such information in perspective of clinical and other molecular findings and of the who diagnosis of the individual patient. for example, all dlgnts typically show loss of chromosome 1p. the cnv profile of the dlgnt depicted in b additionally shows gain of chromosome arm 1q and of chromosome 8, two features that fit very well with the more aggressive ‘methylation class 2’ of these tumors [56]. interestingly, rt-pcr analysis of this dlgnt revealed the 15:09 kiaa1549-braf fusion, i.e. the fusion that, according to ryall s et al, is the primary one seen in plggs with ‘extensive dissemination’ and that is associated with worse progression-free survival. topic 3. gene fusions in infant-type high-grade gliomas [3] in 2019, guerreiro stucklin as, ryall s, et al (with tabori u and hawkins c as last authors) published a study of 171 glioma samples from 150 infants in nature communications in which they identified three main subgroups of infant gliomas: 1) hemispheric rtk-driven tumors, including those showing fusion involving alk, ros1, ntrk, or met, enriched for high-grade gliomas and with an intermediate clinical outcome; 2) hemispheric ras/mapk-driven tumors, characterized by excellent long-term survival post-surgery; and 3) midline ras/mapk-driven tumors, enriched for low-grade glioma with braf alterations and a relatively poor outcome, even after conventional chemotherapeutic approaches [18]. in 2020, clarke m, mackay a, ismer b, et al (last author jones c) published in cancer discovery the findings of a thorough histological and molecular analysis of 241 tumors diagnosed as high-grade and/or diffuse gliomas in children < 4 years of age at the time of diagnosis [3]. after excluding non-gliomas and gliomas matching known subtypes, 130 infant gliomas remained that seemed to be part of an "intrinsic" spectrum of disease specific to the infant population. these included those with targetable mapk pathway alterations. a large proportion of remaining cases harbored gene fusions; these tumors typically occurred in very young children (age at diagnosis less than < 1 year), were located in the cerebral hemispheres, and (like the ‘group 1 tumors’ as reported by guerreiro stucklin as et al) frequently carried fusions involving alk (n = 31), ntrk1/ntrk2/ntrk3 (n = 21), ros1 (n = 9), or met (n = 4) as their driving alterations. importantly, compared to the fusion-negative tumors in this group, these fusion-positive tumors had significantly better outcomes. also, they would be good candidates for targeted therapy. this study is a next example of how in-depth molecular profiling of pediatric brain tumors not only greatly helps to improve prognostication for the children suffering from these neoplasms, but also to discover actionable targets that have the potential to substantially improve their prognosis [19]. topic 4. hypermutation in gliomas [4] while only a limited number of newly diagnosed gliomas are characterized by an inherited mismatch repair (mmr) defect and/or a ‘hypermutator’ phenotype, recurrent gliomas more often show such a phenotype, especially after alkylating chemotherapy [20]. the combination of low tumor mutational burden (tmb) and a highly immunosuppressive microenvironment in most newly diagnosed diffuse gliomas renders (at least so far) effective immunotherapy for these tumors quite challenging. assuming that the hypermutator status leads to an increased expression of neoantigens, gliomas/glioblastomas with a hypermutator phenotype could be better candidates for immune checkpoint blockade. in a paper in nature, touat m and li yy, et al (with beroukhim r, bandopadhayay p, bielle f, and ligon kl as supervising authors) report the results of their comprehensive analysis of the molecular determinants of mutational burden and signatures in 10,294 gliomas [4]. the gliomas were classified into molecular subgroups according to histopathology as well as idh1/idh2 and 1p/19q codeletion status. the median tmb in all samples was 2.6 mutations per mb (range 0.0–781.3). 558 (5.4%) tumors that were designated as being hypermutated (median tmb 50.8 mutations per mb, range 8.8–781.3) were analyzed more in depth. the majority of de novo hypermutated gliomas harbored mutational signatures associated with defects in the mmr pathway (cosmic signatures 6, 15, 26 and 14) or the dna polymerase pole (10 and 14) (2/3 and 1/3 of the samples, respectively), implying that constitutional deficiency in mmr or pole was likely to be the underlying genetic cause of hypermutation. in contrast, almost all gliomas showing high tmb post-treatment had a mutational signature associated with temozolomide exposure (signature 11), and half of these samples showed a co-existing minor mmror pole-deficiency signature component, suggesting that defective dna repair and mutagen exposure cooperate to drive hypermutation in these recurrent gliomas. the observation that bulk analyses of such post-treatment hypermutated glioma did not readily detect microsatellite instabilities (msis), while single-cell whole-genome sequencing analysis did identify microsatellite mutations, can be explained by intra-tumor heterogeneity for this characteristic and a lack of sufficient evolutionary time to select subclonal msi populations. while the therapy-induced snv mutations might not readily elicit effective antitumor responses (because of, for example, the quality of the mutations and/or the subclonal nature of their associated neoantigens), longer treatment exposure or combinatorial strategies may improve the efficacy of checkpoint blockade for such tumors. importantly, and acknowledging that acquired mmr deficiency occurs in the tumors that are considered as being the most temozolomide-sensitive, it is not yet clear whether the acquired mmr deficiency outweighs the positive effects of temozolomide in gliomas. the finding that mmr-deficient cells retain sensitivity to ccnu supports the hypothesis that hypermutation reduces cellular fitness and tolerance to dna-damaging agents other than temozolomide. one of the conclusions of the authors is that longitudinal, molecular analysis of diffuse gliomas can help improve therapeutic management and, ultimately, prognosis as well. topic 5. immunological aspects of the brain tumor microenvironment [5-7] in two papers published back-to-back in cell in june 2020, the authors report on the abundance and heterogeneity of tissue-resident and peripherally recruited leucocytes in glial and metastatic brain tumors [5, 6]. klemm f et al (last author joyce j [5]) comprehensively characterized the tumor microenvironment (tme) of gliomas and brain metastases using flow cytometry, rna sequencing, protein arrays, culture assays, and spatial tissue characterization. they found that ‘education’ of immune cell types in the tme depends on tumor origin and idh mutational status, with pronounced differences in proportional abundance of tissue-resident microglia, infiltrating monocyte-derived macrophages, neutrophils, and t cells. friebel b, kapolou k, et al (last authors neidert mc, becher b [6]) mapped the leukocyte landscape of brain tumors using high-dimensional single-cell profiling (cytof) and found a heterogeneous composition of tissue-resident and invading immune cells within the tme, allowing for a clear distinction between gliomas and brain metastases. gliomas typically showed tissue-resident, reactive microglia, whereas tissue-invading leukocytes accumulated in brain metastases. tissue-invading tumor-associated macrophages showed a distinctive signature trajectory, revealing tumor-driven instruction along with contrasting lymphocyte activation and exhaustion. indeed, these integrated analyses further elucidate the multifaceted immune cell activation within brain tumors and are instrumental for a rational design of more efficacious, targeted immunotherapy strategies. in the study published in cancer discovery, bayik d et al (last author lathia jd) used mouse models to study the presence of monocytic versus granulocytic myeloid-derived suppressor cells (mmdscs/gmdscs) [7]. mdscs are known to be elevated in blood and tumor tissue of patients with glioblastoma and for blocking antitumor immunity. bayik d et al observed that, in their models, mmdscs were enriched in the male tumors, whereas gmdscs were elevated in the blood of females. furthermore, depletion of gmdscs extended survival only in female mice. using gene-expression signatures coupled with network medicine analysis, the authors demonstrated that mmdscs could be targeted with antiproliferative agents in males, whereas gmdsc function could be inhibited by il1β blockade in females. analysis of patient data confirmed that proliferating mmdscs were predominant in male tumors and that a high gmdsc/il1β gene signature correlated with poor prognosis in female patients. interestingly, there is a quite striking sex disparity for glioblastomas at other levels as well. for example, the incidence of glioblastoma is significantly higher in males than in females (incidence rate ratio 1.59:1) (figure 2), and there is a survival advantage for females with glioblastoma independent of treatment, age, karnofsky performance status (kps), or idh mutation status [21, 22]. the work of bayik et al may explain some of such sex differences in patients with glioblastoma and indicate that (immuno)therapeutic approaches should be adapted to the patient’s gender. fig. 2. incidence rate ratios by sex for primary brain tumors. the incidence of most gliomas and embryonal brain tumors is significantly higher in males. for glioblastomas the male:female ratio is 1.59:1 [21]. furthermore, there is a female survival advantage for patients with glioblastoma independent of treatment, age, karnofsky performance status (kps), or idh mutation status [22]. the work of bayik et al may provide support for the hypothesis that differences in immune system functions contribute to these sex differences and that (immuno)therapeutic approaches should be adapted to the patient’s gender. figure based on ostrom qt et al (2020) [21]. * = p<0.05. topic 6. lymphatic drainage enables brain tumor surveillance [8, 9] the central nervous system (cns) has long been considered as lacking lymphatic drainage, a situation which could then contribute to the generally limited immunological response in glioblastomas and other brain tumors. later on, however, it became clear that lymphatic vessels are present in the intracranial meninges along the dural sinuses. these lymphatics drain brain-derived soluble waste to deep cervical lymph nodes, thereby directly connecting the brain with the peripheral immune system. recent studies also demonstrated the presence of meningeal lymphatics along the spinal cord and even suggested that the cerebrospinal fluid (csf) is primarily drained via lymphatics, rather than into the dural sinuses [23]. in a study published in nature, song e et al (last authors thomas jl and iwasaki a) report that in mice orthotopically injected with glioblastoma cells, prophylactic injection of the lymphangiogenesis-promoting protein vegfc in the csf resulted in enhanced priming of cd8 t cells in the draining deep cervical lymph nodes, migration of such cells into the tumor, rapid clearance of the glioblastoma and a long-lasting anti-tumor memory response [8]. transfection of a vegfc-expression mrna construct was found to work synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. furthermore, hu x, deng q, ma l, et al (last author luo j) also published a study last year on the role of meningeal lymphatic vessels in mice with intracranial gliomas or metastatic melanomas [9]. disruption of dorsal meningeal lymphatics alone impaired the dissemination of tumor cells and dendritic cells from the brain tumors to deep cervical lymph nodes, while such trafficking of dendritic cells was found to be increased in mice with enhanced dorsal meningeal lymphangiogenesis. also, disruption of dorsal meningeal lymphatics alone (without affecting basal or nasal meningeal lymphatics) significantly reduced the efficacy of combined anti-pd-1/ctla-4 checkpoint therapy in striatal tumor models, while tumors overexpressing vegfc displayed a better response to such combination therapy. these studies suggest that the immunosurveillance-promoting capacity of vegfc may be exploited to increase the efficacy of immunotherapeutic approaches for brain tumors. topic 7. impact of tumor microenvironment on preclinical glioblastoma models [10] a wide variety of preclinical models for human glioblastomas serve as an important tool for studying these brain tumors. however, no model is perfect, and it is important to determine if/how different models recapitulate different aspects of human glioblastomas. to address this issue, pine ar, cirigliano sm, et al (last author fine ha) performed a complete transcriptomic characterization of tumor cells from 5 patients across four patient-specific glioblastoma–derived model types: 1) glioma spheres (gss), 2) tumor organoids (tos), 3) glioblastoma cerebral organoids (glicos), and 4) patient-derived xenografts (pdxs). they found that glicos and pdxs more closely recapitulated the invasive growth of human glioblastomas. also, the results obtained with bulk and single cell rna sequencing analysis of these models more closely resembled the human tumors. compared to the other models, glicos were enriched for a neural progenitor-like cell subpopulation, showed lower percentages of mesenchymal cells, showed retention of neural and oligodendrocyte progenitor cell populations and recapitulated the cellular states and their plasticity found in the corresponding human tumors best. although glicos thus had a profile most similar to that of the parental glioblastomas, the similarity between glicos and the original glioblastomas was reduced when glico cells were replated in two-dimensional culture conditions. this work underscores the critical impact of the microenvironment in glioblastoma models on the degree to which cellular states, as found in human glioblastomas, are recapitulated. thereby, this study serves as a kind of reality check for those who exploit such preclinical models for elucidation of the pathobiology of and identification of therapeutic approaches for glioblastomas in clinical practice. topic 8. newly discovered medulloblastoma predisposition syndromes [11, 12] medulloblastomas are by far the most frequent of the embryonal cns tumors, most commonly present in childhood and display considerable biological heterogeneity, with distinct molecularly defined groups listed in the who classification. a recent review reports that pathogenic germline variants in established cancer predisposition genes can be identified in about 5% of patients with medulloblastoma [24]. this includes syndromes with a germline defect in sufu or ptch1 (nevoid basal cell carcinoma syndrome/gorlin syndrome), tp53 (li–fraumeni syndrome), apc (familial adenomatous polyposis), crebbp (rubinstein–taybi syndrome), nbs1 (nijmegen breakage syndrome), palb2, and brca2. in 2020, two additional germline mutations were reported that predispose to pediatric medulloblastoma. waszak sm, robinson gw, et al (last authors korbel jo, northcott pa and pfister sm) published a study in nature of 1022 patients with medulloblastoma from whom blood samples and tumor samples were analyzed for germline mutations in 110 cancer predisposition genes [11]. they identified rare germline loss-of-function variants across the elongator complex protein 1 (elp1) gene in 14% of pediatric patients with the sonic hedgehog medulloblastoma subgroup (mbshh). elp1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among pediatric patients in this mbshh subgroup. parent-offspring and pedigree analyses identified two families with a history of pediatric medulloblastoma. most elp1-associated medulloblastomas also exhibited somatic alterations in ptch1. tumors from patients with elp1-associated mbshh were characterized by universal biallelic inactivation of elp1 owing to somatic loss of chromosome arm 9q alterations. the tumor cells were found to show changes consistent with loss of protein homeostasis due to elongator deficiency. the authors conclude that such a genetic predisposition to proteome instability may be a determinant in the pathogenesis of other (pediatric) cancers as well and may provide new targets for therapeutic interference. in another study, begemann m, waszak sm, et al (last authors pfister sm, kontny u, kurth i, and published in the journal of clinical oncology) investigated families with childhood medulloblastoma to identify predisposing germline mutations [12]. initial findings were extended to genomes and epigenomes of 1,044 medulloblastoma cases. the authors identified in six patients with infant-onset medulloblastoma a heterozygous germline mutation in the g protein-coupled receptor 161 (gpr161) gene, which is located on chromosome 1q. gpr161 mutations were exclusively associated with the mbshh subgroup and accounted for 5% of infant cases in these cohorts. molecular profiling revealed loss of heterozygosity (loh) at gpr161 in all affected mbshh tumors without additional somatic driver events. additionally, analysis of 226 mbshh tumors revealed somatic copy-neutral loh of chromosome 1q as a characteristic hallmark and the primary mechanism for biallelic inactivation of gpr161 in the affected mbshh tumors. obviously, the diagnosis of an underlying predisposition syndrome as reported in these papers is important because of its implications for the management of the patients and their families. topic 9. liquid biopsy diagnosis of cns tumors [13] circulating tumor dna (ctdna) in blood is considered to be an easily accessible source of diagnostic, prognostic and/or predictive information that may be very helpful for improving the management of cancer patients. so far, however, for patients with primary brain tumors, the use of csf appeared to be a more promising biosource because of the limited abundance of ctdna in blood of these patients. at the same time, approaches that go beyond dna sequence information, such as analysis of epigenetic signatures in ctdna, can help to boost the use of blood for liquid biopsy diagnostics [25]. in the study published in nature medicine, nassiri f, chakravarthy a, feng s, et al (last authors zadeh g and de carvalho dd) used cell-free methylated dna immunoprecipitation and high-throughput sequencing (cfmedip-seq) to recover and profile methylated dna fragments from plasma [13]. plasma samples of patients with diffuse (idh-wildtype and idh-mutant) gliomas, of patients with other intracranial and extracranial tumors, and of healthy controls were used. these samples were split into training and test sets, and random forest classifiers were trained using the top 300 differentially methylated regions for the different tumor classes. indeed, the classifier was able to accurately distinguish different tumors based on the differences in dna methylation profiles. the potential of such a methylome-based liquid biopsy approach has also already been reported for non-cns tumors and can be applied to other biosources (e.g. urine) as well [26, 27], underscoring the potential of analysis of methylated plasma ctdna to facilitate blood-based diagnosis and monitoring of patients with cancer. topic 10. the glioblastoma microbiome [14] tumor formation involves the co-evolution of neoplastic cells together with extracellular matrix, tumor vasculature and immune cells. evaluating tumors as complete ‘organs’, and not simply as masses of transformed tumor cells, is of paramount importance because heterologous cell types within tumors can actively influence therapeutic response [28]. bacteria were first detected in human tumors more than 100 years ago, raising the possibility that the tumor microbiome may be an additional player in the complex tumor ecosystem. however, the existence of the tumor microbiome remained an issue of debate because of the suspicion of sample contamination. in their study published in science, nejman d, livyatan i, fuks g, et al (last authors shental n, straussman r) characterized the microbiome of 1010 tumor samples from seven human cancer types (breast, lung, ovary, pancreas, melanoma, bone, and brain/glioblastoma) as well as of 516 normal samples (including normal tissue adjacent to the tumor from the same patients) [14]. the authors took multiple measures to minimize and control for contamination. they used pcr sequencing techniques to gain species-level resolution as well as multiple visualization methods and ‘culturomics’ for identification of bacteria. the authors report the presence of bacteria in each tumor type, including in tumors that have no direct connection with the external environment such as glioblastomas. the detection rate ranged from 14.3% in melanomas to >60% in breast, pancreatic, and bone tumors. breast tumors had a richer and more diverse microbiome than all other tumor types tested, and live bacteria could be cultured from these tumors. the bacteria were found to be predominantly localized intracellularly in both cancer cells and immune cells, with different tumor types showing a distinct microbiome composition. the authors also noted correlations between intratumor bacteria and the smoking status and/or the response to immunotherapy of the patients. obviously, these findings immediately raise additional questions, for example: what (if any) is the role of intratumoral bacteria in the development of cancer? and how does the tumor microbiome affect the immune tumor microenvironment and the response to immune therapy? the answers to such questions are crucial for further elucidation of the existence and the importance of the tumor microbiome in cancer [29]. discussion hopefully, this review indeed provides concise, easily digestible information on the topics selected by the author as the top 10 discoveries in the year 2020. of course, during the selection process, quite a few papers were noted that didn’t make it to the top 10 list but that are certainly of great interest as well. for example, it is reassuring to learn that the use of molecular markers for the diagnosis of glioblastoma, idh-wildtype as suggested by cimpact-now update 3 was corroborated in an independent study [30, 31], but it is also good to know that some caution may be warranted for histologically low-grade idh-wildtype diffuse astrocytic tumors with an isolated tert promoter mutation [32]. furthermore, some new tumor types were recognized that will make it to the new who classification of cns tumors (especially diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters (dgonc) and desmoplastic myxoid tumor of the pineal region, smarcb1-mutant [33, 34]), and new insights were obtained in areas such as the oncogenesis of h3.3g34-mutant gliomas [35], posterior fossa group a (pfa) ependymomas [36], subependymal giant cell astrocytomas [37], and in the methylation characteristics of germline-driven replication repair-deficient high-grade gliomas (‘unique hypomethylation patterns’) [38]. very interesting new information was published on how glioblastomas interact with neurons [39], and a roadmap was presented for shaping the emerging field of cancer neuroscience [40]. in depth (epi)genetic analysis of single (brain tumor) cells is ‘hot’ nowadays and indeed provides an enormous amount of new information. a novel tool for exploiting single-cell rna sequencing data, the single-cell tumor-host interaction tool (scthi), was published in a journal with the impressive name gigascience [41]. although not included in the top 10 list in this review, much has been discovered in 2020 about and because of the covid-19 pandemic, with some consequences for neurooncology as well. because of the very infectious and often serious character of the disease, governments had to take drastic actions such as enforcing the wearing of face masks, installing social distancing measures and limiting the free movement of citizens. combined with the finding that cancer patients have an increased risk of dying because of covid-19 infection, this has caused increased stress and anxiety amongst brain tumor patients and their caregivers [42, 43]. neurooncologic care needed to be delivered in an adapted form, minimizing exposures for patients and addressing risks and benefits of all therapeutic interventions, while at the same time ensuring the health of the multi-disciplinary neurooncology workforce as much as possible [44]. interestingly, it appeared that some diagnostic (neuro)pathology services can be reliably provided while working from home when using platforms for whole slide images (wsis). of course, some activities (e.g. tissue sampling, processing, cutting, staining, molecular analyses) still need to be done in the institutes [45-47]. as an emergency response to the pandemic, the uk royal college of pathologists provided guidance for remote reporting of digital pathology slides and the us food and drug administration (fda) granted a waiver for the use of readily available consumer monitors at home [48, 49]. furthermore, the covid-19 pandemic has boosted the exploitation of present-day technology for teaching of medical students and pathology residents (e.g. using digital lectures, videoconferencing, and online resources utilizing collections of wsis) [50, 51] and has drastically changed the way scientific communities interact now that meetings cannot be organized in their traditional format. however, the ‘human touch’ is easily missed in such virtual encounters. finding the right balance between attending web-based meetings, getting (the rest of) our daily job done from home, and maintaining a healthy personal life can be challenging [52]. also, it remains to be seen what the long-term impact of the pandemic is on cancer research [53]. last but not least, ignorance of one’s own ignorance is worrisome, especially so because not infrequently it is combined with overconfidence, a phenomenon called the dunning-kruger effect [54]. improving skills and metacognitive competence helps one to move on from conscious incompetence to a phase of conscious competence that is more in balance with the level of confidence (figure 3). although a review like this only allows for ‘scratching the surface’ of the selected discoveries, it hopefully does help one to move further away from unconscious incompetence, especially so if the review indeed works as an incentive to read and appreciate the original papers that were used as building blocks for the present manuscript. fig. 3. the dunning-kruger effect: a remarkable relationship between competence and confidence. in 1999, david dunning and justin kruger described the phenomenon that people tend to initially hold overly favorable views of their abilities in many social and intellectual domains [54]. indeed, people not infrequently share strong opinions on particular topics without being hindered by their lack of competence (in other words, they’re on top of ‘mount stupid’ without knowing it). improving skills and metacognitive competence helps to move on via the ‘valley of despair’ (conscious incompetence: knowing that you only have very limited knowledge and/or understanding of a particular topic) to the ‘slope of enlightenment’, where competence and confidence are more in balance. acknowledging that competence is often a matter of continuing education and professional development, i doubt if the ultimate goal in medicine is really to strive for the level of unconscious competence (hence the question mark behind that term). meanwhile, this review hopefully helps to climb the ‘slope of enlightenment’ somewhat further. references 1. banan, r., et al., infratentorial idh-mutant astrocytoma is a distinct subtype. acta neuropathol, 2020. 140(4): p. 569-581. 2. ryall, s., et al., integrated molecular and clinical analysis of 1,000 pediatric low-grade gliomas. cancer cell, 2020. 37(4): p. 569-583 e5. 3. clarke, m., et al., infant high-grade gliomas comprise multiple subgroups characterized by novel targetable gene fusions and favorable outcomes. cancer discov, 2020. 10(7): p. 942-963. 4. touat, m., et al., mechanisms and therapeutic implications of hypermutation in gliomas. nature, 2020. 580(7804): p. 517-523. 5. klemm, f., et al., interrogation of the microenvironmental landscape in brain tumors reveals disease-specific alterations of immune cells. cell, 2020. 181(7): p. 1643-1660 e17. 6. friebel, e., et al., single-cell mapping of human brain cancer reveals tumor-specific instruction of tissue-invading leukocytes. cell, 2020. 181(7): p. 1626-1642 e20. 7. bayik, d., et al., myeloid-derived suppressor cell subsets drive glioblastoma growth in a sex-specific manner. cancer discov, 2020. 10(8): p. 1210-1225. 8. song, e., et al., vegf-c-driven lymphatic drainage enables immunosurveillance of brain tumours. nature, 2020. 577(7792): p. 689-694. 9. hu, x., et al., meningeal lymphatic vessels regulate brain tumor drainage and immunity. cell res, 2020. 30(3): p. 229-243. 10. pine, a.r., et al., tumor microenvironment is critical for the maintenance of cellular states found in primary glioblastomas. cancer discov, 2020. 10(7): p. 964-979. 11. waszak, s.m., et al., germline elongator mutations in sonic hedgehog medulloblastoma. nature, 2020. 580(7803): p. 396-401. 12. begemann, m., et al., germline gpr161 mutations predispose to pediatric medulloblastoma. j clin oncol, 2020. 38(1): p. 43-50. 13. nassiri, f., et al., detection and discrimination of intracranial tumors using plasma cell-free dna methylomes. nat med, 2020. 26(7): p. 1044-1047. 14. nejman, d., et al., the human tumor microbiome is composed of tumor type-specific intracellular bacteria. science, 2020. 368(6494): p. 973-980. 15. wesseling, p. and d. capper, who 2016 classification of gliomas. neuropathol appl neurobiol, 2018. 44(2): p. 139-150. 16. brat, d.j., et al., cimpact-now update 5: recommended grading criteria and terminologies for idh-mutant astrocytomas. acta neuropathol, 2020. 139(3): p. 603-608. 17. louis, d.n., et al., cimpact-now update 6: new entity and diagnostic principle recommendations of the cimpact-utrecht meeting on future cns tumor classification and grading. brain pathol, 2020. 30(4): p. 844-856. 18. guerreiro stucklin, a.s., et al., alterations in alk/ros1/ntrk/met drive a group of infantile hemispheric gliomas. nat commun, 2019. 10(1): p. 4343. 19. wong, m., et al., whole genome, transcriptome and methylome profiling enhances actionable target discovery in high-risk pediatric cancer. nat med, 2020. 26(11): p. 1742-1753. 20. barthel, f.p., et al., longitudinal molecular trajectories of diffuse glioma in adults. nature, 2019. 576(7785): p. 112-120. 21. ostrom, q.t., et al., cbtrus statistical report: primary brain and other central nervous system tumors diagnosed in the united states in 2013-2017. neuro oncol, 2020. 22(12 suppl 2): p. iv1-iv96. 22. ostrom, q.t., et al., females have the survival advantage in glioblastoma. neuro oncol, 2018. 20(4): p. 576-577. 23. oliver, g., et al., the lymphatic vasculature in the 21(st) century: novel functional roles in homeostasis and disease. cell, 2020. 182(2): p. 270-296. 24. waszak, s.m., et al., spectrum and prevalence of genetic predisposition in medulloblastoma: a retrospective genetic study and prospective validation in a clinical trial cohort. lancet oncol, 2018. 19(6): p. 785-798. 25. van der pol, y. and f. mouliere, toward the early detection of cancer by decoding the epigenetic and environmental fingerprints of cell-free dna. cancer cell, 2019. 36(4): p. 350-368. 26. shen, s.y., et al., sensitive tumour detection and classification using plasma cell-free dna methylomes. nature, 2018. 563(7732): p. 579-583. 27. nuzzo, p.v., et al., detection of renal cell carcinoma using plasma and urine cell-free dna methylomes. nat med, 2020. 26(7): p. 1041-1043. 28. junttila, m.r. and f.j. de sauvage, influence of tumour micro-environment heterogeneity on therapeutic response. nature, 2013. 501(7467): p. 346-54. 29. atreya, c.e. and p.j. turnbaugh, probing the tumor micro(b)environment. science, 2020. 368(6494): p. 938-939. 30. brat, d.j., et al., cimpact-now update 3: recommended diagnostic criteria for "diffuse astrocytic glioma, idh-wildtype, with molecular features of glioblastoma, who grade iv". acta neuropathol, 2018. 136(5): p. 805-810. 31. tesileanu, c.m.s., et al., survival of diffuse astrocytic glioma, idh1/2 wildtype, with molecular features of glioblastoma, who grade iv: a confirmation of the cimpact-now criteria. neuro oncol, 2020. 22(4): p. 515-523. 32. berzero, g., et al., idh-wildtype lower grade diffuse gliomas: the importance of histological grade and molecular assessment for prognostic stratification. neuro oncol, 2020. 33. deng, m.y., et al., diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (dgonc) a molecularly defined glioneuronal cns tumour class displaying recurrent monosomy 14. neuropathol appl neurobiol, 2020. 46(5): p. 422-430. 34. thomas, c., et al., desmoplastic myxoid tumor, smarcb1-mutant: clinical, histopathological and molecular characterization of a pineal region tumor encountered in adolescents and adults. acta neuropathol, 2020. 139(2): p. 277-286. 35. chen, c.c.l., et al., histone h3.3g34-mutant interneuron progenitors co-opt pdgfra for gliomagenesis. cell, 2020. 183(6): p. 1617-1633 e22. 36. michealraj, k.a., et al., metabolic regulation of the epigenome drives lethal infantile ependymoma. cell, 2020. 181(6): p. 1329-1345 e24. 37. bongaarts, a., et al., dysregulation of the mmp/timp proteolytic system in subependymal giant cell astrocytomas in patients with tuberous sclerosis complex: modulation of mmp by microrna-320d in vitro. j neuropathol exp neurol, 2020. 79(7): p. 777-790. 38. dodgshun, a.j., et al., germline-driven replication repair-deficient high-grade gliomas exhibit unique hypomethylation patterns. acta neuropathol, 2020. 140(5): p. 765-776. 39. yu, k., et al., pik3ca variants selectively initiate brain hyperactivity during gliomagenesis. nature, 2020. 578(7793): p. 166-171. 40. monje, m., et al., roadmap for the emerging field of cancer neuroscience. cell, 2020. 181(2): p. 219-222. 41. caruso, f.p., et al., a map of tumor-host interactions in glioma at single-cell resolution. gigascience, 2020. 9(10). 42. voisin, m.r., et al., brain tumors and covid-19: the patient and caregiver experience. neurooncol adv, 2020. 2(1): p. vdaa104. 43. mehta, v., et al., case fatality rate of cancer patients with covid-19 in a new york hospital system. cancer discov, 2020. 10(7): p. 935-941. 44. mohile, n.a., et al., urgent considerations for the neuro-oncologic treatment of patients with gliomas during the covid-19 pandemic. neuro oncol, 2020. 45. henriksen, j., et al., rapid validation of telepathology by an academic neuropathology practice during the covid-19 pandemic. arch pathol lab med, 2020. 144(11): p. 1311-1320. 46. hanna, m.g., et al., validation of a digital pathology system including remote review during the covid-19 pandemic. mod pathol, 2020. 33(11): p. 2115-2127. 47. stathonikos, n., et al., digital pathology in the time of corona. j clin pathol, 2020. 73(11): p. 706-712. 48. williams, b.j., et al., guidance for remote reporting of digital pathology slides during periods of exceptional service pressure: an emergency response from the uk royal college of pathologists. j pathol inform, 2020. 11: p. 12. 49. cimadamore, a., et al., digital pathology and covid-19 and future crises: pathologists can safely diagnose cases from home using a consumer monitor and a mini pc. j clin pathol, 2020. 73(11): p. 695-696. 50. parker, e.u., o. chang, and l. koch, remote anatomic pathology medical student education in washington state. am j clin pathol, 2020. 154(5): p. 585-591. 51. mukhopadhyay, s., et al., leveraging technology for remote learning in the era of covid-19 and social distancing. arch pathol lab med, 2020. 144(9): p. 1027-1036. 52. albeniz, e., et al., stay connected and up to date. gi meetings and seminars and the covid-19 pandemic. tech innov gastrointest endosc, 2020. 53. bakouny, z., et al., covid-19 and cancer: current challenges and perspectives. cancer cell, 2020. 38(5): p. 629-646. 54. kruger, j. and d. dunning, unskilled and unaware of it: how difficulties in recognizing one's own incompetence lead to inflated self-assessments. j pers soc psychol, 1999. 77(6): p. 1121-34. 55. stichel, d., et al., accurate calling of kiaa1549-braf fusions from dna of human brain tumours using methylation array-based copy number and gene panel sequencing data. neuropathol appl neurobiol, 2020. 56. deng, m.y., et al., molecularly defined diffuse leptomeningeal glioneuronal tumor (dlgnt) comprises two subgroups with distinct clinical and genetic features. acta neuropathol, 2018. 136(2): p. 239-253. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of aβ plaques in alzheimer’s disease. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:20 (2020) original paper clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of aβ plaques in alzheimer’s disease. patrick jarmo paasila 2, danielle suzanne davies 1, greg trevor sutherland 2, claire goldsbury 1 1 discipline of anatomy and histology, school of medical sciences, faculty of medicine and health, the university of sydney, nsw 2006, australia 2 discipline of pathology, school of medical sciences, faculty of medicine and health, the university of sydney, nsw 2006, australia corresponding author: dr claire goldsbury phd · brain and mind centre · 94 mallett street · camperdown, nsw 2050 · australia · tel +61 2 9351 0878 claire.goldsbury@sydney.edu.au submitted: 16 june 2020 accepted: 29 july 2020 copyedited by: jeffrey nirschl published: 04 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2845 keywords: alzheimer’s disease, inferior temporal cortex, microglia, post-mortem human brain tissue, primary motor cortex abstract alzheimer’s disease (ad) is a late-onset disease that has proved difficult to model. microglia are implicated in ad, but reports vary on precisely when and how in the sequence of pathological changes they become involved. here, post-mortem human tissue from two differentially affected regions of the ad brain and from non-demented individuals with a high load of ad-type pathology (high pathology controls) was used to model the disease time course in order to determine how microglial activation relates temporally to the deposition of hallmark amyloid-β (aβ) and hyperphosphorylated microtubule associated protein tau pathology. immunofluorescence against the pan-microglial marker, ionised calcium-binding adapter molecule 1 (iba1), aβ and tau, was performed in the primary motor cortex (pmc), a region relatively spared of ad pathological changes, and compared to the severely affected inferior temporal cortex (itc) in the same cases. unlike the itc, the pmc in the ad cases was spared of any degenerative changes in cortical thickness and the density of betz cells and total neurons. the clustering of activated microglia was greatest in the pmc of ad cases and high pathology controls compared to the itc. this suggests microglial activation is most prominent in the early phases of ad pathophysiology. nascent tau inclusions were found in neuritic plaques in the pmc but were more numerous in the itc of the same case. this shows that tau positive neuritic plaques begin early in ad which is likely of pathogenic importance, however major tau deposition follows the accumulation of aβ and clustering of activated microglia. importantly, findings presented here demonstrate that different states of microglial activation, corresponding to regional accumulations of aβ and tau, are present simultaneously in the same individual; an important factor for consideration if targeting these cells for therapeutic intervention. introduction alzheimer’s disease (ad) is neuropathologically characterised by inclusions of microtubule-associated protein tau (tau) and extracellular deposits of β-amyloid (aβ). intraneuronal tau pathology includes neurofibrillary tangles (nfts) in the cell soma and neuropil threads (nts), which occur mostly in the dendritic compartment, but also in the axonal domain though to a lesser extent (1, 2). nfts and nts are both comprised of paired helical filaments and straight filaments of polymerised hyperphosphorylated tau protein (3, 4). the extent of tau pathology follows a predictable spatiotemporal progression through functionally integrated brain regions (5, 6) and there is an extensive body of literature that demonstrates an inverse correlation between the accumulation of nfts and cognitive status (7) such that the spread and regional level of nfts reflects the severity of dementia with time (8). contrastingly, aβ plaques follow a seemingly more haphazard regional pattern of accumulation throughout the neocortex, indeed plateauing relatively early in the disease time course (9, 10), and therefore correlate poorly with disease status until substantial argyrophilic neuritic tau pathology is also present (5, 7, 11). whilst aβ load may be an unreliable indicator of disease severity, it is generally accepted that the extent of its spread, in combination with tau pathology, is useful for staging purposes (9). aβ plaques can be classified with immunostaining into at least three morphologically distinct categories: diffuse, fibrillar, or dense-cored. diffuse plaques have long been proposed to be a structural precursor of other plaque forms, but whether these categories represent separate entities with independent mechanisms of development or are temporally linked is still unclear. neuritic plaques (nps) are those aβ plaques that also feature dystrophic neurites (dns) with silver staining. dns can occur in all these morphological subtypes of aβ plaques, though more commonly in dense-cored and fibrillar plaques than morphologically diffuse plaques (12). most dns are tau-positive and morphologically similar to nts which are elongated in shape, but may also be globular, and possibly represent swollen presynaptic (axonal) terminals (13). nps that retain sparse dns but show minimal aβ staining have previously been described and were termed ‘remnant plaques’ which were proposed to result from glial phagocytosis of insoluble aβ (14). there remains significant debate as to the sequence of the neuropathological changes that precede the onset of ad symptomatology. the amyloid cascade hypothesis posits aβ, and in particular the soluble, oligomeric, non-fibrillar fraction, as the initiating factor (15-20). a competing view is offered by others who contend that the sequence of pathological events begins with neurofibrillary pathology (21, 22) which precedes the formation of insoluble aβ pathology (23). indeed, there is evidence that tau pathology occurs prior to aβ pathology as it is more common in the brains of non-demented individuals (21) but this might represent a non-ad scenario described as primary age-related tauopathy (part) (24). notwithstanding the order of events, both hypotheses suggest that an activated glial response is an integral component of the pathogenesis of ad. the study of microglia morphology in post-mortem human brain tissue presents a simple method with which to gauge the involvement of microglia in instances of changed physiological conditions or to the development of a disease. microglia with a ramified morphology, characterised by thin, evenly distributed, highly branched processes with a small, spherical soma, represent the healthy cell population (25-27). activated microglia are characterised by reduced morphological complexity including hypertrophy of the soma and processes and may also display the formation of distal phagosomes (23, 25, 28). dystrophic microglia display features consistent with cellular senescence (29), including a loss of processes, tortuosity of remaining processes, and discontinuous iba1-immunolabelling (30-32). lastly, clusters of activated microglia in ad have been noted previously in post-mortem human brain tissue and represent the direct interaction between microglia and aβ and tau pathology (10, 32, 33). by investigating brain regions differentially affected by ad-type pathology in non-demented and demented individuals it may be possible to model the sequence of pathological changes in the disease, in particular the activated microglial response. previously we demonstrated an increased density of activated microglia in the inferior temporal cortex (itc) of non-demented controls with similar levels of ad-type pathology (‘high pathology controls’ – hpcs) as clinically and neuropathologically-confirmed ad cases (26). this suggested that the microglial response occurs in the preclinical phases of ad but we wished to confirm this finding in a belatedly affected region of the ad brain, the primary motor cortex (pmc), compared to an earlier and more severely affected region, the itc, of the same cases. further, we wished to determine the sequence of aβ, tau, and microglia pathological changes that occur in the cortex. the findings here demonstrate that nascent nts and dns in the pmc begin early in the pathogenesis of ad, but follow the clustering of activated microglia. by contrast, looking at the severely affected itc in the same cases, findings suggest that microglial clustering at plaques dissipates once tau and aβ pathology is long established and there is also a substantial loss of iba1-immunoreactivity (26, 34). although an early toxic microglial gain of function cannot be ruled out, these findings appear most consistent with a scenario where microglial activation is neuroprotective early in the pathogenesis of ad. we suggest that this is followed by a gradual exhaustion of microglial function that contributes to cognitive deterioration in the ad brain. table 1. cohort characteristics methods this study was approved by the university of sydney’s human research ethics committee (hrec#2015/477). all tissue samples for this study, as well as demographic and clinical information, were supplied by the new south wales brain tissue resource centre (nswbtrc) and the sydney brain bank (sbb), collectively the new south wales brain banks (nswbb), following approval from their scientific advisory committee. methods for case ascertainment and tissue preparation by nswbb have been previously published (35). the demographic and clinicopathological characteristics of the cohort (table 1) and data from the itc have been previously published (26). immunofluorescence immunofluorescence staining procedures were performed on free-floating 45 μm fixed sections derived from the caudal aspect of the superomedial area of the pmc of controls (n = 10), hpcs (n = 5), and pathologically confirmed ad cases (n = 8) as previously described (26). double-labelled sections using antibodies against aβ, total tau (ttau), and the pan-microglial marker ionised calcium-binding adapter molecule 1 (iba1), were used for the quantification of aβ and tau loads and microglial morphological subtypes. for aβ and iba1 quantification, heat-induced epitope retrieval was performed using a sodium citrate solution (ph 8.5) at 60°c overnight, followed by a 12-minute formic acid (90%) incubation at room temperature. blocking was performed in 10% normal goat serum (gibco #16210072), and primary (mouse aβ, 1:1000, biolegend 803002; rabbit iba1, 1:1000, wako 019-19741) and secondary (1:200; thermo scientific: alexa fluor (af) 488 goat anti-mouse, #a11001; af 568 goat anti-rabbit, #a11011) antibody incubations were performed at 4°c with gentle agitation. nuclear counterstaining was performed in the last 40 minutes of the secondary antibody incubation by the addition of hoechst 33342 dye (1 µg/ml; thermo scientific 62249). sections were mounted using prolong diamond antifade (invitrogen p36961). double-immunolabelling of ttau (rabbit; 1:500; dako k9ja/a0024) and iba1 (mouse; 1:50; millipore mabn92) was carried out as previously described (34). ttau immunostaining of nfts, nts, and dns has previously been demonstrated to give comparable immunostaining to standard phosphotau antibodies (12e8 and at8) (36). briefly, heat-retrieval was performed with sodium citrate (ph 6.0) for 10 minutes at 100°c, before permeabilising, blocking with bsa, and incubating in primary antibodies for three hours at room temperature or overnight at 4°c, and finally incubating in secondary antibodies (1:200; invitrogen: af555 goat anti-mouse, a214424; af647 goat anti-rabbit, a21244). hoechst 33342 was added to counterstain nuclei and sections were mounted in prolong gold antifade. image acquisition and analysis aβ and iba1 double-labelled sections were imaged using a zeiss lsm 800 confocal microscope using the ‘tile scan’ function at the advanced microscopy facility, bosch institute, the university of sydney. a previously validated modified disector sampling approach that utilises one section per individual was used here for the analysis of the aβ and iba1 immunostained sections (37). briefly, a total of three cortical strips per section from areas where the pial surface and the grey-white boundary were strictly in parallel were acquired for the quantification of microglia, aβ plaques and aβ-positive pixels. these cortical strips were constructed of serial images 500 μm in width, 6 μm in z-depth with three z-slices (z-step = 3 μm), spanning all of the cortical laminae of the pmc using a 20×/0.8 numerical aperture (na) objective. clusters of microglia and individual microglia, which were categorised as having either a ramified, activated, or dystrophic morphology as previously described (26), were enumerated in image analysis software (fiji; nih). the terminology used to describe the different populations of morphologically diverse populations of microglia was informed by previous investigations (25, 28, 30). microglia with thin, highly branched processes, and a spherical nucleus were categorised as ‘ramified’. ‘activated’ microglia (previously termed ‘deramified’ (34)) included those cells that displayed hypertrophy of the soma or processes with retraction of secondary or tertiary processes. ‘dystrophic microglia’ were identified on the basis of a loss of processes with the remaining processes displaying significant tortuosities or discontinuous iba1-immunolabelling with or without blebbing or punctate iba1-labelling (previously dystrophic microglia were subcategorised as either ‘punctate’ or ‘discontinuous’ to reflect these observations (34)). a ‘cluster of microglia’ was counted if three or more soma occurred within, or were touching the margins of, a 20 μm2 virtual graticule subregion. larger clusters were counted as one cluster if the graticule subregion could be moved and still incorporate at least three somata. clusters were counted whilst visualising only the 568 nm (iba1-positive) channel to distinguish these from individual microglia and to minimise potential false-positive counts in the presence of either aβor ttau-immunostaining. the total number of microglia counted per section averaged 519 with a coefficient of error (ce) of ≤0.2 for all counts of morphological subtypes, with the exception of microglial clusters (ce ≤0.3) which were relatively rare and displayed significant variance between cases. quantification of ttau was carried out using an olympus vs120 slide scanner at the sydney microscopy and microanalysis, brain and mind centre, the university of sydney. whole section dic and fluorescence overviews of a single section from each case were generated using a 10× objective and were used to systematically map out four representative 500 μm2 regions of interest (= 1mm2 per section) within the mid-cortical laminae (iii-v) for manual counts of nfts and for the quantification of ttau-positive pixels. individual images were captured using a 40×/0.9 na objective and were comprised of seven z-slices with a depth of 6 μm (z-step = 1 μm). image analysis was performed in fiji using manually thresholded images. positive pixel counts were generated for aβ and ttau staining and expressed as a percentage of total pixels (% aβ and % ttau respectively). high resolution imaging of all immunolabelled sections were carried out using a nikon a1r or zeiss lsm 710 confocal microscope (sydney microscopy and microanalysis, charles perkins centre and brain and mind centre, the university of sydney). features were imaged with either a 40×/0.95 na or 100×/1.4 na objective and shown as maximum intensity projections. nissl staining nissl stains of free-floating 45 μm thick formalin-fixed sections were performed for the measurement of cortical thickness and neuronal counts. sections were incubated in 0.1% cresyl violet acetate (0.02% glacial acetic acid; added immediately before use) for 15 minutes at 60°c. differentiation was achieved by sequentially washing sections for three minutes in 70% and 95% ethanol. the final level of staining was adjusted by briefly dipping sections in 100% ethanol and confirmed by light microscopy before clearing in xylene for ten minutes and mounting in dpx. cortical thickness measurements and neuronal counts were performed on three cortical strips from one section per individual using an eyepiece graticule on an olympus bx50 microscope using a 20×/0.75 na objective. a height measure of 45 μm was used in determining density estimates to eliminate artefactual tissue shrinkage during staining; with the microtome accuracy to cut precise sections having been previously validated (37). ces using the modified disector technique outlined above were <0.1 for cortical thickness and <0.15 for the density of betz cells and total neurons. statistical analyses the normality of data was tested using the shapiro-wilk test. equality of variances was tested using brown-forsythe test. group differences were investigated by either welch’s analysis of variance or kruskal-wallis test for non-gaussian distributions, with either games-howell or dunn’s test, respectively, for pairwise comparisons. regional differences to the previously reported itc (26) were investigated by either welch’s t test or wilcoxon rank-sum test for non-gaussian distributions. the pearson correlation coefficient (r) and coefficient of determination (r2), or spearman rho (ρ) for non-gaussian distributions, were calculated for univariate correlations to investigate relationships between ad-type pathology, microglial morphologies, and apoe ε4 status. stepwise regression models which included age, sex, brain ph, post-mortem interval, and fixation period were performed to exclude effects of potential confounders. a p-value <0.05 was considered statistically significant. all statistical analyses were performed using jmp pro 14 (sas institute inc). graphs were produced using microsoft excel. results the alzheimer’s disease primary motor cortex exhibits mild tau and β-amyloid deposition but no evidence of neurodegeneration this study involved 23 autopsy cases that had previously been clinicopathologically characterised as ‘probable ad’ or controls based on abc score and clinical dementia rating (table 1) (39). ad cases (n=8) included individuals with an intermediate–high likelihood of ad dementia following routine neuropathological diagnostic testing (38) and who presented with typical ad dementia prior to death. ‘high pathology controls’ (hpc) (n=5) were grouped as such on the basis of no cognitive impairment but satisfied a diagnosis of intermediate ad likelihood on post-mortem examination. controls (n=10) included individuals with a range of abc scores (a0–3; b0–2; c0–3), though only satisfying a ‘not’ or ‘low’ outcome after diagnostic testing. there was no cortical atrophy (fig. 1a) or neuronal loss (fig. 1b), including the prominent layer vb betz cells (fig. 1c), in the pmc of ad cases or hpcs compared with controls. following a positive pixel analysis, grey matter aβ areal fraction (% aβ) was higher in the pmc of ad cases compared to controls (p = 0.0005), but not hpcs (p = 0.07). the density of total aβ plaques, fibrillar, and dense-cored plaques was also significantly higher in ad cases compared to controls (totalp = 0.003; fibrillarp = 0.002; dense-coredp = 0.004) but not hpcs (totalp = 0.1; fibrillarp = 0.06; dense-coredp = 0.2). total tau areal fraction (% ttau) and the density of nfts was higher in the pmc of ad cases compared to controls (%ttaup = 0.001; nftp = 0.001) and hpcs (%ttaup = 0.007; nftp = 0.01) (table 2). amongst the ad cases, the pmc had significantly reduced % aβ (p = 0.006), total aβ plaques (0.001), and fibrillar plaques (p = 0.0002), but not dense-cored plaques (p = 0.2) compared to the itc. the % ttau (p = 0.02) and the density of nfts (p = 0.02) were also significantly reduced in the pmc compared to the itc of ad cases. amongst controls the % ttau was significantly reduced in the pmc compared to the itc (p = 0.04). there were no other regional differences in terms of pathological load in controls, including amongst hpcs (table 3). in the itc, all of the control, hpc, and ad cases had neuritic tau pathology at varied levels. aβ pathology in the itc occurred in 4/10 controls, and all hpcs and ad cases. the clustering of activated microglia in the itc was apparent in 6/10 controls (including three with only tau pathology), and all of the hpcs and ad cases except one (m12) (itc case data is shown in fig. 1d). in the pmc, neuritic tau pathology was present in 7/10 controls, 4/5 hpcs, and all ad cases, while aβ deposition in the pmc occurred in 6/10 controls, 4/5 hpcs, and all ad cases. microglial clustering in the pmc was observed in 5/10 controls, and all of the hpcs and ad cases, with the exception of one case (m12) (pmc case data is shown in fig. 1e). in controls, the % ttau correlated with age in the pmc (r2 = 0.44, p = 0.04) but not in the itc, which was near significant (r2 = 0.39, p = 0.05). figure 1 characteristics of the pmc and regional neuropathological comparisons. a–c the pmc is spared of ad-related neurodegenerative changes as measured by cortical thickness (a), total neuronal density (b), and the density of layer vb betz cells (inset demonstrates a pyramidal betz cell with multiple asymmetrically distributed perisomatic neurites, a prominent nucleolus, and a dark dense deposit of cytoplasmic lipofuscin) (c). d–e a cohort wide comparison of the percentage of aβ and ttau immunolabelling and microglial clustering in the itc (d) and pmc (e) demonstrates age-related tau build-up in a majority of control brains in both regions and an early build-up of aβ in hpcs (that were scored as a2-3, b2, c0-3 on diagnostic slides) with a concomitant microglial clustering response that is more prevalent in the pmc compared to the itc and which appears to dissipate with severe ad pathology in the itc. scale bar = 50 μm (c) table 2. summary of neuronal and neuropathological data of the primary motor cortexa amean ± standard deviation. banova results; see text for p-values of pairwise comparisons. cinclusive of betz cells. ddensity of betz cells in layer v. table 3. regional neuropathological comparisonsa amean ± standard deviation. clusters of activated microglia are more commonly found in the pmc than the itc of hpcs and ad cases the density of individual activated (fig. 2a–e), ramified (fig. 2f), dystrophic (fig. 2g–h), or total microglia did not differ between any of the three groups in the pmc (table 4). microglial clusters (fig. 2i–j), although a small proportion of total microglia, were significantly more common in hpcs (fig. 2k) and ad (fig. 2l) cases compared to controls in the pmc ( hpc p = 0.01; ad p = 0.04). clusters in the pmc were also higher than in the itc of hpcs (p = 0.02) and ad cases (p = 0.03) (fig. 3a), whilst the density of total (p = 0.04) and ramified microglia (p = 0.002) were reduced in the itc compared to the pmc of ad cases. immunofluorescent double-labelling for aβ and iba1 in the pmc showed that the clusters of activated microglia, occurred preferentially within the boundaries of fibrillar aβ plaques, with a significant correlation in a combined analysis of control and ad brains (spearman ρ = 0.54, p = 0.006; fig. 3a). the percentage of aβ plaques associated with a cluster of activated microglia in the pmc ranged from a mean of 41% in controls and 43% in ad cases to 60% in hpcs, with one control (m15), two hpcs (m14 and m18), and one ad case (m13) having a higher density of microglial clusters than aβ plaques. figure 2 response of microglia to ad pathology. a–e a spectrum of microglial activation can be identified by a series of morphological changes including an enrichment of iba1 labelling of the soma and primary processes (a), hypertrophy of the primary processes (b), retraction of tertiary processes ± the formation of morphological features consistent with phagosomes (arrow heads) (c), further retraction of secondary processes (d), until amoeboid in shape (e). f–h healthy ramified microglia have a small, spherical soma and thin, evenly distributed processes (f), contrasting with dystrophic microglia that have either deramified and tortuous processes (g) or pseudo-fragmentation of processes when marked with iba1 (h). i–j microglia that form a cluster within the boundary of an aβ plaque may be either dystrophic or have reached a phase of early (i) or late/amoeboid (j) activation. k–l mosaics of iba1 staining demonstrating the size and distribution of microglial clusters (arrows), defined as three or more somata occurring within, or touch the boundaries of, a 20 μm2 virtual graticule subregion, in the pmc of an hpc (k; m23) and ad case (l; m13). scale bar in j = 40 μm (a–j); in l = 100 μm (k–l) figure 3 characteristics of microglial clustering in the pmc. a the density of microglial clusters was significantly greater in the pmc of hpcs and ad cases compared to controls. b microglial clusters were more frequently associated with fibrillar neuritic plaques in a combined group analysis; spearman ρ = 0.54, p = 0.006. †significantly reduced compared to pmc of hpcs; ‡significantly reduced compared to pmc of ad cases. *†‡p<0.05. table 4. quantification of the morphological subtypes of microglia in the primary motor cortexa acells/mm2; mean ± standard deviation. banova results; see text for p-values of pairwise comparisons of microglial clusters. a graded extent of neuritic tau pathology and clustering of microglia occurs within nascent aβ plaques, with the persistence of dystrophic neurites and the loss of iba1-immunoreactivity and aβ-immunoreactivity occurring in established neuritic plaques all of the aβ plaques examined in the itc, and a majority in the pmc, contained dns. however, the extent of the accumulated neuritic tau pathology within each plaque was lower in the pmc (fig. 4a) than the itc of the same case (fig. 4b). both aβ deposits and globular dns were seen perivascularly (fig. 4c), consistent with previous observations (39). the density of dns was greatest in the itc of ad cases (fig. 4c), which also had the highest density of remnant plaques characterised by accumulations of dns associated with weak or absent aβ-immunoreactivity (fig. 4d). in the itc a diffuse lattice of elongated nts occurred throughout the neuropil and independently of aβ plaques (fig. 4e). dns appeared radially projecting from aβ plaques (fig. 4e) and showed either elongated or globular morphology (fig. 5a). hoechst dye marked cell nuclei around the periphery of aβ plaques and also partly stained the fibrillar deposits of aβ (fig. 5a). hoechst has previously been reported to stain aβ plaques in transgenic mice (40). immunofluorescent double-labelling showed no colocalisation of aβ and ttau (fig. 5b). figure 4 immunofluorescent double-labelling for aβ and ttau. a–c the majority of aβ plaques examined here were associated tau-positive dystrophic neurites, with a clear gradation visible between the pmc (a) and itc (b) in controls (m01 pictured in a and b) and ad cases (itc of m09 pictured in c). ad cases had the most extensive build-up of dns in fibrillar and dense-cored plaques, as well as perivascular (capillary) aβ deposits (c). d remnant plaques (arrows) are characterised by absent or weak aβ staining and dense accumulations of tau pathology and were much more common in the itc than the pmc (also seen in b). e severely affected regions of the ad brain, such as the itc, showed a diffuse network of elongated nts throughout the parenchyma as well radially projecting dns. scale bar = 20 μm (a–c), 60 μm (d), 40 μm (e) figure 5 high power image of an aβ plaque in ad. a image panel showing a fibrillar neuritic plaque in an ad case (m03) with globular and threadlike dns distributed throughout the plaque, which is also surrounded by a network of nts (dotted box in merged image demonstrates roi shown in b). hoechst staining labelled cell nuclei around the periphery of the plaque as well as the fibrillar aβ component inside the plaque. b colocalisation study showed no coincidence aβ and ttau staining in any of the sections examined here (dotted line represents a 6.5 μm length along which pixel intensities have been compared in this exemplar roi). scale bar = 20 μm (a) microglia cell processes exhibiting evidence of phagocytic activity are interspersed around the periphery and core of aβ plaques microglia that occurred in proximity to diffuse, fibrillar, and cored plaques commonly displayed structures morphologically consistent with phagosomes on distal processes with enriched iba1 immunolabelling that closely associated with the aβ element in the periphery and core of plaques (fig. 6a; fig. 7a). as previously reported for the itc, superior frontal gyrus, and primary visual cortex, the overall density of dystrophic microglia was inversely correlated with brain ph in the pmc (r2 = 0.3, p = 0.01) (26). however, it was also noted that individual plaques with dense dns, that were associated with weak or absent aβ staining which were more abundant in the itc, were associated with dystrophic microglia (fig. 6b) rather than a cluster of activated microglia which more commonly occurred where the extent of dns was not yet fully developed (fig. 6c–d). confocal views showed co-localisation of microglial cell processes with aβ in ad (fig. 7a–b), but no co-localisation with tau pathology (fig. 7c–d). figure 6 responses of microglia to ad neuropathology. a the itc of a control case (m01) demonstrating activated microglia with morphological features consistent with the formation of phagosomes (arrows) responding to peripheral and core elements of an aβ plaque. b in the itc of ad cases (m17 pictured), dystrophic microglia were more commonly associated with plaques that contained dense accumulations of dystrophic neurites, however the overall density of dystrophic microglia was inversely correlated with brain ph and not with ad. c–d conversely, plaques with a lower density of dystrophic neurites were more commonly associated with a cluster of activated microglia (m01 pictured) in both the pmc (c) and itc (d). scale bar = 20 μm (a–d) figure 7 exemplars from an investigation into the potential internalisation of aβ and tau pathology by microglia. a activated microglia in an aβ plaque in the itc of a control case with morphological features consistent with the formation of phagosomes (arrows) (m01; dotted box represents region of interest in b). b coincidence of aβ and iba1 pixel intensities along a 7.8 μm length (dotted line) suggests potential internalisation of aβ by microglia. c activated microglia in close proximity to tau-positive dns in the itc of an ad case (m17; dotted box represents region of interest in d). d there was no evidence of the internalisation of tau pathology by microglia in any of the sections investigated here; exemplar shows aβ and ttau staining intensities over a 7.2 μm length (dotted line). scale bar 20 μm (a, c) discussion ad is a uniquely human disease with a long prodrome and has proved difficult to model. the combination of using different regions of post-mortem brain tissue from individuals with or without dementia and with variable amounts of ad-type pathology may allow the pathological sequence of events to be elucidated. for example, the level of disease severity could be ordered from lowest to highest as follows: pmc-controls < itc-controls < pmc-hpc < itc-hpc < pmc-ad < itc-ad. in particular, regions such as the itc in hpcs and the pmc in ad cases could harbour the pre-symptomatic targets required to therapeutically delay or prevent ad. prior to using this model to understand the role of microglia in ad, a quantitative neuropathological analysis of the pmc was carried out to ensure that it met expectations for being a relatively unaffected region of the ad brain. as expected, the pmc of ad cases had a significantly higher % aβ, aβ plaque count, % ttau, and nft density compared controls. there were no nfts observed in the pmc of controls and hpcs, which also had very similar levels of % ttau (which correlated with increasing age), but differed in their level of % aβ. hpcs were defined according to standard neuropathological diagnostic criteria – having an intermediate abc score (38). incidentally, hpcs and ad cases (intermediate–high abc scores) were similar in their aβ load but differed in their tau levels in both regions. in contrast to the primary visual cortex previously investigated (26), the pmc in ad did have significantly reduced levels of aβ and overall tau pathology, including nfts as well evidenced elsewhere (41-46), compared to the itc of the same cases. overall there was no evidence of neurodegeneration in the pmc of ad cases, unlike the itc, with cortical thickness, number of total neurons, and giant layer vb pyramidal betz cells remaining unchanged as expected (47, 48). examination of the microglial morphologies in the pmc using the previously validated modified disector sampling approach across all cortical laminae yielded no significant differences between controls, hpcs, and ad cases. however, group differences could be seen locally around ad-type pathology within the cortex with an increase in clustering of activated microglia in the pmc of hpcs compared to controls, and in the pmc compared to the itc of ad cases. moreover, a higher percentage of plaques contained clusters of microglia in hpcs than in ad cases and also a portion of microglia clusters that were not spatially associated with aβ. the presence of a strong microglial clustering response in the pmc of ad cases and in the hpcs aligns with pet imaging studies demonstrating early activation of microglia in preclinical ad cases (49). although the presence of clusters unrelated to aβ pathology may be a non-specific observation, it is interesting to note that a previous animal study using a 5×fad model also reported the presence of microgliosis prior to the formation of insoluble aβ plaques (50), with another mouse model also indicating microglial activation in relation to synaptic dysfunction prior to aβ deposition (51). here it is suggested that clusters of activated microglia in the pmc represent a neuroprotective response correlating with the deposition of aβ. we have demonstrated clusters of activated microglia that display phagocytic capabilities in the mildly affected pmc before the development of extensive tau pathology. it is conceivable that once the phagocytic potential of microglia is overwhelmed, a transition to a more neurotoxic proinflammatory phenotype occurs and that this represents a pivotal moment preceding tau-related neurofibrillary degeneration. studies in mouse models of aβ overexpression suggest proinflammatory microglia, which may be induced by the binding of oligomeric and fibrillar aβ species to nlr family pyrin domain containing 3, receptor for advanced glycation endproducts, scavenger receptors, and toll-like receptors, among others (52-58), are associated with poorer cognitive and survival outcomes, have impaired phagocytic capabilities (59), and are capable of secreting an expansive complement of neurotoxic compounds including reactive oxygen species, nitric oxide, peroxynitrite, tumour necrosis factor α, interleukin 1 β, and prostaglandin-e2 (60). however aβ-independent mechanisms of microglia activation or exhaustion in human ad cannot be excluded and require further research considering the so far limited efficacy of the pharmacological clearance of aβ in clinical trials (61, 62). activated microglia tended to be associated with fibrillar nps, and higher resolution confocal photomicrographs showed evidence of aβ internalisation by microglia in both regions. the latter may explain the remnant plaques observed here (that contain weak or absent aβ-immunolabelling; in which higher levels of aβ were associated with activated microglia, but dystrophic microglia where aβ-immunoreactivity was very minimal or absent, particularly where dystrophic neurites were extensive) and described elsewhere (14) and is potentially relevant to the proposed dynamic equilibrium between soluble aβ oligomers and insoluble fibrils (63). however, it will be important to confirm the internalisation of aβ by microglia with super resolution techniques such as direct stochastic optical reconstruction microscopy (dstorm). in contrast, microglia did not specifically cluster around any of the three forms of tau pathology, nfts, nts and dns, nor did they appear to internalise tau in co-localisation studies, although processes of microglia were coincidentally found adjacent to dns. the microglial clustering response dissipated over the modelled disease course with advanced stages, represented by the itc of ad cases, being characterised by reduced iba1 immunoreactivity, as reported elsewhere (31, 64). this suggests a process of microglial incapacitation in the context of increased tau load, a concept which is supported by a growing body of literature (65, 66). from these results it is hypothesised that the activation of microglia coincides with cortical aβ deposition. neuritic inclusions of tau in the cortex are evident early in the disease process, represented here by the pmc of ad cases, but mainly develop after the deposition aβ and the activation of microglia. this sequence of pathological changes is ostensibly consistent with the amyloid cascade hypothesis for ad pathogenesis (19) given the presence of elevated aβ in the pmc of hpcs, however it should be stressed that the levels of aβ and tau pathology were present at similar levels in the pmc of confirmed ad cases. therefore it could be argued that insoluble aβ and tau deposits begin forming concurrently in the cerebral cortex of ad brains. this would be consistent with those arguing in favour of the pathogenetic importance of tau deposition (22, 23, 67, 68) and the possibility that it in fact acts as a causative factor behind ad-related microglial activation (69). certainly, animal models suggest that microglial activation augments tau pathology and specifically tau phosphorylation (70). this scenario would then be consistent with our observation that microglial activation wanes with increased tau deposition and with the idea that ageing impairs the housekeeping functions of microglia (71). finally, even in the presence of extensive tau pathology, the increased presence of remnant plaques in the itc suggests that microglia retain the ability to clear aβ peptides. understanding the functional significance of these dynamic spatiotemporal changes in microglial activity along the time course of ad pathophysiology will be critical before new treatments targeting these cells can be imagined. given that microglia of different brain regions display different activation states simultaneously depending on the graded extent of ad-type pathology present, the implementation of either antior pro-inflammatory microglia-based therapies would presumably be beneficial in one brain region but detrimental in another. in future work the genetic characterisation of subjects investigated here may also provide further insight into how genotype affects individual susceptibility to differential microglial function, represented by the highly variable clustering response of microglia in hpcs and ad cases in particular. overall, findings from the post-mortem model used here suggest that the clustering of activated microglia occurs concomitantly with the formation of aβ plaques, and that tau-related neuritic degeneration follows these changes along with a loss of clustering. acknowledgements the authors would like to thank the donors and their families for their kind gift. brain tissue was received from the nsw brain tissue resource centre and sydney brain bank. these brain banks are supported by the nhmrc of australia, the university of new south wales, neuroscience research australia, and the national institute of alcohol abuse and alcoholism (nih (niaaa) r24aa012725). the authors also acknowledge the facilities used at the bosch institute and microscopy australia at the australian centre for microscopy & microanalysis both at the university of sydney. data availability statement the data that support the findings of this study are available from the corresponding author upon reasonable request. references 1. braak e, braak h, mandelkow em. a sequence of cytoskeleton changes related to the formation of neurofibrillary tangles and neuropil threads. acta neuropathol. 1994;87(6):554-67. 2. perry g, kawai m, tabaton m, onorato m, mulvihill p, richey p, et al. neuropil threads of alzheimer's disease show a marked alteration of the normal cytoskeleton. j neurosci. 1991;11(6):1748-55. 3. barghorn s, davies p, mandelkow e. tau paired helical filaments from alzheimer's disease brain and assembled in vitro are based on beta-structure in the core domain. biochemistry. 2004;43(6):1694-703. 4. fitzpatrick awp, falcon b, he s, murzin ag, murshudov g, garringer hj, et al. cryo-em structures of tau filaments from alzheimer's disease. nature. 2017;547(7662):185-90. 5. braak h, braak e. neuropathological stageing of alzheimer-related changes. acta neuropathol. 1991;82(4):239-59. 6. franzmeier n, neitzel j, rubinski a, smith r, strandberg o, ossenkoppele r, et al. functional brain architecture is associated with the rate of tau accumulation in alzheimer's disease. nat commun. 2020;11(1):347. 7. nelson pt, alafuzoff i, bigio eh, bouras c, braak h, cairns nj, et al. correlation of alzheimer disease neuropathologic changes with cognitive status: a review of the literature. j neuropathol exp neurol. 2012;71. 8. arriagada pv, growdon jh, hedley-whyte et, hyman bt. neurofibrillary tangles but not senile plaques parallel duration and severity of alzheimer's disease. neurology. 1992;42(3 pt 1):631-9. 9. thal dr, rub u, orantes m, braak h. phases of a beta-deposition in the human brain and its relevance for the development of ad. neurology. 2002;58(12):1791-800. 10. serrano-pozo a, mielke ml, gomez-isla t, betensky ra, growdon jh, frosch mp, et al. reactive glia not only associates with plaques but also parallels tangles in alzheimer's disease. am j pathol. 2011;179(3):1373-84. 11. mirra ss, heyman a, mckeel d, sumi sm, crain bj, brownlee lm, et al. the consortium to establish a registry for alzheimer's disease (cerad). part ii. standardization of the neuropathologic assessment of alzheimer's disease. neurology. 1991;41(4):479-86. 12. dickson tc, vickers jc. the morphological phenotype of beta-amyloid plaques and associated neuritic changes in alzheimer's disease. neuroscience. 2001;105(1):99-107. 13. yasuhara o, kawamata t, aimi y, mcgeer eg, mcgeer pl. two types of dystrophic neurites in senile plaques of alzheimer disease and elderly non-demented cases. neurosci lett. 1994;171(1-2):73-6. 14. oide t, kinoshita t, arima k. regression stage senile plaques in the natural course of alzheimer's disease. neuropathol appl neurobiol. 2006;32(5):539-56. 15. yu l, petyuk va, tasaki s, boyle pa, gaiteri c, schneider ja, et al. association of cortical beta-amyloid protein in the absence of insoluble deposits with alzheimer disease. jama neurol. 2019. 16. lesne se, sherman ma, grant m, kuskowski m, schneider ja, bennett da, et al. brain amyloid-beta oligomers in ageing and alzheimer's disease. brain. 2013;136(pt 5):1383-98. 17. lue lf, kuo ym, roher ae, brachova l, shen y, sue l, et al. soluble amyloid beta peptide concentration as a predictor of synaptic change in alzheimer's disease. am j pathol. 1999;155(3):853-62. 18. mclean ca, cherny ra, fraser fw, fuller sj, smith mj, beyreuther k, et al. soluble pool of abeta amyloid as a determinant of severity of neurodegeneration in alzheimer's disease. ann neurol. 1999;46(6):860-6. 19. selkoe dj, hardy j. the amyloid hypothesis of alzheimer's disease at 25 years. embo mol med. 2016;8(6):595-608. 20. hardy j, selkoe dj. the amyloid hypothesis of alzheimer's disease: progress and problems on the road to therapeutics. science. 2002;297(5580):353-6. 21. braak h, del tredici k. the pathological process underlying alzheimer's disease in individuals under thirty. acta neuropathol. 2011;121(2):171-81. 22. braak h, thal dr, ghebremedhin e, del tredici k. stages of the pathologic process in alzheimer disease: age categories from 1 to 100 years. j neuropathol exp neurol. 2011;70(11):960-9. 23. streit wj, braak h, del tredici k, leyh j, lier j, khoshbouei h, et al. microglial activation occurs late during preclinical alzheimer's disease. glia. 2018;66(12):2550-62. 24. crary jf, trojanowski jq, schneider ja, abisambra jf, abner el, alafuzoff i, et al. primary age-related tauopathy (part): a common pathology associated with human aging. acta neuropathol. 2014;128(6):755-66. 25. kettenmann h, hanisch uk, noda m, verkhratsky a. physiology of microglia. physiol rev. 2011;91. 26. paasila pj, davies ds, kril jj, goldsbury c, sutherland gt. the relationship between the morphological subtypes of microglia and alzheimer's disease neuropathology. brain pathol. 2019. 27. bachstetter ad, van eldik lj, schmitt fa, neltner jh, ighodaro et, webster sj, et al. disease-related microglia heterogeneity in the hippocampus of alzheimer's disease, dementia with lewy bodies, and hippocampal sclerosis of aging. acta neuropathol commun. 2015;3:32. 28. streit wj, walter sa, pennell na. reactive microgliosis. prog neurobiol. 1999;57(6):563-81. 29. streit wj, sammons nw, kuhns aj, sparks dl. dystrophic microglia in the aging human brain. glia. 2004;45(2):208-12. 30. streit wj, braak h, xue qs, bechmann i. dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in alzheimer's disease. acta neuropathol. 2009;118(4):475-85. 31. tischer j, krueger m, mueller w, staszewski o, prinz m, streit wj, et al. inhomogeneous distribution of iba-1 characterizes microglial pathology in alzheimer's disease. glia. 2016;64(9):1562-72. 32. sanchez-mejias e, navarro v, jimenez s, sanchez-mico m, sanchez-varo r, nunez-diaz c, et al. soluble phospho-tau from alzheimer's disease hippocampus drives microglial degeneration. acta neuropathol. 2016;132(6):897-916. 33. walker dg, tang tm, mendsaikhan a, tooyama i, serrano ge, sue li, et al. patterns of expression of purinergic receptor p2ry12, a putative marker for non-activated microglia, in aged and alzheimer's disease brains. int j mol sci. 2020;21(2). 34. davies ds, ma j, jegathees t, goldsbury c. microglia show altered morphology and reduced arborization in human brain during aging and alzheimer's disease. brain pathol. 2017;27(6):795-808. 35. sutherland gt, sheedy d, stevens j, mccrossin t, smith cc, van roijen m, et al. the nsw brain tissue resource centre: banking for alcohol and major neuropsychiatric disorders research. alcohol. 2016:33-9. 36. rahman t, davies ds, tannenberg rk, fok s, shepherd c, dodd pr, et al. cofilin rods and aggregates concur with tau pathology and the development of alzheimer's disease. j alzheimers dis. 2014;42(4):1443-60. 37. kril jj, halliday gm, svoboda md, cartwright h. the cerebral cortex is damaged in chronic alcoholics. neuroscience. 1997;79(4):983-98. 38. montine tj, phelps ch, beach tg, bigio eh, cairns nj, dickson dw, et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol. 2012;123(1):1-11. 39. hansra gk, popov g, banaczek po, vogiatzis m, jegathees t, goldsbury ce, et al. the neuritic plaque in alzheimer's disease: perivascular degeneration of neuronal processes. neurobiology of aging. 2019. 40. uchida y, takahashi h. rapid detection of abeta deposits in app transgenic mice by hoechst 33342. neurosci lett. 2008;448(3):279-81. 41. murray me, graff-radford nr, ross oa, petersen rc, duara r, dickson dw. neuropathologically defined subtypes of alzheimer's disease with distinct clinical characteristics: a retrospective study. lancet neurol. 2011;10(9):785-96. 42. arendt t, bruckner mk, gertz hj, marcova l. cortical distribution of neurofibrillary tangles in alzheimer's disease matches the pattern of neurons that retain their capacity of plastic remodelling in the adult brain. neuroscience. 1998;83(4):991-1002. 43. geula c, mesulam mm, saroff dm, wu ck. relationship between plaques, tangles, and loss of cortical cholinergic fibers in alzheimer disease. j neuropathol exp neurol. 1998;57(1):63-75. 44. golaz j, bouras c, hof pr. motor cortex involvement in presenile dementia: report of a case. j geriatr psychiatry neurol. 1992;5(2):85-92. 45. petersen c, nolan al, de paula franca resende e, miller z, ehrenberg aj, gorno-tempini ml, et al. alzheimer's disease clinical variants show distinct regional patterns of neurofibrillary tangle accumulation. acta neuropathol. 2019;138(4):597-612. 46. suva d, favre i, kraftsik r, esteban m, lobrinus a, miklossy j. primary motor cortex involvement in alzheimer disease. j neuropathol exp neurol. 1999;58(11):1125-34. 47. arnold se, hyman bt, flory j, damasio ar, van hoesen gw. the topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with alzheimer's disease. cereb cortex. 1991;1(1):103-16. 48. genç b, jara jh, lagrimas akb, pytel p, roos rp, mesulam mm, et al. apical dendrite degeneration, a novel cellular pathology for betz cells in als. scientific reports. 2017;7:41765-. 49. shen z, bao x, wang r. clinical pet imaging of microglial activation: implications for microglial therapeutics in alzheimer's disease. front aging neurosci. 2018;10:314-. 50. boza-serrano a, yang y, paulus a, deierborg t. innate immune alterations are elicited in microglial cells before plaque deposition in the alzheimer’s disease mouse model 5xfad. scientific reports. 2018;8(1):1550. 51. hong s, beja-glasser vf, nfonoyim bm, frouin a, li s, ramakrishnan s, et al. complement and microglia mediate early synapse loss in alzheimer mouse models. science. 2016;352(6286):712-6. 52. cameron b, tse w, lamb r, li x, lamb bt, landreth ge. loss of interleukin receptor-associated kinase 4 signaling suppresses amyloid pathology and alters microglial phenotype in a mouse model of alzheimer's disease. the journal of neuroscience. 2012; 32(43):15112-23. 53. halle a, hornung v, petzold gc, stewart cr, monks bg, reinheckel t, et al. the nalp3 inflammasome is involved in the innate immune response to amyloid-beta. nat immunol. 2008;9(8):857-65. 54. salminen a, ojala j, kauppinen a, kaarniranta k, suuronen t. inflammation in alzheimer's disease: amyloid-beta oligomers trigger innate immunity defence via pattern recognition receptors. prog neurobiol. 2009;87(3):181-94. 55. heneka mt, kummer mp, stutz a, delekate a, schwartz s, vieira-saecker a, et al. nlrp3 is activated in alzheimer's disease and contributes to pathology in app/ps1 mice. nature. 2013; 493(7434):674-8. 56. heneka mt, golenbock dt, latz e. innate immunity in alzheimer's disease. nat immunol. 2015;16(3):229-36. 57. heneka mt, kummer mp, latz e. innate immune activation in neurodegenerative disease. nat rev immunol. 2014;14(7):463-77. 58. hanisch u-k, kettenmann h. microglia: active sensor and versatile effector cells in the normal and pathologic brain. nat neurosci. 2007;10(11):1387-94. 59. koenigsknecht-talboo j, landreth ge. microglial phagocytosis induced by fibrillar beta-amyloid and iggs are differentially regulated by proinflammatory cytokines. j neurosci. 2005;25(36):8240-9. 60. block ml, zecca l, hong js. microglia-mediated neurotoxicity: uncovering the molecular mechanisms. nat rev neurosci. 2007;8(1):57-69. 61. howard r, liu ky. questions emerge as biogen claims aducanumab turnaround. nature reviews neurology. 2020;16(2):63-4. 62. cummings j, lee g, ritter a, sabbagh m, zhong k. alzheimer's disease drug development pipeline: 2019. alzheimers dement (n y). 2019;5:272-93. 63. wang zx, tan l, liu j, yu jt. the essential role of soluble aβ oligomers in alzheimer's disease. mol neurobiol. 2016;53(3):1905-24. 64. minett t, classey j, matthews fe, fahrenhold m, taga m, brayne c, et al. microglial immunophenotype in dementia with alzheimer's pathology. j neuroinflammation. 2016;13(1):135. 65. streit wj, khoshbouei h, bechmann i. dystrophic microglia in late-onset alzheimer's disease. glia. 2020;68(4):845-54. 66. navarro v, sanchez-mejias e, jimenez s, muñoz-castro c, sanchez-varo r, davila jc, et al. microglia in alzheimer's disease: activated, dysfunctional or degenerative. front aging neurosci. 2018;10:140. 67. iqbal k, liu f, gong cx, alonso adel c, grundke-iqbal i. mechanisms of tau-induced neurodegeneration. acta neuropathol. 2009;118(1):53-69. 68. grinberg lt, rub u, ferretti re, nitrini r, farfel jm, polichiso l, et al. the dorsal raphe nucleus shows phospho-tau neurofibrillary changes before the transentorhinal region in alzheimer's disease. a precocious onset? neuropathol appl neurobiol. 2009;35(4):406-16. 69. felsky d, roostaei t, nho k, risacher sl, bradshaw em, petyuk v, et al. neuropathological correlates and genetic architecture of microglial activation in elderly human brain. nature communications. 2019;10(1):409. 70. ising c, venegas c, zhang s, scheiblich h, schmidt sv, vieira-saecker a, et al. nlrp3 inflammasome activation drives tau pathology. nature. 2019;575(7784):669-73. 71. vogels t, murgoci an, hromadka t. intersection of pathological tau and microglia at the synapse. acta neuropathologica communications. 2019;7(1):109. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. reflections on an unconventional neuropathology career feel free to add comments by clicking these icons on the sidebar free neuropathology 1:18 (2020) reflections reflections on an unconventional neuropathology career margaret miriam esiri nuffield department of clinical neurosciences, oxford university, john radcliffe hospital, oxford, ox3 9du, uk corresponding author: margaret miriam esiri · neuropathology department · level 1, west wing · john radcliffe hospital · oxford ox3 9du · united kingdom margaret.esiri@ndcn.ox.ac.uk submitted: 06 july 2020 accepted: 07 july 2020 copyedited by: deanna fang published: 13 july 2020 https://doi.org/10.17879/freeneuropathology-2020-2903 the author´s cv has been attached as electronic supplementary material: cv supplementary material keywords: neuropathology, oxford university, personal reflections i have no idea if this short account will be of any interest to others but in writing it i have a strong sense of good fortune for having had the career i have had as a neuropathologist. my interest in joining the medical profession emerged in my teens and i found myself in 1960, at the age of 18, commencing a degree in physiology at st hugh’s college, oxford university, as the first stage towards attaining this ambition. this 3-year course was to be followed by three years of clinical training and a supervised year of hospital practice before full registration as a medical doctor. women made up about 10% of the medical students at oxford at that time and, in due course, we were distributed in a wide range of medical specialties. the early stages my professional development was complicated by meeting and marrying a fellow medical student from nigeria and embarking on bringing up a family while still a clinical student. this influenced my choice of specialty because it was almost impossible to specialise part-time and front line clinical specialties demanded very long hours of training which would have been incompatible with bringing up a family. therefore, after completing my clinical studentship, i took up a scholarship from the uk medical research council to have training in research methods as the next stage of my education. the training was undertaken under the supervision of professor graham weddell in the department of anatomy. my brief was to spend a year learning the relatively new technique of electron microscopy and applying it to a study of diseased muscle in mice which had been infected with the leprosy bacillus, mycobacterium leprae, after being immunosuppressed by whole body irradiation. graham weddell was an elderly, avuncular character with a kind but rather vague air. he was an expert in the structure of peripheral nerves, a site that the leprosy bacillus was known to damage. by reducing the immune response to the bacillus by subjecting the experimentally infected mice to irradiation, it was thought that light might be cast on how nerves were damaged in the naturally occurring human disease. this was a completely new field of endeavour for me. i knew nothing about electron microscopy, nor leprosy, nor muscle. my first task was to learn how to prepare the tissues, sent to us from a collaborating laboratory in london, for light and electron microscopy. there were other people in the laboratory who were using the electron microscope for other purposes and they could share their expertise with a newcomer such as myself. it took some time to become familiar with the hugely magnified features of the cells being examined with the electron microscope – it was another world that was being displayed before one’s eyes. i went to the books and scientific papers to get help in deciphering the images i was seeing. i had never felt so much need to follow every letter of the message in papers and books about these ultrastructural features. my attention was much more riveted on what i was reading than it had been when i was reading papers for my tutorials as an undergraduate. now the details were needed to help me make sense of what i could see. some of the muscle samples that i studied had been prepared for examination using a light microscope. this was to enable me to get more of a ‘bird’s eye’ view of what i was studying under the electron microscope. i was frustrated to find that for quite a long time i could not find any leprosy bacilli in my electron microscope preparations despite the fact that i could clearly see clusters of them with the light microscope. this happened because the tissue samples used for electron microscopy were tiny compared with those viewed with the light microscope and it was easy to miss such minute structures as bacilli. i began to wonder if i would ever find any bacilli at the ultrastructural level, but then came a red letter day when a whole cluster of them, appearing large and black, were suddenly there in front of me in a new sample of tissue (fig. 1). i fell on them, photographing them with abandon to preserve a record of their structure and their localization in the muscle fibres. fig. 1. mycobacterium leprae in muscle   colony (c) of mycobacterium leprae in a foot-pad striated-muscle fibre. the bacillary membranes can be clearly distinguishedinthose sectioned transversely. the organisms are lying freely in a sarcoplasmic matrix. myofibrils (mf) and mitochondria (m) are also present. thymectomised-irradiated mouse given 107 myco. leprae i.v. 11 mth previously. ua, lc, em x36,000. published with permission from esiri mm et al. 1972; 106: 73-80. for the electron microscopy work i clearly needed to be present in the anatomy department but for the light microscopy i was given the loan of a microscope to take home so that some of my work could be carried on there at times that were the most convenient to me. it was typical of graham weddell that he went out of his way to enable my work to fit in with my home life. from time to time we would have a discussion about the work so that he could satisfy himself that it was progressing satisfactorily but most of the time he just left me to get on with it. in due course i wrote a short thesis and we prepared a paper describing our findings which was submitted to a scientific journal. i was aware as i carried out my practical work in the anatomy department that professor weddell had regular visits from someone from the local teaching hospital, the radcliffe infirmary, who wished to familiarise himself with the electron microscopy of muscle and nerve. he wished to be able to extend to the ultrastructural level the examination he made of muscle and nerve biopsies from patients that were taken for diagnostic purposes. his name was dr trevor hughes (fig. 2) and he worked at the neuropathology department of the radcliffe infirmary. trevor told me that he was hoping to obtain funds to support a junior research officer post in his department that would be devoted to muscle disease research. would i be interested in applying? i explained that i needed to obtain my full general medical council registration by occupying a year of preregistration house officer posts, but we agreed to keep in touch. fig. 2. dr j trevor hughes while i was carrying out this year of leprosy research, i had kept my hand in at dealing with patients by helping out once a week in an endocrinology clinic run by one of the consultants, dr derek hockaday, whose clinical firm i attended for my second medical attachment as a student. derek was aware of my difficulty in obtaining pre-registration house jobs while responsible for a young family. it was necessary to obtain the full gmc registration to enable me to progress to any subsequent clinical specialty. derek was sympathetic to my plight. he had a wife who worked part-time as a paediatric neurologist and they had 3 young children. he eventually offered me a supernumerary pre-registration house job that would not pay me but would give me the experience necessary to secure full registration. this dealt with the 6 months’ medical experience. to complete the additional 6 months’ surgical experience that i needed, i managed to gain approval to work as a house officer in obstetrics, a specialty that did not have routine pre-registration jobs. the advantage of taking this route was that it entailed a 1-in-3 on call rota at night, whereas in surgery this rota would have been 1-in-2. it was also critical that i had the offer of such an obstetric post from the head of the obstetric service, professor john stallworthy, whereas none of the surgeons was willing to offer me a post. thus, i had secured the possibility of getting my full medical registration, albeit with an unconventional set of posts. i also needed to secure the home situation and here we were very lucky to find a very good young nanny, jenny, who was willing to live in so that i could do my nights on call. i enjoyed this year of clinical work but i knew it would be impossible for me to carry on with this type of work in the future. at the end of the year, i therefore applied for, and obtained, the junior research officer post that trevor hughes had secured. the post was for 3 years and it would enable me to work for a dm research degree while allowing me at the same time to see what i thought about a career in neuropathology. i recognised that this was a very small specialty with only about 20 departments in the uk and those confined to major medical centres. i had always kept at the back of my mind the possibility that i might end up working in nigeria. if i trained in neuropathology i would need to re-train if i were going to nigeria. but since our young family had appeared, nigeria had been experiencing a bloody civil war, ruling it out for settling in for the time being. the prospect of three further years living and working in oxford helped us to decide that it would be best if the children were to be brought up with their roots in the uk. we did not think it would be a good idea to move the children to and fro between nigeria and the uk even if the civil war had ended. so, in 1970, i started as a junior research officer in neuropathology. i needed to decide on a topic to research on muscle disease. perusal of the textbooks about human muscle disease seemed to suggest that although there was a great variety of rare diseases, the two categories of disease that were more common were inflammatory myopathies and muscular dystrophies. i chose to study inflammatory myopathies because i had become interested in the role of the immune system in disease during my experience of studying leprosy. there were also not then the tools readily available to carry out genetic studies of muscular dystrophy that are now very effectively in play. inflammatory myopathies can occur at any age but are most common in late middle age. to make a definitive diagnosis, a small sample of muscle – a biopsy – needs to be removed and studied under the microscope to see if inflammation is present. the cause of the inflammation is not certain but it was thought likely that it was due to an immune reaction to some component of muscle, triggered by division and activation of subsets of lymphocytes. i decided to investigate blood lymphocytes taken from people with recently diagnosed inflammatory myopathies and compare their reactions in culture to a small concentration of homogenised muscle with the reactions of healthy people (myself and my colleagues). to do this i needed to find an immunology laboratory that could accommodate me and my lymphocyte cultures. there was such a laboratory in the nuffield department of medicine. it was headed by dr ian mclellan. although the laboratory was already full of researchers, he very generously allowed me to join them. i carried out my human lymphocyte studies there and also studied rats in which i attempted to produce a similar condition of inflammatory myopathy by injecting them with muscle homogenate combined with an adjuvant. while the lymphocytes were being cultured, i also examined the muscle biopsies from the same patients with light and electron microscopy. although there would be no foreign organisms expected to be present in the muscle if this inflammatory myopathy is an autoimmune disease, it still seemed worthwhile to check for any invading organisms, such as viruses, which might be detectable with an electron microscope. to start with, i used the electron microscope in the anatomy department that i was already familiar with but the radcliffe infirmary soon acquired one of its own in the pathology department where it was particularly useful in diagnosing the nature of kidney disease from studying renal biopsies. i was able to use this electron microscope soon after it was installed and it turned out to be very useful for diagnosing some of the rare muscle diseases. just as i had initiated these studies and satisfied myself they were running well, i was overtaken by an unexpected event – a massive fire in march 1971 that destroyed the whole of the neuropathology department and the adjacent hospital library at the radcliffe infirmary. luckily no one was injured. the fire occurred at night and i came in the next morning to find the department, including all my records, razed to the ground. the hospital authorities had to scurry round searching for somewhere to put us. the professor of surgery generously offered an office and there was some recently vacated animal house accommodation, rather dark and damp, into which we could move. having scavenged a few chairs and a desk or two from sympathetic colleagues, we sat ourselves in the animal house to draw breath when who should appear but a former neuropathologist who had moved a few years earlier to london, dr sabina strich. she had heard the news of the fire on the radio and promptly come to visit and commiserate and to provide us with a new electric kettle, mugs, coffee and chocolate wholemeal biscuits. it was such a welcome gesture! it raised all our spirits and made us determined to make the best of the situation. fortunately, my lymphocyte studies could continue in the immunology laboratory which was in a different part of the hospital, even though the records of the first experiments were lost. there were also negatives from the electron microscope studies that could be retrieved and reprinted. some of the brain specimens from earlier years were still retained in a hut in the grounds of another hospital and those that were most instructive for teaching purposes were retrieved and sections re-cut for microscopy. some second hand equipment such as microscopes and microtomes, needed to cut thin sections, were rapidly acquired and the elements of a re-formed department thus assembled. eventually the whole destroyed floor of the hospital was re-built, though this was not to happen for several years. the three years of the junior research officer post passed quickly with my energies divided between the laboratories and the home. we now had a new nanny, jane, who stayed with us until all the children were at full time school. the combination of research and pathology was much more compatible with family commitments. my husband was pursuing a surgical training and holding a succession of short term posts all over the country as was the usual practice in those days. he returned home whenever he was able to. the lymphocyte studies yielded modest evidence of lymphocyte sensitivity to muscle in inflammatory myopathies. it was not a dramatic effect and could have been the result, rather than the cause, of the condition. it was difficult to provoke any more than a slight inflammatory reaction in the muscle of the sensitised rats. in the electron microscopic studies, there was only one biopsy that contained what might have been virus-like particles. thus, the fruits of my studies were far from conclusive. i believe it remains the case today, more than 40 years after these studies were performed, that inflammatory myopathies are really little better understood. however, the thesis i wrote describing these studies earned me a dm degree and i published a couple of papers summarising the findings. i had found satisfaction from this work but now felt increasingly curious about the central nervous system and its diseases that i had started to become familiar with. however, before i was certain that neuropathology should become my chosen specialty, i needed to become familiar with other branches of pathology as part of my training. what did these two short periods of research teach me? first they reinforced my admiration for science. i particularly admired the way science was prepared to embrace the contributions of people wherever they came from and whatever their background provided they worked with integrity and had innovative ideas. i admired the way science relied on reproducibility of findings to give them credence and not on the advertising ability of their perpetrators to give validity to their findings. i came to appreciate the importance of karl popper’s dictum that scientists should seek to refute their ideas, not confirm them. scientific ‘facts’ are always provisional and can be revised if subsequent experiments are incompatible with them. i learnt to recognise the importance of quantitation in recording results of experiments and the need, in studying disease, to include control samples as well as samples from the disease under investigation. it was important for the investigator, whenever possible, to be unaware of which samples were from controls and which from disease so that there was no chance of bias creeping into the observations being made. it seemed to me that if careful quantitative studies were performed they could almost always yield useful information even if they showed that the ideas that prompted the experiments turned out to be incorrect. in short, science satisfied my urge to ask questions and seek answers to them and having commenced my efforts in that direction i was going to continue if i could. the next stage to become a neuropathologist in the 1970s i needed first to gain experience of other specialties of clinical pathology. i became a trainee registrar with a post that rotated through haematology, bacteriology, virology and histopathology. the longest time (2 years) was spent in histopathology (also known as cellular pathology). there were exams to take for membership of the royal college of pathologists once the practical experience had been obtained. it was essential to gain this qualification before applying for a post as consultant in the nhs. the exam had a heavy component of histopathology, hence the longer time spent in that specialty. i didn’t find the haematology or bacteriology very interesting. the numbers of samples to be analysed in each of these departments were so overwhelming that much of the work had been automated. many samples would typically be sent to each of these departments during the course of each patient’s hospital admission and, in addition, many samples were sent in each day from local general practitioners. the departments were primarily concerned with delivering a diagnostic service and there was relatively little that i, as a newcomer, could contribute. they did not undertake active research. histopathology was of greater interest, being more directly relevant to neuropathology. i learnt how to perform post mortem examinations (in the process giving myself the only nightmares i’ve ever had, in which the corpse came to life while i was busy and i attempted to replace the larynx so that it could speak!) and to report on surgical specimens of tissue. because of the large variety of specimens and of the conditions they exhibited, it took a fair time to be able to make correct judgements on the nature of the disease, often cancer. samples were examined under the microscope, first by myself and then by a consultant who was able to share his or her experience and pass on to me the critical visual clues that yielded the essential evidence about the nature of the disease process. the head of the histopathology department was dr robb-smith, a slightly shy and enigmatic character whom i had encountered as a student at post mortem demonstrations. he was interested in attracting women to his specialty and had secured a trainee post that could be held part-time. this enabled me to have two free afternoons each week. i found the children were all occupied at these times so i used this time to write up my dm thesis, a task i had been putting off while i dealt with more immediate concerns at home and at work. one of the great advantages for me of working in the histopathology department was that i not only gained essential diagnostic experience but i was also able to participate in research. there was a research officer there, dr ian burns, who was experimenting with the use of antibodies to detect specific proteins in tissue sections. if antibodies could be produced towards a purified protein of interest and tagged with a marker substance such as a coloured dye or a fluorescent dye, this could be used to detect specific proteins in tissue sections with much greater specificity than could be provided with the traditional tinctorial stains. this was the principle behind the work that ian burns was carrying out and which has now become the hugely successful branch of pathology, immunohistology or immunocytochemistry. when ian was working in the early 1970s, he was concentrating on the use of antibodies to detect immunoglobulins, themselves antibody proteins produced by plasma cells. i was able to make use of ian’s reagents to study plasma cells containing different types of immunoglobulins in sections of central nervous system tissue affected by different diseases. this was research i undertook when i had moved back from the histopathology department to be a registrar in the neuropathology department. my experience in more general pathology had not seduced me into getting more interested in that field. rather, i thought there were important things i had learnt there, particularly this new subject of immunohistology, that i could usefully apply in neuropathology. back in the neuropathology department, i was also gaining experience of diagnostic aspects of the specialty by examining biopsies removed from brain tumours by the neurosurgeons as well as other biopsies such as those from muscle and peripheral nerves. i was also performing post mortem examinations on cases of neurological disease and brain trauma. this diagnostic work i found still left time for a limited amount of research in which i applied the antibody techniques i had been lucky enough to learn about from ian burns to nervous system diseases. this was an example of the wonderful way that collaboration can be developed in science and i was to become a great advocate of such collaboration, prizing it far above competition which, in some fields or departments of medical science sadly seemed to predominate. i was lucky enough to obtain the modest funds needed to enable me to employ a technician to assist with the application of immunohistology in neuropathological material. i started by studying the lesions or areas damaged in the central nervous system in multiple sclerosis, most generally considered as an autoimmune disease1. this was a condition that was of interest to the other consultant in the neuropathology department, dr david oppenheimer (fig. 3), who had written a dm thesis on it. he had seen many examples of this condition in post mortem studies and the tissues from some of these cases were still available in the department despite the fire that had destroyed much material in 1971. one of the beauties of the immunohistological technique was that it could be used on tissue that had been preserved in the department after being fixed in formalin and then embedded in paraffin wax to enable thin sections to be cut. although some proteins that may be of interest in tissues are destroyed by such treatment, meaning that fresh frozen sections are needed to study them, there are many others that survive this treatment. the advantage of being able to use formalin-fixed, paraffin-embedded sections is that this tissue shows better preservation of its structure than can usually be seen in frozen sections. fig. 3. dr david oppenheimer i followed up the work on multiple sclerosis with studies of diseases that did have a foreign organism in the damaged tissues, starting with poliomyelitis of which we had cases dating from an epidemic of polio in the uk in the late 1950s. later i extended the work to a study of cases of the rare complication of measles, subacute sclerosing panencephalitis. i wanted to know if these diseases showed a similar distribution of plasma cells containing immunoglobulins as i saw in multiple sclerosis. although this turned out to be the case, the difficulty in interpreting the significance of the finding was that the arrangement of plasma cells might be just the same in autoimmune disease as in one with a foreign organism. while these studies were proceeding, more antibodies for use in immunohistology were being produced, including some directed to viruses. i was to make use of these for a later study when i was a research fellow. the neuropathology department when i joined it was a small one. there were 2 consultants, drs hughes and oppenheimer, a registrar (myself), 3 technicians and a laboratory assistant. trevor hughes had a particular interest in muscle disease and spinal cord disease. he had moved into neuropathology from histopathology. david oppenheimer had a less conventional background. he had started his academic career with degrees in philosophy, politics and economics and also music. during the second world war, he had been a conscientious objector and worked with an ambulance team in london rescuing injured people in the blitz. this experience awakened his interest in medicine and he read for his medical degree immediately after the war. initially he worked on neuroanatomy and it was from there that he moved to neuropathology. he was initiated into diagnostic work by his predecessor, sabina strich, over a matter of a few months. he recognised that his lack of experience in histopathology and other pathology specialties was a handicap but his knowledge of neuroanatomy was most valuable. it made him particularly interested in the many important diseases of the nervous system in which there is a neuroanatomical basis for the way the disease is expressed or, in some cases, seems to spread. i had been introduced to the neuropathology department by trevor hughes but as time went on i became more interested in the conditions that interested david oppenheimer, starting with multiple sclerosis. one of the first opportunities that arose from the diagnostic work that i carried out was a chance to study a rare case of herpes zoster (shingles) in an elderly woman who happened to die just after the eruption developed. it wasn’t the herpes zoster that killed her but an advanced state of multiple myeloma which had rendered her susceptible to developing herpes zoster. my patient had herpes zoster affecting the region supplied by part of the trigeminal nerve. herpes zoster is caused by the same virus that causes chickenpox, varicella-zoster virus. the theory had been developed that in the natural history of infection with this virus, chickenpox is the first manifestation of infection. as the virus is overcome by the immune response that enables the person with chickenpox to recover, some virus survives and lies latent in the nerve cells of one or more sensory nerves. circulating antibody which persists after recovery from chickenpox makes any reactivation of the virus uncommon until later in life when the level of antibody wanes or when the person has their immunity compromised by disease. when the virus reactivates in the sensory nerve cells of a particular nerve, it travels along the nerve to the skin where it causes the eruption of herpes zoster. although this theory was widely accepted, the reactivation of the virus in nerve cells had never been demonstrated. it was this reactivation that i was able to show by electron microscopy which revealed the virus particles in the nerve cells of the trigeminal ganglion (fig. 4). a colleague in the virology department, dr albert tomlinson, carried out immunofluorescence using an antibody to the varicella-zoster virus on frozen sections of the ganglion and the nerve and was able to show the virus at both sites. fig. 4. herpes zoster viral particles in the cytoplasm (c) of a trigeminal ganglion neuron.   from esiri mm, tomlinson ah j neurol sci 1982; 15(1); 35—48. the varicella-zoster virus that dr tomlinson was able to show in frozen sections of the nerve and ganglion of our case could not be seen when we tried to detect the virus in formalin fixed tissue – the viral protein was an example of an antigen that could not survive formalin fixation. this observation made me think that there could be more we could learn from our post mortem and surgical specimens if we thought carefully about whether we should preserve a portion of tissue by snap freezing it in liquid nitrogen and then storing it in a -70◦c freezer. snap freezing in liquid nitrogen preserved the tissue structure better than slowly freezing tissue in a freezer but only if the tissue sample was small. the opening of our minds to the potential importance of deep freezing some tissue was to prove important in years to come, not only to enable immunohistology to be performed for detection of antigens that were sensitive to formalin but also to enable biochemical and genetic studies to be carried out. it led to oxford being early in the field of brain banking for research (for a history of brain banking in the uk see 2). the highly trained technicians that we worked with in neuropathology played an absolutely essential role in all that we did. unlike in histopathology, where most tissue sample sections were subjected to a single stain, in neuropathology it was common to have consecutive thin sections from the same block of tissue stained with several different stains, some of which were quite difficult to perform. each technician had their special expertise in carrying out a selection of these stains. some were histochemical stains that enabled enzymes to be displayed in frozen sections. these histochemical stains were mainly used on muscle biopsies. each of the tinctorial stains picked out a particular feature of the nervous system to highlight. nowadays the need for these special tinctorial stains is much reduced because immunohistology can convey the same information using antibodies that are more specific and more straightforward to apply. some of the reagents used in the tinctorial reactions are quite toxic and it is far preferable that technicians can now avoid them. my closest colleagues were those in the neuropathology department. trevor and david took it in turns, several months at a time, to supervise my diagnostic work and i had the close assistance of the technicians in the preparation of the sections i needed to do this work. i liaised closely with the neurosurgeons and neurologists over the results. at times there was another trainee doing a project in the department or a visitor making use of the material we had available for them to study. i remember particularly a very pleasant, recently retired, canadian professor of anatomy, richard saunders, who came to spend a few winters in oxford with his wife to escape the worst of the winter in nova scotia. they had a son who was an oxford rhodes scholar. richard had an intimate knowledge of the structure of the hippocampus which he did his best to impart to me. it turned out to be useful knowledge when i became interested in alzheimer’s disease. i made regular visits to local district general hospitals where i provided a diagnostic service on neurological post mortem cases. i discovered that histopathologists in these hospitals had to deal with quite a lot of neurological cases as the hospitals had consultant neurologists working in them. this visiting service yielded many interesting cases from which i brought back to our department selected samples for expert processing. i would then send reports on these cases back to the histopathologists. once or twice a year there were meetings of the uk neuropathology community. trainee and consultant neuropathologists could apply to join the british neuropathological society, which organised these meetings, after they had delivered one sponsored paper or poster at one of the meetings. some overseas neuropathologists also joined the society. my first paper to the society, sponsored by trevor, described the work i had done on experimental leprosy infection in the anatomy department. it was interesting to get to know neuropathologists working in other centres and to compare notes on our respective research projects. i found them to be a very congenial group. collaboration with some of them became an important asset in devising some research projects for which more cases of a particular disease might be needed than were available in one centre. it was also helpful at times to be able to send a puzzling diagnostic specimen to someone in another centre to obtain a second opinion on the diagnosis. towards the end of my time as a registrar i made the acquaintance of a recently appointed consultant in geriatric medicine, gordon wilcock. gordon had come to enquire if there might be someone interested in collaborating with him to study the neuropathology of dementia, particularly alzheimer’s disease. he was himself interested in learning some neuropathology and he had many patients under his care in the geriatric medicine wards on whom, if they agreed, he had carried out a simple cognitive test to determine whether or not they had dementia. a considerable number of them had dementia but there were others who did not, including quite a number who had had strokes. if any of these patients subsequently died, and their next-of-kin was willing for a post mortem to be performed, we were able to see what abnormalities there were in the brain that contributed to making them demented. it was known that in alzheimer’s disease there were two distinct microscopic abnormalities: the presence of plaques and tangles. gordon and i wanted to see if we could relate either plaques or tangles (or both) to the presence of dementia in his cognitively tested patients. we had the impression that the distribution of tangles in alzheimer’s disease affected parts of the brain that were becoming known to be anatomically linked together. we took to discussing the neuroanatomy of alzheimer’s disease on friday afternoons with experts on these cortico-cortical and cortico-hippocampal connections, drs tom powell and his former dphil student, carl pearson. tom had spent his career on meticulous studies of the cerebral cortex and was greatly pleased to find that some of his work might have relevance to an understanding of such an important disease as alzheimer’s disease. from these discussions came a study of the detailed distribution of plaques and tangles in the different layers of the cerebral cortex3. the findings fitted well with what was known of the manner in which one part of the cortex was connected to others and to the hippocampus. those regions most closely linked to the hippocampus had many tangles while those parts of the cortex which were most remote from the hippocampus had the fewest tangles. this work was my introduction to trying to understand alzheimer’s disease from a neuropathological perspective. another influence in fuelling my new interest in dementia was a publication by a neurochemist at london’s institute of neurology, dr david bowen. in the mid-1970s, he published a paper showing that in post mortem brain specimens that had been frozen at -70◦c immediately after removal from the body there were many enzymes whose activity could be reliably measured. he furthermore showed that in alzheimer’s disease the enzyme, choline acetyl transferase, required for the synthesis of acetyl choline, was deficient. a similar finding was reported from two other laboratories at about the same time. this paper was a real eye-opener for me. i had previously assumed that after death all enzyme activity would cease. gordon and i had discussions with david about providing him with frozen brain samples from the patients gordon had tested for cognition on the wards and we were soon adopting the policy of deep freezing one side of the brain for david’s studies and carrying out our microscopic studies on the other side. this led to a realisation that it is the burden of neurofibrillary tangles that is held within the brain that determines the extent of dementia in alzheimer’s disease, much more so than the amyloid load4. i have watched with dismay the dominance of amyloid in thinking about alzheimer’s disease and how to prevent it that has lasted for decades. hopefully, now that may change5. not long after we had set up this programme to study dementia, gordon was offered a professorship in care of the elderly at bristol university and left oxford but there was an interested consultant in geriatric psychiatry at the large littlemore mental hospital near oxford, dr john robinson, who was able to play a similar role in supplying cases of dementia, though he was not in a position, as gordon had been, to supply cases that did not have dementia. this collaboration with clinicians in our research on dementia was the first time i had had the opportunity to work directly with doctors looking after patients to devise a research strategy. neuropathologists are too remote from direct patient care to be in a position to recruit patients to a study themselves. some neuropathologists developed interests in brain tumours that they could follow using the biopsy specimens they received. others developed experimental models of diseases that could be studied alongside human post mortem material. but i think the most valuable contribution that clinical neuropathologists can make is to work closely with their clinical colleagues to recruit patients into clinic-pathological studies of human disease. by the mid-1970s, i had acquired enough experience to take the royal college of pathologists examination in histopathology slanted to neuropathology. the written examinations were taken in london and for the practical exam i was instructed to go to glasgow. i remember casting my eye hastily over my notes on the long train journey to glasgow and thinking that to be taking examinations at the age of 35 years was too old! fortunately, i passed this last one and was then in a position to think about the next step in my career. for the sake of our children who were happily settled in schools in oxford, i wanted to remain there but there was only one academic post in neuropathology and that would be occupied by david oppenheimer for another few years. one of my colleagues in the histopathology department made a most helpful move at this point. he wrote to the medical research council on my behalf and asked if there might be a prospect of getting support from this source to fund research for a few years in oxford. an encouraging reply to this enquiry led to me applying for a senior clinical fellowship from the medical research council. becoming a senior clinical fellow i put together an application for this senior clinical fellowship to study a disease that had long been of interest to me – herpes simplex encephalitis. this is a rare condition but one that had a considerable mortality at that time and which i had the chance to study post mortem. there were several enigmatic aspects to this disease which intrigued me. it seemed to affect people of a wide range of ages in whom it developed quite unexpectedly. yet it was caused by a virus that infected some 90% of people in some of whom it caused recurrent cold sores. like its cousin, varicella-zoster virus, which i had already encountered, it had the capacity to lie latent in sensory ganglia (for this virus, usually the trigeminal ganglion, because it usually causes a facial infection) from which it could reactivate under a range of conditions such as fever, exposure to strong sunlight or the common cold. what was the process by which it could occasionally cause a devastating encephalitis? i knew i would be able to study the distribution of the virus in the brain using immunohistology and an anti-herpes simplex antibody, and i had tissue from enough cases, which had survived for varying periods of time after disease onset, to enable me to try to piece together the pathological process. armed with this intention i was lucky enough to get the fellowship for 5 years at consultant level salary plus some part-time technical help and costs of consumables. this period of full-time research reinforced my passion for investigating brain diseases of which there seemed so many without an effective treatment, in part a consequence of inadequate understanding of the disease process – hardly surprising given the complexity of the nervous system and the difficulty of accessing brain tissue during the course of many of these diseases. at the time, brain imaging was still rudimentary compared with how it is today and the post mortem examination, for those diseases that were fatal, gave the best lead to increase understanding. herpes simplex encephalitis has a very intriguing distribution of damage in the brain with an emphasis on the temporal lobes but with the damage usually asymmetrically distributed6. the focus of damage overlaps with that in alzheimer’s disease with the hippocampus and sites connected to it badly affected. from my visits to the anatomy department to discuss alzheimer’s disease with tom powell and carl pearson, i knew these areas had close connections with the olfactory system so i studied the olfactory bulbs in these cases of herpes simplex encephalitis because it seemed possible that the virus, with its propensity to travel along axons, might reach the brain along this route. i could indeed detect the virus there but the olfactory bulbs were not as damaged as sites in the amygdala and piriform cortex to which the olfactory tracts projected. thus it seemed possible that the virus reached the olfactory bulbs by moving centrifugally, not centripetally, perhaps from a site of latency within the brain, perhaps the hippocampus. the asymmetry of damage to the temporal lobes, which was more marked in those dying early in the course of the disease, might be explicable if it reactivated from inside one hippocampus and then travelled to the contralateral one via anatomical connections between the two. to take this work further i teamed up again with albert tomlinson, the virologist who had helped me examine the case of herpes zoster. we developed a mouse model of herpes simplex encephalitis and showed that inoculation of the face led to the virus ascending to the brain via both the trigeminal and olfactory routes and the development of both brain stem and temporal lobe inflammation. others have pursued the possibility that latent infection of the brain with herpes simplex virus may predispose to the development of alzheimer’s disease but the opportunity to further investigate the human acute encephalitis was lost on account of the fortunate development of an effective treatment which reduced the fatality of the disease and resulted in many people affected recovering at least partially. a change of direction? while i was intent on this research, my children had grown into their teens and my husband was exploring the possibility of returning to nigeria to provide medical care there. this posed a dilemma for me. should i abandon my neuropathological career and re-train in tropical medicine or could i carry on with my neuropathology in oxford part-time while taking periods of unpaid leave to join my husband in nigeria during school terms when our two younger children were at boarding school? the other alternative to pursue neuropathology in nigeria, in the very remote region in which my husband decided to set up a medical practice would have been impossible. i was able to negotiate to carry on part-time in oxford for the last part of my research project, working full-time while in oxford and visiting nigeria between times. strange though such an arrangement might seem, it actually worked well and, by the time the research was completed, david oppenheimer had retired, giving me the expectation that i might apply for his (now vacant) post. that expectation was thrown into confusion by the government of the day deciding to impose drastic cuts to university financial support. as a result, all re-filling of posts was frozen and many posts were lost. i remained in a form of professional limbo with short-term medical research council support lasting 3 months at a time until a new professor, john newsom-davis, was appointed in the clinical neurology department on the retirement of the previous postholder. john offered to attempt to reinstate the lectureship in neuropathology which had previously been held in the cellular pathology department, but only if it could be held in the clinical neurology department. the post was rescued in this way and i was appointed to it on a part-time basis that allowed me to continue with my visits to nigeria. my husband’s choice of where to establish his clinic was partly determined by his wish to live in a part of the country that is decidedly cooler than the rest. he hated the tropical heat of most of the country and he knew i would too. his clinic was established in a town, gembu, on the mambilla plateau, some 1,500 metres high and not far from the cameroon border which lies to the east of nigeria. the countryside is made up of grass-covered hills, interspersed with deep, fertile, valleys and the climate is really perfect, with temperate, warm, sunny weather and abundant rainfall. the roads were rudimentary in the extreme and getting to the place took some 12-15 hours of uncomfortable land rover motoring after a domestic air flight to the nearest place with an airport, yola. over the years, a tarred road of sorts was created that mounted the steep escarpment to the plateau but it was soon full of potholes that were hardly ever repaired. spending 2 months at a time in gembu in the spring and autumn of each year became my routine for about 20 years from the early 1980s. because i couldn’t continue with neuropathological research while i was away, i decided to turn my attention to writing about the diseases i was interested in. my aim was to inform those coming after me about what i had learnt so far. my first book was written with john booss, a neurologist from yale, who came to oxford on a sabbatical to study multiple sclerosis. together we wrote a book on viral encephalitis7. to write my parts, i took piles of references with me to read and digest in gembu, mainly in the afternoons and evenings after i had helped in the mornings at my husband’s clinic. my offerings there were decidedly amateurish but one felt that in such a place, where medical care barely existed, one was justified in doing one’s best. once i had gained a little confidence from seeing people recover or, at worst, return the next day for a re-think about the diagnosis, i thoroughly enjoyed this experience. with no diversions such as newspapers or television to occupy the time later in the day i was able to think and write without interruption, although, in the evenings, any activity had to be carried out by the dim illumination offered by a ‘tilly’ light, fuelled by kerosene. to follow up after publication of viral encephalitis, i approached david oppenheimer, who had recently retired, to see if he would be willing to write a book on diagnostic neuropathology with me. i felt greatly honoured to have had much tuition from david during my training. he was a truly inspiring person and i felt it would be good if other people could benefit from his teaching even if it had to be in the form of a textbook. i knew he wrote superbly so could convey his knowledge in that way. he thought about it and agreed to write a ‘pamphlet’ in which we would divide up the topics evenly between us. he had kept the photographic slides that he had collected over his career so was able to make use of these and entered into the writing of the ‘pamphlet’ with enthusiasm. i continued to learn from him as we wrote and it was a complete pleasure, at the end of a working day when i was in oxford, to visit him and his wife on my way home and to share the new text he had written during the day. i think he also enjoyed being able to extend his teaching of neuropathology in this way. the result was a textbook of nearly 400 pages that was published as diagnostic neuropathology8. the third book that i initiated was one on the neuropathology of dementia9, which was co-edited with my colleague james morris, who had stepped into the post that trevor hughes had vacated when he retired. that all these three books went in to 2nd editions pleased me as it suggested they had found useful homes among those in the neuroscience community. by this time, a major longitudinal study of ageing and dementia, the oxford project to investigate memory and dementia (optima,) had been started by david smith, professor of pharmacology in oxford, and we were benefitting from the opportunity to study the brains of dementia sufferers and healthy elderly controls who had been followed up in a detailed way during life and had consented to donating their brain for research after their death. this study grew slowly, as such longitudinal studies do, but we eventually collected over 500 brains that helped to increase understanding of dementia, and still continue to do so. forging ahead once i had been appointed to an academic position in neuropathology in the mid-1980s, i started to supervise graduate students who wished to study the brain and its diseases. i always insisted that students should have two co-supervisors because of the periods i spent in nigeria and out of contact with the department. two months was the maximum that i considered possible to remove myself from all the activities that go on in a department that has diagnostic, teaching and research interests. sometimes i would return to find a pile of problems to solve but usually everything had continued smoothly and i had the pleasure of hearing about the new findings that had emerged while i was away. because of these periods of leave i kept my other absences from the department to a minimum which meant attending very few conferences and meetings – a sacrifice that i was willing to make in return for the privilege of being able to continue with my professional work in my rather unusual personal circumstances. the department was expanding at this time (the late 1980s) with the arrival in oxford of professor tim crow, a psychiatrist with a deep interest in biological aspects of psychiatric disease, particularly schizophrenia. we were able to expand into a new small laboratory and recruit an experienced technical manager and a small group of research students to work on psychiatric disease. we were also joined by a research group created by a local academic psychiatrist, dr paul harrison. i was never in any doubt that study of the brain was needed as much to understand these diseases as it was for dementia and ageing and for neurological diseases. in the neuropathology department, i had wonderful collaborations with psychiatrists as well as neurologists and neurosurgeons. it seemed very odd to me that there was little direct communication between clinical neurologists and psychiatrists and little shared training for the two disciplines but this may be changing now. there were also superb opportunities in oxford to collaborate and learn from neuroscientists in the basic science departments from which we benefitted enormously. a great boost to our research on schizophrenia came from the generous provision of a substantial group of brains donated for research in belfast, northern ireland. professor ingrid allen, the senior neuropathologist in belfast, and her colleague in psychiatry, dr steven cooper, were instrumental in creating this initiative with tim crow. tim had a fascinating hypothesis about schizophrenia: that it was somehow related to subtle differences in cerebral asymmetry, that differs normally between men and women, but which he thought might be disturbed in those with schizophrenia. this led to our studies of the brain in schizophrenia being carried out having regard to sex and cerebral hemisphere side. we also studied the corpus callosum and other tracts that connect the two hemispheres. we found tantalising findings on this basis that seemed to indicate, among other things, that schizophrenic males have cerebral hemispheres more akin to normal females than occurs normally in males and vice versa for female schizophrenics. we should not have found these differences if we had studied schizophrenia without distinguishing between the sex of subjects whose brains we studied. although it has become fashionable in psychology to downplay differences in brain structure between males and females i think these differences are important and may have significant roles in psychiatric disease that we are not yet able to understand. while these attempts to understand psychiatric disease were launched, we were also continuing to investigate dementia and ageing with the optima resource. a graduate student, zsuzsa nagy, who remained for a while in the department as a postgraduate research scientist, discovered that neurons expressed antigens indicating they had re-entered the cell division cycle in elderly subjects. this discovery, which has been repeatedly confirmed by others, led to studies of lymphocytes and the suggestion that properties of these cells in culture might have diagnostic value for alzheimer’s disease. although this work has been followed up, we are still without a simple method of diagnosing this disease, particularly in its earliest phase, when symptoms are absent or minimal, but at the best time for any intervention to be made to slow or stop its development. my interest in multiple sclerosis was re-ignited at about this time by two developments. the first was provoked by a paper that came out in the lancet linking episodes of clinical expression at the start and in relapse in multiple sclerosis to episodes of previous sinusitis. the author, frederick gay, was a general practitioner and he made his observations using general practitioner records. he thought it possible that bacterial antigens derived from microorganisms in the paranasal sinuses might reach the cns compartment in some individuals and lead to an immune response that created the demyelinating, inflammatory lesions typical of this condition. we worked together on this idea but, given its unfashionable nature at a time when most interest lay in viruses having a role in the disease (if, indeed, any foreign antigens were involved), funding to make progress was hard to acquire. the idea, however still has great attractions, in my opinion, and it is still being actively pursued. the second development that brought back my active interest in multiple sclerosis was applying immunohistology yet again but this time using an antibody to amyloid precursor protein as a marker of damaged axons. although axons have long been known to be damaged in ms, the extent of that damage, its timing and its role in progression of the disease were little appreciated largely because there had been no sensitive method of assessing its extent. being able to detect damaged axons with great sensitivity, which detection of amyloid precursor protein provided, changed this situation and when we applied the technique to multiple sclerosis lesions of differing age i was greatly surprised by the result. i had expected that we should see damaged axons in chronic lesions which are common in progressive disease but it was actually in the most recent lesions that axon damage was most prolific10 (fig. 5). this was despite the fact that recent lesions are more common at earlier phases of the disease when recovery from relapses is common. initially it seemed hard to understand how damage to axons, which we assumed was irreversible, was compatible with a history of relapse followed often by remission. this pattern of illness seemed more compatible with pathology that was reversible, likely demyelination and the inflammation that accompanied it. but we soon realised that because there is much redundancy in the cns, progression of damage would only occur when a threshold of axon loss and other forms of neurodegeneration was reached, leaving room for recovery between episodes of inflammation before that point was arrived at. fig. 5. graphs to illustrate the profile of axonal injury (app stippled areas) and the number of macrophages (black areas) in a typical acute multiple sclerosis lesion (a), active chronic multiple sclerosis lesion (b) and chronic multiple sclerosis lesion (c).   from ref. 10 this reassessment of multiple sclerosis and, in particular, its neurodegenerative aspects, made me acutely aware of how progress in understanding disease depends on new techniques being applied to enable new knowledge to be acquired. the spectacular developments in neuroimaging over the past few decades have enabled much progress to be made in understanding cns diseases and this will continue. nevertheless, there is still a place for pathological studies because these are still needed to penetrate to the cellular and molecular levels of change that accompany disease. without knowledge at these levels rational treatment or prevention cannot be achieved. the new frontier relating to knowledge that has grown up in the last two decades is about genetics. here there is such a wealth of new information that new ways of managing data are needed but i remain convinced that it is the protein and other structural aspects of cells that will provide the key answers to what happens to the state of the body in disease and, therefore, what can be done to remedy it. fig. 6. margaret m esiri (2000) the future along with new technical developments enabling fresh knowledge to be acquired about disease, it is vital that there are new ideas. these arise from human imagination and ingenuity. ideas can come to a researcher from all sorts of experiences and at any stage of their career but, in the long run, a steady supply of new research students with novel ideas is essential. that is why i have been so fortunate in having a career in an outstanding university in which tutors take extraordinary efforts to select the best students some of whom can then become the generators, along with newcomers from elsewhere, of the next wave of insights. they start off without the baggage of long indoctrination with the previous and current dogmas and bring creativity to a research team. students that i have supervised have repaid my confidence in them in spades and i feel their hands are fully capable of taking forward the fields that came to fascinate me. the person i have kept in closest touch with is gabriele deluca, now a consultant neurologist at oxford with a lively research group, who brings energy, creativity and flare to his research and teaching, making him an inspiration for yet another generation of young medical students and scientists. one thing writing a memoir brings home is the manner in which attitudes and knowledge reach you and which you, in turn, pass on to others. there is a social angle to all this, with an amplifying effect of teaching and example, that, at best, is inspiring and uplifting. at the age of 78, i still find it impossible to abandon my interests in neuropathology and i am incredibly lucky to have a department that still welcomes me into its fold. fig. 7. left: margaret m esiri, right: a composite painting depicting the three buildings in which i worked as a neuropathologist: radcliffe infirmary (left), st hugh’s college (centre), john radcliffe hospital, west wing (right). references 1 esiri mm. multiple sclerosis: a quantitative and qualitative study of immunoglobulin-containing cells in the central nervous system. neuropathol appl neurobiol 1980; 6: 9-21. 2 wellcome trust witness seminar on the development of brain banks in the uk 1970-2010. 3 pearson rc et al. anatomical correlates of the distribution of the pathological changes in the neocortex in alzheimer’s disease. proc natl acad sci 1985; 82: 4531-4. 4 wilcock g, esiri mm. plaques, tangles and dementia: a quantitative study. j neurol sci 1982; 56: 343-56. 5 esiri mm. target tau, not amyloid, to prevent and treat alzheimer’s disease. obm geriatrics 2020; 4, issue 1 doi 10.21926/obm.geriatr2001103 6 esiri mm. herpes simplex encephalitis. an immunohistological study of the distribution of viral antigen within the brain. j neurol sci 1982; 54: 209-26. 7 booss j, esiri mm viral encephalitis: pathology, diagnosis and management. blackwell scientific publications, 1986. 8 esiri mm, oppenheimer dr. diagnostic neuropathology: a practical manual. blackwell scientific publications, 1989. 9 esiri mm, morris jh (eds) the neuropathology of dementia. cambridge university press, 1997. 10 ferguson b et al. axonal damage in acute multiple sclerosis lesions. brain 1997; 120: 393-9. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology, my friend – on the paths of being and non-being feel free to add comments by clicking these icons on the sidebar free neuropathology 1:21 (2020) reflections neuropathology, my friend – on the paths of being and non-being ferenc garzuly markusovszky university teaching hospital, university of pecs, 5 markusovszky street, szombathely, hungary corresponding author: dr. ferenc garzuly · markusovszky university teaching hospital · university of pecs · markusovszky street 5 · szombathely · hungary garzuly@t-online.hu submitted: 02 august 2020 accepted: 03 august 2020 published: 11 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2934 the author´s cv has been attached as electronic supplementary material: cv supplementary material keywords: neuropathology, szombathely, personal reflections contents introduction flight dictatorship and university the mirror of sufferings black water, swirling time neuropathology laboratory – without a boss as chief physician of the department of neurology deus ex machina under the wings of pécs and budapest a young man slips through the iron curtain – the neurologisches institut steps towards rare diseases efforts to diagnose an undiagnosable disease establishment of the hungarian society of clinical neurogenetics, szombathely, 1997 autopsy at night changing times – changing diseases among patients and colleagues – establishment of the west pannon neurological forum, szombathely, 1998 spotlight on rare diseases the music of the 21st century “the dead teach us” (hic mortui vivos docent)   prof. dr. györgy romhányi (1905-1991)   markusovszky hospital szombathely, department of laboratory and pathology (1946-51) and institute of pathology, medical university of pécs (1951-1976) introduction let me start off my recollections by saying that it had never occurred to me that i would ever become a doctor. i was interested in the arts and humanities, history, literature. when i was in high school, back in the early fifties, i participated in a literature competition and wrote a piece called conversation with the moon. it was about our escape from bratislava (preßburg, pozsony). we fled at night by boat on one of the river branches of the danube in august 1945, in the last year of world war ii. i was eight years old. our possessions in bundles on a cart, our destination was a small hungarian town called mosonmagyaróvár. fig. 1. bratislava, 1945 source: tasr archive in my mentioned text conversation with the moon i embedded all my youthful, naive thoughts and emotions from that time. this was in hungary in the early fifties under the rákosi dictatorship. after finishing that story i realized it couldn’t go on like this for long. there was a feeling of looming threat that made my stomach hurt. and now i just had to decide what profession to choose for further learning. both my mother and grandfather were physicians and it became obvious that i, too, would choose this apolitical, strictly fact-based profession, although it felt quite distant for me. flight my paternal grandfather, gyula garzuly, was a ship's captain, he also sailed around the world. later in bratislava (preßburg, pozsony), when it was still a hungarian city, he became the supervisor of the vessels of the hungarian river and shipping company. in 1916 he received a major award, the silver degree of the civil military cross of merit – ships on the danube also took part in the events of the first world war. with this, he earned to be imprisoned in ilava in 1918. he died shortly after his release, i didn’t even know him. my other grandfather, dr. rezső limbacher, the son of a blacksmith in an earldom, was such a skillful blacksmith „assistant” that the count taught him. graduated from university, he became a recognized obstetrician-gynecologist in bratislava (preßburg, pozsony). the first postmortem sectio cesarea in the city was performed by him, an article about this case appeared in the hungarian medical weekly. his son, rezső peéry, as a journalist and writer, was one of those who held the soul with his articles in the hungarians of bratislava after the treaty of trianon. fig. 2. my grandfather’s publication in the hungarian medical weekly, 1910 after the world war ii we had to flee from bratislava. every hungarian escaped who could. the future prime minister, edvard beneš , will announce it shortly: our people cannot live in a common country with the germans and the hungarians. dictatorship and university i was admitted to the medical university of pécs. at that time there were several internationally known professors at the university. one of these was jános szentágothai, the professor of anatomy, whose lectures resembled a grand performance. learning between his posters and drawings of anatomical figures was most interesting and exciting. another professor engraved himself in the hearts of the students with his directness, warm demeanor and his special approach to facts and events. that was györgy romhányi, the professor of pathology. the sentence pathology is the science of human suffering (1) would remain in the memories of his students for a lifetime. during my studies i was most impressed by neurology and psychiatry – at the time these two specializations were not separated yet. professor istván környey was the head of the university clinic in pécs, a well-known and prestigious scientist abroad. neurologist, psychiatrist, neurosurgeon and neuropathologist in one person, a wiry, strict and consistent man. he kept a kind of prussian discipline in the clinic and would not even be broken by the rákosi system. fig. 3. professor dr. istván környey (1901-1988) i decided to become a neurologist and psychiatrist myself. the field of psychiatry was a bit of an exit or detachment from the classical medical professions, although i could only get to know freud’s books under my blanket at that time. neurology, meanwhile, involved the precise mapping and careful evaluation of the patient complaints. particularly the physical examinations were much more complex, such as utilizing tools from internal medicine, palpation, percussion, auscultation and blood pressure measurements. i was ready to take that challenge. after graduation i was eligible to apply for three positions. it was evident that i could not stay at the university in pécs, considering that i did not actively participate in the kisz, the communist youth association. others were much smarter, they knew what needed to be done to stay in pécs and to become future professors. they weren't really friends of mine. i applied for positions of neurology and psychiatry in rural regions. since i did not have any patronage from the comrade secretariat of the university, i was not given the opportunity to do so. i had to choose from pathological positions, because there were usually fewer applicants for these and the vacancies had to be filled with people like me. it was intended as a punishment. this was in 1962 in the times after the failed revolution, not yet in the period of the soft goulash communism, but still in the hard dictatorship of the early sixties – there was no place for complaints. i felt that pathology was the medical field which was farthest from my interests and abilities. i remember my first autopsy practice at the university – when we entered the autopsy room and i saw a female corpse opened up with the wide layer of fat and intestines, i had to hold on to the wall so that i wouldn’t faint. still, i had no choice but to take on the appointed task. the mirror of sufferings i ended up at the hospital of szombathely, now called markusovszky teaching hospital of the university of pécs. the current building complex was built in 1929, and at that time it was the largest hospital in the western part of hungary. after the treaty of trianon, which had tragic consequences for hungary, the university of pozsony (today bratislava) was forced to move 1919 to hungary. szombathely was the only other city after pécs which was considered suitable for the new place of the university (1923). fig. 4. the hospital of county vas and szombathely, 1929 i started working in the pathology department in 1962. slowly i became accustomed to the silent dead and i started to warm up to the colorful histological preparates. i discovered the world that is our internal architecture and the macro-and microscopic war our bodies wage against our ferocious enemies – which, of course, have the same right to live just as we do. yes, it is true what professor romhányi said: pathology is the science of human sufferings and what i see during autopsy is the mirror of sufferings. today i stand at the same autopsy table where romhányi had also worked four years after the war. yes, pathology needs to be approached this way, i understand now. and i began to see the value of analysing medical treatments through the autopsies and also its worth for helping patients in the future. fig. 5. professor dr. györgy romhányi (1905-1991) despite these insights, i still wanted to be a neurologist and psychiatrist in the future. i even told this to the chief physician of pathology and he acknowledged my wishes in good faith. i still would have to wait two more years for this opportunity. looking back i am very grateful for this twist of fate and also to the party-member-packed secretariat of the university which had exiled me to szombathely, where i got to know pathology's world, methods and a point of view. black water, swirling time right at the start of my work at the department of pathology i discovered an old tied-up bundle of autopsy records on the shelves. the oldest ones could be dated back to 1920 and were still handwritten. from 1920 to 1929 autopsies were performed by the chief physician of the department of internal medicine and his doctors. the current complex of hospital buildings was built in 1929 and pathologists have been only working here ever since. fig. 6. a page of an autopsy report, 1923 the reports were interesting and instructive but also informative regarding the history of the pathology department itself and its doctors. many years later, in 2006 i looked back to examine this period. i reviewed the protocols from 1920 to 1956, written before and after world war ii till the hungarian revolution in 1956. they added up to a total of 6000 records. i wrote about it in a book with the title black water, swirling time –the message of autopsy reports (1920-56). fig. 7. black water, swirling time – the message of autopsy reports (1920-1956) during this period a lot of radical changes occurred. interestingly, sifting through the autopsy reports revealed that in the pre-war years every third death protocol was reported on tuberculosis. after the war the rate of infant mortality outstripped these deaths. the reports also included cases of diseases which have since been eradicated such as syphilis, tetanus, scarlet fever, whooping cough, diphtheria or poliomyelitis. they recount the typhus epidemic in the department of psychiatry and the epidemic of hepatitis in a child asylum. heart attacks or lymphoma barely occurred. curiously, patients under the age of 60 died three times as often in the hospital than those over the age of 60. in the meantime, political conditions changed drastically. to illustrate this in my book i have attached excerpts from the local newspaper vasvármegye. in 1940 for example, the chief physician of pathology, dénes görög, could have read news predicting a glorious future: the desertion of england has begun. the english population is in distress about a possible german landing… life in london is paralysed. the prices for a seat on one of the big ocean liners are horrendous... england will either surrender or be destroyed. after the war in 1948 chief physician romhányi might have been reading the local newspaper and be persuaded that the communist party had taken over the role of god in hungary: thank you for my recovery. i am a country woman with five acres. due to my lung problems i was lying in the pulmonology of the public hospital in szombathely. i was operated twice. i owe my recovery to the party and the doctors of the hospital. the events of the time shaped the human destinies severely. dénes görög was taken to auschwitz with his family in 1944. under mengele's supervision he performed autopsies with another hungarian doctor, miklós nyiszli. shortly before his death he said: miklós, with your willpower i am confident that you will make it home. i feel that i won´t survive. my wife and daughter died here in the gas chamber, i have proof that it happened. i hid a little boy with the monks of the monastery in kőszeg. this is my little son sándor, 12 years old. take him with you when you get home. i know for sure that i will die. this is my last wish. this wish was fulfilled and the little boy survived, became a chemist, a pharmaceutical researcher and a member of the hungarian academy of sciences. after his years in szombathely in 1951 györgy romhányi was appointed the head of the pathological institute of the university pécs. his experiences at the hospital in szombathely might have led him to formulate the sentence: the dead teach us. he may have also thought of the fate of dénes görög. neuropathology laboratory – without a boss during the two years i spent in pathology i performed the autopsy of those who died in the care of the department of psychiatry and neurology. i also studied the histological specimens and learned about their diseases. in 1963 this department was divided into two departments, one of them was headed by a new physician dr. lászló baltavári, from the környey-clinic in pécs. his wife worked in the laboratory of that clinic. the new chief physician managed to set up a neuropathology laboratory in the department within a year. at that time there was a vacant medical position in the department, which meant that i was now able to have a transition. since i already had a pathological past, so in addition to my daily medical work on patients, i also received the neuropathology laboratory as a gift. without a boss. i had to stand on my own two feet. i was already able to do the sections of the brains here and through it we were able to evaluate our medical work. i am still doing this work, although the laboratory (with me) has been relocated to the pathology department nearly twenty years ago. the number of brain cuts performed here is close to 6000. fig. 8. department of psychiatry and neurology, szombathely and its physicians, 1965 as chief physician of the department of neurology the departments were transformed into neurology (with 70 beds) and psychiatry (with 250 beds) in the 1980s. i stayed with the former. after twenty years dr. lászló baltavári died and for the next twenty years i became the head of the department. still, i couldn’t leave laboratory work for a minute, i would not give it up. the neuropathology laboratory strongly supported the medical care for patients. at that time there was no ct or mri. only the neuropathology gave us insights into the changes caused by the diseases which was teaching us many lessons even after the death of the patients. it also gave us histological diagnoses which radiological examinations still cannot provide today. because of my medical work both in our and in other departments of the hospital (for example pediatrics, infectiology, intensive care) i often encountered rare diseases. many of them were fatal and difficult to diagnose. i have increasingly used the capabilities of our laboratory to solve these mysterious cases. this was made possible by a number of contacts which i had established through the years, mainly with the institute of neurology (neurologisches institut) in vienna. deus ex machina i have to look back to 1966 when something extraordinary happened. by this time i was already working in the 2nd department of neurology and psychiatry. i received an invitation from professor franz seitelberger, head of the famous institute of neurology in vienna, for a three-month scholarship-funded study trip. beyond the iron curtain, to the land of the capitalist. i was absolutely surprised and only got to know later what happened. the first woman to graduate from the technical university of vienna was my father's sister, margarethe garzuly. she was admitted in 1919 after world war i and graduated in 1923. she moved from bratislava to vienna, became a staff member of the university, habilitated and in the meantime her husband became the head of the department of organic chemistry. when my aunt found out that i was working in neuropathology she called professor seitelberger. it was unbelievable. and it was even more incredible that i received a passport for the trip and could leave the country. at a time when hardly anyone could travel to the west. to this day i still wonder how it could have happened. maybe the party knew (as they knew everything) that i would always come back to my very good friend, a young, nice and very talented ceramic artist, maria geszler. fig. 9. maria geszler, a young and talented ceramic artist and a prince with big nose under the wings of pécs and budapest dr. lászló baltavári advised me not to go to professor seitelberger completely untrained. i should study in pécs for a month under professor környey before i would leave for vienna. shortly afterwards i found myself at the clinic in pécs. in the mornings professor környey was always sitting in a gigantic room behind a very wide baroque desk and about 20 doctors would be standing in front of him. if the floor creaked under one of them he looked up to see, who was that undisciplined one who couldn't stand still just for half an hour? there were three female assistants working in the laboratory and a young doctor, who vanished shortly after. every day i received histological preparates for study from professor környey accompanied by offprints, e. g. about tick-borne encephalitis. in the meantime (or later?) i became acquainted with ferenc gallyas, who was a young chemist. he began to investigate silver impregnations for glial cells at that time. later, this made him world-famous. on such specimens discovered mátyás papp the so called „papp-lantos bodies”. we became friends although i was very surprised that his lunch always consisted of three eggs. from a culinary point of view the everyday life at the clinic was interesting as well, for example lunch time you could choose between soup or bread. rom professor környey i received spielmeyer’s book on neuropathology which included many coloured hand drawn illustrations and i used it to prepare for the journey ahead. in later years környey became my supporter and our relationship deepened especially after he retired and moved to budapest. there he lived in one of the apartments in the park of the national institute of psychiatry and neurology (built in 1868 for mentally ill patients) courtesy of the director professor istván tariska who was also an excellent neuropathologist. the neuropathology laboratory at the national institute of psychiatry and neurology in use here was the leading laboratory in the country. one great advantage of the laboratory was that the well-trained neuropathologist katalin majtényi worked here full-time. i have been there many times with majtényi, tariska and környey to consult histological specimens. környey wrote his book on neuropathology here. to illustrate the chapter about the hallervorden-spatz disease he asked me for some photos. after i handed these to him, he told me that once in berlin hallervorden and spatz faced him on the sidewalk. hallervorden raised his hat and said, guten tag while spatz greeted him with a heil-hitler. after that i always thought about hallervorden as a decent person but later i was very disappointed when i found out about the circumstances of developing the hallervorden collection. in nazi germany, a large number of mentally disabled were killed. the majority of pediatric brains were transferred to the kaiser wilhelm institut für hirnforschung, led by hugo spatz, and was included in the hallervorden collection. i also often met lóránt leel-össy here who headed the neurology department in esztergom but he also had a laboratory in miskolc. he became the first president of the hungarian society of neuropathology. then the years passed, many things changed in the castle-like building, until in 2007 the huge building closed its gates. it is still empty and only owls are hooting in it, missing angry psychiatric patients and clever neuropathologists. fig. 10. the ruins of the national institute of psychiatry and neurology, 2017 source: hu.wikipedia.org a young man slips through the iron curtain – the neurologisches institut at hegyeshalom, on the hungarian-austrian border, i transferred to a train departing for vienna. it carried two wagons, surrounded by four border guards armed with machine guns. i successfully arrived in vienna. i have already mentioned my father’s elder sister, who had two more siblings – both sisters – who fled from bratislava to vienna after the war. they supported me during my stay here. the neurological institute – on the schwarzspanier street – was at the first floor of a huge building. i learned later that sigmund freud, whose writings on decoding of dreams were my favorite readings in those times, had lived on a sloping part of this street not so very long ago. i have found professor seitelberger in his room. he was very friendly with me, he talked a bit to me, and then i got a table in the large lecture hall in front of the benches. i saw him again and again most of the time as he presented the institute to his foreign acquaintances. they also looked into the lecture hall. i greeted everyone politely. there was a constant typewriter-knocking coming from the room next to the lecture hall. kurt jellinger worked here diligently. from the intensity of the knocks, one would think that the number of his publications will at least approach a thousand, if not more. i was very grateful to him, because he took me under his wings immediately: he provided me with tasks and instructions, and i received a case from him for analysis – syringomyelia combined with spinal haemangioblastoma. it was interesting, i was looking for similar disease combinations in the literature, in old and new books and journals in the large library, which always rang from emptiness like a cathedral. unfortunately, i always had to be afraid that someone would lock the iron door of the library from outside and i would have to say goodbye to my young life here, and so i can never see the nice and talented ceramic artist again. i gave a presentation later on the edifications from the analyses at the end of the study tour. since then, i feel bad to have left this area for the sake of rare diseases. but the latter was required by the daily practice. slideshows were not known at that time, so i had to draw my illustrations: the drawings and the related notes could be projected on a stretched canvas with a huge episcope. after the presentation, professor seitelberger invited me to a nearby restaurant for a farewell lunch and i also got a ticket from him to pablo casals’ performance at the musikverein. i last saw professor seitelberger many, many years later, in 1992, in szombathely. i invited him to give a lecture in our hospital library. he chose an interesting title for his performance, i was amazed: the neurobiology of aging – brain aging and cognition. i think he felt that this was a dangerous area for everyone. steps towards rare diseases my trip to vienna was the first initial step, on which further contacts, personal ties and consultations with the institute and its doctors were built. i visited the institute on several occasions for a longer or shorter period of time later, although it was initially difficult due to the iron curtain. but the evaluation, consultation of cases was – however – solvable by sending paraffin blocks too. fig. 11. congress of neuropathology in kyoto, 1990. on the left foto: franz seitelberger, his wife and hans lassmann left, right one of my friends, herbert budka. on the right foto: riki okeda (tokyo medical and dental university), my other friend and i i visited professor kurt jellinger and later his successor, professor herbert budka, and his doctors, first of all ellen gelpi, romana höftberger and later gábor g. kovács with special and rare cases: these meetings also resulted in publications (2-17). neuropathological diagnosis of diseases was a difficult task, especially for the deceased ones at the pediatric department. there has been almost no information or descriptions about the neuropathological abnormalities caused by these diseases, so it was worth dealing with them. we were able to diagnose some of these clinically – just like the family with the lowe-syndrome (2) – but e. g. the disease underlying symmetrical cerebral heterotopias and strange knee calcifications – the zellweger-syndrome (3) – was recognized by kurt jellinger. adult cases were also usually limited to rare diseases such as the foix-alajouanine disease (4), or the jakob-creutzfeldt disease (5). efforts to diagnose an undiagnosable disease of the many rare and interesting cases i have encountered, i would like to highlight only two. in the first, the story began when one of our female hospital assistants became disoriented for half an hour, later not remembering it. we found strange calcification along the sylvian-fissures on the ct scans. the total protein content of the csf was extremely high. the cause of the global transient amnesia was not found. later, it occurred to me that i had seen a similar ct scan from a female patient with suspected multiple sclerosis three years earlier. we have found this recording and it turned out, that this patient also had a very high csf total protein. and we discovered – quite by accident – that these two patients were related to each other, cousins. we mapped the whole family. many years ago, two men from this family passed away at our department: before that, they visited the department many times because of their central nervous system symptoms (migraine like headache, transient global amnesia, nystagmus, spastic paresis, ataxia, hearing loss, incipient dementia). we found the paraffin blocks of the deceased (but only blocks made from the brain). examination of the histological specimens revealed that amyloid was deposited in the central nervous system, primarily subpial. the area next to the deposited amyloid was calcified. it became apparent that these calcifications could be seen on the ct scans. none of the patients had any peripheral nervous system symptoms. however, neither the symptoms, nor the high protein content of the csf was consistent with any of the known central nervous system amyloidoses. and then, one of the two mentioned female patients passed away. after talking to the family, the deceased was brought to the hospital by ambulance and an autopsy was made. it turned out that although the patient had no symptoms of involvement according to the other organs – apart from central nervous system abnormalities – amyloid was found in small amounts in the kidneys, heart, and liver. so the autopsy referred to systemic amyloidosis. but our cases could not be classified here either. our cases were very similar to the very rare, so-called oculoleptomeningeal amyloidosis cases, but the patients had no visual impairment. consultations again and again (neurological institute, university of vienna, max-planck-institut für biochemie, martinsried, departments of pathology and neurology, new york university medical center), immunohistochemical and molecular biological studies revealed a completely unexpected result: the disease was caused by a transthyretin mutation. we summarized findings in our article (authored by vidal r, garzuly f, budka h, lalowski m, linke rp, brittig f, frangione b, wisniewski t) in the american journal of pathology in 1996 as follows: we describe a novel transthyretin mutation at codon 18 where asp is replaced by gly (d18g) in a hungarian kindred. this mutation is associated with meningocerebrovascular amyloidosis. fifty different transthyretin mutations are related to amyloid deposition, typically producing a peripheral neuropathy or cardiac dysfunction… with this report we establish that transthyretin amyloid deposition can also produce central nervous system dysfunction as the major clinical symptom (7). fig. 12. amyloid showing birefringence and the cover of am j pathol with an image from our case in the journal neurology, the patient's clinical symptoms were summarized, naming the disease hungarian type amyloidosis (8). in this form of familiar amyloidosis, the pathological mutant is produced in large amount in the plexus choroideus and – upon entry into the csf – immediately precipitates on the membranes, ependymes, unlike to forms that cause peripheral neuropathy, where mutants remain in the blood (9). now we know that the so-called oculoleptomeningeal amyloidoses are also caused by transthyretin mutations. unfortunately, these "cerebral" forms of amyloidoses are not yet curable, although our neurology department has just admitted the next sick member of our family. establishment of the hungarian society of clinical neurogenetics, szombathely, 1997 five years of efforts made clear to me, that underneath the layer of neuropathology stretches another layer, clinging to it: the neurogenetics of diseases. without any knowledge of this area, we cannot treat the hereditary diseases. without any molecular-biological tests, we can do essentially nothing for diagnosing or researching them, and the key to curing these diseases is also buried here. the recognition that neurogenetics will have a significant development and that the work in hungary should be coordinated, was also evident to other neurologists and neuropathologists. as a result of the joint efforts, we held the first scientific meeting on neurogenetics in hungary: with a significant number of foreign participants, it took place in szombathely on may 9–10, 1997. among the foreign professors of the gathering were george karpati, kurt jellinger, herbert budka, parviz mehraein, hans (hano) bernheimer, jean bénard, manuel b. graeber. the event had a lot of hungarian participants. professors ferenc mechler and mária judit molnár from the clinic of neurology in debrecen also participated at the meeting. they suggested the establishment of the hungarian society of clinical neurogenetics. fig. 13. a group of participants of the first scientific meeting on neurogenetics in hungary. standing row from right: gábor kiss, jean bénard and wife, parviz mehraein and wife, maria geszler, sámuel komoly, in front of them manuel b. graeber and i, george karpati and wife and miklós wenczl are sitting. to this day, the society diligently holds annual scientific meetings and looks to a prosperous future. at the tenth anniversary gathering of the society, professor ferenc mechler and i had the honor to become life members of the association. however, it took more than twenty years to wait for an excellent neurologist and researcher, professor bernadette kálmán – who has spent several decades in the united states – to establish a molecular pathology laboratory at the markusovszky hospital. autopsy at night the other interesting disease was a variant of the autosomal dominantly inherited fabry disease. a patient – who suddenly became unconscious – was brought to our department in the evening. angiography was performed, the images showed an extremely wide basilar artery – a so called megadolichobasilar artery – which was thrombosed. the next morning, during the visit, i lifted the patient's quilt. and then i saw on the skin of the abdomen the characteristic angiokeratomas of fabry disease. i recognized it immediately, because three years ago i saw a similar patient. it was later revealed that the patient seen three years ago was the same. the patient was expected to die within a few days. i called maria mázló, the head of the electron microscope laboratory, of the national institute of psychiatry and neurology in budapest. she said, the investigation would be carried out, but asked us to send fresh samples to the laboratory. i discussed the immediate autopsy with the head physician of our pathological department. the patient died at one o'clock at night. after the obligatory two-hour wait we were standing in the autopsy room, and the autopsy was done at three o'clock. this was the first autopsy in hungary by a patient who died of fabry disease, no previous reports were found. and we believe it was also the first autopsy at night in our country. fig. 14. electron microscopic image of the stored material of our patient with fabry’s disease three years later, a similar case occurred in a neighboring town's hospital: a thrombotized giant basilar artery caused death. we received the formalin-fixed material samples. the lesions were completely similar to our case. and within a short time it turned out that the two patients were related to each other. these cases were also discussed with the colleagues in vienna, meanwhile the molecular pathological examinations were completed at the university of debrecen. the cases were published in the brain with the following title: megadolichobasilar anomaly with thrombosis in a family with fabry's disease and a novel mutation in the alpha-galactosidase a gene (11). shortly after the publication, it became possible to treat fabry-patients, and some of the family members may already be happy with this opportunity. changing times – changing diseases n 2014, our neuropathology laboratory turned 50 years old. on the occasion of the anniversary we published an article in the medical journal orvosi hetilap (18). nearly 5500 autopsy examinations were carried out during the preceding 50 years. the documentation reflects on variations in the occurrence of diseases, and it draws attention to those disorders, which can be prevented or treated today, but may represent diagnostic challenges. measles-related subacute sclerosing panencephalitis caused death in 13 cases, the last occurred in 1991. the mandatory vaccination against the causative morbilli virus has eliminated this severe neurological complication. fourteen lives were lost due to herpes simplex encephalitis, including the last case that occurred in 1999. feasibility of early diagnosis and the availability of acyclovir therapy resulted in better outcome without fatality. tuberculous meningitis still occurred in most recent years, although only sporadically. recognition of this condition is not straightforward due to its rarity, considerations for this disease are often omitted from the routine differential diagnosis. the low mortality rates in tick-borne encephalitis dropped further after the introduction of vaccination, 8 cases were documented altogether. with our colleagues in vienna, we published two reports on tick-borne encephalitis (12, 13). the last fatal cases of neurolues were seen in the 1990s. however, syphilis itself has not disappeared, and the number of cases with newly acquired infection continues to rise. meningosis leukaemica caused the death of 20 patients, mainly children, between 1972 and 1987. it was a sad time for me to witness this as a neurologist. i wrote also my phd dissertation on neuropathological features of leukemias and lymphomas. introduction of intrathecal methotrexate and radiation therapy made the prevention and effective treatment of meningosis leukaemica possible (19, 20). however, a careful coordination of these treatment's modalities is important as nervous system complications may develop in the form of disseminated necrotizing (methotrexate) leukoencephalopathy. we had four such cases, we needed professor herbert budka’s help to diagnose the first one. among patients and colleagues – establishment of the west pannon neurological forum, szombathely, 1998 fifty years is a long time, many things have happened apart from the events in the neuropathology laboratory. i would like to highlight two of these. in the 1980s, the rehabilitation of neurological patients had an increasing emphasis. this was the beginning of the golden era of neurorehabilitation – not only of neuropathology as herbert budka referred to in his recently published reflections (21). in szentgotthárd – a town next to us – a large rehabilitation hospital was established on the site of a former pulmonology sanatorium. together with the hospital's director we visited similar institutes in austria. we contacted professor gerhard barolin, who was the main organizer of the neurological patients' rehabilitation in austria. my friend divided his rehabilitation hospital into three parts and reserved one for neurorehabilitation. in 1988, we organized a two-day scientific meeting on neurorehabilitation entitled current issues in neurorehabilitation, with guest participation of austria, switzerland and germany. in a similar form, the gathering was repeated in 1993. these were the first events in hungary dealing exclusively with neurorehabilitation. maria geszler's ceramic exhibition in the picture galery and the scientific meeting on neurorehabilitation opened at the same day, one after the another in 1993. fig. 15. ceramic statue of maria geszler: falling figure. maria geszler's ceramic exhibition and the scientific meeting on neurorehabilitation opened at the same day. szombathely is located on the western border of hungary, near vienna, but far from the universities of budapest and pécs. it became clear, that our county and the three neighboring counties need to work together. nearly one and a half million people live in this area. this way, we established the west pannon neurological forum with its first meeting in 1998 in szombathely (22), for neurologists, neurosurgeons, and neurorehabilitation physicians. neuropathology was represented consequently by our department. this one-day meeting is held every six months in one of the cities of the four counties. the gatherings are mainly based on case studies with an educational lecture. the event created dialogues among the doctors of these four counties and helps them to solve medical problems. the 50th anniversary meeting will take place next year. fig. 16. one of the west pannon neurological forum's program booklets with crests of the host cities spotlight on rare diseases i retired in 2002 and started to work full-time at the department of pathology. i took the neurohistology laboratory with me: the lab had a better, more spacious place here. sections were always performed in the presence of clinicians and then the evidence was always compared with the results of radiological examinations in the presence of radiologists. we also collaborated with the laboratory of molecular pathology, and several published works resulted from this cooperation (23-27). fig. 17. thrombosis of the basilar artery in a young woman. comparison of the pathological with the radiological findings. the department of pathology organized the clinico-pathological conferences since the early 1950s. these were implemented on a soviet model. professor pavel ivanovich sapochkov said at that time: i work at clinic no. i in moscow and our clinic is extremely restless (sic) when it comes to the patient’s autopsy. pathology examines the relationship between clinical and pathological diagnosis the most accurate way. every two weeks the doctors are called together and the diagnostic differences in each case are talked over, and it’s a shame (sic) for a doctor to be invited to such a hearing, where his fault is discussed. after moving on to the pathology, i was entrusted with organizing these conferences, every three months, with mandatory attendance for hospital physicians. it became clear that the handling of these cases – where an error has occurred – falls within the competence of other forums. we expanded the discussions of cases by talking not only about the deceased ones, but also the healed patients. the conferences no longer featured the doctors who worked poorly, but those who worked well. and we gradually set the focus of the conference on cases that were interesting in some way, predominantly discussing rare diseases. fig. 18. our books about rare diseases the pathology department offers a very good angle to observe to the medical activities of our very large hospital and it is easy to discover interesting cases. over the past nearly twenty years, we have written five books about rare diseases relying on these cases (the sixth one is also in progress) with lots of coloured illustrations. professor romhányi said: one case is not a case. but we relied on 260 cases to present the rare diseases – with the required information and notes – we have experienced during our work. and in addition, at the hospital's lecture hall was named after professor romhányi. fig. 19. the lecture hall of the hospital, named after györgy romhányi, the venue for conferences on rare diseases the music of the 21st century the nice and talented ceramic artist, maria geszler is the one for whom i came back from the bewitching western world. she is my wife now for many-many years. her great-grandmother sang so beautifully as a young girl, that after a concert she was kissed by franz liszt on the forehead. the grandmother, margit tessényi, a student of vianna da motta, was a well-known pianist, her father, györgy geszler a composer and pianist, who gave concerts also with béla bartók. it is no coincidence that her sisters are also musicians. our daughter anna endowed the talent as a gift. she studied in budapest, munich and new york, and became a flutist. she frequently participated in music competitions and was usually among the winners, so she could even give an evening at carnegie hall in new york. her swedish husband, henrik wahlgren is an excellent oboist. they were both members of the gewandhaus orchester in leipzig, but our daughter is currently a professor of chamber music at the musikhochschule in leipzig and a flute professor in weimar. she was a founding member of the quintessenz leipzig flute ensemble, which has now celebrated its 20th anniversary. all three of our grandchildren also play and sing, so music has become a constant companion of our lives. fig. 20. the quintessenz leipzig flute ensemble, in the middle anna garzuly wahlgren we shared a lot of things: good ones and sad ones too. but we are still working. i work in my hospital, as maria does in her studio, or having a lecture somewhere, exhibiting in europe, or elsewhere in the world. she is a ceramic artist well-known everywhere. she won a lot of prizes: last year she was awarded with the grand prix in vallauris, the internationally famous ceramics biennial. fig. 21. maria geszler: arctic meteorite, porcelain object one of her artworks can be seen on the june issue's cover of new ceramic – the most famous magazine for ceramic artists – and a long article about her in the journal. there, she writes following: our nerves are strained like wires, we live among electrical discharges of love and hatred, above our heads there is special buzz of cables, that is the music of the 21st century. we all dream to see something new, to live, to discover, to understand. this experience has pushed mankind since the stone age, even in the millennium of moon travel. who didn’t feel something like this? wandering on the banks of rivers, standing on the sandy shores of the seas, at the foot of the mountains, looking at the peaks? who didn’t feel the attraction of the unknown? to go as far as possible, to see the horizon, to discover the mountain peaks, to reach the skies. references 1. prof. dr. med josef makovitzky: prof. dr. györgy (georg) romhányi (1905-1991) als persönlichkeit, wissenschaftler und lehrer – ein beitrag zur geschichte der polarisationsmikroskopie. isbn:978-3-00-036132-6 druck: druckerei der albert-ludwigs-universität freiburg 2012 2. garzuly f, jellinger k, szabó l, tóth k: morbid changes in lowe’s oculo-cerebro-renal syndrome. neuropadiatrie 1973;4:304-13 3. garzuly f, szabó l, kádas l, jellinger k: disorders of neuronal migration in zellwegers cerebro-hepato-renal syndrome. neuropadiatrie 1974;5:318-28 4. jellinger k, minauf m, garzuly f, neumayer e: angiodysgenetic necrotizing myelopathy (report on 7 cases). arch psychiatr nervenkr 1968;211:377-40 5. garzuly f, jellinger k, pilz p: subacute spongioform encephalopathy (jakob-creutzfeldt syndrome) clinico-pathological evaluation of 9 cases. arch psychiatr nervenkr 1971;214:207-27 6. garzuly f, mészáros e, brittig f, viniczai z, mázló m, budka h: hypernephroma and associated al-amyloidosis with polyneuropathy in monoclonal gammopathy. nervenarzt 1993;64:817-9 7. vidal r, garzuly f, budka h, lalowski m, linke rp, brittig f, frangione b, wisniewski t: meningocerebrovascular amyloidosis associated with a novel transthyretin mis-sense mutation at codon 18 (ttrd18g). am j pathol 1996;148:361-6 8. garzuly f, vidal r, wisniewski t, brittig f, budka h: familial meningocerebrovascular amyloidosis, hungarian type, with mutant transthyretin (ttr asp18gly). neurology 1996;47:1562-7 9. hammarström p, sekijima y, white jt, wiseman rl, lim a, costello ce, altland k, garzuly f, budka h, kelly jw: d18g transthyretin is monomeric, aggregation prone, and not detectable in plasma and cerebral fluid: a presciption for central nervous system amyloidosis? biochemistry 2003;42:6656-63 10. harkány t, garzuly f, csanaky g, luiten pg, nyakas c, linke rp, virágh s: cutaneous lymphatic amyloid deposits in "hungarian-type" familial transthyretin amyloidosis: a case report. br j dermatol 2002;146:674-9. doi: 10.1046/j.1365-2133.2002.04594 11. garzuly f, maródi l, erdös m, grubits j, varga z, gelpi e, rohonyi b, mázló m, molnár a, budka h: megadolichobasilar anomaly with thrombosis in a family with fabry’s disease and a novel mutation in the alpha-galactosidase a gene. brain 2005;128:2078-83 12. gelpi e, preusser m, garzuly f, holzmann h, heinz fx, budka h: visulization of central european tick-borne encephalitis infection in fatal human cases. j neuropathol exp neurol 2005;64:506-12 13. gelpi e, preusser m, laggner u, garzuly f, holzmann h, heinz fx, budka h: inflammatory response in human tick-borne encephalitis: analysis of postmortem brain tissue. j neurovirol 2006;12:322-7 14. höftberger r, garzuly f, dienes hp, grubits j, rohonyi b, fischer g, hanzely z, lassmann h, budka h: fulminant central nervous system demyelinisation associated with interferon-alpha therapy and hepatitis c virus infection. mult scler 2007;13:1100-6 15. höftberger r, kunze m, voigtländer t, unterberger u, regelsberger g, bauer j, aboul-enein f, garzuly f, forss-petter s, bernheimer h, berger j, budka h: peroxisomal localisation of the proopiomelanocortin-derived peptides beta-lipotropin and beta-endorphin endocrinology 2010;151:4801-10 16. garzuly f, hahn k, iványi lj, kereskai l, kovács gg, budka h, kálmán b: association of temporal lobe inflammatory leukoencephalopathy with two b cell malignancies. ideggyogy sz/cli neurosci 2014;67:135-9 17. tolvaj b, hahn k, nagy zs, vadvári a, csomor j, gelpi e, illés zs, garzuly f: life threatening rare lymphomas presenting as longitudinally extensive transverse myelitis: a diagnostic challenge ideggyogy sz/cli neurosci 2020;73:275-285 18. garzuly f, schneider f, iványi jl, nagy z, varga m, sütő k, tolvaj b, kálmán b: changing times – changing diseases. review of the neuropathological autopsy documentations at the markusovszky university teaching hospital (1964-2014). orv hetil 2014;155:1722-8. doi: 10.1556/oh.2014.29996 (in hungarian) 19. garzuly f, brittig f, baltavári l, pócza k, istván l: incidence of various neuroleukemia types in a 5-year record of autopsy cases. orv hetil 1984;125:1685-90 (in hungarian) 20. garzuly f: pathological features of neuroleukemia. changes and lessons. orv hetil 1994;135:1291-5 (in hungarian) 21. budka h: the golden era of neuropathology. free neuropathology 2020;1:1-26 22. garzuly f, grubits j, nikl j: antecedents to the commencement and history of the west-pannonic neurological forum. ideggyogy sz/clin neurosci 2016;69:139-43 (in hungarian) 23. bobest m, tóth c, gyurcsó m, molnár mj, garzuly f: nonsense mutation 193c>t of neurofibromatosis type 2 – a neurosurgical challenge. ideggyogy sz/cli neursci 2007;60:41-5 (in hungarian) 24. kálmán b, szép e, garzuly f, post de: epidermal growth factor receptor as a therapeutic target in glioblastoma. neuromolecular med 2013;152:420-34. doi: 10.1007/s12017-013-8229-y 25. sinko g, garzuly f, kálmán b: striking pathology in leigh syndrome associated with the mtatp6 t8993g mutation. pediatr neurol 2014;51:585-6. doi: 10.1016/j.pediatrneurol.2014.07.015 26. nagy á, garzuly f, kálmán b: pathogenic alterations within the neurofibromin gene in various cancers. magy onkol 2017;61:327-336. (in hungarian) 27. nagy á, garzuly f, padányi g, szűcs i, feldmann á, murnyák b, hortobágyi t, kálmán b: molecular subgroups of glioblastoma – an assessment by immunohistochemical markers. pathol oncol res 2019;25:21-31. doi: 10.1007/s12253-017-0311-6 books on rare diseases (in hungarian) ferenc garzuly: in the labyrinth of diagnostics (2013) györgy pfliegler, ferenc garzuly: rare diseases – diagnostic challenges (2013) ferenc garzuly, csaba tóth, bernadette kálmán: rare diseases, struggle for life (2013) ferenc garzuly, csaba tóth, bernadette kálmán: rare diseases, special forms of diseases (2015) ferenc garzuly, csaba tóth, bernadette kálmán: rare diseases, diagnosis and therapy (2017) ferenc garzuly, csaba tóth, bernadette kálmán: rare and hiding diseases (2020, in progress) other books (in hungarian) garzuly: black water, swirling time – message of autopsy reports (1920-1956) (2006) garzuly: unknown season – irregular diary (2008) garzuly: my father is reading newspapers 1953-1957 (2011) garzuly: good morning, mr. head physician! (2019) copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. the neurovascular unit in diffuse intrinsic pontine gliomas feel free to add comments by clicking these icons on the sidebar free neuropathology 2:17 (2021) original paper the neurovascular unit in diffuse intrinsic pontine gliomas fatma e. el-khouly1,2*, rianne haumann1,2*, marjolein breur3, sophie e.m. veldhuijzen van zanten1,2, gertjan j.l. kaspers1,2, n. harry hendrikse4,5, esther hulleman1,2, dannis g. van vuurden1,2#, marianna bugiani3# 1 emma children’s hospital, amsterdam umc – location vumc, department of pediatric oncology, cancer center amsterdam, de boelelaan 1117, amsterdam, the netherlands 2 princess máxima center for pediatric oncology, utrecht, the netherlands 3 amsterdam umc – location vumc, department of pathology, de boelelaan 1117, amsterdam, the netherlands 4 amsterdam umc – location vumc, department of clinical pharmacology & pharmacy, de boelelaan 1117, amsterdam, the netherlands 5 amsterdam umc – location vumc, department of radiology & nuclear medicine, de boelelaan 1117, amsterdam, the netherlands * co-first authorship # co-last authorship corresponding author: fatma e. el-khouly · amsterdam umc, location vumc · department of pediatric oncology/hematology · de boelelaan 1117 · 1081 hv amsterdam · the netherlands f.el-khouly@amsterdamumc.nl submitted: 24 april 2021 accepted: 27 june 2021 copyedited by: bert m. verheijen published: 5 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3341 additional resources and electronic supplementary material: supplementary material keywords: diffuse intrinsic pontine glioma (dipg), neurovascular unit (nvu), blood-brain barrier (bbb), tight junctions, brainstem, pons abstract aims: diffuse intrinsic pontine glioma (dipg) is a childhood brainstem tumor with a median overall survival of eleven months. lack of chemotherapy efficacy may be related to an intact blood-brain barrier (bbb). in this study we aim to investigate the neurovascular unit (nvu) in dipg patients. methods: dipg biopsy (n = 4) and autopsy samples (n = 6) and age-matched healthy pons samples (n = 20) were immunohistochemically investigated for plasma protein extravasation, and the expression of tight junction proteins claudin-5 and zonula occludens-1 (zo-1), basement membrane component laminin, pericyte marker pdgfr-β, and efflux transporters p-gp and bcrp. the mean vascular density and diameter were also assessed. results: dipgs show a heterogeneity in cell morphology and evidence of bbb leakage. both in tumor biopsy and autopsy samples, expression of claudin-5, zo-1, laminin, pdgfr-β and p-gp was reduced compared to healthy pontine tissues. in dipg autopsy samples, vascular density was lower compared to healthy pons. the density of small vessels (<10 µm) was significantly lower (p<0.001), whereas the density of large vessels (≥10 µm) did not differ between groups (p = 0.404). the median vascular diameter was not significantly different: 6.21 µm in dipg autopsy samples (range 2.25-94.85 µm), and 6.26 µm in controls (range 1.17-264.77 µm). conclusion: our study demonstrates evidence of structural changes in the nvu in dipg patients, both in biopsy and autopsy samples, as well as a reduced vascular density in end-stage disease. adding such a biological perspective may help to better direct future treatment choices for dipg patients. introduction diffuse intrinsic pontine gliomas (dipgs) are rare and aggressive childhood malignancies of the brainstem. these tumors are characterized by a diffuse growth pattern closely interwoven within white matter tracts and grey matter structures, and an intrinsic nature, uttered by hypertrophy of the brainstem often encasing the basilar artery 1,2. with a median overall survival of eleven months, and a two-year survival rate of 10%, dipgs are the leading cause of brain tumor-related deaths in children 3-7. in the recent world health organization (who) classification of tumors of the central nervous system (cns), dipgs were reclassified as h3k27m mutated diffuse midline gliomas (dmg h3k27m) 8. though much research has been dedicated to dipg, its poor outcome has remained unchanged for the past 40 years 9. to date, radiotherapy remains the only (temporarily) effective, albeit palliative treatment, and no chemotherapy regimens prolonging survival have been identified yet. since in vitro and in vivo drug testing on patient-derived tumor cells has shown sensitivity to conventional cytotoxic agents and novel drugs, the lack of efficacy in patients is hypothesized to be related to ineffective drug delivery due to an intact blood-brain barrier (bbb) 10-12. the bbb is formed by endothelial cells interconnected and sealed by tight junctions. the abluminal surface of the endothelium is covered by a basement membrane in which pericytes are embedded. pericytes control the cerebral blood flow by regulating capillary diameter and vessel stability. the basement membrane is enclosed by astrocyte end-feet, also important for brain homeostasis. together, pericytes and astrocyte end-feet induce and maintain the integrity of the bbb 13,14. the bbb regulates transport of essential nutrients to the brain through active transport mechanisms, such as glucose transporters of the glut-family. the efflux of waste products and exogenous compounds is mediated through efflux transporters of the atp-binding cassette family (e.g., p-gp, bcrp, mrp-1) 14,15. additionally, the paracellular barrier capacities of the tight junctions limit transport of circulating monoamines and drugs across the bbb 14-16. the intimate contact and interaction of the bbb complex, formed by endothelial cells, tight junctions, pericytes and astrocyte end-feet, with neurons and perivascular microglia form a dynamic functional unit, called the neurovascular unit (nvu) 13,14,16. some studies report different expression of tight junction proteins throughout the brain, suggestive of regional heterogeneity in bbb permeability 17,18. however, little research has been done on the bbb and nvu in the brainstem and particularly in the pons. yet, better insight into the bbb and the nvu at these sites is needed to develop new treatment strategies for pediatric brainstem tumors. this especially holds true for dipg, where the bbb is thought to be a major contributor to therapeutic inefficacy. in this study, we aim at determining and comparing the histological and immunohistochemical characteristics of the nvu of the pons in children with dipg and age-matched controls. figure 1: characteristics of diffuse intrinsic pontine glioma (dipg). a: t2-weighted mri-image of a dipg patient showing an expanded tumor at the basis of the pons. the arrow indicates the biopsy sampling area; b: gross axial section of the pons and cerebellum showing the presence of a diffuse infiltrating tumor in the pons, reaching the middle cerebellar peduncles. the box indicates the sampling location of autopsy tissue at the non-necrotic tumor site; c-e: hematoxylin and eosin (h&e) staining of the vital tumor bulk showing morphologic heterogeneity compatible with who grade iv tumors (c and d) and grade i tumors (e); f-h: stains against intravascular plasma proteins pre-albumin (f), fibrinogen (g) and igg (h) showing extravasation of these protein into the dipg tumor parenchyma; i-k: hypoxia inducible factor-1α (hif-1α) staining demonstrating a high expression of hif-1α. (scale bar: 5 µm) patients and methods patients and samples dipg pre-treatment biopsy samples (n = 4) were obtained from the biobank of the princess máxima center for pediatric oncology, utrecht, the netherlands, and processed as formalin-fixed paraffin-embedded tissue. end-stage disease dipg autopsy samples (n = 6) were obtained from the ‘vumc brain autopsy in children with dipg’ study 19. this study was approved by the institutional review board of amsterdam umc, location vumc (metc vumc, study number: vumc2009/237) and the scientific committee of the dutch childhood oncology group (dcog). in this study, brain tissue was obtained within a post-mortem interval of less than six hours for dutch patients and less than nine hours for patients from abroad, and was processed as formalin-fixed paraffin-embedded tissue or snap frozen. biopsy samples were mri-guided and taken from the tumor area displaying the highest hyper-intensity on t2-weighted image (figure 1a). autopsy samples were obtained from the non-necrotic tumor core in the pons (figure 1b). age-matched, healthy pontine tissue samples (n = 20) where obtained from the nih neurobiobank, maryland, united states. samples were selected based on (i) brain region (pons), (ii) clinical brain diagnosis (unaffected control/sudden deaths), (iii) post-mortem interval (<17 hours), and (iv) presence of formalin-fixed tissue and frozen tissue. table 1 shows patient and treatment characteristics of the dipg patients. median age at diagnosis was 7.7 years (range 1.3-17.0 years). all patients had a h3k27m mutated dipg. all autopsy patients, except for the youngest, received radiotherapy at diagnosis. of these, at disease progression, three out of six received different chemotherapy regimens and two patients did not proceed to further treatment. median overall survival of patients that were autopsied was 19.5 months (range 5.5-24.0 months). supplementary table 1 shows the characteristics of the control group. median age was 7.0 years (range 1.0-19.0 years). all controls were healthy and had an accidental death. immunohistochemistry air-dried five-μm-thick cryosections were fixed in 2% formaldehyde for 10 min at room temperature (rt). aldehyde groups were blocked in 0.1 g glycine in 100 ml distilled water for 10 min at rt. sections were incubated overnight at rt with a primary antibody: (i) tight junction protein claudin-5 (1:50, invitrogen, carlsbad, ca, usa); (ii) tight junction protein zo-1 (1:50, invitrogen, carlsbad, ca, usa); (iii) basement membrane component laminin (1:500, novus biologicals, abingdon uk); or (iv) pericyte marker pdgfr-β (1:500, abcam, cambridge, uk). the sections were co-stained with glial fibrillary acidic protein (gfap; 1:1000, merck, darmstadt, germany). the following day, the sections were incubated with alexa fluor®-labelled secondary antibodies, background was quenched with 0.1% sudan black b, and sections were mounted in mounting medium (vectashield with 4',6-diamidino-2-fenylindool (dapi); vector laboratories inc., burlingame, ca, usa). five-μm-thick formalin‐fixed paraffin-embedded tissue sections were routinely stained with hematoxylin & eosin (h&e). a gross axial section through the pons and cerebellum was stained with luxol fast blue-periodic acid-schiff. for immunohistochemistry, sections were deparaffinized using xylene, and rehydrated through descending alcohol concentrations. endogenous peroxidase activity was blocked by incubating the slides for 30 min in phosphate buffered saline (pbs) containing 0.3% h2o2. heat-induced antigen retrieval was performed in 0.01 m citrate buffer (ph 6.0). after washing in pbs, the slides were incubated overnight at rt with primary antibodies against p-gp (1:20; millipore, ca, usa), bcrp (1:40; abcam, cambridge, uk), pre-albumin (1:50,000, dako, glostrup, denmark), fibrinogen (1:1,600, dako, glostrup, denmark), igg (1:800, dako, glostrup, denmark) and hypoxia-inducible factor 1α (hif-1α; 1:40, cayman chemical, michigan, usa). the next day, slides were incubated with ready-to-use envision™-hrp (dako, glostrup, denmark) for 1 hour at rt and visualized with 3,3'diaminobenzidine (dab+ dako; 1:50, glostrup, denmark) for 10 min. the slides were counterstained with hematoxylin for 1 min and mounted with quick-d mounting medium (klinipath, duiven, the netherlands). data analysis sections were imaged using a leica dm6000b microscope (400x magnification; leica microsystems bv, rijswijk, the netherlands). from each tissue slide, ten images were made. a semi-quantitative analysis of the bbb staining, comparing dipg samples with control samples, was done by two independent reviewers (fe and rh) using the leica application suite x: las x version 3.1.5.16308. the vascular density was assessed on claudin-5-stained tissue sections by counting the number of blood vessels per mm2. the luminal diameter of the blood vessels was measured with the leica application suite x: las x version 3.1.5.16308. statistics data were analyzed using an independent samples t-test (p-value = 0.05) using ibm spss statistics version 26. the levene’s test of equality of variance was used to first test the assumption of homogeneity or variance between the groups (p-value = 0.05). when equal variances were assumed, pooled estimates were used for the independent t-test statistics. when equal variances were not assumed, unpooled data and an adjustment to the degree of freedom (df) were used for the independent t-test statistics. results immunohistochemistry figure 1 shows typical dipg mri features with enlargement of the pons and contrast enhancement. gross inspection confirms the presence of a partly necrotic and hemorrhagic tumor center in the pons. microscopic examination shows variability of tumor cell morphology, ranging from grade i to grade iv according to the 2016 who classification of cns tumors. tumor areas were recognized based on cell density, the presence of (atypical) mitotic figures and features of high-grade glioma, including necrosis and microvascular proliferation. in these tumors, the integrity of the bbb is compromised, as demonstrated by extravasation of pre-albumin, fibrinogen and igg. this corresponds with expression of hif-1α, indicating tumor hypoxia. notably, as expected for a heterogeneous tumor such as dipg, the density of gfap-expressing astrocytes varied throughout the tumor 20. additionally, tumor cells were differentiated from pre-existing astrocytes by their higher expression levels of gfap, conceivably also related to their less differentiated state 20. figure 2: expression of tight junction proteins claudin-5 and zonula occludens-1 (zo-1) in dipg pre-treatment biopsy and post-mortem autopsy samples. in controls, claudin-5 and zo-1 are sharply defined and have a segmented pattern (a and d). claudin-5 and zo-1 show reduced expression in dipg samples (b, c, e, f). please note the non-activated state of gfap-expressing astrocytes in control tissue. (blue: nuclei; green: astrocytes; red: claudin-5 or zo-1; scale bar: 5 µm) figure 3: expression of basement membrane component laminin in dipg pre-treatment biopsy and post-mortem autopsy samples. in controls (a-c), laminin shows a continuous pattern. laminin expression was reduced at the glial basement membrane in both dipg biopsy (d-f) and autopsy samples (g-i). this was also observed in neovascularization in autopsy samples (j-l). of note: neovascular proliferation was not detected in biopsy samples. (blue: nuclei; green: astrocytes; red: laminin; scale bar: 5 µm) immunohistochemical staining of claudin-5, zo-1, laminin, pdgfr-β, p-gp and bcrp were evaluable in all samples. expression of tight junction proteins claudin-5 and zo-1 was lower at inspection in all dipg biopsy and autopsy samples compared to control samples (figure 2). the expression of basement membrane protein laminin was lower at the glial basement membrane in dipg biopsy and autopsy samples. interestingly, this was observed in both pre-existent vessels within the tumor cells and in neovascular proliferation (figure 3). expression of pericyte marker pdgfr-β was also reduced in both dipg biopsy and autopsy samples (figure 4). efflux transporter p-gp expression was lower in dipg biopsy and autopsy samples, whereas the expression of bcrp was not different in dipg compared to controls (figure 5). figure 4: expression of pericyte marker pdgfr-β in dipg pre-treatment biopsy and post-mortem autopsy samples. in controls (a-c), pdgfr-β shows a continuous pattern. expression of pdgfr-β was reduced in both dipg biopsy (d-f) and autopsy samples (g-i). (blue: nuclei; green: astrocytes; red: pdgfr-β; scale bar: 5 µm). figure 5: expression of efflux transporters p-gp and bcrp in dipg pre-treatment biopsy and post-mortem autopsy samples. p-gp and bcrp are sharply defined and have a segmented pattern in controls (a and d). expression of p-gp was reduced in both dipg samples (b and c). bcrp expression was unchanged in dipg samples (e and f). (blue: nuclei; green: astrocytes; red: p-gp or bcrp; scale bar: 5 µm) vascular density vascular density per mm2 was measured in non-necrotic biopsy and autopsy tissue. it was significantly reduced in dipg autopsy samples compared to controls (1.5±1.2/mm2 versus 17.5±9.5/mm2, respectively; t113,890 = 6.831, p-value <0.001; figure 6a). notably, the density of small blood vessels (<10 µm) was significantly lower in dipg autopsy samples than in controls (t180,609 = -4.303, p-value <0.001), whereas the density of large blood vessels (≥10 µm) did not differ between groups (t597 = -0.835, p-value = 0.404). most blood vessels in dipg autopsy and control samples had a diameter smaller than 10 µm. the median vascular diameter was 6.21 µm in dipg autopsy samples (range 2.25-94.85 µm), versus 6.26 µm in controls (range 1.17-264.77 µm; figure 6b). due to the very small size of the biopsy samples, it was not possible to statistically analyze the vascular density and diameter in these tissue samples. visual inspection of three patients, however, showed a mean vascular density of 7.5 vessels per mm2, and a median vascular diameter of 8.23 µm (data not shown). figure 6: vascular density and diameter in dipg post-mortem autopsy and healthy control samples. a: vascular density in dipg post-mortem autopsy samples and healthy control samples. mean vascular density was 1.5±1.2/mm2 in dipg versus 17.5±9.5/mm2 in controls (red line). b: vascular size distribution in dipg post-mortem samples and healthy control samples. discussion little research has been done to identify the nvu in dipg, while it is hypothesized that treatment failure is caused by an intact bbb. as summarized by figure 7, our study demonstrates structural changes in the nvu of dipg patients that are already present at diagnosis, suggesting these to be tumor-related and not only due to treatment. all studied dipg patients harbored a h3k27m mutation, thus fulfilling the diagnosis of dmg h3k27m according to the revised who classification 8. up to 85% of dipg patients harbor this mutation 21,22. since three out of six dipg autopsy patients were long-term survivors, the median overall survival of the patients in this group was longer than known from literature, 19.5 months versus 11 months, respectively 7. whether neuropathological grading (who ii-iv), tumor location or the presence of a h3k27m mutation have an impact on survival is still not clear 22-24. figure 7: graphical overview of the structural capillary changes observed in dipg pre-treatment biopsy and post-mortem autopsy samples: lower expression of tight junction proteins claudin-5 and zo-1, basement membrane component laminin, pericyte marker pdgfr-β and efflux transporter p-gp in dipg biopsy and autopsy samples; unchanged expression of efflux transporter bcrp-1 in dipg biopsy and autopsy samples. the barrier properties of the nvu strongly depend on the complex interaction between endothelial cells and their tight junctions, pericytes, basement membranes and astrocytes. in physiological conditions, tight junctions are formed by inter-endothelial connections between transmembrane proteins of the claudin-family (claudin-1, 3, 5, and 12), which regulate the function of these tight junctions25,26. claudins are anchored into the endothelial cells by proteins from the zonula occludens-family (zo-1, 2, and 3) that regulate adherens junctions and influence cytoskeletal organization, angiogenic potential and cell migration 27. moreover, zo-1 is responsible for the spatial organization of claudin-5 by linking it to the actin cytoskeleton 25. downregulation of zo-1 can lead to tight junction disruption and a larger intercellular distance between endothelial cells and thus pathologically increased paracellular transport 27. claudin-5 is most abundant in brain vessels (600-times higher expression than other claudins), where it has a heterogeneous distribution 26. the highest claudin-5 expression is seen in capillaries and small post-capillary venules 25,28. in a claudin-5 knockout mouse model, an increased leakage of molecules up to 800 da was observed 29, whereas the permeability of normal bbb only allows passage of molecules up to 500 da 30,31. when additional tight junction proteins are downregulated, a size-dependent increase in paracellular transport is seen of molecules with a size up to 10,000 da 29,32. in our study, a reduced expression of claudin-5 and zo-1 was observed in dipg patients both pre-treatment and post-mortem, indicating a barrier defect, and increasing paracellular transport across the bbb 27. nevertheless, there are more tight junction proteins of the zonula occludens and claudin-family expressed by brain endothelial cells. whether possible downregulation of claudin-5 and zo-1 is compensated by overexpression of other tight junction proteins remains unknown. endothelial cells are surrounded by a basement membrane that contains laminin produced by pericytes and astrocytes 33,34. laminin is essential for basement membrane assembly and maintenance of nvu integrity 35. in our study, employing a panlaminin antibody, we found that expression of laminin was lower at the glial basement membrane in both dipg biopsy and autopsy samples. this was observed in pre-existent vessels amongst the tumor cells and in neovascular proliferation. our results suggest a pathological involvement of pericytes and astrocytes in dipg that could have consequences on the behavior of the endothelial cells, thus also disrupting the integrity of the nvu 34. immunohistochemistry showed also a lower expression of pdgfr-β in dipg biopsy and autopsy samples, suggesting a reduction in pericytic coverage in dipg nvu. besides contributing to secretion of basement membrane components 33,34, pericytes are essential for regulating capillary diameter and vessel stability 13. the possible reduction of pericytic coverage observed in our study may explain the possible downregulation of laminin at the glial basement membrane in dipg patients. under physiological conditions, p-gp and bcrp are the most dominantly expressed efflux transporters in de bbb 36,37. our study shows a decreased p-gp expression and unchanged bcrp expression in dipg pre-treatment and post-mortem samples. this is in line with previous work showing a “moderate expression” of p-gp and intense staining of bcrp in dipg tumor vasculature 10. overall, our results show alterations of the nvu in dipg patients, which could result in or reflect a more leaky nvu. this hypothesis of a leaky nvu is supported by the demonstrated extravasation of some intravascular proteins, such as pre-albumin, fibrinogen and igg. theoretically, this might positively influence influx of chemotherapeutic agents into the tumor, based on passive diffusion. clinical data, however, do not support this possibility 38,39. a possible explanation for this discrepancy might be the markedly reduced vascular density in dipg that could overrule the effects of a leaky nvu. whether the reduction of vascular density is also present at diagnosis remains to be investigated. lack of therapy efficacy in dipg has been linked to an intact bbb. here, we demonstrated structural changes of the nvu together with a lower vascular density in these tumors. these findings have consequences for drug administration, since coverage of the whole tumor, including the migrating/diffusely growing tumor cells, is essential. our findings suggest that drug administration techniques that mostly rely on vascular density for drug distribution, including conventional systemic administration and microbubble mediated focused ultrasound, might show limited efficacy in dipg 12. in contrast, convection-enhanced delivery might be a more suitable technique, in which drug distribution across the tumor relies on a positive pressure gradient instead of passive diffusion 12,40,41. adding such a biological nvu perspective may help to better direct treatment choices for dipg patients in the future. acknowledgement we thank the employees of the expertise center for post-mortem diagnostics of the amsterdam university medical centers, location vumc, for assisting with the autopsies of the dipg patients. we also thank the nih neurobiobank for providing the control tissue. we would also like to thank the semmy foundation (stichting semmy) for financially supporting dipg research in the amsterdam university medical centers, location vumc. ethical approval the study was approved by the medical ethical committee of the amsterdam university medical center, location vumc (metc vumc, study number vumc2009/237). this study was conducted in accordance to the declaration of helsinki. data sharing and data accessibility the data that support the findings of this study are available from the corresponding author upon reasonable request.  references 1. epstein f, mccleary el. intrinsic brain-stem tumors of childhood: surgical indications. j neurosurg 1986; 64(1): 11-5. 2. yoshimura j, onda k, tanaka r, takahashi h. clinicopathological study of diffuse type brainstem gliomas : analysis of 40 autopsy cases. neurol med chir (tokyo) 2003; 43: 375-82. 3. broniscer a, gajjar a. supratentorial high-grade astrocytoma and diffuse brainstem glioma: two challenges for the pediatric oncologist. oncologist 2004; 9(2): 197-206. 4. hargrave d, bartels u, bouffet e. diffuse brainstem glioma in children: critical review of clinical trials. the lancet oncology 2006; 7(3): 241-8. 5. jansen mh, veldhuijzen van zanten se, sanchez aliaga e, et al. survival prediction model of children with diffuse intrinsic pontine glioma based on clinical and radiological criteria. neuro oncol 2015; 17(1): 160-6. 6. veringa sje, biesmans d, van vuurden dg, et al. in vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma. plos one 2013; 8(4): e61512-e. 7. hoffman lm, veldhuijzen van zanten sem, colditz n, et al. clinical, radiologic, pathologic, and molecular characteristics of long-term survivors of diffuse intrinsic pontine glioma (dipg): a collaborative report from the international and european society for pediatric oncology dipg registries. journal of clinical oncology 2018; 36(19): 1963-72. 8. louis dn, perry a, reifenberger g, et al. the 2016 world health organization classification of tumors of the central nervous system: a summary. acta neuropathol 2016; 131(6): 803-20. 9. sewing ac, caretti v, lagerweij t, et al. convection enhanced delivery of carmustine to the murine brainstem: a feasibility study. j neurosci methods 2014; 238: 88-94. 10. veringa sj, biesmans d, van vuurden dg, et al. in vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma. plos one 2013; 8(4): e61512. 11. grasso cs, tang y, truffaux n, et al. functionally defined therapeutic targets in diffuse intrinsic pontine glioma. nat med 2015; 21(6): 555-9. 12. haumann r, videira jc, kaspers gjl, van vuurden dg, hulleman e. overview of current drug delivery methods across the blood-brain barrier for the treatment of primary brain tumors. cns drugs 2020; 34(11): 1121-31. 13. obermeier b, daneman r, ransohoff rm. development, maintenance and disruption of the blood-brain barrier. nat med 2013; 19(12): 1584-96. 14. serlin y, shelef i, knyazer b, friedman a. anatomy and physiology of the blood-brain barrier. semin cell dev biol 2015; 38: 2-6. 15. abbott nj, ronnback l, hansson e. astrocyte-endothelial interactions at the blood-brain barrier. nat rev neurosci 2006; 7(1): 41-53. 16. liebner s, dijkhuizen rm, reiss y, plate kh, agalliu d, constantin g. functional morphology of the blood-brain barrier in health and disease. acta neuropathol 2018; 135(3): 311-36. 17. wilhelm i, nyul-toth a, suciu m, hermenean a, krizbai ia. heterogeneity of the blood-brain barrier. tissue barriers 2016; 4(1): e1143544. 18. villabona-rueda a, erice c, pardo ca, stins mf. the evolving concept of the blood brain barrier (bbb): from a single static barrier to a heterogeneous and dynamic relay center. front cell neurosci 2019; 13: 405. 19. caretti v, jansen mh, van vuurden dg, et al. implementation of a multi-institutional diffuse intrinsic pontine glioma autopsy protocol and characterization of a primary cell culture. neuropathol appl neurobiol 2013; 39(4): 426-36. 20. bugiani m, veldhuijzen van zanten sem, caretti v, et al. deceptive morphologic and epigenetic heterogeneity in diffuse intrinsic pontine glioma. oncotarget 2017; 8(36): 60447-52. 21. castel d, philippe c, calmon r, et al. histone h3f3a and hist1h3b k27m mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes. acta neuropathol 2015; 130(6): 815-27. 22. karremann m, gielen gh, hoffmann m, et al. diffuse high-grade gliomas with h3 k27m mutations carry a dismal prognosis independent of tumor location. neuro oncol 2018; 20(1): 123-31. 23. wagner s, warmuth-metz m, emser a, et al. treatment options in childhood pontine gliomas. j neurooncol 2006; 79(3): 281-7. 24. von bueren ao, karremann m, gielen gh, et al. a suggestion to introduce the diagnosis of "diffuse midline glioma of the pons, h3 k27 wildtype (who grade iv)". acta neuropathol 2018; 136(1): 171-3. 25. greene c, hanley n, campbell m. claudin-5: gatekeeper of neurological function. fluids barriers cns 2019; 16(1): 3. 26. jia w, lu r, martin ta, jiang wg. the role of claudin-5 in blood-brain barrier (bbb) and brain metastases (review). mol med rep 2014; 9(3): 779-85. 27. tornavaca o, chia m, dufton n, et al. zo-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation. j cell biol 2015; 208(6): 821-38. 28. paul d, cowan ae, ge s, pachter js. novel 3d analysis of claudin-5 reveals significant endothelial heterogeneity among cns microvessels. microvasc res 2013; 86: 1-10. 29. nitta t, hata m, gotoh s, et al. size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. j cell biol 2003; 161(3): 653-60. 30. el-khouly fe, van vuurden dg, stroink t, et al. effective drug delivery in diffuse intrinsic pontine glioma: a theoretical model to identify potential candidates. front oncol 2017; 7: 254. 31. pike vw. pet radiotracers: crossing the blood-brain barrier and surviving metabolism. trends in pharmacological sciences 2009; 30(8): 431-40. 32. keaney j, walsh dm, o’malley t, et al. autoregulated paracellular clearance of amyloid-β across the blood-brain barrier. science advances 2015; 1(8): e1500472. 33. gautam j, zhang x, yao y. the role of pericytic laminin in blood brain barrier integrity maintenance. sci rep 2016; 6: 36450. 34. yao y, chen zl, norris eh, strickland s. astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity. nat commun 2014; 5: 3413. 35. givant-horwitz v, davidson b, reich r. laminin-induced signaling in tumor cells. cancer lett 2005; 223(1): 1-10. 36. al-majdoub zm, al feteisi h, achour b, et al. proteomic quantification of human blood-brain barrier slc and abc transporters in healthy individuals and dementia patients. mol pharm 2019; 16(3): 1220-33. 37. uchida y, ohtsuki s, katsukura y, et al. quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. j neurochem 2011; 117(2): 333-45. 38. el-khouly fe, veldhuijzen van zanten sem, santa-maria lopez v, et al. diagnostics and treatment of diffuse intrinsic pontine glioma: where do we stand? j neurooncol 2019; 145(1): 177-84. 39. veldhuijzen van zanten se, jansen mh, sanchez aliaga e, van vuurden dg, vandertop wp, kaspers gj. a twenty-year review of diagnosing and treating children with diffuse intrinsic pontine glioma in the netherlands. expert rev anticancer ther 2015; 15(2): 157-64. 40. barua nu, hopkins k, woolley m, et al. a novel implantable catheter system with transcutaneous port for intermittent convection-enhanced delivery of carboplatin for recurrent glioblastoma. drug deliv 2016; 23(1): 167-73. 41. lewis o, woolley m, johnson d, et al. chronic, intermittent convection-enhanced delivery devices. j neurosci methods 2016; 259: 47-56. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. notes on the career of jacqueline mikol feel free to add comments by clicking these icons on the sidebar free neuropathology 2:25 (2021) reflections notes on the career of jacqueline mikol jacqueline mikol honorary emeritus professor, université de paris, france address for correspondence: jacqueline mikol · université de paris · france jacqueline.mikol@wanadoo.fr submitted: 7 september 2021 accepted: 13 september 2021 copyedited by: jeffrey nirschl published: 20 september 2021 https://doi.org/10.17879/freeneuropathology-2021-3532 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, reflections, autobiography, euro-cns i was born in neuilly sur seine in 1936 but my parents lived in rumaucourt, a small village in the north of france (pas de calais). both were from jewish romanian descent, but they met in france. my father was a family physician and my mother, who had interrupted her medical studies before wwii, resumed them after the end of the war when i was at primary school and became a radiologist. during the war, my father was not present at home. he was first a prisoner in germany, then deported to a concentration camp (auschwitz-monowitz/flossenburg). fortunately, he managed to survive and to come back home in 1945 but after this traumatic experience, life would be never easy again. my parents did not want to have any other children. from 1940 to 1945, my mother and i had lived in beaulieu sur dordogne, in corrèze. at the time, as a little girl, i did not realize that our life was in danger and that i was very protected. this gave me some degree of naivety and the feeling, during my first decades, not to be enough prepared to the ups and downs of private and professional life. in 1956 i got married, then divorced and remarried to françois mikol (1961) who was “interne”, in the department of neurology of professor raymond garcin at salpêtrière hospital, when i was “externe”. i had with françois three children, two boys (1962, 1964) and a girl (1970) and later eight grandchildren. my children often criticized me to spend so much time at work. i had to wait for years, when one of them began to do research, to be told that when you begin an experience, you never know exactly when you are going to finish it. my husband, who became a neurologist, was nominated the head of the department of neurology of the rothschild foundation in paris. this gave me the opportunity, after 1980, to frequently collaborate with him till his retirement. i had been very admirative of my father who resumed his practice, very soon, after coming back from deportation. at the time, a physician took care of medicine treatments, fractures, childbirths, day and night. i was also impressed by the book of balzac, the country physician. so, i decided to study medicine. i began my medical studies in 1954, in paris medicine university, after one year of basic sciences referred to as “physics, chemistry and biology”. i succeeded to become “externe” (1956), “interne” of paris hospitals (1960) then “chef de clinique” in the clinic of neurological diseases in salpêtrière hospital (1966-1969). simultaneously, i was trained in pathology (medicine university: 1963-1966) and histology (science university: 1966-1967), as i had decided to become a neuropathologist. although it seems obvious by now, my training was to investigate the relationships between clinical data and histology to determine the physiopathology of the neurological diseases. having a dual clinical and biological training was then feasible, which unfortunately, is not the case anymore in france. so, i was qualified as a “neuropsychiatre” (1968) and had a research degree in human biology (grade neuropathology-1972). i began my practical training when i was in the laboratory of the department of professor garcin, writing reports on infarcts, then called softenings, and tumors. claude vital and philippe evrard and later jean de recondo were my classmates, under the supervision of professor jean lapresle. i defended my thesis in 1966 on binswanger’s encephalopathy and related forms, directed by professor garcin and professor lapresle. from 1968 to 1983, i became a researcher at inserm (institut national de la santé et de la recherche médicale). the unit was successively directed by doctor jean gruner and professor michel fardeau. during this period, i was responsible for the laboratory of the “clinique neurologique”, at salpêtrière hospital, professor boudin having replaced pr. garcin, to perform day-life practice of neuropathology (1969-1976). afterwards, i moved to groupe hospitalier lariboisière-fernand widal-saint-lazare to set up a neuropathology unit (central nervous system-nerve-muscle), in the department of pathology, at the request of dean raymond houdart, neurosurgeon, professor bernard pepin neurologist and professor jean roujeau, pathologist, my former teacher in faculty. i was alone, in charge, to ensure neuropathology analysis for a long time until 1988 when doctor marc polivka joined me. i was nominated professor of pathology in 1985. this was not an easy journey, as a biologist and a woman. during this period, i particularly studied wilson’s disease, for which professor b. pepin was a clinical expert and also prion diseases which was part of a national project (professor j. j. hauw project leader). i coordinated and participated in the writing of a monograph dedicated to csf. professor annie galian had succeeded to professor roujeau. she was specialized in gastro-enterology diseases. she was the one who encouraged me to study the gut nervous system, which must be included in the training of neuropathology. after her early retirement, i became head of the department, in october 1995. the first part of my career was mostly focused on science and the second part included some management as well. during my residencies, two events have impacted my career. michel fardeau who was responsible of muscle and nerve biopsies in the department of professor garcin went to the united states for a scientific mission. i was required to learn and to practice the surgical technique till he returns. this triggered my interest in muscle pathology. reports on ocular myopathies, carnitine deficiency, inclusion myositis, both with andrew engel, mitochondrial myopathies… will be published years later. i spent my last stage of “internat” in the department of psychiatry, in versailles hospital, headed by professor serge brion. professor brion was qualified in neurology, neuropsychiatry and neuropathology. he had a critical and insightful mind, a great deal of curiosity and a sense of humor: it was the beginning of a long-lasting collaboration. he was really my mentor when i developed my research at inserm, starting from 1968 and beyond. dementia was the main subject. serge brion with gérard guiot and michel fardeau had developed a syringe-trocar to perform cortical biopsies. at the time, to confirm the diagnosis of dementia, biopsies of cerebral cortex were sometimes prescribed. so, in addition to neuropathological cases, i began to learn transmission electron microscopy. fortunately, i was helped by technicians, to prepare grids and to operate the hitachi microscope. in addition, i also had the pleasure and opportunity to work with jacqueline-godet-guillain. i examined the cerebral biopsies of the diseases for which serge brion was recognized as an expert for creutzfeldt-jakob disease, pick’s and alzheimer diseases. the research center where i worked had been created by neurologist professor garcin, psychiatrist professor delay, and biologist professor couteaux. the main aim of this research lab was to gather researchers from different disciplines to together collaborate. pathophysiology of neuro-muscular diseases were one of the main topics of the group. the center was located at pavillon risler, a very old building in hopital de la salpêtrière. the biologists were working on the neuro-muscular junctions of the torpedo fish. the aquarium was always an object of fascination for one of my children when he or she came with me to the laboratory. when the first “open laboratory days” took place (1968), organized by bernard barataud, the president of the “myopathic association” and michel fardeau, the biologists decided that, as a medical doctor, i had to welcome visitors to whom unfortunately we had very little to show that could have a concrete impact on quality of life of patients. this was the first “telethon”. my work was complemented by an experimental and neuropathological study of the connections of the latero-dorsal (ld) nucleus of the thalamus and its relationships with the limbic system. i began to study twelve anatomo-clinical cases of vascular origin in man. serge brion had noted that in some cases of memory disturbances, not only the hippocampo-mammillo-thalamo-cingular network was involved but the ld was also modified. the experimental study was performed in non-human primates in the laboratory of robert naquet in gif sur yvette. it was shown that the ld is part of a large parieto-cingulo-parahippocampic network with multiple direct and reciprocal connections. advice was provided by professor hgjm kuypers, who asked me to come to amsterdam, to discuss the report i had sent to brain pathology. i was very grateful to him. the studies of the limbic studies had shown the prevalence of the lesions of the ld, over those of the dorso-median nucleus of the thalamus. these results were the object of a controversy upon the hypothesis of victor and adams. they were at the origin of anatomical reports on memory disturbances and the relationships between memory and hemispheric functional specialization. it should be noted that these studies were performed before the practice of functional mri which since have shown more complex networks. i also studied tissue cultures of nervous system, made in maximow slides, to study aging and consequences of extracts of pathological specimens of cortex but survival was too short. professor brion sent me to the meeting of the american association of alzheimer’s disease, created by families and chaired by princess yasmina, daughter of rita hayworth. the committee suggested to create the same type of association in france. as i was not any more a clinician, i proposed to professor françoise forette to create and to chair such an association with a scientific comity. i will remain in the committee of france alzheimer for many years. i was in charge of the diploma of normal and pathological aging (1997-2003). before, i had been in charge of the certificate of “master” of pathology (paris university). figure 1. meetings of the french society of neuropathology in tunis. upper part (from left to right): first row: j. gautron is 1st, p. dreyfus is 3rd, a. rouche is 4th, the author is 5th, m. paturneau-jouas is 6th, m. tommasi is 7th, m. m. berard-badier (blue umbrella) is 8th. second row: m. pluot is 1st, m. chevalley is 2nd, f. tomé is 3rd, r. escourolle is 4th, j. f. pellissier is 5th, m. baudrimont is 6th, p. gaspard is 7th, c. duyckaerts is 8th. background: j. f. foncin (pink umbrella) is 1st, j. c. turpin is 5th, n. baumann is 6th, a. privat is 7th, j. j. hauw is 8th. lower part: first row: m. ben hamida is 1st, his wife c. forestier is 2nd, j. flamand is 3rd, c. duyckaerts is 4th, m. baudrimont is 5th, o. robain is 6th, f. dubas is 7th, m. m. rouchoux is 10th, m. coquet is 11th. d. henin is sitting in front in the middle. second row: m. b. delisle is 3rd, f. chapon is 4th, j. lapresle is 6th, c. vedrenne is 7th, m. pluot is 9th, the author is 10th. i was elected member of the french society of neuropathology. founded as a club in 1964 (madeleine bérard-badié, sege brion, raymond escourolle, edith farkas, jean françois foncin, jean gruner, jean lapresle, gilles lyon, maurice toga, michel tommasi), it became a scientific society in 1989. i chaired the society from 1990 to 1992. the meetings of the french society, before the pandemic, took place either in paris in winter or in another city in spring, with international participation or as joint meetings with our european colleagues or extra-european colleagues, especially from canada. these meetings have created productive relationships between members of the society and allow to enjoy social life together. i have participated to concerted actions on aids chaired by professor françoise gray and prion human diseases, chaired by professor herbert budka. i would like to emphasize the importance of these concerted actions. they were the source of scientific progress and collaborative research. each of us strived to show new results. many reports and books were later published, many friendships took place and strong links were established between neuropathologists. each meeting was in a nice location and enabled to socialize in a very pleasant environment. i shall say the same for congresses and conferences. nowadays, for different reasons accelerated by covid-19 pandemic, most meetings are organized with a computer and internet systems. it is difficult to be satisfied by an orwellian evolution of neuropathology. i had the pleasure to see and to listen to the main famous colleagues, to participate in poster sessions extremely rewarding and stimulating. furthermore, to become a member of neuropathological societies, it was necessary to present at least twice. to illustrate the spirit of this community combining scientific excellence and ability to enjoy life, i shall tell a short story. as the international congress was taking place in perth, chaired by professor byron kakulas, i naively asked to professor colin masters, one of the organizers, if bali was far from australia. the answer was “about 2 hours of flight!” inspired by the question he followed up with “great idea! let us organize a small pre-meeting in bali focused on brain tumors and alzheimer’s disease, from neuropathology to molecular biology”. this enabled us to go deep into some specific scientific subjects and provided a unique opportunity to visit indonesia and to discover borobodur temple at sunrise and bali island. our small group of french lady neuropathologists (m. baudrimont, c. dumas, m. n. delisle, c. lacroix and myself) had herbert budka as improvised volunteer bodyguard. figure 2. hiv concerted action, cork, ireland. first row (from left to right): k. majtényi is 2nd, the author is 3nd, f. gray is 4th, k. keohane is 5th, i. elovaara is 6th. second row: j. artigas is 1st, f. scaravilli is 3rd, p. liberski is 4th, f. razavi is 6th. third row: p. trotot is 1st, f. morinet is 2nd, d. boche is 4th, j. bell is 6th. last row: r. gherardi is 1st, h. goebel is 2nd, a. aguzzi is 5th, h. budka is 6th. in april 1991, delegates from the national neuropathological societies of the european community met in paris as the future existence of neuropathology in europe appeared compromised. i had proposed that the meeting took place in my department as paris was easily accessible from different countries. the delegates were the following: professor dr. h. h. goebel, professor dr. w. schlote, professor dr. w. wechsler (germany), professor r. cotrufo, professor n. rizzuto, professor d. schiffer (italy), dr. d. troost (the netherlands), professor d. i. graham, dr. janice r. anderson, professor r. o. weller (united kingdom), professor f. gray, professor j. j. hauw, professor j. f. pelissier (france); dr. s. j. balogiannis (greece) had apologized. professor david graham chaired this first session and asked a representative from each national society to give an account of the practice and status of neuropathology in their country, including training, examinations and accreditation. that was pivotal for the future creation of euro-cns. figure 3. euro-cns meeting. upper part: the author and d. graham lower part (from left to right): h. goebel is 1st, the author is 2nd, n. rizzuto is 3rd. in 1992 david graham was appointed as the representative and the chairman of our group. he went to brussels and obtained to have a representation on the board of pathology. our small group worked a lot and met with other members of the european community who had become eu (european union) and efta countries (european free trade association) including austria, finland and switzerland. the confederation of the neuropathological societies or euro-cns, term proposed by roy weller, was established in june 1993, including delegates and deputies. a constitution of euro-cns was written by david graham and janice anderson in 1993 and multiple sessions involved the research inventory, the rules of training and examination, the clinical practice. a logo was created. in 1994, david graham was elected president of euro-cns to whom i succeeded, when i was organizing the vth congress of neuropathology in paris, under the auspices, for the first time, of euro-cns. to summarize a long process, in march 1997, there was an official recognition of the subspeciality of neuropathology by the management council of uems (union européenne des médecins spécialistes). it needed to be reactivated in 2003 but was rejected in 2004. in 2005, the european parliament voted that recognition must comply with the 2/5 rule, that is in 10 countries out of 25 (25 at the time). we finally never succeeded to obtain the extension of the recognition of neuropathology at european level and it only continued at a national level when previously established. we probably should have had more support from our clinical colleagues. details were reported in an overview on neuropathology in europe (2006). i would like to pay tribute to dirk troost for his instrumental role in the organization of exams and courses as well as hans goebel and to ilja huang, the essential euro-cns secretary. nowadays, neuropathology is recognized only in a few countries but euro-cns is still active, organizes courses and examinations, and patronizes every four years the european congress whose abstracts are published in clinical neuropathology. figure 4. european meeting of neuropathology. upper part (from left to right): p. kleihues is 1st, the author is 2nd, j. artigas is 3rd, g. gosztonyi is 4th. lower part: the author is 1st, roy weller is 2nd, f. gray is 3rd. after my retirement, daily life was not easy. suddenly my timetable had changed. i am not exactly a housewife having been so active professionally. i went to romania with professor h. goebel to teach neuropathology, invited by professor m. alexianu, under the auspices of euro-cns. i started the inventory of the slides of the dejerine foundation, which led me to publish historical reports about this foundation, the life of augusta dejerine-klumpke, the original slides of facioscapulohumeral myopathy and dejerine-roussy syndrome. i have updated the history of pick’s disease. fortunately, dr. j. p. deslys, director of the neuroprion sepia unit, françois jacob institute, at cea proposed to join his group to study experimental prion diseases. i was back to the lab! he wished to have someone with a clinical and experimental experience. thus, i have a very constructive exchange with the members of the team, biochemists, veterinarians, research engineers, technicians, of very different backgrounds and ages from mine. these fruitful intergenerational confrontation of concepts with a wide range of extremely diverse experiences gave me the opportunity to better understand the strains, the transmissions and to publish, recently a paper which, i believe, provides some compelling data to outstanding questions. figure 5. department of pathology hôpital lariboisière paris. the author is in the middle of the front row and m. polivka is in the last row, the 5th from left to right. i feel you cannot do this job if you are not fully engaged in the research. i had the privilege to practice neuropathology at a golden age when there were too many silos. i believe it would be difficult for me to focus exclusively on a specific disease like oncology, although i have studied a lot of glial tumors, lymphomas, pituitary adenomas now almost exclusively dependent on molecular biology. i had chosen serge brion as a mentor because he was not always looking for what was exactly in mainstream thinking. having a deep understanding of the existing knowledge is key but combining different disciplines with new way of thinking often enables to address challenging unresolved biomedical issues and go to the next level. moreover, even if the incents of administration promote overconcentration between hospitals, it is also necessary to avoid any situation of monopoly which leads to a single thought whereas diversity is always a factor of progress. next september, i shall be 85 years old. from my internship until now, i have been practicing neuropathology in a life science laboratory, which i feel a real gift and has brought me a lot of happiness. references mikol j, weller r: neuropathology in europe: an overview. clinical neuropathology: 2006-25;7-13. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. inhibiting the mitochondrial pyruvate carrier does not ameliorate synucleinopathy in the absence of inflammation or metabolic deficits feel free to add comments by clicking these icons on the sidebar free neuropathology 1:33 (2020) original paper inhibiting the mitochondrial pyruvate carrier does not ameliorate synucleinopathy in the absence of inflammation or metabolic deficits wouter peelaerts1,2*, liza bergkvist1,#*, sonia george1, michaela johnson1, lindsay meyerdirk1, emily schulz1, jennifer a. steiner1, zachary madaj3, jiyan ma1, katelyn becker1, k. peter r. nilsson4, jerry r. colca5 and patrik brundin1 1 center for neurodegenerative science, van andel institute, grand rapids, mi, usa. 2 ku leuven, laboratory for neurobiology and gene therapy, dept. of neurosciences, leuven, belgium. 3 bioinformatics and biostatistics core, van andel institute, grand rapids, mi, usa. 4 department of physics, chemistry and biology, linköping university, linköping, sweden. 5 metabolic solutions development company, kalamazoo, mi, usa. # current affiliation: dept. of neurobiology, care sciences and society, karolinska institute, stockholm, sweden. * these authors contributed equally to this work. corresponding author: patrik brundin · center for neurodegenerative science · van andel institute · grand rapids, mi · usa patrik.brundin@vai.org submitted: 01 october 2020 accepted: 21 november 2020 copyedited by: jerry j. lou published: 25 november 2020 https://doi.org/10.17879/freeneuropathology-2020-3049 additional resources and electronic supplementary material: supplementary material keywords: parkinson’s disease, synuclein, neurodegeneration, metabolism, synucleinopathy abstract epidemiological studies suggest a link between type-2 diabetes and parkinson’s disease (pd) risk. treatment of type-2 diabetes with insulin sensitizing drugs lowers the risk of pd. we previously showed that the insulin sensitizing drug, msdc-0160, ameliorates pathogenesis in some animal models of pd. msdc-0160 reversibly binds the mitochondrial pyruvate carrier (mpc) protein complex, which has an anti-inflammatory effect and restores metabolic deficits. since pd is characterized by the deposition of α-synuclein (αsyn), we hypothesized that inhibiting the mpc might directly inhibit αsyn aggregation in vivo in mammals. to answer if modulation of mpc can reduce the development of αsyn assemblies, and reduce neurodegeneration, we treated two chronic and progressive mouse models; a viral vector-based αsyn overexpressing model and a pre-formed fibril (pff) αsyn seeding model with msdc-0160. these two models present distinct types of αsyn pathology but lack inflammatory or autophagy deficits. contrary to our hypothesis, we found that a modulation of mpc in these models did not reduce the accumulation of αsyn aggregates or mitigate neurotoxicity. instead, msdc-0160 changed the post-translational modification and aggregation features of αsyn. these results are consistent with the lack of a direct effect of mpc modulation on synuclein clearance in these models. introduction synucleinopathies are age-related neurodegenerative diseases characterized by α-synuclein (αsyn) protein misfolding, inflammation and metabolic deficits. people with parkinson’s disease (pd), dementia with lewy bodies (dlb) and multiple system atrophy (msa) present with peripheral and central synucleinopathies that coincide with a wide range of non-motor (e.g., hyposmia, constipation and rapid eye movement sleep behavior disorder) and classical motor symptoms1. the disease mechanisms underlying synucleinopathies are not completely understood, but evidence suggests that mitochondrial dysfunction, neuroinflammation and altered protein homeostasis all play a role, as reviewed in detail elsewhere2. epidemiological studies have indicated a link between pd and type 2 diabetes3-5. type 2 diabetes is associated with a 38% increased risk of developing pd3,4. on a molecular level, both people with pd and those with type 2 diabetes have similar metabolic abnormalities such as mitochondrial dysfunction and insulin resistance5. given these links between pd and metabolic dysfunction, clinical trials in pd were initiated to test drugs already approved for the treatment of diabetes. in a randomized, double-blinded placebo-controlled trial with the brain penetrant insulin sensitizing glucagon-like peptide 1 (glp1)-agonist exenatide, it was shown that exenatide treatment was associated with a reduced decline in motor dysfunction in pd patients6. specifically, once-weekly injections of the slow release form of exenatide for 48 weeks resulted in improvements in the mds-updrs (part iii)-defined primary endpoint with effects persisting after a 12-week wash out period6. in a post hoc analysis evaluating non-motor symptoms, secondary end points such as patient and observer-led scales assessing mood also showed positive effects from the treatment7. exenatide is currently in a phase iii trial in 200 pd patients who will be treated for 2 years, with the primary endpoint focused on disease modification (clinicaltrials.gov identifier nct0423296). a second insulin sensitizer that has been associated with a lower risk of pd is pioglitazone3,4. however, when evaluated in a phase 2 clinical pd trial, pioglitazone did not have any significant effect on disease progression as measured via msd-updrs (part iii)8. pioglitazone belongs to a class of anti-diabetic drugs, termed thiazolidinediones or tzds, and was believed to exert its anti-diabetic effects via activation of the transcription factor peroxisome proliferator-activated receptor-γ (pparγ). due to strong binding of pioglitazone to pparγ the drug has several negative side effects. as a result, pparγ-sparing tzds, including msdc-0160 were developed. interestingly, when compared to pioglitazone, msdc-0160 has similar anti-diabetic effects but it does not activate pparγ, indicating that tzds act on a different receptor to elicit effects. the primary target was later found on the inner membrane of mitochondria and was identified as the previously unknown mitochondrial pyruvate carrier (mpc)9. because of the link between tzds and a lowered associated risk of pd, msdc-0160’s limited side effects and its bioavailability in brain, we previously tested it in cell and animal models of pd and found that msdc-0160 is neuroprotective10. specifically, msdc-0160 protects against dopaminergic cell loss in the substantia nigra in neurotoxin-challenged mice and in a genetic mouse model with hemizygous loss of the transcription factor engrailed 1 (en1), which is believed to entail mitochondrial deficits11. we also observed a mitigation of neuron death of msdc-0160 treatment in caenorhabditis elegans (c. elegans) in which we over-expressed αsyn. taken together, we hypothesized that the beneficial effects of msdc-0160 were due to its anti-inflammatory effects and its ability to normalize metabolic deficits. the effects of msdc-0160 on αsyn aggregation in progressive, chronic pd models have previously not been investigated. in this study, we therefore evaluated msdc-0160 in two different α-syn based rodent models of pd; 1) a rat model with adeno-associated virus (aav) vector over-expression of human αsyn and 2) a mouse αsyn pre-formed fibril (pff) seeding model. unexpectedly, we found that msdc-0160 increased the levels of aggregated αsyn in the aav-overexpression model, possibly via increased oxidation of soluble αsyn. in the pff seeding model, msdc-0160 also increased αsyn burden transiently; a significant difference between the treatment groups was observed five weeks after pff injection, but it was not present at the later 13-week timepoint. we examined both models further for inflammatory and lysosomal markers, and found them both to be unaltered, even in the presence of robust αsyn pathology. we conclude that while neuroprotective actions of msdc-0160 may occur via restoring inflammatory and metabolic deficits, we could not demonstrate effects on protein aggregation or protein spreading in these rodent models of synucleinopathies. methods animals twelve-week-old male and female c57bl/6j wildtype (wt) mice were sourced from the jackson laboratory. eight-week old female rats were obtained from charles river. animals were housed with a maximum of four mice or two rats per cage under 12-h light/12-h dark cycles with free access to food and water. for a direct comparison of msdc-0160 and pioglitazone biodistribution of their metabolites in vivo, msdc-0160 and pioglitazone were administered via oral gavage for 3 days for a dose of 30mg/kg/day. collection of plasma, csf and brain mitochondria was performed after day 3. for the chronic administration of msdc-0160, mice and rats were fed a diet of chow formulated to deliver msdc-0160 (30 mg/kg) or control chow starting at 1 week after stereotactic surgery. mice were euthanized at two different time points: one and three months after pbs/pff injections. rat were euthanized four months after viral transduction. the housing of animals and all procedures were performed in accordance with the guide for the care and use of laboratory animals (united states national institutes of health) and were approved by the van andel research institute's animal care and use committee. pff production and ob injections mouse αsyn pffs were produced as described previously12. before surgery, pffs were prepared by the sonication of αsyn aggregates in a water-bath cup-horn sonicator for four min (qsonica, q700 sonicator, 50% power, 120 pulses 1 s on, 1 s off). mice were anesthetized with isoflurane/oxygen and injected unilaterally in the right olfactory bulb with either 0.8 μl of pff (5 μg/μl; n=36, 18 females/males) or 0.8 μl of pbs as a control (n=36, 18 females/males). coordinates from bregma: ap: + 5.4 mm; ml: +/0.75 mm and dv: 1.0 mm from dura. injections were made at a rate of 0.2 μl/min using a glass capillary attached to a 10 μl hamilton syringe. after injection, the capillary was left in place for three min, before being slowly removed. viral vector production and nigral surgeries raav2/5-cmvsyn-human-αsyn and raav2/5-cmvsyn-gfp production was performed as described previously13. genome copies were determined via qpcr and two viral vector titers were used for injection into the rat sn. nigral injections were performed under general anesthesia using an isoflurane/oxygen mixture. for the αsyn low dose and αsyn high dose a total of 1.2x1011gc and 2.5x1011gc were injected, respectively. as a control, 3x1011gc of gfp expressing viral vector or pbs were injected. coordinates from bregma: ap: 5.3 mm, ml: 2.0 mm and dv: 7.2 mm measured from dura14. a volume of 3 μl of viral vector was infused at a rate of 0.25 μl/min using a 30g needle and 10 μl hamilton syringe. msdc-0160 metabolite analysis mice and rats were allowed free access to chow containing either msdc-0160 (30 mg/kg) or placebo for up to four months. for mice, whole blood was collected five and 13 weeks post-surgery from the submandibular vein in 6 ml bd heparinized tubes. for rats, whole blood was collected in identical tubes via cardiac puncture four months after viral vector injection. the samples were centrifuged for 15 min at 4°c (2000 x g), followed by collection of the supernatant (plasma) which was snap frozen and stored at -80°c. for collection of brain mitochondria, brain was isolated and homogenized via dounce homogenization in a sucrose homogenization buffer (225 mm sucrose, 6 mm k2hpo4, 5 mm mgcl2, 20 mm kcl and 2 mm egta). samples were centrifuged at 750g to remove cell debris followed by a second step of centrifugation at 15,800g. the resulting pellet was resuspended in acetone to dry and collected in control plasma to store at -20°c for future analysis. frozen heparinized plasma samples were blind-coded and analyzed by charles river laboratories (mattawan, mi) for the concentrations of the msdc-0160 metabolite msdc-0037 in study protocol 1443-007b. in short, each 25 μl aliquot of standard, qc sample, or study sample was mixed with 10 μl of working internal standard solution (1,250 ng/ml in methanol/water [50/50, v/v]) and 140 μl of water. the samples were vortexed and transferred to an isolute sle plate and eluted with 1.0 ml of mtbe. the samples were evaporated and reconstituted with 100 μl of methanol followed by 100 μl of 5 mm ammonium formate in water. the samples were mixed and transferred to a clean 96-well plate. an aliquot was injected onto an lc ms/ms system for analysis. the liquid chromatography system used a macmod ace 3 c18 column, 2.1 x 50 mm (3 μm particle size) with a gradient flow consisting of 5 mm ammonium formate in water/methanol (75/25, v/v) and 5 mm ammonium formate in methanol/acetonitrile/water (72/18/10, v/v/v) at a flow rate of 500 μl/minute. the analyte, metabolite, and internal standard were detected using a sciex api 5000 triple quadrupole lc ms/ms system equipped with an esi (turboionspray®) ionization source operated in the positive ion mode. the multiple reaction monitoring transitions of the respective [m+h]+ ions were used to monitor msdc-0160 and msdc-0037 as have previously been described for clinical studies15. western blotting rats were euthanized with sodium pentobarbital (130 mg/kg; sigma) and transcardially perfused with pbs to remove blood. brains were isolated and the substantia nigra was dissected on ice using a rat brain matrix. isolated samples were snap frozen and stored at -80°c for later analysis. frozen tissue was weighed in equilibrated eppendorf tubes and homogenized in pbs 10% w/v with protease and phosphatase inhibitors (thermofisher). homogenization was performed by probe sonication at 4°c for 2 rounds of 15 s pulses at 0.5 hz with a 10% amplitude. the whole homogenates were centrifuged for 10 min at 6000 x g at 4°c. the pellet was discarded and supernatant was collected for analysis. to isolate insoluble αsyn, 20% sarkosyl was added to the whole homogenate to result in a 1% sarkosyl pbs sample that was incubated on a rotating shaker for 1 h at room temperature. after incubation, samples were centrifuged for 10 min at 6000 x g at 4°c to remove remaining cell debris. the cleared supernatant was centrifuged at 100,000 x g for 60 min and the resulting pellet was gently washed in pbs after which it was resuspended in 1% sarkosyl in pbs with protease and phosphatase inhibitors. a second centrifugation step at 100,000 x g for 10 min was performed to collect the 1% sarkosyl insoluble pellet. protein concentrations were estimated using a bca kit (thermo fisher). samples were prepared with laemmli buffer containing 2% sds, heated at 95°c for 10 min for denaturation and stored at -80°c. protein samples were separated via 4-15% sds page (biorad) and transferred to a pvdf membrane using the biorad turboblot system. pvdf membranes were blocked with 5% bsa in pbs during 30 min at room temperature. overnight incubation with primary antibody, iba-1 (1:500, wako), human αsyn (4b12, 1:1000, biolegend), pser129-αsyn (ps129, 1:5000, abcam), lamp1 (1:1000, abcam), caspase (1:500, abcam) was followed by incubation with hrp-conjugated secondary antibody (cell signaling technology). signal was detected by chemiluminescence (pico chemiluminescent substrate, thermo fisher) using a biorad imager. western blot bands were quantified using imagej software. behavioral analysis to monitor functional deficits related to nigral dopamine neuron dysfunction and death, rats were subjected to a cylinder test to evaluate spontaneous forelimb use. rats were placed in a clear glass cylinder and were filmed for a total of 30 contacts with the cylinder. percentage of forepaw use was expressed as the percentage of right forepaw touches over total number of touches. non-lesioned rats score around 50%. to examine olfactory function in mice, the buried pellet test was carried out as recently described by our laboratory in johnson et al.16 in short, the mice were fasted overnight starting three days prior to testing. throughout the experiment, their body weight was monitored and mice who lost more than 10% of their original body weight were excluded from further fasting and from the experiment. at the first day of testing, a surface test was performed, where the treat (bio-serv, fruit crunchies) was placed on top of the clean bedding. mice who were not motivated to eat the treat were excluded from further testing (all mice in this study were motivated by the treat and were thus included). for four consecutive days, the treat was buried at different locations 1 cm under the clean bedding and latency for mice to uncover it was recorded. a maximum time was set at 300 s, and if a mouse did not uncover the treat within that time frame, 300 s was recorded as its latency. the average latency to uncover the treat per animal is presented here, with nine to ten animals analyzed per group. immunohistochemistry mice and rats were euthanized with sodium pentobarbital (60 mg/ml; sigma) and transcardially perfused with room temperature 0.9% saline followed by ice-cold 4% paraformaldehyde (pfa) in 0.1 m phosphate buffer. brains were removed, post-fixed overnight at 4°c in 4% pfa and subsequently placed in 30% sucrose. brains were frozen and coronal sections of 40 μm were cut on a microtome (leica) and collected as serial tissue sections. for immunohistochemistry in mice, a series (every 240 μm) of coronal free-floating sections were stained using 1:10000 anti-pser129 αsyn primary antibody (abcam, ab51253), 1:1000 anti-neun primary antibody (millipore, ab377), 1:5000 anti-nitrated αsyn primary antibody (thermo fisher, 35-8300) or 1:800 anti-iba1 primary antibody (wako, 019-19741) over night. for immunohistochemistry in rats, a series (every 240 μm) of coronal free-floating sections of sn was stained using 1:10000 anti-pser129 αsyn primary antibody (abcam, ab51253), 1:5000 anti-nitrated αsyn primary antibody (thermo fisher, 35-8300), 1:2000 anti-th (abcam, ab137869), or 1:800 anti-iba1 primary antibody (wako, 019-19741) overnight. corresponding secondary antibodies (1:500 goat anti-rabbit/goat anti-mouse biotinylated secondary antibody (vector laboratories, ba-1000 and ba-9200) were added. the antibody signal was amplified using a standard peroxidase-based method (vectastain abc kit) and developed using a dab kit (vector laboratories). immunostained tissue sections were mounted onto gelatin coated glass slides. neunand th-immunostained slides were counter stained with cresyl violet (cv). after dehydration, slides were sealed by coverslip with cytoseal 60 mounting medium (thermo fisher scientific). for lamp2 immunostaining, using fluorescence to detect antibody binding, sections were incubated overnight with 1:1000 anti-lamp2 primary antibody (abcam, ab13524) overnight, followed by a 2 h incubation with a fluorescent secondary antibody (thermo fisher/invitrogen, alexa fluor 488). dapi was used to visualize cell nuclei. for the co-stain between h-ftaa and pser129, tissue sections were incubated with 1:1000 anti-pser129 αsyn primary antibody (abcam, ab51253) for 2h at rt followed by a 1h incubation with 1:200 secondary goat anti-rabbit antibody alexa 647 (thermo fisher, a32733) and dapi to visualize cell nuclei. finally, 0.5 μm h-ftaa was added and allowed to incubate with the tissue for 30 min at rt. fluorescently stained tissue was sealed by coverslip using hard-set antifade mounting media (vector laboratories, h-1400). a nikon a1plus-rsi laser scanning confocal microscope (nikon) was used to image these sections. pser129 quantification z-stacks of mounted αsyn pser129-stained tissue sections were captured at 20x magnification using a whole slide scanner (zeiss, axioscan z1) at a 0.22 μm/pixel resolution. extended depth focus (edf) was used to collapse the z-stacks into 2d images as they were collected. tissue thickness was set to 20 μm, z-stacks were collected at 3 μm intervals and the method used was contrast. the images were exported with 85% compression as .jpeg files. the digitized images were then uploaded aiforia™ image processing and management platform (aiforia technologies, helsinki, finland) for analysis with deep learning convolution neural networks (cnns) and supervised learning. a supervised, multi-layered, cnn was trained on annotations from digitized αsyn pser129-stained coronal slices to recognize total αsyn-positive staining. the algorithm was trained on the most diverse and representative images from across multiple pser129 datasets to create a generalizable ai model capable to accurately detecting αsyn pser129-stained profiles in slides collected by multiple investigators. ninety-one images constituted training data. the ground truth, or features of interest used to train the ai model, were annotated for each layer within the aiforia™ cloud platform and constituted input data for each cnn. the first feature layer was annotated using semantic segmentation to distinguish the total tissue from the glass slide. the second feature layer was annotated using semantic segmentation to distinguish total αsyn pser129-positive staining. features that were considered artifact (glass slide, debris) were annotated as background, and constituted additional input training data for the multi-layered cnn. the regions corresponding to the aon and the prh were outlined according to the allen mouse brain atlas (allen institute) and the cnn was used to quantify the area percentage that contained αsyn pser129-positive signal. for each brain region, the average area with αsyn pser129-staining is reported as a percentage of the whole area assayed. for the one-month time point, six animals per group were analyzed and for the three-month time point seven to eight animals per group were included in the analysis. stereology surviving dopamine neurons and total neuronal cell counts were quantified using unbiased sampling and blinded stereology. for the aav αsyn over-expression model, tyrosine hydroxylase (th), αsyn pser129 and nitrated αsyn-positive cells were counted in the snpc and snpr in a series of seven sections at an interval of six sections at 40 μm thickness (240 μm intervals). contours of the regions were drawn at 10x magnification and quantifications were performed at 60x using a 200 μm counting frame. dissector height was 12 μm with a 3 μm guard zone. the gunderson estimated m=1 error was less than 0.1 and a total of eight animals per group was counted. for the αsyn pff seeding model, neunand cv-positive cells were counted in the aon, which was outlined according to the allen mouse brain atlas (allen institute) using a 2.5x objective. the quantification of neun and cv-positive cells was performed on every 6th aon (240 μm) section using the optical fractionator probe in stereo investigator software (mbf bioscience). the counting was accomplished using a 60x oil objective, with the grid set to 200x200 and the frame set to 40x40. the dissector height used was 10 μm, with a 3 μm guard zone. the gunderson estimated m=1 error was less than 0.1 and a total of nine to ten animals per group were counted. assessment of microglia morphology microglia hydraulic radius (area/perimeter ratio) was investigated as previously described13. in short, color (rbg) images were generated using a 60x oil objective. following imaging, a custom matlab script assessed the morphology of the imaged microglia, calculating the area/perimeter ratio. for the pff seeding model, five to six animals per group were analyzed. microglia were imaged from every 6th aon section and a minimum of 30 individual microglia was analyzed per animal, with the average microglia area/perimeter ratio reported. quantification of fluorescent lamp2 signal images corresponding to the aon were captured using a 20x oil objective on a nikon a1plus-rsi laser scanning confocal microscope (nikon) in a blinded manner. the area percentage occupied by lamp2 positive signal was quantified using fiji software (nation institute of health) after an unbiased threshold was applied. the average area percentage calculated for each animal is reported here. for the pff seeding model, a total of six mice per group were analyzed. statistics all aav and pff mouse model data were analyzed using r v3.6.0 (https://cran.r-project.org/) and assumed two-sided hypothesis tests with a significance level <0.05 after benjamini-hochberg multiple testing corrections. in the pff model, neuronal cell count, iba1 and pser129 quantification were done in parallel with another treatment group (pt302, data not shown), meaning they share the same set of placebo treated animals; the multiple testing corrections take into account these additional comparisons. for experiments that analyzed both hemispheres of the brain independently (e.g. ipsilateral versus contralateral) the appropriate mixed-effects model was used (r package lme4 https://cran.r-project.org/web/packages/lme4/index.html unless otherwise noted). count data were analyzed using negative binomial regressions; mixed-effects negative binomial regressions were done using the r package glmmtmb and the ‘nbinom1’ family (https://cran.r-project.org/web/packages/glmmtmb/index.html). time-to-event data (e.g. the buried pellet test) were analyzed using cox proportional hazards regression. all other data types were analyzed via linear regression with log and square-root transformed outcomes as needed based on standard regression diagnostics (i.e. normality of residuals and homoscedasticity) (log: if αsyn pser129 and soluble αsyn; square-root: soluble αsyn pser129, insoluble human αsyn, insoluble αsyn pser129). cook’s distance was used to assess individual observations with high amounts of leverage (set as 4/(n-p-1)). for linear regressions with highly influential observations, robust regressions with mm estimation were used. zero variance data (i.e. dab αsyn pser129+, if αsyn pser129+, soluble human αsyn, and nissl+ measures across both naive and gfp injected animals), logistic regression with a firth correction was used to determine if the odds of detection versus no detection differed between groups. an equivalence interval of +/-10% was used to determine if groups had strong evidence for equivalence based on their 95% confidence intervals. results inhibiting the mpc in a viral vector-based model of human α-syn overexpression to determine the effects of inhibiting the mpc on αsyn aggregation, we used a slowly progressing viral vector-based model that overexpresses αsyn. we selected two doses of viral vector, which from here on will be referred to as ‘αsyn low’ (4.3x1013gc/ml) and ‘αsyn high’ (8.6x1013gc/ml). these two titers yield a 4and 6-fold overexpression of αsyn in rat snpc, respectively (fig. 1a, 3b). fig 1. msdc-0160 treatment does not influence nigral dopaminergic cell viability in a progressive αsyn overexpression model. a) experimental overview of the αsyn viral vector overexpression model in rat sn. b) th expression and nissl+ cells in rat sn for animals treated with placebo or msdc-0160 in control or experimental conditions. scale bar = 500 μm. c) stereological quantification of total dopamine neurons in rat snpc for ipsiand contralateral sides. the contralateral hemisphere had significantly more th cells than the ipsilateral in the αsyn high mice (p = 0.001, ~22.5% more cells ci = 10.3% 36.0%, negative binomial mixed-effect model, n = 8, s.e.m.). in the ipsilateral side, αsyn high had significantly fewer th cells than naïve, gfp and αsyn low (p = 0.031, 14.9% fewer ci = 4.8 – 23.9, *p = 0.05, 13.4% fewer 95% ci 3.1 22.6 and p = 0.0313, 15.6% fewer ci = 24.55.6, respectively, negative binomial mixed-effect model, n = 8, sem). d) stereological quantification of nissl+ cells in rat sn for ipsiand contralateral sides. for the αsyn high group, the ipsilateral side has ~ 13% fewer nissl+ cells across both treatment groups than the contralateral (p < 0.0001, 95% ci 6.4 20.1, negative binomial mixed-effect model, n = 8, sem). αsyn high shows significantly fewer nissl+ cells compared to animals injected with naive, gfp, or low amounts of αsyn (p < 0.001 for all 3, gfp: 15% fewer ci = 8% 21%, naive: 18.4% fewer ci = 12.0% 24.4%, αsyn low: 16% fewer ci = 22.2% 9.3%, negative binomial mixed-effect model, n = 8, sem). female rats were stereotactically injected in snpc at 8 weeks of age with raav2/5 cmv-syn-αsyn or raav2/5 cmv-syn-gfp expressing vector that served as a control (fig. 1a). one week post-surgery, the animals were allowed free access to chow containing either msdc-0160 (30mg/kg) or placebo for four months. in order to evaluate the uptake and distribution of msdc-0160, we analyzed the levels of exposure of msdc-0160, pioglitazone and its metabolites in rat plasma, csf and brain mitochondria 20 hours after administration (supplementary fig. 1). hydroxymitoglitazone, the active metabolite of msdc-0160, was found at higher levels, compared to pioglitazone metabolites, in plasma and csf. in brain mitochondria hydroxymitoglitazone was also found at higher levels then pioglitazone-oh or other metabolites, showing its interaction with brain mitochondria but also suggesting that the interaction of msdc-0160 metabolites with mitochondria is stronger compared to pioglitazone metabolites (supplementary fig. 1). during the time of chow administration, we did not observe any differences in animal body weight or chow consumption (supplementary fig. 2a, b). at the four-month timepoint, plasma was collected and the presence of hydroxymitoglitazone (msdc-0160 metabolite) was quantified (supplementary fig. 2c), showing the presence and metabolization of msdc-0160 at expected levels. motor behavior was examined four months after viral transduction using the cylinder test. no difference in motor behavior was apparent between any of the experimental groups, nor did we observe any statistically significant differences between placebo or msdc-0160 treated groups (supplementary fig. 3). when assessing the total number of thand nissl-positive neurons in the sn via stereology we find a significant decrease in thand nissl-positive cells between the αsyn high group and all other experimental treatment groups (fig. 1a-b), suggesting dose-dependent neurotoxic effects of αsyn expression. no cell loss was observed in the gfp group, showing that no toxic effects are due to the viral vector itself. in addition, when the snpc is separately analyzed for its anterior (bregma -4.8 mm) and posterior (bregma -6.2 mm) areas, th loss in the anterior is much more significant compared to the posterior region (supplementary fig. 4 a-d). this indicates that αsyn expression from the overexpressing αsyn vector is higher in the anterior snpc since it is adjacent to the injection site, and lower in the posterior portion, which again confirms vector-driven αsyn neurotoxic effects with no neurotoxicity observed for the gfp control group. next, we performed stereological counts of nigral cells positive for phosphorylated αsyn (αsyn pser129) (fig. 2a). unexpectedly, in the αsyn low group, animals treated with msdc-0160 show significantly more αsyn pser129+ cells compared to the placebo group (12132 versus 7918 cells, **p = 0.002, negative binomial regression with benjamini-hochberg multiple testing adjustments, sem, n = 8) (fig. 2b). no significant effect was detected in the αsyn high group between msdc-0160 and the placebo group. figure 2. a metabolic switch leads to increased αsyn pathology in rat sn. a) overview of pser129-αsyn positive cells in rat sn. scale bars for higher and lower magnification represents 450 μm and 100 μm, respectively. b) stereological quantification of αsyn pser129 positive cells in rat sn. treatment with msdc-0160 leads to a significant increase in phosphorylated cells in the αsyn low group compared to placebo (n = 8, sem, **p < 0.01 and ***p < 0.001, negative binomial regression with benjamini-hochberg multiple testing adjustments, sem, n = 8)). we further analyzed phosphorylated levels of αsyn via biochemical analyses and found that both soluble αsyn pser129 as well as 1% sarkosyl insoluble αsyn pser129 (1% p129-αsyn) are increased in the msdc-0160 treated animals compared to the placebo group (*p = 0.046, 95% ci 0.039 – 0.471, square-root linear regression, sem, n = 6 for soluble αsyn pser129 and *p = 0.013, 95% ci 0.129 – 0.691, square-root linear regression, sem, n = 6 for 1% sarkosyl insoluble αsyn pser129) (fig. 3e, f). this increase was observed in the αsyn high group between msdc-0160 and placebo animals whereas a similar trend was noticeable in the αsyn low group between treatment conditions (fig. 3e, f). this indicates that administration of msdc-0160 results in increased αsyn aggregation in the slowly progressing viral vector-based model that overexpresses αsyn. figure 3. αsyn aggregation is increased in rat sn after inhibition of the mpc. a) inhibition of the mpc has no effect on viral vector-mediated expression of gfp. b) detection of total levels of αsyn in naïve and experimental conditions. αsyn overexpression leads to a 4and 6-fold increase in αsyn low and high conditions compared to naïve rats. c,d) isolation of sequentially extracted soluble and 1% sarkosyl insoluble human αsyn (1% hu-αsyn) shows no significant differences in aggregated αsyn after treatment with msdc-0160. e,f) inhibition of mpc leads to significantly increased levels of soluble and 1% sarkosyl insoluble αsyn pser129 (1% p129-αsyn) in the αsyn high group (n = 6, sem, *p < 0.05 square-root linear regression, sem, n = 6). g) representative western blot figures for different conditions tested. αsyn has been shown to bind the membrane of mitochondria17. due to its membrane curvature and the presence of the negatively charged cardiolipin in the mitochondrial outer membrane, αsyn has a strong binding affinity for mitochondria18. aggregated αsyn can disrupt mitochondrial membrane integrity and lead to toxicity19. to examine why inhibiting the mpc results in more αsyn aggregation, we investigated if altering mitochondrial function, or changing oxidative environment via msdc-0160 affects αsyn binding or αsyn oxidation status. stereological quantifications of the snpc for nitrated ni-αsyn (ni-αsyn) shows that in the αsyn low group, inhibiting the mpc with msdc-0160 leads to more ni-αsyn (*p = 0.021, 95% ci 11.5% – 111.7% more after negative binomial regression with benjamini-hochberg multiple testing adjustments), indicating that inhibiting the mpc leads to increased oxidation of αsyn (fig. 4). since it has been shown that oxidation of αsyn can affect αsyn aggregation, it raises the possibility that in this αsyn overexpression model a metabolic switch promotes the formation of insoluble assemblies of αsyn by increasing αsyn oxidation. figure 4. ni-αsyn increases upon msdc-0160 treatment. stereological quantification of ni-αsyn positive cells in rat sn. treatment with msdc-0160 leads to a significant increase in nitrated cells in the αsyn low group compared to placebo (*p < 0.05 via negative binomial regression with benjamini-hochberg multiple testing adjustments, n = 8, sem) since our previous work has shown that inhibiting the mpc has strong anti-inflammatory and metabolic effects, we further investigated if our αsyn viral vector model was lacking these features, which could explain the absence of any apparent therapeutic benefits. we examined changes in microglial and lysosomal markers at the four-month timepoint and found no changes in microglial activation (fig. 5a-b, e) and no changes in markers of autophagy (lamp1 and cathepsin d) in any of the groups examined (fig. 5c, f-g). taken together, in absence of metabolic or lysosomal deficits and microglial changes (at least at these later time points), a metabolic switch, by inhibiting the mpc, does not have a beneficial effect on αsyn aggregation. figure 5. absence of inflammatory or autophagy deficits in αsyn overexpressing animals. a) overview of microglial morphology via iba-1 staining in rat sn shows no microglial activation in αsyn overexpressing conditions. scale bar = 50 μm. b) quantification of microglial hydraulic radius as a measure of microglial ramification and activation (n = 8, sem). c) representative western blot images of inflammatory and autophagy markers in sn. d) iba-1 and e) gfap markers show no signs of inflammatory activation (n = 6, sem). d) cathepsin-d and e) lamp1 expression is unaltered in αsyn overexpressing animals (n = 6, sem). inhibiting the mpc in a mouse pff seeding model next, to investigate the effects of inhibiting mpc on αsyn aggregation, we examined the effects of msdc-0160 on αsyn propagation. we used a previously established seeding model where αsyn pffs are unilaterally injected into the ob of 12-week-old wild type mice. over time, αsyn pathology will propagate via intraneuronal connections and cause pathology in upstream regions19,20. one-week post pff or pbs injection into the ob, the mice were allowed free access to chow containing either msdc-0160 or placebo (fig. 6a). during the time of chow administration, we did not observe any differences in animal body weight or chow consumption between groups (supplementary fig. 5a, b). at 5and 13-weeks post-surgery, plasma was collected and the presence of hydroxymitoglitazone (msdc-0160 metabolite) was quantified (supplementary fig. 5c), showing detectable levels of the metabolite in treated groups. figure 6. a metabolic switch increases αsyn pathology load and propagation in mouse aon and prh. a) experimental timeline; at 12 weeks of age c57bl/6j mice were injected with pffs or pbs and starting at one-week post-surgery they had free access to msdc-0160 or placebo containing chow until the 5or 13-week endpoint. quantification of αsyn pser129 positive signal in b) the aon and c) the prh after 5 or 13 weeks of msdc-0160 or placebo treatment. msdc-0160 treated mice had significantly more αsyn pser129 positive stain at 5 weeks post-surgery than the placebo group (1.8 times more; p=0.02, 95% ci 1.176 2.824, beta mixed-effects regression). there is also weak evidence that this persists at the 13-week timepoint (p=0.085, 95% ci 0.986-1.92, beta mixed-effects regression). c) quantification of αsyn pser129 positive signal in the prh after one and three months of msdc-0160 or placebo treatment. d) representative images of αsyn pser129 staining in d) the ipsilateral aon (scalebar = 130 μm) and e) the prh (scalebar = 100 μm). unilateral pff injections have previously been shown to impair olfactory function in mice20. olfactory function was thus examined 13 weeks post-surgery using the buried pellet test. no significant difference was observed between the pbs and pff groups, regardless of whether the mice had been treated with placebo or msdc-0160 (supplementary fig. 6a). when assessing the total number of neun and cv-positive cells in the aon 13 weeks post-surgery via stereological quantification, we found no significant differences between any of the experimental groups (supplementary fig. 6b & c). immunohistological assessment at 5and 13-weeks post-surgery revealed a significantly higher percentage area containing pathological αsyn (αsyn pser129) in the aon than the prh, regardless of time point and treatment group (8.5-16 times higher; all p ≤ 0.001) (fig. 6b-e). the same was seen for the ipsilateral side compared to the contralateral side (65-78% less on the contralateral side; p ≤ 0.001). from 5 to 13 weeks post-surgery, αsyn pser129 increased significantly for msdc-0160 and placebo treated animals, which both gained ~99.2% and 163.2% of their αsyn pser129 immunopositively stained area from week 5 to week 13, respectively in the contralateral aon (p = 0.016 and < 0.001, respectively, 95% ci = 19.2 233.3 and 55.8 344.4). in addition, placebo-treated animals had a significant increase of pathology in the ipsilateral aon from the 5 to 13-week timepoint (p=0.016, 95% ci 18.3% 200%). this was not observed for msdc-0160 treated mice. when comparing treatment groups, msdc-0160 treated mice had significantly more pser129 positive signal at 5 weeks post-surgery than the placebo group (1.8 times more; p=0.02 95% ci 1.16 2.90). there is also weak evidence that this persists at the 13-week timepoint (p=0.085, 95% ci 0.98-1.95). no ni-αsyn was detected in the pff seeding model at the 13-week timepoint (supplementary fig. 7). for detection of potential amyloid (β-sheet rich) structures in the pff seeding model and the aav overexpression model, we used a luminescent conjugated oligothiophene (lco). lcos are small molecules with a flexible thiophene backbone that upon binding to amyloid structures become fluorescent21. one such lco, which has previously been shown to bind aggregates in tissue samples from patients suffering from systemic amyloidosis, as well as a variety of other disease-associated protein aggregates, is h-ftaa21 in the aon at 13 weeks post-surgery we observed ring-like h-ftaa positive structures (fig. 7) in the pff model. these overlapped with αsyn pser129 antibody staining, indicating that the αsyn pathology detected in this model is β-sheet rich with amyloid properties. on the contrary, we did not find a h-ftaa positive signal in the αsyn high group for the aav overexpression model. this highlights differences in αsyn assembly states several weeks after surgery in the two pd rodent models. figure 7. h-ftaa positive protein aggregates detected in the pff seeding model but not the aav overexpression model. representative images of the aon and the sn for the pff model and the aav overexpression model, respectively. the h-ftaa positive signal (green) detected in the aon 13 weeks following surgery co-localizes with the αsyn pser129 signal (pink). dapi was used to visualize the cell nuclei (blue), scale bar = 10 μm and 3 animals per group were analyzed. given the absence of any therapeutic benefits in the pff model, we again investigated the presence of inflammation via measuring the hydraulic radius (area/perimeter ratio) of microglia in the aon 13 weeks post-surgery. when we compared the injected side with the non-injected side, we found no significant change in microglia morphology (fig. 8a, b). furthermore, we did not detect any significant difference between pbs and pffs groups or differences between animals treated with placebo or msdc-0160. in addition, by quantifying lamp2-positive staining we do not observe any lysosomal deficits in the aon 13 weeks post-surgery (fig. 8c, d). taken together, in absence of lysosomal deficits and microglial changes, a metabolic switch, by inhibiting the mpc, does not reduce accumulation of αsyn pser129 in mice injected with pffs in the olfactory bulb. figure 8. absence of inflammatory or autophagy deficits 13-weeks post pff surgery. a) representative images of iba-1 positive staining in the ipsilateral aon, scale bar = 50 μm. b) quantification of microglial hydraulic radius as a measure of microglial ramification and activation (n = 5-6/group, sem), shows no microglial activation following pff injection. c) representative images of lamp2 positive staining (green) in the ipsilateral aon. dapi (blue) was used to visualize the cell nuclei. scale bar = 10 μm d) fluorescent lamp2 quantification after adding adaptive triangle thresholding in the aon (n = 6, sem) discussion improving metabolism, reducing inflammation and inhibiting the aggregation or the spread of pathological αsyn are considered promising therapeutic targets for the treatment of pd. through preclinical and clinical work, it has been suggested that anti-diabetic drugs might hold this promise22. we have previously shown that msdc-0160, a type 2 diabetes insulin sensitizer, can ameliorate pathology in an acute (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, mptp) and slowly progressive, genetic (engrailed1+/-) model of pd and that these effects occurred via modulation of the mpc and its downstream effect involving the mtor pathway10. since the direct effects of inhibiting mpc in chronic models of αsyn aggregation had not been studied, we asked if msdc-0160 could lower the levels of αsyn in two different, chronic and progressive αsyn-based animal models of pd. in our first model, αsyn is expressed via viral vector delivery in the rat sn resulting in aggregation and progressive accumulation of insoluble αsyn. after four months, no significant motor abnormalities with moderate neurodegeneration were observed but, unexpectedly, animals that were treated with msdc-0160 exhibited increased levels of aggregated αsyn. in the second model, pffs were injected in the ob of mice and allowed to seed and spread to connected regions. five weeks after pff injection, we observed an increase in αsyn pathology burden when mice were treated with msdc-0160. this effect was transient and at the later, 13-week timepoint no significant difference between the two treatment groups could be observed. both the αsyn viral vector and the αsyn pff seeding models are based on the introduction of exogenous αsyn that leads to progressive accumulation of phosphorylated and misfolded αsyn over time. the viral vector model results in local accumulation of highly concentrated but soluble αsyn that assembles into insoluble αsyn, whereas the pff model is based on the introduction of aggregated, seeding-potent αsyn that relies on the endogenous protein to template and spread via anatomically connected regions. to date, there are no studies that have directly compared the assembly states of the αsyn in the intraneuronal inclusions that remain after several weeks in an overexpression versus a seeding model, but it appears that inhibiting the mpc has different outcomes in these two models. this tentatively suggests i) that the conformational state or aggregation state of αsyn might be different in the two models and ii) that the effects of inhibiting the mpc depends on the assembly state of the protein. we observed a disparity in αsyn assembly states between the viral vector-based and the seeding model in multiple assays. first, we used detection of nitrated αsyn to determine that αsyn is oxidized in the viral vector-based model. a previous study showed that αsyn can bind directly to the mitochondrial import protein tom20, which leads to increased mitochondrial respiration and ros production17. we now find that oxidized αsyn species are abundant in neurons in the viral vector model, but undetectable in the pff model. we postulate that by altering the tca cycle when treating mice overexpressing αsyn with msdc-0160, the oxidation of αsyn is increased. this suggests that alterations in mitochondrial metabolism might post-translationally modify αsyn and make it more aggregation prone, when the protein is highly concentrated even though it is not known how exactly a metabolic switch would take place in αsyn overexpressing neurons nor how it would affect αsyn aggregation during anaplerosis. furthermore, we propose that elevated levels of soluble αsyn are required for these changes to take place, since we did not observe similar effects in the pff seeding model where the endogenous levels of αsyn are normal and aggregates are formed via an induced seeding mechanism. since the conformational state of αsyn is closely linked to its function23, it is possible that elevated levels of monomeric αsyn lead to changes in assembly state or gain of post-translational modifications resulting in an aberrant interaction with mitochondria. the binding affinity of αsyn is higher for mitochondrial membranes due to the unique presence of cardiolipin in mitochondria18. lipid interaction is an important catalyst for soluble αsyn to convert into aggregated αsyn24 and this conversion has been reported to lead to release of mitochondrial nitric oxide and increased oxidative stress25. αsyn can disrupt membrane integrity and cause mitochondrial damage and degeneration of dopaminergic neurons17,26. recombinant nitrated species of αsyn injected in rat sn have been shown to be more toxic than unmodified αsyn27. in msdc-0160 treated mice, we did indeed observe greater loss of dopamine neurons in the anterior portion of the sn, which was the region where viral vector-mediated expression of αsyn was the highest. multiple molecules of nitrated αsyn have been shown to form soluble assemblies that are off pathway to aggregation and relatively unstructured28. in order to determine if structured amyloid forms of αsyn were present in our animal models, we used the conformation sensitive lco h-ftaa probe29. lcos are amyloid-conformation-specific dyes that specifically bind to amyloid αsyn in brain tissue and csf30. a positive signal therefore indicates the presence of well-structured, β-sheet rich aggregated αsyn. in addition, h-ftaa has been used to distinguish protein inclusion bodies in sporadic inclusion body myositis (s-ibm) and liver diseases21. upon treatment with h-ftaa, we observed a strong positive signal in the aon of animals injected with pffs but no signal in the snpc of animals overexpressing αsyn following viral vector injection. the fluorescent signal was localized to neurons and the morphology was lewy body-like with aggresomal structures that resembled the intracellular distribution we observed when staining for αsyn pser129. this illustrates that the two αsyn animal models also differ in that the αsyn assembly states in these models are distinct. these differences were recently discussed by gómez-benito and colleagues; αsyn species from the aav overexpression model lack seeding activity while the pff model recapitulates αsyn propagation with the accumulation of lewy body-like structures31. in summary, msdc-0160 does not reduce αsyn aggregation in the model where a viral vector is used to overexpress the protein in the nigrostriatal system. on the contrary, the inhibition of mpc results in a slight increase in αsyn protein content. in the model using injection of pffs to trigger a cascade of αsyn aggregate propagation in the olfactory system, msdc-0160 exhibits no effect on the progressive development of synucleinopathy in the olfactory system. in experimental paradigms that involve more acute neuroinflammation, we previously showed that msdc-0160 alleviates the inflammatory response in vitro and in vivo10. however, in the pff and viral vector αsyn models, we did not detect changes in inflammation or evidence for autophagy deficits at the survival times we studied. we conclude that attenuation of the mpc in rodent models that do not exhibit inflammation and limited autophagy does not result in a reduction of introduced αsyn. acknowledgements research reported in this publication was supported by the cure parkinson’s trust award number 42-40384-1 (p.b.). w.p. acknowledges a post-doctoral fellowship from fulbright, idt technologies and fwo flanders. we thank the staff of the vivarium of van andel institute for caring for the mice and rats used in this study. author contributions w.p. performed rat studies (stereotaxic surgery and behavioral, biochemical and immunohistochemical analysis) and wrote the manuscript. l.b. performed mouse olfactory testing, neun immunostaining and stereology, pser129 immunostaining and analysis, ni-αsyn staining, lamp2 immunostaining and analysis, iba-1 immunostaining and analysis, h-ftaa/pser129 co-immunostaining and imaging and wrote the manuscript. s.g. performed pilot studies leading to the final conceptualization of the aav overexpression model study. m.e.j. performed pser129 immunostaining. j.s. organized the manufacturing of the msdc-0160 chow, metabolic detection, plasma, csf and brain mitochondria collection and provided advice. l.m. performed unilateral pff surgery, mouse perfusions and plasma, csf and brain mitochondria collection. e.s. performed unilateral pff surgery, mouse perfusions, plasma, csf and brain mitochondria collection and ni-αsyn staining optimization. z.m. performed the statistical analysis. j.m. provided the pffs. k.b. performed pff manufacturing. j.r.c. provided the compound (msdc-0160), expertise and advice. p.b. conceptualized this study and provided expertise and advice. all authors reviewed and edited the manuscript. disclosure p.b. receives commercial support as a consultant from calico life sciences, curasen, idorsia pharmaceuticals, lundbeck a/s, abbvie, fujifilm-cellular dynamics international, and axial biotherapeutics. he has received commercial support for research from lundbeck a/s and roche. he has ownership interests in acousort ab. j.r.c. is the cofounder and significant owner of metabolic solutions development company (msdc), which is currently developing msdc-0160 as a potential treatment for alzheimer’s disease. msdc-0160 is protected by several patents, including us 8389556 b2 and ep 2001468 b1, which are assigned to msdc. all other authors declare no additional competing financial interests. references 1. stern, m. b., lang, a. & poewe, w. toward a redefinition of parkinson's disease. mov disord. 27, 54–60 (2012). 2. park, j.-s., davis, r. l. & sue, c. m. mitochondrial dysfunction in parkinson’s disease: new mechanistic insights and therapeutic perspectives. 1–11 (2018). doi:10.1007/s11910-018-0829-3 3. brakedal, b. et al. glitazone use associated with reduced risk of parkinson's disease. mov disord. 54, s21–6 (2017). 4. brauer, r. et al. glitazone treatment and incidence of parkinson’s disease among people with diabetes: a retrospective cohort study. plos med 12, e1001854–16 (2015). 5. biosa, a., outeiro, t. f., bubacco, l. & bisaglia, m. diabetes mellitus as a risk factor for parkinson’s disease: a molecular point of view. 1–10 (2018). doi:10.1007/s12035-018-1025-9 6. athauda, d. et al. exenatide once weekly versus placebo in parkinson's disease: a randomized, double-blind, placebo-controlled trial. the lancet 390, 1664–1675 (2017). 7. athauda, d. et al. what effects might exenatide have on non-motor symptoms in parkinson’s disease: a post hoc analysis. jpd 8, 247–258 (2018). 8. ninds exploratory trials in parkinson disease net-pd fs-zone. pioglitazone in early parkinson’s disease: a phase 2, multicenter, double-blind, randomized trial. the lancet neurology 14, 795–803 (2015). 9. chen, z. et al. insulin resistance and metabolic derangements in obese mice are ameliorated by a novel peroxisome proliferator-activated receptor γ-sparing thiazolidinedione. j. biol. chem. 287, 23537–23548 (2012). 10. ghosh, a. et al. mitochondrial pyruvate carrier regulates autophagy, inflammation, and neurodegeneration in experimental models of parkinson’s disease. science translational medicine 1–18 (2016). 11. sonnier, l. et al. progressive loss of dopaminergic neurons in the ventral midbrain of adult mice heterozygote for engrailed1. j. neurosci. 27, 1063–1071 (2007). 12. becker, k. et al. detecting alpha synuclein seeding activity in formaldehyde-fixed msa patient tissue by pmca. mol neurobiol 55, 1–10 (2018). 13. george, s. et al. microglia affect α-synuclein cell-to-cell transfer in a mouse model of parkinson’s disease. mol neurodegeneration 14, 1–22 (2019). 14. van der perren, a. et al. development of an alpha-synuclein based rat model for parkinson's disease via stereotactic injection of a recombinant adeno-associated viral vector. jove 1–8 (2016). doi:10.3791/53670 15. colca, j. r. et al. clinical proof-of-concept study with msdc-0160, a prototype mtot-modulating insulin sensitizer. clin pharmacol ther 93, 352–359 (2013). 16. johnson, m. e. et al. deficits in olfactory sensitivity in a mouse model of parkinson’s disease revealed by plethysmography of odor-evoked sniffing. scientific reports 1–13 (2020). doi:10.1038/s41598-020-66201-8 17. di maio, r. et al. α-synuclein binds to tom20 and inhibits mitochondrial protein import in parkinson's disease. science translational medicine 8, 342ra78–342ra78 (2016). 18. ryan, t. et al. cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein. nature communications 1–17 (2018). doi:10.1038/s41467-018-03241-9 19. winklhofer, k. f. & haass, c. mitochondrial dysfunction in parkinson's disease. bba molecular basis of disease 1802, 29–44 (2010). 20. rey, n. l. et al. widespread transneuronal propagation of α-synucleinopathy triggered in olfactory bulb mimics prodromal parkinson’s disease. the journal of experimental medicine 213, 1759–1778 (2016). 21. klingstedt, t. et al. synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates. org. biomol. chem. 9, 8356–25 (2011). 22. cheong, j. l. y., de pablo-fernandez, e., foltynie, t. & noyce, a. j. the association between type 2 diabetes mellitus and parkinson’s disease. jpd 10, 775–789 (2020). 23. peelaerts, w. & baekelandt, v. α-synuclein strains and the variable pathologies of synucleinopathies. j. neurochem. 256–274 (2016). doi:10.1111/(issn)1471-4159/homepage/special_issues.htm 24. galvagnion, c. et al. lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation. nat chem biol 11, 229–234 (2015). 25. parihar, m. s., parihar, a., fujita, m., hashimoto, m. & ghafourifar, p. mitochondrial association of alpha-synuclein causes oxidative stress. cell. mol. life sci. 65, 1272–1284 (2008). 26. rocha, e. m., de miranda, b. & sanders, l. h. alpha-synuclein: pathology, mitochondrial dysfunction and neuroinflammation in parkinson’s disease. neurobiology of disease 109, 249–257 (2018). 27. yu, z. et al. nitrated α-synuclein induces the loss of dopaminergic neurons in the substantia nigra of rats. plos one 5, e9956–17 (2010). 28. uversky, v. n. et al. effects of nitration on the structure and aggregation of α-synuclein. molecular brain research 134, 84–102 (2005). 29. shirani, h. et al. a palette of fluorescent thiophene-based ligands for the identification of protein aggregates. chem. eur. j. 21, 15133–15137 (2015). 30. shahnawaz, m. et al. discriminating α-synuclein strains in parkinson’s disease and multiple system atrophy. nature 1–23 (2020). doi:10.1038/s41586-020-1984-7 31. gómez-benito, m. et al. modeling parkinson’s disease with the alpha-synuclein protein. front. pharmacol. 11, 1–15 (2020). copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. differentiation of primary cns lymphoma and glioblastoma using raman spectroscopy and machine learning algorithms feel free to add comments by clicking these icons on the sidebar free neuropathology 2:26 (2021) original paper differentiation of primary cns lymphoma and glioblastoma using raman spectroscopy and machine learning algorithms gilbert georg klamminger*1,2,3, karoline klein*1,4, laurent mombaerts*6, finn jelke1,4, giulia mirizzi1,4, rédouane slimani5,7, andreas husch6, michel mittelbronn2,3,6,7,8,9, frank hertel1,4,6, felix b. kleine borgmann3,6,7 1 saarland university medical center and faculty of medicine, homburg, germany 2 national center of pathology (ncp), laboratoire national de santé (lns), dudelange, luxembourg 3 luxembourg center of neuropathology (lcnp), dudelange, luxembourg 4 national center of neurosurgery, centre hospitalier de luxembourg (chl), luxembourg, luxembourg 5 doctoral school in science and engineering (dsse), university of luxembourg (ul), esch-sur-alzette, luxembourg 6 luxembourg centre of systems biomedicine (lcsb), university of luxembourg (ul), esch-sur-alzette, luxembourg 7 department of oncology (donc), luxembourg institute of health (lih), luxembourg, luxembourg 8 department of life sciences and medicine (dlsm), university of luxembourg, esch-sur-alzette, luxembourg 9 faculty of science, technology and medicine, university of luxembourg, esch-sur-alzette, luxembourg * these authors contributed equally to this work. corresponding author: felix b. kleine-borgmann · luxembourg center of neuropathology (lcnp) · department of oncology (donc), luxembourg institute of health (lih) · 84, val fleuri· l-1526 luxembourg · luxembourg felix.kleineborgmann@lih.lu submitted: 04 august 2021 accepted: 27 september 2021 copyedited by: vanessa s. goodwill published: 04 october 2021 https://doi.org/10.17879/freeneuropathology-2021-3458 additional resources and electronic supplementary material: supplementary material keywords: raman spectroscopy, pcnsl, glioblastoma, machine learning abstract objective and methods: timely discrimination between primary cns lymphoma (pcnsl) and glioblastoma is crucial for diagnosis and therapy, but also determines the intraoperative surgical course. advanced radiological methods allow for their distinction to a certain extent but ultimately, biopsies are still necessary for final diagnosis. as an upcoming method that enables tissue analysis by tracking changes in the vibrational state of molecules via inelastic scattered photons, we used raman spectroscopy (rs) as a label free method to examine specimens of both tumor entities intraoperatively, as well as postoperatively in formalin fixed paraffin embedded (ffpe) samples. results: we applied and compared statistical performance of linear and nonlinear machine learning algorithms (logistic regression, random forest and xgboost), and found that random forest classification distinguished the two tumor entities with a balanced accuracy of 82.4% in intraoperative tissue condition and with 94% using measurements of distinct tumor areas on ffpe tissue. taking a deeper insight into the spectral properties of the tumor entities, we describe different tumor-specific raman shifts of interest for classification. conclusions: due to our findings, we propose rs as an additional tool for fast and non-destructive tumor tissue discrimination, which may help to choose the proper treatment option. rs may further serve as a useful additional tool for neuropathological diagnostics with little requirements for tissue integrity. introduction the highly malignant and rare non-hodgkin primary central nervous system lymphoma (pcnsl) accounts for only 3% of all brain tumors1. the common practice to confirm the suspicion of a pcnsl is a stereotactic biopsy, according to which it can be histopathologically diagnosed and the following treatment adjusted2. due to similar initial clinical presentations and imaging properties, an important differential diagnosis of primary cns lymphoma is diffuse high-grade glioma, especially glioblastoma, who grade iv. the clinical management and treatment, however, considerably vary between these entities. while gross surgical resection of the tumor followed by combined radio-chemotherapy is standard of care in the treatment of glioblastoma, surgery plays a subordinate role in pcnsl therapy3-4. it is essential to differentiate between these tumors as early as possible5. recent magnetic resonance imaging (mri) studies have shown potential, using, amongst other parameters, the signal of the tumor blood flow (tbf) and diffusion tensor imaging (dti) characteristics preoperatively5. furthermore, discrimination was described using the levels of myo-inositol concentration measured with magnetic resonance spectroscopy6. nevertheless, stereotactic biopsies remain necessary to confirm the diagnosis in the majority of cases. as a promising method in tumor diagnostics, raman spectroscopy (rs) allows fast and non-destructive, label-free tissue classification even perioperatively. so far, it has been successfully used to distinguish different entities and grades of brain tumors, such as meningiomas and gliomas7,8,9,10. due to their inherent ability of tissue recognition, inelastic scattered photons caused by changes in the vibrational state of molecules are used as a “molecular fingerprint” of the examined tissue. rs has already been shown to be capable of detecting intraocular lymphoma cells11. rs is also able to distinguish between diffuse large b-cells and chronic lymphocytic leukemia in blood samples12, as well as to predict the malignancy status of lymph nodes13. assessment of tumor tissue via rs during diagnostic stereotactic interventions may lead to early changes of the intraoperative surgical course by favoring immediate surgical intervention in the case of glioblastoma or rather to cessation of the procedure in lymphoma patients without waiting for more detailed histopathological analysis. rs may further be applied on processed tissue14, such as on formalin fixed paraffin-embedded (ffpe) tissue in the pathology department to distinguish between pcnsl and malignant glioma. here, we applied rs on freshly resected tissue within the operating room (or) and after histopathological diagnosis of ffpe tissue. we then tested linear and nonlinear machine learning algorithms with the goal to create a classifier that in the future may be useful both in the or and in the pathological diagnostics for the differentiation between pcnsl and glioblastoma. materials and methods patient data 75 measurements from 3 pcnsl patients (due to a low prevalence the number of samples remains limited) and 10 glioblastoma patients were carried out intraoperatively on freshly resected tissue without further processing (2 patients) and after formalin fixation (1 patient). 45 measurements from 3 pcnsl patients and 6 glioblastoma samples were carried out on ffpe tissue. table 1 provides a more detailed overview about conducted measurements, supplemental table 1 and 2 contain additional information about the glioblastoma samples. all tumor samples underwent neuropathological diagnostics (histology, immunohistochemistry, epigenetic and genetic analysis), performed by a board certified neuropathologist at the national center of pathology (ncp) at the laboratoire nationale de santé (lns, luxembourg). the patient data were collected between 2018-2020; all patients were part of the insitu® study (nr. 201804/08), which has been authorized by the ‘comité national d’ethique de recherche’ (cner) and was performed according to the 'eu general data protection regulation' gdpr15, as well as the world medical association (wma) declaration of helsinki. 16 data acquisition and tissue preparation tumor samples of pcnsl biopsies and glioblastoma resections were collected during surgery / biopsy and put into vials with physiological saline solution. this intraoperative standard procedure prevents drying of the tissue and further degradation. for intraoperative data acquisition, we then put the tissue samples directly into an aluminum cup. the insignificant spectral attribution of aluminum allows unimpaired examination of biological tissue and minimizes influencing spectra from the surroundings. for the measurement itself, we used a robotized visualization and spectroscopic acquisition system (solais™, synaptive®, toronto, canada) enabling the determination of exact measuring points. all points were distributed uniformly on the sample; we included data that showed representative coverage of characteristic histopathological features of the subsequent pathologic diagnosis. visible light images of the tissue and the selected measurement spots were acquired for comparison and traceability (figure 1). figure 1. overview about our workflow and the different raman devices used for tissue examination. a) tumor tissue is measured in the or using the solais™ device. b) primary cns lymphoma biopsy with intraoperatively set raman measurement points (colored). c) ffpe tissue blocks are cut using a standard microtome and d) diagnosed by a neuropathologist (corresponding histological sample of the tissue in b). e) consecutive cuts are mounted on a caf2 slide and used for raman measurements on processed tissue with the tsi proraman device. for neuropathological diagnosis, the tumor samples were then fixed in a formalin solution and subsequently embedded in paraffin; resulting ffpe blocks were cut with a standard microtome. the first cuts were used for routine diagnosis (hematoxylin and eosin (he) staining and immunohistochemistry). the consecutive cuts (7 μm) were left unstained and placed on a caf2 (calcium fluoride; crystran, poole, uk) slide, allowing for microscopic examination of the section, which is not possible on aluminum with transmitted light. due to a low amount of spectral background (precisely one single peak at 321 cm-1) the caf2 substrate allows appropriate spectroscopical examination of even thin tissue fragments17. to reduce paraffin signals from residual wax within the tissue18, the slides were dewaxed using our in-house dewaxing protocol. this procedure includes a 60 min incubation period (at 60°c) to melt the paraffin and xylene/ethanol baths afterwards (2 x 15 min xylene; 3 x 2 min ethanol) to dewax the tissue chemically. first, areas with vital tumor cells were identified by means of light microscopy on the diagnostic he slides (surrounding blood and fibrin residuals were excluded), then the corresponding area on the unstained caf2 slides was marked (encircled) with a slide marking pen. the raman measurement of the ffpe tissue was carried out repeatedly at several sites within the marked area using the proraman-l high-performance raman spectrometer (tsi, shoreview, usa). additionally, see figure 1 for an overview of our workflow. raman spectroscopy for data acquisition on intraoperative and ffpe tissue, two different raman spectrometers were used. as our permanent tool in the operating room, the solais™ raman system consists of an automatic data acquisition and visualization system. via a robotized stage the tumor tissue can be exposed to the excitation laser (785 nm). the measuring points can be set individually on the tissue using a camera and an integrated coordination system. acquisition parameters were set to 2 seconds with 6-30 averages for each measuring point. for the raman measurement on ffpe tissue in the neuropathology unit, the portable tsi raman spectrometer was used. it also provides an excitation laser at 785 nm and a ccd sensor; the used lens has a 7 mm working distance and a 100 μm laser spot size. with the proraman reader software version 8.3.6 (tsi, shoreview, usa) acquired spectra can be displayed, baseline correction can be applied, and acquisition parameters can be defined (10 seconds, 30 averages, 90 mw) according to the request of the user. data analysis and machine learning to reduce bias, data acquired at different time points and states of fixation were analyzed separately and divided into two data sets. to reduce the impact of patient dependent clustering, the same number of measurements for each patient was carried out in the majority of cases and all of them were recorded with the same acquisition time in the respective systems. using a custom-made python script, data cleaning was carried out on intraoperatively acquired raman measurements (n = 75), including outliers and trend removal, as well as removal of artifacts and standardization of spectra. the mean spectra and the variance were visualized (figure 2). afterwards, three different machine learning algorithms were applied to distinguish between the two tumor entities and performance statistics were calculated (accuracy/sensitivity/specificity/roc/pr curve). starting with the linear algorithm of logistic regression we continued with random forest classification, which may be more favorable in this study due to non-independent data points within a raman measurement. to further reduce the bias of patient-dependent clustering, random forest classifiers were trained with randomly split data distribution and a distribution split by patients; results were internally checked for comparability and suggested no relevant bias. as the third machine learning algorithm, we used xgboost classification, in which parameters could be set individually. figure 2. mean spectra and variance of the perioperatively acquired rs after standardization. the x-axis displays the wavenumbers (in cm-1) of the inelastic scattered photons (= the raman shift) and the y-axis the corresponding intensity value. figure 3 gives an overview of our data processing and statistical approach. we identified characteristic wavenumbers usable for spectral tumor discrimination and compared our findings with the underlying biochemical composition of the tissue. the raman measurements acquired in the neuropathology department on ffpe tissue (second data set, n total = 45) were classified using a random forest algorithm. due to a low number of patients, the data was not split into a training and an external validation set. in order to create a representative sample of the performance of the algorithms, particular attention was instead made on the internal validation (for example intrinsic re-validation based on bootstrap subsamples). classification was validated using specific parameters of algorithm performance (auroc, aupr), as well as internal comparison of the performance based on randomly shuffling of measurement points with a patient stratified distribution. for a deeper technical insight in our way of machine learning (e.g., way of hyperparameter optimization) and a visualization of sample individual raman spectra, see supplementary material. figure 3. workflow of data selection and machine learning for the intraoperative measurements. results efficient differentiation of distinct tumor entities on freshly resected tissue using linear and nonlinear machine learning algorithms for an accurate overview of the performance of each of our classifiers, see table 2. our trained logistic regression algorithm distinguishes with a balanced accuracy of 79.3% between the intraoperative measured pcnsl versus glioblastoma tumor tissue. our random forest classifier discriminates between these two tumor entities with an overall accuracy of 82.4% (balanced), with a sensitivity of 0.89 ± 0.13 and a specificity of 75 ± 0.12. as an indicator of the classifier performance, we used the roc (receiver operating characteristics) curve, in which each threshold is visualized according to the resulting specificity and sensitivity, and the auc (area under the curve) value. the auc value of the random forest classification for the pcnsl class was 0.86 ± 0.07 (figure 4). since the roc curve may be biased due to an imbalance in the data set, we additionally used the pr (precision – recall) curve, where instead the ratio between ppv (positive predictive value=precision) and recall (=sensitivity) of every possible cut-off is displayed. figure 4 shows our resulting pr curve with the resulting auc value (0.66 ± 0.2) for the pcnsl class. figure 4. left: roc curve of random forest classification with auroc value of 0.86 ± 0.07. right: corresponding pr curve with an aupr value of 0.66 ± 0.2. in order to evaluate our trained models in more detail, sensitivity and specificity were calculated for all algorithms and displayed in table 2 according to the optimal threshold (maximizing sensitivity and specificity). for all resulting plots of statistic performance, see supplementary material. biochemical insights from spectral analysis in order to determine certain wavenumbers of raman shift for more detailed analysis of the underlying biochemical substrates, we arranged wavenumbers of interest in descending order, according to the distinct tumor class and importance as a feature coefficient for the logistic regression. table 3 displays the class-defining wavenumbers (i.e., positive and negative weights). as wavenumbers with highest attribution for glioblastoma classification distinct peaks in the region 2450 to 3000 cm-1 can be detected (2791, 2486, 2650, 2562, 2673, 2971, 2823 cm-1); most of them likely representing ch stretching / oh nh ch stretching motions. in particular, peaks occurring in the region between 2700 to 3000 cm-1 are known to be related to underlying methyl groups from phospholipids19,20,21. on the contrary, the wavenumbers of interest for pcnsl classification cannot be assigned to the high wavenumber region. for pcnsl classification, the most important peak at 1831 cm-1 seems to be related to changes in the vibrational state of c=c compounds22; followed by bins at 374 cm-1 and 977 cm-1, respectively. those latter bins are, according to the literature19-20, related to chain expansions and cc-stretching of n-alkanes. other important peaks for pcnsl classification, notably 1085 cm-1 and 316 cm-1, may also be attributed to n-alkanes, whereas 784 cm-1 is caused by ring vibrations – as the spectral region from 600 to 800 cm-1 is mainly contributed to dna molecules21. figure 5 provides an overview of the wavenumbers with highest impact and their distribution in relation to the mean spectra of the examined tumor entities. figure 5. feature importance for classification between primary cns lymphoma and glioblastoma. the ascending bins are important for pcnsl detection; the descending bins for glioblastoma detection. see table 3 for further explanation of the wavenumbers behind the frequency bins. tumor classification on chemically aggressively treated formalin fixed paraffin-embedded tissue to analyze our measurements of the ffpe tumor tissue (second dataset with n = 45), we chose a random forest algorithm, as it showed the best performance on fresh tumor tissue in our analysis. applying rs on histopathological proven tumor areas, we were able to establish a classifier with a balanced accuracy of 94%, a sensitivity of 0.93 ± 0.09 and a specificity of 0.95 ± 0.05. the auroc value was 0.98 ± 0.03; the aupr was 0.97 ± 0.05. for a deeper understanding of our results, we investigated the wavenumber regions used to classify between glioblastoma and pcnsl. we detected two regions (around 690 700 cm-1 and 2820 2890 cm-1) contributing to the classification performance. pure paraffin wax was described to have spectra with peaks at 1133, 1296, and 1441 cm-1 23,18, but we also saw significant paraffin contribution in higher wavenumber regions, especially around 2820 to 2910 cm-1. therefore, we suppose that, in addition to tissue-originating spectra, different and potentially characteristic amounts of residual paraffin or its reaction products with the tumor tissue may play a role, which, together with the possible influence of other materials related to the dewaxing process and experimental setup, needs to be considered when applying rs on ffpe tissue. discussion in this study, we show that raman spectroscopy can be used as a non-destructive, label-free, fast technique for perioperative tissue classification to address the differential diagnosis between glioblastoma and primary cns lymphoma at an early stage in the diagnostic workflow. we suggest rs as a future additional method in the or. in the current application, the method would complement the biopsy procedure by giving a fast-feedback diagnostic tool that would allow for a direct treatment decision without waiting for lengthy histopathological examination. this could be advantageous, for example by giving the surgeon the opportunity to immediately proceed with surgical resection in the case of a diagnosis of glioblastoma. additionally, the tool could be modified in such a way that an optical probe could replace the biopsy needle and yield an invasive but non-destructive stereotactic assessment of the tissue24-25. with our machine learning algorithms, the spectral data obtained from intraoperative tumor samples could be classified with linear (logistic regression) and nonlinear classifiers (random forest), with the latter displaying the highest potential with an overall balanced accuracy of 82.4%. to our knowledge, we describe for the first-time important wavenumbers to distinguish pcnsl from glioblastoma. the most important numbers for the detection of the who grade iv glioblastoma are mainly arising from the spectral region 2450 to 3000 cm-1, which is not in contradiction with previous findings. zhou et al. 10 described three major peaks (2850, 2885, 2932 cm-1) in that specific region associated with healthy brain tissue, and the mainly phospholipid-derived peaks in the high-wavenumber region also showed the potential to distinguish between different who grades of gliomas. our suggested peaks for primary cns lymphoma most likely arise from vibrational changes in n-alkane molecules (316, 374, 977, 1085 cm-1) and partially also from backbone vibration of nuclei acids (784 cm-1). to our knowledge, these peaks have not yet been used for the differentiation between pcnsl and other tumor entities. so far, shiramizu et al. 26 showed the possibility to discriminate between b-cell lymphoma cells and non-neoplastic lymphocytes by using rs and set the fingerprint wavenumber region to 600 – 1800 cm-1. manago et al. 27 described certain raman peaks of non-neoplastic lymphocytes and lymphoma cells (amongst other peaks in the region 700-800 cm-1). they differentiated between non-neoplastic b-cells and three b-cell lymphoma cell lines and tracked changes in the resulting raman spectra during treatment application27. furthermore, our results demonstrate the capability of rs to distinguish tumor entities on highly processed ffpe tissue in the neuropathology department. here, precise regions of the tissue can be measured at the price of aggressive chemical treatment and spectral contamination by residual paraffin wax, fixation and washing agents. since spectral regions initially attributed to paraffin may even play an influencing role in ffpe tissue classification, standardized dewaxing protocols and tried and tested processes are essential. a potential role for rs in the neuropathological toolbox is the examination of tissue fragments with little requirements for tissue integrity and neuropathological expertise. recent studies investigate this possibility14. future studies will show whether the use of this “spectral molecular fingerprint” holds true, and can also be applied on multiclass differentiation of several tissue entities. due to a low prevalence of primary cns lymphoma, the quantity of measurements in our data set remained limited. to deal with resulting imbalance and to minimize the impact of patient-dependent clustering, several efforts have been made. not only were the number of measurements per patient correlated for both tumor groups to reduce insensitivity on imbalanced data, but we additionally evaluated our statistical algorithms with the balanced accuracy, the pr curve, and the corresponding aupr value. while cross validation and bootstrapping techniques have been used to provide an indicative assessment of the model performance in future patients, external validation would be required to implement such a prediction model in clinical practice. although our classification shows reasonably good results and a distinct separation of classes, for practical application, the number of reads of one single patient (and therefore the number of reads related to tumor tissue and not surrounding brain) is key. therefore, multiple reads from one sample, which is also preferable to further reduce the bias of patient depending clustering, will improve the diagnostic accuracy in a real-world perioperative setting. further studies are required to see if our proposed model of tissue classification holds true when using rs for the differentiation of other tumors, such as metastasis, and non-neoplastic lesions e.g., brain abscess. more extensive knowledge of the amount of tumor cells needed to get a tumor-specific raman signal would aid the surgeon to find the tumor borders28. in a next step, rs may also be able to provide early insights into genetic / epigenetic alterations of tissue29; as well as insights into therapeutic effects of chemoor radiotherapy27. rs might also be used in combination with additional spectral analysis such as ir (infrared) or mass spectroscopy as complementary techniques30-31, and in addition as an image-forming technique32. conclusion in conclusion, our study shows that machine learning algorithms can be successfully applied on spectroscopic data of brain tumor tissue and fulfill the need of early differential diagnosis between primary cns lymphoma and glioblastoma to determine an individual clinical treatment at an early stage. disclosures we thank the fondation cancer luxemburg (grant to frank hertel, michel mittelbronn, andreas husch and felix kleine borgmann) for the generous support. michel mittelbronn would like to thank the luxembourg national research fund (fnr) for the generous support (pearl p16/bm/11192868 grant). the authors declare no conflicts of interest. references kerbauy mn, moraes fy, lok bh, et al. challenges and opportunities in primary cns lymphoma: a systematic review. radiother oncol. 2017;122(3):352-361. https://doi.org/10.1016/j.radonc.2016.12.033 louis dn, ohgaki h, wiestler od, cavenee wk, eds. who classification of tumours of the central nervous system. revised 4t. lyon: international agency for research on cancer; 2016. stupp r, mason wp, van den bent mj, et al. radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. n engl j med. 2005;352(10):987-996. https://doi.org/10.1056/nejmoa043330 han ch, batchelor tt. diagnosis and management of primary central nervous system lymphoma. cancer. 2017;123(22):4314-4324. https://doi.org/10.1002/cncr.30965 razek aaka, el-serougy l, abdelsalam m, gaballa g, talaat m. differentiation of primary central nervous system lymphoma from glioblastoma: quantitative analysis using arterial spin labeling and diffusion tensor imaging. world neurosurg. 2019;123:e303-e309. https://doi.org/10.1016/j.wneu.2018.11.155 nagashima h, sasayama t, tanaka k, et al. myo-inositol concentration in mr spectroscopy for differentiating high grade glioma from primary central nervous system lymphoma. j neurooncol. 2018;136(2):317-326. https://doi.org/10.1007/s11060-017-2655-x auner gw, koya sk, huang c, et al. applications of raman spectroscopy in cancer diagnosis. cancer metastasis rev. 2018;37(4):691-717. https://doi.org/10.1007/s10555-018-9770-9 depaoli d, lemoine é, ember k, et al. rise of raman spectroscopy in neurosurgery: a review. j biomed opt. 2020;25(5):1-36. https://doi.org/10.1117/1.jbo.25.5.050901 zhang j, fan y, he m, et al. accuracy of raman spectroscopy in differentiating brain tumor from normal brain tissue. oncotarget. 2017;8(22):36824-36831. https://doi.org/10.18632/oncotarget.15975 zhou y, liu c-h, wu b, et al. optical biopsy identification and grading of gliomas using label-free visible resonance raman spectroscopy. j biomed opt. 2019;24(9):1-12. https://doi.org/10.1117/1.jbo.24.9.095001 iwasaki y, kawagishi m, takase h, ohno-matsui k. discrimination of dissociated lymphoma cells from leukocytes by raman spectroscopy. sci rep. 2020;10(1):1-12. https://doi.org/10.1038/s41598-020-72762-5 bai y, yu z, yi s, yan y, huang z, qiu l. raman spectroscopy-based biomarker screening by studying the fingerprint characteristics of chronic lymphocytic leukemia and diffuse large b-cell lymphoma. j pharm biomed anal. 2020;190:113514. https://doi.org/10.1016/j.jpba.2020.113514 rau j v., marini f, fosca m, cippitelli c, rocchia m, di napoli a. raman spectroscopy discriminates malignant follicular lymphoma from benign follicular hyperplasia and from tumour metastasis. talanta. 2019;194:763-770. https://doi.org/10.1016/j.talanta.2018.10.086 fullwood lm, clemens g, griffiths d, et al. investigating the use of raman and immersion raman spectroscopy for spectral histopathology of metastatic brain cancer and primary sites of origin. anal methods. 2014;6(12):3948-3961. https://doi.org/10.1039/c3ay42190b eu general data protection regulation. vol 2014.; 2018:45-62. wma declaration of helsinki – ethical principles for medical research involving human subjects – wma – the world medical association. https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/. accessed november 9, 2020. fullwood lm, ashton k, dawson t, et al. effect of substrate choice and tissue type on tissue preparation for spectral histopathology by raman microspectroscopy. analyst. 2014;139(2):446-454. https://doi.org/10.1039/c3an01832f mian s, colley h, thornhill m, rehman i. development of a dewaxing protocol for tissue-engineered models of the oral mucosa used for raman spectroscopic analysis. appl spectrosc rev. 2014;49. https://doi.org/10.1080/05704928.2014.882348 movasaghi z, rehman s, rehman iu. raman spectroscopy of biological tissues. appl spectrosc rev. 2007;42(5):493-541. https://doi.org/10.1080/05704920701551530 edwards hgm. spectra-structure correlations in raman spectroscopy. in: handbook of vibrational spectroscopy. 2006; john wiley & sons ltd. https://doi.org/10.1002/0470027320.s4103 de gelder j, de gussem k, vandenabeele p, moens l. reference database of raman spectra of biological molecules. j raman spectrosc. 2007; 38: 1133–1147. https://doi.org/10.1002/jrs.1734 yvon hj. raman spectroscopy for analysis and monitoring. horiba jobin yvon, raman appl note. 2017:1-2. http://www.horiba.com/fileadmin/uploads/scientific/documents/raman/bands.pdf faoláin eó, hunter mb, byrne jm, et al. raman spectroscopic evaluation of efficacy of current paraffin wax section dewaxing agents. j histochem cytochem. 2005;53(1):121-129. https://doi.org/10.1177/002215540505300114 desroches j, lemoine é, pinto m, et al. development and first in-human use of a raman spectroscopy guidance system integrated with a brain biopsy needle. j biophotonics. 2019;12(3):1-7. https://doi.org/10.1002/jbio.201800396 jermyn m, mok k, mercier j, et al. intraoperative brain cancer detection with raman spectroscopy in humans. sci transl med. 2015;7(274):274ra19. https://doi.org/10.1126/scitranslmed.aaa2384 shiramizu b, oda r, kamada n, et al. unique raman spectroscopic fingerprints of b-cell non-hodgkin lymphoma: implications for diagnosis, prognosis and new therapies. j biol med sci. 2018;2(1):105. managò s, valente c, mirabelli p, et al. a reliable raman-spectroscopy-based approach for diagnosis, classification and follow-up of b-cell acute lymphoblastic leukemia. sci rep. 2016;6:24821. https://doi.org/10.1038/srep24821 jermyn m, desroches j, mercier j, et al. raman spectroscopy detects distant invasive brain cancer cells centimeters beyond mri capability in humans. biomed opt express. 2016;7(12):5129. https://doi.org/10.1364/boe.7.005129 livermore lj, isabelle m, bell i mac, et al. rapid intraoperative molecular genetic classification of gliomas using raman spectroscopy. neuro-oncology adv. 2019;1(1):1-12. https://doi.org/10.1093/noajnl/vdz008 depciuch j, tołpa b, witek p, et al. raman and ftir spectroscopy in determining the chemical changes in healthy brain tissues and glioblastoma tumor tissues. spectrochim acta part a mol biomol spectrosc. 2020;225:117526. https://doi.org/10.1016/j.saa.2019.117526 gajjar k, heppenstall ld, pang w, et al. diagnostic segregation of human brain tumours using fourier-transform infrared and/or raman spectroscopy coupled with discriminant analysis. anal methods. 2013;5(1):89-102. https://doi.org/10.1039/c2ay25544h orringer da, pandian b, niknafs ys, et al. rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated raman scattering microscopy. nat biomed eng. 2017;1:0027. https://doi.org/10.1038/s41551-016-0027 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. an atypical teratoid/rhabdoid tumor (at/rt) with molecular features of pleomorphic xanthoastrocytoma (pxa) in a 62-year-old patient feel free to add comments by clicking these icons on the sidebar free neuropathology 2:31 (2021) case report an atypical teratoid/rhabdoid tumor (at/rt) with molecular features of pleomorphic xanthoastrocytoma (pxa) in a 62-year-old patient matthias dottermusch1,2, ali alomari3, nesrin uksul3, ulrich j. knappe3, julia e. neumann2,1 1 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 2 center for molecular neurobiology (zmnh), university medical center hamburg-eppendorf, hamburg, germany 3 department of neurosurgery, johannes wesling klinikum, university hospital of ruhruniversität bochum, minden, germany corresponding author: julia e. neumann · center for molecular neurobiology (zmnh) · university medical center hamburg-eppendorf · falkenried 94 · 20251 hamburg · germany ju.neumann@uke.de submitted: 20 october 2021 accepted: 09 november 2021 copyedited by: shino magaki published: 15 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3640 additional resources and electronic supplementary material: supplementary material keywords: atypical teratoid/rhabdoid tumor, pleomorphic xanthoastrocytoma, dna methylation atypical teratoid/rhabdoid tumors (at/rt) are aggressively growing malignant embryonal neoplasms of the central nervous system (cns), which mainly affect young children. loss of smarcb1/ini1 (or smarca4/brg1 in rare cases) is recognized as the genetic hallmark of at/rts. furthermore, these tumors can be distinguished into three distinct dna-methylation based molecular subgroups (i.e. -myc, -shh and -tyr) (1–3). while most at/rts are considered to occur de novo, previous studies have recognized secondary smarcb1/ini1-deficient rhabdoid tumors arising from other low grade cns tumors in young patients (4–9). moreover, three at/rts, which harbored epigenetic and mutational characteristics of pleomorphic xanthoastrocytoma (pxa), while being entirely void of nuclear smarcb1/ini1 expression were recently described in older children (10). we here report the first case of an at/rt with molecular features of pxa in an older adult. a 62-year-old woman presented with diffuse headaches since several weeks. medical examination revealed no clinically significant neurological deficits. an mri showed a right-sided tumor in temporomesial location with dimensions of 3.8 x 3.7 x 4.1 cm. the tumor displayed contrast-enhancement, central necrosis und perifocal edema (figure 1a). additionally, a smaller lesion of the infundibulum was identified. preoperative lumbar puncture with examination of cerebrospinal fluid revealed extensive presence of tumor cells. the temporomesial tumor was subtotally resected. figure 1: radiology, histopathology and epigenetic analysis. a) representative axial mri demonstrating the preoperative finding of a large temporomesial tumor (large arrow). contrast enhancement was found partially within the tumor and additionally in the region of the infundibulum (small arrow). t1-weighted image plus contrast medium. b) histomorphology revealed geographical necrosis, prominent rhabdoid morphology and brisk mitotic activity of tumor cells (inset). c) the tumor showed a prominent network of reticular fibres, visible in the gomori silver impregnation. d) immunostaining for vimentin was vastly strongly positive. e) immunostaining for cytokeratins (ae1/3) was vastly strongly positive in the majority of tumor cells. f) immunostaining for gfap was negative in tumor cells while demarcating residual brain tissue. g) ki67 proliferative index amounted to about 40%. h) tumor cells exhibited loss of nuclear smarcb1/ini1 staining. retained nuclear staining was found in blood vessels and inflammatory cells (inset). scale bar in b – h is 150 μm. i) copy number profile of the tumor indicated a homozygous cdkn2a/b (short arrow) and smarcb1/ini1 (long arrow) deletion. j) tsne including a reference set of brain tumors (gse90496, (11)) showed affiliation of the tumor to the group of pxa. clicking the figure will lead you to the full virtual slide (h&e). upon histopathological examination, we saw a highly cellular, partially necrotic tumor, which was entirely composed of pleomorphic rhabdoid cells with eosinophilic cytoplasms (figure 1b). gomori-staining demonstrated a prominent reticulin network (figure 1c). immunohistochemical staining showed positivity for vimentin (figure 1d) and cytokeratin ae1/3 (panck, figure 1e). gfap was negative within the tumor cells (figure 1f). strong proliferation was demonstrated by brisk mitotic activity and a proliferative index of 40% in the ki67-staining (figure 1g). smarcb1/ini1 nuclear signal was consistently lost in tumor cells (figure 1h). further immunohistochemical stainings are displayed in supplementary figure 1. dna was extracted from the tumor and subjected to dna methylation profiling using the illumina epic beadchip array. using the brain tumor methylation classifier (v11b4 and v12.3) (11), the tumor did not match with a defined methylation class (no calibrated score was ≥ 0.3). the copy number profile revealed a homozygous smarcb1/ini1 and cdkn2a/b deletion, gains in chromosomes 2 and 7q as well as losses of chromosomes 9p, 14q, 16 and 22q (figure 1i). t-sne analysis displayed similarity of the case with pxa (figure 1j). dna panel sequencing confirmed a braf v600e mutation (table 1). taking all diagnostic layers together, we saw a malignant rhabdoid tumor, not elsewhere classified, with braf v600e mutation, homozygous smarcb1/ini1 and cdkn2a/b deletion, and a methylation profile with similarity to pxa. in correspondence with the provisional designation of thomas et al. (10), the case was signed out as at/rt with molecular features of pxa. postoperative clinical staging of the patient revealed a smaller lesion of uncertain dignity in the cervical myelon. no further malignancies outside of the cns were found. unfortunately, the patient developed postoperative hemiplegia, infarctions of the basal ganglia and displayed progressive worsening of the clinical status. palliative chemotherapy was administered with intrathecal application of methotrexate. the patient died in the 6th postoperative week. table 1: ngs dna panel results. as it is the case for the herein presented tumor, it remains unclear, if at/rts with molecular features of pxa correspond to secondary rhabdoid tumors, which have emerged from and entirely outgrown preexisting pxas. in contrast, such tumors might also occur independently from precursor lesions and therefore represent a distinct entity. of note, the cns who classification of 2016 has previously recognized high-grade rhabdoid components with loss of smarcb1/ini1 in anaplastic pxas and suggested the term “smarcb1-deficient anaplastic pxa” for such lesions (12). however, since the tumor presented here was entirely void of smarcb1/ini1 retained tumor cells and a preexisting lesion had not been described beforehand, the tumor’s origin is left to speculation. it is moreover important to note that the single genetic and epigenetic alterations we found are not pathognomonic for pxas (13) and the tumor did not match with a defined methylation class of the brain tumor classifier. however, the combination of all molecular findings – including the epigenetic resemblance to pxas via tsne analysis – clearly demonstrates similarity with molecular features of pxas. a complicating aspect of the current and soon to be updated cns who classification of 2016 is that smarcb1/ini1-deficient rhabdoid brain tumors tend to be vastly designated as at/rts. the herein presented case exemplarily demonstrates that especially without molecular investigations this may lead to the subsumption of malignancies with various epigenetic and mutational landscapes as well as different cellular origins. while the preceding summary of the new who classification of 2021 (14) has announced the recognition of epigenetic analyses in at/rts, it is currently still unclear how smarcb1/ini1-deficient brain tumors with rhabdoid morphology and lack of epigenetic features of at/rts are to be classified. future studies should evaluate if predictive and prognostic implications of these tumors call for a more concise classification and terminology. data availability the data that supports the findings of this study is available from the corresponding author upon request. a digital h&e stained slide is deposited here. conflict of interest the authors have no competing interests to declare. acknowledgements we thank celina soltwedel, carolina janko, karin gehlken, ulrike rumpf, tasja lempertz, nicole bernhardt, helena zinn and ulrich schüller (hamburg) for excellent technical support. we thank annika wefers for helpful discussions. we thank martin hasselblatt and christian thomas from the institute of neuropathology of the university hospital münster for confirming the diagnosis. j.n. was supported by the deutsche forschungsgemeinschaft (dfg, emmy noether programme). m.d. was supported by the erich und gertrud roggenbuck-stiftung. ethical statement study approval was obtained from the local ethics committee of the hamburg state chamber of physicians. the patient and/or guardian gave their informed consent for scientific use of the data. author contributions m.d. and j.n. conceived the study and drafted the manuscript. all authors acquired and analyzed data and approved the final version of the manuscript. references johann, p. d. et al. atypical teratoid/rhabdoid tumors are comprised of three epigenetic subgroups with distinct enhancer landscapes. cancer cell 29, 379–393 (2016). frühwald, m. c., biegel, j. a., bourdeaut, f., roberts, c. w. m. & chi, s. n. atypical teratoid/rhabdoid tumors current concepts, advances in biology, and potential future therapies. neuro-oncol. 18, 764–778 (2016). richardson, e. a., ho, b. & huang, a. atypical teratoid rhabdoid tumour: from tumours to therapies. j. korean neurosurg. soc. 61, 302–311 (2018). allen, j. c., judkins, a. r., rosenblum, m. k. & biegel, j. a. atypical teratoid/rhabdoid tumor evolving from an optic pathway ganglioglioma: case study. neuro-oncol. 8, 79 (2006). chacko, g., chacko, a. g., dunham, c. p., judkins, a. r., biegel, j. a. & perry, a. atypical teratoid/rhabdoid tumor arising in the setting of a pleomorphic xanthoastrocytoma. j. neurooncol. 84, 217–222 (2007). kleinschmidt-demasters, b. k., birks, d. k., aisner, d. l., hankinson, t. c. & rosenblum, m. k. atypical teratoid/rhabdoid tumor arising in a ganglioglioma: genetic characterization. am. j. surg. pathol. 35, 1894–1901 (2011). nadi, m., ahmad, t., huang, a., hawkins, c., bouffet, e. & kulkarni, a. v. atypical teratoid rhabdoid tumor diagnosis after partial resection of dysembryoplastic neuroepithelial tumor: case report and review of the literature. pediatr. neurosurg. 51, 191–198 (2016). bertrand, a. et al. rhabdoid component emerging as a subclonal evolution of paediatric glioneuronal tumours. neuropathol. appl. neurobiol. 44, 224–228 (2018). nobusawa, s. et al. secondary ini1-deficient rhabdoid tumors of the central nervous system: analysis of four cases and literature review. virchows arch. 476, 763–772 (2020). thomas, c. et al. atypical teratoid/rhabdoid tumor (at/rt) with molecular features of pleomorphic xanthoastrocytoma. am. j. surg. pathol. 45, 1228–1234 (2021). capper, d. et al. dna methylation-based classification of central nervous system tumours. nature 555, 469–474 (2018). louis, d. n. et al. who classification of tumours of the central nervous system. (international agency for research on cancer, 2016). vaubel, r. et al. biology and grading of pleomorphic xanthoastrocytoma-what have we learned about it? brain pathol. 31, 20–32 (2021). louis, d. n. et al. the 2021 who classification of tumors of the central nervous system: a summary. neuro-oncol. 23, 1231–1251 (2021). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuronal transcriptome from c9orf72 repeat expanded human tissue is associated with loss of c9orf72 function feel free to add comments by clicking these icons on the sidebar free neuropathology 1:23 (2020) original paper neuronal transcriptome from c9orf72 repeat expanded human tissue is associated with loss of c9orf72 function elaine y. liu1, jenny russ1, edward b. lee1 1 translational neuropathology research laboratory, university of pennsylvania, philadelphia, pa, usa corresponding author: edward b. lee, m.d., ph.d. · 613a stellar chance laboratories · 422 curie blvd · philadelphia, pa 9104 · usa · 001 (215) 898-0908 edward.lee@pennmedicine.upenn.edu submitted: 20 july 2020 accepted: 13 august 2020 copyedited by: lauren walker published: 21 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2911 additional resources and electronic supplementary material: supplementary material keywords: dementia, transcriptome, frontotemporal, amyotrophic lateral sclerosis, c9orf72, tdp-43, facs, rna-seq, repeat expansion, denn, frontotemporal lobar degeneration, frontotemporal dementia abstract a hexanucleotide g4c2 repeat expansion in c9orf72 is the most common genetic cause of familial and sporadic cases of amyotrophic lateral sclerosis (als) and frontotemporal degeneration (ftd). the mutation is associated with a reduction of c9orf72 protein and accumulation of toxic rna and dipeptide repeat aggregates. the accumulation of toxic rna has been proposed to sequester rna binding proteins thereby altering rna processing, consistent with previous transcriptome studies that have shown that the c9orf72 repeat expansion is linked to abundant splicing alterations and transcriptome changes. here, we used a subcellular fractionation method and facs to enrich for neuronal nuclei from c9orf72 repeat expanded post-mortem human als/ftd brains, and to remove neuronal nuclei with tdp-43 pathology which are observed in nearly all symptomatic c9orf72 repeat expanded cases. we show that the c9orf72 expansion is associated with relatively mild gene expression changes. dysregulated genes were enriched for vesicle transport pathways, which is consistent with the known functions of c9orf72 protein. further analysis suggests that the c9orf72 transcriptome is not driven by toxic rna but is rather shaped by the depletion of pathologic tdp-43 nuclei and the loss of c9orf72 expression. these findings argue against rna binding protein sequestration in neurons as a major contributor to c9orf72 mediated toxicity. introduction amyotrophic lateral sclerosis (als) and frontotemporal degeneration (ftd) are fatal neurodegenerative diseases with overlapping clinical, pathologic and genetic features. als is a motor neuron disease that primarily affects the upper and lower motor neurons in the motor cortex and spinal cord, respectively, whereas ftd affects the frontal and temporal lobes, thereby affecting cognition. the most common genetic cause of familial and sporadic cases of als and ftd was identified as a hexanucleotide g4c2 repeat expansion found in the gene c9orf72 (1, 2). the repeat expansion is found in the first intron of c9orf72 and can be transcribed into pre-mrna that contains the repeat. c9orf72 protein has been predicted to be a denn rab gtpase, and shown to be involved in vesicular trafficking (3-8). three potential mechanisms of toxicity have been implicated in contributing to disease: haploinsufficiency due to reduction of c9orf72 protein; rna foci and titration of rna binding proteins; and dipeptide repeat protein aggregates (reviewed in (9)). it is currently unclear which mechanism contributes most to toxicity. gain of toxic function via rna foci or dipeptide repeat proteins have been seen in cellular and animal models. indeed, several rna binding proteins have been shown to colocalize with repeat containing rna within rna foci, suggesting that rna binding protein titration may be mediating toxicity (10-12). models overexpressing dipeptide repeat proteins and/or mutant rna in both cell and animal systems have shown that the repeat expansion and dipeptide proteins may confer toxicity via a variety of pathways including nucleolar stress, nucleocytoplasmic transport defects, heterochromatin abnormalities, and dna damage (13-32). however, rna foci and dipeptide repeat proteins do not correlate with neurodegeneration in human tissues and are not toxic in some experimental models (13-16, 33-36), raising questions about the relevance of whether these pathologies contribute to disease. the loss of c9orf72 protein has been substantiated by the fact that c9orf72 expansion carriers have reduced c9orf72 mrna and protein expression (1, 2, 37, 38). thus, it is possible that loss of c9orf72 protein may contribute to disease. however, knockout mouse models of c9orf72 do not show neuronal defects but, rather, exhibit an inflammatory phenotype (39-41), although reduction of c9orf72 protein appears to exacerbate phenotypes in mice overexpressing mutant c9orf72 rna (42). we have shown that a subset of c9orf72 expansion carriers exhibit c9orf72 promoter methylation, and this is associated with reduced c9orf72 associated pathology, later age at death in ftd patients, slower rates of cerebral atrophy, and improved cognitive ability (43-45). moreover, the gnomad human genetics database indicates that missense and loss of function c9orf72 mutations are tolerated and present in control populations (46). consequently, the pathogenic role of the loss of c9orf72 protein in als/ftd is not entirely clear, and overall it remains unclear which mechanism of toxicity contributes most to disease. efforts to better understand these mechanisms include transcriptomic studies to determine whether rna dysregulation can contribute to neurodegeneration. indeed, frontal cortical and motor neuron transcriptome analysis from als patients showed splicing changes, dysregulation of rna processing pathways, and alternative polyadenylation insporadic and c9orf72 expansion carriers (47-49). more recently, a large transcriptome study of c9orf72 cases found changes in genes linked to vesicular transport (8). these studies are sometimes complicated by the complex cellular composition of brain tissue which includes neuronal subtypes, oligodendrocytes, microglia, astrocytes, vascular cells and other cell types. laser capture microdissected tissue studies have been very useful (50) but is labor intensive and often is technically difficult due to relatively low yield and preprocessing steps that can affect rna quality (51). single cell rna sequencing methods have recently addressed the issue of complex cellular mixtures within tissue but suffer from low coverage and data sparsity. we have previously reported the use of fluorescence activated cell sorting (facs) to identify transcriptome signatures in neuronal nuclei without tdp-43 from post-mortem brain in als/ftd patients (52). here, we have used the same method to determine the role of the c9orf72 repeat expansion in post-mortem neurons from als/ftd patients. compared to the profound transcriptome changes associated with the loss of normal nuclear tdp-43 protein (52), we found that the c9orf72 mutation was associated with relatively mild gene expression changes and mild splicing changes. moreover, the molecular signature is largely associated with a loss of c9orf72 function, and not a gain of toxic rna or dipeptide repeat aggregates. materials and methods clinical and pathologic assessment human autopsy tissue was obtained from the university of pennsylvania center for neurodegenerative disease research brain bank as described (53). informed consent from next of kin was obtained for every case. isolation was performed exactly as previously described (52). briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil b (kimble chase) in 0.25m sucrose and adjusted to final molarity of 1.6m sucrose in tkm. the homogenate was spun on a 1.8m sucrose cushion on the beckman coulter xpn-80 ultracentrifuge at 40,000g for 40 minutes at 4°c (beckman coulter inc, indianapolis, in, usa). isolated nuclei were stained with alexa fluor 647 conjugated to 2089 (rabbit polyclonal c-terminal anti-tdp-43 antibody, center for neurodegenerative disease research, university of pennsylvania), alexa fluor 488 conjugated neun (emd millipore, billerica, ma, usa), and dapi (invitrogen, carlsbad, ca, usa). alexa fluor 647 was conjugated to 2089 according to the apex alexa fluor 647 antibody labeling kit protocol (thermo fisher scientific, waltham, ma, usa). stained nuclei were sorted for single cells containing tdp-43 and neun on the bd facsaria ii (bd biosciences, san jose, ca, usa) at 20 psi on 100µm nozzle. rna isolation and rna-seq library generation isolation and library generation was done as previously described (52). briefly, rna was extracted using the standard protocol within the allprep dna/rna micro kit (qiagen, germantown, md, usa). rna quality from sorted nuclear rna was determined based on bioanalyzer picochip analysis (agilent, santa clara, ca, usa). isolated rna was amplified, made into cdna, sheared, and libraries were made. the library was quantified using the qubit dsdna kit (invitrogen) and kapa library quantification kit (kapabiosystems, boston, ma). cdna libraries were pooled, clustered on the cbot and subject to 100 or 125 base pairs paired end reads on the hiseq 2000 or 2500 (illumina, san diego, ca, usa). pre-processing, mapping and filtering of rna-seq data rna-seq analysis was done as previously described (52). briefly, raw sequencing reads were demultiplexed through the upenn functional genomics core and analyzed for quality control using fastqc. reads were mapped to the human genome (grch38, gencode release 22) using star and only uniquely mapping reads were selected for further analysis. ribosomal and mitochondrial reads were removed and sam files were converted to bam files using samtools view and bam files were sorted by coordinate with samtools sort. creation of non-overlapping gene, exon and intron annotations annotations were generated as previously described (52). briefly, annotations were based on the comprehensive gene annotation file of the gencode release 22 (grch38.p2). gtf file was loaded into r (version 3.2.2; r core team (2015): “r: a language and environment for statistical computing”, r foundation for statistical computing, vienna, austria) and converted into a transcriptdb object. from the transcriptdb object, all annotated ensembl genes and their exons were pulled out using exonsby (by=”gene”) and ensembl gene ids were replaced by official gene symbols with biomart (version 2.26.1). genes and introns were defined as previously described in the above chapter. regions shared by overlapping genes were removed to count reads that map to one gene or to genic elements (exon or intron) from one gene. exons and introns annotations were used for subsequent analyses in the r package dexseq (1.16.10). differential gene and genic element expression analysis the genomic annotation used for read counting was done as previously described (52). the mapped, filtered rna sequencing reads were counted using a custom r script including the r packages rsamtools (1.22.0), genomicfeatures (1.22.8) and genomicalignments (1.6.3). briefly, sorted bam files were loaded into r (3.2.2) and the number of reads mapping to genes, exons or introns, was computed. genes were analyzed for differential expression using the r package deseq2 (1.10.1). analysis was used to determine differences between nuclei from both c9orf72 expansion cases and non-diseased controls (option design=~experiment in deseqdatasetfrommatrix tool where experiment is the subject id). sex effects were removed with the r package sva (3.18.0) according to the deseq2 vignette. manual annotation of gene descriptions was performed using gene descriptions on ncbi. differential genic element expression analysis was done using dexseq (1.16.10). briefly, the sorted bam files were used to count the number of reads mapping to exons and introns using the exon and intron annotation generated above and computed using findoverlaps and countsubjecthits. reads mapping to exon-intron junctions were excluded. sex was controlled for by including it in the linear model used in the analysis by adding the term “sex:exon”. the following linear models were used full model = ~sample + exon + sex:exon + experiment:exon and reduced model = ~sample + exon + sex:exon + experiment:exon and reduced model = ~sample + exon + sex:exon, where sample is the sample id and experiment the subject id. in addition, dispersions were estimated using the tool estimatedispersions with option fittype=’local’. changes in expression were significant if bonferroni-hochberg multiple testing adjusted p-values were less than 0.05. alternative splicing analysis splicing analysis was performed as previously described (52). briefly, all fastq files were trimmed to 100bp, aligned to grch38 using star; ribosomal and mitochondrial mapped reads were removed. sam files were converted to sorted bam files and rmats.3.0.9 was run using default parameters with the following options (-t paired –len 100 –c 0.05 – analysis u). significant alternative splicing events were used if bonferroni-hochberg multiple testing adjusted p-values were less than 0.05. rna binding protein clip analysis clip analysis was done as previously described (52) using published tdp-43 iclip data from sh-sy5y cells (54) and hnrnp a, a2b1, f, m, u clip data from hek293 cells (55). briefly, hg18 bowtie files were converted to fastq files, and aligned to hg38. pipeclip was run using the python script. using r, the “genomicranges” package with ‘findoverlaps’ option was used to determine which bins had rna binding protein sites. chi-square analysis was done to determine whether there was a significant enrichment of bins with rna binding protein sites within the significantly differentially used bins linked to the c9orf72 mutation. principal component analysis principal component analysis was done using the log transformed read count dataset in r with the ‘stats’ package and ‘prcomp’ function. the coordinates were retrieved and used to plot principal component 2 (pc2) vs principal component 1. to determine the relationship between pc2 vs c9orf72 expression, the coordinates of pc2 for each sample was plotted against the normalized c9orf72 expression calculated from deseq2 from each sample. pearson’s correlation was performed to determine the correlation between pc2 and c9orf72 expression. gene ontology analysis of the top 1% of all genes that contribute to pc2 was done using webgestaldt (56) with the overrepresentation enrichment analysis using the ‘geneontology’ functional database with a fdr corrected significance level < 0.05. methylation correlation methylation levels for the c9orf72 promoter in patients were determined as previously described (45). briefly, genomic dna from the cerebellum was extracted using the qiagen dneasy blood and tissue kit and subject to overnight digestion with hhai and haeiii (double-digested) or just haeiii (mock) alone. a small aliquot of dna was amplified using primers flanking the hhai cutsite within the c9orf72 promoter region using 2x faststart sybr green master (roche) on the abi steponeplus machine. the difference in cycles to threshold amplification between double and mock digested dna was calculated as methylation values. spearman’s correlation was calculated using gene counts for each gene and methylation values for each c9orf72 mutation case. r package ‘lsr’ with ‘correlate’ function (with options corr.method=”spearman” and p.adjust.method=”fdr”) using the spearman’s correlation and fdr adjusted p-values was used. only correlations from genes with hugo gene symbols were calculated and plotted against gene fold change for each gene that was calculated by deseq2. this was done using both significantly differentially expressed genes linked to the c9orf72 mutation (deseq2 fdr p-value < 0.05) and genome-wide using all expressed genes. results fluorescence activated cell sorting and rna sequencing of sorted neuronal nuclei the c9orf72 mutation leads to tdp-43 pathology which includes the formation of neuronal cytoplasmic inclusions and the loss of normal physiologic nuclear tdp-43 protein. we have previously shown that the loss of normal nuclear tdp-43 protein has large effects on the nuclear transcriptome (52). to determine whether the c9orf72 mutation leads to transcriptomic alterations independent of tdp-43 pathology, we isolated neuronal nuclei with intact tdp-43 expression. mid-frontal neocortex from 7 post-mortem c9orf72 expansion carriers and 6 neurologically normal controls were used to isolate nuclei for transcriptome-wide analysis. the autopsy cohort characteristics can be found in table 1. nuclei were immunostained for neun and tdp-43 and subjected to fac sorting to isolate neun positive, tdp-43 positive nuclei from controls (circled, fig 1a) and c9orf72 expansion cases (circled, fig 1b). rna from equal numbers of tdp-43 positive neuronal nuclei (35,000-100,000) was extracted and amplified to generate barcoded cdna libraries for 100 or 125bp paired end sequencing on the illumina hiseq 2000/2500. sequences were mapped to the human genome (gencode grch38) using star algorithms (version 2.2.4). there were 1.8 billion reads of which 1.065 billion reads mapped uniquely between the 13 libraries, with an average of 90 million uniquely mapped reads per library. these reads were filtered to remove ribosomal and mitochondrial reads and the resulting reads were used for downstream analysis to evaluate differential expression of genes, genic elements and alternative splicing. table 1: autopsy cohort characteristics. als = amyotrophic lateral sclerosis; ftld = frontotemporal lobar degeneration with tdp-43 inclusions; mnd = motor neuron disease; nl = normal; pmi = post-mortem interval; rin = rna integrity number fig 1: c9orf72 repeat expansion contributes to relatively mild transcriptome changes. flow cytometry plots of control (a) and c9orf72 expansion cases (b) with tdp-43 positive neuronal nuclei circled as collected samples. (c) principal component analysis using all expressed genes where shape denotes sex and color denotes condition. (d) principal component 2 strongly correlates with c9orf72 expression (pearson’s r = -0.8205; p=0.0006). (e) ma plot showing gene fold change as a function of mean normalized gene counts with red dots being significantly differentially expressed genes. (f) pie chart distribution of all alternative splicing events between c9orf72 expansion carriers and non-diseased controls. se=skipped exons, mxe=mutually exclusive exons, ri=retained intron, a3ss=alternative 3’ splice site, a5ss=alternative 5’ splice site. functional transcriptomic alterations associated with the c9orf72 hexanucleotide repeat expansion to verify that the variation within the dataset is linked to the mutation status, principal component analysis (pca) was performed using the read counts of the entire dataset (fig 1c). principal component #1 (pc1) explained 21% of the variation whereas pc2 explained 19% of the variation (fig 1c). based on the clustering of the dataset, sex explains the variation in pc1 while the c9orf72 repeat expansion explains the variation in pc2. indeed, pc2 strongly correlated with c9orf72 mrna expression (pearson’s r=-0.8205; p=0.0006), suggesting that c9orf72 expression is the major underlying biological variable that contributes to the variation in pc2 (fig 1d). to identify whether there were enriched pathways within the genes that contribute most to pc2, gene ontology analysis was performed using the top 1% of all expressed genes (318 genes) that contribute to pc2. one pathway that is of interest is the cytoplasmic vesicle part or cytoplasmic vesicle membrane (table 2) and is consistent with the functional role of c9orf72 as a vesicle trafficking protein and rab gtpase (3, 5-7). table 2: gene ontology (go) of genes related to top 1% of all genes that contribute to variation in pc2. previous studies have found that the g4c2 rna foci may colocalize with different rna binding proteins (rbps), suggesting that sequestration of these rbps can result in aberrant rna processing (10-12). differential gene expression analysis showed that there are 323 significantly differentially expressed genes with 202 upregulated and 121 downregulated genes linked to the c9orf72 mutation (fig 1e, supplemental table 1). similar to a previous transcriptome analysis on c9orf72 expansion carriers on the frontal cortex, there were more upregulated genes than downregulated genes within the differentially expressed genes (49). notably, the number of differentially expressed genes linked the c9orf72 repeat expansion was relatively mild compared to the massive transcriptome-wide alterations we previously described associated with tdp-43 pathology in these same cases (5,576 significantly differentially expressed genes, described in (52)). further annotation shows that these genes are generally involved in synaptic vesicle fusion or vesicle formation (table 3). additional genes related to vesicle transport and endosomal trafficking are also dysregulated. interestingly, nine out of these 11 genes are upregulated. additional genes that were upregulated were involved in protein aggregation. dnajb2 is almost exclusively expressed in neurons and has been shown to resolve tdp-43 aggregates by interacting with heat shock protein 70 (57). mgrn1 has been shown to confer cytoprotective effects in an als mouse model and can suppress chaperone associated misfolded protein aggregation and toxicity (58). given that c9orf72 expansion cases exhibit protein aggregates in the form of dipeptide repeat protein and tdp-43 inclusions, it is possible that these genes are upregulated in response to these aggregates. lastly, there were also genes that are involved in dna repair (faap20, fam175a, fancb, hmgn1) (59-62) and response to dna damage (kdm4b and kin) (63-65). annotation of these genes reveal multiple themes relevant to c9orf72 including synaptic vesicle formation, endosomal trafficking, chaperone associated protein aggregation, and dna damage (table 3). c9orf72 mutation is associated with mild splicing alterations previous studies have shown that there are abundant alternative splicing changes that are linked to the c9orf72 mutation (49). the finding of rna binding proteins that colocalize with rna foci also support the idea that rna binding protein sequestration and subsequent splicing alterations may contribute to mutant c9orf72 toxicity. to evaluate splicing changes associated with the c9orf72 mutation, rmats was used to align junction reads to annotated junctions from grch38 assembly. within our dataset, there were a total of 112 events that were significantly alternatively spliced which included 84 skipped exons (se), 9 mutually exclusive exons (mxe), 9 retained introns (ri), 8 alternative 3’ splice sites (a3ss), and 2 alternative 5’ splice site (a5ss) events (fig 1f, supplemental table 2). these events affected 111 genes, of which only 3 were also significantly differentially expressed. gene ontology analysis of 111 genes did not result in any significant enriched pathways. therefore, we observed relatively mild splicing changes associated with the c9orf72 mutation. this may be due in part attributable to technical differences where the use of nuclear rna in our study likely reduces sensitivity in terms of identifying splicing alterations. table 3: annotation of significantly differentially expressed genes with general terms relevant to c9orf72 mutation. fdr = false discovery rate. fig 2: the c9orf72 mutation is associated with differential usage of genic elements with rna binding protein sites. (a) ma plot of dexseq differentially used genic elements with red being significantly differentially used elements. (b) genic elements (5’ utr, exon, intron, 3’ utr) distribution associated with c9orf72 mutation and all expressed bins. (c) differentially used elements that are bound by tdp-43 and hnrnp (χ2=5.617; p=0.0178). c9orf72 mutation is associated with differentially used 3’ utrs altered rna binding protein function has been proposed to contribute to c9orf72 toxicity. rna binding proteins can bind to different genic elements including exons, introns and untranslated regions (utr). if rbp activity is altered via titration of these proteins, it is possible the regions that may be bound by rbps are also differentially expressed. thus, dexseq was used to determine whether the presence of the repeat expansion resulted in differential usage of genic elements. there were a total of 865 significantly differentially used elements linked to the repeat expansion affecting 791 genes. specifically, there were 610 significantly downregulated elements and 255 significantly upregulated elements (fig 2a). when the elements were annotated as either 5’ utr, exon, intron or 3’ utr, there was a significant enrichment of differentially used 3’ utrs due to the c9orf72 mutation (13.35% vs 9.78%, χ2=12.50, p=0.0004) (fig 2b). given the role of rbps in binding to 3’ utrs, we hypothesized that the significantly differentially used elements were enriched for rbp binding sites. thus, hnrnp binding sites, including that of tdp-43 and other hnrnps were identified using pipe-clip algorithms applied to published data sets (54, 55, 66). indeed, there was an enrichment of differentially used genic elements that contain tdp-43 and hnrnp binding sites (χ2=5.617, p=0.0178, fig 2c). thus, the c9orf72 expansion is associated with some differentially used genic elements with rbp binding sites. transcriptomic signature due to depletion of tdp-43 pathologic nuclei given that neurons with tdp-43 inclusions have died during the course of disease and our experimental design includes removal of neurons with tdp-43 pathology via fac sorting before rna sequencing, we next considered the possibility that the transcriptomic alterations associated with the c9orf72 mutation in this dataset would in part reflect the depletion of neurons vulnerable to tdp-43 proteinopathy. indeed, within the neocortex, tdp-43 proteinopathy often preferentially affect neurons in superficial cortical laminae (52, 67). as shown in fig 3a, neurons with tdp-43 pathology including loss of nuclear tdp-43 (white) are depleted in c9orf72 expansion cases, while control brains have the normal complement of neurons (blue, fig 3b). as a result, we hypothesized that the depletion of diseased nuclei would contribute to the transcriptomic alterations associated with the c9orf72 mutation. thus, we expected that common genes that were downregulated in c9orf72 expansion cases would be upregulated in nuclei without tdp-43 and vice versa. indeed, among the 118 common significant differentially expressed genes linked to tdp-43 loss (52) and the c9orf72 mutation, a majority of them were followed this pattern (fig 3c, supplemental table 3). a linear regression analysis showed there was a negative correlation between the gene fold change linked to tdp-43 loss and the gene fold change linked to the c9orf72 mutation (pearson’s r=-0.5708; p<0.0001). furthermore, this relationship was extended transcriptome-wide where we observed a negative correlation in all commonly expressed genes derived from both data sets (fig 3d). for example, igsf11 and pvrl3 expression, preferentially observed in superficial neocortical layers and therefore increased in neuronal nuclei without tdp-43 protein (52), is significantly reduced in the c9orf72 transcriptome. conversely, syt6 and col6a1 expression, preferentially observed in deep neocortical layers and therefore decreased in neuronal nuclei without tdp-43 protein (52), is significantly increased in the c9orf72 transcriptome. this suggested that depletion of the diseased nuclei in c9orf72 expansion cases may explain some of the transcriptomic alterations observed in c9orf72 expansion cases. fig 3: c9orf72 transcriptome is linked to depletion of tdp-43 pathologic nuclei. (a) in c9orf72 expansion cases, there are nuclei with tdp-43 (blue) and those without tdp-43 (white). for our transcriptome analysis, only nuclei with tdp-43 were collected (red box), thereby depleting nuclei without tdp-43. (b) in non-diseased controls, nuclei with tdp-43 were collected (red box). (c) gene fold changes of genes that were common significant differentially expressed linked to tdp-43 loss and the c9orf72 mutation were plotted, showing that genes that were downregulated due to the c9orf72 mutation were upregulated due to tdp-43 loss, suggesting that depletion of diseased nuclei (without tdp-43) can partially explain the transcriptomic changes observed in this dataset (pearson’s r=-0.5708, p<0.0001). (d) genome-wide gene fold changes that were expressed in both datasets linked to tdp-43 loss and c9orf72 mutation showed a similar pattern. global c9orf72 associated transcriptome changes are linked to loss of function of the c9orf72 protein current hypotheses of c9orf72 toxicity include loss of c9orf72 protein or gain of toxic rna foci and dipeptide repeat aggregates (9). while some transcriptome changes which appear to be related to c9orf72 gain of toxic function (genes associated with proteostasis and dna damage, dysregulation of transcripts with hnrnp 3’utr binding sites), other changes appeared to be linked to the loss of c9orf72 protein (genes associated with vesicle membranes and endosomal trafficking). however, at a global transcriptome level, it was unclear whether the overall changes associated with the mutation reflect changes linked to a toxic gain of c9orf72 function or the loss of c9orf72 protein. c9orf72 rna foci and dipeptide repeat protein aggregates are seen in c9orf72 repeat expansion carriers but absent in neurologically normal controls. we have also demonstrated that within c9orf72 mutation carriers, c9orf72 promoter methylation is negatively correlated with the accumulation of rna foci and dipeptide repeat protein aggregates in human brains (43-45). based on this, we developed a theoretical framework (fig 4a-d) wherein statistical analysis was performed within c9orf72 expansion cases versus statistical analysis between c9orf72 expansion cases and controls. as a theoretical example, using c9orf72 itself as a gene that contributes to the loss of c9orf72 protein, we predicted that within c9orf72 expansion carriers, methylation of the c9orf72 promoter would negatively correlate with c9orf72 expression (fig 4a). moreover, we predicted that between-group analysis would show that c9orf72 expression is downregulated compared to controls (fig 4b). this would be an example of a “concordant” gene which provides evidence that the altered gene expression is linked to the loss of c9orf72 protein. conversely, using similar logic, if a gene contributes to the gain of toxic function such as the formation of toxic rna, we would predict that within c9orf72 expansion carriers, methylation of the c9orf72 promoter would negatively correlate with toxic rna (fig 4c). however, between groups, this gene would be upregulated in c9orf72 expansion carriers compared to controls (fig 4d). this would be an example of a “discordant” gene, which provides evidence that altered expression of this gene is associated with a gain of toxic function. fig 4: c9orf72 transcriptome reflects the loss of c9orf72 function. (a) theoretical analysis within c9orf72 mutation carriers, we predict that c9orf72 promoter methylation would negatively correlate with c9orf72 expression. (b) theoretical analysis between c9orf72 mutation carriers and non-diseased controls, we predict that c9orf72 expression is downregulated compared to controls. this would be an example of a “concordant” gene that suggests this gene is linked to loss of c9orf72 protein. (c) theoretical analysis within c9orf72 mutation carriers, we predict that c9orf72 promoter methylation would negative correlate with toxic rna. (d) theoretical analysis between c9orf72 expansion carriers and non-diseased controls, we predict that toxic rna expression would be upregulated. this would be an example of a “discordant” gene that suggests this gene is linked to gain of toxic rna. (e) within the actual data, there was a negative relationship between c9orf72 expression and methylation (pearson r=-0.6958, p=0.0825). (f) c9orf72 expression is downregulated in c9orf72 expansion carriers compared to controls (t-test, p=0.0003) as predicted in genes associated with the loss of c9orf72 protein. (g) within c9orf72 mutation carriers, there was a strong correlation between the expression of syp and c9orf72 methylation (pearson r=0.8835, p=0.0083). (h) between c9orf72 expansion carriers and controls, there was a significant upregulation of syp in expansion carriers (t-test, p=0.03). (i) using significantly differentially expressed genes, the methylation correlation within c9orf72 mutation cases was plotted against gene fold change between normal c9orf72 mutation cases and controls. there were more concordant genes than discordant genes, reflecting that the transcriptome was associated with loss of c9orf72 protein. (h) genome wide analysis of methylation correlation vs gene fold change shows the same trend. using the actual data, a correlation analysis was performed comparing c9orf72 expression and methylation of the c9orf72 promoter within our c9orf72 expansion cohort (pearson’s r=-0.6958; p=0.0825) (fig 4e). moreover, between-group analysis revealed that c9orf72 expression is downregulated in c9orf72 expansion carriers compared to controls as expected (t-test, p=0.0003) (fig 4f). another example of a “concordant” gene is synaptophysin (syp), a synaptic vesicle protein. the same correlation analysis was done which showed a positive correlation within c9orf72 expansion carriers (pearson’s r=0.8835; p=0.0083), and an increase in expression in c9orf72 expansion carriers compared to controls (t-test, p=0.03) (fig 4g-h, supplemental table 4). thus, we are able to show that “concordant” genes tend to reflect genes that may be related to c9orf72 protein function. this analysis was extended to all significantly differentially expressed genes where the “within c9orf72 mutation cases” methylation correlation value was plotted as a function of “between c9orf72 cases and controls” gene fold change values (fig 4i). across all significantly differentially expressed genes, there were 267 genes found within the concordant quadrants (blue circles) compared to only 56 genes found in the discordant quadrants (red circles) (fig 4g). similar results were found when this analysis was further expanded transcriptome-wide, suggesting that the global transcriptome changes linked to the c9orf72 mutation are associated with loss of c9orf72 protein (fig 4h). many “concordant” genes were genes involved in synaptic vesicle formation (i.e. clathrin light chain b, syntaphilin, synaptophysin) and vesicle trafficking (i.e. multivesicular body subunit 12a, rab40b, member ras oncogene family). genes labeled as “discordant” include genes related to inflammatory or apoptotic processes including tec protein tyrosine kinase, tyrosine kinase, and ring finger protein 152. thus two different factors appear to shape this global c9orf72 transcriptome dataset: (1) the depletion of neurons with tdp-43 pathology prior to rna-sequencing and (2) the loss of c9orf72 function. to estimate the relative contributions of these two factors, a multivariate linear regression analysis was performed wherein significant changes in gene expression associated with the c9orf72 mutation were related to (1) the changes in gene expression we previously identified due to tdp-43 pathology and (2) the loss of c9orf72 gene expression due to methylation of the c9orf72 promoter. together, these two factors explained the majority of the variance in this dataset (r2=0.5138; βmethylation=0.838, p<2e-16; βtdp-43=-0.499, p<2e-16). discussion here we report that in using post-mortem neuronal nuclei without tdp-43 pathology from c9orf72 expansion carriers, we were able to identify transcriptome changes that link the c9orf72 mutation with loss of c9orf72 function. we found that the top 1% of genes that contribute most to changes in c9orf72 expression were involved in cytoplasmic vesicle trafficking. significant gene expression changes were linked to the c9orf72 mutation and featured upregulated genes related to synaptic transmission and endosomal/lysosomal trafficking. indeed, c9orf72 has been shown to be involved in endosomal or lysosomal trafficking and may be a rab gtpase, and recent whole tissue transcriptome studies have linked the c9orf72 mutation to changes in vesicular transport genes (3-8). nine of the 11 genes related to trafficking were upregulated, potentially reflecting a compensatory mechanism to counteract the loss of c9orf72 and loss of this trafficking related protein. further analysis correlating methylation and gene expression changes confirmed that the transcriptome changes are linked to loss of c9orf72 expression. to parse the relative contributions of c9orf72 protein loss or gain of toxic rna, a methylation correlation between methylation values and expression data was calculated. this correlation analysis indicated that the transcriptome observed in neuronal nuclei from c9orf72 expansion cases was driven in large part by the loss of c9orf72 protein as opposed to a toxic gain of function. in contrast, we observed minor splicing alterations and relatively subtle changes in terms of alterations in rna binding protein function. thus, the global alterations appear to be most reflective of a loss of c9orf72 function rather than toxic rna mediated effects. however, it should be noted that c9orf72 methylation was used as a proxy for dpr and rna foci levels. a more direct analysis of the effects of dpr or rna foci would require specific isolation of dpr or rna-foci containing neuronal nuclei which has not yet been possible using fac sorting approaches. additional future studies can also include comparisons between als/ftd cases with and without the c9orf72 mutation. genes related to dna repair either as a response to dna damage or important to employ dna repair were also dysregulated. interestingly, recent papers have shown that dna damage is activated by the c9orf72 repeat expansion in both als patients and experimental models (23, 25, 26, 30, 68). dna damage has been a consistent feature among trinucleotide repeat expansion diseases and the dysregulation of dna repair related genes is consistent with dna damage contributing to neurodegeneration (69). furthermore, alterations in proteostasis either by failure to fold proteins properly or failure to degrade proteins likely contribute to protein aggregation and are a common theme among all neurodegenerative diseases (70, 71). indeed, overexpressing proteostasis factors in animal and cell als models prevents protein accumulation and aggregation (57, 72, 73), suggesting that altered proteostasis activity contributes to neurodegeneration. thus, although the global transcriptome appears to be driven by the loss of c9orf72 function, embedded within the c9orf72 transcriptome are changes in gene expression, which may relate to the toxic functions of c9orf72. in comparing the data from previous transcriptome analyses from c9orf72 expansion carriers, several differences can be observed. previous transcriptome analyses have identified pathways involved in inflammation and defense responses among dysregulated genes in the frontal cortices from c9orf72 expansion carriers (49). within our dataset, we did not find enriched inflammatory pathways, which is likely due to the fact that only neurons were being sequenced. it is possible that because c9orf72 is expressed in microglia, c9orf72 protein function may have a larger effect on microglial populations (41). given the evidence supporting that c9orf72 is involved inflammatory processes, further cell-specific analyses may prove beneficial to better understanding the effects of mutant c9orf72 on non-neuronal cells in driving disease pathogenesis. previous studies have found large numbers of splicing changes in c9orf72 expansion carriers and found motifs within these splicing changes that correspond to hnrnp h binding (49). however, we did not detect such large changes and instead found milder splicing changes with motifs that do not correspond to any known rna foci colocalization partners (data not shown). current studies debated over the disease relevance of rna foci as some animal models overexpressing the repeat induce rna foci but do not show motor or cognitive defects (13-16). the lack of splicing changes in our dataset does not support the model wherein neuronal toxicity is linked to the titration of rna binding proteins within rna foci. overall, our findings argue against rna toxicity attributed to sequestration of rna binding proteins. other disease-related pathways, such as protein aggregation and dna repair, were observed in this dataset, supporting a potential role for dipeptide repeat protein aggregates and nuclear dna damage in disease, consistent with experimental studies showing that overexpression of dipeptide repeat proteins are toxic both in cells and in animal models (13-32). while the transcriptome appeared to be shaped in large part by the loss of c9orf72 function, it is unclear whether the loss of c9orf72 function contributes to disease and thus understanding whether these changes are functionally linked to disease pathogenesis requires additional experimental studies and validation. finally, our analysis highlights the complexities associated with molecular studies of post-mortem human tissue wherein issues related to cell identity, experimental design, and admixtures of both gain and loss of function effects can be seen concurrently. contributions and acknowledgements these studies were supported by a grant from the judith & jean pape adams charitable foundation, a clinical scientist development grant from the doris duke charitable foundation, and nih grants (t32 ag000255, r21 ns097749, r01 ns095793, p01 ag017586, and p30 ag010124). we thank paul hallberg from the university of pennsylvania flow cytometry core for technical assistance, and the center for neurodegenerative disease at the university of pennsylvania for their support. we are indebted to the patients, caretakers, families, and physicians, for without their meaningful contributions and dedication to medical research this work would not be possible. references 1. m. dejesus-hernandez et al., expanded ggggcc hexanucleotide repeat in noncoding region of c9orf72 causes chromosome 9p-linked ftd and als. neuron 72, 245 (oct 20, 2011). 2. a. e. renton et al., a hexanucleotide repeat expansion in c9orf72 is the cause of chromosome 9p21-linked als-ftd. neuron 72, 257 (oct 20, 2011). 3. y. aoki et al., c9orf72 and rab7l1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia. brain : a journal of neurology 140, 887 (apr 01, 2017). 4. m. a. farg et al., c9orf72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. hum mol genet 23, 3579 (jul 01, 2014). 5. d. zhang, l. m. iyer, f. he, l. aravind, discovery of novel denn proteins: implications for the evolution of eukaryotic intracellular membrane structures and human disease. front genet 3, 283 (2012). 6. d. tang et al., cryo-em structure of c9orf72-smcr8-wdr41 reveals the role as a gap for rab8a and rab11a. proc natl acad sci u s a 117, 9876 (may 5, 2020). 7. c. p. cali et al., c9orf72 intermediate repeats are associated with corticobasal degeneration, increased c9orf72 expression and disruption of autophagy. acta neuropathologica 138, 795 (nov, 2019). 8. d. w. dickson et al., extensive transcriptomic study emphasizes importance of vesicular transport in c9orf72 expansion carriers. acta neuropathol commun 7, 150 (oct 8, 2019). 9. s. c. ling, m. polymenidou, d. w. cleveland, converging mechanisms in als and ftd: disrupted rna and protein homeostasis. neuron 79, 416 (aug 7, 2013). 10. j. cooper-knock et al., sequestration of multiple rna recognition motif-containing proteins by c9orf72 repeat expansions. brain : a journal of neurology 137, 2040 (jul, 2014). 11. y. b. lee et al., hexanucleotide repeats in als/ftd form length-dependent rna foci, sequester rna binding proteins, and are neurotoxic. cell rep 5, 1178 (dec 12, 2013). 12. e. g. conlon et al., the c9orf72 ggggcc expansion forms rna g-quadruplex inclusions and sequesters hnrnp h to disrupt splicing in als brains. elife 5, (sep 13, 2016). 13. j. g. o'rourke et al., c9orf72 bac transgenic mice display typical pathologic features of als/ftd. neuron 88, 892 (dec 02, 2015). 14. o. m. peters et al., human c9orf72 hexanucleotide expansion reproduces rna foci and dipeptide repeat proteins but not neurodegeneration in bac transgenic mice. neuron 88, 902 (dec 02, 2015). 15. a. jain, r. d. vale, rna phase transitions in repeat expansion disorders. nature 546, 243 (jun 08, 2017). 16. h. tran et al., differential toxicity of nuclear rna foci versus dipeptide repeat proteins in a drosophila model of c9orf72 ftd/als. neuron 87, 1207 (sep 23, 2015). 17. b. d. freibaum et al., ggggcc repeat expansion in c9orf72 compromises nucleocytoplasmic transport. nature 525, 129 (sep 03, 2015). 18. a. jovicic et al., modifiers of c9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to ftd/als. nat neurosci 18, 1226 (sep, 2015). 19. s. may et al., c9orf72 ftld/als-associated gly-ala dipeptide repeat proteins cause neuronal toxicity and unc119 sequestration. acta neuropathol 128, 485 (oct, 2014). 20. z. tao et al., nucleolar stress and impaired stress granule formation contribute to c9orf72 ran translation-induced cytotoxicity. hum mol genet 24, 2426 (may 01, 2015). 21. x. wen et al., antisense proline-arginine ran dipeptides linked to c9orf72-als/ftd form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. neuron 84, 1213 (dec 17, 2014). 22. l. d. goodman et al., toxic expanded ggggcc repeat transcription is mediated by the paf1 complex in c9orf72-associated ftd. nature neuroscience 22, 863 (jun, 2019). 23. y. j. zhang et al., heterochromatin anomalies and double-stranded rna accumulation underlie c9orf72 poly(pr) toxicity. science 363, (feb 15, 2019). 24. y. j. zhang et al., poly(gr) impairs protein translation and stress granule dynamics in c9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. nat med 24, 1136 (aug, 2018). 25. y. nihei et al., poly-glycine-alanine exacerbates c9orf72 repeat expansion-mediated dna damage via sequestration of phosphorylated atm and loss of nuclear hnrnpa3. acta neuropathologica 139, 99 (jan, 2020). 26. r. lopez-gonzalez et al., partial inhibition of the overactivated ku80-dependent dna repair pathway rescues neurodegeneration in c9orf72-als/ftd. proc natl acad sci u s a 116, 9628 (may 7, 2019). 27. t. g. moens et al., c9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eif1a. acta neuropathologica 137, 487 (mar, 2019). 28. a. jovicic et al., modifiers of c9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to ftd/als. nature neuroscience 18, 1226 (sep, 2015). 29. k. zhang et al., the c9orf72 repeat expansion disrupts nucleocytoplasmic transport. nature 525, 56 (sep 3, 2015). 30. r. lopez-gonzalez et al., poly(gr) in c9orf72-related als/ftd compromises mitochondrial function and increases oxidative stress and dna damage in ipsc-derived motor neurons. neuron 92, 383 (oct 19, 2016). 31. s. mizielinska et al., c9orf72 repeat expansions cause neurodegeneration in drosophila through arginine-rich proteins. science 345, 1192 (sep 05, 2014). 32. y. ohki et al., glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of c9orf72 associated neurodegeneration. mol neurodegener 12, 6 (jan 14, 2017). 33. p. e. ash et al., unconventional translation of c9orf72 ggggcc expansion generates insoluble polypeptides specific to c9ftd/als. neuron 77, 639 (feb 20, 2013). 34. m. dejesus-hernandez et al., in-depth clinico-pathological examination of rna foci in a large cohort of c9orf72 expansion carriers. acta neuropathol 134, 255 (aug, 2017). 35. i. r. mackenzie et al., quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in c9orf72 mutation carriers. acta neuropathol 130, 845 (dec, 2015). 36. t. g. moens et al., sense and antisense rna are not toxic in drosophila models of c9orf72-associated als/ftd. acta neuropathologica 135, 445 (mar, 2018). 37. a. j. waite et al., reduced c9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the c9orf72 hexanucleotide repeat expansion. neurobiology of aging 35, 1779 e5 (jul, 2014). 38. p. frick et al., novel antibodies reveal presynaptic localization of c9orf72 protein and reduced protein levels in c9orf72 mutation carriers. acta neuropathol commun 6, 72 (aug 3, 2018). 39. a. burberry et al., loss-of-function mutations in the c9orf72 mouse ortholog cause fatal autoimmune disease. sci transl med 8, 347ra93 (jul 13, 2016). 40. m. koppers et al., c9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits. annals of neurology 78, 426 (sep, 2015). 41. j. g. o'rourke et al., c9orf72 is required for proper macrophage and microglial function in mice. science 351, 1324 (mar 18, 2016). 42. q. zhu et al., reduced c9orf72 function exacerbates gain of toxicity from als/ftd-causing repeat expansion in c9orf72. nature neuroscience 23, 615 (may, 2020). 43. e. y. liu et al., c9orf72 hypermethylation protects against repeat expansion-associated pathology in als/ftd. acta neuropathol 128, 525 (oct, 2014). 44. c. t. mcmillan et al., c9orf72 promoter hypermethylation is neuroprotective: neuroimaging and neuropathologic evidence. neurology 84, 1622 (apr 21, 2015). 45. j. russ et al., hypermethylation of repeat expanded c9orf72 is a clinical and molecular disease modifier. acta neuropathol 129, 39 (jan, 2015). 46. k. j. karczewski et al., the mutational constraint spectrum quantified from variation in 141,456 humans. nature 581, 434 (may, 2020). 47. r. h. batra, k.; vu, a.;rabin, s.j.; baughn, m.w.; libby, r.t.; hoon, s.; ravits, j.; yeo, g.w., gene expression signatures of sporadic als motor neuron populations. biorxiv, (2016). 48. j. cooper-knock et al., c9orf72 ggggcc expanded repeats produce splicing dysregulation which correlates with disease severity in amyotrophic lateral sclerosis. plos one 10, e0127376 (2015). 49. m. prudencio et al., distinct brain transcriptome profiles in c9orf72-associated and sporadic als. nat neurosci 18, 1175 (aug, 2015). 50. f. krach et al., transcriptome-pathology correlation identifies interplay between tdp-43 and the expression of its kinase ck1e in sporadic als. acta neuropathologica 136, 405 (sep, 2018). 51. s. curran, j. a. mckay, h. l. mcleod, g. i. murray, laser capture microscopy. mol pathol 53, 64 (apr, 2000). 52. e. y. liu et al., loss of nuclear tdp-43 is associated with decondensation of line retrotransposons. cell reports 27, 1409 (apr 30, 2019). 53. j. b. toledo et al., a platform for discovery: the university of pennsylvania integrated neurodegenerative disease biobank. alzheimers dement 10, 477 (jul, 2014). 54. j. r. tollervey et al., characterizing the rna targets and position-dependent splicing regulation by tdp-43. nat neurosci 14, 452 (apr, 2011). 55. s. c. huelga et al., integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnrnp proteins. cell rep 1, 167 (feb 23, 2012). 56. j. wang, d. duncan, z. shi, b. zhang, web-based gene set analysis toolkit (webgestalt): update 2013. nucleic acids research 41, w77 (jul, 2013). 57. h. j. chen et al., the heat shock response plays an important role in tdp-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis. brain : a journal of neurology 139, 1417 (may, 2016). 58. d. chhangani et al., mahogunin ring finger 1 confers cytoprotection against mutant sod1 aggresomes and is defective in an als mouse model. neurobiol dis 86, 16 (feb, 2016). 59. a. m. ali et al., faap20: a novel ubiquitin-binding fa nuclear core-complex protein required for functional integrity of the fa-brca dna repair pathway. blood 119, 3285 (apr 05, 2012). 60. y. birger et al., chromosomal protein hmgn1 enhances the rate of dna repair in chromatin. the embo journal 22, 1665 (apr 01, 2003). 61. y. nomura, n. adachi, h. koyama, human mus81 and fancb independently contribute to repair of dna damage during replication. genes cells 12, 1111 (oct, 2007). 62. b. wang et al., abraxas and rap80 form a brca1 protein complex required for the dna damage response. science 316, 1194 (may 25, 2007). 63. l. castellini et al., kdm4b/jmjd2b is a p53 target gene that modulates the amplitude of p53 response after dna damage. nucleic acids research 45, 3674 (apr 20, 2017). 64. l. miccoli et al., selective interactions of human kin17 and rpa proteins with chromatin and the nuclear matrix in a dna damageand cell cycle-regulated manner. nucleic acids research 31, 4162 (jul 15, 2003). 65. l. c. young, d. w. mcdonald, m. j. hendzel, kdm4b histone demethylase is a dna damage response protein and confers a survival advantage following gamma-irradiation. the journal of biological chemistry 288, 21376 (jul 19, 2013). 66. b. chen, j. yun, m. s. kim, j. t. mendell, y. xie, pipe-clip: a comprehensive online tool for clip-seq data analysis. genome biol 15, r18 (jan 22, 2014). 67. e. b. lee et al., expansion of the classification of ftld-tdp: distinct pathology associated with rapidly progressive frontotemporal degeneration. acta neuropathologica 134, 65 (jul, 2017). 68. m. a. farg, a. konopka, k. ying soo, d. ito, j. d. atkin, the dna damage response (ddr) is induced by the c9orf72 repeat expansion in amyotrophic lateral sclerosis. hum mol genet, (may 08, 2017). 69. a. lopez castel, j. d. cleary, c. e. pearson, repeat instability as the basis for human diseases and as a potential target for therapy. nat rev mol cell biol 11, 165 (mar, 2010). 70. p. m. douglas, a. dillin, protein homeostasis and aging in neurodegeneration. j cell biol 190, 719 (sep 06, 2010). 71. o. d. king, a. d. gitler, j. shorter, the tip of the iceberg: rna-binding proteins with prion-like domains in neurodegenerative disease. brain res 1462, 61 (jun 26, 2012). 72. m. e. jackrel et al., potentiated hsp104 variants antagonize diverse proteotoxic misfolding events. cell 156, 170 (jan 16, 2014). 73. p. y. lin et al., heat shock factor 1 over-expression protects against exposure of hydrophobic residues on mutant sod1 and early mortality in a mouse model of amyotrophic lateral sclerosis. mol neurodegener 8, 43 (nov 21, 2013). copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurodevelopmental disorders feel free to add comments by clicking these icons on the sidebar free neuropathology 1:13 (2020) review top ten discoveries of the year: neurodevelopmental disorders mara dierssen centre for genomic regulation (crg); the barcelona institute of science and technology, and universitat pompeu fabra (upf), 08003 barcelona, spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona · spain mara.dierssen@crg.eu submitted: 25 february 2020 accepted: 12 april 2020 copyedited by: cathryn cadwell published: 15 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2672 keywords: in utero diffusion magnetic resonance imaging, in utero diffusion tensor imaging, rna editing, retrotransposons, brain organoids, single cell omics abstract developmental brain disorders, a highly heterogeneous group of disorders with a prevalence of around 3% of worldwide population, represent a growing medical challenge. they are characterized by impaired neurodevelopmental processes leading to deficits in cognition, social interaction, behavior and motor functioning as a result of abnormal development of brain. this can include developmental brain dysfunction, which can manifest as neuropsychiatric problems or impaired motor function, learning, language or non-verbal communication. several of these phenotypes can often co-exist in the same patient and characterize the same disorder. here i discuss some contributions in 2019 that are shaking our basic understanding of the pathogenesis of neurodevelopmental disorders. recent developments in sophisticated in-utero imaging diagnostic tools have raised the possibility of imaging the fetal human brain growth, providing insights into the developing anatomy and improving diagnostics but also allowing a better understanding of antenatal pathology. on the other hand, advances in our understanding of the pathogenetic mechanisms reveal a remarkably complex molecular neuropathology involving a myriad of genetic architectures and regulatory elements that will help establish more rigorous genotype-phenotype correlations. abbreviations 3d three-dimensional, adar adenosine deaminase acting on rna, asd autism spectrum disorder, adhd attention deficit hyperactivity disorder, ca3 cornu ammonis area 3, cen central executive network, cns central nervous system, cnvs copy number variations, dg dentate gyrus, dlpfc dorsolateral prefrontal cortex, dmn default mode network, dsm the diagnostic and statistical manual of mental disorders, dti diffusion tensor imaging, edqtl editing quantitative trait loci, eeg electroencephalography, fa fractional anisotropy, fxs fragile x syndrome, gaba γ-aminobutyric acid, go gene ontology, gwas genome-wide association study, herv human endogenous retroviruses, hpscs human pluripotent stem cells, hescs human embryonic stem cells, hipscs human induced pluripotent stem cells, ipscs induced pluripotent stem cells, l1hs human-specific line-1, line-1 long-interspersed nuclear element-1, mpfc medial prefrontal cortex, mri magnetic resonance imaging, pgc psychiatric genomics consortium, rs-fmri resting-state functional mri, scrna-seq single-cell rna-sequencing, snrna-seq single-nucleus rna-sequencing, tbss tract-based spatial statistics, tes transposable elements, tsa tract-specific analysis introduction human brain structural and functional development occurs over a protracted period compared to many other mammals and primates (watson et al., 2006). this coordinated development provides the architecture for the expansion of behavioral and cognitive abilities, especially rapid in the first years, but also especially vulnerable to genetic and/or environmental insults leading to developmental brain disorders. classically those include intellectual disability, autism spectrum disorder (asd), attention deficit hyperactivity disorder (adhd), specific learning disorder, motor disorder, or epilepsy. however, beyond the traditional concept of neurodevelopmental disorders, studies in humans provide clear evidence that mental disorders such as schizophrenia, drug abuse, or neurodegenerative disorders such as alzheimer’s disease also have a strong developmental component that might be identified by specific neuropathological features (thibaut, 2018). understanding the role that development plays in the expression of these disorders is often overlooked, but definitively needs more attention to fully understand the impact of early life events on the complex neurobiological derangement. from the clinical point of view, there has been a recent move at the diagnostic level from a categorical toward a spectrum-based view. for example, the definition of autism has been highly debated, in several revisions of the diagnostic and statistical manual of mental disorders (dsm; dsm-iii, dsm-iiir, dsm-iv, and dsm-v) criteria (rodgaard et al., 2019). this has led to a steady increase in the heterogeneity of some conditions which could affect the results of autism research. the recent advances in genomic medicine and the development of improved cellular models, will certainly help overcome these difficulties. on the mechanistic side, advances in directed differentiation of human induced pluripotent stem cells (hipscs) and other neural cell preparations (van den ameele et al., 2014), coupled with the application of advanced histological, imaging, molecular, cellular, and genomic techniques, is providing insights into cellular and molecular processes in human neurodevelopmental disorders and also in a wide array of neurological and psychiatric disorders (brennand et al., 2011; lancaster et al., 2013; mariani et al., 2015; pasca et al., 2015). moreover, the recent advancements in single cell functional genomic techniques have enabled comprehensive and unbiased characterization of the molecular processes in human postmortem cns tissues and neural cell culture systems, and will allow us to explore neurodevelopmental neuropathology at a resolution that was not possible before. even so, our understanding of the spatiotemporal landscape of the rna species, and epigenetic features in the developing human brain, and their pathogenicity is still incomplete (qureshi & mehler, 2012). finally, some findings suggest common pathophysiological mechanisms that can be considered as a continuum of developmental brain dysfunctions. these findings revealed molecular pathways that are commonly altered in different forms of developmental brain disorders, both of genetic and environmental origin and new players are starting to be recognized such as genomic regulatory elements. 1. understanding neurodevelopmental disorders through imaging the prenatal brain one of the most crucial questions in neurodevelopmental disorders is what we can really detect from the wide range of developmental processes that take place (and can go wrong) during this critical period, and the extremely rapid pace of structural and functional brain development (geng et al., 2017). the dynamic morphological changes the fetal brain undergoes during early development result from neurogenic events, such as neuronal proliferation, migration, axonal elongation, retraction, and myelination. in the critically sensitive mid-gestational phase of development, structural and functional assessment of the fetal brain opens a window into prenatal diagnostics and prognostics, and would help establish biomarkers for prenatal diagnoses (batalle et al., 2017). conventional t1 and t2 weighted sequences provide anatomic detail of the normally developing brain and can demonstrate lesions, including those associated with preterm birth, hypoxic ischemic encephalopathy, perinatal arterial stroke, infections, and congenital malformations. specialized imaging techniques can also be used to assess cerebral vasculature (magnetic resonance angiography and venography), cerebral metabolism (magnetic resonance spectroscopy), cerebral perfusion (arterial spin labeling), and function (functional magnetic resonance imaging [mri]) (counsell et al., 2019). recent advances in in utero diffusion mri provide unique opportunities to noninvasively study the microstructure of tissue during neurodevelopment and possible mechanisms of how pathologies, maternal, or environmental factors that may interfere with brain development can be potentially detected. one important aspect is that postmortem histologic studies of early childhood cortical development are consistent with imaging studies and provide insights into the neurobiological process that underlie change observed in imaging studies (christiaens et al., 2019). abnormalities in processes, such as white matter and cortical connectivity in preterm babies have already been suggested to derive into late language development and impaired cognitive performance in children. in utero diffusion tensor imaging (dti) provides new windows to monitor the emergence of the connectome. these advances have led to emerging fields of research, such as developmental pathoconnectomics (jakab, 2019) that aim to unravel the development of disrupted brain connectivity. a wealth of quantitative tools, most of which were originally developed for the adult brain, can be applied to study the developing brain in utero and postnatally including measures of tissue microstructure obtained from diffusion mri, morphometric studies to measure whole brain and regional tissue volumes, and automated approaches to study cortical folding. however, the differences in functional organization between the brains of infants and adults call for infant-specific functional atlases for better definition of regions of interest and interpretation of results. thus, the creation of resources such as a dti atlas of the fetal brain is required for reliable detection of major neuronal fiber bundle pathways and for characterization of the fetal brain reorganization in utero. such tools are also useful for detection of normal and abnormal fetal brain development providing normative quantitative and qualitative data. the paper by khan et al. (khan et al., 2019) presents the first dti atlas of the fetal brain computed from in utero diffusion-weighted images. it was built from 67 fetal dti scans acquired from healthy fetuses each scanned at a gestational age between 21 and 39 weeks, addressing a wider gestational age range and larger number of sampled areas than prior studies. the atlas computation method represents the first comprehensive approach to compute motion-robust diffusion tensor maps from noisy in utero fetal measurements and combine individual dti maps into a kernel-regressed template at any given gestational age which would enable statistical voxel-wise analysis, tract-based spatial statistics (tbss), or tract-specific analysis (tsa) based on dti data (pecheva et al., 2017; khan et al., 2018). the neurodevelopmental trends characterized by the atlas in the fetal brain were qualitatively and quantitatively compared with prior ex vivo and in utero studies, and with observations reported in gestational age-equivalent preterm infants. the atlas correctly detected the early presence of limbic fiber bundles followed by the appearance and maturation of projection and association fiber bundles (characterized by an age-related increase in fractal anisotropy) during late 2nd and early 3rd trimesters and the appearance and maturation of projection throughout gestation. during the 3rd trimester association fiber bundles become evident. in parallel with the appearance and maturation of fiber bundles, from 21 to 39 gestational weeks a gradual disappearance of the radial coherence of the telencephalic wall was qualitatively identified. the results presented confirm previous advanced fetal connectome imaging studies indicating increased vulnerability of the human brain during late gestation for pathologies that might lead to impaired connectome development and subsequently interfere with the development of neural substrates serving higher cognition. the atlas complements previous initiatives including recent development of detailed atlases of the fetal (wright et al., 2015) and neonatal (makropoulos et al., 2016) brain that allow robust automated or semi‐automated segmentation of brain regions (makropoulos et al., 2014) and precise delineation of cortical sulcal and gyral development (garcia et al., 2018). together, all these tools allow characterization of the normal trajectories of fetal brain growth and creation of population centile charts (https://www.developingbrain.co.uk/fetalcentiles/ [gousias et al., 2013]). comparison with these typically developing growth charts therefore provides an ideal approach with which to assess, quantify, and identify deviations in regional and whole brain volumes and also in the ontogenesis, architecture, and temporal dynamics of the human brain connectome, and would lead to a more precise understanding of the etiological background of neurodevelopmental and mental disorders. these tools can be used as a reference for dti-based studies on in utero fetal brain development, for groupwise dti studies to investigate normal and abnormal brain development and to enable multi-modality imaging and computer-aided diagnosis from in utero dtis. all these works certainly highlight the significant progresses we have made. however, a lack of specificity between mri signal and neuropathological substrate is reported, as illustrated recently in one study showing that fractional anisotropy (fa) correlated with astrocyte density, a cell type typically not considered in mri studies (stolp et al., 2018). this indicates that the anatomical and histological significance of many “signals” in the brain remain to be investigated and further studies are needed to determine the precise cellular and molecular substrates of abnormal connectivity and gray matter microstructure observed on mri. the new methods of three-dimensional (3d) multiscale histological imaging developed in the last years, including clarity (chung & deisseroth, 2013), map (ku et al., 2016) or shield (park et al., 2018), enable rapid identification of multi-scale functional networks and interrogation of their system-wide, multifactorial interactions and thus, may help in the future for integrative and comprehensive understanding of large-scale complex biological systems. 2. predictive value of childhood neuroimaging as discussed, in utero mri detects fetal brain abnormalities more accurately than ultrasonography and provides additional clinical information in around half of pregnancies. however, it is less accurate when used to predict abnormal developmental outcome, although still better than ultrasonography as shown by hart and colleagues (hart et al., 2020). in their work published in lancet child and adolescent health, they studied the ability of in utero mri to predict developmental outcome and whether performing postnatal neuroimaging after age 6 months changes its diagnostic accuracy. the study was performed in a cohort of children assessed with the bayley scales of infant and toddler development, the ages and stages questionnaire, or both and contrasted with ultrasonography findings. participants' development was categorized as normal, at risk, or abnormal. however, the authors did not find statistically significant differences in infants with abnormal outcome. the authors suggest that although in utero mri remains the optimal tool to identify fetal brain abnormalities, it is not accurate in predicting developmental outcome (hart et al., 2020). further work is needed to determine how the prognostic abilities of in utero mri can be improved to identify putative infant brain markers that might be associated with neurodevelopmental disorders (batalle et al., 2018). instead, some works suggest that mapping the neurodevelopmental trajectories in childhood has the potential to enhance the early identification of risk. whitfield-gabrieli and colleagues analyzed the data from a longitudinal study of 94 children, who underwent resting-state functional mri scans at ages 7 and 11 (whitfield-gabrieli et al., 2019). they explored how specific patterns of brain resting state functional connectivity change during typical development, and how these changes related to behavior. the interesting feature of this study is that children were initially recruited as typically developing, but some began to exhibit clinical symptoms over time. the aim of the study was to identify predictors of anxiety and depression behaviors in children with no familiar risk for these disorders. specifically, the authors explored whether dysregulated top-down control mechanisms can be detected even before behavioral symptoms are evident and can predict individual children’s trajectories of attentional and internalizing problems, given that in adults with depression, anxiety, and adhd a number of studies have shown attenuation or failure of top-down control. the strength of coupling between regions involved in top-down control and their targets can be measured with resting-state functional magnetic resonance imaging (rs-fmri; liu et al., 2018; finn et al., 2015) and reliably characterizes the functional organization of the brain at a systems level (castellanos et al., 2013). the authors focused on functional connectivity involving the default mode network (dmn), a resting-state network associated with internal mentation and self-referential processing, whose key nodes include the medial prefrontal cortex (mpfc). in neurotypical adults, the dmn is negatively correlated (i.e., anticorrelated) with the central executive network (cen; fox et al., 2005), associated with externally focused attention and goal-directed behavior, of which the dorsolateral prefrontal cortex (dlpfc) is a key node. the magnitude of the anticorrelations between the mpfc and the dlpfc is significantly correlated with superior cognitive performance such as working memory capacity (e.g., hampson et al., 2010; keller et al., 2015). in typically developing children, the magnitude of anticorrelations between the mpfc and dlpfc increases with age along with the improvement of top-down control mechanisms. in their study whitfield-gabrieli and colleagues detected individual differences in functional connectivity of the dlpfc that predicted subsequent appearance of symptoms associated with adhd and depression. specifically, weaker positive functional connectivity between the dlpfc and the mpfc at age 7 was associated with a decrease in adhd symptoms by age 11, whereas weaker positive functional connectivity between the dlpfc and the subgenual anterior cingulate cortex was associated with an increase in mood-related symptoms by age 11. in fact, brain connectivity at age 7 predicted mood-related difficulties at age 11 better than baseline clinical symptoms themselves. a limitation of the work is the lack of information about which children eventually developed psychiatric disorders in this sample later on. however, these results suggest the potential utility of connectivity patterns as a biomarker of symptom trajectories. 3. the neurodevelopmental neuropathology of schizophrenia during the last year studies performed in humans have provided clear evidence that mental disorders such as schizophrenia have a strong developmental component that might be identified by specific neuropathological components. earlier neuroimaging works reported gray matter deficits in schizophrenic patients, mostly localized in frontal and temporal lobes, which are present prior to the onset of psychosis and worsen during the first few years of illness (glahn et al., 2008). more recently, widespread white matter decline was shown in whole-brain mri of schizophrenia patients, identifying it as a dysconnectivity syndrome, instead of just a cortical lesion syndrome (fornito et al., 2015). several theories of schizophrenia suggest that structural white matter pathologies may follow developmental (mcgrath et al., 2003; murray et al., 2017), maturational (van haren et al., 2008; french et al., 2015), and/or degenerative (cropley et al., 2017) trajectories. cetin-karayumak et al. (cetin-karayumak et al., 2019) have addressed this question and also studied whether structural white matter pathologies vary among fiber tracts across the brain. to this aim they analyzed the largest sample of harmonized diffusion mri data to comprehensively characterize age-related white matter neuropathology, as measured by fa. the analysis comprised diffusion scans of 600 schizophrenia patients and 492 healthy controls at different illness stages and ages (14–65 years), gathered from 13 sites. the authors cross-sectionally determined the pattern of age-related fa changes associated with schizophrenia. in whole-brain white matter, fa was up to 7% lower across the lifespan and reached peak maturation earlier in patients (27 years) than controls (33 years), and three distinct patterns of neuropathology could be identified. they detected tract-specific early developmental abnormalities in limbic fibers that do not progress over time, suggesting that limbic connections are selectively vulnerable to early developmental anomalies. instead, long-range intra-hemispheric association tracts (including language tracts) displayed abnormal maturation with shorter maturational windows and faster declines consistent with accelerated ageing processes in schizophrenia. finally, the authors report accelerated aging in callosal fibers that exhibited severe deficits from the outset of illness, which became more pronounced with increasing age (reaching a 10% reduction after sixth decade). this reduced anisotropy of the corpus callosum is a well-replicated diffusion imaging finding in schizophrenia, consistent across heterogeneous patient populations and maturational phases. the study makes a timely and important contribution to the field showing that white matter neuropathology in schizophrenia involves lifelong dynamic tract-specific changes. the findings support a developmental perspective, suggesting that widely distributed white matter deficits emerge early or display perturbed maturation. in addition, callosal and long-range association (but not limbic) fibers undergo accelerated aging processes. this work provides an initial benchmark for tract-specific trajectories of white matter abnormalities. 4. disease-in-a-dish developmental neuropathology in recent years there has been a growing emphasis on developing patient-specific cellular models that can be manipulated by the experimenter to understanding the role of different factors in shaping individual brain development and functioning. human pluripotent stem cells (hpscs), including human embryonic stem cells (hescs) and human induced pluripotent stem cells (hipscs), have been revealed as invaluable tools for modeling human disorders, especially those with complex genetic origins (takahashi et al., 2007; takahashi & yamanaka, 2006). hpscs have the potential to differentiate into any cell or tissue type. induced pluripotent stem cells (ipscs) reprogrammed from patient somatic cells also offer an opportunity to recapitulate disease development in relevant cell types, and they provide novel approaches for understanding disease mechanisms. stem cells can be used to generate organoids, organ-like 3d tissue cultures containing multiple cell types that represent accessible systems for modeling organogenesis and developmental disorders (lyon, 2019). hpsc-derived brain organoids self-assemble to form an organized architecture, composed of progenitor, neuronal and glial cell types, resembling the fetal human brain (jo et al., 2016; kadoshima et al., 2017; lancaster et al., 2013). until recently, these in vitro systems had strong limitations. first, organoids develop without the presence of normal embryonic surrounding thus lacking the developmental and patterning cues, which are essential for organ development. additionally, most protocols depend on the ability of stem cells to self-organize into distinct brain structures which can cause inconsistency in producing the desired tissues, resulting in heterogeneity or “batch-effects” in different batches of organoids, which can vary in quality and brain regions they generate, making it difficult to identify real phenotypes. human brain organoids are prone to high organoid-to-organoid variability (quadrato et al., 2016; yoon et al., 2019; grenier et al., 2020). furthermore, the absence of vascularization is probably responsible for the shortage of progenitor populations, making it difficult to replicate cortical plate formation. all of this has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility that is comparable to the endogenous tissue (jabaudon & lancaster, 2018). the last years have been exciting for the field, because some of these limitations are starting to be overcome. recently, mansour et al. (mansour et al., 2018) showed that intracerebral transplantation of brain organoids in mice results in impressive growth of blood vessels into the human tissue, with clear benefits for cell survival and maturation compared with organoids kept in vitro. also, real and colleagues (real et al., 2018) have shown the potential of these methods to model human neuropathology. they transplanted human ipsc-derived cortical neurons from two persons with down syndrome into the adult mouse cortex and observed that those consistently organized into large (up to ~100 mm3) vascularized neuron-glia territories. down syndrome transplants showed increased synaptic stability and reduced oscillations, thus recapitulating in part the patients’ phenotypes. finally velasco and colleagues (velasco et al., 2019) showed that an organoid model of the dorsal forebrain can reliably generate a rich diversity of cell types appropriate for the human cerebral cortex. using single-cell rna-sequencing analysis of 166,242 cells isolated from 21 individual organoids, the authors show that the organoid-to-organoid variability is comparable to that of individual endogenous brains, and 95% of the organoids derived from different stem cell lines generate a virtually indistinguishable compendium of cell types, showing consistent reproducibility in the cell types produced. the authors thus demonstrated that establishment of terminal cell identity is a highly constrained process and that reproducible developmental trajectories of cellular diversity of the cns does not require the context of the embryo. 5. growing networks in a dish most of the current organoid protocols or medium formulations favor progenitor cells, thus not enabling them to mature and mimic some aspects of the human brain development such as dynamic changes in cellular populations during maturation or the formation of long-range connectivity. in a recent work, trujillo and colleagues (trujillo et al., 2019) developed cortical organoids that spontaneously displayed periodic and regular oscillatory network events that are dependent on glutamatergic and γ-aminobutyric acid (gaba)-ergic signaling. they could record consistent increases in electrical activity over the span of several months reflecting the formation of a spontaneous network that displayed periodic and regular oscillatory events. these nested oscillations exhibited cross-frequency coupling, subsequently transitioning to more spatiotemporally irregular patterns, resembling features observed in preterm human electroencephalography (eeg). these results suggest that the development of structured network activity in the human neocortex takes place even in the absence of external or subcortical inputs, and open opportunities for investigating and manipulating the role of network activity in the developing human cortex. two independent groups (cullen et al., 2019; kirihara et al., 2019) have developed a human stem cell-derived model of cerebral tracts and have described the phenotype and connectivity of constrained 3d human axon tracts derived from brain organoids. in the work of kirihara and colleagues they used a microfluidic device, in which two spheroids of cortical neurons derived from hipscs extended axons into a microchannel between the spheroids and spontaneously formed an axon fascicle, mimicking a cortico-cortical tract that connected the two spheroids reciprocally. the axon fascicle was able to communicate electrically between the spheroids with distinct response kinetics. this cerebral tract model should provide a promising platform to study the mechanisms underlying cerebral tract development and related diseases. using their model of cerebral tracts, the authors could recapitulate the agenesis of corpus callosum (edwards et al., 2014), by knocking down l1cam gene (demyanenko et al., 1999; siegenthaler et al., 2015) in the spheroids. axons from the l1cam knockdown cells exhibited significantly lower ratio of axons assembled into a bundle than the control cells, suggesting that this method can be used to model developmental disease related to cerebral tracts. microfluidic devices, involving a two-compartment system connected by narrow grooves, have also been used to reconstruct dentate gyrus (dg)–cornu ammonis area 3 (ca3) circuitry in which dg neurons are cultured in one compartment and ca3 neurons are cultured in the other compartment (sarkar et al., 2018). in this system, axonal growth is allowed through the narrow grooves connecting the two compartments whereas cell migration is restricted. rabies virus infection of the ca3 neurons permits the detection of presynaptic neurons that monosynaptically connect to the postsynaptic ca3 neurons. these engineered neural tissue connectivity models represent a first step toward potentially reconstructing brain circuits by physically replacing neuronal populations and long-range axon tracts in the brain, and might reveal how long-range connections are altered in the brains of people with neurodevelopmental disorders. 6. genes involved in early cortical patterning are at the heart of mental comorbidity over the past decade, genetic studies have been quite successful at identifying rare genetic variations, including inherited and de novo mutations and copy number variations (cnvs), related to specific developmental disorders. however, evidence is mounting to suggest that genetic risk variants identified among individuals with different brain disorders may converge on common genetic pathways. the remarkably complex architecture that embraces genetic mutations of distinct types (chromosomal rearrangements, copy number variants, small indels, and nucleotide substitutions) with distinct frequencies in the population (common, rare, de novo) creates difficulties in establishing rigorous genotype-phenotype correlations (cardoso et al., 2019). moreover, the cumulative effect of multiple common genetic variants, i.e., polygenic risk, is now being recognized as an important indicator of neurodevelopmental and psychiatric disorders (cross-disorder group of the psychiatric genomics consortium, 2013). large-scale genome-wide asd and cross-disorder association studies with enough statistical power to estimate small effects from common genetic variants are only now emerging, and they require combining data sets from multiple, large population samples. this need has been long recognized as testified by initiatives such as the psychiatric genomics consortium (pgc), the largest consortium in the history of psychiatry (sullivan et al., 2018). now, in the largest-ever study of its kind, published in cell, lee et al. (cross-disorder group of the psychiatric genomics consortium, 2019) identified more than 100 genetic variants that affect the risk for more than one mental health condition, indicating that distinct psychiatric diseases share a common genetic structure, as shown. using genome-wide association to analyze genetic data from 494,162 healthy controls and 232,964 individuals diagnosed with at least one of eight common psychiatric disorders, the researchers identified 109 gene variants that affect the risk for more than one psychiatric disorder. certain disorders shared many variants, allowing the researchers to divide the conditions into three groups of genetically-related conditions: disorders characterized by compulsive behaviors (anorexia nervosa, obsessive-compulsive disorder and, to a lesser extent, tourette syndrome); mood and psychotic disorders (bipolar disorder, major depression and schizophrenia); and early-onset neurodevelopmental disorders (asd, adhd and tourette syndrome). importantly, genes associated with multiple disorders showed increased expression beginning in the second trimester of pregnancy and play an important role in brain development. another research team has found the first common genetic risk variants for autism robustly associated with asd (grove et al., 2019) in a genome-wide association meta-analysis carried out in a danish population resource of 18,381 individuals with asd and 27,969 controls. the meta-analysis identified five genome-wide-significant loci. moreover, the study identified several asd gene variants that had especially widespread influence on the risk for a number of psychiatric disorders. concretely, the authors report seven loci shared with other traits, obtained by combining the dataset with genome-wide association study (gwas) results from phenotypes with significantly overlapping genetic architectures (schizophrenia, major depression, and educational attainment). the researchers also uncovered, for the first time, genetic differences between the different clinical subgroups of autism. this will pinpoint genes that separate the diagnostic groups and enable more precise diagnosis, and could orient the search for behavioral, imaging and electrophysiological markers of atypical development in the infant brain. these genetic findings provide an entirely new insight into the cross-disorder biological processes, particularly relating to neuronal function and corticogenesis, and help to triangulate on families of genes active during early cortical patterning (morgan et al., 2019; figure 1), associated with the establishment and maintenance of neuronal connectivity (parikshak et al., 2015) or with the foundation of inhibitory/excitatory balance in early childhood (marin, 2012).   figure 1. left panel: large-cohort genome-wide studies are allowing to triangulate families of genes involved in early brain maldevelopment. middle: early cortical patterning is at the heart of mental comorbidity. the advances in single-cell omics will help understand the pathogenesis of developmental lesions. right panel: critical steps of brain connectivity development have possible links to in utero mri-detectable phenomena. for example, emergence of long-range connectivity may be detected by in-utero diffusion tensor tractography as increasing integration demonstrated using whole-brain diffusion tractography. 7. mechanistic role of rna editing in schizophrenia and autism neuropathology we have progressively realized in recent years that the molecular neuropathology landscape of brain disorders is much more complex than anticipated and new players, such as epigenetic regulation, alternative splicing, post-transcriptional and -translational modifications, and somatic mosaicism of dna, not only in protein-coding sequences but also in large non-coding regions, contribute to developmental neuropathology. transcriptomic analyses of postmortem brains have begun to elucidate some of these new mechanisms in asd and schizophrenia. however, a crucial pathway involved in synaptic development and neuronal function, rna editing, has not yet been studied on a genome-wide scale. rna editing is a major rna processing mechanism, which refers to the alteration of rna sequences through insertion, deletion or substitution of nucleotides. it is thought to constitute one of the molecular mechanisms connecting environmental stimuli and behavioral outputs (lapp & hunter, 2019). adenosine to inosine (a-to-i) editing is the most common form of rna editing (mallela & nishikura, 2012; figure 2), affecting the majority of human genes, and is highly prevalent in the brain (liscovitch et al., 2014; suarez et al., 2018). editing sites in coding regions can be conserved across species and are commonly located in genes involved in neuronal function. site-specific a-to-i rna base conversions, carried out by adenosine deaminase acting on rna (adar) enzymes, exhibit precise regional specificity in the brain and modulate complex behavior in model organisms. examples of transcripts edited by adar are mrnas encoding glutamate receptors, serotonin receptors, and potassium channels and other neuronal signaling functions in the cns (rosenthal & seeburg, 2012; meier et al., 2016; streit & decher, 2011). these sites are tightly and dynamically regulated throughout preand post-natal human cortical development (figure 2). most editing sites reside in non-coding regions, and particularly within sequences called transposons via rna intermediates (retrotransposons, see below) with a possible pathophysiological role (krestel & meier, 2018). aberrant rna editing has been reported in several neurological disorders, including major depression, alzheimer’s disease, and amyotrophic lateral sclerosis.   figure 2. elevated adar2 expression in neuronal nuclei increases a-to-i rna editing during neuronal maturation. left panel: adar enzyme acting on double-stranded rna. adar rna-binding domains act on rna binding sites and convert adenine to inosine. right panel: as neurons mature, a-to-i rna editing increases gradually together with expression of importin-a4. as a result, adar is elevated in the nucleus leading to increased a-to-i rna editing in mature neurons. credits: ilario de toma, adapted from behm et al., 2017 and lorenzini et al., 2018. in asd, recent studies in postmortem samples have revealed transcriptome dysregulation affecting neuronal and glial coding and non-coding gene expression, neuronal splicing including microexons, and microrna targeting (irimia et al., 2014; salloum-asfar et al., 2019; gandal et al., 2018). tran et al. (tran et al., 2019) have now tested the largest cohort of post-mortem asd brain samples, spanning multiple brain regions for abnormalities in rna editing. a previous study had analyzed a few known rna editing sites in synaptic genes and reported altered editing patterns in a small cohort of asd cerebella (eran et al., 2013), but it was not known whether global patterns of rna editing contributed to the neuropathology of asd. now tran and colleagues report global patterns of dysregulated rna editing, with hypoediting across brain regions and involving many synaptic genes in asd brains. the set of genes harboring at least one differential editing site in frontal cortex exhibited significant gene ontology (go) enrichment for ionotropic glutamate receptor activity, glutamate gated ion channel activity, and synaptic transmission. the authors also identified a core set of down-regulated rna editing sites, enriched in asd susceptibility genes. interestingly, a set of these hypoedited sites are related to fragile x syndrome (fxs) proteins. concretely, fmrp and fxr1p interact with adar and modulate a-to-i editing. the authors detected convergent dysregulated patterns of rna editing in fxs and asd patients, consistent with the findings that genes harboring asd risk mutations are enriched in fmrp targets. their findings were corroborated across multiple datasets, including dup15q cases associated with intellectual disability. regarding schizophrenia, breen and colleagues (breen et al., 2019) have analyzed the global landscape and genetic regulation of rna editing across several hundred schizophrenia and control postmortem brain samples from the dorsolateral prefrontal cortex and anterior cingulate cortex. in schizophrenia, rna editing sites in genes encoding ampa-type glutamate receptors and postsynaptic density proteins were less edited, whereas those encoding translation initiation machinery were more edited. these findings were cross-validated in hundreds of non-overlapping dorsolateral prefrontal cortex samples. furthermore, ~30% of rna editing sites associate with cis-regulatory variants (editing quantitative trait loci or edqtls). fine-mapping edqtls with schizophrenia risk loci revealed co-localization of eleven edqtls with six gwas loci. the findings demonstrate widespread altered rna editing in schizophrenia and its genetic regulation, and suggest a causal and mechanistic role of rna editing in schizophrenia neuropathology. overall, both papers provide global insights regarding rna editing in asd and schizophrenia pathogenesis and reveal novel mechanisms underlying these disorders. 8. incomplete silencing of full mutation alleles in males with fragile x syndrome is associated with autistic features the paper by tran et al. (tran et al., 2019) convincingly demonstrated that rna editing acts as a molecular link between fxs and asd. fxs is caused by loss of the fmr1 product (fmrp), but also by mosaicism for active and inactive fmr1 alleles, including alleles termed premutation (55–199 cggs). importantly, both premutation and active full mutation (≥ 200 cggs) alleles often express elevated levels of mrna that are thought to be toxic. two studies published in 2019 report that incomplete silencing of toxic full mutation rna may be associated with autistic features in fxs males. baker and colleagues (baker et al., 2019) studied whether complete fmr1 mrna silencing from full mutation alleles and/or levels of fmr1 mrna (if present) in blood were associated with intellectual functioning and autism features in fxs. the study cohort included 98 participants (70.4% male) with fxs (full mutation-only and mosaic). fmr1 mrna was analyzed against control fmr1 mrna and correlated with intellectual disability and autistic features. fmr1 was completely turned off in some of the patients, while it was partially turned on in the majority of participants. females with fxs had significantly higher levels of fmr1 mrna that were not associated with intellectual functioning nor autistic features. in fxs males decreased levels of fmr1 mrna were associated with decreased intellectual functioning, but not autism features. the authors show for the first time that people with a partially turned-on fmr1 had intellectual disability and more traits of asd, whereas people with the gene completely turned off had intellectual disability and much less severe autism. the authors conclude that abnormally elevated levels of fmr1 mrna may lead to fmr1 mrna-related cellular “toxicity”. these findings may explain why severity of fragile x is not the same between affected individuals. based on this study, some months later field et al. (field et al., 2019) described the case of two young brothers with expanded fmr1 alleles, who were ‘high functioning’. the two brothers presented autistic features and language delay, but a higher non-verbal iq in comparison to typical fxs. both had low-level methylation mosaicism not detected by standard testing in blood, and their fmr1 mrna levels were increased ~5-fold compared to typical developing controls, and significantly above the levels reported from baker’s study described above. the authors speculate that the active unmethylated full mutation and/or premutation alleles lead to the expression of toxic expanded mrna in some cells, in conjunction with possible reduced fmr1 mrna and fmrp levels in other cells with fmr1 methylation. both mechanisms may contribute to the elevated asd symptoms. this hypothesis is also in line with the findings reported by baker et al. showing that males who expressed full mutation fmr1 mrna, had significantly more severe asd symptoms compared to males who had completely silenced fmr1. 9. transposable elements in neurodevelopmental disorders transposable elements (tes) constitute about half of the human genome and are becoming increasingly important to the field of neuroscience as their roles in mammalian development, immune response, and contributions to behavioral and cognitive domains continue to be uncovered (international human genome sequencing consortium, 2001; nandi et al., 2016). transposons are mobile dna elements present in virtually all eukaryotes that can replicate and mobilize from one chromosomal loci to another through either a dna or rna intermediate (levin & moran, 2011). retrotransposons are often called ‘jumping genes’, because the messenger rna transcribed from them can undergo a process called reverse transcription to produce an identical dna sequence that then reinserts into the genome at a different site. they parallel viruses in many ways in their structure and function, as they ensure their own survival by way of reintegration. their propensity to self-propagate has a myriad of consequences and yet their biological significance is not well-understood. although retrotransposons comprise about 42% of the human genome, most carry mutations that render them functionally inactive (cordaux & batzer, 2009). retrotransposons are emerging as potent regulatory elements within the human genome. transcription of those that remain functional must be prevented by proteinor rna-based regulatory mechanisms to prevent the jumping of retrotransposons, which can cause either genetic mutations or genomic instability and might lead to cancer (scott & devine, 2017). moreover, retrotransposons can be reactivated during ageing (de cecco et al., 2013). human endogenous retroviruses (herv) and long-interspersed nuclear element-1 (line-1) are two main classes of retrotransposons, mobilized through a “copy and paste” mechanism. line-1 somatic retrotransposition has been well-demonstrated to occur in neuronal lineage, however the significance of retroelement activity to normal brain function remains uncertain. furthermore, the contribution of these endogenous retroelements to the etiopathogenesis of neurodevelopmental disorders is a topic of recent exploration. roughly 130 pathogenic variants caused by retrotransposon activity have been documented, but the majority of these deleterious events are isolated cases. as such, they are not part of routine clinical sequencing, and thus represent a largely unassessed category of genetic variation in many disorders. human-specific line-1 (l1hs) is the most active autonomous retrotransposon family in the human genome. mounting evidence supports that l1hs retrotransposition occurs postzygotically in the human brain cells, contributing to neuronal genomic diversity. in a paper in 2018 jacob-hirsch et al. (jacob-hirsch et al., 2018) already reported that the number of retrotranspositions is higher in brain tissues than that in non-brain samples and even higher in pathologic vs. normal brains. their findings documented that l1hs elements integrate preferentially into genes associated with neural functions and diseases. the authors propose that pre-existing retrotransposons act as “lightning rods” for novel insertions, which may safeguard from deleterious events and thus, uncontrolled retrotransposition may breach this safeguard and increase the risk of harmful mutagenesis in neurodevelopmental disorders. zhao and colleagues (zhao et al., 2019) profiled genome-wide l1hs insertions among 20 postmortem tissues from rett patients and matched controls. they identified and validated somatic l1hs insertions in both cortical neurons and non-brain tissues, with a higher jumping activity in the brain and concluded that mecp2 dysfunction might alter the genomic pattern of somatic l1hs in rett patients. now gardner et al. (gardner et al., 2019) have identified retrotransposition-derived events in 9738 exome sequenced trios with developmental-affected probands. they encountered 9 de novo retrotransposons and 2 de novo gene retro-duplications. from those, 4 transposons were the likely cause of the symptoms of four patients, three of whom had not had previous diagnoses. these studies open a possibility of “diagnostic retrotransposition events” and are another step along the path to understanding the causes of developmental disorders. 10. single-cell genomics identifies cell type–specific molecular changes in autism i have presented several efforts and advances to disentangle important elements in the molecular neuropathology of neurodevelopmental disorders. one limitation, however, is that most postmortem studies apply bulk omic approximations to a complex tissue, the brain, in which diverse human cells may contribute differently to neurodevelopmental disorders. in asd, for example, previous bulk gene expression studies identified common genes and pathways dysregulated in the neocortex of autism patients that did not explain its clinical and genetic heterogeneity. direct assessment of specific cell types in the brain affected by neurodevelopmental disorders has not been feasible until recent advances in techniques for isolating thousands of intact cells and efficiently sequencing each of them. single-cell rna-sequencing (scrna-seq) has evolved over the past few years as a high-throughput method for transcriptome profiling of thousands of cells (tasic, 2018) and has identified diverse cell types in many brain regions, including neocortex (tasic et al., 2018), hypothalamus (campbell et al., 2017), and retina (shekhar et al., 2016). this will allow scientists to explore neurodevelopmental neuropathology at a resolution that was not possible before. an international consortium called the human cell atlas (https://www.humancellatlas.org/) is an effort to identify every human cell type, where each type is located in the body, and how the cells work together to form tissues and organs. another effort, a collaboration of 53 institutions and 60 companies across europe, called the lifetime initiative (https://lifetime-fetflagship.eu/), is proposing to harness single-cell technologies to understand what happens cell by cell as tissues progress toward diseases. transcriptomic profiling of complex tissues by scrna-seq has unfortunately also limitations, the most important being that neurons, being complex arborizing cells, are vulnerable to mechanical dissociation and sorting. this makes scrna-seq hard to apply to adult brain tissue to tease out individual cells from the elaborately tangled circuitry of the brain. few studies have analyzed neurodevelopmental disorders at a single-cell resolution. a turning point came last year, when velmeshev and colleagues (velmeshev et al., 2019) used newer techniques to extract rna from cell nuclei isolated from samples of brain tissue and then analyzed signature patterns of gene expression of single brain cells, including neurons and glia, from patients with autism. single-nucleus rna-sequencing (snrna-seq) affords some advantages over scrna-seq. in contrast to whole cells, nuclei are more resistant to mechanical assaults and can be isolated from frozen tissue (krishnaswami et al., 2016), so that snrna-seq provides less biased cellular coverage than scrna-seq, since it suffers less cell isolation-related transcriptional artifacts (some cell types are more vulnerable to dissociation process and are thus underrepresented in the final scrna-seq data set (lake et al., 2016)). velmeshev et al. applied this single-nuclei sequencing approach to snap-frozen post-mortem samples of prefrontal cortex and anterior cingulate cortex, two brain regions previously shown to be altered in patients with autism. snrna-seq analysis of more than 100,000 cell nuclei identified a common set of changes in genes involved in synaptic communication as well as neural outgrowth and migration. previous studies suggested convergence of asd on specific cell types during fetal development (parikshak et al., 2013; willsey et al., 2013). furthermore, in patient samples, specific sets of genes enriched in upper-layer cortico-cortical projection neurons and microglia correlated with clinical severity. they also found changes in glial cells that could impact their role in pruning and maintaining healthy neural circuits. many of the differentially expressed genes are known to be widely expressed across the entire brain, but they appeared to be significantly altered only in projection neurons of asd. these findings suggest that molecular changes in cell types with shared developmental lineages exhibit convergent transcriptional changes in adult asd patients, and that the expression of synaptic and neurodevelopmental genes in layer 2/3 cortical neurons is especially affected. this implies that disturbances of gene regulatory programs during development cascade into molecular pathology in specific mature neural cell types, such as upper-layer projection neurons and microglia, which correlate with the clinical severity of asd. one limitation of the study is the small cohort size, since the authors compared brain samples from only 15 people with autism and 16 people who died in the same age range (4-22 years) of non-neurological causes. since almost half of the patients with autism also suffered from seizures, the researchers examined brain samples from a cohort of patients with sporadic epilepsy, to identify brain changes more likely to stem from seizures than primary asd–associated gene expression changes. another general limitation is that, because cells must be removed from the brain, single cell omic techniques alone do not reflect how those cells interact with their neighbors or circuit level changes. future studies involving larger patient cohorts, including whole-exome sequencing and improved single-cell technologies, are needed to allow for precise identification of asd-driven changes and their association with genetic variants. the authors provide an interactive web browser to interrogate their transcriptomic data: https://autism.cells.ucsc.edu. colophon neurodevelopmental disorders are inherently complex, involving multiple components of an intricate network. the multi-dimensional nature of the developmental disorders suggests that no unifying “cause” but instead multi-level perturbation causes complex neurodevelopmental disorders with a host of systemic comorbidities and striking heterogeneity. many research groups are still trying to decipher the genomic complexity of disorders such as autism, and the list of autism “risk” genes grows each year. a number of new unexpected molecular players such as rna editing and retrotransposons have been identified, and their impact on brain development is being examined. because a deep understanding of these underlying mechanisms could prove seminal for personalized medicine, researchers have designed new cellular models with reasonable construct face and predictive validity. of course, other factors such as the contribution of individual lifestyles, cannot be dismissed as they are fueling a large spectrum of gene-environment interactions that have a key role in the genotypes /phenotypes relationships. a more thorough understanding of the intricate processes underlying normal and abnormal human cns development is needed to answer many fundamental questions in biology and medicine and will lead to meaningful clinical biomarkers of developmental neuropathology in humans. funding and acknowledgements this work was supported by the fondation jérôme lejeune, paris, mineco (saf2016-79956-r, h2020 sc1 go-ds21848077, eu (jpnd heroes (the crossroad of dementia syndromes). the crg is a center of excellence severo ochoa sev-2016-0571. the ciber of rare diseases is an initiative of the isciii. the laboratory of mara dierssen is supported by diue de la generalitat de catalunya (grups consolidats 2017 sgr 926). we also acknowledge the support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa and the cerca programme/generalitat de catalunya. references baker, e.k., arpone, m., aliaga, s.m., bretherton, l., kraan, c.m., bui, m., slater, h.r., ling, l., francis, d., hunter, m.f., elliott, j., rogers, c., field, m., cohen, j., cornish, k., santa maria, l., faundes, v., curotto, b., morales, p., trigo, c., salas, i., alliende, a.m., amor, d.j. & godler, d.e. (2019) incomplete silencing of full mutation alleles in males with fragile x syndrome is associated with autistic features. mol autism, 10, 21. batalle, d., edwards, a.d. & o'muircheartaigh, j. (2018) annual research review: not just a small adult brain: understanding later neurodevelopment through imaging the neonatal brain. j child psychol psychiatry, 59, 350-371. batalle, d., hughes, e.j., zhang, h., tournier, j.d., tusor, n., aljabar, p., wali, l., alexander, d.c., hajnal, j.v., nosarti, c., edwards, a.d. & counsell, s.j. (2017) early development of structural networks and the impact of prematurity on brain connectivity. neuroimage, 149, 379-392. behm, m., wahlstedt, h., widmark, a., eriksson, m. & ohman, m. (2017) accumulation of nuclear adar2 regulates adenosine-to-inosine rna editing during neuronal development. j cell sci, 130, 745-753. breen, m.s., dobbyn, a., li, q., roussos, p., hoffman, g.e., stahl, e., chess, a., sklar, p., li, j.b., devlin, b., buxbaum, j.d. & commonmind, c. (2019) global landscape and genetic regulation of rna editing in cortical samples from individuals with schizophrenia. nat neurosci, 22, 1402-1412. brennand, k.j., simone, a., jou, j., gelboin-burkhart, c., tran, n., sangar, s., li, y., mu, y., chen, g., yu, d., mccarthy, s., sebat, j. & gage, f.h. (2011) modelling schizophrenia using human induced pluripotent stem cells. nature, 473, 221-225. campbell, j.n., macosko, e.z., fenselau, h., pers, t.h., lyubetskaya, a., tenen, d., goldman, m., verstegen, a.m., resch, j.m., mccarroll, s.a., rosen, e.d., lowell, b.b. & tsai, l.t. (2017) a molecular census of arcuate hypothalamus and median eminence cell types. nat neurosci, 20, 484-496. cardoso, a.r., lopes-marques, m., silva, r.m., serrano, c., amorim, a., prata, m.j. & azevedo, l. (2019) essential genetic findings in neurodevelopmental disorders. hum genomics, 13, 31. castellanos, f.x., di martino, a., craddock, r.c., mehta, a.d. & milham, m.p. (2013) clinical applications of the functional connectome. neuroimage, 80, 527-540. cetin-karayumak, s., di biase, m.a., chunga, n., reid, b., somes, n., lyall, a.e., kelly, s., solgun, b., pasternak, o., vangel, m., pearlson, g., tamminga, c., sweeney, j.a., clementz, b., schretlen, d., viher, p.v., stegmayer, k., walther, s., lee, j., crow, t., james, a., voineskos, a., buchanan, r.w., szeszko, p.r., malhotra, a.k., hegde, r., mccarley, r., keshavan, m., shenton, m., rathi, y. & kubicki, m. (2019) white matter abnormalities across the lifespan of schizophrenia: a harmonized multi-site diffusion mri study. mol psychiatry, 10.1038/s41380-019-0509-y, online ahead of print. christiaens, d., slator, p.j., cordero-grande, l., price, a.n., deprez, m., alexander, d.c., rutherford, m., hajnal, j.v. & hutter, j. (2019) in utero diffusion mri: challenges, advances, and applications. top magn reson imaging, 28, 255-264. chung, k. & deisseroth, k. (2013) clarity for mapping the nervous system. nat methods, 10, 508-513. cordaux, r. & batzer, m.a. (2009) the impact of retrotransposons on human genome evolution. nat rev genet, 10, 691-703. counsell, s.j., arichi, t., arulkumaran, s. & rutherford, m.a. (2019) fetal and neonatal neuroimaging. handb clin neurol, 162, 67-103. cropley, v.l., klauser, p., lenroot, r.k., bruggemann, j., sundram, s., bousman, c., pereira, a., di biase, m.a., weickert, t.w., weickert, c.s., pantelis, c. & zalesky, a. (2017) accelerated gray and white matter deterioration with age in schizophrenia. am j psychiatry, 174, 286-295. cross-disorder group of the psychiatric genomics consortium. (2013) genetic relationship between five psychiatric disorders estimated from genome-wide snps. nat genet, 45, 984-994. cross-disorder group of the psychiatric genomics consortium. (2019) genomic relationships, novel loci, and pleiotropic mechanisms across eight psychiatric disorders. cell, 179, 1469-1482.e11. cullen, d.k., gordian-velez, w.j., struzyna, l.a., jgamadze, d., lim, j., wofford, k.l., browne, k.d. & chen, h.i. (2019) bundled three-dimensional human axon tracts derived from brain organoids. iscience, 21, 57-67. de cecco, m., criscione, s.w., peterson, a.l., neretti, n., sedivy, j.m. & kreiling, j.a. (2013) transposable elements become active and mobile in the genomes of aging mammalian somatic tissues. aging (albany ny), 5, 867-883. demyanenko, g.p., tsai, a.y. & maness, p.f. (1999) abnormalities in neuronal process extension, hippocampal development, and the ventricular system of l1 knockout mice. j neurosci, 19, 4907-4920. edwards, t.j., sherr, e.h., barkovich, a.j. & richards, l.j. (2014) clinical, genetic and imaging findings identify new causes for corpus callosum development syndromes. brain, 137, 1579-1613. eran, a., li, j.b., vatalaro, k., mccarthy, j., rahimov, f., collins, c., markianos, k., margulies, d.m., brown, e.n., calvo, s.e., kohane, i.s. & kunkel, l.m. (2013) comparative rna editing in autistic and neurotypical cerebella. mol psychiatry, 18, 1041-1048. field, m., dudding-byth, t., arpone, m., baker, e.k., aliaga, s.m., rogers, c., hickerton, c., francis, d., phelan, d.g., palmer, e.e., amor, d.j., slater, h., bretherton, l., ling, l. & godler, d.e. (2019) significantly elevated fmr1 mrna and mosaicism for methylated premutation and full mutation alleles in two brothers with autism features referred for fragile x testing. int j mol sci, 20, 3907. finn, e.s., shen, x., scheinost, d., rosenberg, m.d., huang, j., chun, m.m., papademetris, x. & constable, r.t. (2015) functional connectome fingerprinting: identifying individuals using patterns of brain connectivity. nat neurosci, 18, 1664-1671. fornito, a., zalesky, a. & breakspear, m. (2015) the connectomics of brain disorders. nat rev neurosci, 16, 159-172. fox, m.d., snyder, a.z., vincent, j.l., corbetta, m., van essen, d.c. & raichle, m.e. (2005) the human brain is intrinsically organized into dynamic, anticorrelated functional networks. proc natl acad sci u s a, 102, 9673-9678. french, l., gray, c., leonard, g., perron, m., pike, g.b., richer, l., seguin, j.r., veillette, s., evans, c.j., artiges, e., banaschewski, t., bokde, a.w., bromberg, u., bruehl, r., buchel, c., cattrell, a., conrod, p.j., flor, h., frouin, v., gallinat, j., garavan, h., gowland, p., heinz, a., lemaitre, h., martinot, j.l., nees, f., orfanos, d.p., pangelinan, m.m., poustka, l., rietschel, m., smolka, m.n., walter, h., whelan, r., timpson, n.j., schumann, g., smith, g.d., pausova, z. & paus, t. (2015) early cannabis use, polygenic risk score for schizophrenia and brain maturation in adolescence. jama psychiatry, 72, 1002-1011. gandal, m.j., zhang, p., hadjimichael, e., walker, r.l., chen, c., liu, s., won, h., van bakel, h., varghese, m., wang, y., shieh, a.w., haney, j., parhami, s., belmont, j., kim, m., moran losada, p., khan, z., mleczko, j., xia, y., dai, r., wang, d., yang, y.t., xu, m., fish, k., hof, p.r., warrell, j., fitzgerald, d., white, k., jaffe, a.e., psych, e.c., peters, m.a., gerstein, m., liu, c., iakoucheva, l.m., pinto, d. & geschwind, d.h. (2018) transcriptome-wide isoform-level dysregulation in asd, schizophrenia, and bipolar disorder. science, 362, eaat8127. garcia, k.e., robinson, e.c., alexopoulos, d., dierker, d.l., glasser, m.f., coalson, t.s., ortinau, c.m., rueckert, d., taber, l.a., van essen, d.c., rogers, c.e., smyser, c.d. & bayly, p.v. (2018) dynamic patterns of cortical expansion during folding of the preterm human brain. proc natl acad sci u s a, 115, 3156-3161. gardner, e.j., prigmore, e., gallone, g., danecek, p., samocha, k.e., handsaker, j., gerety, s.s., ironfield, h., short, p.j., sifrim, a., singh, t., chandler, k.e., clement, e., lachlan, k.l., prescott, k., rosser, e., fitzpatrick, d.r., firth, h.v. & hurles, m.e. (2019) contribution of retrotransposition to developmental disorders. nat commun, 10, 4630. geng, x., li, g., lu, z., gao, w., wang, l., shen, d., zhu, h. & gilmore, j.h. (2017) structural and maturational covariance in early childhood brain development. cereb cortex, 27, 1795-1807. glahn, d.c., laird, a.r., ellison-wright, i., thelen, s.m., robinson, j.l., lancaster, j.l., bullmore, e. & fox, p.t. (2008) meta-analysis of gray matter anomalies in schizophrenia: application of anatomic likelihood estimation and network analysis. biol psychiatry, 64, 774-781. gousias, i.s., hammers, a., counsell, s.j., srinivasan, l., rutherford, m.a., heckemann, r.a., hajnal, j.v., rueckert, d. & edwards, a.d. (2013) magnetic resonance imaging of the newborn brain: automatic segmentation of brain images into 50 anatomical regions. plos one, 8, e59990. grenier, k., kao, j. & diamandis, p. (2020) three-dimensional modeling of human neurodegeneration: brain organoids coming of age. mol psychiatry, 25, 254-274. grove, j., ripke, s., als, t.d., mattheisen, m., walters, r.k., won, h., pallesen, j., agerbo, e., andreassen, o.a., anney, r., awashti, s., belliveau, r., bettella, f., buxbaum, j.d., bybjerg-grauholm, j., baekvad-hansen, m., cerrato, f., chambert, k., christensen, j.h., churchhouse, c., dellenvall, k., demontis, d., de rubeis, s., devlin, b., djurovic, s., dumont, a.l., goldstein, j.i., hansen, c.s., hauberg, m.e., hollegaard, m.v., hope, s., howrigan, d.p., huang, h., hultman, c.m., klei, l., maller, j., martin, j., martin, a.r., moran, j.l., nyegaard, m., naerland, t., palmer, d.s., palotie, a., pedersen, c.b., pedersen, m.g., dpoterba, t., poulsen, j.b., pourcain, b.s., qvist, p., rehnstrom, k., reichenberg, a., reichert, j., robinson, e.b., roeder, k., roussos, p., saemundsen, e., sandin, s., satterstrom, f.k., davey smith, g., stefansson, h., steinberg, s., stevens, c.r., sullivan, p.f., turley, p., walters, g.b., xu, x., autism spectrum disorder working group of the psychiatric genomics consortium, bupgen, major depressive disorder working group of the psychiatric genomics consortium, 23andme research team, stefansson, k., geschwind, d.h., nordentoft, m., hougaard, d.m., werge, t., mors, o., mortensen, p.b., neale, b.m., daly, m.j. & borglum, a.d. (2019) identification of common genetic risk variants for autism spectrum disorder. nat genet, 51, 431-444. hampson, m., driesen, n., roth, j.k., gore, j.c. & constable, r.t. (2010) functional connectivity between task-positive and task-negative brain areas and its relation to working memory performance. magn reson imaging, 28, 1051-1057. hart, a.r., embleton, n.d., bradburn, m., connolly, d.j.a., mandefield, l., mooney, c. & griffiths, p.d. (2020) accuracy of in-utero mri to detect fetal brain abnormalities and prognosticate developmental outcome: postnatal follow-up of the meridian cohort. lancet child adolesc health, 4, 131-140. international human genome sequencing consortium (2001) initial sequencing and analysis of the human genome. nature, 409, 860-921. irimia, m., weatheritt, r.j., ellis, j.d., parikshak, n.n., gonatopoulos-pournatzis, t., babor, m., quesnel-vallieres, m., tapial, j., raj, b., o'hanlon, d., barrios-rodiles, m., sternberg, m.j., cordes, s.p., roth, f.p., wrana, j.l., geschwind, d.h. & blencowe, b.j. (2014) a highly conserved program of neuronal microexons is misregulated in autistic brains. cell, 159, 1511-1523. jabaudon, d. & lancaster, m. (2018) exploring landscapes of brain morphogenesis with organoids. development, 145, dev172049. jacob-hirsch, j., eyal, e., knisbacher, b.a., roth, j., cesarkas, k., dor, c., farage-barhom, s., kunik, v., simon, a.j., gal, m., yalon, m., moshitch-moshkovitz, s., tearle, r., constantini, s., levanon, e.y., amariglio, n. & rechavi, g. (2018) whole-genome sequencing reveals principles of brain retrotransposition in neurodevelopmental disorders. cell res, 28, 187-203. jakab, a. (2019) developmental pathoconnectomics and advanced fetal mri. top magn reson imaging, 28, 275-284. jo, j., xiao, y., sun, a.x., cukuroglu, e., tran, h.d., goke, j., tan, z.y., saw, t.y., tan, c.p., lokman, h., lee, y., kim, d., ko, h.s., kim, s.o., park, j.h., cho, n.j., hyde, t.m., kleinman, j.e., shin, j.h., weinberger, d.r., tan, e.k., je, h.s. & ng, h.h. (2016) midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons. cell stem cell, 19, 248-257. kadoshima, t., sakaguchi, h. & eiraku, m. (2017) generation of various telencephalic regions from human embryonic stem cells in three-dimensional culture. methods mol biol, 1597, 1-16. keller, j.b., hedden, t., thompson, t.w., anteraper, s.a., gabrieli, j.d. & whitfield-gabrieli, s. (2015) resting-state anticorrelations between medial and lateral prefrontal cortex: association with working memory, aging, and individual differences. cortex, 64, 271-280. khan, s., hashmi, j.a., mamashli, f., michmizos, k., kitzbichler, m.g., bharadwaj, h., bekhti, y., ganesan, s., garel, k.a., whitfield-gabrieli, s., gollub, r.l., kong, j., vaina, l.m., rana, k.d., stufflebeam, s.m., hamalainen, m.s. & kenet, t. (2018) maturation trajectories of cortical resting-state networks depend on the mediating frequency band. neuroimage, 174, 57-68. khan, s., vasung, l., marami, b., rollins, c.k., afacan, o., ortinau, c.m., yang, e., warfield, s.k. & gholipour, a. (2019) fetal brain growth portrayed by a spatiotemporal diffusion tensor mri atlas computed from in utero images. neuroimage, 185, 593-608. kirihara, t., luo, z., chow, s.y.a., misawa, r., kawada, j., shibata, s., khoyratee, f., vollette, c.a., volz, v., levi, t., fujii, t. & ikeuchi, y. (2019) a human induced pluripotent stem cell-derived tissue model of a cerebral tract connecting two cortical regions. iscience, 14, 301-311. krestel, h. & meier, j.c. (2018) rna editing and retrotransposons in neurology. front mol neurosci, 11, 163. krishnaswami, s.r., grindberg, r.v., novotny, m., venepally, p., lacar, b., bhutani, k., linker, s.b., pham, s., erwin, j.a., miller, j.a., hodge, r., mccarthy, j.k., kelder, m., mccorrison, j., aevermann, b.d., fuertes, f.d., scheuermann, r.h., lee, j., lein, e.s., schork, n., mcconnell, m.j., gage, f.h. & lasken, r.s. (2016) using single nuclei for rna-seq to capture the transcriptome of postmortem neurons. nat protoc, 11, 499-524. ku, t., swaney, j., park, j.y., albanese, a., murray, e., cho, j.h., park, y.g., mangena, v., chen, j. & chung, k. (2016) multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. nat biotechnol, 34, 973-981. lake, b.b., ai, r., kaeser, g.e., salathia, n.s., yung, y.c., liu, r., wildberg, a., gao, d., fung, h.l., chen, s., vijayaraghavan, r., wong, j., chen, a., sheng, x., kaper, f., shen, r., ronaghi, m., fan, j.b., wang, w., chun, j. & zhang, k. (2016) neuronal subtypes and diversity revealed by single-nucleus rna sequencing of the human brain. science, 352, 1586-1590. lancaster, m.a., renner, m., martin, c.a., wenzel, d., bicknell, l.s., hurles, m.e., homfray, t., penninger, j.m., jackson, a.p. & knoblich, j.a. (2013) cerebral organoids model human brain development and microcephaly. nature, 501, 373-379. lapp, h.e. & hunter, r.g. (2019) early life exposures, neurodevelopmental disorders, and transposable elements. neurobiol stress, 11, 100174. levin, h.l. & moran, j.v. (2011) dynamic interactions between transposable elements and their hosts. nat rev genet, 12, 615-627. liscovitch, n., bazak, l., levanon, e.y. & chechik, g. (2014) positive correlation between adar expression and its targets suggests a complex regulation mediated by rna editing in the human brain. rna biol, 11, 1447-1456. liu, j., liao, x., xia, m. & he, y. (2018) chronnectome fingerprinting: identifying individuals and predicting higher cognitive functions using dynamic brain connectivity patterns. hum brain mapp, 39, 902-915. lorenzini, i., moore, s. & sattler, r. (2018) rna editing deficiency in neurodegeneration. adv neurobiol, 20, 63-83. lyon, l. (2019) building brains: using brain organoids to study neural development and disease. brain, 142, e65. makropoulos, a., aljabar, p., wright, r., huning, b., merchant, n., arichi, t., tusor, n., hajnal, j.v., edwards, a.d., counsell, s.j. & rueckert, d. (2016) regional growth and atlasing of the developing human brain. neuroimage, 125, 456-478. makropoulos, a., gousias, i.s., ledig, c., aljabar, p., serag, a., hajnal, j.v., edwards, a.d., counsell, s.j. & rueckert, d. (2014) automatic whole brain mri segmentation of the developing neonatal brain. ieee trans med imaging, 33, 1818-1831. mallela, a. & nishikura, k. (2012) a-to-i editing of protein coding and noncoding rnas. crit rev biochem mol biol, 47, 493-501. mansour, a.a., goncalves, j.t., bloyd, c.w., li, h., fernandes, s., quang, d., johnston, s., parylak, s.l., jin, x. & gage, f.h. (2018) an in vivo model of functional and vascularized human brain organoids. nat biotechnol, 36, 432-441. mariani, j., coppola, g., zhang, p., abyzov, a., provini, l., tomasini, l., amenduni, m., szekely, a., palejev, d., wilson, m., gerstein, m., grigorenko, e.l., chawarska, k., pelphrey, k.a., howe, j.r. & vaccarino, f.m. (2015) foxg1-dependent dysregulation of gaba/glutamate neuron differentiation in autism spectrum disorders. cell, 162, 375-390. marin, o. (2012) interneuron dysfunction in psychiatric disorders. nat rev neurosci, 13, 107-120. mcgrath, j.j., feron, f.p., burne, t.h., mackay-sim, a. & eyles, d.w. (2003) the neurodevelopmental hypothesis of schizophrenia: a review of recent developments. ann med, 35, 86-93. meier, j.c., kankowski, s., krestel, h. & hetsch, f. (2016) rna editing-systemic relevance and clue to disease mechanisms? front mol neurosci, 9, 124. morgan, s.e., seidlitz, j., whitaker, k.j., romero-garcia, r., clifton, n.e., scarpazza, c., van amelsvoort, t., marcelis, m., van os, j., donohoe, g., mothersill, d., corvin, a., pocklington, a., raznahan, a., mcguire, p., vertes, p.e. & bullmore, e.t. (2019) cortical patterning of abnormal morphometric similarity in psychosis is associated with brain expression of schizophrenia-related genes. proc natl acad sci u s a, 116, 9604-9609. murray, r.m., bhavsar, v., tripoli, g. & howes, o. (2017) 30 years on: how the neurodevelopmental hypothesis of schizophrenia morphed into the developmental risk factor model of psychosis. schizophr bull, 43, 1190-1196. nandi, s., chandramohan, d., fioriti, l., melnick, a.m., hebert, j.m., mason, c.e., rajasethupathy, p. & kandel, e.r. (2016) roles for small noncoding rnas in silencing of retrotransposons in the mammalian brain. proc natl acad sci u s a, 113, 12697-12702. parikshak, n.n., gandal, m.j. & geschwind, d.h. (2015) systems biology and gene networks in neurodevelopmental and neurodegenerative disorders. nat rev genet, 16, 441-458. parikshak, n.n., luo, r., zhang, a., won, h., lowe, j.k., chandran, v., horvath, s. & geschwind, d.h. (2013) integrative functional genomic analyses implicate specific molecular pathways and circuits in autism. cell, 155, 1008-1021. park, y.g., sohn, c.h., chen, r., mccue, m., yun, d.h., drummond, g.t., ku, t., evans, n.b., oak, h.c., trieu, w., choi, h., jin, x., lilascharoen, v., wang, j., truttmann, m.c., qi, h.w., ploegh, h.l., golub, t.r., chen, s.c., frosch, m.p., kulik, h.j., lim, b.k. & chung, k. (2018) protection of tissue physicochemical properties using polyfunctional crosslinkers. nat biotechnol, 10.1038/nbt.4281. pasca, a.m., sloan, s.a., clarke, l.e., tian, y., makinson, c.d., huber, n., kim, c.h., park, j.y., o'rourke, n.a., nguyen, k.d., smith, s.j., huguenard, j.r., geschwind, d.h., barres, b.a. & pasca, s.p. (2015) functional cortical neurons and astrocytes from human pluripotent stem cells in 3d culture. nat methods, 12, 671-678. pecheva, d., yushkevich, p., batalle, d., hughes, e., aljabar, p., wurie, j., hajnal, j.v., edwards, a.d., alexander, d.c., counsell, s.j. & zhang, h. (2017) a tract-specific approach to assessing white matter in preterm infants. neuroimage, 157, 675-694. quadrato, g., brown, j. & arlotta, p. (2016) the promises and challenges of human brain organoids as models of neuropsychiatric disease. nat med, 22, 1220-1228. qureshi, i.a. & mehler, m.f. (2012) emerging roles of non-coding rnas in brain evolution, development, plasticity and disease. nat rev neurosci, 13, 528-541. real, r., peter, m., trabalza, a., khan, s., smith, m.a., dopp, j., barnes, s.j., momoh, a., strano, a., volpi, e., knott, g., livesey, f.j. & de paola, v. (2018) in vivo modeling of human neuron dynamics and down syndrome. science, 362, eaau1810. rodgaard, e.m., jensen, k., vergnes, j.n., soulieres, i. & mottron, l. (2019) temporal changes in effect sizes of studies comparing individuals with and without autism: a meta-analysis. jama psychiatry, 76, 1124-1132. rosenthal, j.j. & seeburg, p.h. (2012) a-to-i rna editing: effects on proteins key to neural excitability. neuron, 74, 432-439. salloum-asfar, s., satheesh, n.j. & abdulla, s.a. (2019) circulating mirnas, small but promising biomarkers for autism spectrum disorder. front mol neurosci, 12, 253. sarkar, a., mei, a., paquola, a.c.m., stern, s., bardy, c., klug, j.r., kim, s., neshat, n., kim, h.j., ku, m., shokhirev, m.n., adamowicz, d.h., marchetto, m.c., jappelli, r., erwin, j.a., padmanabhan, k., shtrahman, m., jin, x. & gage, f.h. (2018) efficient generation of ca3 neurons from human pluripotent stem cells enables modeling of hippocampal connectivity in vitro. cell stem cell, 22, 684-697.e9. scott, e.c. & devine, s.e. (2017) the role of somatic l1 retrotransposition in human cancers. viruses, 9, 131. shekhar, k., lapan, s.w., whitney, i.e., tran, n.m., macosko, e.z., kowalczyk, m., adiconis, x., levin, j.z., nemesh, j., goldman, m., mccarroll, s.a., cepko, c.l., regev, a. & sanes, j.r. (2016) comprehensive classification of retinal bipolar neurons by single-cell transcriptomics. cell, 166, 1308-1323.e30. siegenthaler, d., enneking, e.m., moreno, e. & pielage, j. (2015) l1cam/neuroglian controls the axon-axon interactions establishing layered and lobular mushroom body architecture. j cell biol, 208, 1003-1018. stolp, h.b., ball, g., so, p.w., tournier, j.d., jones, m., thornton, c. & edwards, a.d. (2018) voxel-wise comparisons of cellular microstructure and diffusion-mri in mouse hippocampus using 3d bridging of optically-clear histology with neuroimaging data (3d-bond). sci rep, 8, 4011. streit, a.k. & decher, n. (2011) a-to-i rna editing modulates the pharmacology of neuronal ion channels and receptors. biochemistry (mosc), 76, 890-899. suarez, n.a., macia, a. & muotri, a.r. (2018) line-1 retrotransposons in healthy and diseased human brain. dev neurobiol, 78, 434-455. sullivan, p.f., agrawal, a., bulik, c.m., andreassen, o.a., borglum, a.d., breen, g., cichon, s., edenberg, h.j., faraone, s.v., gelernter, j., mathews, c.a., nievergelt, c.m., smoller, j.w., o'donovan, m.c. & psychiatric genomics, c. (2018) psychiatric genomics: an update and an agenda. am j psychiatry, 175, 15-27. takahashi, k., tanabe, k., ohnuki, m., narita, m., ichisaka, t., tomoda, k. & yamanaka, s. (2007) induction of pluripotent stem cells from adult human fibroblasts by defined factors. cell, 131, 861-872. takahashi, k. & yamanaka, s. (2006) induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. cell, 126, 663-676. tasic, b. (2018) single cell transcriptomics in neuroscience: cell classification and beyond. curr opin neurobiol, 50, 242-249. tasic, b., yao, z., graybuck, l.t., smith, k.a., nguyen, t.n., bertagnolli, d., goldy, j., garren, e., economo, m.n., viswanathan, s., penn, o., bakken, t., menon, v., miller, j., fong, o., hirokawa, k.e., lathia, k., rimorin, c., tieu, m., larsen, r., casper, t., barkan, e., kroll, m., parry, s., shapovalova, n.v., hirschstein, d., pendergraft, j., sullivan, h.a., kim, t.k., szafer, a., dee, n., groblewski, p., wickersham, i., cetin, a., harris, j.a., levi, b.p., sunkin, s.m., madisen, l., daigle, t.l., looger, l., bernard, a., phillips, j., lein, e., hawrylycz, m., svoboda, k., jones, a.r., koch, c. & zeng, h. (2018) shared and distinct transcriptomic cell types across neocortical areas. nature, 563, 72-78. thibaut, f. (2018) psychiatric disorders: neurodevelopmental disorders, neurodegenerative disorders, or both? dialogues clin neurosci, 20, 251-252. tran, s.s., jun, h.i., bahn, j.h., azghadi, a., ramaswami, g., van nostrand, e.l., nguyen, t.b., hsiao, y.e., lee, c., pratt, g.a., martinez-cerdeno, v., hagerman, r.j., yeo, g.w., geschwind, d.h. & xiao, x. (2019) widespread rna editing dysregulation in brains from autistic individuals. nat neurosci, 22, 25-36. trujillo, c.a., gao, r., negraes, p.d., gu, j., buchanan, j., preissl, s., wang, a., wu, w., haddad, g.g., chaim, i.a., domissy, a., vandenberghe, m., devor, a., yeo, g.w., voytek, b. & muotri, a.r. (2019) complex oscillatory waves emerging from cortical organoids model early human brain network development. cell stem cell, 25, 558-569.e7. van den ameele, j., tiberi, l., vanderhaeghen, p. & espuny-camacho, i. (2014) thinking out of the dish: what to learn about cortical development using pluripotent stem cells. trends neurosci, 37, 334-342. van haren, n.e., hulshoff pol, h.e., schnack, h.g., cahn, w., brans, r., carati, i., rais, m. & kahn, r.s. (2008) progressive brain volume loss in schizophrenia over the course of the illness: evidence of maturational abnormalities in early adulthood. biol psychiatry, 63, 106-113. velasco, s., kedaigle, a.j., simmons, s.k., nash, a., rocha, m., quadrato, g., paulsen, b., nguyen, l., adiconis, x., regev, a., levin, j.z. & arlotta, p. (2019) individual brain organoids reproducibly form cell diversity of the human cerebral cortex. nature, 570, 523-527. velmeshev, d., schirmer, l., jung, d., haeussler, m., perez, y., mayer, s., bhaduri, a., goyal, n., rowitch, d.h. & kriegstein, a.r. (2019) single-cell genomics identifies cell type-specific molecular changes in autism. science, 364, 685-689. watson, r.e., desesso, j.m., hurtt, m.e. & cappon, g.d. (2006) postnatal growth and morphological development of the brain: a species comparison. birth defects res b dev reprod toxicol, 77, 471-484. whitfield-gabrieli, s., wendelken, c., nieto-castanon, a., bailey, s.k., anteraper, s.a., lee, y.j., chai, x.q., hirshfeld-becker, d.r., biederman, j., cutting, l.e. & bunge, s.a. (2019) association of intrinsic brain architecture with changes in attentional and mood symptoms during development. jama psychiatry, 77, 378-386. willsey, a.j., sanders, s.j., li, m., dong, s., tebbenkamp, a.t., muhle, r.a., reilly, s.k., lin, l., fertuzinhos, s., miller, j.a., murtha, m.t., bichsel, c., niu, w., cotney, j., ercan-sencicek, a.g., gockley, j., gupta, a.r., han, w., he, x., hoffman, e.j., klei, l., lei, j., liu, w., liu, l., lu, c., xu, x., zhu, y., mane, s.m., lein, e.s., wei, l., noonan, j.p., roeder, k., devlin, b., sestan, n. & state, m.w. (2013) coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism. cell, 155, 997-1007. wright, r., makropoulos, a., kyriakopoulou, v., patkee, p.a., koch, l.m., rutherford, m.a., hajnal, j.v., rueckert, d. & aljabar, p. (2015) construction of a fetal spatio-temporal cortical surface atlas from in utero mri: application of spectral surface matching. neuroimage, 120, 467-480. yoon, s.j., elahi, l.s., pasca, a.m., marton, r.m., gordon, a., revah, o., miura, y., walczak, e.m., holdgate, g.m., fan, h.c., huguenard, j.r., geschwind, d.h. & pasca, s.p. (2019) reliability of human cortical organoid generation. nat methods, 16, 75-78. zhao, b., wu, q., ye, a.y., guo, j., zheng, x., yang, x., yan, l., liu, q.r., hyde, t.m., wei, l. & huang, a.y. (2019) somatic line-1 retrotransposition in cortical neurons and non-brain tissues of rett patients and healthy individuals. plos genet, 15, e1008043. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology of covid-19 (neuro-covid): clinicopathological update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:2 (2021) review neuropathology of covid-19 (neuro-covid): clinicopathological update jerry j. lou1,2*, mehrnaz movassaghi1*, dominique gordy1, madeline g. olson1, ting zhang1, maya s. khurana2, zesheng chen1, mari perez-rosendahl2, samasuk thammachantha3, elyse j. singer4, shino d. magaki1, harry v. vinters1,4, william h. yong1,2 1 department of pathology and laboratory medicine, david geffen school of medicine at ucla, usa 2 department of pathology and laboratory medicine, university of california irvine school of medicine, usa 3 department of pathology, prasat neurological institute, thailand 4 department of neurology, david geffen school of medicine at ucla, usa   * authors contributed equally and are listed in alphabetical order corresponding author: william h. yong md · department of pathology and laboratory medicine · university of california irvine school of medicine · usa and department of pathology and laboratory medicine, david geffen school of medicine at ucla · usa yongwh@hs.uci.edu submitted: 03 september 2020 accepted: 14 january 2021 copyedited by: calixto-hope g lucas, jr. published: 18 january 2021 https://doi.org/10.17879/freeneuropathology-2021-2993 additional resources and electronic supplementary material: supplementary material keywords: cns, covid-19, sars-cov-2, brain, pituitary abstract coronavirus disease 2019 (covid-19) is emerging as the greatest public health crisis in the early 21st century. its causative agent, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is an enveloped single-stranded positive-sense ribonucleic acid virus that enters cells via the angiotensin converting enzyme 2 receptor or several other receptors. while covid-19 primarily affects the respiratory system, other organs including the brain can be involved. in western clinical studies, relatively mild neurological dysfunction such as anosmia and dysgeusia is frequent (~70-84%) while severe neurologic disorders such as stroke (~1-6%) and meningoencephalitis are less common. it is unclear how much sars-cov-2 infection contributes to the incidence of stroke given co-morbidities in the affected patient population. rarely, clinically-defined cases of acute disseminated encephalomyelitis, guillain-barré syndrome and acute necrotizing encephalopathy have been reported in covid-19 patients. common neuropathological findings in the 184 patients reviewed include microglial activation (42.9%) with microglial nodules in a subset (33.3%), lymphoid inflammation (37.5%), acute hypoxic-ischemic changes (29.9%), astrogliosis (27.7%), acute/subacute brain infarcts (21.2%), spontaneous hemorrhage (15.8%), and microthrombi (15.2%). in our institutional cases, we also note occasional anterior pituitary infarcts. covid-19 coagulopathy, sepsis, and acute respiratory distress likely contribute to a number of these findings. when present, central nervous system lymphoid inflammation is often minimal to mild, is detected best by immunohistochemistry and, in one study, indistinguishable from control sepsis cases. some cases evince microglial nodules or neuronophagy, strongly supporting viral meningoencephalitis, with a proclivity for involvement of the medulla oblongata. the virus is detectable by reverse transcriptase polymerase chain reaction, immunohistochemistry, or electron microscopy in human cerebrum, cerebellum, cranial nerves, olfactory bulb, as well as in the olfactory epithelium; neurons and endothelium can also be infected. review of the extant cases has limitations including selection bias and limited clinical information in some cases. much remains to be learned about the effects of direct viral infection of brain cells and whether sars-cov-2 persists long-term contributing to chronic symptomatology. 1. introduction in late 2019, a novel infectious disease associated with pneumonia and acute respiratory distress emerged in wuhan, china. the implicated human coronavirus was genetically related to but distinct from the underlying viral agent behind the 2003 severe acute respiratory syndrome (sars) outbreak, sars coronavirus (sars-cov). the international committee of taxonomy of viruses designated this novel coronavirus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2)1 and the world health organization termed the associated disease as coronavirus disease 2019 (covid-19).1 as unsuspecting asymptomatic patients readily spread the disease, the covid-19 pandemic has swept rapidly across the world resulting, by the end of december 2020, in over 79 million confirmed human infections and over 1.7 million deaths.2 these numbers are believed to be substantial underestimates of the true toll. covid-19 is best known for its pulmonary involvement, but other organs are often affected including heart, kidney and nervous system. this review will provide an overview of tissue-based covid-19 neuropathological analyses, almost entirely autopsy derived, as well as a brief discussion of sars-cov-2 virology to provide context for understanding its pathogenesis, and of diagnostic testing limitations that may bias the examined cases. the nervous system is affected both secondarily from systemic complications such as hypoxia and coagulopathy, and also likely from primary infection though much remains to be elucidated regarding viral invasion of the brain, spinal cord and other components of the nervous system. 2. sars-cov-2 virology sars-cov-2 is the latest coronavirus to emerge as a human pathogen. coronaviruses are single-stranded, positive-sensed ribonucleic acid (rna) viruses subdivided into four genera: alpha-coronavirus (α-cov), beta-coronavirus (β-cov), gamma-coronavirus (γ-cov), and delta-coronavirus (δ-cov).3 sars-cov-2 is a beta-coronavirus, typically 60 to 140 nm4–6 in size, that shares genetic similarities to sars-cov (~79% homology) and middle east respiratory syndrome coronavirus (mers-cov) (~50% homology).7 the sars-cov-2 genome encodes 16 non-structural proteins involved in viral replication and four structural proteins consisting of the envelope, membrane, nucleocapsid, and spike glycoprotein (figure 1).3,8,9 the virus is constantly evolving with numerous strains of sars-cov-2 identified, some preferentially localized (at least temporarily) to geographic regions such as europe or north america.10 the mutation rate of sars-cov-2 is estimated to be 0.84-1.12 x 10-3 substitutions per site per year,7,11 which is lower than that of human immunodeficiency virus (hiv)12 or influenza a.13 the genetic spectrum of disparate sars-cov-2 strains undergoing continued mutation could contribute to the variability of neuropathological findings discussed later. antibodies against nucleocapsid and spike proteins have been used for immunohistochemical studies. figure 1: severe acute respiratory coronavirus 2 (sars-cov-2) structure. there are four structural proteins: spike (s) protein (red), envelope (e) protein (violet), membrane (m) protein (blue), and nucleocapsid (n) protein (orange). 3. ace2 and other receptors mediate sars-cov-2 entry the entry of sars-cov-2 into human cells is commonly thought to be mediated by the interaction of the spike protein with the angiotensin converting enzyme 2 (ace2) receptor, an important regulator of the renin-angiotensin system (ras).14 gender, age, lifestyle, smoking, and other patient co-morbidities are implicated in the modulation of ace2 receptor expression in various tissues.15–21 ace2 receptor expression has been reported in the cerebrum, cerebellum, brainstem, retina, and olfactory mucosa.22–24 neurons, vascular pericytes and smooth muscle, and glia express the ace2 receptor.22,23,25 in addition to the ace2 receptor, in vitro studies show that sars-cov-2 may gain entry using other cell receptors, such as basigin (bsg; cd147),26 neuropilin-1 (nrp1),27 transmembrane serine protease 2 and 4 (tmprss2/4),28,29 and cathepsin l (ctsl).9 expression of ace2 receptor is highest in oligodendrocytes, tmprss2/4 in neurons, ctsl in microglia, and nrp1 in endothelial cells.25 in principle therefore, a broad range of cells in the central nervous system (cns) have a variety of receptors that may facilitate infection. 4. laboratory testing for covid-19 has significant limitations in part due to the natural course of disease wherein viral titers rise over time, laboratory testing for sars-cov-2 does not completely exclude patient infection, complicating the neuropathologist’s management of surgical or autopsy cases. there may also be a bias towards evaluating autopsies of covid-19 patients with severe disease and high viral loads. the analytical sensitivity of the commonly used covid-19 reverse transcriptase polymerase chain reaction (rt-pcr) assay is excellent with a limit of detection as low as 6.25 copies/µl in a nasopharyngeal sample.30 in a clinical setting, the sensitivity of rt-pcr may be 83.3% or significantly less, and may be affected by infection phase, sample type, collection procedures, and testing platforms.31,32 covid-19 rt-pcr testing may produce false-negative results in the initial phase of infection. in sequential testing of patients who have symptoms, suspicious chest computed tomography (ct) findings and an initial negative rt-pcr test, covid-19 rt-pcr positivity occurs at a mean of 5.1 ± 1.5 days after the initial test.32 a patient with a negative rt-pcr test result in a nasopharyngeal swab but with a positive sars-cov-2 cerebrospinal fluid (csf) test has been reported.33 while rt-pcr detection of genomic rna is not specific for viable virus, identification of sub-genomic rnas transcribed in infected cells have been used in clinical testing to document the presence of actively replicating virus in lieu of viral cultures.34 although chest ct scans may detect signs of covid-19 days before rt-pcr positivity, ct findings overlap with other viral pneumonias.35 serologic testing has uses for contact tracing, epidemiology, and vaccine studies but is more challenging to use for primary diagnosis given the latency for development of antibodies.36–40 the most common serological assays are the rapid lateral flow assay, enzyme linked immunosorbent assay (elisa), and virus neutralization assay. two-step elisa assays are quantitative and more reliable than flow assays that are easily scalable but are qualitative.40 the virus neutralization assay detects and quantifies antibodies that inhibit viral replication but is technically complex.37 5. systemic pathophysiology of covid-19 in the first stage of infection, sars-cov-2 targets nasal and bronchial epithelial cells as well as pneumocytes.28 as infection progresses, sars-cov-2 infects pulmonary endothelial cells, abrogating the epithelial-endothelial barrier.41 a subsequent ingress of neutrophils and monocytes is followed by pulmonary edema and hyaline membrane formation, a component of early acute respiratory distress syndrome (ards).41,42 in severe covid-19, coagulopathy can occur, reflecting microthrombi formation secondary to endothelial cell inflammation and cytokine storms.41,43 covid-19 associated hypercoagulability induces venous thromboembolism and arterial occlusion.41 the interplay of these systemic derangements likely contributes to the pathologic changes seen in the cns (figure 2). figure 2: flow chart modeling covid-19 pathogenesis and neurological dysfunction. adem= acute disseminated encephalomyelitis; ane= acute necrotizing encephalopathy; gbs= guillain-barré syndrome. 6. covid-19 neurological manifestations covid-19 associated neurological manifestations range from mild symptoms such as dizziness, headache, dysgeusia, or anosmia to severe disorders such as stroke, guillain-barré syndrome (gbs), acute hemorrhagic necrotizing encephalopathy, meningoencephalitis, and cerebral venous thrombosis. the frequency of reported neurological signs and symptoms is variable but substantial regardless. in an early chinese retrospective study, 36.4% of 214 covid-19 patients had neurological symptoms which included dizziness (16.8%), headache (13.1%), impaired consciousness (7.5%), dysgeusia (5.6%), and anosmia (5.1%).44 in western studies, dysgeusia and anosmia are reported in the majority of patients.45,46 a french study reports that 49 out of 58 (84%) covid-19 intensive care unit (icu) patients had neurological signs which included agitation (69%), confusion (65%), corticospinal tract signs (67%), and dysexecutive syndrome (33%).47 a study from a british referral center also describes cases of septic or para-infectious encephalopathy, autoimmune encephalitis including acute disseminated encephalomyelitis (adem), and gbs.48 7. specific neurological disorders associated with covid-19 olfactory and gustatory dysfunction in the aforementioned study from china, covid-19 patients had gustatory dysfunction and olfactory dysfunction at frequencies of less than 6% each.44 in a prospective european study, 88.8% of 385 covid-19 patients had gustatory dysfunction and 85.6% of 417 covid-19 patients had olfactory dysfunction.45 in a california study, 71% of 59 covid-19 patients recorded ageusia and 68% reported anosmia.46 olfactory and gustatory dysfunction in covid-19 typically resolves after 17 to 30 days from initial onset.49,50 although long term follow-up is generally lacking,51 one study reports resolution rates at day 30 in home-quarantined covid-19 patients of 87% for olfactory dysfunction and 82% for gustatory dysfunction.52 presence of sars-cov-2 virions in the olfactory neuroepithelium of the nasal mucosa as well as in the olfactory bulb has been documented53,54 though the exact basis for olfactory dysfunction and recovery remains to be resolved. stroke stroke is the most common debilitating neurological disorder associated with covid-19 and has a predilection for males and the elderly. two retrospective new york studies reported respectively that 1.6% of 1,916 patients with hospitalizations or emergency department visits for covid-19 and 0.9% of 3,556 hospitalized covid-19 patients had radiologically-confirmed ischemic infarcts.55,56 cryptogenic strokes were twice as common in hospitalized covid-19 patients compared to either contemporary or historical controls.56 a third new york retrospective study found 1.1% of 3,218 covid-19 patients had strokes; in the small subset with acute neuroimaging, 68.5% of strokes were ischemic (44.5% large vessel, 24% lacunar) and 24% were hemorrhagic.57 a retrospective chinese study of 214 covid-19 patients reported strokes in six patients (2.8%).44 a small american case series reported large vessel ischemic strokes in five covid-19 patients younger than 50, four of whom had no prior history of stroke.58 for comparison, the average incidence of stroke among patients admitted through a u.s. emergency department prior to the pandemic was approximately 3.2%.59 of patients who visited emergency departments or were hospitalized, only 0.2% of 1486 influenza patients had ischemic infarcts which was significantly less than covid-19 patients even after adjusting for age, sex and, race.55 in another new york study, 33 out of 755 (4.4%) covid-19 patients with neuroimaging had evidence of intracranial hemorrhage not associated with trauma, brain metastases, or tumor resection.60 parenchymal hemorrhages with mass effect and herniation were present in five patients, punctate hemorrhages in seven, small to moderate sized hemorrhages in 17, and large single site hemorrhages without herniation in four.60 twenty-six of the 33 intracranial hemorrhages occurred as a transformation of an ischemic infarct.60 of the five patients with parenchymal hemorrhages, four had high partial thromboplastin time or anti-factor xa in the 72 hours before the intracranial hemorrhage.60 advanced age, cardiovascular disease, cerebrovascular disease, diabetes, chronic respiratory disease, hypertension, obesity, smoking, and cancer are risk factors for severe covid-19 disease or poor outcome.61–67 in a canadian study, critically ill covid-19 patients with blood groups a and ab were more likely to require ventilation, continuous renal replacement therapy, and prolonged intensive care unit admission.68 in contrast, an earlier american covid-19 study did not show an association between specific blood groups and either ventilation or death.69 differences in cohorts including mortality rates may account for the contradictory findings.68 it cannot escape notice that a number of the aforementioned covid-19 prognostic factors are associated with atherosclerosis and arteriolosclerosis that, in the setting of covid-19 respiratory compromise and coagulopathy, may contribute to strokes. guillain-barré syndrome gbs is an uncommon, immune-mediated demyelinating disease of the peripheral nerves that often follows viral infections. common presenting symptoms in covid-19 patients include symmetrical flaccid quadriparesis, ataxia, facial weakness, respiratory failure, and lower paresthesia.70–72 rt-pcr of nasopharyngeal swabs tested positive for sars-cov-2 but csf was negative for tested patients.73–75 gbs symptoms tend to emerge between day five to 10 after covid-19 symptom onset.71 over 30 cases of gbs have been reported.73–76 one proposed mechanism is an autoimmune hyperreaction, triggering release of pro-inflammatory mediators such as interleukin (il)-6, that cause autoimmune demyelination or axonal damage. alternatively in some cases, sars-cov-2 may induce production of antibodies targeting gangliosides, leading to peripheral neuropathy.72,77 covid-19 associated miller fisher syndrome (mfs), a variant of gbs with ophthalmoplegia, areflexia, and ataxia, and polyneuritis cranialis (pnc), a gbs variant with multiple cranial neuropathies, have been reported.78 meningitis and encephalitis covid-19 associated meningitis has only infrequently been reported but one case highlights the need for csf testing if suspected.33 a 24-year-old japanese male presented with headache, generalized fatigue and fever. nine days after onset of typical covid-19 symptoms, the patient had altered mental status, transient generalized seizures, and neck stiffness. brain magnetic resonance imaging (mri) showed hyperintensities along the lateral ventricle and in the medial temporal lobe including the hippocampus. sars-cov-2 rna was not detected by rt-pcr in a nasopharyngeal swab but was in csf.33 autopsies performed on covid-19 patients have suggested meningoencephalitis in some cases and these are discussed in section 8.79 other disorders a brazilian study reported central venous thrombosis (cvt) in three previously healthy covid-19 patients younger than 41 years, underscoring the need for awareness of a hypercoagulable state in covid-19 even in this age group.80 acute hemorrhagic necrotizing encephalopathy, an uncommon complication characterized by multifocal symmetric brain lesions including bilateral thalamic lesions, has been associated with covid-19, though rarely.81,82 this complication may be caused by blood-brain-barrier disruption related to intracranial cytokine storms.81 covid-19 associated cases of adem-like disease and autoimmune encephalitis have been documented.72,83 8. neuropathological findings and neuropathogenesis of covid-19 to examine the spectrum of covid-19 neuropathology, we reviewed 20 papers encompassing 184 patients with tissue-based neuropathological analyses including 101 cases analyzed by rt-pcr for sars-cov-2 and 83 cases by immunohistochemistry (ihc) (table 1).25,53,79,83–99 all cases are autopsies except for four biopsies and one case of unspecified type. the range of histologic findings is broad and in part reflects the heterogeneity of neurological findings. the most frequent findings (table 1) include microglial activation with microglial nodules in a likely underestimated subset; lymphoid inflammation including perivascular lymphocytosis (figure 3a), parenchymal lymphocytic infiltration, and leptomeningeal lymphocytic inflammation; hypoxic-ischemic changes (figure 3b); astrogliosis; acute/subacute brain infarcts; primary hemorrhage; and microthrombi. it should be noted that the prevalence of detected lymphoid inflammation and microglial nodules is high in papers with immunohistochemical studies and low in those without. table 1: neuropathological findings in covid-19 brain tissue table 1: some histologic findings are likely to be under-reported as reviewed studies are variable in their focus. *only studies tabulating the prevalence of microglial nodules are included. studies mentioning microglial nodules in their case series but not enumerating the frequency are excluded. figure 3: representative covid-19 histopathology: a. mild perivascular lymphoid inflammation, 400x (arrow); b. eosinophilia in purkinje cells compatible with acute hypoxic-ischemic change, 200x (arrows); c. subacute infarct of anterior pituitary gland, 100x (arrows); d. alzheimer type ii astrocytes in basal ganglia, 400x (circle). sars-cov-2 is detectable in central nervous system tissue at least three routes of cns infection by sars-cov-2 have been proposed: retrograde transmission via olfactory sensory neurons, infiltration of immune cells, and entry across the blood-brain barrier.100,101 the prevalence of anosmia and ageusia in covid-19 patients led to the theory that sars-cov-2 enters the brain via infection of neurons in the olfactory neuroepithelium (which resides in the mucosa of the nasal cavity) and from there to the olfactory bulb and then to other brain regions. this olfactory route is used by other coronaviruses, such as sars-cov102 and mers-cov.103 covid-19 patients frequently display mri hyperintensity in the olfactory cortex.104 the ace2 receptor, used by sars-cov-2 for cellular entry, is expressed in sustentacular cells and stem cells of the nasal olfactory epithelium.23,105 while one study suggests olfactory neurons themselves do not express the ace2 receptor,23 another report contradicts this finding.106 besides technical sensitivity issues, it may be that the expression levels of the ace2 receptor and other receptors differ under inflammatory conditions as compared to physiologic conditions. viral particles have been detected by electron microscopy and ihc in the olfactory epithelium54 as well as by electron microscopy in olfactory neurons of the nasal mucosa at autopsy.53 morbini and colleagues report ultrastructural evidence of sars-cov-2 particles in the olfactory bulb of a covid-19 patient.54 a second theory posits that sars-cov-2 may infect immune cells that cross the blood-brain barrier and deliver virus into the brain. this mechanism is well described in hiv.107 white blood cells including lymphocytes and monocytes express the ace2 receptor.108,109 infection of immune cells by sars-cov-2 is an active area of study. finally, the third theory extends the well documented behavior of encephalitic blood-borne coronaviruses to enter through the blood-brain barrier via infection of vessel wall cells.110 histologically, on routine hematoxylinand eosin-stained slides, neither viral inclusions nor specific cellular changes recognizable as direct viral infection have been reported. however, sars-cov-2 has been detected in the brain by rt-pcr, ihc, and electron microscopy. as many as 54 out of 101 (53.5%) cases were positive for sars-cov-2 in the brain by rt-pcr and 23 out of 83 (27.7%) cases were positive by ihc (table 1). in some cases, copies of virus detected by rt-pcr were low in number and the detection of virus in blood or blood cells within intracerebral vasculature rather than brain cells was a possibility. however, ihc has confirmed the presence of viral antigens in autopsy brain cells. of note, antibodies against sars-cov-2 spike protein were more effective in detecting viral antigens than those targeted against nucleocapsid protein25,87,97 (supplementary table a). importantly, staining using both anti-spike and anti-nucleocapsid antibodies may be more sensitive than either alone as, in a few cases, nucleocapsid protein was detected while spike protein was not. whether this finding is a technical issue related to the quality of the antibodies or the intrinsic accessibility of the relevant epitopes or both is unresolved. staining of virus localizes in scattered cortical neurons and endothelial cells,87 as well as brainstem cranial nerve roots and isolated cells (cell type unclear) in the medulla oblongata; the images of the isolated cells had a striking lack of attendant chronic inflammation.25 this disconnect between viral infection and inflammation raises the question of immune evasion. one case exhibited viral staining around the edges of subcortical white matter microinfarcts.87 the immunostaining pattern consists of diffuse cytoplasmic and perinuclear positivity with small concentrated foci possibly representing viral inclusion bodies.87 meinhardt and colleagues, using in situ hybridization (ish), identified sars-cov-2 in the olfactory epithelium and mucus.53 lastly, viral particles compatible with sars-cov-2 have been identified by electron microscopy in olfactory bulb and frontal lobe tissue.54,96 to date, regions in which sars-cov-2 have been detected by ihc, rt-pcr, or electron microscopy relevant to the cns include cornea, conjunctiva, olfactory epithelium, olfactory bulb, olfactory tubercle, frontal lobe, cerebellum, medulla oblongata, cranial nerves and trigeminal ganglion. most investigations studied limited areas of brain or did not specify the origin of cerebral cortex tissue. additional studies are needed to better establish the frequency and extent of direct infection in the cns. as some patients have "long covid” wherein they have persistent symptoms for months, whether and how sars-cov-2 may persist in the brain will need to be evaluated in the future. chronic inflammatory and reactive glial changes microgliosis and astrocytosis are common in covid-19 brains including in the olfactory bulb.25 lymphoid inflammation, which tends to be minimal or mild in many cases, is not uncommon particularly in the medulla oblongata and if ihc is used for detection.25 in many studies that did not use ihc, lymphoid inflammation was reported as absent or infrequent; this is concordant with our experience and anecdotally with that of colleagues in asia and in the united states (personal communication). a substantial portion of these reactive changes may be secondary to systemic issues (e.g. sepsis) or other neuropathology (infarcts, hemorrhages, etc.) rather than a response to direct infection. microgliosis and chronic inflammation did not correlate with the severity of covid-19 disease nor were there any discernible neuropathological differences in patients from nursing homes, hospital wards, or intensive care units.25 furthermore, microglial activation, perivascular lymphocytosis, and leptomeningeal lymphocytic infiltration is observed to similar degrees in the control brains of septic (non-covid-19) patients when compared to covid-19 patients.86 some cases present findings compatible with viral meningoencephalitis including parenchymal lymphocytic clustering around microglial nodules, concomitant leptomeningeal lymphocytic inflammation, and even neuronophagy. in the study by matschke and colleagues, four out of 16 cases immunopositive for sars-cov-2 had 10 to 49 cd8+ cytotoxic t lymphocytes per high power field in at least three fields in the medulla oblongata.25 the observed lymphocytes tended to cluster near activated microglial nodules,25 as commonly seen in viral encephalitis. rt-pcr detected sars-cov-2 rna in three out of the four cases.25 three studies report neuronophagy in the medulla associated with histiocytic and lymphocytic parenchymal infiltration.25,84,89 von weyhern and colleagues report six cases of perivascular and parenchymal lymphocytosis with neuronal loss and axon degeneration in the brainstem, which the authors determined to be adequate to diagnose sars-cov-2 viral encephalitis.79 in the same study, five of the six cases had concomitant meningitis.79 respiratory and cardiovascular control centers of the medulla may be affected.53 while the majority of covid-19 patients with neurological manifestations do not have detectable sars-cov-2 rna in csf by rt-pcr,111–113 exceptions to this trend have been reported in at least four covid-19 patients.33,114–116 anti-sars-cov-2 antibodies were detected in the csf of one covid-19 patient suggesting an immune response to viral infection.87 it appears therefore that histologically documentable cases of sars-cov-2 encephalitis and/or meningitis do occur, with a tendency to involve the brainstem. as only a few series report the majority of brainstem microglial encephalitis cases, the question arises as to whether particular viral strains, therapeutic approaches, genetic background or detection methods are responsible for its apparent absence or paucity in the majority of studies. autoimmune mediated inflammation may also occur. autoimmune encephalitis, adem, and acute necrotizing encephalopathy have also been reported clinically though histopathologic evaluation has been limited. adem-like pathology has been reported in one autopsy case.83 microthrombi, infarcts, hemorrhages, and “neutrophilic plugs” sars-cov-2 may induce a cytokine storm, a severe hyperimmune reaction characterized by excessive and rapid release of cytokines such as il-6 and il-1β into the blood.117,118 il-6 activates the coagulation system and increases vascular permeability119 which, in combination with viral endotheliopathy,120 may account for the well documented covid-19 associated coagulopathy.41,121 sars-cov-2 has been detected within cerebral endothelial cells by ihc and electron microscopy.87,96 hypercoagulability, in turn, results in histologic findings of microthrombi, infarcts, and hemorrhages. also, il-1β plays a major role in triggering vascular “neutrophilic plugs” containing neutrophils and/or platelets as well as neutrophil extracellular traps, a mesh of deoxynucleic acid (dna)-rich material coated with antimicrobials that entrap and kill microbes.122 these “neutrophilic plugs” have been found in the brain as well as the lungs, heart, kidneys, and liver of covid-19 patients who come to autopsy.88 approximately 1.6% of cases have “neutrophilic plugs” in the brain (table 1) though this percentage is likely an underestimate as pathologists do not typically evaluate for these structures. pneumonia and ards with resultant hypoxemia as well as pre-existing arteriosclerosis are also likely contributory to the cerebral infarcts. in addition to cerebral infarcts, we have noted occasional cases of pituitary infarcts (figure 3c) in our covid-19 autopsies. numerous extramedullary megakaryocytes were present in subacute cerebral infarcts of one covid-19 patient.84 alzheimer type ii astrocytosis alzheimer type ii astrocytosis (figure 3d) characteristic of hepatic encephalopathy has been reported.89,97 the reports do not specify whether the frequency of the alzheimer type ii astrocytes reaches a threshold of five or more per 20 high power fields, the cutoff suggested by agarwal and colleagues for hepatic encephalopathy.123 in a paper by solomon and colleagues, four out of four cases with alzheimer type ii astrocytes had chronic liver disease or alcohol use disorder.97 the relative contribution of hepatic encephalopathy to cases currently ascribed as septic or para-infectious encephalopathy remains to be seen. 9. conclusions systemic dysfunction and viral infection of the cns can cause a wide range of covid-19 related neuropathological changes. the incidence of neuropathological findings should be viewed with caution given great variability in what tissues were studied and what types of ancillary studies were (or were not) performed. inflammatory changes were reported in a high percentage of cases in some series but not in others, at least in part due to the use of immunostaining; however, the lack of controls in most of these studies limit interpretation of these findings. selection or referral bias, the decedent’s co-morbidities, therapies given, patient’s genetic background, immune status, immunization status and perhaps viral strains of sars-cov-2 may also contribute to differences and will need to be dissected out in the future. whether or how much of the inflammatory changes seen are due to autoimmune phenomena versus direct viral infection or other causes remains to be resolved. the presence of microglial nodules and detectable virus are indicative of viral meningoencephalitis in some cases; a predilection for the medulla perhaps with compromise of respiratory and cardiovascular control centers may compound the covid-19 patient’s often already tenuous cardiorespiratory function. while there is evidence for an olfactory route of infection, it is unclear whether this is the major mechanism of cns infection given that the virus is rarely detectable in the olfactory bulb. cerebral endothelial infection is also present and a hematogenous route of infection is therefore plausible perhaps even common. data on where the virus is detectable in the brain is limited as studies have focused on the frontal lobe and brainstem. it would not be surprising to find a greater extent of infection than currently confirmed. it should be kept in mind that the sensitivity of virus detection is suboptimal given that most samples are derived from autopsies. lastly, it is notable that inflammation does not always coincide with virus localization, raising the concern that the virus may be evading the immune response in the cns. whether the cns is a potential long-term reservoir of virus and a contributor to “long covid” remains to be seen. lastly, increased risk for vascular dementia or neurodegenerative disorders are hypothetical concerns that may need to be addressed at a later time. funding this work was supported in part by nimh 2u24mh10092 and u01 mh083500 (s. magaki, e. singer, w. yong) and in part by the ucla broad stem cell research center covid 19 research award ocrc #20-70 (h.v. vinters, s. magaki, and t. zhang). references 1. naming the coronavirus disease (covid-19) and the virus that causes it. world health organization. accessed october 21, 2020. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)-and-the-virus-that-causes-it 2. weekly epidemiological update 29 december 2020. world health organization. accessed december 30, 2020. https://www.who.int/publications/m/item/weekly-epidemiological-update---29-december-2020 3. ye zw, yuan s, yuen ks, fung sy, chan cp, jin dy. zoonotic origins of human coronaviruses. int j biol sci. 2020;16(10):1686-1697. doi:10.7150/ijbs.45472 4. zhu n, zhang d, wang w, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020;382(8):727-733. doi:10.1056/nejmoa2001017 5. caly l, druce j, roberts j, et al. isolation and rapid sharing of the 2019 novel coronavirus (sars-cov-2) from the first patient diagnosed with covid-19 in australia. med j aust. 2020;212(10):459-462. doi:10.5694/mja2.50569 6. gulholm t, basile k, kok j, chen s, rawlinson w. laboratory diagnosis of severe acute respiratory syndrome coronavirus 2. pathology. 2020;52(7):745-753. doi:10.1016/j.pathol.2020.09.011 7. koyama t, platt d, parida l. variant analysis of sars-cov-2 genomes. bull world health organ. 2020;98(7):495-504. doi:10.2471/blt.20.253591 8. naqvi aat, fatima k, mohammad t, et al. insights into sars-cov-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach. biochim biophys acta mol basis dis. 2020;1866(10):165878. doi:10.1016/j.bbadis.2020.165878 9. ou x, liu y, lei x, et al. characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov. nat commun. 2020;11(1). doi:10.1038/s41467-020-15562-9 10. pachetti m, marini b, benedetti f, et al. emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant. j transl med. 2020;18(1):1-9. doi:10.1186/s12967-020-02344-6 11. day t, gandon s, lion s, otto sp. on the evolutionary epidemiology of sars-cov-2. curr biol. 2020;30(15):r849-r857. doi:10.1016/j.cub.2020.06.031 12. rowland-jones s, andrews sm. recent advances in understanding hiv evolution. f1000research. 2017;6(0):1-7. doi:10.12688/f1000research.10876.1 13. nobusawa e, sato k. comparison of the mutation rates of human influenza a and b viruses. j virol. 2006;80(7):3675-3678. doi:10.1128/jvi.80.7.3675-3678.2006 14. gheblawi m, wang k, viveiros a, et al. angiotensin-converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2. circ res. 2020;126(10):1456-1474. doi:10.1161/circresaha.120.317015 15. wang k, chen w, zhang z, et al. cd147-spike protein is a novel route for sars-cov-2 infection to host cells. signal transduct target ther. 2020;5(1):283. doi:10.1038/s41392-020-00426-x 16. fernández-atucha a, izagirre a, fraile-bermúdez ab, et al. sex differences in the aging pattern of renin-angiotensin system serum peptidases. biol sex differ. 2017;8(1). doi:10.1186/s13293-017-0128-8 17. hu y, li x, wu n, wang n, qui c, li j. study on the correlation among sex, age, and the activity of ace, ace2 and the ratio of ace/ace2. j qiqihar med coll. 2018;39(8):884-887. doi:10.3969/j.issn.1002-1256.2018.08.005 18. liu y, zhao l, zhang q, zhang l, ren g. effect of long-term smoking on expressiong of serum ace and ace2 as well as its significance. j taishan med coll. 2019;40(4):258-260. doi:10.3969/j.issn.1004-7115.2019.04.007 19. smith jc, sausville el, girish v, et al. cigarette smoke exposure and inflammatory signaling increase the expression of the sars-cov-2 receptor ace2 in the respiratory tract. dev cell. 2020;53(5):514-529.e3. doi:https://doi.org/10.1016/j.devcel.2020.05.012 20. bernardi s, toffoli b, zennaro c, et al. high-salt diet increases glomerular ace/ace2 ratio leading to oxidative stress and kidney damage. nephrol dial transplant. 2012;27(5):1793-1800. doi:10.1093/ndt/gfr600 21. lavrentyev en, malik ku. high glucose-induced nox1-derived superoxides downregulate pkc-βii, which subsequently decreases ace2 expression and ang(1-7) formation in rat vsmcs. am j physiol hear circ physiol. 2009;296(1):106-118. doi:10.1152/ajpheart.00239.2008 22. xia h, lazartigues e. angiotensin-converting enzyme 2 in the brain: properties and future directions. j neurochem. 2008;107(6):1482-1494. doi:10.1111/j.1471-4159.2008.05723.x 23. brann d, tsukahara t, weinreb c, et al. non-neuronal expression of sars-cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-associated anosmia. sci adv. 2020;6(31):eabc5801. doi:10.1126/sciadv.abc5801 24. chen m, shen w, rowan nr, et al. elevated ace2 expression in the olfactory neuroepithelium: implications for anosmia and upper respiratory sars-cov-2 entry and replication. eur respir j. 2020;56(3):2001948. doi:10.1183/13993003.01948-2020 25. matschke j, lütgehetmann m, hagel c, et al. neuropathology of patients with covid-19 in germany: a post-mortem case series. lancet neurol. 2020;19(11). doi:10.1016/s1474-4422(20)30308-2 26. wang k, chen w, zhou y sen, et al. sars-cov-2 invades host cells via a novel route: cd147-spike protein. biorxiv. published online january 1, 2020:2020.03.14.988345. doi:10.1101/2020.03.14.988345 27. cantuti-castelvetri l, ojha r, pedro ld, et al. neuropilin-1 facilitates sars-cov-2 cell entry and provides a possible pathway into the central nervous system. biorxiv. published online january 1, 2020:2020.06.07.137802. doi:10.1101/2020.06.07.137802 28. hoffmann m, kleine-weber h, schroeder s, et al. sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor. cell. 2020;181(2):271-280.e8. doi:10.1016/j.cell.2020.02.052 29. zang r, gomez castro mf, mccune bt, et al. tmprss2 and tmprss4 promote sars-cov-2 infection of human small intestinal enterocytes. sci immunol. 2020;5(47):eabc3582. doi:10.1126/sciimmunol.abc3582 30. covid-19 rt-pcr test emergency use authorization (eua) summary. laboratory corporation of america. accessed september 2, 2020. https://www.fda.gov/media/136151/download 31. long c, xu h, shen q, et al. diagnosis of the coronavirus disease (covid-19): rrt-pcr or ct? eur j radiol. 2020;126:108961. doi:10.1016/j.ejrad.2020.108961 32. ai t, yang z, hou h, et al. correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases. radiology. 2020;296(2):e32-e40. doi:10.1148/radiol.2020200642 33. moriguchi t, harii n, goto j, et al. a first case of meningitis/encephalitis associated with sars-coronavirus-2. int j infect dis. 2020;94:55-58. doi:10.1016/j.ijid.2020.03.062 34. perera rapm, tso e, tsang oty, et al. sars-cov-2 virus culture and subgenomic rna for respiratory specimens from patients with mild coronavirus disease. emerg infect dis j. 2020;26(11):2701. doi:10.3201/eid2611.203219 35. dai w-c, zhang hw, yu j, et al. ct imaging and differential diagnosis of covid-19. can assoc radiol j. 2020;71(2):195-200. doi:10.1177/0846537120913033 36. guo l, ren l, yang s, et al. profiling early humoral response to diagnose novel coronavirus disease (covid-19). clin infect dis an off publ infect dis soc am. published online march 2020. doi:10.1093/cid/ciaa310 37. gauger pc, vincent al. serum virus neutralization assay for detection and quantitation of serum neutralizing antibodies to influenza a virus in swine. methods mol biol. 2020;2123:321-333. doi:10.1007/978-1-0716-0346-8_23 38. okba nma, müller ma, li w, et al. severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients. emerg infect dis. 2020;26(7). doi:10.3201/eid2607.200841 39. sun b, feng y, mo x, et al. kinetics of sars-cov-2 specific igm and igg responses in covid-19 patients. emerg microbes infect. 2020;9(1):940-948. doi:10.1080/22221751.2020.1762515 40. krammer f, simon v. serology assays to manage covid-19. science. 2020;368(6495):1060-1061. doi:10.1126/science.abc1227 41. wiersinga wj, rhodes a, cheng ac, peacock sj, prescott hc. pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19): a review. jama. 2020;324(8):782-793. doi:10.1001/jama.2020.12839 42. xu z, shi l, wang y, et al. pathological findings of covid-19 associated with acute respiratory distress syndrome. lancet respir med. 2020;8(4):420-422. doi:10.1016/s2213-2600(20)30076-x 43. jose rj, manuel a. covid-19 cytokine storm: the interplay between inflammation and coagulation. lancet respir med. 2020;8(6):e46-e47. doi:10.1016/s2213-2600(20)30216-2 44. mao l, jin h, wang m, et al. neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china. jama neurol. 2020;77(6):683-690. doi:10.1001/jamaneurol.2020.1127 45. lechien jr, chiesa-estomba cm, de siati dr, et al. olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study. eur arch oto-rhino-laryngology. 2020;277(8):2251-2261. doi:10.1007/s00405-020-05965-1 46. yan ch, faraji f, prajapati dp, boone ce, deconde as. association of chemosensory dysfunction and covid-19 in patients presenting with influenza-like symptoms. int forum allergy rhinol. 2020;10(7):806-813. doi:10.1002/alr.22579 47. helms j, kremer s, merdji h, et al. neurologic features in severe sars-cov-2 infection. n engl j med. 2020;382(21):2268-2270. doi:10.1056/nejmc2008597 48. paterson rw, brown rl, benjamin l, et al. the emerging spectrum of covid-19 neurology: clinical, radiological and laboratory findings. brain. 2020;143(10):3104-3120. doi:10.1093/brain/awaa240 49. paolo g. does covid-19 cause permanent damage to olfactory and gustatory function? med hypotheses. 2020;143:110086. doi:10.1016/j.mehy.2020.110086 50. hopkins c, surda p, whitehead e, kumar bn. early recovery following new onset anosmia during the covid-19 pandemic – an observational cohort study. j otolaryngol head neck surg. 2020;49(1):26. doi:10.1186/s40463-020-00423-8 51. whitcroft kl, hummel t. olfactory dysfunction in covid-19: diagnosis and management. jama. 2020;323(24):2512-2514. doi:10.1001/jama.2020.8391 52. paderno a, mattavelli d, rampinelli v, et al. olfactory and gustatory outcomes in covid-19: a prospective evaluation in nonhospitalized subjects. otolaryngol head neck surg. 2020;163(6):1144-1149. doi:10.1177/0194599820939538 53. meinhardt j, radke j, dittmayer c, et al. olfactory transmucosal sars-cov-2 invasion as a port of central nervous system entry in individuals with covid-19. nat neurosci. published online 2020. doi:10.1038/s41593-020-00758-5 54. morbini p, benazzo m, verga l, et al. ultrastructural evidence of direct viral damage to the olfactory complex in patients testing positive for sars-cov-2. jama otolaryngol neck surg. 2020;146(10):972-973. doi:10.1001/jamaoto.2020.2366 55. merkler ae, parikh ns, mir s, et al. risk of ischemic stroke in patients with coronavirus disease 2019 (covid-19) vs patients with influenza. jama neurol. 2020;77(11):1–7. doi:10.1001/jamaneurol.2020.2730 56. yaghi s, ishida k, torres j, et al. sars-cov-2 and stroke in a new york healthcare system. stroke. 2020;51(7):2002-2011. doi:10.1161/strokeaha.120.030335 57. jain r, young m, dogra s, et al. covid-19 related neuroimaging findings: a signal of thromboembolic complications and a strong prognostic marker of poor patient outcome. j neurol sci. 2020;414:116923. doi:10.1016/j.jns.2020.116923 58. oxley tj, mocco j, majidi s, et al. large-vessel stroke as a presenting feature of covid-19 in the young. n engl j med. 2020;382(20):e60-e60. doi:10.1056/nejmc2009787 59. national hospital ambulatory medical care survey: 2017 emergency department summary tables. centers for disease control and prevention. accessed january 10, 2021. https://www.cdc.gov/nchs/data/nhamcs/web_tables/2017_ed_web_tables-508.pdf 60. dogra s, jain r, cao m, et al. hemorrhagic stroke and anticoagulation in covid-19. j stroke cerebrovasc dis. 2020;29(8):104984. doi:https://doi.org/10.1016/j.jstrokecerebrovasdis.2020.104984 61. wu z, mcgoogan jm. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention. jama. published online february 2020. doi:10.1001/jama.2020.2648 62. yang j, zheng y, gou x, et al. prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis. int j infect dis ijid off publ int soc infect dis. 2020;94:91-95. doi:10.1016/j.ijid.2020.03.017 63. chen t, wu d, chen h, et al. clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study. bmj. 2020;368:m1091. doi:10.1136/bmj.m1091 64. jordan re, adab p, cheng kk. covid-19: risk factors for severe disease and death. bmj. 2020;368:m1198. doi:10.1136/bmj.m1198 65. wang b, li r, lu z, huang y. does comorbidity increase the risk of patients with covid-19: evidence from meta-analysis. aging (albany ny). 2020;12(7):6049-6057. doi:10.18632/aging.103000 66. simonnet a, chetboun m, poissy j, et al. high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation. obesity (silver spring). 2020;28(7):1195-1199. doi:10.1002/oby.22831 67. lighter j, phillips m, hochman s, et al. obesity in patients younger than 60 years is a risk factor for covid-19 hospital admission. clin infect dis. published online april 2020. doi:10.1093/cid/ciaa415 68. hoiland rl, fergusson na, mitra ar, et al. the association of abo blood group with indices of disease severity and multiorgan dysfunction in covid-19. blood adv. 2020;4(20):4981-4989. doi:10.1182/bloodadvances.2020002623 69. latz ca, decarlo c, boitano l, et al. blood type and outcomes in patients with covid-19. ann hematol. 2020;99(9):2113-2118. doi:10.1007/s00277-020-04169-1 70. padroni m, mastrangelo v, asioli gm, et al. guillain-barré syndrome following covid-19: new infection, old complication? j neurol. 2020;(table 1):1-3. doi:10.1007/s00415-020-09849-6 71. toscano g, palmerini f, ravaglia s, et al. guillain–barré syndrome associated with sars-cov-2. n engl j med. 2020;382(26):2574-2576. doi:10.1056/nejmc2009191 72. dalakas mc. guillain-barré syndrome: the first documented covid-19–triggered autoimmune neurologic disease. neurol neuroimmunol neuroinflammation. 2020;7(5):e781. doi:10.1212/nxi.0000000000000781 73. caress jb, castoro rj, simmons z, et al. covid-19-associated guillain-barré syndrome: the early pandemic experience. muscle nerve. 2020;62(4):485-491. doi:10.1002/mus.27024 74. trujillo gittermann lm, valenzuela feris sn, von oetinger giacoman a. relation between covid-19 and guillain-barré syndrome in adults. systematic review tt relación entre covid-19 y síndrome de guillain-barré en adultos. revisión sistemática. neurologia. 2020;35(9):646-654. doi:10.1016/j.nrl.2020.07.004 75. abu-rumeileh s, abdelhak a, foschi m, tumani h, otto m. guillain-barré syndrome spectrum associated with covid-19: an up-to-date systematic review of 73 cases. j neurol. published online august 25, 2020:1-38. doi:10.1007/s00415-020-10124-x 76. rahimi k. guillain-barre syndrome during covid-19 pandemic: an overview of the reports. neurol sci. 2020;41(11):3149-3156. doi:10.1007/s10072-020-04693-y 77. ehrenfeld m, tincani a, andreoli l, et al. covid-19 and autoimmunity. autoimmun rev. 2020;19(8):102597. doi:10.1016/j.autrev.2020.102597 78. gutiérrez-ortiz c, méndez a, rodrigo-rey s, et al. miller fisher syndrome and polyneuritis cranialis in covid-19. neurology. 2020;241:10.1212/wnl.0000000000009619. doi:10.1212/wnl.0000000000009619 79. von weyhern ch, kaufmann i, neff f, kremer m. early evidence of pronounced brain involvement in fatal covid-19 outcomes. lancet (london, england). 2020;395(10241):e109. doi:10.1016/s0140-6736(20)31282-4 80. cavalcanti d, raz e, shapiro m, et al. cerebral venous thrombosis associated with covid-19. am j neuroradiol. 2020;41(8):1-7. doi:10.3174/ajnr.a6644 81. poyiadji n, shahin g, noujaim d, stone m, patel s, griffith b. covid-19-associated acute hemorrhagic necrotizing encephalopathy: imaging features. radiology. 2020;296(2):e119-e120. doi:10.1148/radiol.2020201187 82. dixon l, varley j, gontsarova a, et al. covid-19-related acute necrotizing encephalopathy with brain stem involvement in a patient with aplastic anemia. neurol neuroimmunol neuroinflammation. 2020;7(5):e789. doi:10.1212/nxi.0000000000000789 83. reichard rr, kashani kb, boire na, constantopoulos e, guo y, lucchinetti cf. neuropathology of covid-19: a spectrum of vascular and acute disseminated encephalomyelitis (adem)-like pathology. acta neuropathol. 2020;140(1):1-6. doi:10.1007/s00401-020-02166-2 84. jensen mp, le quesne j, officer-jones l, et al. neuropathological findings in two patients with fatal covid-19. neuropathol appl neurobiol. 2020;n/a(n/a). doi:10.1111/nan.12662 85. jaunmuktane z, mahadeva u, green a, et al. microvascular injury and hypoxic damage: emerging neuropathological signatures in covid-19. acta neuropathol. 2020;140(3):397-400. doi:10.1007/s00401-020-02190-2 86. deigendesch n, sironi l, kutza m, et al. correlates of critical illness-related encephalopathy predominate postmortem covid-19 neuropathology. acta neuropathol. 2020;140(4):583-586. doi:10.1007/s00401-020-02213-y 87. song e, zhang c, israelow b, et al. neuroinvasion of sars-cov-2 in human and mouse brain. biorxiv. published online september 8, 2020:2020.06.25.169946. doi:10.1101/2020.06.25.169946 88. schurink b, roos e, radonic t, et al. viral presence and immunopathology in patients with lethal covid-19: a prospective autopsy cohort study. the lancet microbe. 2020;1(7):e290-e299. doi:10.1016/s2666-5247(20)30144-0 89. al-dalahmah o, thakur kt, nordvig as, et al. neuronophagia and microglial nodules in a sars-cov-2 patient with cerebellar hemorrhage. acta neuropathol commun. 2020;8(1):147. doi:10.1186/s40478-020-01024-2 90. hernández-fernández f, valencia hs, barbella-aponte ra, et al. cerebrovascular disease in patients with covid-19: neuroimaging, histological and clinical description. brain. 2020;143(10):3089-3103. doi:10.1093/brain/awaa239 91. fabbri vp, foschini mp, lazzarotto t, et al. brain ischemic injury in covid-19-infected patients: a series of 10 post-mortem cases. brain pathol. published online october 1, 2020. doi:10.1111/bpa.12901 92. patel sd, kollar r, troy p, et al. malignant cerebral ischemia in a covid-19 infected patient: case review and histopathological findings. j stroke cerebrovasc dis. 2020;29(11):105231. doi:10.1016/j.jstrokecerebrovasdis.2020.105231 93. patel hn, syed a, lobel js, et al. cerebellar infarction requiring surgical decompression in patient with covid 19 pathological analysis and brief review. interdiscip neurosurg adv tech case manag. 2020;22:100850. doi:10.1016/j.inat.2020.100850 94. bryce c, grimes z, pujadas e, et al. pathophysiology of sars-cov-2: targeting of endothelial cells renders a complex disease with thrombotic microangiopathy and aberrant immune response. the mount sinai covid-19 autopsy experience. medrxiv. published online january 1, 2020:2020.05.18.20099960. doi:10.1101/2020.05.18.20099960 95. remmelink m, de mendoca r, d’haene n, et al. unspecific post-mortem findings despite multiorgan 1 viral spread in covid-19 patients. crit care. 2020;21(1):495. doi:10.1186/s13054-020-03218-5 96. paniz-mondolfi a, bryce c, grimes z, et al. central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (sars-cov-2). j med virol. 2020;92(7):699-702. doi:10.1002/jmv.25915 97. solomon ih, normandin e, bhattacharyya s, et al. neuropathological features of covid-19. n engl j med. 2020;383(10):989-992. doi:10.1056/nejmc2019373 98. hanley b, naresh kn, roufosse c, et al. histopathological findings and viral tropism in uk patients with severe fatal covid-19: a post-mortem study. the lancet microbe. 2020;1(6):e245-e253. doi:https://doi.org/10.1016/s2666-5247(20)30115-4 99. kantonen j, mahzabin s, mäyränpää mi, et al. neuropathologic features of four autopsied covid-19 patients. brain pathol. published online august 6, 2020. doi:10.1111/bpa.12889 100. zubair as, mcalpine ls, gardin t, farhadian s, kuruvilla de, spudich s. neuropathogenesis and neurologic manifestations of the coronaviruses in the age of coronavirus disease 2019: a review. jama neurol. 2020;77(8):1018-1027. doi:10.1001/jamaneurol.2020.2065 101. iadecola c, anrather j, kamel h. effects of covid-19 on the nervous system. cell. 2020;183(1):16-27.e1. doi:10.1016/j.cell.2020.08.028 102. netland j, meyerholz dk, moore s, cassell m, perlman s. severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2. j virol. 2008;82(15):7264-7275. doi:10.1128/jvi.00737-08 103. hao xy, lv q, li f di, xu yf, gao h. the characteristics of hdpp4 transgenic mice subjected to aerosol mers coronavirus infection via an animal nose-only exposure device. anim model exp med. 2019;2(4):269-281. doi:10.1002/ame2.12088 104. politi ls, salsano e, grimaldi m. magnetic resonance imaging alteration of the brain in a patient with coronavirus disease 2019 (covid-19) and anosmia. jama neurol. 2020;77(8):1028-1029. doi:10.1001/jamaneurol.2020.2125 105. fodoulian l, tuberosa j, rossier d, et al. sars-cov-2 receptors and entry genes are expressed in the human olfactory neuroepithelium and brain. iscience. 2020;23(12):101839. doi:10.1016/j.isci.2020.101839 106. nampoothiri s, sauve f, ternier g, et al. the hypothalamus as a hub for putative sars-cov-2 brain infection. biorxiv. published online january 1, 2020:2020.06.08.139329. doi:10.1101/2020.06.08.139329 107. kim wk, corey s, alvarez x, williams k. monocyte/macrophage traffic in hiv and siv encephalitis. j leukoc biol. 2003;74(5):650-656. doi:10.1189/jlb.0503207 108. trojanowicz b, ulrich c, kohler f, et al. monocytic angiotensin-converting enzyme 2 relates to atherosclerosis in patients with chronic kidney disease. nephrol dial transplant. 2017;32(2):287-298. doi:10.1093/ndt/gfw206 109. salamanna f, maglio m, landini mp, fini m. body localization of ace-2: on the trail of the keyhole of sars-cov-2. front med. 2020;7:935. https://www.frontiersin.org/article/10.3389/fmed.2020.594495 110. bergmann cc, lane te, stohlman sa. coronavirus infection of the central nervous system: host-virus stand-off. nat rev microbiol. 2006;4(2):121-132. doi:10.1038/nrmicro1343 111. neumann b, schmidbauer ml, dimitriadis k, et al. cerebrospinal fluid findings in covid-19 patients with neurological symptoms. j neurol sci. 2020;418:117090. doi:10.1016/j.jns.2020.117090 112. espíndola o de m, siqueira m, soares cn, et al. patients with covid-19 and neurological manifestations show undetectable sars-cov-2 rna levels in the cerebrospinal fluid. int j infect dis. 2020;96:567-569. doi:10.1016/j.ijid.2020.05.123 113. bellon m, schweblin c, lambeng n, et al. cerebrospinal fluid features in sars-cov-2 rt-pcr positive patients. clin infect dis. published online august 8, 2020:ciaa1165. doi:10.1093/cid/ciaa1165 114. hung ecw, chim ssc, chan pks, et al. detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome. clin chem. 2003;49(12):2108-2109. doi:10.1373/clinchem.2003.025437 115. zhou l, zhang m, wang j, gao j. sars-cov-2: underestimated damage to nervous system. travel med infect dis. 2020;36:101642. doi:10.1016/j.tmaid.2020.101642 116. virhammar j, kumlien e, fällmar d, et al. acute necrotizing encephalopathy with sars-cov-2 rna confirmed in cerebrospinal fluid. neurology. 2020;95(10):445-449. doi:10.1212/wnl.0000000000010250 117. chen g, wu d, guo w, et al. clinical and immunological features of severe and moderate coronavirus disease 2019. j clin invest. 2020;130(5):2620-2629. doi:10.1172/jci137244 118. huang c, wang y, li x, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020;395(10223):497-506. doi:10.1016/s0140-6736(20)30183-5 119. song p, li w, xie j, hou y, you c. cytokine storm induced by sars-cov-2. clin chim acta. 2020;509:280-287. doi:https://doi.org/10.1016/j.cca.2020.06.017 120. goshua g, pine ab, meizlish ml, et al. endotheliopathy in covid-19-associated coagulopathy: evidence from a single-centre, cross-sectional study. lancet haematol. 2020;7(8):e575-e582. doi:10.1016/s2352-3026(20)30216-7 121. thachil j, tang n, gando s, et al. isth interim guidance on recognition and management of coagulopathy in covid-19. j thromb haemost. 2020;18(5):1023-1026. doi:10.1111/jth.14810 122. yaqinuddin a, kvietys p, kashir j. covid-19: role of neutrophil extracellular traps in acute lung injury. respir investig. 2020;58(5):419-420. doi:10.1016/j.resinv.2020.06.001 123. agarwal an, mais dd. sensitivity and specificity of alzheimer type ii astrocytes in hepatic encephalopathy. arch pathol lab med. 2019;143(10):1256-1258. doi:10.5858/arpa.2018-0455-oa copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuroinflammation: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:1 (2021) review neuroinflammation: 2021 update hans lassmann center for brain research, medical university of vienna, austria corresponding author: hans lassmann · center for brain research · medical university of vienna · spitalgasse 4 · 1090 wien · austria hans.lassmann@meduniwien.ac.at submitted: 18 december 2020 accepted: 08 january 2021 copyedited by: aivi nguyen published: 12 january 2021 https://doi.org/10.17879/freeneuropathology-2021-3166 keywords: brain inflammation, covid-19, multiple sclerosis, alzheimer’s disease, mogad abstract key requirements for the validity of a neuropathological study are the inclusion of large numbers of biopsy or autopsy cases and proper controls, the rigorous classification of the basic neuropathology and the selection of the most suitable technologies for investigation. whether the studies are performed with the fanciest, new, and state of the art technology or with rather conventional methodology is of minor importance. following these criteria, a spectrum of neuropathological studies has been published in 2020, which provides new insights on important questions related to neurological disease. they include the pathological substrate of brain disease in covid-19 infected patients, the nature of the adaptive and innate inflammatory response, or the type and mechanisms of tissue injury and repair in multiple sclerosis, and diagnostically relevant or mechanistic new insights into antibody-mediated diseases of the central nervous system. other studies describe in detail the dynamic changes of brain inflammation in patients with trisomy 21 as a disease model for alzheimer’s disease, or the presence and consequences of vascular comorbidities in a chronic inflammatory disease, such as multiple sclerosis. all these contributions have provided important, highly relevant clues for basic and translational neuroscience. 1) what was the brain pathology in patients who died during the course of covid-19? the covid-19 pandemic has been a major challenge for society and particularly for health care institutions during the last year. although covid-19 primarily affects the respiratory system and the major cause of death is pneumonia with respiratory failure, other organs, such as the renal, cardiovascular system, or the digestive tract, are affected (paterson et al. 2020). most relevant for neuropathology is that the nervous system, too, can be a target, reflected for instance by anosmia and ageusia, encephalopathy, focal ischemic stroke, encephalitis, meningitis, or polyneuritis (li et al. 2020, paterson et al. 2020, liu et al. 2020, hernandez-fernandez et al. 2020). to what extent these neurological deficits are due to direct sars-cov2 infection of the nervous tissue, immune-mediated brain damage in the course of a systemic cytokine storm, or are secondary complications of respiratory failure, the intensive care setting, or comorbidities is not clear (liu et al. 2020). neuropathology plays a critical role in answering these questions. the first approach to answer these questions was to analyze whether molecules, which are involved in cellular virus infection or propagation, are expressed within the nervous system. angiotensin concerting enzyme 2 (ace2) is one of the cellular receptors recognized by sars-cov2, when docking to the cell surface. thus, the presence of ace2 on brain cells, such as neurons, glia or cerebral endothelial cells suggests that brain infections is possible (kabbani and olds 2020, kanwar et al. 2020). another docking molecule for sars-cov2 is neuropilin 1 (nrp1) and a high expression of this molecule has been found in virus infected cells in the nasal cavity in covid-19 patients (cantuti-castelvetri et al. 2020). early neuropathological studies in the covid-19 pandemic were restricted to single case reports or investigations in very small patient cohorts, and they were generally restricted to some basic neuropathological investigations (kantonen et al. 2020, reichard et al. 2020, jensen et al. 2020, younger 2020, liu et al. 2020). the results were diverse and controversial, mainly providing evidence for brain damage that was not directly linked to sars-cov2 infection. the first systematic study on this issue appeared on october 2020 in lancet neurology (matschke et al. 2020), and this study combined classical neuropathology with virology and molecular studies on gene expression. in a first set of data the authors describe that different molecules, involved in virus docking and propagation, show a preferential expression in different cells of the central nervous system. for example, ace2 was mainly found in oligodendrocytes. the highest expression of transmembrane protease serine subtype 2 and 4 (tmprss2 / 4) was seen in neurons, and nrp-1 was mainly present in endothelial cells and astrocytes. these data suggest different mechanisms of infection in different brain cells. the neuropathological studies documented the presence of focal and diffuse ischemic lesions and diffuse astrogliosis. microglia activation with microglia nodules and inflammation were mainly seen in the lower brain stem. in about half of the patients, virus was found either by pcr or immunocytochemistry. however, the virus load was very low and by immunohistochemistry only a very small number of virus infected cells became apparent. this study is the first to describe the basic neuropathology seen in covid-19 patients and is, thus, very important and groundbreaking. however, it also has important limitations. it is based on autopsies of 43 patients, which is likely not sufficient to cover the entire neuropathological spectrum of the disease. another limitation is that it does not contain data regarding the neurological status of the patients. thus, it remains unclear, to what extent patients with specific neurological disease manifestations have been included. the third limitation is that it does not include a proper patient control group and, particularly, patients with similarly severe systemic immune activation. this particular question has been addressed in another study on covid-19 neuropathology, which revealed that the degree of inflammation and microglia activation seen in covid-19 patients is similar to that of patients who died under septic conditions (deigendesch et al. 2020), thus suggesting that it may at least in part be a secondary consequence of systemic immune activation. another study, also based on a large sample of autopsies, mainly focused on the possible routes of cns infection (meinhardt et al. 2020). it shows high virus load in the olfactory epithelium, including the olfactory sensory neurons, and in the olfactory pathways within the cns, supporting the view of a neuronal route of virus entry into the cns. additionally, however, virus antigen was also present in cerebral endothelial cells, associated with micro-thrombosis and cerebral microinfarcts. in summary, current pathological studies show that the central nervous system can be infected with sars-cov2, that local infection can be associated with nervous system damage, but the extent of infection is low. a large part of the neuropathological changes seems to be secondary to systemic immune activation and cytokine storm, critical-illness related encephalopathy, or hypoxia and comorbidities. 2) what new insights have emerged into phenotype and disease mechanisms of antibody mediated autoimmune diseases of the central nervous system? the discovery that autoantibodies against neuronal ion channels or neurotransmitter receptors are associated with a spectrum of acute and chronic neurological diseases, has revolutionized neurological disease research (höftberger and lassmann 2017). it has been shown that these diseases, which had before been regarded as functional diseases, neurodegenerative disorders or toxic conditions, are immune-mediated and can be successfully treated with immunosuppression. the number of diseases falling into this category has profoundly increased during the last years (höftberger and lassmann 2017). one reason, which may in part explain the increase in prevalence, is the introduction of immunological checkpoint inhibitor therapies in oncology. immunological checkpoints are critical steps in t-cell activation, which prohibit the development of auto-reactive t-cells and auto-antibodies. when these checkpoints are inhibited, immune reactions against antigens of malignant neoplasms can be triggered, but this may occur at the expense of autoimmunity, which is frequently directed against neuronal antigens (sechi et al. 2020). on this basis it is not surprising that auto-antibody-associated diseases of the central nervous system remained in the focus of research interest in 2020. the respective studies provided novel insights into the function of nmda and glycine receptor directed antibodies in relation to the clinical disease spectrum (matute et al. 2020, carceles-cordon et al. 2020, rauschenberger et al. 2020) or how autoantibodies against iglon 5 may trigger intracellular accumulation of tau-tangles (landa et al. 2020). here, i focus on one study, which deals with an interesting but also controversial aspect of these diseases. pitsch et al. (2020) identified antibodies against the postsynaptic actin binding protein drebrin in patients with severe epileptic seizures, which were associated with encephalopathy and neuropsychiatric symptoms, including depression and cognitive impairment. the antibodies, identified by selective binding to the neuropil and by western blot and sequencing, were specific for these patients, and the specificity for the target antigen was proven by the lack of binding in drebrin knock out mice. drebrin is an intracellular antigen, which is not exposed on the extracellular surface of neurons or synapses. for this reason, the antibodies themselves may not be pathogenic, but just represent a diagnostically useful marker for an autoimmune attack of cytotoxic t-cells, as it occurs in many classical paraneoplastic diseases. the pathology, described in a biopsy of one of the cases, is in line with this assumption, showing infiltrating t-cells in close contact with neurons. however, the study further describes neurophysiological experiments, which show that the antibodies induce altered neuronal activity patterns and increased firing and bursting rates in neuronal networks in vitro. these data suggest that auto-antibodies directed against a cytoplasmic protein in synapses may reach their specific target and induce functional changes. the authors suggest that extensive exocytosis and endocytosis, which takes place in active synapses, facilitates the entry of the antibodies into the intracellular compartment, but this proposed mechanism does not explain how the antibodies leave the endosomal compartment and access the cytosol. this important cell biological question with important disease relevance has to be clarified in future studies. 3) what is the pathological difference between mog antibody associated inflammatory demyelinating disease (mogad) and multiple sclerosis (ms)? focal plaques of demyelination with axonal preservation and reactive gliosis developing in the context of a chronic inflammatory reaction in the central nervous system has been regarded as the specific hallmark of ms pathology. however, a new disease entity has recently emerged, which is an inflammatory demyelinating disease associated with antibodies directed against myelin oligodendrocyte glycoprotein (reindl and waters 2019). not all anti-mog antibodies are pathogenic, but only those that are directed against a conformational epitope, which is accessible on the surface of native oligodendrocytes or on mog-transfected cell lines. using these diagnostic tests, it became clear that mog antibody-associated disease (mogad) differs from ms by its clinical manifestation and course as well as by its response to immunomodulatory treatment (fujihara and cook 2020). to explain these clinical differences, a neuropathological comparison of these diseases is mandatory, but respective knowledge on mogad was restricted to few biopsies with very limited tissue samples. this changed in 2020, with two studies describing the neuropathological changes in large cohorts of mogad patients (höftberger et al. 2020, takai et al. 2020). the lesions in mogad differ from those seen in ms in many aspects, including their topographical distribution in the cns, the type of demyelination, and the nature of the inflammatory response. in ms, new lesions are formed by simultaneous demyelination in large, focal areas or by the peripheral expansion of pre-existing lesions located throughout the brain and spinal cord. in contrast, mogad demyelination occurs by confluence of small perivenous lesions, generally resulting in a demyelination pattern similar to that seen in acute disseminated encephalomyelitis (figure 1). demyelination in mogad is associated with complement deposition at the site of active myelin injury, but the degree of complement activation is much less compared to that seen in patients with aquaporin 4 antibody associated neuromyelitis optica (nmo). in both conditions, ms as well as mogad, the basic lesion is characterized by inflammatory demyelination, partial axonal preservation and reactive astrocytic gliosis. however, the inflammatory reaction is fundamentally different. while in ms the dominant inflammatory reaction is seen around the larger drainage veins in the periventricular tissue and the meninges, in mogad the smaller veins and venules are mainly affected. finally, in mogad, infiltrating lymphocytes are mainly cd4+ t-cells with low numbers of cd8+ t-cells and b-cells; the dominant lymphocytes in active ms lesions are tissue resident cd8+ effector memory t-cells and b-cells / plasma cells. all these data indicate that mogad and ms are fundamentally different diseases. figure 1: key neuropathological features, distinguishing multiple sclerosis (ms) from the inflammatory demyelinating disease, which is associated with auto-antibodies against myelin oligodendrocyte glycoprotein (mogad) in the forebrain. a) in ms, the lesions (blue areas) are located around large cerebral veins with a predilection site around the ventricle and the subcortical white matter. they are sharply demarcated from the surrounding peri-plaque white matter and frequently display finger like perivenous extensions into the deep white matter. in the cortex, band like subpial lesions (red areas) dominate, mainly located in the deep sulci of the cortical ribbon. b) in mogad, lesions are mainly located in the optic nerves and spinal cord, but their structural details have so far not been clearly outlined. when lesions are present in the hemispheres of the forebrain, a type of tissue injury is seen, which resembles acute disseminated encephalomyelitis. it consists of perivenous sleeves of demyelination around small veins and venules (blue dotted areas), which show confluence in the lesion center (dark blue area). when the cortex is affected small perivenous intra-cortical demyelination is seen most frequently (red dotted areas). c) in ms, the lesions form around larger veins with prominent perivascular spaces. the perivascular spaces contain mainly b-cells (red dots) and cd8+ t-lymphocytes (blue dots), the latter also disperse into the plaque parenchyma. cd4+ t-cells (green dots) are very sparse. a characteristic feature of active ms lesions is the radial expansion, reflected by a rim of activated macrophages at the lesion edge (blue dotted area). d) in contrast, in mogad, numerous small perivenous sheaths of demyelination are present, which arise around small veins and venules (blue dotted areas). in the inflammatory infiltrates the cd4+ t-cells dominate, while there is a much lower contribution of cd8+ t-cells (blue dots) or b-cells and plasma cells (red dots). confluence of the perivenous lesions results in larger demyelinated plaques. interestingly, the pathology of mogad closely resembles the pathology seen in experimental models of autoimmune encephalomyelitis (eae), induced by immunization of rats, guinea pigs, or primates with cns tissue myelin or recombinant mog (höftberger et al. 2020). thus, eae appears to be a very suitable model for mogad, but much less for ms. support of this view is provided in a recent study, on archival material from a patient, who died in the 1950s with an acute ms-like inflammatory demyelinating disease after misguided repeated immunization with brain tissue (höftberger et al. 2015). new molecular biology technologies allowed resurrection of a pathogenic auto-antibody from the archival formaldehyde fixed and paraffin embedded tissue, which was found to be directed against a conformational epitope of mog and induced demyelination after transfer in vivo (beltran et al. 2020). 4) is subpial demyelination a unique feature of multiple sclerosis pathology? for a long time, ms has been regarded a demyelinating disease affecting the white matter. however, with the availability of highly sensitive immunocytochemical methods, which allow staining of the very thin myelinated fibers within the grey matter (peterson et al. 2000), it became clear that demyelination in the grey matter in the ms brain is extensive (kutzelnigg et al. 2005) and may even give rise to cortico-spinal or pure cortical variants of the disease in a subset of patients (trapp et al. 2018). three types of cortical lesions have been identified: the cortico-subcortical lesions (type 1), the intracortical lesions (type 2), and the subpial lesions (type 3) (bo et al. 2003). the third type is most abundant in ms and is related to focal inflammatory aggregates in the leptomeninges (magliozzi et al. 2010; figure 2). several previous studies have indicated that subpial demyelinated lesions in the cortex may be specific for ms (moll et al. 2008, fischer et al. 2013), but this view was based on a rather limited sample of neuropathological conditions other than ms. this has now been changed in a very comprehensive study by junker et al. (2020), in which nearly every thinkable neuropathological condition has been investigated for cortical demyelination. interestingly, subpial demyelination was exclusively present in ms and not seen in any of the other conditions. the only key diagnosis missed in this case series was mogad. however, this was also specifically addressed in the systematic studies of mogad pathology outlined above (höftberger et al. 2020, takai et al. 2020), describing intracortical and cortico-subcortical lesions but absent subpial cortical lesions. in rare instances, these intracortical lesions can fuse and give rise to large focal confluent plaques of demyelination. thus, so far it seems that subpial cortical demyelination is a unique feature of ms pathology, but the evidence for its absence in mogad is thus far based on a rather small sample of cases. nevertheless, the presence of a subpial cortical lesion strongly supports a neuropathological diagnosis of ms. figure 2: patterns of cortical demyelination in ms and mogad: a) in ms, the dominant and most specific type of cortical demyelination is the subpial lesion. it is characterized by a band of demyelination spanning over several gyri and sulci (red lesions). the lesions are more extensive in the invaginations of the cortex. active cortical demyelination is associated with meningeal inflammation by t-cells (blue dots) and b-cells (red dots) and is characterized by a band of activated macrophages / microglia at the border between the cortical lesion and the surrounding normal appearing cortex (purple bands). b) in mogad, the dominant cortical pathology is the presence of small perivenous intracortical demyelinated lesions (red circles). they arise around small cortical veins and venules with inflammatory infiltrates. on rare occasions, such intracortical perivenous lesions may give rise to a focal confluent cortical lesion, which frequently also expands into the adjacent subcortical white matter. 5) what immune cells drive inflammation in multiple sclerosis? our understanding of the immune mechanisms driving inflammation in ms is largely biased by immunological studies of experimental autoimmune encephalomyelitis, which now turns out to have more relevance for mogad than for ms. key mechanisms of inflammation, defined from such eae studies, such as the key role of mhc class ii restricted t-cells responses, the involvement of cd4+ th17 cells, or the central role of gm-csf driven myeloid cell activation (schreiner and becher 2015) are not convincingly supported by recent therapeutic trials targeting the respective immunological pathways (baker et al. 2017). in contrast, most effective therapeutic success is seen with drugs that target t-cells and b-cells together or even b-cells alone. thus, it is still an unresolved question, as to what cells and immune mechanisms trigger or drive inflammation in ms. pathology can help clarify this issue by providing a concise account of the types of inflammatory cells seen in different stages in the evolution of ms lesions. until a few years ago, information about the composition of inflammatory infiltrates in ms was restricted to small studies performed on a limited number of patients and lesions. since 2017, however, there are now three large studies available, which performed a phenotyping of inflammatory cells in an overall sample of more than 200 ms patients and which included all stages of the disease (van nierop et al. 2017, machado santos et al. 2018, fransen et al. 2020). all studies reached a similar conclusion that the dominant lymphocytes in the ms lesions are tissue resident cd8+ effector memory t-cells (ttrm), while infiltration with cd4+ t-cells is sparse. this is the case in all types of ms, including fulminant acute ms, relapsing remitting ms as well as primary or secondary progressive ms and is the same in all activity stages of the lesions. in active lesions, a subset of cd8+ cells show focally and temporally restricted activation, and b-cells are much more numerous in early stages of the disease and in active lesions than in inactive lesions at late stages (machado santos et al. 2018). in a recent study, imaging mass cytometry was applied to the biopsy tissue of a single ms patient who suffered from a rebound exacerbation after cessation of natalizumab treatment (ramaglia et al. 2019). also, in this study, the infiltrates contained t-cells and b-cells, but the relative proportion of cd4+ t-cells was higher compared to that seen in the previous studies. however, activation of t-cells, visualized by the expression of the transcription factor nfat or proliferation markers, was seen only in cd8+ t-cells. besides their absolute numbers, the activation state of leukocytes is relevant for driving the inflammatory reaction. similarly as before (machado santos et al. 2018), the most frequent b-cell phenotypes were igg+ cd38+ plasmablasts. thus, overall, the pathological data are in line with the observed effects of anti-inflammatory treatments in ms patients. 6) what do the inflammatory cells in ms lesions recognize? an important open question is what antigen is recognized by the cd8+ tissue resident memory t-cells. since such t-cells acquire their phenotype and persist after effective clearance of their cognate antigen and become reactivated when the antigen re-appears, it is unlikely that these cells recognize a classical auto-antigen, which is present in the cns in excess all the time. in line with this view, no reactivity of cd8+ t-cells from ms brain lesions has been seen against the commonly tested myelin antigens (van nierop et al. 2017). several potential candidate antigens have recently been identified. since accumulating epidemiological evidence associates epstein barr virus infection with ms (bar-or et al. 2020, levin et al. 2010), one hypothesis is that b-cells with latent ebv infection are present within the cns of ms patients (serafini et al. 2007, veroni et al. 2018) together with ebv-reactive cd8+ tissue resident effector memory t-cells (serafini et al. 2020). here, the hypothesis is that inflammatory activity is triggered and propagated when the virus is activated in latently infected cells and recognized by the specific t-cells (serafini et al. 2020). as a note of caution, several other groups have tried to confirm the specific presence of ebv infected b-cells in the ms brain and lesions and have failed (lassmann et al. 2010, van nierop et al. 2017). the reason for these discrepancies is still unresolved. in a similar approach, an interaction between brain resident cd8+ cells and b-cells has been described, where the b-cells did not express ebv (van nierop et al. 2017). this suggests that the cells recognize a b-cell autoantigen. a possible mechanism for how such autoimmunity against b-cells may be induced is provided by jelcic et al. (2018). ms b-cells, possibly in relation to their ebv infection status, expand by auto-proliferation and present a b-cell specific auto-antigen to cd4+ t-cells, which also expand and infiltrate the brain and spinal cord. within the cns, these t-cells are activated by antigen recognition on infiltrating b-cells. in addition, the respective auto-antigen (rasgrp2) is also expressed in neurons, which may further propagate inflammation and tissue injury. whether this mechanism also accounts for activation of cd8+ t-cells, the dominant inflammatory cells in ms lesions, is unknown. a different approach has been followed by konjevic sabolek et al. (2019). in this study, the interaction of cd8+ t-cells with local target cells was identified by the polarized location of cytotoxic granules at the site of contact. when this was seen, the target cell was isolated and its nature was determined in gene expression studies and by immunohistochemistry. in this study, the only cells which interacted with cytotoxic cd8+ t-cells in ms lesions were myeloid cells, expressing markers of macrophages and microglia, suggesting these may harbor the target antigen(s). finally, other studies suggest that the target antigen recognized by t-cells in ms lesions may be the stress protein alpha b crystallin, which is in ms lesions most prominently expressed in active lesions (van noort et al. 2010). in summary, the new data from systematic phenotypical and functional studies on the inflammatory response within ms lesions provide groundbreaking new insights into the pathophysiology of the disease and question the concept of ms being an autoimmune disease against myelin. however, the results are in part very divergent and, to some extent, contradicting. whether this indicates heterogeneous disease mechanisms between individual patients or patient subgroups will be seen in the future. 7) which microglia or macrophage phenotypes are associated with tissue damage in the brain? microglia and recruited macrophages play a major role in the induction of tissue injury, not only in inflammatory brain lesions but also in neurodegenerative diseases. for a long time, it was difficult to distinguish between activated microglia and recruited macrophages within brain lesions. this has changed with the introduction of markers, which are selectively expressed on microglia, such as the membrane protein tmem 119 and the marker for homeostatic microglia p2ry12. these markers have been used in a number of studies on various different inflammatory and neurodegenerative conditions and revealed a surprisingly uniform reaction pattern of myeloid cells in human diseases (zrzavy et al. 2017, 2018, hayashida et al. 2020, jäckle et al. 2020). in essence, at sites of initial tissue injury, resident microglia become activated in a pro-inflammatory pattern. this initial step is followed by recruitment of myeloid cells from the circulation, and the recruited cells also show pro-inflammatory activation or, at later stages of lesion maturation, an intermediate phenotype. the distinction of proversus anti-inflammatory phenotypes is now seen in a much more critical way, since some pro-inflammatory actions, such as the interaction of macrophages with target cells may contribute to tissue damage but may also be essential for neuroprotection and regeneration through the clearance of debris (cignarella et al. 2020). an important pro-inflammatory type of activation, which seems to play a major role in the induction of tissue injury, is the expression of proteins involved in oxidative stress, such as the expression of the nox2 complex (nadph oxidase complex 2), a prominent marker of microglia expressed in active lesions in different inflammatory conditions as well as vascular or neurodegenerative diseases in humans (zrzavy et al. 2017, fischer et al. 2013). to define the patterns of microglia activation in such diseases may finally result in more selective and efficient neuroprotective treatments. this is now possible with new technologies, such as single-cell rna sequencing or immunocytochemical methods, which allow the simultaneous detection of multiple protein antigens within the same section, such as imaging mass cytometry. in some studies of models of autoimmune encephalomyelitis and ms, these techniques have been used and confirmed in an elegant way the patterns of microglia activation and macrophage recruitment, which have been described before with more conventional techniques as summarized above (masuda et al. 2019, ramaglia et al. 2020). however, they additionally showed that microglia and macrophages with different phenotypes are present side-by-side in the same lesion. this may indicate selective activation signals in specific subpopulations of cells. not surprisingly, the data showed that different ms lesions, which were in the same activity stage but still displayed some distinct morphological features, were infiltrated by different subsets of myeloid cells (masuda et al. 2019). however, these studies did not reveal the high expression of molecules involved in oxidative injury, which had been prominent in earlier more conventional neuropathological studies. a possible explanation for these discrepancies is provided by an elegant study, which specifically focused on microglia activation and oxidative injury in autoimmune encephalomyelitis (mendiola et al. 2020). the authors first defined the molecular phenotype of microglia specifically selected from areas of oxidative injury. incorporating this profile in the analysis of single-cell rna sequencing data and immunocytochemistry, they identified specific microglia and macrophage subpopulations, which trigger oxidative stress, and showed that these are selective subtypes of cells within the population of activated myeloid cells. these particular cell types also express molecules that are important in the coagulation cascade and in glutathione metabolism. in addition, they produce molecules, which counteract oxidative stress. one of these molecules acivivin, a glutathione regulating compound, suppresses the inflammatory tissue damage in a model of autoimmune encephalomyelitis. it will be instrumental in the future to validate directly the relevance of these findings in ms lesions and also in vascular or neurodegenerative diseases of the central nervous system. the presence of a subtype of myeloid cells triggering and resolving oxidative stress could reconcile the above discrepancies and provide a mechanism for the excessive oxidative injury in the lesions and the associations of markers for oxidative injury with markers for disturbed blood coagulation in the lesions. in the long run, these data may lead to tools for the therapeutic blockade of one of the most detrimental aspects of microglia activation in human brain disease. 8) how do vascular comorbidities influence multiple sclerosis patients? clinical epidemiology of ms has shown that patients with vascular risk factors, such as diabetes, hypercholesterolaemia, hypertension, or heart disease have a more aggressive disability progression in comparison to patients lacking these co-morbidities (marrie et al. 2010). this is also reflected by lower brain volumes (pichler et al. 2019). furthermore, persistence of inflammatory demyelinating lesions and higher lesion volumes are present in brain areas with a blood perfusion that is lower than in other brain areas (haider et al. 2016). such a synergism in neurodegeneration may in part be explained by shared effector mechanisms of tissue injury between vascular and inflammatory diseases, including microglia activation, oxidative injury, and mitochondrial damage (zrzavy et al. 2017, 2018, mahad et al. 2015). a strategically important piece in the puzzle of vascular comorbidities and ms, which was missing so far, was the lack of a comprehensive neuropathological description of systemic and intracranial vascular pathology in patients versus controls. this information has now been provided by a study, which is based on a unique archival collection of ms and control autopsy cases, where detailed information regarding systemic vascular diseases was recorded, and which was collected prior to the availability of disease modifying treatments (geraldes et al. 2020). the study shows, as expected, that systemic and intracranial vascular abnormalities increase with age in both the ms and the control group. young ms patients appear to have a moderate reduction of systemic vascular co-morbidities compared to controls, but this difference disappears with aging. within the central nervous system, ms patients showed a profound increase in small arteries with increased perivascular space, perivascular hemosiderin depositions and periarteriolar accumulation of inflammatory cells, a type of pathology which correlated in its extent with the degree of ms-related pathology. these results underline the presence of age-related vascular co-morbidities in the brain of ms patients, which may be an amplification factor for neurodegeneration and disease progression in aging patients. additionally, they indicate that, in contrast to the current view, vascular pathology is not restricted to veins but also affects small arteries, and this arterial pathology develops at least in part independently from systemic vascular risk factors. in particular, the mechanisms how inflammatory infiltrates accumulate around arteries in the ms brain requires further attention. 9) what is wrong with remyelination in multiple sclerosis? primary demyelination with preservation of axons is the pathological hallmark of inflammatory demyelinating diseases, such as ms. myelin allows saltatory conduction in axons and also protects the axons from neurodegeneration. thus, major efforts in ms research have been devoted to develop treatments which prevent demyelination or stimulate myelin repair (lubetzki et al. 2020). these repair strategies, mainly designed to provide oligodendrocyte progenitor cells in the lesions and to stimulate their differentiation into myelinating oligodendrocytes, have been developed and tested in experimental models of demyelinating disease and found to be quite effective. however, none of the strategies have yet resulted in clinically meaningful myelin repair in controlled clinical trials in ms patients. in experimental models of demyelinating disease, profound spontaneous remyelination is the rule, and remyelination stimulating therapies in essence accelerate myelin repair. although spontaneous remyelination may also occur in ms patients, in particular in fresh lesions at early disease stages, remyelination is, in general, sparse or absent. several different mechanisms have been suggested to be responsible for this remyelination failure in ms lesions. it may be due to a genuine defect in oligodendrocyte (progenitor) biology. in an elegant study, starost et al. (2020) approached this question by studying oligodendrocytes, induced from pluripotent stem cells from ms patients and controls. they show that there is no difference in the proliferation, differentiation and myelin production between such cells derived from ms patients and from controls, thus strongly suggesting that there is no genuine defect of oligodendrocytes in ms patients, though with the caveat that this was only performed in cells induced to form oligodendrocytes with expression of transcriptional factors from three ms patients and three controls. in a parallel neuropathological approach, the same group analyzed the patterns or remyelination in different disease stages of ms and in different stages of lesion formation (heß et al. 2020). they found that remyelination is highly efficient in a subset of active lesions in the early stage of the disease, and their data suggest that the remyelinating cells may be derived from mature oligodendrocytes that had survived active myelin destruction. this has also been suggested in recent studies, using radiocarbon methods to determine the time of birth of new oligodendrocytes in ms lesions (yeung et al. 2019). in contrast, in chronic active lesions at later stages of ms, very little remyelination was seen and the lack of remyelination was associated with pro-inflammatory activity of the local microglia population (heß et al. 2020). these data suggest that products of activated inflammatory cells in chronic ms lesions may not only lead to their slow expansion but also inhibit the repair of myelin. this finding is also supported by the other study (starost et al. 2020), which provides convincing evidence that pro-inflammatory mediators, in particular gamma-interferon, blocks the differentiation of inducible pluripotent stem cells into myelinating oligodendrocytes. the key importance of these studies is that they document that the process of remyelination failure in ms is complex and not fully reflected in the current experimental models of inflammatory demyelinating disease. thus, for test screening of remyelination-promoting therapies different systems have to be used, which reproduce the inflammation-associated remyelination failure. 10) what can we learn from down syndrome about inflammation and neurodegeneration in alzheimer’s disease? a large spectrum of data coming from experimental studies as well as from neuropathological and genetic investigations suggest an important role of innate immune mechanisms in the pathogenesis of alzheimer’s disease (akiyama et al. 2000). in particular, microglia phenotype and function are associated with progression of cognitive decline. however, information regarding the time course and type of microglia pathology especially in the pre-symptomatic phases of the disease is limited. analyzing down syndrome brain pathology of patients may provide some answers to these questions, since a gene dosage effect on amyloid precursor protein production predictably results in early onset alzheimer’s disease (wisniewski et al. 1985, ballard et al. 2016). this approach was followed in a recent study, in which the patterns of microglia activation and the expression of proand anti-inflammatory cytokines was analyzed in a large sample of brain tissue of patients with trisomy 21 who had died at different ages (flores aguilar et al. 2020). already, in juvenile patients, a long time before the first accumulation of soluble aß or the first deposition of aß-plaques, microglia activation and the production of proinflammatory cytokines was elevated in comparison to age matched controls. in contrast, in older patients, when initial alzheimer’s disease pathology became apparent, microglia activation decreased and was replaced by senescent microglia phenotypes as well as a reduction of the production of pro-inflammatory cytokines. these results further support the view that anti-inflammatory treatment strategies in alzheimer’s disease may be most beneficial when applied in very early (pre-symptomatic) stages of disease evolution. the data further suggest that the inclusion of patients with trisomy 21 in future clinical trials could provide a valuable contribution to improve treatment options in patients with alzheimer’s disease. conclusions neuroimmunology is a highly dynamic field providing fascinating new insights into the pathogenesis of brain inflammation and neurodegeneration. the bulk of data, however, describe experimental models and, thus, an additional step is necessary, which defines the relevance of the data for human disease. for this, neuropathological studies are essential, but they have to be based on systematic analysis of a broad spectrum of cases and controls and have to be performed with the most suitable molecular or immunological technology. in this short review the prime focus was laid on such studies of human disease, which were sufficiently powered to provide definite answers to burning questions of neuroinflammation and inflammatory brain diseases. references akiyama h, barger s, barnum s, bradt b, bauer j, cole gm, et al.. inflammation and alzheimer’s disease. neurobiol aging 2000; 21: 383–421. doi: 10.1016/s0197-4580(00)00124-x. baker d, marta m, pryce g, giovannoni g, schmierer k. memory b cells are major targets for effective immunotherapy in relapsing multiple sclerosis. ebiomedicine. 2017 feb;16:41-50. doi: 10.1016/j.ebiom.2017.01.042. epub 2017 jan 31. pmid: 28161400; pmcid: pmc5474520. ballard c, mobley w, hardy j, williams g, corbett a. dementia in down’s syndrome. lancet neurol 2016; 15: 622–36. doi: 10.1016/s1474-4422(16)00063-6. epub 2016 apr 11. bar-or a, pender mp, khanna r, steinman l, hartung hp, maniar t, croze e, aftab bt, giovannoni g, joshi ma. epstein-barr virus in multiple sclerosis: theory and emerging immunotherapies. trends mol med. 2020 mar;26(3):296-310. doi: 10.1016/j.molmed.2019.11.003. epub 2019 dec 17. pmid: 31862243; pmcid: pmc7106557. beltrán e, paunovic m, gebert d, cesur e, jeitler m, höftberger r, malotka j, mader s, kawakami n, meinl e, bradl m, dornmair k, lassmann h. archeological neuroimmunology: resurrection of a pathogenic immune response from a historical case sheds light on human autoimmune encephalomyelitis and multiple sclerosis. acta neuropathol. 2020 oct 29. doi: 10.1007/s00401-020-02239-2. epub ahead of print. pmid: 33242149. bø l, vedeler ca, nyland hi, trapp bd, mørk sj. subpial demyelination in the cerebral cortex of multiple sclerosis patients. j neuropathol exp neurol. 2003 jul;62(7):723-32. doi: 10.1093/jnen/62.7.723. pmid: 12901699. cantuti-castelvetri l, ojha r, pedro ld, djannatian m, franz j, kuivanen s, van der meer f, kallio k, kaya t, anastasina m, smura t, levanov l, szirovicza l, tobi a, kallio-kokko h, österlund p, joensuu m, meunier fa, butcher sj, winkler ms, mollenhauer b, helenius a, gokce o, teesalu t, hepojoki j, vapalahti o, stadelmann c, balistreri g, simons m. neuropilin-1 facilitates sars-cov-2 cell entry and infectivity. science. 2020 nov 13;370(6518):856-860. doi: 10.1126/science.abd2985. epub 2020 oct 20. pmid: 33082293. carceles-cordon m, mannara f, aguilar e, castellanos a, planagumà j, dalmau j. nmdar antibodies alter dopamine receptors and cause psychotic behavior in mice. ann neurol. 2020 sep;88(3):603-613. doi: 10.1002/ana.25829. epub 2020 jul 11. pmid: 32583480. cignarella f, filipello f, bollman b, cantoni c, locca a, mikesell r, manis m, ibrahim a, deng l, benitez ba, cruchaga c, licastro d, mihindukulasuriya k, harari o, buckland m, holtzman dm, rosenthal a, schwabe t, tassi i, piccio l. trem2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis. acta neuropathol. 2020 oct;140(4):513-534. doi: 10.1007/s00401-020-02193-z. epub 2020 aug 9. pmid: 32772264; pmcid: pmc7498497. deigendesch n, sironi l, kutza m, wischnewski s, fuchs v, hench j, frank a, nienhold r, mertz kd, cathomas g, matter ms, siegemund m, tolnay m, schirmer l, pröbstel ak, tzankov a, frank s. correlates of critical illness-related encephalopathy predominate postmortem covid-19 neuropathology. acta neuropathol. 2020 oct;140(4):583-586. doi: 10.1007/s00401-020-02213-y. epub 2020 aug 26. pmid: 32851506; pmcid: pmc7449525. fischer mt, wimmer i, höftberger r, gerlach s, haider l, zrzavy t, hametner s, mahad d, binder cj, krumbholz m, bauer j, bradl m, lassmann h. disease-specific molecular events in cortical multiple sclerosis lesions. brain. 2013 jun;136(pt 6):1799-815. doi: 10.1093/brain/awt110. epub 2013 may 17. pmid: 23687122; pmcid: pmc3673462. flores-aguilar l, iulita mf, kovecses o, torres md, levi sm, zhang y, askenazi m, wisniewski t, busciglio j, cuello ac. evolution of neuroinflammation across the lifespan of individuals with down syndrome. brain. 2020 nov 18:awaa326. doi: 10.1093/brain/awaa326. epub ahead of print. pmid: 33206953. fransen nl, hsiao cc, van der poel m, engelenburg hj, verdaasdonk k, vincenten mcj, remmerswaal ebm, kuhlmann t, mason mrj, hamann j, smolders j, huitinga i. tissue-resident memory t cells invade the brain parenchyma in multiple sclerosis white matter lesions. brain. 2020 jun 1;143(6):1714-1730. doi: 10.1093/brain/awaa117. pmid: 32400866. fujihara k, cook lj. neuromyelitis optica spectrum disorders and myelin oligodendrocyte glycoprotein antibody-associated disease: current topics. curr opin neurol. 2020 jun;33(3):300-308. doi: 10.1097/wco.0000000000000828. pmid: 32374571. geraldes r, esiri mm, perera r, yee sa, jenkins d, palace j, deluca gc. vascular disease and multiple sclerosis: a post-mortem study exploring their relationships. brain. 2020 oct 1;143(10):2998-3012. doi: 10.1093/brain/awaa255. pmid: 32875311. haider l, zrzavy t, hametner s, höftberger r, bagnato f, grabner g, trattnig s, pfeifenbring s, brück w, lassmann h. the topograpy of demyelination and neurodegeneration in the multiple sclerosis brain. brain. 2016 mar;139(pt 3):807-15. doi: 10.1093/brain/awv398. epub 2016 feb 8. pmid: 26912645; pmcid: pmc4766379. hayashida s, masaki k, suzuki so, yamasaki r, watanabe m, koyama s, isobe n, matsushita t, takahashi k, tabira t, iwaki t, kira ji. distinct microglial and macrophage distribution patterns in the concentric and lamellar lesions in baló's disease and neuromyelitis optica spectrum disorders. brain pathol. 2020 sep 9. doi: 10.1111/bpa.12898. epub ahead of print. pmid: 32902014. hernández-fernández f, sandoval valencia h, barbella-aponte ra, collado-jiménez r, ayo-martín ó, barrena c, molina-nuevo jd, garcía-garcía j, lozano-setién e, alcahut-rodriguez c, martínez-martín á, sánchez-lópez a, segura t. cerebrovascular disease in patients with covid-19: neuroimaging, histological and clinical description. brain. 2020 oct 1;143(10):3089-3103. doi: 10.1093/brain/awaa239. pmid: 32645151; pmcid: pmc7454411. heß k, starost l, kieran nw, thomas c, vincenten mcj, antel j, martino g, huitinga i, healy l, kuhlmann t. lesion stage-dependent causes for impaired remyelination in ms. acta neuropathol. 2020 sep;140(3):359-375. doi: 10.1007/s00401-020-02189-9. epub 2020 jul 24. pmid: 32710244; pmcid: pmc7424408. höftberger r, guo y, flanagan ep, lopez-chiriboga as, endmayr v, hochmeister s, joldic d, pittock sj, tillema jm, gorman m, lassmann h, lucchinetti cf. the pathology of central nervous system inflammatory demyelinating disease accompanying myelin oligodendrocyte glycoprotein autoantibody. acta neuropathol. 2020 may;139(5):875-892. doi: 10.1007/s00401-020-02132-y. epub 2020 feb 11. pmid: 32048003; pmcid: pmc7181560. höftberger r, lassmann h. immune-mediated disorders. handb clin neurol. 2017;145:285-299. doi: 10.1016/b978-0-12-802395-2.00020-1. pmid: 28987176. höftberger r, leisser m, bauer j, lassmann h. autoimmune encephalitis in humans: how closely does it reflect multiple sclerosis ? acta neuropathol commun. 2015 dec 4;3:80. doi: 10.1186/s40478-015-0260-9. pmid: 26637427; pmcid: pmc4670499. jäckle k, zeis t, schaeren-wiemers n, junker a, van der meer f, kramann n, stadelmann c, brück w. molecular signature of slowly expanding lesions in progressive multiple sclerosis. brain. 2020 jul 1;143(7):2073-2088. doi: 10.1093/brain/awaa158. pmid: 32577755. jelcic i, al nimer f, wang j, lentsch v, planas r, jelcic i, madjovski a, ruhrmann s, faigle w, frauenknecht k, pinilla c, santos r, hammer c, ortiz y, opitz l, grönlund h, rogler g, boyman o, reynolds r, lutterotti a, khademi m, olsson t, piehl f, sospedra m, martin r. memory b cells activate brain-homing, autoreactive cd4+ t cells in multiple sclerosis. cell. 2018 sep 20;175(1):85-100.e23. doi: 10.1016/j.cell.2018.08.011. epub 2018 aug 30. pmid: 30173916; pmcid: pmc6191934. jensen mp, le quesne j, officer-jones l, teodòsio a, thaventhiran j, ficken c, goddard m, smith c, menon d, allinson ksj. neuropathological findings in two patients with fatal covid-19. neuropathol appl neurobiol. 2020 sep 8. doi: 10.1111/nan.12662. epub ahead of print. pmid: 32895961. junker a, wozniak j, voigt d, scheidt u, antel j, wegner c, brück w, stadelmann c. extensive subpial cortical demyelination is specific to multiple sclerosis. brain pathol. 2020 may;30(3):641-652. doi: 10.1111/bpa.12813. epub 2020 feb 3. pmid: 31916298. kabbani n, olds jl. does covid19 infect the brain? if so, smokers might be at a higher risk. mol pharmacol. 2020 may;97(5):351-353. doi: 10.1124/molpharm.120.000014. epub 2020 apr 1. pmid: 32238438; pmcid: pmc7237865. kantonen j, mahzabin s, mäyränpää mi, tynninen o, paetau a, andersson n, sajantila a, vapalahti o, carpén o, kekäläinen e, kantele a, myllykangas l. neuropathologic features of four autopsied covid-19 patients. brain pathol. 2020 aug 6:10.1111/bpa.12889. doi: 10.1111/bpa.12889. epub ahead of print. pmid: 32762083; pmcid: pmc7436498. kanwar d, baig am, wasay m. neurological manifestations of covid-19. j pak med assoc. 2020 may;70(suppl 3)(5):s101-s103. doi: 10.5455/jpma.20. pmid: 32515379. konjevic sabolek m, held k, beltrán e, niedl ag, meinl e, hohlfeld r, lassmann h, dornmair k. communication of cd8+ t cells with mononuclear phagocytes in multiple sclerosis. ann clin transl neurol. 2019 jul;6(7):1151-1164. doi: 10.1002/acn3.783. epub 2019 jun 14. pmid: 31353869; pmcid: pmc6649540. kutzelnigg a, lucchinetti cf, stadelmann c, brück w, rauschka h, bergmann m, schmidbauer m, parisi je, lassmann h. cortical demyelination and diffuse white matter injury in multiple sclerosis. brain. 2005 nov;128(pt 11):2705-12. doi: 10.1093/brain/awh641. epub 2005 oct 17. pmid: 16230320. landa j, gaig c, plagumà j, saiz a, antonell a, sanchez-valle r, dalmau j, graus f, sabater l. effects of iglon5 antibodies on neuronal cytoskeleton: a link between autoimmunity and neurodegeneration. ann neurol. 2020 nov;88(5):1023-1027. doi: 10.1002/ana.25857. epub 2020 aug 27. pmid: 32740999. lassmann h, niedobitek g, aloisi f, middeldorp jm; neuropromise ebv working group. epstein-barr virus in the multiple sclerosis brain: a controversial issue--report on a focused workshop held in the centre for brain research of the medical university of vienna, austria. brain. 2011 sep;134(pt 9):2772-86. doi: 10.1093/brain/awr197. epub 2011 aug 16. pmid: 21846731; pmcid: pmc3170536. levin li, munger kl, o'reilly ej, falk ki, ascherio a. primary infection with the epstein-barr virus and risk of multiple sclerosis. ann neurol. 2010 jun;67(6):824-30. doi: 10.1002/ana.21978. pmid: 20517945; pmcid: pmc3089959. li yc, bai wz, hashikawa t. the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients. j med virol. 2020 jun;92(6):552-555. doi: 10.1002/jmv.25728. epub 2020 mar 11. pmid: 32104915; pmcid: pmc7228394. liu jm, tan bh, wu s, gui y, suo jl, li yc. evidence of central nervous system infection and neuroinvasive routes, as well as neurological involvement, in the lethality of sars-cov-2 infection. j med virol. 2020 oct 1:10.1002/jmv.26570. doi: 10.1002/jmv.26570. epub ahead of print. pmid: 33002209; pmcid: pmc7537172. lubetzki c, zalc b, williams a, stadelmann c, stankoff b. remyelination in multiple sclerosis: from basic science to clinical translation. lancet neurol. 2020 aug;19(8):678-688. doi: 10.1016/s1474-4422(20)30140-x. pmid: 32702337. machado-santos j, saji e, tröscher ar, paunovic m, liblau r, gabriely g, bien cg, bauer j, lassmann h. the compartmentalized inflammatory response in the multiple sclerosis brain is composed of tissue-resident cd8+ t lymphocytes and b cells. brain. 2018 jul 1;141(7):2066-2082. doi: 10.1093/brain/awy151. pmid: 29873694; pmcid: pmc6022681. magliozzi r, howell ow, reeves c, roncaroli f, nicholas r, serafini b, aloisi f, reynolds r. a gradient of neuronal loss and meningeal inflammation in multiple sclerosis. ann neurol. 2010 oct;68(4):477-93. doi: 10.1002/ana.22230. pmid: 20976767. mahad dh, trapp bd, lassmann h. pathological mechanisms in progressive multiple sclerosis. lancet neurol. 2015 feb;14(2):183-93. doi: 10.1016/s1474-4422(14)70256-x. pmid: 25772897. marrie ra, rudick r, horwitz r, cutter g, tyry t, campagnolo d, vollmer t. vascular comorbidity is associated with more rapid disability progression in multiple sclerosis. neurology. 2010 mar 30;74(13):1041-7. doi: 10.1212/wnl.0b013e3181d6b125. pmid: 20350978; pmcid: pmc2848107. masuda t, sankowski r, staszewski o, böttcher c, amann l, sagar, scheiwe c, nessler s, kunz p, van loo g, coenen va, reinacher pc, michel a, sure u, gold r, grün d, priller j, stadelmann c, prinz m. spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution. nature. 2019 feb;566(7744):388-392. doi: 10.1038/s41586-019-0924-x. epub 2019 feb 13. erratum in: nature. 2019 apr;568(7751):e4. pmid: 30760929. matschke j, lütgehetmann m, hagel c, sperhake jp, schröder as, edler c, mushumba h, fitzek a, allweiss l, dandri m, dottermusch m, heinemann a, pfefferle s, schwabenland m, sumner magruder d, bonn s, prinz m, gerloff c, püschel k, krasemann s, aepfelbacher m, glatzel m. neuropathology of patients with covid-19 in germany: a post-mortem case series. lancet neurol. 2020 nov;19(11):919-929. doi: 10.1016/s1474-4422(20)30308-2. epub 2020 oct 5. pmid: 33031735; pmcid: pmc7535629. matute c, palma a, serrano-regal mp, maudes e, barman s, sánchez-gómez mv, domercq m, goebels n, dalmau j. n-methyl-d-aspartate receptor antibodies in autoimmune encephalopathy alter oligodendrocyte function. ann neurol. 2020 may;87(5):670-676. doi: 10.1002/ana.25699. epub 2020 feb 24. pmid: 32052483. meinhardt j, radke j, dittmayer c, franz j, thomas c, mothes r, laue m, schneider j, brünink s, greuel s, lehmann m, hassan o, aschman t, schumann e, chua rl, conrad c, eils r, stenzel w, windgassen m, rößler l, goebel hh, gelderblom hr, martin h, nitsche a, schulz-schaeffer wj, hakroush s, winkler ms, tampe b, scheibe f, körtvélyessy p, reinhold d, siegmund b, kühl aa, elezkurtaj s, horst d, oesterhelweg l, tsokos m, ingold-heppner b, stadelmann c, drosten c, corman vm, radbruch h, heppner fl. olfactory transmucosal sars-cov-2 invasion as a port of central nervous system entry in individuals with covid-19. nat neurosci. 2020 nov 30. doi: 10.1038/s41593-020-00758-5. epub ahead of print. pmid: 33257876. mendiola as, ryu jk, bardehle s, meyer-franke a, ang kk, wilson c, baeten km, hanspers k, merlini m, thomas s, petersen ma, williams a, thomas r, rafalski va, meza-acevedo r, tognatta r, yan z, pfaff sj, machado mr, bedard c, rios coronado pe, jiang x, wang j, pleiss ma, green aj, zamvil ss, pico ar, bruneau bg, arkin mr, akassoglou k. transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation. nat immunol. 2020 may;21(5):513-524. doi: 10.1038/s41590-020-0654-0. epub 2020 apr 13. erratum in: nat immunol. 2020 sep;21(9):1135. pmid: 32284594; pmcid: pmc7523413. moll nm, rietsch am, ransohoff aj, cossoy mb, huang d, eichler fs, trapp bd, ransohoff rm. cortical demyelination in pml and ms: similarities and differences. neurology. 2008 jan 29;70(5):336-43. doi: 10.1212/01.wnl.0000284601.54436.e4. pmid: 17914063. paterson rw, brown rl, benjamin l, nortley r, wiethoff s, bharucha t, jayaseelan dl, kumar g, raftopoulos re, zambreanu l, vivekanandam v, khoo a, geraldes r, chinthapalli k, boyd e, tuzlali h, price g, christofi g, morrow j, mcnamara p, mcloughlin b, lim st, mehta pr, levee v, keddie s, yong w, trip sa, foulkes ajm, hotton g, miller td, everitt ad, carswell c, davies nws, yoong m, attwell d, sreedharan j, silber e, schott jm, chandratheva a, perry rj, simister r, checkley a, longley n, farmer sf, carletti f, houlihan c, thom m, lunn mp, spillane j, howard r, vincent a, werring dj, hoskote c, jäger hr, manji h, zandi ms. the emerging spectrum of covid-19 neurology: clinical, radiological and laboratory findings. brain. 2020 oct 1;143(10):3104-3120. doi: 10.1093/brain/awaa240. pmid: 32637987; pmcid: pmc7454352. peterson jw, bö l, mörk s, chang a, trapp bd. transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions. ann neurol. 2001 sep;50(3):389-400. doi: 10.1002/ana.1123. pmid: 11558796. pichler a, khalil m, langkammer c, pinter d, ropele s, fuchs s, bachmaier g, enzinger c, fazekas f. the impact of vascular risk factors on brain volume and lesion load in patients with early multiple sclerosis. mult scler. 2019 jan;25(1):48-54. doi: 10.1177/1352458517736149. epub 2017 oct 13. pmid: 29027843. pitsch j, kamalizade d, braun a, kuehn jc, gulakova pe, rüber t, lubec g, dietrich d, von wrede r, helmstaedter c, surges r, elger ce, hattingen e, vatter h, schoch s, becker aj. drebrin autoantibodies in patients with seizures and suspected encephalitis. ann neurol. 2020 jun;87(6):869-884. doi: 10.1002/ana.25720. epub 2020 apr 10. pmid: 32196746. ramaglia v, sheikh-mohamed s, legg k, park c, rojas ol, zandee s, fu f, ornatsky o, swanson ec, pitt d, prat a, mckee td, gommerman jl. multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry. elife. 2019 aug 1;8:e48051. doi: 10.7554/elife.48051. pmid: 31368890; pmcid: pmc6707785. rauschenberger v, von wardenburg n, schaefer n, ogino k, hirata h, lillesaar c, kluck cj, meinck hm, borrmann m, weishaupt a, doppler k, wickel j, geis c, sommer c, villmann c. glycine receptor autoantibodies impair receptor function and induce motor dysfunction. ann neurol. 2020 sep;88(3):544-561. doi: 10.1002/ana.25832. epub 2020 jul 20. pmid: 32588476. reichard rr, kashani kb, boire na, constantopoulos e, guo y, lucchinetti cf. neuropathology of covid-19: a spectrum of vascular and acute disseminated encephalomyelitis (adem)-like pathology. acta neuropathol. 2020 jul;140(1):1-6. doi: 10.1007/s00401-020-02166-2. epub 2020 may 24. pmid: 32449057; pmcid: pmc7245994. reindl m, waters p. myelin oligodendrocyte glycoprotein antibodies in neurological disease. nat rev neurol. 2019 feb;15(2):89-102. doi: 10.1038/s41582-018-0112-x. pmid: 30559466. schreiner b, becher b. perspectives on cytokine-directed therapies in multiple sclerosis. swiss med wkly. 2015 oct 23;145:w14199. doi: 10.4414/smw.2015.14199. pmid: 26495801. sechi e, markovic sn, mckeon a, dubey d, liewluck t, lennon va, lopez-chiriboga as, klein cj, mauermann m, pittock sj, flanagan ep, zekeridou a. neurologic autoimmunity and immune checkpoint inhibitors: autoantibody profiles and outcomes. neurology. 2020 oct 27;95(17):e2442-e2452. doi: 10.1212/wnl.0000000000010632. epub 2020 aug 13. pmid: 32796130; pmcid: pmc7682911. serafini b, rosicarelli b, franciotta d, magliozzi r, reynolds r, cinque p, andreoni l, trivedi p, salvetti m, faggioni a, aloisi f. dysregulated epstein-barr virus infection in the multiple sclerosis brain. j exp med. 2007 nov 26;204(12):2899-912. doi: 10.1084/jem.20071030. epub 2007 nov 5. pmid: 17984305; pmcid: pmc2118531. serafini b, rosicarelli b, veroni c, mazzola ga, aloisi f. epstein-barr virus-specific cd8 t cells selectively infiltrate the brain in multiple sclerosis and interact locally with virus-infected cells: clue for a virus-driven immunopathological mechanism. j virol. 2019 nov 26;93(24):e00980-19. doi: 10.1128/jvi.00980-19. pmid: 31578295; pmcid: pmc6880158. starost l, lindner m, herold m, xu ykt, drexler hca, heß k, ehrlich m, ottoboni l, ruffini f, stehling m, röpke a, thomas c, schöler hr, antel j, winkler j, martino g, klotz l, kuhlmann t. extrinsic immune cell-derived, but not intrinsic oligodendroglial factors contribute to oligodendroglial differentiation block in multiple sclerosis. acta neuropathol. 2020 nov;140(5):715-736. doi: 10.1007/s00401-020-02217-8. epub 2020 sep 7. pmid: 32894330; pmcid: pmc7547031. takai y, misu t, kaneko k, chihara n, narikawa k, tsuchida s, nishida h, komori t, seki m, komatsu t, nakamagoe k, ikeda t, yoshida m, takahashi t, ono h, nishiyama s, kuroda h, nakashima i, suzuki h, bradl m, lassmann h, fujihara k, aoki m; japan mog-antibody disease consortium. myelin oligodendrocyte glycoprotein antibody-associated disease: an immunopathological study. brain. 2020 may 1;143(5):1431-1446. doi: 10.1093/brain/awaa102. pmid: 32412053. trapp bd, vignos m, dudman j, chang a, fisher e, staugaitis sm, battapady h, mork s, ontaneda d, jones se, fox rj, chen j, nakamura k, rudick ra. cortical neuronal densities and cerebral white matter demyelination in multiple sclerosis: a retrospective study. lancet neurol. 2018 oct;17(10):870-884. doi: 10.1016/s1474-4422(18)30245-x. epub 2018 aug 22. pmid: 30143361; pmcid: pmc6197820. van nierop gp, van luijn mm, michels ss, melief mj, janssen m, langerak aw, ouwendijk wjd, hintzen rq, verjans gmgm. phenotypic and functional characterization of t cells in white matter lesions of multiple sclerosis patients. acta neuropathol. 2017 sep;134(3):383-401. doi: 10.1007/s00401-017-1744-4. epub 2017 jun 17. pmid: 28624961; pmcid: pmc5563341. van noort jm, bsibsi m, gerritsen wh, van der valk p, bajramovic jj, steinman l, amor s. alphab-crystallin is a target for adaptive immune responses and a trigger of innate responses in preactive multiple sclerosis lesions. j neuropathol exp neurol. 2010 jul;69(7):694-703. doi: 10.1097/nen.0b013e3181e4939c. pmid: 20535035. veroni c, serafini b, rosicarelli b, fagnani c, aloisi f. transcriptional profile and epstein-barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis. j neuroinflammation. 2018 jan 16;15(1):18. doi: 10.1186/s12974-017-1049-5. pmid: 29338732; pmcid: pmc5771146. wisniewski ke, wisniewski hm, wen gy. occurrence of neuropathological changes and dementia of alzheimer’s disease in down’s syndrome. ann neurol 1985; 17: 278–82. doi: 10.1002/ana.410170310. yeung msy, djelloul m, steiner e, bernard s, salehpour m, possnert g, brundin l, frisén j. dynamics of oligodendrocyte generation in multiple sclerosis. nature. 2019 feb;566(7745):538-542. doi: 10.1038/s41586-018-0842-3. epub 2019 jan 23. erratum in: nature. 2019 feb 5;: pmid: 30675058; pmcid: pmc6420067. younger ds. postmortem neuropathology in covid-19. brain pathol. 2020 oct 23:e12915. doi: 10.1111/bpa.12915. epub ahead of print. pmid: 33098141; pmcid: pmc7645938. zrzavy t, hametner s, wimmer i, butovsky o, weiner hl, lassmann h. loss of 'homeostatic' microglia and patterns of their activation in active multiple sclerosis. brain. 2017 jul 1;140(7):1900-1913. doi: 10.1093/brain/awx113. pmid: 28541408; pmcid: pmc6057548. zrzavy t, machado-santos j, christine s, baumgartner c, weiner hl, butovsky o, lassmann h. dominant role of microglial and macrophage innate immune responses in human ischemic infarcts. brain pathol. 2018 nov;28(6):791-805. doi: 10.1111/bpa.12583. epub 2017 dec 28. pmid: 29222823; pmcid: pmc6334527. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. treatment of autoimmune encephalomyelitis with a histone deacetylase inhibitor: analyzing the role of immune-response genes feel free to add comments by clicking these icons on the sidebar free neuropathology 1:19 (2020) original paper treatment of autoimmune encephalomyelitis with a histone deacetylase inhibitor analyzing the role of immune-response genes arathi k. jayaraman a, karen avgush a, rashad kulam a, advait soni a, areeb khan a, mourad kerdjoudj a, sundararajan jayaraman a,b a dept. of microbiology & immunology, the university of illinois at chicago, 909 s wolcott avenue, chicago, il 60612, usa b dept. of surgery, the university of illinois college of medicine at peoria, il 61603, usa corresponding author: sundararajan jayaraman · dept. of surgery · the university of illinois college of medicine at peoria · il 61603 · usa anue2468@uic.edu submitted: 28 may 2020 accepted: 08 july 2020 copyedited by: laura peferoen published: 14 july 2020 https://doi.org/10.17879/freeneuropathology-2020-2819 additional resources and electronic supplementary material: supplementary material keywords: autoreactive t cells, epigenetic regulation, gene expression, experimental autoimmune encephalomyelitis, trichostatin a, multiple sclerosis abstract we have previously shown that treatment of female nod mice with a potent nonselective histone deacetylase inhibitor attenuated experimental autoimmune encephalomyelitis, a model for progressive multiple sclerosis. herein we show that immunization with the mog35-55 peptide induced prolonged upregulation of genes encoding interleukin 17a (il-17a), aryl hydrocarbon receptor, and histone deacetylase 11 in the spinal cord whereas the subunits of il-27, il-27p28 and il-27ebi3 were significantly increased in secondary lymphoid organs after a lag period. interestingly, the nitric oxide synthase gene was prominently expressed in both of these anatomic compartments following immunization. treatment with the histone modifier repressed the transcription of all of these genes induced by immunization. moreover, the drug suppressed the steady-state levels of the migration inhibitory factor and cd274 genes in both the spinal cord and peripheral lymphoid tissues. at the same time, the cd39 gene was downregulated only in secondary lymphoid organs. paradoxically, the epigenetic drug enhanced the expression of declin-1 in the spinal cord, suggesting a protective role in neuronal disease. immunization profoundly enhanced transcription of the chemokine ccl2 in the secondary lymphoid tissues without a corresponding increase in the translation of ccl2 protein. histone hyperacetylation neither altered the transcription of ccl2 nor its cognate receptor ccr2 in the central nervous system and peripheral lymphoid tissues. surprisingly, the drug did not exert modulatory influence on most other immune response-related genes previously implicated in encephalomyelitis. nevertheless, our data uncover several potential molecular targets for the intervention of experimental autoimmune encephalomyelitis that have implications for the treatment of progressive multiple sclerosis. 1. introduction multiple sclerosis (ms), an inflammatory disease of the central nervous system (cns), manifests commonly as the relapsing-remitting disease. some patients develop primary and secondary progressive forms of disease (dendrou et al., 2015; lassmann, 2017). the term ‘progressive multiple sclerosis’ has been proposed to encompass the primary and secondary progressive multiple sclerosis since there are more similarities than differences between them (fox and chataway, 2017). although many drugs are effective in reducing relapses, they failed to reverse axonal degeneration and are sometimes associated with adverse side effects, including progressive multifocal leukoencephalopathy (wingerchuk and carter, 2014; tintore et al., 2019). thus, effective drugs for the treatment of progressive multiple sclerosis remain an unmet need. since the early description of the experimental autoimmune encephalomyelitis (eae) in monkeys (rivers et al., 1933), monophasic, self-resolving, ‘classic’ eae, as well as ‘atypical’ eae, have been induced in rats and mice using whole spinal cord homogenates or peptides derived from the cns components such as myelin oligodendrocyte glycoprotein (mog), myelin basic protein (mbp) and proteolipid protein (plp)(dendrou et al., 2015; lassmann and bradl, 2017). autoimmune-prone female non-obese diabetic (nod) mice immunized with the mog35-55 peptide consistently induced severe and long-lasting progressive eae (peae) in 100% of animals characterized by paralysis of fore and hind limbs with (slavin et al., 1998; hidaka et al., 2014; dang et al., 2015) or without discernible remissions (basso et al., 2008; jayaraman et al., 2017; 2018). regardless, peae induced in nod mice mimics features of progressive ms including the life-long disease, prominent demyelination, axonal loss, and astrogliosis (slavin et al., 1998; basso et al., 2008; hidaka et al., 2014; dang et al., 2015; jayaraman et al., 2017; 2018), and hence is ideal for testing the efficacy of new drugs to treat progressive ms. the eae model is amenable for the investigation of the genes critically involved in ms pathogenesis. previous studies analyzed the roles of various immune response-related genes in eae using gene knockout mice and specific neutralizing antibodies in wild-type mice. these studies investigated the role of t helper 1 (th1), th17, and th1/th17 (th17.1) subsets as well as lymphokines such as interferon-γ (ifnγ) (ferber et al., 1996; okuda et al., 1998; hidaka et al., 2014), interleukin-4 (il-4) (falcone et al., 1998; okuda et al., 1998; ponomarev et al., 2007), il-17a (evangelidou et al., 2014), il-10, colony-stimulating factor 1 (borjini et al., 2016), il-22 (kreymborg et al., 2007), transforming growth factor-β (tgf-β (okuda et al., 1998) and il-27 (li et al., 2005) in eae. similarly, the importance of the transcription factors t-bet (o’connor et al., 2013), gata-3 (fernando et al., 2014), and rorγt (martinez et al., 2014) respectively, involved in the transcription of ifn-γ, il-4, and il-17a, was also studied in eae models. moreover, the factors required for the induction of th1 cells such as il-12 (gran et al., 2004), il-18 (lalor et al., 2011), and il-23 (kreymborg et al., 2007; el-behi et al., 2011) critical for the generation of th17 cells were implicated in eae. the roles of the pluripotent cytokines, namely the tumor necrosis factor-α (tnf-α) (hidaka et al., 2014; borjini et al., 2016) and granulocyte-macrophage-colony-stimulating factor (gm-csf/csf2) (mcqualter et al., 2001) were also studied in eae. in addition to the adaptive immune system, the innate immune cells such as neutrophils (jayaraman et al., 2018), and the cns-resident microglia and astrocytes (tran et al., 1997) appear to contribute to eae. interestingly, the first described and phylogenetically conserved pluripotent cytokine, the migration inhibitory factor (mif) (jayaraman and muthukkaruppan, 1977), has been implicated in ms (niino et al., 2000) and eae (powell et al., 2005). other determinants critical for eae include the orphan receptor, aryl hydrocarbon receptor (ahr) (nakahama et al., 2017), the transcription factors eomesodermin (eomes) (raveney et al., 2015) and declin-1 (dec1/bhlhe40) (lin et al., 2016), the immunoregulatory molecule cd39 (mascanfroni et al., 2013), matrix metalloproteinases (mmp) (kandagaddala et al., 2012; rempe et al., 2016), and the chemokine ccl2 (mahad et al., 2003; moreno et al., 2014; hidaka et al., 2014). the caveat is the lack of consensus regarding the critical gene(s) involved in eae. variations including the genetic background of experimental animals (rats and mice), types of eae models studied (short-term, self-resolving ‘classic’ eae, ‘atypical’ eae, relapsing-remitting eae, and peae), the immunogens used for eae elicitation (whole spinal cord homogenates, peptides derived from mog, mbp and plp), tissues (cns vs. peripheral lymphoid cells) and the time points (peak vs. chronic phase) examined can contribute to the uncertainty of the results. hence, investigation of the roles of these various immune response-related genes in a well-characterized model is likely to yield crucial information on the impact of these genes on neurodegeneration. our previous work demonstrated that the treatment of mog35-55 immunized female nod mice with the most potent histone deacetylase (hdac) inhibitor trichostatin a (tsa) (de ruijter et al., 2003) improved the clinical symptoms of peae (jayaraman et al., 2017). protection from peae was accompanied by histone h3 hyperacetylation in the spinal cord (sc) and spleen, reduced influx of t cells, and neutrophils into the cns as well as diminished axonal damage of the neurons in the cns (jayaraman et al., 2017; 2018). to gain insights into the roles of various genes in the peae model, we studied the expression profiles of 41 genes encoding lymphokines, transcription factors, accessory cell-associated determinants, and chemokines in the cns and secondary lymphoid organs (slo) longitudinally during the prolonged course of the disease (27-weeks). surprisingly, only a small set of mostly non-overlapping genes were differentially upregulated in the cns and slo, which were substantially repressed by tsa treatment, indicating their possible roles in peae. these data suggest that similar perturbation of the epigenome of ms patients may facilitate the identification of molecular targets for the development of novel drugs to treat this debilitating disease. 2. material and methods 2.1. eae induction and treatment this study was approved by the institutional animal care and use committee of the university of illinois at chicago and conducted according to the national institutes of health guide for the care and use of laboratory animals (nih publications no. 8023, revised 1978). female nod/shiltj mice were purchased from the jackson laboratories (bar harbor, me) and immunized subcutaneously (s.c) on the flank with 100 µg of mouse mog35-55 peptide (tocris bioscience) emulsified in complete freund's adjuvant and pertussis toxin was administered intravenously (jayaraman et al., 2017; 2018). randomly chosen littermates were injected s.c on the flank with 500 µg of tsa (sigma chemical company, st. louis, mo) per kg body weight three times a week. controls received the same amount of the vehicle, dimethyl sulfoxide (dmso) (sigma) diluted in phosphate-buffered saline (pbs). the body weight, blood glucose levels, (jayaraman et al., 2010; 2013; jayaraman and jayaraman, 2018; patel et al., 2011) and clinical scores (jayaraman et al., 2017; 2018) were recorded three times a week. the eae score was assigned as follows: 0, healthy, 1, limp tail, 2, one hind limb weakness, 3, both hind limb weakness, 4, forelimb weakness, 5, paralysis, moribund or death (jayaraman et al., 2017; 2018). five mice per group were chosen based on our previous investigations (jayaraman et al., 2017; 2018). the data are presented as the mean ± sem for each time point of observation. 2.2. gene expression analysis we analyzed the expression levels of 41 genes in the entire sc and slo (spleen and the draining inguinal, popliteal, axillary and cervical lymph nodes) of mice that were immunized with mog35-55 and treated with dmso or tsa. we have investigated 60 mice (five mice treated with dmso and five mice with tsa at six-time points) for the expression of genes in the sc and slo concurrently. to analyze the effect of the drug on the basal level of gene transcription regardless of immunization, we treated separate groups of 10 unimmunized mice with tsa or dmso and analyzed one day later since tsa acts within hours of treatment (van lint et al., 1996; de ruijter et al., 2003). these data are indicated at the day 1-time point in all figures. on the other hand, treatment groups received dmso or tsa starting from the day of immunization. mice were perfused with pbs before the spinal cord was extracted to avoid peripheral blood contamination (jayaraman et al., 2017; 2018). total rna was isolated from individual mice using trizol (invitrogen, carlsbad, ca). since preliminary experiments indicated similar levels of expression of genes among different mice in each group, the rna from five mice per group at each time point was pooled from identical tissues to minimize individual variability, as described previously (jayaraman et al., 2013). the rna was treated with turbo dnase and converted to cdna using the high-capacity cdna reverse transcription kit (applied biosystems, carlsbad, ca), as described earlier (jayaraman et al., 2010; 2013; 2017; 2018; patel et al., 2011). real-time quantitative reverse-transcriptase mediated polymerase chain reaction (qrt-pcr) was performed using the applied biosystems viia7 real-time pcr system. the cdna equivalent to 100 ng of total rna was used along with the 2x sybr green master mix in the qrt-pcr assay. the primer sets were designed, and specificity validated using the primer3 and blast programs (ncbi.nim.nih.gov) and bisearch web server tool (bisearch.enzim.hu). the mif primer sets were purchased from the integrated dna technologies (coralville, ia), and the custom primers were synthesized at the same facility. whereas the primer sets for gapdh, il4, il17a, il18, ifng, nos2, tnfa, tbet, rorgt, and gata3 were described previously (jayaraman et al., 2010; 2013), other primer sets are listed in supplementary table 1. every cdna sample was analyzed in triplicate at each time point, and the expression level of any given gene was ascertained using gapdh as the normalizer (jayaraman et al., 2010; 2013; 2017; 2018; patel et al., 2011) since it was not altered by tsa treatment in vitro (van lint et al., 1996). the same cdna pool was analyzed for the expression levels of all 41 genes. the gene expression level was determined using the 2-∆∆ct method (jayaraman et al., 2010; 2013; 2017; 2018; patel et al., 2011). the data are represented as the mean ± sem of triplicate technical repeats per time point. the outliers that deviated >10% from other data points were omitted, and the samples reanalyzed for gene expression. 2.3. elisa sera were collected from naïve and immunized mice with or without tsa treatment and assayed for ccl2 using the elisa ready-set-go kit (ebioscience, san diego, ca). samples were pooled from five mice per group, analyzed in duplicate, and expressed per mg of protein. 2.4. statistics the statistical significance of clinical scores between control and tsa-treated groups was determined using the area under the roc curve. the difference in gene expression was calculated using an unpaired two-tailed student's t-test. elisa data were analyzed for significance between groups by two-way anova. the p-value of <0.05 was considered significant. graphpad prism 6.0 software (san diego, ca) was used for all statistical analyses. 3. results 3.1. tsa treatment reduced the clinical manifestation of peae we have reported earlier that after immunization with the mog35-55 peptide the bodyweight declined transiently between 13 and 16 days, which steadily increased after that regardless of treatment with dmso or tsa (jayaraman et al., 2017). since female nod mice develop type 1 diabetes in an age-dependent manner, we routinely monitored blood glucose levels three times a week throughout the experiments. as reported earlier (jayaraman et al., 2010; 2013; jayaraman and jayaraman, 2018; patel et al., 2011), mice that were 9-11 weeks old (fig. 1a, d-e) were normoglycemic at the time of immunization with mog35-55 emulsified in complete freund’s adjuvant and remained diabetes-free throughout the observation, 12-27 weeks of age (fig. 1a, d-e). however, in two experiments, 40 and 60% of 17-week old mice immunized and treated with dmso were found to be diabetic. tsa treatment reduced the diabetes incidence respectively to 0% and 20% when analyzed at 20 and 21 weeks of age (fig. 1b-c). these results are consistent with our previous observations that the administration of complete freund’s adjuvant alone could effectively prevent type 1 diabetes in younger female nod mice. in contrast, similar treatment of older (>13-weeks) mice did not have the same potency in preventing diabetes (jayaraman and jayaraman, 2018). these results indicate that whereas the development of type 1 diabetes is age-dependent, induction of peae by immunization with the mog35-55 peptide emulsified in complete freund’s adjuvant is independent of the age and the glycemic status of mice. fig. 1 tsa treatment improved clinical disease.   randomly chosen littermates of female nod mice were immunized with mog35-55 and treated with tsa or dmso three times a week for the indicated time interval. clinical scores of five mice/group were determined on indicated time intervals and depicted as mean ± sem. the statistical significance between dmso (blank bars) and tsa-treated mice (hatched bars) was determined using the area under the roc curve test and indicated. individual data for dmso (empty circles) and tsa-treated (empty squares) groups are also indicated. mice were killed on day 14 (a), 21 (b), 28 (c), 54 (d), and 115 (e), and their spinal cord and peripheral lymphoid tissues were harvested and used for gene expression analysis. all of the mice immunized with mog35-55 developed peae without discernible remission (fig. 1a-e), consistent with earlier reports (basso et al., 2008; jayaraman et al., 2017; 2018). the overall clinical severity was comparable in most instances except in a single experiment, probably due to the difference in experimental conditions (fig. 1c). treatment with tsa bestowed robust and irreversible protection from peae for the entire period of observation (115 days) even after the cessation of the drug administration on day 45 (fig. 1e), as reported earlier (jayaraman et al., 2017). interestingly, the administration of the drug for one (fig. 1a), two (fig. 1b, c), four (fig. 1d) or six-weeks (fig. 1e) afforded a comparable level of protection against the disease. in previous studies, we have demonstrated that chronic tsa treatment decreased the influx of neutrophils and cd4+ t cells into the sc. drug treatment also reduced the inflammation and particularly axonal damage in the spinal cord, indicating the neuroprotective effect of the hdac inhibitor (jayaraman et al., 2017; 2018). to understand the underlying mechanisms of drug-mediated protection against peae, in the current study, we have concurrently profiled the gene expression in the cns and slo at various time points shown in fig. 1a-e. 3.2. differential impact of the histone modifier on gene expression in the cns the neuronal disease induced in nod mice resembled the ‘classic’ eae except that it lasted longer than in most strains of mice (slavin et al., 1998; basso et al., 2008; hidaka et al., 2014; dang et al., 2015; jayaraman et al., 2017; 2018). since the sc is thought to be the primary target of the ‘classic’ eae and the brain damage was selectively observed in 'atypical’ eae (pierson and goverman, 2017), we profiled the gene expression in the entire sc. expression levels of genes encoding 11 lymphokines, four cytokines, seven accessory cell surface-associated determinants, seven transcription factors, and 11 histone deacetylases (hdacs) were investigated by qrt-pcr. unimmunized mice were separately treated with dmso or tsa to determine the impact of drug treatment on the constitutive level of gene expression. the data are indicated at the day-1 time point in all figures. mice immunized with mog35-55 were treated with dmso or tsa starting from the day of immunization. the entire data sets are presented in supplementary fig. 1-5. only the differences in gene expression that occur consecutively at more than one-time point but not those altered transiently or sporadically are highlighted herein. longitudinal analysis during chronic peae revealed that some immune response-related genes were induced upon mog35-55 immunization after a 7-14-day lag period in both the cns and slo. the th cell-associated il17a, il4, and il22 and the inflammatory cytokine gene nos2 were upregulated for a prolonged period between 21 and 54 days in the cns in different experiments (fig. 2a-d). the epigenetic drug suppressed the expression of these genes that were upregulated by immunization. surprisingly, the constitutive expression of mif, ahr, and cd274 was also notably repressed by tsa treatment during the chronic phase of the disease (fig. 2e-g). unexpectedly, the pan hdac inhibitor upregulated the expression of dec1 (bhlhe40), specifically during the late stage of peae (fig. 2h). fig. 2 differential impact of the histone modifier on gene expression in the cns.   total rna was extracted individually from five mice per group from the spinal cord (sc) at various time points. at each time point, rna pooled from five mice/group was analyzed in triplicate. the expression of indicated genes was normalized to the housekeeping gene, gapdh, and depicted as mean ± sem of triplicate samples. statistical significance (p<0.05) between dmso (blank bars) and drug-treated mice (hatched bars) was determined using an unpaired two-tailed t-test and indicated by asterisks. individual replicates for dmso (empty circles) and tsa-treated samples (empty squares) are also shown. unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. although previous studies implicated many other genes in eae, those encoding il-23, ifn-γ, il-18, il-27p28, il-27ebi3, il-12p35, il-10, gm-csf, tnf-α, and tgf-β were not transcriptionally upregulated in the cns of peae mice (see supplementary fig. 1-2). similarly, the mrna of the accessory cell surface-associated determinants, mmp9, mmp12, and cd74, remained stable in the cns following immunization (supplementary fig. 3). moreover, the transcription factor genes such as tbet, gata3, rorgt, eomes, and foxp3 also remained unchanged in the sc (supplementary fig. 4). notably, the histone modifier failed to alter the transcription of these genes. thus, our comprehensive analysis uncovered the selective repression of genes in the cns without affecting the levels of many other genes previously implicated in neuroinflammation. 3.3. the different patterns of gene regulation in the slo by tsa treatment the expression of the genes encoding the subunits of the heterodimeric lymphokine il-27 such as il-27p28 and il-27ebi3 peaked on day 21 and subsided steadily thereafter in the slo (fig. 3a-b). treatment with tsa reduced the expression of these genes and the inflammatory gene nos2 (fig. 3c). interestingly, the transcription of mif, cd274, and cd39 in the slo was also downregulated by the histone modifier (fig. 3d-f). surprisingly, the genes encoding the lymphokines such as il-4, il-10, il-17a, il-12p35, il-18, il-22, il-23, ifn-γ, tgfβ, gm-csf and tnf-α (supplementary fig. 1-2), as well as the macrophage-associated determinants, mmp9, mmp12, arg-1, and cd74 were neither upregulated during pathogenesis nor repressed by tsa treatment in the slo (supplementary fig. 3). similarly, the transcription of tbet, rorgt, gata3, eomes, dec1, ahr, and foxp3 remained mostly unaffected by the hdac inhibitor treatment (supplementary fig. 4). thus, the antigen-induced transcription of il27p28, il27ebi3, and nos2, as well as the constitutive expression of mif, cd274, and cd39, were selectively impeded by tsa treatment in the slo. these data collectively demonstrate the differential influence of epigenetic modulation on gene expression in the cns and slo in the peae model. fig. 3 different patterns of gene regulation in the slo by tsa treatment.   expression levels of indicated genes were determined in control dmso (blank bars) and tsa-treated mice (hatched bars) using the total rna derived from the spleen and draining lymph nodes (sp) from individual mice at the indicated time points. at each time point, rna pooled from five mice/group was analyzed in triplicate. individual data points for dmso (empty circles) and tsa-treated (empty squares) are shown. gene expression was normalized to the housekeeping gene, gapdh, and depicted as mean ± sem of triplicate samples. statistical significance (p<0.05) between control and drug-treated mice was determined using an unpaired two-tailed t-test and indicated by asterisks. unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. 3.4. epigenetic regulation failed to influence the prominent chemokine system since previous work indicated the nonredundant roles of ccl2 and ccr2 in recruiting the inflammatory cells to the cns (mahad and ransohoff, 2003; moreno et al., 2014), we surmised that tsa-induced neuroprotection could also accompany modulation of these genes. immunization of nod mice with mog35-55 did not transcriptionally upregulate ccl2 in the cns (fig. 4a). on the other hand, ccl2 transcription increased dramatically in the slo between days 14 and 28 (fig. 4b). however, tsa treatment failed to modulate the transcription of ccl2 in the sc or slo. the gene encoding the cognate receptor of ccl2, ccr2, was not distinctively upregulated in either the sc or slo nor perturbed by the histone modifier (fig. 4c-d). in contrast to the robust increase in the ccl2 mrna level in the slo, the amount of ccl2 protein did not increase in circulation during the pre-symptomatic period (up to 16 days) as assessed by elisa, which remained unaffected by tsa treatment (fig. 4e). these data indicate that protection from peae afforded by the cns-permeant tsa (jayaraman et al., 2017; 2018) was not associated with the transcriptional regulation of the prominent chemokine system, ccl2: ccr2 either in the cns or slo. fig. 4 epigenetic regulation failed to influence the prominent chemokine system.   expression levels of indicated genes were determined in dmso (blank bars) and tsa-treated mice (hatched bars) using the total rna derived from the spinal cord (sc) (a, c) and peripheral lymphoid tissues (sp) (b, d) at various time points. at each time point, rna was extracted from five individual mice/group, pooled, and analyzed in triplicate. gene expression was normalized to the housekeeping gene, gapdh, and depicted as mean ± sem of triplicate samples. individual data points are shown for dmso (blank circles) and tsa-treated (empty squares) groups. ccl2 protein was estimated in duplicate from pooled sera of five mice per group using elisa (e). unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. statistical significance (p<0.05) between control and drug-treated mice (n=five/group) was determined using an unpaired two-tailed t-test and indicated by asterisks. 3.5. tsa treatment repressed the transcription of hdac11 selectively in the cns whereas tsa can inhibit the activity of hdac class i, iia, and iib isoenzymes with varying potency in vitro (bradner et al., 2010), its ability to regulate hdac genes in vivo has not been determined. to address this issue, we analyzed the mrna levels of class i (hdac1, hdac2, hdac3, and hdac8), class iia (hdac4, hdac5, hdac7, and hdac9), class iib (hdac6 and hdac10), and class iv (hdac11) hdacs using validated primer sets and qrt-pcr. data shown in fig. 5 indicate that immunization of nod mice with mog35-55 steadily increased the transcription of the class iv hdac11 in the sc, which remained at high levels between 21 and 54 days postimmunization. importantly, tsa treatment reduced the transcript level of hdac11. although hdac1, hdac4, hdac5, hdac6, hdac8, and hdac9 were modestly increased in the cns with different kinetics after immunization, they were relatively insensitive to tsa treatment. surprisingly, the expression levels of hdac genes, including the hdac11, did not increase significantly in the slo of mog35-55 immunized mice nor repressed by tsa treatment (supplementary fig. 5). these results indicate that mog35-55 immunization leads to increased transcription of hdac11 in a cns-specific fashion, which is rendered sensitive to the action of the histone modifier. these results indicate the possibility that hdac11 could represent a novel target for the manipulation of peae. fig. 5 tsa treatment repressed the transcription of hdac11 selectively in the cns.   expression levels of indicated hdac genes were determined in dmso (blank bars) and tsa-treated mice (hatched bars) in the spinal cord as shown. at each time point, rna was isolated from five individual mice/group, pooled and analyzed in triplicate. gene expression was normalized to the housekeeping gene gapdh and depicted as mean ± sem of triplicate samples. individual data points are shown for dmso (blank circles) and tsa-treated (empty squares) groups. statistical significance (p<0.05) between control and the drug-treated group was determined using an unpaired two-tailed t-test and indicated by asterisks. ns, not significant. unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. 4. discussion a cardinal assumption has been that the t lymphocytes play a crucial role in the induction and manifestation of multiple sclerosis, and therefore, induction of antigen-specific t cell tolerance is a reasonable approach to treat this debilitating disease. many attempts, including immunization with various neuronal peptides and t cell vaccinations, have failed to induce t cell tolerance and ameliorate multiple sclerosis symptoms (wingerchuk and carter, 2014; dendrou et al., 2015; steinman, 2015; lassmann, 2017). however, the induction of antigen-specific t cell tolerance using immunomodulatory drugs remains unexplored in patients with ms and other autoimmune disorders. recently, we have demonstrated that treatment with the potent hdac inhibitor tsa not only reduced the frequencies of th1, th17, and th1/th17 cells in the slo and their influx into the cns but also induced mog35-55 peptide-specific t cell tolerance (anergy) in nod mice (jayaraman et al., 2017). although anergy was originally reported in a mouse th1 clone that was suboptimally activated without co-stimulation in vitro (schwartz et al., 1989; jayaraman et al., 1992), the underlying mechanisms have not been fully deciphered. our investigation unraveled a good correlation between tsa-mediated neuroprotection and downregulation of selected immune response-related genes both in the cns and slo. whereas these data may not directly impinge upon histone modifier-facilitated mog35-55 peptide-specific t cell tolerance, they highlight the possible impact of differential gene expression on peae. the epigenetic approach unraveled an inverse relationship between the expression levels of a small set of genes and neuroprotection. in the cns of peae nod mice, il17a expression was upregulated for over a month (fig. 2), unlike its expression at the peak of the monophasic eae (kreymborg et al., 2007; evangelidou et al., 2014; borjini et al., 2016). prolonged expression of nos2 in the cns (fig. 2b) is congruent with the association of inos-positive macrophages and glial cells in demyelinating pathology (tran et al., 1997). in addition to the nos2 (fig. 3c), the subunits of il-27, namely il-27p28, and il-27ebi3, were prominently upregulated at the mrna level in the slo (fig. 3b-c). this is consistent with an encephalitogenic role of il-27 suggested by the suppression of the ongoing eae following administration of the neutralizing antibody against the il-27p28 subunit (goldberg et al., 2004). besides, tsa treatment reduced the steady-state expression of mif in both the cns and slo (fig. 2e, fig. 3e), consistent with a proposed pathogenic role of mif in monophasic eae (powell et al., 2005). we have previously shown that tsa treatment diminished the numbers of splenocytes expressing the co-inhibitory ligand cd274 (pd-l1) (jayaraman et al., 2018). consistently, the cd274 mrna level was also repressed in the cns (fig. 2g) and slo (fig. 3f) of tsa-treated peae mice, suggesting a role for this co-inhibitory ligand in encephalomyelitis, as proposed (jayaraman et al., 2018). also, the expression of ahr, uniquely required for the generation of t cells responsible for the late-onset eae (nakahama et al., 2013) was diminished in mice protected by tsa treatment (fig. 2f). collectively, the tsa-mediated downregulation of both inducible and constitutively expressed genes appears to be inversely proportional to the severity of the neuronal disease. however, it remains to be determined whether the expression levels of these genes may serve as biomarkers for the diagnosis of ms. nevertheless, the information uncovered in the nod mouse model may provide a framework for potential ms treatment, typically diagnosed as clinically isolated syndrome followed by years of asymptomatic period (dendrou et al., 2015). the upregulation of the transcription of hdac11 in a cns-specific fashion in mice immunized with mog35-55 (fig. 5) represents the first report of differential expression of hdac genes in vivo. although the non-selective hdac inhibitor tsa did not diminish the hdac11 enzymatic activity in vitro (bradner et al., 2010), our data demonstrated the control of hdac11 by tsa at the transcriptional level. since hdac11 gene expression was determined in the sc devoid of peripheral blood contamination, its reduction appears to be a direct effect of tsa on the cns resident cells. although we have not identified the cellular source of hdac11 in the spinal cord, previous work suggested that hdac11 knockdown increased il10 expression in peripheral antigen-presenting cells resulting in immunosuppression in vitro (villagra et al., 2009). in contrast, tsa-induced tolerance in mog35-55-specific t lymphocytes (jayaraman et al., 2017) did not accompany the upregulated transcription of il10 in the cns or slo (see supplementary fig. 1). although the deletion of hdac1 selectively in t cells was reported to prevent eae (göschl et al., 2018), immunization with mog35-55 neither upregulated the expression of this gene, nor the epigenetic drug influenced its transcription in the slo or cns (fig. 5, supplementary fig. 5). thus, the histone hyperacetylating drug appears to primarily target the hdac11 gene in the cns of peae mice and the elucidation of the genes that are under the control of hdac11 may provide novel insights into the mechanisms of peae. to our surprise, the epigenetic drug treatment failed to validate the purported roles of the genes critical for the development of encephalitogenic th1 and th17 subsets, including ifng, il12, il18, il23, tbet, rorgt, gata3, and eomes. the disruption of the ifn-γ gene failed to influence eae development (ferber et al., 1995), indicating a lack of ifn-γ-expressing cells in neurodegeneration. consistently, the reduction of clinical symptoms by tsa treatment did not accompany the transcriptional repression of ifn-γ (supplementary fig. 1). besides, the drug-mediated tolerance induction was evident without repressed transcription of tbet, il18, and il12 genes critical for th1 cell development (o'connor et al., 2013; lalor et al., 2011; gran et al., 2004) (see supplementary fig. 3). moreover, the transcription of il23 (kreymborg et al., 2007; el-behi et al., 2011) and rorgt (martinez et al., 2014), respectively, involved in th17 and th2 cell generation was also not diminished by tsa treatment. yet, the histone modifier reduced the overall numbers of th17 and th1/th17 cells in the slo (jayaraman et al., 2017). furthermore, the drug treatment failed to decrease the transcription of csf2 (see supplementary fig. 2) despite the diminished numbers of gm-csf-co-expressing th1 and th17 cells found in the slo of tsa-treated mice (jayaraman et al., 2017). these data indicate that t cell tolerance induction by epigenetic modulation of the genome does not involve selective suppression of genes required for the generation of functionally distinct th cell subsets. further work is necessary to decipher the underlying mechanisms of t cell tolerance. another deviation from the conventional idea of immunoregulation is the lack of the modulation of il10 and foxp3 both in the cns and slo of tsa-treated mice (supplementary fig. 4). although foxp3+ t regulatory cells are considered critical for immunoregulation (hori et al., 2003), their role in eae is less compelling (jayaraman et al., 2017; danikowski et al., 2017; jayaraman and prabhakar, 2019). whereas tsa treatment increased foxp3 expression and promoted t regulatory cell function via the upregulation of hdac9 in a different experimental model (tao et al., 2007), our results contradict these findings. it is also noteworthy that tsa treatment downregulated foxp3 expression and lowered the numbers of cd4+cd25+ t regulatory cells (liu et al., 2010). nevertheless, our data indicate that tsa-mediated protection from peae in autoimmune-prone nod mice is independent of il-10 and foxp3+ t regulatory cells. differential rna display (van lint et al., 1996) and microarray analysis of gene expression (jayaraman et al., 2013) indicated that histone hyperacetylation by tsa treatment could have positive, negative, or no effect on gene transcription. our qrt-pcr analysis showed that out of 41 genes interrogated, 11 were consistently downregulated over multiple time points following tsa treatment (vide supra). rarely, tsa treatment can also increase the transcription of genes due to the transcriptional suppression of repressor complexes that control gene expression. thus, dec1 was selectively upregulated in the cns by the histone modifier, suggesting a role in protection against peae (fig. 2h). this is in contrast to the finding that the genetic deletion of dec1 (bhlhe40-/-) afforded resistance to eae induction (lin et al., 2016). like an earlier study (hidaka et al., 2014), we found that the expression of the ccl2 gene was dramatically increased in the slo of mog35-55 immunized nod mice (fig. 4). however, tsa treatment failed to influence its expression. moreover, there is a disconnect between the ccl2 mrna expression in the slo and the release of the ccl2 protein into the peripheral blood. it has been proposed that ccl2 expressed by the cns resident astrocytes may serve as a target for ms treatment (mahad and ronsohoff, 2003; moreno et al., 2014). however, ccl2 and ccr2 genes are refractory to histone hyperacetylation, and therefore do not appear to be essential for peae induction and manifestation. this observation does not come as a surprise since the level of ccl2 was also reported to be lower in relapsing-remitting ms patients (narikawa et al., 2004; moreira et al., 2006), and the ifn-beta 1a therapy reduced relapses while increasing the level of ccl2 in ms patients (szczuciński and losy, 2004). thus, these data do not support the hypothesis that the ccl2: ccr2 system may impact neurodegeneration. it is noteworthy that tsa administration induced neuroprotection and gene regulation similarly in prediabetic, 9-13 weeks old, and aged (17-weeks old) mice (jayaraman et al., 2017; fig. 1-5). the administration of the epigenetic drug for one to six weeks bestowed comparable neuroprotection and gene regulation irrespective of the age of the mice. circumstantial evidence also indicates the lack of influence of diabetes on peae induction. as expected, the prediabetic (9-13-weeks old) mice were normoglycemic at the time of immunization with mog35-55 and remained non-diabetic as long as 27 weeks of age. the lack of diabetes in immunized mice could be due to the action of the microbial products present in the complete freund’s adjuvant. in fact, we have shown that the administration of complete freund’s adjuvant alone is sufficient to prevent prediabetic nod mice from developing overt diabetes (jayaraman and jayaraman, 2018). although a fraction of the older (17 weeks old) mice developed diabetes, it did not impede peae induction (fig. 1b-c) nor the modulation of gene expression in response to tsa treatment (fig. 2-5). collectively, these data demonstrate that the induction of peae and epigenetic regulation of gene expression is unrelated to the age or the glycemic status of mice. conclusions the analysis of the 41 genes using qrt-pcr unraveled differential regulation of gene expression in the cns and slo by an epigenetic drug. this study has highlighted the roles of il4, hdac11, cd274, and cd39 in the cns in addition to validating previously implicated genes, il17a, il22, inos, ahr, and mif in neuroinflammation. on the other hand, in the slo, the roles of il22, il27, nos2, and mif were confirmed while indicating the participation of cd274 and cd39 in peae. some of these genes are induced by immunization, while others are constitutively expressed. surprisingly, many other genes previously implicated in eae were refractory to histone hyperacetylation mediated transcriptional regulation. hence, it will be difficult to conclude whether these genes do not contribute to neuroinflammation. nevertheless, our data suggest that the drug-induced histone hyperacetylation is a promising strategy to treat demyelination and axonal damage by modifying the expression of selected genes. although the therapeutic potential of hdac inhibitors for the treatment of multiple sclerosis has been entertained (faraco et al., 2011), direct evidence is lacking (göbel et al., 2018). the data generated in the nod mouse model provide a framework for a similar pharmacological approach to treat ms patients. author contributions aj conducted experiments, collected data, edited the graphic and the manuscript. ka, rk, as, ak, and mk performed experiments, collected data, and approved the manuscript. sj conceived the project, designed and implemented experiments, analyzed the data, and wrote the paper. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. acknowledgments mark holterman and bellur prabhakar are acknowledged for the support of this work. references basso, a.s., frenkel, d., quintana, f.j., costa-pinto, f.a., petrovic-stojkovic, s., puckett, l., monsonego, a., bar-shir a., engel y., gozin m., weiner h.l., 2008. reversal of axonal loss and disability in a mouse model of progressive multiple sclerosis. j. clin. invest. 118, 1532-1543. https://doi.org/10.1172/jci33464. borjini, n., fernández, m., giardino l., calzà, l., 2016. cytokine and chemokine alterations in tissue, csf, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (eae), in a rat model of multiple sclerosis. j. neuroinflammation 13, 291. https://doi.org.10.1186/s12974-016-0757-6. bradner, j.e., west, n., grachan, m.l., greenberg, e.f., haggarty, s.j., warnow, t., mazitschek, r., 2010. chemical phylogenetics of histone deacetylases. nat. chem. biol. 6, 238-243. https://doi.org.10.1038/nchembio.313. dang, p.t., bui, q., d'souza, c.s., orian, j.m., 2015. modelling ms: chronic-relapsing eae in the nod/lt mouse strain. curr. top. behav. neurosci. 26, 143-177. https://doi.org.10.1007/7854_2015_378. danikowski, k.m., jayaraman, s., prabhakar, b.s., 2017. regulatory t cells in multiple sclerosis and myasthenia gravis. j. neuroinflammation 14, 117. https://doi.org.10.1186/s12974-017-0892-8. dendrou, c.a., fugger, l., friese, m.a., 2015. immunopathology of multiple sclerosis. nat. rev. immunol. 15, 545-558. https://doi.org.10.1038/nri3871. de ruijter, a.j., van gennip, a.h., caron, h.n., kemp, s., van kuilenburg, a.b., 2003. histone deacetylases (hdacs): characterization of the classical hdac family. biochem. j. 370, 737-749. pmid:12429021. el-behi, m., ciric, b., dai, h., yan, y., cullimore, m., safavi, f., zhang, g.x., dittel, b.n., rostami, a., 2011. the encephalitogenicity of t(h)17 cells is dependent on il-1and il-23-induced production of the cytokine gm-csf. nat. immunol. 12, 568-575. https://doi.org.10.1038/ni.2031. evangelidou, m., karamita, m., vamvakas, s.s., szymkowski, d.e., probert, l., 2014. altered expression of oligodendrocyte and neuronal marker genes predicts the clinical onset of autoimmune encephalomyelitis and indicates the effectiveness of multiple sclerosis-directed therapeutics. j. immunol. 192, 4122-3133. https://doi.org.10.4049/jimmunol.1300633. falcone, m., rajan, a.j., bloom, b.r., brosnan, c.f., 1998. a critical role for il-4 in regulating disease severity in experimental allergic encephalomyelitis as demonstrated in il-4-deficient c57bl/6 mice and balb/c mice. j. immunol. 160, 4822–4830. pmid:9590229. faraco, g., cavone l., chiarugi., 2011. the therapeutic potential of hdac inhibitors in the treatment of multiple sclerosis. mol. med. 17, 442-447. pmid 21373721. ferber, i.a., brocke, s., taylor-edwards, c., ridgway, w., dinisco, c., steinman, l., dalton, d., fathman, c.g., 1996. mice with a disrupted ifn-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (eae). j. immunol. 156, 5-7. pmid:8598493. fernando, v., omura, s., sato, f., kawai, e., martinez, n.e., elliott, s.f., yoh, k., takahashi, s., tsunoda, i., 2014. regulation of an autoimmune model for multiple sclerosis in th2-biased gata3 transgenic mice. int. j. mol. sci. 15, 1700-1708. https://doi.org.10.3390/ijms15021700. fox, r.j., chataway, j., 2017. advancing trial design in progressive multiple sclerosis. mult. scler. 23, 1573-1578. https://doi.org.10.1177/1352458517729768 göbel, k., ruck, t., meuth, s.g., 2018. cytokine signaling in multiple sclerosis: lost in translation. mult. scler. 24, 432-439. https://doi.org.10.1177/1352458518763094. goldberg, r., wildbaum, g., zohar, y., maor, g., karin, n., 2004. suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of il-27. j. immunol. 173, 6465-6471. https://doi.org.10.4049/jimmunol.173.10.6465. göschl, l., preglej, t., hamminger, p., bonelli, m., andersen, l., boucheron, n., gülich, af., müller, l., saferding, v., mufazalov, ia., hirahara, k., seiser, c., matthias, p., penz, t., schuster, m., bock, c., waisman, a., steiner, g., ellmeier, w., 2018. a t cell-specific deletion of hdac1 protects against experimental autoimmune encephalomyelitis. j. autoimmun. 86, 51-61. https://doi.org.10.1016/j.jaut.2017.09.008. gran, b., zhang, g.x., rostami, a., 2004. role of the il-12/il-23 system in the regulation of t-cell responses in central nervous system inflammatory demyelination. crit. rev. immunol. 24, 111-128. pmid:15581393. hidaka, y., inaba, y., matsuda, k., itoh, m., kaneyama, t., nakazawa, y., koh, c.s., ichikawa, m., 2014. cytokine production profiles in chronic relapsing-remitting experimental autoimmune encephalomyelitis: ifn-γ and tnf-α are essential participants in the first attack but not in the relapse. j. neurol. sci. 340, 117-122. https://doi.org/10.1016/j.jns.2014.02.039. hori, s., nomura, t., sakaguchi, s., 2003. control of regulatory t cell development by the transcription factor foxp3. science 299, 1057-1061. https://doi.org.10.1126/science.1079490. jayaraman, s., muthukkaruppan, v.r., 1977. in vitro correlate of transplantation immunity: spleen cell migration inhibition in the lizard, calotes versicolor. dev. comp. immunol. 1, 133-143. https://doi.org.10.1016/s0145-305x(77)80006-2. jayaraman, s., luo, y., dorf, m.e., 1992. tolerance induction in t helper (th1) cells by thymic macrophages. j. immunol. 148, 2672-2681. pmid:1533409. jayaraman, s., patel, t., patel, v., garza, r., ajani, s., jayaraman, a., singh, r., kwon, s., rondelli, d., prabhakar, s., holterman, m., 2010. transfusion of nonobese diabetic mice with allogeneic newborn blood ameliorates autoimmune diabetes and modifies the expression of selected immune response genes. j. immunol. 184, 3008-3015. pmid:20164427. jayaraman, s., patel, a., jayaraman, a., patel, v., holterman, m., prabhakar, b., 2013. transcriptome analysis of epigenetically modulated genome indicates signature genes in manifestation of type 1 diabetes and its prevention in nod mice. plos one 8, e55074. https://doi.org.10.1371/journal.pone.0055074. jayaraman, a., soni, a., prabhakar, b.s., holterman, m., jayaraman, s., 2017. the epigenetic drug trichostatin a ameliorates experimental autoimmune encephalomyelitis via t cell tolerance induction and impaired influx of t cells into the spinal cord. neurobiol. dis. 108, 1-12. https://doi.org.10.1016/j.nbd.2017.07.015. jayaraman, a., sharma, m., prabhakar, b., holterman, m., jayaraman, s., 2018. amelioration of progressive autoimmune encephalomyelitis by epigenetic regulation involves selective repression of mature neutrophils during the preclinical phase. exp. neurol. 304, 14-20. https://doi.org.10.1016/j.expneurol.2018.02.008. jayaraman, s., jayaraman, a., 2018. long-term provision of acidified drinking water fails to influence autoimmune diabetes and encephalomyelitis. j. diab. res. 2018:3424691. pmid:30035128. jayaraman, s., prabhakar, b.s., 2019. immune tolerance in autoimmune central nervous system disorders. in: mitoma, h., manto, m., (eds.) autoimmune central nervous disorders. springer nature, london, pp.143-166. https://doi.org.10.1007/978-3-030-19515-1_5. kandagaddala, l.d., kang, m.j., chung, b.c., patterson, t.a., kwon, o.s., 2012. expression and activation of matrix metalloproteinase-9 and nadph oxidase in tissues and plasma of experimental autoimmune encephalomyelitis in mice. exp. toxicol. pathol. 64, 109-114. https://doi.org.10.1016/j.etp.2010.07.002. kreymborg, k., etzensperger, r., dumoutier, l., haak, s., rebollo, a., buch, t., heppner, f.l., renauld, j.c., becher, b., 2007. il-22 is expressed by th17 cells in an il-23-dependent fashion, but not required for the development of autoimmune encephalomyelitis. j. immunol. 179, 8098-8104. https://doi.org.10.4049/jimmunol.179.12.8098. lassmann, h., 2017. targets of therapy in progressive ms. mult. scler. 23, 1593-1599. https://doi.org/10.1177/1352458517729455. lassmann, h., bradl, m., 2017. multiple sclerosis: experimental models and reality. acta neuropathol. 133, 223-244. https://doi.org/10.1007/s00401-016-1631-4. lalor, s.j., dungan, l.s., sutton, c.e., basdeo, s.a., fletcher, j.m., mills, k.h., 2011. caspase-1-processed cytokines il-1beta and il-18 promote il-17 production by gammadelta and cd4 t cells that mediate autoimmunity. j. immunol. 186, 5738-5748. https://doi.org.10.4049/jimmunol.1003597. li, j., gran, b., zhang, g.x., rostami, a., kamoun, m., 2005. il-27 subunits and its receptor (wsx-1) mrnas are markedly up-regulated in inflammatory cells in the cns during experimental autoimmune encephalomyelitis. j. neurol. sci. 232, 3-9. https://doi.org.10.1016/j.jns.2004.12.013. lin, c.c., bradstreet, t.r., schwarzkopf, e.a., jarjour, n.n., chou, c., archambault, a.s., sim, j., zinselmeyer, b.h., carrero, j.a., wu, g.f., taneja, r., artyomov, m.n., russell, j.h., edelson, b.t., 2016. il-1-induced bhlhe40 identifies pathogenic t helper cells in a model of autoimmune neuroinflammation. j. exp. med. 213, 251-271. https://doi.org.10.1084/jem.20150568. liu, z., zhang, c., sun, j., 2010. deacetylase inhibitor trichostatin a down-regulates foxp3 expression and reduces cd4+cd25+ regulatory t cells. biochem. biophys. res. commun. 400, 409-412. https://doi.org.10.1016/j.bbrc.2010.08.090. mahad, d.j., ransohoff, r.m., 2003. the role of mcp-1 (ccl2) and ccr2 in multiple sclerosis and experimental autoimmune encephalomyelitis (eae). semin. immunol. 15, 23-32. pmid:12495638. martinez, n.e., sato, f., omura, s., kawai, e., takahashi, s., yoh, k., tsunoda, i., 2014. rorγt, but not t-bet, overexpression exacerbates an autoimmune model for multiple sclerosis. j. neuroimmunol. 276, 142-149. https://doi.org.10.1016/j.jneuroim.2014.09.006. mascanfroni, i.d., yeste, a., vieira, s.m., burns, e.j., patel, b., sloma, i., wu, y., mayo, l., ben-hamo, r., efroni, s., kuchroo, v.k., robson, s.c., quintana, f.j., 2013. il-27 acts on dcs to suppress the t cell response and autoimmunity by inducing expression of the immunoregulatory molecule cd39. nat. immunol. 14,1054-1063. https://doi.org.10.1038/ni.2695. mcqualter, j.l., darwiche, r., ewing, c., onuki, m., kay, t.w., hamilton, j.a, reid, h.h., bernard, c.c., 2001. granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis. j. exp. med. 194, 873-882. https://doi.org:10.1084/jem.194.7.873. moreira, m.a., tilbery, c.p., monteiro, l.p., teixeira, m.m., teixeira, a.l., 2006. effect of the treatment with methylprednisolone on the cerebrospinal fluid and serum levels of ccl2 and cxcl10 chemokines in patients with active multiple sclerosis. acta neurol. scand. 114, 109-113. https://doi.org/10.1111/j.1600-0404.2006.00629. moreno, m., bannerman, p., ma, j., guo, f., miers, l., soulika, a.m., pleasure, d., 2014. conditional ablation of astroglial ccl2 suppresses cns accumulation of m1 macrophages and preserves axons in mice with mog peptide eae. j. neurosci. 34, 8175-8185. https://doi.org.10.1523/jneurosci.1137-14.2014. nakahama, t., hanieh, h., nguyen, n.t., chinen, i., ripley, b., millrine, d., lee, s., nyati, k.k., dubey, p.k., chowdhury, k., kawahara, y., kishimoto, t., 2013. aryl hydrocarbon receptor-mediated induction of the microrna-132/212 cluster promotes interleukin-17-producing t-helper cell differentiation. proc. natl. acad. sci. usa 110, 11964-11969. https://doi.org.10.1073/pnas.1311087110. narikawa, k., misu, t., fujihara, k., nakashima, i., sato, s., itoyama, y., 2004. csf chemokine levels in relapsing neuromyelitis optica and multiple sclerosis. j. neuroimmunol. 149, 182-186. https://doi.org/ 10.1016/j.jneuroim.2003.12.010. niino, m., ogata, a., kikuchi, s., tashiro, k., nishihira, j., 2000. macrophage migration inhibitory factor in the cerebrospinal fluid of patients with conventional and optic-spinal forms of multiple sclerosis and neuro-behçet's disease. j. neurol. sci. 179, 127-131. https://doi.org. 10.1016/s0022-510x(00)00397-x. o'connor, r.a., cambrook, h., huettner, k., anderton, s.m., 2013. t-bet is essential for th1-mediated, but not th17-mediated, cns autoimmune disease. eur. j. immunol. 43, 2818-2823. https://doi.org.10.1002/eji.201343689. okuda, y., sakoda, s., yanagihara, t., 1998. the pattern of cytokine gene expression in lymphoid organs and peripheral blood mononuclear cells of mice with experimental allergic encephalomyelitis. j. neuroimmunol. 87, 147-155. pmid:9670856. patel, t., patel, v., singh, r., jayaraman, s., 2011. chromatin remodeling resets the immune system to protect against autoimmune diabetes in mice. immunol. cell biol. 89:640-649. pmid:21321581. pierson, e.r., goverman, j.m., 2017. gm-csf is not essential for experimental autoimmune encephalomyelitis but promotes brain-targeted disease. jci insight 2, e92362. https://doi.org.10.1172/jci.insight.92362. ponomarev, e.d., maresz, k., tan, y., dittel, b.n., 2007. cns-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. j. neurosci. 27, 10714-10721. https://doi.org.10.1523/jneurosci.1922-07.2007. powell, n.d., papenfuss, t.l., mcclain, m.a., gienapp, i.e, shawler, t.m., satoskar, a.r., whitacre, c.c., 2005. cutting edge: macrophage migration inhibitory factor is necessary for progression of experimental autoimmune encephalomyelitis. j. immunol. 175, 5611-5614. https://doi.org.10.4049/jimmunol.175.9.5611. raveney, b.j., oki, s., hohjoh, h., nakamura, m., sato, w., murata, m., yamamura, t., 2015. eomesodermin-expressing t-helper cells are essential for chronic neuroinflammation. nat. commun. 6, 8437. https://doi.org.10.1038/ncomms9437. rempe, r.g., hartz, a.m, bauer, b., 2016. matrix metalloproteinases in the brain and blood-brain barrier: versatile breakers and markers. j. cereb. blood flow metab. 36, 1481-1507. https://doi.org.10.1177/0271678x16655551. rivers, t.m., sprunt, d.h, berry, g.p., 1933. observations on attempts to produce acute disseminated encephalomyelitis in monkeys. j. exp. med. 58, 39-53. https://doi.org.10.1084/jem.58.1.39. schwartz, rh., mueller, dl., jenkins, mk., quill, h., 1989. t-cell clonal anergy. cold spring harb. symp. quant. biol. 54, 605-610. pmid 2534840. slavin, a., ewing, c., liu, j., ichikawa m., slavin j., bernanrd, c.c., 1998. induction of a multiple sclerosis-like disease in mice with an immunodominant epitope of myelin oligodendrocyte glycoprotein. autoimmunity 28, 109-120. https://doi.org.10.3109/08916939809003872. steinman, l., 2015. the re-emergence of antigen-specific tolerance as a potential therapy for ms. mult. scler. 21, 1223-1238. https://doi.org.10.1177/1352458515581441. szczuciński, a., losy, j., 2004. long-term effect of ifn-beta 1a therapy on ccl2 (mcp-1) chemokine in patients with multiple sclerosis. folia neuropathol. 42, 15-18. pmid:15119740. tao, r., de zoeten, e.f., ozkaynak, e., chen, c., wang, l., porrett, p.m., li, b., turka, l.a., olson, e.n., greene, m.i., wells, a.d., hancock, w.w., 2007. deacetylase inhibition promotes the generation and function of regulatory t cells. nat. med. 13, 1299-1307. https://doi.org.10.1038/nm1652. tintore, m., vidal-jordana, a., sastre-garriga, j., 2019. treatment of multiple sclerosis success from bench to bedside. nat. rev. neurol. 15, 53-58. https://doi.org/10.1038/s41582-018-0082-z. tran, e.h., hardin-pouzet, h., verge, g., owens, t., 1997. astrocytes and microglia express inducible nitric oxide synthase in mice with experimental allergic encephalomyelitis. j. neuroimmunol. 74, 121-129. https://doi.org.10.1016/s0165-5728(96)00215-9. van lint, c., emiliani, s., verdin, e., 1996. the expression of a small fraction of cellular genes is changed in response to histone hyperacetylation. gene expr. 5, 245-253. pmid:8723390. villagra, a., cheng, f., wang, h.w., suarez, i., glozak, m., maurin, m., nguyen, d., wright, k.l., atadja, p.w., bhalla, k., pinilla-ibarz, j., seto, e., sotomayor, e.m., 2009. the histone deacetylase hdac11 regulates the expression of interleukin 10 and immune tolerance. nat. immunol. 10, 92-100. https://doi.org.10.1038/ni.1673. wingerchuk, d.m., carter, j.l., 2014. multiple sclerosis: current and emerging disease-modifying therapies and treatment strategies. mayo clin. proc. 89, 225-240. https://doi.org.10.1016/j.mayocp.2013.11.002. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. specific immune modulation of experimental colitis drives enteric alpha-synuclein accumulation and triggers age-related parkinson-like brain pathology feel free to add comments by clicking these icons on the sidebar free neuropathology 2:13 (2021) original paper specific immune modulation of experimental colitis drives enteric alpha-synuclein accumulation and triggers age-related parkinson-like brain pathology stefan grathwohla, emmanuel quansahb, nazia maroofa, jennifer a. steinerb, liz spychera, fethallah benmansourc, gonzalo duran-pachecod, juliane siebourg-polsterd, krisztina oroszlan-szovika, helga remya, markus haenggia, marc stawiskia, matthias selhausend, pierre maliverd, andreas wolferte, thomas emriche, zachary madajb, arel sud, martha l. escobar galvisb, christoph muellerf, annika herrmannd, patrik brundinb*, and markus britschgia* a roche pharma research and early development, neuroscience and rare diseases discovery and translational area, roche innovation center basel, f. hoffmann-la roche ltd, grenzacherstrasse 124, basel, switzerland b parkinson’s disease center, department of neurodegenerative science, van andel institute, 333 bostwick ave. ne, grand rapids, mi, usa c roche pharma research and early development, predi, roche innovation center basel, f. hoffmann-la roche ltd, grenzacherstrasse 124, basel, switzerland d roche pharma research and early development, pharmaceutical sciences, roche innovation center basel, f. hoffmann-la roche ltd, grenzacherstrasse 124, basel, switzerland e roche pharma research and early development, pharmaceutical sciences, roche innovation center munich, roche diagnostics gmbh, nonnenwald 2, penzberg, germany f institute of pathology, university of bern, murtenstrasse 31, bern, switzerland *corresponding authors: markus britschgi · roche pharma research and early development · neuroscience and rare diseases discovery and translational area · roche innovation center basel · f. hoffmann-la roche ltd · grenzacherstrasse 124 · 4070 basel · switzerland markus.britschgi@roche.com patrik brundin · van andel institute · 333 bostwick ave. ne · grand rapids · mi 49503 · usa patrik.brundin@vai.orgs submitted: 07 april 2021 accepted: 08 may 2021 copyedited by: bert m. verheijen published: 18 may 2021 https://doi.org/10.17879/freeneuropathology-2021-3326 additional resources and electronic supplementary material: supplementary material keywords: alpha-synuclein, experimental colitis, enteric nervous system, parkinson’s disease, substantia nigra abstract background: in some people with parkinson’s disease (pd), α-synuclein (αsyn) accumulation may begin in the enteric nervous system (ens) decades before development of brain pathology and disease diagnosis.   objective: to determine how different types and severity of intestinal inflammation could trigger αsyn accumulation in the ens and the subsequent development of αsyn brain pathology.   methods: we assessed the effects of modulating shortand long-term experimental colitis on αsyn accumulation in the gut of αsyn transgenic and wild type mice by immunostaining and gene expression analysis. to determine the long-term effect on the brain, we induced dextran sulfate sodium (dss) colitis in young αsyn transgenic mice and aged them under normal conditions up to 9 or 21 months before tissue analyses.   results: a single strong or sustained mild dss colitis triggered αsyn accumulation in the submucosal plexus of wild type and αsyn transgenic mice, while short-term mild dss colitis or inflammation induced by lipopolysaccharide did not have such an effect. genetic and pharmacological modulation of macrophage-associated pathways modulated the severity of enteric αsyn. remarkably, experimental colitis at three months of age exacerbated the accumulation of aggregated phospho-serine 129 αsyn in the midbrain (including the substantia nigra), in 21but not 9-month-old αsyn transgenic mice. this increase in midbrain αsyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons.   conclusions: our data suggest that specific types and severity of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of pd. introduction parkinson’s disease (pd) is a progressively debilitating neurodegenerative disease affecting 1% of the population above 60 years [1]. typical symptoms are motor impairments including muscle rigidity, tremor, and bradykinesia. neuropathologically, pd is hallmarked by loss of dopaminergic neurons in the substantia nigra (sn), a concomitant reduction of striatal dopaminergic signaling [2], and the presence of intraneuronal inclusions called lewy bodies and neurites [3]. lewy pathology is enriched in α-synuclein (αsyn), a presynaptic protein that tends to aggregate and become phosphorylated at serine 129 under pathological conditions [2]. rare point mutations in αsyn and gene multiplications also cause familial forms of pd and related neurological conditions, and certain single nucleotide polymorphisms close to the αsyn gene (snca) locus are associated with increased risk for sporadic pd [4]. these findings make αsyn a focal point of biomarker and drug development programs for pd. several years before the first appearance of motor symptoms, many patients exhibit a variety of non-motor symptoms including constipation, sleep disorder, depression, and hyposmia [5–7]. indeed, co-occurrence of some of these non-motor symptoms is coupled to elevated pd risk [8–11]. constipation is an important non-motor feature of prodromal pd, with 28-61% of patients having exhibited gastrointestinal dysfunction for several years during the prodrome [7,10,12]. notably, αsyn-immunoreactive inclusions have been found in neurons of the submucosal plexus in people with pd [3,13]. taken together, this converging evidence suggests an early involvement of the enteric nervous system (ens) in the pathogenesis of pd. already over a decade ago, braak and colleagues hypothesized that αsyn-immunoreactive inclusions first appear in the ens and then occur in the parasympathetic (e.g., vagal output neurons in the intestines) and sympathetic (e.g., in the celiac ganglion in the upper abdomen) nervous system and gradually engage the brainstem, including the vagal dorsal motor nucleus and midbrain areas [3,13]. several studies in preclinical models have demonstrated that αsyn pathology in the gut is associated with the development of αsyn pathology in the brain [14–21]. for a better understanding of pd pathogenesis and particularly events happening at preclinical stages of pd, it is critical to determine factors that regulate αsyn accumulation in the ens and to understand whether the process underlying αsyn accumulation in the gut can also lead to αsyn pathology in the brain. inflammation can potentially trigger αsyn pathology in the ens of the gut and in the brain. a recent finding in children with gastrointestinal inflammation suggests an immune regulatory function of αsyn [22]. immune pathways are indeed activated in the brain and colon of pd cases [23,24]. also, several genes associated with an increased pd risk have an immune system-related function [25], and it was recently proposed that pd heritability is not simply due to variation in brain-specific genes, but that several cell types in different tissues are involved [26]. further genetic evidence supporting a role for immune pathways in pd pathogenesis is provided by a genome-wide association study that identified common genetic pathways linking pd and autoimmune disorders [27]. most prominently, lrrk2, a major genetic risk factor for pd [28] also confers increased risk for developing inflammatory bowel disease (ibd) [29]. certain risk alleles are shared between pd and crohn’s disease [30], and lrrk2 is known to modulate the function of monocytes, macrophages and other immune cells [31,32]. intriguingly, ibd is associated with an increased risk for developing pd and specifically blocking the tumor necrosis factor (tnf) pathway reduces this risk [33–37]. recently, it was reported that experimental colitis in αsyn transgenic mice leads to enteric accumulation of αsyn and the development of pd-like brain pathology and symptoms within a few months [38]. converging clinical and nonclinical data suggest that the intestinal immune environment plays a role in triggering pd or facilitating the molecular events involved in the earliest phases of the disease process [39,40]. here, we tested the hypothesis that specific types and severity of intestinal inflammation are required to trigger the accumulation of αsyn in the ens and the subsequent development of αsyn pathology in the brain. experimental forms of colitis in wild type and αsyn transgenic mice demonstrated that the type and degree of inflammation regulates the amount of αsyn accumulation in the colon. macrophage-related signaling limited the extent of αsyn immunoreactivity as demonstrated in a genetic and a pharmacological immune modulation paradigm in the experimental colitis mouse model. most remarkable, when αsyn transgenic mice were exposed to experimental colitis at 3 months of age and then were allowed to age normally up to 9 or 21 months, the accumulation of aggregated αsyn in midbrain, including the sn, was much exacerbated in the 21-month-old group, but not in the 9-month-old group. these 21-month-old mice also exhibited loss of nigral tyrosine hydroxylase-immunoreactive neurons. together, our data provide experimental evidence in mice that certain specific forms of intestinal inflammation might be a relevant upstream trigger that plays a critical role in the initiation of pd pathogenesis and the disease progression. methods aim, design and setting we aimed to combine an αsyn transgenic mouse model of age-dependent development of αsyn pathology with well-established experimental colitis paradigms in order to explore the effect of type and severity of immune activation on the development of αsyn pathology in the colon and the brain. the design and setting of the different studies are illustrated in fig. 1. fig. 1 age-dependent increase of intracellular αsyn accumulation in enteric nervous system of hemizygous (thy1)-h[a30p]αsyn transgenic mice and setup of the experimental colitis paradigms. a confocal microscopy imaging of the inclusions of human αsyn (red, antibody clone 211; human αsyn specific) within the ganglia of the submucosal plexus (green, peripherin; blue, dapi/nuclei) of hemizygous (thy1)-h[a30p]αsyn transgenic mice. arrowhead points to one of the typical irregularly sized and shaped αsyn inclusion bodies visualized in 2d z-stacks of rotated confocal images. scale bar: 100 μm. b stereological quantification of normally occurring human αsyn inclusions in the myenteric and submucosal plexuses of 3and 12-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice (n = 4 per group; mean and s.e.m. are shown; student t-test between the two age groups in each region). c setup of experimental colitis paradigms employing dextran sulfate sodium (dss, per os in drinking water) or bacterial lipopolysaccharide (lps, intraperitoneal injection). except for the ‘chronic dss paradigm, constant dose’, all paradigms were started at the age of 3 months. the ‘chronic dss paradigm, constant dose’ was started in mice aged 5 months and the colon were analyzed right after. for some experiments, we used a ‘chronic dss paradigm, increasing dose’, to mimic better the chronic nature of ibd and the longer water intervals are generally more gentle for the mice from an animal welfare perspective as well. under this paradigm, we induced experimental dss colitis intermittently as indicated over 23 days, then let the mice recover and age for two more months up to the age of 6 months on normal drinking water, and analyzed their colon. in a separate experiment, we aged the mice further up to 9 or 21 months and analyzed their brain pathology. open arrows on time axis indicate that colon was analyzed and stars with closed arrows indicate that brains were analyzed. d hematoxylin staining of 35 μm thick colon sections of 3-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice. organizational layers of the intact colon (left panel). representative images of various severity degrees of dss-driven colitis from weak leukocyte infiltration (top panel of acute dss) to more extensive leukocyte infiltration with mucosal ulceration (lowest panel of acute dss). note the different appearance of enteric inflammation in acute lps-driven peripheral inflammation compared with dss, e.g., confined immune cell clustering and lymphoid hyperplasia, intact mucosal layer. scale bar: 50 μm (intact colon), 100 μm (acute dss), and 200 μm (lps). mice male c57bl/6j wild type mice (jackson laboratories, bar harbor, usa), hemizygous tg(thy1-snca*a30p)18pjk ((thy1)-h[a30p]αsyn) [41] and tg(thy1-snca*a30p)18pjk crossed with cx3cr1tm1litt ((thy1)-h[a30p]αsyn /cx3cr1-def; homozygous for cx3cr1-gfp knock-in allele; [42] transgenic mice were used for the study. (thy1)-h[a30p]αsyn transgenic mice express mutant human αsyn under the neuron selective thy1 promoter. (thy1)-h[a30p]αsyn transgenic mice were crossed to cx3cr1-def transgenic mice which express egfp replacing fractalkine receptor gene expression. all mice were maintained on a c57bl/6j background for more than 10 generations and under specific pathogen-free conditions. to the extent possible, littermates were used in the experiments. health status was monitored daily during experiments. the in vivo experiments were endorsed by a roche internal review board and approved by the local animal welfare authorities of the canton basel-stadt, basel, switzerland. experimental colitis paradigms in mice paradigms for the induction of inflammation were either 1 week (acute) or 3-4 weeks (chronic) with or without an incubation phase under normal conditions of 2-, 6-, or 18-months post application (fig. 1). acute systemic inflammation was induced by intraperitoneal (i.p.) lipopolysaccharide (lps) application [43] of 0.5 mg/kg in 100 µl injection volume on day 0 and 3 (sigma-aldrich chemie gmbh, steinheim, germany, lps 055:b5). acute colitis was induced by application of 36-50kda dextran sulfate sodium (dss) [44] (160110, mp biomedicals, llc, illkirch, france) at 1%, 2.5% or 5% in autoclaved drinking water for 5 continuous days respectively, followed by 2 days of water (1 dss application cycle). chronic colitis was induced by two different dosing protocols: i) in a constant dose of dss (1% or 2.5%) for 5 days and changed to 2 days with normal drinking water and repeated three times (4 application cycles in total); ii) in an increasing dose of dss starting at 1% for 5 days followed for 4 days on normal drinking water, then increased to 1.5% dss for the next 5 days followed by 4 days of water and a final cycle of 2% dss followed by aging the mice on normal drinking water until they were sacrificed. mice from the same littermate group were randomized per cage into ‘exposed to inflammation inducing agent’ (lps or dss, respectively) or ‘unaffected’ (vehicle for the lps paradigm or normal drinking water for the dss paradigms, respectively). for the long-term experiments with the two aging cohorts ‘9 months’ and ‘21 months’, respectively, all mice in that study were simultaneously exposed in one large cohort at the age of about 3 months to the increasing dose chronic dss paradigm (fig. 1) and dss exposure was stopped for all mice on the same day after the 23-day period. the mice were then kept and aged on normal drinking water and under normal housing conditions in the same room until the day they were perfused and tissue was collected. il-10 treatment and exposure measurement two different forms of mouse igg bound murine il-10 (migg(v1)-mil10 and migg(v2)-mil10) were diluted in pre-prepared sterile formulation buffer comprised of 0.5% mouse serum supplemented with 25 mm citrate, 300 mm arginine to a final concentration of 0.75 mg/ml and the ph adjusted to 6.7 on the day of application. each mouse was treated once with 150 µg i.p. concurrently with the initiation of the acute colitis paradigm with 5% dss. the concentrations of migg-mil10 fusion proteins in murine serum samples were determined by enzyme-linked immunosorbent assays (elisa) specific for the fab moiety of the administered migg-mil10 fusion protein. biotinylated migg-mil10-specific target molecules were used for capturing, goat anti-mig igg-hrp conjugate and peroxidase substrate abts were used for quantitative detection of migg-mil10 fusion proteins. immunohistochemistry mice were injected with a lethal dose of pentobarbital (150 mg/kg). upon full anesthesia, mice received transcardial perfusion with room temperature phosphate buffered saline (pbs). for biochemical and immunohistochemical analysis, one section of the proximal colon was either fresh frozen and stored at -80°c or post-fixed in 4% paraformaldehyde (pfa) solution for 24 h. following post-fixation, organs were incubated in 30% sucrose/pbs at 4°c for at least 48 h before further processing. subsequently, enteric tissue was cryotome-sectioned to 35 µm thick longitudinal sections (approx. 1 cm length). the brain was collected and post-fixed for 24 h in 4% pfa followed by 30% sucrose in phosphate buffer until cryo-sectioning of floating sections at 40 μm. histological analysis of mouse colon was performed using standard hematoxylin staining. immunohistochemical staining was accomplished using the vectastain elite abc kits and peroxidase substrate kit sk-4100 (vector laboratories, burlingame, ca, usa) or fluorescently labelled secondary antibodies (alexa 488, 555 or 647, life technologies, zug, switzerland). the following primary antibodies have been used for overnight incubation at a dilution of 1:1000; monoclonal antibody to human αsyn (clone 211, sc-12767, santa cruz biotechnology, heidelberg, germany; specific to human αsyn and binds to normal αsyn as well as abnormal αsyn inclusions which contain the respective epitope), monoclonal antibody generated towards rat αsyn, cross-reactive with murine and human αsyn (syn1/clone 42, bd transduction laboratories, allschwil, switzerland; used for wild type mice), polyclonal antibody to the peripheral neuronal marker peripherin (millipore corporation, billerica, ma, usa), and polyclonal antibody to macrophage marker iba-1 (wako chemical gmbh, neuss, germany). to detect αsyn phosphorylated at serine 129 (pser129-positive inclusions of pathological/abnormal αsyn) in the free-floating brain sections, monoclonal antibody (ab51253, abcam, cambridge, usa) was used at a dilution of 1:10000. prior to the pser129 staining, the free-floating brain sections were incubated for 10 min at room temperature in a phosphate buffered saline solution containing 10 μg/ml proteinase k (cat # 25530015; invitrogen, california, usa). tyrosine hydroxylase (th)-immunoreactive cells were detected using a polyclonal antibody (657012, millipore sigma) at a dilution of 1:1000. to measure the density of nissl-positive cells, the th-stained cells were counter-stained with cresyl violet. the slides were incubated in 0.1% cresyl violet solution for 9 min and then dehydrated in 95% and 100% ethanol and then xylene prior to cover slipping with cytoseal 60 mounting media (thermo fisher scientific). quantifications of the blind-coded th/nissl-stained slides were done using stereo investigator (version 2017.01.1; mbf bioscience, williams, vt, usa) on an imager m2 microscope (zeiss) coupled to a computer. we analyzed 5-7 nigral sections per animal, and a total of 7-8 animals per treatment group. we outlined the substantia nigra pars compacta and counted every th-immunoreactive and nissl-positive cell in that area (using a counting frame of 40 µm x 40 µm, grid size of 140 µm x 140 µm, a guard zone of 2 µm and optical dissector height of 20 µm) and then computed the number of cells per section, generating the average cell count per animal. we then calculated the average count of cells per treatment group and analyzed the data using unpaired student’s t-test after confirming normality and homoscedasticity in prism 7.0 (graphpad software). imaging and stereological quantification of αsyn deposits in enteric nervous system imaging and stereological quantification were performed on a zeiss axio imager z2 fluorescence microscope (carl zeiss ag, jena, germany). leica tcs sp5 confocal system using an hcx pl apo cs 40x 1.3 oil uv or an hcx pl apo lb 63x 1.4 oil uv objective was utilized for image recording. accumulation of αsyn in the ens (i.e., punctate intracellular bodies/features) was assessed on a random set of 3 adjacent 35 µm thick, αsyn-immunostained sections comprising the myenteric and submucosal neuronal plexuses. analysis was performed with the aid of a stereology software (stereo investigator 10, mbf bioscience, williams, vt, usa) as described previously [45]. in the myenteric plexus ganglion, volume was defined by multiple outlined plexuses containing a range of 5-20 neuronal cells and quantified by the optical fraction fractionator technique. in contrast to the myenteric plexus, the submucosa consists of compact plexuses with 1-5 cells including interconnecting neurites. therefore, the entire submucosa was set as region of interest, analyzed with the area fraction fractionator technique. results of the submucosal plexus are displayed by percent area containing αsyn deposits. for the il-10 experiment, αsyn positive inclusions from immunofluorescence images were counted for each image. inclusion body-like features were filtered based on having a size between 12 and 50000 pixels and a minimal intensity value greater than 300. the filtering step was performed to exclude small background features and macrophages (very large spots). the counts were then aggregated to the animal level by summing the inclusion feature counts of all images per animal and then normalizing for (i.e., dividing by) the number of images for a given animal. upon exploratory data analysis two mice were excluded: one mouse because it only had one image (technical outlier, missing data point; repeating the staining for this one mouse would have required re-staining the entire cohort in order to be consistent with staining conditions for quantification; this was unnecessary after statistical analysis) and another due to it being an outlier, based on its infiltration score and image data (i.e., in contrast to the other mice that had received dss, this mouse did not show signs of inflammation or colonic tissue damage that is normally induced by dss; it could not be determined if that mouse was correctly dosed with dss and thus it was excluded from the analysis). quantification of leukocytes infiltration to determine the leukocyte covered area in the colon after lps or dss application, three adjacent hematoxylin-stained sections were quantified. total area of colon sections and localizations of leukocyte assemblies within the tissue architecture were identified and outlined utilizing a stereology software (stereo investigator 6, mbf bioscience, williams, vt, usa). percentage of leukocyte covered area has been set in proportion to total area of the analyzed colon section, e.g., to at least the length of 1 cm of proximal colon. for the il-10 experiment, hematoxylin-stained colon slices were examined by an expert pathologist blinded to treatment conditions. a score of 0-3 was assigned to each section for each of the 3 layers lamina propria, submucosa and muscularis based on the degree of inflammatory infiltration. a score of 0 denoted no inflammation and a score of 3 indicated extensive infiltration. the mean of the values for all 3 layers was taken as the final measure of leukocyte infiltration per mouse. quantification of iba-1/αsyn-double positive macrophages the number of iba-1/αsyn-double positive cells was evaluated by quantification of 10 random regions in 2 adjacent sections of the proximal colon. the region of interest was set to contain the myenteric plexus/circular muscle layer and the submucosal plexus. scoring of pser129 pathology and brain heatmap we evaluated pser129 pathology on a full series of immunostained coronal sections from 10 mice per treatment group (i.e., water vs. dss-treated groups) on blind-coded slides using a previously described method [46]. we visualized pathology from one hemisphere of all brain sections (apart from the olfactory area) using a nikon eclipse ni-u microscope and assigned scores ranging from 0 to 4 to each brain area based on the relative abundance of proteinase k (pk)-resistant pser129-positive inclusions (i.e., cell bodies and neurites). in this case, 0 = no aggregates, 1 = sparse, 2 = mild, 3= dense, 4 = very dense. for the heatmap, we obtained the average score values of each brain area for each treatment group. the average data for each treatment group (n=10 mice/group) was then represented as a heatmap in a sagittal mouse brain background. to create the brain heatmap a postscript file downloaded freely from allen brain atlas (mouse, p56, sagittal, image 15 of 21; -> weblink*) was converted to an xml in r v 3.4.4, and the mean scores were manually assigned to respective brain regions. the remaining brain regions were estimated via the r package ‘akima’, using a pointwise bivariate interpolation algorithm for irregular data on the mean x and y coordinates for each brain region.   *(http://atlas.brain-map.org/atlas?atlas=2#atlas=2&structure=771&resolution=16.75&x=%7755.7470703125&y=3899.625&zoom=-3&plate=100883867&z=5) densitometry of pser129 αsyn brain pathology the density of pser129 pathology in 12 major brain areas (reticular nucleus, pontine reticular nucleus, periaqueductal gray, gray and white layer, reticular formation, substantia nigra, ventral tegmental area, thalamus, hypothalamus, central amygdala, pallidum and striatum) was determined in the water and dss-treated animals. a nikon eclipse ni-u microscope was used to acquire 20x magnification images (without condenser lens) from all the indicated brain areas, using the same exposure time for all images. in all cases, images were acquired on three sections separated by 420 μm intervals (localized between bregma). we then processed the acquired images using image j64 [47], created a mask (to exclude background) that redirects to the original image for analysis, measured the total area and the mean grey value of the area that had inclusions. for brain areas such as periaqueductal gray that do not fill the entirety of the field to be analyzed, we drew a contour of the area and the analysis was performed only within that contoured area. we subsequently calculated the grey value of the area per square pixels for each image (i.e., a.u./px2 = mean grey value x area stained/total area assessed). based on this, we calculated the average grey value per square pixels for each brain area for each animal (n = 6 mice/group), and then extended this calculation to determine the average grey value per square pixels for each treatment group and each of the twelve brain areas of interest. blinding of experimenters for histological and immunohistochemical analyses for analyses of colon and brain tissue on slides, a second individual assigned unique codes to stained slides. therefore, the experimenter conducted the analyses blinded to the identity of the mice. for randomization of treatment groups see above. mrna expression to assess mrna expression levels from the proximal colon, rna was extracted from fresh frozen tissue with magnalyser green beads (roche diagnostics, mannheim, germany) and qiazol lysis (reagent cat.no.79306, hilden, germany) purified on magnapure lc (hp kit no.03542394001, f. hoffmann-la roche ag, rotkreuz, switzerland) and amplified via real-time pcr (4 ng rna/reaction; lightcycler 480, roche diagnostics corporation, indianapolis, usa). amplification of mrna was performed by using taqman probes for human or murine specific α-synuclein and for selected cytokines/chemokines (applied biosystems europe b.v., zug, switzerland). target mrna was normalized to tissue specific murine gapdh levels and displayed as relative expression after 30 amplification cycles. statistics measurements for inflammation and αsyn accumulation in the ens were taken from distinct samples (e.g., in three to six technical replicates per mouse). data from each mouse was used only once, thus no repeated measure of the same sample was performed. statistical analysis of gut pathology and inflammation was performed using graphpad prism 6.04 or 7.0 software (graphpad software, inc. la jolla, ca, usa). the results are expressed as mean values ± standard errors of the mean (s.e.m.). student’s t-test (or welch’s t-test for unequal variances) was used to compare two groups and anova was used for multi-comparison of groups followed by tukey hsd post-hoc analysis. for the statistical analysis of the mrna expression, data quality was assessed by inspecting the distribution of cp values of reference endogenous genes across samples, by inspecting the level of cp variation between technical replicates and by exploring the samples multivariate signal distribution as in a principal component analysis. relative gene expression levels were expressed as 2-(cpgene – cpref). statistical analyses to assess the effect of the experimental conditions on the log2 gene expression levels were done with linear models using the limma package (bioconductor/r, [48]). these analyses were implemented in r v 3.1.1. for the statistical modelling of the effects of the il-10 treatment on αsyn counts, as well as infiltration scores, the levels for igg1(v1)-il10 and igg1(v2)-il10 treatment were compared to the positive (vehicle/dss) control. additionally, since levels of the control antibody treatment (igg1(v1)) were very similar to the positive control, the two groups were pooled in further contrasts in which effects of individual antibodies or control igg was assessed. for αsyn counts in the enteric nervous system, a linear model on the treatment groups with one-degree freedom contrasts was applied. for the infiltration score a kruskal-wallis test, with the same contrasts, was used. all statistical tests were two-tailed with a significance level of p <0.05. for the statistical analysis of the pser129 αsyn brain pathology, zero-inflated negative-binomial mixed-effects models with a random intercept for each sample and variance assumed to increase linearly with the mean (verified against a quadratic increase using akaike information criterion [aic] and bayesian information criterion [bic]) were used to analyze the dataset via the ‘glmmtmb’ package in r v 3.4.4. linear contrasts with false discovery rate (fdr) adjustments were then used to test our hypotheses and account for multiple testing (for brain area and experimental group). results experimental colitis exacerbates αsyn load in the submucosal plexus of αsyn transgenic and wild type mice during the process of further characterizing a (thy1)-h[a30p]αsyn transgenic mouse line [41], we detected by immunohistochemistry human αsyn accumulation in all innervated organs that were analyzed (suppl. fig. 1). to examine the localization of αsyn inclusions in nervous structures in the ens, we performed an immunofluorescent co-staining for human αsyn (clone 211) and peripherin, a specific marker for peripheral nerves. by applying confocal microscopy, we established a process and protocol to identify myenteric and submucosal plexuses in order to quantify the αsyn inclusions found in the nerve cells of the ganglia in the colon. the intracellular presence of the irregularly sized and shaped αsyn inclusion bodies was confirmed in 2d z-stacks of rotated confocal images (fig. 1a). we observed an age-dependent (mice aged 3 months versus 12 months) increase of baseline human αsyn inclusions in both plexuses (fig. 1b). given the clinical and epidemiological link between ibd and pd, we wanted to test whether different types and strengths of ibd-related experimental inflammation in the colon exacerbates this local accumulation of αsyn acutely (e.g., within a few days or weeks) and how the age of the αsyn transgenic mice may influence the outcome. administration of dextran sulfate sodium (dss) in the drinking water in acute or chronic dosing paradigms are well-established mouse models of experimental colitis mimicking aspects of ibd, i.e., by exhibiting infiltration of leukocytes into the submucosa with various degrees of destruction of the colonic mucosa and submucosa [49]. it is well-known that the effects induced by the dss paradigm can vary substantially based on the genetic background of the mice and due to different animal housing environments. thus, in order to establish the dss paradigm in our environment and with our mice, we first tested the effect of dss administration at different doses and durations in the (thy1)-h[a30p]αsyn transgenic mice (fig. 1c). we observed that leukocyte infiltration was appropriately modulated by the acute dose (1% or 2.5% dss for 5 days followed by 2 days drinking water) and chronic constant dose (1% or 2.5% dss alternating with normal drinking water for 28 days, respectively) dss paradigms (fig. 1d and 2a). in the same experiment, we wanted to test for an age effect on a potential aggravation of αsyn accumulation in the ens and applied the acute dose paradigm on 3-month-old mice and the chronic dose paradigm was started at the age of 5 months leading to a final age of 6 months at analysis of the mice. in the acute dss dosing paradigm with mice at the age of 3 months, 2.5%, but not 1%, dss triggered intracellular accumulation of αsyn in peripherin positive nerve cells of the submucosal plexus (fig. 2a, b). in the 28-day chronic constant dss dose paradigm in mice aged 6 months (fig. 1c and 2a), we observed that the 1% constant chronic dss dose showed a similar degree of αsyn inclusions as the 3-month-old mice which were on an acute 2.5% dss dose paradigm. in addition, mice previously exposed to 2.5% constant chronic dss dose presented on average with a slightly but robust increased percent area of αsyn inclusions compared with the 6-month-old mice that were on 1% dss (fig. 2a). together, this demonstrated that different dss paradigms can be established in (thy1)-h[a30p]αsyn transgenic mice and that different dss paradigms can induce robust elevation of αsyn inclusions. this first experiment also provided us with data points to estimate a potential effect size for triggering αsyn inclusions in the different dss paradigms. we observed that the younger (thy1)-h[a30p]αsyn transgenic mice showed a robust increase in αsyn inclusions and better signal-to-noise conditions of the autofluorescent colonic tissue than the older ones, experiments were henceforth continued with mice of the younger age. in this initial experiment, it was also interesting to observe that (thy1)-h[a30p]αsyn transgenic mice exposed to acute 2.5% dss colitis presented with several αsyn-positive cells with a morphology consistent with them being infiltrating leucocytes, which was confirmed by an iba-1 co-staining (suppl. fig. 2). this finding was relevant for the quantification of αsyn inclusions in the myenteric and submucosal plexus, i.e., such features were excluded from the quantification process. fig. 2 experimental dss colitis severity and duration-dependent aggravation of accumulation of αsyn inclusions in the colonic submucosal plexus of hemizygous (thy1)-h[a30p]αsyn transgenic and wild type mice. a administration of dss in drinking water induced a robust increase of leukocyte infiltration in the acute (1% or 2.5% dss for 5 days followed by 2 days of normal drinking water; one group was kept on normal drinking water) and chronic constant dss dose (1% or 2.5% alternating with normal drinking water) paradigm in hemizygous (thy1)-h[a30p]αsyn transgenic mice. the highest acute dose (2.5%) and the two constant chronic doses led to a very robust increase of αsyn inclusions in the submucosal plexus (stereological quantification of αsyn inclusions in the submucosal plexus of all 3and 6-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice; n = 5-7 per group; mean and s.e.m. are shown). b representative 2d z-stacks of confocal images of increasing abundance of αsyn inclusions (red, human-αsyn specific monoclonal antibody clone 211) in a ganglion of the submucosal plexus (green, peripherin) with cellular nuclei in blue (dapi) in the acute dss paradigm. arrow heads point to the typical irregularly sized and shaped αsyn inclusion bodies that accumulate in the highest dss dose. scale bar: 200 µm. c overview of colonic region of 3-month-old wild type mice exposed to water or acute dss (5%) with immunofluorescence analysis of murine αsyn load in the colon performed immediately after colitis. white dotted rectangles in the top row indicate the area that was zoomed-in in the lower panels. in the zoom-ins we show representative images of dapi and αsyn (red, rodent αsyn cross-reactive monoclonal antibody syn1/clone 42) inclusions with and without the peripherin channel (green). the white dotted circled area illustrates the peripherin-positive area that was analyzed for αsyn inclusion bodies (arrow heads in bottom row). scale bar for the lower three panels: 200 μm. d stereological quantification of murine αsyn inclusions in the submucosal plexus of wild type mice right after acute dss colitis (n = 5 per group). note the regularly arranged and smoothly distributed immunoreactivity for the physiological αsyn with barely any inclusion bodies in the intact enteric nerves of the water group. for both panels (a) and (d), statistical analysis for αsyn accumulation was omitted as the noticeable and very robust differences between the means are self-evident (error bars indicate standard error of the mean) and an indication for an estimation for significance would be irrelevant. wild type mice also express endogenous αsyn in innervated organs, but at much lower levels compared with the levels of human αsyn expressed by the hemizygous (thy1)-h[a30p]αsyn transgenic mice (suppl. fig. 1). to confirm that the finding in (thy1)-h[a30p]αsyn transgenic mice was independent of transgenic expression of human αsyn, we applied an acute 5% dss dose paradigm (5 days dss + 2 days water) in wild type mice (fig. 1c). we observed, in the submucosal plexus, small inclusion bodies of endogenous murine αsyn (detected by rodent cross-reactive αsyn-specific monoclonal antibody syn1/clone 42, fig. 2c, d). these features were close to undetectable in the water group that did not experience experimental colitis. a separate experiment also confirmed that the observed effects of elevated αsyn inclusions following acute dss (5% dose) could not be attributed to increased gene expression of murine or the transgenic human αsyn (suppl. fig. 3). together, these results confirmed the validity of this experimental ibd paradigm to test the effect of inflammation on αsyn accumulation in the ens in wild type and (thy1)-h[a30p]αsyn transgenic mice. because the (thy1)-h[a30p]αsyn transgenic mouse model is well-established to analyze human αsyn-related pathology, we focused for the remainder of the study on employing these transgenic mice. colitis induced by peroral dss but not by intraperitoneal administration of lps aggravates αsyn accumulation in colonic submucosal plexus of αsyn transgenic mice different inflammatory agents induce different types of immune stimulation and thus can influence the phenotype of experimental colitis. a well-established experimental immune trigger is the bacterial endotoxin lps. in order to explore the effects of different approaches to induce inflammation in or nearby the gut in (thy1)-h[a30p]αsyn transgenic mice, we compared the outcome of acute (5 days dss + 2 days normal drinking water) 5% dss per os with acute 0.5 mg/kg intraperitoneal lps administration (fig. 1c and 3). to maximize the inflammatory response, we administered both dss and lps at relatively high doses. at day 7, both agents had induced variable degrees of leukocyte infiltration in the submucosa of the colon while a marked destruction of the mucosa was induced when giving only dss (fig. 1d). as before, the dss-exposed mice presented with increased accumulation of αsyn in the ganglia of the submucosal plexus (fig. 3a). in contrast, we detected no change in αsyn load in the myenteric plexus, consistent with lack of leukocyte infiltration in this part of the colonic wall (fig. 3b). despite the high dose, lps-induced inflammation did not increase αsyn accumulation in the colonic nervous plexuses (fig. 3c, d). notably, lps and dss resulted in a differential expression of cytokines, and consistent with leukocyte recruitment, ccl2 was elevated in both (fig. 3f, g). in the lps paradigm, mrna for il-10 was markedly elevated, whereas dss strongly increased il-6 and also il-1β but not il-10. together these results indicate that, in our model, colonic inflammation induced by peroral dss but not intraperitoneal lps increases the accumulation of αsyn in the colon. fig. 3 colitis induced by peroral dss but not peritoneal lps enhances αsyn accumulation in the colonic submucosal plexus of hemizygous (thy1)-h[a30p]αsyn transgenic mice and can be increased by lack of monocyte/macrophage-related cx3cr1 signaling. mice received in an acute paradigm either peroral 5% dss in their drinking water or intraperitoneally 0.5 mg/kg lps. effects of dss and lps in the colon, respectively, were compared to effects induced by vehicle (see figure 1c for timelines). stereological quantification of αsyn inclusions in the submucosal plexus as % area (a, c) and in the mucosal plexus as particle load per ganglion (b, d) (two-way anova with tukey post hoc test; covariates genotype and treatment paradigm). e representative 2d stacks of confocal images of intracellular αsyn inclusions (red, human αsyn specific monoclonal antibody clone 211; arrow heads pointing to some selected inclusions) in a ganglion of the myenteric plexus (green, peripherin) with cellular nuclei in blue (dapi). scale bar: 50 μm. gene expression analysis of selected cytokines in the colon of (thy1)-h[a30p]αsyn transgenic mice that received either acute dss (f) or lps (g) compared to their respective vehicle or water controls. note the strong increase in il-6 and the lack of elevation of il-10 in the dss paradigm compared to the lps paradigm indicating a different inflammatory colonic milieu despite the abundant leukocyte infiltration in both paradigms. n = 5-8 per group; mean and s.e.m.; student’s t-test between inflammatory agent and vehicle for individual cytokines. lack of monocyte/macrophage related cx3cr1 signaling during dss colitis increases αsyn load in the submucosal plexus of αsyn transgenic mice given the role of monocytes/macrophages in ibd and in the related dss paradigm, we hypothesized further that modulating monocytes/macrophages may affect accumulation of αsyn in our dss model as well. in a first set of experiments we manipulated monocytes/macrophages genetically by crossing (thy1)-h[a30p]αsyn transgenic mice with mice that have a deletion for the fractalkine receptor cx3cr1 (cx3cr1-gfp knock-in mice) (fig. 3a, b). the cx3cr1-cx3cl1 axis plays an important role in maintaining the function of the lamina propria macrophage population of the gastrointestinal wall and lack of this signaling pathway in experimental colitis models may either aggravate or ameliorate the induced pathology [50–52]. in our experiment, the area covered by infiltrating leukocytes following exposure to dss was near the mucosa and submucosa and was not significantly higher in the cx3cr1-deficient αsyn transgenic mice than in the cx3cr1-competent mice (suppl. fig. 3a). however, a significantly higher level of αsyn accumulated in the submucosal plexus in αsyn transgenic mice lacking cx3cr1 compared to αsyn transgenic mice expressing cx3cr1 (p = 0.001, two-way anova with tukey hsd post-hoc analysis; fig. 3a). in the myenteric plexus, we found no marked increase in αsyn accumulation in neither the αsyn transgenic mice with normal cx3cr1 nor the αsyn transgenic mice deficient in cx3cr1, indicating as in the experiments above a possible prominent role for the localization of leukocyte infiltration in the process of αsyn accumulation in the submucosa (fig. 3b). collectively, our results in cx3cr1-deficient αsyn transgenic mice provide a potential association between monocyte/macrophage signaling and αsyn accumulation in ens in this experimental ibd model. systemic il-10 reduces dss-induced colitis and associated enteric αsyn accumulation in αsyn transgenic mice to continue testing the hypothesis that modulating monocytes/macrophages may affect accumulation of αsyn in our dss model we moved to a pharmacological modulation of this cellular subset. interleukin-10 (il-10) is an important regulator of monocytes/macrophages, and genetic ablation of il-10 signaling or blocking il-10 with specific antibodies has been reported to enhance dss colitis [53,54]. in the experiments with lps we had also noted an increase of il-10 compared with the dss paradigm and lps inflammation was in contrast to dss colitis not associated with increased αsyn accumulation in the ens (fig. 3). to mimic the effect of higher levels of il-10 in an acute model of dss colitis (5% dss for 5 days + 2 days normal drinking water, fig. 1c), we administered intraperitoneally recombinant murine il-10 (mil10) in this paradigm. the half-life of injected recombinant il-10 protein in blood is very short. to reduce the number of injections, we extended the half-life of mil-10 in circulation by engineering it onto two different murine igg variants (i.e., migg1(v1)-mil10 and migg1(v2)-mil10, respectively). as described above, dss induced a marked increase in leukocyte infiltration and αsyn accumulation, and we found both to be similar in the untreated and control igg treated group (fig. 4a, b). in contrast, both migg1(v1)-mil10 and migg1(v2)-mil10 significantly reduced leukocyte infiltration in mice treated with dss (p<0.0001, one-way anova with tukey hsd post-hoc analysis; fig. 4a, b). a significant down-regulatory effect of an il-10 treatment on dss colitis induced accumulation of human αsyn in the submucosal plexus was only observed with migg1(v2)-mil10 (p=0.02, one-way anova with tukey hsd post-hoc analysis; fig. 4b). this effect by migg1(v2)-mil10 on αsyn levels was accompanied by detectable serum levels of migg1(v2)-mil10 at the end of the in vivo phase, whereas migg1(v1)-mil10 was no longer detectable at that point (fig. 4c). this indicates that although both forms of il-10 have a down-regulatory effect on leukocyte infiltration, a sustained pharmacological exposure of il-10 may be required for reducing αsyn accumulation. these results highlight an important role for the il-10 pathway in keeping αsyn accumulation at a reduced level throughout the course of experimental ibd. together, our observations by genetic (i.e., cx3cr1-cx3cl1 axis) and pharmacological modulation (i.e., il-10) of dss colitis corroborate an important role for monocyte/macrophage pathways in the development of αsyn accumulation in the ens of the colon. fig. 4 systemic il-10 ameliorates dss colitis and slightly reduces associated local αsyn accumulation in (thy1)-h[a30p]αsyn transgenic mice. two different recombinantly engineered and murine igg1-fused forms of murine il-10 (migg1(v1)-mil10 and migg1(v2)-mil10) were administered (150 µg per mouse i.p.) at the beginning of the acute dss paradigm (5%) in (thy1)-h[a30p]αsyn transgenic mice. vehicle and the migg1(v1) alone served as untreated controls. a leukocyte infiltration was assessed by visual scoring and (b) inclusion features of αsyn were stereologically and semi-automatically quantified and result log scaled for statistical analysis. both the vehicle group and the migg1(v1) group had similar levels of leukocyte infiltration and αsyn inclusions and were merged for the statistical analysis to compare with the il-10 treated groups. both forms of il-10 ameliorated leukocyte infiltration whereas migg1(v2)-mil10 also blocked the appearance of αsyn inclusions significantly (n = 3-6 per group; mean and s.e.m.; one-way anova and tukey post hoc test). c persistent exposure migg1(v2)-mil10 versus migg1(v1)-mil10 (lower limit of detection is indicated at <0.234 μg/ml) as measured in serum at the end of the in vivo phase corresponds with beneficial treatment effects on αsyn readout observed above. the migg1(v1) was only measured in two mice. dss colitis-induced submucosal αsyn accumulation at a young age persists for months and is exacerbated by lack of cx3cr1 signaling in humans there is strong epidemiological evidence that ibd increases pd risk [33,35,37] and recent evidence in crohn’s disease [55] indicates that such gut inflammatory conditions are associated with αsyn accumulation in the ens [36]. in these experiments in mice, we have until here established and replicated, in different setups, a link between modulation of inflammation and induction of αsyn accumulation in the ens. because longer exposure to dss (i.e., over several weeks) mimics more closely the chronic nature of ibd, we elected to explore αsyn accumulation in the submucosal plexus of (thy1)-h[a30p]αsyn transgenic mice that were subjected to dss colitis in a 4-week chronic increasing dose paradigm. in addition, the dose increase with longer water intervals is more gentle for the mice from an animal welfare perspective. in order to allow for a full recovery from the chronic inflammation, we aged the mice for two months on normal drinking water and analyzed them at the age of 6 months (fig. 1c). at this point we wanted again to explore the effect of modulating monocytes/macrophages in this chronic setting and added an experimental arm with (thy1)-h[a30p]αsyn transgenic mice lacking cx3cr1. as expected, after 2 months of recovery, the area that is usually extensively covered by leukocytes in the submucosal plexus of the acute dss paradigm had returned to normal levels following the two-month recovery period (suppl. fig. 4a). remarkably, however, the area containing αsyn inclusions in the ganglia of the submucosal plexus was still almost doubled when compared to αsyn transgenic mice that were not exposed to dss, and this was exacerbated in αsyn transgenic mice deficient for cx3cr1 (suppl. fig. 4b). the finding in the αsyn transgenic mice suggests that accumulation of αsyn is not a transient effect or response. in addition, modulation of monocytes/macrophages by down-regulating the cx3cr1-cx3cl1 axis contributes to aggravation of this accumulation. experimental dss colitis-induced at a young age exacerbates αsyn brain pathology and dopaminergic neuron loss in old αsyn transgenic mice at this point, we have established and repeatedly demonstrated that modulation of inflammatory mechanisms in experimental colitis induced by acute and chronic dss administration is causatively linked to induction and persistence of intracellular αsyn inclusions in the ens of young adult mice. the previously highlighted hypothesis by braak and colleagues associates αsyn brain pathology in pd with αsyn pathology in the ens earlier in life [3,56]. to assess development of brain αsyn pathology and to link it to ibd risk, we exposed 3-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice to a chronic increasing dose dss paradigm or normal drinking water and after 23 days returned all mice to normal drinking water until sacrifice several months later (fig. 1c). we chose to use the αsyn transgenic model rather than wild type mice for this study because of two reasons: 1) we knew that the model as hemizygous transgenic mice exhibit some αsyn brain pathology that develops slowly under baseline conditions. importantly, the pathology is much less pronounced than in homozygous (thy1)-h[a30p]αsyn mice [57]; 2) at the time of the experiment, it was not clear whether wild type mice could develop αsyn brain pathology upon dss colitis. thus, we chose hemizygous (thy1)-h[a30p]αsyn transgenic mice to increase the chances for a successful outcome and potentially to aggravate the brain pathology from mild to strong. after exposing the mice either to normal drinking water or a chronic increasing dose dss paradigm, we aged them in two cohorts on normal water and housing conditions to either up to the age of 9 months or 21 months. at these two timepoints we analyzed various brain regions for αsyn inclusions that are generally considered pathological by being proteinase k (pk)-resistant and immunopositive for pser129-αsyn. when we examined the 9-month-old αsyn transgenic mice, we found that both experimental groups (i.e., those who were on dss and those who stayed on normal water throughout their entire life and thus never experienced dss colitis) exhibited extremely low levels of pathological αsyn aggregation in the brain (fig. 5 and suppl. fig. 5). our observation of the level of pathological αsyn aggregations in the brain of these 9-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice (fig. 5a) is indeed consistent with earlier descriptions of the model at the age of 11 months [57]. the 21-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice that only received water during their lifetime showed, in contrast to the 9-month-old cohort, more discernible pk-resistant pser129-αsyn immunoreactive features (fig. 5b) and the abundance of these features was consistent with previous observations in this transgenic line at the age of 24 months [57]. in marked contrast, the 21-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice that were exposed to dss at three months of age presented with pser129-positive αsyn pathology throughout various brain regions in a much more exacerbated fashion than mice that were aged up to 21 months without having experienced dss colitis at young age (fig. 5b-e). the degree and distribution of pk-resistant αsyn in the brain was similar to what was previously described for homozygous (thy1)-h[a30p]αsyn transgenic mice at the age of 8 to 9 months [57]. the significant aggravation of αsyn pathology in the substantia nigra (p ≤ 0.01 in a negative-binomial mixed-effects model adjusting for multiple comparisons performed over all brain areas) was accompanied by a significant loss of tyrosine hydroxylase (th) and nissl-positive cells at 21 months of age (p ≤ 0.05, student’s t-test; fig. 6). together, we found that experimental dss colitis at a young age caused an age-dependent exacerbation of pk-resistant pser129-αsyn pathology and a loss of nigral dopaminergic neurons in the brains of (thy1)-h[a30p]αsyn transgenic mice. fig. 5 a 23-days chronic dss colitis insult at young age causes an age-dependent accumulation of proteinase k-resistant pser129-αsyn in various brain regions of (thy1)-h[a30p]αsyn transgenic mice. a 23-days chronic increasing dose dss paradigm was performed with 3-month-old (thy1)-h[a30p]αsyn transgenic mice. after recovering and further aging, various brain regions were analyzed for proteinase k (pk)-resistant pser129-αsyn immunoreactivity in 9-month (a) and 21-month-old (b) mice, respectively. the dark brown features in (a) (barely any visible in both the water and the dss group) and (b) (strongly visible with typical neuritic and punctated inclusion-type morphology) indicate pk-resistant inclusions with pser129-αsyn immunoreactivity. densitometric quantification of pser129-αsyn immunoreactivity in different brain regions of 21-month-old mice (c, d) (n=6 mice per group). in order to visualize better the differences between the water versus the dss group at 21 months, brain regions with large increase were plotted on an y-axis up to 250 a.u./px2 and small increase on a y-axis up to 40 a.u./px2. the about <5 to 150 a.u./px2 average values in the dss group versus the about 0 to 30 a.u./px2 average values in the water group in several brain regions confirm the visual impression in panel (b). one 21-month-old mouse in the dss group was excluded from analysis due to presumed failed treatment; it is included in the graphs. statistical analyses were performed using negative-binomial mixed-effects models adjusting for multiple comparisons. e representative heatmap of the average distribution scores of pser129-αsyn immunoreactivity for each treatment group in varying brain regions in all the 9-month (a and supplemental figure 5) and 21-month-old (b) mice was generated in a sagittal mouse brain (n=10 mice per group). statistical analyses were performed using linear mixed-effects model adjusting for multiple comparisons. a.u./px2, = mean grey value x area stained/total area assessed. scale bars: 500 μm. discussion currently, there is no therapy for pd available to slow or stop disease progression and an obstacle in the quest to develop one is that we do not understand how the disease develops [58]. abnormal intraneuronal accumulation of αsyn (i.e., in lewy bodies and neurites) is a key neuropathological hallmark and the distribution of lewy pathology in postmortem brain is used for staging in pd [2,59]. accumulation of αsyn has also been observed in the peripheral nervous system in pd, some individuals at risk of developing the disease, and normal individuals [60–62]. similar to this finding in humans, αsyn-immunoreactive inclusions and signs of age-dependent αsyn-related pathological changes have also been detected in the ens of wild type rats [63,64] and several transgenic mouse models prior to development of brain pathology [21,65]. based on preclinical models employing injection of brain extracts or re-combinant αsyn fibrils to different brain regions and intestines [15–17,19,20,46,66,67] together with postmortem brain pathology [56,59,68], it has also been suggested that αsyn pathology propagates temporospatially from cell-to-cell in a prion-like manner [3,59,66,68,69]. however, the initial factors triggering αsyn aggregation in the tissue or organ of origin of the pathology are yet to be established [58] and the involvement of peripheral stimuli in the aggregation and pathogenic spread of αsyn is only beginning to unravel. in this study, we provide evidence that dss colitis, i.e., an experimental ibd-like inflammation, triggers αsyn accumulation in the ens of wild type mice and in a human αsyn transgenic mouse model of pd (fig. 2). we found aggravation of enteric αsyn accumulation in αsyn transgenic mice lacking cx3cr1 signaling and amelioration of inflammation and associated slight reduction of enteric αsyn load by systemic il-10, demonstrating that genetic and pharmacologic modulation of inflammation can influence the degree of αsyn accumulation in the ens (fig. 3 and 4). because il-10 and the cx3cr1-cx3cl1 axis are able to mediate this effect, this suggests that monocytes/macrophages may modulate the process in this model. we further observed that the aggravated αsyn accumulation in the ens persisted even after two months of recovery from dss colitis and was aggravated in the absence of cx3cr1 signaling. this indicates that the accumulation is persistent and this further establishes that monocytes/macrophages play a critical role in this process (suppl. fig. 4). remarkably, at 18 months but not 6 months post induction of dss colitis (thus, at age 21 months but not 9 months, respectively), αsyn transgenic mice had developed massively elevated αsyn brain pathology (fig. 5 and suppl. fig. 5). this elevated pk-resistant pser129-αsyn pathology in the midbrain, including the substantia nigra, and other brain regions coincided with an average decrease of 30-50% of thand nissl-positive cells in the nigra (fig. 6). we chose to perform the long-term experiments in αsyn transgenic mice rather than wild type mice while being aware of the caveats of employing genetic overexpression models, which use a neuron selective thy-1 promoter cassette such as these (thy1)-h[a30p]αsyn transgenic mice. however, these particular αsyn transgenic mice had previously been shown to slowly develop αsyn pathology in the brain on a homozygous genotype [41,57] making them ideal when asking the question of whether transient colonic inflammation can aggravate brain pathology in a genetically predisposed animal such as the hemizygous transgenic mice used in this study. others have recently demonstrated in a more aggressive αsyn transgenic mouse model that mild dss colitis can accelerate αsyn accumulation in the ens and brain [38]. in future long-term studies, we plan to address whether αsyn pathology develops also in the brains of wild type mice if they sustain transient experimental ibd at a young age. in our present study, experimental dss colitis in αsyn transgenic mice recapitulated the accumulation of enteric αsyn which is proposed to occur in humans several years before pd diagnosis [39]. additionally, the subsequent age-related development of αsyn pathology in the brain of αsyn transgenic mice together with the loss of nigral dopaminergic neurons mimicked a progression of the disease similar to what is considered to occur in pd. fig. 6 a 23-days chronic dss colitis insult at young age results in loss of tyrosine hydroxylaseand nissl-positive cells in the substantia nigra of (thy1)-h[a30p]αsyn transgenic mice at 21 months of age. (thy1)-h[a30p]αsyn transgenic mice were exposed to a 23-days chronic increasing dose dss paradigm at the age of 3 months followed by aging on normal drinking water up to the age to 21 months. these mice showed a significant loss of mean count of nissl-positive cells with tyrosine hydroxylase (th) immunoreactivity and cellular nissl staining in the substantia nigra compared to age-matched littermate mice in the group that did not experience dss colitis (water). a representative images of two levels of the substantia nigra in one mouse per group. b stereological quantification of cells positive for th or nissl (n=7-8 mice per group). statistical analyses of the th dataset were performed using student’s t-test, while welch’s t-test was used for the nissl dataset to adjust for unequal variances. scale bar: 500 μm. we established that a mechanism by which a specific type of peripheral inflammation promotes αsyn accumulation in the colon potentially involves monocytes and macrophages. both peroral dss and intraperitoneal lps administration provoked strong local immune reactions resulting in leukocyte infiltration into the submucosa of the colon. the inflamed region of the colon contains the submucosal plexus and is anatomically separated from the myenteric plexus by a thick circular muscle (fig. 1). this discrete localization of inflammation to the submucosa might explain why αsyn only accumulated in the nerves of the submucosal plexus and not in the myenteric plexus of our mice that received dss in both a strong acute and the two chronic paradigms. the mechanism underlying how intraperitoneally administered lps leads to submucosal leukocyte infiltration likely involves the monocyte attractant chemokine ccl2 (fig. 3), but the specifics remain to be clarified [70]. indeed, ccl2 was also upregulated in the colon of our dss model. however, in contrast to intraperitoneal lps, where infiltrating macrophages were present in discrete patches in the colonic wall, dss-related macrophage infiltration was distributed both in small groups and larger randomly distributed patches of cells across the entire colonic submucosa. also, perorally administered dss destroys the mucosa of the colon, similar to some forms of ulcerative colitis, resulting in the transient disintegration of the intestinal epithelial barrier. in our (thy1)-h[a30p]αsyn transgenic mice, the subsequent immune response to the infiltration of commensal bacteria evoked an elevated expression of cytokines such as il-1β and il-6, a phenomenon also observed in the colon of ibd patients [71,72]. this upregulation was absent in the lps paradigm in which the intestinal mucosa remained intact. by acting on tight junctions, il-1β and il-6 can increase intestinal barrier permeability (gut leakiness), facilitating the recruitment of additional immune cells to the site of the inflammation, eventually culminating in widespread immune activation [73,74]. consistent with the breach of barrier permeability in our mouse model, some pd patients exhibit increased colonic cytokines such as il-1β, il-6 and tnf, occurring together with increased intestinal permeability [23,75]. in this context, it is also notable that people with crohn’s disease present with increased enteric αsyn expression [55] and even more striking that ibd patients on anti-tnf therapy have a reduced risk of developing pd compared to ibd patients not given this treatment [35]. notably, mucosal macrophages with intra-lysosomal αsyn content were previously described in the intact human appendix [76]. these macrophages were in close proximity to the axonal varicosities of the vermiform appendix, which showed an enriched staining for αsyn in the mucosal plexus. furthermore, we recently found that the vermiform appendix contains aggregated and truncated αsyn that has the propensity to seed aggregation of recombinant αsyn in vitro [62]. our finding of lps not being able to induce an αsyn phenotype in the colon is in contrast to the reports by kelly and colleagues who injected wild type mice with lps and observed an immediate and progressive increase in αsyn immunoreactivity in the myenteric ganglia of the large intestine but not in the small intestine [77]. the low dose (0.5 mg/kg used in our study versus 2.5 mg/kg used by kelly and colleagues), coupled with the possible usage of different bacterial strains to generate lps affecting potency and pyrogenicity of lps (here strain o55:b5 was administered versus presumably strain o111:b4 used by kelly and colleagues and others [78], and potential effects of different environments and microbiomes [79] may contribute to this discrepancy. it will be interesting to study the mentioned parameters and their role in inducing αsyn pathology further in rodent models and explore how this could be translated to the even more heterogeneous human setting. similarly, others showed that employing a sub-chronic 0.5% dss paradigm for 3 weeks in wild type mice did not trigger an αsyn pathology in the colon [80]. while we have not tested this particular sub-chronic dss colitis paradigm and instead had administered 5% dss acutely to the wild type mice (fig. 2c), the data by garrido-gil and colleagues may corroborate our results which show in αsyn transgenic mice that a certain dss dose and severity of inflammation is required in order to induce accumulation of αsyn in the submucosal plexus (fig. 2a). what could be a functional role of the αsyn species found in abundance in the gut wall? monomeric and oligomeric αsyn species reportedly act as chemoattractants for neutrophils and monocytes, enhancing the maturation of dendritic cells in the ens [22,81]. with such a role in intestinal immunity, it is possible that the tissue destruction induced by dss in the present study led to release of αsyn, which perhaps served as a chemoattractant for monocytes. the increased abundance of αsyn and altered intestinal permeability, along with the dss-evoked inflammatory response may have provided an enabling milieu allowing further αsyn accumulation in the ens of the colon [77]. macrophages and other immune cells are also regulated by several genes including lrrk2, an established risk gene for pd and ibd. it will be interesting to explore how mutations in genes that control autophagy, including the lrrk2 gene, influence the handling of αsyn by macrophages that invade the inflamed colon in our dss colitis paradigm. despite the intriguing translational aspect of our finding in the dss paradigm, others have very recently reported that dss colitis in mice down-regulates the expression of enteric αsyn on protein levels in vivo [82,83]. this is in contrast to our immunofluorescence (i.e., increased accumulation of αsyn in submucosal plexus upon dss colitis; fig. 2, 3, and 4) and gene expression data (e.g., no change in endogenous and transgenic αsyn upon dss colitis; suppl. fig. 3) in the same paradigm and may reflect the well-known lab-to-lab variability that can occur for the dss models [84]. perhaps the most striking finding in our study was that a single period of dss-induced colitis at a young age led to an exacerbation of αsyn pathology in the brain of αsyn transgenic mice much later in life (fig. 5). how does severe αsyn inclusion pathology develop in the brains of these mice? one hypothesis is that the brain αsyn pathology observed in this study could be due to direct effects of peripheral immune activation on the brain and that certain peripheral triggers can directly affect microglial activity. for instance, short-chain fatty acids derived from gut microbiota appear to influence function and maturation of microglia in the mouse brain [85] and inflammatory mediators released by gut microbiota into the bloodstream have been suggested to induce brain pathology and behavioral changes in an αsyn transgenic mouse model [86]. moreover, rats and nematodes have been reported to develop αsyn inclusions after exposure to the bacterial amyloid protein curli, a protein which stimulates microgliosis, astrogliosis, and secretion of il-6 and tnf [87]. intriguingly, a recent study reported that peripherally applied inflammatory stimuli induce acute immune training (that exacerbates β-amyloid pathology) and immune tolerance in the brain that reprograms microglia, an effect which can persist for at least six months [88]. whether this is a relevant mechanism in the dss paradigm needs to be explored. another hypothesis is that the observed brain αsyn pathology may have accumulated as a consequence of the transfer of pathogenic αsyn seeds from the gut via the vagal nerve. several experimental studies have demonstrated that pathogenic αsyn seeds can be transferred from the peripheral to the central nervous system. aggregated recombinant αsyn injected intraperitoneally, intramuscularly or into the gastric wall of certain mouse models of pd results in αsyn inclusions in the brain [16,89]. data from animals injected with recombinant αsyn protein in the gut wall or viral vectors expressing αsyn into the vagal nerve suggest that pathogenic seeds can be transmitted via the vagal nerve [15,19,20,64,67,90–92]. a role for the vagal nerve in pd was also suggested by an epidemiological study indicating that vagotomy in a danish population is associated with decreased pd risk [93], although this association has been challenged [94]. in the present study, αsyn pathology was much more prominent in the reticular nucleus (including the vagal nucleus) and midbrain areas compared to the rostral areas at 18 months post dss colitis. although we did not conduct the definitive experiment of cutting the vagal nerve, our data are consistent with the growing body of evidence that the vagal nerve is involved in the accumulation of αsyn aggregates in the brain. that said, the innervation of the colon occurs via both parasympathetic (e.g., vagal output neurons) and sympathetic (e.g., in the celiac ganglion of the upper abdomen) nerves. the possibility of propagation of αsyn pathology via the two routes is supported by the observation that injection of recombinant αsyn fibrils to the duodenum of certain αsyn transgenic rats leads to accumulation of pathological αsyn in organs innervated by the parasympathetic and the sympathetic nerves [67] and an age-dependent propagation of αsyn pathology from gut-to-brain in wild type rats [64]. clinically, more relevant, αsyn accumulation is present in both parasympathetic and sympathetic vagal nerves in humans as well [95]. thus, propagation may occur through both vagal and spinal routes. aging is considered a major risk factor for neurodegenerative diseases, as failing cellular mechanisms are proposed to be unable to efficiently clear pathologically accumulating proteins and organelles [96]. intriguingly, in the current study, while both cohorts were exposed to the same peripheral dss colitis insult at 3 months of age, αsyn pathology only developed in the brain of aged (21-month-old) but not in the relatively younger (9-month-old) mice (fig. 5). while the mechanisms involved in this phenomenon are still unclear, a recent study in mice reports an age-dependent association of development of αsyn pathology in the brain and development of motor deficits after inoculating the gut wall with recombinant αsyn fibrils [20]. investigating this further, the study observed a reduction of the lysosomal enzyme glucocerebrosidase in the gut of aged but not young mice [20]. viral vector-based overexpression of glucocerebrosidase partially rescued the ens network connectivity with concomitant variable downregulation of pser129-αsyn levels [20]. although other age-related factors are likely involved, the finding of challis and colleagues, together with the well-known genetic risk for glucocerebrosidase and several other lysosomal genes in pd [97,98], suggest a critical role of the lysosomal pathway in the long-term persistence of pathology in the ens and the progression of αsyn pathology from the gut to the brain of aged mice. upcoming studies in human tissue may shed more light on the mechanisms on how autophagy-lysosomal pathways are regulated and how they, together with aging, may contribute to the pathogenesis in pd. in summary, we report that αsyn accumulates in the colon of αsyn transgenic and wild type mice subjected to experimental dss colitis and that this process can be modulated by genetically and pharmacologically modifying pathways related to monocyte/macrophage signaling. we further demonstrate that chronic but transient dss colitis in young αsyn transgenic mice leads to a markedly exacerbated accumulation of αsyn aggregates in the brain of aged mice. in the same aged mice, the numbers of thand nissl-positive neurons in the substantia nigra are reduced, suggestive of a neurodegenerative process. together, our findings are in consonance with studies demonstrating a link between ibd and pd [33,35,99] and suggest a critical role for specific types of intestinal inflammation and αsyn accumulation in the initiation and progression of pd. acknowledgments including sources of support we thank drs. l. ozmen and a. bergadano for their tremendous support in maintaining the mouse colony and we are grateful to the animal caretakers, veterinarians and many unnamed staff at roche for their valuable work with the mice in this study. in addition, at roche we thank dr. k.g. lassen for critical input to the paper, dr. c. ullmer for co-mentoring s.g. and providing scientific input, dr. l. collin for helping with confocal imaging and we are grateful to dr. t. kremer, n. haenggi, d. mona, a. girardeau, and j. messer for providing support in tissue dissections and g. walker and r. lauria for technical support. ms. e. schulz from vai assisted with immunostaining of the brain tissue. we thank the contract research organization frimorfo for carefully sectioning the brains for this study. we acknowledge drs. l. gaudimier (née chicha) and f. pan-montojo for scientific discussions early in the project, dr. w. zago from prothena for valuable scientific input throughout the project, and drs. m. and p. derkinderen for critical input on the link between ibd and the enteric αsyn accumulation in humans. p.b. acknowledges the van andel institute and the many individuals and corporations that supported financially the neurodegenerative research at van andel institute. research at van andel institute reported in this publication was also financially supported by roche through a research collaboration with p.b. s.g and n.m. were supported by a grant from roche under the roche postdoctoral fellowship (rpf) program. conflict of interest at the time of the study s.g. and n.m. were roche postdoctoral fellows employed by roche and l.s., f.b., g.d.p., j.s.p., k.o.s., h.r., m.h., m.se. m.st., p.m., a.w., t.e., a.h. and m.b. are or were fulltime employees or trainees at roche and they may additionally hold roche stock/stock options. s.g. and l.s. are currently employees of neurimmune ag, schlieren, switzerland. p.b. has received commercial support as a consultant from axial biotherapeutics, calico life sciences, curasen, fujifilm-cellular dynamics international, h. lundbeck a/s, and idorsia pharmaceuticals ltd. he has received commercial support for grants/research from h. lundbeck a/s and roche. he has ownership interests in acousort ab. the other authors declare that they have no competing interest with regard to this research. author contributions s.g., n.m. and l.s. planned and performed the in vivo experiments, colon immunostaining, analysis, and quantification; s.g. and n.m. drafted a first version of the manuscript; e.q. performed, imaged, quantitated pser129, th and nissl staining in the brain sections, and drafted a more advanced version of the manuscript with j.a.s., who also provided helpful discussion. both j.a.s. and e.q. were critical in revising the manuscript. f.b. and k.o.s. supported the image acquisition and image analysis for the colon samples; m.st. performed imaging and data analysis of experiments with wild type mice; g.d.p. and j.s.p. performed statistical analysis of the dss experiments; h.r. and m.h. performed mrna analyses; m.se. trained s.g. and l.s. on mouse necropsy and supported their work; p.m. performed expert pathology staging on leukocyte infiltration; t.e. and a.w. provided migg-mil-10 fusion proteins and measured serum exposure; z.m. performed statistical analysis for the pser129 αsyn immunohistochemistry data. a.s. contributed with scientific and veterinary expert input for implementation and analysis of the dss colitis model at roche. m.l.e.g. provided helpful discussion and project planning. a.h. co-mentored s.g. and n.m., performed expert pathology staging on leukocyte infiltration and contributed to experimental planning. c.m. trained s.g. on the colitis model and provided expert input on the experimental ibd model. m.b. and p.b. co-mentored roche postdoctoral fellows s.g. and n.m., conceived and oversaw the study, and performed experimental planning; m.b., p.b. j.a.s. and e.q. wrote the final version of the manuscript. all authors read and approved the final manuscript. references 1. tysnes o-b, storstein a. epidemiology of parkinson’s disease. j neural transm (vienna). 2017;124:901–5. 2. spillantini mg, goedert m. neurodegeneration and the ordered assembly of α-synuclein. cell tissue res. 2017;373:137–48. 3. del tredici k, braak h. lewy pathology and neurodegeneration in premotor parkinson’s disease. mov disord. 2012;27:597–607. 4. polymeropoulos mh, lavedan c, leroy e, ide se, dehejia a, dutra a, et al. mutation in the alpha-synuclein gene identified in families with parkinson’s disease. science. 1997;276:2045–7. 5. schrag a, horsfall l, walters k, noyce a, petersen i. prediagnostic presentations of parkinson’s disease in primary care: a case-control study. lancet neurol. 2015;14:57–64. 6. gaenslen a, swid i, liepelt-scarfone i, godau j, berg d. the patients’ perception of prodromal symptoms before the initial diagnosis of parkinson’s disease. mov disord. 2011;26:653–8. 7. pont-sunyer c, hotter a, gaig c, seppi k, compta y, katzenschlager r, et al. the onset of nonmotor symptoms in parkinson’s disease (the onset pd study). mov disord. 2015;30:229–37. 8. berg d, godau j, seppi k, behnke s, liepelt-scarfone i, lerche s, et al. the prips study: screening battery for subjects at risk for parkinson’s disease. eur j neurol. 2013;20:102–8. 9. postuma rb, gagnon j-f, bertrand j-a, génier marchand d, montplaisir jy. parkinson risk in idiopathic rem sleep behavior disorder: preparing for neuroprotective trials. neurology. 2015;84:1104–13. 10. abbott rd, petrovitch h, white lr, masaki kh, tanner cm, curb jd, et al. frequency of bowel movements and the future risk of parkinson’s disease. neurology. 2001;57:456–62. 11. savica r, carlin jm, grossardt br, bower jh, ahlskog je, maraganore dm, et al. medical records documentation of constipation preceding parkinson disease: a case-control study. neurology. 2009;73:1752–8. 12. mahlknecht p, seppi k, poewe w. the concept of prodromal parkinson’s disease. j parkinsons dis. 5:681–97. 13. braak h, de vos rai, bohl j, del tredici k. gastric alpha-synuclein immunoreactive inclusions in meissner’s and auerbach’s plexuses in cases staged for parkinson’s disease-related brain pathology. neurosci lett. 2006;396:67–72. 14. phillips rj, walter gc, wilder sl, baronowsky ea, powley tl. alpha-synuclein-immunopositive myenteric neurons and vagal preganglionic terminals: autonomic pathway implicated in parkinson’s disease? neuroscience. 2008;153:733–50. 15. holmqvist s, chutna o, bousset l, aldrin-kirk p, li w, björklund t, et al. direct evidence of parkinson pathology spread from the gastrointestinal tract to the brain in rats. acta neuropathol. 2014;128:805–20. 16. breid s, bernis me, babila jt, garza mc, wille h, tamgüney g. neuroinvasion of α-synuclein prionoids after intraperitoneal and intraglossal inoculation. j virol. 2016;90:9182–93. 17. sargent d, verchère j, lazizzera c, gaillard d, lakhdar l, streichenberger n, et al. “prion-like” propagation of the synucleinopathy of m83 transgenic mice depends on the mouse genotype and type of inoculum. j neurochem. 2017;143:126–35. 18. manfredsson fp, luk kc, benskey mj, gezer a, garcia j, kuhn nc, et al. induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient cns pathology. neurobiol dis. 2018;112:106–18. 19. arotcarena m-l, dovero s, prigent a, bourdenx m, camus s, porras g, et al. bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates. brain. oxford academic; 2020;143:1462–75. 20. challis c, hori a, sampson tr, yoo bb, challis rc, hamilton am, et al. gut-seeded α-synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice. nature neuroscience. nature publishing group; 2020;23:327–36. 21. bencsik a, muselli l, leboidre m, lakhdar l, baron t. early and persistent expression of phosphorylated α-synuclein in the enteric nervous system of a53t mutant human α-synuclein transgenic mice. journal of neuropathology & experimental neurology. 2014;73:1144–51. 22. stolzenberg e, berry d, yang d, lee ey, kroemer a, kaufman s, et al. a role for neuronal alpha-synuclein in gastrointestinal immunity. jin. 2017;9:456–63. 23. devos d, lebouvier t, lardeux b, biraud m, rouaud t, pouclet h, et al. colonic inflammation in parkinson’s disease. neurobiol dis. 2013;50:42–8. 24. mogi m, harada m, kondo t, riederer p, inagaki h, minami m, et al. interleukin-1 beta, interleukin-6, epidermal growth factor and transforming growth factor-alpha are elevated in the brain from parkinsonian patients. neurosci lett. 1994;180:147–50. 25. brás j, guerreiro r, hardy j. snapshot: genetics of parkinson’s disease. cell. 2015;160:570-570.e1. 26. reynolds rh, botía j, nalls ma, hardy j, taliun sag, ryten m. moving beyond neurons: the role of cell type-specific gene regulation in parkinson’s disease heritability. npj parkinson’s disease. 2019;5:6. 27. witoelar a, jansen ie, wang y, desikan rs, gibbs jr, blauwendraat c, et al. genome-wide pleiotropy between parkinson disease and autoimmune diseases. jama neurol. 2017;74:780–92. 28. foo jn, chung sj, tan lc, liany h, ryu h-s, hong m, et al. linking a genome-wide association study signal to a lrrk2 coding variant in parkinson’s disease. mov disord. 2016;31:484–7. 29. umeno j, asano k, matsushita t, matsumoto t, kiyohara y, iida m, et al. meta-analysis of published studies identified eight additional common susceptibility loci for crohn’s disease and ulcerative colitis. inflamm bowel dis. 2011;17:2407–15. 30. hui ky, fernandez-hernandez h, hu j, schaffner a, pankratz n, hsu n-y, et al. functional variants in the lrrk2 gene confer shared effects on risk for crohn’s disease and parkinson’s disease. science translational medicine. 2018;10:eaai7795. 31. gardet a, benita y, li c, sands be, ballester i, stevens c, et al. lrrk2 is involved in the ifn-gamma response and host response to pathogens. j immunol. 2010;185:5577–85. 32. hakimi m, selvanantham t, swinton e, padmore rf, tong y, kabbach g, et al. parkinson’s disease-linked lrrk2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures. j neural transm (vienna). 2011;118:795–808. 33. lin j-c, lin c-s, hsu c-w, lin c-l, kao c-h. association between parkinson’s disease and inflammatory bowel disease: a nationwide taiwanese retrospective cohort study. inflamm bowel dis. 2016;22:1049–55. 34. nerius m, doblhammer g, tamgüney g. gi infections are associated with an increased risk of parkinson’s disease. gut. 2019;gutjnl-2019-318822. 35. peter i, dubinsky m, bressman s, park a, lu c, chen n, et al. anti-tumor necrosis factor therapy and incidence of parkinson disease among patients with inflammatory bowel disease. jama neurol. 2018;75:939–46. 36. rolli-derkinderen m, leclair-visonneau l, bourreille a, coron e, neunlist m, derkinderen p. is parkinson’s disease a chronic low-grade inflammatory bowel disease? j neurol. 2019;1–7. 37. wan q-y, zhao r, wu x-t. older patients with ibd might have higher risk of parkinson’s disease. gut. 2018;gutjnl-2018-317103. 38. kishimoto y, zhu w, hosoda w, sen jm, mattson mp. chronic mild gut inflammation accelerates brain neuropathology and motor dysfunction in α-synuclein mutant mice. neuromolecular med. 2019;21:239-249. 39. houser mc, tansey mg. the gut-brain axis: is intestinal inflammation a silent driver of parkinson’s disease pathogenesis? npj parkinson’s disease. 2017;3:3. 40. joers v, masilamoni g, kempf d, weiss ar, rotterman tm, murray b, et al. microglia, inflammation and gut microbiota responses in a progressive monkey model of parkinson’s disease: a case series. neurobiology of disease. 2020;144:105027. 41. kahle pj, neumann m, ozmen l, muller v, jacobsen h, schindzielorz a, et al. subcellular localization of wild-type and parkinson’s disease-associated mutant alpha -synuclein in human and transgenic mouse brain. j neurosci. 2000;20:6365–73. 42. jung s, aliberti j, graemmel p, sunshine mj, kreutzberg gw, sher a, et al. analysis of fractalkine receptor cx(3)cr1 function by targeted deletion and green fluorescent protein reporter gene insertion. mol cell biol. 2000;20:4106–14. 43. kitazawa m, oddo s, yamasaki tr, green kn, laferla fm. lipopolysaccharide-induced inflammation exacerbates tau pathology by a cyclin-dependent kinase 5-mediated pathway in a transgenic model of alzheimer’s disease. j neurosci. 2005;25:8843–53. 44. schenk m, bouchon a, seibold f, mueller c. trem-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases. j clin invest. 2007;117:3097–106. 45. grathwohl sa, kälin re, bolmont t, prokop s, winkelmann g, kaeser sa, et al. formation and maintenance of alzheimer’s disease β-amyloid plaques in the absence of microglia. nat neurosci. 2009;12:1361–3. 46. rey nl, george s, steiner ja, madaj z, luk kc, trojanowski jq, et al. spread of aggregates after olfactory bulb injection of α-synuclein fibrils is associated with early neuronal loss and is reduced long term. acta neuropathol. 2017;1–19. 47. schneider ca, rasband ws, eliceiri kw. nih image to imagej: 25 years of image analysis. nat methods. 2012;9:671–5. 48. smyth gk. limma: linear models for microarray data. in: gentleman r, carey vj, huber w, irizarry ra, dudoit s, editors. bioinformatics and computational biology solutions using r and bioconductor [internet]. new york, ny: springer new york; 2005 [cited 2019 may 1]. p. 397–420. available from: https://doi.org/10.1007/0-387-29362-0_23 49. chassaing b, aitken jd, malleshappa m, vijay-kumar m. dextran sulfate sodium (dss)-induced colitis in mice. curr protoc immunol. 2014;104:unit 15.25. 50. weber b, saurer l, schenk m, dickgreber n, mueller c. cx3cr1 defines functionally distinct intestinal mononuclear phagocyte subsets which maintain their respective functions during homeostatic and inflammatory conditions. eur j immunol. 2011;41:773–9. 51. medina-contreras o, geem d, laur o, williams ir, lira sa, nusrat a, et al. cx3cr1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic th17 responses in mice. j clin invest. 2011;121:4787–95. 52. kostadinova fi, baba t, ishida y, kondo t, popivanova bk, mukaida n. crucial involvement of the cx3cr1-cx3cl1 axis in dextran sulfate sodium-mediated acute colitis in mice. j leukoc biol. 2010;88:133–43. 53. kang s, okuno t, takegahara n, takamatsu h, nojima s, kimura t, et al. intestinal epithelial cell-derived semaphorin 7a negatively regulates development of colitis via αvβ1 integrin. j immunol. 2012;188:1108–16. 54. li b, alli r, vogel p, geiger tl. il-10 modulates dss-induced colitis through a macrophage-ros-no axis. mucosal immunol. 2014;7:869–78. 55. prigent a, lionnet a, durieu e, chapelet g, bourreille a, neunlist m, et al. enteric alpha-synuclein expression is increased in crohn’s disease. acta neuropathol. 2019;137:359–61. 56. braak h, del tredici k, rüb u, de vos rai, jansen steur enh, braak e. staging of brain pathology related to sporadic parkinson’s disease. neurobiol aging. 2003;24:197–211. 57. neumann m, kahle pj, giasson bi, ozmen l, borroni e, spooren w, et al. misfolded proteinase k-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies. j clin invest. 2002;110:1429–39. 58. johnson me, stecher b, labrie v, brundin l, brundin p. triggers, facilitators, and aggravators: redefining parkinson’s disease pathogenesis. trends neurosci. 2018;s0166-2236:30253–4. 59. braak h, del tredici k, bratzke h, hamm-clement j, sandmann-keil d, rüb u. staging of the intracerebral inclusion body pathology associated with idiopathic parkinson’s disease (preclinical and clinical stages). j neurol. 2002;249 suppl 3:iii/1-5. 60. shannon km, keshavarzian a, dodiya hb, jakate s, kordower jh. is alpha-synuclein in the colon a biomarker for premotor parkinson’s disease? evidence from 3 cases. mov disord. 2012;27:716–9. 61. lebouvier t, neunlist m, bruley des varannes s, coron e, drouard a, n’guyen j-m, et al. colonic biopsies to assess the neuropathology of parkinson’s disease and its relationship with symptoms. plos one. 2010;5:e12728. 62. killinger ba, madaj z, sikora jw, rey n, haas aj, vepa y, et al. the vermiform appendix impacts the risk of developing parkinson’s disease. sci transl med. 2018;10:eaar5280. 63. phillips rj, walter gc, ringer be, higgs km, powley tl. alpha-synuclein immunopositive aggregates in the myenteric plexus of the aging fischer 344 rat. exp neurol. 2009;220:109–19. 64. van den berge n, ferreira n, mikkelsen tw, alstrup ako, tamgüney g, karlsson p, et al. ageing promotes pathological alpha-synuclein propagation and autonomic dysfunction in wild-type rats. brain. 2021;awab061 65. kuo y-m, li z, jiao y, gaborit n, pani ak, orrison bm, et al. extensive enteric nervous system abnormalities in mice transgenic for artificial chromosomes containing parkinson disease-associated α-synuclein gene mutations precede central nervous system changes. hum mol genet. 2010;19:1633–50. 66. luk kc, kehm v, carroll j, zhang b, o’brien p, trojanowski jq, et al. pathological α-synuclein transmission initiates parkinson-like neurodegeneration in nontransgenic mice. science. 2012;338:949–53. 67. van den berge n, ferreira n, gram h, mikkelsen tw, alstrup ako, casadei n, et al. evidence for bidirectional and trans-synaptic parasympathetic and sympathetic propagation of alpha-synuclein in rats. acta neuropathol. 2019;138:535-550. 68. spillantini mg, schmidt ml, lee vm, trojanowski jq, jakes r, goedert m. alpha-synuclein in lewy bodies. nature. 1997;388:839–40. 69. rey nl, steiner ja, maroof n, luk kc, madaj z, trojanowski jq, et al. widespread transneuronal propagation of α-synucleinopathy triggered in olfactory bulb mimics prodromal parkinson’s disease. j exp med. 2016;213:1759–78. 70. puntambekar ss, davis ds, hawel l, crane j, byus cv, carson mj. lps-induced ccl2 expression and macrophage influx into the murine central nervous system is polyamine-dependent. brain behav immun. 2011;25:629–39. 71. andus t, daig r, vogl d, aschenbrenner e, lock g, hollerbach s, et al. imbalance of the interleukin 1 system in colonic mucosa—association with intestinal inflammation and interleukin 1 receptor agonist genotype 2. gut. 1997;41:651–7. 72. sanchez-muñoz f, dominguez-lopez a, yamamoto-furusho jk. role of cytokines in inflammatory bowel disease. world j gastroenterol. 2008;14:4280–8. 73. al-sadi rm, ma ty. il-1beta causes an increase in intestinal epithelial tight junction permeability. j immunol. 2007;178:4641–9. 74. capaldo ct, nusrat a. cytokine regulation of tight junctions. biochim biophys acta. 2009;1788:864–71. 75. forsyth cb, shannon km, kordower jh, voigt rm, shaikh m, jaglin ja, et al. increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early parkinson’s disease. plos one. 2011;6:e28032. 76. gray mt, munoz dg, gray da, schlossmacher mg, woulfe jm. alpha-synuclein in the appendiceal mucosa of neurologically intact subjects. mov disord. 2014;29:991–8. 77. kelly lp, carvey pm, keshavarzian a, shannon km, shaikh m, bakay rae, et al. progression of intestinal permeability changes and alpha-synuclein expression in a mouse model of parkinson’s disease. mov disord. 2014;29:999–1009. 78. akarsu es, mamuk s. escherichia coli lipopolysaccharides produce serotype-specific hypothermic response in biotelemetered rats. am j physiol regul integr comp physiol. 2007;292:r1846-1850. 79. dodiya hb, forsyth cb, voigt rm, engen pa, patel j, shaikh m, et al. chronic stress-induced gut dysfunction exacerbates parkinson’s disease phenotype and pathology in a rotenone-induced mouse model of parkinson’s disease. neurobiol dis. 2020;135:104352. 80. garrido-gil p, rodriguez-perez ai, dominguez-meijide a, guerra mj, labandeira-garcia jl. bidirectional neural interaction between central dopaminergic and gut lesions in parkinson’s disease models. mol neurobiol. 2018;55:7297-7316 81. labrie v, brundin p. alpha-synuclein to the rescue: immune cell recruitment by alpha-synuclein during gastrointestinal infection. jin. 2017;9:437–40. 82. prigent a, gonzales j, durand t, le berre-scoul c, rolli-derkinderen m, neunlist m, et al. acute inflammation down-regulates alpha-synuclein expression in enteric neurons. j neurochem. 2019;148:746–60. 83. resnikoff h, metzger jm, lopez m, bondarenko v, mejia a, simmons ha, et al. colonic inflammation affects myenteric alpha-synuclein in nonhuman primates. j inflamm res. 2019;12:113–26. 84. whittem cg, williams ad, williams cs. murine colitis modeling using dextran sulfate sodium (dss). j vis exp [internet]. 2010 [cited 2019 apr 27]; available from: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2841571/ 85. erny d, hrabě de angelis al, jaitin d, wieghofer p, staszewski o, david e, et al. host microbiota constantly control maturation and function of microglia in the cns. nat neurosci. 2015;18:965–77. 86. sampson tr, debelius jw, thron t, janssen s, shastri gg, ilhan ze, et al. gut microbiota regulate motor deficits and neuroinflammation in a model of parkinson’s disease. cell. 2016;167:1469-1480.e12. 87. chen sg, stribinskis v, rane mj, demuth dr, gozal e, roberts am, et al. exposure to the functional bacterial amyloid protein curli enhances alpha-synuclein aggregation in aged fischer 344 rats and caenorhabditis elegans. scientific reports. 2016;6:34477. 88. wendeln a-c, degenhardt k, kaurani l, gertig m, ulas t, jain g, et al. innate immune memory in the brain shapes neurological disease hallmarks. nature. 2018;556:332–8. 89. sacino an, brooks m, thomas ma, mckinney ab, lee s, regenhardt rw, et al. intramuscular injection of α-synuclein induces cns α-synuclein pathology and a rapid-onset motor phenotype in transgenic mice. proc natl acad sci u s a. 2014;111:10732–7. 90. kim s, kwon s-h, kam t-i, panicker n, karuppagounder ss, lee s, et al. transneuronal propagation of pathologic α-synuclein from the gut to the brain models parkinson’s disease. neuron [internet]. 2019 [cited 2019 jul 25];0. available from: https://www.cell.com/neuron/abstract/s0896-6273(19)30488-x 91. lohmann s, bernis me, tachu bj, ziemski a, grigoletto j, tamgüney g. oral and intravenous transmission of α-synuclein fibrils to mice. acta neuropathol. 2019;138(4):515-533. 92. uemura n, yagi h, uemura mt, hatanaka y, yamakado h, takahashi r. inoculation of α-synuclein preformed fibrils into the mouse gastrointestinal tract induces lewy body-like aggregates in the brainstem via the vagus nerve. molecular neurodegeneration. 2018;13:21. 93. svensson e, horváth-puhó e, thomsen rw, djurhuus jc, pedersen l, borghammer p, et al. vagotomy and subsequent risk of parkinson’s disease. ann neurol. 2015;78:522–9. 94. tysnes o-b, kenborg l, herlofson k, steding-jessen m, horn a, olsen jh, et al. does vagotomy reduce the risk of parkinson’s disease? ann neurol. 2015;78:1011–2. 95. braak h, sastre m, bohl jre, de vos rai, del tredici k. parkinson’s disease: lesions in dorsal horn layer i, involvement of parasympathetic and sympathetic preand postganglionic neurons. acta neuropathol. 2007;113:421–9. 96. reeve a, simcox e, turnbull d. ageing and parkinson’s disease: why is advancing age the biggest risk factor? ageing research reviews. 2014;14:19–30. 97. neumann j, bras j, deas e, o’sullivan ss, parkkinen l, lachmann rh, et al. glucocerebrosidase mutations in clinical and pathologically proven parkinson’s disease. brain. 2009;132:1783–94. 98. nalls ma, pankratz n, lill cm, do cb, hernandez dg, saad m, et al. large-scale meta-analysis of genome-wide association data identifies six new risk loci for parkinson’s disease. nat genet. 2014;46:989–93. 99. villumsen m, aznar s, pakkenberg b, jess t, brudek t. inflammatory bowel disease increases the risk of parkinson’s disease: a danish nationwide cohort study 1977-2014. gut. 2018;68:18–24. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. dementia with lewy bodies – a clinicopathological update feel free to add comments by clicking these icons on the sidebar free neuropathology 1:7 (2020) review dementia with lewy bodies – a clinicopathological update jános bencze1,2, woosung seo3, abdul hye4,5, dag aarsland5,6,7, tibor hortobágyi2,6,7,8 1 department of pathology, faculty of medicine, university of debrecen, debrecen, hungary 2 mta-de cerebrovascular and neurodegenerative research group, department of neurology, university of debrecen, debrecen, hungary 3 department of surgical sciences, radiology, uppsala university, uppsala, sweden 4 maurice wohl clinical neuroscience institute, institute of psychiatry, psychology and neuroscience, king’s college london, london, uk 5 nihr biomedical research centre for mental health & biomedical research unit for dementia at south london & maudsley nhs foundation, london, uk 6 department of old age psychiatry, institute of psychiatry psychology and neuroscience, king’s college london, london, uk 7 centre for age-related medicine, sesam, stavanger university hospital, stavanger, norway 8 institute of pathology, faculty of medicine, university of szeged, szeged, hungary corresponding author: tibor hortobágyi md phd dsc frcpath efn · institute of pathology · faculty of medicine · university of szeged · szeged · állomás utca 1 · h-6725 · hungary hortobagyi.tibor@med.u-szeged.hu submitted: 31 december 2019 accepted: 06 february 2020 published: 18 february 2020 https://doi.org/10.17879/freeneuropathology-2020-2613 keywords: α-synuclein, biomarkers, diagnostic criteria, clinico-pathological correlation, dementia with lewy bodies abstract dementia is one of the major burdens of our aging society. according to certain predictions, the number of patients will double every 20 years. although alzheimer’s disease (ad), as the most frequent neurodegenerative dementia, has been extensively analysed, less is known about dementia with lewy bodies (dlb). neuropathological hallmarks of dlb are the deposition of intracellular lewy bodies (lb) and lewy neurites (ln). dlb belongs to the α-synucleinopathies, as the major component of these inclusions is pathologically aggregated α-synuclein. depending on the localisation of lbs and lns in the central nervous system cognitive and motor symptoms can occur. in our work, we will systematically review the possible etiology and epidemiology, pathological (both macroscopic and microscopic) features, structural and functional imaging findings, with a special emphasis on the clinico-pathological correlations. finally, we summarize the latest clinical symptoms-based diagnostic criteria and the novel therapeutic approaches. since dlb is frequently accompanied with ad pathology, highlighting possible differential diagnostic approaches is an integral part of our paper. although our present knowledge is insufficient, the rapid development of diagnostic and research methods provide hope for better diagnosis and more efficient treatment, contributing to a better quality of life. abbreviations aa, alzheimer’s association; ad, alzheimer’s disease; aβ, amyloid-beta; apoe, apolipoprotein e gene; bne, brainnet europe consortium; bold, blood oxygen level dependent; chat, choline-acetyltransferase; chat-i, acetyl-cholinesterase inhibitors; cr, creatinine; csf, cerebrospinal fluid; dlb, dementia with lewy bodies; dmn, default mode network; dsm, diagnostic statistical manual; fmri, functional mri; fp-cit, [123i] 2ß-carbomethoxy-3b-(4-iodophenyl)-n-(3-fluoropropyl) nortropane; gba, glucosylceramidase-beta gene; ilbd, incidental lewy body disease; lb, lewy body; lbda, lewy body dementia association; ln, lewy neurite; mapt, microtubule associated protein tau gene; mds, movement disorders society; mri, magnetic resonance imaging; naa, n-acetyl aspartate; ncd, neurocognitive disorder; nf, neurofilament; nia, national institute on aging; nmdar, n-methyl-d-aspartate receptor; pd, parkinson’s disease; pdd, parkinson’s disease dementia; pet, positron emission tomography; psd95, density protein 95; rem, rapid-eye-movement; scarb2, scavenger receptor class b member 2; snare, snap (soluble nsf attachment protein) receptor; snca, α-synuclein gene; spect, single-photon emission computed tomography; ssri, selective serotonin reuptake inhibitors; upr, unfolded protein response; wml, white matter lesions; znt3, zinc transporter 3 epidemiology and etiology dementia with lewy bodies (dlb) is the second most common primary neurodegenerative dementia. the etiology is mainly unknown, however, in certain cases there is strong evidence of genetic background. a few papers have reported families with accumulating occurrence of cognitive impairment throughout generations1. the vast majority of the familial cases are traced to α-synuclein (snca) gene alterations, particularly to e46k mutation2. five candidate genes, including apoe, gba, mapt, snca and scarb2, are considered as a significant risk factor for dlb, nevertheless, further genetic studies are needed3. although it mostly appears in older age, dementia is not strictly associated with aging. rarely it may occur under the age of 65 and even in young adulthood. surprisingly in the case report of a teenager, the clinical symptoms and post-mortem pathological findings were consistent with those of in dlb4. the prevalence of the disease is not clearly established. according to a comprehensive analysis of epidemiological data, the prevalence is 4.2% in community based and 7.5% in clinical studies, while the incidence is 3.8% and grows linearly with aging5 and the prevalence of dlb among patients with dementia is probably around 15%6. pathologic background 1. macroscopic observation many of the general pathologic features resemble those in parkinson’s disease (pd). the brain weight is often within the normal limits, mild cortical atrophy of the frontal lobe, neuromelanin pigment loss in the substantia nigra and locus coeruleus are noted. in the case of severe concomitant ad pathology, atrophy of the temporal and parietal regions is more evident. in contrast to ad, the general brain atrophy is less prominent in dlb along with the relatively preserved temporal lobe and hippocampus7. 2. microscopic feature  lewy bodies (lbs), the key pathological findings in dlb, were initially described by friedrich lewy in 19128. however, lbs are characteristic of other neurodegenerative diseases including pd. their typical appearance, stained with conventional hematoxylin-eosin, is a central spherical eosinophilic core surrounded by a peripheral halo situated intracellularly, causing dislocation of subcellular organs. these features are regularly found in brainstem predominant lb formation. the rest of the brain expresses rather irregular lbs without the typical peripheral halo (figure 1). figure 1: dementia with lewy bodies (dlb) specific pathological changes are shown with hematoxylin and eosin (he) staining (panel a) and α-synuclein immunohistochemistry (ich) (panel b). cortical-type lewy bodies (lbs) are eosinophilic intracellar neuronal inclusions which dislocate the nucleus (panel a, black star). α-synuclein ihc highlights lbs (white star, panel b) and lns (arrowhead, panel b). histopathologically, the dominant component of the central core is α-synuclein, while the peripheral halo consists of several ubiquitinated proteins9. lewy neurites (lns), as an additional hallmark of α-synucleinopathies, are abnormal thickened neurites containing filaments corresponding to those in lbs10. interestingly, in experimental mouse models mutant snca inoculation of wild type mouse causes lb/ln-like pathology, supporting the pathognomonic role of the protein11. the background of lb genesis is still undiscovered, although numerous hypotheses have been proposed trying to explain the proper mechanism. according to the aggresome hypothesis, lb formation is originally a neuroprotective process, facilitating the cells to remove harmful proteins12. however, failure of the aggresome formation may occur, leading to excessive lb development13. other authors presume that autophagy dysfunction is responsible for the neuronal loss14. a recent study suggests that the increased unfolded protein response (upr) activation has an important role in the lb pathology15. nevertheless, according to tompkins et al. neurons burdened with lbs are less apoptotic16. moreover, the downregulation of tyrosine hydroxylase enzymes protects against toxic products of dopamine oxidation17. these findings confirm the theory that α-synuclein is physiologically involved in the maintenance of cell homeostasis. the first standardized criterion assessing the connections between pathological findings and dlb was made by kosaka et al. in 1984. based on the anatomical distribution of pathology they determined three different subtypes: i) brainstem predominant lbs (commonly in pd) ii) limbic (transitional) lbs and iii) diffuse cortical lbs18. later the consortium on dlb international workshop has improved the original assignment and published a more detailed instruction emphasizing the importance of the diagnostic procedure. the widely used immunohistochemical markers, α-synuclein (the most specific) and p62 serve the accurate pathological diagnosis19. the latest mckeith diagnostic consensus criteria recommends a semiquantitative grading of 10 different brain regions (dorsal motor nucleus of vagus, locus coeruleus, substantia nigra, nucleus basalis of meynert, amygdala, transentorhinal and cingulate gyri, temporal-, frontaland parietal lobes) based on the lesion density instead of the previously used lb counting method. moreover, it suggests two additional categories, the amygdala-predominant and the olfactory bulb only dlb. according to the distribution of lb pathology on α-synuclein immunostained slides, the scoring system distinguishes four stages: 1 – mild (sparse lbs or lns); 2 – moderate (1< lbs in a low power field and sparse lns); 3 severe (4≤ lbs and scattered lns in a low power field); 4 – very severe (numerous lbs and numerous lns). finally, as a synthesis of score and localization it ranks the seen pathology into one out of the three previously mentioned subtypes20. besides the mckeith staging and subtyping, the other widely used evaluating technique is the braak staging21. the authors proposed to assess the severity of lewy-type pathology labelled by α-synuclein immunostaining in 13 different brain areas (dorsal motor nucleus of vagus, locus coeruleus, raphe, substantia nigra, ca2 region of hippocampus, nucleus basalis of meynert, transentorhinal-, cingulateand insular gyri, temporo-occipital-, temporal-, frontaland parietal lobes). considering that neither mckeith nor braak protocol could reach more than 80% inter-observer agreement; the original methods have been modified by leverenz et al.22 and müller et al.23, respectively. however, their results did not lead to a significant improvement in the scoring systems. thus, in 2009 the brainnet europe consortium (bne) revised their former assignments and suggested modifications, to reach a better inter-observer agreement. using the original mckeith and braak staging, but eliminating their major pitfalls and obstacles as well as introducing the amygdala predominant category, bne’s novel strategy resulted in above 80% agreement in both typing and staging of α-synuclein pathology24. beach et al. also published their unified staging system with the aim of dividing every subject with lewy-type α-synuclein pathology into a well-defined neuropathological group25. in 2009 they used mckeith26 and braak21 staging systems and failed to categorize individuals with olfactory bulb or limbic-predominant lb pathology. investigating 10 standard brain regions they could classify all patients with pd, dlb, incidental lewy body disease (ilbd) and ad with concomitant lb pathology into one of the following stages: i olfactory bulb only; iia brainstem-predominant; iib limbic-predominant; iii brainstem and limbic; iv neocortical. moreover, they found strong correlation between the progression through these stages and the severity of nigrostriatal degeneration, cognitive impairment and motor dysfunction. it should be noted that olfactory bulb only and amygdala-predominant subtypes are also included in the current mckeith criteria20. as mentioned above, ad pathology is frequently encountered in dlb. approximately 80% of patients have diffuse amyloid-beta (aβ) plaques and 60% have neurofibrillary tangles with varying severity in the entorhinal cortex and rarely in the neocortex. some of these cases meet the pathological criteria of ad27. in contrast, “pure” neuropathological form of dlb is less common. autopsy series suggest that the frequency of this entity is approximately 25% of all dlb cases28,29. theoretically, the likelihood to manifest dlb clinical syndrome is directly proportional to the severity of lb pathology and inversely proportional to the severity of ad pathology. the mckeith classification integrates the assessment of concomitant ad pathology by national institute on aging and alzheimer’s association (nia-aa)30, the braak criteria and the type of lb pathology20. table 1 shows the probability of pathological findings in relation to dlb clinical syndrome. it is likely that not only the α-synuclein pathology is responsible for the cognitive decline, but plaques and phosphorylated tau proteins also contribute to the overall deficit31. table 1: likelihood of dementia with lewy bodies (dlb) clinical syndrome resulting from the assessment of alzheimer's-type and lewy body pathology (lb= lewy body; dlb=dementia with lewy bodies; nia-aa = national institute on aging – alzheimer’s association) [modified from mckeith et al.20] cognitive impairment is also a common hallmark of dlb and parkinson’s disease dementia (pdd). these two neurocognitive disorders share several clinical and neuropathological features, i.e. they are both characterized by cortical and subcortical α-synuclein/lb, β-amyloid and tau pathologies32. according to jellinger et al., a common pathophysiology in dlb and pdd is synaptic dysfunction due to aggregation of α-synuclein in the presynapses. this results in disruption of axonal transport and neurotransmitter deprivation leading to neurodegeneration32. interestingly, there are some morphologic differences as well. dlb seems to show higher load of β-amyloid and tau in several brain regions primarily in striatum, cortex, claustrum, amygdala and putamen compared to pdd32. jellinger et al. also describes that α-synuclein distribution is different in dlb and pdd where α-synuclein load was highest in hippocampal subarea ca2 and in amygdala in dlb, whereas in pd it is highest in the cingulate cortex. moreover, nigral neuronal loss is more marked in pdd which ultimately results in dopaminergic upregulation32. a simple interpretation of above findings could support the current diagnostic criteria where dlb is associated with early cognitive impairment including memory problems, which differs from pdd with mainly motor impairment in the early phase. in reality, it is not easy to define dlb based upon only the histological findings, thus in uncertain cases, anamnestic clinical data has invaluable support for the diagnostics. it is important to highlight that the listed strategies are not absolute or perfect diagnostic criteria, but rather useful schemes to predict the clinical syndrome of dlb based on the observed pathological findings. as mentioned above, a major weakness of current guidelines is the low inter-observer agreement; therefore further research is needed to assess the accuracy of the current clinical diagnostic criteria versus neuropathology similar to studies assessing the 2005 criteria in this respect33. 3. synaptic alterations in connection with the dopaminergic neuronal loss of substantia nigra, dopamine transporter level is decreased in the striatum in dlb, although not to the same extent observed in pd34. the choline-acetyltransferase (chat) levels are lower than in patients with ad, who suffer from similarly severe dementia35,36. interestingly, dynamin-1, which takes part in the regulation of synaptic transmission, shows significantly decreased levels in the prefrontal cortex in parallel with the severity of cognitive decline37. in addition, the amount of zinc transporter 3 (znt3) and postsynaptic density protein 95 (psd95), are significantly reduced in dlb compared to aged-controls and patients with ad38,39. it should be noted that at the beginning of the disease, an upregulation of soluble nsf attachment protein receptors (snare) complex is observed, probably as a compensatory response to the synaptic loss. however, during the progression of dlb synaptotagmin, synapsin and synaptophysin, proteins involved in the snare complex steadily disintegrate, contributing further to synaptic dysfunction40,41. recent studies have revealed that more than 90% of α-synuclein aggregates are located at the presynapses leading to neurotransmitter deficits42,43. these findings suggest that degeneration of postsynaptic neurons may result from the loss of their inputs. this theory serves as a potential explanation for the dlb-specific clinical symptoms as well as raises the possibility of future curative treatments by pharmaceutical modification of neurotransmission. 4. incidental lewy-body disease (ilbd) in ilbd, patients have histologically detectable α-synuclein deposits in their brain, without presenting any clinical symptoms. researchers accept that ilbd, which appears in approximately 8-17% of the clinically normal 60+ years old patients, is the early stage of pd or dlb44,45. confirming this hypothesis, it is frequently observed in patients who represent the prodromes of pd, including olfactory dysfunction or bowel frequency46,47. the presence of neuronal loss is usually minimal in these cases contrary to pd or dlb48. distribution of α-synuclein pathology in ilbd is particularly predictive, i.e. the brainstem predominant subtype frequently results in pd, while the cortical predominant subtype often leads to dlb44. clinical symptoms the new diagnostic statistical manual (dsm-5) (published in 2013) created a new neurocognitive disorder (ncd) group, replacing the former ‘dementia, delirium, amnestic and other cognitive disorders’ category, that were used in the previous dsm-iv handbook. the definition emphasizes that ncd is a progressive, acquired decline; which implies that disorders occurring at birth or at the early stage of cognitive development are excluded from this category. as an advantage, the new handbook removed the stigmatizing debilitating term, dementia. within the ncd there are two subcategories: major and mild neurocognitive disorders based upon the severity of decline. for the diagnosis, six main fields are examined by dsm-5: complex attention, executive function, learning and memory, language, perceptual motor or social cognition49. in the case of dlb, patients show both cortical and subcortical progressive dementia symptoms. characteristic features comprise of attention and spatial perception disorder, dysexecutive syndrome, fluctuating cognitive performance lasting from minutes to days. among the psychiatric alteration the most frequent is the visual hallucination, although anxiety, apathy or organised delusions could also be detected50,51. interestingly, advanced cerebral amyloid angiopathy and small vessel disease are associated with psychosis in ad and not in dlb52. depression is more common in patients with dlb than in patients with ad. it may accompany with mild dementia, although more frequent in cases of advanced dlb53,54. the vast majority of the patients, from the beginning or during the progression, show extrapyramidal signs, such as action tremor, gait disturbances, rigidity or changes in facial expressions55. rapid-eye-movement (rem) parasomnia is frequently diagnosed in dlb, characterised by vivid, often frightening dreams and complex purposeful motor activity. since it often appears in different neurodegenerative disorders, rem parasomnia might be an early herald of these diseases56. in dlb the autonomic nervous system dysfunction is more severe than in ad. patients usually complain of dizziness, falls or loss of consciousness. orthostatic hypotension, cardioinhibitory carotid sinushypersensitivity and urinary incontinency often accompany with dlb57. pathognomonic feature is the neuroleptic hypersensitivity, triggered by even a small dose of drug, leading to severe parkinsonism20. a 5-year prospective cohort study published by rongve et al. has reported that the progression of cognitive decline from mild to severe stage is more rapid in patients with dlb than in patients with ad58. many clinical symptoms of dlb cannot be explained only by the intracerebral localization of lbs and lns33. neurotransmitter depletion, synaptic alterations, concomitant ad-type pathology and metabolic changes highly influence the clinical presentation of dlb, and correlate with the severity of cognitive decline6,42,59,60. moreover, a recent paper has reported association of serum potassium levels with cognitive decline in dlb, specifically in patients not using any medications that affect serum k+ levels61. reduced perfusion and dysconnectivity of the occipital lobe may contribute to visual hallucinations and disturbance of visuospatial orientation62,63. rem sleep behavior disorder rbd associates with striatal dopamine depletion64. vegetative dysfunctions (i.e. orthostatic hypotension) may result from the deposition of lbs in the autonomic ganglia65. decreased znt3 levels were also identified in patients with depression, providing a novel therapeutic target38,66. interestingly, several clinical symptoms of dlb are transient in nature, however lbs and lns permanently occur in the brain. probably, metabolic disturbances67, hormonal alterations68, circadian rhythm69 and comorbidities such as high blood pressure70 can affect the clinical appearance of dlb. radiologic feature besides the clinical symptoms, the most significant diagnostic tool in the identification of dlb is the rapidly developing imaging techniques. the numerous reachable modalities allow both structural and functional examination. magnetic resonance imaging (mri) examination regarding the structural changes, the literature is not consistent: some studies have noted significant atrophy of the insular, frontal, inferior parietal, temporal or occipital cortex71, whereas others have found only a minimal volumetric decrease in the frontal and parietal lobe, or in the territory of hypothalamus, basal forebrain and midbrain72. however, these alterations are not specific to dlb and might appear in ad. although differentiation from ad is usually based on the absence of medial temporal atrophy, its presence cannot rule out the diagnosis of dlb73. sabattoli et al. have found atrophic changes in the anterior ca1, ca2/3 hippocampus, subiculum and presubiculum in dlb. this pattern differs significantly from those in ad74. according to an mri study, the annual progression rate of cortical atrophy is approximately 2x higher in patients with ad72. the role of white matter lesions (wml) in dlb, including loss of myelin, axonal damage and gliosis is not consistent. presumably, wmls are implicated in vascular dementia rather than being specific feature of neurodegenerative disorders75. mri spectroscopy provides a chance to indirectly assess the neuronal and glial function by measuring the key metabolites. n-acetyl aspartate (naa) and creatinine (cr) are two widely used markers in the characterization of central nervous system metabolism. watson et al. have found that the naa/cr ratio is relatively preserved in dlb in comparison to in ad76. functional imaging although, task-related functional mri (fmri) has been performed in very few cases of dlb, a paper has reported reduced activity in response to movement activity compared with aged controls and patients with ad77. alternatively, researchers have examined the functional activity during rest. the resting-state network shows increased activity during rest and decreased activity during cognitive tasks. the most investigated resting-state network to date is the default mode network (dmn), including prefrontal cortex, posterior cingulate gyrus, medial temporal lobe and precuneus. in these areas the task-related deactivation during the colour and motion tasks were decreased in dlb, but it was not significantly different from ad78. a technique for evaluation of functional resting state connectivity is based on the functional alterations in blood oxygen level dependent (bold) activity. galvin et al. have noted increased resting state connectivity between precuneus seeding regions, inferior parietal cortex and putamen, with decreased connectivity between medial prefrontal, frontoparietal operculum and visual cortex79. taking into account that the results in dlb significantly differed from those of in ad, the method can be considered in the in vivo differential diagnosis. the real value of perfusion imaging techniques in the identification of dlb is not fully consistent. the vast majority of the authors described occipital hypoperfusion as a hallmark of the disease on single-photon emission computed tomography (spect) images, whereas the specificity of the method is variable depending on the study80. [123i] 2ß-carbomethoxy-3b-(4-iodophenyl)-n-(3-fluoropropyl) nortropane (fp-cit) could be useful to discover dlb in early stages before the full spectrum of clinical symptoms evolve, moreover it reliably identifies neurobiological changes in the dopaminergic system81. in dlb there is markedly reduced tracer uptake in the regions of caudate and putamen reflecting the severely affected dopaminergic system. strong connection has been found between striatal fp-cit uptake and certain clinical symptoms, such as depression, anxiety, apathy and daytime somnolence82. positron emission tomography (pet) is probably a more sensitive functional imaging technique than spect in different dementias. ishii et al. have compared the two methods and found that pet is more reliable in the detection of occipital and parietal lobe hypometabolism83. besides the dopaminergic system, cholinergic transmission also has a crucial role in the pathomechanism of dlb, for instance in the alteration of memory, attention or arousal. significant cholinergic neuronal loss and reduced chat activity are noted extensively in the cortical and subcortical regions, including both nicotinergic and muscarinergic systems, compared to ad36. shimada et al. have shown reduced chat enzymatic activity in the medial occipital cortex with relatively preserved temporal activity in dlb, in contrast to ad84. the fact that eeg is involved in the mckeith criterion further justifies its diagnostic relevance. compared to ad the detectable slower background activity and more diffuse slow-wave activity probably reflect the severe cholinergic deficit in dlb85. biomarkers it is difficult to find specific biomarkers of dlb that would allow us to discriminate neuropathologically ‘pure’ forms of the disease from cases with concomitant ad pathology86, as that majority of dlb cases show a combination of α-synuclein, tau and β-amyloid pathologies87,88. however, novel biomarkers such as reduced electroencephalography activity89 and the detection of rbd90 also have some value. recent dlb biomarker research has focused on targets that are primarily related to ad. as in ad, csf levels of aβ1-42 are decreased in dlb91, although tau seems to show an opposite relationship from ad92. that said tau has been shown to be higher in dlb when compared to pd and pdd87,93,94. dlb is considered α-synucleinopathy alongside with pd and pdd. when α-synuclein was discovered as a major component of lb this initiated a string of studies investigating α-synuclein as a biomarker in csf95,96. in general, some groups have reported reduced α-synuclein97,98 while others have shown contradictory results99,100. these discrepancies may arise from the nature of α-synuclein expression. α-synuclein appears in four isoforms, (α-syn98, α-syn112, α-syn126 and α-syn140), based on alternative splicing of exon 3 and 5, and the largest isoform, α-syn140, is the most abundant isoform in the brain101,102. nonetheless, to overcome this issue several groups have tried using a combination of antibodies to quantify ‘total’ α-synuclein103. in addition to this many are convinced that oligomeric α-synuclein is potentially a more pathogenic form104 and efforts have been made to measure this in both csf and in plasma105–107. in addition to α-synuclein and ad biomarkers, other potential biomarkers such as neurofilaments (nf) have been investigated108. nfs are components within a cell that assists in maintaining the structural integrity. it has been shown that nfs are associated with ad and other degenerative disorders109–111, however that is not the case with dlb. nfs seem to provide only a general hint of neuronal and axonal dysfunction without providing any differential value to separate dlb from other disorders. on the other hand, other isoforms of nfs112 do exist and would have to be further investigated in dlb. since there is greater involvement of the dopaminergic and serotonergic neurotransmitters in dlb compared to ad, several groups have investigated metabolites from these pathways. in combination with csf aβ1-42, reduced levels of 5hydroxyindolacetic acid and 3methoxy-4-hydroxyphenylethyleneglycol have been found in dlb compared with ad104,113. in summary, current studies suggest that dlb is intermediate to ad and pd, such that biomarkers from ad and pd have been tested in dlb with moderate success114. likewise, new diagnostic proteins may be discovered in the future with further proteomic studies which could provide a better differentiation of dlb from other closely related neurodegenerative disorders115–117. diagnostic criteria considering the heterogeneous phenotype and the frequently associated ad pathology, the clinical symptom-based diagnosis of dlb is rather difficult. to date, there is no consensus on the guidelines for assessing clinical symptoms of dlb. a recent paper58 recommends the following rating strategies: for evaluating cognitive decline – clinical dementia rating scale118; for estimating fluctuating cognition – clinician assessment of cognitive fluctuations119 or mayo fluctuation questionnaire120; for investigating rem parasomnia mayo sleep questionnaire121; for rating parkinsonism – unified parkinson’s rating scale122; for testing disability rapid disability rating scale-2123; for diagnosing visual hallucinations or other psychiatric disorders – neuropsychiatric inventory124; for measuring effects of comorbidities – cumulative illness rating scale118. additional difficulty is to make a distinction, if it exists, between dlb and parkinson’s disease dementia. the question, whether dlb and pdd are different entities or the same one, is still debated. although there are a few morphologic differences between them (e.g. cortical spreading of lbs or rate of neuronal loss in sn)125, differential diagnosis is rather based on the temporal sequence of symptoms. if dementia occurs 1 year after the onset of extrapyramidal motor signs, it is considered as pdd, otherwise if dementia has proceeded or presented within 1 year after movement disorder, the diagnosis should be dlb126. the latest mckeith diagnostic criteria define probable and possible dlb based on the clinical symptoms and biomarkers (figure 2)20. the presence of dementia, memory impairment and deficit of attention, executive functions and visuospatial orientation is essential for diagnosis. core symptoms are frequently observed in dlb clinical syndrome; in the lack of at least one core feature, the probable dlb diagnosis cannot be established. the development of diagnostic techniques revealed that rem sleep behaviour disorder is more characteristic of lbd than it was previously thought. therefore, mckeith et al. recategorized rbd from supportive to core features20. supportive features may help the clinicians; however, these symptoms are not specific to dlb and their presence is not required for the diagnosis of neither probable nor possible dlb. the refreshed criteria emphasize the importance of disease-specific biomarkers. one indicative biomarker itself is sufficient for possible dlb diagnosis. if one indicative biomarker associates with one core symptom, the diagnosis is probable dlb. figure 2: symptoms and biomarkers contribute to the diagnosis of probable or possible dementia with lewy bodies (dlb). essential symptoms are mandatory for diagnosis of dlb. core clinical symptoms are characteristic to dlb, while supportive symptoms can confirm the decision by clinicians. indicative biomarkers are frequently observed in dlb, while supportive biomarkers can facilitate the decision-making procedure. important to note that clinicians can not establish the diagnosis of probable dlb based on the biomarkers. (recategorized or newly added features are highlighted in blue colour; rem = rapid eye movement; dat = dopamine transporter; mibg = metaiodobenzylguanidine; psg = polysomnography; rbd = rem sleep behavior disorder; spect = single-photon emission computed tomography; pet = positron-emission tomography; eeg = electroencephalography; ↓ = decrease). [adapted from mckeith et al.20] therapy there are two different therapeutic approaches: pharmacologic and non-pharmacologic. although there is no curative treatment strategy, the adequate therapy may slow the disease progression with the chance of a better quality of life. non-pharmacologic interventions at the first signs of dlb or mild ncd, installation of the below mentioned interventions are recommended. changes in the dietary habit, mediterranean diet, and regularly performed social and mental tasks as well as physical activities could reduce the rate of progression127. personalized cognitive rehabilitation trainings with focus on the declined field, improve both quality of life and memory128. pharmacologic treatments the frequently noted extrapyramidal signs should be treated with the smallest effective dose of levodopa, to avoid the worsening of psychiatric symptoms129. selective serotonin reuptake inhibitors (ssris) are widely-used and considered as effective medications for depression130. in the case of rem sleep behaviour disorder, clonazepam and melatonin also have beneficial effect131 acetyl-cholinesterase inhibitors (chat-i) have benefits in the treatment of psychiatric disturbances such as visual hallucination, delusions, behaviour disorders or apathy. despite gastrointestinal side effects (i.e. vomiting, diarrhoea) these drugs are usually well tolerated20. if chat-is (rivastigmine, donepezil) are not effective, the use of atypical neuroleptic drugs (i.e. clozapine) might be inevitable, but typical neuroleptics should be avoided to minimize the possibility of severe neuroleptic hypersensitivity reaction132. chat-is are more effective in patients with dlb compared to patients with ad. these drugs improve the cognitive functions, reduce fluctuation, decrease the progression and in addition patients score better on neuropsychological tests133. n-methyl-d-aspartate receptor (nmda-r) antagonist memantine is also useful in the treatment of cognitive decline134. lucza et al. have suggested treatment with chat-is in mild or mid-severe dementia and memantine combined with high dose of rivastigmine (13.5 mg) in severe dementia135. summary despite the continuously growing incidence of dementia, which could be the most prevalent disease within 20 years in the industrialized world, our present knowledge is still insufficient to fully comprehend the underlying pathomechanism. this is particularly true for dlb, which is the second most common neurodegenerative dementia. etiology of the disorder, its connection with aging, underlying pathomechanisms of clinical signs and imaging findings are still in need for further elucidation. unfortunately, the definitive diagnosis is possible only by post-mortem histopathological examination and not by in vivo techniques (apart from a low percentage of cases). the concomitant alzheimer’s-type and vascular pathology raises issues regarding diagnostic clarity and accuracy136. future research should be multidisciplinary and should include pathological, proteomic, genetic and epigenetic approaches to identify the key factors of the disease and reveal the correlation between clinical symptoms, radiological alterations and pathological findings. development of imaging techniques brings the possibility of in vivo diagnosis, which implies adequate treatments in early stages, a crucial advancement to ensure better quality of life. longitudinal cohort studies are also required to provide detailed prognostic information that are essential for planning a more effective and cost-efficient treatment protocol. author’s contribution authors contributed equally to the work. funding supported by the únkp-19-3 new national excellence program of the ministry of innovation and technology and efop-3.6.3-vekop-16-2017-00009 (j.b.); ginop-2.3.2-15-2016-00043, hungarian brain research program (2017-1.2.1-nkp-2017-00002), nkfih snn 132999, szte áok-kka no. 5s 567 (a202) and de áok research fund (t.h.). this paper represents independent study partly funded by the national institute for health research (nihr) biomedical research centre at south london and maudsley nhs foundation trust and king’s college london. the views expressed are those of the author(s) and not necessarily those of the nhs, the nihr or the department of health and social care. references 1. tsuang, d. w. et al. familial dementia with lewy bodies: a clinical and neuropathological study of 2 families. arch. neurol. 59, 1622–30 (2002). 2. zarranz, j. j. et al. the new mutation, e46k, of alpha-synuclein causes parkinson and lewy body dementia. ann. neurol. 55, 164–73 (2004). 3. bras, j. et al. genetic analysis implicates apoe, snca and suggests lysosomal dysfunction in the etiology of dementia with lewy bodies. hum. mol. genet. 23, 6139–46 (2014). 4. takao, m. et al. early-onset dementia with lewy bodies. brain pathol. 14, 137–147 (2004). 5. hogan, d. b. et al. the prevalence and incidence of dementia with lewy bodies: a systematic review. can. j. neurol. sci. 43, s83–s95 (2016). 6. aarsland, d. cognitive impairment in parkinson’s disease and dementia with lewy bodies. park. relat. disord. 22, s144–s148 (2016). 7. love, j., kalaria r. dementia. in: love, s. et al. greenfield’s neuropathology. 9th ed., pp. 858-973, crc press (2015). 8. lewy, f. h. paralysis agitans. i. pathologische anatomie. in: handb. der neurol. 920–958, springer, berlin (1912). 9. wakabayashi, k. et al. the lewy body in parkinson’s disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates. neuropathology 27, 494–506 (2007). 10. spillantini, m. g. et al. alpha-synuclein in filamentous inclusions of lewy bodies from parkinson’s disease and dementia with lewy bodies. proc. natl. acad. sci. u. s. a. 95, 6469–73 (1998). 11. luk, k. c. et al. modeling lewy pathology propagation in parkinson’s disease. park. relat. disord. 20, s85-7 (2014). 12. olanow, c. w. et al. lewy-body formation is an aggresome-related process: a hypothesis. lancet neurol. 3, 496–503 (2004). 13. alghamdi, a. et al. reduction of rpt6/s8 (a proteasome component) and proteasome activity in the cortex is associated with cognitive impairment in lewy body dementia. j. alzheimers dis. 57, 373–386 (2017). 14. cuervo, a. m. et al. impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. science 305, 1292–5 (2004). 15. baek, j. h. et al. unfolded protein response is activated in lewy body dementias. neuropathol. appl. neurobiol. 42, 352–365 (2016). 16. tompkins, m. m. et al. contribution of somal lewy bodies to neuronal death. brain res. 775, 24–9 (1997). 17. mori, f. et al. relationship among alpha-synuclein accumulation, dopamine synthesis, and neurodegeneration in parkinson disease substantia nigra. j. neuropathol. exp. neurol. 65, 808–15 (2006). 18. kosaka, k. et al. diffuse type of lewy body disease: progressive dementia with abundant cortical lewy bodies and senile changes of varying degree--a new disease? clin. neuropathol. 3, 185–92 (1984). 19. kuusisto, e. et al. morphogenesis of lewy bodies: dissimilar incorporation of alpha-synuclein, ubiquitin, and p62. j. neuropathol. exp. neurol. 62, 1241–53 (2003). 20. mckeith, i. g. et al. diagnosis and management of dementia with lewy bodies. neurology 89, 88–100 (2017). 21. braak, h. et al. staging of brain pathology related to sporadic parkinson’s disease. neurobiol. aging 24, 197–211 (2003). 22. leverenz, j. b. et al. empiric refinement of the pathologic assessment of lewy-related pathology in the dementia patient. brain pathol. 18, 220–4 (2008). 23. müller, c. m. et al. staging of sporadic parkinson disease-related alpha-synuclein pathology: interand intra-rater reliability. j. neuropathol. exp. neurol. 64, 623–8 (2005). 24. alafuzoff, i. et al. staging/typing of lewy body related α-synuclein pathology: a study of the brainnet europe consortium. acta neuropathol. 117, 635–652 (2009). 25. beach, t. g. et al. unified staging system for lewy body disorders: correlation with nigrostriatal degeneration, cognitive impairment and motor dysfunction. acta neuropathol. 117, 613–634 (2009). 26. mckeith, i. g. et al. diagnosis and management of dementia with lewy bodies: third report of the dlb consortium. neurology 65, 1863–1872 (2005). 27. jellinger, k. a. et al. impact of coexistent alzheimer pathology on the natural history of parkinson’s disease. j. neural transm. 109, 329–39 (2002). 28. barker, w. w. et al. relative frequencies of alzheimer disease, lewy body, vascular and frontotemporal dementia, and hippocampal sclerosis in the state of florida brain bank. alzheimer dis. assoc. disord. 16, 203–212 (2002). 29. kosaka, k. diffuse lewy body disease in japan. j. neurol. 237, 197–204 (1990). 30. hyman, b. t. et al. longitudinal assessment of ab and cognition in aging and alzheimer disease. ann. neurol. 69, 181–192 (2011). 31. howlett, d. r. et al. regional multiple pathology scores are associated with cognitive decline in lewy body dementias. brain pathol. 25, 401–408 (2015). 32. jellinger, k. a. et al. are dementia with lewy bodies and parkinson’s disease dementia the same disease? bmc med. 16, (2018). 33. skogseth, r. et al. accuracy of clinical diagnosis of dementia with lewy bodies versus neuropathology. j. alzheimer’s dis. 59, 1139–1152 (2017). 34. piggott, m. a. et al. striatal dopaminergic markers in dementia with lewy bodies, alzheimer’s and parkinson’s diseases: rostrocaudal distribution. brain 122, 1449–1468 (1999). 35. perry, e. k. et al. cholinergic correlates of cognitive impairment in parkinson’s disease: comparisons with alzheimer’s disease. j. neurol. neurosurg. psychiatry 48, 413–21 (1985). 36. tiraboschi, p. et al. cholinergic dysfunction in diseases with lewy bodies. neurology 54, 407–411 (2000). 37. vallortigara, j. et al. dynamin1 concentration in the prefrontal cortex is associated with cognitive impairment in lewy body dementia. f1000research 3, 108 (2014). 38. whitfield, d. r. et al. assessment of znt3 and psd95 protein levels in lewy body dementias and alzheimer’s disease: association with cognitive impairment. neurobiol. aging 35, 2836–2844 (2014). 39. bereczki, e. et al. synaptic proteins predict cognitive decline in alzheimer’s disease and lewy body dementia. alzheimer’s dement. 12, 1149–1158 (2016). 40. vallortigara, j. et al. decreased levels of vamp2 and monomeric alpha-synuclein correlate with duration of dementia. j. alzheimer’s dis. 50, 101–110 (2015). 41. bereczki, e. et al. synaptic markers of cognitive decline in neurodegenerative diseases: a proteomic approach. brain 141, 582–595 (2018). 42. schulz-schaeffer, w. j. the synaptic pathology of α-synuclein aggregation in dementia with lewy bodies, parkinson’s disease and parkinson’s disease dementia. acta neuropathol. 120, 131–143 (2010). 43. bridi, j. c. et al. mechanisms of α-synuclein induced synaptopathy in parkinson’s disease. front. neurosci. 12, (2018). 44. frigerio, r. et al. incidental lewy body disease: do some cases represent a preclinical stage of dementia with lewy bodies? neurobiol. aging 32, 857–63 (2011). 45. auning, e. et al. early and presenting symptoms of dementia with lewy bodies. dement. geriatr. cogn. disord. 32, 202–208 (2011). 46. ross, g. w. et al. association of olfactory dysfunction with incidental lewy bodies. mov. disord. 21, 2062–2067 (2006). 47. abbott, r. d. et al. bowel movement frequency in late-life and incidental lewy bodies. mov. disord. 22, 1581–6 (2007). 48. delledonne, a. et al. incidental lewy body disease and preclinical parkinson disease. arch. neurol. 65, 1074–80 (2008). 49. american psychiatric association: diagnostic and statistical manual of mental disorders. american psychiatric publishing, arlington, usa. (2013). 50. walker, z. et al. lewy body dementias. lancet 386, 1683–1697 (2015). 51. majer, r. et al. behavioural and psychological symptoms in neurocognitive disorders: specific patterns in dementia subtypes. open med. 14, 307–316 (2019). 52. vik-mo, a. o. et al. advanced cerebral amyloid angiopathy and small vessel disease are associated with psychosis in alzheimer’s disease. j. neurol. neurosurg. psychiatry 90, 728–730 (2019). 53. fritze, f. et al. depressive symptoms in alzheimer’s disease and lewy body dementia: a one-year follow-up study. dement. geriatr. cogn. disord. 32, 143–149 (2011). 54. fritze, f. et al. depression in mild dementia: associations with diagnosis, apoe genotype and clinical features. int. j. geriatr. psychiatry 26, 1054–1061 (2011). 55. aarsland, d. et al. comparison of extrapyramidal signs in dementia with lewy bodies and parkinson’s disease. j. neuropsychiatry clin. neurosci. 13, 374–379 (2001). 56. ferini-strambi, l. et al. rem sleep behavior disorder (rbd) as a marker of neurodegenerative disorders. arch. ital. biol. 152, 129–146 (2014). 57. ballard, c. et al. high prevalence of neurovascular instability in neurodegenerative dementias. neurology 51, 1760–2 (1998). 58. rongve, a. et al. cognitive decline in dementia with lewy bodies: a 5-year prospective cohort study. bmj open 6, e010357 (2016). 59. perry, e. k. et al. neocortical cholinergic activities differentiate lewy body dementia from classical alzheimer’s disease. neuroreport 5, 747–749 (1994). 60. bereczki, e. et al. synaptic markers of cognitive decline in neurodegenerative diseases: a proteomic approach. brain 141, 582–595 (2018). 61. giil, l. m. et al. serum potassium is associated with cognitive decline in patients with lewy body dementia. j. alzheimer’s dis. 68, 239–253 (2019). 62. ishii, k. et al. regional cerebral blood flow difference between dementia with lewy bodies and ad. neurology 53, 413–6 (1999). 63. mak, e. et al. neuroimaging characteristics of dementia with lewy bodies. alzheimers. res. ther. 6, 18 (2014). 64. eisensehr, i. et al. reduced striatal dopamine transporters in idiopathic rapid eye movement sleep behaviour disorder. comparison with parkinson’s disease and controls. brain 123, 1155–1160 (2000). 65. gelpi, e. et al. multiple organ involvement by alpha-synuclein pathology in lewy body disorders. mov. disord. 29, 1010–1018 (2014). 66. whitfield, d. r. et al. depression and synaptic zinc regulation in alzheimer’s disease, dementia with lewy bodies and parkinson’s disease dementia. am. j. geriatr. psychiatry 23, 1–8 (2014). 67. huber, m. et al. metabolic correlates of dopaminergic loss in dementia with lewy bodies. mov. disord. mds.27945 (2019) doi:10.1002/mds.27945. 68. lessig, s. et al. reduced hypocretin (orexin) levels in dementia with lewy bodies. neuroreport 21, 756–760 (2010). 69. ferman, t. j. et al. sleep in parkinson’s disease and dementia with lewy bodies. psychiatry park. dis. 27, 61–70 (2012). 70. borda, m. g. et al. association of depressive symptoms and subjective memory complaints with the incidence of cognitive impairment in older adults with high blood pressure. eur. geriatr. med. 10, 413–420 (2019). 71. beyer, m. k. et al. gray matter atrophy in parkinson disease with dementia and dementia with lewy bodies. neurology 69, 747–54 (2007). 72. whitwell, j. l. et al. focal atrophy in dementia with lewy bodies on mri: a distinct pattern from alzheimer’s disease. brain 130, 708–19 (2007). 73. barkhof, f. et al. the significance of medial temporal lobe atrophy: a postmortem mri study in the very old. neurology 69, 1521–7 (2007). 74. sabattoli, f. et al. hippocampal shape differences in dementia with lewy bodies. neuroimage 41, 699–705 (2008). 75. mcaleese, k. e. et al. post-mortem assessment in vascular dementia: advances and aspirations. bmc med. 14, 129 (2016). 76. watson, r. et al. magnetic resonance imaging in lewy body dementias. dement. geriatr. cogn. disord. 28, 493–506 (2009). 77. sauer, j. et al. differences between alzheimer’s disease and dementia with lewy bodies: an fmri study of task-related brain activity. brain 129, 1780–1788 (2006). 78. stebbins, g. t. et al. altered cortical visual processing in pd with hallucinations: an fmri study. neurology 63, 1409–16 (2004). 79. galvin, j. e. et al. resting bold fmri differentiates dementia with lewy bodies vs alzheimer disease. neurology 76, 1797–803 (2011). 80. taylor, j. p. et al. neuroimaging of dementia with lewy bodies. neuroimaging clin. n. am. 22, 67–81 (2012). 81. siepel, f. j. et al. (123i)fp-cit spect in suspected dementia with lewy bodies: a longitudinal case study. bmj open 3, e002642 (2013). 82. murai, t. et al. in vivo evidence for differential association of striatal dopamine and midbrain serotonin systems with neuropsychiatric symptoms in parkinson’s disease. j. neuropsychiatry clin. neurosci. 13, 222–8 (2001). 83. ishii, k. et al. comparison of fdg-pet and imp-spect in patients with dementia with lewy bodies. ann. nucl. med. 18, 447–451 (2004). 84. shimada, h. et al. mapping of brain acetylcholinesterase alterations in lewy body disease by pet. neurology 73, 273–8 (2009). 85. roks, g. et al. the use of eeg in the diagnosis of dementia with lewy bodies. j. neurol. neurosurg. psychiatry 79, 377–380 (2008). 86. dickson, d. w. introduction to neurodegeneration: the molecular pathology of dementia and movement disorders. in: neurodegeneration: the molecular pathology of dementia and movement disorders: 2nd ed. pp. 1–5, wiley-blackwell (2011). 87. schade, s. et al. biomarkers in biological fluids for dementia with lewy bodies. alzheimers. res. ther. 6, 72 (2014). 88. jendroska, k. et al. amyloid β peptide and precursor protein (app) in mild and severe brain ischemia. ann. n. y. acad. sci. 826, 401–405 (1997). 89. bonanni, l. et al. quantitative electroencephalogram utility in predicting conversion of mild cognitive impairment to dementia with lewy bodies. neurobiol. aging 36, 434–445 (2015). 90. terzaghi, m. et al. analysis of video-polysomnographic sleep findings in dementia with lewy bodies. mov. disord. 28, 1416–1423 (2013). 91. kanemaru, k. et al. decreased csf amyloid beta42 and normal tau levels in dementia with lewy bodies. neurology 54, 1875–1876 (2000). 92. clark, c. m. et al. cerebrospinal fluid tau and betaamyloid. arch neurol 60, 1696–1702 (2003). 93. mollenhauer, b. et al. tau protein, abeta42 and s-100b protein in cerebrospinal fluid of patients with dementia with lewy bodies. dement. geriatr. cogn. disord. 19, 164–170 (2005). 94. llorens, f. et al. cerebrospinal α-synuclein in α-synuclein aggregation disorders: tau/α-synuclein ratio as potential biomarker for dementia with lewy bodies. j. neurol. 263, 2271–2277 (2016). 95. tredici, k. del et al. lewy pathology in the submandibular gland of individuals with incidental lewy body disease and sporadic parkinson’s disease. acta neuropathol. 119, 703–713 (2010). 96. spillantini, m. g. et al. alpha-synuclein in filamentous inclusions of lewy bodies from parkinson’s disease and dementia with lewy bodies. proc. natl. acad. sci. u. s. a. 95, 6469–73 (1998). 97. lim, x. et al. the diagnostic utility of cerebrospinal fluid alpha-synuclein analysis in dementia with lewy bodies a systematic review and meta-analysis. park. relat. disord. 19, 851–858 (2013). 98. mollenhauer, b. et al. direct quantification of csf α-synuclein by elisa and first cross-sectional study in patients with neurodegeneration. exp. neurol. 213, 315–325 (2008). 99. noguchi-shinohara, m. et al. csf α-synuclein levels in dementia with lewy bodies and alzheimer’s disease. brain res. 1251, 1–6 (2009). 100. spies, p. e. et al. cerebrospinal fluid α-synuclein does not discriminate between dementia disorders. j. alzheimer’s dis. 16, 363–369 (2009). 101. campion, d. et al. mutations of the presenilin i gene in families with early-onset alzheimer’s disease. hum. mol. genet. 4, 2373–2377 (1995). 102. uéda, k. et al. tissue-dependent alternative splicing of mrna for nacp, the precursor of non-a beta component of alzheimer’s disease amyloid. biochem. biophys. res. commun. 205, 1366–72 (1994). 103. tokuda, t. et al. decreased α-synuclein in cerebrospinal fluid of aged individuals and subjects with parkinson’s disease. biochem. biophys. res. commun. 349, 162–166 (2006). 104. el-agnaf, o. m. a. et al. α-synuclein implicated in parkinson’s disease is present in extracellular biological fluids, including human plasma. faseb j. 17, 1945–1947 (2003). 105. tokuda, t. et al. detection of elevated levels of α-synuclein oligomers in csf from patients with parkinson disease. neurology 75, 1766–1772 (2010). 106. el-agnaf, o. m. a. et al. detection of oligomeric forms of α-synuclein protein in human plasma as a potential biomarker for parkinson’s disease. faseb j. 20, 419–425 (2006). 107. simonsen, a. h. et al. the utility of α-synuclein as biofluid marker in neurodegenerative diseases: a systematic review of the literature. biomark. med. 10, 19–34 (2016). 108. ashton, n. j. et al. increased plasma neurofilament light chain concentration correlates with severity of post-mortem neurofibrillary tangle pathology and neurodegeneration. acta neuropathol. commun. 7, 5 (2019). 109. zetterberg, h. et al. association of cerebrospinal fluid neurofila-ment light concentration with alzheimer disease progression. jama neurol. 73, 60 (2016). 110. gaiottino, j. et al. increased neurofilament light chain blood levels in neurodegenerative neurological diseases. plos one 8, e75091 (2013). 111. xu, z. et al. neurofilaments as biomarkers for amyotrophic lateral sclerosis: a systematic review and meta-analysis. plos one 11, e0164625 (2016). 112. schmidt, m. l. et al. epitope map of neurofilament protein domains in cortical and peripheral nervous system lewy bodies. am. j. pathol. 139, 53–65 (1991). 113. aerts, m. b. et al. csf α-synuclein does not differentiate between parkinsonian disorders. neurobiol. aging 33, 430.e1–3 (2012). 114. ashton, n. j. et al. an update on blood-based biomarkers for non-alzheimer neurodegenerative dementias. nat. rev. neurol. (in press). 115. bencze, j. et al. biological function of lemur tyrosine kinase 2 (lmtk2): implications in neurodegeneration. mol. brain 11, 20 (2018). 116. bencze, j. et al. neuropathological characterization of lemur tyrosine kinase 2 (lmtk2) in alzheimer’s disease and neocortical lewy body disease. sci. rep. 9, 17222 (2019). 117. bencze et al. lemur tyrosine kinase 2 (lmtk2) level inversely correlates with phospho-tau in neuropathological stages of alzheimer’s disease. brain sci. 10, 68 (2020). 118. morris, j. c. clinical dementia rating: a reliable and valid diagnostic and staging measure for dementia of the alzheimer type. int. psychogeriatrics 9, 173–176 (1997). 119. walker, m. p. et al. the clinician assessment of fluctuation and the one day fluctuation assessment scale: two methods to assess fluctuating confusion in dementia. br. j. psychiatry 177, 252–256 (2000). 120. ferman, t. j. et al. dlb fluctuations: specific features that reliably differentiate dlb from ad and normal aging. neurology 62, 181–7 (2004). 121. boeve, b. f. et al. validation of the mayo sleep questionnaire to screen for rem sleep behavior disorder in an aging and dementia cohort. sleep med. 12, 445–453 (2011). 122. goetz, c. c. the unified parkinson’s disease rating scale (updrs): status and recommendations. mov. disord. 18, 738–750 (2003). 123. linn, m. w. et al. the rapid disability rating scale—2. j. am. geriatr. soc. 30, 378–382 (1982). 124. cummings, j. l. et al. the neuropsychiatric inventory: comprehensive assessment of psychopathology in dementia. neurology 44, 2308–14 (1994). 125. tsuboi, y. et al. dementia with lewy bodies and parkinson’s disease with dementia: are they different? park. relat. disord. 11, s47-51 (2005). 126. mckeith, i. commentary: dlb and pdd: the same or different? is there a debate? int. psychogeriatrics 21, 220 (2009). 127. grande, g. et al. physical activity reduces the risk of dementia in mild cognitive impairment subjects: a cohort study. j. alzheimers. dis. 39, 833–9 (2014). 128. kalbe, e. et al. task force wanted: many reasons to promote research on cognitive rehabilitation to prevent, delay, and treat cognitive dysfunctions in patients with parkinson’s disease. parkinsonism relat. disord. 21, 166–167 (2015). 129. molloy, s. et al. the role of levodopa in the management of dementia with lewy bodies. j. neurol. neurosurg. psychiatry 76, 1200–3 (2005). 130. boot, b. p. et al. treatment of dementia with lewy bodies. curr. treat. options neurol. 15, 738–64 (2013). 131. boeve, b. f. et al. rem sleep behavior disorder in parkinson’s disease and dementia with lewy bodies. j. geriatr. psychiatry neurol. 17, 146–57 (2004). 132. aarsland, d. et al. neuroleptic sensitivity in parkinson’s disease and parkinsonian dementias. j. clin. psychiatry 66, 633–7 (2005). 133. aarsland, d. et al. role of cholinesterase inhibitors in parkinson’s disease and dementia with lewy bodies. j. geriatr. psychiatry neurol. 17, 164–71 (2004). 134. stinton, c. et al. pharmacological management of lewy body dementia: a systematic review and meta-analysis. am. j. psychiatry 172, 731–742 (2015). 135. lucza, t. et al. neurocognitive disorders in parkinson’s disease. orv. hetil. 156, 915–926 (2015). 136. skrobot, o. a. et al. vascular cognitive impairment neuropathology guidelines (vcing): the contribution of cerebrovascular pathology to cognitive impairment. brain 139, 2957–2969 (2016). copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. medulloblastoma and cowden syndrome: further evidence of an association feel free to add comments by clicking these icons on the sidebar free neuropathology 3:1 (2022) case report medulloblastoma and cowden syndrome: further evidence of an association steffen albrecht1,*, barbara miedzybrodzki2, laura palma3,4, van hung nguyen1, roy w.r. dudley5, torsten pietsch6, tobias goschzik6, nada jabado3,7,8, catherine goudie8,9, william d. foulkes3,10,11,12 1 department of pathology, mcgill university, montreal, qc, canada 2 division of dermatology, department of pediatrics, mcgill university health centre, montreal children's hospital, montreal, qc, canada 3 department of human genetics, mcgill university, montreal, qc, canada 4 division of medical genetics, department of specialized medicine, mcgill university health centre, montreal, qc, canada 5 division of neurosurgery, department of pediatric surgery, montreal children's hospital, mcgill university health centre, montreal, qc, canada 6 department of neuropathology, dgnn brain tumor reference center, university of bonn medical center, bonn, germany 7 department of pediatrics, mcgill university, montreal, qc, canada 8 the research institute of the mcgill university health centre, child health and human development program, montreal, qc, canada 9 division of hematology-oncology, montreal children's hospital, department of pediatrics, mcgill university, montreal, qc, canada 10 cancer axis, lady davis institute, the jewish general hospital, montreal, qc, canada 11 cancer research program, the research institute of the mcgill university health centre, montreal, qc, canada 12 gerald bronfman department of oncology, mcgill university, montreal, qc, canada * retired since aug. 2021 corresponding author: steffen albrecht · 511-244 sherbrooke street east · montreal, qc · h2x 1e1 · canada steffen.albrecht@hotmail.com submitted: 02 december 2021 accepted: 02 january 2022 copyedited by: jeffrey nirschl published: 11 january 2022 https://doi.org/10.17879/freeneuropathology-2022-3684 keywords: medulloblastoma, cowden syndrome, pten abstract cowden syndrome (cs) is an autosomal dominant hamartoma and tumor predisposition syndrome caused by heterozygous pathogenic germline variants in pten in most affected individuals. major features include macrocrania, multiple facial tricholemmomas, acral and oral keratoses and papillomas, as well as mammary, non-medullary thyroid, renal, and endometrial carcinomas. lhermitte-duclos disease (ldd), or dysplastic gangliocytoma of the cerebellum, is the typical brain tumor associated with cs; the lifetime risk for ldd in cs patients has been estimated to be as high as 30%. in contrast, medulloblastoma is much rarer in cs, with only 4 reported cases in the literature. we report a 5th such patient. all 5 patients were diagnosed between 1 and 2 years of age and not all showed the pathognomonic clinical stigmata of cs at the time of their medulloblastoma diagnosis. where detailed information was available, the medulloblastoma was of the shh-subtype, in keeping with the observation that in sporadic medulloblastomas, pten-alterations are usually encountered in the shh-subtype. medulloblastomas can be associated with several tumor-predisposition syndromes and of the 4 medulloblastoma subtypes, shh-medulloblastomas in children have the highest prevalence of predisposing germline variants (approx. 40%). cs should be added to the list of shh-medulloblastoma-associated syndromes. germline analysis of pten should be performed in infants with shh-medulloblastomas, regardless of their clinical phenotype, especially if they do not carry pathogenic germline variants in ptch1 or sufu, the most commonly altered predisposing genes in this age-group. in addition, these cases show that cs has a biphasic brain tumor distribution, both in regards to the age of onset and the tumor type: a small number of cs patients develop a medulloblastoma in infancy while many more develop ldd in adulthood. case history the patient was born at term after an uneventful pregnancy; however, she was noted to have pronounced frontal bossing and macrocrania, (head circumference > 98th percentile). she presented at age 15 months because of rapidly increasing head circumference. mri showed an extra-axial posterior fossa mass within the cisterna magna, measuring 5.3 x 4.4 x 3.2 cm. the tumor was resected; post-operative mri confirmed gross total resection. histologically, it was a medulloblastoma with extensive nodularity (mben). she was treated according to the ccg-99703 protocol, which consists of 3 cycles of induction chemotherapy followed by 3 cycles of marrow-ablative consolidation chemotherapy with autologous stem-cell rescue [cohen et al. 2015]; however, she only received 2 of the latter due to hematological toxicity. during follow-up, some keratotic papules on the fingers and buttocks were noted and therefore gorlin syndrome (nevoid basal cell carcinoma syndrome; online mendelian inheritance in man (omim) #109400) was suspected clinically. some of these cutaneous lesions were biopsied but consisted of non-specific keratoses or warts; there were no basal carcinomas. initial germline genetic testing at age 2 consisted of deletion analysis and sanger sequencing of ptch1, which were negative. sanger sequencing of sufu was done at age 5 and was negative. at age 8, a firm papule was noted on the neck; histologically, it was a sclerotic fibroma. (a similar lesion was also noted on the left thigh, but not excised.) at age 9, four palmar pits were noted on the right hand. although the combination of palmar pits, mben, and macrocrania was suggestive of gorlin syndrome, sclerotic fibroma has been associated with cowden syndrome (cs; omim #158350) [kieselova et al. 2017]. therefore, next-generation germline sequencing of ptch1, ptch2, sufu, and pten was performed. a previously reported pathogenic variant in pten, c.388c>t, p.r130* (cosv64288463), was identified. no variants were found in the other genes. parental studies were negative, suggesting most likely a de novo mutation, although gonadal mosaicism in one of the parents cannot be ruled out. at age 10, a posterior fossa dural arterio-venous fistula (davf) was suspected on a routine follow-up mri and confirmed by cerebral angiogram to be a borden type 1 davf draining into an ectopic pouch of the right transverse sinus; it was fed by branches of the right and left external carotid arteries and the right posterior cerebral artery. although hemorrhage from intracranial avfs has been reported in patients with cs [prats-sánchez et al. 2016], borden type 1 davfs have a very low risk of hemorrhage [gandhi et al. 2012] and the lesion was completely asymptomatic. it was therefore not treated. it was no longer seen on repeat imaging a year later, suggesting spontaneous involution-resolution. at age 12, multiple thyroid nodules were noted bilaterally on ultrasound; fine needle aspiration cytology was consistent with benign follicular nodules. now age 13 (i.e., 12 years post-diagnosis), the patient is alive and well without evidence of recurrent medulloblastoma. she does not have facial or oral lesions. figure 1. representative images of the cerebellar tumor’s histology and immunophenotype. individual images are labelled with the respective stain. on low power, the tumor is biphasic and distinctly nodular. most of the tumor is made up of neurocytic to gangliocytic cells (high power inset) that are strongly neun immunoreactive and embedded in a synaptophysin-positive neuropil-like matrix that contains bundles of neurites. the highly cellular desmoplastic component shows some staining for gfap and is strongly positive for gab1 and yap1. ki-67 labelling is seen almost exclusively in the desmoplastic component. both components lack expression of pten, which is retained in endothelial cells. clicking the figure will lead you to the full virtual slides. pathology representative images of the cerebellar tumor are shown in figure 1. the tumor was biphasic and had a distinctly nodular growth pattern. most of the tumor consisted of large confluent islands of isomorphic cells with a neurocytic to gangliocytic appearance, embedded in a finely fibrillary neuropil-like matrix. these cells were strongly positive for neun and the neuropil stained intensely for synaptophysin and showed bundles of neurites on a neurofilament immunostain. there was virtually no ki-67 labelling in this component and it was negative for yap1 and gab1. the remainder of the tumor was made up of a very cellular "small blue cell" component, which was rich in reticulin fibers, had a high mitotic rate with approximately 50% ki-67 labelling, and focal immunoreactivity for gfap along with strong staining for yap1 and gab1. the features were typical of an mben, shh-activated. immunostaining for pten and genetic analysis of the tumor were performed after the pten germline variant was identified. expression of pten was retained in vessels but lost in both tumor components. the tumor carried the pten p.r130* variant, as expected. in addition, using a truseq custom amplicon (illumina) for 13 genes frequently mutated in shh-medulloblastomas [goschzik et al. 2021], a known pathogenic variant in smo, c.1234c>t, p.l412f (cosv50824425), was identified, which was confirmed by sanger sequencing. molecular inversion probe technology was used to perform high-resolution genome-wide copy number analysis as described previously [wang et al. 2012]. it showed a stable genome with copy-neutral allelic loss on chromosome arm 10q. there was no amplification of myc or mycn. discussion cowden syndrome (omim #158350) is an autosomal dominant hamartoma and tumor predisposition syndrome caused by heterozygous pathogenic germline variants in pten in about 80% of affected individuals [yehia & eng 2001/2021]. major features include macrocrania, multiple facial tricholemmomas, acral and oral keratoses and papillomas, as well as mammary, non-medullary thyroid, renal, and endometrial carcinomas [yehia & eng 2001/2021]. lhermitte-duclos disease (ldd), or dysplastic gangliocytoma of the cerebellum, is the typical brain tumor associated with cs; in fact, in adults, ldd is considered pathognomonic of cs [yehia & eng 2001/2021]. conversely, the lifetime risk for ldd in cs patients has been reported to be as high as 30% [riegert-johnson et al. 2010]. in contrast, the association between cs and medulloblastoma is very rare. in a study of 914 children and adolescents with cancer, including 227 with medulloblastoma, no pten germline variants were identified [gröbner et al. 2018]. similarly, among 1022 medulloblastoma patients screened for germline variants in 110 cancer predisposition genes, only one pathogenic variant was reported in pten [waszak et al. 2018]. in a clinical cohort of 368 patients with cowden syndrome and pathogenic pten germline variants (including 98 patients under the age of 18 years) recruited and followed prospectively by an international consortium between 2000 and 2010, no cases of medulloblastoma were reported [tan et al. 2012]. on the other hand, including this case, five patients with cs and medulloblastoma have now been reported in the literature; they are summarized in table 1. patient 1 was reported before the genetic defect in cs was known [bagan et al. 1989], hence the diagnosis of cs is based on the clinical findings [eng 2000]; he has since been lost to follow-up (j.v. bagan, personal communication). the four other patients carried a pathogenic germline variant in pten. given the rarity of cs, with an estimated prevalence of 1 in 200,000 [yehia & eng 2001/2021], this is unlikely to be a coincidence. table 1: patients with cowden syndrome and medulloblastoma. case sex/age at diagnosis (case id if applicable) mb subtype (histology if known) germline pten variant additional variants and/or other genetic changes in tumor status reference               1 m/2 y (case 2) na na na ned at 19 y bagan et al. 1989               2 m/2 y ns c.697c>t, p.r233* nd ned at 14 y patini et al. 2016               3 f/1 y (icgc_mb217) shh c.856dela, p.t286pfs*5 loss of heterozygosity ns waszak et al. 2018               4 f/14 mo shh (dn¶) c.18dup, p.e7rfs*4 sufu c.412delinscc, p.a138pfs*32 kdr c.787c>a, p.p263t ned at 5½ y¶ tolonen et al. 2020               5 f/15 mo shh (mben) c.388c>t, p.r130* smo c.1234c>t, p.l412f copy-neutral allelic loss on 10q ned at 13 y current report abbreviations: dn, desmoplastic nodular; mb, medulloblastoma; mben, medulloblastoma with extensive nodularity; mo, month; na, not applicable; nd, not done; ned, no evidence of disease; ns, not stated; y, year ¶ r. niinimäki, personal communication interestingly, in the three cases in which a detailed analysis of the medulloblastoma was performed, it was an shh-activated medulloblastoma. two of them also had an additional pathogenic variant in a component of the shh-pathway and 2 showed allelic loss involving pten/10q. this mirrors findings in sporadic medulloblastomas, where mutations or deletions of pten are more frequent in shh-medulloblastomas than in other medulloblastoma subtypes [northcott et al. 2012]. furthermore, in mouse models of shh-medulloblastomas driven either by inactivation of ptch1 [metcalfe et al. 2013] or activation of smo [castellino et al. 2010], additional loss of pten changed the tumor histology from tumors resembling classic medulloblastomas to tumors resembling mben. using similarity network fusion applied to genome-wide dna methylation and gene expression data, shh-medulloblastomas can be divided into 4 distinct subtypes [cavalli et al. 2017]; we did not perform this type of analysis on our patient’s tumor and based on the published data, neither did the authors of the two other cases where the tumor was analyzed (cases 3 and 4). brain-specific suppression of pten in mice produces cerebellar lesions resembling ldd, but not medulloblastomas [backman et al. 2001; kwon et al. 2001]. in human shh-medulloblastomas, loss of pten almost never occurs in isolation. in a detailed analysis of 196 sporadic shh-medulloblastomas [skowron et al. 2021, figure 2a], nine had a deletion and/or mutation of pten. eight of these tumors had additional, often multiple, genetic alterations, typically involving a gene of the shh-pathway, loss of 9q (which deletes ptch1), loss of 10q (which deletes pten and sufu), or mutations and fusions in other genes. only one tumor had an isolated variant in pten, but the analyses may not have captured all possible genetic alterations (p. skowron and m. taylor, personal communications). these findings indicate that in most instances, loss of pten alone is insufficient to produce medulloblastomas. shh-activated medulloblastomas are thought to arise from granule cell precursors that form the external granular layer (egl) of the cerebellar cortex; their proliferation and inward migration to form the internal granule cell layer is driven by purkinje cell-derived shh [tamayo-orreo & charron 2019]. in humans, the egl persists after birth. it starts to involute by the 2nd to 4th post-natal month and usually has disappeared by 1 year, although rare cases of persistence until 2 years have been reported [friede 1989]. in a patient with cs, all granule cell precursors in the egl are haploinsufficient for pten, but this is not sufficient to produce a medulloblastoma. however, if one of these cells acquires additional alterations of the type described by skowron et al. [skowron et al. 2021], it can give rise to an shh-medulloblastoma. this would be a rare event, resulting in a low frequency of medulloblastomas in cs patients. furthermore, given the rapid disappearance of the egl after birth, the "window of opportunity" for the development of a medulloblastoma in cs is brief and explains why they only occur in infants in these patients. cs may be under-recognized in medulloblastoma patients. while 90% of patients with cs will have some features of cs by age 20 [yehia & eng 2002/2021], infants and young children may not show some of the pathognomonic features, such as multiple facial tricholemmomas, and present instead with non-specific findings, like macrocrania, autism, or developmental delay [busa et al. 2015]. among the 5 patients with cs and medulloblastoma, patient 1 had oral papillomas at the time of his medulloblastoma diagnosis [bagan et al. 1989]. patient 2 developed gastro-intestinal polyps after his medulloblastoma and sought medical attention for oral and cutaneous lesions at age 14, at which time the diagnosis of cs was made [palatini et al. 2016]. no clinical details are available on patient 3 [waszak et al. 2018]. patient 4 showed only macrocrania at the time her medulloblastoma was diagnosed [tolonen et al. 2020] and still does not have other stigmata of cs (r. niinimäki, personal communication). in our patient, sequencing of pten was only undertaken at age 9, after the diagnosis of the sclerotic fibroma. thus, in some of these young patients, the diagnosis of cs was not clinically obvious at the time they presented with a medulloblastoma; we therefore suspect that cs may be underdiagnosed in medulloblastoma patients. a high prevalence of tumor predisposition syndromes in children with shh-medulloblastomas, especially infants, has long been known. for instance, in a single-institution series of 82 medulloblastoma patients, 6 of 12 patients with an mben had a genetic syndrome (5 gorlin, 1 fragile-x); 5 of these 6 were less than 24 months of age [garrè et al. 2009]. in the aforementioned series of 1022 medulloblastomas, predisposing germline variants in apc, brca2, palpb2, ptch1, sufu, and tp53 occurred in 6% of patients overall, and in 20% of those with shh-medulloblastomas [waszak et al. 2018]. a further analysis by the same group [waszak et al. 2020] identified in addition pathogenic germline variants in elp1 in 14% of pediatric patients with shh-medulloblastomas; all in all, 77 of 202 children (38%) with an shh-medulloblastoma had a predisposing germline variant in one of 7 genes. here, we present evidence that cs, too, can be associated with shh-medulloblastomas in infants. therefore, pten should be analyzed in these patients, regardless of their clinical phenotype, especially if they do not carry pathogenic germline variants in ptch1 or sufu, the most commonly altered predisposing genes in infant shh-medulloblastomas [waszak et al. 2020]. whether cs also predisposes to other types of medulloblastoma (i.e., wnt, group 3, or group 4) remains to be seen. in addition, these cases show that cowden syndrome has a biphasic brain tumor distribution, both in regards to the age of onset and the tumor type: a small number of cowden patients develop a medulloblastoma in infancy while many more develop ldd in adulthood. acknowledgements we thank j.v. bagan, r. niinimäki, s. pfister, p. skowron, and m.d. taylor for kindly providing additional information on previously published cases. w. foulkes receives funding from the canadian institutes of health research (grant fdn-148390) and so does n. jabado (grant mop-286756 and fdn-154307). references backman sa et al. deletion of pten in mouse brain causes seizures, ataxia and defects in soma size resembling lhermitte-duclos disease. nat genet 29: 396–403, 2001. https://doi.org/10.1038/ng782 bagan jv et al. cowden syndrome: clinical and pathological consideration in two new cases. j oral maxillofac surg 47: 291–4, 1989. https://doi.org/10.1016/0278-2391(89)90234-6 busa t et al. clinical presentation of pten mutations in childhood in the absence of family history of cowden syndrome. eur j paediatr neurol 19: 188–92, 2015. https://doi.org/10.1016/j.ejpn.2014.11.012 castellino rc et al. heterozygosity for pten promotes tumorigenesis in a mouse model of medulloblastoma. plos one 5: e10849, 2010. https://doi.org/10.1371/journal.pone.0010849 cavalli fmg et al. intertumoral heterogeneity within medulloblastoma subgroups. cancer cell 31: 737–54.e6, 2017. https://doi.org/10.1016/j.ccell.2017.05.005 cohen bh et al. pilot study of intensive chemotherapy with peripheral hematopoietic cell support for children less than 3 years of age with malignant brain tumors, the ccg-99703 phase i/ii study. a report from the children's oncology group. pediatr neurol 53: 31–46, 2015. https://doi.org/10.1016/j.pediatrneurol.2015.03.019 eng c. will the real cowden syndrome please stand up: revised diagnostic criteria. j med genet. 37: 828–30, 2000. https://doi.org/10.1136/jmg.37.11.828 friede rl. developmental neuropathology. 2nd ed, springer, 1989, p. 15–6. gandhi d et al. intracranial dural arteriovenous fistulas: classification, imaging findings, and treatment. ajnr am j neuroradiol. 33: 1007–13, 2012. https://doi.org/10.3174/ajnr.a2798 garrè ml et al. medulloblastoma variants: age-dependent occurrence and relation to gorlin syndrome—a new clinical perspective. clin cancer res 15: 2463–71, 2009. https://doi.org/10.1158/1078-0432.ccr-08-2023 goschzik t et al. medulloblastoma in adults: cytogenetic phenotypes identify prognostic subgroups. j neuropathol exp neurol 80: 419–30, 2021. https://doi.org/10.1093/jnen/nlab020 gröbner sn et al. the landscape of genomic alterations across childhood cancers. nature 555: 321–27, 2018. https://doi.org/10.1038/nature25480 kieselova k et al. multiple sclerotic fibromas of the skin: an important clue for the diagnosis of cowden syndrome. bmj case rep 2017. https://doi.org/10.1136/bcr-2017-221695 kwon ch et al. pten regulates neuronal soma size: a mouse model of lhermitte-duclos disease. nat genet 29: 404–11, 2001. https://doi.org/10.1038/ng781 metcalfe c et al. pten loss mitigates the response of medulloblastoma to hedgehog pathway inhibition. cancer res 73: 7034–42, 2013. https://doi.org/10.1158/0008-5472.can-13-1222 northcott pa et al. subgroup-specific structural variation across 1,000 medulloblastoma genomes. nature 488: 49–56, 2012. https://doi.org/10.1038/nature11327 prats-sánchez la et al. multiple intracranial arteriovenous fistulas in cowden syndrome. j stroke cerebrovasc dis 25: e93–4, 2016. https://doi.org/10.1016/j.jstrokecerebrovasdis.2016.03.048 riegert-johnson dl et al. cancer and lhermitte-duclos disease are common in cowden syndrome patients. hered cancer clin pract 8: 6, 2010. https://doi.org/10.1186/1897-4287-8-6 skowron p et al. the transcriptional landscape of shh medulloblastoma. nat commun 12: 1749, 2021. https://doi.org/10.1038/s41467-021-21883-0 tamayo-orrego l, charron f. recent advances in shh medulloblastoma progression: tumor suppressor mechanisms and the tumor microenvironment. f1000res 8: f1000 faculty rev-1823, 2019. https://doi.org/10.12688/f1000research.20013.1 tan mh et al. lifetime cancer risks in individuals with germline pten mutations. clin cancer res 18: 400–7, 2012. https://doi.org/10.1158/1078-0432.ccr-11-2283 tolonen jp et al. medulloblastoma, macrocephaly, and a pathogenic germline pten variant: cause or coincidence? molec genet genomic med 8: e1302, 2020. https://doi.org/10.1002/mgg3.1302 waszak sm et al. spectrum and prevalence of genetic predisposition in medulloblastoma: a retrospective genetic study and prospective validation in a clinical trial cohort. lancet. oncol 19: 785–98, 2018. https://doi.org/10.1016/s1470-2045(18)30242-0 waszak sm et al. germline elongator mutations in sonic hedgehog medulloblastoma. nature 580: 396–401, 2020. https://doi.org/10.1038/s41586-020-2164-5 yehia l, eng c. pten hamartoma tumor syndrome. 2001 nov 29 [updated 2021 feb 11]. in: adam mp, ardinger hh, pagon ra, et al., editors. genereviews® [internet]. seattle (wa): university of washington, seattle; 1993-2021. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a varied neuropathology career turning west and east feel free to add comments by clicking these icons on the sidebar free neuropathology 2:20 (2021) reflections a varied neuropathology career turning west and east ralf schober st. georg teaching hospital of the university of leipzig, leipzig corresponding author: ralf schober · simsonstr. 7 · 04107 leipzig · germany ralf_schober@t-online.de submitted: 10 july 2021 accepted: 19 july 2021 copyedited by: bert m. verheijen published: 29 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3448 keywords: neuropathology, reflections, autobiography i feel very honored to be included in the list of authors for the “reflections” series in free neuropathology, being that i do not really consider myself as a protagonist of our profession. my curriculum is rather variegated and adjusted to the circumstances, resilient yet as it has been tradition in my ancestry. origin and childhood my mother, while just being in labor in an upper floor of a house in bad oeynhausen/ lower saxony in 1944, refused the usual demand to take refuge in the basement when bombers were approaching. thus, the world received me with sunshine, and i believe that i had this perception ever since. my mother’s father, author of the “atlas metallographicus”, saved his position at the university of berlin during the nazi-era by falsifying his passport with the aid of a distant relative who was major in a small rural town and had the suitable seals. my father, having received a degree in chemistry, in world war ii suffered a bullet wound with temporal lobe injury and severe meningitis resulting in epilepsy and partial hearing loss, yet he resumed his beloved piano playing and later on even gave regular house concerts. survival in the post-war famine was not easy, but we were lucky to be assigned as refugees to the farm of some distant relatives (fig. 1). my father, before being able to take up a position in chemistry again, gave private school instructions to the children whilst all public schools were still closed. fig. 1. rs, showing his love for nature early on, with no fears of cjd. education being thus born in a family of the “bildungsbürgertum”, it was only natural for me to attend a “humanistisches gymnasium”, a high school with latin and greek as major subjects. but i have also good memories of the elementary school: in periods of very warm weather the teacher gave his lessons in the nearby park – i am regarding this a good example for the current covid-19 period. the high school was situated in hannover, at that time british occupation zone. the school principal, von drygalski, son of a famous polar explorer, was very proud that we were the first school class permitted to start with latin instead of english, and that the high school was named after the last german emperor. to our amusement, the name placed at the entrance repeatedly changed from “kaiser wilhelms gymnasium” to “kaiser-wilhelm-gymnasium”, due to different opinions of the city officials about the nature of this association. retrospectively, cancel-culture does not appear to be an entirely new phenomenon. we received a broad-based education and did not really suffer from gender segregation, being that the girls’ school was located in the vicinity. sports was also promoted. i spent hours in a rowing boat on the city’s “maschsee” almost daily, and our team received a number of medals. even in much later years we still could be seen as a rowing crew, exploring the landscape of the danube and other rivers on holiday occasions (fig. 2). similarly, for my school class the “abitur” was not the final event, we are still trying to have a get-together each year. fig. 2. rowing trip on the danube river with a crew of old school comrades (rs 4th from left). studies to study medicine was a family decision, especially supported by an aunt of mine who had spent her training in internal medicine in the united states. the emphasis on basic medical sciences in the german curriculum was appealing to me, and so was the relative freedom to arrange the order of courses. i was thus able to change the university three times, from marburg to berlin to göttingen. i was also able to attend voluntary general courses, for example an introduction to informatics given by the computer pioneer konrad zuse. besides i became a member of “marchia”, one of the oldest german fraternities, founded 1810 in berlin concomitant with the operative initiation of the humboldt university. in göttingen, i had the good luck to get a position as “studentischer hilfsassistent” at the university institute for pathology. this was very advantageous for several reasons. first, i could partially unburden my parents from their monthly financial support. second, i became familiar with pathological anatomy, since one of my tasks was to prepare and to display organs with characteristic changes suited to the lectures of prof. johannes linzbach, head of the institute. apparently satisfied with my work, he soon included me in his invitations of assistants and associates for the occasional barbecues in his private garden. i kept this habit in memory as a good example during my own future career. third, i had no problems to secure tissue material for the investigations of my doctoral thesis, the histochemical detection of trace elements in the organs of newborns. this work was done at the max-planck-institute for experimental medicine under the supervision of prof. friedrich timm, in neuropathology well known in connection with the histochemical timm stain for zinc in the hippocampus. graduation in medicine was in 1969, passing one written examination and then oral examinations for each medical discipline at intervals of one week. candidates were grouped into four, and one of them i was the one had to ask each of the chosen professors for the examination date. our whole group passed summa cum laude. at the end we happily invited all professors for dinner in a renowned restaurant, and to our pleasant surprise quite a number of them took part in this rather special farewell. such a ceremony would certainly not have been possible in times where virtually everything is restricted to video contacts. following graduation, a year of practical medicine was required to obtain the medical license. i completed this, the final portion at the department of internal medicine of the university of göttingen under werner creutzfeldt, son of the neuropathologist hans georg creutzfeldt. he backed my plans for a year of pathology in the united states. prerequisites were in existence, by previous medical clerkships in london and in st. louis as well as by the ecfmg certification. i was fortunate in that my successful applications included stanford university, and i also obtained a travel grant of the fulbright commission. the travel was very comfortable, and on the “ms bremen” i assisted the ship’s doctor since i was one of the few passengers that did not become seasick. professional training the planned one year stay at stanford, as it turned out, finally extended to four years, mostly due to the support and encouragement of klaus bensch. originally coming from eastern germany with rather bad experiences over there, he was one of the professors of anatomic pathology and later on followed david korn as head of the department. i was trained in electron microscopy for a project of membrane and cell fusion. we did not finally succeed in the aim of introduction of foreign material, but this research was an excellent basis for membrane research later in my career. my living came from my employment as intern and resident, the last two years in the department of neuropathology headed by lucien rubinstein, and i was working at the university hospital and in turn at the veterans administration hospital. here, lysia forno taught me both the meticulous brain cutting and the very meticulous look at h&e-stained slides, making most special stains confirmatory only. careful observation was then also the basis for a paper with mary herman, the wife of lucien rubinstein, describing brain changes following heart transplantation. it was pleasant to note that with this project we had the unrestricted support of the cardiac surgeon, norman shumway. while working in surgical pathology, i made a fine structural analysis of amyloid in a pituitary adenoma, pointing out a possible pathogenetic correlation with beta-amyloid in senile plaques. upon acceptance of the paper, i was told by the faculty that it had found the interest of bob terry. this signified to me that there were no further problems with the extension of my contract. the academic atmosphere was generally very liberal, with staff from many different countries. coming from germany i took it as a great privilege to take part. my contemporary fellows were, amongst others, samuel ludwin, william langston, bernd scheithauer, and scott vandenberg. of course, we all also enjoyed california, despite lucien rubinstein’s saying that he would not accept people that evidently came because of the good weather. with my käfer-volkswagen, imported when i became chief resident, i repeatedly made tours to big sur, to the sierra nevada and also to the christmas tree farm of jon kosek, professor of pathology at the va hospital. jon used to make this tour by bike, just as he did every day for his way to work. environmental concerns were already well developed these days, and this was also exemplified by louis fajardo, the other va-professor of pathology. he used to supply the whole department with the giant zucchinis raised in his garden. i also enjoyed a sailing course starting in sausalito and finally finished with a high-seas sailing certificate. more important, however, were other obtained certificates: the american board of pathology and neuropathology and the california medical license. furthermore, membership in the association of military surgeons of the united states, later on helping me to follow my professional career without interruption. at the end it was a difficult decision to leave for a position that was offered to me in germany, yet i kept all these certificates valid until now by implementing the continuing medical education requirements. early professional position the decision to take up a position at the max planck institute for brain research in frankfurt in 1974 was a fortunate one. not least so since in the city of frankfurt i met emilia ferrero, daughter of a piemontese family that established a new business extension there. the meeting was arranged by virgilio rolleri, a member of my fraternity and lawyer of reputation, on occasion of a grand ball. being married, we continued to enjoy dancing ever since. at the max planck institute i was in good company of international scholarship holders, coming from turkey, poland, japan, mexico, and other countries. founder of the institute in 1962 and head of neuropathology was wilhelm krücke, in conjunction with his directory of the ludwig edinger institute of neurology of the university. i was in charge of the electron microscopy group, succeeding j. michael schröder. the instrument was the zeiss em9, having good optics but requiring all adjustments to be made by hand and to photographically develop the glass plates. thus, it was good to collaborate with a japanese scholar, yasuhiro yamamura, on the experimental project, nerve regeneration of the muscle spindle after denervation. we were not successful yet to finish this project in time, and only several years later i reported the differences between motor and sensory nerves in two joint papers. meanwhile, trying to improve my skills in more up-to-date techniques, i took an embo-course on subcellular fractionation at the max planck institute for biophysical chemistry in göttingen. the ensuing collaboration resulted in several papers on membrane fusion. a paper in “science” resulted from additional collaboration with the other department of the max planck institute in frankfurt, headed by rolf hassler. diagnostic neuropathology was falling in the competence of the edinger-institute and was conducted collectively by helge gräfin vitzthum, ekkehard thomas and me. prof. krücke, apart from signing out the cases, also rejoiced in some brain cutting personally. this was always a special event for the clinicians, associates and assistants sitting around his table. while dissecting the formalin-fixed organs and laying out the brain slices, he used to tell anecdotes and life stories, often acknowledging the merits of persons that were instrumental in his career. for example, the teacher of latin at his school in dillenburg, being very popular but suddenly disappearing due to his jewish origin and having been saved only by hiding away with some neighbors. or hugo spatz and julius hallervorden, both coming from the team of walther spielmeyer but having very different character traits, one rather expansive and dominant, the other scrupulous at work, rather humble and a person of integrity. or webb haymayker, while with the nasa at moffett field, kindly providing scientific literature that at that time could not be obtained in germany. the autopsy material to be dissected and sampled for histology did not only derive from the university clinics but also from neurologic and psychiatric institutions in the vicinity of frankfurt. i spent many days performing these external autopsies and hold good contacts with the respective clinicians. a favorite destination was the landesheilanstalt eichberg, offering a very pleasant atmosphere irrespective of the dark side of its history, and also the opportunity for me and the autopsy diener to buy some bottles of rhein-wine nearby. in the context of these activities, on the other hand, i also had a very startling experience. one of the dieners that usually accompanied me was skillful at work, reliable and well disposed toward me. it was somewhat conspicuous that he was always dressed extremely well. one day he did not appear without notice, and we did not see him again. we were then informed by the police that he had died in an exchange of fire, following the discovery of his thefts in a store of elegant menswear. all dissected brains and spinal cords of interest were permanently saved at the max planck institute, adding to the ones that were already stored in the basement and up to many decades old. someday i had a request from heiko braak for slices that he would embed and cut with his new technique for large sections in the institute of anatomy. following the selection of cases according to the old documents that to a large part were also still existent as well, i willingly went with him through the dark and dusty rooms to find the right buckets. this action, i believe, may have been helpful not only for heiko braak’s further career but also for the further scientific elucidation of neurodegenerative diseases. when in later years the max planck society decided to bury all this material in a ceremony burdened with guilt, many colleagues were rather bewildered. it was also regarded as fortunate that wilhelm krücke, continuing to work for some time in the edinger institute as an emeritus together with his successor wolfgang schlote, did not live to see this anymore. i thought, in contrast, that he would not have been surprised. he used to recount a special event from his previous time in bavaria. at the end of the war, when the allied forces were approaching, one could see some formalin-fixed brains floating down the isar river. they had been discarded by his colleagues in fear of accusations, despite no guiltiness at all. my time at the max planck institute for brain research then came to an end since the max planck society closed the department of neuropathology in favor of two neuroscience departments. i was very grateful to obtain a grant for a half-year stay abroad in order to facilitate the search for a new job. advanced professional training i was successful with an application to the national institutes of health to study the pathogenesis of idiopathic polyneuritis on patient material, using the myelin protein antibodies against p0, p1, p2 and mag that were newly developed there. in 1979 i thus had the privilege to work with henry de forest webster in the laboratory of neuropathology and neuroanatomical sciences at ninds. with the permission of prof. krücke i collected appropriate paraffin blocks prior to my trip. among these was a huge one containing a furled entire sciatic nerve. i personally made sections from this block with an old tetrander in the max planck institute, anticipating that such technical facilities would not be present in bethesda. harry webster supported the study by asking for additional material from harvard university and the university of pennsylvania. in the end we did not find a pathogenetic association, but the nevertheless ensuing paper was thus co-authored by edward p. richardson and arthur k. asbury. another co-author was yasuto itoyama, a young japanese scientist staying there for a longer period. both of us were well fit for the demanded very careful work, too much so in some european eyes, for example to clean all objectives before taking photos at the zeiss axiomat microscope. harry webster also invited both of us in turn to accompany him on his frequent if not regular weekend sailings. the boat was specially designed with retractable keels on each side to suit the rather shallow banks of chesapeake bay, a challenge even for an experienced yachtsman. back in germany i found a new job in frankfurt at the department of pathology of the st. markus city hospital. though not directly subserving my career, the two years that i spent there were required to obtain the pathology license in germany since my american certificates were not fully acknowledged. due to my continuing activity in external autopsies, i could nevertheless publish a paper in virchow’s archives, a case report of a lymphoma involving the nervous system. preparing the manuscript, i gratefully acknowledged the hand-written support of ronald f. dorfman, my former teacher of surgical pathology at stanford university and an internationally recognized lymphoma expert. professional maturity i returned to neuropathology in 1982 at the university of düsseldorf, serving as assistant physician in the institute of wolfgang wechsler. in tradition with wechsler’s training by klaus joachim zülch, the research focus in the institute was neuro-oncology. this topic was strongly picked up by my companion assistant guido reifenberger, transiently scholarship holder at the sahlgrenska-hospitals in göteborg and in the later succession becoming head of the institute in düsseldorf. my own contributions, resulting from my continuing clinical-diagnostic work, were mostly case reports in collaboration with my very cooperative young colleagues. to name just a few: eva neuen-jacob, specializing in myopathology and later on being in charge of all related diagnostics at the institute; martina deckert, later on head of neuropathology in cologne; thomas bilzer, veterinary neuropathologist and specialist in borna disease; reinhard prior, later on working in italy and being coordinator of european science projects; jürgen kiwit, later on chief physician of neurosurgery in berlin-buch; hiroshi himuro, fellow in neurosurgery and later on returning to fukuoka; stan krajewski, of polish origin and later on together with his wife working at the sanford-burnham medical research institute in la jolla. a most welcome collaborator belonging to a different lifespan was henry urich, serving as locum tenens for prof. wechsler as he had done before in many other places. we signed out all cases together and published several of them, and i profited from his great experience especially in the pathology of peripheral nervous system tumors. i furthermore had a very good collaboration with the institute of pathology just some stairs below. consultations were frequently made with franz borchard, assistant medical director and later on chief physician of pathology in aschaffenburg. one of the assistants was karl-friedrich bürrig, later on chief physician of pathology in hildesheim and president of the bundesverband deutscher pathologen e.v. the director of the institute of pathology was waldemar hort, already known to me from my time as a student in göttingen. hort and wechsler were quite different characters, easily to be seen when on the way to work. the former one bicycling along the rhine river, the big front basket and the side bags fully loaden with medical papers and documents. the latter one in a porsche sports-car for the rather long trip from his home in bergisch-gladbach, and whenever the sun was shining, with open roof and a white cap on his head. in some nice and hot summer days we sometimes also saw him similarly capped, climbing up the ladder to the roof of the institute. my own primary research went in a different direction, the investigation of laser-induced tissue changes in the central and peripheral nervous system. this was a truly collaborative venture, initiated by hans-joachim schwarzmaier at the department of laser medicine. utilized was a new type of laser, the 1,32 μm nd:yag laser, in service by stefan hessel at mbb-medizintechnik gmbh münchen. the operative procedures on experimental animals were done by frank ulrich and other colleagues at the department of neurosurgery. in charge of neuroradiology was thomas kahn, continuously collaborating with me in later years in leipzig as well. the primary goal of the project was to treat brain tumors by laser-induced interstitial thermotherapy in a mini mally invasive way. after completion of the experimental phase frank ulrich indeed was successful with clinical applications, performed under stereotactic or “open” magnetic resonance imaging guidance. my histologic investigations were acknowledged as an important contribution, allowing me to participate in the gordon-research-conference on lasers in biology and medicine, meriden/usa. yet with presentations at some other conferences, i heard rather different commentaries the mission of neuropathology should be the analysis and not the destruction of tissue. i did not quite agree but in fact i saw the major utility of this laser in tissue welding, especially for peripheral nerve anastomoses. a paper in “science”, elucidating the fine structural basis for this procedure, was a major module for my habilitation and postdoctoral lecture qualification at the university of düsseldorf in 1987. not entirely with pleasure since i lost my job, due to university regulations on fixed-term contracts. consequently, i pondered an offer of my aunt to take over her practice of general medicine in her nice house in bad essen, an idyllic small town at the foot of the teutoburger wald. i had already gained some insight there from occasional holiday locums in order to supplement the budget of my family. some of the good memories were that i never again have eaten such fresh and tasty bread, butter, eggs, and bacon gifted by the grateful farmer-patients. nevertheless, i declined the offer of my aunt. this turned out to be the right decision since half a year later i could continue at the university department in a superior position, as deputy medical head. eventually, applications for an independent position were also successful. i got a very cheerful farewell ceremony when i left to the university of leipzig in 1994 (fig. 3). fig. 3. my favorite poster, compiled by my friends and colleagues in düsseldorf. the format of my neuropathology reports is remaining unchanged up to now. [please click on the figure to download a high-resolution version] independent professional position in leipzig, much of what i encountered was unusual at first, but i nevertheless liked it from the beginning. my presentation as a candidate took place in the old bologna-style auditorium of the institute of pathology, originally designed for autopsy demonstrations. speaking while standing behind the desk at the floor, i was rather confident when i saw hans hilmar göbel and jorge cervos-navarro as members of the committee, sitting at short distance in the first row. my wife, however, was sitting in the top row and became dizzy when looking down. but stepping down after my presentation, she was welcomed and accompanied by the local professors in a very warm and polite way, not always her experience elsewhere. similarly, when i started with my work, it was customary in the morning meeting to first shake hands with all colleagues. i was also very fortunate with the staff that was either assigned to me or chosen by me: my secretary annelies krob, prudent and internationally experienced from her previous work at the trade mission of the german democratic republic in japan. my laboratory technicians, starting work in their usual habit at 6:30 a.m., thus having finished some urgent tasks already when i arrived considerably later. my assistant physicians steffen kellermann and dietmar thal, greatly contributing to establish neuropathology as an independent department, the first one in the area of eastern germany. steffen kellermann had already worked in the institute of pathology and continued in my department to organize the “rätselecken”, diagnostic slide sessions that now were addressing neuropathology. to our surprise and pleasure the auditorium was filled with numerous participants, including well established colleagues coming from distant institutions, such as werner jänisch together with marlis günther from brandenburg, or rolf warzok together with silke vogelgesang from greifswald. regarding other participants coming from nearby, we were not always sure about their motives since we heard frequent praises of the dishes and cakes prepared by my wife, served in the break and afterwards. dietmar thal soon made contact with thomas arendt at the paul-flechsig-institute for brain research and with other local researchers, establishing a fruitful collaboration and the basis for his later achievements in the classification of alzheimer’s disease. quite helpful for the development of my department was furthermore a half-year stay of henry def. webster, financed by the senior scientist award of the alexander von humboldt foundation. i could only offer him a very small room as workplace, a transit room to the secretary’s office. when i tried repeatedly and in vain to get a larger room in the institute, i received the ground plan of the institute together with a small figure from a well-meaning pathology colleague, thomas friedrich, later on pathologist in zurich. my wife, sawing this, integrated it in an amusing cartoon (fig. 4). fig. 4. a unique cartoon of a problem known to many neuropathologists (“prof. schober strides through the pathology institute, searching for new rooms”). but harry webster was content with a chair and desk for his own laptop since he primarily intended to finish some publications. his stay was greatly acknowledged by the team (fig. 5).the weekends were usually reserved for excursions together with my family, in return of the hospitality that i had received in bethesda. harry and his wife marion, coming back from stays in her home country hungary, sat in the in the back row of my van, in front of them our three small children celestina, giulio and giovanni. when exploring old castles and other historical sites we soon noticed that harry was the only person having a real interest in these, and thus we spent more time in country festivals, markets, and restaurants. it was reassuring to find no pejorative words about this period in harry’s book of memoirs that he sent me 10 years later. fig. 5. the neuropathology team in leipzig confers an award to henry def. webster (upper left and lower right). professional leadership a major event in my professional life was the “international symposium and 45th annual meeting of the dgnn” in the spring of 2000, organized by my team and me when i was the elected president of the german society for neuropathology and neuro anatomy. with the generous support of the dfg, bmbf, smwk, and avhf, conveyed by otmar wiestler and other colleagues, we could invite numerous internationally leading neuropathologists and scientists as speakers and chairmen for the symposium. with some of them i hold long-lasting friendships, e.g., with davide schiffer, janus szymas, and alfonso escobar. the congress took place in the mendelssohn-hall of the gewandhaus and in representative lecture halls of the university, facilitated by the generous support of industrial and other companies that was gained by our pertinacious personal canvassing. addresses of welcome were made by volker bigl, rector of the university and previously head of the paul-flechsig-institute for brain research, and by gottfried geiler, member of the leopoldina and previously head of the institute for pathology. in the following ceremonial act, the alfons-maria-jacob-medaille was awarded to jürgen peiffer by dimitrios stavrou. refreshments were offered generously throughout, and at the social evening a free dinner was served in the “ratskeller”. the congress finished with a satellite symposium on east-west-connections in neuropathology, organized in collaboration with lothar pickenhain and with ortrun riha, director of the karl-sudhoff-institute for the history of medicine and natural sciences at the medical faculty of the university of leipzig. referees were jürgen peiffer, loránt leel-össy, irina n. bogolepova, hans-dieter mennel, ingrid kästner, igor klatzo, arcadiu petrescu, and a.o. sapetskii. in retrospect we could hear from many participants that they would not have expected such an event to take place in eastern germany 10 years after the fall of the wall. the neuropathology team in leipzig was always complemented by younger people, either research students working for their doctorate or student assistants paid with a small stipend. their selection and supervision were mostly made by vera ogunlade, a phd always good humored, quite necessary for this task. she originated from the caucasus area, was married in africa, became a member of the arnold-sommerfeld-society in leipzig, and following her job in my department she later worked with hans kretzschmar in munich. a very good graduate student was christian schulze. originating from zwickau and studying in leipzig, he successfully engaged in my continuing laser project, resulting in several publications. he later worked at harvard medical school, then in new york at columbia university medical center, and is now head of cardiology at the university of jena. other publications of my department resulted from cooperations with various clinical colleagues, especially with the neurosurgeon juergen meixensberger. with his support, my associate manfred bauer initiated a long-standing collaboration with the neuro-oncology group headed by heidrun holland, using up-to-date cytogenetic and molecular genetic methods. i personally, in addition to neuro-oncological and neuromuscular diagnostics as well as brain cutting for legal and other expert reports (fig. 6), made some investigations of historical sites and about physicians in saxony, resulting in contributions to the periodical of the dggn (german society for the history of neurology). an article about richard arwed pfeifer was made in cooperation with wolfgang schlote, another article about hugo kufs in cooperation with hans hilmar goebel. fig. 6. persistent activity of brain cutting, as usual in a standing position. additional professional obligations included frequent meetings as a member of the faculty council and of the graduation committee as well as yearly meetings in brussels as an eu expert evaluator. this activity has continued without interruption, now however in an online format. although i see the necessity for video communications, i have to admit that i am missing the personal contacts with the other experts and the concluding dinners together with the staff. attending the restaurants in brussels i was usually accompanied by my wife. we usually also went together to the social evenings of various society meetings when held in european countries and not requiring transcontinental flights. i may just name a few such social events, of which we or i have nice and lasting memories. episodes 1. at the 2nd joint meeting of hungarian-german neuropathologists, organized by samuel komoly and katalin majtényi in budapest 1999, we enjoyed a barbecue and open dance in the nice setting of a park. at the end there was a dancing competition, with emphasis on viennese waltz. and who was the winner? not surprisingly, somebody from vienna. herbert budka stood out by his extraordinary elegance and vigor with switching clockwise and counterclockwise turnings. 2. at the sixth ibro world congress of neuroscience in prague 2003, the social dinner took place in the zofin-palace, a highly representative historical building on a moldova river island. the evening was opened by eva syková, appearing on the stage in a magnificent long dress with a fur stole. the curtain swept back, and we heard some czech music very nicely played on a grand piano. the dinner came in exquisite dishes. starting with the soup, the plate at each place was covered with a high porcelain cap to facilitate a collective beginning, and the following courses of the delicious meal were all individually served just like at court. 3. the 2005 meeting of the peripheral nerve society and the italian peripheral nerve study group took place at il ciocco, a very scenic resort in tuscany. we came by car together with our youngest son and with my parents in law from torino as additional guests. having transcended the apennine mountains on a very narrow and curvy road, we were hungry and rather happy to see a great variety of delicious dishes lying out as finger food at numerous tables in the big courtyard. all participants of the meeting ate with pleasure, and the dishes as well as wines and soft drinks were replenished repeatedly. then, to our surprise, the opening ceremony ended with an invitation to walk into the building and to have dinner. this was sumptuous and was served in the traditional sequence of pasta, main dish and fruits and liquors. 4. in contrast to professional trips in europe, those to overseas destinations i usually made alone. once a year i applied for a travel grant of the daad for a presentation overseas, thus regularly meeting my american friends and colleagues again at the annual meetings of aanp and/or asip in various cities. i usually booked the flight at a considerably reduced price for a whole week, giving me the opportunity to make new friends at local rotary clubs and to explore further localities. 5. special mention deserve some very nice experiences that i made in japan. at the 39th annual meeting of the japanese society of neuropathology in fukuoka 1998, president masashi fukui invited some speakers and special guests for dinner, amongst others henry def. webster, bernd scheithauer, peter j. dyck, dimitrios stavrou, akira hori, and me. the main dish was shark fins, a highly valued delicacy. we were all seated at a round table and thus could well observe how different the skills were to eat with chopsticks and to use the small bowls with soy sauce, rather a challenge with this dish. several guests gave up and changed over to forks, but nobody was in a hurry. at the end of this cheerful company with a lot of sake, we had the choice to either take a taxi to the hotel or to continue the party in the rear of the building. looking at each other we exactly knew in advance what the choices were. the taxi was a big cab, allowing further conversations. harry webster soon spoke out and elucidated the values of the family. those of us that were neuropathologists recollected that he even in his publications had used this term allegorically, for the description of the groups of outgrowing axons. 6. another most memorable journey was to the 50th annual meeting of the japanese society of neuropathology, organized by kiyomitsu oyanagi in takamatsu 2009. this time i made a round trip, and i was accompanied by two persons. first, by my aunt, retired from general practice and at that time 87 years old but still very adventurous. second, by my older son giulio, student in theoretical physics and at that time in tokyo as an awardee of the japanese ministry of education, culture, sports, science and technology. he had asked his professor, naoto nagaosa, for an interview in his institute at the hongo campus, and we were kindly received in his office. he gave us an introduction to the new class of topological insulators, drawing a möbius strip at the blackboard and also acknowledging the origin of möbius from leipzig. at the end of this interesting but intellectually demanding recourse, my aunt drew a large old photograph out of her handbag. it was dated 1926 and showed a ceremony in japan, where her uncle walter lwowski received a golden sake-bowl engraved with the emblem of the tenno, acknowledging of the construction of the first sheet mills at kawasaki and at yawata over a period of five years. consecutively we made some further talk, and we expressed our thanks to prof. nagaosa for the long time that he had spent with this interview. we then made a sightseeing tour on the hongo campus and were fortunate to be guided very competently by masaya oda, still fluent in german from his stay at the max planck institute for brain research in frankfurt long years ago. after further sightseeing in tokyo, we took the shinkansen to takamatsu, a fast and comfortable ride. 7. the closer we came to takamatsu, the brighter blue was the sky. on our arrival at the most scenic landscape, the splendid seaside location, and the very comfortable hotel at walking distance, it almost seemed to be a pity to spend the time in conference rooms. but the meeting then proved to be extremely well organized and interesting. following the presidential lecture by kiyomitsu oyanagi and the plenary lectures by john trojanowski and dennis dickson, presentations were scheduled in several different rooms, but all participants could enjoy a bento (japanese lunch) together. a major event was the memorial celebration party. at the end, after several addresses by dignitaries and notabilities, i dared to ask kiyomitsu if i could say a few sentences in japanese that i had prepared together with my son. my japanese sentences were apparently understood, to judge by the applause. finally, to our surprise and without having been prepared, my aunt was cited to the stage. she climbed up without hesitation and reported, translated by my son, some episodes of her life as well as the connections of her family with japan, and she got even more applause. upon returning back to tokyo, accompanied by akira hori in the shinkansen and following his explanations regarding the history of the landscapes and monuments passing by, we had another very nice farewell. masaya oda invited us for dinner in a top-class restaurant, together with his family. the cook prepared the meal on hot plates just in front of each guest, separately yet perfectly synchronized (fig. 7). fig. 7. invitation for dinner in a fine restaurant in tokyo (from left: my son giulio, my aunt gisela rohlmann, rs, masaya oda and his daughter). retirement and beyond in 2010 at the age of 65, i did not feel like retiring, as it is usually required for civil servants in germany. therefore, i personally went to my employer, the smwk (saxonian ministry for science and arts) in dresden, and i asked for special permit to continue as head of the neuropathology department in leipzig. the answer of the officer in charge was affirmative. not so, however, the answer of the dean of the university in leipzig. fortunately, i soon got an offer to work as a neuropathology consultant elsewhere in leipzig, in the institute of pathology and tumor diagnostics at the municipal hospital st. georg. the director there, volker wiechmann, as well as the chief of the associated pathology practice, andreas plötner, were known to me as previous participants of my slide seminars. i was sympathetically accepted as a member of the team, and i have always been invited for social gatherings such as the annual christmas dinner party. for more than 10 years up to now i thus have continued to make diagnostic work for the neurosurgery and other departments as well as all routine brain cutting. in a rather comfortable way for several hours daily, usually in the afternoon except once a week at 7 a.m., a time that is scheduled for the tumor board. i enjoy instructing the younger colleagues at a multi-viewer microscope, and there is also the opportunity to give some lectures to students since st. georg is a teaching hospital of the university. furthermore, there are some ongoing clinical research projects in collaboration with max holzer from the paul-flechsig-institute for brain research and with other scientists. on occasion i am pleased to get visits by external colleagues and old friends, giving testimony that leipzig has become an attractive location again (fig. 8). the institute of pathology of the st. georg hospital, founded in 1913, is one example of the many historical and well restored buildings (fig. 9). fig. 8. kiyomitsu oyanagi and rs in front of dr. faustus and mephisto at the auerbach’s keller restaurant in leipzig. at the university clinics i am still participating in academic events and i am still a member in a graduation committee. asked about my hobbies and proclivities, i can say that there is no boredom. trying to stay up to date, i am reading two daily newspapers and i am browsing through the serial issues of about 10 journals including science, jnen, biospektrum, and rotary magazin. one particular field of interest is astronomy, and my collection of the pertinent literature has resulted in several presentations on galaxy evolution to a lay public. another one is history, especially in relation to the origin of religions or ideologies. i am also fond of fine arts, though not personally active anymore in painting and in piano playing. last not least i am enjoying fine cuisine, being spoiled already at home by my italian wife. my mobility is somewhat limited by a peripheral neuropathy that is associated with a protein well known to me. however, there is no change both in my general view of pathology as well as in relation to myself: a molecular detail may be important and have far-reaching implications, but it does not carry the weight of an integration of all macroscopic and microscopic aspects in correlation with the individual history. altogether it is thus a pleasure to continue with my professional activities, comprising 50 years in neuropathology. cv (short): to be found at my homepage http://www.schober-info.com/ fig. 9. the pathology staff at the st. georg hospital in leipzig (upper row from right: director volker wiechmann, rs, chief pathologist andreas plötner). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. hypothesis: entrapment of lipoprotein particles in the brain causes alzheimer’s disease feel free to add comments by clicking these icons on the sidebar free neuropathology 2:30 (2021) opinion piece hypothesis: entrapment of lipoprotein particles in the brain causes alzheimer’s disease delphine boche1, james ar nicoll1,2 1 clinical neurosciences, clinical and experimental science, faculty of medicine, university of southampton, southampton, united kingdom 2 department of cellular pathology, university hospital southampton nhs foundation trust, southampton, united kingdom corresponding author: delphine boche · clinical neurosciences, clinical and experimental sciences academic unit · faculty of medicine · university of southampton · southampton general hospital · mailpoint 806 · southampton so16 6yd · united kingdom d.boche@soton.ac.uk submitted: 05 august 2021 accepted: 21 october 2021 copyedited by: cinthya agüero published: 02 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3459 keywords: lipoprotein particles, extracellular matrix, cholesterol transport, apolipoprotein e, alzheimer’s disease abstract we present for consideration a hypothesis that impaired movement of lipoprotein particles in the extracellular space in the brain in ageing is central to and causes all the key pathophysiological features of alzheimer’s disease (ad). the role of lipoprotein particles is to transport cholesterol from glial cells, where it is synthesised, to neurons, which require cholesterol for synaptic plasticity. the lipoprotein particles have a cholesterol-containing hydrophobic core, in which amyloid-β (aβ) can be solubilised. the core is surrounded by a hydrophilic surface containing apolipoprotein e (apoe) which, as neurons bear receptors for apoe, determines the destination of the particles. the problem arises because the extracellular space is a narrow cleft, barely wider than the lipoprotein particles themselves, which they have to navigate in order to perform their crucial cholesterol-transporting function. we explain how lipoprotein particles could become trapped in the ageing extracellular matrix and that this primary abnormality results in reduced delivery of cholesterol to neurons leading to impaired synaptic plasticity, crucial for learning and memory. it can also explain extracellular aβ accumulation, to which a microglial response generates a neurotoxic reaction, and intraneuronal tau aggregation, each of which exacerbate the problem. all these players have been known for many years to be important in alzheimer’s pathogenesis but a single unifying mechanism to explain how they are linked has been lacking. this proposed mechanism, with entrapment of lipoproteins particles as key to the development of ad, can explain the failure of so many clinical trials and points out new directions to be taken. genetics shows the overriding importance of apolipoprotein e in a complex multifaceted disease process, such as alzheimer’s disease (ad), genetic factors are important in highlighting key elements relevant to the onset of the disease. by far the major genetic risk factor for ad, in terms of scale of effect, is the apolipoprotein e genotype (gene: apoe, protein: apoe). there are 3 common apoe gene alleles (ε2, ε3 and ε4); as each person inherits one allele from either parent there is a total of 6 apoe genotypes (ε2/ε2, ε2/ε3, ε3/ε3, ε3/ε4 and ε4/ε4). the apoe ε4 allele carriage rate (i.e. the proportion of people in a population who possess one or two ε4 alleles) is typically about 25% of people of european ancestry [1], but varies considerably around the world among different populations [2]. a single copy of the apoe ε4 allele confers a two to fourfold increased risk of developing ad, with ε4 homozygotes at fourteen times increased risk of developing ad, whereas the less common ε2 allele confers relative protection, approximately halving the risk [3]. the effect is so substantial that a person who is ε4 homozygous and lives to 85 years of age has a lifetime risk of ad of more than 50%, comparable to the risk associated with brca1 mutation in breast cancer [4]. on the other hand, the protective effect of apoe ε2 is so substantial that ε2 homozygotes have a very low likelihood of developing ad; ε2 homozygotes have a 87% lower odds ratio than ε3 homozygotes and a 99.6% lower odds ratio than ε4 homozygotes [5]. the apoe gene polymorphism is lacking from non-human primates [6, 7] although, interestingly, rhesus monkeys which have an ε4-like apoe sequence develop aβ plaques as they age [8]. the fact that there are more than 200 animal models of ad highlights the difficulty in mimicking the complexity of the human disease in experimental animals. a complete understanding of the pathophysiology of ad ideally would explain all aspects of the human disease, and in particular incorporate the role of the apoe protein; how it causes and interacts with the accumulation of amyloid-β (aβ) and tau proteins, the glial cell activity and most importantly, the neuronal and synaptic dysfunction and loss. the function of apoe is to deliver cholesterol, packaged in lipoprotein particles, to neurons apoe is the principal cholesterol carrier in the brain, acting as a detergent with hydrophobic and hydrophilic moieties. its main role is to solubilise cholesterol and other lipids and lipid-soluble substances to enable them to be transported in the aqueous extracellular environment of the brain. the importance of cholesterol to the brain is highlighted by the fact that 25% of the body’s cholesterol is contained within the brain, despite the brain representing only 2% of the body weight [9]. cholesterol forms about 30% of the lipid bilayer of the membrane of all cells and is important in maintaining membrane fluidity. in the brain, the cell membrane is essential for conducting the action potential and for communication between neurons at synapses. however, despite its importance to neuronal function, neurons do not synthesise their own cholesterol, but rely on cholesterol which is synthesised by glial cells and then transported to neurons [10]. delivery of cholesterol to neurons is in particular demand when the neuron changes, as in synaptic plasticity which underpins learning and memory which are particularly affected in ad [11]. of note, genome-wide association studies have highlighted polymorphisms in genes in addition to apoe that are involved in cholesterol handling (clu, picalm, bin1 and abca7) as risk factors for ad [12, 13], supporting an important role for cholesterol handling in the disease mechanism [14, 15]. apoe is located in the shell of lipoprotein particles, with hydrophobic substances including cholesterol, which is loaded onto the lipoprotein particle by the enzyme abca1, being transported in the core [16]. the destination of the lipoprotein particles is determined by the receptors for the proteins on the surface of the lipoprotein shell. apoe, on the outer surface of the lipoprotein particles, binds to receptors of the low-density lipoprotein (ldl) receptor family (principally lrp1/apoe receptor) present on neuronal cell membranes and, by this mechanism, the cholesterol is internalised within the neurons. some cns lipoprotein particles also bear apoj (clusterin) on their surface; ependymal cells, but not neurons or other glia, bear apoj receptors so it is unlikely to be relevant for cholesterol delivery to neurons [17]. however, it is notable that in genome-wide association studies, polymorphism of apoj/clusterin gene has also been shown to influence risk for ad [18], raising the possible importance of clearance of lipoprotein particles and cholesterol to the csf. outside the brain, in the rest of the body, there are several other lipoproteins that can fulfil the function of cholesterol transport in addition to apoe, including apoa which is synthesised in the liver [19]. apoa-containing lipoprotein particles are detectable in the csf but appear to be excluded from the brain parenchyma by the blood-brain barrier. consequently, the cholesterol required by neurons is synthesised within the brain and delivered to neurons by apoe-containing lipoprotein particles [20] [9, 21]. lipoprotein particles become entrapped in the ageing extracellular matrix the lipoprotein particles, which resemble the high-density lipoproteins (hdl) present in the bloodstream, are in the region of 11-20 nm in diameter [22]. in order to transport cholesterol to neurons, they have to travel in the extracellular space in the brain which itself measures only about 40 nm between adjacent cells [23], below the resolution of light microscopy. small molecules can pass readily, by diffusion and potentially by active flow, through the extracellular space but diffusion even of conventional macromolecules much smaller that lipoprotein particles is hindered, particularly in pathological processes such as gliosis (activation of glial cells) [24], which occurs in association with ageing and the neurodegeneration in ad. an additional complexity is that the extracellular space resembles a sponge, containing the extracellular matrix (ecm). in the brain, the ecm is a complex multimolecular three-dimensional structure consisting of proteoglycans/glycosaminoglycans, proteins, proteinases, and cytokines [25]. expression of collagen iv, laminin and fibronectin is upregulated in the cerebral cortex in early ad. with ageing-related changes to the extracellular matrix, exacerbated by other risk factors for ad (hypertension, diabetes, obesity, inflammation and physical inactivity), the narrow extracellular space likely becomes compromised (‘fibrosed’), impeding the movement of, and trapping, lipoprotein particles in between cells. interestingly, oxidative modification of plasma lipoproteins is one of the earliest steps in the development of atherosclerosis, involving accumulation of lipoprotein particles in the vessel wall provoking inflammatory reaction [26]. in ad patients, there is evidence of greater oxidation of plasma and csf lipoproteins [26] and this could conceivably further impair the passage of lipoprotein particles through the extracellular space in the brain. consequently, the apoe-mediated system for transporting cholesterol and lipids from glial cells to neurons in the brain is unique, crucial for neuronal function and plasticity and vulnerable to age-related failure (figure 1). it is proposed that this results in a number of consequences leading to the key features of ad pathophysiology as follows. figure 1. entrapment of lipoprotein particles in the brain causes alzheimer’s disease. lipoprotein particles transport cholesterol from glial cells, where it is synthesised, to neurons which require cholesterol for synaptic plasticity. the lipoprotein particles have a cholesterol-containing hydrophobic core, in which aβ is solubilised, and a hydrophilic surface containing apolipoprotein e. receptors for apoe on the neuronal cell membrane determine the destination of the particles. the lipoprotein particles must navigate the extracellular space to reach the neurons, a narrow cleft barely wider than the particles themselves. as the extracellular matrix ages, lipoprotein particles become trapped between cells and this primary abnormality results in reduced delivery of cholesterol to neurons leading to impaired synaptic plasticity, crucial for learning and memory. impaired movement of lipoprotein particles in the extracellular space in the brain in ageing is central to and causes all the key pathophysiological features of alzheimer’s disease: degeneration of entrapped lipoprotein particles releases aβ which aggregates in the aqueous environment of the extracellular space; a microglial reaction to the aβ results in secretion of neurotoxic substances; neuronal cholesterol deficiency causes intraneuronal tau accumulation. [artwork by dr jennifer m dewing] entrapment of lipoprotein particles can explain the key features of alzheimer’s disease aβ deposition in the parenchyma as plaques: aβ peptide is notoriously insoluble in an aqueous environment, but it is lipid soluble and is transported in the hydrophobic core of lipoprotein particles [27-30]. therefore, trapping lipoprotein particles would immobilise aβ in the extracellular space. as the trapped particles degrade and rupture, we propose they release aβ peptide into the surrounding aqueous environment where it aggregates, initiating the formation of extracellular aβ plaques. co-localisation of apoe with aβ plaques is consistent with the origin of the aβ as being from apoe-containing lipoprotein particles [31]. immunohistochemistry for aβ on semi-thin sections (1μm) shows that diffuse plaques, usually interpreted as the earliest stage of aβ deposition, are formed from a cluster of dot-like structures [32], and electron microscopy shows small vesicles associated with amyloid fibrils in the extracellular space [32]. in the context of the current hypothesis, it is intriguing to speculate that these might be entrapped lipoprotein particles from which the amyloid has originated. app transgenic mice, over-expressing the v717f human amyloid precursor protein, develop aβ plaques as they age. interestingly, when crossed with apoe knock out mice so that they lack apoe, these mice do not accumulate plaques [33]. this finding indicates that apoe is essential for the deposition of aβ plaques, supporting the mechanism we propose. with ageing, the primary risk factor for ad, the human brain decreases in weight and size with an overall decline in cortical cholesterol that accelerates from the age of 80 [34, 35]. this is consistent with age-associated entrapment of the lipoprotein particles impairing delivery of cholesterol to neurons and resulting in neuronal cholesterol deficiency [36]. this has further effects on aβ as cholesterol deficiency leads to thinning of the cell membrane, shifting the site for secretase cleavage of the amyloid protein precursor (app) from producing predominantly the shorter form of aβ (aβ40) to the longer form (aβ42) which is relatively even more insoluble and prone to aggregation [37, 38]. this exacerbates the effect of trapped lipoprotein particles as the aβ42 then coalesces onto the initial plaque seeds facilitating their growth. in physiological conditions, aβ is suggested to act as a regulator of cholesterol homeostasis as observed in experimental models [39]. in humans, the familial forms of alzheimer’s disease are due to an imbalance in aβ production and are associated with increased cholesterol levels [40]. the binding of apoe to lipoprotein receptor-related protein (lrp) [41], the major apoe receptor on neurons, is also involved in the cellular uptake of cholesterol and aβ, consistent with apoe, cholesterol and aβ all being components of the lipoprotein particle. in the blood vessel walls as cerebral amyloid angiopathy (caa): aβ also colocalises with apoe in the walls of cerebral blood vessels suggesting that lipoprotein particles become trapped in the extracellular space of the vessel wall basement membrane as a consequence of age-related changes. the blood vessel dysfunction caused by the presence of caa further compromises brain function by haemorrhage, ischaemia and paralysing autoregulation of cerebral blood flow [42]. impaired neuronal and synaptic function deficiency of neuronal cholesterol results in impaired communication between neurons at the synapse, and particularly it interferes with the alterations in neurons which underpin learning and memory (i.e. synaptic plasticity) [43-46]. synaptic plasticity requires neuronal cell membrane to be synthesised to form and re-form synapses as they are remodelled. in neuronal cultures, the presence of glial cells enhances the formation and function of synapses and the essential factor mediating this effect has been identified as glia-derived cholesterol, delivered to the neurons by apoe-containing lipoprotein particles binding to the neuronal ldl receptors [11, 47, 48]. interestingly, in a number of clinical studies of traumatic brain injury, the possession of apoe ε4 is associated with a worse outcome and severe neurologic deficits [49-51]. this seems particularly so in young people in whom neuronal plasticity after injury might otherwise be more pronounced [50]. experimental studies in apoe ε4 transgenic mice confirmed impaired neuronal plasticity after brain injury [52-56]. further evidence comes from experimental models of global cerebral ischaemia in which apoe-deficient mice have increased neuronal damage which is ameliorated by intraventricular infusion of lipoprotein particles [57]. in addition, agonists of liver x receptors (lxr), which act as cholesterol sensors and promote lipidation of apoe by atp-binding cassette transporter a1 (abca1), ameliorate neuronal injury in experimental models of trauma [58] and reverse deficits in mouse models of ad [59]. the effects of neuronal and synaptic dysfunction due to lack of cholesterol [60] would be expected to be most pronounced in neuroanatomical locations in which plasticity is greatest (i.e. hippocampus involved in memory, association cortex involved in interpretation of sensations) and less severe or absent where plasticity is least (i.e. primary motor and sensory cortex, cerebellum, spinal cord). this is consistent with the clinical observations that the hierarchical sequence of loss of functions over time in ad follows the distribution of neuroplasticity [61]. tau protein accumulation we propose that neuronal cholesterol deficiency, resulting from the entrapment of lipoprotein particles, leads to the intracellular aggregation of tau. tau is involved in maintaining the cytoskeletal structure of neurons and, in particular, the transport of proteins from the cell body along axons to the synapses. accumulation of hyperphosphorylated tau occurs early in regions where the cholesterol is most in demand, that is where the rate of plasticity and synaptic remodelling is greatest (i.e. hippocampus, association cortex) and later or not at all where plasticity is least (i.e. primary motor and sensory cortex, cerebellum, spinal cord). a difficulty in trying to explain a direct link between aβ and tau pathology in ad has been that they appear to arise in different neuroanatomical locations; tau accumulation occurs earlier in the hippocampus and associated structures, only spreading later to the cerebral neocortex, whereas aβ accumulation occurs early in the cerebral neocortex. the hypothesis proposed here that there is not a direct link between aβ and tau pathology, but rather that they are each driven by cholesterol deficiency, resolves this conundrum. direct experimental support comes from cholesterol depletion in neuronal cultures which induces tau hyperphosphorylation that can be prevented by treatment with lipoproteins and cholesterol [62]. the link between neuronal cholesterol deficiency and tau accumulation is further highlighted by the occurrence of tangles at a young age in niemann-pick type disease type c, a rare progressive genetic disorder characterised by the inability of the body to transport cholesterol and lipids [63-64]. additional circumstantial evidence supporting a link between cholesterol deficiency and tangle formation potentially comes from the study of chronic traumatic encephalopathy (cte). cte is a condition caused by repeated blows to the head, typically in boxing and other sports, and is associated with the development of dementia with the formation of tangles as the key pathological feature [65]. it has been found that after a head injury, levels of apoe and cholesterol-containing lipoproteins in the cerebrospinal fluid plummet, just at the time when there is an increased demand for cholesterol for neuronal repair [66-68]. this relative deficiency of cholesterol, when recurrent over time with repeated blows to the head, could explain the development of tangles in cte [51-56] [65] studies using cerebral organoids derived from ad patients highlight an association of tau pathology with apoe ε4 carriage [69]. glial cell dysfunction astrocytes and microglia have a major role in supporting neurons and activation of glial cells is a hallmark of ad. in particular, they play an important role in the cycling of lipoprotein particles, scavenging lipid debris from degenerating neuron/synapses, lipidation the particles and releasing them for transport and uptake by neurons [70]. in apoe ε4 carriers, the brain is relatively deficient in apoe and the lipoprotein particles are smaller [71], more prone to aggregate and carry less cholesterol, rendering them particularly vulnerable to the effects of age-related cholesterol deficiency. human induced pluripotent cell (ipsc)-derived astrocytes from ε4 homozygotes produce lipoprotein particles that are smaller, carry less cholesterol and support neurons less well in terms of viability and expression of synaptic proteins compared with those from ε3 carriers [72]. microglia are the immune cells of the brain and are markedly activated in ad [73, 74]. many of the genes identified in genome-wide association studies are expressed by microglia, indicating that they play an important role in the development and/or progression of the disease [75]. the presence of extracellular aβ expelled by trapped and degraded lipoprotein particles is putatively recognised by the microglial pattern recognition receptors (pprs) evolved to detect molecular patterns associated with the bacterial cell walls. the consequent pro-inflammatory state provoked by this response causes release of cytotoxic substance evolved to destroy invading micro-organisms, which inadvertently has a harmful effect on neurons, compounding the effects of cholesterol deficiency [73, 76]. weaknesses/limitations of the hypothesis the ideas presented here form a hypothesis to explain the development of ad which is coherent and explains many facets of the disease. it is important to emphasise that a hypothesis is also an extrapolation of what is currently known and so some of the statements above are not firmly established but are potentially controversial and remain to be explored in the testing of the hypothesis. potential thorns in the side of this hypothesis include: localisation of aβ within apoe-containing lipoprotein particles has not been directly demonstrated in the human brain. this needs to be explored and represents an important gap in our knowledge. available technology is a limitation because the lipoprotein particles are below the resolution of light/confocal microscopy but are potentially amenable to study by novel 3d electron microscopy methods combined with multilabel immunostaining. direct evidence that lipoprotein particles become trapped in the extracellular space, disintegrate and release aβ is currently lacking. studies of immunohistochemistry for aβ and apoe identify apoe in some, but not all plaques. apoe seems to be present particularly in cored plaques, which are interpreted as later stage plaques, rather than early diffuse plaques as might be predicted from the hypothesis. the presence of apoe in later stage plaques could possibly reflect involvement of apoe-containing lipoprotein particles in attempted removal of aβ, in addition to a role in plaque formation. evidence that high peripheral cholesterol levels are associated with increased risk of ad and that statins, which reduce circulating cholesterol by inhibiting the cholesterol-synthesising enzyme hmg-coa reductase, may reduce risk for ad [77] might seem to contradict the hypothesis presented here. it seems that statins do reduce cholesterol levels in the brain [78]. however, it may be that the relative amounts of cholesterol, aβ and apoe are important i.e., that there is sufficient cholesterol as required by neurons, sufficient apoe to solubilise the cholesterol and a sufficient volume of lipophilic core within the lipoprotein particles to transport aβ and prevent its escape into the aqueous environment of the extracellular space where it is prone to aggregate. therapeutic consequences over the past decades, human clinical trials of new therapies for ad have been unrelentingly disappointing. this is despite there being no shortage of endeavour and expenditure, and no lack of successful therapeutic studies in animal models of specific aspects of ad. why is this? we suggest it is because the wrong targets have been addressed. whereas each of the major suspects listed above (aβ, tau, glial cells) may exacerbate the neurodegeneration and set up self-perpetuating vicious cycles, we propose they are not the initiating factor in sporadic ad, either singly or in combination. the pathophysiological scheme outlined above indicates that, according to this hypothesis, aβ and tau are not the cause of ad but are a consequence of the primary abnormality, which is trapping of lipoprotein particles in the extracellular space resulting in disruption of the normal system of delivery of cholesterol to neurons. this implies that even if, for example, the ad brain can be completely cleared of aβ plaques, this will not halt the neurodegenerative decline as observed first in the long-term follow up of ad patients immunised against aβ using an1792 [79] and also in subsequent trials. instead, therapy for ad must be directed at the health of the extracellular space, cholesterol and apoe, lubricating the passage of cholesterol-containing lipoprotein particles. references wolters fj, yang q, biggs ml, jakobsdottir j, li s, evans ds, et al. the impact of apoe genotype on survival: results of 38,537 participants from six population-based cohorts (e2-charge). plos one. 2019;14(7):e0219668. https://doi.org/10.1371/journal.pone.0219668 corbo rm, scacchi r. apolipoprotein e (apoe) allele distribution in the world. is apoe*4 a 'thrifty' allele? ann hum genet. 1999;63(pt 4):301-10. https://doi.org/10.1046/j.1469-1809.1999.6340301.x saunders am, strittmatter wj, schmechel d, george-hyslop ph, pericak-vance ma, joo sh, et al. association of apolipoprotein e allele epsilon 4 with late-onset familial and sporadic alzheimer's disease. neurology. 1993;43(8):1467-72. genin e, hannequin d, wallon d, sleegers k, hiltunen m, combarros o, et al. apoe and alzheimer disease: a major gene with semi-dominant inheritance. mol psychiatry. 2011;16(9):903-7. https://doi.org/10.1038/mp.2011.52 reiman em, arboleda-velasquez jf, quiroz yt, huentelman mj, beach tg, caselli rj, et al. exceptionally low likelihood of alzheimer's dementia in apoe2 homozygotes from a 5,000-person neuropathological study. nat commun. 2020;11(1):667. https://doi.org/10.1038/s41467-019-14279-8 zannis vi, nicolosi rj, jensen e, breslow jl, hayes kc. plasma and hepatic apoe isoproteins of nonhuman primates. differences in apoe among humans, apes, and new and old world monkeys. j lipid res. 1985;26(12):1421-30. mcintosh am, bennett c, dickson d, anestis sf, watts dp, webster th, et al. the apolipoprotein e (apoe) gene appears functionally monomorphic in chimpanzees (pan troglodytes). plos one. 2012;7(10):e47760. https://doi.org/10.1371/journal.pone.0047760 poduri a, gearing m, rebeck gw, mirra ss, tigges j, hyman bt. apolipoprotein e4 and beta amyloid in senile plaques and cerebral blood vessels of aged rhesus monkeys. am j pathol. 1994;144(6):1183-7. dietschy jm, turley sd. thematic review series: brain lipids. cholesterol metabolism in the central nervous system during early development and in the mature animal. j lipid res. 2004;45(8):1375-97. https://doi.org/10.1194/jlr.r400004-jlr200 poirier j. apolipoprotein e and cholesterol metabolism in the pathogenesis and treatment of alzheimer's disease. trends in molecular medicine. 2003;9(3):94-101. https://doi.org/10.1016/s1471-4914(03)00007-8 pfrieger fw. cholesterol homeostasis and function in neurons of the central nervous system. cell mol life sci. 2003;60(6):1158-71. https://doi.org/10.1007/s00018-003-3018-7 jones l, harold d, williams j. genetic evidence for the involvement of lipid metabolism in alzheimer's disease. biochim biophys acta. 2010;1801(8):754-61. https://doi.org/10.1016/j.bbalip.2010.04.005 hollingworth p, harold d, sims r, gerrish a, lambert jc, carrasquillo mm, et al. common variants at abca7, ms4a6a/ms4a4e, epha1, cd33 and cd2ap are associated with alzheimer's disease. nat genet. 2011;43(5):429-35. https://doi.org/10.1038/ng.803 lamartiniere y, boucau mc, dehouck l, krohn m, pahnke j, candela p, et al. abca7 downregulation modifies cellular cholesterol homeostasis and decreases amyloid-beta peptide efflux in an in vitro model of the blood-brain barrier. j alzheimers dis. 2018;64(4):1195-211. https://doi.org/10.3233/jad-170883 dib s, pahnke j, gosselet f. role of abca7 in human health and in alzheimer's disease. int j mol sci. 2021;22(9) :4603. https://doi.org/10.3390/ijms22094603 wahrle se, jiang h, parsadanian m, legleiter j, han x, fryer jd, et al. abca1 is required for normal central nervous system apoe levels and for lipidation of astrocyte-secreted apoe. j biol chem. 2004;279(39):40987-93. https://doi.org/10.1074/jbc.m407963200 ladu mj, gilligan sm, lukens jr, cabana vg, reardon ca, van eldik lj, et al. nascent astrocyte particles differ from lipoproteins in csf. j neurochem. 1998;70(5):2070-81. https://doi.org/10.1046/j.1471-4159.1998.70052070.x lambert jc, heath s, even g, campion d, sleegers k, hiltunen m, et al. genome-wide association study identifies variants at clu and cr1 associated with alzheimer's disease. nat genet. 2009;41(10):1094-9. https://doi.org/10.1038/ng.439 koch s, donarski n, goetze k, kreckel m, stuerenburg h-j, buhmann c, et al. characterization of four lipoprotein classes in human cerebrospinal fluid. journal of lipid research. 2001;42(7):1143-51. https://doi.org/10.1016/s0022-2275(20)31605-9 mahley rw. central nervous system lipoproteins: apoe and regulation of cholesterol metabolism. arterioscler thromb vasc biol. 2016;36(7):1305-15. https://doi.org/10.1161/atvbaha.116.307023 linton mf, gish r, hubl st, bütler e, esquivel c, bry wi, et al. phenotypes of apolipoprotein b and apolipoprotein e after liver transplantation. j clin invest. 1991;88(1):270-81. https://doi.org/10.1172/jci115288 stukas s, kulis i, zareyan s, wellington cl. lipids and lipoproteins in alzheimer’s disease. in: zerr i, editor. alzheimer's disease challenges for the future: intechopen; 2015. nicholson c, hrabetova s. brain extracellular space: the final frontier of neuroscience. biophys j. 2017;113(10):2133-42. https://doi.org/10.1016/j.bpj.2017.06.052 nicholson c, sykova e. extracellular space structure revealed by diffusion analysis. trends neurosci. 1998;21(5):207-15. https://doi.org/10.1016/s0166-2236(98)01261-2 ma j, ma c, li j, sun y, ye f, liu k, et al. extracellular matrix proteins involved in alzheimer's disease. chemistry. 2020;26(53):12101-10. https://doi.org/10.1002/chem.202000782 martins ij, berger t, sharman mj, verdile g, fuller sj, martins rn. cholesterol metabolism and transport in the pathogenesis of alzheimer's disease. j neurochem. 2009;111(6):1275-308. https://doi.org/10.1111/j.1471-4159.2009.06408.x beffert u, poirier j. apolipoprotein e, plaques, tangles and cholinergic dysfunction in alzheimer's disease. ann n y acad sci. 1996;777:166-74. https://doi.org/10.1111/j.1749-6632.1996.tb34415.x biere al, ostaszewski b, stimson er, hyman bt, maggio je, selkoe dj. amyloid beta-peptide is transported on lipoproteins and albumin in human plasma. j biol chem. 1996;271(51):32916-22. https://doi.org/10.1074/jbc.271.51.32916 cole gm, ard md. influence of lipoproteins on microglial degradation of alzheimer's amyloid beta-protein. microsc res tech. 2000;50(4):316-24. https://doi.org/10.1002/1097-0029(20000815)50:4%3c316::aid-jemt11%3e3.0.co;2-e tamamizu-kato s, cohen jk, drake cb, kosaraju mg, drury j, narayanaswami v. interaction with amyloid beta peptide compromises the lipid binding function of apolipoprotein e. biochemistry. 2008;47(18):5225-34. https://doi.org/10.1021/bi702097s arold s, sullivan p, bilousova t, teng e, miller ca, poon ww, et al. apolipoprotein e level and cholesterol are associated with reduced synaptic amyloid beta in alzheimer's disease and apoe tr mouse cortex. acta neuropathol. 2012;123(1):39-52. https://doi.org/10.1007/s00401-011-0892-1 yamaguchi h, nakazato y, hirai s, shoji m, harigaya y. electron micrograph of diffuse plaques. initial stage of senile plaque formation in the alzheimer brain. am j pathol. 1989;135(4):593-7. bales kr, verina t, cummins dj, du y, dodel rc, saura j, et al. apolipoprotein e is essential for amyloid deposition in the app(v717f) transgenic mouse model of alzheimer's disease. proc natl acad sci u s a. 1999;96(26):15233-8. https://doi.org/10.1073/pnas.96.26.15233 soderberg m, edlund c, kristensson k, dallner g. lipid compositions of different regions of the human brain during aging. j neurochem. 1990;54(2):415-23. https://doi.org/10.1111/j.1471-4159.1990.tb01889.x svennerholm l, bostrom k, jungbjer b, olsson l. membrane lipids of adult human brain: lipid composition of frontal and temporal lobe in subjects of age 20 to 100 years. j neurochem. 1994;63(5):1802-11. https://doi.org/10.1046/j.1471-4159.1994.63051802.x de chaves ep, narayanaswami v. apolipoprotein e and cholesterol in aging and disease in the brain. future lipidol. 2008;3(5):505-30. https://doi.org/10.2217/17460875.3.5.505 welander h, frånberg j, graff c, sundström e, winblad b, tjernberg lo. abeta43 is more frequent than abeta40 in amyloid plaque cores from alzheimer disease brains. j neurochem. 2009;110(2):697-706. https://doi.org/10.1111/j.1471-4159.2009.06170.x saito t, suemoto t, brouwers n, sleegers k, funamoto s, mihira n, et al. potent amyloidogenicity and pathogenicity of abeta43. nat neurosci. 2011;14(8):1023-32. https://doi.org/10.1038/nn.2858 grimm mo, grimm hs, hartmann t. amyloid beta as a regulator of lipid homeostasis. trends mol med. 2007;13(8):337-44. https://doi.org/10.1016/j.molmed.2007.06.004 grimm mo, grimm hs, patzold aj, zinser eg, halonen r, duering m, et al. regulation of cholesterol and sphingomyelin metabolism by amyloid-beta and presenilin. nat cell biol. 2005;7(11):1118-23. https://doi.org/10.1038/ncb1313 tachibana m, holm ml, liu cc, shinohara m, aikawa t, oue h, et al. apoe4-mediated amyloid-beta pathology depends on its neuronal receptor lrp1. j clin invest. 2019;129(3):1272-7. https://doi.org/10.1172/jci124853 dumas a, dierksen ga, gurol me, halpin a, martinez-ramirez s, schwab k, et al. functional magnetic resonance imaging detection of vascular reactivity in cerebral amyloid angiopathy. ann neurol. 2012;72(1):76-81. https://doi.org/10.1002/ana.23566 frank c, rufini s, tancredi v, forcina r, grossi d, d'arcangelo g. cholesterol depletion inhibits synaptic transmission and synaptic plasticity in rat hippocampus. exp neurol. 2008;212(2):407-14. https://doi.org/10.1016/j.expneurol.2008.04.019 maggo s, ashton jc. effects of hmg-coa reductase inhibitors on learning and memory in the guinea pig. european journal of pharmacology. 2014;723:294-304. https://doi.org/10.1016/j.ejphar.2013.11.018 korinek m, gonzalez-gonzalez im, smejkalova t, hajdukovic d, skrenkova k, krusek j, et al. cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission. scientific reports. 2020;10(1):12651. https://doi.org/10.1038/s41598-020-69454-5 yujun g, guichang z, jin j, lei y, keke q, yang p, et al. simvastatin impairs hippocampal synaptic plasticity and cognitive function in mice. molecular brain. 2020. https://doi.org/10.21203/rs.3.rs-76680/v1 mauch dh, nagler k, schumacher s, goritz c, muller ec, otto a, et al. cns synaptogenesis promoted by glia-derived cholesterol. science. 2001;294(5545):1354-7. https://doi.org/10.1126/science.294.5545.1354 pfrieger fw. role of cholesterol in synapse formation and function. biochim biophys acta. 2003;1610(2):271-80. https://doi.org/10.1016/s0005-2736(03)00024-5 teasdale gm, nicoll ja, murray g, fiddes m. association of apolipoprotein e polymorphism with outcome after head injury. lancet. 1997;350(9084):1069-71. https://doi.org/10.1016/s0140-6736(97)04318-3 teasdale gm, murray gd, nicoll ja. the association between apoe epsilon4, age and outcome after head injury: a prospective cohort study. brain. 2005;128(pt 11):2556-61. https://doi.org/10.1093/brain/awh595 deng h, ordaz a, upadhyayula ps, gillis-buck em, suen cg, melhado cg, et al. apolipoprotein e epsilon 4 genotype, mild traumatic brain injury, and the development of chronic traumatic encephalopathy. med sci (basel). 2018;6(3):78. https://doi.org/10.3390/medsci6030078 poirier j. apolipoprotein e in animal models of cns injury and in alzheimer's disease. trends neurosci. 1994;17(12):525-30. https://doi.org/10.1016/0166-2236(94)90156-2 sabo t, lomnitski l, nyska a, beni s, maronpot rr, shohami e, et al. susceptibility of transgenic mice expressing human apolipoprotein e to closed head injury: the allele e3 is neuroprotective whereas e4 increases fatalities. neuroscience. 2000;101(4):879-84. https://doi.org/10.1016/s0306-4522(00)00438-3 white f, nicoll ja, roses ad, horsburgh k. impaired neuronal plasticity in transgenic mice expressing human apolipoprotein e4 compared to e3 in a model of entorhinal cortex lesion. neurobiol dis. 2001;8(4):611-25. https://doi.org/10.1006/nbdi.2001.0401 mannix rc, zhang j, park j, zhang x, bilal k, walker k, et al. age-dependent effect of apolipoprotein e4 on functional outcome after controlled cortical impact in mice. j cereb blood flow metab. 2011;31(1):351-61. https://doi.org/10.1038/jcbfm.2010.99 tensaouti y, yu t-s, kernie sg. apolipoprotein e regulates the maturation of injury-induced adult-born hippocampal neurons following traumatic brain injury. plos one. 2020;15(3):e0229240. https://doi.org/10.1371/journal.pone.0229240 horsburgh k, mcculloch j, nilsen m, mccracken e, large c, roses ad, et al. intraventricular infusion of apolipoprotein e ameliorates acute neuronal damage after global cerebral ischemia in mice. j cereb blood flow metab. 2000;20(3):458-62. https://doi.org/10.1097/00004647-200003000-00003 báez-becerra c, filipello f, sandoval-hernández a, arboleda h, arboleda g. liver x receptor agonist gw3965 regulates synaptic function upon amyloid beta exposure in hippocampal neurons. neurotox res. 2018;33(3):569-79. https://doi.org/10.1007/s12640-017-9845-3 cramer pe, cirrito jr, wesson dw, lee cyd, karlo jc, zinn ae, et al. apoe-directed therapeutics rapidly clear β-amyloid and reverse deficits in ad mouse models. science (new york, ny). 2012;335(6075):1503-6. https://doi.org/10.1126/science.1217697 koudinov ar, koudinova nv. cholesterol homeostasis failure as a unifying cause of synaptic degeneration. j neurol sci. 2005;229-230:233-40. https://doi.org/10.1016/j.jns.2004.11.036 mesulam mm. a plasticity-based theory of the pathogenesis of alzheimer's disease. ann n y acad sci. 2000;924:42-52. https://doi.org/10.1111/j.1749-6632.2000.tb05559.x fan qw, yu w, senda t, yanagisawa k, michikawa m. cholesterol-dependent modulation of tau phosphorylation in cultured neurons. j neurochem. 2001;76(2):391-400. https://doi.org/10.1046/j.1471-4159.2001.00063.x love s, bridges lr, case cp. neurofibrillary tangles in niemann-pick disease type c. brain. 1995;118 (pt 1):119-29. https://doi.org/10.1093/brain/118.1.119 suzuki k, parker cc, pentchev pg, katz d, ghetti b, d'agostino an, et al. neurofibrillary tangles in niemann-pick disease type c. acta neuropathol. 1995;89(3):227-38. https://doi.org/10.1007/bf00309338 mckee ac, cairns nj, dickson dw, folkerth rd, keene cd, litvan i, et al. the first ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. acta neuropathol. 2016;131(1):75-86. https://doi.org/10.1007/s00401-015-1515-z kay ad, petzold a, kerr m, keir g, thompson ej, nicoll ja. cerebrospinal fluid apolipoprotein e concentration decreases after traumatic brain injury. j neurotrauma. 2003;20(3):243-50. https://doi.org/10.1089/089771503321532824 kay ad, day sp, kerr m, nicoll ja, packard cj, caslake mj. remodeling of cerebrospinal fluid lipoprotein particles after human traumatic brain injury. j neurotrauma. 2003;20(8):717-23. https://doi.org/10.1089/089771503767869953 kay ad, petzold a, kerr m, keir g, thompson e, nicoll ja. alterations in cerebrospinal fluid apolipoprotein e and amyloid beta-protein after traumatic brain injury. j neurotrauma. 2003;20(10):943-52. https://doi.org/10.1089/089771503770195795 zhao j, fu y, yamazaki y, ren y, davis md, liu c-c, et al. apoe4 exacerbates synapse loss and neurodegeneration in alzheimer’s disease patient ipsc-derived cerebral organoids. nature communications. 2020;11(1):5540. https://doi.org/10.1038/s41467-020-19264-0 horsburgh k, mccarron mo, white f, nicoll ja. the role of apolipoprotein e in alzheimer's disease, acute brain injury and cerebrovascular disease: evidence of common mechanisms and utility of animal models. neurobiol aging. 2000;21(2):245-55. https://doi.org/10.1016/s0197-4580(00)00097-x heinsinger nm, gachechiladze ma, rebeck gw. apolipoprotein e genotype affects size of apoe complexes in cerebrospinal fluid. journal of neuropathology & experimental neurology. 2016;75(10):918-24. https://doi.org/10.1093/jnen/nlw067 zhao j, davis md, martens ya, shinohara m, graff-radford nr, younkin sg, et al. apoe epsilon4/epsilon4 diminishes neurotrophic function of human ipsc-derived astrocytes. hum mol genet. 2017;26(14):2690-700. https://doi.org/10.1093/hmg/ddx155 boche d, nicoll jar. invited review understanding cause and effect in alzheimer's pathophysiology: implications for clinical trials. neuropathol appl neurobiol. 2020;46(7):623-40. https://doi.org/10.1111/nan.12642 franco-bocanegra dk, gourari y, mcauley c, chatelet ds, johnston da, nicoll jar, et al. microglial morphology in alzheimer's disease and after abeta immunotherapy. sci rep. 2021;11(1):15955. https://doi.org/10.1038/s41598-021-95535-0 jones l, holmans pa, hamshere ml, harold d, moskvina v, ivanov d, et al. genetic evidence implicates the immune system and cholesterol metabolism in the aetiology of alzheimer's disease. plos one. 2010;5(11):e13950. https://doi.org/10.1371/journal.pone.0013950 boche d, perry vh, nicoll jar. review: activation patterns of microglia and their identification in the human brain. neuropathology and applied neurobiology. 2013;39(1):3-18. https://doi.org/10.1111/nan.12011 torrandell-haro g, branigan gl, vitali f, geifman n, zissimopoulos jm, brinton rd. statin therapy and risk of alzheimer's and age-related neurodegenerative diseases. alzheimers dement (n y). 2020;6(1):e12108. https://doi.org/10.1002/trc2.12108 cibičková l. statins and their influence on brain cholesterol. j clin lipidol. 2011;5(5):373-9. https://doi.org/10.1016/j.jacl.2011.06.007 holmes c, boche d, wilkinson d, yadegarfar g, hopkins v, bayer a, et al. long-term effects of abeta42 immunisation in alzheimer's disease: follow-up of a randomised, placebo-controlled phase i trial. lancet. 2008;372(9634):216-23. https://doi.org/10.1016/s0140-6736(08)61075-2 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. the ’accidental neuropathologist' – on 40 years in neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:24 (2020) reflections the ’accidental neuropathologist' – on 40 years in neuropathology harry v. vinters pathology & laboratory medicine & neurology, david geffen school of medicine at ucla, los angeles, ca, usa corresponding author: professor harry v. vinters · laboratory medicine & pathology · university of alberta · faculty of medicine & dentistry · edmonton, alberta · canada hvinters@mednet.ucla.edu submitted: 14 august 2020 accepted: 16 august 2020 copyedited by: christian thomas published: 25 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2956 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, ucla, personal reflections this article is dedicated to the memory of my brother raymond john vinters (1952-2020). beginnings in canada my family were latvians who, by the end of world war ii, had become refugees from the soviet union. for the repressive communist regime that controlled the ussr, my family (on both sides) were or would soon become ‘criminals’ who owned land and were involved in commerce. they saw no future under a communist regime, and the fate of relatives who remained in latvia after world war ii confirmed the wisdom of their decision and choice – some were exiled to siberia for many years, others simply had their property confiscated. my family – grandparents, their two children (one my father) and assorted household staff who joined them – fled the country as the red army moved in to ‘liberate’ its people and begin a brutal and merciless tyranny that would last until the early 1990s. during that happy time in the early 1990s the ussr thankfully collapsed under the weight of grotesque corruption and reliable, predictable, frequently comical soviet incompetence. members of my family lived in various refugee camps in europe between 1944 and 1949, at which time they were sponsored by distant relatives to relocate to canada; some of their lasting and most intimate friendships were made in the camps during those postwar years. my parents had wed in lübeck, germany. it was their (and my) good fortune that our family ended up on the northwest shore of lake superior, in the small city of port arthur (now integrated into a larger municipality, thunder bay), where i was born. if one envisions lake superior as having the shape of a wolf’s head, thunder bay is about where its eyes would be located. this beautiful small city is situated on rolling hills overlooking the lake, and is home to one of the harshest (winter) climates of any metropolitan area in canada. it provided me with an outstanding primary and high school education at the hands of, and with the mentorship of, dedicated and devoted teachers who inspired a love of learning and a first-rate work ethic, of which they themselves were emblematic. very lucky break for me! winters were long and brutal in port arthur. i well remember sitting in my high school classes, looking out onto the harbor – port arthur was a port city thanks to the st. lawrence seaway (opened in the late 1950s, queen elizabeth ii herself came to our fair city for the official opening), which allowed ocean-going vessels to come into the great lakes. the harbor was still frozen in april and may, and i longed for warmer days – never dreaming then that i would spend most of my working career in a climate that can safely be described as endless summer. i was also fortunate to have support and encouragement from a wonderful father [figure 1] who, as a single parent, raised me and my brother after our mother left in the early 1960s to begin a new life in the usa and my parents divorced. he died in 1980 but never a day goes by that i don’t think – with much love and gratitude – of him and his dedication to his sons and family, his generosity, support and kindness. our loving and resourceful family included grandparents, aunts and uncles, eventually cousins, all trying to find their way in the brave new world into which they had been thrust by world war ii and its aftermath and sequelae. as with many immigrant families trying to establish life in a foreign land, barely speaking english and with limited finances, extended family members (usually from three generations) occupied small apartments and houses, then eventually spread their wings and left to make lives of their own in their new homes, when they could finally afford them. my favorite home in those early days was a ramshackle house – little more than a cottage – just outside the port arthur city limits, with two tiny bedrooms and one miniscule bathroom (bath, no shower), situated on the banks of a small meandering river and on a gorgeous piece of land. the property was also home to several massive pine trees and came to be known (by the latvian expatriate community) as eglaines (loosely translated, the home of tall pine trees). seven of us called this home for many years. it had an old coal furnace in which fuel needed to be replenished during the icy winter nights, a task shared by my father and grandfather. my grandparents, who had been ‘saimnieks and saimniece’ (the master and mistress of their household) and oversaw a large farm in latvia, lovingly cultivated their gardens of flowers, berries and vegetables on the two acres that eglaines occupied. i often wondered what it would be like to start one’s life over, at the age of 49 or 50, in a foreign land and culture, as my resilient grandparents had; the task was easier – though no less challenging! – for my father and mother, who later became a medical technologist. my father and grandfather worked in the grain elevators that surrounded the harbor (the grain coming from canada’s prairie provinces and destined to be loaded onto seaway-bound ships) until my dad became a real estate agent, then subsequently a real estate broker. my paternal grandmother worked as a domestic. while i loved port arthur (that affection continues to this day) i yearned to pursue higher education in a larger center, and a variety of scholarships allowed me to do so at the university of toronto (u of t), another lucky twist of fate. being a toronto resident introduced me to the joys (and challenges…) of big city metropolitan life, and i have enjoyed urban living ever since. i adore the buzz of large cities, almost without exception. idyllic country life, a dream for some, is not for me. fig 1: my father gunnar (gunars) vinters, 1925-1980 during the summer between my graduation from high school and the beginning of university (1968) i had the marvelous opportunity to work in the laboratory of a family friend (and my aunt’s godmother), irene grodums, who after immigrating to canada had completed her ph.d. and become a virologist at the university of saskatchewan in saskatoon. prof. grodums instilled in me a respect for the importance of careful observation in tissue studies, whether done by light or electron microscopy (she worked on coxsackie virus effects on the heart) and respect for experimental animals used in biological research. in the 1970s medical school was increasingly regarded as a place of graduate studies, following upon an undergraduate experience. my first degree was in biology and most of my courses given in the botany department (these days, plant sciences). u of t undergraduate instruction was loosely based in one of the constituent colleges, and my academic (and residential) home was university college. i thrived on living on an historic campus situated in the middle of a world class city, one with a great sense of history. one of my first summer research jobs was in the botany department, studying the kinetics of water movement into and out of giant algae with profs. jack dainty and mel tyree (both accomplished biophysicists); this resulted in my first publication [1]. while in medical school at the university of toronto (1972-76) i developed an abiding affection for the neurosciences, though i realized i would never have the skill set, insights, patience or scientific acumen to be a solid basic neuroscientist. our inspiring professors included the legendary neuropharmacologists phil seeman and oleh hornykiewicz; dr. seeman (probably one of the best lecturers i have ever encountered) had previously discovered how anesthetics and tranquilizers interact with cell membranes [2] and subsequently contributed to the molecular understanding of complex genes that control dopamine receptor function, while dr. hornykiewicz was the discoverer of the dopamine deficiency [3] that leads to parkinson disease. the other option was to choose a clinical neuroscience in which i would have both clinical responsibilities and an opportunity to do research and teaching. i decided to pursue postgraduate training in neurology, having eliminated neurosurgery as a career path – i lacked the required stamina and manual dexterity, both (in addition to excellent clinical judgement) requirements of a first-rate neurosurgeon. fortunately, throughout my career i have had the opportunity to work with some of the very best in the latter field, in numerous productive collaborations and diagnostic challenges (see below). how neuropathology chose me neuropathology was a largely invisible specialty while i was in medical school. admittedly, in the early 1970s it was itself a fledgling discipline in the clinical neuroscience firmament, certainly in canada. we received wonderful lectures from neuropathologists – including dr. barry rewcastle – but were never really apprised of how a neuropathologist spends her/his time and contributes to the diagnosis and understanding of neurologic disease. indeed, we hardly knew that such individuals existed or how one went about becoming a neuropathologist, this despite several significant neuropathologic discoveries having been made at u of t’s banting institute on college street, then home of the research components of the pathology department. (frederick banting, the discoverer of insulin, was the first and is still one of the few canadians to be awarded the nobel prize in medicine or physiology). the initial report of progressive supranuclear palsy [4] was co-authored by the great jerzy olszewski, though he had unfortunately passed on by the time i was in medical school. i spent my internship year at the university of alberta hospital in edmonton, in retrospect a wise choice because it introduced me to the beauty of the rugged canadian west (stunning prairie skies and amazing geography) and facilitated my meeting people who remain steadfast friends to this day. of course that year also provided a terrific opportunity to learn important fundamentals of medicine from energetic and knowledgeable instructors. the university of alberta hospital would, many years later, become a place where i would do an extended locum. i began training in neurology in london, ontario in 1977 in the department of clinical neurological sciences (cns) at the university of western ontario / uwo (subsequently renamed western university). this department represented a logical fusion of talent from neurosurgery, neurology, neuroradiology, and neuropathology. its leadership included two legends of 20th century neurosurgery (charles g. drake) and neurology (henry j.m. barnett). london was also a major center for stroke research; the clinical specimens we were shown in surgical pathology and at autopsy conferences inspired an appreciation and respect for the importance of cerebrovascular disease in neurology. the weekly 3-hour city-wide clinical neuroscience rounds included presentations from all four of the sub-disciplines, including a clinicopathologic conference weekly or every two weeks. i was coming to an important, somewhat uneasy career juncture: neurology felt like perhaps not quite the optimal choice of specialization for me. however… an inspirational figure during that first year of neurology training came to be dr. john c.e. kaufmann, chief of neuropathology at the (recently built) university hospital on the main uwo campus in the north end of the city. dr. kaufmann (or jcek, as his trainees knew him, figure 2) had been recruited from south africa to be the first neuropathologist at uwo, and one of only a handful in canada at the time. more than being a marvelous diagnostician, jcek was the essence of a ‘gentleman and scholar’, someone who took responsibility for the wellbeing and mental and physical health of his trainees. i daresay most of us lucky to be in that multi-year cohort try to emulate jcek in our own careers. he and his wife suzanne kaufmann loved art (they had an inspiring collection which included an andy warhol), entertaining on a grand scale, fine (exquisite) cuisine usually prepared in their own kitchen, good and witty conversation and travel. the presentations jcek oversaw at the weekly clinical neuroscience rounds were marvels of clarity and insights into neurologic disease. throughout that year of training in neurology, i came to the conclusion that neuropathology might be a more appropriate career path for me. fortunately, dr. barnett was very passionate about the vital importance of neuropathology in neurodiagnostics. when i requested the possibility of doing a full year of neuropathology training (then configured as part of my neurology residency) dr. barnett was favorable to the idea, as was jcek. i embarked on the road to my career as a neuropathologist with some trepidation, but a passion for the study of the anatomic basis of neurologic diseases using a multitude of special stains, which in those days (late 1970s) were, together with electron microscopy, the ‘gold standard’ for neuropathologic investigations. oh yes, things have changed! fig 2: dr. john c.e. kaufmann, chief of neuropathology, university hospital, london, ontario, canada my other mentors in london were dr. melvyn j. ball, who was already a prominent alzheimer researcher, and dr. joseph gilbert [figure 3], who had trained in neurology and neuropathology in cleveland, though practiced the latter. neuropathology in canada is configured as a specialty rather than a ‘subspecialty’ requiring fellowship training. jcek and joe gilbert were in charge of np training at the two major uwo teaching hospitals, university and victoria, and possessed complementary, mutually reinforcing didactic skills. jcek was an experienced, meticulous and insightful neurohistologist. joe gilbert was a master of providing clinicopathologic insights based upon careful correlation of neuropathologic findings with clinical signs and symptoms (structural brain imaging in the late 1970s was still in its infancy). the experience of working with them both and learning from them was exhilarating, humbling and yes – a little daunting. fig 3: dr. joseph j. gilbert, neuropathology mentor, london, ontario joe gilbert also suggested to me a translational research project that would determine and define my research interest for the coming decades: diseases of the cerebral microvasculature. as a stroke referral center, uwo was encountering many patients with lobar intracerebral hemorrhages caused by, or at least associated with, cerebral amyloid angiopathy (caa). joe suggested that we collect our cases of (putative) caa-related brain hemorrhage and write a descriptive paper on this association; a companion study would assess the topography and severity of caa in relation to brain aging. in the late 1970s and early 1980s (well before the discovery of beta-amyloid protein in 1984, see below) caa was evaluated by the use of congo red staining and polarization microscopy (alternatively using thioflavin stains and fluorescence microscopy). the ‘topography/severity of caa with age’ study was performed simply by staining a large number of brain sections, from uniformly sampled autopsy brains, with congo red and evaluating caa in these. the two resultant manuscripts went through several drafts, were eventually submitted to the journal stroke as ‘back-to-back papers’, and published in 1983 [5,6]. it is gratifying that the findings we described using fairly primitive (by today’s standards) technology have been replicated using beta-amyloid immunohistochemistry in later studies; it is perhaps especially satisfying that the papers are still being cited 37 years after their publication (combined total citations of 500+ per the web of science). examining the pathogenesis and consequences of caa and other microangiopathies in the brain would constitute a major theme of my subsequent research endeavours. my np residency was spent feasting on the instruction and support of my mentors and the remarkable and stimulating milieu created by the clinical neurologists, neuroradiologists and neurosurgeons at uwo. this showed and highlighted the importance of collaborative activities in understanding complex neurologic diseases. luminaries in neurology and neurosurgery (fred plum, raymond adams among them) were frequent visitors to this small but ‘neurologically important’ city half way between toronto and detroit. one of my interests was in the effects on cerebral arteriovenous malformations (avms) of agents used in novel embolization therapies that were just being developed, many of them at uwo. two skilled endovascular specialists (drs. fernando vinuela and allen fox) were pioneering new agents and approaches to non-surgical therapies for vascular brain lesions, and continued to do so throughout their careers. when these lesions (usually avms) were subsequently resected, they showed unique and sometimes dramatic reactive changes caused by the embolotherapy agents (about which more later…). many of my research interests have been driven by clinical or clinicopathologic questions. here were two large and important ones: why (and how) do embolotherapy agents work through their interactions with vessel walls in vascular malformations? what might be some neuropathologic clues to why they sometimes fail? they were also questions that could be effectively addressed by simple observation (using routine stains) of previously embolized lesions when they made their way to the surgical neuropathology laboratory. there was discussion of my returning on faculty to uwo, but only after i had spent some time developing basic research skills pertinent to the study of cerebrovascular disease. the university would provide or facilitate funding for my research endeavours, but i had the freedom to choose a laboratory in which i might develop appropriate experimental techniques and skills that i could repatriate to uwo. at that time, some of the most exciting work on cerebral microvascular biology (especially the blood-brain barrier/ bbb) was being done using cell biologic techniques in the laboratories of drs. pasquale (pat) cancilla [figure 4] and michael n. hart [figure 5] at the university of iowa (u of i) [7,8]. it was arranged that i would spend two years working with them, after a 6-month period refining my diagnostic skills in pediatric neuropathology with dr. margaret norman in vancouver; marg became and remained a good friend for the rest of her life. fig 4: dr. pasquale (pat) and enid cancilla, undated photograph trying my hand at (basic) research i arrived in iowa city (in which the university of iowa is located) in january 1982, in the midst of a nasty midwestern blizzard. during a lonely weekend in a slightly seedy motel in coralville (a suburb of iowa city) i frequently thought: ‘what on earth have i done?’ during my first day in the new laboratory environment at the u of i, pat cancilla asked if i could go with him for a cup of coffee. then came the bombshell: he had just accepted the chair of pathology position at university of california los angeles (ucla). he suggested that, since i had independent funding that was not tied to a specific institution, i might either stay at the u of i and work with michael hart, or move with him to ucla. as i looked outside at the heavy snowfall and felt the subzero temperature en route back to the lab, i had essentially made my decision, and moved with dr. cancilla to ucla that summer. during the six months i resided and worked there, however, i developed an affection for iowa city, sometimes described as the ‘athens of the midwest’—famous for its world class medical center (including brilliant translational neuroscience and behavioral neurology) and the iowa writers workshop. my colleagues there included drs. jonathan fratkin and david beck, the latter an outstanding neurosurgeon (then still a resident) bursting with excellent research ideas that might optimize the use of cultured cells in understanding bbb physiology and abnormalities [9]. fig 5: dr. michael noel hart ucla, destined to become my academic home for most of the remainder of my career (though that decision was still in the future), was research heaven for a young scientist. it was fun helping to set up pat cancilla’s laboratory (our senior technician at the time was jim bready) with top-of-the-line equipment and facilities that were the perks of being a newly recruited chair and entering the academic milieu of a major research university. our ‘lab warming’ party was a smash hit throughout the medical center. pioneering studies of cerebral microvessels (especially the bbb) were ongoing in the laboratories of two ‘bills’ – william pardridge and william oldendorf. dr. oldendorf had previously (1975) won the lasker award, and probably deserved the nobel, for suggesting the principles that led to computerized tomographic (ct) scanning, but was also an innovative and creative investigator looking at the physiology of the bbb, especially using isotope tracer techniques. he was also bill pardridge’s mentor. pat cancilla’s lab became part of an informal consortium of investigators (also including drs. judith a. berliner, an experienced cell biologist, and eain cornford at the west la va medical center) interested in cerebral microvasculature (especially its endothelium) and optimal ways to study it. our laboratory was mainly intrigued by the potential use of tissue culture approaches and had been able to isolate and culture endothelial and smooth muscle cells from brain microvessels, initially from mouse brains but eventually (some years later) even those derived from human corticectomies for intractable epilepsy. i was also smitten by the city of angels, los angeles. it represented a far different world from the one in which i grew up. l.a. was (and is…) a city with a near perfect climate (summer and winter), far cleaner air than is widely believed, stunning geography with canyons, hills and flatlands, a brilliant seaside location on santa monica bay, and remarkable ethnic and cultural diversity. it reminds itself almost daily that it is the ‘world capital of the entertainment industry’, which is probably true, and ‘the creative center of the planet’ – the latter a claim that london (england), amsterdam, tokyo, paris and a few other cities might dispute. i remember reading that the german novelist gunther grass had once described berlin as representing a city that ‘faced the realities of the 20th century’ (this may be an erroneous recollection…), and thinking that would probably apply quite accurately to l.a. in the late 20th and early 21st centuries. though i at first lived near the ucla campus, i found myself spending many weekends at venice beach, where i have now had a home for the past 30+ years. (rationale: if you live near the pacific ocean, why not at one of the most famous beaches in the world?) as i was nearing the end of my postdoctoral fellowship, i was working in the lab on a saturday morning. pat cancilla called me into his office (immediately adjacent to the laboratory) and rather directly asked if i might be interested in a ucla faculty position – one had just come open in neuropathology because of the retirement of dr. jann brown. we discussed the possibility briefly. this was the second time in my career that a casual chat with pat cancilla would alter the course of my career. return to london, ontario and uwo (briefly) because of visa issues and funding, i was committed to return to london, ontario, but my faculty job there was a ‘soft money’ position, unlike the tenure track post for which i was being considered at ucla. i had formally applied for the ucla faculty position while still a postdoc there, interviewed for it, and eventually (a few weeks after returning to london, ontario) was offered and decided to accept the job. it was a decision made over an agonizing weekend during which i weighed the many pros and cons – the university of western ontario would have made a wonderful academic home for me, and london a welcoming and very livable city (i had already bought a home there). john kaufmann and joe gilbert were amazing mentors and colleagues. yet i had enjoyed my postdoc at ucla so much… that i felt i had to ‘give it [the faculty position] a try’. having heard that american academic medicine could be rather brutal and fiercely competitive (though i had seen little evidence of this during my postdoc), i began to wonder whether i had the stomach for this. i rationalized the decision by thinking i would remain at ucla for 2-3 years, then return to london, ontario (assuming a position was still open there…). at the appointed time, i considered this move back, and decided to remain at ucla, where i am (part time, semi-retired) today. my 18 months in london (pending receipt of the green card / permanent resident status that would allow me to take up life in the usa) were delightful and rewarding both professionally and personally – so much so that i began having some doubts as to whether i had made the correct decision in accepting the ucla job. (timing is everything. were i making this same career-changing deliberation in the current unwelcoming usa immigration climate, i would almost certainly have stayed in london!) i managed to set up a small tissue culture laboratory at university hospital – thanks to the fact that a fully equipped laboratory with appropriate equipment had just been made available by a departing or retiring faculty member. we were able to successfully culture endothelial and smooth muscle cells from human cerebral microvessels [10]. i continued to have an interest in ‘embolized avms’. as a result of the neurosurgical skills and expertise of drs. charles drake and sidney (‘skip’) peerless, brilliant endovascular therapy experts vinuela and fox (see above) and their respective teams, london and uwo attracted complex avm cases from around the world that might be candidates for this unique therapy. if one is going to do translational research, it helps to have a unique collection of abundant clinical and pathologic specimens to study – and our neuropathology laboratory had just such an array of treated avms, an amazing experiment of nature, though one admittedly manipulated by physicians. with the help of jcek and a very industrious undergraduate student (mark lundie) i set about a careful examination of avms that had been treated by embolization with isobutyl cyanoacrylate, one of the widely used embolotherapy agents at that time. (it is an agent that is injected through a catheter in liquid form, but polymerizes at various rates depending upon how much contrast medium is in the mixture. with this agent, timing of the polymerization process was of paramount importance – if it polymerized too quickly, the catheter could become stuck to the intima of the avm ‘feeding’ vessel; if too slowly, it would end up passing through the avm and into a treated patient’s lungs.) we had vascular malformations with a wide range of post-embolization intervals that allowed for inferences about how this polymer was interacting with abnormal vessel walls in avms. we put together a significant (and rather unique) data set, which i brought with me to ucla when i returned there in july, 1985, hoping i would have the time to analyze it in detail and prepare a manuscript. fig 6: dr. michael a. farrell, friend, colleague and ‘partner in crime’ i also had the good fortune to meet, during that 1 ½ years back at uwo, dr. michael farrell [figure 6], a newly arrived neuropathology trainee from dublin, ireland. michael became a great friend and colleague during that time and after he returned to a consultant post in dublin. over the years, we have written many papers (see below) and co-authored a textbook, he has often visited l.a. (even doing a mini-sabbatical in my laboratory during the early 1990s), and i have gone to dublin dozens of times; it is on my list of favorite cities around the globe. great and generous friend. through michael i eventually came to know the small but vibrant irish neuropathology community, including drs. katie keohane and francesca brett. on faculty at ucla how does one summarize 35 years of academic and diagnostic work at one institution? there are many things i love about ucla: the physical beauty and location of the campus in westwood (the ucla medical center, now ronald reagan ucla medical center, is at the southern edge of the main campus), the collegial and collaborative nature of the faculty, the wealth of expertise in all aspects of basic and clinical science, especially neuroscience, talented and intelligent students and trainees, and the cultural and ethnic diversity of both students and faculty who are committed to scholarly work. yes, there are things not to like (but one endures them…): the numbing, frequently obstructive bureaucracy, the ‘bend over backwards’ approach to being politically correct are among them. i returned to l.a. and the ucla family the first week of july, 1985. i used the ‘down time’ in setting up a small research laboratory, to finesse two projects. one was collating the embolized avm data into a coherent story. as i wrote the manuscript and put the results into perspective, i thought the material was sufficiently novel that it might be worthy of submission to the prestigious new england journal of medicine, with the thought that ‘nothing ventured, nothing gained’. as i put the manuscript in the mail (yes, what we now call snail mail...) i was already reformatting the paper for a lower level, less prestigious journal because of the certainty of rejection. then a few days later i received a surprising phone call from the nejm telling me the manuscript had been accepted, with minimal revisions; they were calling me to verify that there was no overlap with a review article on cyanoacrylates we had recently written – i assured them there was none [11]. the nejm paper was published in 1986 [12] and represents one of my two appearances as an author of a paper in this high-profile venue, the second coming almost exactly 20 years later as a co-authored contribution on a totally different topic [13]. my other project was to write a review article on (what else?) caa – i suggested this to henry barnett, then executive editor of stroke, and he liked the idea. that paper eventually appeared (after a few revisions) in 1987 [14] and is still frequently quoted today. in the meantime, i resumed my tissue culture studies but decided to embark on other scientific adventures. while i was impressed by the simple questions that could be addressed (and answered) using in vitro methods, i was (like many investigators) concerned about how these phenomena could be understood in the context of complex multi-cellular tissues and organs, especially in the brain. being a junior faculty member at ucla was the academic equivalent of being a child at a candy store. colleagues were always approaching one about working on any variety of projects, some utilizing human cns specimens, others exploiting interesting animal models, transgenic and knockout mice, etc. neuropathologists were and are in the unique position of understanding the structure of the nervous system (normal and abnormal), and how best to study it. i re-established a close collaboration with william pardridge (see above), an endocrinologist in the dept. of medicine who was interested in the biochemistry (and eventually molecular biology) of transport mechanisms through the bbb, but was now also looking for a way to study abnormalities (diseases?) of small blood vessels. we had many discussions about cerebral amyloid angiopathy (caa), which of course affects arterioles and capillaries and, together with arteriolosclerosis, is one of the two most common microvasculopathies found in the brain. in 1984, glenner and wong [15] had isolated the ‘a4’ protein from amyloid-laden meningeal vessels of an alzheimer patient. bill pardridge was amazingly skilled at making antibodies and proceeded to make a polyclonal antibody to ‘a4’, using the partial sequence of the peptide provided by glenner and wong in their paper. it worked magnificently in immunohistochemical protocols to demonstrate both senile plaques and caa, resulting in several publications [16,17]. bill pardridge was also the best person in the school of medicine to review the draft of a grant proposal; i am eternally grateful for his merciless critique of one of my first independent national institutes of health (nih) proposals, which through his intervention helped me transform an unfocused and improbable set of studies into a fundable application. my chief of neuropathology was m. anthony verity, and we divided the service and diagnostic commitments equally – each of us covered 6 months of the year. my interests were obviously in cerebrovascular disease and dementia, tony’s in neurotoxicology and neuromuscular diseases. when one of our outstanding neurologists, dr. jeffrey cummings, decided to submit an nih (national institute on aging) grant that, if successful, would fund an alzheimer disease center (adc) at ucla in the late 1980s/early 1990s, i seemed the logical choice to head the neuropathology core and brain bank. we were funded on the first submission and i was thrilled to formally become part of the alzheimer research enterprise at ucla, even though my main academic focus (caa and related microvascular diseases) were at the periphery of most debates on ad pathogenesis. this situation has changed drastically in recent years. it is also worth noting that glenner and wong had isolated a4 from amyloid-laden meningeal microvessels (‘a novel cerebrovascular amyloid protein’) rather than senile plaques (sps); as is now well known, the anti-a4 antibody indeed labels sps as well as caa, but not neurofibrillary tangles, despite initial claims that it might. over the years until his departure from ucla, jeff cummings was an amazing leader: a superb diagnostician and organizer who supported basic research efforts and consistently brought out the strengths in investigators and his staff. and oh yes, he was a masterful fund raiser, successful in obtaining private donations that were crucial to supporting the adc when nih funding lapsed or became thin. he eventually secured an endowment that led to re-naming of the adc as the mary easton alzheimer center. i had obtained nih and other funding for my own projects within 3-4 years of arriving at ucla, but have always felt most at home scientifically in the environment of a collaborative center or program project grant; pieces of these have constituted most of my research efforts and funding support while on faculty at ucla. of course, in the early to mid-1980s other events were taking center stage in the world of medicine: the disease that was eventually named acquired immune deficiency syndrome (aids) presented in a handful of gay men, initially studied in new york city and l.a. (please also see herbert budka’s thorough and excellent description of the beginnings of this epidemic in his reflections piece published earlier this year.) as pathologists, we were witnessing the evolution of a feared illness that, at the time and for many years to come, had no effective treatment and was almost uniformly fatal. by the mid-1980s it was known to be caused by the retrovirus hiv-1 (initially named htlv-iii). in that same time frame, thanks to the work of many investigators including george shaw, beatrice hahn, clayton wiley, leroy sharer and others, it became appreciated that hiv frequently infected the brain in both children and adults, causing a syndrome initially described as aids dementia complex (in adults) though later re-named several times. but i’m getting ahead of myself… pathologists have now (and always have had) a key role in providing morphologic evidence for unique disease mechanisms – some of them based simply upon careful observation of novel but convincing histopathologic changes within tissues. consider, as one example outside the nervous system, the astute observation by robin warren (pathologist) and barry marshall (gastroenterologist) of ‘unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration’. this description of helicobacter pylori, initially published in lancet (in 1984) eventually led to their being awarded the nobel prize in 2005 because the observation led to a complete re-examination of the rationale for ways to treat peptic ulcer disease, a major paradigm shift. moving back into the cns, neuropathologists have made countless key observations (often in human biopsy or autopsy specimens) pertinent to the pathogenesis of neurodegenerative disease, intractable epilepsy, stroke, developmental disorders, tumors, and the list goes on. with the beginning of the aids epidemic, here was another opportunity to see a new infectious disease evolving before our eyes. this might be (often is…) called ‘opportunistic research’, with the slight implication that it is not as challenging or important as ‘basic research’. however, when unique opportunities to carefully study a human disease present themselves, one would be foolish not to take advantage. at ucla center for the health sciences (chs), one of the bright young residents from canada, karl anders [figure 7], suggested we review the neuropathologic features of our large and growing autopsy collection of brains from aids patients. karl and i, both recent arrivals from canada, bonded as soon as i arrived at ucla. we had both grown up in two cities located about 400 miles apart (karl in winnipeg), were raised in the households of immigrant families, and we both liked looking at slides – a lot of slides. fig 7: dr. karl h. anders, pathologist and frequent collaborator on projects, currently in calgary, alberta, canada we also engaged in this neuropathologic enterprise the collaboration of dr. uwamie tomiyasu [figure 8], an amazingly insightful, organized, helpful and collegial neuropathologist at the west l.a. va medical center (who over the years became a close friend), and involved a promising young ucla medical student wayne guerra in the project. karl anders had been the first author on the report of our initial series of aids autopsy cases (less than 30) written while i was still a research fellow at ucla. we had submitted this detailed report to clinical neuropathology. the manuscript had been almost immediately accepted for publication in late 1983 or early 1984. then, to our amazement and shock, clinical neuropathology ‘sat on’ this very topical manuscript for over 2 years before publishing it – in the meantime printing (arguably) less impactful and timely studies that had clearly been accepted after our paper [18]. by 1986 we had compiled data on almost 90 aids autopsy cases, and submitted this updated report to the american journal of pathology, where it was accepted with minimal revisions, and published a few short months after the much delayed clinical neuropathology paper [19]. karl and i wondered whether someone reading both papers (published in 1986) would question how we accumulated 60 new aids cases in a few short months, which of course we had not. the neuro-aids work took some interesting twists and turns, largely as the result of collaborations with some of my formidably talented colleagues in virology and neurology interested in viral neuropathogenesis. i co-authored two papers together with david ho and his research group while he was briefly on staff at cedars sinai medical center in los angeles [20,21]; these are the only two publications i have written with a physician-scientist who would eventually become time magazine’s man/person of the year in 1996. (i very much enjoyed basking in the reflected glow of david’s richly deserved fame.) i was also approached by a brilliant and productive retrovirologist, dr. irvin s.y. chen, who had trained with one of the discoverers of reverse transcriptase (howard temin at the university of wisconsin) before becoming a faculty member at ucla at about the same time as me. irvin was (and remains) keenly interested in the molecular pathogenesis of interactions between hiv and the brain. his laboratory used autopsy brain and csf specimens from neuro-aids subjects to characterize a neurotropic strain of hiv, and exploited pcr to quantify hiv viral load in the cns of affected patients [22,23]. it seemed an ideal collaboration between a talented basic virologist and someone (hvv) interested in morphologic brain changes induced by hiv. at the same time, my laboratory remained fascinated by the interesting opportunistic infections (ois) – especially other viral agents, that can cause puzzling varieties of devastating neurologic diseases in immunosuppressed aids patients. cytomegalovirus was one of the viral ois capable of producing variable cns disease in aids, including (at its worst extreme) severe encephalitis, ventriculitis and myeloradiculitis, yet minimal abnormalities in others. contributors to these studies included karl anders, a postdoctoral fellow from germany, dr. regina von einsiedel and marcia cornford, who was one of our first trainees in neuropathology and is still a faculty member at harbor-ucla medical center in torrance, california [24,25]. i became the pathologist of record for the ucla site of the multi-center aids cohort study (macs, pi dr. roger detels), one of the longest funded nih projects in history. karl anders and i co-authored a monograph on the neuropathologic features of aids [26]. fig 8: dr. uwamie tomiyasu, (dec 2004), former neuropathologist, west l.a. va medical center and ucla faculty member, friend by the midto late 1980s and into the 1990s there were other interesting developments in ucla’s active epilepsy surgery programs. it already had a longstanding research effort on temporal lobectomy for intractable temporal lobe seizures, under the capable and inspiring direction of jerome (‘pete’) engel (neurologist) and paul crandall (neurosurgeon). pete was, and continues to be, one of the great advocates for bringing the possibility of curative temporal lobe surgery or ‘neocorticectomy’ to epilepsy patients throughout the world. paul was a meticulous neurosurgeon who provided us with anatomically intact (including the hippocampus) temporal lobes. the arrival at ucla of pediatric neurosurgeon dr. warwick peacock from south africa in the mid-1980s ushered in a new era of pediatric epilepsy surgery. using high resolution neuroimaging (including structural and metabolic/positron emission tomographic imaging) modalities linked with electrocorticography, warwick and the pediatric epilepsy group (under the visionary leadership of drs. donald shields and raman sankar) developed the concept that generalized epilepsies in infants and children (e.g. leading to infantile spasms) might have a focal origin – a ‘zone of cortical abnormality (zca)’ within the cerebral cortex – and that removing such a focus might effectively treat the seizures. at the time, this was considered a radical, almost heretical – possibly even dangerous! – approach, but our clinicians were certain they had convincing evidence for the zca and proceeded with corticectomies for intractable infantile and pediatric epilepsy, sometimes removing a significant portion of one cerebral hemisphere in an infant. the challenge for neuropathologists was to identify what the underlying structural abnormality (abnormalities) causing the seizures might be. again, we were handed the opportunity to study unique specimens that might explain important clinical signs and symptoms. by the late 1980s and early 1990s we had accumulated a significant experience with these specimens. fortuitously, my friend and colleague dr. michael farrell came over from dublin in 1991 to do a mini-sabbatical at ucla, and set about collating the data on our collection of pediatric epilepsy surgery cases. the most common neuropathologic change (especially among the youngest symptomatic patients) was a malformation of cortical development that had first been described by david taylor and colleagues in the 1970s. it had a striking resemblance to the tubers found within the brains of patients with tuberous sclerosis complex (tsc) even though affected individuals showed none of the visceral or cutaneous stigmata of this neurocutaneous disorder [27]. ironically, ‘tuberectomy’ has now become an accepted treatment for some tsc patients with intractable seizures. a much less common finding among corticectomies was an inflammatory disorder first described in 1958, rasmussen encephalitis (re). dr. farrell was first author on our initial ‘pediatric corticectomy’ paper [28] and we continued to collaborate on other projects, including one aimed at elucidating the detailed neuropathologic changes of re [29]. paul mischel, then a neuropathology fellow and now a leading neuro-oncology researcher at uc san diego, was a lead author on a paper in which we proposed a ‘grading’ scheme for the (as yet poorly understood) condition of cortical dysplasia (cd) [30] – a scheme that served its purpose in the mid-1990s and has been superseded by others, as it should be. the team of clinicians and basic researchers who collaborated in the pediatric epilepsy surgery program was a pleasure to work with. this was team science at its finest – everyone in the group respected (and utilized) the unique skills of all other basic and clinical neuroscientists, and the resulting multi-disciplinary studies benefited from this synergy. experts in linguistics, neuropsychology, neuroradiology, neurophysiology and psychiatry interacted productively with a simple neuropathologist. our goal was not only to understand what structural changes in the brain may have contributed to the catastrophic seizure disorder, but how corticectomy might impact a child’s behavior and language development. dr. gary mathern, a ucla-trained neurosurgeon and protégé of paul crandall and warwick peacock, eventually became the surgeon-of-record for most of the pediatric epilepsy cases. dr. mathern also has the ‘soul of an investigator’, is possessed of enviable organizational skills and leadership qualities, and fostered new and exciting directions in which the ucla pediatric epilepsy surgery program might move, places where it might thrive. dr. carlos cepeda, michael levine, (the late) robin fisher, veronique andre and others became integral to the study of electrophysiological and morphological properties of the epileptogenic tissue originating from infants and children with intractable seizures [31-33]. dr. noriko salamon in neuroradiology contributed her powerful diagnostic insights and observations [34,35]. in 2006, and based upon our morpho-anatomical and electrophysiologic studies, we proposed the ‘dysmature cerebral developmental hypothesis’ as a possible etiology for cd [32]. at the same time, we continued to explore the possible relationship of sporadic cd to tsc tubers. the genes that are mutated to cause tsc (first tsc2, then tsc1) had been discovered in the 1990s; we made probes to the relevant gene transcripts and eventually antibodies to the gene products (tuberin and hamartin), using these to study gene expression in normal brain, tubers of tsc and sporadic cortical dysplasia [36-39]. this led to a valuable collaboration with leading tsc researcher dr. david kwiatkowski and his associates in boston – which included sophisticated molecular genetic analysis of many of our corticectomies and even allowed us to examine a novel animal model of tsc, one of the first to be developed by his group [40,41]. i became ucla’s chief of neuropathology in 1993, when tony verity decided to take advantage of the university’s ‘voluntary early retirement incentive plan, verip’, though thankfully he remained active as an emeritus faculty for many years, sharing his expertise in neuromuscular diagnosis (dr. negar khanlou became his protégé and successor) and his passion for cricket. tony was also an excellent artist and i am proud to have one of his paintings on the wall of my living room in venice. i served in the chief of neuropathology post until i myself took ‘semi-retirement’ in 2016, and had the pleasure to mentor junior colleagues who went on to highly successful careers: cynthia welsh, dennis chute, jeff twiss, paul mischel among them. dr. william yong, my current chief, was a medical student who i tutored in the 1980s, later a neuropathology fellow in our program, and has been a marvelous colleague – alas, recently departed for a position at university of california irvine medical center. for several years, i was honored to hold the sarkaria chair in diagnostic medicine. in the mid-1990s i was asked by university of southern california (usc) neurologist dr. helena chui to become part of a california-wide study she was planning, examining the clinical, neuroimaging and neuropathologic substrates of individuals with documented (or at risk for) cerebrovascular disease. the overarching (and perhaps overly ambitious) theme and goal of the program project grant was to highlight the neurobehavioral, imaging and neuropathologic features of ischemic vascular dementia (ivd), on the assumption that such an entity exists. helena was an inspiring and superbly organized leader, and an insightful judge of character, the consummate solver of problems. nih program project grants (ppgs), including the one that funded this study, involve the interactions of widely differing personalities and the principal investigator of such an endeavor (i.e. helena chui) needs to manage those people and their disagreements as well as their productive interactions. our group involved neuropsychologists, epidemiologists, neurologists, and experts in neuroimaging: bruce reed, dan mungas, mike weiner, bill jagust, ling zheng, wendy mack and many others. my fellow investigators in the neuropathology core included william ellis (uc davis), ewa borys (now in chicago) and chris zarow (then a talented neuroanatomist at usc). our studies, over the long duration of the ivd-ppg, showed that ‘pure’ ivd was a comparatively rare phenomenon, but that risk factors for cerebrovascular disease probably contribute to accelerated brain aging, and even some components of alzheimer disease (ad), a view that is gaining traction in the neuroscientific community and a subject that has generated much research interest [42-44]. two papers from the ivd group focused on the ‘neuropathologic substrates’ of cognitive impairment resulting from ischemic brain lesions [45,46], though crucial neuropathologic data were integrated into many other publications. for several years, i have had an enjoyable collaboration with dr. wolff kirsch, a neurosurgeon at loma linda university medical center (approximately 100 miles east of l.a.) who, rather than quietly retire after a stellar career in neurosurgery, decided to become a researcher with an interest in, among other things, brain aging and cerebral amyloid angiopathy (see table 1). dr. kirsch is devoted to the idea of eventually preventing or treating this microangiopathy and has enlisted materials from the easton center brain bank in studies with that goal. very entrepreneurial and creative neuroscientist and much fun to work with in joint projects. as the above summary indicates, much of my research has been collaborative and translational. grant reviewing agencies have, i am quite certain, considered it ‘scattershot’ and unfocused. ‘can vinters not focus on a single topic?’ – the answer is clearly ‘no’. i’ve enjoyed all of the disparate research endeavours in which i have been involved. they have included interactions with investigators possessed of unique talents and abilities, providing the possibility of forming irresistible, tantalizing scientific alliances. table 1 summarizes the key individuals with whom i have worked and published most intensely over the years; it is by no means a complete list. my advice to people entering neuropathology would be to choose your collaborators carefully, wisely. consider individuals who can bring projects to completion in a timely fashion and make effective use of a neuropathologist’s unique skill set. have i myself been successful in following that mandate for timely completion of projects? well… not always… but often enough. work with people who will criticize your work and writing, sometimes brutally. i’ve been fortunate to co-author a manuscript with a pharmacologist, dr. louis ignarro, destined to win the 1998 nobel prize in medicine/physiology for his discovery of nitric oxide [47]. (disclosure: dr. ignarro did not win his nobel on the basis of our study!) my favorite projects and papers have been those that translated basic science observations to address and inform important clinicopathologic issues – ones that might eventually be used to help patients avoid or recover from devastating neurologic illnesses. in 2009, dr. michael sofroniew in ucla’s dept. of neurobiology approached me with the idea to write a review of basic astrocyte biology (his expertise), and how astrocytic dysfunction might mediate neurologic disease (my interest) [48]. it resulted in my most cited publication and led to further productive collaborations that continue now – studies of astrocyte derangement in certain seizure conditions. i benefit daily from my colleagues (drs. william yong, shino magaki, negar khanlou), fellows (currently drs. zesheng chen and josh byers) and technically super-skilled histotechnologists and staff research associates (e.g. kazu williams, current laboratory manager of the easton center brain bank, nelly vehabedian, jennifer yi, cecilia choi). part of the group is shown in figures 9 and 10, masked and ‘social distancing’ (at least in figure 10) as appropriate for the pandemic. when one of the technical staff contributes significantly to a research project and the resultant publication, i include her/him as a co-author on the manuscript. table 2 lists key senior research technicians and lab managers who have each co-authored several publications, many of which have been cited 100+ times (per the web of science). i take pride that many of these skilled technicians have a publication record that surpasses (in both quantity and quality) those of some junior faculty. sadly, one of the individuals on that list (justine pomakian) passed earlier this year after a long battle with leukemia. another (spencer tung) will graduate this year as a physician. a huge bonus or perk of academic life is the possibility to teach, train and influence others – including medical students, basic science colleagues, graduate students, postdoctoral fellows, residents and clinical fellows. i am especially proud of the many neuropathology fellows who have graduated from the ucla training program and have gone out to initiate clinical and/or research programs of their own. our goal as teachers and mentors should be to foster the creation of individuals who are better than we are – more creative, more energetic, better and more insightful diagnosticians. i flatter myself to think that overseas visitors who spent significant time at ucla neuropathology – drs. sung hye park from korea, remco natte from the netherlands, orestes solis from the philippines, hajime miyata from japan, regina von einsiedel from germany, many others – brought back to their home countries skills and knowledge gained in los angeles in ucla’s laboratory of neuropathology. fig 9: team neuropathology at ucla, 2020 (briefly suspending social distancing rules during the covid 19 epidemic) the joy of scientific meetings, sabatticals, visiting professorships and academic visits academics can be fun, terrific fun! most of us enjoy scientific meetings – well, used to enjoy them before they devolved into sitting in front of a computer screen, asking ‘should i share my screen? unmute yourself… can everyone hear me? interesting living room décor, or is it an artificial background? oh no, black screen again’, etc. i have a nagging fear that the trainees of today will come to think: this really isn’t so bad! perhaps it is acceptable as a temporary substitute, but of course there is nothing to compare with the give and take of in person conferences, the body language and passion that are evident in heated or even subdued scientific debates, the stimulating discussions – and yes, gossip! – that occur over cocktails and meals. i recall an american association of neuropathologists meeting (in the early days of my career) during which two senior prominent academics who both worked on neurodegenerative diseases (names withheld to protect the guilty… though alas, both are now deceased…) almost came to blows in front of a poster focusing on the pathogenesis and significance of senile plaques. a little embarrassing, but also somewhat gratifying that each should care so completely and enthusiastically about the significance of these tiny (though controversial) brain lesions – whose importance and relevance to alzheimer’s disease are still debated today. fig 10: team neuropathology at ucla, practicing social distancing what would academic life be without sabbaticals? i spent a marvelous year in the early 1990s with dr. clayton a. wiley at uc san diego, learning the basics of molecular biology and diagnostics as applied to human brain specimens, especially those originating from corticectomies for intractable seizures [49]. i had the opportunity to work with an outstanding technician, rebecca wang (who sadly died of metastatic melanoma a few years later) and dr. cris achim, who remains a friend and collaborator while now on faculty at ucsd. in 1993, i was kindly and generously funded by the daad (german academic exchange service) to work for several weeks with heiko and eva braak in frankfurt – learning how elegant neuroanatomic methods that they had perfected could provide insights into alzheimer’s disease. i’ve had the opportunity to speak and study in countries i would probably not have visited, save for an invitation to lecture or work there – usually at the expense of the host nation/city/academic institution, for which generosity i am sincerely grateful. i’ve even given a lecture in riga, latvia (2015) and was interviewed in latvian (of course i speak the language, in fact english is my second language) on their national radio network. in 1997 (for 6 months) and later in 2004 (for a shorter time interval), i had the chance to do research and teaching as a visiting professor (‘hoogleraar’) at the leiden university medical center, located in an historic city that is home to an equally ancient and revered university – what might be described as a college town in the usa. leiden is situated between amsterdam and the hauge (closer to the latter) in the netherlands and is a short bicycle ride to katwijk on the north sea coast. i shared an office, and many stimulating discussions (about neuropathology, politics, the dutch royal family and other non-scientific matters) with dr. marion maat-schieman [figure 11]. i had the privilege to study, in great detail and with marion’s assistance and much input from dr. remco natte (now a successful pathologist in holland), the fascinating and unique dutch form of hereditary cerebral amyloid angiopathy that results from a distinctive app codon 693 mutation. this causes perhaps the purest form of vascular dementia resulting from microvascular disease, at least beta-amyloid caa [50]. remco later spent several productive months performing complex tissue culture experiments (using vascular-derived smooth muscle cells) in my lab at ucla. in 1998, burroughs wellcome generously provided me with funds to work for a month with dr. michael farrell at the beaumont hospital in dublin. for several years in the early 2010s, i had the privilege to travel to manila in the philippines to lecture at the university of santo tomas and several national and international meetings held in that fascinating country. manila itself is full of paradoxes – great wealth and grinding poverty among them, sometimes existing in close physical proximity – and is one of the most welcoming and hospitable nations in the world. fig 11: dr. marion maat-schieman, retired neuropathologist, leiden university medical center, the netherlands i am proud to be both canadian and american, hold two passports and even have a home in vancouver, canada – which i hope to see again some day after the covid19-related canadian/usa travel restrictions are lifted or at least decreased. for several summers from 2013-2017, i spent a 1-2 week micro-sabbatical in vancouver, working with dr. ian mackenzie and his associates at the vancouver hospital, refining my diagnostic skills in the area of frontotemporal dementia and lobar degenerations, enjoying this almost impossibly beautiful city, and ian’s amazing knowledge of this complex group of disorders. one of the great tragic consequences of covid 19 is the impact it has had on international travel. joining in the life of the university ucla medical center (for many years now the ronald reagan ucla medical center/ rrumc) is located at the southern part of the larger ucla campus. unlike many metropolitan medical centers, it is thus closely linked not only to the non-medical scientific departments with which we interact (including most notably chemistry and biochemistry, biomedical engineering) but also the arts and humanities. while i have been at ucla, two faculty members in the department of chemistry/biochemistry (donald cram and paul boyer) have been awarded nobel prizes; excellent resource to have when one is contemplating experiments with a biochemical component. since arriving at ucla as a junior faculty member, i’ve taken advantage of this proximity, having served for several years on the campus-wide graduate council and, more recently, the university’s council on academic personnel (cap). cap is a committee of 12-15 senior faculty members representing all (most) segments of ucla, who review the dossiers of other ucla academic faculty for initial appointment, major academic promotions (e.g. to the rank of distinguished professor) or appointment to an endowed chair. serving on cap provided a remarkable overview of the diversity of talent and skills at the university. it also gave an interesting perspective into the heterogeneous activities that qualify as creative accomplishment in ucla departments such as near eastern languages and culture, english, philosophy and digital media, to name four among many. life does not end with retirement – nor should it… and closing comments i formally retired from my full time equivalent (fte) in july 2016, and have been on ‘recall’ for clinical and academic work since then, continuing to the present time. for some unknown reason, ucla allows recall faculty to work 43% of time (??!!), though i probably spend more time than that in my office and lab. in 2017, i was honored by the british neuropathological society with the alfred meyer medal and lectureship, received and delivered in london; the british surely are the masters at doing this sort of festive event, and i was suitably ‘chuffed’ and grateful for the honor. that same year i received the award for meritorious contributions to neuropathology, from the american association of neuropathologists. agreed – awards are not that important, but they are a lovely symbol of regard from one’s colleagues. over many years, i have established the gunnar vinters bursary at lakehead university in my home town of thunder bay. last year, i also endowed the elise and arnold vinters scholarship, in memory of my grandparents, at the northern ontario medical school in the same university. i enjoy building on the endowment for each academic award, annually and encouraging others to contribute. since 2018 i have been doing locum work at the university of alberta, in a unit known as alberta precision laboratories (apl). my wonderful colleagues there are drs. laura schmitt, frank van landeghem and sumit das [figure 12]. the work is currently on hiatus (since february, 2020), pending resumption of travel between canada and the usa, but i hope that happens soon. i recently co-authored my first paper with colleagues at apl [51]. it is a form of coming full circle, since i interned at the old university of alberta hospital in 1976-77. fig 12: my neuropathology colleagues at alberta precision laboratories, edmonton, alberta, 2020 (l to r drs. laura schmitt, frank van landeghem, sumit das) i love travel, including (and especially) air travel… but as of this writing have not been on an airplane or near an airport for 5+ months. however, there is much still to do at ucla, projects that – so i hope – benefit from my input, complex cases on which i can shed some light. in my spare time, i revel in the delights of venice beach, california, where there is – shall we say – never a dull moment. it is a beachside region of los angeles founded in the early 1900s by abbot kinney, a tobacco heir who was so impressed with venice, italy, that he thought it would be fun to replicate it in southern california. i have great friends there, throughout l.a. and in canada and five accomplished godchildren whose antics keep life interesting. in my spare time, which seems sometimes not to be terribly abundant, i write short stories and oped pieces, none of which is destined for publication, though i’ve published a few ‘back in the day’. now it is time to end this reflection, which seems to have expanded to the length of a novella! when i was in medical school, i never dreamed i would spend most of my adult life in southern california. as i heard or read somewhere, ‘life is what happens to you while you are making plans…’ – how true. i appreciate free neuropathology giving me the opportunity to write this piece. acknowledgements kazu williams assisted with preparation of figures. dr. shino magaki and kazu williams assisted with critical reading of the manuscript. references 1. vinters h, dainty j, tyree mt. cell-wall elastic properties of chara corallina. canadian journal of botany. 1977; 55: 1933-1939 2. seeman p. membrane actions of anesthetics and tranquilizers. pharmacological reviews. 1972; 24: 583-655 3. hornykiewicz o. dopamine (3-hydroxytyramine) and brain function. pharmacological reviews. 1966; 18: 925-964 4. steele jc, richardson jc, olszewski j. progressive supranuclear palsy. a heterogeneous degeneration involving the brain stem, basal ganglia and cerebellum with vertical gaze and pseudobulbar palsy, nuchal dystonia and dementia. archives of neurology. 1964; 10: 333-359 5. gilbert jj, vinters hv. cerebral amyloid angiopathy—incidence and complications in the aging brain. 1. cerebral hemorrhage. stroke. 1983; 14: 915-923 6. vinters hv, gilbert gg. cerebral amyloid angiopathy—incidence and complications in the aging brain. 2. the distribution of amyloid vascular changes. stroke. 1983; 14: 924-934 7. debault le, cancilla pa. gamma-glutamyl transpeptidase in isolated brain endothelial cells: induction by glial cells in vitro. science. 1980; 207:653-655 8. debault le, cancilla pa. induction of gamma-glutamyl transpeptidase in isolated cerebral endothelial cells. advances in experimental medicine biology. 1980; 131: 79-88 9. beck dw, vinters hv, hart mn, cancilla pa. glial cells influence polarity of the blood-brain barrier. journal of neuropathology & experimental neurology. 1984; 43: 219-214 10. vinters hv, reave s, costello p, girvin jp, moore sa. isolation and culture of cells derived from human cerebral microvessels. cell tissue research. 1987; 249: 657-667 11. vinters hv, galil ka, lundie mj, kaufmann jce. the histotoxicity of cyanoacrylates. a selective review. neuroradiology. 1985; 27: 279-291 12. vinters hv, lundie mj, kaufmann jce. long-term pathological follow-up of cerebral arteriovenous malformations treated by embolization with bucrylate. new england journal of medicine. 1986; 314: 477-483 13. small gw, kepe v, ercoli lm, prabha s, bookheimer sy, miller kj, lavretsky h, burggren ac, cole gm, vinters hv, thompson pm, huang s-c, satyamurthy n, phelps me, barrio jr. pet of brain amyloid and tau in mild cognitive impairment. new england journal of medicine 2006; 355: 2652-2663 14. vinters hv. cerebral amyloid angiopathy. a critical review. stroke 1987; 18: 311-324 15. glenner gg, wong cw. alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. biochemical and biophysical research communications 1984; 120: 885-890 16. vinters hv, pardridge wm, yang j. immunohistochemical study of cerebral amyloid angiopathy: use of an antiserum to a synthetic 28-amino-acid peptide fragment of the alzheimer’s disease amyloid precursor. human pathology 1988; 19: 214-222 17. vinters hv, nishimura gs, secor dl, pardridge wm. immunoreactive a4 and gamma-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with alzheimer’s disease. american journal of pathology 1990; 137: 233-240 18. anders k, steinsapir kd, iverson dj, glasgow bj, layfield lj, brown wj, cancilla pa, verity ma, vinters hv. neuropathologic findings in the acquired immunodeficiency syndrome (aids). clinical neuropathology 1986; 5: 1-20 19. anders kh, guerra wf, tomiyasu u, verity ma, vinters hv. the neuropathology of aids. ucla experience and review. american journal of pathology 1986; 124: 537-558 20. li xl, moudgil t, vinters hv, ho dd. cd4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1. journal of virology 1990; 64: 1383-1387 21. ho dd, bredesen de, vinters hv, daar es. the acquired immunodeficiency syndrome (aids) dementia complex. annals of internal medicine 1989; 111: 400-410 22. koyanagi y, miles s, mitsuyasu rt, merrill je, vinters hv, chen isy. dual infection by of the central nervous system by aids viruses with distinct cellular tropisms. science 1987; 236: 819-822 23. pang s, koyanagi y, miles s, wiley c, vinters hv, chen isy. high levels of unintegrated hiv-1 dna in brain tissue of aids dementia patients. nature 1990; 343: 85-89 24. vinters hv, kwok mk, ho hw, anders kh, tomiyasu u, wolfson wl, robert f. cytomegalovirus in the nervous system of patients with acquired immune deficiency syndrome. brain 1989; 112 (pt 1): 245-268 25. von einsiedel rw, fife td, aksamit aj, cornford me, secor dl, tomiyasu u, itabashi hh, vinters hv. progressive multifocal leukoencephalopathy in aids. a clinicopathological study and review of the literature. journal of neurology 1993; 240: 391-406 26. vinters hv, anders kh. neuropathology of aids. boca raton, florida: crc press, 1990; 229 pp 27. taylor dc, falconer ma, bruton cj, corsellis ja. focal dysplasia of the cerebral cortex in epilepsy. journal of neurology, neurosurgery and psychiatry 1971; 34: 369-387 28. farrell ma, derosa mj, curran jg, secor dl, cornford me, comair yg, peacock wj, shields wd, vinters hv. neuropathologic findings in cortical resections (including hemispherectomies) for the treatment of intractable childhood epilepsy. acta neuropathologica 1992; 83: 246-259 29. farrell ma, droogan o, secor dl, poukens v, quinn b, vinters hv. chronic encephalitis associated with epilepsy. immunohistochemical and ultrastructural studies. acta neuropathologica 1995; 89: 313-321 30. mischel ps, nguyen lp, vinters hv. cerebral cortical dysplasia associated with pediatric epilepsy. review of neuropathologic features and proposal for a grading system. journal of neuropathology and experimental neurology 1995; 54:137-153 31. cepeda c, hurst rs, flores-hernandez j, hernandez-echeagaray e, klapstein gj, boylan mk, calvert cr, jocoy el, et al. morphological and electrophysiological characterization of abnormal cell types in pediatric cortical dysplasia. journal of neuroscience research 2003; 72: 472-486 32. cepeda c, andre vm, levine ms, salamon n, miyata h, vinters hv, mathern gw. epileptogenesis in pediatric cortical dysplasia: the dysmature cerebral developmental hypothesis. epilepsy & behavior 2006; 219-235 33. cepeda c, chen jy, wu jy, fisher rs, vinters hv, mathern gw, levine ms. pacemaker gaba synaptic activity may contribute to network synchronization in pediatric cortical dysplasia. neurobiology of disease 2014; 62: 208-217 34. chandra ps, salamon n, nguyen st, chang jw, huynh mn, cepeda c, leite jp, neder l, koh s, vinters hv, et al. infantile spasm-associated microencephaly in tuberous sclerosis complex and cortical dysplasia. neurology 2007; 438-445 35. salamon n, andres m, chute dj, nguyen st, chang jw, huynh mn, chandra ps, andre vm, cepeda c, levine ms, et al. contralateral hemimicrencephaly and clinical-pathological correlations in children with hemimegalencephaly. brain 2006; 129: 352-365 (part 2) 36. menchine m, emelin jk, mischel ps, haag ta, norman mg, pepkowitz sh, welsh ct, townsend jt, vinters hv. tissue and cell-type specific expression of the tuberous sclerosis gene, tsc2, in human tissues. modern pathology 1996; 1071-1080 37. vinters hv, park sh, johnson mw, mischel ps, catania m, kerfoot c. c. cortical dysplasia, genetic abnormalities and neurocutaneous syndromes. developmental neuroscience 1999; 21 (3-5): 248-259 38. johnson mw, miyata h, vinters hv. ezrin and moesin expression within the developing human cerebrum and tuberous sclerosis-associated cortical tubers. acta neuropathologica 2002; 104: 188-196 39. johnson mw, emelin jk, park sh, vinters hv. co-localization of tsc1 and tsc2 gene products in tubers of patients with tuberous sclerosis. brain pathology 1999; 9: 45-54 40. qin w, chan ja, vinters hv, mathern gw, franz dn, taillon be, bouffard p, kwiatkowski dj. analysis of tsc cortical tubers by deep sequencing of tsc1, tsc2 and kras demonstrates that small second-hit mutations in these genes are rare events. brain pathology 2010; 20: 1096-1105 41. goto j, talos dm, klein p, qin w, chekaluk yi, anderl s, malinowska ia, di nardo a, et al. regulable neural progenitor-specific tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis complex. proceedings of the national academy of sciences of the usa 2011; 108: e1070-e1079 42. reed br, mungas dm, kramer jh, ellis w, vinters hv, zarow c, jagust wj, chui hc. profiles of neuropsychological impairment in autopsy-defined alzheimer’s disease and cerebrovascular disease. brain 2007; 130 (pt 3): 731-739 43. jagust wj, zheng l, harvey dj, mack wj, vinters hv, weiner mw, ellis wg, zarow c, mungas d, reed br, et al. neuropathological basis of magnetic resonance images in aging and dementia. annals of neurology 2008; 63: 72-80 44. chui hc, zarow c, mack wj, ellis wg, zheng l, jagust wj, mungas d, reed br, kramer jh, decarli cc, et al. cognitive impact of subcortical vascular and alzheimer’s disease pathology. annals of neurology 2006; 60: 677-687 45. vinters hv, ellis wg, zarow c, zaias bw, jagust wj, mack wj, chui hc. neuropathologic substrates of ischemic vascular dementia. journal of neuropathology and experimental neurology 2000; 59: 931-945 46. vinters hv, zarow c, borys e, whitman jd, tung s, ellis wg, zheng l, chui hc. review: vascular dementia: clinicopathologic and genetic considerations. neuropathology and applied neurobiology 2018; 44: 247-266 47. mitrovic b, ignarro lj, vinters hv, akers ma, schmid j, uittenbogaart c, merrill je. nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes. neuroscience 1995; 65: 531-539 48. sofroniew mv, vinters hv. astrocytes: biology and pathology. acta neuropathologica 2010; 119: 7-35 49. vinters hv, wang r, wiley ca. herpesviruses in chronic encephalitis associated with intractable childhood epilepsy. human pathology 1993; 24: 871-879 50. vinters hv, natte r, maat-schieman mlc, van duinen sg, hegeman-kleinn i, welling-graafland c, haan j, roos rac. secondary microvascular degeneration in amyloid angiopathy of patients with hereditary cerebral hemorrhage with amyloidosis, dutch type (hchwa-d). acta neuropathologica 1998; 95: 235-244 51. chan j, magaki s, zhang xr, chin c, greenspan s, linetsky m, kattar m, vinters hv. intravascular carcinomatosis of the brain: a report of two cases. brain tumor pathology 2020; 37: 118-125 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. ex vivo mri facilitates localization of cerebral microbleeds of different ages during neuropathology assessment feel free to add comments by clicking these icons on the sidebar free neuropathology 2:35 (2021) original paper ex vivo mri facilitates localization of cerebral microbleeds of different ages during neuropathology assessment sukriti nag1,2, er-yun chen2, ryan johnson2, ashish tamhane2, konstantinos arfanakis2,3, julie a. schneider1,2,4 1 department of pathology (neuropathology), rush university medical center, chicago, il, usa 2 rush alzheimer’s disease center, rush university medical center, chicago, il, usa 3 department of biomedical engineering, illinois institute of technology, chicago, il, usa 4 department of neurological sciences, rush university medical center, chicago, il, usa corresponding author: sukriti nag · rush alzheimer disease center · suite 1000 · rush university medical center · 1750 w harrison street · chicago, il 60612 · usa sukriti_nag@rush.edu submitted: 20 october 2021 accepted: 12 december 2021 copyedited by: christian thomas published: 20 december 2021 https://doi.org/10.17879/freeneuropathology-2021-3638 keywords: alzheimer’s dementia, cerebral microbleeds, ex vivo magnetic resonance imaging, hemosiderin, microhemorrhages abstract cerebral microbleeds (cmbs) identified by in vivo magnetic resonance imaging (mri) of brains of older persons may have clinical relevance due to their association with cognitive impairment and other adverse neurologic outcomes, but are often not detected in routine neuropathology evaluations. in this study, the utility of ex vivo mri in the neuropathological identification, localization, and frequency of cmbs was investigated. the study included 3 community dwelling elders with alzheimer’s dementia, and mild to severe small vessel disease (svd). ex vivo mri was performed on the fixed hemisphere to identify cmbs, blinded to the neuropathology diagnoses. the hemibrains were then sliced at 1 cm intervals and 2, 1 or 0 microhemorrhages (mh) were detected on the cut surfaces of brain slabs using the routine neuropathology protocol. ex vivo imaging detected 15, 14 and 9 possible cmbs in cases 1, 2 and 3, respectively. to obtain histological confirmation of the cmbs detected by ex vivo mri, the 1 cm brain slabs were dissected further and mhs or areas corresponding to the cmbs detected by ex vivo mri were blocked and serially sectioned at 6 μm intervals. macroscopic examination followed by microscopy post ex vivo mri resulted in detection of 35 mhs and therefore, about 12 times as many mhs were detected compared to routine neuropathology assessment without ex vivo mri. while microscopy identified previously unrecognized chronic mhs, it also showed that mhs were acute or subacute and therefore may represent perimortem events. ex vivo mri detected cmbs not otherwise identified on routine neuropathological examination of brains of older persons and histologic evaluation of the cmbs is necessary to determine the age and clinical relevance of each hemorrhage. introduction cerebral microbleeds (cmbs) are defined as rounded or ovoid, hypointense foci that are best demonstrated in susceptibility-weighted and gradient echo magnetic resonance imaging (mri). in most studies, the upper limit of the diameter of cmbs is 5-5.7 mm [1-3], although a diameter of 10 mm has also been reported [4]. neuroimaging studies show that cmbs tend to increase with age being observed in 5% of healthy adults [5, 6], and 7% of volunteers having a mean age of 62.1 ±7.4 years who were enrolled in the uk biobank study [7]. cmbs are reported in 18% of subjects between 60 to 69 years, while in those older than 80 years prevalence increases to 38% [6]. cmbs have also been associated with increased risk of stroke in the general population [8]. prevalence is higher in those with ischemic stroke and with non-traumatic intracerebral hemorrhage, being 34% and 60%, respectively [5]. in the latter study, cmbs were associated with hypertension or diabetes mellitus in otherwise healthy adults, while in adults with cerebrovascular disease they were only associated with hypertension. the frequency of cmbs is 84.9% in those with subcortical vascular dementia and in these subjects cmbs were related to cognitive impairment in multiple domains [9]. in most studies, cmbs were identified by in vivo mri alone [8-12], while in a few studies by neuropathology assessment alone in which they are referred to as microhemorrhages (mhs) [13]. the development of ex vivo mri led to studies correlating ex vivo mri findings with neuropathology in selected brain slabs [1, 2, 14-16]. in these studies, cmbs were considered to be markers of focal hemosiderin deposition that remained in macrophages for years following a mh [1, 11]. there is increasing evidence that the pathologic correlate of cmbs is more varied than just mhs since a significant association was observed between hemosiderin collections in the putamen and indices of small vessel ischemia such as microinfarcts, arteriolosclerosis and lacunes in any of the brain regions examined [2, 14]. the aim of the present study was to characterize the morphologic correlate of cmbs identified by ex vivo mri of a single cerebral hemisphere of 3 brains with and without mhs identified on initial neuropathology assessment. this was followed by detailed neuropathology assessment to determine the spectrum of pathology associated with the cmbs identified by ex vivo mri. since one in five patients with alzheimer’s disease (ad) has cmbs [17], and the frequency of cmbs is higher in association with cerebral amyloid angiopathy (caa) [2, 18], and arteriolosclerosis [1, 19], 3 community dwelling elders with a clinical diagnosis of alzheimer’s dementia and varying degrees of small vessel disease (svd) were selected for assessment. materials and methods the criteria for selection of the three autopsied cases for this study include a clinical diagnosis of alzheimer’s dementia and pathologic evidence of caa and arteriolosclerosis as well as absence of a clinical history of traumatic brain injury with loss of consciousness since diffuse axonal injury following head trauma is another potential secondary cause of cmb [11]. cases were from the memory and aging project (map), a large clinical-pathology study of aging and dementia with community dwelling participants. the protocols used in this study were approved by the institutional review board of rush university medical center. a signed informed consent was obtained from each participant for an annual clinical evaluation and for brain donation. participants underwent uniform clinical evaluations at baseline and annually thereafter for odor testing, parkinsonism signs and cognitive function as described previously [20]. ex vivo imaging following removal, the brain was hemisected and one hemisphere with visible gross pathology or one hemisphere with absence of pathology was selected arbitrarily and submerged medial side up in 4 % paraformaldehyde in a container which was rocked back and forth until no more air bubbles escaped from the lateral ventricles. the hemisphere was then placed medial side down and refrigerated. mri was done on average 1 month postmortem on hemibrains returned to room temperature, immersed in 10% formalin and held in place in a container using a plastic divider. ex vivo mri was done using a siemens 3tesla mri scanner and a three-dimensional multi-echo gradient echo (gre) sequence with 6 echoes (te = 5 + n×5 ms (n=0-5), tr = 35 ms), an acquired voxel-size of 1×1×1 mm3, and a scan time of about 8 min. microbleeds were identified by a trained reader who was blinded to clinical and pathology data. cmbs were diagnosed when a relatively round, small, hypointense region of few mm in diameter was observed on the last echo of the gre data. at least half of the hypointensity had to be surrounded by brain parenchyma to qualify. elongated, vessel-like hypointensities and potential mimics, including lesions in the subarachnoid space, calcifications, or vascular malformations were excluded. neuropathology assessment the mean postmortem interval for cases 1-3 was 22, 8 and 6 hours, respectively (table 1). a standard protocol was used to evaluate brains which included cutting each hemibrain into 1 cm coronal slabs guided by a plexiglass jig and brain blocks from eleven brain regions and any mh noted on the cut surfaces of the slabs were blocked for microscopy as described previously [21]. pathological diagnosis of ad, hippocampal sclerosis (hs), limbic predominant age-related tdp-43 encephalopathy neuropathologic change (late-nc), macroscopic and microscopic infarcts were made as described previously [21]. caa was assessed in meningeal and intracortical vessels in sections from the midfrontal, midtemporal, inferior parietal and occipital cortices which were immunostained for β-amyloid and graded as described previously [22]. arteriolosclerosis was assessed in the basal ganglia and graded using a semiquantitative scale from 0 (none) to 6 (severe) as described previously [21]. table 1: clinical and pathological findings in 3 cases with a clinical diagnosis of alzheimer’s dementia.   case 1 case 2 case 3 age, years 91.4 96.2 79.2 sex female female male education, years 16 12 16 blood pressure, mm hg (last valid) 96/54a 171/96b m mmse score (last valid) 11a 5b 9c total cmbs detected by ex vivo mri 15 14 9   post mortem interval, hours 22 8 6   pathological diagnoses from blocks taken routinely alzheimer’s disease + + cerad probable probable definite braak stage i iii iv thal stage 3 3 5 adnc low intermediate intermediate hippocampal sclerosis + late-nc stage 3 3 0 cerebral amyloid angiopathy, mild-severe mild mild severe arteriolosclerosis mild mod-severe mild atherosclerosis possible mild mild-mod macroscopic infarcts, chronic 4 1 microscopic infarcts, chronic 7 1 total mhs using the routine protocol (no ex vivo mri) 2 1 0 total mhs post ex vivo mri acute 5 2 5 subacute 9 0 0 chronic 1 12 1 additional pathologies in blocks taken post ex vivo mri microinfarct, chronic 1 1 microinfarct, subacute 0 1 0 abbreviations: adnc alzheimer’s disease neuropathologic change; cerad consortium to establish a registry for alzheimer’s disease; cmbs cerebral microbleeds; late-nc limbic predominant age-related tdp-43 neuropathologic change; m missing; mhs microhemorrhages; mod moderate. values obtained 3a, 7b and 4c months prior to death. microhemorrhages the ex vivo mri images were superimposed on photomicrographs of the brain slabs to identify the region of the suspected cmb. in this protocol, each 1 cm slab was further dissected to detect the cmbs observed in the ex vivo mr images and brain blocks containing these cmbs were obtained. in instances where a mh was not identified on the cut surface of the brain slab, a block was obtained in the region containing the cmb identified by ex vivo mri. all brain blocks were processed using standard techniques and embedded in paraffin. each block was serially sectioned at 6μm thickness to obtain 200-250 serial sections from each block. every 10th section was stained with hematoxylin and eosin (he) and the adjacent section was stained by the perl’s prussian blue stain for hemosiderin. in select cases, additional sections were stained by he and for hemosiderin. immunohistochemistry to detect β-amyloid was done in all cmb’s as described previously [22]. the size of mhs was measured using a 1 mm graticule at a magnification of x100. results the age of the three decedents was 91.4, 96.2 and 79.2 years and in keeping with the clinical diagnosis of alzheimer’s dementia, all cases had a low mini-mental state examination score which varied from 5-11 (table 1). blood pressure data was only available for 2 cases and only case 2 was hypertensive. on routine neuropathology assessment, (table 1) two of the three cases had intermediate likelihood of ad by the nia-reagan and adnc criteria while the third case with low likelihood of ad had hippocampal sclerosis with late-nc (stage 3) implying involvement of the amygdala, limbic and neocortical areas by tdp-43 neuronal and glial cytoplasmic inclusions. regarding vascular changes, cases 1 and 2 had mild caa while case 3 had severe caa. arteriolosclerosis was mild in cases 1 and 3 and moderate-severe in case 2. ex vivo imaging detected 15,14 and 9 possible cmbs in cases 1, 2 and 3, respectively (table 1). mr images showed hypointense, rounded areas in the gray or white matter which were interpreted as cmbs. (fig. 1a, e, 2a) the diameter of cmbs measured in the same echo as used for their visualization, varied from 1.56 – 4.20 mm. most of the cmbs observed in this study were located in the subcortical white matter near the grey/white junction of the frontal, temporal or occipital lobes, although, involvement of gray matter structures such as the midtemporal cortex, thalamus and caudate nucleus were also noted. figure 1: ex vivo mri scans, corresponding brain slabs and microscopy of acute and subacute hemorrhages are shown. (a) the mri scan shows a cmb in the white matter of the temporal lobe (arrowhead). (b) the corresponding brain slab was further sliced in the area of the cmb shown in the ex vivo mr scan and delineated by the rectangle to detect the mh which is shown in the inset (case 1, mh#6). (a, b) also present is another cmb at the interface of the cortex and white matter of the superior temporal gyrus (case 1, mh#8). (c) microscopy of a hematoxylin and eosin (he) stained section from mh#6 shows an acute hemorrhage with extravasation of red blood cells through a venular wall into the surrounding brain. (d) this mh shows no macrophages or hemosiderin by the perl’s prussian blue stain at this level or in serial sections. (e) ex vivo mri shows a cmb in the white matter of the inferior parietal lobule (arrowhead). (f) further slices of the corresponding brain slab in the area delineated by a rectangle shows a mh (case1, mh#14) in the white matter which is shown in the inset. (g) an he stained section of the mh shows significant extravasation of red blood cells through an arteriolar wall (arrowhead) into the neuropil. a few pigment-laden macrophages are identified at the periphery of the hemorrhage (inset). (h) the increased number of macrophages are clearly evident by the perl’s prussian blue stain. higher magnification of the hemosiderin positive macrophages is shown in the inset. c, d, g, h scale bar = 50 μm; insets g, h scale bar = 10 μm macroscopic findings the appearance of the mhs on macroscopic examination of the cut surfaces of brain slabs whether using the routine or the extended protocol was similar. mhs appeared as rounded, reddish areas, 1-2 mm in diameter resembling the mh shown in the inset of fig. 1b. in a few cases, there was a focal area of bluish discoloration suggesting presence of a hemorrhage below the cut surface of the brain slab while in other cases no hemorrhage was noted on the cut surface of the slab and was only identified following serial sectioning in the regions showing cmbs by ex vivo imaging (fig. 1f). microscopic findings a total of 12 acute, 9 subacute and 14 mhs were identified by microscopy of the 3 cases which include the 3 mhs observed using routine neuropathology assessment. most of the hemorrhages (74%) were located in the white matter while the remainder were in grey matter structures such as one in the midtemporal cortex, one in the caudate nucleus and others in the thalamus (table 2). microscopy of all 35 mhs showed that their diameter varied from 0.2-0.8 mm. most mhs were associated with arterioles. among the acute hemorrhages, 3 were associated with venules and 1 with multiple capillaries. in the case of chronic hemorrhages, only one was associated with a venule, while another chronic hemorrhage was associated with both an arteriole and venule which were adjacent to each other, while others were associated with arterioles. on serial sectioning, mhs involved variable lengths of the vessel with some mhs involving short 125-300 μm segments of arterioles while others involved long segments of arterioles varying from 480-780 μm in length. there was no evidence of calcification or vascular malformations, such as a cavernous hemangioma in any of the sections examined. most blocks showed a single mh correlating with the cmb seen on ex vivo mri, however, there were 3 instances when the block showed additional mhs which were not identified by ex vivo mri (table 2). table 2: location and age of microhemorrhages (mh) detected without mri and pathological features of those detected following ex vivo mri. mh diagnosis by routine np cmbs diagnosed following ex vivo mri microhemorrhages (mh) histological findings other microscopic findings location, age location age perl’s no. of mhs case 1 1 superior frontal, wm acute 1 2 subcortical wm, between superior and middle frontal gyri acute 1 3 inferior frontal gyrus, wm subacute + 1 4 anterior temporal tip, wm subacute + 1 5 midtemporal gyrus, wm subacute + 2 6 midtemporal gyrus, wm acute 1 7 superior temporal gyrus wm, acute superior temporal gyrus, wm subacute + 1 8 superior temporal gyrus, wm subacute + 1 9 superior temporal gyrus, wm subacute + 1 10 postcentral gyrus, wm subacute + 1 11 inferior temporal gyrus wm, acute inferior temp gyrus, wm acute 1 12 temporal periventricular wm no mh 0 13 superior parietal lobule, wm acute 1 14 inferior parietal lobule, wm subacute + 1 15 anterior insula, wm chronic + 1 case 2 1 occipital wm, posterior no mh 0 dilated vein with luminal blood 2 occipital wm, anterior chronic + 1 3 midtemporal cortex, chronic midtemporal cortex chronic + 1 4 thalamus, (ventral posterior lateral nucleus) no mh 0 5 thalamus, (lateral, posterior nucleus) chronic + 2 subacute microinfarct-1 6 thalamus (dorsomedial nucleus) no mh 0 7 thalamus (dorsomedial nucleus near midline) no mh 0 dilated vessels with luminal blood 8 wm near ventro-lateral nucleus of thalamus chronic + 2 9 thalamus (ventro-lateral nucleus) chronic + 4 10 thalamus (ventro-lateral nucleus) chronic 1 chronic microinfarct-1 11 wm ventral to putamen (posterior) no mh 0 12 wm adjacent to lateral putamen chronic + 1 perivascular hemosiderin 13 wm ventral to putamen (anterior) acute 1 14 caudate nucleus acute 1 case 3 1 no cmb diagnosed superior frontal gyrus no mh 0 2 wm lateral to putamen no mh 0 dilated vessels with luminal blood 3 frontal lobe wm acute 1 4 parietal lobe wm acute 1 5 occipitotemporal gyrus,wm acute 1 6 occipitotemporal gyrus, wm acute 1 7 occipital lobe wm acute 1 8 occipital lobe wm no mh 0 9 occipital lobe wm chronic + 1 chronic microinfarct-1 abbreviations: cmbs cerebral microbleeds; mhs microhemorrhages; wm white matter. mhs were acute, subacute or chronic based on the cellular response present. acute mhs showed extravasation of erythrocytes through the walls of mainly arterioles and fewer venules and extended into the surrounding neuropil (fig. 1c). serial sectioning of the entire block did not show inflammatory cells or hemosiderin either on he or the perl’s prussian blue stain thus their age was not altered (fig. 1d). an additional finding in subacute mhs was the extension of red blood cells further from vessel walls than observed in the acute hemorrhages (fig. 1g). a few intravascular and a variable number of perivascular macrophages were present and many contained faint brown hemosiderin pigment. the adjacent perl’s-stained section accentuated the macrophage numbers and their hemosiderin content (fig. 1h). the neuropil surrounding the hemorrhage showed vacuolation and sparse gemistocytes. in chronic mhs, a cavity was present containing or surrounded by a variable number of macrophages which showed intense brown cytoplasmic staining due to hemosiderin (fig. 2c, d). the neuropil surrounding the hemorrhage showed fibrillary astrocytes. adjacent arterioles showed acellular mural thickening with absence of smooth muscle cells and failed to show β-amyloid positivity (fig. 2e). in all mhs, vessels failed to show mural β-amyloid positivity with the exception of one chronic mh (case 2, mh #3) in the midtemporal cortex which showed a disrupted vessel associated with hemosiderin-laden macrophages (fig. 2f) and focal β-amyloid positivity at the periphery of the vessel (fig. 2g). another pattern of chronic mh was the presence of scattered hemosiderin-laden macrophages in a focal area of the white matter (fig. 3a). serial sectioning showed that these areas extended for depths of 120-180 μm and were related to terminal arterioles and capillaries (fig. 3b). in some mhs, arterioles with sclerosed, hyaline walls and collapsed lumina were present surrounded by variable numbers of hemosiderin containing macrophages in the neuropil. in one instance, only perivascular collections of few macrophages were present in multifocal areas of the putamen. figure 2: ex vivo mri scan, corresponding brain slab and microscopy of chronic grey matter hemorrhages are shown. (a) ex vivo mri shows a cmb in the lateral thalamus. (b) the corresponding brain slab shows an area of reddish discoloration (arrowhead) which is shown at higher magnification in the inset (case 2 mh#8). (c) the corresponding he stained section shows a cavity containing hemosiderin-laden macrophages adjacent to an arteriole which shows focal mural disruption and double-barreling. a nearby arteriole shows perivascular hemosiderin-laden macrophages. (d) the adjacent section shows strong prussian blue staining of the hemosiderin collections. (e) immunostaining of the arteriole shown in c and d fails to show mural β-amyloid although the double barreling is highly suggestive of caa. (f) a focal hemorrhage in layer three of the superior temporal cortex shows disruption of a vessel associated with hemosiderin-laden macrophages which are also present in the surrounding neuropil along with gemistocytes (arrowhead) (g) the adjacent section shows focal β-amyloid immunostaining at the periphery of the vessel (arrowhead). c, d scale bar = 50 μm; e, g scale bar = 50 μm, f scale bar = 50 μm. in addition to the mhs which correlated with the hypointense areas detected by ex vivo mri, serial sectioning showed mhs measuring < 0.4 mm in their greatest dimension (fig. 3c, d). these mhs were much smaller than the cmbs identified by ex vivo mri and were present in 1-3 consecutive sections per block. these small mhs were not included in the total mh counts shown in table 1, since they did not correlate with any of the reported hypointensities observed by mri and since they were too small to be visualized by a 3t scanner. although 38 cmbs were described in the ex vivo mr scans, in 8 instances microscopy did not show mhs despite additional slicing of brain slabs and microscopy of serially sectioned blocks. on microscopy, 3 of these presumed cmbs showed 2-3 large dilated vessels with luminal blood (table 2). in 2 presumed cmbs, the area of hypointensity represented the site of entry of penetrating arterioles in the lateral and inferior portion of the putamen and in 2 presumed cmbs the area of hypointensity probably represented the depths of sulci and only in one case no pathology was detected. additional pathology identified by serial sectioning were chronic microinfarcts in 2 cases and a subacute microinfarct in 1 case (table 2) and microinfarcts measuring < 0.4 mm in their greatest dimension (fig. 3e, f). figure 3: microscopy of chronic hemorrhages and chronic microinfarcts. (a) an example of a focal chronic white matter hemorrhage (600 x 300 μm) showing scattered hemosiderin-laden macrophages (arrowheads). (b) the adjacent section shows a terminal arteriole (arrowhead) and capillary (below the asterisk) with all macrophages showing positivity by the perls’s prussian blue stain. serial sections show two small mhs (c, d) measuring 200 (c) and 300 (d) μm in their longest dimension and two chronic microinfarcts (e, f) measuring 300 (e) and 200 (f) μm which do not correlate with any cmbs observed in the ex vivo scans. the microinfarcts show disruption of the neuropil and many macrophages some of which appeared pale brown suggesting hemosiderin but were negative by the perl’s prussian blue stain. a-c scale bar = 50 μm; d-f scale bar = 50 μm. discussion this study demonstrates the utility of ex vivo mri in the identification of mhs by neuropathology assessment. routine macroscopic assessment showed a total of 3 mhs while both macroscopic and microscopic assessment after ex vivo mri showed about 12 times as many mhs. in cases 1 and 2, the number of mhs was increased from that seen on routine neuropathology assessment, while in case 3, no mhs were identified on routine neuropathology assessment but were detected by ex vivo mri followed by neuropathology assessment. thus, routine neuropathology assessment alone is unreliable in detecting the presence or the total number of mhs in a specific brain. the mhs identified in this study by microscopy were smaller than indicated by their mr appearance being < 1 mm in diameter. the finding that cmbs detected by mri appear larger on gradient-echo sequences compared with the actual tissue lesions is attributed to the “blooming effect” of the mr signal at the border of these lesions [11, 23]. also, further shrinkage of brain blocks during the processing schedule for paraffin embedding may contribute to the reduced diameter observed on microscopy. diameters measured in ex vivo scans were reported to be on average 1.6 ±0.75 times [2] or 4 times [16] higher than the diameter identified by pathology assessment. however, since the size of a cmb on mri depends on imaging parameters, such as field strength and pulse sequence, comparison of cmb size across studies is probably not valid due to the difficulty in implementing identical imaging parameters on different mri systems [11]. the localization of mhs in the current study in both the grey and the subcortical white matter near the grey/white junction is similar to previous observations [1, 2, 13]. in addition, involvement of mainly arterioles and fewer capillaries and intracerebral venules in mhs has also been reported in a prior study [13]. in the current study, 38 blocks were examined by serial sectioning and 35 mhs were identified in the 3 cases with some blocks having more mhs than identified by ex vivo mri. the very small mhs observed only by histology and not by ex vivo mri in the present and previous studies [1, 13], are probably beyond the sensitivity of a 3t mri scanner. ex vivo imaging using a 7t scanner and high resolution (200 um isotropic resolution) can detect 2.6 times as many cmbs as compared to an in vivo 7t mri scanner while ultrahigh resolution detects additional hypointensities which on microscopy show vessel pathology but no additional hemorrhages [16]. the criteria used in the current study for aging mhs as acute, subacute or chronic is supported by previous experimental studies of rodent [24, 25] and rabbit [26] brains with mhs in which the time course of the cellular reactions was documented. during the acute phase up to 48 hours, the hematoma is surrounded by edematous neuropil and no inflammatory cells. the initial peri-hemorrhage inflammatory response at 48 hours consists of few blood-borne neutrophils and brain-resident microglia (cx3cr1 positive) which migrate to the lesion site from the surrounding tissue. astrocytic activation is reported to occur a few days after the onset of the microglial response. conversion to hemosiderin begins at day 5 after the injection of either blood or red blood cells into brain [26]. by day 8, macrophages (brain-resident microglia and fewer blood-derived macrophages) are abundant and show evidence of proliferation at the lesion site. by 2 weeks, the inflammatory response resolves. therefore, subacute mhs are up to 2 weeks or few weeks of age. changes between few weeks and 3 months are not reported, however, at 3 months cavities are observed at the mh site with variable numbers of macrophages showing abundant hemosiderin. fibrillary gliosis is present and arterioles in the area showed mural thickening which is not related to amyloid as noted in the current and a previous study [13], while in another study mural β-amyloid was detected in a bleeding vessel [2]. therefore, chronic mhs are 3 months of age or older. the frequency of acute mhs in the current study was 34%, while other studies have reported percentages of 26% [2] and 38% [16]. since serial sections were done in the current study, the possibility of focal hemosiderin deposits along the vessel length, that could elicit mr changes was excluded. the acute mhs were likely agonal as suggested previously [2]. none of the other studies have characterized mhs as subacute and there seems little justification for characterizing all hemosiderin-positive hemorrhages with edema of the neuropil and lack of inflammatory changes as chronic [2, 16]. the histology of chronic mhs observed in the current study was similar to previous reports [1, 2, 11]. although cmbs are generally considered to be markers of focal hemosiderin deposits [1, 11], other vascular pathologies have been observed in the areas of mri hypointensity. two hypointensities observed by susceptibility-weighted imaging were found to be due to a dissection in the wall of a grossly distended vessel and in the second case, a microaneurysm was present [2]. the finding that a distended vessel correlates with a hypointensity explains why the three hypointensities (table 2, case 2 #1 and #7, case 3 #2), observed in the current study were related to a single or several large vessel profiles with luminal blood. in a single study of subjects aged 65 and older, hemosiderin deposits which were considered to be the residua of mhs in the putamen, were associated with putaminal microinfarcts suggesting that cmbs detected by mri may be a surrogate for ischemic pathology rather than exclusively a hemorrhagic diathesis [14]. supporting these findings is the observation that three of the hypointensities (table 2, case 2 #5 and #10, case 3 #9) observed in this study showed additional microinfarcts by microscopy. in a prior study, a pathologic correlate for 38% of cmbs was not detected [1], while in the present study, no pathologic correlate was found for 13% of cmbs. most of the mhs in this study and another large hospital-based autopsy study [27] were located in the white matter or near the grey/white junction where caa is uncommon, suggesting that caa may not be an etiological factor in these white matter mhs. other studies [13, 27, 28] have reported lack of β-amyloid at the site of mhs. however, the findings of focal β-amyloid immunostaining at the bleeding site of one cortical arteriole in the present study, arterioles in a prior study [2] and increased pittsburg compound b retention at the sites of caa-related cmbs [29] do not rule out caa in the etiology of mhs. possibly, alteration of arteriolar morphology by mural β-amyloid deposition, predisposes to vessel disruption and release of β-amyloid into the bloodstream hence the lack of β-amyloid immunostaining in vessels in remote chronic mhs. this concept explains the presence of β-amyloid upstream or downstream of a bleeding vessel but not at the hemorrhage site [28]. vascular risk factors such as hypertension are associated with cmbs in otherwise healthy adults [5, 7, 13, 19] and in adults with cerebrovascular diseases [5]. another vascular risk factor, severe arteriolosclerosis, is reported to correlate with increased numbers of cerebellar mhs [19]. many of these risk factors were present in the 3 cases in the present study as listed in table 1. further studies with larger samples are warranted to determine the interplay of the stated factors and possibly additional factors in the pathogenesis of mhs. cmbs are clinically important in assessing the risk of cognitive impairment and high cmb counts are associated with an increased risk for cognitive deterioration and dementia [30]. another study while agreeing that cmbs are associated with cognitive impairment in the form of poorer executive function and decline in visuospatial ability, did not observe significant differences in incident dementia rates [31]. in both these studies, cmbs were diagnosed by in vivo mri only. the limitations of using only in vivo mri to diagnose cmbs is that mhs of different ages as well as hemorrhagic microinfarcts cannot easily be distinguished. in the present study, ex vivo imaging following by histology facilitated the detection of mhs of different ages and microinfarcts. the limitation of a small sample size in the present study with only 3 cases of alzheimer’s dementia and varying degrees of svd was offset by neuropathology assessment of 35 cmbs by serial sections and prussian blue staining for hemosiderin. hypointensities produced by anatomical landmarks such as the depths of sulci or the entry sites of basal ganglionic vessels can be excluded by making the mri reader aware of these pitfalls. in summary, in spite of a few limitations, these data suggest that ex vivo imaging is useful to supplement routine neuropathology examination for clinical-pathological correlations in relevant autopsy cases and for research purposes. acknowledgements the authors thank the participants of the rush memory and aging project and the staff of rush alzheimer’s disease center. this work was supported by national institute on aging grants r01ag064233, r01ag067482, p30ag010161, p30ag072975 and national disorders and stroke grants uh2-uh3ns100599 and uf1ns100599. references fazekas f, kleinert r, roob g, kleinert g, kapeller p, schmidt r, hartung hp: histopathologic analysis of foci of signal loss on gradient-echo t2*-weighted mr images in patients with spontaneous intracerebral hemorrhage: evidence of microangiopathy-related microbleeds. ajnr am j neuroradiol 1999, 20(4):637-642. schrag m, mcauley g, pomakian j, jiffry a, tung s, mueller c, vinters hv, haacke em, holshouser b, kido d et al: correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem mri study. acta neuropathol 2010, 119(3):291-302. tanaka a, ueno y, nakayama y, takano k, takebayashi s: small chronic hemorrhages and ischemic lesions in association with spontaneous intracerebral hematomas. stroke 1999, 30(8):1637-1642. koennecke hc: cerebral microbleeds on mri: prevalence, associations, and potential clinical implications. neurology 2006, 66(2):165-171. cordonnier c, al-shahi salman r, wardlaw j: spontaneous brain microbleeds: systematic review, subgroup analyses and standards for study design and reporting. brain 2007, 130(pt 8):1988-2003. vernooij mw, van der lugt a, ikram ma, wielopolski pa, niessen wj, hofman a, krestin gp, breteler mm: prevalence and risk factors of cerebral microbleeds: the rotterdam scan study. neurology 2008, 70(14):1208-1214. lu d, liu j, mackinnon ad, tozer dj, markus hs: prevalence and risk factors of cerebral microbleeds: an analysis from the uk biobank. neurology 2021, 97(15):e1493-e1502. akoudad s, portegies ml, koudstaal pj, hofman a, van der lugt a, ikram ma, vernooij mw: cerebral microbleeds are associated with an increased risk of stroke: the rotterdam study. circulation 2015, 132(6):509-516. seo sw, hwa lee b, kim ej, chin j, sun cho y, yoon u, na dl: clinical significance of microbleeds in subcortical vascular dementia. stroke 2007, 38(6):1949-1951. de reuck jl, cordonnier c, deramecourt v, auger f, durieux n, bordet r, maurage ca, leys d, pasquier f: microbleeds in postmortem brains of patients with alzheimer disease: a t2*-weighted gradient-echo 7.0 t magnetic resonance imaging study. alzheimer dis assoc disord 2013, 27(2):162-167. greenberg sm, vernooij mw, cordonnier c, viswanathan a, al-shahi salman r, warach s, launer lj, van buchem ma, breteler mm, microbleed study g: cerebral microbleeds: a guide to detection and interpretation. lancet neurol 2009, 8(2):165-174. kirsch w, mcauley g, holshouser b, petersen f, ayaz m, vinters hv, dickson c, haacke em, britt w, 3rd, larseng j et al: serial susceptibility weighted mri measures brain iron and microbleeds in dementia. j alzheimers dis 2009, 17(3):599-609. fisher m, french s, ji p, kim rc: cerebral microbleeds in the elderly: a pathological analysis. stroke 2010, 41(12):2782-2785. janaway bm, simpson je, hoggard n, highley jr, forster g, drew d, gebril oh, matthews fe, brayne c, wharton sb et al: brain haemosiderin in older people: pathological evidence for an ischaemic origin of magnetic resonance imaging (mri) microbleeds. neuropathol appl neurobiol 2014, 40(3):258-269. tatsumi s, shinohara m, yamamoto t: direct comparison of histology of microbleeds with postmortem mr images: a case report. cerebrovasc dis 2008, 26(2):142-146. van veluw sj, charidimou a, van der kouwe aj, lauer a, reijmer yd, costantino i, gurol me, biessels gj, frosch mp, viswanathan a et al: microbleed and microinfarct detection in amyloid angiopathy: a high-resolution mri-histopathology study. brain 2016, 139(pt 12):3151-3162. cordonnier c: brain microbleeds. pract neurol 2010, 10(2):94-100. de reuck j, deramecourt v, cordonnier c, leys d, pasquier f, maurage ca: prevalence of small cerebral bleeds in patients with a neurodegenerative dementia: a neuropathological study. j neurol sci 2011, 300(1-2):63-66. de reuck jl, deramecourt v, auger f, durieux n, cordonnier c, devos d, defebvre l, moreau c, capparos-lefebvre d, pasquier f et al: the significance of cortical cerebellar microbleeds and microinfarcts in neurodegenerative and cerebrovascular diseases. a post-mortem 7.0-tesla magnetic resonance study with neuropathological correlates. cerebrovasc dis 2015, 39(2):138-143. nag s, barnes ll, yu l, buchman as, bennett da, schneider ja, wilson rs: association of lewy bodies with age-related clinical characteristics in black and white decedents. neurology 2021, 97(8):e825-e835. nag s, yu l, boyle pa, leurgans se, bennett da, schneider ja: tdp-43 pathology in anterior temporal pole cortex in aging and alzheimer's disease. acta neuropathol commun 2018, 6(1):33. yu l, boyle pa, nag s, leurgans s, buchman as, wilson rs, arvanitakis z, farfel jm, de jager pl, bennett da et al: apoe and cerebral amyloid angiopathy in community-dwelling older persons. neurobiol aging 2015, 36(11):2946-2953. alemany ripoll m, stenborg a, sonninen p, terent a, raininko r: detection and appearance of intraparenchymal haematomas of the brain at 1.5 t with spin-echo, flair and ge sequences: poor relationship to the age of the haematoma. neuroradiology 2004, 46(6):435-443. ahn sj, anrather j, nishimura n, schaffer cb: diverse inflammatory response after cerebral microbleeds includes coordinated microglial migration and proliferation. stroke 2018, 49(7):1719-1726. jenkins a, maxwell wl, graham di: experimental intracerebral haematoma in the rat: sequential light microscopical changes. neuropathol appl neurobiol 1989, 15(5):477-486. koeppen ah, dickson ac, mcevoy ja: the cellular reactions to experimental intracerebral hemorrhage. j neurol sci 1995, 134 suppl:102-112. kovari e, charidimou a, herrmann fr, giannakopoulos p, bouras c, gold g: no neuropathological evidence for a direct topographical relation between microbleeds and cerebral amyloid angiopathy. acta neuropathol commun 2015, 3:49. van veluw sj, scherlek aa, freeze wm, ter telgte a, van der kouwe aj, bacskai bj, frosch mp, greenberg sm: different microvascular alterations underlie microbleeds and microinfarcts. ann neurol 2019, 86(2):279-292. dierksen ga, skehan me, khan ma, jeng j, nandigam rn, becker ja, kumar a, neal kl, betensky ra, frosch mp et al: spatial relation between microbleeds and amyloid deposits in amyloid angiopathy. ann neurol 2010, 68(4):545-548. akoudad s, wolters fj, viswanathan a, de bruijn rf, van der lugt a, hofman a, koudstaal pj, ikram ma, vernooij mw: association of cerebral microbleeds with cognitive decline and dementia. jama neurol 2016, 73(8):934-943. paradise m, seruga a, crawford jd, chaganti j, thalamuthu a, kochan na, brodaty h, wen w, sachdev ps: the relationship of cerebral microbleeds to cognition and incident dementia in non-demented older individuals. brain imaging behav 2019, 13(3):750-761. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cerebrovascular disease lesions are additive and tied to vascular risk factors and cognitive impairment feel free to add comments by clicking these icons on the sidebar free neuropathology 3:7 (2022) original paper cerebrovascular disease lesions are additive and tied to vascular risk factors and cognitive impairment john l robinson1, hayley richardson3, sharon x xie1,3, brian alfaro1, nicholas loh1, virginia m-y lee1, edward b lee2, john q trojanowski✝ 1 center for neurodegenerative disease research, department of pathology and laboratory medicine, institute on aging, university of pennsylvania perelman school of medicine, philadelphia, pa, usa. 2 translational neuropathology research laboratory, department of pathology and laboratory medicine, university of pennsylvania perelman school of medicine, philadelphia, pa, usa. 3 department of biostatistics, epidemiology and informatics, university of pennsylvania perelman school of medicine, philadelphia, pa, usa. ✝ passed away on 08 february 2022. corresponding author: edward b. lee · 613a stellar chance laboratories · 422 curie blvd · philadelphia, pa 19104 · usa edward.lee@pennmedicine.upenn.edu submitted: 24 january 2022 accepted: 04 march 2022 copyedited by: lauren walker published: 14 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3792 keywords: cerebrovascular disease, alzheimer’s disease, infarcts, amyloid angiopathy, arteriolosclerosis, vascular risk factors, ageing abstract cerebrovascular lesions are prevalent in late life and frequently co-occur but the relationship to cognitive impairment is complicated by the lack of consensus around which lesions represent hallmark pathologies for vascular impairment, particularly in the presence of alzheimer’s disease (ad). we developed an easily applicable model of cerebrovascular disease (cvd), defined as the presence of two or more lesions: moderate to severe cerebral amyloid angiopathy, moderate to severe arteriolosclerosis, infarcts (large, lacunar, or micro), and/or hemorrhages. ad was defined as intermediate or high ad neuropathologic change. the contribution of vascular risk factors such as atherosclerosis and/or a health history of heart disease, hyperlipidemia, stroke events, diabetes, or hypertension was also assessed. logistic regression analysis reported the association of cvd with increasing age, vascular risk factors, ad, and cognitive impairment in this study of 1,485 autopsied individuals. cerebrovascular lesions were present in 48% and 16% had cvd. increasing age associated with all lesions (p<0.001), except hemorrhages (p=0.41). cvd was more likely in individuals with vascular risk factors or ad (p<0.01). cvd, but not individual cerebrovascular lesions, associated with impairment in cases without ad (p<0.01), but not in cases with ad (p>0.61). from this, we conclude that a simple, additive model of cvd is 1) age and ad-associated, 2) is associated with vascular risk factors, and 3) clinically correlates with cognitive decline independent of ad. introduction alzheimer’s disease (ad) and cerebrovascular disease (cvd) are the two most common causes of late-life dementia (1). ad is best described as the widespread distribution of β-amyloid deposits, tau-positive neurofibrillary tangles, and cortical neuritic plaques as primary pathologies along with the cerebral amyloid angiopathy (caa), lewy bodies, and tdp-43 inclusions also prevalent as secondary pathologies (2,3). cvd is also considered the accumulation of multiple pathologies including infarcts (large, lacunar, and micro), arteriolosclerosis, perivascular space dilation, perivascular hemosiderin leakage, myelin loss, caa (leptomeningeal, parenchymal and capillary), hemorrhages (micro and larger), fibrinoid necrosis, and microaneurysms (4). unfortunately, while consensus criteria exists to determine the level of ad neuropathologic change by the examination of three hallmark pathologies, it is not clear which cerebrovascular lesions relate to impairment and which accumulate due to conditions such as cardiac atherosclerosis, monogenic stroke disorders, embolic disease, vasculopathies, haematological disorders, or metabolic disorders or simply ‘brain ageing’ (4–7). to address this, the vcing study reported seven pathologies that reproducibly correlate with cognitive impairment to varying degrees in a study of 113 individuals (55 to 100 years) including leptomeningeal caa, large infarcts, lacunar infarcts, microinfarcts, arteriolosclerosis, perivascular space dilation and myelin loss (8). a simplified model was proposed highlighting just three cerebrovascular lesions: large infarcts, moderate/severe occipital leptomeningeal caa, and moderate/severe arteriolosclerosis in the occipital white matter. inspired by this attempt at consensus, we screened for three lesions in the occipital cortex – moderate to severe leptomeningeal caa, moderate to severe white matter arteriolosclerosis, and microinfarcts – in a cohort of 1,485 individuals (31 to 103 years old). we then compiled available autopsy data for the presence of moderate to severe caa in other cortical areas, global infarcts (large, lacunar and micro), and large hemorrhages. we describe an additive model of cvd, defined as a presence of multiple cerebrovascular lesions. the goal of this study is to test the hypothesis that our easily replicable, histological definition of cvd associates with increasing age, with cognitive impairment and/or dementia, and with vascular risk factors such as atherosclerosis and/or a health history of heart disease, hyperlipidemia, stroke events, diabetes, or hypertension. materials and methods cohort the cohort consisted of individuals with known ages at death of ≥30 years, and available cerebrovascular and ad pathology measures who participated in the autopsy program at the center for neurodegenerative disease research (cndr) at the university of pennsylvania. of 1,785 individuals age ≥30 at time of death, ad pathology measures were known for 1,659. as of december 2020, cerebrovascular measures were assessed in 1,512 of these individuals. 7 were excluded for having rare clinical diagnoses and 20 were excluded for rare neuropathological diagnoses, resulting in a cohort of 1,485 individuals. clinical diagnoses included no impairment (ni) for individuals without a neurodegenerative disease, ad (probable and possible), amyotrophic lateral sclerosis (suspected, possible, probable, and definite), corticobasal syndrome, cognitive impairment (including amnestic and non-amnestic mild cognitive impairment), frontotemporal degeneration (including behavioral variant, primary progressive aphasias, and not otherwise specified), multiple system atrophy, parkinson’s disease (with and without cognitive impairment), parkinson’s disease dementia or dementia with lewy bodies, progressive supranuclear palsy, schizophrenia, and vascular dementia. the frequency of each is listed in table 1. neuropathologically, the cohort includes neuropathologic ad (intermediate or high level of ad neuropathologic change), low ad (low level of ad neuropathologic change), not ad (including primary age related tauopathy and unremarkable cases), amyotrophic lateral sclerosis, corticobasal degeneration, frontotemporal lobar degeneration, lewy body disease, pick disease, and progressive supranuclear palsy, with the frequency of each is listed in table 2. since our brain bank consists of such a diverse population of neurodegenerative diseases, two subgroups were defined to assess the clinical impact of ad and cerebrovascular pathology (table 3). the ad spectrum subgroup (n=743) is comprised of cases with underlying ad neuropathology (from not ad to high ad) as the primary neuropathological diagnosis regardless of clinical symptoms. the ni to ad subgroup (n=566) is comprised of cases in the ad spectrum subgroup but excludes clinical frontotemporal syndromes and other atypical ad presentations. informed consent for autopsy was obtained in accordance with state laws and protocols approved by the university of pennsylvania. vascular risk factors vascular risk factors included atherosclerosis and/or a history of heart disease, hyperlipidemia, stroke events, diabetes, or hypertension, extracted from health records spanning three decades. heart disease (n=867) included diagnoses of atrial fibrillation, aortic stenosis, cardiac arrhythmia, coronary artery disease, and/or cardiac murmur. stroke events (n=845) included diagnoses of transient ischemic attacks and/or cerebrovascular accidents. hyperlipidemia (n=577) included diagnoses of hyperlipidemia or hypercholesterolemia. diabetes (n=867) and hypertension (n=846) status were also obtained. atherosclerosis was defined as moderate to severe atherosclerosis observable in the circle of willis at autopsy and was available for the majority of cases (n=1,480). 846 cases had enough health history data to define them as having any single risk factor or multiple risk factors. pathology sixteen brain regions are routinely examined in the cndr neuropathology evaluations (9). each region was assigned a semi-quantitative score, i.e. none, rare, mild, moderate, or severe for neurofibrillary tangles based on immunohistochemistry against tau (mouse antibody phf1, a gift from dr. peter davies), or for plaques and caa based on immunohistochemistry against amyloid-β (mouse antibody nab228, generated in cndr). furthermore, arteriolosclerosis scores were determined based on hematoxylin and eosin histology. moderate to severe caa and arteriolosclerosis was assessed as per deramecourt et al (10). all cases were reviewed by a board-certified neuropathologist (jqt and/or ebl) for quality assurance and accurate grading. the level of ad neuropathology was assessed by the amount of tau-positive neurofibrillary tangles, tau-positive neuritic plaques, and amyloid-β positive plaques according to consensus criteria (2). neuropathologic ad was defined as an intermediate or high level of ad neuropathologic change. cerebrovascular lesions were assessed by examination of histological and immunohistochemically stained slides and compiled from post-autopsy gross and microscopic reports and as described below. data on large infarcts were collected from reports detailing infarcts ≥1 cm in diameter visible at autopsy. lacunar infarcts were also determined from records that reported grossly visible infarcts <1 cm in diameter or that were described as lacunar infarcts at time of autopsy. microinfarcts were defined as infarcts <0.5cm in diameter that were not observed grossly and were collected from microscopic reports. in addition, the occipital hematoxylin and eosin slides were examined for all cases for the presence of microinfarcts. hemorrhages refers to large hemorrhages described grossly. arteriosclerosis in the occipital white matter was assessed for all cases. caa severity was primarily assessed in the occipital cortex and was reported as a maximum of scores from three additional neocortical regions if available: middle frontal cortex, superior temporal cortex, and angular gyrus. four neocortical caa scores were available for the majority of cases (n=1,258). statistical analysis r 3.6.2 was used for all regression analyses. univariate analysis was performed to quantify the association between variables. logistic regression analysis was used to build a model of dementia to assess each measure’s contribution. odds ratios and effect sizes for age were assessed for each 5-year increase with confidence intervals reported at 95%. likelihood ratio tests for nested models were used to assess the effect of variables with multiple categories, such as clinical diagnosis. we investigated the effect of missing vascular risk factor data through inverse probability weighting (11). this procedure aims to correct potential bias incurred by considering only subjects with available risk factor data. inverse probability weighting results were very similar to those using available data, illustrating the robustness of results. as such, complete case analyses are presented. unadjusted analysis of cases with cognitive impairment or dementia, and with no cvd, with a single cerebrovascular lesion and with cvd, between participants with and without dementia was performed using pearson’s χ2 tests using two degrees of freedom. all statistical tests were two-sided with statistical significance set at <0.05 level. results full cohort demographics demographic and neuropathological data was available for 1,485 cases (table 3). ni individuals, comprising 9% of the cohort, were without a history of clinical neurodegenerative disease. of the remaining individuals, 51% were clinically late-onset cases with an average age of onset of 64.9 years and an average age of death of 74.5 years. 66% had cognitive impairment or dementia including ad, frontotemporal syndrome, and parkinson’s disease dementia or dementia with lewy bodies (see table 1 for the breakdown by primary clinical diagnosis). pathologically, over ten primary neuropathological diagnoses were represented, with ad and lewy body disease representing the most frequent underlying neurodegenerative diseases (see table 2 for the breakdown by primary neurodegenerative disease). vascular risk factors – such as the presence of moderate to severe atherosclerosis or a health history that included diabetes, heart problems, hypertension, high levels of lipids, or stroke events – were present in 64% of individuals. atherosclerosis, hypertension, and high lipids were the most common risk factors. these risk factors frequently co-occurred. individually, diabetes, heart disease, and stroke had a >90% co-occurrence with other vascular risk factors while atherosclerosis, hypertension, and high lipids had a >60% co-occurrence with other vascular factors. overall, multiple risk factors were present in 35% of individuals. neuropathologic ad, defined as the presence of intermediate or high ad neuropathologic change, occurred in 48% of all cases, representing 28% with ad dementia, 8% with atypical ad clinical phenotypes such as frontotemporal degeneration, 10% of cases as a co-pathology, and 1% of ni cases. fig. 1 representative cerebrovascular lesions moderate to severe (a) leptomeningeal caa was frequently observed in the occipital cortex. (b) severe white matter arteriolosclerosis was not uncommon particularly in the occipital white matter adjacent to the ventricle. (c) infarcts were observed in all subcortical and cortical regions, including this large infarct affecting occipital cortex. scale bar is 100um in (a) and (b) and 1mm in (c). cerebrovascular lesions the presence and severity of cerebrovascular lesions were histologically assessed (figure 1, table 4). the most common lesions affected blood vessels including the presence of moderate to severe caa or arteriolosclerosis which affected 29% and 18% of the overall cohort, respectively. infarcts and hemorrhages were less common, but when present, the majority occurred when concurrent cerebrovascular lesions were described. large infarcts, for instance, were noted in only 5% of all cases, but 86% of cases with large infarcts had other cerebrovascular lesions. similarly, the majority of cerebrovascular lesions co-occurred 52-86% of the time, with only caa occurring with other lesions in a minority of cases 38% of the time. given the preponderance of concurrent cerebrovascular lesions, cvd was defined when more than one lesion was present. overall, cerebrovascular lesions were observed in 48% (n=720) of cases, including 32% (n=482) with a single lesion and 16% (n=238) with multiple lesions. we next assessed, which, if any, neuropathological or clinical groups had elevated frequencies of cvd. as age at death significantly associated with the presence of either single or multiple cerebrovascular lesions (or 1.27, 1.21-1.34, p<0.001), we adjusted for age in our analysis. neuropathologically, the cases with a primary diagnosis of amyotrophic lateral sclerosis, corticobasal degeneration, frontotemporal lobar degeneration, lewy body disease, pick disease, and progressive supranuclear palsy, low ad , and neuropathologic ad were compared to the cases with ‘not ad’ as their primary diagnosis. only neuropathologic ad had an increased prevalence of cvd (or 2.59, 1.44-4.67, p<0.01). other neurodegenerative diseases were not more likely to have cvd (table 2). similarly, clinical ad dementia cases were twice as likely as ni cases to have either single or multiple cerebrovascular lesions (or 2.24, 1.50 3.36, p<0.001). other clinical groups were not more likely to have cvd (table 1) except for corticobasal syndrome cases (or 1.90, 1.05-3.41, p=0.03). since many of the corticobasal syndrome cases were neuropathologically diagnosed as ad, to further assess the impact of clinical diagnoses, we performed a likelihood ratio test comparing the fit of a model including both age and neuropathologic ad to a model including age, neuropathologic ad and clinical diagnoses. in this analysis, the model including clinical diagnoses did not improve the model fit (p=0.33). thus, it appears that only cases with neuropathologic ad had an increased likelihood for cvd. age and ad associations with cvd since an increased incidence of cvd only occurred with neuropathologic ad and the level of ad neuropathologic change was assessed in all cases, we next fit logistic regression models using neuropathologic ad and age as predictors for each cerebrovascular lesion (table 5). in this analysis, increasing age associated with an increasing likelihood of each lesion except hemorrhage (p<0.001) and neuropathologic ad associated with an increasing likelihood of the presence of caa (p<0.001) but not the other cerebrovascular lesions (p>0.07). overall, single cerebrovascular lesions were not age associated (p=0.09), but were neuropathologic ad-associated (p<0.001), while cvd associated with both increasing age and neuropathologic ad (p<0.001). we conclude that infarcts and arteriolosclerosis are more likely with increasing age, while caa is more likely when ad pathology is present, and cvd is associated with both increasing age and neuropathologic ad. vascular risk factors and cvd vascular risk factor information was available for a majority of the cohort (57%; n=846). unsurprisingly, the presence of any or multiple vascular factors increased with age (any risk factor = or 1.48, 1.37-1.59, p<0.01; multiple risk factors = or 1.32, 1.23-1.42, p<0.01). to better understand the role of vascular risk factors in the development of cerebrovascular pathology, we next performed logistic regression analysis incorporating vascular risk factors, age and ad in a model of cvd (table 6). when any vascular risk factors are present, there is an association with cvd (p<0.001) but not single cerebrovascular lesions (p=0.37). vascular risk factors had the strongest association with the presence of infarcts (p<0.001), and also associated with arteriolosclerosis (p<0.01), but did not associate with caa (p=0.55). in these analyses, age consistently associated with all cerebrovascular lesions (p<0.01), while neuropathologic ad strongly associated with caa (p<0.001), arteriolosclerosis (p=0.001), but not infarcts (p=0.23). in all cases, multiple risk factors were not significantly more likely to associate with cerebrovascular lesions in the presence of any risk factor. ad spectrum and ni to ad demographics because cognitive impairment or dementia due to causes other than neuropathologic ad was common in our cohort, we hypothesized that the clinical impact of cvd would only be apparent in cases with ad or cerebrovascular pathology as their primary pathology. these cases are the ad spectrum and ni to ad subgroups (table 3). the ad spectrum subgroup includes approximately half of the full cohort (n=743) including 56% with ad dementia, 17% with atypical dementias and 3% with neuropathologic ad in the absence of cognitive impairment. the ni to ad subgroup (n=566) is the ad spectrum subgroup excluding the atypical ad cases and includes 74% with ad dementia. the ad spectrum and ni to ad groups shared similar demographics. both were older at onset (68.1 and 69.1 respectively) and at death (77.0 and 77.3 respectively) compared to the overall cohort. more women were present in the ad spectrum and ni to ad groups (53% and 54% compared to 44%). cvd was elevated in these groups. cvd was present at 22% and 23% respectively compared to 16% for the overall cohort. the presence of one or more risk factors was also higher in the ad spectrum and ni to ad groups. any vascular risk factor was present at 73% and 77% respectively compared to 64% for the full cohort, while multiple risk factors were present in 46% and 50% respectively compared to 35% for the overall cohort. clinical relevance of cvd we next asked if the presence of cvd associated with cognitive impairment in the ad spectrum and ni to ad subgroups (table 7). in the full cohort, cvd did not correlate with a higher incidence of impairment in cases with (p=0.13) or without (p=0.77) neuropathologic ad. in the ad spectrum subgroup, neuropathologic ad cases with cvd did not differ by their incidence of impairment (p=0.90) compared to neuropathologic ad cases without cvd. however, cases without neuropathologic ad but with cvd were substantially more likely to have impairment than cases without neuropathologic ad or cvd (p<0.001). in the absence of neuropathologic ad, the incidence of impairment was 56% if definite cvd was present, but only 19% if limited cvd was present, and 12% if cvd was absent. a similar result was observed in the ni to ad cohort. neuropathologic ad cases, with and without cvd, did not differ by their incidence of impairment (p=0.61), but cases without neuropathologic ad, but with cvd, were substantially more likely to have impairment than cases without cvd (p<0.01). in the ni to ad cohort, in the absence of neuropathologic ad, the incidence of impairment was 30% if cvd was present, but only 6% if a single cerebrovascular lesion was present, and 5% if no cerebrovascular lesions were observed. discussion that cerebrovascular disease is the second most common cause of dementia after alzheimer’s disease is well known (12), but the term “cerebrovascular disease” has various meanings depending on the clinical, neuroimaging or neuropathological context. even within the field of neuropathology, dozens of lesions have been reported, many of which are dependent on laboratory-specific sampling and histological techniques (4,6). furthermore, while understanding of individual lesions has improved (13–17), the co-occurrence of lesions confounds efforts to correlate the lesion-specific effects with cognitive impairment (8,18). thus, it is important to define a model of cvd that is straightforward and easily reproduced (figure 2). fig. 2 cvd associates with cognitive impairmentthe possibility of either ad (or 1.11, 95% ci 1.02-1.20, p<0.01) or vascular co-morbidities (or 1.46, 95% ci 1.30-1.64, p<0.01) is more likely with increasing age. individual cvd lesions frequently occur when ad or vascular risk factors are present (see tables 3 and 4). vascular risk factors also increase the likelihood of multiple cvd lesions (or 1.94, 95% ci 1.07-3.52, p=0.03) as does ageing itself (or 1.15, 95% ci 1.02-1.30, p = 0.03). multiple cvd lesions (or 2.36, 95% ci 1.23-4.52, p<0.01), but not single lesions (or 0.93, 95% ci 0.54-1.60, p=0.80), associate with cognitive impairment or dementia. logistic regression modelling the effect of age, ad pathology, vascular risk factors and cvd on cognitive impairment for ad spectrum cases with available vascular risk factors (n=421). similar results were obtained when modelling for dementia (not shown). our approach examined six cvd pathologies including moderate/severe cortical caa, moderate/severe white matter arteriolosclerosis, large infarcts, lacunar infarcts, microinfarcts and hemorrhages, and defined a moderate to high level of cvd as the presence of two or more of these pathologies. the vcing study’s model of three pathologies weighted large infarcts twice as strongly as moderate/severe occipital leptomeningeal caa or moderate/severe occipital white matter arteriolosclerosis and defined a moderate to high level of cvd as the presence of either large infarcts, both caa and arteriolosclerosis, or large infarcts with one or both other lesions. since large infarcts were associated with other cvd lesions 86% of the time, our additive model of cvd is functionally similar to the vcing model that defines all cases with large infarcts as having a moderate level of cvd. indeed, the correspondence between the vcing model and our study was high (pearson’s correlation coefficient = 0.803). cases with a moderate or high level of cvd by vcing were also defined as a moderate to high level of cvd by cndr with 91-100% agreement (table 8). however, with the inclusion of additional lesions in our model, many more cases have cerebrovascular pathology than are defined by the vcing model, so 36-44% of our moderate to high level cvd cases would not be captured by the vcing model (table 8). defining cvd as the presence of multiple cerebrovascular lesions correlated well with cognitive impairment in the absence of definite ad, while single cerebrovascular lesions did not (table 7). other studies have also reported that a moderate or high burden of cerebrovascular lesions is a better correlate of cognitive decline than low levels (17,19,20). by age 90, for instance, close to 50% of individuals without dementia have microinfarcts, while just over 50% of individuals with dementia have infarcts (19). however, three or more microinfarcts were present in 26% of those with dementia compared to 7% without, indicating that it is the presence of multiple infarcts that associated with dementia, while one or two were tolerated without concomitant dementia. similar results have been reported for younger cohorts (17,20), suggesting that a more severe cvd burden is a better correlate of impairment. it should also be pointed out that an additive model of cvd is functionally similar to the consensus criteria for ad (2). in ad, an individual with only β-amyloid plaques has a low level of ad neuropathologic change, but an individual with additional tau-positive neurofibrillary tangles who also develop tau-positive cortical neuritic plaques is more likely to have an intermediate or high level of ad neuropathologic change. most older individuals without cognitive impairment have a low level of ad neuropathologic change but most individuals with dementia have a high level of ad neuropathologic change (3). our study attempted to disentangle the contribution of ageing itself or ad pathology to the development of cerebrovascular lesions (table 5). individual lesions such as infarcts, moderate/severe arteriolosclerosis, and moderate/severe caa associated with increasing age, but only moderate/severe caa associated with the presence of ad pathology. however, it should be noted that ad and cvd rarely occur in isolation. not only is the common co-morbid lesion frequency estimated at >90% for caa, ~50% for arteriolosclerosis, >30% for microinfarcts, >10% for larger infarcts and >10% for hemorrhages (18), but both ad and cerebrovascular pathologies may accumulate a decade or even decades before clinical symptoms (21–24). recent imaging studies have even suggested that hypoperfusion (25) and blood brain barrier dysfunction (26) are early events in the development of ad, much as they are speculated to be in the development of cvd. nonetheless, our data is consistent with ageing as a risk factor for infarcts and arteriolosclerosis, and ad as a risk factor for caa. cvd also associated with vascular risk factors in our study (table 6). both cvd and ad are ageing-related diseases, but cerebrovascular lesions such as infarcts or moderate/severe arteriolosclerosis were more likely when co-morbidities such as hypertension, diabetes or atherosclerosis were present. while these associations between vascular risk factors, cerebrovascular pathology, and cognitive impairment are widely assumed to exist, they are rarely reported [7, 11, 18]. there are multiple reasons for this, but the largest confound may be the overlap between risk factors associated with both cvd and ad. indeed, several ‘vascular’ risk factors are also ad risk factors and previous studies have suggested that up to 35% of late-life dementia may be preventable by targeting multiple risk factors such as diabetes, hypertension, obesity, smoking, depression, cognitive inactivity, and physical inactivity (1,27,28). hypertension and diabetes are potentially modifiable risk factors for clinical ad [17], atherosclerosis is increasingly seen as contributing to dementia in a mode similar to ad [28], and vascular risk factors have correlated with increasing ad biomarkers in preclinical ad [3]. in our study, multiple vascular risk factors were often present in the same individual, but, surprisingly, the presence of multiple risk factors was not more likely than the presence of a single risk factor to associate with cvd pathology. the present work is best understood in the context of the strengths and limitations of our study design. for this study, as recommended in the preferred model of the vcing study (8), we screened the occipital cortex for caa, arteriolosclerosis and micro-infarcts. it should be noted that our model is minimalistic, and other models of cerebrovascular disease also deserve closer attention for their relevance to the study of dementia in older adults. strozyk et al examined a 6-point vascular score (involving large infarcts, lacunar infarcts and leukoencephalopathy) that associated with dementia (29); deramecourt et al reported a 20-point vascular score (involving arteriolosclerosis; amyloid angiopathy, perivascular hemosiderin leakage; perivascular space dilation, myelin loss and cortical micro-infarcts and large infarcts in multiple regions) that correlated well with vascular and mixed dementia (10); the newcastle categorization describes cerebrovascular disease as heterogeneous and proposes six subtypes to best describe the presence and distribution of cerebrovascular lesions (30). one obvious limitation is that we graded leptomeningeal, parenchymal and capillary caa as a single cortical caa score when each type of caa may have differential effects in a cvd model of cognitive impairment (32,33). relatedly, reporting infarcts by subtype – i.e. strategic infarcts versus multiple small lacunes versus cortical infarcts – may have revealed which type of infarct most correlated with cognitive impairment (29, 30). the primary benefit of screening the occipital cortex for arteriolosclerosis, micro-infarcts and caa is that it appears to be susceptible to both caa (5) and micro-infarcts (19). nonetheless, the inclusion of additional regions or stains may have also affected the overall picture of cvd in our cohort, as the differences in postmortem sampling and staining methods is a well-known problem in the field (4). in our analysis of vascular risk factors, we describe atherosclerosis in the circle of willis as a risk factor, while other studies report atherosclerosis as a neuropathologic change (23, 31). in fact, both views on atherosclerosis are valid. atherosclerosis as an intracranial pathologic change may be a consequence of other risk factors such as hyperlipidemia, and atherosclerosis as a risk factor may contribute to the intracerebral parenchymal lesions in our study. finally, the majority of cases in our brain bank are composed of individuals with a neurodegenerative disease who were enrolled for brain tissue donation through tertiary referral clinics, and therefore our cohort has a younger age at death than many communityor population-based cohorts, with an overall lower incidence of cerebrovascular lesions than is observed in older cohorts (34). as a consequence, the number of ad-associated cvd lesions is perhaps overrepresented compared to the number of age-associated cvd lesions. in conclusion, we report an additive model of cvd that accounts for the presence of global infarcts, moderate/severe caa, and moderate/severe arteriolosclerosis. when multiple cerebrovascular lesions are present, cvd associates with cognitive impairment or dementia in the absence of other neurodegenerative diseases and, importantly, cvd also associates with vascular risk factors. as a straightforward to apply definition of cvd, our model could be easily reproduced in community‐based cohorts for a more complete understanding of the frequency of cvd in the population. future studies may be better able to disentangle the overlap between risk factors, ageing, and ad pathology with cerebrovascular pathology and thus improve the detection and treatment of vascular cognitive impairment and dementia. author contributions jqt conceived and designed the study. jlr and ebl also contributed to the study design. jlr, ba and nl performed immunohistochemistry and semi-quantitative grading which were verified by jqt and ebl. hr and sxx performed the statistical analysis. jqt, hr, sxx, vmyl, ebl and jlr contributed to data analysis and interpretation. jlr wrote the initial draft of the manuscript, with all authors contributing to the final version. all authors read and approved the final manuscript. potential conflicts of interest ebl is an editorial board member but was not involved in the editorial handling of this manuscript. jlr (no conflict); hr (no conflict); sxx (no conflict); ba ((no conflict), nl (no conflict); vmyl (no conflict); jqt (no conflict) funding us national institute on aging (national institutes of health u19 ag062418, p30 ag010124, pi: jqt; p01 ag017586, pi: vmyl; p01 ag066597, p30 ag072979, pi: ebl). acknowledgements we are indebted to patients and families who donated tissue to the cndr. we also thank all past and current referring neurologists, pathology assistants and pathologists for making this research possible. in addition, we would like to thank theresa schuck for helping us procure and stain all samples. references livingston g, sommerlad a, orgeta v, costafreda sg, huntley j, ames d, et al. dementia prevention, intervention, and care. lancet. 2017 dec;390(10113):2673–734. montine tj, phelps ch, beach tg, bigio eh, cairns nj, dickson dw, et al. national institute on aging-alzheimer’s association guidelines for the neuropathologic assessment of alzheimer’s disease: a practical approach. acta neuropathol. 2012 jan;123(1):1–11. robinson jl, richardson h, xie sx, suh e, van deerlin vm, alfaro b, et al. the development and convergence of co-pathologies in alzheimer’s disease. brain. 2021 apr;144(3):953–62. kalaria rn. neuropathological diagnosis of vascular cognitive impairment and vascular dementia with implications for alzheimer’s disease. acta neuropathol. 2016 may;131(5):659–85. vinters hv, zarow c, borys e, whitman jd, tung s, ellis wg, et al. review: vascular dementia: clinicopathologic and genetic considerations. neuropathol appl neurobiol. 2018 apr;44(3):247–66. pantoni l, sarti c, alafuzoff i, jellinger k, munoz dg, ogata j, et al. postmortem examination of vascular lesions in cognitive impairment: a survey among neuropathological services. stroke. 2006 apr;37(4):1005–9. wardlaw jm, smith c, dichgans m. small vessel disease: mechanisms and clinical implications. lancet neurol. 2019 jul;18(7):684–96. skrobot oa, attems j, esiri m, hortobágyi t, ironside jw, kalaria rn, et al. vascular cognitive impairment neuropathology guidelines (vcing): the contribution of cerebrovascular pathology to cognitive impairment. brain j neurol. 2016 nov;139(11):2957–69. toledo jb, van deerlin vm, lee eb, suh e, baek y, robinson jl, et al. a platform for discovery: the university of pennsylvania integrated neurodegenerative disease biobank. alzheimers dement j alzheimers assoc. 2014 jul;10(4):477–84.e1. deramecourt v, slade jy, oakley ae, perry rh, ince pg, maurage c-a, et al. staging and natural history of cerebrovascular pathology in dementia. neurology. 2012 apr;78(14):1043–50. seaman sr, white ir. review of inverse probability weighting for dealing with missing data. stat methods med res. 2013 jun;22(3):278–95. o’brien jt, thomas a. vascular dementia. lancet. 2015 oct;386(10004):1698–706. blevins bl, vinters hv, love s, wilcock dm, grinberg lt, schneider ja, et al. brain arteriolosclerosis. acta neuropathol. 2021 jan;141(1):1–24. gutierrez j, gil-guevara a, ramaswamy s, derosa j, di tullio mr, cheung k, et al. classification of covert brain infarct subtype and risk of death and vascular events. stroke. 2020 jan;51(1):90–8. chui hc, zheng l, reed br, vinters hv, mack wj. vascular risk factors and alzheimer’s disease: are these risk factors for plaques and tangles or for concomitant vascular pathology that increases the likelihood of dementia? an evidence-based review. alzheimers res ther. 2012 jan;4(1):1–13. cannistraro rj, badi m, eidelman bh, dickson dw, middlebrooks eh, meschia jf. cns small vessel disease: a clinical review. neurology. 2019 jun;92(24):1146–56. kapasi a, leurgans se, james bd, boyle pa, arvanitakis z, nag s, et al. watershed microinfarct pathology and cognition in older persons. neurobiol aging. 2018 oct;70:10–7. attems j, jellinger ka. the overlap between vascular disease and alzheimer’s disease--lessons from pathology. bmc med. 2014 nov;12:206. corrada mm, sonnen ja, kim rc, kawas ch. microinfarcts are common and strongly related to dementia in the oldest-old: the 90+ study. alzheimers dement j alzheimers assoc. 2016 aug;12(8):900–8. sonnen ja, larson eb, crane pk, haneuse s, li g, schellenberg gd, et al. pathological correlates of dementia in a longitudinal, population-based sample of aging. ann neurol. 2007 oct;62(4):406–13. jack cr jr, knopman ds, jagust wj, shaw lm, aisen ps, weiner mw, et al. hypothetical model of dynamic biomarkers of the alzheimer’s pathological cascade. lancet neurol. 2010 jan;9(1):119–28. brundel m, de bresser j, van dillen jj, kappelle lj, biessels gj. cerebral microinfarcts: a systematic review of neuropathological studies. j cereb blood flow metab off j int soc cereb blood flow metab. 2012 mar;32(3):425–36. kapasi a, schneider ja. vascular contributions to cognitive impairment, clinical alzheimer’s disease, and dementia in older persons. biochim biophys acta. 2016 may;1862(5):878–86. tanskanen m, mäkelä m, myllykangas l, notkola i-l, polvikoski t, sulkava r, et al. prevalence and severity of cerebral amyloid angiopathy: a population-based study on very elderly finns (vantaa 85+). neuropathol appl neurobiol. 2012 jun;38(4):329–36. love s, miners js. cerebrovascular disease in ageing and alzheimer’s disease. acta neuropathol. 2016 may;131(5):645–58. montagne a, nation da, sagare ap, barisano g, sweeney md, chakhoyan a, et al. apoe4 leads to blood-brain barrier dysfunction predicting cognitive decline. nature. 2020 may;581(7806):71–6. selby jv, peng t, karter aj, alexander m, sidney s, lian j, et al. high rates of co-occurrence of hypertension, elevated low-density lipoprotein cholesterol, and diabetes mellitus in a large managed care population. am j manag care. 2004 feb;10(2 pt 2):163–70. barnes de, yaffe k. the projected effect of risk factor reduction on alzheimer’s disease prevalence. lancet neurol. 2011 sep;10(9):819–28. strozyk d, dickson dw, lipton rb, et al. contribution of vascular pathology to the clinical expression of dementia. neurobiol aging. 2010 oct;31(10):1710–20. kalaria rn. neuropathological diagnosis of vascular cognitive impairment and vascular dementia with implications for alzheimer’s disease. acta neuropathol. 2016 may;131(5):659–85. yarchoan m, xie sx, kling ma, et al. cerebrovascular atherosclerosis correlates with alzheimer pathology in neurodegenerative dementias. brain j neurol. 2012 dec;135(pt 12):3749–56. thal dr, griffin wst, braak h. parenchymal and vascular abeta-deposition and its effects on the degeneration of neurons and cognition in alzheimer’s disease. j cell mol med. 2008 oct;12(5b):1848–62. graff-radford j, lesnick tg, mielke mm, constantopoulos e, rabinstein aa, przybelski sa, et al. cerebral amyloid angiopathy burden and cerebral microbleeds: pathological evidence for distinct phenotypes. j alzheimers dis jad. 2021 may;81(1):113–22. dodge hh, zhu j, woltjer r, nelson pt, bennett da, cairns nj, et al. risk of incident clinical diagnosis of alzheimer’s disease-type dementia attributable to pathology-confirmed vascular disease. alzheimers dement j alzheimers assoc. 2017 jun;13(6):613–23. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology as a life-task feel free to add comments by clicking these icons on the sidebar free neuropathology 1:29 (2020) reflections neuropathology as a life-task werner jänisch address for correspondence: werner jänisch · heinrich-heine-str. 13 a · 14547 fichtenwalde · germany werner.jaenisch@googlemail.com submitted: 12 september 2020 accepted: 13 october 2020 copyedited by: henry robbert and biswarathan ramani published: 16 october 2020 https://doi.org/10.17879/freeneuropathology-2020-3052 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, personal reflections, gdr, charité 1. prologue neuropathology as a special field in medicine saw its development not until the second half of the twentieth century. its roots are in neurology and pathology. its significance has risen because of the emergence of neurosurgery. the validity of neuropathological diagnoses greatly increased especially because of the application of immunohistochemistry and genetics. this may have been the rationale for the editor of this journal to have neuropathologists’ paths of life delineated for future generations under the heading “reflections.” this includes the various pathways and detours to neuropathology and the ways research findings have been generated, which may eventually prove important or useless. it has been the desire of the editor that authors not only reflect on their professional activities and experiences, but also present themselves as persons. this paper should not be intended as an autobiography, but the development and experiences of the author be presented in appropriate length. i think of this as a good approach, which i will attempt to pursue. 2. social origin and education i was born in 1932 in the german reich, specifically in the industrial city of chemnitz, saxony. my parents married in 1928 and lost employment shortly after due to a hard-hitting economic crisis. even as my father successfully found work as a truck driver the income of my parents was so low and the living conditions so confined that i remained their only child. my childhood was overshadowed by the reign of national socialism in germany. in 1938, i entered the volksschule (i.e., the prevalent type of elementary school at that time). in the beginning, i felt enthusiastic about a young, skilled teacher. when he did not return after the summer break of the year 1939, i and the other kids were very disappointed. the teacher had been drafted as a soldier by the wehrmacht. he was replaced by an old teacher who approached school lessons by using a cane for whipping and punching, thus taking away our joy in studying. in the volksschule we learned how to read, write, basic math and adolf hitler’s curriculum vitae. many classes were replaced by foot drills in the schoolyard. they were directed by a teacher who had suffered a brain injury in the beginning of the war, disqualifying him from military service. by the end of 1943, when i, a mediocre student, had reached sixth grade, classes were suspended in the larger cities of the german reich. this was due to the alleged danger posed by anglo-american air strikes. my father was drafted as an ordinary soldier at the beginning of the war in 1939, finally became an unteroffizier (i.e., roughly on the level of a sergeant) and died as a prisoner of war in a military hospital in september of 1946. my hometown chemnitz was spared from air strikes until the beginning of 1945, but was largely destroyed on march 5, 1945 by anglo-american bomber squadrons. our two-room apartment, which was located in a big apartment complex, was affected by that, as well. my mother and i survived, became homeless and possessed only what we had on our bodies. we were placed in an emergency shelter in the small town of mittweida, 18 km north of chemnitz. in mid-april of 1945, this area was occupied by american troops without a fight. these troops left after about two weeks and a few days later troops of the red army took over the town, because it was part of the designated soviet occupation zone. a soviet military administration had control over this zone. one of its first actions was the order to vaccinate the entire population. this was a life-saving measure because of the aftermath of the war and the refugee streams infectious diseases such as typhus, diphtheria and tuberculosis were spreading out quickly. for me personally there was another order of the soviet military administration that was of great importance: it was said that beginning on october 15, 1945, classes were to resume. this meant that i, because of my age at that point, was able to get into eighth grade of the volksschule. together with 44 other boys, i joined class led by a teacher who was an elevator installer but had been prepared for teaching in a crash course. my knowledge gaps were vast, which was also the case with other students who went through similar experiences as i had. my teacher tried to make up for this with remarkable patience and a lot of empathy. however, it should be said that it helped that most students were very eager to study. our thirst for knowledge was insatiable. i am grateful to this teacher for providing us with a solid base of knowledge and teaching us important basic principles in ethics. unfortunately, eighth grade was the last grade of the volksschule. graduates were instructed in a profession, or they entered professional life without any instruction. as a child, i wanted to become engine driver of a steam locomotive. the requirement for this was the professional instruction in a railroad facility. a few weeks before graduation, i applied for such an apprenticeship and got a rejection. deeply disappointed, i started looking for an alternative. mittweida was part of the textile industry. workers were able to work in spinning mills, weaving mills and sewing rooms without instruction (unlike their superiors). these places also offered no apprenticeships. the only choices i had were to become instructed as a painter, carpenter, metal worker, chimney sweeper, baker or butcher. none of these prospects was desirable. in this situation suddenly a new perspective opened up due to another order of the soviet military administration: it was said that skilled graduates of the volksschule were able to enter the oberschule (roughly equal to high school). until then parents were only granted to put their children into oberschule (which are called gymnasium nowadays) after they had finished fourth grade – but only in case they were able to pay tuition. this was not the case for me. my teacher made arrangements so that i could continue my studies without having to pay tuition! my mother and i were thrilled. i initially joined a class for students who had to adapt, which means that lessons were conceptualized as to make sure students could gain the knowledge the other students had been building on since fifth grade of the oberschule. i was an enthusiastic student. latin, biology, chemistry, history, german literature and russian became my favorite subjects. math was not one of those, although we had a good teacher. i also encountered problems in english. i was not aware that english words were pronounced differently as could be assumed from the way they were written (for example, it was not clear to me how to pronounce the “u” in a word like “must” the way it sounds in english as opposed to a “u” in german). the english teacher, a former protestant minister, never imparted this knowledge to us. hence, i eagerly studied the vocabulary from the book but still had bad grades. after a couple of months the teacher left the school, so english lessons were suspended because there were not many english teachers left in the soviet occupation zone. because unemployment was widespread in the other occupation zones in the years 1946–1948, workers came to the soviet occupation zone, including english teachers. one of them came to our school and with him his unconventional methods. we have been studying walter scott’s novel “ivanhoe” for a full school year. he distributed copies of the novel’s german translation, told us to read it chapter by chapter, and demanded summaries and interpretations of its content in german. this became the basis for our grades. this way i was able to study an important work of english literature and got good grades. however, i did not learn a single word of english and i was aware of this. i became friend with a student of a higher grade who had english classes prior to 1945. he agreed to teach me some of his knowledge. this way i was able to improve my pronunciation of the vocabulary and learn some basic grammar. but still my english skills remained utterly mediocre. i was able to read and understand simple texts, but i was lacking an active command of the language. because i took great joy in studying, my time in the oberschule went by in a flash. a few months before the abitur (i.e., the high school diploma), we were asked to decide if we planned to continue studying, which subject we wanted to major in, and at which university we would try to apply. since i had been an eager student of biology in school and extended my knowledge reading zoological books and spending time in nature, i knew early that i wanted to study biology. but suddenly doubts arose: what would i do if biology would not meet my expectations, if chemistry and physics were more interesting to me? this led to the idea of studying medicine. it includes morphological subjects, microbiology, genetics, biochemistry, pharmacology, applied radiophysics and i could choose which scientific area i wanted to pursue later in my career! the next question was concerning the place i wanted to study. then, in the gdr (german democratic republic, ddr), there were medical schools at the universities in berlin, leipzig, jena, rostock and greifswald. because of my interest in biology i was leaning towards jena early on, even though i had never visited the city. the reputation of the biologist ernst haeckel and the anatomist christian loder made that university so attractive to me, which was why i applied to this school. since i graduated from the oberschule with the final grade “sehr gut” (i.e., the best overall grade) in 1951, i was promptly enrolled as a student of medicine and received a monthly stipend of 260 mark. it should be noted that in the gdr every student could apply for a scholarship, which was not a loan and did not have to be paid back later. the sum of the stipend was dependent on the final grade in high school and the grades a student got at the university and could vary between 180 and 260 mark. the sum of the stipend i received was sizable for me personally (my mother, who was full-time employed, earned much less than that). it should be noted that i paid my landlady 20 mark per month, that 1 kg of bread cost 30 pfennige (pennies) and one meal in the dining hall 70 pfennige. after my first year i completed my exams in physics, zoology and general botany with the best grade (“sehr gut”) and was ordered to the dean’s office. i was offered the possibility to continue my studies at a university in the soviet union. i was selected because of my academic performance so far, and my high school grade in russian. i asked for some time to think because i wanted my mother to agree. this was necessary, because she would be by herself for a long time if i went to study abroad. studying in jena, i was able to visit her for a weekend every two months. without any hesitation she recommended me to take the offer. so in october of 1952 i went to moscow with about 200 other students of various disciplines and from there went to leningrad to study medicine. there were three major civil medical schools with different instructional foci and a military medical school. the different foci of the schools can be explained by the fact that most graduates are sent to different parts of the country without receiving a specialist medical instruction. in remote places of the country (siberia, north of the polar circle, etc.) it was not uncommon for them to be the solely responsible physician. there was no telemedicine back then. i arrived at a medical school for future pediatricians. today this is the saint petersburg academy of pediatrics and maternity. its students came from all republics of the soviet union and many foreign countries. all special areas of medicine were taught, as was the case in german universities as well. what was special about this medical school was that there was a special focus on children. this concerned infectious diseases, nutritional disorders, and skin diseases, among others. but this also concerned theoretical subjects such as physiology, pathology (pediatric pathology was taught very extensively) and even forensic medicine. the instruction of the students took top priority, was very thorough and frequently individualized. interested students were offered “academic student circles” in different special areas and could thus learn about and participate in professors’ research activities. i took part in this as well, first in anatomy and later in pathology. in 1958, my studies concluded with a state exam “with distinction” (“ausgezeichnet”), and i returned to the gdr. fig. 1: in the reading hall of the dormitory in 1953. 3. memories of leningrad after arriving in leningrad, i was lodged in a dorm room. the dormitory’s construction began before world war ii and was finished after the end of the war, partly with the help of german prisoners of war. it was clean and cozy. on the ground floor there was a library with a reading room offering literature relevant for studies and the belles lettres that could be borrowed and studied (fig. 1). all newly arrived foreign students could take part in a tour of the city. since the city underwent restorations shortly before, the historical buildings were shiny and the parks well maintained. we were shown the memorials of tsar peter the great and tsar catherine the great and various cultural sites. i used to go to the theater frequently to enjoy the works of pushkin and chekhov in the beauty of their language, to opera performances and the ballet of leningrad, and occasionally, symphonies, as well. the latter was directed by kurt sanderling, who during the reign of national socialism emigrated to the soviet union and later acted as chief conductor in the gdr. leningrad presented itself as a vibrant big city with a dense net of buses, trolleybuses, and streetcars. back then underground railways did not exist yet. as a german student i was warmly welcomed and offered help by the university and the general population. never was i or, as far as i know, any other students from the gdr blamed for the drastic crimes that happened during the german blockade of leningrad. we received a monthly stipend of 800 rubles. (later the ruble, and the stipends, was devalued 10 to 1.) the dorm room cost 15 rubles per month. it should be noted that the monthly salary of our professors were between 1,200 and 1,600 rubles. hence, we could dedicate ourselves to our studies without having any material worries. my fellow students welcomed me with friendliness. they were eager to get to know how we were living in germany and how we experienced the war. the first question i was asked was: “is hitler still alive?” because of my imperfect command of the russian language, i thought i misunderstood that question, because for us germans it was clear without a doubt that hitler was dead. until some back and forth it became clear to me that even in 1952 the people still worried that hitler might have escaped and could become dangerous again. i asked many questions, too, and got to know a lot about the reality of everyday life and life during the war. successively i learned to understand the russian people’s way of thinking. one example from the first days of my stay might make this clear: i asked a russian student, whom i shared a dorm room with the question: “valentin, where is it you are from?” he replied: “prigorod”, which translates to suburb, i.e. close to leningrad. later i found out that the distance between the two places was 800 km. i got further insight when i was invited to the homes of university staff. i was introduced to russian hospitality and their difficult living conditions. one of the families, which consisted of two parents and two small daughters, lived in a room of about 40 m² in what was called “communal quarter”. these were large apartments consisting of eight or nine rooms in houses spanning four stories constructed during the reign of the tsar. because of the scarcity of housing, these large apartments were divided and rented roomwise. the former kitchen that was accessible to all families was equipped with a big boiling pot for everyone and a small gas stove for each family. at the apartment’s entrance, there was a doorbell and a list with the family names of the renters accompanied by a number of ringtones. my hosts had the number seven. when the ringtone occurred, somebody in the family answered the door. during my stay with them and during intense conversations the doorbell rang frequently. when i said, “this is for you” they replied, calmly, “no, it was only six times” and continued talking to each other. i mention this because this situation was unusual to me and gloomy, but they showed to me that there are more important things, namely practicing hospitality, which they did extensively. this had an influence on me, so for my wife and me it was clear that we were hospitable to visiting students and fellow researchers, regardless from which country they came. i also feel deep gratitude for my professors and their assistants. they trained me how to do my first pathological/anatomical dissections. they introduced me to the great public library of leningrad and showed me how to navigate the scientific catalogs. with their support i learned how to make microscopic preparations and use for my first academic publications. saying farewell to leningrad was hard for me. 4. becoming a pathologist and my journey to neuropathology in medical school i was especially interested in morphological subjects: first anatomy, then pathology. as a student i conducted more than 100 autopsies by myself, as well as writing the dissection reports and participating in clinicopathological conferences. this was an occasion for me to delve deep into scientific literature. for example, performing a postmortem of a patient with an oligodendroglioma motivated me to buy and study k. j. zülch’s book on brain tumors (23) in 1957. back then, i was already intent on becoming active as a physician in the field of pathological anatomy. it was my desire to get instructed in this field at a university institute. in order to be granted a full medical license in germany, one had to have practiced as an assistant in a clinical subject for at least one year. therefore, in 1958 i applied to the medical academy erfurt to be instructed for six months each in the clinics for internal medicine and surgery. in the clinic for surgery i was working in the department of neurosurgery, which was headed by professor usbeck. the department mostly treated patients with brain tumors and trigeminal neuralgia. at the end of this training professor usbeck offered me to train me as a neurosurgeon. i declined by explaining that i had decided to receive my specialized training at the institute for pathological anatomy of the medical academy erfurt. to this, he responded: “then go ahead and take care of histopathological diagnostics of glioma. almost every diagnosis i receive from the pathologists is astrocytoma. this cannot be right, because since baily and cushing (1) we know more about glioma and need exact diagnoses for our treatment strategies.” i took this suggestion seriously. first, because i knew from my own experience that at most german facilities for pathological anatomy – regardless if university or municipal – neuropathological questions did not get much attention. it was mostly neurologists and psychiatrists who were dealing with neuropathology. secondly, i had realized that the field of pathology was so extensive and diverse that it was not possible to gain profound knowledge in every subfield. furthermore, i desired to do research. therefore i decided to pay special attention to the area of neuropathology during my training in pathology. this began with in-depth studies of the literature. the institute for pathology at the medical academy erfurt, founded in 1954, had a well-equipped special library. also, the academy had a central library, which allowed to order any missing scientific literature from all major libraries in the country and abroad for free. thus, there was nothing in the way of further specialization. however, i needed professor harry güthert to agree to my endeavors (fig. 2). he became prosector of the municipal clinic in erfurt in 1946. he became the director of the newly built institute for pathology when the medical academy erfurt was founded in 1954. the institute also had a large barn for test animals. the institute’s staff comprised about 40 people, including two chief physicians (oberärzte) and nine assistant physicians (assistenzärzte). about six months after starting this activity i suggested to the director that i wanted to specialize in neuropathology. after a couple of minutes of thinking he replied: “agreed. i will send at the university of leipzig for a couple of weeks. there you can become familiar with the basics of neuropathology and study histopathological preparations.” he had his secretary immediately connect him to the director of that institute to arrange a date for my visit. i used to see again and again that this kind of rapid and focused reaction was typical of professor güthert’s style of leadership: when something was suggested to him that furthered the development of the staff and the institute he got behind those suggestions with all means that were at his disposal. fig. 2: professor harry güthert, director of the institute of pathological anatomy of the medical academy erfurt in the year of his retirement. after returning from this stay i suggested to open a neuropathological laboratory at the institute for pathology in erfurt. in leipzig i was able to get to know the advantages of microscopic large sections for brain research and the meticulous photographic documentation of medical findings. therefore, i asked professor güthert to acquire a microtome for large sections. back then in germany, the only producer of such devices was the company jung in heidelberg. the devices and the necessary equipment for preparing paraffin sections were rather expensive. since the universities in the gdr were wholly financed by the state, the necessary foreign currency (west-german deutsche mark) had to be provided by the ministry of education. this did not pose a major challenge for the director, either. in the fall of 1960, he sent me to heidelberg to the company jung so i could select and order a device tailored to our needs. furthermore, he arranged for me to get to know how to use the device at the institute of neuropathology at the university of gießen during the same trip. he also granted me the support of a photographer (who was on the institute’s staff) whenever i needed informative macro shots of neuropathological findings to be produced with a plate camera. hence, already at the beginning of my neuropathological activities, scientifically valuable documentations of medical findings could be generated in black and white, which i could later use for publications in books and journals. back then, many diseases of the central nervous system (e.g., tumors, aneurysms, abscesses) were not surgically operated, because the diagnostics and, in part, surgical procedures had not been developed well enough (there were no special imaging techniques except for pneumencephalography and arteriography, and no microsurgical methods). hence, at that time pathological findings of the brain without iatrogenic changes were not rare. furthermore, in the gdr there was an obligation to autopsy people who died with suspected or evidenced malignant tumors, infectious diseases, stillborn and babies, people who died without explainable cause of death, suspected unnatural death, or when physicians had a scientific interest in the autopsy findings. hence, autopsies could be conducted relatively often. for example, the staff of the institute for pathology at the medical academy erfurt conducted between 2,000 and 3,000 autopsies annually. the share of autopsies with neuropathological findings was accordingly large. i did not conduct every single of these neuropathologically relevant autopsies; however, the decision to photographically document the findings and the diagnostic treatment of neuropathological findings was reserved for me. this way, after a very short period of time i had a relatively large, diverse wealth of material at my disposal, which could be used for in-depth scientific analysis and the training of students. i was involved in the latter fairly early, first in courses and seminars, then in lectures on general pathology (tumors, inflammations) and neuropathology, as well. this material was also used in the regular clinico-neuropathological conferences with neurosurgeons and neurologists. my neuropathological training benefitted from the fact that professor usbeck regularly invited specialists from the federal republic of germany and other european countries, among them neuropathologists, to an annual symposium on neurosurgical issues. this way i got in touch with dr. jürgen peiffer in 1961 (later director of the institute for brain research at the university of tübingen), with whom i discussed subject-specific and societal problems. it has been decisive for my training as a pathologist that professor güthert made it possible for his staff to acquire knowledge outside the gdr. in 1961 i was allowed to take part in the annual conference of the german society of pathology in münster, which was succeeded by a two-week visit at the department of neuropathology at the university hospital hamburg-eppendorf. this was followed by multi-week study trips to university hospitals for nervous diseases in pécs (headed by the renowned neurologist, neurosurgeon and neuropathologist professor környey) and to the institute for pathology at charles university in prague with professor bednar, who also was a renowned researcher in the area of neuropathology. furthermore, i was offered the opportunity to take part with a convention of polish neuropathologists in krakow, as well as an international convention of neuropathologists in zurich. all these trips were funded by the state. this way i was able to acquire special knowledge and establish professional contacts to researchers abroad and neuropathologists active in the federal republic of germany. this led me, with dr. schnabel from the medical academy magdeburg and other interested physicians and scientists in the gdr, to found our own society of neuropathology. the history of the founding and activities of this society is subject of a doctoral dissertation, which was successfully defended by antonia stahl at the charité berlin in 2017. 5. neuropathological research endeavors my specific scientific interest was focused on tumors of the central nervous system, especially concerning their etiology. therefore questions about the experimental induction and the epidemiology of these tumors became the focus of my life’s work. 5.1 experimental neurooncology at the beginning of my engagement with the neuropathological literature i encountered statements about the etiology of glioma that i had doubts about. from general pathology i knew that the following factors may be responsible for the emergence of malignant tumors: ionizing radiation, chemical carcinogens, some virus types and chronic inflammation. the relevant publications said that this cannot apply to brain tumors, because the brain is protected by cranial bone from ionizing radiation, and that chemical carcinogens cannot reach the brain because of the blood-brain barrier. also, there were no cases documented where the emergence of brain tumors could be attributed to sequelae of chronic inflammation or trauma (2). instead, the hypothesis was that genetically determined hereditary dispositions were the main causal factor for the emergence of neuroectodermal tumors in the central nervous system. a prominent advocate of this hypothesis was berthold ostertag in his publications from 1936 and 1941. in that period of time in the german reich, forced sterilization of people with “sick genes” was conducted for ideological reasons. i had doubts about the validity of these arguments, which led to the decision to contribute to the solution of these etiological problems through intense studies of the literature and my own experimental research. as early as 1961 i was able to get dr. schreiber (fig. 3), an assistant physician who had just arrived at the institute, interested in neuropathology and research endeavors. the first result of our cooperation, which endured for decades, was the monography “experimental tumors of the central nervous system,” published in 1969 by veb gustav fischer verlag jena (7). its subtitle is “induction, morphology, transplantation and application,” which shows the range of its content. our aim was to consider all publications on the topic in the world, even if they were hard to access or not in one of the world languages. these studies of the literature offered various inspirations for our own subsequent examinations, which we critically discussed. soon the effort paid out. we were requested to have the book translated to english and published in the u.s. the publisher and us, the authors, agreed. professors d. d. bigner and j. a. swenburg arranged the translation and supplemented the text with new insights, especially in the area of viral tumor induction (8). our work on the topic continued when i was appointed full professor (ordinarius) for pathology at martin luther university in halle/wittenberg in 1970, where dr. schreiber followed with me as a docent. conducting further experiments, we were able to show that tumors in the central nervous system could be induced by nitroso compounds in various animal species regularly and in significant percentages, whereas other animal species turned out to be resistant. we were concerned with the question which of these two groups humans belong to, because if there were evidence showing that we are vulnerable, preventive measures would have to be introduced, for example regarding work life or through modifications of our eating habits. it goes without saying that experiments with humans are impossible, so we included rhesus monkeys into our research. it became apparent that intravenous injections of different doses of alkyl nitroso compounds did not lead to the emergence of tumors. this applied to adult, as well as to newborn animals. these results on rhesus monkeys could be regarded as indication, but not as evidence of a resistance of the human central nervous system against these substances. following this, we started looking for chemical differences between the brains of the different species. this work was conducted in a research group, which included dr. rath and dr. felicetti from the institute for pathology in halle. it showed that especially one enzyme, the zinc-activated acid phosphatase, is present in the normal brain tissue of different animal species in different quantities: in the brain of species where neuroectodermal tumors could be induced by alkyl nitroso compounds, it was high. in the brain of species that did not develop brain tumors, it was missing. in addition, this enzyme could not be found in human autopsy or biopsy brain tissue. we concluded that the danger of brain tumor induction by alkyl nitroso compounds is probably low for humans. however, this does not mean that other chemical substances, which have not been identified yet, could cause primary brain tumors in humans. fig. 3: professor dieter schreiber (second from left) in a seminar in madrid, 1980. 5.2 primary tumors of the central nervous system in fetuses and babies animal experiments showed that tumors in the central nervous system could also be induced transplacentally. this shifted our interest to brain tumors in human fetuses and babies. we derived from studies of the literature documenting that case studies existed, but that systematic examinations on this topic were yet missing. hence, we initiated a collection and analysis of as much material as possible. the aim was to find out which primary tumors occurred in this age group and which symptoms they exhibited. there were good conditions for this kind of research in the gdr: first, a mandatory national tumor register had been existing since 1952. all physicians were legally obligated to report suspected and evidenced malignant tumors. in 1956, this duty to report was extended to all tumors of the central nervous system, regardless of grade of malignancy. second, in the gdr there were special consulting centers for all pregnant women and women with babies. through these, all medical records on the pregnancy and birth including its development and diseases were accessible. third, autopsy of stillborns and dead babies was mandatory. this way we could access all necessary information and request pathology reports and microscopic preparations. additionally, our colleagues from abroad reported relevant cases to us and provided us with material to be examined. the support of this endeavor by our colleagues in poland and czechoslovakia deserves special thanks. on this basis dr. schreiber, dr. gerlach and i authored the monography “tumors of the central nervous system in fetuses and babies” (9). as a supplement to the morphological records, we obtained records on the development of the newborns and babies. we aimed to identify events that might have led to transplacental tumor inductions, and to detect the early symptoms of tumors in the central nervous system of newborns and babies. we intended to include this knowledge into the (continued) training of physicians. we achieved this (13), but the search for transplacental causes for tumors was suspended in 1990. the central tumor register of the gdr was not continued, and with it our access to new data. further inquiries into the data already collected was foiled by the changes in my professional career, leaving the emerging findings hypothetical and unpublished. 5.3 epidemiology of primary tumors of the central nervous system the fundamental works on the pathology of tumors of the central nervous system by zülch (23), folke henschen (2), and russell and rubinstein (19) give hints on the age and gender distribution of different types of tumors. these are composed of summaries of their own analyses and those of other researchers. however, they do not allow conclusions about the actual frequency in the population; they are not epidemiological findings. an epidemiology of tumors can only be realized if all cases in a predefined population group are recorded over time. statistics on the material analyzed in some institutions for pathology or some clinics do not amount to epidemiological findings. the existence of the central tumor register of the gdr described above provided us with the opportunity to generate epidemiological findings on primary tumors of the central nervous system in a population of 17 million people. with the help of dr. joanna haas, a well-trained epidemiologist from the u.s., and other staff from the tumor register we were able to generate robust findings. these were published in single publications (5, 10), as well as in books (9, 11). 5.4 neuroectodermal stem cell tumors studying tumors of the central nervous system of children sparked a scientific interest in a group of tumors that i summed up using the term “neuroectodermal stem cell tumors.” hart and earle (6) called them “primitive neuroectodermal tumors” (pnets). the term “stem cell tumor” is based on the idea that its cells are at the start of a process that will spawn highly differentiated cells through cell maturation. such maturation processes can be morphologically demonstrated (15). the cell maturation in stem cell tumors (neuroepithelioma, neuroblastoma, medulloblastoma, medullomyoblastoma etc.) can lead to a decrease of tumor growth and in some cases even to spontaneous recovery of the patients (11, 22). this has been seen with neuroblastoma in the sympathetic nervous system and very rarely also with stem cell tumors in the central nervous system. in many cases, the differentiation process remains on a low level and only gets to part of the tumor tissue, hence having no effect on the course of the disease without improving the prognosis. furthermore, together with my staff, i intended to research how these maturation processes are controlled, and to find out which therapeutic possibilities might emerge from this. this research produced first results, but had to be suspended in 1994, leaving the intended tasks unfinished. it is my hope that the next generation of researchers will take up this topic and perhaps win the nobel prize in medicine. fig. 4: participants of the international symposium on brain tumors in madrid, 1990. fig. 5: with professor paul kleihues at the rudolf virchow haus of the charité in berlin, 1991. 6. the importance of medical societies and personal contacts it is essential for every physician and professor involved in scientific research to keep up with research findings and the growth of knowledge in the field. this information can be learned from scientific writing, conventions and personal contacts. as a member of the international society of neuropathology i took part in several of the meetings and was able to gain many valuable inspirations for my own work; this was also the case for the congresses of the european confederation of neuropathological societies (euro-cns). i have been interested in informative presentations and panel discussions, as well as in the opportunity to get to know and discuss specific problems with renowned and next generation researchers from abroad. for example, at the 1970 international congress of neuropathology in paris, i met the well-known veterinary pathologist professor fankhauser, co-author of a work on comparative neuropathology (3). i used this opportunity to learn something from him about brain tumors in animals. he then invited me to a multi-week stay in bern where i had the chance to study his extensive collection of histological preparations of brain tumors of animals. there i also discussed problems of comparative neuropathology with him and professor frauchiger. but the exchange of scientific experiences and personal contacts were not limited to international events (fig. 4); the conventions of national societies of neuropathology and symposia proved valuable, as well. in the gdr, there were the annual conventions of the society of neuropathology and symposia with international attendance (20). the proceedings of these meetings were usually published in the journal “zentralblatt für allgemeine pathologie und pathologische anatomie” (21). several of these events were dedicated to tumors of the central nervous system. first they took place in erfurt, then in halle/saale. they offered neuropathologists in the gdr the chance to personally get in touch with researchers from many european countries and countries outside of europe. among the frequent guests were professor zülch, professor wechsler, professor kleihues (fig. 5), professor mennel from the federal republic of germany, professor cervos-navarro from west berlin, professor jellinger from vienna, professor mossakowski from warsaw, and dr. jablonowskaja from moscow, for instance. in exchange, my staff and i were invited to the national neuropathological events in their home countries. sometimes there were invitations from researchers that i had not met before. professor katsuo ogawa (fig. 6) from japan invited me together with lucy rorke (philadelphia) and umberto cravioto (new york) to the convention of the japanese society of neuropathology in okayama in 1985 (fig. 7). that society had more than 900 members back then (the society of neuropathology in the gdr had 34 members.) after that convention i had been invited to talks at different universities and research facilities in japan, among them tokyo, niigata, maebashi, nagasaki, and hiroshima. from this, professional and familial contacts with japanese pathologists and neuropathologists emerged. in 1992, on the occasion of the congress of neuropathology in niigata, my wife and i were invited to the house of professor ikuta (fig. 8), where we enjoyed japanese hospitality together with professor hirano (fig. 9). then again, my wife and i hosted many peers when they stayed in our hometown (figures 10 and 11). fig. 6: with professor katsuo ogawa in okayama, 1985. fig. 7: discussion with professor lucy b. rorke in okayama, 1985. 7. professorship in iraq in 1967, the government of the republic of iraq headed by president aref asked the government of the gdr to name qualified professors to be temporarily sent to work at iraqi universities. this was an unusual step, because at this time the gdr had not been internationally acknowledged as a sovereign state. the claim to sole representation by the federal republic of germany (hallstein doctrine) was still in place. it said that the federal republic of germany is the only successor state of the german reich and that foreign countries can have diplomatic relations to no other german state. any violations of this doctrine would be met with extensive sanctions, including the suspension of diplomatic relations and drastic trade restrictions. the only countries excluded from the hallstein doctrine were those in the socialist realm, which at this point already had the usual diplomatic and other intergovernmental relations with both the gdr and the federal republic of germany in place. the ministry of higher education of the gdr announced the iraqi government’s request to the country’s professors. i learned about this and expressed my interest in such an activity, however stating that my english skills at that point would not be sufficient for such an appointment. hence, i was invited to a language course that was offered in small groups for three months under the instruction of experienced simultaneous translators, which turned out to be very effective. in may of 1968, i flew to baghdad with my wife and kids. we were welcomed by the trade mission of the gdr. i was appointed to act as director of the chair for pathology at the university of mosul. the trade mission had rented a spacious house on the outskirts of the city, not far from ninive. since it was so far away from the medical school, the trade mission provided me with a car of the soviet brand lada. the advantage of this car was that it was very sturdy, and therefore always ready to be used in the summer heat and resistant to driving on desert roads. since this car was the only one of its type in the whole city everyone knew who was coming along when i was going somewhere in it. despite the lack of any traffic rules, i was never involved in an accident. the department of pathology was housed on the fourth floor of a new building for theoretical medical disciplines and had four large rooms and its own lecture hall for 150 students, but no room for conducting autopsies. the staff consisted of an assistant of kurdish nationality who had studied veterinary medicine, an english-speaking manager and four male staffers who only spoke in arabic. one of them was able to prepare paraffin sections and preparations with the usual aniline colorations. the other three assisted by fetching things, cleaning up and making tea. in an adjacent chamber, there was an electron microscope, which had been ordered by one of my predecessors, and it was still unused. there were no tools available for ultrathin sectioning. for the training of the students there were some commercial pathological/anatomical preparations of organs and histological preparations which had been purchased in england. these had been used in classes by the assistant physician. doing lectures was aided by a blackboard with chalk and a slide projector, but there were no lantern slides. fortunately, i had brought a large chunk of my collection of diapositives pertaining to general and specific pathology, which could be used to visualize the lectures. additionally, i demonstrated pathological findings to the students using native organ parts, which came from surgical operations. among them, echinococcus cysts from the dog tapeworm were frequent, which had been removed by surgeons from various regions of the body such as the liver, kidney and even from the ear lobe. echinococcus was a frequent disease in mosul, which was due to the breeding of sheep and the many stray dogs. there were no neurosurgeons active in mosul. therefore, i could demonstrate neuropathological findings to the students only in the autopsy room. however, the problem was that patients who had died did not undergo autopsy in the university hospitals. people who had died were usually picked up by their relatives shortly after death and buried before dusk. there was a small, simply equipped autopsy room in an adjacent building on the clinic’s premises, but this had only been used by forensic doctors. these autopsies had been ordered by the court and conducted against the will of the dead person’s relatives. the chair of forensic medicine was held by an iraqi who had been trained in england. i got in touch with him and we arranged that i would conduct autopsies if the circumstances of the death would allow it. in addition, there was a military hospital in mosul. they also sometimes requested me to do autopsies. every time, the building where the autopsy took place was guarded by the military in order to prevent relatives from entering the room, taking the dead person with them, or altercations. it was a problem for me that i could not schedule autopsies for demonstrations with the students, because they had to happen immediately. we solved this problem by sending out my staffers into the clinic building and informing randomly encountered students that there was going to be an autopsy in an hour. this information circulated quickly among the students, leading to crowds in the autopsy room. this way i was able to conduct and analyze more than 20 autopsies together with the students during my work at the university of mosul. among them there were heart attacks, fatal lung embolisms, but also neuropathological causes of death, such as hypertensive cerebral hemorrhage or a large tuberculoma in the cerebellum. i would like to stress that the students (nine female and 152 male) were disciplined, curious and grateful. fig. 8: break-time talk with professor ikuta, director of the institute for brain research at the university of niigata during the 33rd convention of the japanese society of neuropathology, 1992. fig. 9: staying at professor ikuta’s house. standing between my wife and me: mrs. ikuta and professor hirano (new york). 8. deputy health minister and who consultant back then, the minister of health of the gdr was a physician by the name of professor ludwig mecklinger. he had five deputies. in 1978, the government of the gdr was informed that the soviet union would meet its wish to drastically increase oil deliveries. energy generation through the unprofitable and environmentally harmful combustion of lignite could then be abandoned, leading to massive financial gains for the state. the government, headed by prime minister willy stoph, planned to allocate a large amount of these gains to medical research. mecklinger was ordered to lay out plans for this. he asked for a sixth deputy health minister whose main job would be the further development of medical research in the gdr. he wanted somebody who was a scientist himself and was experienced enough to evaluate research endeavors. this is how my name came into play. i declined to take this job because we were making great progress with our research at the department of pathology in halle. i also stated that i was lacking the necessary expertise in some fields of medicine. i was then assured that a “council for medical research” was to be founded and that renowned scientists from all fields of medicine would constitute this council in order to consult me. this reasoning as well as the significance of medical research for the further development of the health care system of the gdr convinced me to accept the assignment in 1979. i was not influenced by material incentives because even the salary of the minister of health of the gdr was below that of a university institute director. we started working, but by the end of 1980 were informed that the soviet union would eventually not deliver the amount of oil they had promised before. the power plants, which in the meantime had been adapted to the combustion of oil, had to be quickly reconfigured back to the combustion of lignite. this cost a lot of money, leading to not more but less investments in medical research. mecklinger was a smart and warmhearted person. he accepted that my position was not necessary anymore and let me leave the ministry in order to return to the university. this was how i came to the charité berlin in 1983. the “council for medical research” continued to exist and i belonged to it until its dissolution by the last minister of health, professor kleditzsch, in 1990. from 1986 to 1990, i was also acting as consultant to the who, where i was involved in the revision of the international classification of diseases (icd-10). therefore, i spent multiple months per year in geneva. this way i gained insight into the way a united nations agency works and got to know many professionals from several countries, which allowed me to expand my worldview. 9. the end of my academic career and research activities the integration of the gdr into the federal republic of germany brought along changes for many citizens of the gdr, and for me as well. in march of 1991 i was told that the senate administration for internal affairs in west berlin had ordered a vetting of all employees working in the public sector in east berlin (formerly gdr). it was to be decided who would be able to transition into the public sector of the federal republic of germany and who would have to be let go. the focus was on personal aptitude, and on whether or not somebody had worked for the ministry of state security (mfs). this also concerned the professors of the humboldt university. hence, i was asked to answer an extensive questionnaire. on august 27, 1991 i was informed that “no indications of formal or informal activities for the mfs were found and that for this reason no measures concerning employment law [were] to be taken.” i was allowed to continue my activities in the training of students, supervision of graduates and doctoral students, diagnostics of biopsies and autopsies, and heading the department of neuropathology. based on provisions in the unification treaty i was allowed to continue to bear the title “full professor” (“ordentlicher professor”), which i was awarded in 1970 by the ministry of higher education of the gdr. however, the title did not imply an according salary. in the federal republic of germany, professors were paid according to the salary groups “c3” or “c4”. my salary group equaled that of an assistant physician. it was my ethical belief as a professional that i would continue teaching and do my work in neuropathology until my retirement regardless of my salary. however, i was later informed that i would be able to apply as a professor. my position was advertised for applicants from the whole country under the heading “c3 professorship of pathology with emphasis on neuropathology.” i was allowed to apply to that, as well. i and four other applicants from west berlin and west germany were asked to give a talk in front of a commission. after that, we were interviewed in person. during my interview, the chairman of the commission, professor detlev ganden, was especially interested in my research on neuroectodermal stem cell tumors. i have never been notified about the result of the selection process. on december 2, 1993 i was notified by the personnel office of the charité that the selection process became unnecessary “because the advertised position was not part of the legally binding employment plan of humboldt university.” in the same letter, i was told that i would be terminated effective february 2, 1994, because “there [were] concerns regarding my personal aptitude for continued employment in the public sector.” i only received this letter after returning from vacation in indonesia that i had spent with my wife from december 1–22, 1993. i refrained from filing a lawsuit because i believed (and still do believe) that i would not force myself on anybody if my special knowledge and my efforts in the (continued) training of the next generation of physicians was not deemed necessary. i have explained this opinion to the institute’s staff when they tried to encourage me to take steps pertaining to employment law. apparently, the administrative director of the charité heard about this, too, because in mid-january of 1994 he offered me an annulment contract instead of a termination if i refrained from suing them. the benefit was that the contract would not be effective february 2, but february 28. this was indeed of great significance to me, because i had been storing large collections of scientific literature, photographic documentations, case studies and correspondence (i was still editor of the zentralblatt) at the institute. because i was given more time, i was able to rent a large storage facility, which my son helped me to store all the irreplaceable material in. we saved it from destruction, because the newly appointed director of the institute threatened to order the removing of everything in the building as soon as i had been dismissed. i was surprised that the annulment contract did not mention the “insufficient personal aptitude” as a reason for the threatened termination. fig. 10: professor jorge cervos-navarro, director of the institute of neuropathology at free university berlin, at our apartment, 2002. fig. 11: visitor from east asia in the rudolf virchow house of the charité berlin, 1992 (note the virchow bust in the background). 10. restarting my career shortly after the announcement of my separation from the charité the medical director of the landesklinik brandenburg offered me to become director of their department of neuropathology. it had five staffers, was well-equipped and had its own electron microscope (fig. 12). my main tasks would be to examine the brains of patients who had died (autopsies would be conducted at the institute of pathology of the municipal clinic brandenburg) and examining nerve and muscle biopsies. in addition, i was told that in a few months neurosurgery would be opened at the municipal clinic brandenburg and that we would be given tissue samples for examination. this was the reason for me to accept the job offer. then the prime minister of brandenburg converted the position of the head of department into that of a chief physician, thus putting it on the same level with the chief physicians of the clinics for neurology and psychiatry. one of the staffers in the department was dr. hermann, who had special skills in the area of morphological diagnostics of muscle and nerve biopsies. he undertook this part of the department’s tasks, which was good for me, as i did not have deep knowledge of this special area. working in this department and the cooperation between the clinics was good. clinicopathological conferences were introduced and occurred on a regular basis. physicians from the landesklinik as well as the municipal clinic for neurosurgery participated. the exchange of opinion that took place furthered the cooperation of the different disciplines to the advantage of the patients. since the chief physicians participated themselves in these conferences, they served as continued training for all participants. there also emerged a close cooperation with the brandenburg state institute for forensic medicine in potsdam. the forensic doctors asked for our help when examinations of the brain and spinal cord of patients who had died were of forensic significance. we were given these organs in toto, sectioned them, and photographically documented macroscopic findings. after microscopic examinations were finished, the forensic doctors received a detailed report of the findings, which included an epicritic evaluation. we were never present in court ourselves and only rarely corresponded with the prosecution. we also had neuropathological conferences involving the forensic doctors. when i reached retirement age the director of the landesklinik dismissed me, but shortly after dr. pauli, chief physician of the pathological institute at the municipal clinic brandenburg, offered me to work with him as a pathologist and neuropathologist. this way the cooperation with the neurosurgeons and forensic doctors could be continued. back when i was working at the landesklinik, an assistant at the institute for pathology in brandenburg who was in special training, dr. marlies günther (fig. 13), asked me to help her become acquainted with the field of neuropathology. hence, we could continue our cooperation until the point where she could conduct neuropathological diagnostics herself. i took pleasure in this cooperation because of her thirst for knowledge. my presence at the institute in brandenburg was usually limited to three days per week. therefore, it was possible for me to accept an offer to work at the institute of neuropathology at free university medical school berlin. the dean asked for my temporary help because the former director of the institute, professor cervos-navarro, had left after reaching retirement age, and his replacement, professor gisela stoltenburg-didinger, was on sick leave. i agreed to help on two days per week. the job was mainly concerned with the diagnostics of neurosurgical material and postmortal neuropathology. i was not asked to be involved with the training of students. a good cooperation with the director of neurosurgery, prof. brock, and his staffers quickly emerged. intraoperative frozen sections which were prepared in a room next to the operating room and diagnosed by me, fostered this cooperation. this way i was able to communicate and discuss the diagnosis with the surgeon instantly. he then demonstrated the intraoperative situs to me on a display and told me details that would be relevant for my decisions. fig. 12: working in the department of neuropathology of the landesklinik brandenburg, 1995. fig. 13: working with dr. marlies günther. 11. as a neuropathologist in thailand after my dismissal by the directors of the charité, i continued to receive invitations for conventions in the field of neuropathology. one of them became a special memory: it came from the medical school of the university in khon kaen in northeastern thailand. i was asked to organize a workshop for physicians consisting of presentations, autopsy seminars and case study discussions about tumors, inflammations and parasitoses in the central nervous system over the span of several days. the discussions were supposed to give participants the opportunity to present cases of their own choosing, request my opinion on them and discuss them together. i was also asked to give a lecture on neuropathology and answer medical students’ questions. the expenses of this ten-day journey were covered by a japanese sponsor of the university. the offer was appealing, but also somewhat risky as i did not have any information on how knowledgeable the participants would be, what their cases might be about, and if the students would even be interested in neuropathology. furthermore, the events was planned to take place in english and i was not sure if my presentations would be comprehensible. therefore, i prepared for the events using my extensive collection of diapositives because my previous teaching activity in mosul showed to me that language barriers could be overcome using illustrative and expressive diapositives. about 60 physicians took part with the workshops i offered for postgraduates. the physicians and students were attentive and engaged in sober discussions. the lecture hall was filled with students up to the last seat. my wife took a seat in the last row and later reported to me how attentively everyone was listening to my delineations. i felt this myself when, after a one-hour lecture, i invited listeners to ask questions. for more than two hours, i replied to profound inquiries, which were proof of the good knowledge basis and intellect of the thai students. this experience was of great joy to me and made the extensive preparations for these events pay off. 12. epilogue my activities in neuropathology teaching and research came to an abrupt halt in the spring of 1994. in 2012, i decided to conclude my activities as a pathologist and neuropathologist. as of this writing, i am living in the 89th year of my life. if someone asked me if would choose this career again, i would clearly reply “yes!” however, i have to advise younger readers that they cannot expect material wealth from working in this area. i am not the only person who had to make the experience of working for an hourly wage of two or three cents (co-)authoring neuropathological books (11, 12). i emphasized this in the prologue: this is not an autobiography. however, i still would like to briefly express my commitment to my philosophy of life: my childhood experiences made me a staunch enemy of national socialist ways of governing and the horrors of world war ii made me an opponent of war. i did not enroll in the army. the experiences of the japanese people taught me that nuclear armament poses a threat to civilization. therefore i joined the organization “international physicians for the prevention of nuclear war (ippnw)” in 1980 and worked for their cause. this worldview determines the way i live until this day. references 1. bailey p., cushing h.: gewebsverschiedenheit der hirngliome. gustav fischer-verlag, jena 1930 2. henschen, f.: tumoren des zentralnervensystems und seiner hüllen. handbuch der speziellen pathologischen anatomie und histologie, herausgeg von o. lubarsch, f. henke, r.rössle. band 13, teil 3; springer-verlag, berlin-göttingen-heidelberg, 1955 3. frauchiger e., fankhhauser r.: vergleichende neuropathologie des menschen und der tiere. springer-verlag, berlin-göttingen-heidelberg 1957 4. gerlach h., jänisch w., schreiber d.: perinatale teratome des zentralnervensystems. wiss. z. ernst-moritz-arndt-univ. greifswald 32: 112-114; 1983 5. haas j.f., jänisch w., staneczek w.: newly diagnosed primary intracranial neoplasms in pregnant woman: a population-based assessment. j. neurol. neurosurg. psychiat. 49: 874-800; 1986 6. hart m.n., earle k.m.: primitive neuroectodermal tumors of the brain in children. cancer 32: 890.897; 1973 7. jänisch w., schreiber d.: experimentelle geschwülste des zentralnervensystems. veb gustav fischer-verlag, jena 1969 8. jänisch w., schreiber d.: experimental tumors of the central nervous system. edited by d.d.bigner and j. swenberg; upjohn comp., kalamazoo/mich.; 1977 9. jänisch w., schreiber d., gerlach h.: tumoren des zentralnervensystems bei feten und säuglingen. veb gustav fischer-verlag, jena 1980 10. jänisch w.: zur epidemiologie der primären geschwülste des zentralnervensystems im ersten lebensjahr. arch. geschwulstforsch. 55: 489-494; 1985 11. jänisch w., schreiber d., güthert h.: neuropathologie (band 1), tumoren des nervensystems. veb gustav fischer-verlag, jena 1988 12. jänisch w., schreiber d., warzok r.: neuropathologie (band 2), pathomorphologie und pathogenese neurologischer krankheiten. veb gustav fischer-verlag, jena 1990 13. jänisch w.: hirngeschwülste bei säuglingen. z. allg. med. 65: 484-489; 1989 14. jänisch w: pathologie der geschwülste des nervensystems. in: klinische neuropathologie. herausgegeben von j. cervos-navarro u. r. ferszt: georg thieme-verlag, stuttgart-new york; 1989 15. jänisch w., grieshammer t.: expression von immunhistochemischen differenzierungsmarkern i n normalen und neoplastisch transformierten neuroektodermalen stammzellen. acta histochemica suppl.-band xlii: 139-142; 1992 16. jänisch w.: nocardiose. in: die entzündlichen erkrankungen des nervensystems, band 2; herausgeg. von h. henkes u. h.w. kölmel; ecomed-verlag, landsberg/lech 1993 17. jänisch w.: mykosen des zentralnervensystems. in: die entzündlichen erkrankungen des nervensystems, band 5; herausgeg. von h. henkes u. h.w. kölmel; ecomed-verlag, landsberg/lech 1993 18. paulus w., jänisch w.: clinicopathologic correlations in epithelial choroid plexus neoplasms: a study of 52 cases. acta neuropathol. 80: 635-641; 1990 19. russell d.s., rubinstein l.j.: pathology of tumours of the nervous system. edward arnold , london 1959 20. schreiber d. und jänisch w. (herausgeber): experimentelle neuroonkologie. wissenschaftliche beiträge der martin-luther –universität halle-wittenberg; heft 7, 1974 21. schreiber d., güthert h.: bericht über die 2. tagung der gesellschaft für neuropathologie der ddr am 1. und 2. oktober 1970 in erfurt. zbl. allg. path. 114: 276-285, 1971 22. warzok r., jänisch w., lang g.: morphology and biology of cerebellar neuroblastomas. j. neuro-oncol. 1: 373-379; 1983 23. zülch k.j.: die hirngeschwülste in biologischer und morphologischer darstellung. barth-verlag, leipzig 1956 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. differential gene expression in the cortical sulcus compared to the gyral crest within the early stages of chronic traumatic encephalopathy feel free to add comments by clicking these icons on the sidebar free neuropathology 2:21 (2021) original paper differential gene expression in the cortical sulcus compared to the gyral crest within the early stages of chronic traumatic encephalopathy jonathan d. cherry*1,2,3,4, filisia agus2,5, erin dixon3,4, bertrand huber2,3,4,6, victor e. alvarez2,3,4,7, jesse mez2,3, ann c. mckee1,2,3,4,7, adam labadorf2,5,6, thor d. stein1,3,4,7 1 department of pathology and laboratory medicine, boston university school of medicine, boston ma, usa 2 department of neurology, boston university school of medicine, boston ma, usa 3 boston university alzheimer’s disease and cte centers, boston university school of medicine, boston ma, usa 4 va boston healthcare system, jamaica plain ma, usa 5 bioinformatics program, boston university, boston ma, usa 6 national center for ptsd, va boston healthcare system, boston ma, usa 7 va bedford healthcare system, bedford ma, usa * corresponding authors: jonathan d. cherry, phd · va boston healthcare system · 150 s huntington ave · boston, ma 02130 · usa jdcherry@bu.edu thor d. stein, md. phd · va boston healthcare system · 150 s huntington ave · boston, ma 02130 · usa tdstein@bu.edu submitted: 21 july 2021 accepted: 12 august 2021 copyedited by: lauren walker published: 17 august 2021 https://doi.org/10.17879/freeneuropathology-2021-3453 additional resources and electronic supplementary material: download supplementary material keywords: cte, rna-seq, repetitive head trauma, inflammation, tau, tbi abstract chronic traumatic encephalopathy (cte) is a progressive neurodegenerative tauopathy found in individuals with a history of repetitive head impacts (rhi). previous work has demonstrated that neuroinflammation is involved in cte pathogenesis, however, the specific inflammatory mechanisms are still unclear. here, using rna-sequencing and gene set enrichment analysis (gsea), we investigated the genetic changes found in tissue taken from the region cte pathology is first found, the cortical sulcus, and compared it to neighboring gryal crest tissue to identify what pathways were directly related to initial hyperphosphorylated tau (p-tau) deposition. 21 cases were chosen for analysis: 6 cases had no exposure to rhi or presence of neurodegenerative disease (control), 5 cases had exposure to rhi but no presence of neurodegenerative disease (rhi), and 10 cases had exposure to rhi and low stage cte (cte). two sets of genes were identified: genes that changed in both the sulcus and crest and genes that changed specifically in the sulcus relative to the crest. when examining genes that changed in both the sulcus and crest, gsea demonstrated an increase in immune related processes and a decrease in neuronal processes in rhi and cte groups. sulcal specific alterations were observed to be driven by three mechanisms: anatomy, rhi, or p-tau. first, we observed consistent sulcal specific alterations in immune, extracellular matrix, vascular, neuronal, and endocytosis/exocytosis categories across all groups, suggesting the sulcus has a unique molecular signature compared to the neighboring crest independent of pathology. second, individuals with a history of rhi demonstrated impairment in metabolic and mitochondrial related processes. finally, in individuals with cte, we observed impairment of immune and phagocytic related processes. overall, this work provides the first observation of biological processes specifically altered in the sulcus that could be directly implicated in cte pathogenesis and provide novel targets for biomarkers and therapies. introduction chronic traumatic encephalopathy (cte) is a progressive neurodegenerative disease found in individuals with a history of exposure to repetitive head impacts (rhi) typically received through playing contact sports such as american football, hockey, soccer, boxing, or rugby, or through military service-related injuries like blasts [3, 23, 24]. currently, cte can only be definitively diagnosed after death through neuropathologic autopsy. the pathognomonic lesion that defines the disease consists of perivascular hyperphosphorylated tau (p-tau) found in neurons and sometimes astrocytes, initially at the depths of the cortical sulcus in the frontal cortex [3]. through computational modeling and helmet sensor data, it was observed that the sulcus and blood vessels receive the greatest amount of damage after head trauma, directly linking rhi induced trauma to cte pathogenesis [11]. however, the specific biological response after trauma that drives cte pathology is still unclear. under normal circumstances, inflammation is a necessary part of the injury cascade. in the brain, neuroinflammation has been observed to be necessary for wound healing responses after damage, triggering an innate and adaptive immune response to fight infectious agents, and recruit beneficial immune cells to areas of need [6]. however, prolonged or chronic inflammation can be harmful and can result in tissue damage and long-term pathology. rhi has been shown to induce a chronic neuroinflammatory environment in both mouse and human studies [9, 10, 32]. additionally, previous work from our laboratory has demonstrated that elevated microglial recruitment via the chemokine ccl2, and glial activation was related to greater years playing contact sports and is a prominent feature in cte [4, 7]. we observed that microglia-mediated neuroinflammation may exist in a feedback loop with p-tau pathology. neuroinflammation induces p-tau deposition, which in turn induces more neuroinflammation leading to a vicious cycle, similar to the inflammatory cascade hypothesis in alzheimer’s disease (ad) [12, 16]. however, the concept of neuroinflammation is broad, encompassing inflammatory, anti-inflammatory, senescent, and immunoregulatory features that previous histology-based studies were unable to easily segregate [6]. therefore, there is a critical need to understand the individual inflammatory phenotypes that emerge during rhi to have more complete insight into the various mechanisms which may directly result in cte pathogenesis. to help fill in knowledge gaps, high level bioinformatic analyses are needed. previous studies have demonstrated that high throughput genomic techniques and bioinformatic pipelines are an optimal method to investigate large scale changes in the brain and determine specific pathways and effects in a more efficient manner [1, 17]. herein, we utilized bulk tissue rna-sequencing targeted to cortical anatomy, in order to investigate the spectrum of inflammatory and neurodegenerative phenotypes that occur as a result of rhi and during early stage cte. as the earliest neuropathologic feature of cte is p-tau pathology found at the depth of the cortical sulcus, we chose to examine what genetic changes occur specifically in the sulcus compared to the neighboring gyral crest. by directly comparing between the sulcus and crest within each case, we will have unique insights into which genes or biological processes are specifically altered in the sulcus as opposed to genes that are part of a more tissue wide response. we will utilize three separate groups, individuals without any rhi or cte (controls), individuals with rhi but no cte (rhi), and individuals with early stage cte (cte). comparison of these three groups will allow direct analysis of different stages of disease. furthermore, the results will provide new understandings and insight into what genes and biological processes might be directly related to initial cte p-tau deposition as opposed to a general cns response to trauma. finally, genes that are found to be specific to cte related pathology will become targets for future biomarker or therapeutic intervention studies. methods subjects a convenience sample of 15 brain donors with a history of rhi exposure from contact sports were selected from the understanding neurological injury and traumatic encephalopathy (unite) brain bank. selection was based on availability of frozen frontal cortex tissue and if the cases were male and between the ages of 35-65 years old. samples were excluded from the study if they carried a neuropathologic diagnosis of ad, neocortical lewy bodies, frontotemporal lobar degeneration (ftld), or motor neuron disease (mnd) based on established neuropathologic criteria for each disease [19, 20, 25, 26]. an additional 6 donors were obtained from the national post-traumatic stress disorder (ptsd) brain bank (national center for ptsd, va boston healthcare system, boston ma). these 6 donors were selected from a group that lacked a history of rhi, did not have evidence of a neurodegenerative disease at autopsy, and were not diagnosed with ptsd in life. samples were grouped into three categories: 6 cases with no history of repetitive head trauma or neurodegenerative disease (control), 5 cases with a history of repetitive head trauma but no neurodegenerative disease (rhi), and 10 cases with a history of repetitive head trauma and a diagnosis of low stage cte (cte). there was no difference in the mean age of death of each group: control51.3 years old ± 8.2, rhi51.4 years old ± 6.8, cte49.5 years old ± 8.1. all cases were male and had a history of playing american football. individual descriptive statistics for the age at death, years of exposure, sport played, and rna integrity number (rin), are provided in table 1. next-of-kin provided written consent for participation and donation. institutional review board approval for brain donation was obtained through the boston university alzheimer’s disease and cte center, human subjects institutional review board of the boston university school of medicine, and edith nourse rogers memorial veterans hospital (bedford, ma). neuropathological assessment post-mortem fresh frozen tissue from the dorsolateral frontal cortex was obtained using previously described procedures [4]. neuropathologic diagnosis was obtained from formalin fixed paraffin embedded tissue as previously described [21, 24]. briefly, 22 sections of paraffin embedded tissue were stained for hyperphosphorylated tau (p-tau), alpha-synuclein, amyloid beta, tdp-43, luxol fast blue, and hematoxylin and eosin using previously described methods [22]. a neuropathologic diagnosis of cte was made using national institute of neurological disorders and stroke (ninds) consensus criteria [3, 21]. cte cases could be subdivided based on the presence, extent, and severity of p-tau deposition through the brain. as the focus of the study was to examine early changes, only cases that met the “low stage cte” criteria were used for analysis. the low stage cte designation encompasses cte stage i&ii [2, 21] and shows strong agreement with the recent ninds consensus staging for low stage cte [3]. neuropathologic examination occurred blinded to clinical results. all evaluations were reviewed by four neuropathologists (va, bh, ts, am); discrepancies in the diagnosis were resolved by consensus conference. demographics, athletic history (type of sports played, level, position, age of first exposure to sports and years playing contact sports), military history (branch, location of service and duration of combat exposure), and traumatic brain injury (tbi) history (including number of concussions) were queried during a telephone interview as detailed previously [30]. rna-sequencing 50µg of fresh frozen tissue was taken from the dorsolateral frontal cortex grey matter at two locations for each case: the depth of the superior frontal cortical sulcus (defined as the bottom third of the sulcus) and the associated neighboring gryal crest at the level of the anterior caudate nucleus. frozen tissue was process using a mortar and pestle, and mrna was extracted and isolated using a maxwell rna extraction kit (promega) as per manufactures instruction. an ion apliseq transcriptome human gene expression kit (thermofisher scientific) was then used to convert mrna to cdna and establish human transcriptome cdna libraries as per manufactures instructions. human transcriptome libraries were then sequenced using an ion torrent s5 next generation sequencer (thermofisher scientific). samples were processed and run across two batches. batch one contained 6 control and 6 cte cases, and batch two contained 5 rhi and 4 cte cases and were compared separately to avoid batch effects. normalized counts for each sample were determined using the ion torrent suit software 5.10 and concatenated together into genes by sample count matrix. the normalized counts were then transformed with rlog for principal component analysis (pca). differential expression analysis genes with more than 50% zero counts within each group were filtered out. pairwise differential expression (de) analyses were conducted separately for cte vs rhi and cte vs control using deseq2 bioconductor package, modeling counts as a function of case status adjusting for age at death [18]. to analyze changes between the sulcus and crest within each group, a ratio between the sulcus and crest genes were calculated using the concatenated normalized counts matrix. genes that could not be detected in all samples (genes with 0 counts for all samples) were filtered out. a pseudocount of 0.01 was added to the counts to prevent infinity values from zero divisions. the ratio was then calculated as log 2 of sulcus counts divided by crest counts for each gene within each sample. to attenuate extreme ratios due to small count values in either tissue, the ratios were arcsin transformed by first dividing by 0.5 and then applying the arcsin function using the numpy python package. this procedure results in a log2 fold change of sulcus vs crest for each gene in each sample. to identify genes that exhibited significantly different expression between tissues within each case status, one-sample, two-tailed t-tests comparing with an expected mean log2 fold change of zero were performed for the fold changes of each gene. p-values were adjusted for multiple hypotheses using the benjamini-hochberg procedure. similarly, two-sample t-tests were run to compare the sulcus vs crest fold changes for cte vs rhi, cte vs control, and rhi vs control. gene set enrichment analysis (gsea) gene set enrichment analysis for all de gene lists were performed using the fgsea r package in bioconductor and gene ontology (go) geneset annotations from goatools python package [14, 29]. gsea statistics were calculated using gene list sorted by descending log2 fold change (from de) or log 2 ratio t-statistics, and significance was assessed for gene sets at fdr < 0.1. the resulting normalized enrichment scores (nes) were then used to create heatmaps. gsea biological process were grouped into 1 out of 11 biological categories by hand, based on expert opinion. categories were discussed and confirmed among 3 authors (jc, al, ts). any differences were resolved through consensus conference. the biological categories were: immune, vascular, neuronal, extracellular matrix (ecm), mitochondrial/metabolism, signaling, cytoskeleton, transcription/translation, endocytosis/exocytosis, protein processing, and any process that didn’t fit the previous 10 category was assigned to “other”. finally, gene ontology analysis from common or unique genes groups was carried out using metascape [33]. all code and supplementary information needed to reproduce this analysis is available at https://osf.io/exsfa/. results overall, 3 differential expression analyses were carried out. a summary of each comparison is outlined in figure 1. the first analysis was to directly compare the differentially expressed genes in the sulcus or crest to the matching region across control, rhi, or cte groups (figure 1a, comparison 1). the second analysis was to directly compare the sulcus vs. crest within each group (figure 1b, comparison 2). the final analysis was to compare how the sulcus vs. crest relative ratios of each gene changed across each disease group (figure 1c, comparison 3). figure 1. summary of differential expression analysis used for comparisonsrepresentative images demonstrating how which regions were being used for comparison. a) comparison 1 compares the differentially expressed genes in the sulcus and crest to the corresponding region between control, rhi, and cte. b) comparison 2 directly compares the sulcus to the crest within each group. c) comparison 3 uses the ratio created from comparison 2 and compares that sulcus vs crest ratio from rhi and cte groups to the control group to determine how the sulcal specific genes changes over the course of disease. differentially expressed genes had similar directions of effect in the sulcus and crest we first examined the differentially expressed (de) genes found in the sulcus and crest to identify the genes most affected in early cte compared with control and rhi groups (table 2) (figure 1a, comparison 1). the relative sulcus and crest de genes that were significantly altered (nominal p < 0.05) in at least one region were plotted against each other to examine if there were similar direction changes occurring in the sulcus and crest (figure 2). changes that were consistent across both sulcus and crest could be interpreted as a general frontal cortex wide response. first, we compared cte to control (figure 2a). we observed that 95% of genes had the same direction of effect in both the sulcus and crest. only 109 (5.0% total de genes) genes had an opposite direction of effect (figure 2a). when examining the changes between cte and rhi, we observed fewer genes were altered compared to cte vs control (table 2). almost all of the genes had the same direction of effect (figure 2b). only 2 genes (0.4% total de genes), jmjd6 and vat1l, were elevated in the sulcus but decreased in the crest. the genes with the largest change, in addition to specific genes of interest were highlighted. a full list of de genes for the cte vs control and cte vs rhi analysis can be found in supplementary file 1. figure 2. comparative analysis of the differentially expressed genes found in the sulcus and crestanalysis of de genes from the sulcus and crest plotted against each other comparing (a) cte vs control and (b) cte vs rhi. genes were plotted if they were significantly altered (p<0.05) in at least one region. each dot represents one gene. genes of interest and the top expressed have been annotated. gene set enrichment analysis (gsea) was then performed on each set of genes to characterize biological processes that are altered in cte (table 3). each gsea process was then assigned to 1 of 11 overall biological categories to better contextualize changes (figure 3). when comparing cte vs control (figure 3a), 81 processes were observed to have an fdr adjusted p < 0.1 in the sulcus or the crest. 41 processes were positively enriched and 34 were negatively enriched in the same direction in both the sulcus and crest. a complete breakdown of group assignments can be found in table 3. the majority of positively enriched processes were involved the immune category with 70.7% of total processes. conversely, only 14.7% of the negatively enriched processes were immune processes. neuronal processes were the largest group of negatively enriched processes with 35.5%. there were fewer positively enriched neuronal processes (4.9%). most signaling processes were negatively enriched (20.6%) compared to positively enriched (4.9%). mitochondria processes were only found to be negatively enriched, while vascular processes were only found in the positively enriched group (table 3). 6 processes were found to have a mixed direction of effect. of those 6 processes, 2 were immune and 4 were extra-cellular matrix (ecm) related (table 3). for cte vs rhi (figure 3b), 44 processes were observed to have an fdr adjusted p < 0.1 in the sulcus or crest. 36 processes were positively enriched, and 7 processes were negatively enriched in the same direction. the largest positively enriched category was immune processes with 38.9%. no immune processes were negatively enriched. ecm processes were the second largest positively enriched category with 30.6% of processes. no ecm processes were negatively enriched. neuronal processes were the majority of negatively enriched processes (85.7%) and were higher than positively enriched neuronal processes. the only other negatively enriched process was transcription/translation (14.3%). vascular, signaling, cytoskeletal, endo/exocytosis, and other processes were all similarly expressed. one process, “signaling receptor activity”, from the signaling category, was decreased in the crest and increased in the sulcus (figure 3). figure 3. heatmap of sulcus and crest gsea biological processesusing the de genes from the sulcus and crest, gsea biological processes were derived and plotted in a heatmap comparing (a) cte vs control and (b) cte vs rhi. processes were included if they were significantly enriched (fdr adjusted p < 0.1) in at least one region. each gsea biological process was assigned to 1 of 11 biological categories to better identify overall changes. [please click on the figure to download a high-resolution version] there were significant differences in the sulcus compared to the crest we next chose to directly compare the sulcus and crest within the controls, rhi, and cte groups to determine which genes were specifically altered in the sulcus compared with the crest by computing an arcsin transformed log2 ratio of sulcus vs crest normalized counts within individuals (see methods) (table 4) (figure 1b, comparison 2). alterations within each group could be related to the unique aspects of each condition. changes in the control sulcus are related to general anatomy, rhi sulcal changes are related to repetitive head trauma induced damage, and cte sulcal changes were related to p-tau deposition. within controls, we observed 671 genes had an fdr adjusted p < 0.1. of the fdr corrected genes, 579 were increased in the sulcus and 92 were decreased in the sulcus compared to the crest (figure 4a). for cases in the rhi group, 1926 genes were found to have a nominal p < 0.05. however, only 1 gene, manea, reached fdr corrected significance. of the nominally significant genes, 1127 were increased and 799 were decreased in the sulcus compared to the crest (figure 4b). in subjects with cte, 1147 genes reached fdr adjusted p < 0.1. 603 genes were elevated in the sulcus and 433 genes were decreased in the sulcus compared to the crest (figure 4c). the top 10 increased and decreased genes by log2 fold change were annotated in each plot. a full list of sulcus vs. crest gene changes can be found in supplementary file 2. figure 4. genes that are differentially expressed in the sulcus compared to the crestthe sulcus was directly compared to the crest across (a) control, (b) rhi, and (c) cte and sulcal specific de genes were calculated. each dot represents one gene. dots that are red represent genes that met fdr adjusted p < 0.1 significance. the top 10 positive and negatively enriched genes were annotated. gsea analysis clarifies biological response processes that are occurring in the sulcus in each condition to further explore how the sulcal specific response contributed to the larger biological response, gsea was performed using the genes that were significantly altered in the sulcus compared to the crest. using fdr adjusted p < 0.1, 301 significant processes were observed in controls, 78 in rhi, and 129 in cte. the observed processes represented biological changes specific to the sulcus compared to the crest in each relative group. we then wanted to compare enrichment of each biological process across groups and determine if there were similar or different directions of effect (figure 5). for added clarity and biological relevance, each process was assigned to 1 of 11 greater biological categories similar to figure 3. a heatmap was then created using processes that were significantly enriched in at least one category (figure 5). the immune category had 51 (13.7%) processes. the majority of processes were observed to be elevated in the sulcus compared to the crest across all 3 sample groups. however, among the cte group there was an increase in the number of processes that had a negative enrichment in the sulcus compare to the crest (figure 5a). next, there were 30 (8.0%) transcription/translation processes, the majority of which were positively enriched in the sulcus compared to the crest (figure 5b). the category with the most assigned processes was the neuronal category with 99 (26.5%) processes. almost all of the neuronal processes demonstrated a negative enrichment in the sulcus. of the few processes that did have a positive enrichment, there was a trend towards decreasing after rhi and during cte (figure 4c). next, there were 9 (2.4%) cytoskeletal processes. the ecm category had 34 (6.4%) processes. all the processes were positively enriched in control cases. however, there was a possible progressive increase in negatively enriched processes through rhi and cte (figure 5e). mitochondria had 24 (6.4%) associated processes. the rhi group only had 1 negatively enriched pathway while control and cte had a mixed split of positive and negative processes (figure 5f). the signaling category had 56 (15.0%) processes and was evenly split between positive and negative enrichment (figure 5g). the vascular category had 14 (8.0%) processes. the control group was entirely positively enriched in the sulcus but there was a switch to more negatively associated processes in rhi and cte (figure 5h). the endocytosis/exocytosis category had 13 (3.5%) processes and was primarily negatively enriched in the sulcus (figure 5i). the protein processing category had 16 (4.3%) processes and was a mix of positive and negative processes. there appeared to be an increase in negative processes associated with cte (figure 5j). finally, the 27 processes that did not fit into the previous 10 categories were placed into “other” (figure 5k). overall, throughout all 11 categories, there was a high agreement in the direction of effect in all cases. 73 (21.2%) processes did not have the same direction of effect. figure 5. heatmap of sulcal specific gsea biological processesusing the genes that were significantly altered in the sulcus compared to the crest, we used gsea to identify biological processes. each process was then assigned to a greater overall biological category: a) immune, b) transcriptional/translational, c) neuronal, d) cytoskeletal, e) extracellular matrix, f) mitochondria/metabolism, g) signaling, h) vascular, i) endocytosis/exocytosis, j) protein processing, k) other. [please click on the figure to download a high-resolution version] next, using the ratio that was created by directly comparing the differentially expressed genes in the sulcus to crest within each group, we compared how those ratios changed in rhi and cte relative to control (figure 1c, comparison 3). this analysis provided additional information into how genes which were increased in the sulcus compared to the crest change after exposure to head trauma and p-tau pathology. 87 processes were found to be significantly altered in rhi vs control, and 127 processes were altered in cte vs control. the same 11 greater biological processes were used to group gsea processes and all processes that were significant for at least one of the two comparisons were included in a heatmap (figure 6a-k). there were 54 immune processes altered (figure 6a). this was the largest number of altered processes compared to the other 10 categories. the majority of immune processes were observed to be decreased compared to controls. transcription/translation was the second largest with 46 processes being altered (figure 6b). the neuronal category had 31 processes and the majority were observed to be increased compared to control (figure 6c). cytoskeletal had 16 processes and had more processes that decreased compared to increased (figure 6d). ecm had 25 processes and all of them were decreased compared to control (figure 6e). mitochondria had 21 processes and the majority were increased compared to controls (figure 6f). signaling had 44, with the majority being decreased compared to control (figure 6g). vascular had 16 processes with all but one observed to decrease compared to controls (figure 6h). endocytosis/exocytosis had 9 processes with an even split of changes (figure 6i). protein processing had 22 processes with an even split of changes (figure 6j). finally, the 12 processes that didn’t fall in the previous 10 categories were assigned to an “other” category and were all decreased compared to control (figure 6k). overall, rhi and cte had similar changes compared to controls. for rhi-control and cte-control comparisons that did not have the same direction of effect, cte-control comparisons always had reduced enrichment of those processes. figure 6. heatmap of sulcal specific gsea biological processes that change in rhi and cte compared to controlthe rhi and cte gsea processes that were found to be specifically altered in the sulcus were compared to the control. each process was again assigned to a greater overall biological category: a) immune, b) transcriptional/translational, c) neuronal, d) cytoskeletal, e) extracellular matrix, f) mitochondria/metabolism, g) signaling, h) vascular, i) endocytosis/exocytosis, j) protein processing, k) other. [please click on the figure to download a high-resolution version] common and unique genes found in the sulcus across disease groups finally, using the differentially expressed gene results comparing the sulcus to the crest (figure 1b, comparison 2), we wanted to examine if there were any sulcal specific alterations that were unique or common across each group (figure 7). first, we examined what sulcal specific genes were commonly altered in all three groups. 333 genes were differentially expressed in the sulcus compared with the crest among all three groups (figure 7a). 331 of 333 genes had the same direction of effect among all three diagnostic groups (figure 7b). only 2 genes, kiaa2022 (up in rhi) and plekhh2 (down in rhi), had a conflicting direction of effect in rhi compared to control and cte. to better understand the biological relevance of those common genes, gene ontology (go) analysis was performed (figure 7c). the most highly significant go term of the common genes was “gliogenesis”, suggesting an altered glial response is an innate feature of the sulcus compared to other brain regions. when examining the rest of the top 20 significant processes, many processes were observed to encompass development, homeostasis, and organization. we then wanted to determine which differentially expressed genes in the sulcus compared with the crest were unique to each diagnostic group. a stringent criteria was used to avoid including any gene that was significant in one group but also had p values close to significant in the other two suggesting they could be common genes if power were increased. therefore, only genes that had a nominal p < 0.05 in one group and a p > 0.25 in all others were marked to be “uniquely” altered. 1074 genes were found to be uniquely altered in the control sulcus with 806 being increased and 268 being decreased compared to the crest (figure 7d). 168 genes had an fdr < 0.1. when observing the go processes of the unique control genes, developmental and immune regulation processes appeared to be the most prominent (figure 7e). 389 genes were found to be unique in the rhi group. 192 genes were increased, and 197 genes were decreased in the rhi sulcus (figure 7f). no rhi unique gene met fdr corrected significance. using go analysis, it was observed that many of the processes were metabolic or transcriptionally related (figure 6g). finally, 531 genes were uniquely altered in the cte sulcus. 239 genes were increased, and 292 genes were decreased in the sulcus relative to the crest (figure 7h). 136 genes met frd corrected significance < 0.1. go analysis of cte specific biological processes demonstrated immune and cell damage biological processes (figure 7i). figure 7. analysis of common and unique genes and biological process found in the sulcususing the genes that were found to be significantly altered in the sulcus compared to the crest, we compared which genes were common to all groups and which were unique to each condition. a stringent criteria was used to avoid including any genes that were significant in one group but also had p values close to significant others suggesting they could be common genes if power was increased. therefore, only genes that had a p < 0.05 in one group and a p > 0.25 in all others were marked to be “uniquely” altered. a) a venn diagram of the common and unique genes found among all three groups. b) a heatmap of the common genes showing similar directions of effect of the majority of genes. c) go analysis of the common genes. d) a plot of the unique sulcal genes found in control cases. e) go analysis of the unique control genes. f) a plot of the unique sulcal genes found in rhi cases. g) go analysis of unique rhi genes. h) a plot of the unique sulcal genes found in cte cases. i) go analysis of unique cte genes. in all plots, red dots denote genes that met fdr adjusted p < 0.1. go analysis was performed using any gene that met nominal p < 0.05 significance. the top 5 positive and negative genes for each group were annotated. [please click on the figure to download a high-resolution version] discussion building on previous work suggesting that altered neuroinflammation is an early event in cte, here we have demonstrated that there is a complex inflammatory response encompassing several unique biological categories occurring specifically in the sulcus and might be directly related to cte pathogenesis. when comparing the sulcus to the neighboring gyral crest we observed that although there was pronounced gene expression related changes present in both regions, there was a further amplified and specific sulcal alterations that might be related to head trauma and cte pathogenesis. when comparing what genes and biological processes were altered in the sulcus compared to the crest, several higher order biological categories were observed to consistently change across control, rhi, and cte. immune, transcriptional/translation, ecm, and vascular categories all were positively enriched in the sulcus while neuronal and endocytosis/exocytosis processes were commonly negatively enriched in the sulcus. these findings demonstrate that even in the absence of rhi exposure or cte pathology, the sulcus had a unique expression pattern compared to the gryal crest and might help partly explain the susceptibility to head trauma and tau deposition. when examining how the sulcal specific alterations might change in response to trauma and disease, in the rhi and cte group there was a complex mixed increase and decrease of inflammatory processes such as astrocyte development, scavenger receptor activity, regulation of immune response, leukocyte migration, and antigen processing compared to the controls. additionally, neuronal processes were elevated after rhi and during cte relative to control cases, possibly suggesting injured neurons might induce compensatory mechanisms in efforts to mitigate rhi induced damage. these complex alterations could also be observed in transcriptional/translational, cytoskeletal, ecm, mitochondria/metabolism, signaling, vascular, endocytosis/exocytosis, and protein processing processes as well. finally, we observed that each of the three groups had a set of genes and processes that were uniquely altered in the sulcus. go analysis demonstrated that within the control group, specifically altered genes were related to development and homeostasis; within rhi, altered genes were metabolic and mitochondria related; and within cte, altered genes were immune and inflammation related. overall, we have built on previous work and expanded our understanding of the complex neurodegenerative response that occurs after rhi and during cte. since p-tau deposition in the crest is less likely in early cte, genes that were found to be differentially expressed in both the sulcus and the crest are possibly less specific to cte sulcal p-tau deposition and more related to a general response to head trauma. consistent with previous reports on how repetitive head trauma affects the cns, the most common genes that were altered in both the sulcus and crest in cte were immune and inflammatory related (figures 2&3). we observed that there was a significant upregulation in complement genes c4a and c4b suggesting a persistent innate immune response and activation of the complement cascade was present in the frontal cortex. complement has a wide variety of functions, but in the context of neurodegeneration, it has been associated with increased synaptic pruning and synaptic loss [27]. it is possible the c4 activity is related to the psd-95 synaptic protein loss that has been reported in previous cte studies [5]. c4 has been suggested to play a role in synaptic pruning in schizophrenia [28], and c4 gene expression and protein levels have recently been linked to tau phosphatases and the development of ad suggesting a possible mechanism in the development of tau pathology [15]. additionally, microglia related genes, such as cxcr1, were elevated in cte, which is in agreement with previous studies examining microglia related changes in cte [4, 7]. astrocytic genes, gfap and aqp1, were also found to be increased in both the sulcus and crest. it has been suggested that there is impairment of aqp4 after trauma, and it is possible that aqp1 was upregulated in compensation [31]. interestingly, there also appeared to be a significant neutrophil response found across the sulcus and the crest. several neutrophil related genes such as cd177, s100a8, s100a9, bpi, and lcn2, and multiple neutrophil gsea processes were highly increased. however, it is possible that the observed neutrophil response could be related to various causes of death as opposed to contact sport exposure. future work will be needed to closely examine if neutrophil markers could be observed using biomarkers from living individuals to better explore the possible effects of tbi and a long-term neutrophil response. altogether, these results demonstrate that there is a prominent neuroinflammatory response found in the frontal cortex of subjects who experience repetitive head trauma. however, it is likely that changes that occur uniformly in both the sulcus and crest do not fully explain the mechanisms behind p-tau deposition and are only part of the equation of cte mechanisms. therefore, it was crucial that we expand our analysis to focusing on what changes could be observed uniquely in the sulcus and might directly relate to cte pathogenesis. interestingly, we found that multiple factors might be contributing to sulcal specific alterations. our findings suggested that sulcal specific changes were the result of either 1) anatomy, 2) repetitive head trauma, and 3) p-tau pathology. although the sulcus and crest are only separated by centimeters, our current result suggest that each region has a unique environment even in the absence on trauma or neurodegenerative disease. when comparing the sulcus to the crest across each group, there was high agreement in the direction of effect observed in all the processes between control, rhi, and cte. these results provide novel information on basic neuroanatomy of how closely related tissue regions might be significantly different and result in unique neuropathologic features. to our knowledge, this is the first study to report such findings. the largest category of processes was found to be neuronal, and was downregulated in the sulcus compared to the crest. there are two possible explanations for this, either the sulcus has less neurons and supporting cells or there is a persistent neuronal impairment found in the sulcus across all groups. regardless which is correct, it is possible that an impaired or reduced neuronal response might predispose the sulcus to elevated damage post head trauma. the next largest category of processes were immune related. interestingly, the majority of these processes were elevated compared to the crest. this suggests that the sulcus might already be “primed” to have an exaggerated immune reaction. the primed phenotype could be consistent with having more glial present or the current glia might be predisposed to have a more severe immune reaction. additionally, the sulcus was observed to be positively enriched in ecm and vascular processes compared to the crest, further demonstrating the sulcus is comprised of a unique environment that responds differently during pathology compared to other neighboring regions. although it is likely the physics of head trauma induced damage and sulcal force concentration are the main driver of sulcal specific pathology [11], the findings reported here could describe additional mechanisms explaining how the sulcus is preferentially affected after repetitive head trauma [3]. consistent with our hypothesis and previous observations, repetitive head trauma was also a significant mechanism driving sulcal specific genetic alterations. the cte and rhi group were observed to be very similar in many genes and biological processes that were altered in the sulcus compared to controls (figure 5). the changes that were observed to be consistent between the rhi and cte group could be interpreted as trauma related alterations, as a common feature of both groups is they share a history of repetitive head trauma received from playing american football. it was unexpected to observe more than half of the of immune biological responses in rhi and cte were decreased in the sulcus compared to control. previous work had suggested that neuroinflammation was a prominent feature of head trauma and likely related to initial p-tau deposition [7]. when examining which biological processes were altered in the sulcus, it was observed that the upregulated processes were more related to foreign object recognition, antigen presentation, and lysosomal function, while the downregulated processes were related to inflammatory cytokine production and other elements related to neurodegeneration. this is consistent with our previous study demonstrating that after repetitive head trauma there was relatively unchanged numbers of total microglia, but increased levels of cells positive for cd68, a lysosomal marker that is elevated during increased phagocytosis and innate immune activation [8] furthermore, tmem106b genotype, which is related to lysosomal and phagocytic alterations, has been also found to relate to cte severity [5]. overall, the surprising downregulation of multiple immune-related processes may suggest that an impaired immune response following rhi predisposes individuals to developing initial tau pathology cte. future studies examining cell type specific alterations and the effects of immune-related genetic polymorphisms will be necessary to test this hypothesis. although the cte and rhi group did overlap for many alterations, we observed that the predominant unique rhi genes and go processes were related to mitochondrial and metabolic activity. rhi is a group of cases that is defined by having exposure to repetitive head trauma, but no neurodegenerative pathology. therefore, it is intriguing to observe that there was a strong metabolic deficit present even before pathology occurs. mitochondrial activity is heavily tied to the health of the cell and any impairments could directly impact cell function. these results suggest that after a history of repetitive head trauma, mitochondrial dysfunction might be the main element driving early symptoms and pathology. additionally, there have been several reports of mitochondria related dysfunction occurring as a consequence of head trauma [13]. it is unclear if there are a specific cell type that is most affected or what might be upstream of the mitochondrial dysfunction, but these results point towards another possible therapeutic target to treat disease before p-tau deposition even begins. finally, we investigated the contribution of p-tau pathology to the sulcal specific genetic alterations. when directly comparing sulcal specific changes found in rhi and cte, very few differences were observed. this was unsurprising as our cte group was restricted to cases with low stage cte and had minimal tau pathology. therefore, it is likely that exposure to repetitive head trauma was a stronger driving force for sulcal specific changes than p-tau pathology in these individuals. it is also likely that many genes related to repetitive head trauma are part of the p-tau deposition mechanisms and are not necessarily unique to cte. rhi and cte might not be district pathologic groups, rather they exist in a spectrum. however, we were still able to identify unique genes found altered only in the cte sulcus and therefore, likely related to p-tau deposition. the main cte unique genes and go processes were related to immune and cell damage further highlighting the possible connection between p-tau deposition and the immune system. it is unclear if these unique cte genes are a consequence of p-tau deposition or act as a mechanism that, in addition to other trauma related pathways, could lead to p-tau deposition. however, these observed unique cte biological processes are novel targets for future biomarkers studies to help identify early stage disease. there are several limitations in this study. rna-sequencing was done on bulk tissue so no comments on the contribution of individual cells can be made at this time. future work utilizing single cell technologies will be needed to determine the specific effects of each cell on the sulcal environment during cte. furthermore, it is difficult to determine the full mechanistic effect in studies using post-mortem human tissue, as we can only look at a single point in time at the end of the donor’s life. mechanistic studies using mouse models or cell culture systems will be needed to determine if the observed genes are mechanistically related to pathologic or healthy physiologic responses. additionally, due to the inherent variation that is present in human studies, a larger sample size is needed to identify more subtle changes. as the present study only focused on early disease, future work is also needed to compare the changes found in low stage disease to high stage cte to determine if processes are similar. finally, as all of the observed changes were based on mrna, proteomic and histologic studies will be needed to verify if the results are also found at the protein level. in conclusion, these results provide clear evidence that there is a complex molecular response occurring in the sulcus compared to the gyral crest. even without exposure to repetitive head trauma, the sulcus had a persistent immune response. in response to rhi, there was immune alteration, metabolic/mitochondria dysfunction, vascular, and ecm impairment. during cte pathogenesis, the immune response found in low stage cte was not observed to be tissue destruction, cell loss, overt neuroinflammation, or other neurodegenerative related responses, rather, it was more focused on antigen presentation, phagocytosis, and wound repair responses. in addition to complex immune changes, early mitochondrial, vascular, ecm, and neuronal activity are all affected during repetitive head trauma induced damage and early tau deposition, and represent possible avenues to halt cte pathogenesis and prevent disease. these results help refine our understanding of the neuroinflammatory environment present during disease and will help guide the discovery of future therapeutic strategies and novel biomarkers. future studies will be crucial to further dissect and study the observed biological processes provided here to better understand the full spectrum of early disease. acknowledgements this work was supported by grant funding from: nih (u19-ag068753, ag08122, ag054076), nia (ag057902, ag06234, rf1ag054156, rf1ag057768), ninds (u54ns115266, u01ns086659, and k23ns102399), national institute of aging boston university ad center (p30ag013846; supplement 0572063345-5); department of veterans affairs biorepository (bx002466) department of veterans affairs merit award (i01-cx001038), department of veterans affairs career development award (bx004349), and the nick and lynn buoniconti foundation. the views, opinions, and/or findings contained in this article are those of the authors and should not be construed as an official veterans affairs or department of defense position, policy, or decision, unless so designated by other official documentation. funders did not have a role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication. references 1. agus f, crespo d, myers rh, labadorf a (2019) the caudate nucleus undergoes dramatic and unique transcriptional changes in human prodromal huntington's disease brain. bmc med genomics 12: 137 https://doi.org/10.1186/s12920-019-0581-9 2. alosco ml, cherry jd, huber br, tripodis y, baucom z, kowall nw et al (2020) characterizing tau deposition in chronic traumatic encephalopathy (cte): utility of the mckee cte staging scheme. acta neuropathol 140: 495-512 https://doi.org/10.1007/s00401-020-02197-9 3. bieniek kf, cairns nj, crary jf, dickson dw, folkerth rd, keene cd et al (2021) the second ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. j neuropathol exp neurol 80: 210-219 https://doi.org/10.1093/jnen/nlab001 4. cherry jd, meng g, daley s, xia w, svirsky s, alvarez ve et al (2020) ccl2 is associated with microglia and macrophage recruitment in chronic traumatic encephalopathy. j neuroinflammation 17: 370 https://doi.org/10.1186/s12974-020-02036-4 5. cherry jd, mez j, crary jf, tripodis y, alvarez ve, mahar i et al (2018) variation in tmem106b in chronic traumatic encephalopathy. acta neuropathologica communications 6: 115 https://doi.org/10.1186/s40478-018-0619-9 6. cherry jd, olschowka ja, o'banion mk (2014) neuroinflammation and m2 microglia: the good, the bad, and the inflamed. j neuroinflammation 11: 98 https://doi.org/10.1186/1742-2094-11-98 7. cherry jd, tripodis y, alvarez ve, huber b, kiernan pt, daneshvar dh et al (2016) microglial neuroinflammation contributes to tau accumulation in chronic traumatic encephalopathy. acta neuropathol commun 4: 112 https://doi.org/10.1186/s40478-016-0382-8 8. chistiakov da, killingsworth mc, myasoedova va, orekhov an, bobryshev yv (2017) cd68/macrosialin: not just a histochemical marker. lab invest 97: 4-13 https://doi.org/10.1038/labinvest.2016.116 9. coughlin jm, wang y, minn i, bienko n, ambinder eb, xu x et al (2017) imaging of glial cell activation and white matter integrity in brains of active and recently retired national football league players. jama neurol 74: 67-74 https://doi.org/10.1001/jamaneurol.2016.3764 10. coughlin jm, wang y, munro ca, ma s, yue c, chen s et al (2015) neuroinflammation and brain atrophy in former nfl players: an in vivo multimodal imaging pilot study. neurobiol dis 74: 58-65 https://doi.org/10.1016/j.nbd.2014.10.019 11. ghajari m, hellyer pj, sharp dj (2017) computational modelling of traumatic brain injury predicts the location of chronic traumatic encephalopathy pathology. brain 140: 333-343 https://doi.org/10.1093/brain/aww317 12. hardy ja, higgins ga (1992) alzheimer's disease: the amyloid cascade hypothesis. science 256: 184-185 https://doi.org/10.1126/science.1566067 13. hiebert jb, shen q, thimmesch ar, pierce jd (2015) traumatic brain injury and mitochondrial dysfunction. am j med sci 350: 132-138 https://doi.org/10.1097/maj.0000000000000506 14. huber w, carey vj, gentleman r, anders s, carlson m, carvalho bs et al (2015) orchestrating high-throughput genomic analysis with bioconductor. nat methods 12: 115-121 https://doi.org/10.1038/nmeth.3252 15. jun gr, you y, zhu c, meng g, chung j, panitch r et al (2020) protein phosphatase 2a, complement component 4, and apoe genotype linked to alzheimer’s disease using a systems biology approach. medrxiv: https://doi.org/10.1101/2020.11.20.20235051 16. karran e, mercken m, de strooper b (2011) the amyloid cascade hypothesis for alzheimer's disease: an appraisal for the development of therapeutics. nat rev drug discov 10: 698-712 https://doi.org/10.1038/nrd3505 17. labadorf a, choi sh, myers rh (2017) evidence for a pan-neurodegenerative disease response in huntington's and parkinson's disease expression profiles. front mol neurosci 10: 430 https://doi.org/10.3389/fnmol.2017.00430 18. love mi, huber w, anders s (2014) moderated estimation of fold change and dispersion for rna-seq data with deseq2. genome biol 15: 550 https://doi.org/10.1186/s13059-014-0550-8 19. love s, louis d, ellison dw (2008) greenfield’s neuropathology, 2-volume set. crc press 20. mackenzie ir, neumann m, bigio eh, cairns nj, alafuzoff i, kril j et al (2010) nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an update. acta neuropathol 119: 1-4 https://doi.org/10.1007/s00401-009-0612-2 21. mckee ac, cairns nj, dickson dw, folkerth rd, dirk keene c, litvan i et al (2016) the first ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. acta neuropathol 131: 75-86 https://doi.org/10.1007/s00401-015-1515-z 22. mckee ac, cantu rc, nowinski cj, hedley-whyte et, gavett be, budson ae et al (2009) chronic traumatic encephalopathy in athletes: progressive tauopathy after repetitive head injury. j neuropathol exp neurol 68: 709-735 https://doi.org/10.1097/nen.0b013e3181a9d503 23. mckee ac, daneshvar dh, alvarez ve, stein td (2014) the neuropathology of sport. acta neuropathol 127: 29-51 https://doi.org/10.1007/s00401-013-1230-6 24. mckee ac, stern ra, nowinski cj, stein td, alvarez ve, daneshvar dh et al (2013) the spectrum of disease in chronic traumatic encephalopathy. brain 136: 43-64 https://doi.org/10.1093/brain/aws307 25. mckeith ig (2006) consensus guidelines for the clinical and pathologic diagnosis of dementia with lewy bodies (dlb): report of the consortium on dlb international workshop. j alzheimers dis 9: 417-423. https://doi.org/10.3233/jad-2006-9s347 26. montine tj, phelps ch, beach tg, bigio eh, cairns nj, dickson dw et al (2012) national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol 123: 1-11 https://doi.org/10.1007/s00401-011-0910-3 27. schartz nd, tenner aj (2020) the good, the bad, and the opportunities of the complement system in neurodegenerative disease. j neuroinflammation 17: 354 https://doi.org/10.1186/s12974-020-02024-8 28. sekar a, bialas ar, de rivera h, davis a, hammond tr, kamitaki n et al (2016) schizophrenia risk from complex variation of complement component 4. nature 530: 177-183 https://doi.org/10.1038/nature16549 29. sergushichev aa (2016) an algorithm for fast preranked gene set enrichment analysis using cumulative statistic calculation. biorxiv: https://doi.org/doi.org/10.1101/060012 30. stern ra, daneshvar dh, baugh cm, seichepine dr, montenigro ph, riley do et al (2013) clinical presentation of chronic traumatic encephalopathy. neurology 81: 1122-1129 https://doi.org/10.1212/wnl.0b013e3182a55f7f 31. szu ji, chaturvedi s, patel dd, binder dk (2020) aquaporin-4 dysregulation in a controlled cortical impact injury model of posttraumatic epilepsy. neuroscience 428: 140-153 https://doi.org/10.1016/j.neuroscience.2019.12.006 32. tagge ca, fisher am, minaeva ov, gaudreau-balderrama a, moncaster ja, zhang xl et al (2018) concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model. brain 141: 422-458 https://doi.org/10.1093/brain/awx350 33. zhou y, zhou b, pache l, chang m, khodabakhshi ah, tanaseichuk o et al (2019) metascape provides a biologist-oriented resource for the analysis of systems-level datasets. nat. commun 10: 1523 https://doi.org/10.1038/s41467-019-09234-6 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a potential diagnostic pitfall: primary synovial sarcoma of the central nervous system feel free to add comments by clicking these icons on the sidebar free neuropathology 3:11 (2022) letter a potential diagnostic pitfall: primary synovial sarcoma of the central nervous system arnault tauziède-espariat1,2, nicolas macagno3, daniel pissaloux3, dominique figarella-branger4,5, romain appay4,5, dorian bochaton6, sanaa tazi7, paul kauv8, lauren hasty1, alice métais1,2, fabrice chrétien1, pascale varlet1,2 1 department of neuropathology, ghu paris psychiatry and neuroscience, sainte-anne hospital, paris, france 2 inserm, ima-brain, institute of psychiatry and neurosciences of paris, université de paris, umr s1266, paris, france 3 department of biopathology, léon bérard cancer center, lyon, france 4 department of pathology, toulouse university hospital, toulouse, france 5 inserm u1037, cancer research center of toulouse (crct), toulouse, france 6 institut curie hospital, laboratory of somatic genetics, paris, france 7 department of neurosurgery, henri mondor hospital, créteil, france 8 department of radiology, henri mondor hospital, créteil, france corresponding author: arnault tauziède-espariat · department of neuropathology · ghu paris psychiatry and neuroscience, sainte-anne hospital · 1 rue cabanis, 75014 paris · france a.tauziede-espariat@ghu-paris.fr submitted: 14 march 2022 accepted: 25 april 2022 copyedited by: cinthya agüero published: 26 april 2022 https://doi.org/10.17879/freeneuropathology-2022-3811 keywords: ss18, ssx, synovial sarcoma, bcor introduction synovial sarcoma (ss) is an aggressive soft tissue sarcoma that occurs primarily in the juxta-articular extremital regions of young adults, followed by the trunk and the head and neck. they are universally defined by a fusion of ss18 with one of the ssx genes (predominantly ssx1). primary ss of the central nervous system (cns) have been rarely reported in the literature (n=22 molecularly confirmed cases) and have not yet been added to the mesenchymal non-meningothelial chapter of the world health organization (who) classification of cns tumors. here, we report one case of ss18-rearranged cns-ss, with a review of the literature to determine its clinical, radiological, and histopathological features and its main differential diagnoses. case presentation a 62-year-old woman presented with an intraventricular hemorrhage (figure 1 a-b). the patient had a history of a hemorrhagic lesion diagnosed as a central neurocytoma and was treated by surgery 3 years earlier. the tumor exhibited histologically dense, solid, hemorrhagic, and basophilic cell proliferation (figure 1c), composed of monotonous spindle cells, with round to oval nuclei lacking any pleomorphism or mitotic activity. the cytoplasm was scant or more abundant and vacuolated (figure 1d). there was no collagenous stroma, myxoid changes, “hemangiopericytoma-like” vasculature, whorls, psammoma bodies, neurocytic rosettes, papillae, rhabdoid, or glandular formations. necrosis and microvascular proliferation were absent, and the ki67 labeling index was low (1%) (figure 1e). tumor cells were immunonegative for ae1/ae3, ck7, ck20, ck18, sstr2a, ema, cd34, stat6, cd45, cd99, nut, olig2, gfap, sox10, ps100, hmb45, chromogranin a, synaptophysin, neun, neurofilament, desmin, and smooth muscle actin. the expression of h3k27me3 and brg1 was retained. ini1 was maintained with a diminished expression in comparison to the endothelium (figure 1f). the tumor expressed vimentin, cd56, bcl2, tle1, and bcor (but with less intense staining compared to our control, a molecularly confirmed cns tumor with internal tandem duplication of bcor) (figure 1g-j). histopathological characteristics were like those of the previous surgical sample. the initial tumor was diagnosed as a neurocytoma due to its intraventricular location, its expression of cd56 and because of its absence of any sign of malignancy (particularly no mitosis). initial and recent whole-body imaging did not show other tumor locations. digital droplet pcr failed to reveal an internal tandem duplication of bcor, and whole-exome rna sequencing [1] demonstrated a ss18::ssx1 gene fusion (figure 1k). an additional immunostaining targeting the chimeric protein ss18::ssx was secondarily performed and revealed a strong and diffuse expression in the primary tumor and its recurrence (figure 1k). using dna-methylation analysis, the tumor was not classifiable by the heidelberg brain tumor classifier (v12.5) and the sarcoma classifier (v12.2). a final diagnosis of primary cns-ss was suggested. forty months after the initial diagnosis, the patient was alive without a tumor residue. figure 1. radiological, histological, and molecular features of the recurrent tumor. (a) axial ct scan showing a voluminous left intraventricular mass with hemorrhage. (b) axial ct scan after contrast injection showing a heterogeneous enhancement of the mass. (c) densely cellular proliferation with hemorrhagic changes (hps, magnification x100). (d) monotonous basophilic spindle cells with round to oval nuclei and vacuolated cytoplasm (hps, magnification x400). (e) low ki67 labeling index (magnification x400). (f) retained expression of ini1 with low intensity (magnification x400). (g) diffuse but moderate immunoexpression of bcor (magnification x400). (h) diffuse immunoexpression of vimentin (magnification x400). (i) diffuse immunoexpression of cd56 (magnification x400). (j) diffuse immunoexpression of bcl2 (magnification x400). (k) whole exome rna-seq analysis highlights a fusion between ss18 (pink) and ssx1 (blue) genes, respectively located on chr.18 and chrx. immunopositivity for the chimeric protein between ss18 and ssx with retained protein domain of ss18 detected by immunohistochemistry (insert magnification x400). black scale bars represent 50 μm for figures d-j; and 250 μm for figure c. hps: hematoxylin phloxin saffron. clicking the figure will lead you to a virtual slide (h&e). discussion and conclusion molecularly confirmed examples of primary cns-ss are very rarely reported in the literature (n=22 cases, table s1) [2–8]. like their soft tissue counterpart, they affect young adults (median age 21 years with ranges from 1 to 81) or children (32% of cases) [2,3] with a male predominance (sex ratio male to female: 1.75) [2–8]. they are mainly supratentorial (19/22, 86% of cases) [2–8] and 9% of them are intraventricular as was the case for our patient [2]. clinical symptoms depend on the location of the tumor, but interestingly, 4/22 cases (18%) are revealed by an intracerebral hematoma [2,5,8]. radiologically, they present as a solid (13/22 cases) or cystic mass (8/22 cases) that is well defined from the parenchyma (11/16 cases) [2–4,8]. some of them present a dural attachment (4/22 cases) [4–7]. histopathologically, ss is classically divided into three subtypes within the soft tissue: biphasic (composed of a mixture of spindle cells and epithelial component), monophasic (only composed of spindle cells), and poorly differentiated (composed of round or spindle cells with a high mitotic index and pronounced atypia). rare ss examples can exhibit a round cell morphology [9–11]. in the cns, all these variants have been reported, the monophasic subtype (as our case) being the most frequent (10/21 cases compared to 9/21 biphasic and 2/21 poorly differentiated cases) [2,4–7]. recently, a highly specific and sensitive antibody targeting the ss18::ssx fusion protein has been developed independently of the ss18 fusion gene partner among ssx genes [12], which can serve as a surrogate to molecular testing [12,13]. like their soft tissue counterparts, ss frequently and strongly express bcl2, vimentin, and cd99, which are not specific. the classical phenotype includes a variable, usually focal aberrant epithelial phenotype, diffuse and intense nuclear expression of tle1 [14], and reduced expression of ini1 [15]. the glial markers, sstr2a, cd34, and stat6, are not expressed, which allows one to rule out differential diagnoses (gliosarcoma, meningioma, and solitary fibrous tumor). however, the occasional expression of bcor may be a confusing finding [16], but additional molecular analyses did not confirm the presence of bcor alterations and found a ss18::ssx1 fusion, the most frequent fusion transcript described in soft tissue ss. in the cns, no detailed fusion has been reported, except in one case (ss18::ssx2) because only fish analyses for ss18 have been performed [2–8]. like many fusion-driven neoplasms, the cellular origin of ss remains unknown. a mesenchymal hypothesis was suggested with respect to its preferential location in soft tissue. because meningeal involvement has been reported in a part of cns cases, it is reasonable to suggest a possible mesenchymal dural origin. recently, dna-methylation-based classification of cns tumors has been shown to be a robust tool for molecular tumor classification, most likely because individual tumor types maintain an epigenetic ‘memory’ of their distinct cell of origin [17]. in v12.2 of the sarcoma classifier, there is a methylation class of synovial sarcoma [18]. however, due to the low cellularity in our hemorrhagic sample, the dna methylation profiling did not properly cluster and did not allow us to classify this tumor. the prognosis of soft tissue ss depends on the fnclcc grade [19] and genomic index [20]. cns-ss is associated with a unfavorable outcome, high rates of recurrence (18/21 cases, 86%), and death (16/21 cases, 76%) with a median overall survival of 14 months [2–5,7,8]. our case represents an exception with the longest overall survival ever described without adjuvant treatment (40 months), possibly due to the low histological grade of the tumor. in conclusion, cns ss with a ss18::ssx1 fusion represents a rare mesenchymal non-meningothelial tumor, not yet included in the who classification of cns tumors. although its diagnosis remains challenging, different diagnostic tools (immunohistochemistry, fish, rna sequencing) exist to identify ss. further studies are needed to elucidate its cellular origin in the cns. declarations ethics approval this study was approved by the ghu paris psychiatry neurosciences, sainte-anne hospital’s local ethic committee. consent for publication the patient signed informed consent forms. competing interests the authors declare that they have no conflicts of interest directly related to the topic of this article. funding the authors declare that they have not received any funding. authors’ contributions ate, pk and st compiled the mri and clinical records; ate, am, fc and pv conducted the neuropathological examinations; nc, dp, dfb, ra and db conducted the molecular studies; ate, lh, and pv drafted the manuscript. all authors reviewed the manuscript. acknowledgements we would like to thank the laboratory technicians at the ghu paris neuro sainte-anne for their assistance. references macagno n, pissaloux d, de la fouchardière a, karanian m, lantuejoul s, galateau salle f, et al. wholistic approach: transcriptomic analysis and beyond using archival material for molecular diagnosis. genes chromosomes cancer. 2022; 29:1–12. zhang g, xiao b, huang h, zhang y, zhang x, zhang j, et al. intracranial synovial sarcoma: a clinical, radiological and pathological study of 16 cases. eur j surg oncol j. 2019; 45:2379–85. xiao g, pan b, tian x, li y, li b, li z. synovial sarcoma in cerebellum: a case report and literature review. brain tumor pathol. 2014; 31:68–75. lin y-j, yang q-x, tian x-y, li b, li z. unusual primary intracranial dural-based poorly differentiated synovial sarcoma with t(x; 18)(p11; q11). neuropathology. 2013; 33:75–82. aggad m, gkasdaris g, rousselot c, destrieux c, françois p, velut s, et al. intracranial primary synovial sarcoma mimicking a spontaneous cerebral hematoma-a case report and review of the literature. neurochirurgie. 2021; s0028-3770(21)00200-9. sharma s, sharma a, lobo g, nayak m, pradhan d, samriti, et al. primary dura-based synovial sarcoma of the parafalcine region of brain. pathol res pract. 2017; 213:868–71. horbinski c, cieply k, bejjani gk, mcfadden k. primary intracranial dural-based synovial sarcoma with an unusual syt fluorescence in situ hybridization pattern. j neurosurg. 2008; 109:897–903. patel m, li l, nguyen hs, doan n, sinson g, mueller w. primary intracranial synovial sarcoma. case rep neurol med. 2016; 2016:5608315. silverman jf, landreneau rj, sturgis cd, raab ss, fox kr, jasnosz km, et al. small-cell variant of synovial sarcoma: fine-needle aspiration with ancillary features and potential diagnostic pitfalls. diagn cytopathol. 2000; 23:118–23. cole p, ladanyi m, gerald wl, cheung nk, kramer k, laquaglia mp, et al. synovial sarcoma mimicking desmoplastic small round-cell tumor: critical role for molecular diagnosis. med pediatr oncol. 1999; 32:97–101. cohen ij, stark b, avigad s. synovial sarcoma mimicking desmoplastic small round-cell tumor: critical role for molecular diagnosis. med pediatr oncol. 2000; 34:234. baranov e, mcbride mj, bellizzi am, ligon ah, fletcher cdm, kadoch c, et al. a novel ss18-ssx fusion-specific antibody for the diagnosis of synovial sarcoma. am j surg pathol. 2020; 44:922–33. perret r, velasco v, le guellec s, coindre j-m, le loarer f. the ss18-ssx antibody has perfect specificity for the ss18-ssx fusion protein: a validation study of 609 neoplasms including 2 unclassified tumors with ss18-non-ssx fusions. am j surg pathol. 2021; 45:582–4. el beaino m, jupiter dc, assi t, rassy e, lazar aj, araujo dm, et al. diagnostic value of tle1 in synovial sarcoma: a systematic review and meta-analysis. sarcoma. 2020; 2020:7192347. kohashi k, oda y, yamamoto h, tamiya s, matono h, iwamoto y, et al. reduced expression of smarcb1/ini1 protein in synovial sarcoma. mod pathol off j u s can acad pathol inc. 2010; 23:981–90. kao y-c, sung y-s, zhang l, kenan s, singer s, tap wd, et al. bcor upregulation in a poorly differentiated synovial sarcoma with ss18l1-ssx1 fusion-a pathologic and molecular pitfall. genes chromosomes cancer. 2017; 56:296–302. capper d, jones dtw, sill m, hovestadt v, schrimpf d, sturm d, et al. dna methylation-based classification of central nervous system tumours. nature. 2018; 555:469–74. koelsche c, schrimpf d, stichel d, sill m, sahm f, reuss de, et al. sarcoma classification by dna methylation profiling. nat commun. 2021; 12:498. trassard m, le doussal v, hacène k, terrier p, ranchère d, guillou l, et al. prognostic factors in localized primary synovial sarcoma: a multicenter study of 128 adult patients. j clin oncol off j am soc clin oncol. 2001; 19:525–34. orbach d, mosseri v, pissaloux d, pierron g, brennan b, ferrari a, et al. genomic complexity in pediatric synovial sarcomas (synobio study): the european pediatric soft tissue sarcoma group (epssg) experience. cancer med. 2018; 7:1384–93. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. pigmented ependymoma, a tumor with predilection for the middle-aged adult: case report with methylation classification and review of 16 literature cases feel free to add comments by clicking these icons on the sidebar free neuropathology 3:16 (2022) review pigmented ependymoma, a tumor with predilection for the middle-aged adult: case report with methylation classification and review of 16 literature cases alexander s. himstead, bs1,2, mari perez-rosendahl, md2, gianna m. fote, phd1, angie zhang, md1, michael g. kim, md1, david floriolli, md3, martha quezado, md4, kenneth aldape, md4, drew pratt, md4, zied abdullaev, phd4, edwin s. monuki, md, phd2, frank p. k. hsu, md, phd1, william h. yong, md, fcap2 1 department of neurosurgery, university of california, irvine school of medicine, orange, ca, usa 2 department of pathology and laboratory medicine, university of california, irvine school of medicine, orange, ca, usa 3 department of radiology, university of california, irvine school of medicine, orange, ca, usa 4 laboratory of pathology, center for cancer research, national cancer institute, national institutes of health, bethesda, md, usa corresponding author: william h. yong, md, fcap · department of pathology and laboratory medicine · uc irvine health, school of medicine · 101 the city drive south · orange, ca 92868 · usa yongwh@hs.uci.edu submitted: 09 june 2022 accepted: 21 june 2022 copyedited by: mónica miranda published: 08 july 2022 https://doi.org/10.17879/freeneuropathology-2022-4076 additional resources and electronic supplementary material: supplementary material keywords: pigmented; ependymoma; lipofuscin; fourth ventricle; case report; posterior fossa; methylation abstract ependymomas have rarely been described to contain pigment other than melanin, neuromelanin, lipofuscin or a combination. in this case report, we present a pigmented ependymoma in the fourth ventricle of an adult patient and review 16 additional cases of pigmented ependymoma from the literature. a 46-year-old female showed up with hearing loss, headaches, and nausea. magnetic resonance imaging revealed a 2.5 cm contrast-enhancing cystic mass in the fourth ventricle, which was resected. intraoperatively, the tumor appeared grey-brown, cystic, and was adherent to the brainstem. routine histology revealed a tumor with true rosettes, perivascular pseudorosettes and ependymal canals consistent with ependymoma, but also showed chronic inflammation and abundant distended pigmented tumor cells that mimicked macrophages in frozen and permanent sections. the pigmented cells were positive for gfap and negative for cd163 consonant with glial tumor cells. the pigment was negative for fontana-masson, positive for periodic-acid schiff and autofluorescent, which coincide with characteristics of lipofuscin. proliferation indices were low and h3k27me3 showed partial loss. h3k27me 3 is an epigenetic modification to the dna packaging protein histone h3 that indicates the tri-methylation of lysine 27 on histone h3 protein. this methylation classification was compatible with a posterior fossa group b ependymoma (epn_pfb). the patient was clinically well without recurrence at three-month post-operative follow-up appointment. our analysis of all 17 cases, including the one presented, shows that pigmented ependymomas are most common in the middle-aged with a median age of 42 years and most have a favorable outcome. however, one patient that also developed secondary leptomeningeal melanin accumulations died. most (58.8%) arise in the 4th ventricle, while spinal cord (17.6%) and supratentorial locations (17.6%) were less common. the age of presentation and generally good prognosis raise the question of whether most other posterior fossa pigmented ependymomas may also fall into the epn_pfb group, but additional study is required to address that question. introduction primary pigmented tumors of the central nervous system (cns) are rare entities and often contain melanin.1 primary pigmented intracranial tumors include melanoma, melanocytoma, schwannomas,2 meningiomas,1 and, less commonly, gliomas,3 neurocytomas,4 ependymomas and choroid plexus tumors.2 ependymomas are glial neoplasms found, most commonly, intracranially in the posterior fossa of children and within the spinal cord of adults.5 prognosis primarily depends on location, with intracranial ependymomas harboring worse outcomes than spinal variants.6,7 pigmented ependymomas are most commonly melanotic,8 although these neoplasms are often incompletely characterized in the literature. furthermore, cases of pigmentation with neuromelanin and lipofuscin have been previously described.2,9 nowadays, subtyping of ependymomas includes genetic and methylation profiling, therefore several methylation profiles have emerged which may correlate with clinical prognosis.10 here, we describe a pigmented ependymoma of the fourth ventricle that contains lipofuscin and has a methylation profile consistent with a posterior fossa group b ependymoma (epn_pfb). we also review the available published cases pertaining to pigmented ependymomas by characterizing their age distribution, location, clinical features and outcomes. case history a 46-year-old hispanic female presented with hearing loss for one year and worsening headaches and nausea for two months. her physical examination revealed no focal neurological deficits. magnetic resonance imaging (mri) revealed a 2.2 x 1.5 x 2.6 cm mass involving the floor of the fourth ventricle, which showed marked intrinsic t1 hyperintensity, variable t2 signal (with areas of both hyperintensity and hypointensity), predominantly hyperintense fluid-attenuated inversion recovery (flair) signal and areas of contrast-enhancement (fig. 1). after informed consent to surgery and involvement in research (irb ethics approval deferred as university of california, irvine policy permits reports of up to 3 individuals), she underwent a suboccipital craniotomy for resection of the mass. intraoperatively, the tumor appeared as a grey-brown cystic mass within the fourth ventricle that was tightly adherent to the posterior inferior cerebellar arteries and lower brainstem (fig. 2). radiographic gross-total resection (gtr) was achieved. the patient’s postoperative course was uncomplicated, and she was without radiographic recurrence and satisfied with her treatment at oneand three-month follow-up appointments (fig. 3). figure 1. a. sagittal pre-contrast t1 demonstrates an intraventricular mass involving the floor of the fourth ventricle and heterogenous t1 signal with areas of prominent intrinsic t1 hyperintensity (blue arrow), areas isointense to brain (red arrows), and scattered areas of low signal. b. fat saturated sagittal post-contrast t1 demonstrates enhancement of the previously isointense portions (red arrows) with persistent areas of low signal. c. axial fat saturated t2 at the level of the lesion demonstrates cystic areas of high t2 signal (red arrow) which correspond to low intensity of pre-contrast t1. d. a prominent low signal area on t2 is intrinsically bright on t1 (blue arrow), suggesting high protein and/or lipid contents. e. axial flair demonstrates predominantly isoto hyperintense signal. figure 2. intraoperative photographs of the tumor demonstrating: a. dissection of arachnoid bands tethering the tumor to the floor of the fourth ventricle and b. the macroscopic appearance of the lesion with notable grey-brown color (blue arrows). figure 3. patient care timeline. pathology intraoperative smears showed pigmented cells often with swollen granular cytoplasm mimicking macrophages (fig. 4a), as well as a population of cells with ovoid nuclei and a moderate amount of cytoplasm with variably epithelioid features (fig. 4b). intraoperative frozen sections showed perivascular chronic inflammation, abundant pigmented cells and sheets of tumor cells with epithelial surfaces mimicking glands, but reflecting ependymal rosettes or canals (arrowhead) (fig. 4c). the pigment was fine and grey-brown in micrographs of the frozen section (fig. 4d). figure 4. staining is with hematoxylin and eosin (h&e) unless otherwise specified. a. (400x) smearswollen pigmented tumor cells. b. (400x) smearunpigmented and pigmented tumor cells. c. (100x) frozen section“epithelial” surfaces resembling glands (red arrowhead). d. (400x) frozen sectionfine, grey-brown pigment. figures e-i are from permanent sections. e. (40x) cellular tumor (right fragment) and areas reminiscent of subependymoma gliopil (left fragment). f. (100x) ependymal canal (blue arrowhead) and pseudopapillary configuration (red arrowhead). g. (200x) perivascular pseudorosette (red arrowhead). h. (100x), i. (400x) swollen pigmented cells. j. (200x) fontana-masson staintumor cells negative; few perivascular macrophages positive (blue arrowhead). k. (600x) pas stain. l. (600x) fluorescence microscopy shows autofluorescence. m. (200x), n. (400x) gfappigmented cells immunopositive (red arrowheads). o. (200x) cd163tumor cells negative; scattered microglia immunopositive. p. (200x) h3k27me3loss in subset of tumor cells. clicking into the figure will lead you to the full virtual slide. permanent sections showed tumor tissue that was mostly cellular (right fragment) with minor areas where the background consisted of an eosinophilic gliopil (left fragment) (fig. 4e). ependymal canals (blue arrowhead) were focally prominent, sometimes suggesting a papillary configuration (red arrowhead) (fig. 4f). perivascular pseudorosettes could also be seen (fig. 4g). swollen cells with grey-brown pigment were abundant in some areas (fig. 4h, i). the pigmented cells were negative for melanin and neuromelanin by fontana-masson (fig. 4j); only scant focal often coarser and/or larger black-staining pigment was seen extracellularly and in a few perivascular macrophages (arrowhead). these cells stained strongly with pas (fig. 4k) and were autofluorescent with fluorescence microscopy (fig. 4l). concerning the gfap staining, the tumor was diffusely and strongly positive (fig. 4m), as were the swollen pigmented cells, which showed gfap staining at the cytoplasmic periphery (arrowheads) consistent with a glial origin (fig. 4n). both unpigmented and pigmented tumor cells were negative for the macrophage marker cd163; with only scattered, interspersed, small darkly staining microglia (fig. 4o). collectively, these findings indicated a lipofuscin-pigmented ependymoma. an h3k27me3 immunostain showed immunonegativity in approximately 20-30% of tumor cells, but did not show global loss (fig. 4p). whole slide digital images of frozen h&e (sfig. 1), permanent h&e (sfig. 2), pas (sfig. 3), gfap (sfig. 4) and h3k27me3 (sfig. 5) slides are available in the supplement section. dna methylation arrays and analysis methylation profiling was performed at the national institutes of health (nih), bethesda, md, usa. genomic dna was extracted from formalin-fixed paraffin-embedded (ffpe) tissue sections on slides using an allprep dna/rna ffpe kit (qiagen, venlo, netherlands). the dna was bisulfite-converted using the ez dna methylation kit (zymo research, irvine, ca, usa), and processed to create a beadchip (850k methylation sites) using the infinium methylation epic kit (illumina, san diego, ca, usa). the beadchip was then scanned on the iscan reader (illumina). methylation profiling was carried out through an nih pipeline using umap, the nci-epic methylation classifier, and the heidelberg classifier (brain, versions 11b6 and 12.5), obtained from the german cancer research center in heidelberg, germany. the sample was classified as ependymoma, posterior fossa group b with a score of 0.47 on version 11b6, and the same methylation class with a higher score of 0.98 on version 12.5 of the heidelberg classifier. on the umap, this sample embedded with the epn_pfb cluster (fig. 5). the consensus methylation profile, in the context of the clinical and histopathologic features, supported epn_pfb, cns world health organization (who) grade ii. figure 5. methylation analysis results. a. genome-wide copy number profile generated from dna methylation array signal intensities. the copy number profile shows numeric whole chromosome changes, which are typical for epn_pfb group. b. the umap embedding of dna methylation array data for select tumor groups. sample tumor embeds with epn_pfb tumor cluster. discussion ependymomas are glial neoplasms with a slight male predominance that represent, approximately, 2-6% of all intracranial tumors and 50% of intramedullary spinal tumors.11,12 much less common are pigmented ependymomas.2 these rare tumors may contain three types of pigment: melanin, neuromelanin, or lipofuscin.13 the most common pigment in pigmented ependymomas is melanin, a tyrosine derivative present in the choroid of the eye and, sometimes, in the meninges.13 rarely, pigmented ependymomas contain neuromelanin, which is likely produced by nonenzymatic oxidation of dopamine or lipofuscin. lipofuscin arises from iron-catalyzed peroxidation of membrane lipids and lipoproteins within lysosomes, and tends to accumulate with cellular aging.2,13 a recent case report and literature review on pigmented ependymomas describes only one previous case, in which lipofuscin was discovered to be the sole pigment contained within a pigmented ependymoma in a 16-year-old male.2 here, we report on an additional case of lipofuscin-pigmented ependymoma of the fourth ventricle in a 46-year-old female with the epn_pfb subtype. given the rarity of this entity, a literature review was performed using the pubmed and scopus search engines to identify previously described cases of pigmented ependymoma. keywords used included “pigment”, “melanin”, “lipofuscin”, “neuromelanin” and “ependymoma”. this yielded 8 total studies2,8,9,14-19 reporting on 16 total patients with pathologically confirmed pigmented ependymoma (table 1). melanin was the most common pigment found in these ependymomas (n=12, 70.6%), followed by lipofuscin and neuromelanin (n=2, 11.8%) and lipofuscin alone (n=2, 11.8%). in one case, the pigment composition was not reported.16 only two studies reported the tumor grade using the who classification.8,15 six cases were who grade ii, and two were who grade iii anaplastic ependymoma according to the who schema at that time. pigmented ependymomas tend to occur in young adults, most commonly in the 4th ventricle, and present with symptoms caused by local mass effect. the primary treatment described was resection variably with adjuvant radiation therapy, and gross-total resection was reported in all but 5 cases (29.4%). the average age at time of diagnosis was 38.6 years and the median age was 42 years (range 13-52) (fig. 6). there was a slight male predominance with 9 out of 17 male patients (52.9%). the most common location was the fourth ventricle (n=10, 58.8%), followed by the cervical spine (n=2, 11.8%), the thoracic spine, temporal lobe, frontoparietal lobe, and sella turcica (n=1, 5.9% each) (table 2). figure 6. histogram demonstrating age distribution of patients with pigmented ependymomas. the primary clinical presentation was based on mass effect from the tumor location. several patients with fourth ventricular tumors presented with symptoms of obstructive hydrocephalus (n=4). one patient was asymptomatic. another developed right upper extremity numbness and right lower extremity weakness. only one noteworthy case described a posterior fossa melanotic ependymoma complicated by secondary pial accumulation of melanin, resulting in dysarthria, hearing loss and death of the patient twelve days after subtotal resection.14 this was the only death reported in the literature and may be related to the secondary leptomeningeal pigment accumulation. the typical treatment for these tumors was attempted gtr, with adjuvant radiation therapy in six cases. there was one case where the patient was intact after resection, but developed hydrocephalus in the setting of adjuvant radiotherapy and devolved into a coma.8 in all other cases, the patients were alive without recurrence at last follow-up, with a mean follow up of 43.7 months. there are three distinct molecular variants of posterior fossa ependymoma, including posterior fossa group a ependymoma (epn_pfa), epn_pfb, and subependymoma, distinguished by their dna methylation profile. patients with epn_pfb are typically adults, whereas epn_pfa is more likely to be diagnosed in infants. given the age of onset (adult) and typical lack of recurrence, we hypothesize that the methylation profiles of the posterior fossa pigmented ependymomas described in the literature are likely to be epn_pfb in most cases. the molecular subgroup has been demonstrated to be a powerful independent predictor of outcomes.10 a recent study found that epn_pfb was associated with an excellent 10-year overall survival (os) rate of 96.1% after gtr, whereas epn_pfa, the other posterior fossa variant, had worse outcomes; 5-year os after gtr was 65.2% in females and 45.5% in males. furthermore, epn_pfa was a highly significant predictor of poor progression-free survival (hazard ratio [hr] 2.14; 95% confidence interval [ci] 1.31 to 3.49; p=0.002) and overall survival (hr 4.30; 95% ci 1.88 to 9.87; p<0.001) in this cohort.10 in addition to differences in methylation profile, epn_pfa and epn_pfb differ by protein expression of trimethylated histone h3k27 (h3k27me3).20,21 a study of 112 childhood ependymomas showed that 100% of epn_pfa ependymomas had h3k27me3 loss defined as staining in less than 80% of cells.20 in contrast, only 1 out of 40 epn_pfb ependymomas (2.5%) had less than 80% h3k27me3 immunopositivity.20 in a later study, two out of 15 epn_pfb tumors (13.3%) showed h3k27me3 loss with immunopositivity in 10% and 60% of cells respectively.22 the specificity for epn_pfb is 100%, but sensitivity is only 86.7% using the 80% immunopositivity cutoff.22 while all 29 epn_pfa tumors showed h3k27me3 loss, the amount of “loss” was quite variable. a majority (62%) of epn_pfa tumors showed h3k27me3 immunopositivity that ranged from 5-50%; another 38% of epn_pfa tumors expressed h3k27me3 in less than 5% of cells.22 our case, which demonstrated an estimated 70-80% h3k27me3 immunopositivity, is in the vicinity of the original 80% cutoff for epn_pfb and is at a level of expression above that of most epn_pfa tumors. the only previous case of ependymoma containing only lipofuscin pigment was reported by malhotra et al. in 2021.2 they describe a fourth ventricular mass in a 16-year-old male that demonstrated perivascular pseudorosettes, ependymal canals, and tumor cells with plump cytoplasm filled with a greyish granular pigment and a high mitotic count (5 in 10 high-power fields in hotspots). similar to the present case, the cells stained positively for pas, but were negative for fontana-masson. in addition, the cells autofluoresced. two 4th ventricle tumors demonstrated the presence of both lipofuscin and neuromelanin. the basis of the excessive intracellular lipofuscin accumulation is unclear. ultimately, the prognostic significance of lipofuscin-pigmented ependymomas has yet to be determined given the small number of cases and limited long-term follow-up. nonetheless, this literature review suggests a generally good prognosis in pigmented ependymoma, especially if gtr is achieved. conclusion here, we present an additional case of lipofuscin-pigmented ependymoma of the fourth ventricle and review the literature pertaining to these unique, uncommon neoplasms. pigmented ependymomas occur most commonly in the 4th ventricle and in middle-aged patients, typically contain melanin, and seem to be associated with good long-term outcomes, especially when gross-total resec-tion is achieved. secondary leptomeningeal melanin accumulations may portend a worse prognosis. further study, molecular profiling, and long-term follow-up are required to better understand the natural history and recommended treatment for this rare central nervous system neoplasm. acknowledgments we thank dr. jack reid for assistance in creating whole slide digital images. references 1. smith ab, rushing ej, smirniotopoulos jg. pigmented lesions of the central nervous system: radiologic-pathologic correlation. radiographics. 2009;29(5): 1503-1524. https://doi.org/10.1148/rg.295095109. 2. malhotra a, rao s, santhoshkumar r, muralidharan n, mitra s, shetty s. pigmented ependymoma of the fourth ventricle-a curious entity: report of a rare case with review of literature. international journal of surgical pathology. 2021;29(1): 80-84. https://doi.org/10.1177/1066896920926700. 3. vajtai i, yonekawa y, schäuble b, paulus w. melanotic astrocytoma. acta neuropathol. 1996;91(5): 549-553. https://doi.org/10.1007/s004010050465. 4. ng th, wong ay, boadle r, compton js. pigmented central neurocytoma: case report and literature review. the american journal of surgical pathology. 1999;23(9): 1136-1140. https://doi.org/10.1097/00000478-199909000-00019. 5. brown nj, wilson b, lien bv, et al. citation analysis of the most influential ependymoma research articles illustrates improved knowledge of the molecular biology of ependymoma. neurosurgical review. 2021;45: 1041-1088. https://doi.org/10.1007/s10143-021-01579-1. 6. boström a, von lehe m, hartmann w, et al. surgery for spinal cord ependymomas: outcome and prognostic factors. neurosurgery. 2011;68(2): 302-308; discussion 309. https://doi.org/10.1227/neu.0b013e3182004c1e. 7. cage ta, clark aj, aranda d, et al. a systematic review of treatment outcomes in pediatric patients with intracranial ependymomas. journal of neurosurgery: pediatrics. 2013;11(6): 673-681. https://doi.org/10.3171/2013.2.peds12345. 8. yang c, li g, fang j, wu l, deng x, xu y. clinical analysis of primary melanotic ependymoma in the central nervous system: case series and literature review. acta neurochirurgica. 2013;155(10): 1839-1847. https://doi.org/10.1007/s00701-013-1810-1. 9. chan ac, ho lc, yip ww, cheung fc. pigmented ependymoma with lipofuscin and neuromelanin production. archives of pathology & laboratory medicine. 2003;127(7): 872-875. https://doi.org/10.5858/2003-127-872-pewlan. 10. ramaswamy v, hielscher t, mack sc, et al. therapeutic impact of cytoreductive surgery and irradiation of posterior fossa ependymoma in the molecular era: a retrospective multicohort analysis. journal of clinical oncology. 2016;34(21): 2468-2477. https://doi.org/10.1200/jco.2015.65.7825. 11. iwasaki y, hida k, sawamura y, abe h. spinal intramedullary ependymomas: surgical results and immunohistochemical analysis of tumour proliferation activity. british journal of neurosurgery. 2000;14(4): 331-336. https://doi.org/10.1080/026886900417315. 12. rawlings ce, 3rd, giangaspero f, burger pc, bullard de. ependymomas: a clinicopathologic study. surgical neurology. 1988;29(4): 271-281. https://doi.org/10.1016/0090-3019(88)90158-9. 13. double kl, dedov vn, fedorow h, et al. the comparative biology of neuromelanin and lipofuscin in the human brain. cellular and molecular life sciences. 2008;65(11): 1669-1682. https://doi.org/10.1007/s00018-008-7581-9. 14. albayram s, urger e, oz b, kafadar a, islak c, kocer n. mr imaging of pial melanosis secondary to a posterior fossa melanotic ependymoma. american journal of neuroradiology. 2005;26(4): 804-808. https://doi.org/15814924. 15. ertan y, sarsik b, ozgiray e, kitis o, dalbasti t, akalin t. pigmented ependymoma with signet-ring cells and rosenthal fibers: a rare variant of ependymoma. neuropathology. 2010;30(1): 71-75. https://doi.org/10.1111/j.1440-1789.2009.01031.x. 16. lyu l, zou l, jiang y, et al. clinical characteristics and treatment outcomes of pigmented tumors in central nervous system: focusing on melanocytic tumors. journal of clinical neuroscience. 2020;81: 83-89. https://doi.org/10.1016/j.jocn.2020.09.009. 17. mccloskey jj, parker jc, jr., brooks wh, blacker hm. melanin as a component of cerebral gliomas: the melanotic cerebral ependymoma. cancer. 1976;37(5): 2373-2379. https://doi.org/10.1002/1097-0142(197605)37:5<2373::aid-cncr2820370529>>3.0.co;2-2 18. ogawa y, watanabe m, jokura h, tominaga t. primary intrasellar melanotic ependymoma successfully treated by combined transsphenoidal and gamma knife surgeries. international journal of clinical and experimental medicine. 2016;9 (1): 371-375. 19. rosenblum mk, erlandson ra, aleksic sn, budzilovich gn. melanotic ependymoma and subependymoma. the american journal of surgical pathology. 1990;14(8): 729-736. https://doi.org/10.1097/00000478-199008000-00005. 20. panwalkar p, clark j, ramaswamy v, et al. immunohistochemical analysis of h3k27me3 demonstrates global reduction in group-a childhood posterior fossa ependymoma and is a powerful predictor of outcome. acta neuropathologica. 2017;134(5): 705-714. https://doi.org/10.1007/s00401-017-1752-4. 21. bayliss j, mukherjee p, lu c, et al. lowered h3k27me3 and dna hypomethylation define poorly prognostic pediatric posterior fossa ependymomas. science translational medicine. 2016;8(366): 366ra161. https://doi.org/10.1126/scitranslmed.aah6904. 22. fukuoka k, kanemura y, shofuda t, et al. significance of molecular classification of ependymomas: c11orf95-rela fusion-negative supratentorial ependymomas are a heterogeneous group of tumors. acta neuropathologica communications. 2018;6(1): 134. https://doi.org/10.1186/s40478-018-0630-1. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. amygdala granular fuzzy astrocytes as lesions preceding development of argyrophilic grains: data from 239 autopsy cases feel free to add comments by clicking these icons on the sidebar free neuropathology 3:18 (2022) letter amygdala granular fuzzy astrocytes as lesions preceding development of argyrophilic grains: data from 239 autopsy cases osamu yokota1,2,3,4, tomoko miki1,2,3,4, chikako ikeda2,3, hideki ishizu3, takashi haraguchi4, akinori miyashita5, takeshi ikeuchi5, shintaro takenoshita6, seishi terada2 1 department of psychiatry, kinoko espoir hospital, kasaoka, japan 2 department of neuropsychiatry, okayama university graduate school of medicine, dentistry and pharmaceutical sciences, okayama, japan 3 department of laboratory medicine and pathology, zikei institute of psychiatry, okayama, japan 4 department of neurology, national hospital organization minami-okayama medical center, okayama, japan 5 department of molecular genetics, brain research institute, niigata university, niigata, japan 6 department of neuropsychiatry, okayama university hospital, okayama, japan corresponding author: osamu yokota · department of neuropsychiatry · okayama university graduate school of medicine · dentistry and pharmaceutical sciences · 2-5-1 shikata-cho · okayama 700-8558 · japan oyokota1@yahoo.co.jp submitted: 15 june 2022 accepted: 21 july 2022 copyedited by: irati bastero published: 27 july 2022 https://doi.org/10.17879/freeneuropathology-2022-4285 additional resources and electronic supplementary material: supplementary material keywords: amygdala, argyrophilic grain disease, artag, blood vessel, granular fuzzy astrocyte, tau cases of argyrophilic grain disease (agd) frequently have astrocytes containing non-argyrophilic tau-positive fine granules in the foot processes sprouting from cell bodies in the amygdala. they were first called bush-like astrocytes by botez et al. [1], and later they were classified as one of the age-related tau astrogliopathies and re-named granular fuzzy astrocytes (gfas) [2]. in our previous studies, the formation of gfas was not simply associated with age alone, and their distributions were influenced by underlying tauopathies [3,4]. especially in agd cases, gfas preferentially develop in the amygdala rather than the frontal cortex and striatum [3]. kovacs et al. previously noted the possibility that gfas in the amygdala might be a forerunner of argyrophilic grains [5]. however, there is no comprehensive pathological data regarding the relationship between the frequencies and severities of agd and gfas in the amygdala, and whether argyrophilic grains develop in the absence of amygdala gfas remains unclear. to address these issues, first, we analyzed the relationship between the presence or absence of agd and the severity of amygdala gfas in a case series. then, their spatial relationship was examined using double staining with the gallyas method and anti-phospho-tau (at8) immunohistochemistry. among a total of 1,166 autopsy cases registered in our database, 474 cases that were evaluated using modern standardized methods, including a panel of immunohistochemistry, modified bielschowsky silver stain, and gallyas-braak silver stain from 2001 to 2022, were selected (supplementary file 1). all cases died in psychiatric hospitals or neurological departments of general hospitals. of these cases, we extracted 263 cases for which semiquantitative data of the at8-positive gfas, as well as the saito agd stage [6] (the distribution of argyrophilic grains in the cerebrum) assessed using the gallyas method, are available. then, 24 cases having progressive supranuclear palsy, corticobasal degeneration, pick’s disease, globular glial tauopathies, post-encephalitic parkinsonism, and myotonic dystrophy were excluded because distinct tau-positive astrocytic lesions were noted. finally, 239 cases were included in the present study. in all of these cases, gfas were semiquantitatively assessed in the frontal cortex, caudate nucleus, putamen, and amygdala on at8-stained sections, using the following grading system (gfa stage): stage 0, no lesion in the anatomical region (i.e., the superior frontal gyrus, caudate nucleus, putamen, and amygdala); stage 1, more than one lesion in the anatomical region but less than one lesion per ×200 visual field; stage 2, one lesion per ×200 visual field; or stage 3, two or more lesions per ×200 visual field. among the 239 cases, 112 had at least one gfa in the amygdala, while 127 lacked amygdala gfas (amygdala gfa stage 0) (table 1). the age at death was significantly higher in the amygdala gfa stage 1, 2, and 3 groups than in the amygdala gfa stage 0 group (p<0.001, 0.0025, and 0.0023, kruskal-wallis and steel-dwass tests). however, the sex ratio, brain weight, braak stage, thal phase, proportions of lewy body disease (lbd) pathological subtypes, and proportions of tar dna-binding protein 43 (tdp-43) proteinopathy pathological subtypes did not significantly differ between the four amygdala gfa stage groups. table 1. demographic data in all subjects stratified by severity of gfas gfa: granular fuzzy astrocyte, agd: argyrophilic grain disease, n: number of cases, nft: neurofibrillary tangles, lbd: lewy body disease, late-nc: limbic-predominant age-related tdp-43 encephalopathy neuropathological change, als-tdp: amyotrophic lateral sclerosis with tdp-43-positive inclusions, ftld-tdp: frontotemporal lobar degeneration with tdp-43-positive inclusions. agd was noted only when a case had at least one gfa in the amygdala; that is, there was no case that lacked amygdala gfas but had agd (figure 1a). on the other hand, cases having amygdala gfas did not always have agd: of all 112 cases having amygdala gfas, 40 cases (35.7%) had argyrophilic grains in the amygdala (figure 1a). the frequency of cases with agd increased parallel to the amygdala gfa stage: 18.7% in gfa stage 1, 61.5% in gfa stage 2, and 75.0% in gfa stage 3 groups (figure 1b). the saito agd stage was significantly correlated with the amygdala gfa stage (ρ=0.5878, p<0.001, spearman rank-order correlation test). the binomial logistic regression analysis was used to evaluate whether the age at death, braak stage, thal phase, frontal lobe gfa stage, caudate nucleus gfa stage, putamen gfa stage, or amygdala gfa stage could be used as possible predictors of the development of agd, and it demonstrated that the amygdala gfa stage (the standardized partial regression coefficient (β), 1.79; odds ratio, 6.63; 95% confidence interval (ci), 3.57–12.32; p<0.001), the age at death (β, 1.34; odds ratio, 1.09; 95% ci, 1.02–1.16; p<0.001), the frontal lobe gfa stage (β, 0.62; odds ratio, 3.47; 95% ci, 1.06–11.33; p=0.0391), and the putamen gfa stage (β, 0.64; odds ratio, 3.10; 95% ci, 1.005–9.56; p=0.0490) were significant independent predictors of the occurrence of argyrophilic grains. figure 1. relationship between the formation of agd and amygdala gfas (a) the relationship between the occurrence of agd and the presence or absence of amygdala gfas. agd was noted only in cases having amygdala gfas. (b) the severity of agd by amygdala gfa stage. the frequency and severity of agd increase with the amygdala gfa stage in presence of amygdala gfas. (c-e) gfas in the amygdala. at8 immunohistochemistry. gfas are often in contact with vessel walls (asterisks). further, argyrophilic grains are scattered around gfas (arrows). (f-h) gallyas method. the amygdala. argyrophilic grains (arrows) are scattered at a distance around blood vessels (asterisks). (i-k) gallyas (black)-at8 (light blue) double staining. the amygdala. argyrophilic grains (black) are scattered around gfas (light blue) contacting the vessel walls (asterisks). all scale bars: 30 μm as previously reported [3,4], gfas with tau-positive astrocytic endfeet were often in contact with blood vessel walls on at8-immunostained sections of the amygdala (figures 1c-1e). in addition, a small number of at8-positive argyrophilic grains were often scattered around the gfas (figures 1c-1e). although gfas are not stained by the gallyas method, a potential association between argyrophilic grains and vessels was also noted on gallyas-stained sections: a few argyrophilic grains are scattered at a distance around vessels (figures 1f-1h). likewise, double staining with the gallyas method and at8 immunohistochemistry showed the proximity of argyrophilic grains, gfas, and blood vessels (figures 1i-1k). noteworthy, these phenomena could be observed only in regions where argyrophilic grains were relatively scarce rather than abundant. the findings presented here support the notion that amygdala gfas may develop prior to the formation of argyrophilic grains in the amygdala [5] and suggest that amygdala gfas may play some role in the occurrence of argyrophilic grains, which are preferably formed in the dendrospinal portion of neurons [7]. these findings also suggest that at least some cases having amygdala gfas but lacking argyrophilic grains may be in the earliest ‘pre-grain’ stage in the pathological process of agd (supplementary file 2). whether amygdala gfas affect cognitive function and psychiatric status, especially in elderly people, also needs to be explored. finally, table 1 shows that some cases had gfas in the frontal cortex, caudate nucleus, and/or putamen even when the case lacked amygdala gfas and argyrophilic grains (amygdala gfa stage 0). based on our previously reported findings [3], this fronto-striatal-predominant distribution pattern of gfas led us to consider the possibility that the case has an early pathological process of progressive supranuclear palsy (psp). for a better understanding of the relationship between argyrophilic grains, gfas, subcortical neuronal tau accumulation, and psp as the pathological disease entity, the sequence of the appearance of these lesions and pathological conditions needs to be further examined. declarations ethics approval autopsy was carried out after written informed consent was obtained from family members, and all experiments in this study were approved by the ethical committees of the okayama university graduate school of medicine, dentistry and pharmaceutical sciences, national hospital organization minami-okayama medical center, niigata university, and zikei hospital. competing interests oy is an editorial board member but was not involved in the editorial handling of this manuscript. the other authors declare that they have no conflicts of interest. funding this work was supported by grants from the strategic research program for brain sciences from the japan agency for medical research and development (amed, jp22wm0425019, jp22dk020704) and grants from the zikei institute of psychiatry. authors’ contributions oy, tm, sht, and set macroscopically and histopathologically assessed all subjects; ci, hi, and th conducted the autopsies; nh, am, and ti administered the brain bank; oy and tm drafted the manuscript. all authors reviewed the manuscript. acknowledgements we thank ms. y. matsuo for her technical assistance. references 1. botez g, probst a, ipsen s, et al.: astrocytes expressing hyperphosphorylated tau protein without glial fibrillary tangles in argyrophilic grain disease. acta neuropathol 1999;98:251-6. https://doi.org/10.1007/s004010051077. 2. kovacs gg, ferrer i, grinberg lt, et al.: aging-related tau astrogliopathy (artag): harmonized evaluation strategy. acta neuropathol 2016;131:87–102. https://doi.org/10.1007/s00401-015-1509-x. 3. miki t, yokota o, haraguchi t, et al.: factors associated with development and distribution of granular/fuzzy astrocytes in neurodegenerative diseases. brain pathol 2020;30:811-830. https://doi.org/10.1111/bpa.12843. 4. ikeda c, yokota o, nagao s, et al.: the relationship between development of neuronal and astrocytic tau pathologies in subcortical nuclei and progression of argyrophilic grain disease. brain pathol 2016;26:488-505. https://doi.org/10.1111/bpa.12319. 5. kovacs gg, xie sx, robinson jl, et al.: sequential stages and distribution patterns of aging-related tau astrogliopathy (artag) in the human brain. acta neuropathol commun 2018;6:50. https://doi.org/10.1186/s40478-018-0552-y. 6. saito y, ruberu nn, sawabe m, et al.: staging of argyrophilic grains: an age-associated tauopathy. j neuropathol exp neurol 2004;63:911-918. https://doi.org/10.1093/jnen/63.9.911. 7. ikeda k, akiyama h, kondo h, et al.: a study of dementia with argyrophilic grains. possible cytoskeletal abnormality in dendrospinal portion of neurons and oligodendroglia. acta neuropathol 1995;89:409-414. https://doi.org/10.1007/bf00307644. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology through the ages – personal reflections: the golden era of neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:15 (2020) reflections neuropathology through the ages – personal reflections: the golden era of neuropathology herbert budka medical university vienna corresponding author: herbert budka · medical university vienna · department of neurology · division of neuropathology and neurochemistry · währinger gürtel 18-20 · 1090 vienna, austria herbert.budka@meduniwien.ac.at submitted: 19 may 2020 accepted: 25 may 2020 copyedited by: biswa ramani published: 04 june 2020 https://doi.org/10.17879/freeneuropathology-2020-2817 the author´s cv has been attached as electronic supplementary material: cv supplementary material keywords: neuropathology, institute of neurology vienna, personal reflections contents introduction the first family schooling why medicine? why neuropathology? the early years the second family going international and something other than work the hiv onslaught a midlife crisis national and international neuropathology the prion decades success and recognition the third family zurich retirement and aftermath epilogue references addendum if i have seen farther than others, it is because i was standing on the shoulders of giants. ---isaac newton if i have not seen as far as others, it is because giants were standing on my shoulders. ---hal abelson introduction at my current age of 73, the ability to see – somewhere into the mid-distance between the quotations above – has been shaped by mentors, colleagues, students and trainees, friends and family. i started to write this since the second week of the covid-19-related lockdown. virtually nobody was seen outside, a bizarre experience that reminds me of my first memories as an infant in post-war occupied vienna when people tried to avoid public encounters, particularly with patrolling soviet soldiers. in mid-march 2020, nobody could foresee how the sars-cov2 pandemic would evolve. as my wife, an active hospital nurse, and our 12-year-old daughter at school had and have some risk to get infected, we agreed to temporarily separate. now i stay in self-imposed isolation with our dog gorry in a small rented apartment in the beautiful vienna woods. like others who, during the present lockdown, have a chance to re-consider their way of life, i have ample time to reflect on my life and on neuropathology. i write this just based on my memories, as most of my written documents are either back in my home or have been destroyed after i retired from my directorship of the (clinical) institute of neurology, formerly neurological institute (ni, obersteiner institute) in vienna. without doubt, the reader will detect in these memoirs the characteristic reminiscence bump of psychology, i. e. the strongest memories date back to adolescence and early adulthood, and emotionally positive memories dominate. however, i consider my whole professional life as extraordinary privilege to have done what i enjoyed most, having made many friends and met great personalities including true giants in medicine, science and research. moreover, i believe to have witnessed the golden era of neuropathology, spanning from rather subjective interpretation of classical morphology to unprecedentedly detailed molecular diagnoses and fascinating understanding of aetiologies and pathogenesis of diseases of the nervous system. i will keep this fascination forever. the first family my family had the almost obligatory migrant background of viennese. probably my later commitment for refugees and human rights was the subconscious recognition of that fact. my father’s budka (meaning small hut or kiosk in slavic languages) family originated from the double town of cieszyn (in polish) / těšín (in czech) or teschen (in german), at the polish-czech border in silesia, then part of the austrian empire. my grandfather migrated to vienna where my father was born. i have only faint memories of my grandfather – the most vivid one is an old photograph depicting him, with a huge moustache and in a typical knee-long body jersey next to a huge and heavy iron bike, as winner of an austrian bicycling championship. another memory is my first visit with him to the prater entertainment park that had just re-opened. in a haunted house train taking us around many horrifying corners that i passed by firmly closing my eyes, a ghost took grandpa’s hat away. grandpa became very angry and tried to hit the culprit ghost with his stick. i was greatly impressed by such courage – even against ghosts! – also resulting in getting the hat back at the entrance. my mother’s panoš family had lived in olomouc (in czech) or olmütz (in german) where she was born. her father was a recognised expert of beer brewing, then as now a classical czech talent, who became employed by the then well-known nussdorf brewery in vienna. my fondness for beer seems to be another subconscious recognition of my heritage. i was born in 1946 in a small village in upper austria where my parents had to relocate after wwii bombs had destroyed their home in vienna. however, we returned to vienna when i was 3, and rented an apartment in an area where most buildings had been either razed to the ground or heavily damaged. for a boy, this was paradise: the building debris of ruins just across the street was an ideal adventure playground for all kids of the neighbourhood, including slopes for first skiing attempts and sledging, hiding places to play “doctor” and have your first smoke – not cigarettes, of course, but twigs of elder bush. retrospectively, i think we were extremely lucky not to have found one of the many explosive war relics. my father worked as a salesman, and compared to others we did relatively well in the 1950s. he was a highly talented piano and violin player, and i still retain recordings of his usual training in bach and beethoven sonatas that accompanied my school homework. moreover, we had weekly visits of friends who played string quartets or piano trios with my father. at the end of his life, he suffered from alzheimer’s disease (ad), and one striking experiment confirmed more recent findings in brain research that the musical reminiscence bump survives longest in ad and arises strong emotions: in the chronic care institution where my father finally lived, a piano was existing, and once i took my father, then already in a very advanced disease stage, had him sit down and put his hands on the keys. as in the old times, he started immediately to play flawlessly, starting with bach, then beethoven and finally switching into a more popular viennese waltz. all other patients and visitors in the hall immediately stopped to talk and listened. after some two to three minutes, my father suddenly stopped and appeared to be confused what was going on. there was thundering applause, a final salute to this great musician, and tears were running down his face. he died soon thereafter. unfortunately, i was much less gifted in musical instruments, but my father forced me to exercise every day at least for one hour, hoping to have me become a famous pianist that was his own but failed life vision. always thinking of something else more interesting during my daily practicing lessons at the piano, i can still hear him shouting corrections like “c sharp, not c” at the doorstep when he entered the house after work. it took me several years to convince him to allow me to stop playing, a decision that i regret very much now. i would love to play mozart or beethoven like i did as a child, but the rigid enforcement by my father erased or blocked all former training imprints in my brain. according to the traditional role model, my mother stayed at home and took care of my elder sister monika and me (fig. 1). moreover, she was a housewife in the famous bohemian tradition, with a cookbook that she wrote by hand after recipes told by her mother, an apparently outstanding cook. i am still hunting for that old bohemian cookbook in our family records, as i have become more and more interested in perfecting my own cooking. fig. 1. the family in the mid-1950s. my father was also a good tennis player, and with tennis he was more successful to get me interested than with the piano. indeed, i became a quite good junior player who could play competitively in a tennis club and even in the austrian boy championship (age bracket 12-14) where i was able to get into the quarterfinals. the tennis league tournament between clubs always included one slot for a junior (up to an age of 18). unfortunately, i was the only junior of my club interested to play competitively, and this already at an age between 12 and 14. at that early age, i already had to compete with 18-year-old players. i mostly lost such matches, as my competitors were much taller and shot much stronger serves than i was able to do. this was one, but not the only, reason why i increasingly lost interest in tennis; the other was my awakening interest in girls that kept me more and more occupied in the later teenage years. schooling my primary school, then of course gender-separated, was also in the heavily damaged quarter where we lived first in vienna, and i was fortunate to get a highly motivated and skilled teacher, mr. pokorny. i had no problems at all at school; already in kindergarten i had learnt unintentionally everything by listening and looking how my sister in her first class learnt to read and write. later, i just listened during school lessons and remembered everything, without a need to repeat. at my age of 11, after the first year in a municipal high school, my father was so proud of my school performance to have me switching to an élite gymnasium, the theresianum that was founded in the 18th century by empress maria theresia for the higher education of kids (of course only boys!) of aristocrats and public officials. at the time, it just re-opened again after wwii with a newly composed team of qualified teachers. it was a private boarding school where about one half of the kids – the interns – lived and slept there and had only occasional permission to visit their families elsewhere in the country. the other half including me – the half-interns – stayed there for the day and went back to the family in the evening, then a highly unusual schooling schedule. focus was on languages: starting with english at age 10, latin was added after two years, french after two more years, and finally russian after one more year. apparently subconsciously, i decided that three foreign languages should be enough – my schoolmates obviously felt the same –, and took russian quite lightly. now i would be most happy if i could read tolstoy in the original version, but all what has remained is the cyrillic alphabet and a beautiful poem by lermontov that we had to learn by heart. we had opportunities to practice all types of sports, including soccer and athletics fields in the huge park, and swimming in an indoor pool. so every early afternoon we rushed out for about two hours, mostly for playing soccer; this was a feeder for my lifelong love for sports and physical exercise in general. however, we also had some 2 or 3 hours of obligatory study time in the late afternoon. we were about 25 boys in our class. while most teachers were real experts in their field, some were less competent in paedagogic terms and even helpless in front of a bunch of merciless juveniles. the same went for the social knit among classmates – there were the usual fluctuating cliques that bullied outsiders. now, after more than five decades, i still feel ashamed not to have intervened then and would want to apologise to bullied classmates and teachers. personally, i tried to steer clear of cliques without becoming an outsider, a habit of independence that i have cherished through my whole life. in 1964, at 18, we had our final exam (matura, abitur) where i got excellent marks. our class quickly distributed to all corners of the world, and i met most classmates again only later at reunions after 50 and 55 years. some proved to have learnt little from life, playing the same clique stereotypes as during their teenage years. astonishing but probably not so unusual. why medicine? we had nobody with a medical background in the family. however, since i was some 10 years old, it was absolutely clear to me that i would become a medical doctor. i cannot remember a reason or triggering event, it was a matter of course. my career in (neuro)pathology started with the section of a sparrow who broke its neck by flying against a glass window, and that of a goldfish found dead in an aquarium. i just wanted to know how the inside looked like, and what was the very reason for death. this hunger for knowledge also accompanied my medical studies. i was totally immersed and – although i consider myself a true product of the 1968 generation in attitudes and spirit – in fact did not take much note of, nor participated in, the upheavals and demonstrations that paralysed university life in 1968. i kept this no-commitment tradition in politics for long, with one notable exception: having just graduated as an md, i participated in a demonstration by doctors for better pay and working conditions. i felt it somehow obligatory to participate, now that i was a doctor myself. a few hundred white-coated people ceremoniously marched down the famous ringstrasse with banners and slogans, but increasingly my attention was drawn to what happened between demonstrators and onlookers. it was impossible to overlook the strong emotions elicited in surrounding spectators, shouting curses at doctors who were considered privileged over common people and earning too much anyway. it was frightening indeed: a spirit of outright hatred had seized many. with a feeling how easy control might be lost, i decided to never again go to the streets, valid until today. medical study at the time had a relatively free schedule; you just had to pass some practical exercises and a defined sequence of about 20 rigorous oral examinations, rigorosa. it was up to you how quickly you proceed. an important first step was the practical course in anatomy, with careful dissection of conserved corpses donated for teaching and scientific purposes. a group of 6 to 8 students worked collectively on one corpse during most of the first year, dissecting muscles, nerves and blood vessels in the smelly atmosphere of the phenol-formol fixative. we also had a special brain anatomy course with some dissection work that i found drab and uninspiring. thank god, this impression did not last. sitting for long daily hours around the corpses to study and dissect, it was the place to get familiar and make lifelong friends like i did, or, for many, to find partners to marry. the second year included histology, and i soon realised that i had some talent for that. i even gave a crash course to my best friend who initially was unable to recognise anything in microscopic preparations other than coloured spots. later he became a radiologist – maybe he unconsciously fled into a predominantly black-and-white world. after two and a half year, students switched to the clinical phase, starting with pathologic anatomy, pharmacology and then all major clinical disciplines. i became aware that i had no favourite field at the time. in contrast, many mates were confident where to specialise. at exams, there were only three marks given, excellent, sufficient, and fail, and it was good or bad luck which examiner you got. i can remember almost every single rigorosum. the first one was in medical physics, after the first year. we were a group of no less than 12 examinees, and it was surprising to me that i got a “sufficient” already after a single question that i answered satisfactorily. i did not consider it relevant enough to make an effort to improve the mark. the next exam was in chemistry, with a professor who was probably the most feared examiner of the medical faculty. he was reportedly proud of having a low pass rate, particularly with female students, in my memory the only gender bias that i encountered when studying. my first question was about coenzyme a, a large molecule that i could describe quite well. then it was me who asked whether i should draw the formula – the professor reacted surprised and agreed. i did it perfectly but was unsure if the professor liked it or not. after quite some more demanding questions, he gave me an “excellent”, my first one in a row until the end, as i could manage to pass all subsequent rigorosa with the best mark. this appeared to be relevant; i had been told that i could become a candidate for a “promotio sub auspiciis praesidentis rei publicae” and be rewarded with employment in a public institution of my choice, at a time when many medical graduates were working as “guest doctors” without salary. unfortunately it finally turned out that the single lower mark in the initial physics exam did not allow such a distinction. however, i made my way without it. at summer holidays, i had some 3 months to spend until autumn when university life would start again. i had bought a small rubber boat with outboard motor and took unforgettable weeks with a group of friends on the greek island of thassos. days were filled with sunbathing, retsina and ouzo drinking, tavli playing, swimming, snorkelling and fish hunting with a handy harpoon, then still allowed in the mediterranean. we even vowed to ourselves to live only on what we would be able to hunt. however, fish were mostly lucky, as we said, in fact much too fast for us, and escaped our clumsy attempts. once we mistook a seagull for a tasty duck and were disgusted to finally have an after-feathering tiny bird to feed four hungry pals. thus the vow had to be broken soon after arrival, and we raided the next inn to engulf mountains of souvlaki meat with tsatsiki. in addition to these summer delights, i worked as tour guide during other holiday breaks. usually i had a busload of austrian tourists, mostly high school teachers, a rather demanding clientele. i was required to execute by myself the local travel organisation with hotels and restaurants, as well as to provide the explanations at sightseeing spots. of course i had to make detailed preparations and wrote my own manuscript for the whole travel schedule; thus i learnt a lot about countries like italy, france, spain, portugal, morocco, usa, mexico and guatemala. a great personal interest in everything connected with history has emerged from these activities. my favourite trips were those to the absolutely stunning sites of pre-columbian cultures in central america, in particular places like the then recently discovered palenque, tikal or teotihuacán, and the unbelievable archaeological museum in mexico city. something else that i learnt during the several weeks of staying together with up to 50 people was group dynamics and how to handle critical situations and conflicts that emerged quite regularly. christmas, easter and parts of summer holidays were spent that way over subsequent years, and i could earn enough money to buy my own car, first a fiat cinquecento and later a volkswagen beetle, something quite unusual for a student at the time. why neuropathology? while it was obvious to me since childhood to have medicine as my lifeblood, the choice of neuropathology was just by chance. my exam in neurology and psychiatry, then still combined and scheduled about half a year before graduation, was decisive for my further career. the professor, a highly respected psychiatrist with a remarkable empathic style, seemed to be pleased by my performance and asked about my future plans; if i were interested in his field, i should meet him again after the end of my study. of course i was more than delighted, as officially recognised training positions in a given speciality were then almost impossible to get, and i envisaged myself already as renowned psychiatrist. soberingly, when i came back half a year later with high expectations, he did not appear to remember and emphasized the existence of a long cue of candidates lining up for still unpaid positions. however, i was categorical in that i would never, never work without pay, as i was still living with my parents and wanted desperately to escape from that crowded setting. hearing that, he asked for my further interests and recommended to make a visit to the neurological institute (ni) where my preference should be met best, in his opinion. the ni of the medical faculty occupied a full floor in a building shared with histology at schwarzspanierstrasse, in a block where most pre-clinical institutes were located, in about mid-distance between the main university building at ringstrasse and the general hospital (akh). since ni was then not involved in teaching to medical students, i had passed by its huge glass-and-white wooden doorway and large lettering of its name many times without entering, when i was on my way to attend histology courses upstairs. none of us students had any idea what was going on behind its doors. anyway, i made an appointment with its director, professor franz seitelberger, and he welcomed me in his office, with his desk at the very end of an impressively large room. he asked about my interests, and i told him that at the moment i was reading a booklet about biological psychiatry. i read there the description of a (chromatographic, i learnt later) “pink spot” in urine that was claimed as specific for schizophrenia. wow, this was it! i was absolutely fascinated – an extremely complex psychopathology condensed into a single spot. what could be more exciting to study?! prof. seitelberger listened patiently and asked me about many additional private topics like family, hobbies, sports etc. that i considered completely irrelevant. finally he said, yes, there is a position available. while he mentioned that my pink spot fascination was in neurochemistry, an area also covered by the institute, he emphasised that i would be required to start in neuropathology, a discipline that analyses structural changes in the nervous system during disease, something absolutely basic to all studies of nervous system disorders. of course i had nothing to object, and we agreed upon my entry to the institute later that autumn. surely, i remembered to have already liked histology during my study, but wasn’t aware then which chance i would get from the very start: it would become an absolutely perfect fit for my interests and abilities – or did neuropathology choose me? looking back on my professional life, i cannot imagine something better suited to satisfy a hunger for knowledge and understanding, to experience case histories to evolve like a thriller, to feed any curiosity, to keep your inner fire burning and to help transmitting scientific fascination to others. and the best thing was that it was not only much fun and satisfaction but – very important for me as a young guy who would soon have his own family – you are even paid for it! the early years fig. 2. bust of heinrich obersteiner, having its traditional place immediately behind the glass-wooden doorway of the neurological institute (ni). my small kids occasionally visited the ni and were greatly impressed by this bust. after asking once to be allowed to tweak the stony cold nose, they did it again every time they visited. after ni’s move to the general university hospital (akh) in 1993, the statue was re-located to the honour courtyard of the university of vienna building at ringstrasse. when i started to work in the ni on oct. 1st, 1971, nobody could foresee that i would stay there uninterruptedly until my retirement, on the day exactly 40 years later, a straightforward but highly unusual curriculum vitae for a scientific researcher. i realised only later the eminence of the ni as the first multidisciplinary institution of the neurosciences in the world. founded in 1882 by heinrich obersteiner (fig. 2) and blossoming under otto marburg until the collapse of the medical faculty by the advent of nazism, it had become a model for such institutes elsewhere, from fukuoka in japan to montreal, new york and philadelphia. absolutely unique was ni’s library that contained an unrivalled treasure of neuroscience books, in particular from the 19th century. it was franz seitelberger’s merit to have re-built the ni after wwii, from practically scratch with only a single half-time position, to a nationally and internationally recognised centre of excellence when he became professor emeritus in 1986. at my entry to the ni, it was a composite of several disciplines led by distinguished scientists (fig. 3). tumour and autopsy neuropathology was led by kurt jellinger; neuromuscular neuropathology by elfriede sluga who mainly worked the ni’s own electron microscope; neuropathology of the vegetative nervous system by gustav lassmann; neurochemistry by hans (hanno) bernheimer; neurophysiology by hellmuth petsche; and neurolinguistics by karl gloning. soon another young aspiring researcher arrived, hans lassmann, to establish a lab of experimental neuropathology. fig. 3. people working at the ni in the early 1970s. 1st row, sitting from left: lassmann sen., petsche, seitelberger, jellinger, gloning; 2nd row: 4th from right, sluga; 3rd row at left auff; last row, 2nd from left budka, 4th bernheimer. unnamed persons include the indispensable workforce of technicians and secretaries. note the distinctive traditional hierarchy, as evident from the arrangement of this photograph, that then imbibed ni’s atmosphere everywhere. in the ni, i was supposed to become kurt jellinger’s assistant and, over some 3 years, was given a unique chance to familiarise with neuropathology in my very personal way, by re-examining the huge histopathological collection and compare my fresh impressions with the available written reports. this autodidactic learning style was well in agreement with both my and kurt’s self-absorbed approach to work. for about half a year, i did not really know how to appropriately re-examine an autopsy case with very large-sized microscopic sections. it took me half a day to microscope such a section, as i mainly used high magnifications and even the oil immersion objective! no wonder that i saw an astonishing new universe of structures of amazing shapes, sizes and colours. after about a couple of weeks of such microscoping, i saw something really bizarre, a rounded body with a strange and multicoloured structure. consulting not only oil immersion but also textbooks, i could not find anything that resembled this discovery. i got very excited and immediately informed prof. seitelberger who, after a shared microscoping session in his office, agreed about the novel character of this strange body. he even suggested to publish it together, and advised me to take microphotographs. of course i had no idea about microphotographing and admitted that. no problem, he replied and pointed to a small mountain of something hidden under a huge dusty cover in one corner of his room. he would do it himself, as that photomicroscope was the very best in the world. i should come back in about one week. so i asked after one week and weeks thereafter, but he always answered to have been too busy to do it. then, there was no need anymore to document the strange body: i found out by myself that it was a corpus amylaceum, an extremely common structure produced by astroglia, occasionally looking a bit unusual. as working space was scarce in and between the lab rooms crammed with instruments and equipment, i was given a desk and microscope within the lecture hall, a huge room with large windows and old-fashioned seating rows (fig. 4). already a postdoc from japan had arrived, riki okeda, a new friend who later became professor of neuropathology at tokyo medical and dental university. another close friend, ferenc garzuly who became head neurologist in szombathely, hungary, completed our trio there. it was much fun – we had small parties, riki used to play the violin, and we took everyday a nap after lunchtime snoring in the seat rows. when i recently told ferenc that i am writing up my memories, he obliged me to mention my first day in the lecture hall when i drew onto the chalkboard a cell nucleus to demonstrate a “wonderful” change, but both ferenc and riki could not detect anything unusual. yes, already on my first day i thought to see something special and difficult for others to see. moreover, according to ferenc, i must mention to have had a green metal box with some gadgets that made enough noise to prevent both of them from sleeping in the seat rows. fig. 4. the lecture hall of the ni in 1991 during an informal meeting. hans lassmann at far left, the author at right in front, thomas berger, the present director of the neurological clinic of the medical university vienna, in rows at right, 3rd from front. indeed, it was a great – and by then rather unusual – tradition to always have several postdocs at the ni, in particular from japan. this started already in the early 20th century when the famous poet saitṓ mokichi stayed for 2 years at the ni and wrote some haiku poems there, including so-called tankas that are limited to 31 syllables. one tanka was written about prof. obersteiner (translated from german to english by myself): "unexpectedly stepped the old teacher next to me with great simplicity he encouraged me." "unerwartet trat der alte lehrer neben mich mit großer einfachheit munterte er mich auf." another tanka was written about prof. marburg: "in front of my eyes stands professor marburg, whom with trembling heart i express my thanks.” "vor meinen augen steht professor marburg, dem ich mit bebendem herzen meinen dank ausdrücke" over several decades, numerous postdocs have been trained in neuropathology in the ni (a long list of names that i can remember is attached at the end of this article). in addition to many japanese colleagues (who have organised a “vienna party” whenever i made a visit to japan), we intentionally kept traditional connections to our neighbouring countries, in particular to those that were behind the iron curtain at the time, such as hungary, poland and yugoslavia. my first student was a bearded guy always wearing black spectacles, pawel p. liberski from łodz, poland, who was interested to study a rare disease named after creutzfeldt and jakob since his first visit in 1979; he became a friend and collaborator during his regular subsequent visits. two more persons to mention are takeshi kurata, professor at the nih in tokyo, with whom i published work on virus detection in brain and who also became a friend and regular visitor until today, and ichiro akiguchi, professor of neurology at the university of kyoto, another regular visitor and collaborator on the neuropathological work-up of the community-based vita study on ageing. he became a friend and invited me several times to stay for up to two weeks in kyoto, in addition to several trips that i made to all parts of that captivating country. this was to pour oil onto my fire for traditional japanese lifestyle and culture such as food and onsen baths, as well as buddhist temples, shinto shrines and amazing japanese gardens. after a couple of years, i was allowed to write histopathological reports on neurosurgical biopsies in kurt’s absence, and autopsy reports on less complicated cases, almost exclusively strokes. i believe there are few neuropathologists in the world (of course with the exception of charles miller fisher) who had the opportunity and gusto to study stroke cases in great detail from the start of their career, and my first book chapter publication was on the neuropathology of cerebrovascular diseases. i am still proud of that early article written in german in the pre-mri era, now forgotten since decades, because it comprises interesting and original data on the infarct patterns resulting from verified occlusions in the carotid-media system (fig. 5), demonstrating high interindividual variability and lack of any predictable correlation between site of infarct(s) and site of occlusion [1]. at that time, i was unaware of the importance 1) to publish in english, 2) to publish in a peer-reviewed journal rather than in an obscure multi-author book, and 3) of the relevance to consider bibliometric data such as the impact factor. fig. 5. topographical patterns of brain infarcts in 196 autopsies with verified occlusions in the carotid-media vascular supply. among the 23 patterns that highlight the eminent individual variability by site and extent of brain infarction, the percentage of the four most frequent patterns (total, subtotal, central and peripheral media infarcts, in sum 71%) are indicated in red. vascular areas: a = anterior cerebral artery, m = media, p = posterior, cha = a. choriodea anterior, cp= a. communicans posterior. modified from ref. [1]. in that early time, one of my few responsibilities was taking macroscopic photographs during brain cutting sessions, taking place every wednesday morning (fig. 6). i made several experiments to optimise that type of documentation: the best background for colour contrast was finally settled as a dark blue, and whole brains and brain slices were arranged on a glass plate some 10 cm above the background cloth, in order not to have any interfering shadows when the illumination was from both sides. fig. 6. brain cutting session with prof. seitelberger in the 1980s. note the numerous fixed brains in glass jars in the back – and the formal ties everybody wore then during work. one more responsibility was to visit the department of pathology every early morning, in order to determine which autopsy brains would be interesting and important enough to be fixed for neuropathology. at that time, autopsies still numbered more than 2000 per year, and not every brain could be fixed and examined. i still remember vividly the boss of the autopsy suite, prof. kucsko, in the beautiful old building of the institute of pathology (now the institute of brain research) where already carl von rokitansky had worked. kucsko was a gaunt chain smoker always with a cigarette butt on his lips during a whole section. he was an extremely experienced pathologist who taught me great respect for the autopsy as golden standard and one of the most difficult tasks in medicine, less with regard to skills but more to generalised knowledge, experience and “feeling”, a talent somewhat difficult to explain by words. another lesson i learnt from him is the importance of a very detailed macroscopic examination – what you do not see or at least suspect at gross dissection, you have little chance to find by microscopy. initially he had little consideration for that young guy interested only in the brain. however, once i told him that i found small cancer metastases in the brain when the body section by him had not found anything. can’t be, he snapped, and went back to the inner organs that fortunately had been fixed and were still available. he called me back in the afternoon to confirm that, indeed, no tumour was found in the body. when i stayed firm with my metastases, he went back again to the fixed organs and finally found, after 3 days of cutting almost every millimetre, a tiny scar carcinoma of the lung. after that i had some reputation with him and was free to express my wishes for neuropathology. this came in handy when kurt ordered me to freeze fresh brain slices from then relatively many creutzfeldt-jakob disease cases autopsied in vienna. i had to send them on dry ice to d. carleton gajdusek at the nih in bethesda, for transmission experiments into non-human primates. i did that without any special safety precautions that then were unheard of, and retrospectively i am not sure whether i touched these brains by bare hand or not. i was later very proud to have contributed some part, even when minuscule, to carleton’s nobel prize in 1976. kurt jellinger left the ni in 1975 for a position of head of a department of neurology in the municipal lainz hospital in vienna where he could do both clinical neurology and neuropathology. of course, this was a severe blow for the ni, but in particular for me, as suddenly all responsibilities for diagnostic neuropathology were mine. kurt was the collector type of neuropathologist who always had large case numbers available for his research. during my years with him, the number of neuropathological autopsies was as high as 600 per year, and neurosurgical biopsies numbered around 1000. only he could handle such a volume alone or with one assistant, and when he left, i – a newcomer with only 3.5 years in business – was almost alone to shoulder such tremendous quantities. i decided quickly to give priority to tumour pathology, and for several years i was unable to keep up with the flow of autopsy cases even when, by necessity, they had to be reduced to a significant extent. to lose kurt at that time was still more painful to me, as i just had entered into clinical training. traditionally all mds of the ni had training in a clinical speciality (to become facharzt or consultant), usually neurology rather than pathology. neuropathology became a speciality of its own in austria only later, a development that i could push forward to a significant extent. to become a specialist in neurology and psychiatry, i started a year of internal medicine in 1974, psychiatry in 1975, and neurology in the following 4 years. it had been agreed beforehand with the directors of the respective clinics that i would do clinical training in the mornings until noon or early afternoon, and then continue my work in the ni where i kept my official position. under these circumstances, it is not difficult to imagine what workloads i had to face when returning to the ni in the afternoon and working into the night. i soon became unsure whether i could, and should, carry on this double burden at all, a feeling of uneasiness and uncertainty that dominated the second half of my 1970s. even under these extremely difficult circumstances, i soon realised that research was pre-eminent in importance for the career. as a relative newcomer, i was not sure in which topic i should invest, in preparation for the next step in career building, the habilitation (lectureship, to become dozent). i had spent most of my year in internal medicine in haemato-oncology, then led by the very amicable prof. josef kühböck, and could witness the successes as well as complications of then modern aggressive and even prophylactic chemotherapy of leukaemia and lymphoma. in parallel, i could collect a small but very interesting and then timely series of brains of patients who unfortunately had died with such complications. thus i published “the pathology of encephalopathies induced by treatment or prophylaxis of neoplastic lesions of the nervous system”, again as a book chapter that i was proud of but became quickly forgotten; however, this time i wrote in english [2]. another potential topic that i considered for habilitation was the neuropathological substrate of collagen vascular disease, again a publication that i was very happy with but went practically unnoticed, even in an american journal [3]. finally, publication of the first description of the adult type of adrenoleukodystrophy (ald), what is now called adrenomyeloneuropathy (amn), became a highly cited paper for the first time [4]. in order to be successful, it would be particularly important to introduce to neuropathology, as performed at the ni, new investigations, such as csf cytology, and techniques such as immunohistochemistry (ihc), in addition to an interesting topic and adequate material to study. i developed csf cytology with a special sedimentation device, examining csf samples that i brought to the ni from patients with leukaemia or lymphoma. we counted the number of cells, sedimented them and made a giemsa stain, later also occasional ihc labellings. for ihc, i started with visualisation of immunoglobulins (igs), because good antibodies were then available, to be used in direct or indirect immunofluorescence. astrocytes were usually full of igs, but soon i realised that no really interesting problem was to be solved with these antibodies. fortunately, at that time larry eng had just purified glial fibrillary acidic protein (gfap) as astroglia-specific marker and raised antibodies against it, and lucien rubinstein – then with larry in stanford – had successfully made first immunostainings on brain tumours. thus the opportunity emerged to introduce ihc for gfap to the institute, at the start still with immunofluorescence, soon thereafter using ludwig sternberger’s elegant peroxidase-antiperoxidase (pap) technique. over the next decades, ihc (and to a lesser degree nucleic acid in situ hybridisation, ish) would dominate most of, if not all, my diagnostic and research work in neuropathology, in particular in neural tumours, infections and neurodegeneration. of course ihc was also essential for neuromuscular biopsies, but in the old ni they were elfriede sluga’s domain, and i entered that field only after she moved, like kurt earlier, to a position of head of a department of neurology in the municipal wilhelminen hospital in vienna. much of this work on new techniques must be credited to the indispensable help of our amazing laboratory technicians who patiently and persistently developed perfect (immuno)visualising systems in pioneer experiments: mrs. helga flicker, ni’s all-time good spirit and helping hand who virtually accompanied my whole career in vienna, mrs. irene leisser who arrived only shortly later, and from the millennial years onwards mrs. gerda ricken. while they kindly agreed to co-author the publication of some of their successes, in fact they should have been on the author ticket of all respective papers. having said this, any thanks to them would be incomplete without the recognition of work that was accomplished also by other technicians, secretaries and support staff – i was extremely lucky to have such people to work with. despite all troubles, my early years in neuropathology ended successfully, in 1980 with recognition as clinical specialist (facharzt) in neurology and psychiatry, a step that also promoted me from assistant to consultant (oberarzt), and further on in 1982 with my habilitation (lectureship) in neuropathology. the title of my habilitation thesis was “viral diseases of the nervous system” and combined 3 original papers [5-7] on viral antigens in herpes simplex virus (hsv) encephalitis, rabies and subacute sclerosing panencephalitis (sspe). i was more than happy that the exhausting time of riding two horses at the same time has ended, even when i liked clinical work very much. so it was only later, during my midlife crisis, that i considered to go back to clinical medicine. the second family the turbulent first decade of my professional life coincided with establishment of my own new family. i met christine at the csf lab of the neurological clinic where she worked as technician, and we married one year later. philipp was born in 1974, julia in 1977, daniel in 1979, and rafael in 1980. with these kids born in a short period, my wife had stayed at home and it was not that easy for me, as university assistant with a very mediocre salary, to sustain a family of 6. our financial situation improved somewhat when prof. seitelberger allowed me to make forensic expertises that are remunerated additionally. that activity turned out to be demanding but most rewarding intellectually and scientifically, and i have continued it up to this day – something that always has enabled me to keep my fascination for neuropathology alive. with all my obligations, it was very difficult to spend time with the family. i devoted all weekends and holidays to them, but during the week i used to come home from work when most if not all kids were already asleep. we tried to remedy the situation by establishment of a weekly family day when i would come home earlier, play, do storytelling and reading with the kids. nevertheless, i used to have a guilty conscience about my inability to dedicate enough time that a family with children would deserve. moreover, we always had dogs that became full family members as well, the wire-haired dachshund ilko at start, followed by polski owcarek nizinny (polish lowland sheepdog) betsy and later hovawart (“farm watcher”) debbie. although dogs snip away further bits from your time, they are beautiful and most grateful creatures enriching and rounding up any family; they love you unconditionally, whether you deserve it or not. all my kids grew up to become successful professionals and, more importantly of course, happy, amicable and easygoing personalities to be proud of: philipp became an established lecturer and researcher in social and cultural anthropology at the university of vienna and free university berlin, specialising in the sociocultural consequences of digital media and technologies, living in a harmonious partnership with julia, an online media editor. my daughter julia became an internationally highly respected egyptologist who repeatedly has performed excavating campaigns in egypt and sudan and has won two extremely competitive erc research grants; she is now professor of egyptology at the ludwig-maximilian-university munich and in a partnership with tanja, a kindergarten supervisor. i admire and envy her, as she is rapidly running up to the number of entries on the internet that i have. daniel became an expert, art-prize-winning graphic designer and video producer, happily married to caroline, a hospital physician; they have delightful ellie and are expecting their second child soon. rafael succeeded in establishing his own media and marketing company to organise various types of events, from business celebrations to pop concerts, living in partnership with nathalie, a journalist, and their two gorgeous girls lori and noemi. looking back when getting old, it is most gratifying to see a sequence of generations that carry on the spirit and ideas that you have valued highly during your life, even when my small wish, to have one of my kids enter medicine, has not realised (but i still have such hopes for my fifth child, natalia). the difficult family years had one casualty, as it turned out only later. my marriage broke apart, fortunately only when all kids had already left our home. certainly a divorce, and mine was a bitter and battled one, is a sad event in any life. however, to quote paul watzlawik, there is nothing bad in life that doesn’t have something good as well: my longstanding guilty conscience about the inability to dedicate enough time to the family fell apart quickly, and i suddenly felt completely free, a feeling of happiness and contentment that i had missed since decades. of course it is a very selfish thing to be back to independence, but my advice to everybody is to listen to one’s very gut feelings how you really sense your being. i discussed the situation that i went through with friends, and some of them had similar constellations; a couple of them did the same as i – and these made identical experiences. in the following years i was free to work as i wanted, to travel around as i wanted, and to have no private commitments at all. the most important matter, however, was continuation of my good relation with all children despite the divorce. going international and something other than work totally focused on work and a growing family, i had neither time nor interest for the many distractions young people had at the time. a somewhat more relaxed schedule was possible when i went to scientific meetings and congresses outside of vienna. from early on, i tried to attend international congresses of neuropathology (icns), the biggest event in our field. my first icn was in 1974 in budapest, and the technique that dominated most investigations was electron microscopy, with “virus-like structures” almost everywhere. brain tumour pathology, for the very last time, had retained a strong influence by spanish and latin american researchers using sophisticated gold and silver techniques as basis for classification – we dubbed a symposium chaired by the famous professor moises polak in budapest as “metal mining” meeting. within the few following years, the centre of gravity shifted to the anglo-american tumour tradition, mostly represented by the landmark book editions by dorothy s. russel and lucien j. rubinstein, then the bible and only comparable in impact to the present who classification of tumours of the central nervous system. when the icn in 1982 was held in vienna, i was charged with organising a tumour slide seminar together with lucien. it was an unbelievable luck and privilege to meet, discuss and work with such a giant. i visited him in charlottesville, va, well before the icn in order to select interesting candidate cases to present. what impressed me most was his memory with the incredibly huge collection of ”similar” cases that he had at his fingertips – whatever you showed and asked, he immediately answered convincingly with demonstration at the microscope of a similar case. by the way, he was equally impressive as an impeccable british gentleman (although originally from belgium), almost seeming misplaced in the us (he could have been the topic of sting’s song ”englishman in new york”, in particular the quote “if manners maketh man, as someone said, he's that hero of the day”), as a true polyhistor with amazing knowledge of art (he decorated his home with stunning huge tapestries by jean lurçat), and as husband to a most charming and warm-hearted wife, mary m. herman, also a neuropathologist and his closest collaborator, with particular em expertise. in addition to icns that are only held every 3 or 4 years, i regularly attended annual meetings in austria’s great neighbour, germany, i.e. those of the german society of neuropathology and neuroanatomy. in addition to a much broader scientific scope than what we had in small austria, those meetings were nice social events to meet new and old friends, in a relaxed atmosphere that sometimes culminated in rather hilarious sessions, unexpectedly in business sessions of society members. i remember some colleagues with unbelievable humour, such as an extremely witty guy from frankfurt, prof. thomas. everybody was already holding back laughter when he started to speak and made bizarre suggestions. one of the early german meetings that i attended, in 1979, was held together with the polish society of neuropathology in cracow. i went there by car, then quite a trip across countries behind the iron curtain, and highways were still non-existing on that route. i went there also to have a glimpse of cieszyn / těšín / teschen from where my grandfather had migrated to vienna. i arrived there late in the evening, and the impression was appalling, as the town appeared completely dark, depopulated and dilapidated; in my thoughts i congratulated my grandfather to have escaped from there. for fairness: when i came back 40 years later, it was a very nice and well-maintained central european town with a huge historical main square, deserving a visit. the meeting in cracow was nice, the city very beautiful and full of history, but i looked also elsewhere and soon found out that auschwitz was close. i thought that it would be obvious, even obligatory, to make a visit to such a historical site nearby. therefore i asked at the german society members’ business meeting about the tour that certainly would be organised there and whether a place was free for me to join. totally naïve as a youngster who was spared from the horrors of the nazi period by the blessing of late birth, i did not expect that my question would raise any nerve, but it did. there was icy silence, and nobody wanted, dared, or was interested, to answer. my god, i thought, this was then at least 34 years ago. good lesson for my naïvety. it was only later that i learnt of the involvement of some austrian and german neuropathologists in nazi atrocities, but also of the admirable efforts by some, in particular by prof. jürgen peiffer from tübingen, to elucidate and document the disastrous deviation of our discipline during that period. twenty years later, after i became director of the ni, i had to re-visit and clean up such involvement in our own backyard. to some degree, the visit to auschwitz changed my life. the main camp, auschwitz i, had been restored as a museum that was well maintained and diligently documented, with all the horrors of gallows, execution walls, mountains of hairs, spectacles or children’s shoes, all difficult or impossible to stomach. however, the extermination camp proper, auschwitz ii – birkenau, was different; it was kept as it was, without any restoration, additions or changes, giving it a surreal atmosphere of the still present, villainous evil as well as persistent agony. i spent the whole day there, wandering around completely distraught, aimlessly and disoriented, sometimes hallucinating victims’ whispers. it took me a few days to get over this experience, and i decided for myself to contribute my very own, very personal, however small, part to have that never to happen again. back to vienna, i identified amnesty international (ai) as perfect fit for my abilities and intentions, and founded the first ai medical doctors’ group there. ai’s remit is to fight human rights abuses, torture and death penalty everywhere in the world. then it was still cold war – the fight against lawbreakers and offending countries was three-tiered, politically balanced between the west, countries behind the iron curtain, and those in the developing world. medical doctors had a definite role in that fight. as examples, our group documented objective signs of torture in victims who were lucky to have escaped to austria during and after the iranian revolution or from latin american dictatorships. i also participated in missions that investigated and reported on conditions of prisons in countries accused of abuses in jail, another disturbing personal experience. since ai does not provide direct treatment to affected people, siroos mirzaei, a radiologist and amicable friend with iranian roots, and i decided later to proceed with an organisation called hemayat (meaning help or protection in arab and persian) that was to provide free psychological and physical treatment for victims of torture in vienna. the balkans war of the 1990s created many, often too many, victims in need of help. in this year, hemayat celebrated its 25th anniversary – and is still necessary to exist, maybe now more than ever. the hiv onslaught by the first half of the 1980s, i had investigated the regional and cellular tropism in a range of viral infections involving the nervous system, but nobody had previously assumed that this knowledge would become very helpful when, since 1981 in the usa and then in europe, a new disease appeared apparently out of the blue, acquired immune deficiency syndrome (aids). in austria, we had our first autopsies from early 1983, and we were well aware that appropriate safety measures had to be applied, although only later that year luc montagnier (with françoise barré-sinoussi, also sharing with her the nobel prize in 2008) isolated the culprit as lymphadenopathy-associated virus (lav), or htlv-iii as bob gallo called it, or human immunodeficiency virus (hiv) since 1986. i could meet and listen to these two eminent researchers, both of whom gave the immediate impression to be characters as different and irreconcilable as possible to imagine, maybe one of the reasons for their bitter fight for recognition as hiv discoverers and exploiters. when i had examined a small series of aids brains, i realised there was something going on in addition to the by then well known opportunistic infections, an inflammatory process with multinucleated haematogenous cells as hallmark. i published that in 1986 [8], after having described with paul kleihues from zurich another characteristic type of pathology, a progressive diffuse leukoencephalopathy [9]. in those years, i was busy with presenting my hiv data at lectures and meetings. after such a meeting in germany, a well-known professor took me aside and asked: “well, i also have a series of hiv brains but have never found the multinucleated cells you describe!”. i replied by suggesting to show me slides of whatever case he wanted to discuss, and it was perfect luck for me to find almost immediately these cells in his own slides. “oh, they look like this, i didn’t expect that!” was his reaction. yes indeed, these cells may look somewhat strange occasionally and differ from other types of multinucleated cells. one lesson that i took from that and similar situations was my absolute belief in the old wisdom that there is a lot to see when you look, and in old-fashioned, analogue microscoping for training, when one can play around with focus over several planes, something that is difficult to impossible to experience with simple pictures or even more advanced digital slide processing. so up to today, i refuse to give diagnoses just on cabled images. exactly at that time i met gianriccardo trabattoni, an extremely big-hearted neurologist from parma, italy, having striking similarities in physiognomy and humour with woody allen. he asked whether i would be interested to study autopsy brains from the aids epidemic in italy. an enormous tragedy was then occurring in milan, with many deaths every day. now this sad history repeats itself there, with the difference that sars-cov-2 kills preferentially old people, whereas hiv killed indiscriminately, including many youngsters. yes, no question, i was interested to study those brains, and gianriccardo spent subsequent weeks in smuggling caseloads of formol-fixed brains in jam glass jars by rail across the brenner frontier from italy into austria. at that time, there were still border controls, and i wondered what would happen if he would have had his luggage controlled – i had nightmares of press headlines roaring “band of brain smugglers arrested” or, still worse, “scientists uncovered as graverobbers”. almost immediately we had several dozens of brains, and in 1987 we published our combined neuropathological experience from 100 aids autopsies, including most beautiful em photographs of hiv virions provided by silvia cristina, a very gifted pathologist [10]. the next step was visualisation of hiv products in the brain, as i had done previously with other viral infections, and detailed determination of cell tropism in the brain [11]. as already suggested in my work on the multinucleated giant cells of hiv encephalitis, productive infection was seen exclusively in microglia and macrophages, a new paradigm of infection and inflammation in the brain, with neural elements proper not participating in virus replication and production. however, neurons are secondarily affected: in collaboration with prof. haug’s neuroanatomy group in lübeck, we demonstrated by morphometry neuronal loss in aids brains [12], in addition to hiv encephalitis and leukoencephalopathy another substrate of hiv-associated neurological disorders (hand). a midlife crisis my career in neuropathology was successfully under way when, almost exactly at midterm of my period in the ni and at the age of 44, the confidence in my future was shattered. in 1990, i had two invitations for meetings in india, one to speak at the world neurology congress in new delhi on hiv and the nervous system, and another to contribute to a specialised satellite symposium on sspe at the famous christian medical college in vellore, a more than 100 years old foundation in a city in the south, originally for women only, with an affiliated hospital, by now one of the premier such institutions in india. whenever participating at scientific meetings, i always try to squeeze off a few days for my own sightseeing, on and off the beaten track, in particular when it was the first time for me in that area. then it was my first time in india, and i was shocked to verify myself the amount of poverty and suffering on the streets, with people lying and dying on crammed sidewalks in the north. to some contrast, the south seemed better off, with less pressure by sheer numbers of people and a friendly tropical climate. the meeting in vellore was organised by dr. jacob john, already then an eminent virologist who fortunately makes his expert opinion still heard now on covid-19. i was much impressed, but still more when he took us foreign participants to the hospital ward to meet patients with infections in india. as neuropathologists, usually we do not see individual fates and histories behind our fascinating stains and slides; so first-hand experience by meeting patients with diseases of our study topics is a completely different dimension. they had several boys with sspe in the ward, for most of us now an interesting but extremely rare if not exotic condition; however, i would wish that anti-vaccinationists could see the devastating and deadly disease that is preventable by measles vaccination. other interesting cases included what i discussed with dr. john as tropical spastic paraparesis, then a new retrovirus-mediated disorder, tuberculoma and many unclear conditions. i became speechless seeing all that. apparently this experience well coincided with what was emotionally my midlife crisis – i spent the following nights and days seriously considering to drop out of my safe harbour of academic work, career and family life and start again from scratch with clinical work in a place like southern india, where your personal contribution to alleviate sufferings could really make a difference. i turned pros and cons in my mind, realising that the youngest of my kids was already 10 years old, so it would be not that damaging for their upbringing if i would leave, whereas their economic future might become uncertain. the longer i pondered these considerations, the paler the memory of south india became, and finally these ideas faded away. apparently i had no guts for a complete new start, and it was much more convenient to follow in the prepared footsteps of one’s comfortable life. do i regret it? i am not sure, even now when i am very happy with my life track, and probably any present thoughts on what-would-have-been are more coquetry than deep-hearted wishes. national and international neuropathology the 1990s were an important period for the old ni. after more than 100 years, its facilities moved in 1993 from the old building at schwarspanierstrasse to the newly built university hospital, the new allgemeines krankenhaus (akh) where ni was renamed as clinical institute of neurology (kin) and located in the ground floor at the southeast corner of the enormous main building. i had been involved in planning for the new location since more than a decade and was proud to have achieved a consented plan offering unique facilities for the future. the move itself was one of my worst experiences, and i commuted for weeks from the old to the new building. kin was provided in the new quarters with generous space and top-class laboratories featuring most modern equipments for neuropathology (including an own em and tissue culture), neurochemistry and molecular neurobiology. what was still missing i could manage to have added in later years, such as a dedicated prion bsl-3** lab, laser scanning and confocal microscopes and laser microdissecting device. the downside was that the other multidisciplinary but non-clinical parts of the old ni were not transferred as well, but moved to a brand-new institute of brain research in the completely refurbished building of the former institute of pathology, with hans lassmann appointed as professor of neuroimmunology and first director. for the clinically working parts of the old ni, now the kin, the environment of a modern university hospital turned out to be most beneficial, not only for diagnostic service, but also for research and teaching. after i became full professor and director of kin, i followed the research output by publications year by year, and from very modest scores in the early 1990s, a regular increase of cumulative impact factors per year became evident, surpassing 100 in the last years that i served as director until 2011. the move to the akh was also favourable for teaching. although ni had been involved in some practical courses to undergraduates, kin became increasingly involved with teaching lessons for clinics of neurology, psychiatry and the institute of pathology. usually selected topics of the neuropathological spectrum of diseases of the nervous system were lectured, but i always tried to include some of my very own concerns for future mds. one general was functional neuroanatomy that i found rather underrepresented in students’ knowledge and understanding, and another specific was brain trauma, in particular by voluntary activities such as boxing, with the aim to inflict – or at least tolerate – brain damage to others, an issue that should prevent its performance as sport. such new success did not happen without shadows. when describing above my early experience at a german congress in poland with regard to a visit to auschwitz, i mentioned also the need to clean up the institute’s backyard. it was only very late, around the turn of the millennium, that efforts were made by the university of vienna and its medical faculty (from 2004 the new medical university of vienna) to finally clarify, document and purge any remains of the nazi past. for the institute, it turned out that the neuropathological archive contained brain samples, mostly processed histological slides and blocks, of “handicapped” persons in mental institutions, mostly kids, who had been murdered within the nazi “euthanasia” program. in vienna, the psychiatric institution “am spiegelgrund” was the place where these killings occurred, and brains of victims were collected and neuropathologically examined – later unbelievably supported by public funds – by heinrich gross who had also a role in the killings. for further research, some samples were later brought by gross to the ni, and much later i had to identify and de-archive such specimens that were then ceremoniously laid to rest in a dedicated memorial tomb at the vienna central cemetery. additional data by jürgen peiffer from tübingen revealed that brains with a peculiar neuropathological disease from three brothers, murdered in the landesanstalt görden in brandenburg, germany, were first described by franz seitelberger as connatal type of pelizaeus-merzbacher disease (known today to be due to missense mutations in the plp1 gene) in his thesis for habilitation. the samples had originally been archived in the max-planck-institute for brain research in gießen, germany, headed by julius hallervorden who had been heavily involved in the neuropathological work-up of brains of victims of nazi “euthanasia”. seitelberger had spent a postdoc stint in gießen and brought these samples to vienna and the ni. again, i identified these samples in the archive and sent them to hamburg where they were ceremoniously laid to final rest. as a small discipline, neuropathology relies very much on international contacts, interdisciplinary discussions, connections with colleagues and friends, and communication at meetings. having realised this soon, i have fostered such contacts by personal visits, invitations and correspondence. in addition to diagnostic matters, it was research that was, and still is, most in need of international networking. an overlapping field encompassing both diagnostic work and research like neuropathology has classification of disease as a necessary but also possibly arbitrary component of biomedical science and practice. nowhere this was more evident than in the field of pathology of tumours of the nervous system that has been based on histogenetic concepts since long but clouded by complexity over generations. during medical studies, most of my colleagues just skipped the several pages of brain tumour types in our learning documents, as the classification given there was considered counterintuitive, intricate, confusing in places and simply too cumbersome to absorb. this fact has been recognised by the who that reacted by publication of the blue book series on tumour pathology. the 1st blue book on “histological typing of tumours of the central nervous system” was edited in 1979 by klaus joachim zülch, an eminent neurologist-neuropathologist in cologne-merheim, and appeared, at least to outsiders, as compromise between zülch’s classical german tradition of neuropathology and the anglo-american school represented by lucien rubinstein. as example, it included the monstrocellular sarcoma, one of zülch’s dearest entities as sarcoma because of a distinctive network of reticulin fibres, as well as the monstrocellular glioblastoma championed by rubinstein because of gfap immunopositivity, both the very same tumour type. thus most of the complexity remained. as i had to diagnose large numbers of neurosurgical biopsies, i applied my immunohistochemical expertise from my studies on viruses to cns tumours and always tried to keep abreast of current trends. much credit is due to paul kleihues, then professor of neuropathology in zurich, to make an effort to renew brain tumour classification in a modern system that took account of molecular and genetic characteristics and did not only rely on morphology. a meeting was convened in zurich in 1990, with 26 distinguished experts in neurooncological pathology, almost all male, including me, and only two renowned female researchers, lucy b. rorke and ana lia taratuto. one giant was notably missing – lucien rubinstein had died from brain haemorrhage briefly before. the result of intense deliberations – to include new entities, in particular the concept of primitive neuroectodermal tumour (pnet), and delete obsolete ones like the monstrocellular sarcoma – was finally published in 1993 as 2nd edition of the blue book. the 3rd edition in 1997 and its update of 2000 followed meetings in lyon in 1997 and 1999, were re-titled as “pathology and genetics of tumours of the nervous system”, had expansion on genetics and were published by the international society of neuropathology (isn) together with the international agency for research on cancer (iarc) that was then directed by paul kleihues. the 4th edition in 2007 and its revision in 2016 returned to the title “who classification of tumours of the central nervous system” of the old blue books. all these editions proved to be an indispensable tool for neuropathologists not only in their daily diagnostic work, but also in use of a common language with clinical colleagues and basic researchers. i have contributed to all editions and was mostly happy with the outcome, with one exception: i remained sceptical about the scientific rigour and value of the who grading system that applied four uniform prognostic categories across all types of tumours and was carried on unchanged since the very start in 1979. we had heated discussions at all meetings on that issue, but paul was a passionate and dominant defender of who grading. moreover, he was clever enough to put discussions on this topic to the very end of the agenda – usually there was not enough time left for debates in depth. based on my experience with tumour neuropathology, i realised the importance of working with established national and international bodies with regard to diagnostic matters, research and fostering of the discipline. thus i have served for several periods as president of the austrian society of neuropathology. in europe, the supranational european confederation of neuropathological societies (euro-cns) is the contact for professional matters, and i became president in the early 2000s. the international society of neuropathology (isn) is the global representative of our discipline, and i have served as national councillor, as vice-president and finally as president from 2010 to 2014. many scientists have disgust for such officer honours and “committology” in general, as they feel it much more rewarding to spend time in the lab than in discussions at formal administrative meetings. indeed, such meetings may produce little more than hot air, but my experience tells it depends on you – yes, on you in whatever function you attend – how productive or not your participation will be. moreover, don't forget: if you don’t commit yourself to meetings, there is always a possibility of non-transparent agreements negotiated in back rooms by parties attempting to over-rule others, something nobody wishes (or claims so…). the prion decades in the early 1990s, i realised that the type of hiv neuropathological studies, as i had done for almost a decade, was unlikely to contribute much novelty to our future understanding. so where to look next? in the field of infections, a new paradigm was emerging that overlapped with neurodegeneration – something called prion, an infectious proteinaceous particle, a name coined by the later nobel laureate stan prusiner for agents causing transmissible spongiform encephalopathies, then still hotly debated and regarded by many as viruses or virus-like agents. in austria, we had a disease dubbed as “austrian kuru” by franz seitelberger who studied the neuropathology of several cases of a family condition first described by josef gerstmann, ernst sträussler and i. scheinker in 1936, now usually abbreviated as gss. scheinker's first name initial would mean isaac, as i found out, a name not appropriate in germany under nazi rule. featuring prominent amyloid plaques, the pathology indeed resembled that of kuru, the new papuan disease that d. carleton gajdusek transmitted to chimpanzees, like he did later also with creutzfeldt-jakob disease (cjd). again, i stepped into that research field more or less by chance – or by another good luck? fortunately, i had some reputation with the then professor of neurology in the university hospital vienna, herbert reisner. he sometimes called for my opinion when he suspected strange and rare clinical cases. as example, i could diagnose the first clinical case of progressive supranuclear palsy, a disease then practically unheard of by clinicians in vienna. once he even took me as company when he got a call from the veterinary university to consult on a horse with a strange gait disorder. we came to the stable holding the horse, to have a look how the animal moved. the horse was there, but prof. reisner not – he had immediately hidden behind the door and told me to examine the horse in his absence. i learnt later that, possibly as sequel of war trauma, he hated horses and was terrified by them. the horse suffered from what veterinarians called a “wobbler”, a yet rather unspecified ataxic condition, and i had to decide myself what to recommend. guess what my, a neuropathologist’s, recommendation was? euthanasia and neuropathological examination to get a final diagnosis, something that turned out not to be feasible because of severe squeezing artefactual damage of the thick equine spinal cord when the mortuary assistants pulled it out like a rope. in 1990 prof. reisner asked me for an opinion on a rare case that some considered as cjd. the middle-aged lady had indeed symptoms and signs indicative of cjd, but additional spinal long tract signs were unusual, and i had a yet unspecified feeling of something very special. normally, i never think of it, but here i looked for the maiden name of the patient. bingo! it was “h”, the name of a family that i knew very well from earlier studies of important cases in the archive of the ni – i had discovered a new case of the original austrian gss kindred! it had been completely lost to follow up since some 30 years, as family members were dissatisfied by their experience with medical doctors who were unable to help them; some even told them to belong to a “syphilitic family”, since spinal long tract signs are characteristic of tabes dorsalis. in the small villages south of vienna where most of the family lived, this was not something to like to hear or speak about. anyway, the re-discovery enabled us to complete the full pedigree to 221 members in nine generations from the late 18th century onward, with at least 20 patients suffering from definite gss, our last proposita presenting a switch from classical gss to a cjd-like phenotype [13]. we tried over the following years to find out why and how a disease with the very same genetic aberration in the same family was able to change the phenotype, but this has remained elusive. anyway, it was reassuring to find the salient p102l mutation in the prion protein gene prnp also in the original gss family, in collaboration with hans kretzschmar from munich [14]. from 1994 onwards, initially well before the advent of variant cjd (vcjd), i succeeded in coordinating a series of large european networking projects funded by the european commission (ec). the aim was to establish and apply diagnostic criteria in the neuropathological assessment of human prion disease, to determine their clinicopathological phenotypic spectrum and work on specific research issue related to tissue pathology. these projects perfectly complemented other ec projects coordinated by bob will from edinburgh on clinical surveillance of human prion diseases. it was a pleasure and privilege to collaborate with bob for many years on these challenging issues, and to make many good friends in the prion surveillance system, including heino diringer, paul brown, james ironside, maurizio pocchiari, fabrizio tagliavini, inga zerr, pawel liberski…. the scientific collaboration within such networks was strengthened by meetings at regular intervals; my own ec projects had their meetings usually in the small town of baden near vienna, a historical spa type of place popular already in the austro-hungarian empire. the huge bonus of these meetings were the limited number of participants (usually between 50 and 100), lectures on timely and emerging prion issues by top researchers followed by enough time for detailed discussions, and half a day reserved for socialising, walking and hiking. following these simple rules, these meetings have become almost legendary in retrospect – whenever i meet people who had attended, it becomes a nostalgic exchange of memories. baden is amidst a wonderful, wine-growing landscape at the very eastern end of the alps, where pleasant hills of the viennese woods come down to the pannonian planes, so hiking there was always a most enjoyable activity – and sometimes a true escapade to remember. as the baden area was not too familiar to me and we had to keep our time schedule, i wanted somebody to guide us on our hiking tours. i have a friend who lives in the area, erich, a retired high school teacher who described himself as knowing every rock in the vicinity and was enthusiastic to take over the organisation and guidance of our hike. it was an unusually hot day in early june, and the first part of the track was steeply uphill for about 45 minutes. erich calmed down early wishes for a break by promising a mountain lodge on top. when we arrived, thirst was significant, as was disappointment: the lodge was closed for restauration. no problem for erich: as he knew all hidden details of that part of the forest, he promised to get us to a spring with wonderful fresh water. of course, in order to get there, we had to leave the marked trail and step into the dark forest. i had advised everybody in the 40-plus-numbering group of hikers to wear strong walking shoes, but some ladies apparently misunderstood that as invitation to test the most modern and glitzy slippers then available. as to expect, it was not that easy for such a poorly equipped party to walk across thickly covered forest floor. finally arriving exhausted at the spring, another disappointment and still more reason for thirst: the spring had dried up, and erich had some trouble to explain why he had not been aware of an unusually prolonged dry period in the immediate past. feelings of despair were palpable, and i decided in a revolutionary mood to take command. i phoned our bus driver, ordered him to buy a supply of boxes with hectolitres of water bottles and come to the nearest parking spot where a bus could go. it was still some more 45 minutes to walk there, but fortunately downhill below shady trees, so a full rebellion by the party could be avoided by repeated announcements how near the bus parking already was. when we arrived at the bus, exsiccated and with ragged clothes, everybody offered a fortune for a drop of water. i threw away my chances to get such a fortune, like always, and gave the water for free. finally, whatever worries people had, they disappeared suddenly and were replaced by everlasting memories of an exciting adventure. the work on human prion diseases coincided with emergence since the late 1980s of a new animal disease, bovine spongiform encephalopathy (bse), aka mad cow disease. hundreds of thousands of bovines were affected, had to be slaughtered and disposed of in the uk. this economic disaster accompanied loss of public confidence in politics, a phenomenon that spilled over also to continental europe where bse became a problem only delayed, from around 2000. in 1996, the high water mark broke when uk government officials had to concede that bse had transmitted to humans in form of another new disease, variant cjd (vcjd). proof for that, of course, came by neuropathology: james ironside in bob will’s group demonstrated characteristics of a new prion neuropathology. immediately, some hysterical press releases forecasted an armageddon of millions of victims, and feverishly reactions and solutions were deliberated, in the uk as well as in the european union (eu). this was the hour of experts – however, at the time few were there, as prion diseases had been the domain of a small group of basic researchers in biochemistry, of veterinarians working on scrapie, the prototype prion disease in small ruminants, and of medical specialists in epidemiology, neurology and – you are right again in guessing – neuropathology. surveillance for human prion diseases became quickly established throughout europe and needed neurologists, laboratory specialists and neuropathologists. in austria, the austrian reference centre for human prion diseases (örpe) was formally established at the ni and funded by the ministry of health. örpe served as national focal point as well as representative for international contacts in the quickly enlarging prion arena. i was head of örpe from 1996 until 2012 when i left to work in zurich. at the time of the bse/vcjd meltdown, the top scientific advisory body in the eu was the scientific steering committee (ssc) that quickly established its own prion advisory panel, the tse/bse ad hoc group with about 20 members with experience in all aspects of these diseases. i had the privilege to be chosen as member, and from 1997 we had meetings in brussels every month or even more often. for preparation of these meetings we were sent exhaustive documents, sometimes numbering hundreds of pages. the pressure was considerable, but the panel was highly successful to recommend a series of comprehensive measures to mitigate the risk, most notably with regard to the safety of animal-derived products, identification for destruction of specified risk materials in the food and feed chains, quantification of the residual bse risk in food, and country-wise evaluation of the geographical bse risk according to a standardised set of parameters [15]. it was a most interesting experience, as everything had not only public health and economic, but also political implications. in contrast to some members of the ssc, the tse/bse ad hoc group appeared to me more reluctant and cautious, and in the group i worked most closely with dominique dormont and hans kretzschmar. they were not only wonderful scientists and friends, but had the very same cautious approach to a situation with many unknowns. we even insisted on a minority report when we disagreed with the ssc on a pivotal issue. now, years after they have died, i still miss them. when it became clear in 2000 that bse was present in continental europe, we also had in austria our first bse cases that put the government into a panic-stricken work mode. restaurants in vienna were particularly affected by the bse crisis, as boiled beef from tafelspitz, a peculiar meat cut, is a traditional delicacy that, as the saying goes, was never missing at francis joseph’s imperial table (* note to gourmets: my personal preference is not the famous tafelspitz that i find too dry, but some cuts with slightly fatter composition like schulterscherzel or really fat beinfleisch, or kruspelspitz with a small cartilage. this must be served within a beef broth garnished with slices of bone marrow tasting like paradise on toasted dark bread). for a few weeks, i was interviewed many times by tv and radio, and in the midst of this turmoil i got a phone call from a governmental secretary to invite me to a “beef dinner” with the austrian federal chancellor, the head of government, the federal minster of agriculture – and me. although having never been a member of a political party or movement, i smelled that the aim was to stage a televised public event at which the safety of austrian beef should be verified beyond doubts by shared politico-scientific consumption of tafelspitz. i respectfully declined the invitation and explained that i loved boiled beef but that i was in science and medicine but not in political activism. retrospectively, some might argue that i missed a chance to climb up the career ladder, but i was happy with what i got – and then indeed did not rise higher. after 2003, scientific advice and risk assessments were continued by the newly established european food safety authority (efsa); prion-related issues were handled by its biohazard panel. again, i became a member, later its vice-chairman, until 2012. monthly two-day meetings were held, at first in brussels and, after completion of the new efsa headquarters, in parma, italy. numerous opinions on various aspects of food safety were the outcome of our work there. with regard to bse, vcjd and other human prion diseases, it became clear that scientific advice on risk mitigation has resulted in almost complete disappearance of classical bse, a real success story, whereas other prion problems persist [16]. during the long years with efsa the beautiful and historical city of parma became dear to me, as did the colleagues in the panel – it was a constructive and open atmosphere that i enjoyed very much. as parma is the very food capital of italy, we had of course splendid dinners there. my favourite was il bollito misto de parma, a famous dish with meats from cheek, tongue, tail, belly and head, mostly beef, some stuffed but all just boiled, sounding like components of scottish haggis, but reminding me more of the typical viennese tafelspitz. as the habsburgs used to export princesses as monarchic wives all over europe, maria luisa, the daughter of austrian emperor francis ii, who had been napoleon’s second wife and thus empress of france, was later made duchess of parma. maria luisa had great impact on parma, and there are still austrian souvenirs in parma, with the bollito probably as one of the most prominent. upon request, i can advise on the best ristorante for bollito. success and recognition how do you measure success in science? depends. as always, money counts also in research, no question. thus it is essential, already early in a career, to acquire research funds from whatever source possible, something that needs to be trained, as amounts of funds collected for research, patents etc. have become one hard currency of measuring success in science. the other is publications; there are now bibliometric parameters that determine work, career and even the very existence of scientists. this can be considered good or bad, but it definitely is not all what counts. in the present world of multi-authorships, sometimes diluted up to 100 co-authors or more, i am proud to have been one of the very few who wrote many articles as single author, without the safety net of helping hands. of course, this has become different nowadays – present science by necessity has become teamwork, even a world-spanning network. another aspect is the range of research, whether it is narrow and limited to a single problem or wider. for an academic career – and i have witnessed that from sitting on a plethora of search committees for academic positions – it is important to prove both depth and breadth in research. as a now almost extinct species in this regard, i am also proud of having published in virtually every field of the discipline, from developmental neuropathology over metabolic and toxic, inflammatory, infectious and vascular diseases to neurodegeneration, tumours and neuromuscular disorders. many papers have been highly cited, and i have achieved a high hirsch index – the beauty with that, in my view, is its steady growing, even when you publish little at present, as i do. however, what is really important is to have made new contributions to the general body of knowledge, such as i did with first observations or conceptual outlines, as our report in collaboration with kenji kosaka about a case series with neocortical lewy bodies characterising a new disease, now called lewy body dementia [17]; or neuropathological diagnostic criteria for cjd [18], neuropathological features [19] and other aspects of prion diseases [13, 14 and many others] including the subcellular localisation of disease-associated prion protein [20] and characterisation of a new disease in wild-type animals by synthetic prions [21]; the neuropathology of hiv infection [10, 22 and many others], viral products in tick-borne encephalitis [23]; gfap in oligodendrogliomas [24], the clinical relevance of meningioma subtypes [25]; a new glial globular tauopathy [26], nigral burden of α–synuclein as a correlate of striatal dopamine deficit in parkinson’s disease [27], morphological evidence of α–synuclein propagation in the human brain [28], the basis of biomarker diagnostics in neurodegeneration [29], the frequent mixture of neurodegenerative pathologies in the aging community [30], and transmission of aβ by dural grafting [31]. after transfer of the old ni into the akh, we established several clinical routine processes to support our daily work in an expanded environment, such as weekly conferences with the whole staff, research reporting meetings and time-fixed daily microscoping sessions around a multi-ocular device that was able to accommodate up to 10 viewers (fig. 7). it was where we had most of our diagnostic and scientific discussions, in particular when we welcomed distinguished visitors such as john j. kepes (fig. 7) or bob d. terry (fig. 8). and it was always fun and a pleasure, as well as a place where to preserve the good working atmosphere. fig. 7. daily microscoping sessions were always held around the multi-head viewing microscope, here in the second half of the 1990s when an old and cherished friend and neuropathological giant, john kepes with wife (in foreground) was visiting from kansas city. sitting behind them, haberler at left and budka at right, standing from left, hainfellner, hussun (a pathologist from yemen), jarius, wanschitz. john had briefly stayed at the ni after the failed hungarian revolution of 1956. fig. 8. bob terry, another giant of neuropathology, with the author at an alzheimer conference in southern styria, a wine-growing region, nov. 2007. kurt jellinger in the centre at back. in the early 2000s, a young clinical neurologist arrived from budapest, gabor g. kovacs, to learn some neuropathology. his boss at semmelweis university, prof. szirmai who had stayed as postdoc at the ni two decades before, was reluctant to let him go. soon i found out why: gabor became the very best trainee i ever had, with an unbelievable eye for morphology, a sound biological understanding and intelligent translation of neuropathological features into clinical significance, extreme diligence and last but not least, in agreement with albert einstein’s dictum that it is character that makes the scientist excellent, an open, warm-hearted nature that made him soon everybody’s darling at the institute. it was gabor who soon covered most of the research work, most prominently in the prion field but also elsewhere in his broad interests. it is sad for me to realise that he left – in my belief was forced to leave because of the circumstances – two years ago to become full professor in toronto, canada, a brain-drain consequence of the turmoils suffered by the institute in recent years, but definitely a deserved recognition for this fine man and scientist. in the years of my directorship, i was indeed lucky to have an excellent staff that was about equally balanced between neuropathology and neurochemistry (fig. 9). around the same time when gabor arrived, ellen gelpi, another young researcher, joined from barcelona. with her warm-hearted mediterranean-style approach to all matters, irrespective of how important or not they were, she soon became an indispensable hand in diagnosis and research, from prion diseases to tick-borne encephalitis. we lost her for some years to an attractive position as head of a brain bank in her home city, but fortunately she is now back and remains the very soul of the institute. another indispensable mind and soul is romana höftberger who trained with hans lassmann in neuroimmunology in vienna, and with josep dalmau and francesc graus in barcelona. she has high reputation in research on antibody-mediated disorders of the cns, actually relevant for both neuropathology and neurochemistry, and thus is now the no-longer-missing-link between the two major areas of work in the institute. last but not least, christine haberler has made her name known in the field of paediatric tumour neuropathology, something that needs very specialised expertise. for me, aging as an “old white man”, it was a pleasure to see a highly successful female touch in achieving the most recent accomplishments of the institute. fig. 9. the kin staff in 2006. 1st row, from left: hainfellner, regelsberger, budka, ströbel, gelpi, pipp, haberler, höftberger, bernheimer, trabattoni, preusser; behind, the dedicated laboratory and secretariat staff committed to the success of the institute. with increasing age, and success of kin in general, i was awarded some prizes that recognised our work in some specific areas. already as far back as 1982, i had received the moore award for the best paper on clinico-pathological correlation at the american association of neuropathologists (aanp) meeting; it was for my work on ihc in pml, and i was proud of it because i received the award despite the session chairman’s attempts to disturb my presentation. he insisted to limit the number of slides at my talk; so this was a very distinguished virologist who arbitrarily decided that a youngster had two slides too many than what he personally considered appropriate. no, sir, i explained politely but firmly to him that i would definitely show all my slides, that numbers of slides don’t matter as long the allotted time frame is kept, but that he could, of course, stop me if i would go overtime. so good arguments do help youngsters against forceful professors. for explanation of the then highly publicised bse and prion situation, i frequently gave scientific statements to the public by press, tv and radio. these were obviously well received, as i was awarded with the title of “austrian scientist of the year 1998”; the prize included also the naming of a star after the awardee in one of the far corners of the universe. in 2004 i received an honorary doctorate from the medical academy łodz in poland, and in 2008 i had the honour to give the dorothy russell memorial lecture at the british neuropathological society meeting in london (fig. 10). fig. 10. lecture at the award ceremony to receive the dorothy s. russel prize by the british society of neuropathology in london 2008, in front of a memorizing image of my all-time hero, lucien j. rubinstein (photo courtesy of bns). in 2007, we celebrated the 125th anniversary of the institute by a dedicated symposium [32]. many friends, including some from overseas, came to follow a series of lectures and posters on ni/kin’s history and our more recent achievements. it appeared then that the future of kin was safe and expected to prosper, and i even speculated whether i would live to see the institute’s 150th anniversary in 2032. one of my concerns was to maintain and foster a good relation to the non-medical and non-scientific world that is funding everything we do. one possibility was the children’s university when kids who visited kin were told about our work and, most importantly, even could touch a human brain if they dared to (fig. 11). fig. 11. since about 2010, kin participated in the “children’s university” with demonstrations of brain anatomy to primary schoolchildren. all are exited to see a real brain – some even do not dare to look. the third family i met my second wife ivana in 2003 by chance, during a summer most europeans probably remember, as it was extremely hot and beautiful. after my divorce i used weekends and holidays to make long trips by bike (or by car when it was too hot) and stop in between for a refreshing swim, once even in the big danube river when a most beautiful young lady caught my eyes. i was immediately hooked. she is czech, worked as a nurse in mobile care in an area northwest of vienna, and then took a dip in between like i did. she soon decided to take a job offer in a big municipal hospital in the post-surgery recovery room according to her training in anaesthesia. our daughter natalia was born in 2007, and this time circumstances were much more family-friendly than with my earlier family. i had more time to spend with the family, of course never enough in a job such as mine, and it was – and is – an absolute delight to see natalia growing up. when i went to work in zurich, ivana wanted to accompany me with natalia, but i was unsure how it would work out in a completely different setting, with a foreseeable deadline and much higher costs for schooling and living than in vienna. so we arranged that the family would stay in vienna, and in the “guest-worker” tradition of europe i would live over the week in switzerland, with commuting flights every friday afternoon to vienna, and early monday morning from vienna. after i came back from zurich at the end of 2016, i had of course plenty of time to spend with the family. ivana was ambitious to do a master’s study of three years and is now a recognised specialist for pain nursing. two years ago we gave in to natalia’s dearest wish, and gorry joined our family, a female portuguese water dog, which is admired not only by us, but also by practically everybody we meet when walking her (fig. 12). fig. 12. the family in dec. 2019 at a dog exhibition when gorry became austrian junior champion. zurich at an age of 65, i felt too active to do little to nothing after retirement in vienna, so i looked elsewhere and found an announcement for a position of consultant at the institute of neuropathology of the university hospital zurich, headed by prof. adriano aguzzi. i knew adriano from several meetings, as he is a renowned prion researcher. adriano was delighted when i phoned him, and we quickly agreed on my start in zurich from may 2012. i commuted between vienna and zurich by flights on mondays and fridays, almost always with the same bunch of expats in the plane. i tried a few times to drive by car, but it is some 750 km and more than 7 hours driving time, a rather exhausting adventure. i lived for my first two years in a hostel tower for university visitors, students and postdocs, high enough to have a spectacular panoramic view of the city, before renting a small apartment near the limmat river. it was wonderful to walk every morning from there alongside the river to the hospital and back in the evening, watching a lot of waterbirds like cranes, encountering youngsters jumping into the water or balancing on slacklines, and passing by still existing public river baths made entirely of wood, cherished by the locals as “badis”. in comparison with vienna, zurich has a well preserved medieval centre, but is much smaller, much is within walking or biking distance (adriano turned out to be a passionate mountain biker daily climbing up the ütliberg overlooking the city), the river is in the very centre, and the lakeside part of town has an almost mediterranean feeling. so it is a gorgeous place to live indeed. i must admit that i came to switzerland with some trepidation, as i was unsure how the relation with adriano, who was well known as strong personality, would work out. in fact, it turned out to be an extremely pleasant and convenient situation: since an eternity, i had suddenly no or few administrative duties but could concentrate on what i like most (and probably do best), studying and diagnosing interesting neuropathological cases. as adriano was busy with his big experimental lab that comprised some 30-plus postdocs and graduate students in addition to a basic staff of researchers and technicians, he was happy to leave the clinical diagnostic service to me and elisabeth rushing, a very friendly and easygoing colleague and most experienced neuropathologist with whom it was a delight to harmoniously share supervision of a young team of motivated and gifted trainees. elisabeth had specialised in tumour and muscle neuropathology and had been the last head of neuropathology at the legendary armed forces institute of pathology (afip) in washington, dc, before its closure in 2011. together with adriano, i was also responsible for the swiss national reference centre for human prion diseases (nrpe). overall, my time in zurich was the very best indeed that could happen to an aging guy at the end of his career. i am most grateful to adriano – who might have had also some trepidation at start, as i have been considered by some as strong personality as well – to have given me such an opportunity. i learnt a lot there, most notably in experimental neuropathology where i had little experience, but also how to organise and run a successful institute in a way differing from what i was used to do. in fact, after passing the age of 70, it was with a heavy heart that i had to stop my stint in zurich. retirement and aftermath for a long time, i thought that formal retirement in vienna, due for oct 1st, 2011 (fig. 13), was still a long shot in the future. big error, it came quicker than i had imagined. i had made some plans that would be options for that remote time, but in fact, believe it or not, it took me almost by surprise. somehow lost, i suddenly had to look for attractive possibilities to continue at least some of my activities. so i made a visit as honorary professor to the naval general hospital in beijing, on invitation by prof. xiaokun qi, a neurologist with keen interest in neuropathology. in addition, i visited the brain bank, karolinska institutet, huddinge hospital, stockholm, sweden, as guest consultant for several times, on invitation by prof. bengt winblad, caroline graff and inger nennesmo, the local neuropathologist. fig. 13. commemorating plaque given to me by the kin personnel at my farewell party in 2011. it is in the style of a shop sign in the habsburg tradition. its text means “highly laudable neuropathologist in non-retirement”. however, i hoped most to be able to contribute in some function for kin in the future. for a few months i was given a room that was soon converted into a secretariat, and i could keep only a desk elsewhere that, again, was taken away after a short time. after these disappointments, i looked for something else and found the position in zurich described above. after coming back to vienna from 2017, i continued to work on a few forensic cases every year, giving detailed reports to state attorneys and courts, a good possibility to keep active and in touch with neuropathology. of course the number of my publications has dwindled down, and at the end of my scientific career i came back to my neuropathological love affair, viruses: most recently, i wrote a chapter on flaviviruses and tick-borne encephalitis (tbe) in an isn-sponsored book on infections of the nervous system [33]. in view of the present unique global challenge by sars-cov2, i hope to be able still to contribute my experience with neuroviral pathology to research on covid-19. with regard to the future of kin, i expected that a search committee would be appointed soon after my retirement by the rector and senate of the medical university vienna, to fill the vacant position of full professor of neuropathology and institute director. such a committee is common practice in most if not all universities when a professor retires, and its aim is to find the best candidate; my personal favourite was gabor, of course. for unclear reasons – at least for me – this did not happen, and year by year went by while i was in zurich. my former deputy, hans hainfellner, was keen to head the institute but remained formally only deputy director until 2019 when, till the end of that year, the director of the institute of pathology was intermittently appointed to head kin. reasons to appoint an external director were, as i was told, financial frictions between the medical university vienna (muv) and akh about charging for kin diagnostic services; in a hybrid fashion, kin has been funded by both muv and akh. finally and sadly, it was decided to formally degrade the traditionally independent institute on jan. 1st, 2020, after a highly successful history of 137 years, to a division of neuropathology and neurochemistry of the neurological clinic, as a muv-only organisational unit. this was only the second best option to avoid the worst, i.e. complete disbanding when neuropathology would become part of pathology, and neurochemistry would be incorporated into laboratory medicine. why the by far very best option was not pursued, i.e. the further existence of such a renowned and successful institution, has remained a mystery, not only for me, but also for other muv professors. fortunately romana höftberger, an associate professor of kin with excellent knowledge of both neuropathology and neurochemistry, was made head of the new division; i am sure she will do her best to steer it into calm waters. similarly, prof. thomas berger, the director of the neurological clinic and now responsible for the old institute becoming new division, promises to keep it alive as much as possible. epilogue at the end, it is time to balance. was all indeed as positive as these reflections appear, or is it just due to an extended version of the reminiscence bump? for my professional and family lives, there is definitely nothing to regret. with regard to the role of my beloved neuropathology, i cannot find a better summary definition than what lucien rubinstein wrote: “it is that neuropathology is an exquisitely enjoyable pursuit”. the only bitter pill, however, is my personal finale – i never dreamed of becoming the very last director of the original institution that has shaped my whole life. what about the future? for a long time, i used to give a special farewell gift to postdocs and graduate students who trained with me, santiago ramón y cajal’s “advice to a young investigator”. it is an interesting and amusing reading, not only because he was one of the most eminent neuroscientists in history, but also because his advice penetrates virtually every relevant issue of life. a hilarious example is cajal’s criteria for selecting the best wife for a scientist: she should be unattractive, because otherwise she would be too distractive, and she should be rich, to make the husband financially independent in his research. i don’t have such down to earth advice (and i did not follow that advice). however, i believe in an attitude that seems to imbibe also ramón y cajal’s writing and can be put in a nutshell of three words: competence, solidarity and trust. then, now and forever, they are as essential for a scientist or medical practitioner as for any individual’s life. still more, they are pivotal in the fight against crises, including what we are now enduring. if we respect and follow them, there is no reason to await the future with trepidation. references 1. budka h: morphologische aspekte zerebrovaskulärer erkrankungen. in: die zerebrale apoplexie, hrsg. g. s. barolin, pp. 62-79. stuttgart : enke 1980 (1. auflage, 2. auflage 1983, 3. auflage 1985). 2. budka h: pathology of encephalopathies induced by treatment or prophylaxis of neoplastic lesions of the nervous system. in: treatment of neoplastic lesions of the nervous system. j. hildebrand, d. gangji, eds. (europ. j. cancer clin. oncol. suppl. 3) pp. 45-50. pergamon press, oxford-new york 1982. 3. budka h: brain pathology in the collagen vascular diseases. angiology 32: 365-372 (1981). 4. budka h, sluga e, heiss w-d: spastic paraplegia associated with addison's disease: adult variant of adreno-leukodystrophy. j. neurol. 213: 237-250 (1976) 5. budka h, popow-kraupp th: immunohistological studies in viral encephalitis. acta neuropathol. suppl. vii: 142-144 (1981) 6. budka h, popow-kraupp th: rabies and herpes simplex virus encephalitis. an immunohistological study on site and distribution of viral antigens. virchows arch a 390: 353-364 (1981) 7. budka h, popow-kraupp th: immunmorphologische untersuchungen viraler antigene bei verschiedenen enzephalitiden. verh. dtsch. ges. pathol. 65: 181-185 (1981) 8. budka h: multinucleated giant cells in brain: a hallmark of the acquired immune deficiency syndrome (aids). acta neuropathol. 69: 253-258 (1986) 9. kleihues p, lang w, burger pc, budka h, vogt m, maurer r, lüthy r, siegenthaler w: progressive diffuse leukoencephalopathy in patients with acquired immune deficiency syndrome (aids). acta neuropathol. 68: 333-339 (1985) 10. budka h, costanzi g, cristina s, lechi a, parravicini c, trabattoni r, vago l: brain pathology induced by infection with the human immunodeficiency virus (hiv). a histological, immunocytochemical, and electron microscopical study of 100 autopsy cases. acta neuropathol. 75: 185-198 (1987) 11. budka h: human immunodeficiency virus (hiv) envelope and core proteins in cns tissues of patients with the acquired immune deficiency syndrome (aids). acta neuropathol 79: 611-619 (1990) 12. ketzler s, weis s, haug h, budka h: loss of neurons in the frontal cortex in aids brains. acta neuropathol 80: 92-94 (1990) 13. hainfellner ja, brantner-inthaler s, cervenáková l, brown p, kitamoto t, tateishi j, diringer h, liberski pp, regele h, feucht m, mayr n, wessely p, summer k, seitelberger f, budka h: the original gerstmann-sträussler-scheinker family of austria: divergent clinicopathological phenotypes but constant prp genotype. brain pathol 5: 201-211 (1995) 14. kretzschmar ha, honold g, seitelberger f, feucht m, wessely p, mehraein p, budka h: prion protein mutation in family first reported by gerstmann, sträussler, and scheinker. lancet 337: 1160 (1991) 15. vossen p, kreysa j, goll m (eds): overview of the bse risk assessments of the european commission’s scientific steering committee (ssc) and its tse/bse ad hoc group adopted between september 1997 and april 2003. pp. 18-34. european commission, published online june 5, 2003; https://ec.europa.eu/food/sites/food/files/safety/docs/sci-com_ssc_out364_en.pdf 16. budka h, will rg: the end of the bse saga: do we still need surveillance for human prion diseases? swiss med wkly 145:w14212 (2015) 17. kosaka k., m. yoshimura, k. ikeda, h. budka: diffuse type of lewy body disease: a progressive dementia with numerous cortical lewy bodies and senile changes of varying degree a new disease? clin. neuropathol. 3: 185-192 (1984) 18. budka h, aguzzi a, brown p, brucher j-m, bugiani o, gullotta f, haltia m, hauw j-j, ironside jw, jellinger k, kretzschmar ha, lantos pl, masullo c, schlote w, tateishi j, weller ro: neuropathological diagnostic criteria for creutzfeldt-jakob disease (cjd) and other human spongiform encephalopathies (prion diseases). brain pathol 5: 459-466 (1995) 19. budka h: neuropathology of prion diseases. brit med bull 66: 121-130 & plate x (2003) 20. kovács gg, preusser m, strohschneider m, budka h: subcellular localization of disease associated prion protein in the human brain. am j pathol 166: 287-294 (2005) 21. makarava n, kovacs gg, bocharova o, savtchenko r, alexeeva i, budka h, rohwer rg, baskakov iv: recombinant prion protein induces a new transmissible prion disease in wild type animals. acta neuropathol 119/2: 177-187 (2010) 22. budka h: neuropathology of human immunodeficiency virus infection. brain pathol 1: 163-175 (1991) 23. gelpi e, preusser m, garzuly f, holzmann h, heinz fx, budka h: visualization of central european tick borne encephalitis infection in fatal human cases. j neuropathol exp neurol 64: 506-512 (2005) 24. herpers mjhm, budka h: glial fibrillary acidic protein (gfap) in oligodendroglial tumors: gliofibrillary oligodendroglioma and transitional oligoastrocytoma as subtypes of oligodendroglioma. acta neuropathol. 64: 265-272 (1984) 25. maier h, öfner d, hittmair a, kitz k, budka h: classical, "atypical" and anaplastic meningioma: three histopathological subtypes of clinical relevance. j neurosurg 77: 616-623 (1992) 26. kovacs gg, majtenyi k, spina s, murrell jr, gelpi e, hoftberger r, fraser g, crowther ra, goedert m, budka h, ghetti b: white matter tauopathy with globular glial inclusions: a distinct entity of sporadic frontotemporal lobar degeneration. j neuropathol exp neurol 67: 963-975 (2008) 27. kovacs gg, milenkovic ij, preusser m, budka h: nigral burden of a-synuclein correlates with striatal dopamine deficit. mov disord 23 (11): 1608-1612 (2008) 28. kovacs gg, breydo l, green r, kis v, puska g, lőrincz p, perju-dumbrava l, giera r, pirker w, lutz m, lachmann i, budka h, uversky vn, molnár k, lászló l: intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread. neurobiol dis 69: 76-92 (2014) 29. kovacs gg, botond g, budka h: protein coding of neurodegenerative dementias: the neuropathological basis of biomarker diagnostics. acta neuropathol 119: 389-408 (2010) 30. kovacs gg, milenkovic i, wöhrer a, höftberger r, gelpi e, haberler c, hönigschnabl s, reiner-concin a, heinzl h, jungwirth s, krampla w, fischer p, budka h: non-alzheimer neurodegenerative pathologies and their combinations are more frequent than commonly believed in the elderly brain: a community-based autopsy series. acta neuropathol 126: 365-384 (2013) 31. frontzek k, lutz mi, aguzzi a, kovacs gg, budka h: amyloid-β pathology and cerebral amyloid angiopathy are frequent in iatrogenic creutzfeldt-jakob disease after dural grafting. swiss med wkly 146:w14287 (2016) 32. kreft g, kovacs gg, voigtländer t, haberler c, hainfellner ja, bernheimer h, budka h: 125th anniversary of the institute of neurology (obersteiner institute) in vienna. “germ cell” of interdisciplinary neuroscience. clin neuropathol 27: 439-443 (2008) 33. budka h: flaviviruses 1. general introduction and tick borne encephalitis. in: chrétien f, wong kt, sharer lr, keohane c, gray f (eds) infections of the central nervous system: neuropathology and genetics. international society of neuropathology (isn) book series pathology and genetics, chapter 14, pp 131-146. wiley, hoboken, nj 2020 addendum list of neuropathological research visitors and postdocs who trained in neuropathology in the ni/kin: masanori tomonaga, professor in tokyo, riki okeda, professor in tokyo, takeshi kurata, professor at the nih tokyo, ichiro akiguchi, professor in kyoto, toshihiko suenaga, yoshitomo shirakashi, yasuhiro kawamoto, hiroshi sugiyama, toru kimura, kyoko ozawa, all kyoto, masahiro yoshimura-yasuhara, hitoshi yamanouchi, both tokyo, noburo kochi, tatsuo morimura, both hyogo, kazuko sato-matsumura, sapporo, sami khoshyomn, burlington vt, silvia cristina, milano, gianriccardo trabattoni, parma, carlos lima, lisboa, mara popovic, professor in ljubljana, raf sciot, louvain, marcel jhm herpers, professor in maastricht, ferenc garzuly, szombathely, tibor hortobágyi, professor in szeged, felicia slowik, budapest, andras guseo, székesfehérvár, pawel p. liberski, professor in łodz, beata sikorska, professor in łodz, radek kordek, łodz, maria barcikowska, łodz, radek matej, professor in prague, marin guentchev, professor in sofia, ognan kalev, sofia, later linz, yervand karapetyan, armenia, hussun, yemen, sashine tolunay, bursa, karl rössler, professor in vienna, christian bancher, horn, eduard auff, professor in vienna, stefan klöppel, professor in freiburg i. br, peter pilz, salzburg, hans maier, innsbruck, christine wüstinger, johannes preiser, elisabeth wondrusch, claudia radbauer, peter mazal, oskar koperek, christa jarius, elisabeth lindeck-pozza, anna c rudnay, roland sedivy, ute laggner, irene pipp, ivan j milenkovic, harald stefanits, all vienna, michael huemer, schwarzach-st. veit, reza yassari, chicago il. i apologise to those whom i missed, as i could not cross-check the list with documents in the institute. past and present neuropathological researchers who have worked for prolonged periods in the ni/kin include ellen gelpi, johannes hainfellner, christine haberler, romana höftberger, gabor g. kovacs, matthias preusser, manfred schmidbauer, sabine urbanits, julia wanschitz, adelheid wöhrer. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 63rd meeting of the french society of neuropathology meeting abstracts , december 3rd. 2021 feel free to add comments by clicking these icons on the sidebar free neuropathology 2:32 (2021) meeting abstracts 63rd meeting of the french society of neuropathology meeting abstracts december 3rd, 2021   the meeting will take place online at: https://us02web.zoom.us/s/87471519403 the french society of neuropathology was created in 1989, succeeding the french club of neuropathology set up in 1965.   submitted: 19 november 2021 accepted: 19 november 2021 published: 23 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3682 keywords: french society of neuropathology, sfnp, meeting abstracts, 63rd meeting dec. 2021   9:00 introduction       short communications 9:00 deciphering the genetic and epigenetic landscape of pediatric bithalamic tumors 9:15 hedgehog-activated anterior skull base meningiomas overexpress gab1 9:30 1p deletion and fgfr1 alterations as specific diagnostic tools to discriminate dlgnt from pa in medullary location: an integrative radiological and histomolecular series of 28 cases 9:45 classification of pituitary neuroendocrine tumors: genomic data on cell lineage 10:00 large brain vessel vasculitis in immunocompromised patients 10:15 cerebellar ataxia with neuropathy and vestibular areflexia syndrome (canvas): a case report 10:30 neuropathological and amyloid peptide differences between down syndrome and familial alzheimer’s disease with duplications and missense mutations in app gene 10:45 characterization of the endolysosomal compartment of noradrenergic neurons of the locus cœruleus in neurodegenerative diseases 11:00 neuropathology analysis as a precious tool to ascertain the pathogenicity of nearsplice / intronic variants in amyotrophic lateral sclerosis: example of a sod1 nearsplice / intronic mutation       conferences 11:15 implication of microglial cells and peripheral macrophages in amyotrophic lateral sclerosis (als) 14:15 the neuro-ceb brainbank: a brief history (15 years! ) of neuropathology 14:30 human brain imaging with stochastic optical reconstruction microscopy (storm) 14:45 ai applied in neuropathology: automated detection of intraepidermal nerve fibers for the diagnosis of small-fiber neuropathies 15:00 histological description of myopathy related to chronic graft versus host disease and characterization of inflammatory infiltrate by imaging mass cytometry 15:15 highlighting autophagy in a fatal case of pompe’s disease 15:30 diagnosis and classification of peripheral nerve vasculitis: attempt at simplification bases on a retrospective study at limoges university hospital between 2014 and 2020 15:45 experimental evaluation of myosuppressive effects of ifn-gamma       general assembly (members only) short communications:   free neuropathol 2:32:3 deciphering the genetic and epigenetic landscape of pediatric bithalamic tumors arnault tauziède-espariat1, marie-anne debily2,3, david castel2,4, jacques grill2,4, stéphanie puget5, alexandre roux6, raphaël saffroy7, guillaume gauchotte8, ellen wahler1, lauren hasty1, fabrice chrétien1, emmanuèle lechapt1, volodia dangouloff-ros9, nathalie boddaert9, philipp sievers10,11#, pascale varlet1# 1 department of neuropathology, ghu paris-psychiatrie et neurosciences, sainte-anne hospital, 75014 paris, france 2 u981, molecular predictors and new targets in oncology, inserm, gustave roussy, université paris-saclay, 94805 villejuif, france 3 université evry, université paris-saclay, 91000 evry, france 4 department of pediatric oncology, gustave roussy, université paris-saclay, 94805 villejuif, france 5 department of pediatric neurosurgery, necker hospital, aphp, université paris descartes, sorbonne paris cite, 75015 paris, france 6 department of neurosurgery, ghu paris-psychiatrie et neurosciences, sainte-anne hospital, 75014 paris, france 7 department of biochemistry and oncogenetic, paul brousse hospital, 94804 villejuif, france 8 department of pathology, chru, nancy, france 9 pediatric radiology department, hôpital necker enfants malades, ap-hp, university de paris, france 10 department of neuropathology, institute of pathology, university hospital heidelberg, heidelberg, germany 11 clinical cooperation unit neuropathology, german consortium for translational cancer research (dktk), german cancer research center dkfz, heidelberg, germany # these authors contributed equally to this work. over the past several years, based on the results of the literature: 1) diffuse midline gliomas (dmg) were defined as a new tumoral type in the 2016 who classification, and 2) four different subtypes are now defined depending on their molecular characteristics, and/or locations: dmg h3.3 k27–mutant, dmg h3.1 or h3.2 k27–mutant, dmg h3-wildtype with ezhip overexpression, and dmg egfr-altered. however, in this rapidly evolving field, a more comprehensive analysis of pediatric bithalamic gliomas is needed. we investigated retrospectively data from 19 pediatric bithalamic gliomas, confirmed by a central radiological review. we also performed a comprehensive clinical, histopathological and molecular evaluation, as well as dna methylation profiling.     free neuropathol 2:32:4 hedgehog-activated anterior skull base meningiomas overexpress gab1 julien boetto1,2, julie lerond2,3, matthieu peyre2,4,5, suzanne tran5,6, pauline marijon2,4, michel kalamarides2,4,5, franck bielle2,3,5,6,7 1 department of neurosurgery, gui de chauliac hospital, montpellier university hospital center, montpellier, france 2 icm inserm u1127 cnrs umr 7225, paris brain institute, paris, france 3 siric curamus (cancer united research associating medicine, university & society) site de recherche intégrée sur le cancer iuc aphp.6 sorbonne université, paris, france 4 department of neurosurgery, ap-hp, hôpital pitié-salpêtrière, paris, france 5 sorbonne université, upmc université paris 06, paris, france 6 department of neuropathology, ap-hp, hôpital pitié salpêtrière, paris, france 7 onconeurotek, ap-hp, hôpital pitié-salpêtrière, paris, france anterior skull base meningiomas were previously showed to have mutations activating the hedgehog (hh) signalling pathway. however, identification of hh-activated tumours is hampered by the lack of a reliable immunohistochemical marker. we report gab1 as a potential immunohistochemical marker of hh-activated meningiomas. gab1 immunolabeling was compared to smo mutation detection with sanger and ngs techniques as well as hh pathway activation study through mrna expression level analyses in a discovery set of 110 anterior skull base meningiomas. we showed that a cut-off score of 250 for the gab1 expression score (from 0 to 400) lead to excellent detection of hh pathway mutations (sensitivity 100%, specificity 86%). the prospective validation set of 21 meningiomas confirmed the excellent negative predictive value of gab1 expression score. gab1 immunohistochemistry is a fast and cost-efficient tool to screen anterior skull base meningiomas and to facilitate the identification of candidate tumours for targeted therapy.     free neuropathol 2:32:5 1p deletion and fgfr1 alterations as specific diagnostic tools to discriminate dlgnt from pa in medullary location: an integrative radiological and histomolecular series of 28 cases alice métais1, arnault tauziède-espariat1, wael yacoub2, thomas kergrohan3, raphael saffroy4, dominique figarella-branger5, emmanuele uro-coste6, annick sevely7, delphine larrieu-cirron8,9, alexandre roux10, sandro benichi11, lila saidoun3, yassine bouchoucha12, françois doz12, volodia dangoulof-ros2, jacques grill3, pascale varlet1 1 department of neuropathology, ghu paris-psychiatrie and neurosciences, sainte-anne hospital, paris, france 2 department of pediatric radiology, hôpital necker-enfants malades, paris, france 3 département de cancérologie de l'enfant et de l'adolescent, institut gustave roussy, université paris-sud, villejuif, france 4 department of biochemistry, paul-brousse hospital, villejuif, france 5 department of pathology and neuropathology, timone hospital, marseille, france 6 département d'anatomie et cytologie pathologiques, chu de toulouse, iuct-oncopole, toulouse, france 7 department of radiology, purpan university hospital, toulouse, france 8 department of neurology, toulouse university hospital, toulouse, france 9 department of medical oncology, iuct-oncopole, toulouse, france 10 department of neurosurgery, ghu paris-psychiatrie et neurosciences sainte-anne hospital, paris, france 11 department of pediatric neurosurgery, hôpital necker-enfants malades, paris, france 12 siredo center (care, innovation, research in, children, adolescent and young adults oncology), institut curie, paris, france new tumor types such as diffuse leptomeningeal glioneuronal tumors (dlgnt) have been added to the 2016 who. their distinction from others intramedullary gliomas is poorly defined. we retrospectively studied a cohort of 28 children with low-grade intramedullary tumors. the radiological, histopathological and molecular portrait including methylation profiling was established as well oncological treatments data. we observed two main tumor types: dlgnt and pilocytic astrocytomas (pa) that were hardly distinguishable by neuroradiology or histopathology alone. the major criteria to segregate these two tumor types presenting a mapkinase alteration was the 1p deletion. we observed that these tumors interested different populations (infants for pa, school children for dlgnt). these tumors evolved insidiously during a long period of time, remission was rarely achieved (5/28). two cases had a deadly evolution. we recommend to systematically assess 1p deletion in intramedullary low-grade glioma. improving the management of these diseases remains a major challenge.     free neuropathol 2:32:6 classification of pituitary neuroendocrine tumors: genomic data on cell lineage villa c.1,2, armignacco r.2, jouinot a.2, perlemoine k.2, baussart b.3, bertherat j.4,2, gaillard s.3, assié g. 4,2 1 department of neuropathology, hôpital de la pitié-salpêtrière aphp sorbonne université, 47-83 bd de l’hôpital 75651 paris, france 2 inserm u1016, institut cochin, 75014 paris, france; cnrs umr 8104, 75014 paris, france; université paris descartes-université de paris, 75006 paris, france 3 department of neurosurgery, hôpital de la pitié-salpêtrière aphp sorbonne université, 76561 paris, france 4 department of endocrinology, center for rare adrenal diseases, assistance publique-hôpitaux de paris, hôpital cochin, 75014 paris, france the 2017 world health organization (who) classification of pituitary adenomas is based on cell lineage and transcription factors (tfs). transcriptome of 134 pitnets (rna sequencing) was used to determine tfs expression at mrna level, and to provide a canonical transcriptome signature for each cell-types. pathological study of the present serie of pitnets included the histological examination and the immunohistochemical tests for all pituitary hormones, proliferation markers and tfs including gata3. pit1 lineage : based on transcriptome classification, accurate thresholds of immunoexpression for gh, prl and tsh were established in order to define the different pit-1 subtypes. t-pit lineage : t-pit mrna showed the expected expression in corticotroph pitnets (35/35), lower in silent ones (wilcoxon p<10-5). gonadotroph lineage : sf1 mrna expression showed the expected high expression in gonadotroph pitnets (29/29), but also in a subset of somatotroph pitnets (9/21). sf1 immunopositivity was confirmed in this somatotroph subgroup.     free neuropathol 2:32:7 large brain vessel vasculitis in immunocompromised patients isabelle plu1, elie haddad2, arnaud fekkar3, sophie bonnin4, natalia shor5, valérie touitou4, véronique leblond6, nicolas weiss7, eric caumes2, sophie demeret7, valérie pourcher2, danielle seilhean1 sorbonne université, ap-hp, hôpitaux universitaires pitié-salpêtrière charles foix, 75013 paris, france 1 département de neuropathologie 2 service de maladies infectieuses et tropicales 3 service de parasitologie-mycologie 4 service d’ophtalmologie 5 département de neuroradiology 6 service d’hématologie 7 service de neurologie inflammation of the large arteries of the brain is a relatively rare condition whose best known mechanism is hyperimmunity with granulomatous reaction or circulating immune complexes. some vasculitides are secondary to a systemic hyperimmune disease or to an infectious disease (purulent or tuberculous meningitis). we describe here the occurrence of cerebral infarcts in three immunocompromised patients with neutrophilic aseptic meningitis. the mri appearance was vasculitis of the anterior cerebral arteries (one case) or basilar arteries (two cases). pcr found aspergillus fumigatus in the csf two days before a positive cavum biopsy in one case. in the other two cases, aspergillus fumigatus was found postmortem. the neuropathologic appearance was that of filament-rich necrotizing arteritis. there was no other organ involvement. these observations highlight a probably underestimated cause of central nervous system arteritis whose curability relies on early diagnosis based on repeated and extensive mycological testing in csf.     free neuropathol 2:32:8 cerebellar ataxia with neuropathy and vestibular areflexia syndrome (canvas): a case report susana boluda1, david grabli2, vincent huin3, giulia coarelli4, alexandra durr4, danielle seilhean1 1 sorbonne université, institut du cerveau paris brain institute icm, inserm, cnrs, aphp, laboratoire neuropathologie raymond escourolle, hôpital de la pitié salpêtrière, paris, france 2 ap-hp, pitié salpêtrière university hospital, department of neurology, sorbonne university, paris, france 3 sorbonne université, institut du cerveau paris brain instituteicm, inserm, cnrs; university of lille, univ. lille, inserm, chu lille, u1172 lilncog (jparc) lille neuroscience & cognition, lille, france 4 sorbonne université, institut du cerveau paris brain institute icm, inserm, cnrs, aphp, genetics department, hôpital de la pitié salpêtrière, paris, france canvas is a rare disease characterized by an intronic biallelic aaggg expansion in rfc1. we report the case of a 74-year-old man who presented with cramps, mixed sensory and cerebellar ataxia, vestibular areflexia and neuropathic pain in addition to parkinsonian symptoms. macroscopic examination showed atrophy of the cerebellar vermis and pallor and atrophy of the posterior columns. microscopic examination revealed, in addition to diffuse lewy body pathology, axonal loss in the dorsal columns and a moderate loss of purkinje cells in the cerebellum. there was marked astrocytic gliosis in contact with the dendrites in the spinal cord and molecular layer of the cerebellum as well as disorganization of the radial glia in the cerebellum. we report the first complete neuropathological examination of the brain and spinal cord of an rfc1 patient and describe astrocytic abnormalities that would explain the severe clinical phenotype in the absence of severe neuronal loss.     free neuropathol 2:32:9 neuropathological and amyloid peptide differences between down syndrome and familial alzheimer’s disease with duplications and missense mutations in app gene amal kasri1, léa durix1, susana boluda1,2, lev stimmer1, eleni. gkanatsiou3, gunnar brinkmalm3, yannick vermeiren4,5, sarah e. pape6, peter p. de deyn4,5, charles duyckaerts1,2, henrik zetterberg3,7, andre strydom6, marie-claude potier1 1 paris brain institute, icm, cnrs umr7225 inserm u1127 – upmc, paris, france 2 laboratoire de neuropathologie r escourolle, hôpital de la pitié-salpêtrière, ap-hp, paris, france 3 institute of neuroscience and physiology, the sahlgrenska academy at the university of gothenburg, gothenburg, sweden 4 department of biomedical sciences, neurochemistry and behavior, institute born-bunge, university of antwerp, antwerp, belgium 5 department of neurology and alzheimer center, university of groningen, university medical center groningen (umcg), groningen, netherlands 6 institute of psychology and neuroscience, king’s college london, 16 de crespigny park, london, united kingdom 7 sahlgrenska university hospital, clinical neurochemistry laboratory, mölndal, sweden cerebral amyloid angiopathy (caa) is present in 80% of alzheimer's disease (ad) patients. however, caa is more prominent in familial cases with app mutations or duplications (dupapp) and in individuals with down syndrome (ds). in order to explain these differences, we investigated the alterations in the endo-lysosomal pathway, aβ species and caa grading in post-mortem human brain tissues. using immunohistochemistry, we identified increased rab5 puncta size in all cases except those with app mutations compared to controls. moreover, we found elevated levels of aβ40 but not aβ42 in dupapp and in ds correlating with caa grading. altogether, these results suggest that caa arises from specific accumulation of aβ40 species or, alternatively, that aβ40 is the principal aβ species produced in dupapp and ds and is more prone to aggregation in blood vessels. the ongoing aβ and caa grading analysis aim to unravel pathophysiological mechanisms involved in specific aβ production and deposits.     free neuropathol 2:32:10 characterization of the endolysosomal compartment of noradrenergic neurons of the locus cœruleus in neurodegenerative diseases marta fructuoso1, susana boluda1,2, lev stimmer1, yannick vermeiren3, peter p. de deyn3, debby van dam3, andre strydom4, charles duyckaerts1,2, marie-claude potier1 1 icm institut du cerveau et de la moelle épinière, cnrs umr7225, inserm u1127, upmc, hôpital de la pitié-salpêtrière, 47 bd de l’hôpital, paris, france 2 laboratoire de neuropathologie r escourolle, hôpital de la pitié-salpêtrière, ap-hp, 75013 paris, france 3 department of neurology and alzheimer center, university medical center groningen, university of groningen, groningen, the netherlands. laboratory of neurochemistry and behavior, department of biomedical sciences, institute born-bunge, university of antwerp, antwerp, belgium 4 department of forensic and neurodevelopmental sciences, institute of psychiatry, psychology & neuroscience, king's college london, london, uk degeneration of the locus coeruleus (lc) is a common feature in parkinson's disease (pd), alzheimer's disease (ad), and down syndrome (ds). lc is the main source of noradrenaline in the brain, and neuronal loss in this nucleus is associated with dementia. endolysosomal abnormalities may contribute to the accumulation of toxic proteins. we hypothesize that this could be one mechanism of lc neurons vulnerability (jpnd-heroes, www.heroes-jpnd.eu). using post-mortem paraffin fixed lc of control and pathological cases, we confirm the presence of aβ plaques and p-tau in ad and ds brains, and lewy bodies in pd samples. analysis of confocal microscopy images of immunofluorescence revealed that area of the rab5 positive puncta of endosomes was increased in ad whereas the area cathepsin b positive puncta of lysosomes was reduced in pd. our results suggest that endolysosomal alterations could be a mechanism implicated in the degeneration of noradrenergic neurons, and might be disorder-specific.     free neuropathol 2:32:11-12 neuropathology analysis as a precious tool to ascertain the pathogenicity of nearsplice / intronic variants in amyotrophic lateral sclerosis: example of a sod1 nearsplice / intronic mutation françois muratet1, elisa teyssou1, aude chiot1, séverine boillée1, christian s. lobsiger1, delphine bohl1, beata gyorgy1, justine guegan1, yannick marie1, maria-del-mar amador1,2, françois salachas1,2, vincent meininger3, emilien bernard4,5, jean-christophe antoine6, jean-philippe camdessanché6, william camu7, cécile cazeneuve8, anne-laure fauret-amsellem8, eric leguern1,8, kevin mouzat9, claire guissart9, serge lumbroso9, philippe corcia10,11, patrick vourc’h11,12, aude-marie grapperon13, shahram attarian13, annie verschueren13, danielle seilhean1,14* and stéphanie millecamps1* 1 cm, institut du cerveau, inserm u1127, cnrs umr7225, sorbonne université, paris, france 2 ap-hp, département de neurologie, centre de référence sla ile de france, hôpital de la pitié-salpêtrière, paris, france 3 hôpital des peupliers, ramsay générale de santé, paris, france 4 centre de référence sla, hôpital neurologique pierre wertheimer, hospices civils de lyon, université de lyon, bron, france 5 institut neuromyogène, cnrs umr5310, inserm u1217, faculté de médecine rockefeller, université claude bernard lyon i, lyon, france 6 service de neurologie, centre de ressource et de compétence sla, hôpital nord, chu de saint-etienne, saint-etienne, france 7 centre de référence sla, hôpital gui de chauliac, chu et université de montpellier, montpellier, france 8 département de génétique et cytogénétique, unité fonctionnelle de neurogénétique moléculaire et cellulaire, aphp, hôpital pitié-salpêtrière, paris, france 9 laboratoire de biochimie et biologie moleculaire, chu nimes, nîmes, motoneuron disease: pathophysiology and therapy, inm, inserm, univ. montpellier, montpellier, france 10 centre sla, chu tours, tours, france 11 umr 1253, université de tours, inserm, tours, france 12 service de biochimie et biologie moléculaire, chu tours, tours, france 13 centre de référence pour les maladies neuromusculaire et la sla, chu de marseille, hôpital de la timone, marseille, france 14 ap-hp, département de neuropathologie, hôpital pitié-salpêtrière, paris, france * equal contribution among etiologic factors assumed to be responsible of amyotrophic lateral sclerosis (als), several plausible causative genes have emerged. it becomes crucial to determine the pathogenicity of any genetic variant identified through whole exome/genome sequencing analysis, including those located in non-coding regions. we describe a c.358-10t>g nearsplice/intronic variant in the sod1 gene, encoding superoxide dismutase 1, as the second prominent mutation among the sod1 related-french als families. this variant leads to the addition of three amino acids in the protein sequence and impairs the protein secondary structure. biochemical and neuropathological analyses performed on patient tissue revealed massive cytoplasmic sod1 and neurofilament accumulation in spinal motor neurons, similar to those observed in spinal cord of patients with d83g or g93d sod1 mutations. these neuropathology findings ascertain this variant pathogenicity, which is a crucial information in the context of patient enrolment in ongoing clinical trials targeting sod1 by antisense oligonucleotides     conferences:   free neuropathol 2:32:13 implication of microglial cells and peripheral macrophages in amyotrophic lateral sclerosis (als) aude chiot1,2 , félix berriat f1, matthieu ribon1, sakina zaïdi1, charlène iltis1,3, michel mallat1, delphine bohl1, stéphanie millecamps1, danielle seilhean1,4, christian s lobsiger1, séverine boillée1 1 sorbonne université, institut du cerveau – paris brain institute icm, inserm, cnrs ap-hp, hôpital de la pitié-salpêtrière, paris, france 2 current address: department of molecular microbiology and immunology, oregon health and science university, portland, or 97239, usa 3 current address: cancer biology & genetics program, memorial sloan kettering cancer center, new york, ny 10065, usa 4 département de neuropathologie, assistance publique hôpitaux de paris (ap-hp), hôpital pitié-salpêtrière, paris, france in mouse models of amyotrophic lateral sclerosis (als), the most common motor neuron (mn) disease of the adult, microglial cells/ macrophages have been shown to participate to disease progression. however, the respective contributions of microglial cells (mg) and peripheral macrophages (pm) were not documented. we have now shown that pm were present around mn axons of als patients, both in motor roots and peripheral nerves. in als mice, pm were progressively activated and their infiltration into the spinal cord was very limited and disease-length dependent. transcriptomics analysis showed that mg and pm reacted differently to mn degeneration. replacing pm by macrophages more neurotrophic, at disease onset, increased als mouse survival and downregulated not only pm activation in peripheral nerves but also suppressed proinflammatory responses of mg in the spinal cord, with a switch toward neuronal support. thus, targeting pm, directly at the periphery, could be of therapeutic value in als.     free neuropathol 2:32:14 the neuro-ceb brainbank: a brief history (15 years! ) of neuropathology marie-claire artaud-botté1, sabrina leclère-turbant2, charles duyckaerts2, marie laure martin-négrier3, franck letournel4, neuro-ceb neuropathologist’ network5, isabelle plu6, susana boluda2,6, danielle seilhean2,6 1 association neuro-ceb (paris, france): arsla, association france dft, association france parkinson, csc, fondation arsep, fondation vaincre alzheimer 2 icm-paris brain institute cnrs umr7225 inserm u1127 – sorbonne university gh pitié-salpêtrière, paris france 3 gh pellegrin, bordeaux, france 4 chu d’angers, france 5 neuro-ceb neuropathologist’ network: susana boluda (gh pitié-salpêtrière, paris, france), jean boutonnat (hôpital michalon, grenoble, france), fanny burel-vandenbos (hôpital pasteur, nice, france), françoise chapon (hôpital côte de nacre, caen, france), dan-christian chiforeanu (hôpital pontchaillou, rennes, france), vincent deramecourt (chu salengro, lille, france), charles duyckaerts (gh pitié-salpêtrière, paris, france), maxime faisant (hôpital côte de nacre, caen, france), catherine godfraind (chu gabriel montpied, clermont-ferrand, france), béatrice lannes (hôpital de hautepierre, strasbourg, france), annie laquerrière (chu charles nicolle, rouen, france), franck letournel (chu d’angers, france), benoit lhermitte (hôpital de hautepierre, strasbourg, france), florent marguet (chu charles nicolle, rouen, france), marie-laure martin-négrier (gh pellegrin, bordeaux, france), andré maues de paula (chu timone 2, marseille, france), claude-alain maurage (chu salengro, lille, france), david meyronet (hôpital pierre wertheimer, lyon, france), isabelle plu (gh pitié-salpêtrière, paris, france), valérie rigau (hôpital gui de chauliac, (montpellier, france) 6 laboratoire de neuropathologie, gh pitié-salpêtrière, ap-hp-sorbonne université, paris, france the neuro-ceb national brainbank was founded in 2006 at the initiative of patients’ associations, to support a post mortem brain donation program. the missions are to collect, sample, prepare and store nervous tissues (brain and spinal cord), taken post mortem from "control" subjects (not suffering from neurological diseases) and from patients suffering from alzheimer or parkinson disease, multiple sclerosis, ataxia, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, or cadasil. the biological ressources are provided to research teams presenting a suitable project reviewed by a scientific committee. the organization relies on a network of neuropathologists from 14 hospitals. they are in charge of brain sampling and neuropathological diagnosis. the expertise of these neuropathologists ensures a high quality of the samples. their involvement over the past 15 years alongside research teams, has led to the publication of more than 100 scientific articles. this illustrates the impact of the neuro-ceb biobank at an international level.     free neuropathol 2:32:15 human brain imaging with stochastic optical reconstruction microscopy (storm) philippe codron1,2,3, franck letournel1,2, guy lenaers1,3, arnaud chevrollier3 1 service de neurologie, centre hospitalier universitaire d’angers, angers, france 2 service de neurobiologie et neuropathologie, centre hospitalier universitaire d’angers, angers, france 3 équipe mitolab, institut mitovasc, inserm u1083, cnrs 6015, université d'angers, angers, france the recent development of stochastic optical reconstruction microscopy (storm) has contributed to major advances in neuroscience. however this technique is restricted to cultured cells and rodent brain, and no experiment on human samples has been reported so far. to this end, we combined cellular microscopy protocols with neuropathology tissue preparation techniques to characterize physiological and pathological structures in brain samples with 2d-, 3dand two-color storm. this approach proved to be particularly effective at visualizing the organization of dense protein inclusions in samples from patients affected with neurodegenerative diseases. these very first results open further gates to a more comprehensive understanding of the human brain organization and revelations about the underlying mechanisms responsible for neurodegenerative disorders.     free neuropathol 2:32:16 ai applied in neuropathology: automated detection of intraepidermal nerve fibers for the diagnosis of small-fiber neuropathies labeyrie c1,2,3, adam c.2,3, lorenzo a.4, brunel n.4, blot v.4, sevillia h.4, hervault d.4, trassard o.3, morassi o.1, adams d.1,4, guettier c2,3 1 service de neurologie adulte, crmr neuropathie amyloïde familiale et autres neuropathies périphériques rares (nnerf), chu bicêtre, 78 rue du gal leclerc, 94275 le kremlin-bicêre, aphp, france 2 service d’anatomopathologie, chu bicêtre, 78 rue du gal leclerc, 94275 le kremlin-bicêre, aphp, france 3 institut biomédical de bicêtre, ums32, chu bicêtre, 78 rue du gal leclerc, 94275 le kremlin-bicêre, aphp, france 4 quantmetry, 52 rue d’anjou, 75008 paris, france 5 umr1195, université paris sud, faculté de médecine bicêtre, 80 rue du gal leclerc, 94276 le kremlin-bicêre, aphp, france the measurement of intra-epidermal nerve fiber density (ienfd) in skin biopsy is the gold standard for the diagnosis of small fiber neuropathy (sfn). this recently described entity causes neuropathic pain and dysautonomia. numerous etiologies are described, although the majority of them remain idiopathic. the classic dfnie reading method, derived from a 3mm punch biopsy and 50 micron sections, is based on the anti-pgp9.5 labelling with a visual count related to the analyzed length, a long process thus slowing down its development. thanks to the digitization of the double-labeled slides in immunofluorescence anti-pgp9.5 and anti-coll iv since 2012 allowing the computer labeling of the traversing fibers, we were able to train an automated reading algorithm which obtains an accuracy of 71% and a recall of 77%.     free neuropathol 2:32:17 histological description of myopathy related to chronic graft versus host disease and characterization of inflammatory infiltrate by imaging mass cytometry amélie bourhis1,2, baptiste hervier3,4, arnaud uguen1,2, pascale marcorelles1, patrice hemon2, sarah léonard-louis5,6 1 pathology department, hospital morvan, brest, france 2 lbai, umr1227, univ brest, inserm, labex igo, brest, france 3 internal medicine and clinical immunology department, french referral centre for rare neuromuscular disorders, hôpital pitié-salpêtrière, aphp, paris, france 4 inserm umr-s 1135, cimi-paris, upmc & sorbonne université, paris, france 5 neuropathology department, aphp, pitié salpétrière, sorbonne university, paris, france 6 institute of myology, neuromuscular reference center nord/est/ile de france, ap-hp, pitié-salpêtrière hospital, sorbonne university, paris, france chronic graft versus host disease (cgvhd) is a long-term side effect of allogenic hematopoietic cells transplantation, affecting muscles in 3,4 to 7,7% of patients. this study proposes a complete histological and immunohistological description of myopathy in the context of cgvhd and a characterization of the inflammatory infiltrate by imaging mass cytometry technology (imc). histological description was performed on 19 patients and divided patients into three groups: 10 patients with no fiber necrosis and no or very low inflammatory infiltrate, 6 patients with fiber necrosis and high inflammatory infiltrate and 3 patients with fasciitis. all patients had variations in fiber size, diffuse mch-i and perifascicular mch-ii immunostaining. 7 patients were analyzed by imc. the main inflammatory population was macrophage cells, but eosinophils represented more than 5% of inflammatory cells in 5 cases and seemed to be specific of cgvhd induced myopathy as they were already described in other cgvhd locations.     free neuropathol 2:32:18 highlighting autophagy in a fatal case of pompe’s disease julien gouju1, philippe codron1,2, caroline savary1, aleksandra nadaj-pakleza3, marco spinazzi1,2, franck letournel1 1 département de pathologie – chu angers, angers, france 2 département de neurologie – chu angers, angers, france 3 centre de référence des maladies neuromusculaires nord-est-ile de france, service de neurologie, hôpitaux universitaires de strasbourg, strasbourg, france pompe’s disease is a rare autosomal recessive disorder caused by mutations in the acid α-glucosidase (gaa) gene. alpha-glucosidase deficit leads to lysosomal and non-lysosomal accumulation of glycogen with different forms of the disease, ranging from early-onset to late-onset form (lof). we report the case of a lof treated by enzyme replacement therapy (ert), who died of myocardial infarct. we describe expression of several lysosomal and autophagy markers in several tissues. we observed a glycogen-accumulation in brain, associated to increased expression of p62, lc3a/b and lamp-2 localized in the neocortex and hippocampus. both neurons and glial cells were affected, however cerebral vessels were normal. similar results were observed in muscle, heart and liver. our data suggest that autophagy impairment could be an important pathogenic mechanism in the muscles and the nervous system of patients with pompe’s disease     free neuropathol 2:32:19 diagnosis and classification of peripheral nerve vasculitis: attempt at simplification bases on a retrospective study at limoges university hospital between 2014 and 2020 camille guibert1, laurent magy2, mathilde duchesne3 1 service d’anatomie pathologique, chu de dijon, france 2 service de neurologie, chu de limoges, france 3 service d’anatomie pathologique et laboratoire de neurologie, chu de limoges, france peripheral nerve vasculitis remain rare and challenging to diagnose. we tried to simplify criteria of the pns 2010 by searching for those strongly associated with certain and probable vasculitis, and to propose a management of the biopsy. this retrospective study included 46 patients with definite and probable vasculitis according to the criteria of the pns 2010 who underwent neuromuscular biopsy and 21 controls (neurolymphomatosis and cidp). clinical, biological and pathological features were collected. criteria discriminating vasculitis from controls were asymmetry, axonal involvement, biological inflammation, t cells in vessel wall, and nerve haemosiderin deposits. two criteria distinguish definite and probable vasculitis: ovoid and endoneurial haemosiderin deposits. the techniques useful for pathological diagnosis are serial cuts, typing of inflammatory elements and perls staining. we proposed a slightly simplified classification of vasculitis according to the pns 2010 and a management of neuromuscular biopsy in this indication.     free neuropathol 2:32:20 experimental evaluation of myosuppressive effects of ifn-gamma cyrielle hou1; baptiste périou1, marianne gervais1, juliette berthier1, yasmine baba-amer1, sarah souvannanorath1,2, edoardo malfatti1,2, frédéric relaix1,2, françois jérôme authier1,2 1 inserm u955-eq. relaix, faculté de santé, université paris est-créteil; créteil, france 2 hu henri mondor (aphp), centre de référence des maladies neuromusculaires, département de pathologie, créteil, france dysimmune and inflammatory myopathies (dims) are acquired idiopathic myopathy associated with immune response dysregulation. inclusion body myositis (ibm), the most common dims, is characterized by endomysial infiltrates of cytotoxic t lymphocytes cd8, muscle type ii-interferon (ifnγ) signature, and by the lack of response to immunomodulatory therapies. we showed that ibm differs from other myopathies by the presence of chronic degenerative myopathic features including the altered functions of skeletal muscle stem cells. here, we demonstrated that, in vitro, protracted ifnγ treatment inhibits the activation, proliferation, migration, differentiation, and fusion of myogenic progenitor cells and promotes their senescence through jak-stat-dependent activation. jak-stat inhibitor, ruxolitinib abrogates the deleterious effects of ifnγ, in conclusion, our results indicate that ifnγ could impair muscle regeneration in the context of inflammatory myopathies and that jak inhibitors could represent interesting therapies for immune myopathies with ifnγ signature.   copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. title feel free to add comments by clicking these icons on the sidebar free neuropathology 4:11 (2023) obituary vale emeritus professor byron a kakulas ao, 1932 – 2023 clive harper1, colin l masters2 neuropathology, dept. of health sciences, charles perkins centre, university of sydney, australia florey institute and the university of melbourne, australia corresponding author: colin l masters md · laureate professor of dementia research · florey institute and the university of melbourne, australia c.masters@unimelb.edu.au submitted: 17 may 2023 accepted: 17 may 2023 published: 22 june 2023 https://doi.org/10.17879/freeneuropathology-2023-4820 keywords: obituary, neuropathology australia, inclusion body myositis, muscular dystrophy vale emeritus professor byron a kakulas ao, 1932 2023. it is with great sadness that we reflect on the recent passing of our friend and colleague, emeritus professor byron kakulas ao. byron kakulas was born on march 29, 1932 in perth, western australia. like many west australians seeking to join the medical profession at that time, he completed his of bachelor of medicine, bachelor of science at the university of adelaide in 1956. he commenced his medical career as a resident medical officer at royal perth hospital in 1957 and went on to complete his specialist clinical neurology training in 1963. byron married valerie patsoyannis in 1961 before they moved to boston. they have three children: arthur phillip, felice anne and carolyn rose. professor kakulas' move to boston was to continue his training in neurology/neuropathology at the massachusetts general hospital, harvard medical school (1963-1965). he worked closely with professors raymond d adams and e pearson richardson. they remained friends and colleagues for a lifetime and visited perth on several occasions. living in perth, byron had the opportunity of visiting the lovely island of rottnest, famous for its small marsupial, the rottnest island quokka. he observed that many of these cute little animals developed a paralytic disease when placed in captivity. he decided to make this the subject of his doctoral thesis and found that the disorder was due to breakdown of muscle resulting from vitamin e deficiency. by treating this deficiency in the quokkas, he showed for the first time that skeletal muscle had the ability to regenerate. this was a momentous breakthrough since it demonstrated the potential for some muscle diseases including muscular dystrophy to be curable. byron communicated his studies very effectively to the wider community, and became well known on television with the annual fundraising activities of telethon. this original work eventually led to a treatment for sufferers of duchenne muscular dystrophy, which received accelerated approval by the fda in 2016. his research contributions were very extensive covering a variety of fields especially childhood and adult muscle disease. he showed that paralysis in muscular dystrophy was the result of continuous cycles of necrosis and regeneration. he was the first to identify inclusion body myositis as an entity. in the early 1990s, he introduced molecular genetics and dna technology in the investigation of muscle diseases. other highlights were the development of in vitro models for polymyositis (in collaboration with roger l dawkins) and the pathology of kuru and experimentally transmitted prion infections (in collaboration with carleton d gajdusek and michael p alpers). the neuropathology of spinal cord injuries was a major lifelong interest, for which his pioneer work gained much international acclaim, setting the scientific basis for better treatments. in 1967, he founded the muscular dystrophy association of wa and, soon after, the australian neuromuscular research institute (anri) becoming the medical director of both. he held these posts until 2010. in 2017, the anri became the perron institute for neurological and translational science. professor kakulas was appointed as head of the department of neuropathology at the royal perth hospital in 1967. he was made the foundation professor of neuropathology at the university of western australia in 1971 and was dean of the faculty of medicine from 1976-1978. clive harper (emeritus professor of neuropathology, university of sydney) recalls that he met byron at the international society of neuropathology in budapest in 1974. byron asked clive what he planned to do after working in switzerland in neuropathology. byron said, "i can offer you the position of assistant neuropathologist in perth". clive flew into perth from switzerland in 1975. he started work at the royal perth hospital immediately. byron had set up the biggest and best neuropathology department in australia. it was big, even by international standards. clive recalls: "there were excellent facilities and large numbers of secretarial and technical staff. there were two neuropathology registrars and several overseas trainees. the registrars, peter blumbergs and tony tannenberg and myself worked closely together for several years until they took up senior neuropathology positions in adelaide and brisbane, respectively". all three have remained good friends to this day. colin l masters started with byron's research team in 1967 when, as a medical student, he helped with the analysis of the brains of prion-inoculated non-human primates sent to perth by carleton gadjusek. he continued postgraduate studies in neuro-virology in byron's department. colin later took up the position of professor of pathology at the university of melbourne in 1989. clive worked with byron until 1985 when he was invited to take up the foundation chair in neuropathology at the university of sydney and royal prince alfred hospital. hence, byron was responsible for the training of most of the senior neuropathology staff in queensland, nsw, victoria and south australia. the royal perth hospital department of neuropathology was responsible for most of the forensic and hospital neuropathology studies and, each week there were reviews of cases in the laboratory. these were attended by many of the hospital clinicians and were considered by trainees and senior staff as great learning experiences. professor kakulas held many important positions in a range of scientific, medical and other societies. these included membership of the science committee for the international spinal research trust (1983). he was a fellow of the royal australasian college of physicians, the royal college pathologists australia and the royal college of pathologists (united kingdom). he was a founding member of the australian and new zealand society for neuropathology and president from 1985 to 1988. professor kakulas played an important role in many national and international neuroscience societies and conferences. he was on the executive committee of the world federation of neurology and was vice president for the international congress in muscle disease in 1971, 1974 and 1978. byron was vice president for the fifth international congress on neuromuscular diseases in 1982. he was on the board of management at sir charles gairdner hospital from 1977-1980. byron was president of the australian brain foundation from 1981-1985. professor kakulas received many honours and awards including officer of the order of australia, an honorary doctorate of the university of athens, the gaetano conte prize of the naples conte academy and a lifetime achievement award by the world federation of neurology. he was also a paul harris fellow in rotary. written by prof clive harper & prof colin l masters on behalf of anzsnp. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 61st annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 2:29 (2021) meeting abstracts 61st annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts october 14–16, 2021 submitted: 26 october 2021 accepted: 27 october 2021 published: 29 october 2021   the canadian association of neuropathologist – association canadienne des neuropathologistes (canp-acnp) held their 61st annual meeting via zoom from october 14th to 16th, 2021, under the leadership of dr. peter gould, president of the canp-acnp, dr. peter schutz, secretary treasurer of the canp-acnp, and with technical support from canp administrators heather dow and colleen fifield. the academic program comprised 17 scientific abstracts, 8 unknown cases, the presidential symposium on epilepsy and neoplasms, and a neuropathology in practice session on the implementation of the new who classification of cns neoplasms in canada. digital pathology images from the 8 unknown cases are available for viewing online (www.canp.ca). the unknown case sessions were moderated by dr. andrew gao. the presidential symposium 2021 on epilepsy and neoplasms featured the david robertson lecture given by dr. eleonora aronica entitled evolving classification of epilepsy-associated tumors: understanding epileptogenesis, the gordon mathieson lecture delivered by dr. harvey sarnat on why are some cerebral malformations more epileptogenic than others? clues from developmental neuropathology, and the jerzy olszewsky lecture presented by dr. maria thom on neuropathology in sudden and unexpected death in epilepsy and future directions. the program was completed dr. george ibrahim’s lecture on epilepsy as a network disorder. the mary tom award for best clinical science presentation by a trainee went to dr. suzy kosteniuk (supervisor dr. s. das), and the morrison h. finlayson award for best basic science presentation by a trainee was won by dr. nicole schwab (supervisor dr. l.n. hazrati). the following abstracts were presented at the 61st annual meeting of the canadian association of neuropathologists – association candienne des neuropathologistes (canp-acnp) in october 2021. https://doi.org/10.17879/freeneuropathology-2021-3671 keywords: canadian association of neuropathologists, canp, meeting abstracts, 61st meeting oct. 2021 contents abstract 1: histologic and clinical spectrum of brain tumours harbouring fgfr3-tacc3 fusions abstract 2: ganglion cell maturational markers in peripheral neuroblastic tumours of children abstract 3: long term disease-free survival after surgical resection only: malignant idh-mutated astrocytoma versus ganglioglioma abstract 4: diagnostic challenges in unusual high-grade gliomas abstract 5: regional variability of α-synuclein inclusion size in multiple system atrophy abstract 6: cognitive resilience in individuals with severe alzheimer’s disease neuropathology abstract 7: linking iron homeostasis to vulnerability patterns in progressive supranuclear palsy abstract 8: friedreich cardiomyopathy is a secondary desminopathy abstract 9: glial senescence (not tau) is the driver of post-concussive symptoms abstract 10: hail to astrogliosis! the unsung hero of the cns tissue reaction to the spinal cord injury abstract 11: ischemic-like pathology in aberrant white matter tracts of fetal holoprosencephaly: a case series abstract 12: brain pathology in patients with congenital heart disease abstract 13: characterizing the hippocampal dentate gyrus involvement in temporal lobe epilepsy abstract 14: deep learning approaches to deciphering intra-tumoural heterogeneity in glioblastoma abstract 15: integrating morphologic and molecular histopathological features through whole slide image registration and deep learning abstract 16: neuropathology of eight cases of the new brunswick cluster of neurological syndrome of unknown cause (nsuc) abstract 17: covid-19 pandemic impact on surgical neuropathology services at london health sciences centre     abstract 1 free neuropathol 2:29:4 histologic and clinical spectrum of brain tumours harbouring fgfr3-tacc3 fusions francesca gianno1, jennifer a. chan2, phedias diamandis3,4, john a. maguire5, qi zhang6, cynthia hawkins1, lili-naz hazrati1 1 department of laboratory medicine and pathobiology, university of toronto, toronto, ontario, canada; department of pathology, the hospital for sick children, toronto, ontario, canada 2 university of calgary, calgary, alberta, canada 3 princess margaret cancer centre/laboratory medicine program, university health network, toronto, ontario, canada 4 departments of laboratory medicine and pathobiology and medical biophysics, university of toronto, toronto, ontario, canada 5 department of pathology & laboratory medicine, costal health-vancouver general hospital, canada 6 department of pathology, london health sciences centre, london, canada fibroblast growth factor receptor (fgfr) gene family alterations, including point mutations and fusions, have been described in brain tumours as oncogenic drivers (ng 2013; 45(6): 602–612). fgfr3-tacc3 fusions were initially described in idh wildtype glioblastomas (science 2012; 337:1231–1235) and are most frequently seen in morphologic or molecular gbm although occasional who grade 2 astrocytomas may harbour this alteration. the significance of this alteration as a single driver in low-grade glioma is unclear. in order to better understand the clinical implications of finding this alteration, a series of low-grade gliomas underwent molecular testing at sickkids. the series includes six samples of diffuse low-grade gliomas, with a mean age of 24.8 years (range 1 to 44 years), three males and three females. five out of six tumours are localized in the supratentorial region, and one in the spinal cord. three patients presented with seizures, one was asymptomatic. morphologic appearance showed different aspects, from cells with oligo-like features in a neuropil-rich stroma to spindle cells with fibrillary cytoplasm in a loose microcystic background. single nucleotide polymorphism (snp) array was performed on two samples and one of them shows the gain of chromosome 7 and loss of chromosome 10, indicating a molecular profile of high grade glioma. follow-up was available for four patients, all are alive without progressive disease with follow-up ranging from six months to two years. longer follow-up will be required to determine the significance of this alteration in low grade glioma.   abstract 2 free neuropathol 2:29:5 ganglion cell maturational markers in peripheral neuroblastic tumours of children harvey b. sarnat1 1 university of calgary & alberta children's hospital, calgary, alberta, canada peripheral neuroblastic tumours of neural crest origin are the most frequent solid neoplasms outside the cns in children. neuroblastoma/ganglioneuroblastoma have a natural evolution of histological differentiation over time. together with mitosis-karyorrhexis index and patient age (international neuroblastoma pathology classification criteria), ganglion cell maturation determines grading and prognosis. maturation presently is assessed only by h&e histology. methods: immunocytochemical markers of neuroblast maturation in fetal cns were applied to peripheral neuroblastic tumours arising in adrenal medulla or sympathetic chain. resected tumours of 4 toddlers were examined using antibodies demonstrating neuronal identity and maturation: map2; synaptophysin; chromogranin-a; neun; keratan sulfate (ks); glutamate receptor antibody (glur2). others: ki67; s-100β protein; vimentin; nestin; α-b-crystallin; neuroblastoma marker phox2b. results: degrees of neuroblastic maturation were demonstrated by map2, chromogranin, synaptophysin, ks and glur2; neun was negative consistent with sympathetic neural crest lineage. ks was sparsely distributed within the tumours adherent to somata and proximal neuritic trunks. phox2b did not distinguish maturational stages. ki67 was expressed in scattered primitive cells that also expressed vimentin and nestin, but not in differentiated neoplastic neurons. s-100β protein and α-b-crystallin labeled schwann cells, especially schwannian ganglioneuroma. conclusions: immunocytochemical markers of neuroblast maturation in fetal brain also are useful in peripheral neuroblastic tumours, providing greater precision than histology alone. the most practical are map2 and synaptophysin. prognosis and choice of treatment including chemotherapy might be influenced.   abstract 3 free neuropathol 2:29:6 long term disease-free survival after surgical resection only: malignant idh-mutated astrocytoma versus ganglioglioma roland n. auer1,5, egiroh omene2, sarah e. edwards1, kotoo meguro1, daryl r. fourney1, jose f. tellez-zenteno2, gary r. w. hunter2, kyle moulton3, vijayananda kundapur4 1 department of surgery (neurosurgery) , university of saskatchewan, saskatoon, saskatchewan, canada 2 department of neurology, university of saskatchewan, saskatoon, saskatchewan, canada 3 department of radiology, university of saskatchewan, saskatoon, saskatchewan, canada 4 department of oncology, university of saskatchewan, saskatoon, saskatchewan, canada 5 department of pathology and laboratory medicine, university of saskatchewan, saskatoon, saskatchewan, canada gangliogliomas are important to distinguish from anaplastic astrocytoma and glioblastoma multiforme, to avoid potentially damaging radiation and for accurate prognostication. two patients with a seizure presentation at age 31 and 30 had large, well demarcated brain tumors each 4.3 cm with no suggestion of diffuse glioma on imaging. both patients requested tumor resection. the first patient intra-operatively was found to have the tumor in close proximity to the motor cortex, and resection was thus incomplete, leaving residual tumor in situ. the second patient had a gross total resection. an initial diagnosis of ganglioglioma, was changed to malignant astrocytoma based on idh1 mutation, loss of atrx, abundant mitoses and a high ki67 labelling index. conventional histology revealed glial, neuronal and intermediate forms in both tumors. both patients declined treatment following neurosurgery and remain without tumor growth on neuroimaging at 6 and 3 years later, respectively. the findings illustrate the conundrum of tumors diagnosed as malignant astrocytomas based on molecular profile, but showing no growth after no treatment, as well as the superiority of resection over needle biopsy, as the first tumor was called a diffuse astrocytoma on a needle biopsy prior to resection. without diffuse astrocytoma appearing on long-term follow-up in the absence of radiochemotherapy, a diagnosis of diffuse, malignant astrocytoma is increasingly untenable as years pass. both patients and their clinicians question the malignant diagnosis. gangliogliomas have been reported with idh mutation (brain pathol 2011; 21: 564-574). ganglioglioma versus diffuse astrocytoma remains a difficult differential diagnosis in neuropathology.   abstract 4 free neuropathol 2:29:7 diagnostic challenges in unusual high-grade gliomas namita sinha1, arie perry2 1 department of pathology, health sciences center, university of manitoba, winnipeg, manitoba, canada 2 department of pathology, university of california, san francisco, california, usa diagnosis of most of adult high-grade gliomas is largely based on morphology and immunohistochemistry, with an increasing role of molecular studies to aid proper classification (idh wildtype and idh-mutant gliomas, h3k27m mutant diffuse midline glioma). on occasions, despite all the work-up, the results do not allow for a definite diagnosis that are established in the most recent who classification. some glial tumor shows dedifferentiation and looses gfap expression. in these cases, methylation profiling is a power tool in establishing the diagnosis. therefore, it is very important that the tissue should be utilized optimally and carefully especially when the biopsies are very small and extensive work may be required. on rare occasions, definitive diagnosis can be challenging despite complete histological and molecular characterization. some variants of glioblastoma may show epithelial or primitive neuronal differentiation and some variants may have increased predilection for csf metastasis and therefore it is important to subclassify these variants. these challenges will be discussed with some examples of high-grade gliomas that we received in our institution at health sciences center, university of manitoba.   abstract 5 free neuropathol 2:29:8 regional variability of α-synuclein inclusion size in multiple system atrophy ain kim1,2,3, ivan martinez-valbuena2,3, gabor g. kovacs1,2,3* 1 department of laboratory medicine & pathobiology, university of toronto, ontario, canada 2 tanz centre for research in neurodegenerative disease, university of toronto, ontario, canada 3 krembil research institute, university health network, ontario, canada relevance: multiple system atrophy (msa) patients show variable duration of illness and distinct constellation of clinical symptoms suggestive of different pathological α-synuclein strains. objective: based on this, our aim is to investigate whether the seeding and aggregation process of α-synuclein are different in msa brains, leading to variability of size and detectability of oligodendrocytic inclusions using different anti-α-synuclein antibodies. methods: the putamen and cerebellum of 6 msa cases were immunostained using nitrated, truncated, phosphorylated and disease-specific α-synuclein (clone 5g4) antibodies. sections were scanned using tissuescopetm and images were processed using photoshop (v.21.0.3). 5g4-immunoreactive oligodendrocytes with visible nucleus were optically dissected and inclusion sizes were evaluated using algorithms that we developed using the software image j. results: the size of 5g4-reactive inclusions were significantly larger in the cerebellum than in the putamen in 4 out of 6 cases. in one case, the inclusion size was significantly larger in the putamen than in the cerebellum, while a single case showed similar sizes. in addition, case-wise comparison revealed significant variability between the size of inclusions in both regions. in addition, substantial differences were noted between the number of inclusions stained by the different α-synuclein antibodies. conclusions: we developed a novel computer-based method to measure the size and number of α-synuclein inclusions in msa. our observations on the variability of size and number of inclusions, detected by different α-synuclein epitopes between brain regions and cases, support the notion of distinct subtypes of disease. our study further provides a first step to developing ai-based evaluation strategies for large scale comparative studies.   abstract 6 free neuropathol 2:29:9 cognitive resilience in individuals with severe alzheimer’s disease neuropathology narges ahangari1, corinne e. fischer2,3,4, tom a. schweizer2,4,5, david g. munoz1,2,6 1 division of pathology, st. michael’s hospital, toronto, ontario, canada 2 keenan research centre for biomedical research, the li ka shing knowledge institute, st.michael’s hospital, toronto, ontario, canada 3 department of psychiatry, faculty of medicine, university of toronto, ontario, canada 4 institute of medical sciences, university of toronto, toronto, ontario, canada 5 division of neurosurgery, faculty of medicine, university of toronto, ontario, canada 6 department of laboratory medicine and pathobiology, university of toronto, ontario, canada we sought to identify demographic, clinical, genetic, and neuropathological features associated with cognitive resilience in subjects with severe alzheimer’s disease (ad) neuropathology. data for this study was obtained from a national alzheimer’s coordinating centre (nacc) dataset. study inclusion criteria are as follows: severe ad pathology, i.e., frequent neuritic plaques and braak & braak stage v/vi pathology, interval between last visit and death ≤ 2 years, and absence of other primary neuropathology diagnoses. cognition was assessed by the mini-mental status examination (mmse) score. a total of 654 cases met our criteria. of these, 59 (9%) persons had mmse scores ≥24 at their last visit and were categorized as cognitively resilient. first, bivariate analysis was done to compare resilient with non-resilient groups. then, variables with significant results were entered into the multivariable model. based on our binary logistic regression model, resilient subjects were older (odds ratio [or]=1.03; 95% confidence interval [ci]=1–1.07), had more years of education (or=1.16; 95% ci=1.04-1.29), had lower bmi (or=0.91; 95% ci=0.85-0.99), were more likely to be a smoker (or=2.78; 95% ci=1.45-5.34), and were more likely to use an anticoagulant/antiplatelet at last visit compared with subjects with impaired cognition (or=1.87; 95% ci=1.01-3.48). in addition to expected protective factors such as higher education and lower bmi, our results showed that more smoking and frequent use of an anticoagulant/antiplatelet at last visit could also be possibly associated with resilience to clinical expression of ad severe pathology. pharmacological approaches that mimic the effects of nicotine may be useful in amelioration of ad symptoms.   abstract 7 free neuropathol 2:29:10 linking iron homeostasis to vulnerability patterns in progressive supranuclear palsy seojin lee1, ivan martinez-valbuena1, suganthini ilaalagan1,3, naomi p. visanji2,4, gabor g. kovacs1,2,3,4 1 tanz centre for research in neurodegenerative disease, university of toronto, toronto, ontario, canada 2 department of laboratory medicine and pathobiology and department of medicine, university of toronto, toronto, ontario, canada 3 laboratory medicine program & krembil brain institute, university health network, toronto, ontario, canada 4 edmund j. safra program in parkinson’s disease and rossy program in progressive supranuclear palsy, toronto western hospital, toronto, ontario, canada progressive supranuclear palsy (psp) is a 4-repeat tauopathy in which pathology starts in select subcortical areas including the globus pallidus (gp) and the substantia nigra (sn), regions also associated with age-related iron accumulation. toxic iron burden in these regions of psp brains have been examined but given the differences in cellular iron homeostasis across cell types and the heterogeneity in psp tau cytopathology, we aim to examine the possible role of iron accumulation on the cellular selective vulnerability of tau pathology in the vulnerable anatomical regions of psp brains. using human post-mortem brain tissue of the early-affected psp brain regions (gp, sn, and putamen), we visualized iron deposition in the neurons, astroglia, oligodendrocytes, and microglia using a combination of dab-enhanced perl’s (ferric) and turnbull (ferrous) iron staining with immunohistochemistry of cell type-specific markers. iron deposition was also examined in relation to their tau cytopathologies using at8-immunohistochemistry. in all three regions, astrocytes and microglia were seen to predominantly accumulate both species of iron. moreover, tau-positive astrocytes showed the highest frequency of cellular iron deposition compared to neurofibrillary tangles and oligodendroglial coiled bodies. on the other hand, association of iron burden with different cellular tau pathologies was species-specific in the same regions, suggesting iron functions in psp tau cytopathology may be distinct by species. our mapping of cellular iron burden in relation to pathology in psp brains suggests a selective cellular vulnerability to iron deposition in diseased brains, and further supports the role of pathological iron in the early pathogenesis of psp.   abstract 8 free neuropathol 2:29:11 friedreich cardiomyopathy is a secondary desminopathy arnulf h. koeppen1,6, rahman f. rafique1, joseph e. mazurkiewicz2, steven pelech3,4, catherine sutter3, qishan lin5 , jiang qian6 1 research service, veterans affairs medical center, albany, new york, usa 2 department of neuroscience and experimental therapeutics, albany medical college, albany, new york, usa 3 kinexus bioinformatics corporation, vancouver, british columbia, canada 4 division of neurology, department of medicine, university of british columbia, vancouver, british columbia, canada 5 rna epitranscriptomics & proteomics resource, university at albany, albany, new york, usa 6 department of pathology, albany medical college, albany, new york, usa hypertrophic cardiomyopathy with or without arrhythmia is the predominant cause of death in friedreich ataxia (fa). the clinical and pathological phenotypes of fa are diverse. the pathogenesis of the fa-related lesions in the central and peripheral nervous systems, heart, skeleton, and endocrine pancreas is incompletely understood. the hypothesis in this research is that frataxin deficiency affects the cellular proteome in downstream mechanisms. antibody microarrays of pooled fa heart lysates showed upregulation in several proteins, including alpha b crystallin, a desmin chaperone. western blots of individual and pooled heart lysates revealed a prominent extra desmin band at 47 kda that was absent or under-expressed in control samples. mass spectrometry confirmed the origin of this band from canonical desmin (53 kda). co-immunoprecipitation of fa heart lysates with anti-desmin, recovery of the antigen-antibody complex with protein a-sepharose, sodium dodecylsulfate polyacrylamide gel electrophoresis, and western blotting confirmed the interaction of desmin and αb crystallin. slide techniques, including immunohistochemistry and double-label immunofluorescence, disclosed desmin and alpha b crystallin aggregation near intercalated discs and z-discs. congo red fluorescence microscopy did not confirm the formation of amyloid. we suggest that accumulation of a truncated desmin and aggregation with αb crystallin cause accelerated heart disease in fa.   abstract 9 free neuropathol 2:29:12 glial senescence (not tau) is the driver of post-concussive symptoms lili-naz hazrati1,2, nicole schwab1,2 1 the hospital for sick children, toronto, ontario, canada 2 department of laboratory medicine and pathobiology, university of toronto, toronto, ontario, canada mild traumatic brain injury (mtbi) leads to chronic symptoms in some patients. pathologically, mtbi is associated with chronic traumatic encephalopathy (cte), a neurodegenerative disease characterized by hyperphosphorylated tau (p-tau) in neurons and astrocytes at the depths of cortical sulci. cte has been proposed as the driver of these symptoms, despite many patients presenting with severe symptoms yet minimal pathology. it is known that low levels of p-tau are present in cognitively intact individuals, including ageing-related tau astrogliopathy (artag), and that p-tau accumulates prior to a diagnosis of alzheimer’s disease. we propose that examining changes beyond p-tau is critical to understand mtbi pathology. the objective of this study is to identify novel pathological markers of mtbi which may explain clinical symptoms and allow for the development of therapeutic targets. using a brain bank of former professional athletes with mtbi history we used immunohistochemistry and gene expression analysis to show cellular senescence as a mechanism driving brain dysfunction after mtbi. compared to controls, mtbi brains show widespread dna damage (γh2ax) and cellular senescence in ependymal cells, astrocytes, and oligodendrocytes even in cases with no neuropathology. gene expression analysis revealed upregulation of the senescence-associated secretory phenotype (sasp), a form of chronic low-level inflammation which has been proposed to drive cognitive dysfunction and proteinopathy. in a mouse model of mtbi, we have recapitulated these results and shown that eliminating senescent cells with senolytic therapy is beneficial. mtbi brains are characterized by early ageing through cellular senescence, which may drive clinical symptoms and p-tau pathology.   abstract 10 free neuropathol 2:29:13 hail to astrogliosis! the unsung hero of the cns tissue reaction to the spinal cord injury. jacek m. kwiecien1 1 department of pathology and molecular medicine, faculty of health sciences, mcmaster university, hamilton, ontario, canada spinal trauma results in a localized spinal cord injury (sci) with an area of hemorrhage and necrosis surrounded by edema in acute phase that lasts 2 days followed by an inflammatory phase beginning on day 3 with infiltration of necrosis by inflammatory cd68+/cd163macrophages whose numbers rapidly increase and persist at high levels for 4 weeks and then decline but are still present in low numbers at 16 weeks. this destructive inflammation is fuelled by myelin-rich necrotic debris and the macrophage activation causes ongoing damage to the surrounding spinal cord and further damaged myelin to sustain a mechanism of vicious cycle. the severity of post-sci inflammation becomes inhibited and eliminated by a spinal cord tissue response in the 3rd phase, resolution. astrogliosis is a hallmark of reaction to sci. in the 1st week astrocytes surrounding the lesion hypertrophy and define a cavity of injury (coi) containing macrophage-rich inflammation and accumulation of water. progression of severity of astrogliosis around the coi coincides with the reduction of numbers of macrophages and leads to formation of quiescent syrinx. while the inflammation in the coi leads to damage of blood vessel in the surrounding spinal cord resulting in vasogenic edema beyond 16 weeks post-sci, astrocytes equipped with aquaporin-4 pass excess edema water to 4 extra-spinal spaces; (1) the sub-arachnoid, (2) the central canal, (3) the blood vessels, and (4) the coi, a novel mechanism. astrogliosis is a beneficial element in neuroplasticity following the sci and should not be inhibited in anti-inflammatory treatments effecting neuroprotection.   abstract 11 free neuropathol 2:29:14 ischemic-like pathology in aberrant white matter tracts of fetal holoprosencephaly: a case series sumit das1, jessica saunders2, sarah nikkel3, lindsay brown4, christopher dunham2 1 department of pathology and lab medicine, university hospital, edmonton, alberta, canada 2 division of anatomical pathology, bc children’s hospital, vancouver, british columbia, canada 3 department of medical genetics, bc children’s hospital, vancouver, british columbia, canada 4 division of genome diagnostics, bc children’s hospital, vancouver, british columbia, canada holoprosencephaly (hpe) is due to defective early forebrain induction resulting in a “lack of cleavage” of the primitive prosencephalon into paired cerebral hemispheres. hpe is generally considered sporadic, although clear environmental and genetic factors have been recognized. from a genetic perspective, an autosomal dominant inheritance pattern is most commonly noted. chromosomal abnormalities are common in hpe, seen in 25-50% of affected individuals, with trisomy 13 being the most frequent. a number of structural chromosomal abnormalities (e.g., 21q22.3 deletion) and pathogenic copy number variations have also been described, as have syndromic associations. pertinent biologic pathways include shh, nodal and bmp; in turn, several single gene mutations related to these pathways have been uncovered, including shh, zic2, and six3. although not frequently emphasized in neuropathologic descriptions of hpe, abnormal white matter has been reported, mainly in the form of aberrant tracts (e.g., absence of the corpus callosum and hypoplasia of the corticospinal pathway). in this study, we detail the clinicopathologic features of 8 fetal bcch cases bearing a seemingly unique dual form of white pathology. all 8 cases demonstrated ischemic-like pathology, with 7 of 8 exhibiting such pathology in a similar location – ventral to the fused deep grey nuclei. diffusion tensor imaging (dti) studies suggest that the neuroanatomic substrate of this aberrant white matter is fused superior and inferior occipito-frontal fasciculi, with the resultant fibers crossing the midline. while the etiology of this ischemic-like pathology is unclear, the literature raises the possibility of a role for the shh pathway.   abstract 12 free neuropathol 2:29:15 brain pathology in patients with congenital heart disease murad a. alturkustani1,2,3, linda j. szymanski2 1 king abdulaziz university, jeddah, saudi arabia 2 children’s hospital los angeles, los angeles, california, usa 3 western university, london, ontario, canada brain pathology in patients with congenital heart disease (chd) are associated with neurodevelopmental delay. imaging studies support vascular etiology for both white and gray matter lesions. in this retrospective study, we describe the pathological changes in the brains of patients with chd. the last 20 autopsy cases in patients with chd at our institution were retrieved and the clinical and the autopsy report were reviewed. the available h&e, special, and immunostains were evaluated and at least one section from each case was stained with gfap, app, and hla-dr. five control cases included telencephalic leukoencephalopathy (3) and no significant pathological changes (2). the following histological features were assessed: necrotic cells in cortex, hippocampus and the cerebellum; app and gfap staining pattern, and presence of focal lesions and amphophilic globules. twenty patients (10 males, 10 females) were identified with age range of 2 weeks – 19 years. the pathological findings are as follows: 8 cases had changes consistent with acute global hypoperfusion, 5 cases showed features consistent with chronic global hypoperfusion, 3 cases showed focal white matter lesions (1 secondary, to septic emboli), 4 cases with only widespread gliosis, one case with only old focal neuronal loss in dentate gyrus, and one with no significant changes. subarachnoid hemorrhages were present in 5 cases and germinal matrix hemorrhage in 3 cases. in conclusion, most of the pathological changes could be explained by cerebral hypoperfusion and better techniques to improve the cerebral perfusion are warranted in the management of these patients.   abstract 13 free neuropathol 2:29:16 characterizing the hippocampal dentate gyrus involvement in temporal lobe epilepsy carolyn twible1, qi zhang1,2 1 department of pathology and laboratory medicine, schulich school of medicine and dentistry, western university, london, ontario, canada 2 department of pathology and laboratory medicine, london health sciences centre, london, ontario, canada hippocampal sclerosis (hs) is the most common pathology finding for drug resistant temporal lobe epilepsy (tle) and is characterized by neuronal loss and gliotic cornu ammonis. nearly 20% of surgical specimens from drug-resistant tle surgery contain “normal” populations of neurons, termed no-hs, yet still benefit from surgical resection. this observation suggests a neuropathological explanation for the epileptogenic focus in the resected tissue that is not detected through standard diagnostic practices. the goals of this project are to increase the neuropathological understanding of drug resistant tle, and to elucidate the structural changes of hs and no-hs, focusing on the granule cell layer (gcl). in this study, 21 tle surgical resection cases were examined, including 14 hs and 7 no-hs cases, to investigate gcl morphometry. information on histopathological diagnosis and post-operative outcome were collected. the digital image analysis software qupath was used to perform cell detection analysis on the gcl. measures including delaunay mean cell density were analyzed. hs patients show a significant increase in granule cell spacing and decrease in granule cell density within the gcl compared to no-hs patients. regardless of the histological diagnosis, patients who achieved seizure freedom post-operatively demonstrated an increase in granule cell spacing and decrease in granule cell density in comparison to those who did not achieve seizure freedom post-operatively. hs and no-hs diagnosis groups have diseaseand post-operative outcome dependent morphometry differences. the post-operative outcome dependent morphometry observed in hs and no-hs patients presents a potential additional method to evaluate post-operative prognosis for tle surgical-resection patients.   abstract 14 free neuropathol 2:29:17 deep learning approaches to deciphering intra-tumoural heterogeneity in glioblastoma anglin dent1, brian lam1, kevin faust1, alberto j. leon3, phedias diamandis1 1 princess margaret cancer centre, university health network, toronto, ontario, canada background: emerging evidence strongly implicates intra-tumoral heterogeneous biology in treatment resistance and disease progression across many cancer types (1). using glioblastoma (gbm) as a prototype, i have aimed to leverage the computational power of artificial intelligence (ai) and deep learning (2) to develop an autonomous workflow for the objective definition and quantification of biologically distinct tumour subpopulations. objectives: i hypothesize that ai-defined tumoral clusters predict spatially distinct molecular profiles and therapeutic targets. methods: i apply our developed image clustering workflows (3) to quantify ai-defined subregions within a clinical cohort of 10 gbm patient tumors and use laser capture microdissection and mass spectrometry-based proteomics to address if ai-defined subregions show intra-tumoral molecular variation. further, i leverage existing pharmacogenomic databases (4) and carry out drug sensitivity and transcriptional clustering to define biomarkers and validate their intra-tumoral expression in my clinical gbm cohort. results: preliminary data shows that region-to-region heterogeneity can be found in idh wild-type gbm using our unbiased omics approach, in addition to predicting different pharmacogenomic sensitivities. conclusions: this project aims to develop the first ai-driven tool to guide the routine and systematic molecular analysis of spatial morphogenomic heterogeneity. further, this tool may have the potential to provide novel approaches for personalized care by selecting drug combinations that target a larger fraction of a tumor’s true biology.   abstract 15 free neuropathol 2:29:18 integrating morphologic and molecular histopathological features through whole slide image registration and deep learning michael k. lee1, kevin faust2,3, anglin dent1, clare fiala3, alessia portante1, madhu rabindranath1, noor alsafwani3,4, andrew gao1,3, ugljesa djuric5, phedias diamandis1,3,5,6 1 department of laboratory medicine and pathobiology, university of toronto, toronto, ontario, canada 2 department of computer science, university of toronto, toronto, ontario, canada 3 laboratory medicine program, department of pathology, university health network, toronto, ontario, canada 4 department of pathology, college of medicine, imam abdulrahman bin faisal university, dammam, saudi arabia 5 princess margaret cancer centre, toronto, ontario, canada 6 department of medical biophysics, university of toronto, toronto, ontario, canada background and objective: despite recent innovations in deep learning, integrating histomorphologic and molecular information found on respective h&e and ihc-stained tissue sections still remains a challenge. while human observers can easily align these different tissue sections, routine computational approaches for image registration of giga-byte sized wsis are still needed. here, we aim to address this issue by incorporating another computer vision tool, scale-invariant feature transform (sift), to align h&e-stained sections with accompanying ihc studies for automated subclassification of gliomas. method: to test the workflow, we first trained a vgg19 convolutional neural network (cnn) using pathologist-annotated h&e wsis to recognize histological patterns of 16 common tissue and lesion classes. afterwards, we optimized a different set of cnns to recognize various ihc markers, such as idh1-r132h and atrx, which are relevant for molecular subclassification of gliomas. for the integrated analysis, we employed sift to find features for image matching which were used to align lesional regions of h&e and ihc slides. results: the histomorphologic classifier (cnnh&e) excelled at classification with accuracies of 100% for glioma, meningioma and metastatic carcinoma, and 93% for schwannoma (n = 125). using the newly developed cnnh&e and sift-based alignment, the quantitative analysis by the ihc classifiers significantly improved for atrx retained and idh1-r132h positive wsis when compared to that of unaligned wsis. conclusion: sift can work in concert with deep learning tools to provide a pathologist-inspired workflow to help automate advanced immunohistochemically diagnostic tasks, such as subclassification of glioma.   abstract 16 free neuropathol 2:29:19 neuropathology of eight cases of the new brunswick cluster of neurological syndrome of unknown cause (nsuc) gerard h. jansen1, sidney e. croul2, angela k. miller3, alexander s. easton2, john m. j. woulfe1 1 department of pathology and laboratory medicine, university of ottawa, ottawa, ontario, canada 2 department of pathology, qeii health science centre, halifax, nova scotia, canada 3 department of pathology, health sciences centre, winnipeg, manitoba, canada (previously department of pathology, moncton hospital, moncton, new brunswick, canada) on march 17, 2021 a press conference aired featuring a moncton neurologist and new brunswick’s chief medical health officer, regarding a cluster of patients in new brunswick who had symptoms reminiscent of cjd, and were claimed to have a novel neurological syndrome. the onset of the disease was between 2015 and 2021. all patients in that cluster had at that time been reported by one neurologist, although subsequently a few incidental cases were reported by other neurologists. the size of this cluster has been reported at around 50 cases. further news publications have suggested that an environmental factor is the causative factor for this cluster. this news has significantly disturbed the medical community. since 2019 eight patients have died that belonged to this cluster. we report their neuropathological findings and hope this will bring some clarity. there was one case of metastatic carcinoma, one case of ftld-tdp43, one case of neocortical lewy body pathology, one case of neocortical lewy body pathology and ad, 2 cases of ad with vascular pathology, one case of mainly vascular pathology, and one case without significant pathology (consistent with the patient’s previous history). in these 8 patients no evidence for a prion disease was found, nor novel pathology. we suggest that these 8 patients represent a group of misclassified clinical diagnoses. since june 3, 2021 the oversight committee was established in new brunswick reviewing the clinical and epidemiological data of the patients in this cluster. we hope that our findings are useful to them.   abstract 17 free neuropathol 2:29:20 covid-19 pandemic impact on surgical neuropathology services at london health sciences centre shervin pejhan1, chris tran1, david driman1, robert hammond1,2, lee cyn ang1,2, qi zhang1,2 1 department of pathology & lab medicine, london health sciences centre, london, ontario, canada 2 department of clinical neurological sciences, london health sciences centre, london, ontario, canada the covid-19 pandemic has had a significant impact on medical services. many jurisdictions postponed non-urgent procedures to balance individual patient care with public health precautions. surgical backlogs caused by the covid-19 pandemic have been evaluated by different groups. however, the impact of this pandemic on pathology and specifically neuropathology (np) services has received limited attention. in this study, we reviewed all surgical np reports of our centre from march 2018 (two years before the pandemic declaration) through july 2021. patient demographic information and pathological variables were collected. for tumours, site, type, and who grade were analyzed. within the period under study, the total number of np samples was lowest in april 2020, corresponding to the first ontario provincial lockdown. in comparison to the dramatic decrease in other surgical pathology subspecialties (camj 2021;193(10): e343), the np surgical specimen initially had a minimal volume reduction, with a rapid return to baseline. among the different types of np surgical specimens, muscle biopsy and epilepsy-related specimens showed a more significant reduction. there was a slight increase in higher-grade tumours. interestingly, a gradual increase of brain biopsies for inflammatory conditions was noticed. our results show that the neuropathology service volume reduction due to the covid-19 pandemic has not been as significant as other pathology subspecialties. studying the variations in histopathological diagnoses in pandemic years could be helpful for future planning in both clinical and pathological sectors, especially when the data is strengthened by the experiences of other medical centers.   copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuromuscular disease: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:3 (2021) review neuromuscular disease: 2021 update marta margeta department of pathology, university of california, san francisco, ca, usa corresponding author: marta margeta · ucsf pathology, box 0511 · 513 parnassus ave., hsw-514 · san francisco, ca 94143 · usa marta.margeta@ucsf.edu submitted: 5 february 2021 accepted: 22 february 2021 copyedited by: henry robbert published: 23 february 2021 https://doi.org/10.17879/freeneuropathology-2021-3236 keywords: costameres, sarcomeres, satellite cell activation, covid-19, guillain-barré syndrome, 5q-sma, smaled, lobulated fibers, supervillin, vacuolar myopathy, perilipin 4, sarcolemmal repair, trim72, vasculitis, amyloidosis, gene therapy, large-scale electron microscopy abstract this review highlights ten important advances in the neuromuscular disease field that were first reported in 2020. the overarching topics include (i) advances in understanding of fundamental neuromuscular biology; (ii) new / emerging diseases; (iii) advances in understanding of disease etiology and pathogenesis; (iv) diagnostic advances; and (v) therapeutic advances. within this broad framework, the individual disease entities that are discussed in more detail include neuromuscular complications of covid-19, supervillin-deficient myopathy, 19p13.3-linked distal myopathy, vasculitic neuropathy due to eosinophilic granulomatosis with polyangiitis, spinal muscular atrophy, idiopathic inflammatory myopathies, and transthyretin neuropathy/myopathy. in addition, the review highlights several other advances (such as the revised view of the myofibrillar architecture, new insights into molecular and cellular mechanisms of muscle regeneration, and development of new electron microscopy tools) that will likely have a significant impact on the overall neuromuscular disease field going forward. abbreviations aav, adeno-associated virus; anca, antineutrophil cytoplasmic antibody; asm, antisynthetase syndrome-associated myositis; attr, transthyretin amyloidosis; cgrp, calcitonin-gene related peptide; covid-19, coronavirus disease 2019; ck, creatine kinase; dm, dermatomyositis; fesem, field-emission scanning electron microscopy; glud1, glutamate dehydrogenase 1; egpa, eosinophilic granulomatosis with polyangiitis; gbs, guillain-barré syndrome(s); hmgcr, 3-hydroxy-3-methylglutaryl-coa reductase; iim, idiopathic inflammatory myopathy; imnm, immune-mediated necrotizing myopathy; lmn, lower motor neuron; mmsc, muscle mesenchymal stromal cells; nmd, neuromuscular disease; sars, severe acute respiratory syndrome; sibm, sporadic inclusion body myositis; sma, spinal muscular atrophy; smaled, spinal muscular atrophy lower extremity dominant; srp, signal recognition particle; stem, scanning transmission electron microscopy; svv, small vessel vasculitis; tem, transmission electron microscopy; tregs, regulatory t cells; ttr, transthyretin. in this update, i will briefly describe ten 2020 neuromuscular field advances that i consider to be most important and/or interesting; as in the prior update (margeta, 2020b), these advances will be grouped into several different “discovery clusters” and listed in no particular order. advances in fundamental neuromuscular biology with implications for neuromuscular disease 1. new understanding of the myofibrillar architecture based on numerous studies that used traditional electron microcopy methods for evaluation of skeletal muscle ultrastructure, the myofibrillar apparatus has long been understood as consisting of independent tube-like myofibrils that are arranged in parallel and span the entire length of a fiber, with each myofibril composed of sarcomeres that are serially connected through z-discs. this traditional model of the myofibrillar apparatus was effective in explaining how the contractile forces are transmitted down the length of the fiber towards the myotendinous junction, but could not readily explain how these forces were transmitted laterally, towards the sarcolemma and the extracellular matrix (into which myofibers are anchored through costameres, the sarcolemma-associated supramolecular structures that form membrane skeleton and mediate fiber adhesion). a recent groundbreaking study by willingham et al. has used a new method (focused ion beam-scanning electron microscopy) to image muscle fibers in three dimensions at high resolution, providing a new view of the myofibrillar architecture (willingham et al., 2020). to their surprise, the authors found that sarcomeres are not organized into independent parallel myofibrils as traditionally thought, but instead form the unified, non-linear myofibrillar matrix that spans both the length and the width of a fiber and is interconnected through extensive and bidirectional sarcomere branching (fig. 1). this branching occurs in what appears to be a random pattern, with branch points that are not restricted to any specific region of the sarcomere; however, the frequency of branches is fiber-type specific, with branches approximately three times more frequent in slow twitch than fast twitch fibers (and approximately two times more frequent in slow twitch fibers than cardiac myocytes). interestingly, sarcomere branching is developmentally regulated: while present at birth, it is downregulated in the period of rapid postnatal muscle growth and then upregulated again late in development. three different mechanisms of sarcomere branching were identified: sarcomere splitting (where some of the myofilaments from one sarcomere separated from the rest, forming two distinct myofibrillar segments), myofilament transfer (where some of the myofilaments were redistributed between sarcomeres, separating from one myofibrillar segment and joining the adjacent one) and myofilament trade (where myofilament transfer between two adjacent myofibrillar segments was reciprocal/bilateral). while most of the work was performed on murine samples, the few human samples that were evaluated in this study showed essentially identical findings, except for a slightly higher branching frequency in human compared to murine fast-twitch fibers (2.4 ± 0.2 vs. 1.6 ± 0.2 branches per 10 sarcomeres); however, a more thorough investigation of inter-species differences remains to be done. figure 1. 3d renderings of the interconnected sarcomeres that form the unified myofibrillar matrix. a. 3d rendering of 115 directly connected sarcomeres within the myofibrillar matrix of a fast-twitch muscle. individual colors represent different myofibrillar segments linked by branching sarcomeres. b-d. 3d renderings at different perspectives highlight the nonlinearity and connections between myofibrillar segments. representative of 7,707 sarcomeres from four volumes from four mice. scale bars, 2 µm. (this figure and its legend were adopted from figure 1 in willingham et al., 2020; this use is permitted under the creative commons attribution 4.0 international license.) what is the relevance of this new understanding of the myofibrillar architecture for neuromuscular disease (nmd)? that must be experimentally addressed and therefore remains to be seen, but it is very likely that abnormal branching of sarcomeres contributes to poor contractile force generation in various congenital / structural myopathies, which show different degrees of organelle malpositioning and myofilament disorganization. in addition, this new discovery may have important implications for pathogenesis of muscular dystrophies: given that the myofibrillar matrix is directly connected to the costameric membrane skeleton (discussed further in advance #4 below), it is possible that disruption of these connections may result in inappropriate transfer of lateral mechanical forces from the interior of the fiber to the extracellular matrix, thereby contributing to the likelihood of segmental fiber necrosis that over time leads to exhaustion of the muscle regenerative capacity and muscle wasting. 2. deepening insights into molecular and cellular mechanisms of muscle regeneration over the past decade, much has been learned about the mechanisms of skeletal muscle regeneration; the key role of satellite cells (muscle stem cells) in this process was first demonstrated in 2011, and the work that followed since then has showed that satellite cell activation requires cytokines and growth factors released by foxp3-expressing regulatory t cells (tregs) and macrophages. two studies published last year build further on these discoveries by elucidating the intercellular crosstalk and chemical signals that mediate muscle regenerative response. the first study focused on the interplay between intramuscular peripheral nerve twigs, muscle mesenchymal stromal cells (mmscs), and tregs in skeletal muscle regeneration (wang et al., 2020). following muscle injury, mmscs are the main source of interleukin (il)-33, which drives subsequent accumulation of tregs required for satellite cell activation and muscle repair. wang et al. have found that il-33+ mmscs are located in close proximity to peripheral nerve bundles and that they titrate their il-33 production in response to calcitonin-gene related peptide (cgrp), which plays a role in pain perception and is released by sensory peripheral nerve terminals in response to tissue damage. in agreement with these findings, systemic administration of cgrp led to an increase in the muscle il-33 level and recruitment of tregs into the muscle compartment. however, it remains to be shown whether cgrp treatment will enhance muscle regeneration and – conversely – whether inhibition of cgrp signaling will attenuate effective repair following muscle injury; the latter question is particularly pressing given that anti-cgrp antibodies and cgrp antagonists have recently been approved for prevention of migraine headaches. in addition, it remains to be seen whether effective muscle repair requires only signaling from sensory nerve terminals, or whether motor nerve twigs also play a role in this process. the second and more comprehensive of the two studies centered on the nature of chemical crosstalk between macrophages and satellite cells that is required for muscle repair (shang et al., 2020). the authors of that study modulated glutamine secretion from macrophages via genetic or pharmacologic manipulation of glutamine synthetase (which generates glutamine by condensation of glutamate and ammonia) or glutamate dehydrogenase 1 (glud1, which catalyzes oxidative deamination of glutamate and thereby reduces the amount of glutamate available for glutamine synthesis); in addition, they blocked satellite cell glutamine uptake by knocking down glutamine transporter slc1a5 selectively in those cells. using these experimental approaches both in vitro and in vivo, the authors demonstrated that satellite cells take up glutamine that is secreted by monocyte-derived macrophages recruited into skeletal muscle following injury; this glutamate uptake, which leads to activation of mtor signaling, is required for satellite cell proliferation and differentiation. excitingly, shang et al. showed that increased glutamine bioavailability in injured or aged muscle translates into increased muscle regenerative capacity: mice with decreased glud1 activity showed faster and more effective fiber regeneration following acute myotoxic injury when young, as well as increased muscle mass and improved baseline physical performance when aged. while these findings need to be replicated in other model systems and ultimately human clinical trials, they hold promise not only for treatment of muscular dystrophies and other skeletal myopathies characterized by recurring muscle fiber necrosis, but also for treatment of the age-associated sarcopenia that leads to significant morbidity and mortality in elderly populations. newly defined / emerging neuromuscular diseases 3. neuromuscular complications of covid-19 2020 has been dramatically shaped by the covid-19 pandemic, which has affected the nmd field in addition to all other aspects of life. covid-19 is caused by infection with sars-cov-2, an rna virus from the coronavirus family; the disease is asymptomatic in up to 50% of patients, but can also cause severe pneumonia, acute respiratory distress syndrome, and death. neurologic complications of covid-19 were noted very early in the pandemic (mao et al., 2020) and include cns, pns, and skeletal muscle manifestations (cagnazzo et al., 2020); in this update, two forms of potential neuromuscular involvement [guillain-barré syndromes (gbs) and rhabdomyolysis / myopathy] will be discussed in more detail. gbs are a group of acute immune-mediated polyneuropathies that are characterized by ascending weakness, mild-moderate sensory abnormalities, and pain; the most common subtypes include acute demyelinating inflammatory polyradiculoneuropathy, acute motor axonal neuropathy, and the miller-fischer syndrome (dalakas, 2020). in approximately 70% of gbs patients, a flu-like illness precedes neurologic symptoms by ~1-3 weeks, and all subtypes of gbs show association with different infectious agents (both viruses and bacteria). perhaps not surprisingly, gbs was also documented in patients with covid-19, first through case reports and small case series (reviewed in caress et al., 2020; dalakas, 2020; de sanctis et al., 2020) and later through small retrospective case-control studies, one from italy (filosto et al., 2020) and another from spain (fragiel et al., 2020). based on these early reports, covid-19-associated gbs was shown to have typical clinical characteristics, with most cases falling within the acute demyelinating subtype and showing onset of neurologic symptoms a median of 10-23 days after the covid-19 diagnosis. the two retrospective case-control studies found 3to 10-fold higher gbs incidence during the study period (the 2-month pandemic peak in each country) compared to the equivalent period in 2019; however, the conclusions were limited by relatively small sample sizes and a high potential for confounding due to selection and ascertainment bias. indeed, the most comprehensive investigation of the potential link between gbs and covid-19 to date (a combined retrospective epidemiologic and prospective cohort study performed in the uk and published at the end of 2020) found no association between these two conditions (keddie et al., 2020). in fact, and somewhat counterintuitively, the authors of that study found a decrease (rather than increase) in the number of gbs cases that required ivig treatment during the 2-month pandemic peak compared to prior years; while reasons for this decrease are not entirely clear, one possible explanation is a decrease in the number of infections with known gbs-triggering pathogens as a result of drastic public health actions that were undertaken to control the pandemic. in addition to a lack of increase in gbs incidence, keddie et al. found no temporal or spatial association between covid-19 and gbs cases in the uk during the study period, further weakening the likelihood of true association between these two diseases. given these contradictory findings, additional studies should be done; however, the preponderance of current evidence suggests that the sars-cov-2 infection is not a major trigger of autoimmune polyneuropathy. what about skeletal muscle involvement in covid-19? as with other flu-like illnesses, myalgias are a common symptom of this viral disease; however, actual skeletal muscle injury [as evidenced by elevated creatine kinase (ck) levels] is seen only in a subset (~10-30%) of patients (dalakas, 2020; manzano et al., 2020). in some of these “covid-19 myopathy” cases, muscle injury is very severe, with ck levels greater than 10,000 u/l (buckholz et al., 2020; dalakas, 2020; and a number of individual case reports); the most severe case was documented in a patient on rosuvastatin therapy who was infected with sars-cov-2 and developed fatal rhabdomyolysis, with the ck level of ~1,000,000 u/l (anklesaria et al., 2020). however, very few muscle biopsies or autopsy muscle samples from covid-19 patients have been comprehensively evaluated to date; as a result, very little is known about the mechanisms that underlie covid 19-associated muscle injury. skeletal muscle expresses angiotensin-converting enzyme 2, which serves as the cellular receptor for sars-cov-2 (ferrandi et al., 2020); thus, it is conceivable that this virus can invade and directly injure muscle fibers. however, limited studies published thus far have not provided evidence for viral presence in the injured muscle (manzano et al., 2020; rosato et al., 2020; zhang et al., 2020). [one case report noted the presence of coronavirus-like particles in a postmortem muscle sample from a patient who died from covid-19 but had no symptoms of skeletal muscle injury (hooper et al., 2020); however, ultrastructural detection of coronaviruses can be challenging (dittmayer et al., 2020a) and the virus-like particles shown in that paper more closely resemble clathrin-coated vesicles or parts of the rough endoplasmic reticulum than true coronaviruses.] instead, the currently available data suggest that covid 19-associated muscle injury is most likely para-infectious / immune-mediated, and can manifest as dermatomyositis-like “type i interferonopathy” (manzano et al., 2020), myositis (zhang et al., 2020), or immune-mediated necrotizing myopathy (fig. 2). this is in agreement with the work done during the sars epidemic in early 2000s, which suggested that vasculitis or immune-mediated mechanisms, rather than direct viral infection, were a cause of sars-associated myopathy (ding et al., 2003; leung et al., 2005). given that the available studies are very limited in scope, much additional work is needed to fully elucidate the mechanisms of skeletal muscle injury in covid-19. however, an intriguing possibility is that both mechanisms play a role, as recently shown for the chikungunya infection-associated myopathy: in that emerging viral disease, muscle pathology is due to activation of the host immune response but is triggered by viral replication in muscle fibers (lentscher et al., 2020). figure 2. immune-mediated necrotizing myopathy in a patient with covid-19. a representative h&e-stained cryosection (a) shows frequent, randomly distributed degenerating/regenerating muscle fibers and focal myophagocytosis in the absence of a significant lymphocytic inflammatory infiltrate; unusual dystrophic calcifications are seen in a subset of necrotic fibers (arrows), confirmed by von kossa calcium stain (not shown). deposition of the complement membrane attack complex (c5b9-immunostained cryosection; b) is seen in the sarcoplasm of necrotic fibers as well as the sarcolemma of many intact-appearing fibers. immunohistochemical stains for lc3 (c) and p62 (d; both performed on formalin-fixed, paraffin embedded tissue) show frequent fibers with densely packed fine puncta. immunohistochemical stain for sars-cov-2 was negative and no coronavirus particles were detected on ultrastructural evaluation (not shown). the patient had a history of hepatitis c, diabetes mellitus, hypertension, and hyperlipidemia treated with atorvastatin. he was diagnosed with covid-19 based on a positive sars-cov-2 pcr test performed following a close exposure; he was asymptomatic at the time, but subsequently developed progressive lower extremity weakness and dysphagia. approximately 1 month after the first weakness symptoms, he lost his sense of smell, developed shortness of breath, and was hospitalized. at admission, his ck level was 33,000 u/l and he was still positive for sars-cov-2; muscle biopsy was performed 10 days into his hospital course (~ 2 months following initial covid-19 diagnosis). the patient was ultimately treated with prednisone, which led to improvement of his neuromuscular symptoms; he was discharged from the hospital never requiring mechanical ventilation for his covid-19 pneumonia. testing for anti-hmgcr and anti-srp antibodies was not performed. scale bar, 50 µm. 4. structural myopathy caused by supervillin deficiency the presence of occasional lobulated fibers is a relatively common and therefore nonspecific finding in muscle biopsies. in some cases, however, frequent lobulated fibers dominate the overall histopathologic picture, resulting in a descriptive diagnosis of a “lobular” (or “trabecular”) myopathy. in general, this descriptive diagnosis does not correspond to a specific, well-defined clinicopathologic entity; however, a newly defined structural myopathy caused by loss-of-function mutations in supervillin (hedberg-oldfors et al., 2020) is an exciting exception to this rule and, while probably rare, should be on the radar screen of every neuromuscular pathologist going forward. supervillin is a large, differentially spliced and ubiquitously expressed actin-binding protein that is particularly abundant in striated muscle; the 250 kda muscle isoform (also called archvillin) interacts and/or co-localizes with costameric proteins such as dystrophin and dystrophin-associated proteins, integrin/vinculin/talin complex, α-actinin, desmin, and caveolin 3 (oh et al., 2003). (as mentioned in advance #1, costameres are membrane-associated supramolecular structures that mediate muscle fiber adhesion to the extracellular matrix.) during skeletal muscle development, supervillin is located at the ends of differentiating myotubes, where it is thought to contribute to the myotendinous junction formation (oh et al., 2003). in addition, supervillin plays a role in the myofibril assembly (lee et al., 2007) and helps anchor peripheral myofibrils to the sarcolemma by connecting a z-disc protein nebulin to costameric proteins dystrophin and γ-sarcoglycan (lee et al., 2008; spinazzola et al., 2015). (highlighting the critical importance of myofibril anchoring, z-discs of peripheral myofibrils are also linked to the sarcolemma through binding of a z-disc protein filamin c to costameric proteins γ-sarcoglycan and integrin; reviewed in peter et al., 2011.) mutations in genes that encode costameric proteins mostly lead to muscular dystrophies, while mutations in myofibrillar and cytoskeletal proteins generally result in congenital myopathies; loss-of-function mutations in supervillin, which have previously not been associated with any muscle disease, are now known to cause a unique structural myopathy (hedberg-oldfors et al., 2020). the authors described four patients from two unrelated consanguineous families (two affected siblings in each family) that were ultimately shown to carry homozygous truncating mutations in supervillin, resulting in a complete loss of supervillin protein. while there were subtle clinical differences between these two families, shared clinical findings included onset in childhood/adolescence, muscle pain and stiffness without significant muscle weakness, wide neck and hypertrophy of back muscles, progressive contractures, moderately elevated ck levels, and mild cardiac involvement (mainly left ventricular hypertrophy). muscle biopsies from all four patients showed frequent lobulated type 1 fibers, myofibrillar disarray (including occasional z-band streaming and nemaline rods), subsarcolemmal protein aggregates, and autophagic vacuoles (fig. 3; see also hedberg-oldfors et al., 2020). aside from frequent lobulated fibers, these histopathologic features show some overlap with myofibrillar myopathies, a group of muscle disorders characterized by z-disc instability and defects in the chaperone-assisted selective autophagy (reviewed in margeta, 2020a); this histopathologic overlap may reflect the role of supervillin in the z-disc stabilization / sarcomere anchoring. interestingly, the currently known supervillin mutations are expected to abolish expression of all five supervillin isoforms (not just the archvillin isoform expressed in striated muscle); while these four patients did not show clinical involvement of any non-muscle tissues, the disease spectrum is likely to widen as additional patients (and mutations) are identified in the future. going forward, it will be particularly interesting to elucidate how the loss of supervillin leads to fiber lobulation and to establish whether similar mechanisms operate in other myopathies with frequent lobulated fibers. figure 3. histopathology of “supervillinopathy”. biopsy from the deltoid muscle of patient iii:2 in family 2, as visualized by light (a–c) and electron microscopy (d–g). a. a considerable number of partially atrophic lobulated muscle fibers with pointed, occasionally cap-like subsarcolemmal deposits (arrows) (h&e). b. prominent oxidative enzyme activity in these fibers (arrows) (nadh). c. atrophic lobulated fibers are almost exclusively type 1 fibers (arrows); no neurogenic pattern is observed (matpase at ph 9.4). d. subsarcolemmal accumulation of degraded myofibrils, glycogen, pleomorphic material and autophagy-associated (arrows) organelles. e. heterogeneous lipoprotein deposits including maturing lipofuscin (arrows). f. sporadically, nemaline rods are seen (arrow). g. three rods in transverse section (arrows). (this figure and its legend were adopted from figure 5 in hedberg-oldfors et al., 2020; this use is permitted under the creative commons attribution 4.0 international license.) advances in understanding of etiology and pathogenesis of neuromuscular diseases 5. genetic vacuolar myopathies: expanding etiology spectrum vacuolar myopathies are pathologically defined by the presence of autophagic vacuoles that can be seen on the light microscopic level (“rimmed vacuoles”), electron microscopic level, or both. vacuolar myopathies have varied etiologies that include myotoxic drugs, chronic inflammation, and genetic mutations; genetic forms can occur either as stand-alone myopathies or together with amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and/or paget’s disease of the bone as a component of multiple system proteinopathies (reviewed in margeta, 2020a). unsurprisingly, then, the differential diagnosis of a vacuolar myopathy remains broad, and the final diagnosis can be difficult to establish even in the era of widely available genetic testing; this complexity was highlighted by a 2020 study (mair et al., 2020) that performed thorough clinicopathologic and genetic evaluation of 32 adult vacuolar myopathy cases from 30 unrelated families, and which includes a very comprehensive table of non-inflammatory causes that will be a useful resource for every practicing muscle pathologist. however, and highlighting the rapid pace of advancement in the nmd field, this table is already incomplete: a very exciting work published last year (ruggieri et al., 2020) demonstrated that a repeat expansion in the lipid droplet-coating protein perilipin 4 is the cause of another rimmed vacuolar myopathy the autosomal dominant distal myopathy that was previously linked to chromosome 19p13.3 (di blasi et al., 2004). the age at diagnosis in the originally published kindred of 19 individuals varied from 26 to 73 years, but was earlier in the later generations; the symptoms were variable, but never included dysphagia or dysphonia. pathologically, the biopsies showed some unique features: vacuoles were mainly subsarcolemmal, membrane-limited, and sometimes connected to the cell surface and/or bordered by the basal lamina, but did not include filamentous inclusions and were not congo redor thioflavin s-positive (fig. 4; see also di blasi et al., 2004; ruggieri et al., 2020). in spite of early genetic linkage of this disease to chromosome 19p, the causative mutation proved challenging to elucidate; ultimately, ruggieri et al. honed in on the correct target by performing quantitative mass spectroscopy of microdissected vacuoles, which identified perilipin 4 as the most enriched among 700 identified proteins. the subsequent analysis showed that the gene encoding perilipin 4, plin4, maps to 19p13.3; in affected patients, this gene contains a 891-nucelotide expansion of the repetitive nucleotide sequence in exon 4, resulting in extra 297 amino acids in the amphipathic domain of perilipin 4 protein. figure 4. histopathology of “perilipinopathy” (19p13.3-linked distal myopathy). a-b. gomori trichrome and h&e staining, respectively, in patients iv:3 and v:3, showing vacuoles (arrows) [rimmed, empty, or containing granular or basophilic material (arrowheads), mainly located in the subsarcolemmal region of fibers], fiber size variability, central nuclei, and mildly increased endomysial spaces. note minimal changes in v:13. c. electron micrographs unveiling vacuoles located in the subsarcolemmal region or deep in the sarcoplasm, containing small vesicles, membranous bodies, and granular debris; inset shows granular debris within a small subsarcolemmal vacuole (arrow) opening to the fiber’s surface and sarcolemmal interruption (arrowheads). (this figure and its legend were adopted from figure 1 in ruggieri et al., 2020; this use is permitted under the creative commons attribution 4.0 international license.) perilipins are proteins that localize to the surface of lipid droplets, where they interact with lipid enzymes and other regulators of lipid droplet metabolism; the five known isoforms all contain an 11-mer repeat sequence that repeats 3 times to form a 33-mer amphipathic helix required for lipid droplet localization (copic et al., 2018). perilipin 4 [the isoform most highly expressed in skeletal muscle, where it mainly localizes to the subsarcolemmal regions of type 1 fibers (pourteymour et al., 2015)] has a very long amphipathic domain that normally consists of 29-31 33-mer repeats but is extended by 9 additional 33-mers in patients with 19p13.3 distal myopathy (ruggieri et al., 2020). it is currently not understood how this expansion of the perilipin 4 amphipathic domain leads to muscle disease; however, the defect almost certainly does not involve metabolism of lipid droplets, which were similar in size and number in control and patient samples (ruggieri et al., 2020). on the other hand, the patient samples showed an increase in subsarcolemmal perilipin 4 expression and co-localization of this protein with p62/sqstm1, nbr1 and wdfy3 [autophagic adapters that play a key role in aggrephagy and granulophagy (selective autophagy of protein aggregates and stress granules, respectively)]. this finding provides a potential link between 19p13.3 distal myopathy and multiple system proteinopathies, which all show a defect in granulophagy (reviewed in margeta, 2020a). interestingly, expression of perilipin 4 in the cns is regulated by a stress granule rna-binding protein tia1 (heck et al., 2014), the gain-of-function mutations in which lead to welander distal myopathy (hackman et al., 2013; klar et al., 2013). it remains to be shown whether tia1 also regulates perilipin 4 expression in skeletal muscle and whether this regulation plays a role in pathogenesis of these two (and potentially other) distal vacuolar myopathies. 6. vasculitic neuropathy secondary to eosinophilic granulomatosis with polyangiitis: two pathogenetic mechanisms vasculitic neuropathy, the most common indication for nerve biopsy in the current neuromuscular pathology practice, can be secondary to many different forms of vasculitis. based on the most recent consensus nomenclature (jennette et al., 2013), small vessel vasculitis (svv; the category of vasculitis that is most likely to involve peripheral nerves) is divided into two subcategories – immune complex svv (which shows abundant immunoglobulin deposition in the walls of affected vessels) and anca (antineutrophil cytoplasmic antibody)-associated svv (where affected vessel walls show a paucity of immunoglobulin deposits). systemic anca-associated svv includes three distinct subcategories: microscopic polyangiitis, granulomatosis with polyangiitis (previously known as wegener’s granulomatosis), and eosinophilic granulomatosis with polyangiitis (egpa; previously known as churg-strauss syndrome). interestingly, some patients with anca-associated svv do not actually have detectable anca; it is not entirely clear whether that is because they have anca that cannot be detected with current methods, because they have novel, yet-to-be discovered anca, or because pathogenesis of their vasculitis actually does not involve anca at all (jennette et al., 2013). a large clinicopathologic study of egpa-associated neuropathy published last year (nishi et al., 2020) sheds some light on this question by providing evidence for different pathogenic mechanisms in anca-positive and anca-negative forms of egpa-associated neuropathy. egpa is a form of systemic svv that is associated with asthma, eosinophilia, and peripheral neuropathy; neuropathy is common and affects 50-75% of patients. among the three subtypes of systemic anca-associated svv, egpa is least likely to show anca positivity: just 30-40% of patients have anti-myeloperoxidase anca, and almost none have anti-proteinase 3 anca (nishi et al., 2020). to explore differences between anca-positive and anca-negative forms of egpa, nishi and co-authors have retrospectively investigated 82 consecutive patients who were diagnosed with egpa-associated neuropathy and underwent sural nerve biopsy; 33% of study subjects were positive for anti-myeloperoxidase anca and none were positive for anti-proteinase 3 anca. clinically, anca-positive and anca-negative egpa patients were quite similar, although anca-positive patients were more likely to show involvement of the upper extremities than the anca-negative ones. pathologically, both anca-positive and anca-negative cases showed predominantly axonal form of nerve damage with a focal or multifocal pattern of axon loss suggestive of ischemic injury; however, the vascular involvement differed between the two groups. nerve biopsies from anca-positive patients were more likely to show frank vasculitis and/or destruction of vascular structures, with the mean diameter of affected vessels ~170 µm (fig. 5a); in contrast, nerve biopsies from anca-negative cases were more likely to show a large number of eosinophils in the epineurial vessel lumina, epineurial vessels occluded by intraluminal eosinophils (with vessels smaller than 13 µm typically showing more than 50% occlusion; fig. 5b), and extravasation of eosinophils into the endoneurium. interestingly, the pattern of axon loss in anca-negative cases showed no topographic correlation with the location of endonurial eosinophils; rather, it was patchy (focal or multifocal), suggestive of nerve ischemia. taken together, these findings suggest that there are two distinct pathogenetic mechanisms for ischemic injury in egpa-associated neuropathy: frank vessel destruction in anca-positive cases and vessel occlusion by eosinophils in anca-negative cases (the latter mechanism is conceptually similar to vessel occlusion by intravascular large b-cell lymphoma, which causes tissue ischemia without directly destroying vascular structures). while both patterns of vessel involvement can be seen in skeletal muscle (fig. 5) in addition to peripheral nerves of patients with egpa, it remains to be seen whether differences between anca-positive and anca-negative egpa cases will be conserved across all affected organ systems. in addition, it needs to be established whether similar mechanistic differences distinguish anca-positive and anca-negative cases in the other two types of anca-associated svv. finally, given the almost complete lack of vessel wall inflammation in anca-negative egpa cases, it needs to be re-evaluated whether this clinicopathologic syndrome should remain classified as a vasculitis or whether a different disease category would be more appropriate. figure 5. two patterns of vascular involvement in eosinophilic granulomatosis with polyangiitis. a. muscle biopsy from a patient with anca-positive egpa shows necrotizing vasculitis affecting small intramuscular artery; the inflammatory infiltrate includes eosinophils. b. the lumen of a very small perimysial artery (arrow) is nearly completely occluded by intraluminal eosinophils in a muscle biopsy from a patient with egpa; anca status was not reported. representative images from h&e-stained sections of formalin-fixed, paraffin-embedded tissue are shown for both cases. scale bar, 20 µm. 7. spinal muscular atrophies: skeletal muscle involvement spinal muscular atrophies (smas) are a group of genetic disorders in which the loss of lower motor neurons (lmns) in the anterior horn of the spinal cord, which typically occurs early in life, results in neurogenic muscle atrophy and weakness. the most common sma subtype, 5q-sma, accounts for ~95% of sma cases and is caused by autosomal recessive, loss-of-function mutations in both copies of smn1 (survival motor neuron 1) gene; the clinical phenotype is modified by the presence of the paralog gene, smn2, which yields only a small number of full-length transcripts and is present in a variable number of copies in different individuals, with more copies resulting in a milder clinical phenotype and better survival. a different sma subtype, known as smaled (“spinal muscular atrophy lower extremity predominant”), is caused by autosomal dominant mutations in dync1h1 and bicd2 genes, which impair centripetal microtubular transport that moves intracellular cargo from the cell periphery to the perinuclear region [dync1h1 encodes a subunit of the dynamin/dynactin retrograde transport complex, while bicd2 encodes a cargo adapter protein that directly binds to dync1h1 (koboldt et al., 2020)]. pathogenesis of both 5q-sma and smaled was thought to involve loss of lmns that occurs in a cell-autonomous manner; however, two studies published last year (kim et al., 2020; rossor et al., 2020) highlight the important and somewhat unexpected role of skeletal muscle in these disease processes. in addition to the lmn loss that is a defining feature of the sma phenotype, bicd2 deficiency causes cerebellar hypoplasia through a pathway that involves bergmann glia in a non-cell autonomous manner. to investigate whether the lmn loss in smaled also involves non-cell autonomous mechanisms, rossor et al. selectively deleted bicd2 gene from either lmns or skeletal muscle (rossor et al., 2020). while neither mouse model showed altered gait or decreased life expectancy, a subtle loss of small diameter (gamma) motor axons was observed in the mice lacking bicd2 in skeletal muscle; in contrast, there was no evident axon loss (or any other mn phenotype) in the mice lacking bicd2 expression in lmns. further experiments demonstrated that skeletal muscle from bicd2-/mice showed a reduction in the number of muscle spindles but no neurogenic changes indicative of the loss of alpha lmns; similar findings were seen in mice lacking dync1h1. finally, the authors used an in vitro assay to show a decreased secretory activity in fibroblasts from a smaled patient with a bicd2 mutation when compared to fibroblasts from a control patient. based on these findings, rossor et al. proposed that the lmn loss in smaled is non-cell autonomous and involves reduced neurotrophin secretion from skeletal muscle, which limits neurotrophin availability and leads to excess lmn apoptosis during development. while this hypothesis is intriguing, it is currently not clear whether a similar pathogenetic mechanism operates in human disease; in particular, biopsies from smaled patients show a mixture of neurogenic and myopathic changes that is not well replicated by the existing mouse models. in addition, it is not clear why diminished neurotrophin availability would preferentially affect lmns that innervate lower extremities. nonetheless, the results of this study are sufficiently compelling to warrant further investigation. kim et al. have used a similar experimental approach to explore the role of skeletal muscle in the 5q-sma pathogenesis (kim et al., 2020). intriguingly, they found that the lack of smn expression in skeletal muscle results in an age-dependent myopathy that is usually obscured by the concurrent neurogenic process driven by the lmn loss. in the presence of a single copy of smn2 gene, smn1 deficiency in skeletal muscle led to an early-onset myopathy and severely shortened life span, with most mice dying by the postnatal day 25. a milder phenotype was seen in mice deficient for smn1 that carried two copies of snm2 gene: at a young age, their skeletal muscles appeared normal but showed impaired regeneration following a myotoxic insult; by 6-7 months of age, outright myopathic changes and impaired functional muscle performance were observed, leading to early mortality with only 14% of mutants (but 95% of controls) viable at 18 months. importantly, restoration of muscle smn levels in 7-month-old mice mitigated the observed muscle pathology. conceptually, this study reinforces the paradigm observed with many genetic diseases: a treatment that is effective but targets only the most severely affected organ system typically unmasks the effects of the same mutation on other organ systems that were previously not apparent, but now need to be addressed. for patients with 5q-sma, the implications are more practical: nusinersen treatment, which is given intrathecally and therefore increases smn expression in the spinal cord but not in skeletal muscle, may need to be combined with (or replaced by) a treatment that also increases smn levels in peripheral tissues; one candidate for such therapy is aav9-smn, an aav9 (adeno-associated virus 9) vector carrying human smn transgene that was approved for 5q-sma treatment in 2019, but is currently restricted to patients younger than 2 years of age [for more information on emerging treatments for 5q-sma, see the prior update in this article series (margeta, 2020b)]. additional clinical trials that specifically focus on myopathic pathology in 5q-sma will be needed to address this clinically important issue. moreover, it will be important to determine how smn deficiency leads to the failure of muscle maintenance, which seems to underpin this late-onset muscle pathology. 8. idiopathic inflammatory myopathies: the role of autoantibodies targeting sarcolemmal repair proteins based on the current clinicoseropathologic criteria, idiopathic inflammatory myopathies (iims) are divided into four distinct subcategories [dermatomyositis (dm), anti-synthetase syndrome-associated myositis (asm), immune-mediated necrotizing myopathy (imnm), and sporadic inclusion body myositis (sibm); for a brief overview of the diagnostic criteria and other recent advances in the iim field, see last year’s update in this article series (margeta, 2020b)]. iim pathogenesis is complex and only partly understood, but an interesting study published last year uncovered a feed-forward mechanism that contributes to iim progression by linking development of autoantibodies against trim72 (also known as mitsugumin 53) with defective sarcolemmal repair and ongoing exposure of intracellular autoantigens (mcelhanon et al., 2020). trim72 is a 53 kda member of the trim family of proteins; it is highly expressed in striated muscle and has many different cellular functions, including a key role in the plasma membrane repair. following sarcolemmal injury, trim72 nucleates and then coats intracellular lipid vesicles that accumulate at the site of the membrane break; fusion of these vesicles with damaged sarcolemma creates a “patch” that seals the membrane defect in a process that requires ca2+ as well as interaction of trim72 with dysferlin and caveolin 3 (reviewed in benissan-messan et al., 2020). while mutations in dysferlin and caveolin 3 lead to limb-girdle muscular dystrophies 2b and 1c, respectively, mutations in trim72 have not yet been linked to any human disease; however, the myocardium of trim72-deficient mice is more vulnerable to ischemia (cao et al., 2010), highlighting the importance of this protein for striated muscle integrity. building on that body of work, mcelhanon et al. have now implicated trim72 in the iim pathogenesis by using an adaptive transfer animal model of iim. in this model, lymph node cells from syt7-/foxp3-/γ double mutant mice (which have defective membrane repair and are prone to autoimmunity because they lack tregs) are transferred into immunologically naïve rag1-/mice, who one week after transfer develop an inflammatory myopathy. intriguingly, endomysial inflammatory infiltrates composed of t cells and macrophages were present only in the proximal muscles of the recipient mice, while “leaky” muscle fibers with compromised sarcolemma were identified in both proximal and distal muscles. the presence of myofiber damage in non-inflamed distal muscles raised the possibility that humoral rather than cellular autoimmunity caused impaired sarcolemmal repair in this mouse model, leading the authors to investigate whether the recipient mice developed autoantibodies against one or more proteins involved in this process. indeed, anti-trim72 autoantibodies were detectable in the sera of the recipient mice as well as in 10-30% (depending on the chosen cutoff) of sera from 103 human iim patients. importantly, the authors showed that anti-trim72 antibodies were pathogenic: treatment of isolated normal muscle fibers by either purified anti-trim72 antibodies or by human iim sera positive for these antibodies caused a membrane repair defect (as measured by an in vitro membrane resealing assay), while depletion of anti-trim72 antibodies from human iim sera strongly attenuated this effect. based on these findings, the authors proposed a new model of iim pathogenesis, in which sarcolemmal disruption caused by any number of events (including a viral or bacterial infection, trauma, or overexertion) results in exposure of sarcolemmal repair proteins to the immune system; in susceptible individuals, this exposure leads to development of autoantibodies that target trim72 and/or other repair proteins, compromising membrane resealing and leading to continuous autoantigen exposure in a feed-forward loop. while this model needs to be experimentally tested, it does provide possible explanation for association of covid-19 (and other viral illnesses) with immune-mediated muscle pathology (discussed in advance #3). one limitation of this otherwise very nicely done study is the outdated classification of the patient iim sera used for experiments (they were classified into dm and polymyositis categories, presumably because they were obtained from a specimen bank that performed specimen collection before introduction of the current iim diagnostic criteria). nevertheless, the study included information about the status of a subset of myositis-specific antibodies in the same iim specimen set, providing at least partial clues to more up-to-date classification. based on this limited information, anti-trim72 antibodies were present in a significant fraction of iim patients with dm-associated antibodies (anti-mi-2, anti-tif1γ, and anti-sea), as well as one of two patients with imnm-associated anti-srp antibodies; in contrast, they were not detected in any of patients with the asm-associated anti-jo-1 antibodies. while these findings need to be replicated and extended by evaluation of patients from all currently defined iim subcategories (including sibm and other subtypes of imnm), they do raise the possibility that this new pathomechanism plays a bigger role in pathogenesis of dm and imnm than asm. in that context, it is intriguing that tif1γ / trim33 – which is targeted by one of the dm-associated myositis-specific antibodies – is another member of the trim protein family, although in contrast to trim72 it does not seem to play a significant role in skeletal muscle repair (parks et al., 2019). advances in neuromuscular disease diagnostics 9. large-scale electron microscopy transmission electron microscopy (tem) remains an important diagnostic tool in the contemporary neuromuscular pathology workflow, but some practical limitations diminish its potential usefulness. in particular, it is typically necessary to maximize the use of these expensive instruments while minimizing the number of people who must obtain specialized training required to operate them; as a result, diagnostic ultrastructural evaluation is typically performed on static images of select areas of interest that were previously captured by the trained laboratory staff. while reasonably effective, this approach makes it challenging to relate the captured nanoscale images to the microscale tissue architecture appreciated by light microscopy; in addition, depending on the staff expertise level, there is always a possibility that the captured images are not fully representative of the underlying pathology, leading to diagnostic errors. to circumvent some of these limitations, large-scale em [also known as “virtual em” or nanotomy (short for nanometer-scale anatomy)] has been developed over the last decade; while the first use of this technique in the nmd field was reported in 2003 (sullivan et al., 2003), a wider adoption did not happen until last year (dittmayer et al., 2020b; dittmayer et al., 2020c; rocha et al., 2020). in large-scale em, the entire ultrathin sections are digitized by automated acquisition of multiple overlapping image tiles that are then stitched into a single large image; the resulting high-resolution digital images can then be evaluated on a standard computer workstation, enabling easy navigation between micrometer and nanometer scales across the entire ultrathin section. initial applications of this technology were based on stitching of multiple traditionally acquired tem images (sullivan et al., 2003; faas et al., 2012; lee and mak, 2011); while providing a proof-of-principle for virtual em as a diagnostic and research tool, this approach had two major limitations (1) imperfect image quality due to interference from the specimen support grids and various preparation flaws, and (2) a long time required to capture and stich a very large number of standard tem images. in the years that followed, these limitations were addressed by replacing traditional tem by field-emission scanning em (fesem); one fesem image captures the field of view that is equivalent to ~100 tem images, thus significantly reducing the amount of required image stitching and accelerating the entire process (kuipers et al., 2016). in fesem, imaging of ultrathin sections can be performed using a conductive silicon substrate that does not cause any visual interference, producing very sharp tem-like images through detection of backscattered electrons (dittmayer et al., 2018); this solves problem 1, but not entirely problem 2 because imaging speed remains slow (kuipers et al., 2016). alternatively, fesem can be used for digitization of sections prepared with conventional tem slot grids and imaged in a transmission mode with a scanning transmission electron microscopy (stem) detector (kuipers et al., 2016); this solves problem 2 due to faster imaging, but preparation of samples with minimal flaws remains challenging. despite remaining limitations, the capabilities of this new technology are already impressive: if stem is combined with conventional modern systems, automated digitization of entire ultrathin sections at a 7 nm-pixel resolution can be performed in about 12 h (dittmayer et al., 2020c), while specialized high-throughput imaging systems based on fesem and tem technology can reduce the imaging time to about 10-20 min. when will large-scale em become available for routine diagnostic use? that is difficult to predict, given that the most sophisticated version of the technique requires purchase of new, more advanced em instruments; however, the cost could be justified by anticipated improvements in diagnostic accuracy, opportunities for digital pathology research, and reduction in the time spent on image acquisition by the laboratory staff. in the meantime, one can get a taste of this exciting new technology by viewing open-source datasets available at http://www.nanotomy.org/ advances in neuromuscular disease treatment 10. long-term efficacy and safety: gene-silencing therapies for transthyretin amyloid neuropathy transthyretin amyloidosis (attr) is caused by deposition of either wild-type or mutant transthyretin (ttr) in different tissues throughout the body; these two disease forms are referred to as attrwt and attrv (“v” for variant), respectively. ttr functions as a carrier for thyroxin and retinol/vitamin a and is primarily synthetized by the liver; clinical symptoms of attr are largely due to accumulation of ttr amyloid in the heart and peripheral nerves, but skeletal muscle can also be affected in rare cases (fig. 6 and pinto et al., 2020; see also fig. 1 in lam et al., 2015). interestingly, recent work has shown that tissue damage in attr is caused not only by the amyloid fibril deposition but also by toxicity of nonfibrillar transthyretin oligomers, which directly damage endothelial cells and nerve fibers (reviewed in koike and katsuno, 2020). figure 6. transthyretin amyloid deposition in a peripheral nerve and skeletal muscle of a patient with attrv. sural nerve biopsy (a-d) shows accumulation of congo red-positive amyloid substance in the arterial walls (a); the deposits demonstrate apple-green birefringence under the polarized light (b) and are immunoreactive with anti-transthyretin antibody (c). marked loss of myelinated axons is evident on toluidine blue stain (d). sections from a gastrocnemius muscle biopsy (e-j) show deposition of ttr amyloid in the arterial walls (e-g) and fiber sarcoplasm (h-j); well-developed neurogenic changes were also present (not shown). subsequent to the biopsy diagnosis of ttr amyloid neuropathy and myopathy, the patient was found to carry a pathogenic ttr p.phe84leu mutation and was diagnosed with attrv; he was treated by inotersen and initially stabilized, but then continued to progress and will likely be switched to the patisiran therapy going forward. panels a-c and e-j, formalin-fixed, paraffin embedded tissue; panel d, epon-embedded tissue. panels a-b, e-f, and h-i, congo red stain; panels c, g and j, transthyretin immunoperoxidase stain; panel d, toluidine blue stain. scale bars, 50 µm. fully assembled ttr is a conformationally stable homotetramer; in contrast, individual ttr monomers are prone to misfolding and aggregation. thus, the currently approved attrv therapies aim either to stabilize the tetrameric quaternary structure of ttr (tafimidis or diflunisal; approved in the early 2010s) or to inhibit ttr synthesis through gene silencing approaches (inotersen or patisiran; both approved in 2018). untreated attrv is a rapidly progressive disease with a poor prognosis, and the availability of several disease-modifying treatments was an incredible breakthrough for the nmd field. at the same time, many important treatment-related questions remained open, and had to be addressed through post-approval studies. for example, at the time of approval little was known about the long-term efficacy and safety of ttr-silencing drugs. in addition, it was not clear how different attrv treatment options compare to each other. while much work still remains to be done, at least some of these questions have been decisively answered by studies published in 2020. inotersen is an antisense oligonucleotide that inhibits ttr production and thereby slows the progression of attrv polyneuropathy; based on a 2-year update from the open-label extension of the neuro-ttr clinical trial (which led to its initial approval), no new safety concerns were identified and the drug continued to demonstrate therapeutic benefits. however, the outcomes were significantly better in study subjects who were originally placed in the experimental arm than in those who were originally in the placebo arm, indicating that early treatment is critical for inotersen efficacy (brannagan et al., 2020). similar results were seen with patisiran, which is an rna-interference therapeutic that also inhibits ttr synthesis; a 12-month update from the open-label extension of the apollo clinical trial (which led to initial approval of this drug) also demonstrated no new safety concerns and continued efficacy, with sustained benefit in patients who were originally part of the patisiran arm and a new improvement in patients who were originally part of the placebo arm. interestingly, the frequency of deaths was higher in the initial placebo group than in the initial patisiran group, again highlighting the benefits of early treatment (adams et al., 2021). finally, an indirect comparison of the data from neuro-ttr and apollo clinical trials demonstrated that patisiran was generally more effective than inotersen, with a greater treatment benefit observed on all polyneuropathy and quality of life measures evaluated by both trials (gorevic et al., 2021). taken together with a prior study that showed a greater efficacy of inotersen compared to tafimidis in patients with attrv polyneuropathy (plante-bordeneuve et al., 2019), these data suggest that patisiran is the best currently available treatment for attrv polyneuropathy. it needs to be noted that both these indirect comparison studies were funded by alnylam pharmaceuticals, the manufacturer of patisiran; however, given the magnitude and robustness of the observed therapeutic differences, it is unlikely that the results were significantly confounded by the sponsor’s financial interests. despite these advances, a lot remains to be explored. for example, it needs to be established whether ttr-silencing therapies will also be effective for treatment of attrwt polyneuropathy, and if they are whether their therapeutic benefit will be superior to that of tafimidis (which has been approved for attrwt treatment). similarly, both ttr-silencing drugs still need to be evaluated as treatments for attrv (and attrwt) cardiomyopathy: initial exploratory studies showed that inotersen led to no improvement in cardiac function while patisiran stopped (and even reversed) the progression of cardiac disease in attrv patients (reviewed in adams and slama, 2020), but randomized clinical trials addressing the cardiac aspect of attr still need to be done. disclosure statement the author receives research support from audentes therapeutics as a member of the muscle biopsy review committee for the aspiro clinical trial (nct03199469), which is evaluating the safety and efficacy of gene transfer therapy for x-linked myotubular myopathy. acknowledgements i am grateful to ms. christine lin for assistance with figure preparation. references adams, d., polydefkis, m., gonzalez-duarte, a., wixner, j., kristen, a.v., schmidt, h.h., berk, j.l., losada lopez, i.a., dispenzieri, a., quan, d., et al. (2021). long-term safety and efficacy of patisiran for hereditary transthyretin-mediated amyloidosis with polyneuropathy: 12-month results of an open-label extension study. lancet neurol 20, 49-59. adams, d., and slama, m. (2020). hereditary transthyretin amyloidosis: current treatment. curr opin neurol 33, 553-561. anklesaria, z., frankman, j., gordin, j., zhan, j., and liu, a.k. (2020). fatal rhabdomyolysis in a covid-19 patient on rosuvastatin. cureus 12, e11186. benissan-messan, d.z., zhu, h., zhong, w., tan, t., ma, j., and lee, p.h.u. (2020). multi-cellular functions of mg53 in muscle calcium signaling and regeneration. front physiol 11, 583393. brannagan, t.h., wang, a.k., coelho, t., waddington cruz, m., polydefkis, m.j., dyck, p.j., plante-bordeneuve, v., berk, j.l., barroso, f., merlini, g., et al. (2020). early data on long-term efficacy and safety of inotersen in patients with hereditary transthyretin amyloidosis: a 2-year update from the open-label extension of the neuro-ttr trial. eur j neurol 27, 1374-1381. buckholz, a.p., kaplan, a., rosenblatt, r.e., and wan, d. (2020). clinical characteristics, diagnosis, and outcomes of 6 patients with covid-19 infection and rhabdomyolysis. mayo clin proc 95, 2557-2559. cagnazzo, f., arquizan, c., derraz, i., dargazanli, c., lefevre, p.h., riquelme, c., gaillard, n., mourand, i., gascou, g., bonafe, a., et al. (2020). neurological manifestations of patients infected with the sars-cov-2: a systematic review of the literature. j neurol, epub ahead of print. cao, c.m., zhang, y., weisleder, n., ferrante, c., wang, x., lv, f., zhang, y., song, r., hwang, m., jin, l., et al. (2010). mg53 constitutes a primary determinant of cardiac ischemic preconditioning. circulation 121, 2565-2574. caress, j.b., castoro, r.j., simmons, z., scelsa, s.n., lewis, r.a., ahlawat, a., and narayanaswami, p. (2020). covid-19-associated guillain-barre syndrome: the early pandemic experience. muscle nerve 62, 485-491. copic, a., antoine-bally, s., gimenez-andres, m., la torre garay, c., antonny, b., manni, m.m., pagnotta, s., guihot, j., and jackson, c.l. (2018). a giant amphipathic helix from a perilipin that is adapted for coating lipid droplets. nat commun 9, 1332. dalakas, m.c. (2020). guillain-barre syndrome: the first documented covid-19-triggered autoimmune neurologic disease: more to come with myositis in the offing. neurol neuroimmunol neuroinflamm 7, e781. de sanctis, p., doneddu, p.e., vigano, l., selmi, c., and nobile-orazio, e. (2020). guillain-barre syndrome associated with sars-cov-2 infection. a systematic review. eur j neurol 27, 2361-2370. di blasi, c., moghadaszadeh, b., ciano, c., negri, t., giavazzi, a., cornelio, f., morandi, l., and mora, m. (2004). abnormal lysosomal and ubiquitin-proteasome pathways in 19p13.3 distal myopathy. ann neurol 56, 133-138. ding, y., wang, h., shen, h., li, z., geng, j., han, h., cai, j., li, x., kang, w., weng, d., et al. (2003). the clinical pathology of severe acute respiratory syndrome (sars): a report from china. j pathol 200, 282-289. dittmayer, c., meinhardt, j., radbruch, h., radke, j., heppner, b.i., heppner, f.l., stenzel, w., holland, g., and laue, m. (2020a). why misinterpretation of electron micrographs in sars-cov-2-infected tissue goes viral. lancet 396, e64-e65. dittmayer, c., siegert, e., uruha, a., goebel, h., and stenzel, w. (2020b). large-scale electron microscopy reveals capillary pathology in muscle samples of patients with systemic sclerosis. neuromuscul disord 30, s150. dittmayer, c., stenzel, w., goebel, h.h., krusche, m., schneider, u., uruha, a., and englert, b. (2020c). morphological characteristics of the transition from juvenile to adult dermatomyositis. neuropathol appl neurobiol 46, 790-794. dittmayer, c., volcker, e., wacker, i., schroder, r.r., and bachmann, s. (2018). modern field emission scanning electron microscopy provides new perspectives for imaging kidney ultrastructure. kidney int 94, 625-631. faas, f.g., avramut, m.c., van den berg, b.m., mommaas, a.m., koster, a.j., and ravelli, r.b. (2012). virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps. j cell biol 198, 457-469. ferrandi, p.j., alway, s.e., and mohamed, j.s. (2020). the interaction between sars-cov-2 and ace2 may have consequences for skeletal muscle viral susceptibility and myopathies. j appl physiol (1985) 129, 864-867. filosto, m., cotti piccinelli, s., gazzina, s., foresti, c., frigeni, b., servalli, m.c., sessa, m., cosentino, g., marchioni, e., ravaglia, s., et al. (2020). guillain-barre syndrome and covid-19: an observational multicentre study from two italian hotspot regions. j neurol neurosurg psychiatry, epub ahead of print. fragiel, m., miro, o., llorens, p., jimenez, s., pinera, p., burillo, g., martin, a., martin-sanchez, f.j., garcia-lamberechts, e.j., jacob, j., et al. (2020). incidence, clinical, risk factors and outcomes of guillain-barre in covid-19. ann neurol, epub ahead of print. gorevic, p., franklin, j., chen, j., sajeev, g., wang, j.c.h., and lin, h. (2021). indirect treatment comparison of the efficacy of patisiran and inotersen for hereditary transthyretin-mediated amyloidosis with polyneuropathy. expert opin pharmacother 22, 121-129. hackman, p., sarparanta, j., lehtinen, s., vihola, a., evila, a., jonson, p.h., luque, h., kere, j., screen, m., chinnery, p.f., et al. (2013). welander distal myopathy is caused by a mutation in the rna-binding protein tia1. ann neurol 73, 500-509. heck, m.v., azizov, m., stehning, t., walter, m., kedersha, n., and auburger, g. (2014). dysregulated expression of lipid storage and membrane dynamics factors in tia1 knockout mouse nervous tissue. neurogenetics 15, 135-144. hedberg-oldfors, c., meyer, r., nolte, k., abdul rahim, y., lindberg, c., karason, k., thuestad, i.j., visuttijai, k., geijer, m., begemann, m., et al. (2020). loss of supervillin causes myopathy with myofibrillar disorganization and autophagic vacuoles. brain 143, 2406-2420. hooper, j.e., uner, m., priemer, d.s., rosenberg, a., and chen, l. (2020). muscle biopsy findings in a case of sars-cov-2-associated muscle injury. j neuropathol exp neurol, epub ahead of print. jennette, j.c., falk, r.j., bacon, p.a., basu, n., cid, m.c., ferrario, f., flores-suarez, l.f., gross, w.l., guillevin, l., hagen, e.c., et al. (2013). 2012 revised international chapel hill consensus conference nomenclature of vasculitides. arthritis rheum 65, 1-11. keddie, s., pakpoor, j., mousele, c., pipis, m., machado, p.m., foster, m., record, c.j., keh, r.y.s., fehmi, j., paterson, r.w., et al. (2020). epidemiological and cohort study finds no association between covid-19 and guillain-barre syndrome. brain, epub ahead of print. kim, j.k., jha, n.n., feng, z., faleiro, m.r., chiriboga, c.a., wei-lapierre, l., dirksen, r.t., ko, c.p., and monani, u.r. (2020). muscle-specific smn reduction reveals motor neuron-independent disease in spinal muscular atrophy models. j clin invest 130, 1271-1287. klar, j., sobol, m., melberg, a., mabert, k., ameur, a., johansson, a.c., feuk, l., entesarian, m., orlen, h., casar-borota, o., et al. (2013). welander distal myopathy caused by an ancient founder mutation in tia1 associated with perturbed splicing. hum mutat 34, 572-577. koboldt, d.c., waldrop, m.a., wilson, r.k., and flanigan, k.m. (2020). the genotypic and phenotypic spectrum of bicd2 variants in spinal muscular atrophy. ann neurol 87, 487-496. koike, h., and katsuno, m. (2020). transthyretin amyloidosis: update on the clinical spectrum, pathogenesis, and disease-modifying therapies. neurol ther 9, 317-333. kuipers, j., kalicharan, r.d., wolters, a.h., van ham, t.j., and giepmans, b.n. (2016). large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain. j vis exp 111, 53635. lam, l., margeta, m., and layzer, r. (2015). amyloid polyneuropathy caused by wild-type transthyretin. muscle nerve 52, 146-149. lee, k.c., and mak, l.s. (2011). virtual electron microscopy: a simple implementation creating a new paradigm in ultrastructural examination. int j surg pathol 19, 570-575. lee, m.a., joo, y.m., lee, y.m., kim, h.s., kim, j.h., choi, j.k., ahn, s.j., min, b.i., and kim, c.r. (2008). archvillin anchors in the z-line of skeletal muscle via the nebulin c-terminus. biochem biophys res commun 374, 320-324. lee, y.m., kim, h.s., choi, j.h., choi, j.k., joo, y.m., ahn, s.j., min, b.i., and kim, c.r. (2007). knockdown of archvillin by sirna inhibits myofibril assembly in cultured skeletal myoblast. j exp biomed sci 13, 251-261. lentscher, a.j., mccarthy, m.k., may, n.a., davenport, b.j., montgomery, s.a., raghunathan, k., mcallister, n., silva, l.a., morrison, t.e., and dermody, t.s. (2020). chikungunya virus replication in skeletal muscle cells is required for disease development. j clin invest 130, 1466-1478. leung, t.w., wong, k.s., hui, a.c., to, k.f., lai, s.t., ng, w.f., and ng, h.k. (2005). myopathic changes associated with severe acute respiratory syndrome: a postmortem case series. arch neurol 62, 1113-1117. mair, d., biskup, s., kress, w., abicht, a., bruck, w., zechel, s., knop, k.c., koenig, f.b., tey, s., nikolin, s., et al. (2020). differential diagnosis of vacuolar myopathies in the ngs era. brain pathol 30, 877-896. manzano, g.s., woods, j.k., and amato, a.a. (2020). covid-19-associated myopathy caused by type i interferonopathy. n engl j med 383, 2389-2390. mao, l., jin, h., wang, m., hu, y., chen, s., he, q., chang, j., hong, c., zhou, y., wang, d., et al. (2020). neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china. jama neurol 77, 683-690. margeta, m. (2020a). autophagy defects in skeletal myopathies. annu rev pathol 15, 261-285. margeta, m. (2020b). top ten discoveries of the year: neuromuscular disease. free neuropathol 1, 4. mcelhanon, k.e., young, n., hampton, j., paleo, b.j., kwiatkowski, t.a., beck, e.x., capati, a., jablonski, k., gurney, t., perez, m.a.l., et al. (2020). autoantibodies targeting trim72 compromise membrane repair and contribute to inflammatory myopathy. j clin invest 130, 4440-4455. nishi, r., koike, h., ohyama, k., fukami, y., ikeda, s., kawagashira, y., iijima, m., katsuno, m., and sobue, g. (2020). differential clinicopathologic features of egpa-associated neuropathy with and without anca. neurology 94, e1726-e1737. oh, s.w., pope, r.k., smith, k.p., crowley, j.l., nebl, t., lawrence, j.b., and luna, e.j. (2003). archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton. j cell sci 116, 2261-2275. parks, c.a., pak, k., pinal-fernandez, i., huang, w., derfoul, a., and mammen, a.l. (2019). trim33 (tif1gamma) is not required for skeletal muscle development or regeneration but suppresses cholecystokinin expression. sci rep 9, 18507. peter, a.k., cheng, h., ross, r.s., knowlton, k.u., and chen, j. (2011). the costamere bridges sarcomeres to the sarcolemma in striated muscle. prog pediatr cardiol 31, 83-88. pinto, m.v., milone, m., mauermann, m.l., dyck, p.j.b., alhammad, r., mcphail, e.d., grogan, m., and liewluck, t. (2020). transthyretin amyloidosis: putting myopathy on the map. muscle nerve 61, 95-100. plante-bordeneuve, v., lin, h., gollob, j., agarwal, s., betts, m., fahrbach, k., chitnis, m., and polydefkis, m. (2019). an indirect treatment comparison of the efficacy of patisiran and tafamidis for the treatment of hereditary transthyretin-mediated amyloidosis with polyneuropathy. expert opin pharmacother 20, 473-481. pourteymour, s., lee, s., langleite, t.m., eckardt, k., hjorth, m., bindesboll, c., dalen, k.t., birkeland, k.i., drevon, c.a., holen, t., et al. (2015). perilipin 4 in human skeletal muscle: localization and effect of physical activity. physiol rep 3, e12481. rocha, m.l., dittmayer, c., uruha, a., korinth, d., chaoui, r., schlembach, d., rossi, r., pelin, k., suk, e.k., schmid, s., et al. (2020). a novel mutation in neb causing foetal nemaline myopathy with arthrogryposis during early gestation. neuromuscul disord, epub ahead of print. rosato, c., bolondi, g., russo, e., oliva, a., scognamiglio, g., mambelli, e., longoni, m., rossi, g., and agnoletti, v. (2020). clinical, electromyographical, histopathological characteristics of covid-19 related rhabdomyolysis. sage open med case rep 8, 2050313x20983132. rossor, a.m., sleigh, j.n., groves, m., muntoni, f., reilly, m.m., hoogenraad, c.c., and schiavo, g. (2020). loss of bicd2 in muscle drives motor neuron loss in a developmental form of spinal muscular atrophy. acta neuropathol commun 8, 34. ruggieri, a., naumenko, s., smith, m.a., iannibelli, e., blasevich, f., bragato, c., gibertini, s., barton, k., vorgerd, m., marcus, k., et al. (2020). multiomic elucidation of a coding 99-mer repeat-expansion skeletal muscle disease. acta neuropathol 140, 231-235. shang, m., cappellesso, f., amorim, r., serneels, j., virga, f., eelen, g., carobbio, s., rincon, m.y., maechler, p., de bock, k., et al. (2020). macrophage-derived glutamine boosts satellite cells and muscle regeneration. nature 587, 626-631. spinazzola, j.m., smith, t.c., liu, m., luna, e.j., and barton, e.r. (2015). gamma-sarcoglycan is required for the response of archvillin to mechanical stimulation in skeletal muscle. hum mol genet 24, 2470-2481. sullivan, k.a., brown, m.s., harmon, l., and greene, d.a. (2003). digital electron microscopic examination of human sural nerve biopsies. j peripher nerv syst 8, 260-270. wang, k., yaghi, o.k., spallanzani, r.g., chen, x., zemmour, d., lai, n., chiu, i.m., benoist, c., and mathis, d. (2020). neuronal, stromal, and t-regulatory cell crosstalk in murine skeletal muscle. proc natl acad sci u s a 117, 5402-5408. willingham, t.b., kim, y., lindberg, e., bleck, c.k.e., and glancy, b. (2020). the unified myofibrillar matrix for force generation in muscle. nat commun 11, 3722. zhang, h., charmchi, z., seidman, r.j., anziska, y., velayudhan, v., and perk, j. (2020). covid-19-associated myositis with severe proximal and bulbar weakness. muscle nerve 62, e57-e60. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. malignant melanotic nerve sheath tumor with prkar1a, kmt2c and gnaq mutations feel free to add comments by clicking these icons on the sidebar free neuropathology 3:21 (2022) case report malignant melanotic nerve sheath tumor with prkar1a, kmt2c and gnaq mutations merryl terry1, kristina wakeman1, brian j. williams2,4, donald m. miller3,4, müge sak5, zied abdullaev6, marwil c. pacheco1, kenneth aldape6, norman l. lehman1,4,5 1 department of pathology and laboratory medicine, university of louisville, louisville, ky, usa 2 department of neurological surgery, university of louisville, louisville, ky, usa 3 department of internal medicine, university of louisville, louisville, ky, usa 4 the brown cancer center, university of louisville, louisville, ky, usa 5 department of biochemistry and molecular genetics, university of louisville, louisville, ky, usa 6 laboratory of pathology, center for cancer research, national cancer institute, bethesda, md, usa corresponding author: norman l. lehman · department of pathology and laboratory medicine · brown cancer center · department of biochemistry and molecular genetics · university of louisville · 505 south hancock street · louisville, ky 40202 · usa nllehman1@gmail.com submitted: 03 april 2022 accepted: 12 august 2022 copyedited by: jeffrey nirschl published: 26 august 2022 https://doi.org/10.17879/freeneuropathology-2022-3864 keywords: malignant melanotic nerve sheath tumor, mmnst, psammomatous melanotic schwannoma, nerve sheath tumor, pigmented epithelioid melanocytoma, prkar1a, kmt2c, gnaq abstract malignant melanotic nerve sheath tumor (mmnst) is a rare and potentially aggressive lesion defined in the 2021 who classification of tumors of the central nervous system. mmnst demonstrate overlapping histologic and clinical features of schwannoma and melanoma. mmnst often harbor prkar1a mutations, especially within the carney complex. we present a case of aggressive mmnst of the sacral region in a 48-year-old woman. the tumor contained prkar1a frame shift pr352hfs*89, kmt2c splice site c.7443-1g>t and gnaq p.r183l missense mutations, as well as braf and myc gains. genomic dna methylation analysis using the illumina 850k epicbead chip revealed that the lesion did not match an established methylation class; however, uniform manifold approximation and projection (umap) placed the tumor very near schwannomas. the tumor expressed pd-l1, and the patient was treated with radiation and immune checkpoint inhibitors following en bloc resection. although she had symptomatic improvement, she suffered early disease progression with local recurrence, and distant metastases, and died 18 months after resection. it has been suggested that the presence of gnaq mutations can differentiate leptomeningeal melanocytic neoplasms and uveal melanoma from mmnst. this case and others demonstrate that gnaq mutations may exist in malignant nerve sheath tumors; that gnaq and prkar1a mutations are not always mutually exclusive and that neither can be used to differentiate mmnst or mpnst from all melanocytic lesions. introduction malignant melanotic nerve sheath tumors (mmnsts) are rare neoplasms formerly known as melanotic schwannoma [1] and sometimes melanotic malignant peripheral nerve sheath tumor (mpnst). they are renamed in the 2021 who cns tumor classification [2] to better reflect their potentially aggressive nature and align with the 2020 who soft tissue nomenclature [3]. mmnst often carry an inactivating mutation in the protein kinase a inhibitory subunit gene protein kinase camp-dependent type i regulatory subunit alpha (prkar1a) on chromosome 17p22-24, which acts as a tumor suppressor [3, 4]. mmnst occur in some patients with carney complex, a rare syndrome associated with prkar1a mutation, cardiac and skin myxomas, endocrine and gonadal neoplasms, variable endocrine manifestations such as cushing syndrome or acromegaly, increased lentigines, and a type of blue nevus known as pigmented epithelioid melanocytoma [5, 6]. we present a case of an aggressive sacral mmnst in a woman which demonstrated prkar1a, kmt2c, and gnaq mutations, braf and myc copy number gains, and pd-l1 overexpression. we also compare histologic, genetic, and clinical features of conventional schwannomas, mmnst, mpnst, and melanoma. case report a 48-year-old female developed severe lower back and right buttock pain with right thigh numbness over a period of one year. she also reported difficulty urinating and defecating. her past medical history included endometriosis, hypothyroidism, and multinodular goiter. on examination, she had saddle anesthesia and urinary retention. computed tomography (ct) demonstrated a right sacral mass with posterior bony destruction. no local regional lymphadenopathy was appreciated. magnetic resonance imaging (mri) revealed a 7.8 x 8.6 cm heterogeneously enhancing mass with a large soft tissue component (figure 1). figure 1: tumor imaging. (a). axial and (b). sagittal t2 mri of the pelvis demonstrating an intermediate to brightly heterogeneously enhancing mass in the right sacrum and soft tissue with boney destruction. ct-guided core needle biopsy showed a pigmented malignant spindle cell neoplasm. the main histological differential diagnosis was metastatic melanoma, primary cns melanoma and mmnst. positron-emission computed tomography (pet/ct) demonstrated that the sacral mass was centered on the right s2 nerve root, with anterior and posterior soft tissue extensions, and a large lytic component in the right posterior ilium. there was no evidence of metastatic disease. a trans-abdominal, stage i procedure for high sacrectomy, with colectomy and colostomy was performed. this was followed one week later by en bloc resection and sacrectomy with l3-pelvis posterior spinal instrumentation and fusion, and flap reconstruction. intraoperative pathology revealed a pigmented spindle cell, malignant tumor involving the surgical margin. additional tissue was excised with grossly negative margins. figure 2: gross sacrectomy specimen. (a). lateral view, displaying a well-circumscribed tumor involving soft tissue and bone. (b). after inking the specimen blue, sectioning the tumor revealed the typical black “dried tar” appearance of cut surfaces. pathology the resected tumor was 9.0 x 8.5 x 7.0 cm in dimension, centered in soft tissue, and eroded into adjacent bone. it was relatively circumscribed, heavily pigmented with a “black tar” appearance, and partly covered by a thin fibrous membrane. (figure 2a-b). microscopically, the cells were arranged in sheets, lobules, and fascicles (figure 3a-d). click here to view the full virtual slide. large areas of necrosis were present (figure 3a). most tumor cells were spindleto epithelioid-shaped with oval nuclei, vesicular chromatin, and prominent nucleoli (figure 3e). up to 18 mitotic figures were counted in ten 400x fields. scattered melanin was identified within tumor cells and melanophages (figure 3b and 3f). focal areas showed verocay-like nuclear palisading. some areas contained highly pleomorphic cells demonstrating eosinophilic intranuclear pseudoinclusions (figure 3f). the tumor was surrounded by a pseudocapsule containing neurofilament-immunoreactive normal ganglion cells and nerve tissue (figures 3b-c and 4a). multiple areas of pseudocapsular and lymphovascular invasion were noted. occasional psammoma bodies were seen within the pseudocapsule adjacent to the tumor. figure 3: tumor histology. (a). geographic necrosis comprised approximately 30-40% of sampled tumor areas. (b). the tumor was lobular, pigmented, and surrounded by a pseudocapsule containing benign nerve elements. (c). the pseudocapsule incorporated benign ganglion cells (left). some tumor areas were deceptively bland (right). (d). most tumor areas contained spindled cells in a fascicular and vaguely nodular arrangement. (e). nucleoli were prominent and mitoses were abundant. (f). some areas were highly pleomorphic and/or demonstrated nuclear pseudoinclusions. magnification: a-b, 20x; c-d, 100x; e-f 400x. click here to view the full virtual slide. tumor cells were diffusely immunopositive for sox10, hmb-45, and melan-a (figure 4b). s100 was positive in scattered cells. cytokeratins ae1/ae3, braf-v600e, and pax-8 immunostains were negative. ini1 (smarcb1) was positive in tumor cell nuclei. reticulin stain diffusely highlighted spindle tumor cell-associated basement membranes and showed focal lobular staining of small groups of epithelioid cells (figure 4c). ki-67 labeling was 5-10% in most areas and up to 20% focally (figure 4d). pd-l1 was detected by immunohistochemistry (clone zr3; pd-l1 expression score 2, detection cut-off score ≥ 1). figure 4: tumor immunohistochemistry and reticulin stain. (a). neurofilamentimmunoreactive entrapped nerve elements within the tumor (upper left) and ganglion cells and axons within the pseudocapsule (lower right). (b). diffuse positivity for melan-a. (c). pericellular and lobular reticulin staining. (d). ki-67 immunohistochemical staining. magnification: a, 40x; b-d, 200x. next generation sequencing (neogenomics neotype analysis discovery profile) and fluorescence in situ hybridization (neotype discovery solid tumor fish panel) were performed on formalin-fixed paraffin-embedded (ffpe) tumor. two predicted inactivating mutations were detected: a lysine methyltransferase 2c (kmt2c) splice site mutation (c.7443-1g>t) and a prkar1a frameshift mutation (c.1055del, p.r352hfs*89). additionally, a pathogenic missense mutation (c.548g>t, p.r183l) was identified in the g protein subunit alpha q (gnaq) [7]. missense variants of unknown significance were detected in akt2 (c.1013t>g, p.v338g), kmt2c (c.7015a>g, p.n2339d) and setd2 (c.65c>g, p.t22s). nf1 gene mutation was not detected. fish demonstrated additional copies of braf (>2f, 52.0%; negative <25.1%) and myc (3f, 30.0%; negative <16.2%), consistent with gains of braf (7q34) or chromosome 7 and myc (8q24) or chromosome 8. genomic dna methylation analysis was performed on ffpe tissue using the illumina infinium methylation epic beadchip 850k microarray [8]. methylation data was uploaded to the german cancer research center (dkfz) methylation-based classification platform for central nervous system tumors (www.molecularneuropathology.org). results did not match any existing tumor methylation class (calibration scores < 0.9). chromosome copy number analysis confirmed chromosome 7 and 8 gains, as well as gains of 6p, 6q, 11, 12, 18, and 20p, and loss of 1p, 16q, 17, 20q, and 22q, among other changes (figure 5a). uniform manifold approximation and projection (umap) dimensional reduction analysis comparing the tumor methylation data to the dkfz brain tumor reference cohort [8] placed the tumor very near, or within, the schwannoma/melanotic schwannoma group (figure 5b). figure 5: genomic dna analysis. (a). chromosomal copy number data. (b). unsupervised umap dimension reduction analysis of tumor genomic dna methylation data. although the tumor did not match a specific tumor methylation class using the dkfz classifier, it grouped near, or with, schwannoma/melanotic schwannoma by umap (inset). adjuvant therapy and follow-up: postoperatively, the patient received 5940 cgy radiation in 33 fractions to her pelvis. she then received three cycles of pembrolizumab. the fourth cycle was withheld due to autoimmune hepatitis. the patient had a dramatic improvement in pain from a visual analogue scale (vas) pain score of 8-10/10 to 2-3/10. her karnofsky performance score was 90. ct scans five months after surgery demonstrated a new enhancing lesion of the pericardium, new gluteal mass, and new lytic lesions in the t2 and l2 vertebra and the left acetabulum. positron-emission tomography (pet) showed these and multiple rib lesions. core needle biopsy of the right gluteal muscle confirmed tumor recurrence. six months after surgery, immunotherapy with ipilimumab and nivolumab was initiated. she completed her fourth cycle three months later. ct showed new and worsening osseous metastases, as well as innumerable sub-centimeter pulmonary metastases, and enlargement of the pericardial mass. spinal mri revealed a pathologic fracture of t2. the patient was hospitalized with right-sided radiculopathy and underwent surgical stabilization of t2 followed by radiation to residual t2 tumor. she also developed a 3.6 cm right frontal calvarial lesion, which was irradiated. the patient was then treated with doxorubicin. she had further progression at c2 three months after spinal surgery. the patient elected for hospice care and died one month later (18 mo after primary resection). discussion mmnst are slightly more common in females (1.4:1 female to male) [3]. they are most often located paraspinally or in the gastrointestinal tract, but also occur at other sites [9, 10]. their average age of presentation is 33.2 years for sporadic tumors and 22.5 years within the carney complex. grossly, mmnst are typically solitary, partially circumscribed or encapsulated, and heavily pigmented [2]. they range from 0.5 cm to 25 cm in diameter, but most exceed 5 cm [10]. mmnst are associated with nerves or soft tissue and may erode bone. true intraosseous examples are rare [11]. microscopically, they are comprised of short fascicles or sheets of polygonal or spindled cells with a syncytial appearance. vague tumor cell palisading or whorling may be evident [2]. pigment is variable. nuclei are typically round to ovoid with nuclear grooves, pseudoinclusions, and/or prominent nucleoli. marked pleomorphism and nuclear hyperchromasia may be present [12]. associated vessels are usually capillary-like. psammoma bodies are present in ~50% of cases. psammomatous variants may contain lipoma-like fat accumulation and occur more often in carney complex [6, 11]. residual ganglion cells may be identified in paraspinal examples. mmnst usually strongly express s100 and melanocyte markers (sox10, hmb45, melan-a, and tyrosinase) [13]. although, examples of s100 patchy-positivity or s100-negative tumors have been reported [2, 13, 14]. ultrastructurally, tumor cells exhibit both schwann cell and melanocyte features, i.e., elaborate cytoplasmic processes, premelanosomes, and melanosomes [2, 10, 12]. mitotic rate is the only histologic feature found to be predictive of clinical outcome. in one study, a mitotic rate of >2/10 hpf correlated with metastases (p=0.008) [34]. however, >50% of tumors that eventually metastasized did not show increased mitoses. mmnst are biologically distinct from conventional schwannoma. mmnst tend to occur in posterior spinal nerves and ganglia, while schwannomas additionally may involve other nerves, including cranial nerves. the peak age of mmnst onset is a decade younger than for schwannoma [12]. unlike the latter, mmnst generally lack antoni a and b regions and hyalinized blood vessels histologically. gene expression profiling has shown significant differences between mmnst, melanoma, and schwannoma [13]. mmnst exhibited downregulation of genes involved in schwann cell function, e.g., pmp22, pmp2, and mpz, while genes related to melanin synthesis were upregulated in mmnst compared to schwannoma. in contrast to mmnst, conventional mpnst usually arise from peripheral nerves or extraneural soft tissue and are commonly seen in neurofibromatosis type 1 (nf1) [2], while mmnst only rarely occurs in nf1 [10, 15]. mpnst histologic features include monomorphic spindle cells with broad, intersecting, herringbone fascicles, alternating hyperand hypocellular areas, a high mitotic count, and geographic necrosis [2]. mpnst also generally lack pigment and are negative for melanotic markers. reported gene alterations in mpnst include nf1, cdkn2a, cdkn2b, tp53, tyk2, eed, and suz12 mutations and deletions, and egfr, pdgfra, and met amplification [2, 16]. the uncommon epithelioid variant of mpnst arises from a pre-existing schwannoma and harbors smarcb1 mutations [9, 11], whereas smarcb1 has been reported to be intact in mmnst [13]. distinguishing mmnst from melanoma can be difficult. both may demonstrate spindled or epithelioid cells with pigment, violaceous macronucleoli, and positivity for s100 and melanocytic markers [9]. a lower ki-67 labeling index may be suggestive of mmnst [2]. extensive collagen iv or reticulin basement membrane staining supports schwannian differentiation. a lobular/clustered tumor cell arrangement with syncytial-like cytology, psammoma bodies, and fat accumulation may also help distinguish mmnst from melanoma [9]. the genetics of mmnst are not completely defined. the majority of tumors are sporadic. prkar1a mutations and loss of prkar1a protein expression are seen in most cases and should prompt a search for other findings of the carney complex [3, 4]. a complex karyotype including 22q monosomy, recurrent losses involving chromosomes 1, 2, 21, and 17p, trisomy 6p, and ring chromosome 11 have been described in mmnst [3, 4, 10, 17]. melanoma typically shows 6p and 8q gains and 11q loss [17, 18]. mpnst exhibits gains of chromosomes 2, 7p, 8q, 14 and 17q, and loss of 9p, 11q, 13q, 17p, 18 and 22q [11, 19]. schwannoma also shows 17p and 22q loss. this case thus exhibits reported karyotypic changes overlapping mmnst, mpnst, and schwannoma (8q gain, and 1p, 17p, and 22q loss), and mmnst and melanoma (6p and 8q gains) (figure 5a). the case also demonstrates gene aberrations not previously ascribed to mmnst, namely, braf copy number gain and kmt2c splice site, and gnaq r183l missense mutations. braf v600e mutations, not gains, are found in cutaneous melanoma and pigmented epithelioid melanocytoma [20]. notably, the latter also may harbor prkar1a aberrations [21] or loss of heterozygosity [22]. gnaq codon q209 mutations are reported in leptomeningeal melanocytic neoplasms, uveal melanoma, and pigmented epithelioid melanocytoma [20, 21, 23, 24]. gnaq variants have also been described in a malignantly transformed schwannoma (gnaq t96s) [25] and an nf1-associated mpnst (gnaq y101x stop gain) [16] (angela hirbe, personal communication, july 2022). therefore, unlike previously suggested for mmnst [2, 23], neither gnaq nor prkar1a mutations can be relied on to differentiate mmnst from melanocytic lesions. although, prkar1a and gnaq mutations coexist in the mmnst described herein, we found no other documentation of their coexistence in the entities discussed above [16, 20, 21, 23, 24, 25]. however, in an important study comparing mmnsts to melanocytic lesions, only exon 5 of gnaq containing the q209 codon was sequenced [23]. thus, in that study gnaq codon t96, y101, and r183 hotspot mutations within exons 2 and 4, respectively, could not have been detected. gnaq mrna tissue distribution is greatest in the brain [26]. a propensity for gnaq mutations in neoplasms of other neuroectoderm/neural crest-derived tissues, i.e., melanocytes and nerves, is thus not surprising. however, gnaq is also important in vascular development. gnaq r183l occurs in brain endothelial cells of sturge-weber syndrome [27]. gnaq r183l and r183q mutations have been identified in capillary malformations [7, 28, 29] and gnaq q209 mutations in other benign vascular lesions [30]. additional tissues also highly express gnaq and gnaq mutations may occur in endocrine tumors and other neoplasms [31]. achieving long-term survival in mmnst is hampered by its tendency to metastasize and recur, and its propensity to involve the spine or other structures, which preclude complete surgical resection [32]. one study reports a 20-year remission rate of 67% with total resection [33]. however, torres-mora et al [13] reported mmnst local recurrence and metastatic rates of 35% and 44%, respectively (mean follow-up 55 mo), with 73% of metastases occurring in <4 years. metastases are typically to the lung and pleura. radical resection with or without radiation and/or chemotherapy is the mainstay of treatment. although, radiation has not shown a clear benefit [12]. a few reports using traditional chemotherapeutics, e.g., carboplatin and etoposide, showed low response rates and no survival benefit [14]. while immune checkpoint inhibitors are commonly used in cutaneous melanoma, their use in mmnst is not well-established. this patient with documented pd-l1 expression was treated with the checkpoint inhibitors pembrolizumab, ipilimumab, and nivolumab with pain improvement, but no objective response in disease progression. bajpai et al., however, described a patient with recurrent and metastatic mmnst who underwent multiple surgeries, external beam radiation, and nivolumab treatment [34]. ipilimumab was added after disease recurrence. the authors reported a survival of 51 months from initial diagnosis and 35 months after the start of checkpoint inhibitor therapy. vining et al. described a patient with retrocaval mmnst who experienced decreased pain and disease stabilization with pembrolizumab prior to resection [35]. thus, the use of immune checkpoint inhibitors in mmnst is limited to three reported cases, nevertheless, they suggest some evidence that they may provide symptomatic improvement and clinical response in some pd-l1-positive mmnst patients. references louis dn, ohgaki h, wiestler od, cavenee wk, world health organization, international agency for research on cancer (2016) tumours of the cranial and paraspinal nerves. who classification of tumours of the central nervous system. revised 4th edn. international agency for research on cancer, lyon, 213-230 who classification of tumours editorial board (2021) cranial and paraspinal nerve tumours. who classification of tumours: central nervous system tumours. 5th edn. international agency for research on cancer, lyon, france, 259-279 who classification of tumours editorial board (2020) peripheral nerve sheath tumours. who classification of tumours: soft tissue and bone tumours. 5th edn. international agency for research on cancer, lyon, france, 226-260 wang l, zehir a, sadowska j, zhou n, rosenblum m, busam k, agaram n, travis w, arcila m, dogan s, berger mf, cheng dt, ladanyi m, nafa k, hameed m (2015) consistent copy number changes and recurrent prkar1a mutations distinguish melanotic schwannomas from melanomas: snp-array and next generation sequencing analysis. genes chromosomes cancer 54:463-471. https://doi.org/10.1002/gcc.22254 louis dn, perry a, wesseling p, brat dj, cree ia, figarella-branger d, hawkins c, ng hk, pfister sm, reifenberger g, soffietti r, von deimling a, ellison dw (2021) the 2021 who classification of tumors of the central nervous system: a summary. neuro oncol 23:1231-1251. https://doi.org/10.1093/neuonc/noab106 carney ja (1990) psammomatous melanotic schwannoma. a distinctive, heritable tumor with special associations, including cardiac myxoma and the cushing syndrome. am j surg pathol 14:206-222. https://doi.org/10.1097/00000478-199003000-00002 national center for biotechnology information. clinvar; [vcv001285390.1], https://www.ncbi.nlm.nih.gov/clinvar/variation/vcv001285390.1 (accessed april 22, 2022). capper d, jones dtw, sill m, hovestadt v, schrimpf d, sturm d, koelsche c, sahm f, chavez l, reuss de, kratz a, wefers ak, huang k, pajtler kw, schweizer l, stichel d, olar a, engel nw, lindenberg k, harter pn, braczynski ak, plate kh, dohmen h, garvalov bk, coras r, holsken a, hewer e, bewerunge-hudler m, schick m, fischer r, beschorner r, schittenhelm j, staszewski o, wani k, varlet p, pages m, temming p, lohmann d, selt f, witt h, milde t, witt o, aronica e, giangaspero f, rushing e, scheurlen w, geisenberger c, rodriguez fj, becker a, preusser m, haberler c, bjerkvig r, cryan j, farrell m, deckert m, hench j, frank s, serrano j, kannan k, tsirigos a, bruck w, hofer s, brehmer s, seiz-rosenhagen m, hanggi d, hans v, rozsnoki s, hansford jr, kohlhof p, kristensen bw, lechner m, lopes b, mawrin c, ketter r, kulozik a, khatib z, heppner f, koch a, jouvet a, keohane c, muhleisen h, mueller w, pohl u, prinz m, benner a, zapatka m, gottardo ng, driever ph, kramm cm, muller hl, rutkowski s, von hoff k, fruhwald mc, gnekow a, fleischhack g, tippelt s, calaminus g, monoranu cm, perry a, jones c, jacques ts, radlwimmer b, gessi m, pietsch t, schramm j, schackert g, westphal m, reifenberger g, wesseling p, weller m, collins vp, blumcke i, bendszus m, debus j, huang a, jabado n, northcott pa, paulus w, gajjar a, robinson gw, taylor md, jaunmuktane z, ryzhova m, platten m, unterberg a, wick w, karajannis ma, mittelbronn m, acker t, hartmann c, aldape k, schuller u, buslei r, lichter p, kool m, herold-mende c, ellison dw, hasselblatt m, snuderl m, brandner s, korshunov a, von deimling a, pfister sm (2018) dna methylation-based classification of central nervous system tumours. nature 555:469-474. https://doi.org/10.1038/nature26000 perry a, brat dj (2018) practical surgical neuropathology : a diagnostic approach. elsevier, philadelphia, pa alexiev ba, chou pm, jennings lj (2018) pathology of melanotic schwannoma. arch pathol lab med 142:1517-1523. https://doi.org/10.5858/arpa.2017-0162-ra rodriguez fj, folpe al, giannini c, perry a (2012) pathology of peripheral nerve sheath tumors: diagnostic overview and update on selected diagnostic problems. acta neuropathol 123:295-319. https://doi.org/10.1007/s00401-012-0954-z mees st, spieker t, eltze e, brockmann j, senninger n, bruewer m (2008) intrathoracic psammomatous melanotic schwannoma associated with the carney complex. ann thorac surg 86:657-660. https://doi.org/10.1016/j.athoracsur.2008.02.007 torres-mora j, dry s, li x, binder s, amin m, folpe al (2014) malignant melanotic schwannian tumor: a clinicopathologic, immunohistochemical, and gene expression profiling study of 40 cases, with a proposal for the reclassification of "melanotic schwannoma". am j surg pathol 38:94-105. https://doi.org/10.1097/pas.0b013e3182a0a150 watson jc, stratakis ca, bryant-greenwood pk, koch ca, kirschner ls, nguyen t, carney ja, oldfield eh (2000) neurosurgical implications of carney complex. j neurosurg 92:413-418. https://doi.org/10.3171/jns.2000.92.3.0413 rodriguez fj, stratakis ca, evans dg (2012) genetic predisposition to peripheral nerve neoplasia: diagnostic criteria and pathogenesis of neurofibromatoses, carney complex, and related syndromes. acta neuropathol 123:349-367. https://doi.org/10.1007/s00401-011-0935-7 hirbe ac, kaushal m, sharma mk, dahiya s, pekmezci m, perry a, gutmann dh (2017) clinical genomic profiling identifies tyk2 mutation and overexpression in patients with neurofibromatosis type 1-associated malignant peripheral nerve sheath tumors. cancer 123:1194-1201. https://doi.org/10.1002/cncr.30455 italiano a, michalak s, soulie p, peyron ac, pedeutour f (2011) trisomy 6p and ring chromosome 11 in a melanotic schwannoma suggest relation to malignant melanoma rather than conventional schwannoma. acta neuropathol 121:669-670. https://doi.org/10.1007/s00401-011-0820-4 hoglund m, gisselsson d, hansen gb, white va, sall t, mitelman f, horsman d (2004) dissecting karyotypic patterns in malignant melanomas: temporal clustering of losses and gains in melanoma karyotypic evolution. int j cancer 108:57-65. https://doi.org/10.1002/ijc.11558 mertens f, dal cin p, de wever i, fletcher cd, mandahl n, mitelman f, rosai j, rydholm a, sciot r, tallini g, van den berghe h, vanni r, willen h (2000) cytogenetic characterization of peripheral nerve sheath tumours: a report of the champ study group. j pathol 190:31-38. https://doi.org/10.1002/(sici)1096-9896(200001)190:1<31::aid-path505>3.0.co;2-# isales mc, mohan ls, quan vl, garfield em, zhang b, shi k, arva n, beaubier n, yazdan p, white k, taxter tj, gerami p (2019) distinct genomic patterns in pigmented epithelioid melanocytoma: a molecular and histologic analysis of 16 cases. am j surg pathol 43:480-488. https://doi.org/10.1097/pas.0000000000001195 cohen jn, joseph nm, north jp, onodera c, zembowicz a, leboit pe (2017) genomic analysis of pigmented epithelioid melanocytomas reveals recurrent alterations in prkar1a, and prkca genes. am j surg pathol 41:1333-1346. https://doi.org/10.1097/pas.0000000000000902 zembowicz a, knoepp sm, bei t, stergiopoulos s, eng c, mihm mc, stratakis ca (2007) loss of expression of protein kinase a regulatory subunit 1alpha in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions. am j surg pathol 31:1764-1775. https://doi.org/10.1097/pas.0b013e318057faa7 kusters-vandevelde hv, van engen-van grunsven ia, kusters b, van dijk mr, groenen pj, wesseling p, blokx wa (2010) improved discrimination of melanotic schwannoma from melanocytic lesions by combined morphological and gnaq mutational analysis. acta neuropathol 120:755-764. https://doi.org/10.1007/s00401-010-0749-z onken md, worley la, long md, duan s, council ml, bowcock am, harbour jw (2008) oncogenic mutations in gnaq occur early in uveal melanoma. invest ophthalmol vis sci 49:5230-5234. https://doi.org/10.1167/iovs.08-2145 havik al, bruland o, miletic h, poulsgaard l, scheie d, fugleholm k, lund-johansen m, knappskog pm (2022) genetic alterations associated with malignant transformation of sporadic vestibular schwannoma. acta neurochir (wien) 164:343-352. https://doi.org/10.1007/s00701-021-05062-0 uhlen m, fagerberg l, hallstrom bm, lindskog c, oksvold p, mardinoglu a, sivertsson a, kampf c, sjostedt e, asplund a, olsson i, edlund k, lundberg e, navani s, szigyarto ca, odeberg j, djureinovic d, takanen jo, hober s, alm t, edqvist ph, berling h, tegel h, mulder j, rockberg j, nilsson p, schwenk jm, hamsten m, von feilitzen k, forsberg m, persson l, johansson f, zwahlen m, von heijne g, nielsen j, ponten f (2015) proteomics. tissue-based map of the human proteome. science 347:1260419. https://doi.org/10.1126/science.1260419 huang l, couto ja, pinto a, alexandrescu s, madsen jr, greene ak, sahin m, bischoff j (2017) somatic gnaq mutation is enriched in brain endothelial cells in sturge-weber syndrome. pediatr neurol 67:59-63. https://doi.org/10.1016/j.pediatrneurol.2016.10.010 couto ja, huang l, vivero mp, kamitaki n, maclellan ra, mulliken jb, bischoff j, warman ml, greene ak (2016) endothelial cells from capillary malformations are enriched for somatic gnaq mutations. plast reconstr surg 137:77e-82e. https://doi.org/10.1097/prs.0000000000001868 diociaiuti a, rotunno r, pisaneschi e, cesario c, carnevale c, condorelli ag, rollo m, di cecca s, quintarelli c, novelli a, zambruno g, el hachem m (2022) clinical and molecular spectrum of sporadic vascular malformations: a single-center study. biomedicines 10. https://doi.org/10.3390/biomedicines10061460 jansen p, muller h, lodde gc, zaremba a, moller i, sucker a, paschen a, esser s, schaller j, gunzer m, standl f, bauer s, schadendorf d, mentzel t, hadaschik e, griewank kg (2021) gna14, gna11, and gnaq mutations are frequent in benign but not malignant cutaneous vascular tumors. front genet 12:663272. https://doi.org/10.3389/fgene.2021.663272 parish aj, nguyen v, goodman am, murugesan k, frampton gm, kurzrock r (2018) gnas, gnaq, and gna11 alterations in patients with diverse cancers. cancer 124:4080-4089. https://doi.org/10.1002/cncr.31724 vallat-decouvelaere av, wassef m, lot g, catala m, moussalam m, caruel n, mikol j (1999) spinal melanotic schwannoma: a tumour with poor prognosis. histopathology 35:558-566. https://doi.org/10.1046/j.1365-2559.1999.00786.x siordia ja, golden tr (2016) current discoveries and management of psammomatous melanotic schwannoma. journal of cancer and tumor international 3:1-7. https://doi.org/10.9734/jcti/2016/23786 bajpai j, kapoor a, jalali r, gounder mm (2021) checkpoint inhibitors and radiotherapy in refractory malignant melanocytic schwannoma with carney complex: first evidence of efficacy. bmj case rep 14. https://doi.org/10.1136/bcr-2020-240296 vining cc, hsu pj, miller a, olson dj, gajewski tf, pytel p, bauer bs, millis mj, roggin kk (2021) novel response to neoadjuvant anti-pd1 therapy for a patient with retrocaval melanotic schwannoma. melanoma res 31:92-97. https://doi.org/10.1097/cmr.0000000000000711 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. multiple system atrophy a clinicopathological update feel free to add comments by clicking these icons on the sidebar free neuropathology 1:17 (2020) review multiple system atrophy a clinicopathological update kurt a. jellinger institute of clinical neurobiology, vienna, austria corresponding author: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13, a-1150 vienna, austria kurt.jellinger@univie.ac.at submitted: 25 may 2020 accepted: 24 june 2020 copyedited by: nicole schwab published: 3 july 2020 https://doi.org/10.17879/freeneuropathology-2020-2813 keywords: multiple system atrophy, α-synuclein, glio-neuronal degeneration, animal models, etiopathogenesis, prion-like seeding, biomarkers, experimental therapeutics abstract multiple system atrophy (msa) is a fatal, adult-onset neurodegenerative disorder of uncertain etiology, clinically characterized by various combinations of levo-dopa-unresponsive parkinsonism, and cerebellar, motor, and autonomic dysfunctions. msa is an α-synucleinopathy with specific glioneuronal degeneration involving striatonigral, olivopontocerebellar, autonomic and peripheral nervous systems. the pathologic hallmark of this unique proteinopathy is the deposition of aberrant α-synuclein (αsyn) in both glia (mainly oligodendroglia) and neurons forming pathological inclusions that cause cell dysfunction and demise. the major variants are striatonigral degeneration (msa with predominant parkinsonism / msa-p) and olivopontocerebellar atrophy (msa with prominent cerebellar ataxia / msa-c). however, the clinical and pathological features of msa are broader than previously considered. studies in various mouse models and human patients have helped to better understand the molecular mechanisms that underlie the progression of the disease. the pathogenesis of msa is characterized by propagation of disease-specific strains of αsyn from neurons to oligodendroglia and cell-to-cell spreading in a "prion-like" manner, oxidative stress, proteasomal and mitochondrial dysfunctions, myelin dysregulation, neuroinflammation, decreased neurotrophic factors, and energy failure. the combination of these mechanisms results in neurodegeneration with widespread demyelination and a multisystem involvement that is specific for msa. clinical diagnostic accuracy and differential diagnosis of msa have improved by using combined biomarkers. cognitive impairment, which has been a non-supporting feature of msa, is not uncommon, while severe dementia is rare. despite several pharmacological approaches in msa models, no effective disease-modifying therapeutic strategies are currently available, although many clinical trials targeting disease modification, including immunotherapy and combined approaches, are under way. multidisciplinary research to elucidate the genetic and molecular background of the noxious processes as the basis for development of an effective treatment of the hitherto incurable disorder are urgently needed. abbreviations αsyn α-synuclein, adnc alzheimer disease neuropathological changes, bg basal ganglia, caa cerebral amyloid angiopathy, ci cognitive im-pairment, cn caudate nucleus, cns central nervous system, csf cerebrospinal fluid, dat dopamine transporter, ftld frontotemporal lobar degeneration gci glial cytoplasmic inclu-sion, gdnf glia-derived neurotrophic factors, gp globus pallidus, gwas genome-wide association study, hpr hyperintensive putaminal rim, lbd lewy body disease, lbs lewy bodies, mbp myelin basic protein, mci mild cognitive impairment, msa multiple system atrophy, msa-c msa with prominent cerebellar ataxia, msa-p msa with predominant parkinsonism, opc olivopontocerebellar, opca olivo¬pontocerebellar atrophy, os oxidative stress, part primary age-related tauopathy, pd parkinson disease, pet positron emission tomography, prpc cellular prion protein, psp progressive supranuclear palsy, sn substantia nigra, snd striatonigral degeneration, tg transgenic, tppp tubulin polymerization-promoting protein, wt wild type introduction multiple system atrophy (msa) is a rare adult-onset and lethal neurodegenerative disorder clinically characterized by rapidly progressing autonomic and motor dysfunctions. the pathological hallmark of msa, a specific form of α-synucleinopathy, is abnormal accumulation of fibrillar α-synuclein (αsyn) in oligodendrocytes as glial cytoplasmic inclusions (gci) [1], which may represent a primary pathologic event [2]. degeneration of many neuronal pathways causes multifaceted clinical phenotypes: a parkinsonian variant (msa-p), associated with striatonigral degeneration (snd), and a cerebellar (msa-c) variant with olivopontocerebellar atrophy (opca) [3]. in addition to combined or "mixed" msa, there are several disease variants [4-6]. the underlying molecular mechanisms are poorly understood, but converging evidence suggests that a "prion-like" spreading of disease-specific αsyn strains is involved in the pathogenic cascade leading to a specific multisystem neurodegeneration in this (oligodendro)glioneuronal proteinopathy [4, 7-12]. the aim of the present review is to describe recent advances in msa neuropathology, clinical diagnosis, neuroimaging, and candidate biomarkers. it further provides an overview of the mechanisms underlying msa pathogenesis and of possible novel therapeutic targets that have emerged from animal studies and preclinical interventional trials [13-16]. epidemiology msa is a rare disease with an estimated incidence of 0.6-0.7/100,000 person-years [17], although studies from russia and northern sweden have reported incidences of 0.1 and 2.4/100,000 person-years, respectively [18, 19]. prevalence estimates range from 1.9 to 4.9/100,000 [20] but may reach up to 7.8 after the age of 40 years [21], and up to 12/100,000 after the age of 70 [22]. msa-p accounts for 70-80% of cases in the western world [23], whereas msa-c is more frequent in asian populations (67-84%) with rather frequent mixed phenotypes [24-27], probably due to genetic and environmental factors [5]. etiology and genetics msa is generally considered a sporadic disease [17], but msa pedigrees with both autosomal dominant and autosomal recessive inheritance patterns have been reported in europe and asia [28-33]. a genome-wide association study (gwas) found an estimated heritability of 2-7% [34], but unlike parkinson disease (pd), no single gene mutations linked to familial forms and no definite environmental risk factors have been identified [35]. screening for pd causal genes (mapt, pdyn, parkin, pink1, and several single nucleotide polymorphisms/snps) did not reveal any association with msa [36-38], while lrrk2 exon variants may contribute to its susceptibility [39]. glucocerebrosidase (gba) variants were associated with autopsy-proven msa [40, 41], particularly with msa-c [42], while others have found no association [43]. furthermore, c9orf72 repeat expansions [44] and snca polymorphisms as risk factors of msa [45, 46] have not been confirmed [47-49]. no significant associations of the apoe locus nor the prion prnp with risk of msa was observed [50, 51], and there is no evidence of autosomal dominant msa or of de novo mutations in this disorder [52]. a british family with snca mutation showed neuropathologic features of both pd and msa [53], but they are distinct from pd patients carrying the h50q or snca duplication [54]. none of the nucleotide polymorphisms (fbxo47, elovl7, edn1, etc.) reached genome-wide significance [34], and polymorphisms of the lingo1 and lingo2 (nogo receptor interacting protein-1 and -2) do not decrease the risk of msa [55]. the possible involvement of the snca, coq2, mapt, gba1, lrrk2 and c9orf72 genes in msa pathogenesis was examined recently [56]. the link between v393a mutations and the coq2 gene, encoding the coenzyme q10 (coq10), and familial or sporadic msa in japanese and other asian populations [44, 57-61] has not been confirmed in other populations [34, 62-64]. thus, coq2 polymorphisms may be region-specific and may not represent common genetic factors for msa. decreased levels of coq10 in cerebellum and plasma of msa patients [65, 66] suggest that its deficiency may contribute to pathogenesis due to decreased electron transport in the mitochondria and increased vulnerability to oxidative stress (os) [67]. rna analyses of msa brain tissue revealed alterations in αand β-immunoglobuline [68], dysregulations of micrornas that regulate gene expression in the pons and cerebellum [69, 70], and disruption of long intervening non-coding rnas (lincrna) in the frontal cortex along with protein coding genes related to iron metabolism and immune response regulation [71, 72]. epidemiological studies suggested that epigenetic factors or environmental toxins may be associated with the risk for msa [73], but there are no convincing data correlating increased risk of msa with exposure to pesticides, solvents, other toxins, or alcohol consumption [74, 75]. pathogenesis although our understanding of msa remains incomplete, evidence from animal models and human post mortem studies indicates that the accumulation of misfolded αsyn, particularly in oligodendrocytes but also in neurons, plays an essential role in the disease process [10, 76-78]. the impact of the neuronal endosomal-lysosomal system in the processing of αsyn in pd is well established, while lysosomes contribute to the pathogenesis of msa, enabling oligodendroglial and neuronal uptakt of αsyn [79]. reduced oligodendrocyte-derived enriched microvesicles (oemvs) could be an important mechanism related to pathological αsyn aggregation in oligodendrocytes [80]. although it has been speculated that primary neuronal pathology leads to secondary oligodendroglial degeneration, as suggested by the widespread occurrence of ncis even in areas lacking gcis [77], the distribution and severity of neurodegeneration reflects subregional gci density and supports the assumption that msa is a primary oligodendrogliopathy [2, 81]. the role of oligodendroglia in introducing the neurodegenerative process was confirmed experimentally in transgenic (tg) mice overexpressing αsyn in oligodendrocytes [10, 13, 82-84]. these and other results highlight the role of endogenous αsyn and p25α in the formation of αsyn assemblies in oligodendrocytes and provide in vivo evidence of the role of oligodendroglial αsyn in the establishment of αsyn pathology in msa [85]. early events are an ectopic appearance of αsyn in oligodendrocytes, loss of the camp-regulated phosphoprotein of 32kda (darpp-32), and calbindin indicating calcium toxicity and disturbance of phosphorylated proteins [86]. recent findings suggest the possibility of endogenous αsyn accumulation in oligodendrocyte precursor cells that contribute to gci formation and perturbation of neuronal/glia support in msa brain [86a]. reduced oemvs could be an important mechanism related to pathological αsyn aggregates in oligodendroglia, inducing dysfunction of the snare protein complex, which regulates membran fusion in eukaryotic cells. the concentrations of oemvs in msa were significantly reduced compared to those in pd [80]. decreased expression of glia-derived neurotrophic factors (gdnf) in msa brains [87] indicates that αsyn aggregation in oligodendrocytes impacts their trophic transport to neurons. oligodendroglial changes are more widespread than αsyn positive gcis, suggesting that oligodendroglial pathology induces degeneration of the oligodendroglia-myelin-axon-neuron complex [2, 26]. the selectivity of the neurodegeneration in msa is determined by the interaction of multiple noxious factors. some of these factors include: ectopic αsyn accumulation in oligodendrocytes and neurons, "prion-like" propagation of disease-specific strains of misfolded αsyn [88], targeting distinct brain regions and cell types [89, 90], impaired protein degradation, proteasomal and mitochondrial dysfunctions [91, 92], alterations of the autophagic pathway [91, 93, 94], perturbed iron homeostasis [95], lipid dysfunction involved in myelin synthesis [96-98], genetic polymorphism [55], microglial activation [97, 99], neuroinflammation [100], proteolytic disturbance, autophagy [101], and microrna dysregulation [102] driving inflammation, disrupting myelin, and contributing αsyn accumulation via the dysregulation of autophagy and prion mechanisms [103]. these and other factors are contributing to os, which is suggested to be a major pathogenic factor in msa and related diseases [104]. these multiple mechanisms interact to result in the system-specific pattern of neurodegeneration in msa (fig. 1). tnfα-dependent neuroinflammation may play a key role in msa pathogenesis, and its relevance has been underlined in various models of the disease [105]. αsyn, which shows specific conformational strains [88, 106] that are primarily generated by neurons, can be toxic once released to the extracellular environment [107] and can spread throughout the brain in a "prion-like" manner [9, 108-111]. extracellular αsyn, interacting with neuronal and non-neuronal cell types, mediates neuroinflammation and cell-to-cell spread [112, 113]. neuron-to-oligodendrocyte transport of misfolded αsyn plays a major role in the pathogenesis of msa [114, 115]. msa and pd show different phosphorylation signatures of αsyn and distinct seed characteristics, indicating that distinct strains underlie these diseases [90, 116, 117]. after propagation in tgm83 tg mice, strain-specific phenotypic differences are maintained after serial transmission, providing evidence that disease heterogeneity among the synucleinopathies is caused by distinct αsyn strains [89]. msa strains show several similarities to pd strains, and less so with dlb strains, but more potently induce motor deficits, nigrostriatal degeneration, αsyn spreading, and inflammation, reflecting the aggressive nature of this disease [118]. fig. 1. pathogenetical features of msa causing neurodegeneration. spontaneous misfolding of αsyn results in formation of abnormally folded dimers and further assembly results in oligomers and amyloid formations. αsyn-rich gcis involving oligodendroglia result in demyelization and neurodegeneration. the red arrow shows the “prion-like” cell-to-cell transfer of misfolded αsyn. courtesy of victoria sidoroff, md, dept. of neurology, medical university of innsbruck, innsbruck, austria recent animal model studies that only partially replicate the human disorder have provided some progress in our understanding of msa pathogenesis [13, 15, 84, 119, 120]. early accumulation of p25α (tppp), a potent stimulator of αsyn aggregation, may decrease myelin basic protein (mbp), favoring both the deposition and fibrillation of αsyn and changing myelin metabolism [121]. relocation of p25α from the myelin sheaths to the oligodendroglial soma, due to mitochondrial dysfunction, and the formation of cytoplasmic p25α inclusions precedes the aggregation of transformed αsyn in oligodendrocytes. endogenous αsyn and p25α induce the formation of pathological αsyn assemblies in oligodendrocytes and provide in vivo evidence of their contribution to the pathogenesis of msa [85]. although large inclusions appear in a later disease states, small, soluble assemblies of αsyn, promoted by p25α, are pathogenic [122]. the source of αsyn in oligodendroglia is unclear, but it contains αsyn mrna expression and αsyn may be secreted by neurons and taken up by oligodendrocytes, which is facilitated by protein cx32 via direct protein-protein interaction in both neurons and oligodendroglia [115]. 21% of proteins found consistently in gcis and lbs are synaptic vesicle-related, suggesting that misfolded αsyn may be targeted via vesicle-mediated transport, and may explain the presence of this neuronal protein within gcis [123]. thus, msa represents a specific form of oligodendroglial proteinopathies [124], while others suggest that it is a primary neuronal disease with secondary accumulation of αsyn in oligodendrocytes [77]. induced pluripotent stem cell (ipsc) studies indicate a pathological phenotype of msa neurons, independently from oligodendrocytes. these data together with findings in animal models suggest that both neurons and oligodendrocytes are affected in msa [91]. the disease is currently viewed as a primary synucleinopathy with specific glio-neuronal degeneration developing via the oligo-myelin-axon-neuron complex [2, 4]. histopathology and molecular pathology the histological core features of msa encompass the following types of different severity: (1)specific αsyn-immunoreactive inclusion pathology with five types of inclusions: gcis within oligodendrocytes (also referred to as papp-lantos bodies [125], the presence of which is mandatory for the post mortem diagnosis of definite msa [1]) and less frequently glial nuclear inclusions, neuronal nuclear inclusions, astroglial cytoplasmic inclusions, and neuronal threads, also composed of αsyn [126]; (2) selective neuronal loss and axonal degeneration involving multiple regions of the nervous system; (3) extensive myelin degeneration with pallor and reduction in mbp with astrogliosis; and (4) widespread microglial activation [127] and neuroinflammation [128, 129], with extensive cd4 and cd8 t-cell infiltration [130]. gcis and the resulting neurodegeneration show a characteristic distribution, involving not only the striatonigral and opc systems, but also cortical regions, autonomic and motor nuclei in the brainstem, spinal cord, preganglionic autonomic nerve structures [131-134], and the peripheral nervous system [135-138], characterizing msa as a multi-system/-organ disorder [2, 77, 139]. phosphorylated αsyn is accumulated in subpial and periventricular astrocytes after long disease duration [140]. however, αsyn-positive astrocytes in subpial and perivascular regions are seen in both msa and lewy body disease (lbd), suggesting that this pathology is not a specific feature of msa [141]. inclusion pathology gcis are argyrophilic, triangular, sickle-/half moon-shaped or oval cytoplasmic aggregations, composed of fibrillar αsyn, ubiquitin and various multifunctional proteins, including 14-3-3 protein, lrrk2, aggressomal proteins, etc. [125] (fig. 2). they form a meshwork of loosely packed filaments or tubules 15-30 nm in diameter with a periodicity of 70-90 nm and straight filaments, both composed of polymerized αsyn granular material and other filaments. the central core contains phosphorylated (ser129) αsyn cryo-em showed that αsyn inclusions from msa are made of two types of filaments, each of which consists of two different protofilaments. each type contains non-proteinaceous molecules at the interface of the two proteofilaments. thus, they differ from those in dlb brain, which suggests that distinct conformations/strains are characteristic for specific synucleinopathies. in addition, αsyn filament extracts from msa tissue differ from those formed in vitro using recombinant proteins, which may have implications for the mechanisms of protein aggregation and neurodegeneration [142]. soluble αsyn in gcis differs from the insoluble form in lewy bodies (lbs) [143]. purification of αsyn containing gcis revealed 11.9% αsyn, 2.8% α-β-crystallin, and 1.7% 14-3-3 protein compared to 8.5%, 2.0% and 1.5% in lbs [144]. in the msa brain, αsyn 140 and 122 isoform levels are increased, whereas αsyn 126 is decreased, in the substantia nigra (sn), striatum, and cerebellum. in early disease states, diffuse αsyn staining in neuronal nuclei and cytoplasm occurs in many gray matter areas, indicating that aggregation of non-fibrillary αsyn occurs early in neurons [26]. recent studies using a proximity ligation assay revealed a wide distribution of αsyn oligomers not only in oligodendrocytes but also in neocortical neurons and purkinje cells, suggesting that αsyn oligomer-mediated toxicity is an early event in msa, inducing neuronal loss in msa [145]. fig. 2. (a–c) glial cytoplamic inclusions in msa: (a) in globus pallidus (gallyas silver impregnation), (b) in pontine basis (α-synuclein) and (c) in frontal white matter, anti-ubiquitin. (d) neuronal cytoplasmic inclusion and neurites in pontine basis (α-synuclein). (a–d) original magnification 34,000. from [126]. on the other side, interactions exist between extracellular αsyn and each of the major central nervous system (cns) cell types. this has thepotential to contribute to secondary disease processes such as neuroinflammation, synaptic dysfunc-tion, and cell-to-cell spread, with vehicles such as microglia and exosomes that mediate spread of αsyn pathology to peripheral brain regions [113]. cathepsin-d, calpain-1 and kallikrein-6 are elevated in the putamen, pontine basis, and cerebellar white matter, indicating that αsyn accumulation is not due to reduced activity of these proteases, but rather that their upregulation is compensatory to increased αsyn [146]. iron levels in basal ganglia (bg) and sn are higher in msa than in pd and controls, indicating perturbed iron homeostasis as a potential pathogenic factor in msa neurodegeneration [95]. quantitative analyses of neuronal death and gci density showed a positive correlation with each other, indicating the pivotal role of gcis in neuronal death [81, 147], and additionally, both lesions increase with disease duration [148-150]. in the sn, severe neuronal loss is accompanied by low gci density, indicating that this and other areas affected in early disease have been burned out [139]. glial nuclear inclusions show a distinct distribution from gcis (fig. 2d), and similarly the density of neuronal cytoplasmic inclusions (ncis) and neuronal nuclear inclusions are unrelated to that of gcis [151]. ncis are more widespread and show a hierarchical pattern related to the duration of disease but are independent of neuronal destruction, suggesting that other factors may induce the subtype-dependent neuronal loss [77]. region-specific astrogliosis is positively correlated with αsyn pathology in msa, in contrast to pd [152], and in general parallels the severity of neurodegeneration [148]. microglial activation in degenerated regions accompanies gci pathology and is most abundant in white matter areas with mild to moderate demyelination [153]. in msa-c, the cerebellar subcortical white matter and cerebellar brainstem projections are the earliest involved, followed by other cns regions. distribution of lesions a grading system for snd was proposed based on semiquantitative assessment of atrophy, neuronal loss, and the presence of gcis [154]: neuronal loss in the sn pars compacta is grade 1; extension to the putamen is grade 2; further involvement of the caudate and globus pallidus (gp) is grade 3. subsequently, the grading system was extended for both snd and opca [155]. of 42 patients, 22 were assigned as msa-p and 20 as msa-c, but none displayed "pure" opca pathology or more severe opca pathology than snd (i.e., opca iii+snd i/ii). these clinicopathological subtypes correlated with initial symptoms and clinical features of both types. post mortem mri changes in the putamen (type 1, mild atrophy and isointensity; type 2, atrophy and diffuse hypointensity with a hyperintensive putaminal rim/hpr; type 3, putaminal atrophy and isoor hypointensity with hpr) reflect various degrees of brain damage [156]. in two large series from the uk and japan, another grading system for msa was proposed [148]: each case of snd and opca was divided into three grades based on semiquantitative assessment of neuronal loss in regions of interest: for snd, the putamen, gp and sn; and for opca the pontine nucleus, cerebellar hemisphere and vermis, inferior olivary nucleus and sn. this classification showed significant clinicopathological correlations. snd phenotypes showed more severe bradykinesia, and the opca phenotype more frequently showed cerebellar signs. no patients showed "pure" snd or "pure" opca. however, there is an increasing overlap of αsyn pathology with increased duration of the disease the extent of αsyn pathology [157]. damage to the striatonigral system is most severe in the dorsolateral caudal putamen and lateral sn, suggesting transsynaptic degeneration of the striatonigral fibers. consistently and severely affected areas are the putamen, cn, sn, pontine and medullary tegmental nuclei, inferior olives, and cerebellar white matter; moderately affected areas are the motor cortex and gp, and mild lesions involve the cingular cortex, hypothalamus, nucleus basalis of meynert, thalamus, subthalamus, and pontine tegmentum [158]. degeneration of the gp and sn leads to dysfunction of these inhibitory nuclei projecting to the motor thalamus, but the sn loss is of dopamine, not gaba (gamma aminobutyric acid), neurons. stereological studies of the bg revealed a substantial loss of neurons in the sn, putamen, and gp, whereas astrocytes were more frequent in the putamen and caudate nucleus (cn). microglia were found in all cns regions with greatest frequency in the, otherwise unaffected, red nucleus. these data support the region-specific pattern of pathological changes in msa [159]. another neuropathological study showed that the striatonigral region was most severely affected in 34% of snd and in 17% in opca cases, while in almost half of them both regions were equally affected [133]. in view of the frequent overlap and mixed forms, the value of grading systems for evaluation of msa is under discussion [139]. there is widespread involvement of the neocortex with significant loss of neurons and increase of astrocytes and microglia in the frontal and parietal areas, but no change in the total number of oligodendrocytes [160]. early degeneration of the bg drives late onset cortical atrophy due to fronto-striatal degeneration [161, 162]. reduced neuronal numbers in the anterior olfactory nucleus and intrabulbar part of the primary olfactory (pyriform) cortex may underlie olfactory dysfunction in msa [163]. limbic tdp-43 pathology is rare in msa, but co-localization with αsyn suggests an interaction between the two molecules [164-167]; tdp-43 positive cases showed significantly older age at death than negative ones, suggesting that tdp-43 pathology in msa is an age-related phenomenon rather than a disease-specific change [141]. demyelination of variable intensity affecting all parts of the nervous system [168] is associated with reduction of mbp by about 50% [96]. gcis and microglial burden are greatest in mild to moderate white matter lesions and decrease with progression of myelin damage that increases with disease duration [169]. the regional vulnerability of the white matter to msa pathology is poorly understood, but recent gwass revealed dysregulation of various methylated loci, including hip1, lman2, mobp, and others, giving the first evidence that dna methylation changes contribute to the molecular processes altered in msa [170]. early msa stages show increased microglia (about 100%) in the white matter [127], without concomitant astrogliosis or oligodendroglial degeneration [171]. both microglial activation and αsyn-containing oligodendrocytes trigger neuroinflammation in the white matter [128]. the loss of tubulin polymerization-promoting protein (tppp)/p25α immunoreactivity correlated significantly with the degree of microglial reaction and loss of mbp density as a marker of tract degeneration [124]. white matter degeneration causes degeneration of neuronal loops, leading to dysfunction of cerebral autoregulation [172]. gliosis in the degenerated areas of the msa brain usually correlates with αsyn pathology and the severity of neurodegeneration [153, 173], which is in contrast to pd [174]. significant increase of monoaminoxidase b (mao-b), a biomarker of astrogliosis, in the degenerated putamen (+83%) was associated with astrogliosis and showed a positive correlation with αsyn accumulation [175]. microglial activation accompanying αsyn pathology and phagocytosing degenerating myelin is prominent in all degenerating regions [176], particularly in white matter input tracts to the extrapyramidal system and cerebellum [177]. stereological studies revealed a significant increase of microglia in the white matter without concomitant astrogliosis and with absence of significant oligodendroglial degeneration [171], suggesting that microglia cells play an important role in the initiation and progression of neurodegeneration in msa [100, 178]. this is supported by tg mouse models indicating an active contribution of microglial activation by triggering neuroinflammatory responses in the msa brain [179]. in msa-c, gcis are most prominent in the cerebellum, pons, and medulla [169]. the cerebellar purkinje cells are more severely affected in the vermis, with atrophy of olivary nucleus, cerebellopontine fibers, and pontine basis, causing interruption of specific cerebellocortical circuits [180]. the motor subnetwork in msa-c is significantly altered in both bg and cerebellar connectivity [181], with hyperintensity of the middle cerebellar peduncle [182]. involvement of autonomic and peripheral nervous systems degeneration of preganglionic autonomic neurons of the brain stem and spinal cord cause multidomain autonomic failures in msa [133, 183, 184]. supraspinal lesions involve cholinergic neurons of the ventrolateral nucleus ambiguous [185, 186], tegmental nuclei [187], ventral periaqueductal dopaminergic neurons [188], medullary and arcuate nucleus, noradrenergic locus ceruleus [134], serotonergic medullary groups, ventrolateral medulla [189], caudal raphe neurons [190, 191], catecholaminergic neurons of rostral ventral medulla, and noradrenergic neurons of the caudal ventrolateral medulla [185, 192]. the medullary serotonergic and catecholaminergic systems are involved in early stages of msa [193]. other involved areas are the dorsal vagal nucleus [185], periaqueductal gray [132], the westphal-edinger nucleus and posterior hypothalamus, the tuberomamillary and suprachiasmatic nuclei [194], and the pontomedullary reticular formation [149]. the density of αsyn pathology did not correlate with neuronal loss, and there was no correlation between the αsyn burden and disease duration in these regions, indicating that the loss of monoaminergic neurons may progress independently from αsyn accumulation [195]. sympathetic preganglionic neurons in the intermediolateral cell columns of the thoracolumbar spinal cord [26, 134, 196] and sympathetic ganglia and schwann cells in autonomic nerves are involved [197]. neuronal loss affects onuf's nucleus in the sacral region [198], with minor loss of upper and lower motor neurons [26] and variable involvement of anterior horn cells [134]. mild degeneration of cardiac sympathetic innervation has been reported in some cases of msa [199, 200], which accounts for a mild to moderate decrease in the number of tyrosine hydroxylase, but not of neurofilament-immunoreactive nerve fibers in the epicardium. however, depletion of cardiac sympathetic nerves is closely related to the presence of αsyn pathology in the sympathetic ganglia of the cns [200, 201]. the peripheral nervous system shows αsyn deposits in sympathetic ganglia, skin nerve fibers [138, 202, 203], and schwann cells [204], but lack of αsyn immunoreactivity in dermal fibers in contrast to pd [203, 205]. filamentous αsyn aggregates involve the cytoplasm of schwann cells in cranial, spinal and autonomic nerves in msa [141, 197, 206]. clinical features the onset of motor symptoms is 56±9 (mean ± sd) years, with both sexes equally affected [207], however 20-75% of msa patients have a prodromal/preclinical phase with non-motor symptoms. this phase includes cardiovascular and other autonomic failures (urogenital and sexual dysfunctions, orthostatic hypotension, and rem sleep behavior disorder (rbd), which occurs in 88% or more [208, 209]), which may precede the motor presentation by months to years [210, 211] and indicates more rapid progression of the disease [212, 213]. average age at disease onset is earlier in msa-c compared to msa-p, the latter leading to more severe disability [214-216]. average duration after clinical diagnosis is 6-10 (mean 9.5) years [12, 23], with few patients surviving more than 15 years [217]. others have reported a 5 year survival of 78% [218] and a 43% death rate during 3 years of follow-up [135]. a pan-american multicenter study reported that 68% of the participants presenting as msa-p showed an age at onset of 61.5 years, and those as msa-c of 57.4 years [219], while a prospective cohort in the usa reported a median survival of 9.8 (95% ci 8.8-10.7) years [220]. early autonomic dysfunctions and severity of orthostatic hypertension have negative impact on both disease progression and survival [221] and more than triples the risk of shorter survival [222, 223], and a meta-analysis identified severe dysautonomia, early combined autonomic and motor failure, and early falls as unfavorable predictors of survival, whereas msa phenotype and sex did not predict survival [224]. parkinsonism with rigidity, slowness of movements, postural instability, gait disability, and a tendency to fall, characterize the motor presentation of msa-p [12]. parkinsonism is rapidly progressing to wheelchair confinement within 5 to 10 years from symptom onset, poorly responsive to l-dopa, and is often associated with atypical features [17]. unilateral parkinsonism occurs in 40% of msa patients [220] and typical tremor in 4-10% [225]. early postural instability and gait difficulties with recurrent falls are also seen in msa [35]. polyminimyoclonus, not included in the current diagnostic criteria of msa, has now been recognized as a specific clinical feature of msa. among motor and non-motor symptoms in early msa, dysarthria was the most prevalent feature (98.4%), followed by sexual dysfunction (95%), rbd (90.2%), constipation (82%), snoring (70.5%), dysphagia (69%), and stridor (42.6%), which was more common in msa-c than in msa-p [226]. a resting tremor is rare, whereas irregular postural and action tremor may occur [227, 228]. cerebellar ataxia, widespread gait, uncoordinated limb movements, action tremor, and spontaneous or gaze invoked nystagmus predominate msa-c [35]. hyperreflexia and a babinski sign occur in 30-50% of patients, while abnormal postures, such as bent spine, antecollis, and hand or foot dystonia are rare [229]. early generalized and rapidly progressive autonomic failure is typical of msa [230] and, in the absence of parkinsonism or cerebellar signs, indicating pure autonomic failure, which converts to msa within a few years in about 28% [231-233]. among non-motor symptoms observed in 75-95% of patients [234], urinary urgency and increased frequency are common in early disease stages [35]. in a subset of msa patients with early urinary retention, the disease may begin in the sacral spinal cord and then spread to other regions [235]. orthostatic hypotension with recurrent syncope, which occurs after the onset of urogenital symptoms, is a hallmark feature of msa; less specific are dizziness and nausea. other symptoms are anhydrosis, gastrointestinal dysfunction with early dysphagia and constipation [225], pupillary autonomic involvement with blurred vision and dry eyes. [236]. dysproportional antecollis and pisa syndrome are common postural deformities in msa [35]. about 50% of patients with msa-p develop cerebellar signs and even a higher proportion of msa-c cases develop parkinsonian features [23, 220]. dystonia, repeated falls, drooling, dysphagia, dysphonia, and pain occur in advanced stages of the disease [237]. laryngeal stridor is rare [210]. respiratory disturbances including diurnal or nocturnal inspiratory stridor and sleep apnea are frequent [238, 239]. diagnostic biomarkers despite numerous studies, to date there are no reliable diagnostic and prognostic biomarkers available. while multimodal imaging of structural and functional brain changes gave insight into the pathophysiology and may evaluate disease progression, recent studies suggest that the combination of neuroimaging and fluid biomarkers may be more successful than using single markers to increase the accuracy of the clinical (differential) diagnosis of msa [240]. fluid and tissue biomarkers studies of αsyn levels in cerebrospinal fluid (csf) and plasma have been shown to not be useful in the discrimination between msa and pd or psp [5, 241, 242]. a recent meta-analysis of available csf data showed that reduction of p-tau, αsyn, aβ-42 and total tau and elevated nfl are indicators for msa [243]. currently, the most promising approach is a combination of csf dj-1, phospho-tau, light chain neurofilament protein (nfl) and aβ-42 that may be helpful in the differential diagnosis between msa and other parkinsonian disorders [5, 240, 243, 244] (fig. 3). other studies have shown increased csf levels of cytokines such as mcp-3, mdc, fractalkine, and mip-1β [246]. phosphorylated αsyn in red blood cells may be a potential diagnostic biomarker for msa [247]. the results of proteomics for biomarker discovery and mrna expression need further elucidation [248]. fig. 3. candidate biomarkers of multiple system atrophy compared to parkinson’s disease and controls. msa: multiple system atrophy; pd: parkinson’s disease; nfl: neurofilament light chain; fh: complement factor h; c3: complement 3; mhpg: 3-methoxy-4-hydroxyphenylethyleneglycol; igf-i: insulin-like growth factor i; uch-l1: ubiquitin carboxy-terminal hydrolase l1; oxdj: oxidized dj-1 protein; mirna: microrna. modified from [245]. molecular and functional imaging a cardiac sympathetic postganglionic denervation distinguishes pd from msa, showing intact innervation. i-123 mibg (metaiodobenzylguanidine) scintigraphy can help differentiate the two diseases with a pooled specificity of 77% (95% ci: 68-84%) [199]. recent meta-analyses suggest that mibg imaging is useful to discriminate pd from msa in moderate to advanced disease stages, but unreliable in early stages [199, 249]. however, interactions with many drugs limit the value of this method [250]. the anteroposterior diameter of the medulla oblongata is a potential imaging marker of parasympathetic dysfunction in msa [251]. in recent years, several brain magnetic resonance imaging (mri) features have been described as helpful in the differential diagnosis of parkinsonian syndromes. they include atrophy of the putamen, pons, cerebellum, and middle cerebellar peduncle, a dilated fourth ventricle, and various signal intensity variations on mri [252]. mri abnormalities including the "hot-cross bun" sign, a cruciform hyperintensity in the pons [253], and the "putaminal rim sign", which marks hyperintensive bordering of the dorsolateral margins of the puta men in t2-weighted mri reflecting degeneration and iron deposition, may differentiate msa-p from pd [254-258]. they are, however, non-specific signs and therefore not included in the recent consensus criteria [3], in contrast to putaminal atrophy which shows 92.3% specificity but low sensitivity (44.4%) [259, 260]. putaminal atrophy together with hypointense putaminal signal changes on iron-sensitive routine sequences seem to be specific for msa-p [252]. others showed significantly increased putaminal diffusivity volumes in the small anterior region of interest in msa-p versus pd [261]. another distinguishing feature is the extensive and widespread volume loss across the entire brain in msa-p [262]. in quantitative mri studies, the bilateral r2* increase in the putamen best separated msa-p from pd [263]. putaminal and infratentorial volume information classified 96.8% of msa cases [260]. diffusion tensor imaging permits differentiation between pd and msa-p, the latter showing higher values of the diffusion coefficient in the inner capsule, corona radiata, and lateral periputaminal white matter [264], while a meta-analysis of putaminal diffusivity measurements showed sensitivity of 90% and specificity of 93% in distinguishing msa-p from pd based on putaminal diffusivity [265]. combined use of diffusion ratios and magnetic susceptibility values/quantitative susceptibility mapping allowed differentiation of msa-p and msa-c from other parkinsonian syndromes with sensitivities and specificities of 81-100% [266]. hyperintensity of the middle cerebellar peduncle and hot cross bun sign should be added into the list of additional neuroimaging features of possible msa-c [182]. several studies assessed the diagnostic potential of multimodal mri [267-270]. in conclusion, the sensitivity of conventional mri findings in msa compared to pd and healthy controls is inconsistent (36-83%), the specificity of mri abnormalities differentiating msa from pd is high (88-100%). automated imaging differentiation in parkinsonism (aid-p) and magnetic resonance parkinsonism index (mrpi) are robust biomarkers for pd and msa [271]. diffusion weighted images, t2* weighted images and proton density weighted images are useful for diagnosis msa-p in early stages [272]. fluorodeoxyglucose-positron emission tomography (fdg-pet) can distinguish msa-p from pd, showing different patterns of decreased glucose metabolism with a positive predictive value of 97% [273, 274]. targeting postsynaptic dopaminergic functions using 123fβcit spect differentiates pd (normal or increased signal) from msa (normal or increased signal) [275]. dopamine transporter (dat) imaging showed more prominent and earlier dat loss in the anterior caudate and ventral putamen in msa than in pd [276], although normal dat imaging does not exclude msa [277]. in autopsy-confirmed cases a greater asymmetry of striatal binding was seen in msa than in pd [278], but it is highly correlated with sn cell loss [279]. 18f-dopa-pet showed more widespread bg dysfunction in msa than in pd without evidence of early compensatory increase in dopa uptake [280]. future studies will be needed to determine the usefulness of tau-pet imaging for the characterization of αsyn filaments and the differential diagnosis of atypical parkisonian disorders. interpretation of tau-pet should be done cautiously, since some msa cases with severe gci pathology may be false-positive [281, 282], even though the affinity of pbb3 is 10 to 50 times less than αsyn [283]. 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-3-isoquinoline carboxamide (pk11195) for imaging microglia-mediated processes showed elevated tracer binding in many areas of the msa brain, consistent with the known neuropathologic distribution [284]. diagnostic accuracy and differential diagnosis revised consensus guidelines define 3 degrees of certainty of clinical diagnosis of msa: definite, probable and possible [3] (table 1, fig. 4). fig. 4. diagnostic scheme for msa according to the current consensus diagnostic criteria. definite msa requires post mortem evidence of widespread αsyn inclusions with concomitant snd or opca [1]. probable msa is defined as a sporadic, progressive disorder in adults, clinically characterized by severe autonomic failure, urinary dysfunction and poor l-dopa-responsive parkinsonism or cerebellar ataxia. a diagnosis of probable msa is based on clinical features and ancillary diagnostic tests. possible msa can be diagnosed when a sporadic progressive adult-onset disorder with parkinsonism or cerebellar ataxia is accompanied by at least one of the following additional features within 3 years of motor onset: dysphagia, gait ataxia and other cerebellar symptoms (table 1). table 1. diagnostic clinical markers for msa. modified from [240]. msa, multiple system atrophy; msa-c, msa with cerebellar features; msa-p, msa with predominant parkinsonism. "red flag" diagnostic features the presence of "red flag" (warning sign) features highly specific for msa may provide important clues for a correct and early diagnosis. they include orofacial dystonia; inspiratory signs, contractures of hands and feet, jerky myoclonic postural/action tremor, polyminimyoclonus, severe dysphonia and dysarthria, pathological laughter and crying, snoring, disproportional antecollis, camptocormia and/or pisa syndrome, and cold hands and feet [225, 229] (table 2). in addition, severe disability milestones include: frequent falls, use of urinary catheters, wheelchair dependence, unintelligible speech, cognitive impairment, severe dysphagia, and residential care. in a recent clinicopathological study of 203 clinically diagnosed msa patients, a lifetime recorded number of red flags in both msa-p and msa-c was compared to lbd and psp [225]. recognition of patients with early or possible msa may be supported by one or more red flags, and two or more out of six had a specificity of 98.3% and a sensitivity of 84.2% [228, 229], while no differences were found in the frequencies of red flags within 3 years from disease onset between msa and msa look-alikes [225]. recent studies confirmed the validity of an eight-item pilot scale for the assessment of early msa [285]. table 2. clinical features supporting and non-supporting a diagnosis of multiple system atrophy. modified from [240]. due to the heterogeneity of clinical phenotypes and lack of specific biomarkers, it is a challenge to make a correct antemortem diagnosis of msa [286]. the sensitivity of the second consensus criteria was 41% for possible and 18% for probable msa at first clinical visit and 92% and 63% at last clinical visit, respectively [287]. in two recent brain bank studies, among patients diagnosed with msa during life, only 62% and 79% met the pathological criteria [225, 286], while 25% of patients with the diagnosis of "possible" msa had different pathological diagnoses, including pd and psp [225]. the most common misdiagnoses were dlb (13 and 14%, respectively), psp (6 and 11%) and pd (6%). autonomic failure was the leading cause of misdiagnosis in pd and dlb, and cerebellar ataxia that of misdiagnosis in psp [286]. sporadic spinocerebellar ataxia (sca) with autonomic failure can masquerade as msa-c. a study reported that 7% of patients with clinically diagnosed msa had mutations in sca genes [288]. fragile x tremor-ataxia syndrome and x-linked adrenoleukodystrophy can also be misdiagnosed as msa-c [61]. the possible explanations for the suboptimal diagnostic accuracy of the current consensus criteria for msa that saw a positive predictive diagnosis even in later disease stages from 60 to 90% [286, 287] have been recently discussed [289]. atypical msa almost all cases of msa display neuronal loss in both striatonigral and opc structures [24, 148], with only 11 of 42 cases assigned to the category of "pure" snd [155]. however, msa has a wider range of presentations, which expands the list of differential diagnoses. several subtypes of msa do not fit into the current classification [290]. "minimal change" msa is a rare aggressive form with gcis and neurodegeneration almost restricted to the sn, putamen, and locus coeruleus, thus representing "pure" snd [291-294], suggesting that gci formation is an early event and may precede neuronal loss. one patient with "minimal" msa-c showed abundant gcis in pontine nuclei, middle cerebellar peduncle and cerebellar white matter, with ncis and neuronal nuclear inclusions restricted to the pontine basis, cerebellar vermis, and inferior olivary nuclei, which were associated with neuronal loss indicating a link between both lesions in early disease [295]. neurologically normal individuals are rarely found to have gcis at autopsy as coincidental or incidental findings limited to the pons and inferior olivary nuclei with mild neuronal loss restricted to the sn, suggesting that these regions may be afflicted first in msa-p [296, 297]. the presence of gcis may represent an age-related phenomenon not necessarily progressing to overt clinical disease, classifying these cases as "incidental" or "prodromal/preclinical" msa, similar to incidental lbd [298]. young-onset msa with a mean age of 36.4 years shows more l-dopa-induced dyskinesia but less common myoclonus and pyramidal signs compared to late-onset cases. on post mortem analysis, the "minimal change" variant was more common in young-onset msa [299]. the other extreme are "benign" msa cases with prolonged survival up to 15 years in about 2-3% of patients [217, 300]. most of them showed slowly progressing parkinsonism with subsequent rapid deterioration after development of autonomic failure [301]. many of them developed motor fluctuation and l-dopa-induced choreiform dyskinesias [302, 303]. other cases of survival up to 18 years revealed extensive distribution of gcis in the cns [304]. another variant of pathological confirmed msa showed neither parkinsonism nor cerebellar symptoms [305]. an atypical case of frontotemporal lobar degeneration (ftld)-tdp type a with msa phenocopy syndrome showed severe striatal degeneration and cerebellar involvement [306], while four cases with clinical features of ftld, but without autonomic dysfunction, showed frontotemporal atrophy and severe limbic αsyn neuronal pathology with pick body-like, but tau-negative, inclusions. these cases were suggested to represent a novel subtype of ftld associated with αsyn (ftld-αsyn) [307]. rare cases in a family with pathologic hexanucleotide repeat expansions in c9orf72, a gene linked to amyotrophic lateral sclerosis, demonstrated clinical and neuroimaging features indistinguishable from msa [308], and a cerebello-brainstem dominant form of x-linked adrenoleukodystrophy presented as msa [61]. recently, rare cases of msa with transitional or diffuse dlb developing clinical features of pdd or dlb have been reported. those with neuronal loss in sn but not in striatal or opc systems with widespread gcis were considered "minimal change" msa, in which lbd was considered the primary pathology and msa as coincidental. apoe allele frequency was not different between these forms [309]. these and other subtypes should be considered in establishing a correct diagnosis of msa. cognitive impairment in msa unlike other synucleinopathies, msa has not been associated with significant cognitive impairment (ci), which has been considered an exclusion criterion for the diagnosis of msa [3]. however, a recent position statement by the neuropathology task force of the movement disorder society indicated that ci may be an under recognized feature in msa occurring in 17-47% of msa patients, while severe dementia is rare [310]. because ci has been underestimated in msa, not all patients have undergone formal cognitive assessments and, therefore, the frequency could be higher than reported in several studies. the degree of ci in msa patients ranges from mild to moderate decline and affect executive, attentional and visuospatial functions, while memory is less often impaired [197, 310-312]. ci may occur in early stages of msa, but it is generally common in advanced cases [313] and often correlates with disease duration [314]. mild cognitive impairment (mci) has been reported in up to 40% of msa-p patients, mainly characterized by frontal dysfunction [310, 315]. mild or moderate ci has been reported in 14-37% of pathologically proven msa cases [134, 286, 302, 316]. more severe and widespread cognitive dysfunction was seen in msa-p than in msa-c patients [317], probably due to prefrontal impairment [315], whereas others saw no differences in cognitive variables between the two groups [318] or more severe cognitive dysfunctions in msa-c [319]. ci has been regarded as a result of cortical and subcortical structural changes [320], frontal lobe dysfunction [321, 322], cortical dysfunction driven by focal frontostriatal degeneration [162], alterations in the corpus callosum [323], the dorsolateral prefrontal cortex network [324], or neocortical neuronal loss [159], while others have not found any differences in the severity of pathological findings between cases with and without ci [325]. recent studies indicated that nci burden in the hippocampus and parahippocampal gyrus is associated with memory impairment in msa [326]. alzheimer’s disease neuropathological changes (adnc), cerebral amyloid angiopathy (caa), and cerebrovascular lesions did not differ between cases with and without ci [325], whereas others showed a greater burden of ncis in medial temporal regions, the hippocampus or perirhinal regions [77, 197, 316, 326, 327]. adnc has been reported in only 2/35 (7%) autopsy-proven cases of msa [134], whereas two cases of combined msa and ad (braak stages iii and vi) have been reported, in which only a few neurons shared αsyn and tau [328]. a recent retrospective clinicopathological study of 48 msa patients (33 msa-p and 15 msa-c) with a mean age at death of 60.5±7.8 (range 46-82) years, reported mci in 10 cases (20.8%), in which three had associated moderate cortical tau pathology (braak i-ii), and moderate ci in seven patients (14.5%), for which six had associated cortical amyloid plaques and moderate cortical tau pathology (braak ii-iii), one had probable primary age-related tauopathy (part), and one female aged 82 years with severe dementia showed fully developed ad. cortical lewy pathology, observed in four cases, was not associated with clinical ci. 77.1% of the msa cases were free of adnc, compared to 42% in controls, while lewy pathology was higher than in the control groups (8.4%) [329]. in view of the limited data on the molecular basis of ci (and other neuropsychiatric symptoms) in msa, further studies on the pathological basis of ci in msa are needed. msa a prion-like or prion disease? the spread of αsyn pathology from one cell to another and even from one nervous structure to another has been demonstrated in vivo [11, 330-335]. this pattern, resembling prion spreading, has led to the concept of prion-like propagation of αsyn and tau [110]. self-propagation of αsyn oligomers, however, is not sufficient to declare them as prions, because they show "seeding" activity rather than infectivity of αsyn [336]. however, the applicability of the prion hypothesis in α-synucleinopathies and, in particular, msa remains controversial, since injections of brain lysated from msa patients failed to replicate the oligodendroglial αsyn pathology that is typical for msa. while studies in wild type (wt) mice provided insights into the mechanisms of oligodendroglial αsyn aggregations in msa, intracerebral inoculation studies in non-human primates to the best of our knowledge have not been performed yet. there are other challenges to the hypothesis that msa is a prion disease. first, endogenous wt αsyn is insufficient to propagate αsyn pathology; mutant αsyn is needed as a template. the transmission of αsyn "prions" to a second synucleinopathy model and their ability to propagate between two distinct mouse cell lines while retaining strain-specific properties was suggested to provide evidence that msa is a prion disease [337]. however, these and other mouse experiments have not yet explained why in msa αsyn pathology predominantly accumulates in oligodendroglia, as msa-derived αsyn does not appear to have the ability to induce strain-like cell-specific aggregates. this demonstrates that the intrinsic properties of a53t αsyn in the m83 mouse model dominate over any strain features harbored by misfolded αsyn in msa brains [9]. furthermore, gcis have never been identified in wt mouse brains inoculated with msa-derived αsyn [338]. hence, αsyn aggregates ("prionoids") derived from msa patients created a neurodegenerative pattern that is atypical for msa [336]. moreover, αsyn aggregates, the morphological hallmarks of msa, were not detected in msa-inoculated tgm83+/mice [339, 340], and no study has definitely propagated patient-derived seeds from cell-to-cell or mouse-to-mouse, or fully characterized αsyn strains from msa vs. pd [117]. the variety of seeds, animal models, and methodologies currently prevents clear conclusions regarding αsyn-related spreading and toxicity, as well as translation of preclinical findings to human disease [341]. a recent study found no evidence of binding between cellular prion protein (prpc) and αsyn oligomers, while prpc neither binds to αsyn oligomers nor mediates their detrimental effects [342]. however, there may be different species of αsyn oligomers, which have different binding capacity with prpc, and it remains possible that future studies could demonstrate that both prpc-dependent and -independent pathways could play a role in the pathogenesis of synucleinopathies [343]. accordingly, it could be possible that aggregated αsyn is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-propagating states that can lead to prion diseases upon serial passing in wt animals [344]. however, recent studies showed that abnormal misfolded cellular prion protein was able to efficiently propagate in the brain of animals even in the absence of αsyn, suggesting that this protein may not act as a key modulator of prion propagation. thus, αsyn may take part in this process of self-propagation but is not specifically required for sustaining prion conversion and propagation [345]. finally, gene analyses have shown that the homozygous state of positions 129 in the prnp gene is not a risk factor for msa and no variants of the prnp gene were associated with increased risk for msa [50]. review of clinical notes from patients who had died of msa showed no evidence of neurosurgical transmission [346], and studies of couples whose spouses had autopsy-confirmed pd, psp, or msa, did not suggest an increased risk of synucleinopathy development in the other spouses [347, 348]. although there is no evidence of iatrogenic or direct transmission in autopsy-confirmed msa cases, this is no evidence of absence of human transmission or misfolded proteins other than prions and β-amyloid, and further research is necessary before any conclusion can be drawn [349]. in conclusion, it seems reasonable to postulate that even if prion-like spreading in experimental systems may justify the view that the progression of neurodegeneration in msa reflects a cell-to-cell spread of pathological αsyn, this is not sufficient to define msa as classical prion disease [336]. new therapies so far there are no causative or disease-modifying treatments available for msa and symptomatic therapies are limited [35, 350]. the first-line treatment of a hypokinetic-rigid syndrome is dopaminergic treatment with l-dopa, the initial responsiveness to which has been reported in 83% of msa patients [228], but its effect is usually transient, and only 31% showed a response for a period of 3.5 years [23]. l-dopa response was observed in 42-57% of msa-p and in 13-25% of msa-c patients [220]. recent animal studies suggest that l-dopa failure can be induced by restricted lateral striatal lesions combined with dopaminergic denervation [351]. in some patients, motor fluctuations with wearing-off phenomena or off-bound dystonia were observed [352]. l-dopa-induced dyskinesias were reported in 24.7% of definite msa patients [23]. dopamine agonists are not considered a therapeutic option, as they show poor efficacy and may involve severe side effects, particularly the worsening of orthostatic hypotension [353]. for cerebellar symptoms, no efficient drug treatments are available. deep brain stimulation in msa patients showed only transient improvement of motor symptoms, but was rapidly counteracted by the occurrence of disabling symptoms [303]. non-pharmacological treatment options such as physiotherapy and occupational therapy play an important role in improving symptoms and patients' quality of life, and should be integrated into the therapeutic concept [354]. translational and novel therapeutic approaches based on the current knowledge about the pathogenesis of msa and the different findings in animal models, a number of therapeutic strategies have been proposed to target disease progression in msa [5, 15, 16]. based on the ability of αsyn to be transferred from cell to cell and to spread through the brain in a prion-like manner, inhibition of αsyn oligomerization and aggregation may constitute a promising therapeutic strategy for disease modification, and interesting efforts have been made in this direction. these include (1) αsyn inhibition, (2) αsyn degradation enhancement, (3) intervening neuroinflammation, and (4) neuronal loss. numerous randomized, placebo-controlled trials of putative disease-modifying agents have been performed including riluzole, minocyline, lithium, rifampicin, fluoxetine, rasagilin, neuroprotective mesenchymal stem cells, epigallocatechin gallate, intravenous immunoglobulins and others. although most of these treatments were efficient in cellular or animal models of msa, in human patients they showed no clinical effects [16, 341]. among drugs targeting αsyn aggregation, promesa studies on the effect of epigallocatechin gallate, a polyphenol found in green tea which reduces aggregation and toxicity of αsyn oligomers [329], did not modify disease progression [355]. among αsyn degradation enhancing compounds, rapamycin, an autophagy enhancer, showed a reduction of αsyn aggregates in some brain areas [356] in preclinical studies, and is now under clinical trial [357]. another approach concerns the possible involvement of toll-like receptor 4 (tlr4) and its selective antagonist monophosphoryl lipid a (mpla) that reduced gcis and motor deficits in mice [358]. targeting neuroinflammation, the inhibition of myeloperoxidase as well as the reduction of tnfα-dependent reactions are promising disease-modifying targets and are being clinically tested in msa patients [16]. the use of microglia inhibitors, such as minocycline, that rescues dopaminergic neurons in msa mice, and the anti-inflammatory substance fluoxetine, however, fail to change disease progression. an alternative approach was used in msa patients to target neuroinflammation by delivering intravenous immunoglobulin, but the results were inconclusive [359]. the compound fty720-mitoxy, an fda-approved immunosuppressive for multiple sclerosis, reduced parkinsonism by increasing brain-derived neurotrophic factor (bdnf), and protected movement and mitochondria in wt and cnp-αsyn mice [360]. numerous efforts have been undertaken to address neuronal loss, including bone marrow-derived mesenchymal stem cells [361, 362] and the antioxidant target of rapamycin (mtor) receptor [363], however all of these efforts have failed to slow or halt disease progression [16]. several studies have successfully proven the therapeutic potential of anti-αsyn immunotherapy by preventing αsyn spreading [5, 15]. based on the fact that active immunization of mbp mice reduced αsyn accumulation and neurodegeneration [364], two αsyn vaccines (pd03a, pd01a) were evaluated in phase i studies with msa and pd patients and showed good safety and tolerability [365, 366]. active immunization against αsyn and combination with anti-inflammatory treatment may also be promising therapeutic strategies [367, 368]. gene therapy may constitute another feasible approach to diminish os excitotoxicity and subsequent neuronal loss, but none of the used compounds demonstrated effects on disease progression and the underlying neurodegeneration [16]. new strategies targeting αsyn are in progress [16, 280, 369], based on completed or ongoing interventional trials by the msa coalition [12]. therefore, there is a strong need to clarify the pathogenic mechanisms of msa in order to develop new therapeutic strategy options, including combined approaches by targeting different msa-specific pathogenetic effects. conclusions and further outlook current evidence supports the hypothesis that misfolded αsyn contributes to os that induces a cascade of deleterious events, including proteasomal and mitochondrial dysfunctions, neuroinflammation, and energy failure that is associated with deposition of aberrant αsyn in both glia (mainly oligodendroglia) and neurons resulting in neurodegeneration and demyelination. currently, the cascade of events that underlies the pathogenesis of msa is not completely understood. recent studies using animal models that only partially replicate human pathology and the molecular dynamics of the neurodegenerative process have provided progress in our understanding of msa pathogenesis. the disease is viewed as a primary synucleinopathy with specific (oligodendro)glial-neuronal degeneration developing secondarily via the oligo-myelin-axon-neuron complex [2, 4, 370]. strong evidence against a primary neuronal pathology with the formation of gcis, resulting from secondary accumulation of pathological αsyn that may be of neuronal origin [371], is the fact that gcis are the hallmark of msa and not of pd, a disease with similar patterns of αsyn inclusions (lbs) but resulting from different strains of αsyn, differentiating the two disorders [88, 89, 106, 372]. the source of αsyn in msa and the pathogenic cascade leading to "prion-like" spreading of its strains contributing to progression of the disease need further elucidation, and there is no convincing evidence for the suggestion that msa is a prion disease. although disease-modifying treatments are currently not available, better knowledge about the molecular pathogenesis of msa derived from animal models and human post mortem experience have contributed to the development of future therapeutic strategies to target disease progression in msa. acknowledgements the author thanks mr. e. mitter-ferstl, phd, for secretarial and editorial work. funding the study was partially funded by the society for the promotion of research in experimental neurology, vienna, austria. references [1] trojanowski jq, revesz t (2007) proposed neuropathological criteria for the post mortem diagnosis of multiple system atrophy, neuropathol appl neurobiol 33:615-20. [2] jellinger ka, wenning gk (2016) multiple system atrophy: pathogenic mechanisms and biomarkers, j neural transm (vienna) 123:555-72. [3] gilman s et al. (2008) second consensus statement on the diagnosis of multiple system atrophy, neurology 71:670-6. [4] jellinger ka (2018) multiple system atrophy: an oligodendroglioneural synucleinopathy, j alzheimers dis 62:1141-79. [5] koga s, dickson dw (2017) recent advances in neuropathology, biomarkers and therapeutic approach of multiple system atrophy, j neurol neurosurg psychiatry online aug 31: doi 10.1136/jnnp-2017-315813. [6] shimohata t (2020) [diagnosis of multiple system atrophy for establishing disease-modifying therapies] (japanese), brain nerve 72:131-6. [7] goedert m et al. (2017) the synucleinopathies: twenty years on, j parkinsons dis 7:s53-s71. [8] monzio compagnoni g, di fonzo a (2019) understanding the pathogenesis of multiple system atrophy: state of the art and future perspectives, acta neuropathol commun 7:113. [9] dhillon js et al. (2019) dissecting alpha-synuclein inclusion pathology diversity in multiple system atrophy: implications for the prion-like transmission hypothesis, lab invest online feb 8: doi 10.1038/s41374-019-0198-9. [10] kaji s et al. (2020) insights into the pathogenesis of multiple system atrophy: focus on glial cytoplasmic inclusions, transl neurodegener 9:7. [11] woerman al et al. (2018) alpha-synuclein: multiple system atrophy prions, cold spring harb perspect med 8. [12] krismer f, wenning gk (2017) multiple system atrophy: insights into a rare and debilitating movement disorder, nat rev neurol 13:232-43. [13] refolo v et al. (2018) progressive striatonigral degeneration in a transgenic mouse model of multiple system atrophy: translational implications for interventional therapies, acta neuropathol commun 6:2. [14] overk c et al. (2018) multiple system atrophy: experimental models and reality, acta neuropathol 135:33-47. [15] heras-garvin a, stefanova n (2020) msa: from basic mechanisms to experimental therapeutics, parkinsonism relat disord. [16] meszaros l et al. (2020) current symptomatic and disease-modifying treatments in multiple system atrophy, int j mol sci 21. [17] fanciulli a, wenning gk (2015) multiple-system atrophy, n engl j med 372:249-63. [18] linder j et al. (2010) incidence of parkinson's disease and parkinsonism in northern sweden: a population-based study, mov disord 25:341-8. [19] winter y et al. (2010) incidence of parkinson's disease and atypical parkinsonism: russian population-based study, mov disord 25:349-56. [20] tison f et al. (2000) prevalence of multiple system atrophy, lancet 355:495-6. [21] schrag a et al. (1999) prevalence of progressive supranuclear palsy and multiple system atrophy: a cross-sectional study, lancet 354:1771-5. [22] chrysostome v et al. (2004) epidemiology of multiple system atrophy: a prevalence and pilot risk factor study in aquitaine, france, neuroepidemiology 23:201-8. [23] wenning gk et al. (2013) the natural history of multiple system atrophy: a prospective european cohort study, lancet neurol 12:264-74. [24] ozawa t, onodera o (2017) multiple system atrophy: clinicopathological characteristics in japanese patients, proc jpn acad ser b phys biol sci 93:251-8. [25] yabe i et al. (2006) msa-c is the predominant clinical phenotype of msa in japan: analysis of 142 patients with probable msa, j neurol sci 249:115-21. [26] yoshida m (2007) multiple system atrophy: alpha-synuclein and neuronal degeneration, neuropathology 27:484-93. [27] lyoo ch et al. (2008) effects of disease duration on the clinical features and brain glucose metabolism in patients with mixed type multiple system atrophy, brain 131:438-46. [28] itoh k et al. (2014) definite familial multiple system atrophy with unknown genetics, neuropathology 34:309-13. [29] hara k et al. (2007) multiplex families with multiple system atrophy, arch neurol 64:545-51. [30] hohler ad, singh vj (2012) probable hereditary multiple system atrophy-autonomic (msa-a) in a family in the united states, j clin neurosci 19:479-80. [31] vidal js et al. (2010) familial aggregation in atypical parkinson's disease: a case control study in multiple system atrophy and progressive supranuclear palsy, j neurol 257:1388-93. [32] wüllner u et al. (2004) probable multiple system atrophy in a german family, j neurol neurosurg psychiatry 75:924-5. [33] fujioka s et al. (2014) update on novel familial forms of parkinson's disease and multiple system atrophy, parkinsonism relat disord 20 suppl 1:s29-34. [34] sailer a et al. (2016) a genome-wide association study in multiple system atrophy, neurology 87:1591-8. [35] fanciulli a et al. (2019) multiple system atrophy, int rev neurobiol 149:137-92. [36] brooks ja et al. (2011) mutational analysis of parkin and pink1 in multiple system atrophy, neurobiol aging 32:548 e5-7. [37] gu x et al. (2018) analysis of gwas-linked variants in multiple system atrophy, neurobiol aging 67:201 e1e4. [38] yuan x et al. (2015) an association analysis of the r1628p and g2385r polymorphisms of the lrrk2 gene in multiple system atrophy in a chinese population, parkinsonism relat disord 21:147-9. [39] heckman mg et al. (2014) lrrk2 exonic variants and risk of multiple system atrophy, neurology 83:2256-61. [40] sklerov m et al. (2017) frequency of gba variants in autopsy-proven multiple system atrophy, mov disord clin pract 4:574-81. [41] sun qy et al. (2013) genetic association study of glucocerebrosidase gene l444p mutation in essential tremor and multiple system atrophy in mainland china, j clin neurosci 20:217-9. [42] mitsui j et al. (2015) variants associated with gaucher disease in multiple system atrophy, ann clin transl neurol 2:417-26. [43] srulijes k et al. (2013) no association of gba mutations and multiple system atrophy, eur j neurol 20:e61-2. [44] chen x et al. (2016) c9orf72 repeat expansions in chinese patients with parkinson's disease and multiple system atrophy, j neural transm (vienna) 123:1341-5. [45] al-chalabi a et al. (2009) genetic variants of the alpha-synuclein gene snca are associated with multiple system atrophy, plos one 4:e7114. doi:10.1371/journal.pone.0007114. [46] scholz sw et al. (2009) snca variants are associated with increased risk for multiple system atrophy, ann neurol 65:610-4. [47] federoff m et al. (2016) genome-wide estimate of the heritability of multiple system atrophy, parkinsonism relat disord 22:35-41. [48] nussbaum rl (2018) (2017) genetics of synucleinopathies, cold spring harb perspect med 8. [49] sun z et al. (2015) snp rs11931074 of the snca gene may not be associated with multiple system atrophy in chinese population, int j neurosci 125:612-5. [50] chelban v et al. (2017) analysis of the prion protein gene in multiple system atrophy, neurobiol aging 49:216 e15e18. [51] ogaki k et al. (2018) multiple system atrophy and apolipoprotein e, mov disord 33:647-50. [52] laurens b et al. (2017) multiple system atrophy state of the art, curr neurol neurosci rep 17:41. [53] kiely ap et al. (2013) alpha-synucleinopathy associated with g51d snca mutation: a link between parkinson's disease and multiple system atrophy?, acta neuropathol 125:753-69. [54] kiely ap et al. (2015) distinct clinical and neuropathological features of g51d snca mutation cases compared with snca duplication and h50q mutation, mol neurodegener 10:41. [55] chen y et al. (2015) analysis and meta-analysis of five polymorphisms of the lingo1 and lingo2 genes in parkinson's disease and multiple system atrophy in a chinese population, j neurol 262:2478-83. [56] katzeff js et al. (2019) cross-examining candidate genes implicated in multiple system atrophy, acta neuropathol commun 7:117. [57] lin ch et al. (2015) coq2 gene variants associate with cerebellar subtype of multiple system atrophy in chinese, mov disord 30:436-7. [58] multiple-system atrophy research collaboration (2013) mutations in coq2 in familial and sporadic multiple-system atrophy, n engl j med 369:233-44. [59] zhao q et al. (2016) association of the coq2 v393a variant with risk of multiple system atrophy in east asians: a case-control study and meta-analysis of the literature, neurol sci 37:423-30. [60] quinzii cm et al. (2014) mutant coq2 in multiple-system atrophy, n engl j med 371:80-3. doi:10.1056/nejmc1311763. [61] ogaki k et al. (2016) adult-onset cerebello-brainstem dominant form of x-linked adrenoleukodystrophy presenting as multiple system atrophy: case report and literature review, neuropathology 36:64-76. [62] sharma m et al. (2014) mutant coq2 in multiple-system atrophy, n engl j med 371:80-3. [63] ronchi d et al. (2016) mutational analysis of coq2 in patients with msa in italy, neurobiol aging 45:213 e1-2. [64] ferguson mc et al. (2014) shc2 gene copy number in multiple system atrophy (msa), clin auton res 24:25-30. [65] barca e et al. (2016) decreased coenzyme q10 levels in multiple system atrophy cerebellum, j neuropathol exp neurol 75:663-72. [66] schottlaender lv et al. (2016) coenzyme q10 levels are decreased in the cerebellum of multiple-system atrophy patients, plos one 11:e0149557. [67] nakamoto fk et al. (2018) the pathogenesis linked to coenzyme q10 insufficiency in ipsc-derived neurons from patients with multiple-system atrophy, sci rep 8:14215. [68] mills jd et al. (2015) transcriptome analysis of grey and white matter cortical tissue in multiple system atrophy, neurogenetics 16:107-22. [69] lee st et al. (2015) altered expression of mir-202 in cerebellum of multiple-system atrophy, mol neurobiol 51:180-6. [70] wakabayashi k et al. (2016) microrna expression profiles of multiple system atrophy from formalin-fixed paraffin-embedded samples, neurosci lett 635:117-22. [71] mills jd et al. (2016) strand-specific rna-sequencing analysis of multiple system atrophy brain transcriptome, neuroscience 322:234-50. [72] wan p et al. (2017) the role of long noncoding rnas in neurodegenerative diseases, mol neurobiol 54:2012-21. [73] sturm e, stefanova n (2014) multiple system atrophy: genetic or epigenetic?, exp neurobiol 23:277-91. [74] vidal js et al. (2008) risk factors of multiple system atrophy: a case-control study in french patients, mov disord 23:797-803. [75] vanacore n et al. (2005) case-control study of multiple system atrophy, mov disord 20:158-63. [76] bleasel jm et al. (2016) animal modeling an oligodendrogliopathy multiple system atrophy, acta neuropathol commun 4:12. [77] cykowski md et al. (2015) expanding the spectrum of neuronal pathology in multiple system atrophy, brain 138:2293-309. [78] valera e, masliah e (2018) the neuropathology of multiple system atrophy and its therapeutic implications, auton neurosci 211:1-6. [79] puska g et al. (2018) lysosomal response in relation to alpha-synuclein pathology differs between parkinson's disease and multiple system atrophy, neurobiol dis 114:140-52. [80] yu z et al. (2020) reduced oligodendrocyte exosome secretion in multiple system atrophy involves snare dysfunction, brain. [81] wenning gk et al. (2008) multiple system atrophy: a primary oligodendrogliopathy, ann neurol 64:239-46. [82] fellner l et al. (2015) models of multiple system atrophy, curr top behav neurosci 22:369-93. [83] stemberger s et al. (2010) targeted overexpression of human alpha-synuclein in oligodendroglia induces lesions linked to msa-like progressive autonomic failure, exp neurol 224:459-64. [84] tanji k et al. (2019) a mouse model of adult-onset multiple system atrophy, neurobiol dis 127:339-49. [85] mavroeidi p et al. (2019) endogenous oligodendroglial alpha-synuclein and tppp/p25alpha orchestrate alpha-synuclein pathology in experimental multiple system atrophy models, acta neuropathol online apr 22: doi 10.1007/s00401-019-2014-y. [86] hayakawa h et al. (2013) loss of darpp-32 and calbindin in multiple system atrophy, j neural transm 120:1689-98. [86a] kaji s et al. (2018) pathological endogenous alpha-synuclein accumulation in oligodendrocyte precursor cells potentially induces inclusions in multiple system atrophy, stem cell reports 10:356-65. [87] ubhi k et al. (2010) neurodegeneration in a transgenic mouse model of multiple system atrophy is associated with altered expression of oligodendroglial-derived neurotrophic factors, j neurosci 30:6236-46. [88] shahnawaz m et al. (2020) discriminating alpha-synuclein strains in parkinson's disease and multiple system atrophy, nature 578:273-7. [89] lau a et al. (2020) alpha-synuclein strains target distinct brain regions and cell types, nat neurosci 23:21-31. [90] morgan sa et al. (2020) alpha-synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species, j biol chem. [91] monzio compagnoni g et al. (2018) mitochondrial dysregulation and impaired autophagy in ipsc-derived dopaminergic neurons of multiple system atrophy, stem cell reports 11:1185-98. [92] ubhi k et al. (2014) widespread microrna dysregulation in multiple system atrophy disease-related alteration in mir-96, eur j neurosci 39:1026-41. [93] valera e et al. (2017) microrna-101 modulates autophagy and oligodendroglial alpha-synuclein accumulation in multiple system atrophy, front mol neurosci 10:329. [94] tanji k et al. (2013) alteration of autophagosomal proteins in the brain of multiple system atrophy, neurobiol dis 49:190-8. [95] kaindlstorfer c et al. (2018) the relevance of iron in the pathogenesis of multiple system atrophy: a viewpoint, j alzheimers dis 61:1253-73. [96] don as et al. (2014) altered lipid levels provide evidence for myelin dysfunction in multiple system atrophy, acta neuropathol commun 2:150. [97] bleasel jm et al. (2014) lipid dysfunction and pathogenesis of multiple system atrophy, acta neuropathol commun 2:15. [98] grigoletto j et al. (2017) higher levels of myelin phospholipids in brains of neuronal alpha-synuclein transgenic mice precede myelin loss, acta neuropathol commun 5:37. [99] stefanova n et al. (2007) microglial activation mediates neurodegeneration related to oligodendroglial alpha-synucleinopathy: implications for multiple system atrophy, mov disord 22:2196-203. [100] von bernhardi r et al. (2015) microglial cell dysregulation in brain aging and neurodegeneration, front aging neurosci 7:124. [101] miki y et al. (2017) ambra1, a novel alpha-synuclein-binding protein, is implicated in the pathogenesis of multiple system atrophy, brain pathol online 2017 march 15: doi 10.1111/bpa.12461. [102] xiang c et al. (2019) micrornas dysregulation and metabolism in multiple system atrophy, front neurosci 13:1103. [103] kim t et al. (2019) alterations in striatal microrna-mrna networks contribute to neuroinflammation in multiple system atrophy, mol neurobiol 56:7003-21. [104] ubhi k et al. (2009) mitochondrial inhibitor 3-nitroproprionic acid enhances oxidative modification of alpha-synuclein in a transgenic mouse model of multiple system atrophy, j neurosci res 87:2728-39. [105] ndayisaba a et al. (2019) tnfalpha inhibitors as targets for protective therapies in msa: a viewpoint, j neuroinflammation 16:80. [106] fearon c, farrell ma (2020) disease-specific strains of a-synuclein in multiple system atrophy and parkinson's disease: but why?, mov disord 35:756-7. [107] guo jl, lee vm (2014) cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases, nat med 20:130-8. [108] dhillon js et al. (2019) comparative analyses of the in vivo induction and transmission of alpha-synuclein pathology in transgenic mice by msa brain lysate and recombinant alpha-synuclein fibrils, acta neuropathol commun 7:80. [109] duyckaerts c et al. (2019) the prion-like propagation hypothesis in alzheimer's and parkinson's disease, curr opin neurol 32:266-71. [110] goedert m et al. (2017) like prions: the propagation of aggregated tau and alpha-synuclein in neurodegeneration, brain 140:266-78. [111] karpowicz rj, jr. et al. (2019) transmission of alpha-synuclein seeds in neurodegenerative disease: recent developments, lab invest online feb 13: doi 10.1038/s41374-019-0195-z. [112] davis aa et al. (2018) intercellular spread of protein aggregates in neurodegenerative disease, annu rev cell dev biol 34:545-68. [113] valdinocci d et al. (2018) extracellular interactions of alpha-synuclein in multiple system atrophy, int j mol sci 19. [114] reyes jf et al. (2014) alpha-synuclein transfers from neurons to oligodendrocytes, glia 62:387-98. [115] reyes jf et al. (2019) binding of alpha-synuclein oligomers to cx32 facilitates protein uptake and transfer in neurons and oligodendrocytes, acta neuropathol 138:23-47. [116] rossi m et al. (2020) ultrasensitive rt-quic assay with high sensitivity and specificity for lewy body-associated synucleinopathies, acta neuropathol. [117] yamasaki tr et al. (2019) parkinson's disease and multiple system atrophy have distinct alpha-synuclein seed characteristics, j biol chem 294:1045-58. [118] van der perren a et al. (2020) the structural differences between patient-derived alpha-synuclein strains dictate characteristics of parkinson's disease, multiple system atrophy and dementia with lewy bodies, acta neuropathol. [119] bassil f et al. (2017) viral-mediated oligodendroglial alpha-synuclein expression models multiple system atrophy, mov disord 32:1230-9. [120] heras-garvin a et al. (2019) anle138b modulates alpha-synuclein oligomerization and prevents motor decline and neurodegeneration in a mouse model of multiple system atrophy, mov disord 34:255-63. [121] olah j et al. (2017) role of the microtubule-associated tppp/p25 in parkinson's and related diseases and its therapeutic potential, expert rev proteomics 14:301-9. [122] olah j, ovadi j (2019) pharmacological targeting of alpha-synuclein and tppp/p25 in parkinson's disease: challenges and opportunities in a nutshell, febs lett online may 31: doi 10.1002/873-3468.13464. [123] mccormack a et al. (2019) abundance of synaptic vesicle-related proteins in alpha-synuclein-containing protein inclusions suggests a targeted formation mechanism, neurotox res 35:883-97. [124] rohan z et al. (2016) shared and distinct patterns of oligodendroglial response in alpha-synucleinopathies and tauopathies, j neuropathol exp neurol 75:1100-9. [125] jellinger ka, lantos pl (2010) papp-lantos inclusions and the pathogenesis of multiple system atrophy: an update, acta neuropathol 119:657-67. [126] wenning gk, jellinger ka (2005) the role of alpha-synuclein in the pathogenesis of multiple system atrophy, acta neuropathol 109:129-40. [127] kübler d et al. (2019) widespread microglial activation in multiple system atrophy, mov disord 34:564-8. [128] hoffmann a et al. (2019) oligodendroglial a-synucleinopathy-driven neuroinflammation in multiple system atrophy, brain pathol 29:380-96. [129] kiely ap et al. (2018) immunohistochemical and molecular investigations show alteration in the inflammatory profile of multiple system atrophy brain, j neuropathol exp neurol 77:598-607. [130] williams gp et al. (2020) t cell infiltration in both human multiple system atrophy and a novel mouse model of the disease, acta neuropathol 139:855-74. [131] vanderhorst vg et al. (2015) alpha-synuclein pathology accumulates in sacral spinal visceral sensory pathways, ann neurol 78:142-9. [132] benarroch ee et al. (2010) differential involvement of the periaqueductal gray in multiple system atrophy, auton neurosci 158:111-7. [133] ozawa t (2007) morphological substrate of autonomic failure and neurohormonal dysfunction in multiple system atrophy: impact on determining phenotype spectrum, acta neuropathol (berl) 114:201-11. [134] wenning gk et al. (1997) multiple system atrophy: a review of 203 pathologically proven cases, mov disord 12:133-47. [135] bensimon g et al. (2009) riluzole treatment, survival and diagnostic criteria in parkinson plus disorders: the nnipps study, brain 132:156-71. [136] wakabayashi k et al. (2010) involvement of the peripheral nervous system in synucleinopathies, tauopathies and other neurodegenerative proteinopathies of the brain, acta neuropathol 120:1-12. [137] kuzdas-wood d et al. (2015) involvement of peripheral nerves in the transgenic plp-alpha-syn model of multiple system atrophy: extending the phenotype, plos one 10:e0136575. [138] donadio v et al. (2020) skin biopsy may help to distinguishing msa-p from parkinson’s disease with orthostatic hypotension, mov disord in print. [139] ahmed z et al. (2012) the neuropathology, pathophysiology and genetics of multiple system atrophy, neuropathol appl neurobiol 38:4-24. [140] nakamura k et al. (2015) accumulation of phosphorylated alpha-synuclein in subpial and periventricular astrocytes in multiple system atrophy of long duration, neuropathology 36:157-67. [141] koga s et al. (2017) alpha-synuclein astrogliopathy: a possible specific feature in alpha-synucleinopathy, neuropathology 37:379-81. [142] schweighauser m et al. (2020) structures of alpha-synuclein filaments from multiple system atrophy, nature, online may 27: doi 10.1038/s41586-020-2317-6. [143] campbell bc et al. (2001) the solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with lewy bodies and parkinson's disease, j neurochem 76:87-96. [144] mccormack a et al. (2016) purification of alpha-synuclein containing inclusions from human post mortem brain tissue, j neurosci methods 266:141-50. [145] sekiya h et al. (2019) wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation, acta neuropathol 137:455-66. [146] kiely ap et al. (2019) exploring the putative role of kallikrein-6, calpain-1 and cathepsin-d in the proteolytic degradation of alpha-synuclein in multiple system atrophy, neuropathol appl neurobiol 45:347-60. [147] ozawa t (2006) pathology and genetics of multiple system atrophy: an approach to determining genetic susceptibility spectrum, acta neuropathol 112:531-8. [148] ozawa t et al. (2004) the spectrum of pathological involvement of the striatonigral and olivopontocerebellar systems in multiple system atrophy: clinicopathological correlations, brain 127:2657-71. [149] papp mi, lantos pl (1994) the distribution of oligodendroglial inclusions in multiple system atrophy and its relevance to clinical symptomatology, brain 117:235-43. [150] inoue m et al. (1997) the distribution and dynamic density of oligodendroglial cytoplasmic inclusions (gcis) in multiple system atrophy: a correlation between the density of gcis and the degree of involvement of striatonigral and olivopontocerebellar systems, acta neuropathol 93:585-91. [151] armstrong ra et al. (2004) a quantitative study of the pathological changes in ten patients with multiple system atrophy (msa), j neural transm (vienna) 111:485-95. [152] tong j et al. (2015) low levels of astroglial markers in parkinson's disease: relationship to alpha-synuclein accumulation, neurobiol dis 82:243-53. [153] ishizawa k et al. (2008) glial cytoplasmic inclusions and tissue injury in multiple system atrophy: a quantitative study in white matter (olivopontocerebellar system) and gray matter (nigrostriatal system), neuropathology 28:249-57. [154] wenning gk et al. (2002) a novel grading scale for striatonigral degeneration (multiple system atrophy), j neural transm (vienna) 109:307-20. [155] jellinger ka et al. (2005) grading of neuropathology in multiple system atrophy: proposal for a novel scale, mov disord 20 suppl 12:s29-36. [156] matsusue e et al. (2008) putaminal lesion in multiple system atrophy: postmortem mr-pathological correlations, neuroradiology 50:559-67. [157] brettschneider j et al. (2018) converging patterns of alpha-synuclein pathology in multiple system atrophy, j neuropathol exp neurol 77:1005-16. [158] dickson dw (2012) parkinson's disease and parkinsonism: neuropathology, cold spring harb perspect med 2: a009258. [159] salvesen l et al. (2015) changes in total cell numbers of the basal ganglia in patients with multiple system atrophy a stereological study, neurobiol dis 74:104-13. [160] salvesen l et al. (2017) neocortical neuronal loss in patients with multiple system atrophy: a stereological study, cereb cortex 27:400-10. [161] brenneis c et al. (2007) progression of brain atrophy in multiple system atrophy. a longitudinal vbm study, j neurol 254:191-6. [162] fiorenzato e et al. (2017) brain structural profile of multiple system atrophy patients with cognitive impairment, j neural transm (vienna) 124:293-302. [163] kovacs t et al. (2003) olfactory bulb in multiple system atrophy, mov disord 18:938-42. [164] koga s, dickson dw (2018) recent advances in neuropathology, biomarkers and therapeutic approach of multiple system atrophy, j neurol neurosurg psychiatry 89:175-84. [165] koga s et al. (2018) corticobasal degeneration with tdp-43 pathology presenting with progressive supranuclear palsy syndrome: a distinct clinicopathologic subtype, acta neuropathol 136:389-404. [166] koga s et al. (2018) tdp-43 pathology in multiple system atrophy: colocalization of tdp-43 and alpha-synuclein in glial cytoplasmic inclusions, neuropathol appl neurobiol 44:707-21. [167] robinson jl et al. (2018) neurodegenerative disease concomitant proteinopathies are prevalent, age-related and apoe4-associated, brain 141:2181-93. [168] matsusue e et al. (2009) cerebellar lesions in multiple system atrophy: postmortem mr imaging-pathologic correlations, ajnr am j neuroradiol 30:1725-30. [169] brettschneider j et al. (2017) progression of alpha-synuclein pathology in multiple system atrophy of the cerebellar type, neuropathol appl neurobiol 43:315-29. [170] bettencourt c et al. (2020) white matter dna methylation profiling reveals deregulation of hip1, lman2, mobp, and other loci in multiple system atrophy, acta neuropathol 139:135-56. [171] nykjaer c et al. (2017) changes in the cell population in brain white matter in multiple system atrophy, mov disord online 2017 apr 10: doi: 10.1002/mds.26979. [172] indelicato e et al. (2015) cerebral autoregulation and white matter lesions in parkinson's disease and multiple system atrophy, parkinsonism relat disord 21:1393-7. [173] song yj et al. (2009) degeneration in different parkinsonian syndromes relates to astrocyte type and astrocyte protein expression, j neuropathol exp neurol 68:1073-83. [174] tong j et al. (2010) brain alpha-synuclein accumulation in multiple system atrophy, parkinson's disease and progressive supranuclear palsy: a comparative investigation, brain 133:172-88. [175] tong j et al. (2017) brain monoamine oxidase b and a in human parkinsonian dopamine deficiency disorders, brain 140:2460–74. [176] gerhard a et al. (2006) in vivo imaging of microglial activation with [11c](r)-pk11195 pet in idiopathic parkinson's disease, neurobiol dis 21:404-12. [177] ishizawa k et al. (2004) microglial activation parallels system degeneration in multiple system atrophy, j neuropathol exp neurol 63:43-52. [178] fellner l, stefanova n (2013) the role of glia in alpha-synucleinopathies, mol neurobiol 47:575-86. [179] stefanova n et al. (2011) toll-like receptor 4 promotes alpha-synuclein clearance and survival of nigral dopaminergic neurons, am j pathol 179:954-63. [180] ren s et al. (2019) altered functional connectivity of cerebello-cortical circuit in multiple system atrophy (cerebellar-type), front neurosci 12:996. [181] shah a et al. (2019) altered structural connectivity of the motor subnetwork in multiple system atrophy with cerebellar features, eur radiol 29:2783-91. [182] carré g et al. (2020) brain mri of multiple system atrophy of cerebellar type: a prospective study with implications for diagnosis criteria, j neurol 267:1269-77. [183] coon e et al. (2017) neuropathology of autonomic dysfunction in synucleinopathies, mov disord accepted for publication. [184] iodice v et al. (2012) autopsy confirmed multiple system atrophy cases: mayo experience and role of autonomic function tests, j neurol neurosurg psychiatry 83:453-9. [185] benarroch ee et al. (2006) involvement of vagal autonomic nuclei in multiple system atrophy and lewy body disease, neurology 66:378-83. [186] kuzdas d et al. (2013) oligodendroglial alpha-synucleinopathy and msa-like cardiovascular autonomic failure: experimental evidence, exp neurol 247:531-6. [187] schmeichel am et al. (2008) mesopontine cholinergic neuron involvement in lewy body dementia and multiple system atrophy, neurology 70:368-73. [188] benarroch ee et al. (2008) loss of a5 noradrenergic neurons in multiple system atrophy, acta neuropathol 115:629-34. [189] benarroch ee et al. (2004) involvement of medullary serotonergic groups in multiple system atrophy, ann neurol 55:418-22. [190] benarroch ee et al. (2002) depletion of mesopontine cholinergic and sparing of raphe neurons in multiple system atrophy, neurology 59:944-6. [191] benarroch ee et al. (2007) depletion of putative chemosensitive respiratory neurons in the ventral medullary surface in multiple system atrophy, brain 130:469-75. [192] benarroch ee et al. (2015) putative neuropathological interactions in msa: focus in the rostral ventrolateral medulla, clin auton res 25:77-80. [193] tada m et al. (2009) depletion of medullary serotonergic neurons in patients with multiple system atrophy who succumbed to sudden death, brain 132:1810-9. [194] benarroch ee et al. (2015) histaminergic tuberomammillary neuron loss in multiple system atrophy and dementia with lewy bodies, mov disord 30:1133-9. [195] coon ea et al. (2016) medullary neuronal loss is not associated with alpha-synuclein burden in multiple system atrophy, mov disord 31:1802-9. [196] gray f et al. (1988) quantitative study of lateral horn cells in 15 cases of multiple system atrophy, acta neuropathol 75:513-8. [197] koga s et al. (2017) profile of cognitive impairment and underlying pathology in multiple system atrophy, mov disord 32:405-13. [198] yamamoto t et al. (2005) when is onuf's nucleus involved in multiple system atrophy? a sphincter electromyography study, j neurol neurosurg psychiatry 76:1645-8. [199] treglia g et al. (2011) diagnostic performance of iodine-123-metaiodobenzylguanidine scintigraphy in differential diagnosis between parkinson's disease and multiple-system atrophy: a systematic review and a meta-analysis, clin neurol neurosurg 113:823-9. [200] orimo s et al. (2007) degeneration of cardiac sympathetic nerve can occur in multiple system atrophy, acta neuropathol 113:81-6. [201] sone m et al. (2005) alpha-synuclein-immunoreactive structure formation is enhanced in sympathetic ganglia of patients with multiple system atrophy, acta neuropathol 110:19-26. [202] doppler k et al. (2015) distinctive distribution of phospho-alpha-synuclein in dermal nerves in multiple system atrophy, mov disord 30:1688-92. [203] zange l et al. (2015) phosphorylated alpha-synuclein in skin nerve fibres differentiates parkinson's disease from multiple system atrophy, brain 138:2310-21. [204] nakamura k et al. (2015) filamentous aggregations of phosphorylated alpha-synuclein in schwann cells (schwann cell cytoplasmic inclusions) in multiple system atrophy, acta neuropathol commun 3:29. [205] haga r et al. (2015) clinical utility of skin biopsy in differentiating between parkinson's disease and multiple system atrophy, parkinsons dis 2015:167038. [206] mori f et al. (2002) alpha-synuclein immunoreactivity in normal and neoplastic schwann cells, acta neuropathol 103:145-51. [207] quinn n (1989) multiple system atrophy--the nature of the beast, j neurol neurosurg psychiatry suppl:78-89. [208] iranzo a et al. (2005) characteristics of idiopathic rem sleep behavior disorder and that associated with msa and pd, neurology 65:247-52. [209] palma ja et al. (2015) prevalence of rem sleep behavior disorder in multiple system atrophy: a multicenter study and meta-analysis, clin auton res 25:69-75. [210] jecmenica-lukic m et al. (2012) premotor signs and symptoms of multiple system atrophy, lancet neurol 11:361-8. [211] xie t et al. (2015) comparison of clinical features in pathologically confirmed psp and msa patients followed at a tertiary center, npj parkinson's dis 1:15007. [212] giannini g et al. (2020) progression and prognosis in multiple system atrophy presenting with rem behavior disorder, neurology 94:e1828-e34. [213] postuma rb et al. (2009) quantifying the risk of neurodegenerative disease in idiopathic rem sleep behavior disorder, neurology 72:1296-300. [214] coon ea et al. (2015) clinical features and autonomic testing predict survival in multiple system atrophy, brain 138:3623-31. [215] sakushima k et al. (2015) epidemiology of multiple system atrophy in hokkaido, the northernmost island of japan, cerebellum 14:682-7. [216] lee sw, koh sb (2012) clinical features and disability milestones in multiple system atrophy and progressive supranuclear palsy, j mov disord 5:42-7. [217] petrovic in et al. (2012) multiple system atrophy-parkinsonism with slow progression and prolonged survival: a diagnostic catch, mov disord 27:1186-90. [218] jecmenica-lukic m et al. (2014) clinical outcomes of two main variants of progressive supranuclear palsy and multiple system atrophy: a prospective natural history study, j neurol 261:1575-83. [219] gatto e et al. (2014) pan-american consortium of multiple system atrophy (panmsa). a pan-american multicentre cohort study of multiple system atrophy, j parkinsons dis 4:693-8. [220] low pa et al. (2015) natural history of multiple system atrophy in the usa: a prospective cohort study, lancet neurol 14:710-9. [221] foubert-samier a et al. (2020) disease progression and prognostic factors in multiple system atrophy: a prospective cohort study, neurobiol dis 139:104813. [222] figueroa jj et al. (2014) multiple system atrophy: prognostic indicators of survival, mov disord 29:1151-7. [223] tada m et al. (2007) early development of autonomic dysfunction may predict poor prognosis in patients with multiple system atrophy, arch neurol 64:256-60. [224] glasmacher sa et al. (2017) predictors of survival in progressive supranuclear palsy and multiple system atrophy: a systematic review and meta-analysis, j neurol neurosurg psychiatry 88:402-11. [225] miki y et al. (2019) improving diagnostic accuracy of multiple system atrophy: a clinicopathological study, brain 142:2813-27. [226] jung yj et al. (2020) various motor and non-motor symptoms in early multiple system atrophy, neurodegener dis:1-6. [227] kaindlstorfer c et al. (2013) tremor in multiple system atrophy a review, tremor other hyperkinet mov (n y) 3:pii: tre-03-165-4252-1, doi: 10.7916/d8nv9gz9. [228] köllensperger m et al. (2010) presentation, diagnosis, and management of multiple system atrophy in europe: final analysis of the european multiple system atrophy registry, mov disord 25:2604-12. [229] köllensperger m et al. (2008) red flags for multiple system atrophy, mov disord 23:1093-9. [230] mckay jh, cheshire wp (2018) first symptoms in multiple system atrophy, clin auton res 28:215-21. [231] giannini g et al. (2018) the natural history of idiopathic autonomic failure: the iaf-bo cohort study, neurology 91:e1245-e54. [232] kaufmann h et al. (2017) natural history of pure autonomic failure: a united states prospective cohort, ann neurol 81:287-97. [233] singer w et al. (2017) pure autonomic failure: predictors of conversion to clinical cns involvement, neurology 88:1129-36. [234] eschlböck s et al. (2017) non-motor symptoms and gender differences in multiple system atrophy (abstr.), neurologisch suppl 1:8. [235] panicker jn et al. (2020) early presentation of urinary retention in multiple system atrophy: can the disease begin in the sacral spinal cord?, j neurol 267:659-64. [236] garcia md et al. (2018) ocular features of multiple system atrophy, j clin neurosci 47:234-9. [237] wenning gk et al. (2000) what clinical features are most useful to distinguish definite multiple system atrophy from parkinson's disease?, j neurol neurosurg psychiatry 68:434-40. [238] ghorayeb i et al. (2004) relationship between stridor and sleep apnoea syndrome: is it as simple as that?, j neurol neurosurg psychiatry 75:512-3; author reply 3. [239] ohshima y et al. (2017) natural course and potential prognostic factors for sleep-disordered breathing in multiple system atrophy, sleep med 34:13-7. [240] jellinger ka (2017) potential clinical utility of multiple system atrophy biomarkers, expert rev neurother 17:1189-208. [241] aerts mb et al. (2012) csf alpha-synuclein does not differentiate between parkinsonian disorders, neurobiol aging 33:430 e1-3. [242] mollenhauer b et al. (2011) alpha-synuclein and tau concentrations in cerebrospinal fluid of patients presenting with parkinsonism: a cohort study, lancet neurol 10:230-40. [243] cong s et al. (2020) diagnostic utility of fluid biomarkers in multiple system atrophy: a systematic review and meta-analysis, j neurol. [244] laurens b et al. (2015) fluid biomarkers in multiple system atrophy: a review of the msa biomarker initiative, neurobiol dis 80:29-41. [245] zhang f et al. (2014) candidate biomarkers of multiple system atrophy in cerebrospinal fluid, rev neurosci 25:653-62. [246] compta y et al. (2019) cerebrospinal fluid cytokines in multiple system atrophy: a cross-sectional catalan msa registry study, parkinsonism relat disord 65:3-12. [247] li xy et al. (2020) phosphorylated alpha-synuclein in red blood cells as a potential diagnostic biomarker for multiple system atrophy: a pilot study, parkinsons dis 2020:8740419. [248] magdalinou nk et al. (2017) identification of candidate cerebrospinal fluid biomarkers in parkinsonism using quantitative proteomics, parkinsonism relat disord 37:65-71. [249] orimo s et al. (2012) 123i-mibg myocardial scintigraphy for differentiating parkinson's disease from other neurodegenerative parkinsonism: a systematic review and meta-analysis, parkinsonism relat disord 18:494-500. [250] baschieri f et al. (2017) iodine-123-meta-iodobenzylguanidine myocardial scintigraphy in isolated autonomic failure: potential red flag for future multiple system atrophy, front neurol 8:225. [251] suzuki m et al. (2020) relationship between cardiac parasympathetic dysfunction and the anteroposterior diameter of the medulla oblongata in multiple system atrophy, clin auton res. [252] heim b et al. (2018) structural imaging in atypical parkinsonism, int rev neurobiol 142:67-148. [253] schrag a et al. (2000) differentiation of atypical parkinsonian syndromes with routine mri, neurology 54:697-702. [254] lee jh et al. (2015) progression of subcortical atrophy and iron deposition in multiple system atrophy: a comparison between clinical subtypes, j neurol 262:1876-82. [255] hwang i et al. (2015) differentiation of parkinsonism-predominant multiple system atrophy from idiopathic parkinson disease using 3t susceptibility-weighted mr imaging, focusing on putaminal change and lesion asymmetry, ajnr am j neuroradiol 36:2227-34. [256] ramli n et al. (2015) differentiating multiple-system atrophy from parkinson's disease, clin radiol 70:555-64. [257] sugiyama a et al. (2015) putaminal hypointensity on t2*-weighted mr imaging is the most practically useful sign in diagnosing multiple system atrophy: a preliminary study, j neurol sci 349:174-8. [258] deguchi k et al. (2015) significance of the hot-cross bun sign on t2*-weighted mri for the diagnosis of multiple system atrophy, j neurol 262:1433-9. [259] feng jy et al. (2015) the putaminal abnormalities on 3.0t magnetic resonance imaging: can they separate parkinsonism-predominant multiple system atrophy from parkinson's disease?, acta radiol 56:322-8. [260] krismer f et al. (2019) morphometric mri profiles of multiple system atrophy variants and implications for differential diagnosis, mov disord 34:1041-8. [261] de marzi r et al. (2017) putaminal diffusion imaging for the differential diagnosis of the parkinsoniao variant of multiple system atrophy from parkinson's disease: impact of segmentation accuracy, neurologisch suppl 1:8-9. [262] planetta pj et al. (2015) distinct functional and macrostructural brain changes in parkinson's disease and multiple system atrophy, hum brain mapp 36:1165-79. [263] focke nk et al. (2011) differentiation of typical and atypical parkinson syndromes by quantitative mr imaging, ajnr am j neuroradiol 32:2087-92. [264] cnyrim cd et al. (2014) diffusion tensor imaging in idiopathic parkinson's disease and multisystem atrophy (parkinsonian type), neurodegener dis 13:1-8. [265] bajaj s et al. (2017) diffusion-weighted mri distinguishes parkinson disease from the parkinsonian variant of multiple system atrophy: a systematic review and meta-analysis, plos one 12:e0189897. [266] ito k et al. (2017) differential diagnosis of parkinsonism by a combined use of diffusion kurtosis imaging and quantitative susceptibility mapping, neuroradiology 59:759-69. [267] péran p et al. (2018) mri supervised and unsupervised classification of parkinson's disease and multiple system atrophy, mov disord 33:600-8. [268] zanigni s et al. (2017) white matter and cortical changes in atypical parkinsonisms: a multimodal quantitative mr study, parkinsonism relat disord 39:44-51. [269] lee k et al. (2018) lrrk2 p.ile1371val mutation in a case with neuropathologically confirmed multi-system atrophy, j parkinsons dis 8:93-100. [270] chelban v et al. (2019) an update on advances in magnetic resonance imaging of multiple system atrophy, j neurol 266:1036-45. [271] archer db et al. (2020) magnetic resonance imaging and neurofilament light in the differentiation of parkinsonism, mov disord. [272] yamawaki t (2020) [diagnosis of msa-p and psp-p in early stage], brain nerve 72:331-43. [273] tang cc, eidelberg d (2010) abnormal metabolic brain networks in parkinson's disease from blackboard to bedside, prog brain res 184:161-76. [274] grimaldi s et al. (2019) multiple system atrophy: phenotypic spectrum approach coupled with brain 18-fdg pet, parkinsonism relat disord 67:3-9. [275] brooks dj, seppi k (2009) proposed neuroimaging criteria for the diagnosis of multiple system atrophy, mov disord 24:949-64. [276] nocker m et al. (2012) progression of dopamine transporter decline in patients with the parkinson variant of multiple system atrophy: a voxel-based analysis of [123i]beta-cit spect, eur j nucl med mol imaging 39:1012-20. [277] mckinley j et al. (2014) normal dopamine transporter imaging does not exclude multiple system atrophy, parkinsonism relat disord 20:933-4. [278] perju-dumbrava ld et al. (2012) dopamine transporter imaging in autopsy-confirmed parkinson's disease and multiple system atrophy, mov disord 27:65-71. [279] kraemmer j et al. (2014) correlation of striatal dopamine transporter imaging with post mortem substantia nigra cell counts, mov disord 29:1767-73. [280] levin j et al. (2017) multiple system atrophy. in: falup-pecurariu c, ferreira j, martinez-martin p, chaudhuri kr (eds) movement disorders curricula. springer wien, pp 183-92. [281] perez-soriano a et al. (2017) pbb3 imaging in parkinsonian disorders: evidence for binding to tau and other proteins, mov disord 32:1016-24. [282] cho h et al. (2017) (18) f-av-1451 binds to putamen in multiple system atrophy, mov disord 32:171-3. [283] ono m et al. (2017) distinct binding of pet ligands pbb3 and av-1451 to tau fibril strains in neurodegenerative tauopathies, brain 140:764-80. [284] gerhard a et al. (2003) [11c](r)-pk11195 pet imaging of microglial activation in multiple system atrophy, neurology 61:686-9. [285] matsushima m et al. (2017) validity and reliability of a pilot scale for assessment of multiple system atrophy symptoms, cerebellum ataxias 4:11. [286] koga s et al. (2015) when dlb, pd, and psp masquerade as msa: an autopsy study of 134 patients, neurology 85:404-12. [287] osaki y et al. (2009) a validation exercise on the new consensus criteria for multiple system atrophy, mov disord 24:2272-6. [288] kim hj et al. (2014) should genetic testing for scas be included in the diagnostic workup for msa?, neurology 83:1733-8. [289] stankovic i et al. (2019) a critique of the second consensus criteria for multiple system atrophy, mov disord 34:975-84. [290] watanabe h et al. (2016) expanding concept of clinical conditions and symptoms in multiple system atrophy, rinsho shinkeigaku 56:457-64. [291] berciano j et al. (2002) presynaptic parkinsonism in multiple system atrophy mimicking parkinson's disease: a clinicopathological case study, mov disord 17:812-6. [292] kon t et al. (2013) an autopsy case of preclinical multiple system atrophy (msa-c), neuropathology 33:667-72. [293] wenning gk et al. (1994) clinical features and natural history of multiple system atrophy. an analysis of 100 cases, brain 117:835-45. [294] ling h et al. (2015) minimal change multiple system atrophy: an aggressive variant?, mov disord 30:960-7. [295] wakabayashi k et al. (2005) an autopsy case of early ("minimal change") olivopontocerebellar atrophy (multiple system atrophy-cerebellar), acta neuropathol 110:185-90. [296] fujishiro h et al. (2008) glial cytoplasmic inclusions in neurologically normal elderly: prodromal multiple system atrophy?, acta neuropathol 116:269-75. [297] parkkinen l et al. (2007) abundant glial alpha-synuclein pathology in a case without overt clinical symptoms, clin neuropathol 26:276-83. [298] delledonne a et al. (2008) incidental lewy body disease and preclinical parkinson disease, arch neurol 65:1074-80. [299] batla a et al. (2018) young-onset multiple system atrophy: clinical and pathological features, mov disord 33:1099-107. [300] kim hj, jeon bs (2012) multiple system atrophy with prolonged survival, mov disord 27:1834. [301] calandra-buonaura g et al. (2013) multiple system atrophy with prolonged survival: is late onset of dysautonomia the clue?, neurol sci 34:1875-8. [302] kim hj et al. (2012) young-onset multiple system atrophy, j neurol sci 319:168-70. [303] meissner wg et al. (2016) outcome of deep brain stimulation in slowly progressive multiple system atrophy: a clinico-pathological series and review of the literature, parkinsonism relat disord 24:69-75. [304] masui k et al. (2012) extensive distribution of glial cytoplasmic inclusions in an autopsied case of multiple system atrophy with a prolonged 18-year clinical course, neuropathology 32:69-76. [305] gaig c et al. (2008) pathological description of a non-motor variant of multiple system atrophy, j neurol neurosurg psychiatry 79:1399-400. [306] sousa al et al. (2017) frontotemporal lobar degeneration-tdp with 'multiple system atrophy phenocopy syndrome', neuropathol appl neurobiol online 2017 feb 9: doi 10.1111/nan.12391. [307] aoki n et al. (2015) atypical multiple system atrophy is a new subtype of frontotemporal lobar degeneration: frontotemporal lobar degeneration associated with alpha-synuclein, acta neuropathol 130:93-105. [308] goldman js et al. (2014) multiple system atrophy and amyotrophic lateral sclerosis in a family with hexanucleotide repeat expansions in c9orf72, jama neurol 71:771-4. [309] koga s et al. (2020) clinicopathologic and genetic features of multiple system atrophy with lewy body disease, brain pathol. [310] stankovic i et al. (2014) cognitive impairment in multiple system atrophy: a position statement by the neuropsychology task force of the mds multiple system atrophy (modimsa) study group, mov disord 29:857-67. [311] abrahao a et al. (2011) cognitive impairment in multiple system atrophy: changing concepts, dement neuropsychol 5:303-9. [312] siri c et al. (2013) a cross-sectional multicenter study of cognitive and behavioural features in multiple system atrophy patients of the parkinsonian and cerebellar type, j neural transm (vienna) 120:613-8. [313] brown rg et al. (2010) cognitive impairment in patients with multiple system atrophy and progressive supranuclear palsy, brain 133:2382-93. [314] hatakeyama m et al. (2018) predictors of cognitive impairment in multiple system atrophy, j neurol sci 388:128-32. [315] kawai y et al. (2008) cognitive impairments in multiple system atrophy: msa-c vs msa-p, neurology 70:1390-6. [316] homma t et al. (2016) frequent globular neuronal cytoplasmic inclusions in the medial temporal region as a possible characteristic feature in multiple system atrophy with dementia, neuropathology 36:421-31. [317] gatto e et al. (2014) cognition in a multiple system atrophy series of cases from argentina, arq neuropsiquiatr 72:773-6. [318] santangelo g et al. (2020) evolution of neuropsychological profile in motor subtypes of multiple system atrophy, parkinsonism relat disord 70:67-73. [319] hong hj et al. (2011) cognitive impairments in multiple system atrophy of the cerebellar type, j mov disord 4:41-5. [320] caso f et al. (2020) cognitive impairment and structural brain damage in multiple system atrophy-parkinsonian variant, j neurol 267:87-94. [321] cao b et al. (2015) the global cognition, frontal lobe dysfunction and behavior changes in chinese patients with multiple system atrophy, plos one 10:e0139773. [322] chang cc et al. (2009) cognitive deficits in multiple system atrophy correlate with frontal atrophy and disease duration, eur j neurol 16:1144-50. [323] hara k et al. (2018) corpus callosal involvement is correlated with cognitive impairment in multiple system atrophy, j neurol 265:2079-87. [324] yang h et al. (2020) altered resting-state voxel-level whole-brain functional connectivity in multiple system atrophy patients with cognitive impairment, clin neurophysiol 131:54-62. [325] asi yt et al. (2014) neuropathological features of multiple system atrophy with cognitive impairment, mov disord 29:884-8. [326] miki y et al. (2020) hippocampal a-synuclein pathology correlates with memory impairment in multiple system atrophy, brain. [327] saito m et al. (2017) perirhinal accumulation of neuronal alpha-synuclein in a multiple system atrophy patient with dementia, neuropathology 37:431-40. [328] terni b et al. (2007) mutant ubiquitin and p62 immunoreactivity in cases of combined multiple system atrophy and alzheimer's disease, acta neuropathol 113:403-16. [329] jellinger ka (2020) neuropathological findings in multiple system atrophy with cognitive impairment, j neural transm (vienna). [330] hasegawa m et al. (2017) prion-like mechanisms and potential therapeutic targets in neurodegenerative disorders, pharmacol ther 172:22-33. [331] dehay b et al. (2016) alpha-synuclein propagation: new insights from animal models, mov disord 31:161-8. [332] peelaerts w et al. (2018) a-synuclein strains and seeding in parkinson's disease, incidental lewy body disease, dementia with lewy bodies and multiple system atrophy: similarities and differences, cell tissue res 373:195-212. [333] steiner ja et al. (2018) the concept of alpha-synuclein as a prion-like protein: ten years after, cell tissue res 373:161-73. [334] stopschinski be, diamond mi (2017) the prion model for progression and diversity of neurodegenerative diseases, lancet neurol 16:323-32. [335] valdinocci d et al. (2017) potential modes of intercellular alpha-synuclein transmission, int j mol sci 18:469. [336] wenning g et al. (2018) is multiple system atrophy an infectious disease?, ann neurol 83:10-2. [337] woerman al et al. (2019) multiple system atrophy prions retain strain specificity after serial propagation in two different tg(snca*a53t) mouse lines, acta neuropathol 137:437-54. [338] watts jc et al. (2013) transmission of multiple system atrophy prions to transgenic mice, proc natl acad sci u s a 110:19555-60. [339] bernis me et al. (2015) prion-like propagation of human brain-derived alpha-synuclein in transgenic mice expressing human wild-type alpha-synuclein, acta neuropathol commun 3:75. [340] prusiner sb et al. (2015) evidence for alpha-synuclein prions causing multiple system atrophy in humans with parkinsonism, proc natl acad sci u s a 112:e5308-17. [341] meissner wg et al. (2019) multiple system atrophy: recent developments and future perspectives, mov disord. [342] la vitola p et al. (2019) cellular prion protein neither binds to alpha-synuclein oligomers nor mediates their detrimental effects, brain 142:249-54. [343] gelpi e, colom-cadena m (2019) oligomers: a hot topic for neurodegeneration and a note of caution for experimental models, brain 142:228-30. [344] visanji np et al. (2019) beyond the synucleinopathies: alpha synuclein as a driving force in neurodegenerative comorbidities, transl neurodegener 8:28. [345] bistaffa e et al. (2019) prion efficiently replicates in alpha-synuclein knockout mice, mol neurobiol 56:7448-57. [346] de pablo-fernandez e et al. (2018) no evidence of iatrogenic human transmission in autopsy confirmed multiple system atrophy, mov disord 33:1183-4. [347] coon ea et al. (2019) conjugal multiple system atrophy: chance, shared risk factors, or evidence of transmissibility?, parkinsonism relat disord 67:10-3. [348] rajput ah et al. (2016) conjugal parkinsonism clinical, pathology and genetic study. no evidence of person-to-person transmission, parkinsonism relat disord 31:87-90. [349] jaunmuktane z, brandner s (2019) the role of prion-like mechanisms in neurodegenerative diseases, neuropathol appl neurobiol. [350] maass s et al. (2016) current treatment of multiple system atrophy, curr treat options neurol 18:51. [351] kaindlstorfer c et al. (2019) l-dopa response pattern in a rat model of mild striatonigral degeneration, plos one 14:e0218130. [352] boesch sm et al. (2002) dystonia in multiple system atrophy, j neurol neurosurg psychiatry 72:300-3. [353] flabeau o et al. (2010) multiple system atrophy: current and future approaches to management, ther adv neurol disord 3:249-63. [354] raccagni c et al. (2019) physiotherapy improves motor function in patients with the parkinson variant of multiple system atrophy: a prospective trial, parkinsonism relat disord 67:60-5. [355] levin j et al. (2019) safety and efficacy of epigallocatechin gallate in multiple system atrophy (promesa): a randomised, double-blind, placebo-controlled trial, lancet neurol 18:724-35. [356] lopez-cuina mg et al. (2018) rapamycin for treating msa: a preclinical proof of concept study [abstract], international congress of parkinson disease and movement disorders., mov disord 33 (suppl 2). [357] palma j-a et al. (2019) a futility trial of sirolimus in multiple system atrophy: protocol, recruitment and preliminary adverse event profile (abstr.), neurology 92 (15 suppl): p3.8-019. [358] venezia s et al. (2017) toll-like receptor 4 stimulation with monophosphoryl lipid a ameliorates motor deficits and nigral neurodegeneration triggered by extraneuronal alpha-synucleinopathy, mol neurodegener 12:52. [359] novak p et al. (2012) treatment of multiple system atrophy using intravenous immunoglobulin, bmc neurol 12:131. [360] vidal-martinez g et al. (2020) fty720-mitoxy reduces synucleinopathy and neuroinflammation, restores behavior and mitochondria function, and increases gdnf expression in multiple system atrophy mouse models, exp neurol 325:113120. [361] park kr et al. (2020) prevention of multiple system atrophy using human bone marrow-derived mesenchymal stem cells by reducing polyamine and cholesterol-induced neural damages, stem cell res ther 11:63. [362] singer w et al. (2019) intrathecal administration of autologous mesenchymal stem cells in multiple system atrophy, neurology 93:e77-e87. [363] ndayisaba a, wenning gk (2020) inhibition of the mammalian target or rapamycin (mtor): a potential therapeutic strategy for multiple system atrophy, clin auton res 30:7-8. [364] mandler m et al. (2015) active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy, mol neurodegener 10:10. [365] lemos m et al. (2020) targeting a-synuclein by pd03 affitope® and anle138b rescues neurodegenerative pathology in a transgenic mouse: clinical relevance, transl neurodegener in press. [366] affiris ag (2018) affiris announces encouraging long-term data from a series of first-in-human studies using affitope® pd01a targeting oligomeric alpha-synuclein in early parkinson's disease patients, affiris press release https://affiris.com/investors/#: press release 14 may 2018. [367] valera e et al. (2016) review: novel treatment strategies targeting alpha-synuclein in multiple system atrophy as a model of synucleinopathy, neuropathol appl neurobiol 42:95-106. [368] valera e et al. (2017) combination of alpha-synuclein immunotherapy with anti-inflammatory treatment in a transgenic mouse model of multiple system atrophy, acta neuropathol commun 5:2. [369] schenk db et al. (2017) first-in-human assessment of prx002, an anti-alpha-synuclein monoclonal antibody, in healthy volunteers, mov disord 32:211-8. [370] jellinger ka, wenning gk (2016) overlaps between multiple system atrophy and multiple sclerosis: a novel perspective, mov disord 31:1767-71. [371] ubhi k et al. (2011) multiple system atrophy: a clinical and neuropathological perspective, trends neurosci 34:581-90. [372] halliday gm (2015) re-evaluating the glio-centric view of multiple system atrophy by highlighting the neuronal involvement, brain 138:2116-9. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. friedreich cardiomyopathy is a desminopathy feel free to add comments by clicking these icons on the sidebar free neuropathology 2:34 (2021) original paper friedreich cardiomyopathy is a desminopathy arnulf h. koeppen1,2, rahman f. rafique1, joseph e. mazurkiewicz3, steven pelech4,5, catherine sutter4, qishan lin6, jiang qian7 1 research service, veterans affairs medical center, albany, ny, usa 2 departments of neurology and pathology, albany medical college, albany, ny, usa 3 department of neuroscience and experimental therapeutics, albany medical college, albany, ny, usa 4 kinexus bioinformatics corporation, vancouver, bc, canada 5 department of medicine, university of british columbia, vancouver, bc, canada 6 rna epitranscriptomic & proteomics resource, university at albany, albany, ny, usa 7 department of pathology, albany medical college, albany, ny, usa corresponding author: arnulf h koeppen, md · research service (151) · va medical center · 113 holland ave · albany, ny · 12208, usa arnulf.koeppen@med.va.gov submitted: 11 november 2021 accepted: 08 december 2021 copyedited by: vanessa s. goodwill published: 13 december 2021 https://doi.org/10.17879/freeneuropathology-2021-3679 keywords: friedreich ataxia, cardiomyopathy, proteomics, desmin, αb-crystallin, desminopathy abstract heart disease is an integral part of friedreich ataxia (fa) and the most common cause of death in this autosomal recessive disease. the result of the mutation is lack of frataxin, a small mitochondrial protein. the clinical and pathological phenotypes of fa are complex, involving brain, spinal cord, dorsal root ganglia, sensory nerves, heart, and endocrine pancreas. the hypothesis is that frataxin deficiency causes downstream changes in the proteome of the affected tissues, including the heart. a proteomic analysis of heart proteins in fa cardiomyopathy by antibody microarray, western blots, immunohistochemistry, and double-label laser scanning confocal immunofluorescence microscopy revealed upregulation of desmin and its chaperone protein, αb-crystallin. in normal hearts, these two proteins are co-localized at intercalated discs and z discs. in fa, desmin and αb-crystallin aggregate, causing chaotic modification of intercalated discs, clustering of mitochondria, and destruction of the contractile apparatus of cardiomyocytes. western blots of tissue lysates in fa cardiomyopathy reveal a truncated desmin isoprotein that migrates at a lower molecular weight range than wild type desmin. while desmin and αb-crystallin are not mutated in fa, the accumulation of these proteins in fa hearts allows the conclusion that fa cardiomyopathy is a desminopathy akin to desmin myopathy of skeletal muscle. introduction heart disease in friedreich ataxia (fa), hereafter called fa cardiomyopathy, is the most common cause of death in this otherwise rather neurological disease. fa cardiomyopathy was introduced as an integral part of fa by russell (1946), and there is no doubt that the cardiac lesion is due to frataxin deficiency (perdomini et al., 2014). under the microscope, sections of fa heart show multiple abnormalities, including fiber hypertrophy (koeppen et al., 2015), fiber necrosis, fibrosis, inflammatory infiltration, chaotic disorganization of intercalated discs, and accumulation of iron in a small percentage of cardiomyocytes without iron excess in the whole heart (ramirez et al., 2012; kruger et al., 2016). the reason for the highly variable pathological phenotype of fa as it affects heart, central and peripheral nervous systems, eyes, and endocrine pancreas has not been fully established. the current hypothesis is that structural and signaling proteins are affected downstream from cellular frataxin deficiency. on antibody microarrays and western blots, desmin and its chaperone, αb-crystallin, are upregulated in fa cardiomyopathy. desminopathy is well known from the study of desmin-related skeletal myopathies, in which cardiomyopathy is frequent (dalakas et al., 2000; goldfarb et al., 2004; hnia et al., 2015). desmin mutations cause aggregation in skeletal muscle, and some experiments have suggested that posttranslational modifications of desmin, such as phosphorylation and proteolytic fragmentation, make the protein amyloidogenic (agnetti et al., 2014; kedia et al., 2015). based on the observations detailed in this report, the authors propose that fa cardiomyopathy is a protein aggregation myopathy involving desmin and αb-crystallin. in its biogrid data base of protein, genetic, and chemical interactions (version 4.3.196), uniprot (2021) lists 96 desmin-interacting proteins. among them, αb-crystallin is prominently recognized as a desmin partner or “chaperone”. indeed, αb-crystallin mutations cause “desmin” myopathy (goldfarb and dalakas, 2009). while desmin is an abundant cytoskeletal protein, it also has signaling properties, and the term “mechanochemical” may be applicable (hnia et al., 2015). material and methods autopsy specimens: the institutional review board of the veterans affairs medical center in albany, ny, usa has approved this investigation. heart autopsy specimens were obtained from 35 patients with fa through a national fa tissue repository in albany, ny. thirty-three patients had homozygous guanine-adenine-adenine (gaa) trinucleotide repeat expansions, and two were compound heterozygotes (becker et al., 2017). thirty-four patients had clinical heart disease or fa cardiomyopathy by histology. one fa patient had no heart disease during life or under the microscope. normal hearts were made available by national disease research interchange (philadelphia, pa, usa). at the time of autopsy, a transverse slice weighing approximately 50 g, was made midway between the apex and atrioventricular groove, and immediately frozen at -20°c or, if available, at -80°c. the remainder of the heart was fixed in cold phosphate buffered formalin (ph 7.4). samples of formalin-fixed left ventricular wall (lvw) were embedded in paraffin and sectioned at 6 µm for routine stains, immunohistochemistry, laser scanning confocal congo red fluorescence microscopy, and double-label immunofluorescence with antibodies to desmin and αb-crystallin, or desmin and atp synthase f1 subunit β (atp5b), a mitochondrial marker. antibody microarray: the principles of this proteomic method, advantages, and pitfalls were described previously in detail (yue and pelech, 2018). tissue samples were homogenized by sonication at ice temperatures in a lysis buffer containing (concentrations in parentheses) 3-morpholinopropane-1-sulfonic acid buffer ph 7.2 (20 mm), ethylene glycol-bis (β-aminoethyl ether), n,n,n’,n’-tetra acetic acid (2 mm), ethylenediaminetetraacetic acid (2 mm), sodium fluoride (50 mm), β-glycerophosphate (60 mm), sodium pyrophosphate (25 mm), sodium orthovanadate (5 mm), phenyl arsine phosphate (50 nm), triton x-100 (1%), and sodium dodecylsulfate (0.05%). the lysis buffer also contained a protease inhibitor cocktail of aprotinin (0.5 µm), benzamidine (3 mm), 4(2-aminoethyl) benzene sulfonyl fluoride hydrochloric acid (1 mm), leupeptin (10 µm); and dithiothreitol (1 mm) to disrupt disulfide bonds. protein concentrations were determined by bradford’s method (1976). proteins were cleaved at cysteine residues by the addition of tris (2-carboxyethyl) phosphine hydrochloride (10 mm) to the lysis buffer, followed by 2-nitro-5-thiocyanatobenzoic acid (100 mm) after sonication. pooled lysates were biotinylated (yue et al., 2017) and layered over 2 microarrays displaying, respectively, 900 and 1150 antibodies to a total of 2,050 antibodies (kinexus bioinformatics, vancouver, bc, canada). proteins bound to the antibody spots were detected by incubation with a 1:1 mixture of cy3and alexa 546-labeled anti-biotin. the fluorescent signals from the scanned microarrays were captured in a tif file image. signals were then quantified with imagene 9.0 microarray image analysis software (biodiscovery, el segundo, ca, usa) and normalized according to the sum of all the recorded signals per field. the values for duplicate measurements were averaged, and the mean value for the difference in duplicates from the average was typically ~10%. processing lysates of fa and control hearts in triplicate allowed statistical determination of significant upor down-regulation of proteins in fa by the student t-test. frataxin assay by enzyme-linked immunosorbent assay (elisa): details of frataxin elisa were published before (koeppen et al., 2015), but the amplifying steps with biotinylated anti-rabbit igg and horse radish peroxidase-labeled streptavidin were omitted. the reason for the change was the presence of biotin in non-fat dry milk that was used as a suppressor protein. briefly, heart lysates obtained by the method of condò et al. (2006) were diluted 10-fold in phosphate-buffered saline (ph 7.2) (pbs) to reduce the concentration of nonionic detergents to less than 0.1%. the diluted lysates were centrifuged through membrane filter units with a molecular mass cutoff of 30 kda (millipore sigma, burlington, ma, usa). wells of polystyrene elisa plates were coated with a layer of monoclonal anti-frataxin (abcam, cambridge, ma, usa, cat. no. ab268063), followed by aliquots of filtered lysates (corresponding to 4 mg original tissue) or human recombinant frataxin (10 pg to 1 ng; abcam, cat. no. ab95502). the sequence of subsequent additions (washing steps omitted) was polyclonal anti-frataxin (abcam, cat. no. ab175402) and horseradish peroxidase-labeled goat anti-rabbit gamma globulin (sigma, st. louis, mo, usa, cat. no. a4914). the chromogenic solution contained ortho-phenylenediamine (2 mm) and 0.01% hydrogen peroxide in sodium phosphate-citric acid buffer (ph 5). absorbances at 492 nm were determined in an elisa plate reader manufactured by molecular devices (san josé, ca, usa). frataxin levels were expressed as ng/g wet weight. coimmunoprecipitation: pooled tissue lysate of 6 fa hearts, corresponding to 150 mg wet tissue in a volume of 150 µl, were mixed with 5 µg rabbit polyclonal anti-desmin and 50 µl of a slurry of protein a on sepharose beads (thermo scientific, rockford, il, usa, cat. no. 29333). after overnight incubation at 4 ͦc, the sepharose particles were collected by brief centrifugation and washed with phosphate-buffered saline (pbs). bound proteins were released from protein a-sepharose by laemmli’s sample buffer and heating at 100 ͦc for 15 min. after cooling, the remaining sepharose particles were precipitated by brief centrifugation, and 24 µl aliquots were electrophoresed on 10% polyacrylamide gels. at the end of the electrophoresis, proteins were transferred by electroblotting onto polyvinyledenefluoride (pvdf) membranes. the pvdf membranes were cut into longitudinal strips for the detection of established or putative desmin-associated proteins (frataxin, αb-crystallin; α-actinin, ankyrin 1, α-spectrin, plectin, and zasp [cypher]). sources of antibodies were as follows: anti-desmin (polyclonal, invitrogen, rockford, il, usa. cat no. pa5-16705); anti-αb-crystallin (monoclonal, santa cruz biotechnology, dallas, tx, usa. cat. no. sc-137129); anti-α-actinin (monoclonal, abcam, waltham, ma, usa, cat. no. ab62298); anti-ankyrin 1 (monoclonal, santa cruz biotechnology, cat. no. sc-12719); anti-α-spectrin (monoclonal, santa cruz, cat no. sc-48382); anti-plectin (monoclonal, santa cruz biotechnology, cat. no. 37649); anti-zasp (cypher) (monoclonal, santa cruz biotechnology, cat. no. sc-374359); and anti-frataxin (monoclonal, abcam, branford, ct, usa; cat. no. ab268063). mass spectrometry: protein bands of interest on coomassie blue-stained sds-page gels were manually excised and minced. the pieces were dehydrated with acetonitrile for 10 min, vacuum dried, rehydrated with 5 mm triphosphine hydrochloride in 25 mm ammonium bicarbonate (ph 8.5) at 37 °c for 1 h and then alkylated with 100 mm iodoacetamide in 25 mm ammonium bicarbonate (ph 8.5) at room temperature for 1 h. the pieces were washed twice with 50% acetonitrile in 25 mm ammonium bicarbonate (ph 8.5) for 15 min, dehydrated with acetonitrile for 10 min, dried, and digested in sequencing grade modified trypsin (25 ng/µl; sigma, st. louis, mo, usa, cat. no. t8658) in 25 mm ammonium bicarbonate (ph 8.5) at 37 °c overnight. following digestion, tryptic peptides were extracted three times with 50% acetonitrile containing 5% formic acid for 15 min each time while being vortexed. the extracted solutions were pooled and evaporated to dryness under vacuum. liquid chromatography-tandem mass spectrometry (lc-ms/ms) was performed on an integrated micro-lc-orbitrap velos system (thermo), comprising a waters caplc microscale chromatography system (waters corp., milford, ma) with an auto-sampler, a stream-select module configured for pre-column plus analytical capillary column, and an orbitrap velos mass spectrometer fitted with an h-electrospray ionizer probe operated under xcalibur 2.2 control. injected samples (30 µl) were first trapped and desalted isocratically on a home-packed c18 pre-column cartridge (everest c18, 5 µm particle diameter; column dimensions: internal diameter, 500 µm; length, 15 mm [grace, deerfield, il]) for 6 min with 0.1% formic acid delivered by an auxiliary pump at 40 μl/min. the peptides were then eluted from the pre-column and separated on an ace c18 capillary column (15 cm x 500 μm internal diameter) packed with ace c18-300 particles (5 µm resin, advanced chromatography technologies, aberdeen, scotland). the c18 column was connected in-line with the mass spectrometer and eluted at a flow rate of 20 µl/min with a 40-min gradient of 5-to-80% acetonitrile in 0.1% formic acid. tandem spectrum data was processed using mascot 2.7 (matrix science, boston, ma). the lists of peaks were used to query a human protein database downloaded from national center of biotechnology information by setting the following parameters: peptide mass tolerance, 10 ppm; ms/ms ion mass tolerance, 0.1 da; allowing up to one missed cleavage; considering variable modifications such as methionine oxidation, cysteine carboxyamidomethylation, and deamidation. only significant hits as defined by mascot probability analysis was considered for a positive protein identification. immunohistochemistry: to visualize desmin, its chaperone protein αb-crystallin, and known desmin-linked intermediate filament proteins (α-spectrin; ankyrin-1; plectin; zasp [cypher]; and α-actinin), 6-µm thick paraffin sections were rehydrated by sequential incubation in a series of xylene, absolute ethanol, 95% ethanol, 80% ethanol, and deionized water. endogenous peroxidase activity was suppressed by incubation in methanol containing 3% aqueous hydrogen peroxide. antigen retrieval, background suppression, incubation with polyclonal or monoclonal antibodies, secondary biotinylated anti-rabbit igg (for rabbit polyclonal primary antibodies) or anti-mouse igg (for primary mouse monoclonal antibodies), and chromogenic solutions of diaminobenzidine and hydrogen peroxide were as described (koeppen et al., 2020). double-label fluorescence and immunofluorescence: to examine possible amyloidogenicity of desmin in fa cardiomyopathy, immunofluorescence of desmin and fluorescence of congo red were combined. slide processing for immunofluorescence was as described before (koeppen et al., 2020), and the fluorophore for desmin was alexa fluor 488-labeled goat anti-mouse igg (jackson immunoresearch, west grove, pa, usa, cat. no. 115-545-003). after washing, the sections were incubated in a solution of high ph congo red, as described by the supplier (ihc world, woodstock, md, usa), washed in water, covered by pbs containing 50% glycerol (by vol), and cover-slipped. excitation by laser light at 488 nm was suitable for the fluorescence of both alexa fluor 488 (desmin) and congo red. images were collected in a zeiss lsm 880 laser scanning confocal microscope (oberkochen, germany) set to a band pass of 507-544 nm for alexa fluor 488 and 560-600 nm for congo red. double-label immunofluorescence of the pairs desmin/αb crystallin and desmin/mitochondrial atp5b followed a published protocol (koeppen et al., 2020). atp5b, the β subunit of atp synthase f1, was detected by a polyclonal antibody (santa cruz biotechnology, dallas, tx, usa, cat. no. sc 33618) and cy3-labeled secondary goat anti-rabbit igg (jackson immunoresearch, cat. no. 111-165-003). results antibody microarray of heart lysates: each dot in the panels of figure 1 represents a unique validated antibody, and the legend describes how fluorescent signals of fa and controls were superimposed and analyzed. thirteen proteins were upregulated to variable degrees on the 900-antibody microarray. when expressed as percent change from control (%cfc), the following proteins stood out: adducin, %cfc 210; αb-crystallin, %cfc 194; insulin receptor substrate, %cfc 324; serine/threonine kinase type 4, %cfc 103; ribosomal protein s kinase alpha 3, %cfc 149. the 1150-antibody microarray of kinexus (yue, et al, 2018) yielded modest upregulation of 23 other proteins that were not further analyzed. the up-regulation of αb-crystallin prompted a more detailed study of this protein and its partner, desmin, by sds-page, western blots, mass spectrometry, and double-label immunofluorescence (figs. 2-8). figure 1. antibody microarray of pooled heart lysates of 9 fa patients (left upper panel) and 9 normal controls (left lower panel). in the smaller panels, pseudocolors represent high fluorescence (red) and lower fluorescence (blue). intermediate fluorescence intensity is represented by green and yellow, in that order. the larger image was created by overlaying the fa and control images (fa on top). all spots of the control image were made blue, and all fa spots were made red. using adobe photoshop technology, the fa images on top were rendered 50% transparent so that the bottom spots are white. if fa intensity was higher than that of the matching control, the spot appears as red. if the composite signal is blue, the signal in fa was lower than that of the control. pooling of lysates was intended to control for between-samples variability, and performance in triplicate allowed statistical analysis by t-testing. the next step in the experiment was sds-page and western blotting of proteins in pooled and individual lysates. frataxin levels: the frataxin level in 12 fa heart lysates (mean ± standard deviation) was 23.3±3.5 ng/g wet weight. in 4 control heart lysates the level was 522.5 ± 35 ng/g wet weight. the difference was significant at p=0.022. western blots: figure 2 shows a western blot of desmin and frataxin in pooled fa heart lysates. the pooled lysates represent equal additions of the first 6 fa cases shown in figure 3. the polyclonal antibody to desmin revealed a low intensity band at 53 kda which represents the canonical protein. the strongest desmin signal occurred from a fragment of approximately 45 kda. mass spectrometric analysis of this fragment (fig. 2) confirmed its derivation from desmin. while pooling of 12 heart lysates was useful in the control of inter-sample variability in fa, the western blot in figure 3 shows that the desmin fragment is shared by all fa samples. the qualitative intensity of the desmin band did not correlate with the length of the gaa trinucleotide repeat expansions in the shorter allele of the frataxin gene (fig. 3). a comparable desmin isoprotein is also present in one normal control (fig. 2, left panel, subject f,62). figure 2. western blot of desmin and frataxin in 6 pooled fa heart lysates and mass spectrometry of a proteolytic desmin fragment. lane 1: prestained proteins of known mass. lane 2: western blot of frataxin (fxn). pooling of heart lysates allows the detection of residual frataxin in fa. lane 3: western blot of desmin. the canonical desmin migrates at 53 kda (des), but a second, strongly reactive desmin band (arrow) is located at approximately 45 kda. lane 4: mass spectrometry of the truncated desmin fragment in lane 3 (arrow). the amino acids in red were identified by liquid chromatography-tandem mass spectrometry. the sequences represent signature peptides of desmin. figure 3. western blots of desmin in 6 normal control and 12 fa heart lysates. all wells received a total protein load of 50 µg. the arrows show the location of canonical desmin at 53 kda, a desmin fragment, and gapdh (as a loading control). the pvdf membranes were cut horizontally to generate two segments of the blot and allow visualization of gapdh. sex and ages are shown for the lanes of control lysates (left); in the right panel, of fa lysates, the entries include sex, age, and gaa trinucleotide repeats. in 8 of 12 fa lysates, the blots show prominent expression of a proteolytic desmin fragments at approximately 45 kda. figure 4. desmin co-immunoprecipitation. desmin in pooled lysates of 6 fa hearts was precipitated by anti-desmin, and the protein-antibody complex was recovered on protein a sepharose. desmin and other proteins were released from protein a by heating in laemmli’s sample buffer containing β-mercaptoethanol. aliquots were electrophoresed by sds-page, electroblotted onto pvdf membranes, and visualized by immunochemistry with antibodies to the stated proteins. the western blot shows desmin bands at approximately 53 kda and between 40 and 45 kda, respectively. a distinct band of αb-crystallin is also present. other lighter bands at 45 kda, 25 kda and 15 kda are considered preparative artifacts. immunohistochemistry: in normal hearts, antibodies to desmin and αb-crystallin revealed reaction product in intercalated discs and z-discs. anti-αb-crystallin also showed m bands on longitudinal sections of heart fibers that were desmin negative (fig. 5f). in fa, desmin and αb-crystallin reaction product showed abnormally wide, thickened, and hyperconvoluted intercalated discs and aggregates in the sarcoplasm (fig. 5). desmin and αb-crystallin reaction product in the two compound heterozygous fa cases showed a similar degree of protein aggregate myopathy. reaction product for β-spectrin, ankyrin, plectin, and zasp (cypher) was less informative, though α-spectrin immunohistochemistry displayed the chaotic disorganization of intercalated discs. α-actinin reaction product showed the striation of cardiomyocytes by reacting with z-discs but was not useful to assess the state of intercalated discs. figure 5. immunohistochemistry of desmin and αb-crystallin in left ventricular wall. a-b, desmin; c-f, αb-crystallin. a and c, normal left ventricular wall; b, d-f, fa. in normal myocardium, desmin (a) and αb-crystallin (c) are localized in intercalated and z discs. in fa, desmin reaction product reveals accumulation in or near disorganized intercalated discs (b, arrow). αb-crystallin immunohistochemistry shows wide and fragmented intercalated discs (d and e). enlargement of the square in e displays αb-crystallin reaction product in m bands (f, arrow, m). m bands are desmin-negative. bars; a-e, 10 µm; f, 3 µm. fa patients: b, f, 67 gaa 621/766; d-f, f, 69, gaa 560/560. double-label laser scanning confocal immunofluorescence: figure 6 shows the colocalization of desmin and αb-crystallin in z-discs and intercalated discs. in fa, intercalated discs are wider at approximately 50 µm than normal (approximately 23 µm) and have undergone chaotic disorganization (fig. 6d). a more advanced fiber destruction is illustrated in figure 7. two adjacent fibers display accumulation of desmin and αb-crystallin, obliterating the contractile apparatus of the cardiomyocytes. a search for amyloid by polarizing light for apple-green birefringence and congo red fluorescence did not reveal amyloid deposits. figure 8 shows double-label immunofluorescence of desmin and atp5b. all fibers in fa contain clusters of mitochondria, visualized by green fluorescence of atp5b, and one cluster is surrounded by a rim of desmin. in normal cardiomyocytes, mitochondria occur in rows of delicate granules between heart fibrils (fig. 8, top row). figure 6. double-label laser scanning confocal immunofluorescence microscopy of desmin and αb-crystallin. desmin immunofluorescence (a, d) is alexa fluor 488 green, αb-crystallin (b,e) cy3 red. merged images appear in c and f. the top panel (a-c) represents a normal control, the bottom panel (d-f) fa (man, 24, gaa 700/1050). desmin and αb-crystallin fluorescence colocalize at intercalated discs (arrows) and z discs. in fa, an intercalated disc has undergone chaotic modification. bars, 10 µm. figure 7. desmin and αb-crystallin overload in fa cardiomyopathy (same patient as in fig. 6). desmin is shown as alexa fluor 488 green, αb-crystallin as cy3 red, the merged image on the right shows extensive colocalization of these protein and destructive aggregation in two fibers (arrows). bars, 10 μm. figure 8. double-label laser scanning confocal immunofluorescence microscopy of desmin and atp5b. top row, normal control; bottom row, fa (same patient as in fig. 5). desmin is shown as cy3 red, atp5b as alexa fluor 488 green. in the normal control, mitochondria appear as rows of punctate green organelles aligned along fibrils. in fa, green mitochondria appear in clusters (arrows). the upper cluster is surrounded by a desmin deposit. bars, 20 µm. discussion frataxin levels in fa hearts: the detection limit of the described elisa is 10 pg. we followed biochemical tradition by expressing frataxin levels as mass per gram wet weight, which is at variance with the current practice of fa investigators to express frataxin as ng/total protein. the data must be interpreted with some caution because we assumed that the wet weight of normal and fa heart tissues was close to its water content. tissue lysates contained inhibitory substances that interfered with elisa. filtration through membranes with a molecular mass cutoff of 30 kda removed the interference but may have introduced an additional error in the calculation of corresponding wet weight. desmin in fa cardiomyopathy: uniprot (2021) identifies 36 natural variants of human desmin, of which some are pathogenic mutations. none are listed that could account for the deletion of an estimated 80 amino acids that would explain the high signal desmin isoprotein of 45 kda in fa hearts (figs. 2 and 3). clemen et al. (2008) attributed pathogenicity in patients with skeletal muscle desminopathy to the presence of a low-abundance truncated desmin. it is unknown whether this pathological mechanism is analogous to the prominent desmin band on our western blots of fa patients (fig.3). desmin is not mutated in fa, and fa patients have no skeletal myopathy. arbustini et al. (1998) presented western blots of heart and muscle lysates from patients with desmin-related cardiomyopathy that showed two isoforms of desmin of about the same intensity at masses of 55 and 53 kda, respectively. a truncated muscle desmin of the cases of skeletal myopathy reported by clemen et al. (2008) migrated at 54 kda, rather than the 45 kda in the lysates of fa hearts reported here (figs. 2 and 3). we assume that the truncated desmin in fa cardiomyopathy derives from normal desmin by proteolysis (fig. 2). the functional role of desmin-containing intermediate filaments in skeletal and cardiac muscle is complex. authors who published three-dimensional renditions of desmin in skeletal muscle (goldfarb and dalakas, 2009; clemen et al., 2013) and heart muscle (capetanaki et al., 2015) emphasized the localization of the protein at z discs and intercalated discs (heart only), but also near the nuclear and sarcoplasmic membranes, and surrounding mitochondria. the role of αb-crystallin in possible proteolysis and aggregation of desmin in fa cardiomyopathy is uncertain, but αb-crystallin is viewed as essential for the proper assembly of mature desmin filaments (ghosh et al., 2007; sharma et al., 2017). peculiarly, mutations of αb-crystallin cause myopathy with a phenotype that is very similar to desmin myopathy, including desmin accumulation in muscle tissue (goldfarb et al., 2004). autosomal dominant mutations in the desmin gene cause desmin myopathy that may be followed by cardiomyopathy later in the course of the disease. in contrast, the desmin cardiomyopathy reported here is not followed by skeletal myopathy. desmin and mitochondria in fa cardiomyopathy: frataxin is a mitochondrial protein, and fa is a mitochondrial disease. figure 8 illustrates the clustering of mitochondria near desmin aggregates, but it is unknown whether this seeming entrapment impairs mitochondrial function in fa. references agnetti g, halperin vl, kirek ja, chakire k, guo yj, lund l, nicolini f, gherli t, guarnieri c, caldarera cm, tomaselli gf, kass da, van eyk je. desmin modifications associate with amyloid-like oligomers deposition in heart failure. cardiovasc res 2014; 102:24-34. https://doi.org/10.1093/cvr/cvu003 arbustini e, morbini p, grasso m, fasani r, verga l, bellini o, dal bello b, campana c, piccolo g, febo o, opasich c, gabazzi a, ferrans vj. restrictive cardiomyopathy, atrioventricular block and mild subclinical myopathy in patients with desmin-immunoractive material deposits. j am coll cardiol 1998; 3:645-53. https://doi.org/10.1016/s0735-1097(98)00026-6 becker ab, qian j, gelman bb, yang m, bauer p, koeppen ah. heart and nervous system pathology in compound heterozygous friedreich ataxia. j neuropathol exp neurol 2017; 76: 665-75. https://doi.org/10.1093/jnen/nlx047 bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal biochem 1976; 72:348-54. https://doi.org/10.1006/abio.1976.9999 capetanaki y, papathanasiou s, diokmetzidou a, vatswellas g, tsikitis m. desmin related disease: a matter of cell survival failure. curr opin cell biol 2015; 32:113-20. https://doi.org/10.1016/j.ceb.2015.01.004 clemen cs, fischer d, heimann j, eichinger l, müller cr, müller hd, goebel hh, schröder r. how much mutant protein is needed to cause a protein aggregate myopathy in vivo? lessons from an exceptional desminopathy. hum mutat 2008; 30:e490-e499. https://doi.org/10.1002/humu.20941 clemen cs, herrmann h, strelkov sv, schröder r. desminopathies: pathology and mechanisms. acta neuropathol 2013; 125:47-75. https://doi.org/10.1007/s00401-012-1057-6 condò i, ventura n, malisan f, tomassini b, testi re. a pool of extramitochondrial frataxin that promotes cell survival. j biol chem 2006; 281:16750-6. https://doi.org/10.1074/jbc.m511960200 dalakas, mc, park k-y, semino-mora c, lee, hs, sivakumar k, goldfarb lg. desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene. new engl j med 2000; 342:770-80. https://doi.org/10.1056/nejm200003163421104 ghosh jg, houck sa, clark ji. interactive sequences in the stress protein and molecular chaperone human αb crystallin recognize and modulate the assembly of filaments. int j biochem cell biol 2007; 39:1804-15. https://doi.org/10.1016/j.biocel.2007.04.027 goldfarb lg, vicart p, goebel hh, dalakas mc. desmin myopathy. brain 2004; 127(pt 4):723-34. https://doi.org/10.1093/brain/awh033 goldfarb lg, dalakas mc. tragedy in a heartbeat: malfunctioning desmin causes skeletal and cardiac muscle disease. j clin invest 2009; 119:1806-13. https://doi.org/10.1172/jci38027 hnia k, rumspacher c, vermot j, laporte j. desmin in muscle and associated disease: beyond the structural function. cell tiss res 2015; 360:591-608. https://doi.org/10.1007/s00441-014-2016-4 kedia n, arhzaouy k, pittman sk, sun y, batchelor m, weihl cc, bieschke j. desmin forms toxic, seeding-competent amyloid aggregates that persist in muscle fibers. proc natl acad sci usa 2019; 116(34):16835-4. https://doi.org/10.1073/pnas.1908263116 koeppen ah, ramirez rl, becker ab, bjork st, levi s, santambrogio p, parsons pj, kruger pc, yang kx, feustel pj, mazurkiewicz je. the pathogenesis of cardiomyopathy in friedreich ataxia. plos one 2015; 10:e116396. https://doi.org/10.1371/journal.pone.0116396 koeppen ah, qian j, travis am, sossei ab, feustel pj, mazurkiewicz je. microvascular pathology in friedreich cardiomyopathy. histol histopathol 2020; 35:39-46. https://doi.org/10.14670/hh-18-132 kruger pc, yang kx, parsons pj, becker ab, feustel pj, koeppen ah. abundance and significance of iron, zinc, copper, and calcium in the hearts of patients with friedreich ataxia. am j cardiol 2016; 18:127-31. https://doi.org/10.1016/j.amjcard.2016.04.024 perdomini m, belbellaa b, monassier l, reutenauer l, messaddeq n, cartier n, crystal reg, aubourg p, puccio h. prevention, and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of friedreich’s ataxia. nat med 2014; 20:542-7. https://doi.org/10.1038/nm.3510 ramirez rl, qian j, santambrogio p, levi s, koeppen ah. relation of cytosolic iron excess to the cardiomyopathy of friedreich's ataxia. am j cardiol 2012; 110:1820-7. https://doi.org/10.1016/j.amjcard.2012.08.018 russell ds. myocarditis in friedreich’s ataxia. j path bact 1946; 63:739-48. https://doi.org/10.1002/path.1700580414 sharma s, conover gm, elliott jl, der perng m, herrmann h, quinlan ra. αb-crystallin is a sensor for assembly intermediates and for the subunit topology of desmin intermediate filaments. cell stress chaperones 2017; 22:613-26. https://doi.org/10.1007/s12192-017-0788-7 uniprot: the universal protein knowledge base. nucleic acids res 2021; 49(d1):d480-d489. https://doi.org/10.1093/nar/gkaa1100 uniprot. biogrid biological general repository for interaction datasets 2021. https://doi.org/10.25504/fairsharing.9d5f5r yue l, sam c, arora n, winkler dfh, pelech s. antibody microarray and immunoblotting analyses of the egf signaling phosphorylation network in human a431 epidermoid carcinoma cells. clin proteom bioinformat 2017; 2:1-10. https://doi.org/10.15761/cpb.1000119 yue l, pelech s. application of high content antibody microarrays for biomarker discovery and tracking cellular signaling. adv proteom bioinformat 2018:1-24. https://doi.org/10.29011/apbi-107.100007 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuroinflammation: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:3 (2022) review neuroinflammation: 2022 update hans lassmann1 1 center for brain research, medical university of vienna, austria address for correspondence: hans lassmann · center for brain research · medical university of vienna · spitalgasse 4 · 1090 wien · austria hans.lassmann@meduniwien.ac.at submitted: 20 january 2022 accepted: 08 february 2022 copyedited by: shino magaki published: 10 february 2022 https://doi.org/10.17879/freeneuropathology-2022-3790 keywords: covid-19, multiple sclerosis, alzheimer’s disease, microglia, neurodegeneration abstract besides important progress in the understanding of the pathological substrate of covid-19-associated brain disease, major insights into mechanisms of neurodegeneration in human disease have been provided in neuropathological studies published in 2021. recently developed techniques, which allow the simultaneous detection of a large battery of different molecules within single cells, have proven useful in the analysis of disease mechanisms in experimental and human neuroinflammatory conditions. they have elucidated protective and detrimental effects of activated microglia, which act in a stage and context-dependent manner in the induction and propagation of neurodegeneration. in addition, they emphasize the importance of synaptic damage and of selective neuronal vulnerability in the respective diseases. the results provide important new insights with high clinical relevance. introduction many manuscripts published in 2021 focused on neuropathological aspects of brain diseases, and it is thus difficult to select studies for a review article providing an update on their immunopathology. the manuscripts were identified by the frequent screening of the journals of neuropathology, neuroimmunology and clinical neurology, supplemented by the search for articles in pubmed, dealing with immunopathology of human diseases. obviously, all aspects related to the covid-19 pandemic raised major attention, and key elements of this work deserve to be presented here. in addition, i have focused this year mainly on aspects of neurodegeneration, which not only describe a specific feature in a single disease, but have relevance for a broad spectrum of different disorders. covid-19 and the brain not quite unexpected, a major focus in neuropathological research in 2021 focused on the changes in the central nervous system (cns) present in patients with covid-19. insight into the nature of the respective damage in the nervous system is of critical importance to develop strategies to prevent neurological disease complications. in addition, it may provide a new understanding of the pathogenetic substrate of the long covid syndrome. consensus was reached during 2021 that two different pathologies dominate in patients who died in the course of covid-19: vascular hypoxic/ischemic brain lesions and global microglia activation (matschke et al. 2020, maiese et al. 2021, pajo et al. 2021, al-sarraj et al. 2021). vascular pathology included endotheliitis, associated with a variable degree of focal ischemic or hemorrhagic lesions and diffuse or focal hypoxic alterations of neurons and glia (thakur et al. 2021, lee et al. 2021, fabbri et al. 2021). microglia activation was most prominent in the brain stem and the hippocampus. it consisted of a diffuse increase of microglia density with cellular activation and the appearance of microglia nodules (thakur et al. 2021, maiese et al. 2021). overall, neither the vascular changes nor the patterns of microglia activation are considered specific for sars-cov-2 infection, but can also be found in other virus infections of the cns, in conditions of systemic immune activation or sepsis and in patients dying in intensive care units (thakur et al. 2021, al-sarraj et al. 2021). thus, systemic cytokine storm in the course of the disease together with episodes of general hypoxia or metabolic derangement are likely to be at least in part responsible for the respective damage in the nervous system. since the mean age of the patients in the larger autopsy series was beyond seventy years, it was not a surprise that about half of them exhibited pathologies related to age-related neurodegenerative diseases. all these pathological changes are also in line with the overall clinical appearance of neurological complications of covid-19 (thakur et al. 2021). the prevalence and potential role of direct sars-cov-2 infection in the cns is still controversial (al-sarraj et al. 2021). in the majority of cases no virus rna or antigen in the cns has been detected, and when it was present, the virus titers were low even in cases with persistent active infection in the respiratory organs (maiese et al. 2021, thakur et al. 2021, fabbri et al. 2021). this, however, does not exclude a transient endothelial infection, which triggers endotheliitis and vascular occlusion at early lesion stages (kirschenbaum et al. 2021, nalugo et al. 2021). another potential route of virus invasion of the brain may occur through the olfactory system. however, a recent very systematic analysis of experimental and clinical data on this topic came to the conclusion that brain infection via olfactory nerves and a trans-neuronal spreading of infection through the olfactory system is unlikely (butowt et al. 2021). yet, neuronal spread of the virus by retrograde axonal transport in sensory nerves from the infected mucosa is still an option in some cases. importantly, this study also pinpoints to the limits of experimental research in this topic, since those models, which mimic human disease best, are induced in animal species, which are only poorly suitable for currently available immunological and molecular research tools (butowt et al. 2021). an important question is whether structural brain damage in the disease course of covid-19 could in part explain long-term sequelae of the disease (long covid; baig 2021). ischemic changes and microglial activation were seen in 40% and 80% of brains, respectively. however, only 17% of these patients had neurological manifestations that were severe enough to involve a neurological consultant during life (thakur et al. 2021). this suggests that brain damage in the course of covid-19 is more frequent and widespread than reflected in clinically overt neurological disease. this is in line with the results of a very large clinical study on a cohort of more than 10,000 covid-19 patients from the philippines, which describes a high prevalence of mild neurological deficits (espiritu et al. 2021). clinically, mild cognitive impairment, encephalopathy and cerebrovascular disease were the most common manifestations. the high incidence of microglia activation in the hippocampus and brain stem may be related to neuropsychiatric symptoms in covid-19 survivors, including memory disturbances, somnolence and fatigue (thakur et al. 2021). the immunopathology of the covid-19 brain the studies discussed above describe the basic neuropathology of the central nervous system of patients with covid-19, but they have not addressed the phenotype of the immune reaction within the brain, and its relation to a possible infection with sars-cov-2. this gap has recently been filled by a broad and systematic study, which was performed on a large sample of brain autopsies from covid-19 patients, in comparison to patients who died in intensive care units under conditions of respiratory distress or to patients with multiple sclerosis (schwabenland et al. 2021). the study used imaging mass spectrometry, which allows simultaneous detection of multiple antigens, their spatial distribution in relation to lesion architecture and their vicinity to other immune or resident cells within the tissue. searching for sars-cov-2 antigen within the brain tissue, the study reached similar conclusions, as described above, by showing only sparse virus antigen in the central nervous system. when present, it was mainly seen in endothelial cells of cerebral vessels. displaying leukocyte-related molecules revealed a remarkable inflammatory reaction in the brain. it was characterized by cell clusters, representing different stages of microglia activation and perivascular myeloid cells recruited from the circulation. most profound pro-inflammatory microglia activation was seen in the microglia nodules. microglia and myeloid cell activation was associated with infiltration of the tissue by t-lymphocytes with a dominance of cd8+ cells, but a very low number of b-cells and plasma cells. in comparison to other diseases, neuroinflammation was more pronounced than in patients who died in intensive care units under respiratory distress only, but the inflammatory reaction in patients with multiple sclerosis was much more severe. considering the immensely rich spectrum of immunological details described in this study, a number of conclusions have been reached. there is apparent evidence for a sars-cov-2 infection in the vessel walls, in particular affecting endothelial cells, and this is associated with a perivascular inflammatory reaction with blood brain barrier damage, mainly composed of cd8+ t-lymphocytes and recruited myeloid cells. however, in contrast to other previous studies thrombotic changes in inflamed vessels were absent. the inflammatory process concurred with global microglia activation and the formation of microglia nodules. the latter were the sites for most intense microglia activation and within the nodules cd8+ t-cells closely interacted with activated microglia, possibly providing pro-inflammatory as well as immunoregulatory signals. whether such microglia nodules are a secondary phenomenon of the profound inflammatory reaction in the tissue or drive the inflammation through (virus) antigen recognition remains unresolved. analysis of virus antigen expression did not provide evidence for virus entry into the brain through retrograde axonal transport from mucosal sites or through olfactory nerves. despite the ground-breaking nature of this study, there are still a number of limitations. the major open question relates to the mechanisms of microglia activation. although the authors did not find positive evidence, a potential role of direct sars-cov-2 infection in the brain tissue is not completely excluded. to finally prove or disprove a neuronal transport of virus into the brain and its direct role in microglia activation and neurodegeneration, the analysis of much earlier stages in the infectious process, disease stages which are rarely encountered in human autopsies, may be required. similarly, a final view on similarities and differences of the inflammatory process between covid-19 and multiple sclerosis requires the analysis of much larger numbers of samples from different disease and lesion stages. finally, there are still some technical issues which have to be resolved in the future. the technology used here depicts phenotypic cell clusters, but whether these separations are functionally meaningful has yet to be shown. furthermore, when the expression profiles of the different markers in imaging mass spectrometry, as shown for instance in the supplementary figure 3b in this study, are compared with the experience obtained during the last decades with conventional immunocytochemistry, there are some obvious discrepancies, which raise some questions regarding specificity controls for each individual marker used in the imaging mass spectrometry panel. selective vulnerability of neuron subpopulations in inflammatory or demyelinating conditions acute and partially reversible neurological deficit is a common feature of inflammatory brain diseases. this can be mediated through disturbance of electrical conduction or through demyelination or synaptic loss, and can be compensated by remyelination or synaptic plasticity. in contrast, neuronal damage and loss is the prime substrate of permanent and irreversible neurological deficit. an interesting but controversial issue is whether there is a selective vulnerability of certain neuronal subpopulations, and when this is the case, what its mechanisms and consequences are. as an example, in cortical lesions in multiple sclerosis (ms) several groups have provided evidence that the gabaergic interneurons are prime targets for neurodegeneration (dutta et al. 2006, clements et al. 2008), while in another study a predominant degeneration of cux2 positive excitatory neurons was observed and gabaergic interneurons were unaffected (schirmer et al. 2019). these contradicting data need to be resolved. two large and very well-performed studies have re-addressed the question of selective vulnerability of cortical neurons in inflammatory demyelinating disease in 2021, and both came to the conclusion that the specific subpopulation of gabaergic parvalbumin-containing inhibitory interneurons is lost preferentially in the ms cortex (magliozzi et al. 2021, zoupi et al. 2021). this is in agreement with previous human and experimental studies, which also showed a preferential loss of parvalbumin positive interneurons in autoimmune encephalomyelitis and in toxic demyelination (falco et al. 2014, lapato et al. 2017). importantly, the loss of these neurons was associated with a significant and selective reduction of gabaergic presynaptic terminals in the cortex (zoupi et al. 2021). in an attempt to clarify the underlying mechanisms, the authors also analyzed an experimental rodent model with focal primary cortical demyelination, induced by a chronic application of a lysolecithin-containing hydrogel. the authors conclude that demyelination triggers the loss of these particular neurons, and this may be related to their particular features of axonal myelination (zoupi et al. 2021). in the other study (magliozzi et al. 2021) it was shown that the extent of loss of parvalbumin-containing neurons correlated with the extent of meningeal inflammation. since meningeal inflammation also determines the extent of cortical demyelination in ms lesions, demyelination may be the driving force of neurodegeneration also in this study. the observation that parvalbumin (pv) containing interneurons are preferentially destroyed in inflammatory and/or demyelinating diseases has clinical implications. parvalbumin is liberated during cell death and reaches the cerebrospinal fluid, where it may serve as a paraclinical marker for active neurodegeneration (magliozzi et al. 2021). furthermore, loss of gabaergic interneurons in the course of inflammatory demyelination results in an altered excitatory/inhibitory balance, clinically manifested by epileptic seizures (potter et al. 2016, lapato et al. 2017). thus, the selective vulnerability of inhibitory interneurons may be one of the explanations for the occurrence of epileptic seizures in multiple sclerosis and other inflammatory brain diseases (koch et al. 2008, nicholas et al. 2016, langenbruch et al. 2019). loss of gabaergic interneurons involves complement and results in specific brain region dependent functional consequences a similar selective loss of gabaergic interneurons was observed in the hippocampus of multiple sclerosis patients, mainly affecting the ca2 subfield. in line with the results discussed above this was also associated with an even more prominent loss of gabaergic synaptic terminals. since excitatory glutamatergic terminals were unaffected, a dysbalance between excitatory and inhibitory synaptic input was noted (ramaglia et al. 2021). essentially similar findings were obtained in the demyelinated hippocampus in the rodent cuprizone model (ramaglia et al. 2021). this allowed a detailed analysis of neurobiological mechanisms of neurodegeneration and its functional consequences. synaptic degeneration was associated with profound precipitation of the complement component c1q, which significantly correlated with synaptic pathology and the extent of demyelination, but was not associated with the presence of pathogenic autoantibodies. a key role of complement in synaptic plasticity and degeneration has been observed in a variety of studies before (schartz and tenner 2020). early complement components, such as c1q or c3, are produced in part in neurons and their production is enhanced in conditions of neurodegeneration. these proteins are transported in the axons to the terminals and focally accumulate at sites of axonal or synaptic damage (michailidou et al. 2016). their removal by microglia is a prerequisite for synaptic plasticity and axonal regeneration. in pathological conditions this mechanism is enhanced (michailidou et al. 2016) and the subsequent complement/microglia interaction may result in substantial axonal and synaptic loss, followed by retrograde neuronal degeneration (schartz and tenner 2020). the neurophysiological consequences of the loss of gabaergic interneurons and synapses in the ca2 region was a disturbance of the excitatory/inhibitor balance and a loss of feed-forward inhibition of pyramidal neurons. in concomitant behavioral investigations it turned out that these functional changes were mainly reflected in a disturbance of social memory tasks, which were not accompanied by a general learning impairment or by changes of motor activities (ramaglia et al. 2021). in summary, this study, too, shows a selective vulnerability of gabaergic interneurons in ms and cuprizone lesions and suggests that these changes are a potential consequence of demyelination. however, no loss of pv-containing gabaergic interneurons has been seen in another study in hippocampal ms lesions despite extensive demyelination (kiljan et al. 2019), suggesting that demyelination alone is unlikely to be responsible. a similar preferential loss of parvalbumin reactive neurons has also been seen in a mouse model of autoimmune encephalomyelitis, which is driven by inflammation, but largely lacks primary demyelination in the cortex (falco et al. 2014). furthermore, hypoxic/ischemic damage is associated with a preferential loss of pv-containing gabaergic interneurons (fowke et al. 2018, stolp et al. 2019, vaes et al. 2020, povysheva et al. 2019) and a similar selective vulnerability has also been observed in a mouse model of alzheimer’s disease (petrache et al. 2019) thus, the key question arises whether there are common mechanisms of brain injury shared between these different conditions. all of these conditions are associated with microglia activation, reflected by the expression of nadph oxidase in activated microglia (zrzavy et al. 2017, 2018). the beneficial effect of its pharmacological blockade indicates a role of oxidative injury in the induction of neuronal damage (yuan et al. 2015) by mitochondrial injury and subsequent energy deficiency (mahad et al. 2015). in such a state of “virtual hypoxia” (dutta et al. 2013) additional demyelination amplifies damage due to the increased energy demand of denuded axons. microglia phenotype in relation to alzheimer’s disease (ad) pathology research performed during the last decade brought microglia into a central position in the cascade of events triggering neurodegeneration. in alzheimer’s disease the concept was originally proposed following the observation that activated microglia cumulate in amyloid plaques (mcgeer et al. 1988) and that a variety of different pro-inflammatory cytokines and mediators are expressed in the ad cortex (eikelenboom and stam 1982, mcgeer et al. 1988, dickson and rogers 1992). more recently, systematic genome wide association studies (gwas) identified numerous risk genes for ad susceptibility, and many of these genes code for molecules involved in microglia function (lambert et al. 2013, lewcock et al. 2020). these results encouraged new studies on the phenotype of microglia in alzheimer’s disease using the new opportunities offered by single cell transcriptomics and proteomics (mathys et al. 2019, lewcock et al. 2020). this allowed the identification of a disease-associated microglia phenotype (dam), which was characterized by the loss of homeostatic phenotypic markers and the expression of a large array of different activation markers (figure 1). figure 1: microglia activation in alzheimer’s disease: microglia in the cortex of ad patients are activated within the amyloid plaques and also in surrounding cortical parenchyma. within the plaques (labeled with *) the activation is more advanced showing an ameboid microglia phenotype and a higher expression of markers associated with phagocytosis (cd68), antigen presentation (mhc), oxidative injury (nadph oxidase and iron storage (ferritin, iron)), and proteolysis (tpp2). a key step in this activation is the induction of the trem2/apoe pathway, which occurs in response to programed neuronal cell death (krasemann et al. 2017). a variety of different molecules, identified by gwas in ad patients, are related to this pathway, and loss of function mutations of trem2 are associated with earlier onset and more severe disease. functional studies revealed that loss of trem2 function arrests microglia in a homeostatic state, reduces phagocytosis and inhibits the focal stabilization of aβ deposits (lewcock et al. 2020). all these data support the view that activated microglia within plaques play a neuroprotective role by inhibiting the spread of soluble aβ oligomers. however, microglia in the ad cortex are also activated outside of aβ plaques, where they are also associated with degeneration and loss of neurons and synapses. whether these microglia cells have other activation profiles compared to the plaque-associated microglia has been addressed in a recent systematic study using single nuclei transcriptomics (gerrits et al. 2021). the authors confirmed the phenotype of the plaque-associated microglia. in addition, however, they identified several new phenotypic microglia clusters, which were associated with the severity of cortical tau-pathology. this shows for the first time that the activation patterns of microglia are heterogeneous in the ad cortex in a context-dependent manner. these data indicate that tau-related neurodegeneration is triggered or associated with microglia activation steps, which differ from those responsible for their protective role in plaques. only a minority of the molecules identified in these neurodegeneration-associated clusters are so far functionally characterized, some of them, however, seem to be related to mechanisms of tissue plasticity and repair. thus, the final role of this new neurodegeneration-associated microglia phenotype is currently unresolved. despite the highly innovative approach of recent studies using single cell transcriptomics and proteomics, such studies have major limitations. they identify a large battery of cluster-associated molecules, but only a small fraction of them is functionally defined at the present time. their functional characterization has to be performed in experimental models, but this approach has limitations. although basic activation steps of microglia are similar between different species, there are major differences in the molecular phenotypes of activated microglia between rodents and humans (wimmer et al. 2018), and further differences in their patterns of activation are seen in human disease in comparison to those in ad models (sobue et al. 2021). another problem is related to the annotation of specific molecules to specific cell types. as an example, molecules of the nadph oxidase complex, which are prominently expressed in human microglia and are clearly associated with oxidative injury in a variety of different diseases (fischer et al. 2012), are surprisingly absent from the microglia activation list in recent single cells transcriptomic studies. why this is the case is not clear. it may be that these molecules are not included in the list of microglia genes, since they are prominently expressed in granulocytes and thus annotated as granulocyte markers. the other possibility is that they are not included, since the gene lists have originally been defined in mouse models, and nadph oxidase, in contrast to humans, is not prominently expressed in mouse microglia. protective or detrimental action of microglia in alzheimer’s disease: context or lesion stage dependent? as discussed above, heterogeneous clusters of activated microglia are prominent in the ad cortex and they are associated with deposits of aβ in the plaques (lewcock et al. 2020). whether activated microglia promote or ameliorate disease is unclear and can only partly be addressed in humans. the identification of trem2 loss of function mutations as risk genes in ad suggests that microglia activation is protective by inhibiting aβ spread into the cortical parenchyma. however, experimental models suggest a more complex role of microglia. thus, microglia depletion before the onset of amyloid deposition in ad mouse models reduces plaque formation, possibly by inhibiting synapse degeneration, the liberation of amyloid precursor protein (app) and its cleavage into aβ fragments in the course of synapse degeneration (delizannis et al. 2021). in addition, microglia seem to play a fundamentally different role in neurodegeneration in the ad brain. this was recently observed in transgenic mice expressing familial alzheimer’s disease and human tau mutations (5xfad/ps19 mice), in which tau tangles were precipitated by stereotactic seed injections of synthetic preformed tau fibrils into the hippocampus (lodder et al. 2021). these animals developed severe aβ plaque pathology in addition to profound tau-tangle pathology in the hippocampus and cerebral cortex, and both pathologies were associated with a disease-associated microglia phenotype (dam). microglia in the central nervous system can be depleted by chronic treatment of the animals with plx 3397, a drug which selectively inhibits the colony stimulating factor 1 receptor (csf1r). interestingly, this treatment had a differential effect on plaque or tangle-associated microglia. while the diffuse microglia infiltration, which was associated with tau-pathology, was completely eliminated, the plaque-associated activated microglia was unaffected. this selective elimination of diffuse microglia in the cortical parenchyma resulted in a significant attenuation of the propagation of tau-pathology and neurodegeneration. from all these data one can conclude that microglia in the ad brain exert different functions, depending upon the stage of the cortical lesions and the nature of the damage. the initial stage of aβ plaque formation is propagated, but the spread of mature plaques into the surrounding tissue is ameliorated by activated microglia. when there is a predisposition for tau phosphorylation, aβ deposition propagates tau-tangles, and this process is further enhanced by activated microglia. this heterogenous scenario of microglia function in ad pathogenesis is a challenge for the development of anti-inflammatory treatment strategies. iron and microglia in alzheimer’s disease microglia activation is seen in human ad as well as in mouse ad models and their basic activation pattern is similar, but not identical between these two conditions (krasemann et al. 2017, keren-shaul et al. 2017, mathys et al. 2019). most importantly, most rodent models of ad differ from human disease in the absence or the extent of neurodegeneration. one difference between human and rodent neuropathology is the age-related accumulation of iron in the brain (connor et al. 1992, hametner et al. 2013), which is largely absent in rodents, at least at ages of the animals generally investigated in ad models. iron accumulation in human microglia is increased in neurodegenerative diseases, and it is associated with a pro-inflammatory activation of the cells (mehta et al. 2013, hametner et al. 2013, van duijn et al. 2017), possibly related to its pro-oxidative function. furthermore, the phenotype of dystrophic microglia, present in human ad but largely absent in rodent models, is restricted to iron and ferritin-loaded cells (lopes et al. 2008, hametner et al. 2013; figure 2). figure 2: iron accumulation and microglia senescence: microglia, identified with the panmicroglia marker iba1 in the cortex of ad patients may display a homeostatic phenotype, expressing p2ry12 and showing a normal ramification of their cell processes. another fraction of microglia contains high levels of iron stored together with ferritin. these cells frequently show a dystrophic phenotype with loss and clumping of cell processes. the insert in the iron figure shows double staining for iron (brown) and the microglia/macrophage marker cd68 (blue). an important missing piece of knowledge was a detailed phenotypic and functional characterization of different microglia populations in the ad brain in relation to their iron content. this information is now provided by a recent study, describing the microglia activation profile in the cortex of ad patients using multispectral immunofluorescence (kenkhuis et al. 2021). as described before, the study confirmed the increased microglia infiltration and activation in the ad cortex, the association of activated microglia with aβ-plaques and the ferritin and iron accumulation in the microglia cytoplasm. fine mapping of microglia was performed by cluster analysis. this revealed microglia clusters, which were mainly associated with ad lesions, and these clusters had in common the high ferritin content and the downregulation of various markers of the homeostatic microglia phenotype. the high ferritin content was associated with a phenotypic change of the cells towards dystrophic microglia. in contrast, microglia in the surrounding normal-appearing brain tissue were grouped in clusters with high or variable expression of homeostatic markers and low expression of ferritin. thus, this study identifies disease-associated microglia through their iron and ferritin content. this was also reflected by the association of this microglia phenotype with disease severity, apo-e genotype and the accumulation of aβ plaques and neurofibrillary tangles in the brain. the major limitation of this study is the selection of ad cases, which is dominated by patients at later disease stage (braak stage v to vi). thus, it remains unclear whether iron accumulation and its binding to ferritin is a secondary phenomenon in the formation of aβ plaques. thus, iron uptake in microglia at the expense of microglia dystrophy may reflect removal of free iron from the extracellular space in an attempt to ameliorate oxidative injury. however, iron in microglia may activate microglia in a pro-inflammatory manner, which contributes to oxidative damage in the lesions. the iron content in the brain can be visualized and quantified in magnetic resonance imaging (mri), using specific sequences such as r2* or quantitative susceptibility mapping (qsm) (bulk et al. 2018). using this technology, the authors also showed that the increased iron content in the sections is also correlated with an increased mri signal (kenkhuis et al. 2021). dynamics of chronic microglia-associated neurodegeneration the possibility to detect accumulation of iron-loaded microglia in vivo by magnetic resonance imaging offers a very attractive tool to monitor the dynamics of active lesions and neurodegeneration in human disease. this is exemplified in multiple sclerosis, where the presence of an iron rim around the lesion identifies it as chronic active (bagnato et al. 2011, mehta et al. 2013, hametner et al. 2013; figure 3). furthermore, such lesions show more pronounced tissue damage and neurodegeneration in comparison to those lacking an iron ring, and the presence of such lesions is associated with an aggressive clinical disease course (absinta et al. 2016, weber et al. 2021). pathological data on the distribution of tissue degradation products in such iron lesions suggested that they expand very slowly, but the time course of their dynamic evolution was unknown. this has changed when prospective longitudinal mri studies using iron sensitive sequences were performed, which provide data on dynamic changes in ms lesions over a time period of up to 7 years (dal bianco et al. 2017, 2021). brain lesions without an iron rim shrank during the first 3 years and then their volume stabilized. in contrast, iron ring lesions slowly expanded over 3 to 5 years, before their size stabilized. these data indicate that chronic neurodegeneration in progressive multiple sclerosis is a very slow process, gradually developing over years, while demyelination and neurodegeneration in acute relapses develop within weeks. figure 3: slowly expanding iron ring lesions in the ms brain can be visualized on mri with iron sensitive imaging sequences. the typical presentation of such lesions reveals high iron signal in one or several central veins and in a rim at the lesion edge, reflecting iron-containing activated microglia. the iron rim (r) demarcates the demyelinated plaque (pl); the abnormal mri signal around the iron rim is due to wallerian degeneration (wd). direct neuropathological analysis of mri scanned tissue blocks showed that the iron ring seen in mri always reflects the iron-loaded microglia at the edge of active plaques, while the t2 signal of the lesions gradually expands outside the ring (dal bianco et al. 2017, 2021). these lesion changes reflect an expanding halo of secondary wallerian degeneration around a destructive lesion (figure 3). thus, the quantitative lesion load, determined on mri, not necessarily indicated lesion load, but also wallerian degeneration in the normal appearing white matter. all these data highlight the importance of studies, directly correlating mri changes with neuropathology and integrating this information into long term longitudinal prospective studies. it is expected that a similar approach will also provide significant new information for age-related neurodegenerative diseases. is there evidence for involvement of t-cells in neurodegenerative disease? neuropathological studies have provided evidence that cells of the adaptive immune system infiltrate the brain, in particular at sites of ongoing neurodegeneration in diseases such as alzheimer’s disease or parkinson’s disease (togo et al. 2002, brochard et al. 2009, evans et al. 2019). these cells are mainly t-cells, including mhc class ii restricted cd4+ and mhc class i restricted cd8+ cells. the key question, which is currently unresolved, is whether these cells actively contribute to tissue injury, are neuroprotective or are just passively recruited bystanders. this question has recently been addressed in an experimental model of parkinson’s disease, induced by local injection of an adeno-associated virus vector, inducing the over-expression of α-synuclein in the substantia nigra of mice (williams et al. 2021). in this model cd4+ and cd8+ t-cells infiltrate the midbrain, and this is associated with activation of myeloid cells, including microglia, and the degeneration and loss of tyrosine hydroxylase positive nigral neurons. the t-cell infiltrates are associated with the local expression of pro-inflammatory and in part also regulatory cytokines. most importantly, t-cell receptor or cd4 deficient mice revealed a reduced activation of immune effector cells and also an ameliorated neurodegeneration in the substantia nigra. this study thus provides evidence that adaptive immune reactions, mainly mediated by mhc class ii restricted cd4+ t-cells, contribute to the propagation of neurodegeneration in this model. the results support the view that attempts to therapeutically modulate the t-cell immune response (weiner et al. 2011, mayo et al. 2016) could be effective. direct support is provided by the observation that fingolimod, a drug which reduces the infiltration of immune cells into the central nervous system and is effective in the treatment of multiple sclerosis patients, ameliorates disease in the respective parkinson’s disease model (williams et al. 2021). despite these findings, which suggest a pathogenic role of t-cells in parkinson’s disease and other neurodegenerative diseases, it remains unresolved whether this is mediated by autoimmunity. no proof is provided that the t-cell immune reaction is directed against a specific autoantigen, such as for instance α-synuclein (williams et al. 2021). thus, local overexpression of α-synuclein may attract non-specifically t-cells into the brain. since the normal immune repertoire contains t-cells against a variety of different brain auto-antigens (wekerle et al. 1996), their activation in the lesions may just represent a non-specific consequence of the inflammatory response. also, the mechanism of damage of neurons in the substantia nigra remains unresolved. involvement of microglia and macrophages, which are activated by the t-cell mediated immune response, is likely to amplify neurodegeneration, but innate immunity alone will not specifically and selectively target dopaminergic neurons (lassmann and van horssen 2011). finally, the amelioration of neuronal damage in animals treated with fingolimod does not necessarily prove immune mediated damage, since the modulation of sphingosine 1 phosphate receptors may also have direct neuroprotective effects (colombo and farina 2022). thus, the study by williams et al. (2021) supports a possible involvement of t-cell mediated mechanisms in neurodegeneration in parkinson’s disease, but further research is necessary to clarify the underlying mechanisms. the local inflammatory response in gliomas: good or bad or irrelevant? it is generally accepted that local inflammation is present in the malignant gliomas, but it is largely unresolved whether this is beneficial or detrimental for the patients (jack and lu 2015). overall, there is an immunosuppressive environment within gliomas due to the production of anti-inflammatory cytokines, such as transforming growth factor β (tgf-β) in the tumor cells (binnewies et al. 2018, brown et al. 2018). the extent of tgf-β expression is, however, variable between patients and one expects that lower levels of immunosuppression is associated with inflammation, immune attack of tumor cells and good clinical prognosis in patients. alternatively, high density of inflammatory cells within the tumor may be secondary to more aggressive tissue damage and associated with a less favorable prognosis. in addition, the inflammatory response itself may be dominated by pro-inflammatory activated cytotoxic effector cells or by regulatory t-cells and myeloid-derived suppressor cells, and this, too, may influence tumor progression and functional outcome (kmiecik et al. 2013, 2014, gabriely et al. 2017). several studies have so far analyzed the phenotype of tumor-infiltrating inflammatory cells and its relation to clinical outcome, but the results are variable and in part contradictory (charles et al. 2012, szulzewsky et al. 2015, orrego et al. 2018, martinez et al. 2009, zhang et al. 2019). this is mainly due to the fact that the studies were performed by immunocytochemistry, which does not allow the simultaneous analysis of a large spectrum of different immune cells. this problem was addressed in a recent study by applying flow cytometry of dissociated cells from a large number of biopsy samples from carefully characterized glioblastoma patients (gonzales-tablas pimenta et al. 2021). by using novel state-of-the-art techniques of multi-antibody and multi-color labeling, a simultaneous quantitative evaluation of all different leukocyte populations was possible. as shown before, the study revealed that on average only a quarter of the total cell population were cd45+ immune cells, two thirds of them being tumor-associated macrophages of hematogenous or microglia origin and granulocytes. the lymphocyte population mainly consisted of mhc class i restricted cd8+ and mhc class ii restricted cd4+ t-cells. the infiltration by regulatory t-cells, b-lymphocytes and natural killer cells (nk cells) was very low. immunosuppressive cytokines, such as tgf-β and interleukin 10 (il10) were mainly seen within tumor cells and in low incidence in myeloid derived suppressor cells. the broad spectrum of markers used simultaneously in this study allowed to define three patient clusters with distinct immune infiltration. in the first, immune cell infiltration was sparse and dominated by tumor-associated macrophages. in the second, the tissue infiltration was profound, but consisted mainly of myeloid cells with little contribution of lymphocytes. in the third, the immune infiltration, too, was high containing high amounts of tumor-associated macrophages, granulocytes and t-lymphocytes. the latter patient cohort showed a significantly worse clinical prognosis compared to the other groups. this study represents an important first step for a comprehensive characterization of the local immune response in gliomas in correlation to clinical prognosis and tumor genotype. this approach will also be essential to interpret the effects of new immunotherapies in gliomas. conclusions mechanisms of adaptive and innate immunity appear to be involved in the propagation of neurodegeneration in many different diseases, including inflammatory conditions such as multiple sclerosis or classical neurodegenerative disorders. these mechanisms can now be studied in a holistic approach with modern technologies of single cell transcriptomics and proteomics. microglia plays a central role. depending upon the type and stage of their activation they have beneficial or detrimental roles. the effects are in part reproduced in experimental animal models, but there are major differences in microglia function between humans and rodents. importantly, microglia-associated neuronal damage results in a preferential vulnerability of gabaergic interneurons, which results in an altered excitatory/inhibitory balance and this may in part explain the association of brain inflammation with seizures. references absinta m, sati p, schindler m, leibovitch ec, ohayon j, wu t, meani a, filippi m, jacobson s, cortese ic, reich ds. persistent 7-tesla phase rim predicts poor outcome in new multiple sclerosis patient lesions. j clin invest. 2016;126:2597-2609. https://doi.org/10.1172/jci86198 epub 2016 jun 6. pmid: 27270171; pmcid: pmc4922708. al-sarraj s, troakes c, hanley b, osborn m, richardson mp, hotopf m, bullmore e, everall ip. invited review: the spectrum of neuropathology in covid-19. neuropathol appl neurobiol. 2021;47:3-16. https://doi.org/10.1111/nan.12667 epub 2020 oct 20. pmid: 32935873. bagnato f, hametner s, yao b, van gelderen p, merkle h, cantor fk, lassmann h, duyn jh. tracking iron in multiple sclerosis: a combined imaging and histopathological study at 7 tesla. brain. 2011;134:3602-3615. https://doi.org/10.1093/brain/awr278 pmid: 22171355; pmcid: pmc3235560. baig am. chronic long-covid syndrome: a protracted covid-19 illness with neurological dysfunctions. cns neurosci ther. 2021;27:1433-1436. https://doi.org/10.1111/cns.13737 epub 2021 oct 9. pmid: 34626096; pmcid: pmc8611765. binnewies m, roberts ew, kersten k, chan v, fearon df, merad m, coussens lm, gabrilovich di, ostrand-rosenberg s, hedrick cc, vonderheide rh, pittet mj, jain rk, zou w, howcroft tk, woodhouse ec, weinberg ra, krummel, mf. understanding the tumor immune microenvironment (time) for effective therapy. nat med. 2018;24:541–550. https://doi.org/10.1038/s41591-018-0014-x epub 2018 apr 23. pmid: 29686425; pmcid: pmc5998822 brochard v, combadière b, prigent a, laouar y, perrin a, beray-berthat v, bonduelle o, alvarez-fischer d, callebert j, launay jm, duyckaerts c, flavell ra, hirsch ec, hunot s. infiltration of cd4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of parkinson disease. j clin invest. 2009;119:182-192. https://doi.org/10.1172/jci36470 epub 2008 dec 22. pmid: 19104149; pmcid: pmc2613467. brown nf, carter tj, ottaviani d, mulholland p. harnessing the immune system in glioblastoma. br j cancer. 2018;119:1171–1181. https://doi.org/10.1038/s41416-018-0258-8 pmid: 30393372; pmcid: pmc6251037. bulk m, kenkhuis b, van der graaf lm, goeman jj, natté r, van der weerd l.postmortem t2*weighted mri imaging of cortical iron reflects severity of alzheimer’s disease. j alzheimers dis. 2018;65:1125–1137. https://doi.org/10.3233/jad-180317 pmid: 30103327; pmcid: pmc6218127. butowt r, meunier n, bryche b, von bartheld cs. the olfactory nerve is not a likely route to brain infection in covid-19: a critical review of data from humans and animal models. acta neuropathol. 2021;141:809-822. https://doi.org/10.1007/s00401-021-02314-2 epub 2021 apr 26. pmid: 33903954; pmcid: pmc8075028. charles na, holland ec, gilbertson r, glass r, kettenmann h. the brain tumor microenvironment. glia. 2012;60:502-514. https://doi.org/10.1002/glia.21264 pmid: 22379614. clements rj, mcdonough j, freeman ej. distribution of parvalbumin and calretinin immunoreactive interneurons in motor cortex from multiple sclerosis post-mortem tissue. exp brain res. 2008;187:459-465. https://doi.org/10.1007/s00221-008-1317-9 epub 2008 feb 23. pmid: 18297277. colombo e, farina c. lessons from s1p receptor targeting in multiple sclerosis. pharmacol ther. 2022;230:107971. https://doi.org/10.1016/j.pharmthera.2021.107971 epub 2021 aug 25. pmid: 34450231. connor jr, menzies sl, st. martin sm, mufson ej. a histochemical study of iron, transferrin, and ferritin in alzheimer’s diseased brains. j neurosci res. 1992;31:75–83. https://doi.org/10.1002/jnr.490310111 pmid: 1613823. dal-bianco a, grabner g, kronnerwetter c, weber m, höftberger r, berger t, auff e, leutmezer f, trattnig s, lassmann h, bagnato f, hametner s. slow expansion of multiple sclerosis iron rim lesions: pathology and 7 t magnetic resonance imaging. acta neuropathol. 2017;133:25-42. https://doi.org/10.1007/s00401-016-1636-z epub 2016 oct 27. pmid: 27796537; pmcid: pmc5209400. dal-bianco a, grabner g, kronnerwetter c, weber m, kornek b, kasprian g, berger t, leutmezer f, rommer ps, trattnig s, lassmann h, hametner s. long-term evolution of multiple sclerosis iron rim lesions in 7 t mri. brain. 2021;144:833-847. https://doi.org/10.1093/brain/awaa436 pmid: 33484118. delizannis at, nonneman a, tsering w, de bondt a, van den wyngaert i, zhang b, meymand e, olufemi mf, koivula p, maimaiti s, trojanowski jq, lee vm, brunden kr. effects of microglial depletion and trem2 deficiency on aβ plaque burden and neuritic plaque tau pathology in 5xfad mice. acta neuropathol commun. 2021;9:150. https://doi.org/10.1186/s40478-021-01251-1 pmid: 34503586; pmcid: pmc8428059. dickson dw, rogers j. neuroimmunology of alzheimer's disease: a conference report. neurobiol aging. 1992;13:793-798. https://doi.org/10.1016/0197-4580(92)90104-6 pmid: 1491745. dutta r, chomyk am, chang a, ribaudo mv, deckard sa, doud mk, edberg dd, bai b, li m, baranzini se, fox rj, staugaitis sm, macklin wb, trapp bd. hippocampal demyelination and memory dysfunction are associated with increased levels of the neuronal microrna mir-124 and reduced ampa receptors. ann neurol. 2013;73:637-645. https://doi.org/10.1002/ana.23860 epub 2013 apr 17. pmid: 23595422; pmcid: pmc3679350. dutta r, mcdonough j, yin x, peterson j, chang a, torres t, gudz t, macklin wb, lewis da, fox rj, rudick r, mirnics k, trapp bd. mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. ann neurol. 2006;59:478-489. https://doi.org/10.1002/ana.20736 pmid: 16392116. eikelenboom p, stam fc. immunoglobulins and complement factors in senile plaques. an immunoperoxidase study. acta neuropathol. 1982;57:239-242. https://doi.org/10.1007/bf00685397 pmid: 6812382. espiritu ai, sy mcc, anlacan vmm, jamora rdg; philippine corona study group investigators. covid-19 outcomes of 10,881 patients: retrospective study of neurological symptoms and associated manifestations (philippine corona study). j neural transm (vienna). 2021;128:1687-1703. https://doi.org/10.1007/s00702-021-02400-5 epub 2021 aug 27. pmid: 34448930; pmcid: pmc8391861. evans fl, dittmer m, de la fuente ag, fitzgerald dc. protective and regenerative roles of t cells in central nervous system disorders. front immunol. 2019;10:2171. https://doi.org/10.3389/fimmu.2019.02171 pmid: 31572381; pmcid: pmc6751344. fabbri vp, foschini mp, lazzarotto t, gabrielli l, cenacchi g, gallo c, aspide r, frascaroli g, cortelli p, riefolo m, giannini c, d'errico a. brain ischemic injury in covid-19-infected patients: a series of 10 post-mortem cases. brain pathol. 2021;31:205-210. https://doi.org/10.1111/bpa.12901 epub 2020 nov 2. pmid: 33002281; pmcid: pmc7536900. falco a, pennucci r, brambilla e, de curtis i. reduction in parvalbumin-positive interneurons and inhibitory input in the cortex of mice with experimental autoimmune encephalomyelitis. exp brain res. 2014;232:2439-2449. https://doi.org/10.1007/s00221-014-3944-7 epub 2014 apr 26. pmid: 24770856; pmcid: pmc4055863. fischer mt, sharma r, lim jl, haider l, frischer jm, drexhage j, mahad d, bradl m, van horssen j, lassmann h. nadph oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. brain. 2012;135:886-899. https://doi.org/10.1093/brain/aws012 pmid: 22366799; pmcid: pmc3286337. fowke tm, galinsky r, davidson jo, wassink g, karunasinghe rn, prasad jd, bennet l, gunn aj, dean jm. loss of interneurons and disruption of perineuronal nets in the cerebral cortex following hypoxia-ischaemia in near-term fetal sheep. sci rep. 2018;8:17686. https://doi.org/10.1038/s41598-018-36083-y pmid: 30523273; pmcid: pmc6283845. gabriely g, da cunha ap, rezende rm, kenyon b, madi a, vandeventer t, skillin n, rubino s, garo l, mazzola ma, kolypetri p, lanser aj, moreira t, faria amc, lassmann h, kuchroo v, murugaiyan g, weiner hl. targeting latency-associated peptide promotes antitumor immunity. sci immunol. 2017;2:eaaj1738. https://doi.org/10.1126/sciimmunol.aaj1738 pmid: 28763794; pmcid: pmc5657397. gerrits e, brouwer n, kooistra sm, woodbury me, vermeiren y, lambourne m, mulder j, kummer m, möller t, biber k, dunnen wfad, de deyn pp, eggen bjl, boddeke ewgm. distinct amyloid-β and tau-associated microglia profiles in alzheimer's disease. acta neuropathol. 2021;141:681-696. https://doi.org/10.1007/s00401-021-02263-w epub 2021 feb 20. pmid: 33609158; pmcid: pmc8043951. gonzález-tablas pimenta m, otero á, arandia guzman da, pascual-argente d, ruíz martín l, sousa-casasnovas p, garcía-martin a, roa montes de oca jc, villaseñor-ledezma j, torres carretero l, almeida m, ortiz j, nieto a, orfao a, tabernero md. tumor cell and immune cell profiles in primary human glioblastoma: impact on patient outcome. brain pathol. 2021;31:365-380. https://doi.org/10.1111/bpa.12927 epub 2021 feb 13. pmid: 33314398; pmcid: pmc8018082. hametner s, wimmer i, haider l, pfeifenbring s, brück w, lassmann h. iron and neurodegeneration in the multiple sclerosis brain. ann neurol. 2013;74:848-861. https://doi.org/10.1002/ana.23974 epub 2013 oct 7. pmid: 23868451; pmcid: pmc4223935. jack as, lu j-q. immune cell infiltrates in the central nervous system tumors. austin neurosurg open access. 2015;2:1024. kenkhuis b, somarakis a, de haan l, dzyubachyk o, ijsselsteijn me, de miranda nfcc, lelieveldt bpf, dijkstra j, van roon-mom wmc, höllt t, van der weerd l. iron loading is a prominent feature of activated microglia in alzheimer's disease patients. acta neuropathol commun. 2021;9:27. https://doi.org/10.1186/s40478-021-01126-5 pmid: 33597025; pmcid: pmc7887813. keren-shaul h, spinrad a, weiner a, matcovitch-natan o, dvir-szternfeld r, ulland tk, david e, baruch k, lara-astaiso d, toth b, itzkovitz s, colonna m, schwartz m, amit i. a unique microglia type associated with restricting development of alzheimer's disease. cell. 2017;169:1276-1290.e17. https://doi.org/10.1016/j.cell.2017.05.018 epub 2017 jun 8. pmid: 28602351. kiljan s, prins m, baselmans bm, bol jgjm, schenk gj, van dam am. enhanced gabaergic immunoreactivity in hippocampal neurons and astroglia of multiple sclerosis patients. j neuropathol exp neurol. 2019;78:480-491. https://doi.org/10.1093/jnen/nlz028 pmid: 31100147; pmcid: pmc6524632. kirschenbaum d, imbach ll, rushing ej, frauenknecht kbm, gascho d, ineichen bv, keller e, kohler s, lichtblau m, reimann rr, schreib k, ulrich s, steiger p, aguzzi a, frontzek k. intracerebral endotheliitis and microbleeds are neuropathological features of covid-19. neuropathol appl neurobiol. 2021;47:454-459. https://doi.org/10.1111/nan.12677 epub 2020 dec 14. pmid: 33249605; pmcid: pmc7753688. kmiecik j, poli a, brons nhc, waha a, eide ge, enger pø, zimmer j, chekenya m. elevated cd3+ and cd8+ tumor-infiltrating immune cells correlate with prolonged survival in glioblastoma patients despite integrated immunosuppressive mechanisms in the tumor microenvironment and at the systemic level. j neuroimmunol. 2013;264:71–83. https://doi.org/10.1016/j.jneuroim.2013.08.013 epub 2013 aug 31. pmid: 24045166. kmiecik j, zimmer j, chekenya m. natural killer cells in intracranial neoplasms: presence and therapeutic efficacy against brain tumours. j neurooncol. 2014;116:1–9. https://doi.org/10.1007/s11060-013-1265-5 epub 2013 oct 2. pmid: 24085644; pmcid: pmc3889498. koch m, uyttenboogaart m, polman s, de keyser j. seizures in multiple sclerosis. epilepsia. 2008;49:948-953. https://doi.org/10.1111/j.1528-1167.2008.01565.x epub 2008 mar 11. pmid: 18336559. krasemann s, madore c, cialic r, baufeld c, calcagno n, el fatimy r, beckers l, o'loughlin e, xu y, fanek z, greco dj, smith st, tweet g, humulock z, zrzavy t, conde-sanroman p, gacias m, weng z, chen h, tjon e, mazaheri f, hartmann k, madi a, ulrich jd, glatzel m, worthmann a, heeren j, budnik b, lemere c, ikezu t, heppner fl, litvak v, holtzman dm, lassmann h, weiner hl, ochando j, haass c, butovsky o. the trem2-apoe pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. immunity. 2017;47:566-581.e9. https://doi.org/10.1016/j.immuni.2017.08.008 pmid: 28930663; pmcid: pmc5719893. lambert jc, ibrahim-verbaas ca, harold d, naj ac, sims r, bellenguez c, destafano al, bis jc, beecham gw, grenier-boley b, russo g, thorton-wells ta, jones n, smith av, chouraki v, thomas c, ikram ma, zelenika d, vardarajan bn, kamatani y, lin cf, gerrish a, schmidt h, kunkle b, dunstan ml, ruiz a, bihoreau mt, choi sh, reitz c, pasquier f, cruchaga c, craig d, amin n, berr c, lopez ol, de jager pl, deramecourt v, johnston ja, evans d, lovestone s, letenneur l, morón fj, rubinsztein dc, eiriksdottir g, sleegers k, goate am, fiévet n, huentelman mw, gill m, brown k, kamboh mi, keller l, barberger-gateau p, mcguiness b, larson eb, green r, myers aj, dufouil c, todd s, wallon d, love s, rogaeva e, gallacher j, st george-hyslop p, clarimon j, lleo a, bayer a, tsuang dw, yu l, tsolaki m, bossù p, spalletta g, proitsi p, collinge j, sorbi s, sanchez-garcia f, fox nc, hardy j, deniz naranjo mc, bosco p, clarke r, brayne c, galimberti d, mancuso m, matthews f; european alzheimer's disease initiative (eadi); genetic and environmental risk in alzheimer's disease; alzheimer's disease genetic consortium; cohorts for heart and aging research in genomic epidemiology, moebus s, mecocci p, del zompo m, maier w, hampel h, pilotto a, bullido m, panza f, caffarra p, nacmias b, gilbert jr, mayhaus m, lannefelt l, hakonarson h, pichler s, carrasquillo mm, ingelsson m, beekly d, alvarez v, zou f, valladares o, younkin sg, coto e, hamilton-nelson kl, gu w, razquin c, pastor p, mateo i, owen mj, faber km, jonsson pv, combarros o, o'donovan mc, cantwell lb, soininen h, blacker d, mead s, mosley th jr, bennett da, harris tb, fratiglioni l, holmes c, de bruijn rf, passmore p, montine tj, bettens k, rotter ji, brice a, morgan k, foroud tm, kukull wa, hannequin d, powell jf, nalls ma, ritchie k, lunetta kl, kauwe js, boerwinkle e, riemenschneider m, boada m, hiltuenen m, martin er, schmidt r, rujescu d, wang ls, dartigues jf, mayeux r, tzourio c, hofman a, nöthen mm, graff c, psaty bm, jones l, haines jl, holmans pa, lathrop m, pericak-vance ma, launer lj, farrer la, van duijn cm, van broeckhoven c, moskvina v, seshadri s, williams j, schellenberg gd, amouyel p. meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for alzheimer’s disease. nat genet. 2013;45:1452–1458. https://doi.org/10.1038/ng.2802 epub 2013 oct 27. pmid: 24162737; pmcid: pmc3896259. langenbruch l, krämer j, güler s, möddel g, geβner s, melzer n, elger ce, wiendl h, budde t, meuth sg, kovac s. seizures and epilepsy in multiple sclerosis: epidemiology and prognosis in a large tertiary referral center. j neurol. 2019;266:1789-1795. https://doi.org/10.1007/s00415-019-09332-x epub 2019 may 8. pmid: 31069528. lapato as, szu ji, hasselmann jpc, khalaj aj, binder dk, tiwari-woodruff sk. chronic demyelination-induced seizures. neuroscience. 2017;346:409-422. https://doi.org/10.1016/j.neuroscience.2017.01.035 epub 2017 jan 30. pmid: 28153692; pmcid: pmc5394933. lassmann h, van horssen j. the molecular basis of neurodegeneration in multiple sclerosis. febs lett. 2011;585:3715-3723. https://doi.org/10.1016/j.febslet.2011.08.004 epub 2011 aug 16. pmid: 21854776. lee mh, perl dp, nair g, li w, maric d, murray h, dodd sj, koretsky ap, watts ja, cheung v, masliah e, horkayne-szakaly i, jones r, stram mn, moncur j, hefti m, folkerth rd, nath a. microvascular injury in the brains of patients with covid-19. n engl j med. 2021;384:481-483. https://doi.org/10.1056/nejmc2033369 epub 2020 dec 30. pmid: 33378608; pmcid: pmc7787217. lewcock jw, schlepckow k, di paolo g, tahirovic s, monroe km, haass c. emerging microglia biology defines novel therapeutic approaches for alzheimer's disease. neuron. 2020;108:801-821. https://doi.org/10.1016/j.neuron.2020.09.029 epub 2020 oct 22. pmid: 33096024. lodder c, scheyltjens i, stancu ic, botella lucena p, gutiérrez de ravé m, vanherle s, vanmierlo t, cremers n, vanrusselt h, brône b, hanseeuw b, octave jn, bottelbergs a, movahedi k, dewachter i. csf1r inhibition rescues tau pathology and neurodegeneration in an a/t/n model with combined ad pathologies, while preserving plaque associated microglia. acta neuropathol commun. 2021;9:108. https://doi.org/10.1186/s40478-021-01204-8 pmid: 34103079; pmcid: pmc8188790. lopes ko, sparks dl, streit wj. microglial dystrophy in the aged and alzheimer’s disease brain is associated with ferritin immunoreactivity. glia. 2008;56:1048–1060. https://doi.org/10.1002/glia.20678 pmid: 18442088. magliozzi r, pitteri m, ziccardi s, pisani ai, montibeller l, marastoni d, rossi s, mazziotti v, guandalini m, dapor c, schiavi g, tamanti a, nicholas r, reynolds r, calabrese m. csf parvalbumin levels reflect interneuron loss linked with cortical pathology in multiple sclerosis. ann clin transl neurol. 2021;8:534-547. https://doi.org/10.1002/acn3.51298 epub 2021 jan 23. pmid: 33484486; pmcid: pmc7951111. mahad dh, trapp bd, lassmann h. pathological mechanisms in progressive multiple sclerosis. lancet neurol. 2015;14:183-193. https://doi.org/10.1016/s1474-4422(14)70256-x pmid: 25772897. maiese a, manetti ac, bosetti c, del duca f, la russa r, frati p, di paolo m, turillazzi e, fineschi v. sars-cov-2 and the brain: a review of the current knowledge on neuropathology in covid-19. brain pathol. 2021;31:e13013. https://doi.org/10.1111/bpa.13013 epub 2021 aug 13. pmid: 34390282; pmcid: pmc8420197. martinez fo, helming l, gordon s. alternative activation of macrophages: an immunologic functional perspective. annu rev immunol. 2009;27:451–483. https://doi.org/10.1146/annurev.immunol.021908.132532 pmid: 19105661. mathys h, davila-velderrain j, peng z, gao f, mohammadi s, young jz, menon m, he l, abdurrob f, jiang x, martorell aj, ransohoff rm, hafler bp, bennett da, kellis m, tsai lh. single-cell transcriptomic analysis of alzheimer's disease. nature. 2019;570:332-337. https://doi.org/10.1038/s41586-019-1195-2 epub 2019 may 1. erratum in: nature. 2019;571:e1. pmid: 31042697; pmcid: pmc6865822. matschke j, lütgehetmann m, hagel c, sperhake jp, schröder as, edler c, mushumba h, fitzek a, allweiss l, dandri m, dottermusch m, heinemann a, pfefferle s, schwabenland m, sumner magruder d, bonn s, prinz m, gerloff c, püschel k, krasemann s, aepfelbacher m, glatzel m. neuropathology of patients with covid-19 in germany: a post-mortem case series. lancet neurol. 2020;19:919-929. https://doi.org/10.1016/s1474-4422(20)30308-2 epub 2020 oct 5. pmid: 33031735; pmcid: pmc7535629. mayo l, cunha ap, madi a, beynon v, yang z, alvarez ji, prat a, sobel ra, kobzik l, lassmann h, quintana fj, weiner hl. il-10-dependent tr1 cells attenuate astrocyte activation and ameliorate chronic central nervous system inflammation. brain. 2016;139:1939-1957. https://doi.org/10.1093/brain/aww113 epub 2016 may 31. pmid: 27246324; pmcid: pmc4939696. mcgeer pl, itagaki s, mcgeer eg. expression of the histocompatibility glycoprotein hla-dr in neurological disease. acta neuropathol. 1988;76:550-557. https://doi.org/10.1007/bf00689592 pmid: 2974227. mehta v, pei w, yang g, li s, swamy e, boster a, schmalbrock p, pitt d. iron is a sensitive biomarker for inflammation in multiple sclerosis lesions. plos one. 2013;8:e57573. https://doi.org/10.1371/journal.pone.0057573 epub 2013 mar 14. pmid: 23516409; pmcid: pmc3597727. michailidou i, naessens dm, hametner s, guldenaar w, kooi ej, geurts jj, baas f, lassmann h, ramaglia v. complement c3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: implication for disease pathogenesis. glia. 2017;65:264-277. https://doi.org/10.1002/glia.23090 epub 2016 oct 25. pmid: 27778395; pmcid: pmc5215693. nalugo m, schulte lj, masood mf, zayed ma. microvascular angiopathic consequences of covid-19. front cardiovasc med. 2021;8:636843. https://doi.org/10.3389/fcvm.2021.636843 pmid: 33604359; pmcid: pmc7884319. nicholas r, magliozzi r, campbell g, mahad d, reynolds r. temporal lobe cortical pathology and inhibitory gaba interneuron cell loss are associated with seizures in multiple sclerosis. mult scler. 2016;22:25-35. https://doi.org/10.1177/1352458515579445 epub 2015 apr 28. pmid: 25921040; pmcid: pmc4702245. orrego e, castaneda ca, castillo m, bernabe la, casavilca s, chakravarti a, meng w, garcia-corrochano p, villa-robles mr, zevallos r, mejia o, deza p, belmar-lopez c, ojeda l. distribution of tumor-infiltrating immune cells in glioblastoma. cns oncol. 2018;7:cns21. https://doi.org/10.2217/cns-2017-0037 epub 2018 oct 9. pmid: 30299157; pmcid: pmc6331699. pajo at, espiritu ai, apor adao, jamora rdg. neuropathologic findings of patients with covid-19: a systematic review. neurol sci. 2021;42:1255-1266. https://doi.org/10.1007/s10072-021-05068-7 epub 2021 jan 22. pmid: 33483885; pmcid: pmc7822400. petrache al, rajulawalla a, shi a, wetzel a, saito t, saido tc, harvey k, ali ab. aberrant excitatory-inhibitory synaptic mechanisms in entorhinal cortex microcircuits during the pathogenesis of alzheimer's disease. cereb cortex. 2019;29:1834-1850. https://doi.org/10.1093/cercor/bhz016 pmid: 30766992; pmcid: pmc6418384. potter le, paylor jw, suh js, tenorio g, caliaperumal j, colbourne f, baker g, winship i, kerr bj. altered excitatory-inhibitory balance within somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a mouse model of multiple sclerosis. j neuroinflammation. 2016;13:142. https://doi.org/10.1186/s12974-016-0609-4 pmid: 27282914; pmcid: pmc4901403. povysheva n, nigam a, brisbin ak, johnson jw, barrionuevo g. oxygen-glucose deprivation differentially affects neocortical pyramidal neurons and parvalbumin-positive interneurons. neuroscience. 2019;412:72-82. https://doi.org/10.1016/j.neuroscience.2019.05.042 epub 2019 may 30. pmid: 31152933; pmcid: pmc6818263. ramaglia v, dubey m, malpede ma, petersen n, de vries si, ahmed sm, lee dsw, schenk gj, gold sm, huitinga i, gommerman jl, geurts jjg, kole mhp. complement-associated loss of ca2 inhibitory synapses in the demyelinated hippocampus impairs memory. acta neuropathol. 2021;142:643-667. https://doi.org/10.1007/s00401-021-02338-8 epub 2021 jun 25. pmid: 34170374; pmcid: pmc8423657. schartz nd, tenner aj. the good, the bad, and the opportunities of the complement system in neurodegenerative disease. j neuroinflammation. 2020;17:354. https://doi.org/10.1186/s12974-020-02024-8 pmid: 33239010; pmcid: pmc7690210. schirmer l, velmeshev d, holmqvist s, kaufmann m, werneburg s, jung d, vistnes s, stockley jh, young a, steindel m, tung b, goyal n, bhaduri a, mayer s, engler jb, bayraktar oa, franklin rjm, haeussler m, reynolds r, schafer dp, friese ma, shiow lr, kriegstein ar, rowitch dh. neuronal vulnerability and multilineage diversity in multiple sclerosis. nature. 2019;573:75-82. https://doi.org/10.1038/s41586-019-1404-z epub 2019 jul 17. pmid: 31316211; pmcid: pmc6731122. schwabenland m, salié h, tanevski j, killmer s, lago ms, schlaak ae, mayer l, matschke j, püschel k, fitzek a, ondruschka b, mei he, boettler t, neumann-haefelin c, hofmann m, breithaupt a, genc n, stadelmann c, saez-rodriguez j, bronsert p, knobeloch kp, blank t, thimme r, glatzel m, prinz m, bengsch b. deep spatial profiling of human covid-19 brains reveals neuroinflammation with distinct microanatomical microglia-t-cell interactions. immunity. 2021;54:1594-1610.e11. https://doi.org/10.1016/j.immuni.2021.06.002 epub 2021 jun 9. pmid: 34174183; pmcid: pmc8188302. sobue a, komine o, hara y, endo f, mizoguchi h, watanabe s, murayama s, saito t, saido tc, sahara n, higuchi m, ogi t, yamanaka k. microglial gene signature reveals loss of homeostatic microglia associated with neurodegeneration of alzheimer's disease. acta neuropathol commun. 2021;9:1. https://doi.org/10.1186/s40478-020-01099-x pmid: 33402227; pmcid: pmc7786928. stolp hb, fleiss b, arai y, supramaniam v, vontell r, birtles s, yates ag, baburamani aa, thornton c, rutherford m, edwards ad, gressens p. interneuron development is disrupted in preterm brains with diffuse white matter injury: observations in mouse and human. front physiol. 2019;10:955. https://doi.org/10.3389/fphys.2019.00955 pmid: 31417418; pmcid: pmc6683859. szulzewsky f, pelz a, feng x, synowitz m, markovic d, langmann t, holtman ir, wang x, eggen bj, boddeke hw, hambardzumyan d, wolf sa, kettenmann h.. glioma-associated microglia/macrophages display an expression profile different from m1 and m2 polarization and highly express gpnmb and spp1. plos one. 2015;10:e0116644. https://doi.org/10.1371/journal.pone.0116644 pmid: 25658639; pmcid: pmc4320099. thakur kt, miller eh, glendinning md, al-dalahmah o, banu ma, boehme ak, boubour al, bruce ss, chong am, claassen j, faust pl, hargus g, hickman ra, jambawalikar s, khandji ag, kim cy, klein rs, lignelli-dipple a, lin cc, liu y, miller ml, moonis g, nordvig as, overdevest jb, prust ml, przedborski s, roth wh, soung a, tanji k, teich af, agalliu d, uhlemann ac, goldman je, canoll p. covid-19 neuropathology at columbia university irving medical center/new york presbyterian hospital. brain. 2021;144:2696-2708. https://doi.org/10.1093/brain/awab148 pmid: 33856027; pmcid: pmc8083258. togo t, akiyama h, iseki e, kondo h, ikeda k, kato m, oda t, tsuchiya k, kosaka k. occurrence of t cells in the brain of alzheimer's disease and other neurological diseases. j neuroimmunol. 2002;124:83-92. https://doi.org/10.1016/s0165-5728(01)00496-9 pmid: 11958825. vaes jeg, kosmeijer cm, kaal m, van vliet r, brandt mjv, benders mjnl, nijboer ch. regenerative therapies to restore interneuron disturbances in experimental models of encephalopathy of prematurity. int j mol sci. 2020;22:211. https://doi.org/10.3390/ijms22010211 pmid: 33379239; pmcid: pmc7795049. van duijn s, bulk m, van duinen sg, nabuurs rja, van buchem ma, van der weerd l, natté r. cortical iron reflects severity of alzheimer’s disease. j alzheimers dis. 2017;60:1533–1545. https://doi.org/10.3233/jad-161143 pmid: 29081415; pmcid: pmc5676973. weber ce, krämer j, wittayer m, gregori j, randoll s, weiler f, heldmann s, roβmanith c, platten m, gass a, eisele p. association of iron rim lesions with brain and cervical cord volume in relapsing multiple sclerosis. eur radiol. 2021 sep 22. https://doi.org/10.1007/s00330-021-08233-w epub ahead of print. pmid: 34549326. weiner hl, da cunha ap, quintana f, wu h. oral tolerance. immunol rev. 2011;241:241-259. https://doi.org/10.1111/j.1600-065x.2011.01017.x pmid: 21488901; pmcid: pmc3296283. wekerle h, bradl m, linington c, kääb g, kojima k. the shaping of the brain-specific t lymphocyte repertoire in the thymus. immunol rev. 1996;149:231-243. https://doi.org/10.1111/j.1600-065x.1996.tb00907.x pmid: 9005217. williams gp, schonhoff am, jurkuvenaite a, gallups nj, standaert dg, harms as. cd4 t cells mediate brain inflammation and neurodegeneration in a mouse model of parkinson's disease. brain. 2021;144:2047-2059. https://doi.org/10.1093/brain/awab103 pmid: 33704423; pmcid: pmc8370411. wimmer i, zrzavy t, lassmann h. neuroinflammatory responses in experimental and human stroke lesions. j neuroimmunol. 2018;323:10-18. https://doi.org/10.1016/j.jneuroim.2018.07.003 epub 2018 jul 6. pmid: 30196821. yuan l, wu j, liu j, li g, liang d. intermittent hypoxia-induced parvalbumin-immunoreactive interneurons loss and neurobehavioral impairment is mediated by nadph-oxidase-2. neurochem res. 2015;40:1232-1242. https://doi.org/10.1007/s11064-015-1586-1 epub 2015 apr 25. pmid: 25911467. zhang b, shen r, cheng s, feng l. immune microenvironments differ in immune characteristics and outcome of glioblastoma multiforme. cancer med. 2019;8:2897–2907. https://doi.org/10.1002/cam4.2192 epub 2019 apr 30. pmid: 31038851; pmcid: pmc6558448. zoupi l, booker sa, eigel d, werner c, kind pc, spires-jones tl, newland b, williams ac. selective vulnerability of inhibitory networks in multiple sclerosis. acta neuropathol. 2021;141:415-429. https://doi.org/10.1007/s00401-020-02258-z epub 2021 jan 15. pmid: 33449171; pmcid: pmc7882577. zrzavy t, hametner s, wimmer i, butovsky o, weiner hl, lassmann h. loss of 'homeostatic' microglia and patterns of their activation in active multiple sclerosis. brain. 2017;140:1900-1913. https://doi.org/10.1093/brain/awx113 pmid: 28541408; pmcid: pmc6057548. zrzavy t, machado-santos j, christine s, baumgartner c, weiner hl, butovsky o, lassmann h. dominant role of microglial and macrophage innate immune responses in human ischemic infarcts. brain pathol. 2018;28:791-805. https://doi.org/10.1111/bpa.12583 epub 2017 dec 28. pmid: 29222823; pmcid: pmc6334527. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology through the ages my life between neurology and neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:25 (2020) reflections neuropathology through the ages my life between neurology and neuropathology kurt a. jellinger neurobiology, vienna, austria corresponding author: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13 · a-1150 vienna · austria kurt.jellinger@univie.ac.at submitted: 12 august 2020 accepted: 12 august 2020 copyedited by: katy lawson published: 27 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2945 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, vienna, personal reflections introduction when the editors of free neuropathology, werner and tibor, approached me about writing an autobiography for the journal, similar in scope to sam ludwin’s excellent piece two years ago [1], i confess that i had reservations about writing an autobiography, because i never wanted to talk about myself. however, after a lengthy hesitation and eager discussions with my wife and my former scholar and friend, hans lassmann, i realized with a certain degree of doubt that, on the threshold of age 90 years, i could perhaps provide some thoughts about the bridges between clinical neurology and neuropathology for those who might be interested in the experiences of an old neuroscientist. thus, in lieu of concentrating on my career as neuropathologist, i wish to instead write about my life in the context of unifying these intrinsically linked but disparate areas within the rapidly progressing scientific world: neurology and neuropathology. this long, and not always easy, path through the wide field of practical and theoretical neuroscience, as well as other interests, was influenced by my late mother, my beloved wife elisabeth, colleagues, patients, friends, and students whom i met during my professional and private life. this essay is intended to encourage young colleagues to not concentrate exclusively on experimental neuropathology without also considering the practical part of the neurosciences since there is an urgent need to inspire young neurologists to pursue a double career as both a clinician and neuroscientist in order to promote progress in the neurosciences. why the appeal of clinical neurology and neuropathology? there are three principal reasons to try to unify these two closely connected fields of neuroscience. first and foremost is the aim to help patients suffering from disorders of the nervous system by trying to make an early diagnosis, provide effective treatment, and promote preventive measures. second, is the ongoing need to apply ever-evolving basic scientific approaches to diseases of the nervous system in order to support the clinical neurologist in elucidating the pathogenesis of disorders afflicting their patients. neuropathology should be pursued not just for its own sake but to aid the clinician with the mutual goal to improve diagnoses and enable successful therapeutic strategies for many hitherto incurable disorders. lastly, there is inherent fascination and fulfillment in using our knowledge to elucidate the structure and function of the nervous system in the healthy and diseased human individual. based on this knowledge, the neuropathologist is able to elucidate much of the background of disorders of the nervous system using immunohistochemical, molecular, biological, and ultrastructural methods not only in autopsy material but also tissues from living patients, including biopsies, cerebrospinal fluid, and blood. often, these images are comparable to modern art (fig. 1). the selective and interdisciplinary application of these methods may enable the neuropathologist to not only make a reasonable diagnosis of many, though not all, disorders of the nervous system but may also provide insight into the complex pathogenetic cascades that are responsible for the onset and progression of such disorders. in this respect, the neuropathologist is privileged to act as both a physician and a basic scientist. this retrospective will consider highlights and drawbacks in my 63 years of life between clinical neurology, neuropathology, and private activities. given my many years of work and limited space herein, may my friends, scholars, colleagues, and co-workers excuse that i regrettably could not consider all of them in this review. early life and schooling i was born may 28, 1931 in vienna, as the only child to rosa and alois jellinger, both with roots from upper austria and moravia (near olomouc). my father was an official in the government of lower austria; my mother, a trained dressmaker, was a housewife. in the prewar years, i had a wonderful and well-kept childhood filled with summer vacations in the beautiful surroundings of vienna and in the idyllic countryside, while in the city there were increasing tensions, poverty, and changes in the political atmosphere. i remember kurt schuschnigg's radio speech of march 11, 1938, with his concluding sentence “may god protect austria,” after which my father commented, “this will be the beginning of the end.” after austria's “anschluss” to the german reich, i attended a public primary school in vienna and later, during world war ii, a public secondary school (realgymnasium, also in vienna), where i received basic education in languages, natural sciences, geography, and history. in 1943, to escape from air raids on the city, many children were sent to live with farmers in the batschka, then southern hungary, now serbia. once there, we had a wonderful and peaceful time, though the atrocity of witnessing the pogroms of jewish people was never far from sight or mind. on our way home we passed the then peaceful city of budapest, which i would see again during the hungarian revolution in 1956. once returned to vienna, in 1944, we had to change school buildings due to bombing damages. air raids were still so frequent that i often did not make it home in the evenings but had to instead seek shelter along the way. in september of 1944, we lost our home due to an air raid and had to wait for a new flat. around this time, i attended one of the last performances of schiller's wallenstein trilogy of plays featuring the famous actor werner krauss in the vienna burgtheater. soon afterwards, all theaters were closed. on april 13, 1945, during the battle between german and russian armies in the city of vienna, i had my first medical “experience” by cleaning up brain tissue of russian soldiers who were killed by the projectile from a german tank. after my father died in a russian pow camp in 1947, my mother and i were left alone to overcome the difficult times after the end of the war. she worked in her former profession as a dressmaker and i became tutor of children. in 1949, i graduated high school summa cum laude. in that final year, our class contained 18 boys and, when we had our final exams, i got excellent marks (fig. 2). after graduation, our class, led by our latin teacher, traveled to rome and naples, where we enjoyed arts, cuisine, and life in italy. to this day, i remain grateful to my many teachers who encouraged me to liberally approach history and natural sciences. years later, one of my favorite high school teachers became a patient in my clinic with myasthenia gravis, which we treated with success. over the years, my classmates and i met several times at reunions to remember and discuss previous experiences. the last meeting was 2009 to celebrate the 50th anniversary of our graduation. fig. 1. (a) ms-like autoimmune encephalitis after repeated subcutaneous injections of lyophilized calf brain cells in male aged 51 years with hemi-parkinson syndrome. left hemisphere with prominent periventrivular demyelinated lesion and multiple demyelinated plaques in cortex and subcortical nuclei. from [2] (b) em image of focal (core) plaque in hippocampus showing dense accumulation of thick amyloid fiber bundles, surrounded by dystrophic neuritic endings and hyperintense structure of myelinated axon. (x 4000). photo: k.a. jellinger. (c) major histopathological changes in alzheimer’s disease. (c1) amyloid deposits in the neuropil (plaques) and vasculature (caa) (congo red stain). (c2) neuritic plaque with dystrophic neurites (bodian stain). (c3) neurofibrillary tangles (large arrows) and neuropil threads (small arrows) (bodian stain). from [3]. medical school and beyond in 1949, i entered the medical faculty of the university of vienna, where the circumstances of medical education were made far more difficult due to the sequelae of the war. for example, we worked in the anatomy theater in winter with glassless windows and without warm water. despite this, and with eight of us were working on a corpse, we succeeded thanks to the influence of our professor, alfred gisel, a wonderful teacher and enthusiastic anatomist, who, in addition, was medical chief of the austrian red cross. the curriculum had basic subjects like anatomy, chemistry, physics, and physiology for 2 years, followed by 3 years of theoretical and practical training, beginning with pathological anatomy, physiology, pharmacology, and clinical specialties. most of our teachers were outstanding, some of them scientists of high reputation, such as: prof. h. tuppy, chemistry (nobel price candidate); prof. f.t. brücke, pharmacology; prof. g. schubert, physiology; and prof. h. chiari, head of pathological anatomy. during my studies, and despite regular grants, i supported myself with work as a paramedic for physically disabled people, as a tutor for children and students, and as a writer of short stories for newspapers and magazines. our clinical teachers were very dedicated and regularly organized seminars and clinical practicums, but the majority of teaching was much less practical but more formal and didactic than in the new curricula. in december 1955, i finished my medical studies, but had to wait for my graduation due to administrative delay since mine was the first “promotio sub auspiciis praesidentis rei publicae,” the highest possible distinction for academic achievements in austria, in medicine since the end of world war ii. in the meantime, i worked as guest doctor without salary in the franz josef hospital in vienna, within the department of internal medicine and surgery, directed by prof. herbert kraus, later chairman of the first department of neurosurgery of vienna medical school. on april 14, 1956, i was promoted to doctor of medicine “sub auspiciis praesidentis rei publicae,” which i received in the uniform of an officer of the austrian red cross (fig. 3). hospital years and first experiences in 1956, once the soviet army left budapest subsequent to the bloody uprising of the hungarian people, i went with a red cross convoy to deliver medical supplies and blood bottles to the liberated citizens of budapest. we were lucky to return to vienna before the russian army reconquered budapest. i enjoyed clinical medicine but my major interest was neuropathology. on january 5, 1957, i began as a postdoctoral fellow (wissenschaftliche hilfskraft) at the neurological institute (ni) a.k.a. obersteiner institute of the vienna medical school headed by hans hoff, who was also chairman of the neuropsychiatric clinic. founded by heinrich obersteiner in 1882 as a box that was subsequently expanded by obersteiner's personal expenses, the ni became the first interdisciplinary institute of neurosciences and was visited by many scientists from japan, the usa, and other countries. obersteiner edited a series of publications under the title “arbeiten aus dem obersteinerschen institut” and books about basic neuroanatomy and pathology. together with the edinger institute in frankfurt, the ni became one of the leading centers of neurosciences in the world until the beginning of world war ii, when it was directed by oskar gagel, an experienced neurooncologist. the ni, together with the institute of histology, occupied the first floor in a building of preclinical institutes, next to the general hospital of the vienna medical school (allgemeines krankenhaus/akh). parts of this area had been bomb-damaged during the war. fig. 2. our graduation class 1949 together with h.r. klieba, principal of the realgymnasium wien viii, and dr. seifert, our class teacher. kj second left in the first row. my first venture into neuropathology was to save and reorganize the large collection of slides that included material from major brain diseases. it was through this work that i learned the basics of neuroanatomy and neuropathology. the ni had a huge library, originally compiled by obersteiner himself, which was a treasure trove of rare and antique books, including an original edition of thomas willis' “circulus arteriosus cerebri,” one of the first descriptions of brain vasculature. in 1958, prof. franz seitelberger became director of the ni (fig. 4). in the same year, i met and immediately fell in love with elisabeth, a beautiful and clever girl aged 21 years, born in brno (then cssr), who studied english, russian, and history of arts. we attended theaters, operas, concerts, and frequently met in the university library. elisabeth and i married on august 17, 1960. fig. 3. promotion to doctor of medicine 1956 in the uniform of a red cross officer, with dr. heinrich drimmel, federal minister of education and science. training progress during the following years, i was trained by franz seitelberger, an internationally renowned neuropathologist with particular interest in metabolic and neurodegenerative diseases of the nervous system, who later became dean and rector of the university of vienna. i was in charge of brain autopsies (600 to 1000 per year) and diagnostic workup of neurosurgical biopsies. my early scientific work concerned acute demyelinating encephalitis following injections of lyophilized calf brain cells in a patient suffering from parkinson's disease (pd) [2] (fig. 1a). this particular case, which closely resembled those of patients who received rabies vaccinations in japan, was later re-published as acute autoimmune demyelinating encephalitis [4]. other projects included studying the neuropathological side effects of the 1958 influenza epidemic and the neuropathology of spring-summer (tick-borne) encephalitis in austria, which we described in detail. in parallel to my work at the ni, i underwent specialization in neurology and psychiatry as a guest doctor at the university clinic of neurology and psychiatry in vienna, headed by hans hoff, between january 1958 and december 1963. among my tutors were klara weingarten, an excellent clinical neurologist, and franz gerstenbrand, later chairman of the clinic of neurology, medical university innsbruck. we examined patients, both clinically and neuropathologically, with chronic coma or apallic syndrome (now unresponsive wakefulness syndrome) following head injury and those who suffered hypoxic brain damage and found a close relationship between the pattern of brainstem lesions due to intracranial pressure, the state of consciousness, and clinical outcome. in addition to hospital work, i was assistant to prof. hoff's lectures in neurology, which was often difficult since he wanted the patients' histories and clinical statuses to be in exact accordance with his neurology book. i also heard lectures by prof. herbert reisner, an excellent neurologist and psychiatrist, who later became chairman of the clinic of neurology of the university of vienna. after training in neurology, i worked in the division of psychiatry, where i got experience in all kinds of psychiatric disorders. on free weekends, i worked as a voluntary health officer with the red cross. on january 1st, 1960, i was promoted from assistant to consultant (oberarzt) at the ni. i studied encephalitis and myelitis following poliomyelitis vaccination and movement disorders (progressive pallidum atrophy and striatonigral degeneration) with erwin neumeyer, who unfortunately died early. in 1961, elisabeth and i attended the international congress of neuropathology in munich where we met all the top scientists in this field. in 1962, after a lecture on chronic vascular myelopathy at the 25th reunion neurologique in paris, i became membre associé à titre ètranger (associate foreign member) of the french neurology association. during that time, many excellent neuroscientists visited the ni and lectured there, such as hugo spatz, hans jacob, wilhelm krücke, chief of the max planck institute of brain research in frankfurt, webb haymaker, chief of the armed forces institute of pathology (afip), and ludo van bogaert of bunge institute in antwerp, belgium, for whom i had the honor of translating his lectures. a number of postdocs and research fellows, particularly from japan, were trained in the ni, such as mansori tomonaga, professor at tokyo university (his charming wife was a wonderful soprano singer), riki okeda (who counted cells in the pons during the the night while listening to classical music), and many others (see [5]). it was an exciting time in the international neuroscience community! between february and august 1963, i worked part-time as guest at the institute of pathology, vienna medical university, chaired by hans chiari, where i performed general autopsies and brain cutting. i detected the first case of clinically-suspected creutzfeldt-jakob disease (cjd), which i “stole” in order to study it histologically, with the consequence that i was dismissed from the morgue. this was the end of my training in general pathology, but the relations with this department remained excellent. between 1963 and 1974, i additionally worked two afternoons each week as chief of the outpatient service of the anton proksch institute, a therapy facility for alcoholism and addiction, in order to supplement my low salary as a university assistant. with wichard kryspin-exner, later chairman of the department of psychiatry, medical university of innsbruck, a number of papers about the course and prevention of alcoholism were published. full-time neuropathology in the following years, in addition to routine postmortem neuropathology and diagnostic biopsy work, i examined vascular spinal cord pathology and commented on zülch's vascular “borderline zones.” other subjects included spinal cord injuries, in particular, cervical hyperextension trauma with vascular complications. in january 1963, i earned board certification as specialist (facharzt) in neurology and psychiatry and returned as a full-time consultant to the ni. fig. 4. briefing with prof. f. seitelberger (left) and dr. h. hoff (right) in the neurological institute, 1958. in 1965, a postmortem study of a series of anoxic-vascular brain lesions resulting from complications of open heart surgery caused quite an outcry in the surgical community, but, as a consequence, the incidence of complications was reduced due to changed modalities and techniques in cardiac and vascular surgery. at the international congress of neuropathology in zurich, i gave a lecture on the neuropathology of coma and postcomatose encephalopathies. then, i began experimental studies of spinal cord vasculature in cats and other animals, working together with the neurosurgeon heinrich brenner in the experimental laboratory of the department of surgery. these studies, together with large human postmortem material, were the basis for my phd thesis “zur onkologie und pathologie der rückenmarksdurchblutung” (“on the oncology and pathology of spinal cord blood circulation”), published in 1966 by springer verlag wien. on january 19, 1966, i was granted the venia legendi (dozentur, lecturer) in neurology, neuroanatomy, and neuropathology, and became chief of the division of neuropathology of the ni vienna. in addition to routine diagnostic work, i lectured about general and specific neuropathology, and performed regular brain cutting sessions for students and colleagues who were interested in neuropathology (fig. 5). in the same year i received the kardinal innitzer award for promotion of medical research. atypical encephalitides and spinal cord circulation disorders were presented at the 1st meeting of the cssr neuropathology meeting in prague in september 1966, where prof. bednar, chairman of die institute of pathology of prague medical school, was a charming host. 1967, with w. sturm, a series of radiation injuries of the cervical spinal cord with relation to the applied radiation doses was published. at that time of progressing international congresses, publications and oral presentations were increasingly presented in english, and i published many articles in acta neuropathologica, founded in 1961 by franz seitelberger, who was editor-in-chief until 1987. the focus of clinical work and research were brain tumors, which were discussed in weekly clinico-pathological conferences with the department of neurosurgery. tumor cell imprints for rapid diagnosis were introduced, which were confirmed by histology with up to 95% concordance. in 1970, invited by m. mossakowski, head of the polish academy of sciences, i lectured about various neuropathological subjects in warsaw, poznań, and kraków, and was impressed by the lifestyle of the political upper class. at the 6th international congress of neuropathology in paris, where i spoke about delayed radiation lesions of the spinal cord, elisabeth and i were overwhelmed by the charm of france's capital. in verona, the neuropathology of the “apallic syndrome” after head trauma was presented. in the same year, seitelberger and igor klatzo, from the nih, bethesda, usa, organized an international symposium on axons and axonal flow, where i discussed neuroaxonal dystrophy in humans. fig. 5. brain cutting with (left to right) hans lassmann, gernot wöber (neurosurgeon), kj, and georg spiel (later pediatric neurologist) in the neurological institute, 1966. note the numerous fixed brains in the glass jars in the back. in october 1971 herbert budka joined us and became my assistant for more than three years. we shared routine diagnostic neuropathology. herbert recently described the situation and further fate of the ni, and became director of the clinical institute of neurology, the successor of the ni after its transfer to the allgemeine krankenhaus (see [5]). in 1972, i lectured in baltimore, kansas city (j.j. kepes' invitation), and new york. heinz regele, later chief of pathology in linz, and i observed peculiar things that were described later by others. this included, in patients with brain death, finding cerebellar material around the cervical and thoracic spinal cord, which dropped down as sequelae of cerebellar incarceration due to increased intracerebral pressure, later reported as a characteristic finding in brain death (vita reducta). with lothar kucsko, a highly experienced pathologist and heavy smoker, a postmortem transorbital puncture of the brain in a patient with clinically-suspected cjd was performed. under the electron microscope, i saw ruptured cell membranes, which i initially interpreted as postmortem artifact, but peter lampert from the afip published these findings as typical lesions in cjd [6] so we were too late! in the course of routine examination of brain tumor biopsies, i detected a large b cell lymphoma in the parietal lobe of a famous conductor. erwin deutsch, chairman of the department of internal medicine, did not believe in my diagnosis, since whole body examination did not show any malignancy. so i sent slides to harry zimmerman, chairman of neuropathology at montefiore hospital in new york, one of the world’s best neuropathologists and mentor to generations of neuropathologists, who confirmed my diagnosis. this patient survived for more than 12 years. autopsy revealed a generalized immunocytoma, but no tumor residual in the brain. together with harry and seitelberger, in 1974, an international symposium on malignant lymphomas of the central nervous system (cns) in vienna was organized, where experts discussed the classification of primary lymphomas of the cns. in 1972, traumatic vascular diseases of the spinal cord were reviewed for the handbook of clinical neurology. with t. radaszkiewicz, an excellent expert in lymphomas who unfortunately died too soon, a large collection of primary and secondary lymphomas of the cns was published. in 1973, hanno bernheimer, oleh hornykiewicz (who died may 26, 2020), walther birkmayer, franz seitelberger, and i published a highly-cited paper (currently 3,005 citations) about dopamine in the syndromes of parkinson and huntington. it was one if the first studies that correlated the neuropathological findings in the striatonigral system (semiquantitative assessment of neuronal loss in substantia nigra) and the biochemical data (dopamine and its metabolites) in a large number of autopsy cases [7]. during my time at the ni and afterwards, i extended brain autopsy service to several institutions outside of vienna, including the neurological hospital in linz. since i had no car, i traveled by train. once, on the return trip to vienna, while transporting sacks with freshly-dissected brains soaked with formalin to be examined in the ni, an acute train stop resulted some brains falling out of the sacks. i was almost arrested as a suspected mass murderer but the awful smell of formalin explained the reality. later career in 1973, i was offered the position of chairman of the division of neuropathology at the institute of pathology in zürich, but as i was number two on the list, reinhard friede was instead appointed. in february 1974, i was offered the position of professor and chairman of the department of neuropathology at the medical university düsseldorf, germany, which after 2 years of unsuccessful negotiations and for family reasons, i had to decline. wolfgang wechsler, then at the max planck institute at cologne, got this position which he held until his retirement in 1998. in 1975, in collaboration with andras guseo, a young neurologist from székesfehérvár, hungary and guest in the ni, the patterns of inflammatory perivascular infiltrates and their impact on the type and prognosis of multiple sclerosis were published. this was the beginning of a fruitful cooperation with the group of hans lassmann, then chief of the neuroimmunology division of the austrian academy of sciences and later of the department of neuroimmunology at the institute for brain research in vienna. in the same year, together with felicia slowik, a young neurooncologist from budapest and guest in the ni, a large collection of biopsy and autopsy-proven meningiomas was examined with impact on the histological specificities and prognostic relevance of various subtypes. in between, i was offered the position of head of the division of neuropathology at miami school of medicine, fl. on this occasion, elisabeth and i toured through the usa and i lectured at various universities. we got a deep impression about the way of living in the usa, which differed considerably from ours. a few months later i was offered the position of head of the division of neuropathology at case western medical school of medicine, cleveland, oh (successorship of r. friede). however, due to personal and family reasons, i declined all these positions. since it was impossible to get an independent position in vienna,, i finally decided to accept the position as head of the department of neurology at lainz hospital, one of the oldest municipal and teaching hospitals in vienna. it was built between 1908 and 1913 together with a large geriatric centre on the occasion of the 60th anniversary of emperor franz josef's regency and was equipped with all facilities of modern medicine. during julius tandler's time, a famous anatomist and counselor of health of the city of vienna, lainz hospital became “a second university.” in 2006 it was fused with the nearby neurological hospital rosenhügel as a neurological center and is currently named clinic hietzing. on october 12, 1976, i was appointed chief of the department of neurology and became director of the ludwig boltzmann institute of clinical neurobiology in january 1977, as successor of walter birkmayer, one of the “fathers” of research in pd. the ludwig boltzmann society, named after the austrian physicist ludwig boltzmann, was founded 1960 as an austrian network of specialized research institutes in the fields of medicine, humanities, and social sciences, all of which were sponsored by the city of vienna and the austrian government. in 2002, the society was reorganized and the number of institutes greatly reduced. so, one career ended and a new area of neuroscience began, although many within the vienna neurological community reacted with skepticism whether a neuropathologist would be able to manage a new clinical department. and, indeed, the beginning was difficult. the department had 75 beds, part of them still occupied by geriatric patients, a waterbed ward, two trained nurses, and some auxiliary personnel but only a minimum of technical equipment. i had to find coworkers, some of whom needed a training position, which was not easy to organize. in addition to the clinical department, which occupied the ground floor of the building, the histological and neurochemical labs on the second floor had to be equipped and prepared for functioning. my first coworkers from the previous neurogeriatric department were m. podiwinsky, an elderly neuropsychiatrist, and roda weiss, a young armenian doctor born in odessa and married to a viennese lawyer. soon afterwards, herbert flament and hannes schmidt, two young neurologists in training from the nearby rosenhügel hospital, and two specialists, peter kothbauer, nephew of the former minister of health, ingrid leodolter, and edith sunder-plassmann, wife of a neurosurgeon, completed my first crew. kothbauer later had to leave the department. within a few years, i finally gathered a crew of 12 neurologists, most of them becoming excellent clinicians. for the neuropathological lab, mrs. veronika rappelsberger followed me from the ni. without her help, i would never have been able to do my routine work and research in neuropathology. i had to perform brain autopsies and biopsies for the whole hospital and geriatric center and biopsies for a large neurosurgical department. since i received no funding from the city, i had to pay my lab assistant from my own pocket. the chief of the neurochemistry lab was peter riederer, an experienced neurochemist who had graduated from the technical university vienna and, since 1971, had worked together with walter birkmayer in the ludwig boltzmann institute (lbi) of neurochemistry, the predecessor to the lbi of clinical neurobiology. later, a number of students and postdocs joined us. in addition to my new double functions, in 1976, i was appointed editor-in-chief of acta neuropathologica, after having acted as supplementary editor for several years. between 1976 and 1981 i also edited the series “current topics in neuropathology” with selected papers from meetings of the austrian society of neuropathology. in total, i edited or co-edited 10 books. with peter riederer, wolf-dieter rausch, a dedicated post-doc who is currently professor of neurochemistry at the veterinary university of vienna, paul kruzik, and others, we examined the biochemistry of hepatic encephalopathy and the mode of action of l-deprenyl in the human cns. peter left vienna in 1986 and became professor of neurochemistry and head of clinical neurochemistry at the psychiatric clinic of würzburg medical university until his retirement in 2008. we had a successful scientific cooperation and became close friends with him and his wife, inge. graduate engineer ildiko wichart, a young neurochemist from budapest, also joined our group and worked with us for many years even after her retirement. in addition to clinical service, with 1,700 to 2,000 in-patients and an outpatient service with more than 15,000 consultancies per year, our department had to perform the neurological and psychiatric consultant service for the whole hospital complex. the clinical crew published about brain tumors, intramedullary metastases, and lymphomas; while the neurochemistry group wrote about neurotransmitters in metabolic encephalopathies, hepatic coma, brain infarcts, and deprenyl in the human brain. in june 1976, i gave lectures about cns involvement by malignant lymphomas/leukemias and radiation-induced lesions of the cns in new york, philadelphia, and baltimore. in 1977, i received the award of the vienna university foundation, and in 1979 the award of the medical scientific foundation of the city of vienna. we organized the first neurooncology group in vienna, in which the preoperative examinations were done by my department, neurosurgery by the hospital rudolfstiftung (head h. brenner), biopsy diagnosis, combined radiation, and polychemotherapy according to the comt scheme, and postoperative care by our department. we introduced modern csf cytology, and studied cytological changes of gliomas caused by chemotherapy. the clinical crew was organized in a way that everyone had their own specialty: clinical neurology (edith sunder-plassmann, herbert flament, roda weiss, andrea vass, maria grisold, dieter volc); and neurophysiology (rembert vollmer, wolfgang grisold). with alexander meng, son of a chinese professor of pediatrics and a viennese mother, and a specialist in acupuncture and traditional chinese medicine (tcm), a successful multidisciplinary pain clinic was organized. later, we performed the first biomarker screenings (total tau and β-amyloid-42) in csf for the early diagnosis of alzheimer disease (ad). the department got the first transmission zeiss em 9 microscope in a city hospital, transferred from the lbi of hematology (chief alois stacher, both an experienced hematologist and dedicated politician). in 1978, our neurochemistry group studied the activity of tyrosine hydroxylase (th), the key enzyme of the dopaminergic system in the brain and adrenal medulla. we found that the th activity was not only reduced in the striatonigral system by up to 90% but also in the adrenal medulla by about 74% [8], a fact that questioned later research about transplantation of adrenal glands in patients with pd and would go on to save billions of dollars. in the background of this research, a laboratory for muscle diseases and electromyography was opened with wolfgang grisold, one of the best clinical neurologists and neurophsiologists. he later became chief of the neurology department of franz josef hospital, the lbi for neurooncology, and is currently secretary of the world federation of neurology. between 1975 and 1980, i wrote the chapter of pathology of the nervous system for several editions of the working book of pathology, edited by j.h. holzner, successor of m. chiari as chief of pathology of the vienna medical university. in 1978, reviews about vascular tumors and malformations were published, and lectures were given in new york, bethesda, and washington (international congress of neuropathology). again in collaboration with peter riederer, the first austrian-german brain bank was organized and we collected a host of neurodegenerative diseases, ad, pd, and normal controls, all of which was later transferred to peter's lab in würzburg. in 1980, with filippo gullotta, first chairman of the department of neuropathology at münster medical school, germany, and miroslaw mossakowski from warsaw, the first european congress of neuropathology in vienna was organized, bringing together many neuropathologists in our city. in the following years, many new associates joined our group. christian bancher was active in both clinical and neuropathological research, with a focus on neurodegenerative and senile brain diseases, and werner paulus, a postdoc from germany, worked on brain tumors, lymphomas, and iron and ferritin in pd brains. he later became chairman of the department of neuropathology at münster medical school and editor-in-chief of acta neuropathologica until 2019, and is the current editor-in-chief of free neuropathology since 2020. my working day was full and split. often, in the mornings, i went to pick up fresh brains from the nearby mortuary and carried them over the yard to the laboratory to dissect them for the brain bank, with one half being deep frozen for neurochemistry and the other fixed in formalin for histological examination. the first part of the day was dedicated to clinical work and, after the morning conference with my coworkers, i went on clinical rounds twice a week and every saturday. in between, i saw patients in the outpatient department or had conferences with other clinicians or neuroscientists. meals were eaten together with doctors and nurses in the kitchen of the ward. the afternoons were dedicated to neuropathology, brain cutting, and sign out (about 700 brains per year), or research in the em lab (fig. 1b). in addition, i prepared a catalogue of all in-patients and their diagnoses on cards intended to be computerized later, which unfortunately was not possible at that time. i routinely read the letters of dismission (discharge) of patients, since all of my associates had to prepare these letters spontaneously – “trust is good but control is better.” this usually lasted until night. besides the clinic and the institute, i ran a small private praxis once or twice a week, together with elisabeth, who was an ideal and helpful companion, loved by most of our patients. in addition, i served as court-certified expert witness for insurance companies or courts. so, my clinical duties were as broad as my scientific interests, which suited me very well and was tolerated by elisabeth with great patience. international activities: the roaring eigthies in 1980, i was invited to speak by prof. hoshino, chairman of the department of neurosurgery, teikyo university, tokyo, one of the founders of high dose irradiation of brain tumors. in addition to lecturing, i reclassified the tumor collection of this department. during my 3-week stay in japan, i got deep insights into the japanese lifestyle, culture, and cuisine, not only in big cities but also in the countryside. also around this time, i presented holoprosencephaly and agenesis of the corpus callosum at the meeting of the american association of neuropathology in vancouver, canada. in 1982, together with f. seitelberger, and h. lassmann, the 9th international congress of neuropathology in vienna was organized, one of the most important meetings of this specialty in vienna that was visited by about 700 specialists from all over the world. at a meeting about advances in neurotraumatology in milan, october 1982, i presented brain stem involvement in blunt head injuries and elisabeth and i were welcomed by our italian colleagues and we thoroughly enjoyed their hospitality and the flair of the city. again with peter riederer, metabolic brain diseases, dopa and tryptamine binding, selegiline effects, and lisuride in the treatment of pd were studied, thus broadening my experience beyond neuropathology. at a 1984 symposium on parkinsonism in bermuda, i was impressed by this wonderful island. between clinical work, congresses, routine neuropathology, and publishing scientific papers, my editorial work for acta neuropathologica occupied much of my private time. since we had no computers or internet and i often read articles and proofs during bathing in our home, the journal caused some tension in our private life. many of the clinical and clinicopathological presentations were performed together with members of my team, in particular with wolfgang grisold, one of the best neurologists i ever met, dieter volc, later chief of the parkinson service in a private clinic, and mrs. elisabeth kienzl, student of the technical university vienna and one of my best assistants in the laboratory. with andreas rett, an experienced neuropediatrist, a large number of brain malformations were studied. franz seitelberger, dawna armstrong, an outstanding pediatric neuropathologist from houston, texas, and i were among the first to extensively study the morphology and neurochemistry of rett syndrome. in 1985, i attended workshops in baltimore and bethesda, and was regularly invited to the annual meeting of the british neuropathological society. in addition to neurodegenerative disorders, the morphology and biochemistry of schizophrenia was studied with my cousin eberhart gabriel, director of the vienna psychiatric otto wagner hospital and renowned historian in psychiatry. in 1986, i edited the book “therapy of malignant brain tumours” (springer verlag, vienna), in which international experts reviewed pathology, imaging, neurosurgery, radiation, and chemotherapy of brain neoplasms. between 1986 and 1999, i published chapters about neurodegenerative diseases, brain tumors, and spinal circulation disorders in the book series “neurology in clinic and practice” and, in 1987, about pallidal degeneration in the “handbook of neurology.” in september 1986, elisabeth and i visited stockholm for the 10th international congress of neuropathology, where i presented rett syndrome and changes of subcortical nuclei in ad and pd. we were overwhelmed by this charming city and the hospitality of its citizens. in september 1986 i attended the workshop “histological classification of tumors of the nervous system” in houston, texas, which initiated a new classification of brain neoplasms (fig. 6). in may 1988, i participated in the preparation of the 11th international congress of neuropathology 1990 in kyoto and gave lectures in tokyo, sendai, and niigata, where elisabeth and i were hosted by professor iikuta, head of the institute of brain research in niigata and his wonderful wife, who spoke perfect english. here, we saw the other side of the japanese island (figs. 7 and 8). fig. 6. brain tumor meeting in houston, texas, 1988, with (left to right) lucien rubinstein, kj, lucy rorke-adams and paul kleihues. in march 1989, we had a wonderful austrian neuroscience winter meeting at kitzbühel, and in april 1989 i discussed lewy bodies at a parkinson symposium in chicago. the pathology of parkinson syndrome was published in the handbook of experimental pharmacology, edited by donald calne, a pioneer of pd research. between 1989 and 1991, felicia slowik, a highly experienced neurooncologist in budapest worked with me on hemangiopericytomas, the ultrastructure of malignant lymphomas, and cerebral sarcomas. with werner paulus, the neuropathological basis of different clinical subgroups of pd and their clinical relevance were studied. in 1991, i received the city of vienna prize for medical sciences. the next year, together with e. kienzl and emin sofic, now professor of pharmacology, faculty of sciences, university of sarajevo, selective increase of iron in the sn of pd and changes in the iron-melanin complex in the sn of pd brains with x-ray microanalysis were studied and presented at the meeting of the canadian association of neuropathology, september 1992 in toronto, canada. fig. 7. visit to yashiko shrine hiigata, may 1988 with prof. kreutzberg and prof. ikuta. fig. 8. neuropathology and hospitality in niigate, 1988. more work, more congresses in april 1990, i participated in the who meeting on histological classification of tumors of the cns, organized by my friend paul kleihues, chairman of neuropathology in zürich and later director of the who international agency for research on cancer in lyon, france. werner paulus and i, together with 25 other distinguished brain tumor experts, participated in the meeting of the blue book’s classification of brain tumours. the 2nd edition followed in 1993, the 3rd edition in 1997, and another in 2000, following a meeting in lyon in 1997, where i also participated. in its update, retitled “pathology and genetics of tumours of the nervous system,” edited by p. kleihues and w. cavanee, with werner paulus, malignant lymphomas, histiocytic tumors, and melanotic lesions were reviewed. in august 1990, i presented primary cerebral lymphomas at the international cancer congress in hamburg, and in september 1990, elisabeth and i visited the 12th international congress of neuropathology in kyoto, where we were hosted by prof. yonezawa, who is not only an excellent neuropathologist but also a gourmet and friend of the good life. further highlights were the 5th world congress of biological psychiatry in florence in june 1991, where i presented pathomorphological aspects of schizophrenia, and the 10th international symposium of parkinson's disease in tokyo in october 1991. at the paneuropean congress of neurology in vienna, december 1991, the results of the vienna longitudinal study of dementia were presented, which showed correlations between cognitive impairment and quantitative alzheimer pathology. with christian bancher, the neuropathology of progressive supranuclear palsy (psp) was reviewed in a book edited by irene litvan and yves agid. at this time, the clinical department was fully developed and successful, including a new laboratory for ultrasound examination of the cerebral vasculature. upon the 80th anniversary of lainz hospital, a festschrift was published in 1993 that documented the progress of our department. in april 1993, the neuropathology and classification of psp was discussed in bethesda, md, and i received the eloise troxel memorial grant of the society for progressive supranuclear palsy, baltimore, md. at meetings in tokyo in april 1994, i lectured on new developments in ad and the pathogenesis of pd. a further highlight was the 12th international congress of neuropathology in toronto in september 1994, where our group presented new data about cell death detected by dna fragmentation and phospholipids in the alzheimer brain. in 1994, together in a working group of hans lassmann, pathogenetic problems of multiple sclerosis were studied. that same year, with werner paulus, advances in the pathology of primary cns lymphomas was also studied. a number of neurodegenerative disorders, like psp, multiple system atrophy (msa), and corticobasal degeneration (cbd) were studied in cooperation with an international group headed by irene litvan, now at university of california, san diego, usa. in 1995, christian bancher and i described the neurofibrillary predominant form of senile dementia, a rare subtype with low apoe ε4 genotype, which recently was renamed primary age-related tauopathy (part) [9]. in 1995, the pathology of aids encephalopathy and, together with herbert's group, the neuropathology and diagnostic criteria of cjd and other spongiform prion diseases were studied. in october 1995, i attended the workshop on dementia with lewy bodies (dlb) in newcastle/tyne, a dementing disorder hardly known at that time, and the guidelines for its diagnosis were published in the journal “neurology” in 1996. organizations and committees through the years, i was elected to various national and international scientific organizations and committees. i served as vice president of the international society of neuropathology between 1981 and 1984; was chairman of the eu working group from 1996 to 1999; secretary of the european society of psychopharmacology from 1994 to 2000; member of the fsn study group of neuropathology from 1987 to 2000; vice president of the austrian alzheimer society from 1997 to 2002; president of the viennese association of psychiatry and neurology in 1986/87; and president of the austrian society of neurology and psychiatry from 1990-1992. from 1994 to 2000, i was a member of a committee reporting on neuroscience in austria, on behalf of the austrian ministry of sciences. based on these data, a national program for neuroscientific research for austria was prepared in 1966, coordinated by the lbi of clinical neurobiology as a basis for austria's entry into the european union. with increasing age, i became member and honorary member of national and international scientific societies, including the american, british, german, hungarian, and austrian societies of neuropathology, the german societies of neurology and pathology, the austrian neuroscience association, honorary member of the austrian and german parkinson societies, the scientific commission of the german hirnliga, among many others. during these years, i reviewed articles for multiple national and international journals and became editor or member of the editorial board of many of them. the great adventure: first austrian neurologist in central china in march 1996, a delegation from the austrian society of neurology was invited by the chinese ministry of health to visit medical institutions and hospitals in china. this included 6 persons: hans and jutta lassmann, alexander meng, christian bancher, barbara zeman, and myself. the tour was organized and guided by mrs. zhou quin, program officer of the department of international cooperation of the chinese ministry of health (fig. 9). we visited china from march 31 to april 14, 1996. in beijing, we were informed about the health organization in the people’s republic of china. during the weekend, we visited the great wall, the ming graves, and the imperial palace. we then toured the neurological clinics of beijing hospital and of capital medical college, the two largest hospitals in the city, where hans and i gave lectures about multiple sclerosis (ms) and pd. we flew to chengdu, the capital of sichuan, the largest province of china, where we visited the medical college for traditional chinese medicine, which consisted of traditional and westernized parts, the first specializing in acupuncture and other traditional methods. fig. 9. first delegation of austrian neurologists in central china, with hans lassmann, kj, zhou quin, jutta lassmann, barbara zeman, christian bancher (left to right). we visited the largest herbal pharmacy of china and were invited to an herb dinner. on april 6, a sino-austrian symposium on neurosciences was organized in which lectures about ms, pd, and other topics were presented. by invitation of the minister of health of sichuan, we visited the min river and the buddhist grove sanctuaries in baodingshan and beishan, which impressed us very much. after a short visit to chongqing, a city with many respiratory diseases and lung cancers, we traveled by ship down the jangtze river to wuhan and visited the largest hospital of hubei province, which had modern technical equipment and many german-trained professors. the journey on the jangtse river, with the three gorges, was one of the most impressive adventures in our life. we experienced china's culture, landscape, and people, and passed the largest hydroelectric power station in china spanning the yangtse river, which at that time was in construction and considered one of the most impressive technical projects. for reasons of time, a visit to shanghai was not possible, and we returned to vienna. in conclusion, all of us were deeply impressed by the landscape, the historical relics, culture, health institutions, life in the countryside, and, in particular, by the friendly hospitality of our chinese colleagues. difficult times and new challenges in 1996, the ludwig boltzmann society decided to dismiss heinrich gross, who had been involved in the scandal about the euthanasia of handicapped children in the vienna psychiatric institution “am spiegelgrund” during nazi times, and i was asked to take over his lbi for the research of malformations of the cns, located in the psychiatric hospital baumgartner höhe, which was now combined with the lbi of clinical neurobiology. while the formalin-fixed brains of these children were stored in the cellar of the institute of pathology of this hospital, blocks of those brains had been secretly stored somewhere in the premises of these former lbis and i had the difficult task to find these materials from the archives so that they may be buried in a special memorial, which took place in 2002. this required much time to locate the specific material, to which i never had access before, while other specimens had been stored in the archives of the ni (see [5]). this affair was discussed in the popular media and had unforeseen political consequences, which hurt me, but i will not go into details. in 1996, together with norbert rösler, a postdoc from germany, the validity of csf biomarkers (total tau and amyloid-β-42) in the diagnosis of ad was studied. christian bancher and i visited an alzheimer congress in osaka, japan, and reported about the structural basis of dementia in ad and pd. the next year was of great importance for the department and myself, since i had reached the age of retirement from active service. however, my contract was prolonged for an extra year, during which we opened a stroke unit with 4 beds for surveillance and intensive treatment of patients with acute stroke. furthermore, christian bancher got the lectureship (dozentur) in neurology and neuropathology. in the years before, two other associates, rembert vollmer (neurology and neurophysiology) and wolfgang grisold (neurology) had got their dozentur. on september 30, 1997, i retired from my position as director of the department of neurology at lainz hospital, but remained chief the lbi of clinical neurobiology, which was transferred to a building in the psychiatrical otto wagner hospital. together with my lab assistant, mrs. rappelsberger, i continued neuropathological routine work examining brains from a large psychiatric and geriatric hospital. unfortunately, the archives of all the in-patients during the last 22 years, including the neuropathological protocols and brain material, was not available any more. my successor was dozent manfred schmidbauer, an excellent neurologist interested in neuropathology, book author, and painter. after the fusion of lainz hospital with the nearby neurological hospital rosenhügel, schmidbauer had to succeed the retired gernot schnabert as chief of both neurological departments. in addition, he continued routine neuropathology with focus on peripheral nerve biopsies. he retired in 2016 and became professor of neurodegenerative diseases at the sigmund freud private university in vienna. he was succeeded by christoph baumgartner, a highly-qualified epileptologist. however, this marked the end of neuropathology, which i had initiated and continued for 22 years. but i overcame this blow and continued scientific work. in addition to a small private praxis, i worked as a court-certified expert and still write second opinion expertises about difficult neurological problems. in october 1998, i presented data about movement disorders, malignant lymphomas, lewy body disease, and tangle dementia in new york, washington, bethesda, and montreal. at the 6th conference on ad and related disorders, in amsterdam in july 1998, i described vascular dementia and its relation with ad and received the alzheimer obelisk (fig. 10). in 1999 and 2000, the foci of my interests were pd and related disorders, in particular msa, as well as mechanisms of neuronal cell death, including apoptosis. with hallinah baran, an experienced neurochemist, later at the veterinarian university vienna, we studied kynurenine metabolism in ad, and with christine stadelmann, then assistant in lassmann's institute and now director of the institute of neuropathology, university göttingen, we studied cell death mechanisms in neurodegenerative diseases. at a traditional medicine symposium in vienna, i discussed the basis and impact of acupuncture in neurology. the same year, i was invited as guest professor of neuropathology at the medical university innsbruck, where i gave block lectures and seminars for students and colleagues. since the innsbruck university had no neuropathology at that time, i examined a number of autopsy cases of rare movement disorders, such as msa and psp. the symposium was hosted by prof. werner poewe, chairman of neurology, and gregor wenning, later chief of the division of clinical neurobiology, with whom a long-lasting and fruitful cooperation and deep friendship began. in 2000, i received the burda award of the austrian parkinson society. in the same year, i was invited as guest professor at the medical university of graz, where i lectured about many subjects of clinical and theoretical neuropathology in the clinic of neurology under chairmen erwin ott and later hans-peter hartung, now chairman of neurology in düsseldorf, germany. it was a wonderful time in the beautiful city of graz and its surroundings, which i enjoyed very much. it was a leisure living compared to vienna. in 2000, we described cell death mechanisms in pd and, in 2001, aids-related pathologies before and after antiretroviral therapy. since 2002, i helped the pathological bacteriological institute of the otto wagner hospital in the diagnostic workup of postmortem brains, mainly from elderly demented and non-demented individuals. i performed brain cuttings once a week, and instructed colleagues how to dissect brains, which were examined in the lbi. my trainee and friend johannes attems, then assistant at the local institute of pathology, examined the cases and we discussed them together between 2000 and 2008, thus giving him an excellent basis for his future career and a fruitful cooperation resulting in a number of shared publications. since 2009, johannes is professor of neuropathology and head of the brain bank at newcastle university in england and, since recently, editor-in-chief of acta neuropathological as successor of werner paulus. in 2002, i received the prestigious award for meritorious contribution to neuropathology by the american association of neuropathology, together with s. horoupian, professor emeritus at stanford university in the usa and fusahiro iikuta, director of the brain research center at niigata university, japan. the laudation of my award was written by robert d. terry, one of the most important neuropathologists of the 20th century and co-founder of us alzheimer research (addendum). we met on several occasions, including a visit to his home in california approximately one year before his death in may 2017. fig. 10. presentation of the alzheimer obelisk with dick swab (left) and henry wisniewski (center) at the 6th internat. conference on ad, amsterdam, july 1998. end of an institution and a new beginning in 2002, the lbi of clinical neurobiology was closed despite it being one of the most active scientific institutions outside the university with more than 1,400 publications until 2000. since that year, we received no budget and i had to find other sponsors. the equipment was transferred to the new institute of clinical neurobiology, which was run by the society for the support of research in experimental neurology in vienna, sponsored by donations and previous clinical trials. since 1999, my assistant has been dr. erich mitter-ferstl, phd, who studied at the zurich technical university and is an excellent computer specialist, without whom i hardly would have succeeded in this latest chapter of my career. time went on with both continuing examination of brain material at the wagner-jauregg hospital, evaluating old material, and continuing research. in 2003, i wrote a chapter about the neuropathology of pd in the book “neurodegeneration” (isn neuropath press, basel) edited by the international society of neuropathology and again for the 2nd edition, edited by d.w. dickson and r.o. weller in 2011. in 2007, a review of lewy body disorders appeared in abel lajtha's book “degenerative diseases of the nervous system” (springer, vienna) and since 2011, i continue to write the chapter on neuropathology of movement disorders in youman’s neurological surgery (elsevier saunders, philadelphia, editor w.r. winn), 6th edition, of which the 8th edition is currently in press. in 2014, i contributed the chapters on neuropathology and pathogenesis for the book “multiple system atrophy” (springer, wien) edited by g. wenning and a.fanciulli. lastly, with johannes attems, the chapter on neuropathology for the “textbook of old age psychiatry,” (oxford univ. press, oxford, uk) the 2nd edition of which is now in press. we had a co-operation with the vienna transdanube aging (vita) study, a long-term inter-institutional clinical, neuropsychological, genetic, neuroimaging, and neuropathological assessment of a large group of individuals aged 75 years in the eastern part of vienna, which was performed by the lbi of aging under the direction of hans tragl. for this group, with ildiko wichart, who worked at the university of veterinary medicine, vienna, aβ-42 protein in plasma was studied and correlated as valid marker with many clinical and neuropsychological data, which resulted in a large number of publications and congress reports between 2005 and 2010. unfortunately, the vita study ended for financial reasons, although it had been the first population-based long-term study about risks and development of cognitive changes in the oldest of old people. in 2003, at the 3rd german parkinson congress in dresden, heiko braak, an outstanding neuroscientist, who, among others, introduced the staging of pd and ad pathologies, and i received the lundbeck award for parkinson research (fig. 11). in the same year, i received the karl maria jakob medal of the german association of neuropathology in hamburg. the co-operation with johannes attems studying olfactory involvement and multimorbidity of the aging brain and with gregor wenning’s team on lewy body pathology was successfully continued. furthermore, with mrs. e. kienzl and a research group of the neurological clinic of the technical university of dresden (chief heinz reichmann), the alzheimer associated (alzas) gene was studied as a possible marker for ad. in 2005, i received the istván környey tarscosag award on the occasion of the 100th birthday celebration of this eminent hungarian neurologist, for whom i had the honor to present the laudation. in the same year, i contributed the chapter on synucleinopathies for the polish mossakowski memorial book of neuropathology (wydawnictwo czelej sp. z o.o., lublin, edited by p.d. liberski and w. papierz). since 2006, i lecture on the neuropathology of stroke and vascular dementia at the danube university in krems. the year 2006 was particularly busy. on the occasion of the 100th anniversary of aloys alzheimer's description of the disease, several books appeared. in one edited by george perry, the clinicopathological results of a large series of demented elderly person were presented, and i published a book about the hallmarks of 100 years of scientific research on ad presenting the most important publications between 1938 and 1999 in german (translated by myself). in a book edited by h. herholtz et al., i reviewed the early diagnosis of dementias from the neuropathological point of view based on 1,500 consecutive autopsy cases in comparison to the clinical diagnoses, where i emphasized the impact of multi-morbidity. in october 2007, i lectured about parkinson dementia in south korea, vascular dementia in budapest, and biological ad markers in wroclaw, poland. in 2008, together with lassmann's group, the relations between ms and ad from the neuroimmunological point of view were examined, and, at a parkinson symposium in tokyo, the formation and role of lewy bodies were presented. between 2005 and 2008, the kurt jellinger prize for outstanding scientific writing in neuropathology was sponsored by acta neuropathologica, for which i had served as executive editor for 29 years. the last recipient was goran simic (2008) from the brain research institute in zagreb, who had been guest scientist in my lbi a few years before. in 2008, i found myself among the 20 highly-cited scientists in austria in the isi webb of knowledge. since johannes attems went to newcastle upontyne in great britain, i stopped lab work and concentrated on specific neuroscience problems, reviewing manuscripts, and writing invited review papers. fig. 11. with heiko braak receiving the lundbeck award for parkinson research at the german parkinson congress 2003 in dresden. in 2011, i received the g. cotzias award of the movement study group of the spanish society of neuropathology in barcelona. in honor of my 80th birthday, four of my former scholars and now renowned neuroscientists, hans lassmann, christian bancher, johannes attems and werner paulus, dedicated a “festschrift” of acta neuropathologica [10], for which i am very grateful to them. on the same occasion, my friends and former associates organized a festivity, which all of us enjoyed very much. together with johannes attems and a. thomas, the correlations between cortical and subcortical tau pathologies and their relations with braak tau stages were reviewed, in addition to the pathological correlates of behavioral disorders in dementias with m.f. casanova and s.e. starkstein, and the problem of vascular depression with an international group. the role of α-synuclein in pd was presented in the book “emerging drugs and targets for parkinson's disease,” (the royal society of chemistry, cambridge, uk) edited by a. martinez and c. gil in 2013. invited in february 2013 by g. logroscino, an us-trained italian neurologist and neuroepidemiologist, i gave the lectio magistralis about the role of neuropathology in neurodegenerative diseases at the university of bari, italy. in june 2013, i discussed the role of α-synuclein as the gateway to neurodegeneration and demonstrated brain cutting to the members of the division of clinical neurobiology (head gregor wenning), with whom we celebrated our cooperation since 1993, which still persists. in march 2014, elisabeth and i were invited to a series of lectures at the department of neurology at seoul national university, where we were hosted by prof. beomseok jeon, a wonderful neuroscientist, who had overcome serious injuries in an alpine accident with utmost energy. we were overwhelmed by the megacity of seoul that had been completely destroyed during the civil war, and were deeply impressed by the lifestyle and culture of the korean people. we never had experienced such a warm-hearted hospitality before. after this tour, i was invited for the position of professor of neuropathology at seoul, but i had to decline for age reasons. i like small and intimate meetings, such as those by the society for amelioration of the quality of life, organized by stavros baloyannis, chairman of neurology at thessaloniki medical university, and sponsored by the orthodox church. these meetings occurred in marseille 2008, thessaloniki 2009, delphi 2010, and catania, sicily, in 2009, where franz, mrs. gerstenbrand and i climbed the aetna mountain. other favorite meetings were the “neurology spring” in the austrian waldviertel, organized by christian bancher, now chief of neurology at horn, and the world congresses on controversies in neurology (cony), organized by amos korczyn, professor emeritus in tel aviv, where problems in neurology are discussed by international experts. in may 2015, i presented the pathogenesis of msa at the α-synuclein symposium in innsbruck then once more at the congress on vascular dementia in ljubljana in october 2015. postmortem assessment in vascular dementia, a consensus report on vascular depression, and the relations of diabetes mellitus and dementia were published in collaboration with international experts. together with amos korczyn, an opinion paper discussing whether dementia with lewy bodies and parkinson dementia are the same disease was published in 2018. fig. 12. workshop on neuropathological diagnosis of ad at the ad/pd confernece in lisbon, march 2019, with johannes attems, irina alafuzoff, kj, melissa murray, dietmar thal (left to right). together with johannes attems, dietmar thal, and irina alafuzoff, i helped lead a workshop on neuropathological diagnosis of ad, which was organized by johannes at the aaic congresses in copenhagen 2012, washington 2015, and london 2017; and again at the ad/pd congress in nice 2015 and vienna 2017; and finally, with johannes attems, irina alafuzoff, dietmar thal, and melissa murray at the ad/pd conference in lisbon, march 2019 (fig. 12). my last congressional activities were the annual meeting of the austrian parkinson society in vienna in speaking about parkinson dementia and dementia with lewy bodies, and a biological definition of the alzheimer spectrum at a course about cns proteinopathies in milan, italy in november 29, 2019. now, at the age of 89, i still find it impossible to abandon my interest in neuropathology, and i try to continue scientific work as long and as well as i can. in the last year, i discussed whether braak staging is valid for all types of pd, and, together with gregor wenning's group, protective therapies in msa. after two extensive reviews about the neuropathology and pathogenesis of extrapyramidal disorders, the neuropathology of cognitive impairment in msa and an update of msa were published. two reviews of the pathobiological definition of alzheimer continuum, including the neuropathology of its pathobiological subtypes, are in press. life beyond neurology/neuropathology although i “was married to neuropathology,” together with elisabeth, we had an intensive and happy private life, as busy as it has been. before our wedding in august 1960, we rented a flat that took years to get completely refurnished. elisabeth was a student, and i a poorly-paid university assistant. in summer 1958, she was an au pair with a family in london, while i was paramedic in a summer camp for students in italy. in 1959, we spent a splendid summer vacation on the island of maly losinj at the croatian coast, where i had helped to reestablish the school. in 1960, we spent our honeymoon in lignano and venice, and the next year we went to the greek island of mykonos, where we were among the very few tourists on this beautiful island, which later became a hotspot of elegant tourism. elisabeth finished her studies in english with a thesis about the medieval towneley plays, which i typed. later, she became administrator in the institute of immunology of vienna medical faculty, run by my friend prof. martha eibl. afterwards, elisabeth joined me in literature research and as an assistant in my private medical practice, where she was loved by all my patients. in springtime, we traveled to southern countries, in particular toscana, umbria, the marches, romne, gubbio, urbino, arezzo, parma, sicily, corsica, burgundy, bretagne, and many other interesting regions, where we enjoyed the lifestyles, local gastronomy, cultures, and traditions. in summer we had regular vacations on many greek islands, but also in sicily, mallorca, and sardinia. in summer 1986, we spent our vacation in a beach house of my friend nenad grčević, an excellent croatian neuropathologist, in umag, a small town on the istrian peninsula, when the “prague spring” was suppressed by the soviet army. in 1996, after a peaceful stay at syros, we escaped from the island, afraid of a possible war between greece and turkey, and had to wait in athens for a flight home. later, many summers were spent on the beautiful island of chios, which is next to the turkish border, in a small hotel run by a friendly greek family, but during the last years we could not go there because of the refugee crisis and lack of direct flights. at christmas time and new year, we used to spend some weeks in paris, which we loved for its culture and way of life. unfortunately, during this past year we could not go to our beloved hotel de la bretonnerie in the old marais district because of the yellow jacket riots and the novel coronavirus pandemic. during the rest of the year, we regularly attend the vienna opera and concerts. for years, we have had subscriptions to the vienna philharmonic orchestra and many other chamber music presentations. in previous times, we regularly visited the salzburg festivals, where we met friends, many of them being members of the vienna philharmonic orchestra. during leisure time, i regularly read historical books and try to inform myself about austrian and international history. we like to walk in the idyllic landscape and, in order to exercise our brains, elisabeth and i take regular italian lessons with a nice italian interpreter. in 1969, my late father-in-law built a small house in maiersdorf, a small village at the foot of the hohe wand, a mountain of 1,200 meters, about 65 km south of vienna, which we visited nearly every weekend in summertime and around christmas. since march 13, 2020, and with few interruptions, we have lived in this house during the covid-19 lockdown, and it is here, in the tranquil landscape and beautiful springtime, that i wrote the major part of this report. once the lockdown ends, we hope that “normal life” will resume, although this virus is still an enormous and expanding danger for the whole world. however, we don't agree with the slogan in a humorist vienna journal in july 1918: “the spanish influenza finishes us all,” and, despite all warnings, we are looking optimistically to the future. we just celebrated the 60th anniversary of our wedding not on a greek island but in our rural home. acknowledgment the author thanks mr. erich mitter-ferstl phd, for his excellent secretarial and editorial work, without which this paper could not have been prepared. references 1. ludwin sk (2018) in search of knowledge and joy: my life as a neuropathologist. j neuropathol exp neurol 77:162–175 2. jellinger k, seitelberger f (1958) [acute fatal demyelinizing encephalitis after repeated injections of dry brain cells]. klin wochenschr 36:437-441 3. jellinger ka (2007) alzheimer's disease. in: gilman s (ed) neurobiology of disease. elsevier academic press amsterdam, pp 69-82 4. höftberger r, leisser m, bauer j, lassmann h (2015) autoimmune encephalitis in humans: how closely does it reflect multiple sclerosis ? acta neuropathol commun 3:80 5. budka h (2020) neuropathology through the ages – personal reflections. free neuropathol 1:15. https://doi.org/10.17879/freeneuropathology-12020-12817 6. lampert pw, gajdusek dc, gibbs cj, jr. (1972) subacute spongiform virus encephalopathies. scrapie, kuru and creutzfeldt-jakob disease: a review. am j pathol 68:626-652 7. bernheimer h, birkmayer w, hornykiewicz o, jellinger k, seitelberger f (1973) brain dopamine and the syndromes of parkinson and huntington. clinical, morphological and neurochemical correlations. j neurol sci 20:415-455 8. riederer p, rausch wd, birkmayer w, jellinger k, seemann d (1978) cns modulation of adrenal tyrosine hydroxylase in parkinson's disease and metabolic encephalopathies. j neural transm suppl:121-131 9. crary jf, trojanowski jq, schneider ja, abisambra jf, abner el, alafuzoff i, arnold se, attems j, beach tg, bigio eh, cairns nj, dickson dw, gearing m, grinberg lt, hof pr, hyman bt, jellinger k, jicha ga, kovacs gg, knopman ds, kofler j, kukull wa, mackenzie ir, masliah e, mckee a, montine tj, murray me, neltner jh, santa-maria i, seeley ww, serrano-pozo a, shelanski ml, stein t, takao m, thal dr, toledo jb, troncoso jc, vonsattel jp, white cl, 3rd, wisniewski t, woltjer rl, yamada m, nelson pt (2014) primary age-related tauopathy (part): a common pathology associated with human aging. acta neuropathol 128:755-766 10. lassmann h, bancher c, attems j, paulus w (2011) in honor of the 80th birthday of kurt jellinger: a living legend in neuropathology. acta neuropathol 121:565-568 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology studies of dementia in us persons other than non-hispanic whites feel free to add comments by clicking these icons on the sidebar free neuropathology 3:6 (2022) review neuropathology studies of dementia in us persons other than non-hispanic whites my-le nguyen1, emily z. huie1, rachel a. whitmer2, kristen m. george2, brittany n. dugger1 1 department of pathology and laboratory medicine, university of california, davis, usa 2 department of public health sciences, university of california, davis, usa corresponding author: brittany n. dugger · department of pathology and laboratory medicine · university of california, davis · 4645 2nd ave. · 3400a research building iii · sacramento, ca 95817 · usa bndugger@ucdavis.edu submitted: 26 january 2022 accepted: 04 march 2022 copyedited by: biswarathan ramani published: 10 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3795 additional resources and electronic supplementary material: supplementary material keywords: disparities, dementia, neuropathology, hispanic, latino, asian, african american, brain abstract alzheimer’s disease (ad) and vascular dementia are two of the most prevalent dementias that afflict the aging population in the united states (us). studies have made great strides in understanding the neuropathology of these diseases; however, many studies are conducted in the context of non-hispanic whites (nhws), and few include the rapidly growing underrepresented populations that reside in the us. we sought to characterize current knowledge of the neuropathologic landscape of ad and vascular dementia of the largest growing us minority groups, namely latinos/hispanics, black americans, and asian americans, compared with nhws being the majority group. it is vital to note these historic categories are social constructs and cultural and social associations may underlie differences. we conducted a literature search utilizing specific criteria to yield neuropathology papers that addressed the demographics and neuropathologies of relevance, then collated the findings into this review. we reveal that while there has been much progress in neuropathological research involving latinos/hispanics and black americans in the past decade, no cohesive conclusions could be extrapolated from the existing data due to the dearth of minority participants and even smaller amount of information related to the heterogeneity within each minority group, especially latinos/hispanics. furthermore, we reveal an even greater scarcity in neuropathological studies involving asian americans, also a very heterogeneous group. we hope the presented findings will illuminate the paucity of minority representation in not just neuropathological research but the field of clinical research overall and serve to inspire clinicians and researchers to help reduce the health disparities underrepresented groups in the us face. introduction clinically, alzheimer’s disease (ad) is defined as a type of dementia distinguished by neurodegeneration that results in memory loss and deterioration of cognitive functions24, 38. neuropathologically, ad is defined by aggregations of the amyloid-β (aβ) protein, in the form of aβ plaques, and tau protein, in the form of neurofibrillary tangles (nfts)13, 25, 97. in addition to aβ plaques and nfts, neuropathologies associated with vascular dementia can also be concomitant57, 78, 101. vascular dementia is the second most common cause of dementia following ad and neuropathologically can manifest as infarcts and hemorrhages as well as other vascular pathologies such as cerebral amyloid angiopathy (caa) and arteriolosclerosis9, 17, 23, 63, 90, 101. while there has been progress with understanding disease phenotype (for review see 89) and progress with therapies having evidence of targeting and reducing aβ plaques in the brain, there have yet to be treatments that fully cure or stop the progression of the disease20, 22. although great strides have been made to combat this disease, most studies have focused on select populations or cohorts, specifically composed of persons identifying as non-hispanic whites (nhws)28, 43, 73, 97. the population of persons age 65 and over in the united states (us) has significantly increased in the past decade—as much as a 36% increase2. us ethnoracial minorities made up 20% of this age demographic at the beginning of the decade, and increased to 24% by the end of it2. the nhw population of age 65 and over is projected to increase 29% by 2040 in comparison to the 115% increase of the ethnoracial minority population2. the largest us minority population is latinos/hispanics, followed by black americans, then asian americans, which are the fastest growing demographic18, 53. other notable underrepresented groups are american indians (native americans), native hawaiians, and pacific islanders4. it is important to note in this paper we will utilize historic terms (such as those within the us census) and these categories are social constructs and cultural and social associations may underlie differences. diversity in studies maximizes variability in risk and protective factors. this can aid in studying clinically relevant transitions across the spectrum of a disease with the goal of identifying modifiable pathways to support maintenance of normal functions. as racial/ethnic groups in the us continue to grow in addition to longer life expectancies, more diverse elderly individuals will be seen for diagnosis and treatment of neurodegenerative diseases; thus, more research reflecting the population diversity is needed so that prevention, treatment, and prognosis strategies encompass all who are affected by the devastating impact of dementia. heterogeneity of race and ethnic categories race and ethnicity are two terms fundamentally distinct from one another. race historically has been used to describe the physical traits of an individual such as their eyes, hair, and skin, whereas ethnicity has been used to describe an individual’s cultural identity74. it is essential to remember these terms are socially constructed and hold no bearing on an individual’s biology. the categorizations for race and ethnicity in the us have evolved over time as self-identification shifts, immigration, and mixed racial heritage became more prevalent1.the term “race” has regretfully implied a sole focus on an individual’s morphology, and historically has not accounted for other background variables such as geographic origin, environmental factors, and sociocultural characteristics that influence these differences43, 98. while the historic terms employed in this review are not optimal given how restrictive and tentative they are, the u.s. census uses them, which many neuropathology studies have utilized as well, and thus are presented as such in this review. thankfully, there is a growing awareness that these historic terms alone do not aptly stratify these individuals in a scientific context and new approaches are being encouraged1, 6, 43, 49. furthermore, it is important to recognize there is heterogeneity within racial/ethnic categories. the terms latinos and hispanics are sometimes used interchangeably65. the term hispanics historically has been used to describe those who are from spain or other spanish-speaking country, while latino signifies those who originate from latin america, regardless of their spoken language62. moreover, it is noted the term caribbean hispanics is used to describe the latino population that resides in puerto rico, dominican republic, and cuba96. to further expand on distinctions within the latino/hispanic population of the us, some studies have partitioned this ethnicity based on area of decedents’ self-reported origin: mexican, south american, central american, and caribbean99, 100. these categories are standardized by the us census; hence studies have followed the same format64. the mexican descendent population is the largest latino/hispanic group in the us and is spread out through the country, with more density in the southwest27. individuals from south and central america also follow this distribution pattern, whereas caribbean latinos/hispanics are more concentrated in eastern states3. these geographical distributions when examining ad cohorts can be immensely important as there have been reported socioeconomic and cultural differences within these groups that are associated with risk factors for ad77. there is also heterogeneity within the black american population. the largest subgroup within the us black population is single-race non-hispanic, comprising of 87% of the total us black population95. the following largest is the multiracial non-hispanic population, constituting 8% of the overall us black population, with black hispanics making up the remainder 5%95. the majority of black americans are of west/central african and european heritage, and some also have native american roots34. an estimated 90% of the total black population was born in the us in which most are descendants of enslaved people84, 94. despite the great migration where the black population had dispersed to areas in the us away from the south, the distribution in the south has begun to grow in the past few decades92. another observed migration pattern in the last few decades has been a substantial increase among the foreign-born us black population, making up 10% of the current us black population in which the majority of foreign-born us black persons, 88%, were born in african or caribbean nations94. similarly, there is immense diversity within the asian american population. there are 21 distinct asian subgroups living in the us according to gathered data from the census american community survey, with the largest being chinese (including taiwanese), followed by asian indian, filipino, vietnamese, korean, and japanese15, 16. almost half (45%) of asian americans live in the western us, with nearly one third of the nation’s population living in california alone (30%), while 24% reside in southern states, 19% in the northeast, and 12% in the midwest15. data have shown a substantial proportion of chinese, filipino, vietnamese, korean, and japanese inhabit the western us, amounting to at least 43% distribution across all groups46. on the other hand, asian indians occupied the northeastern and southern regions of the us more frequently, accounting for over 29% distribution for each region46. by addressing the geographic, ethnic, and cultural variations that exist among race and ethnic subgroups, ad research can be refined to yield more precise methodology and analyses. differences in clinical ad based on race and ethnicity differences in the prevalence and incidence of clinically defined ad and related dementias (adrd) have been observed when comparing across race/ethnicity. ad prevalence is highest in black americans and latinos/hispanics, followed by nhws, and then asian americans21, 33, 40, 41, 61, 66-68, 70, 72, 79, 86, 96. notably, one study showed us-born nhws, hispanics, and other uncategorized races had lower frequency of dementia compared to their immigrant counterparts, except for non-hispanic blacks (nhbs), where it was an inverse correlation72. studies have also shown differences in dementia prevalence between latino subgroups. one study revealed a 4.8% prevalence of dementia among mexican americans ≥65 years residing in sacramento county, california, with up to 31% prevalence in those aged 85 years or older42. in contrast, a study of caribbean hispanics residing in north manhattan estimated a dementia prevalence for individuals aged 65-74 of 7.5%, 27.9% for those 75-84, and 62.9% for those 85 and older41. latinos/hispanics have been shown to have an earlier age of onset of ad when compared to nhws and black americans, though the difference was marginal for the latter in some cohorts21, 33, 42, 61, 72. both latinos/hispanics and black americans have a high prevalence of cardiovascular risk factors associated with adrd, while prevalence of these risk factors is lower among nhws followed by asian americans 40, 42, 61, 67, 96, 102. in a california study that examined dementia incidence in asian american subgroups, filipino americans had the highest incidence rate at 17.3 per 1000 person-years, and south asian americans (i.e., asian indian, pakistani, bangladeshi, sri lankan, or nepalese) had the lowest rate at 12.1 per 1000 person-years67. while many observable trends have been correlated with ad from a clinical perspective across different race/ethnicities, they do not confirm the presence of the hallmark protein aggregates that are currently are the gold standard for diagnoses of adrd, in other words, the neuropathology. do the neuropathological profiles of individuals with adrd of the three largest minority groups in the us differ, as there have been noted cultural and geographic differences in these groups? neuropathology studies have been conducted on predominantly nhw cohorts, so little is known about adrd in these other racial/ethnic groups6, 28, 43, 73. for this review, we will discuss the current landscape of neuropathological findings in ad and vascular dementia in latinos/hispanics, black americans, and asian americans. we seek a more comprehensive understanding of the disease profile in these groups of individuals to provide improved diagnoses and develop effective countermeasure therapies or methods to allay the risk of ad and vascular dementia. methods inclusion and exclusion criteria all literature in this review focused on signature pathologies associated with ad and vascular dementia in latino/hispanic, black american, and asian american cohorts. studies meeting the inclusion criteria were published 1995 and onwards, peer-reviewed, specifically presented ad or vascular dementia neuropathological findings, and were conducted in the us. neuropathologic evaluations of interest included but were not limited to braak neurofibrillary tangle (nft) stage, consortium to establish a registry for alzheimer’s disease (cerad) neuritic plaque score; thal amyloid phase; and national institute of aging and alzheimer’s association (nia-aa) criteria of overall ad diagnosis based on neuropathologic changes13, 48, 71, 97. for vascular dementia, pathologies of interest included, but were not limited to, infarcts, hemorrhages, arteriolosclerosis, atherosclerosis, and caa. as for staging schemes and creating a consensus for diagnosing vascular dementia, there have been multiple attempts23, 55, 83, 90, 92; however, there is no universally used system in place. exclusion criteria included papers that contained only living cohorts (i.e., cohorts not having neuropathologic evaluation of persons after death) presented data with no mention of ad neuropathologies specifically (e.g., genotyping, neuropsychological tests, etc.), studies that were not conducted on human subjects, and articles that were not sourced from peer-reviewed journals. search strategy the literature review was conducted by searching specific mesh terms in pubmed, scopus, and web of science to yield the peer-reviewed articles investigating ad and vascular neuropathology among latinos/hispanics, african americans/black americans, and asian americans on november 19, 2021. the mesh search term refinement process for these databases was guided by a uc davis health medical librarian. the full list of search terms can be found in the supplemental methods at the end of this review. results neuropathologic findings on us minority groups were often compared to nhws. for the purposes of this review, we will use latinos/hispanics as the collective term to describe this demographic unless a mentioned study further partitions out subgroups, in which the terms that are consistent with the study will be used. this same principle applies to the term black americans being used for mentions of this group unless the study denotes otherwise. details of selected main papers are within table 1 relating to neuropathological data of latinos/hispanics and/or black americans as many of these studies compared these groups. additional studies on asian americans are in table 2. click here to view a large version of this table. ad and vascular dementia neuropathology in latino/hispanics one observed neuropathological trend in latinos/hispanics was cerebrovascular pathologies, such as infarcts, caa, arteriolosclerosis, and atherosclerosis, that were typically associated with dementia and/or ad diagnoses (see table 1). a 2010 study using the national alzheimer’s coordinating center (nacc) database showed latinos/hispanics were more likely to have neurovascular pathology compared to nhws70. in persons with dementia during life, studies conducted at the alzheimer’s disease research centers (adrcs) at both university of california, davis (ucd) and the university of california, san diego (ucsd) reported a higher frequency of concomitant neurovascular pathologies compared to nhws29, 102. one comparison of interest between these studies with respect to cerebrovascular disease (cvd) pathologies—specifically microinfarcts and macroinfarcts—is filshtein et al. found occurrence was higher in latinos/hispanics compared to nhws in the ucd cohort29, while weissburger et al. discovered there were no significant differences between the two groups in their ucsd cohort102. this contradiction may be because persons with evidence of in-vivo hemorrhages, strokes, and other major agonal infarcts were excluded from the ucsd study102. a study also using the national alzheimer’s coordinating center database illustrated this theme in the specific context of caa, in which latino individuals with neuropathologically confirmed ad were more probable to have severe caa than nhws81. the study done by weissburger et al. also supported this trend102. another study by ucsd further validated this pattern by finding higher caa burden in the ad group compared to the no pathology group, which was defined as not having significant brain pathologies, and the non-ad pathology group was defined as only have tauopathies, frontotemporal dementia (ftd), progressive supranuclear palsy (psp), dementia with lewy bodies (dlb), or parkinson’s disease (pd) with neocortical lewy bodies, in an all-latino cohort91. as for hallmark ad pathologies, aβ plaques and nfts, there were more inconsistent patterns between latinos/hispanics and nhws. the study done by filshtein et al. utilizing demented cases from the alzheimer’s disease center at ucd, a california based cohort, revealed latinos/hispanics had the lowest occurrence of ad clinicopathological diagnosis without the involvement of cvd, including lower frequencies of persons at higher braak nft stage compared to nhws and black americans29. this is consistent with literature where latino cohorts tended to exhibit concomitant neurovascular pathologies with their ad diagnoses29, 102. conversely, santos et al., in a florida based cohort, demonstrated latinos/hispanics were twice as likely to have a higher braak nft stage than nhws86, while a study conducted by mehta et al., including cases with a clinical possible/probable diagnoses of ad, revealed braak nft stage did not differ significantly between latinos/hispanics and nhws70. the study by weissburger et al. also showed both groups (nhws and latinos/hispanics) had similar braak nft stage102. regarding plaques, which can include neuritic plaques (amyloid plaques containing dystrophic neurites) in some literature, mehta et al. found neuritic plaques were more frequent in latinos/hispanics compared to nhws70. however, the results by filshtein et al. revealed latinos/hispanics had the lowest proportion of cerad frequent neuritic plaque score, implying that neuritic plaques may not be as much of a major contributing pathology to their dementia29. santos et al., excluding persons that did not have autopsy confirmed ad and cases with known mutations, opted to use thal amyloid phase to categorize plaque presence, in which the phases did not differ between latinos/hispanics and nhws86. notably, latino/hispanic participants in these studies may represent diverse ethnic groups in terms of geography and nation of origin leading to seemingly contradictory findings. for instance, santos et al. had utilized the florida autopsies multi-ethnic (flame) cohort located at the mayo clinic of florida for their study, which consisted of individuals primarily from the caribbean origin for their latino group, whereas weissburger et al. and soria et al. had utilized cohorts from the ucsd adrc, which comprised of individuals primarily of mexican descent for their latino/hispanic group86, 91, 102. furthermore, these studies also had slightly different inclusion and exclusion criteria, as outlined in table 1, that may also contribute to discrepancies. ad and vascular dementia neuropathology in black americans like latinos/hispanics, cerebrovascular pathologies are also commonly observed in black americans, but the pattern is not completely consistent which may be due to cohort inclusion/exclusion criteria, demographic locations, and recruitment strategies. a study at the rush alzheimer’s disease clinical core, based in the chicago illinois area, revealed black decedents had significantly greater severity in both atherosclerosis and arteriolosclerosis when compared to nhws7. the study by filshtein et al. corroborates this finding, in which their results of persons with dementia demonstrated black participants had a higher proportion of cvd compared to nhws29. another study utilizing patient data from over 30 alzheimer’s disease centers across the country also found black americans were more likely to have had a contributing diagnosis of vascular dementia than nhws, although this study was based on small group numbers and did not account for center biases40. interestingly, mehta et al. revealed black americans had similar neurovascular pathology rates as nhws on autopsy, in contrast to the consensus of the other studies70. likewise, the results from a study on a cohort based in washington university’s alzheimer’s disease research center (adrc) also showed no differences in cerebrovascular infarcts between the nhw and black american participants107. multiple studies found caa burden did not differ significantly between black americans and nhws56, 81, 82. in contrast, graff-radford et al. observed black americans had significantly greater frequencies of caa in addition to the other vascular neuropathologies (i.e., infarcts, hemorrhages, arteriolosclerosis, atherosclerosis) in comparison to nhws40; however this study utilized data from multiple cohorts and did not control for center biases. for ad pathologies, there were also contradictions in the literature. findings from barnes et al. revealed black decedents were less likely to have ad-only pathology, defined by neuritic plaques and nfts as the single contributing pathology to their dementia diagnosis compared to nhw decedents7. along similar conclusions, filshtein et al. demonstrated mixed pathologies were more common in black decedents than in nhw decedents29. with respect to hallmark ad proteinopathy comparisons, such as neuritic and diffuse plaque counts, thal amyloid phase, and likelihood of higher braak nft stage, multiple studies showed no significant neuropathological differences in both categories for either patient demographic82, 85, 86, 107. in contrast, more than one study demonstrated black american decedents were more likely to exhibit higher braak nft stage29, 40, 70. graff-radford et al. also indicated black american participants had greater cerad-frequent scores for neuritic and diffuse plaques40, whereas mehta et al. revealed black americans had similar neuritic and diffuse plaque counts as nhws70. both studies had utilized data from the nacc database, but this conflict in findings may be due to the fact the mehta et al. study had a larger sample size from the longitudinal window from 1984 through 200570, compared to the graff-radford et al. study which recruited data from a smaller sample size from 2005 to 201540. as with the previous section, discrepancies may lie within cohort selection criteria as highlighted in table 1. ad and vascular dementia neuropathology in asian americans ad and vascular dementia pathological trends for asian americans compared to nhws are largely unexplored for all pathological categories as literature for this minority group in this specific context is still sparse. of the studies conducted, most have focused on japanese americans, specifically men, through the honolulu asian aging study (haas) which includes very few, if any other subgroups. the haas was established in 1991 and comprised surviving participants of the honolulu heart program, a prospective, community-based cohort study of heart disease and stroke established in 196536, 54, 93, 105. for neurovascular pathologies, a study revealed microinfarcts were significantly more common in japanese american men in haas compared to caucasian women in the nun study (ns)106. another finding from the haas showed that the frequency of microvascular lesions as the contributing dementia pathology was nearly the same as ad pathologies104; however, a later paper relative to this one showed that microvascular infarcts as the dominant or exclusive contributing lesion to dementia were the most frequent among decedents, then followed by ad lesions103. there were additional findings on the haas revealing dementia frequency increased with neuritic plaques in decedents with nfts and even further with cvd lesions76. interestingly, one analysis showed microinfarcts were strongly associated with poor cognitive function score in non-demented individuals, whereas nfts were strongly associated with poor cognitive function score in demented individuals59. for neuropathologic change involving ad proteinopathies, a more recent study showed that the haas was more resistant to aβ accumulation, but the ns was more resistant to neurofibrillary degeneration for individuals without aβ accumulation58. click here to view a large version of this table. native american, alaska native, native hawaiian, other groups, and points of further research the scope of this paper had focused on the neuropathology of the three largest minority groups of the us, as those were the demographics that offered adequate findings to collate into a cohesive and purposeful review. the current presented literature offers a foundation for ad and vascular dementia research in underrepresented us groups and seems to only expand each year (see figure 1), with more research being conducted on a greater variety of cohorts and sites. the existing findings are concentrated and substantial enough to serve as preliminary data for comparison of future findings, depending on the objective demographic. nonetheless, despite the upward trends of more ad and vascular dementia research being conducted in diverse cohorts, there are still many gaps that need to be filled and other demographics that need to be considered. for example, alaskan natives and american indians (native americans) constitute the fourth largest distinct (i.e. one race) population of the us4, yet there is a paucity of medical studies on persons of these backgrounds. this may be due to cultural aspects, where those who valued tradition (including religious beliefs) strongly advocated for the body to buried whole52. this dearth of information also applies to native hawaiians and other pacific islanders, despite being the fifth largest single race population and second fastest growing race in the country behind asian americans4, 45. the lack of neuropathology literature that captures the diversity of asian americans also highlights imbalances in research, as it is most probable the neuropathological trends of japanese americans would not accurately encompass the depth and breadth of diversity of persons across the asian continent. an overall paucity of literature presently exists in comprehensive studies centering on these mentioned groups and is not limited to specifically neuropathology studies. figure 1. number of papers found on pubmed as of november 19, 2021 by year using the search terms “alzheimer’s brain pathology” along with the demographic term (categories in legend, see supplemental section for further information). while there has been advances in neuropathology literature focused on latinos/hispanics and black americans in recent years compared to other minority groups, there are still limited participants in these cohorts. the existing literature is limited in sample size, which varies widely between studies and typically with the minority groups representing a small fraction of the cohort (see tables 1 and 2). furthermore, studies can have certain inclusion and exclusion criteria that may hinder participation in select groups; for example, exclusion of cvd for ad studies may decrease frequencies of certain minority groups with higher frequencies of cvd50. these constraints from low minority group recruitment may be due to numerous factors, including lack of access to healthcare, historical abuses of minority groups for medical research, mistrust of the healthcare system making participants less likely to agree to participate in clinical trials or autopsy programs, and language barriers5, 8, 10, 11, 30, 31, 37, 49, 75, 100. with respect to retention, a systematic review highlights a lack of literature that examines retention exclusively from recruitment39. socioeconomic circumstance was shown to be the most powerful contributor to the absence of participants for longitudinal studies involving ethnoracial minorities26. low socioeconomic status largely impacts access to health care resources such as regular visits to a health professional as a result of being uninsured10, 87, 88, in which patients may not only lack the direct care they need but also the general awareness of clinical study enrollment opportunities. flexible scheduling played a substantial role in participation as many individuals were restrained by work or childcare obligations for their appointments as well as transportation26, 32, 69. financial compensation was a major influencer in recruitment amongst ethnoracial minorities51, 69; it has been reported that members of the latino/hispanic community were motivated by monetary compensation for their time because they experienced economic hardships69. patients may also feel discrimination in the process of seeking care, especially among non-whites for their race, color, and/or ethnicity35. half of black americans report they have faced healthcare discrimination, and one third of asian americans and latino americans similarly report having experienced healthcare discrimination as well35. a vast majority of non-white americans believe that in the importance of having ad and dementia care providers to understand their ethnic/racial backgrounds, such as native americans, black americans, latinos/hispanics, and asian americans35. however, less than half of black and native americans are confident there are culturally competent providers, and only roughly 3 in 5 asian americans and latinos/hispanics are confident35. a few studies have attempted to understand barriers and willingness for brain donation across major us racial ethnic groups: nhws, latinos/hispanics, black americans, and asian americans11, 12. while conducting focus groups, the first study revealed concerns, attitudes and beliefs around brain donation that fell into three categories: 1) religious beliefs 2) concerns and misconceptions about brain research and 3) the role of the family7. a follow up study surveying nhws as well as 169 african americans, 50 asians, and 61 hispanics revealed older age, latino ethnicity and understanding of brain use by researchers and what participants need to do to ensure brain is donated were positive predictive factors, while the belief that the body should remain whole at burial, african/african american race, and concern researchers might not be respectful of the body during autopsy were negative predictive factors11, 12. the belief that the body should remain whole was shared amongst latinos/hispanics, african americans, and asian americans, which was a similar sentiment of alaskan natives and american indians mentioned earlier52. this further illustrates the substantial role cultural barriers may play in cohort participation from us minority groups. knowledge, stigma, and apprehension of adrd also differ across ethnic/racial groups. for example, one study revealed that nhws tended to have greater knowledge about ad compared to black americans, and black americans had same or greater levels of concern about getting ad as nhws depending on their geographic location47. another study discovered concern about developing adrd in native americans, black americans, and latinos/hispanics is noticeably lower compared to nhws35, which contradicts the finding about black americans in the aforementioned study, possibly due to region differences where the data was taken. it has also been shown that asian americans do not exhibit a strong concern of adrd as many believed it was a natural occurrence for aging people14. multiple papers have denoted that asian americans had beliefs of stigma of persons with ad, which played a significant role in seeking care from primary care providers for ad14, 19, 60. limited knowledge on not only adrd but also the brain removal process poses some hesitance on minority subject participation8, 11. as stated above, some themes that subjects or family members of subjects shared skepticism on were understanding the purpose of studying a decedent’s brain, misconceptions on how the brain is used or collected for research, and overall knowledge about the brain donation procedure11. it is important to recognize the existing inadequacies and confines of the study recruitment process for us minority groups to further advance the representation of these populations in biomedical research. fortunately, there has been progress to minimize these barriers. the uc davis adc utilized many avenues to increase diversity in enrollment in research cohorts, such as satellite clinic sites, increasing face to face screening at community events, options of in-home visits, compensation for transportation to clinic visits, dedicated drivers to transport participants to visits, and employing bicultural and bilingual individuals with proficiency of the involved populations44. these methods facilitated a substantial increase in the number of ethnic minority participants, as much as a four-fold increase44; this approach also led to more diversity in other variables as well, such as educational background44. a later study showed that mailing recruitment letters was the most successful method in a multi-modal recruitment approach in enrolling more ethnoracial minorities for adrd cohorts80. as these issues get addressed on a more widespread scale, significant advancements can be made not only in the field of neuropathology, but all fields of clinical research. acknowledgements this work was supported by the national institute on aging of the national institutes of health under award numbers ag062517, ag052132, ag050782 and ag056519, and supported by the california department of public health alzheimer’s disease program (grant # 19-10611) with partial funding from the 2019 california budget act. the views and opinions expressed in this manuscript are those of the author and do not necessarily reflect the official policy or position of any public health agency of california or of the united states government. references in: anderson nb, bulatao ra, cohen b, eds. critical perspectives on racial and ethnic differences in health in late life. washington (dc)2004. 2020 profile of older americans: administration for community living, may 2021. hispanic population and origin in select u.s. metropolitan areas, 2014 [online]. available at: https://www.pewresearch.org/hispanic/interactives/hispanic-population-in-select-u-s-metropolitan-areas/. accessed 19 november 2021. race and ethnicity in the united states: 2010 census and 2020 census [online]. available at: https://www.census.gov/library/visualizations/interactive/race-and-ethnicity-in-the-united-state-2010-and-2020-census.html. accessed 19 november 2021. amorrortu rp, arevalo m, vernon sw, et al. recruitment of racial and ethnic minorities to clinical trials conducted within specialty clinics: an intervention mapping approach. trials 2018;19:115. babulal gm, quiroz yt, albensi bc, et al. perspectives on ethnic and racial disparities in alzheimer's disease and related dementias: update and areas of immediate need. alzheimers dement 2019;15:292-312. barnes ll, leurgans s, aggarwal nt, et al. mixed pathology is more likely in black than white decedents with alzheimer dementia. neurology 2015;85:528-534. bilbrey ac, humber mb, plowey ed, et al. the impact of latino values and cultural beliefs on brain donation: results of a pilot study to develop culturally appropriate materials and methods to increase rates of brain donation in this under-studied patient group. clin gerontol 2018;41:237-248. blevins bl, vinters hv, love s, et al. brain arteriolosclerosis. acta neuropathol 2021;141:1-24. blustein j, weiss lj. visits to specialists under medicare: socioeconomic advantage and access to care. j health care poor underserved 1998;9:153-169. boise l, hinton l, rosen hj, ruhl m. will my soul go to heaven if they take my brain? beliefs and worries about brain donation among four ethnic groups. gerontologist 2017;57:719-734. boise l, hinton l, rosen hj, et al. willingness to be a brain donor: a survey of research volunteers from 4 racial/ethnic groups. alzheimer dis assoc disord 2017;31:135-140. braak h, braak e. neuropathological stageing of alzheimer-related changes. acta neuropathol 1991;82:239-259. braun kl, browne cv. perceptions of dementia, caregiving, and help seeking among asian and pacific islander americans. health soc work 1998;23:262-274. budiman a, ruiz ng. key facts about asian americans, a diverse and growing population [online]. available at: https://www.pewresearch.org/fact-tank/2021/04/29/key-facts-about-asian-americans/. accessed 30 november 2021. budiman a, ruiz ng. key facts about asian origin groups in the u.s. [online]. available at: https://www.pewresearch.org/fact-tank/2021/04/29/key-facts-about-asian-origin-groups-in-the-u-s/. accessed 25 january 2022. burns a, iliffe s. dementia. bmj 2009;338:b75. bustamante ln, hugo-lopez m, krogstad jm. u.s. hispanic population surpassed 60 million in 2019, but growth has slowed [online]. available at: https://www.pewresearch.org/fact-tank/2020/07/07/u-s-hispanic-population-surpassed-60-million-in-2019-but-growth-has-slowed/. accessed october 17 2020. casado bl, hong m, lee se. attitudes toward alzheimer's care-seeking among korean americans: effects of knowledge, stigma, and subjective norm. gerontologist 2018;58:e25-e34. cavazzoni p. fda's decision to approve new treatment for alzheimer's disease [online]. available at: https://www.fda.gov/drugs/news-events-human-drugs/fdas-decision-approve-new-treatment-alzheimers-disease. accessed 7 june 2021. clark cm, decarli c, mungas d, et al. earlier onset of alzheimer disease symptoms in latino individuals compared with anglo individuals. arch neurol 2005;62:774-778. cummings j, ritter a, zhong k. clinical trials for disease-modifying therapies in alzheimer's disease: a primer, lessons learned, and a blueprint for the future. j alzheimers dis 2018;64:s3-s22. deramecourt v, slade jy, oakley ae, et al. staging and natural history of cerebrovascular pathology in dementia. neurology 2012;78:1043-1050. dubois b, feldman hh, jacova c, et al. revising the definition of alzheimer's disease: a new lexicon. lancet neurol 2010;9:1118-1127. dugger bn, dickson dw. pathology of neurodegenerative diseases. cold spring harb perspect biol 2017;9. ejiogu n, norbeck jh, mason ma, cromwell bc, zonderman ab, evans mk. recruitment and retention strategies for minority or poor clinical research participants: lessons from the healthy aging in neighborhoods of diversity across the life span study. gerontologist 2011;51 suppl 1:s33-45. ennis sr, rios-vargas m, albert ng. the hispanic population: 2010: u.s. census bureau, 2011 may. filshtein tj, brenowitz wd, mayeda er, et al. reserve and alzheimer's disease genetic risk: effects on hospitalization and mortality. alzheimers dement 2019;15:907-916. filshtein tj, dugger bn, jin lw, et al. neuropathological diagnoses of demented hispanic, black, and non-hispanic white decedents seen at an alzheimer's disease center. j alzheimers dis 2019;68:145-158. frates j, garcia bohrer g. hispanic perceptions of organ donation. prog transplant 2002;12:169-175. gamble vn. under the shadow of tuskegee: african americans and health care. am j public health 1997;87:1773-1778. gamboa cj, julion wa. proactive recruitment of older african-americans for alzheimer's research with brain donation: a cohort case study of success. j racial ethn health disparities 2021;8:463-474. garcia ma, saenz j, downer b, wong r. the role of education in the association between race/ethnicity/nativity, cognitive impairment, and dementia among older adults in the united states. demogr res 2018;38:155-168. gates hlj. in search of our roots: how 19 extraordinary african americans reclaimed their past. new york: crown publishing, 2009. gaugler j jb, johnson t, reimer j, and weuve j. . 2021 alzheimer's disease facts and figures. alzheimers dement 2021;17:327-406. gelber rp, launer lj, white lr. the honolulu-asia aging study: epidemiologic and neuropathologic research on cognitive impairment. curr alzheimer res 2012;9:664-672. george s, duran n, norris k. a systematic review of barriers and facilitators to minority research participation among african americans, latinos, asian americans, and pacific islanders. am j public health 2014;104:e16-31. giaccone g, arzberger t, alafuzoff i, et al. new lexicon and criteria for the diagnosis of alzheimer's disease. lancet neurol 2011;10:298-299; author reply 300-291. gilmore-bykovskyi al, jin y, gleason c, et al. recruitment and retention of underrepresented populations in alzheimer's disease research: a systematic review. alzheimers dement (n y) 2019;5:751-770. graff-radford nr, besser lm, crook je, kukull wa, dickson dw. neuropathologic differences by race from the national alzheimer's coordinating center. alzheimers dement 2016;12:669-677. gurland bj, wilder de, lantigua r, et al. rates of dementia in three ethnoracial groups. int j geriatr psychiatry 1999;14:481-493. haan mn, mungas dm, gonzalez hm, ortiz ta, acharya a, jagust wj. prevalence of dementia in older latinos: the influence of type 2 diabetes mellitus, stroke and genetic factors. j am geriatr soc 2003;51:169-177. hill cv, perez-stable ej, anderson na, bernard ma. the national institute on aging health disparities research framework. ethn dis 2015;25:245-254. hinton l, carter k, reed br, et al. recruitment of a community-based cohort for research on diversity and risk of dementia. alzheimer dis assoc disord 2010;24:234-241. hixson l, hepler b, kim mo. native hawaiian and other pacific islander population: u.s. census bureau, 2012 may. hoeffel em, rastogi s, kim mo, shahid h. the asian population 2010: u.s. census bureau, 2012 march. howell jc, soyinka o, parker m, et al. knowledge and attitudes in alzheimer's disease in a cohort of older african americans and caucasians. am j alzheimers dis other demen 2016;31:361-367. hyman bt, phelps ch, beach tg, et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease. alzheimers dement 2012;8:1-13. ighodaro et, nelson pt, kukull wa, et al. challenges and considerations related to studying dementia in blacks/african americans. j alzheimers dis 2017;60:1-10. indorewalla kk, o'connor mk, budson ae, guess diterlizzi c, jackson j. modifiable barriers for recruitment and retention of older adults participants from underrepresented minorities in alzheimer's disease research. j alzheimers dis 2021;80:927-940. jefferson al, lambe s, chaisson c, palmisano j, horvath kj, karlawish j. clinical research participation among aging adults enrolled in an alzheimer's disease center research registry. j alzheimers dis 2011;23:443-452. jernigan m, fahrenwald n, harris r, tsosie u, baker lo, buchwald d. knowledge, beliefs, and behaviors regarding organ and tissue donation in selected tribal college communities. j community health 2013;38:734-740. jones n, marks r, ramirez r, rios-vargas m. 2020 census illuminates racial and ethnic composition of the country [online]. available at: https://www.census.gov/library/stories/2021/08/improved-race-ethnicity-measures-reveal-united-states-population-much-more-multiracial.html. accessed 9 november 2021. kagan a. the honolulu heart program : an epidemiological study of coronary heart disease and stroke. amsterdam: harwood academic, 1996. kalaria rn, kenny ra, ballard cg, perry r, ince p, polvikoski t. towards defining the neuropathological substrates of vascular dementia. j neurol sci 2004;226:75-80. kamara dm, gangishetti u, gearing m, et al. cerebral amyloid angiopathy: similarity in african-americans and caucasians with alzheimer's disease. j alzheimers dis 2018;62:1815-1826. kapasi a, decarli c, schneider ja. impact of multiple pathologies on the threshold for clinically overt dementia. acta neuropathol 2017;134:171-186. latimer cs, keene cd, flanagan me, et al. resistance to alzheimer disease neuropathologic changes and apparent cognitive resilience in the nun and honolulu-asia aging studies. j neuropathol exp neurol 2017;76:458-466. launer lj, hughes tm, white lr. microinfarcts, brain atrophy, and cognitive function: the honolulu asia aging study autopsy study. ann neurol 2011;70:774-780. liu d, hinton l, tran c, hinton d, barker jc. reexamining the relationships among dementia, stigma, and aging in immigrant chinese and vietnamese family caregivers. j cross cult gerontol 2008;23:283-299. livney mg, clark cm, karlawish jh, et al. ethnoracial differences in the clinical characteristics of alzheimer's disease at initial presentation at an urban alzheimer's disease center. am j geriatr psychiatry 2011;19:430-439. lopez mh, krogstad jm, passel js. who is hispanic? [online]. available at: https://www.pewresearch.org/fact-tank/2021/09/23/who-is-hispanic/. accessed 17 october 2021. love s, chalmers k, ince p, et al. development, appraisal, validation and implementation of a consensus protocol for the assessment of cerebral amyloid angiopathy in post-mortem brain tissue. am j neurodegener dis 2014;3:19-32. marks r, jones n. collecting and tabulating ethnicity and race responses in 2020 census: u.s. census bureau, 2020. martinez de, gonzalez ke. “latino” or “hispanic”? the sociodemographic correlates of panethnic label preferences among u.s. latinos/hispanics. sociological perspectives 2021;64:365-386. matthews ka, xu w, gaglioti ah, et al. racial and ethnic estimates of alzheimer's disease and related dementias in the united states (2015-2060) in adults aged >/=65 years. alzheimers dement 2019;15:17-24. mayeda er, glymour mm, quesenberry cp, jr., whitmer ra. heterogeneity in 14-year dementia incidence between asian american subgroups. alzheimer dis assoc disord 2017;31:181-186. mayeda er, glymour mm, quesenberry cp, whitmer ra. inequalities in dementia incidence between six racial and ethnic groups over 14 years. alzheimers dement 2016;12:216-224. mcdougall gj, jr., simpson g, friend ml. strategies for research recruitment and retention of older adults of racial and ethnic minorities. j gerontol nurs 2015;41:14-23; quiz 24-15. mehta km, yaffe k, perez-stable ej, et al. race/ethnic differences in ad survival in us alzheimer's disease centers. neurology 2008;70:1163-1170. mirra ss, heyman a, mckeel d, et al. the consortium to establish a registry for alzheimer's disease (cerad). part ii. standardization of the neuropathologic assessment of alzheimer's disease. neurology 1991;41:479-486. moon h, badana ans, hwang sy, sears js, haley we. dementia prevalence in older adults: variation by race/ethnicity and immigrant status. am j geriatr psychiatry 2019;27:241-250. napoles am, chadiha la, resource centers for minority aging r. advancing the science of recruitment and retention of ethnically diverse populations. gerontologist 2011;51 suppl 1:s142-146. pariona a. what is the difference between race and ethnicity? [online]. available at: https://www.worldatlas.com/articles/what-is-the-difference-between-race-and-ethnicity.html. accessed 14 june 2021. peters tg, kittur ds, mcgaw lj, roy mrs, nelson ew. organ donors and nondonors. an american dilemma. arch intern med 1996;156:2419-2424. petrovitch h, ross gw, steinhorn sc, et al. ad lesions and infarcts in demented and non-demented japanese-american men. ann neurol 2005;57:98-103. proctor bd, semega jl, kollar ma. income and poverty in the united states: 2015. us census bureau: u.s. census bureau, 2016 september. querfurth hw, laferla fm. alzheimer's disease. n engl j med 2010;362:329-344. rajan kb, weuve j, barnes ll, wilson rs, evans da. prevalence and incidence of clinically diagnosed alzheimer's disease dementia from 1994 to 2012 in a population study. alzheimers dement 2019;15:1-7. reuland m, sloan d, antonsdottir im, spliedt m, johnston mcd, samus q. recruitment of a diverse research cohort in a large metropolitan area for dementia intervention studies. contemp clin trials 2022;112:106622. ringman jm, sachs mc, zhou y, monsell se, saver jl, vinters hv. clinical predictors of severe cerebral amyloid angiopathy and influence of apoe genotype in persons with pathologically verified alzheimer disease. jama neurol 2014;71:878-883. riudavets ma, rubio a, cox c, rudow g, fowler d, troncoso jc. the prevalence of alzheimer neuropathologic lesions is similar in blacks and whites. j neuropathol exp neurol 2006;65:1143-1148. roman gc, tatemichi tk, erkinjuntti t, et al. vascular dementia: diagnostic criteria for research studies. report of the ninds-airen international workshop. neurology 1993;43:250-260. rucker wc. the river flows on: black resistance, culture, and identity formation in early america: lsu press., 2006. sandberg g, stewart w, smialek j, troncoso jc. the prevalence of the neuropathological lesions of alzheimer's disease is independent of race and gender. neurobiol aging 2001;22:169-175. santos oa, pedraza o, lucas ja, et al. ethnoracial differences in alzheimer's disease from the florida autopsied multi-ethnic (flame) cohort. alzheimers dement 2019;15:635-643. schiller js, lucas jw, ward bw, peregoy ja. summary health statistics for u.s. adults: national health interview survey, 2010. vital health stat 10 2012:1-207. schneider ls. drug development, clinical trials, cultural heterogeneity in alzheimer disease: the need for pro-active recruitment. alzheimer dis assoc disord 2005;19:279-283. shakir mn, dugger bn. advances in deep neuropathological phenotyping of alzheimer disease: past, present, and future. j neuropathol exp neurol 2022;81:2-15. skrobot oa, black se, chen c, et al. progress toward standardized diagnosis of vascular cognitive impairment: guidelines from the vascular impairment of cognition classification consensus study. alzheimers dement 2018;14:280-292. soria ja, huisa bn, edland sd, et al. clinical-neuropathological correlations of alzheimer's disease and related dementias in latino volunteers. j alzheimers dis 2018;66:1539-1548. strozyk d, dickson dw, lipton rb, et al. contribution of vascular pathology to the clinical expression of dementia. neurobiol aging 2010;31:1710-1720. syme sl, marmot mg, kagan a, kato h, rhoads g. epidemiologic studies of coronary heart disease and stroke in japanese men living in japan, hawaii and california: introduction. am j epidemiol 1975;102:477-480. tamir c. key findings about black america [online]. available at: https://www.pewresearch.org/fact-tank/2021/03/25/key-findings-about-black-america/. accessed 14 june 2021. tamir c, budiman a, noe-bustamante l, mora l. facts about the u.s. black population [online]. available at: https://www.pewresearch.org/social-trends/fact-sheet/facts-about-the-us-black-population/. accessed 14 june 2021. tang mx, cross p, andrews h, et al. incidence of ad in african-americans, caribbean hispanics, and caucasians in northern manhattan. neurology 2001;56:49-56. thal dr, rub u, orantes m, braak h. phases of a beta-deposition in the human brain and its relevance for the development of ad. neurology 2002;58:1791-1800. tishkoff sa, kidd kk. implications of biogeography of human populations for 'race' and medicine. nat genet 2004;36:s21-27. vargas bustamante a, chen j, rodriguez hp, rizzo ja, ortega an. use of preventive care services among latino subgroups. am j prev med 2010;38:610-619. vega ie, cabrera ly, wygant cm, velez-ortiz d, counts se. alzheimer's disease in the latino community: intersection of genetics and social determinants of health. j alzheimers dis 2017;58:979-992. vinters hv, zarow c, borys e, et al. review: vascular dementia: clinicopathologic and genetic considerations. neuropathol appl neurobiol 2018;44:247-266. weissberger gh, gollan th, bondi mw, et al. neuropsychological deficit profiles, vascular risk factors, and neuropathological findings in hispanic older adults with autopsy-confirmed alzheimer's disease. j alzheimers dis 2019;67:291-302. white l. brain lesions at autopsy in older japanese-american men as related to cognitive impairment and dementia in the final years of life: a summary report from the honolulu-asia aging study. j alzheimers dis 2009;18:713-725. white l, petrovitch h, hardman j, et al. cerebrovascular pathology and dementia in autopsied honolulu-asia aging study participants. ann n y acad sci 2002;977:9-23. white l, petrovitch h, ross gw, et al. prevalence of dementia in older japanese-american men in hawaii: the honolulu-asia aging study. jama 1996;276:955-960. white lr, edland sd, hemmy ls, et al. neuropathologic comorbidity and cognitive impairment in the nun and honolulu-asia aging studies. neurology 2016;86:1000-1008. wilkins ch, grant ea, schmitt se, mckeel dw, morris jc. the neuropathology of alzheimer disease in african american and white individuals. arch neurol 2006;63:87-90. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurovascular disease: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:15 (2022) review neurovascular disease: 2022 update louise d. mccullough department of neurology, mcgovern medical school, uthealth houston and memorial hermann hospital, houston, texas, usa corresponding author: louise d. mccullough · department of neurology · mcgovern medical school · uthealth houston and memorial hermann hospital · houston, texas · usa louise.d.mccullough@uth.tmc.edu submitted: 25 april 2022 accepted: 07 june 2022 copyedited by: félicia jeannelle published: 14 june 2022 https://doi.org/10.17879/freeneuropathology-2022-3910 keywords: stroke, vascular malformations, microglia, t cells, neuroinflammation abstract in this update we present a series of papers focused on topics that have emerged in vascular disease over the prior year. the first two papers focus on the pathogenesis of vascular malformations, the first on brain arteriovenous malformations, and the second on cerebral cavernous malformations. these disorders can lead to significant brain injuries from intracerebral hemorrhage (if they rupture) or other neurological complications, including seizures. the next set of papers reflects work that has advanced our understanding of how the brain and the immune system “communicate” after brain injury, including stroke (papers 3-6). the first of these shows that t cells are involved in white matter repair after ischemic injury, an effect dependent on microglia, demonstrating the important cross-talk between innate and adaptive immunity. the next two papers focus on b cells, which have been relatively understudied in the context of brain injury. the contribution of antigen-experienced b cells from the meninges and skull bone marrow, rather than blood-derived b cells in neuroinflammation opens up a very novel area of investigation. the possibility that antibody secreting b cells may contribute to vascular dementia will certainly be an active area for future investigations. similarly, in paper 6, investigators found that cns-infiltrating myeloid cells can originate from brain borders tissues. these cells have unique transcriptional signatures that are distinct from their blood-derived counterparts, and likely contribute to myeloid cell infiltration from bone-marrow niches in close proximity to the brain. the contribution of microglia, the primary innate immune cell of the brain, to amyloid deposition and propagation is then discussed, followed by work on how perivascular aβ is potentially cleared along the cerebral vessels in patients with cerebral amyloid angiopathy. the final two papers focus on the contribution of senescent endothelial cells and pericytes. the first used a model of accelerated senescence (hutchinson-gilford progeria syndrome; hgps) and shows the translational potential of an approach to reduce telomere shortening to slow aging. the final paper demonstrates how capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain. interestingly, several of the papers identified therapeutic strategies that could be potentially translated into clinical populations. introduction this “neurovascular disease: 2022 update” presents topics that were selected as the top 10 papers and discoveries published in the field of neurovascular disorders in 2021. these papers were selected based on their broad appeal, those with new methods or discoveries, and those that have a direct link to neurovascular inflammation, vascular abnormalities and neuroimmunology. it is important to note that these top ten papers were subjectively selected by many individuals when queried regarding what papers they felt were most impactful in 2021 (individual contributors are listed in the acknowledgement section). thus, there is certainly considerable “selection bias”. these papers were chosen by a large group of neurosurgery and neurology faculty, and are not ranked in any specific order or by number of citations. overall, the theme of the selected papers reflects our growing understanding of how immunological events, both in the brain and in the periphery, alter the response to vascular and degenerative diseases. the field of neuroimmunology has seen tremendous growth over the past decade, and this is reflected in the papers selected. we apologize to all the authors we could not highlight due to space limitations. topic 1: selective endothelial hyperactivation of oncogenic kras induces brain arteriovenous malformations in mice brain arteriovenous malformations (bavms) are a challenging cerebrovascular disease, characterized by tangled/enlarged vessels occurred by direct connections between arteries and veins without intervening capillary bed. bavms are a major cause of the most debilitating spontaneous intracerebral hemorrhage in children and young adults. however, the pathogenesis of the disease is largely unknown, and the treatment options for bavm patients are severely limited mostly relying on risky surgical methods. recently, a clinical study has reported that somatic activating kras mutations were detected in the endothelium of ~60% of human sporadic bavms (1). in addition, mosaic variants have also been found in the braf and map2k1/mek in intracranial or extracranial avms, especially in high-flow vascular malformations (2). these observations suggest the critical role of kras signaling, especially raf/mek/erk kinase cascade in avm pathogenesis. based on this evidence, the authors tested the role of kras mutation in bavm development using an adeno-associated virus (aav)-br1 specifically targeting brain vascular endothelial cells. strikingly, the systemic administration of aav-br1 encoding human krasg12v gene (aav-br1-krasg12v) induced vascular malformations in the mouse brains that recapitulates salient features of human bavms including the tangled nidus connected with feeding arteries and draining veins, multi-focal spontaneous intracerebral hemorrhages, ambiguous arterial/venous identity and irregular/incomplete mural cell coverage, and enhanced proliferating endothelial cells and angiogenic factors. the accumulation of activated microglia/macrophages, enhanced inflammatory response, and neuronal death were observed around bavms, and mice with bavm showed impaired behavioral functions. the study found the erk activation, but not pakt and p38, in the bavms suggesting that mek/erk signaling is the key mediator for the mutant kras-induced bavm development, and finally revealed that trametinib (an fda-approved mek inhibitor for cancer) attenuates the growth of krasg12v-induced bavms (figure 1). the novel approach by park et al. (3) clearly confirmed the causative role of kras mutations in bavm development, and generated a novel/clinically-relevant mouse model of bavms that will be useful to define the underlying pathomechanism in bavms. this is the first preclinical study showing the efficacy of pharmacological intervention in sporadic bavms associated with kras mutation providing a potential therapeutic strategy for bavm patients by repurposing a cancer drug. figure 1. the characteristics of mouse bavms induced by brain endothelial krasg12v overexpression. systemic injection of aav-br1 carrying krasg12v (aav-br1-krasg12v)-induced abnormal vasculatures that recapitulate the salient features of human bavms including tangled nidus, irregular mural cell coverages, and spontaneous hemorrhages. enhanced endogenous vascular endothelial growth factor (vegf) signaling and endothelial cell proliferation were observed in the bavms. accumulating activated microglia were seen around bavms and accompanied with increased expression of inflammatory factors (e.g., cytokines, matrix metalloproteinases) and apoptotic neurons. mice with bavm showed significantly impaired behavioral function. prolonged treatment with trametinib, an fda-approved mek inhibitor (for six weeks from one day after the aav-br1-krasg12v) inhibited bavm growth suggesting the mek/erk signaling is critical in bavm development induced by kras mutation (park et al., 2021). topic 2: pik3ca and ccm mutations fuel cavernomas through a cancer-like mechanism another interesting work, also on the topic of brain vascular malformations, comes from the laboratory of dr. khan (4). in this work the authors focused on cerebral cavernous malformations (ccms) rather than the avms explored in the prior manuscript. ccms develop due to inactivation of the endothelial ccm protein complex, which is needed to decrease the activity of the kinase mekk3. these malformations can lead to intracerebral hemorrhages and seizures, and often affect younger individuals. the clinical spectrum of disease presentation, including growth and rupture, varies between individuals. some ccms are clinically silent, whereas others grow rapidly and are at risk for repeated hemorrhages. identifying which subset of ccms are more likely to become symptomatic is important, as surgical resection may be warranted. however, surgery has its own risks, and patients often have multiple lesions. genetic studies have implicated a monogenic basis for ccm disease associated with biallelic loss of function mutations in three genes, krit1, ccm2, and pdcd10, that encode the components of a heterotrimeric ccm protein complex, as described by others. in this work, the authors demonstrate that the growth of ccms requires increased signaling through the phosphatidylinositol-3-kinase (pi3k)-mtor pathway as well as loss of function of the ccm complex. this work identified that symptomatic ccm disease arises through a cancer-like program, with the accumulation of multiple somatic mutations in the same cell. this results in both the loss of a vascular malformation suppressor gene (i.e., the ccm gene) and the gain of vascular malformation growth gene (i.e. pik3ca). the authors found somatic gain-of-function mutations in pik3ca and loss-of-function mutations in the ccm complex in the same cells in the majority of human ccms. they then moved to mouse models and found that the growth of ccms requires both pi3k gain of function (gof) and ccm loss of function (lof) in endothelial cells, as both were required for ccm development. both ccm loss of function and increased expression of the transcription factor klf4 (a downstream effector of mekk3) increased mtor signaling in endothelial cells. importantly, the authors then went on to utilize rapamycin, a mtorc1 inhibitor in mice. as single dose given at p2 reduced ccm formation by 75%. in adult mice, chronic administration of rapamycin effectively blocked the formation of ccms in mouse models. this work suggests that there is a “triple-hit” mechanism involving the acquisition of as many as three distinct genetic mutations that culminate in ccm lof and pik3ca gof and contribute to rapidly growing, clinically symptomatic ccms. this three-hit mechanism is analogous to cancer, in which aggressive vascular malformations arise through both the loss of vascular “suppressor genes” that limit vessel growth and gain of function of a vascular “oncogene” that then stimulates excess vascular growth. importantly, these findings suggest that aggressive ccms may be treatable using clinically approved mtorc1 inhibitors. topic 3: treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke in this paper (5) the role of regulatory t cells (treg) in stroke recovery was examined. shi et al. sorted infiltrating immune cells from the ischemic brain at 5 and 14 days after transient middle cerebral artery occlusion (mcao). the numbers of t cells increased robustly from 5 to 14 days after stroke, suggesting a possible role of these cells in stroke pathophysiology and recovery. the authors subclustered and more deeply assessed the 14-day t cell population. seven subclusters were identified, including cd4+ treg cells, cd4+ memory t cells, natural killer t (nkt) cells, macrophage-like t cells, and populations of cd8+ t effector cells. the distinct treg cell subcluster expressed transcripts of canonical treg cell markers such as cd4, il2ra (cd25), foxp3, and ikzf2 (helios), which is essential for t cell regulatory function. temporal changes in the cd3+ cd4+ cd25+foxp3+ treg cell population were assessed in the ischemic brain using flow cytometry at different time points (3, 5, 7, 14, and 35 days). minimal treg cell infiltration was observed 3 and 5 days after mcao. the number of infiltrating treg cells significantly increased from 7 days onward, increasing until at least 35 days post stroke. the visualization of t-distributed stochastic neighbor embedding (visne) map of flow cytometry data revealed a prominent treg cell population among all cd4+ t cells in single-cell brain suspensions collected 14 days after stroke. these cells expressed cd25 and foxp3. consistent with recent studies, the majority of brain-infiltrating treg cells were found to be helios+, a population recently identified as phenotypically active treg cells. these cells accumulated near white matter. higher percentages of infiltrating treg cells in the ischemic brain expressed osteopontin (opn) compared with circulating treg cells from sham or stroke mice. bioinformatic analyses predicted strong interactions between treg cells and microglia through opn and integrin receptors, which were then confirmed experimentally by a series of in vivo and in vitro studies. the authors then selectively depleted tregs using a diphtheria toxin method and found that loss of these cells impaired motor recovery and reduced myelin coverage, which was confirmed by whole brain ex vivo diffusion tensor imaging and electron microscopy. the beneficial effects of treg cells were linked to oligodendrogenesis that was dependent on microglia. microglia depletion, but not t cell lymphopenia, mitigated the beneficial effects of transferred treg cells on white matter regeneration. mechanistically, treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. these results confirm that treg cell-microglia interactions enhance a reparative microglial phenotype that promotes opc differentiation. therefore, treg cells appear to contribute to brain repair via both indirect immunomodulation of microglial responses as well as direct trophic effects. boosting treg cell numbers could be a practical approach to improve wm repair and functional recovery after stroke. topic 4: heterogeneity of meningeal b cells reveals a lymphopoietic niche at the cns borders meningeal immune cells are thought to originate from the systemic blood circulation. however, recent findings of developmentally immature b cells in the mouse central nervous system question this assumption. brioschi et al. (6) used single cell analysis from parabiotic and chimeric mouse models to demonstrate that the skull bone marrow contains a hematopoietic niche from which extravascular meningeal b cells can arise to participate in the local immune response. meningeal b cells are capable of trafficking through meningeal lymphatics to the cervical lymph nodes, enabling them to participate in antigen presentation linking brain and systemic immunity. these meningeal b cells are composed of a heterogeneous population with respect to their developmental and maturity stage, with late (b220lowcd43low), and mature (b220highcd43−) subsets representing over 80% of the total b cell population. this was confirmed by single cell rna sequencing (scrna-seq) and cytof mass spectrometry. scrna-seq analysis showed that the meningeal and skull bone marrow b cells exhibited overlapping transcriptomic phenotypes, and that these were distinct from the blood-sourced b cells. targeted gamma irradiation of the skull followed by reconstitution with donor bone marrow cells was used to generate a skull bone marrow transplantation model to investigate the origin of the meningeal b cells. the skull bone marrow chimera experiments confirmed that the meningeal b cells originate from the skull. taken together, this study showed that skull bone marrow provides an accessible source of b cells that are locally educated by cns-derived antigens. lastly, scrnaseq data showed an age-dependent accumulation of blood-derived antigen-experienced b cells (identified as b220highcd23−cd2+sca1+ cells) in the meninges. meninges from aged animals (20-25 months of age) housed a significant population of igm+ and igg+ plasma cells, indicating their antigen-experienced status. these specific “age-associated b cells” (abcs) accumulating in the meninges with aging were found to originate from the peripheral circulation, and represent a separate pool of b cells from those derived from the calvaria. given the importance of b cells in the regulation of neuroinflammation and autoimmunity, meningeal b cells may be a critical subset of the adaptive immune cells, uniquely positioned to contribute to the pool of self-reactive b cells seen with aging (see next paper). topic 5: single-cell profiling of cns border compartment leukocytes reveals that b cells and their progenitors reside in non-diseased meninges central nervous system (cns) meninges (dura, arachnoid, and pia) surround the cns tissue at its critical borders. recent studies examining the dural lymphatics have highlighted the importance of these cns interfaces and their immune landscape. schafflick et al. (7) used scrna-seq of sorted leukocytes from rat meninges, blood, and cerebrospinal fluid (csf) to identify unique cell compositions within the dura. intravenous immune labeling using fluorophore-labeled cd45 (common leukocyte marker) antibody was used to exclude intravascular immune cells. comparing scrna-seq data from different tissues revealed that the dura contained a large cluster of b cells. these dural b cells were subclustered into cxcr4high (signifying immature bone marrow b cells) versus ccl2highcxcl1/2highccl4highitgb2high (signifying chemotactically active b cells). using myelin oligodendrocyte glycoprotein (mog)35–55 peptide-induced experimental autoimmune encephalomyelitis (eae) models of neuroinflammation, the authors showed that inflammation drives phenotypic changes of dural b cells by favoring class-switched plasma cells with higher expression of iga than igg. dural b cells exhibited higher mhc class ii and other markers, indicating an increased antigen-presenting capacity under inflammatory conditions. location of meningeal b cells under homeostatic conditions was then investigated using immunofluorescence of whole mount dura preparations after intravenous injections with a fluorophore-labeled cd45 antibody. results showed that dural b cells are located both within and outside of blood and lymphatic vessels, and interestingly, the extravascular b cells were particularly abundant along the skull sutures. next, the authors confirmed the presence of meningeal b cells in human autopsy samples using cd19+cd20+cd79a+ stainings, and found that human dura also houses a relevant population of b cells. using parabiotic mouse models with different cd45 haplotypes, the precursor dural b cells were shown to be tissue resident with capacity for local proliferation, and the authors claimed their skull labeling approach challenges the presumptive skull-to-dura immune cell flux. this study provided elegant data regarding the presence of a previously-neglected, strategically-located population of meningeal b cells with high potency for antigen-presentation, chemotaxis, and regulation of cns-antigen-specific neuroinflammation. topic 6: skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and cns parenchyma a third paper (8) that integrates well with the theme of the prior two, also examined skull and vertebral bone marrow, but this focused on the myeloid compartment. meninges as evidenced from this triad of papers, contain a large pool of immune cells. however, unlike the prior papers, this paper focused on innate immune cells rather than adaptive b cells. myeloid cells are some of the earlies responders to brain injury. it is well known that these cells can migrate from the periphery to invade the cns. this paper shows that these are not only “blood-derived” but can also come into the cns, rapidly, from the skull bone marrow. the perivascular spaces and the meningeal membranes that cover the cns border zones are also populated by a variety of myeloid cells. in this work, cugarra and others show that cns-associated bone marrow niches in the skull and vertebrae are also myeloid reservoirs for the meninges and cns parenchyma. under homeostasis, these bone marrow pools supply the brain and spinal dural meninges with monocytes and neutrophils via direct dural-bone marrow connections. using elegant fate mapping, dural ly6c+ monocytes and neutrophils were suspected to be from a local source rather than the blood. a calvaria bone-flap with accompanying bone marrow reservoir from gfp+ mice was transplanted into wild-type (wt) mice with an intact dura. gfp+ cells including ccr2+ monocytes, iba1+ macrophages, and cd31+ vasculature was seen in the underlying dura of the transplanted calvaria flap. thus, under homeostatic conditions, the myeloid niche distributed along the brain borders receives a substantial input from the skull bone marrow, which appears to act as a critical dispenser of myeloid cells. this provides an example of a healthy tissue hosting myeloid cells that are continuously replenished by a source that does not use blood as a major route. the authors then went one step further to determine if these cells were involved in an early response to injury using parabiotic models in three injury paradigms, experimental autoimmune encephalitis, spinal cord injury, and optic nerve crush. after injury and in neuroinflammatory diseases, the authors found that these cells can mobilize to infiltrate the cns parenchyma, and display distinct phenotypes from their blood-derived counterparts. cns-infiltrating ly6c+ monocytes (but not neutrophils, cd4 t cells, or ly6c− monocytes) were supplied not from the blood, but by adjacent skull and vertebral bone marrow. it is becoming increasingly clear that under pathological conditions, cns-infiltrating myeloid cells can originate from brain borders and display transcriptional signatures distinct from their blood-derived counterparts. these myeloid cells from bone-marrow niches in close proximity to the brain can supply innate immune cells under both homeostatic and pathological conditions. topic 7: microglia contribute to the propagation of aβ into unaffected brain tissue this very recent study revealed that microglia may act as aβ carriers in the brain of alzheimer’s disease (ad) model mice (9). d’errico and colleagues transplanted embryonic neuronal cells from wt mice into the neocortex of pre-symptomatic 5xfad transgenic mice, a mouse model recapitulating ad-related pathology. interestingly, they found that aβ plaques developed as early as 4 weeks within the grafted neuronal cells and continually increase over time. they confirmed that this was not due to anterograde transport, as very few host processes entered the graft using thy1-gfp/5xfad mice, confirming that the spread of aβ is independent of axonal transport. the greatest amyloid accumulation was along the graft border, an area with higher microglia density. therefore, the authors next investigated whether host microglia are able to invade the wt graft using cx3cr1+/−/5xfad mice. even as early as 2 weeks after transplantation, migration of host cx3cr1+ microglia from cx3cr1+/-/5xfad mice was evident within the wt graft, indicating the ability of microglia to act as an aβ carrier leading to graft invasion. using a variety of methods, the authors confirmed that the microglia were not host derived, or peripheral infiltrating monocytes. they also confirmed using both in vitro and in vivo models that a decline in microglial phagocytosis of aβ occurs in both old wt and 5xfad mice. several additional models including aged 5xfad mice (with less microglial phagocytic activity), irf8-/-/5xfad mice (with less microglial branching), and microglia-depleted 5xfad mice using csf-1r inhibition exhibited a reduction of aβ plaques in the wt grafts, further corroborating the specific role of microglia in regulating aβ propagation. this work confirms that microglia are involved in the pathogenic spread of aβ. they also went one step further in a final experiment. the authors used a model of laser-induced focal tissue injury and two-photon microscopy, and confirmed that aβ can be transported by microglia and form plaques in the lesioned tissue (figure 2). in conclusion, this study suggests a novel role of microglia in the propagation of aβ pathology in neurodegenerative and neural injury models. figure 2. embryonic neuronal cells from wild-type (wt) mice were implanted into the neocortex of pre-symptomatic 5xfad transgenic mice. aβ plaques developed as early as 4 weeks. aβ can be transported by microglia and form plaques in the lesioned tissue. microglia were involved in the propagation of aβ pathology. created with biorender.com topic 8: perivascular space dilation is associated with vascular amyloid-β accumulation in the overlying cortex this work examined the enlargement of the perivascular space (pvs) in patients with cerebral amyloid angiopathy (caa), an increasingly common form of cerebral small vessel disease. caa is a common cause of lobar hemorrhage, cortical microhemorrhages and white matter disease in the elderly (figure 3). in caa, aβ accumulates in the walls of cortical and leptomeningeal arteries, resulting in a loss of smooth muscle cells and impaired vascular function and can lead to lobar intracerebral hemorrhage in the elderly. the presence of cerebral vascular amyloid is a common neuropathological feature in patients with ad. pvs are compartments surrounding cerebral blood vessels that become visible on mri when enlarged. it has been suggested that these spaces enlarge when there are deficits in dysfunctional perivascular clearance of waste products from the brain, including amyloid. the presence of enlarged pvs (epvs) on mri is associated with aging, high blood pressure, stroke and cognitive decline in other studies. figure 3. mri and ct scan imaging of a 73-year-old woman with pathogenically confirmed cerebral amyloid angiopathy. panel a shows innumerable cortical microhemorrhages, panel b demonstrates cortical siderosis (gre), and panel c (flair) shows extensive white matter disease and cerebral atrophy. panel d is a ct scan taken 3 months after her prior mri, when she presented with left hemiparesis and mental status changes. a large lobal hemorrhage is seen in the right frontal area, along with mass effect and cerebral edema. in this paper (10) the authors used 3t and 7t ex vivo mri, semi-automatic segmentation and validated deep-learning-based models to quantify epvs and associated histopathological abnormalities in 19 caa cases and 5 controls. severity of mri-visible pvs during life was significantly associated with the severity of mri-visible pvs on ex vivo mri. this also corresponded with pvs enlargement on histopathology in the same areas. epvs were located mainly around the white matter of perforating cortical arterioles. the amount of epvs was associated with caa severity in the overlying cortex. in addition, the authors found markedly reduced smooth muscle cells and increased vascular aβ accumulation, extending into the wm, was seen in individual affected vessels with an epvs. the presence of aβ was also observed in the wm portion of the same perforating vessels, decreasing in intensity with increasing distance from the cortex. this could reflect blockage of aβ clearance towards the brain surface, consistent with the decreasing severity of caa between superficial and deep cortical layers, as previously shown histologically. these findings were confirmed in a patient with hereditary amyloid that was much younger (dutch mutation). these findings are consistent with the concept that the development of epvs reflects impaired outward flow along arterioles. this has implications for other small vessel diseases and suggests the importance of perivascular clearance mechanisms in human brain, which play an important role in the pathophysiology of both caa and ad. these results support the concept of outward flow along the walls of cortical arterioles and suggest that impaired perivascular clearance of aβ might lead to fluid stagnation and pvs enlargement around upstream portions of connected perivascular compartments. interestingly, removal of aβ plaques with anti-aβ immunotherapy can worsen cortical and leptomeningeal caa (11), suggesting that after plaque disaggregation aβ is cleared along the cerebral vessels. considering the currently unknown role of epvs and white matter hyper-intensities (wmhs) and what they signify in humans both with and without ad/caa, this paper has important clinical and pathophysiological relevance. topic 9: telomerase therapy reverses vascular senescence and extends lifespan in progeria mice aging is a major risk factor for cardiovascular disease. with aging, the vasculature is altered, and molecular and structural changes occur in endothelial cells. senescent endothelial cells show enhanced oxidative stress and dna damages, aberrant chromatin changes, alteration in the nuclear envelope, telomere erosion, and increased production and secretion of pro-inflammatory molecules that negatively affect neighboring cells. given that endothelial function regulates organ perfusion, inflammation and accommodates resident stem cells responsible for tissue repair, it is expected that restoring vascular homeostasis in aged organisms would improve organ function and extend lifespan. hutchinson-gilford progeria syndrome (hgps) is a pediatric genetic disease characterized by accelerated aging. 90% of hgps patients manifest cardiovascular disease early in life, in their teenage years. hgps is caused by a mutation in a nuclear envelop protein (lamin a or progerin), which leads to deleterious consequences in nuclear morphology and gene expression. unfortunately, treatments to mitigate this cardiovascular pathology are limited. the authors (11) used induced pluripotent stem cells (ipscs) derived from hgps patients, in addition to a hgps mouse model to determine if reversing the senescence-associated phenotype in the vasculature had beneficial effects on lifespan and behavior in mice. they found that transient delivery of modified mrna encoding for an enzyme that prevents telomere shortening (human telomerase, htert), which is a distinct feature of cell senescence, restored endothelial proliferation and function, upregulated lamin a, mitigated dna damage, and reversed the inflammatory secretome seen in cultured hgps endothelial cells (figure 4). in addition, they delivered lentiviral mouse tert (mtert) to hgps mice, and found that the aorta of treated mtert mice showed reduced levels of vcam-1 expression (an endothelial adhesion molecule that is increased with vascular inflammation), compared with control progeroid mice. these treated mice also exhibited reduced dna damages in the endothelium of different tissues (aorta, lung, and liver), and their lifespan were significantly extended, compared with hgps mice. this study demonstrates that tert treatment restores senescence-associated phenotype in human hgps endothelial cells, and expands life expectancy in a mouse model of accelerated aging. its relevance lies in the importance of specifically targeting the endothelium to prevent vascular aging and mitigates cardiovascular disease associated with aging. figure 4. telomerase (tert) therapy reverses senescence-associated phenotypes in endothelial cells differentiated from hutchinson-gilford (hgps) human ipsc lines and in a hgps mouse model. human tert (htert) mrna was delivered into hipsc-derived (control and hgps) endothelial cells by lipofectamine transfection, and mouse tert (mtert) lentivirus construct was injected in 3 and 6 months old wild-type and hgps mice via tail vein. created with biorender.com. topic 10: brain capillary pericytes exert a substantial but slow influence on blood flow the role of capillary pericytes, cells that line the capillary networks in the brain, on cerebral blood flow was investigated in this work from dr. shih’s group (12). the capillary bed has the highest flow resistance in the cerebral vasculature given their small diameter, and these vessels are lined with pericytes, unlike the larger arterioles of the brain in which neurovascular coupling is controlled largely by smooth muscle cells due to rapid blood flow demand and consist of α-sma-positive mural cells. two-photon microscopy and optogenetic stimulation was used to directly observe and manipulate brain capillary pericytes in vivo. to directly assess the contractile ability of capillary pericytes in vivo, the authors crossed pdgfrβ-cre mice with reporter mice for the light-gated ion channel, chr2-yfp. the authors confirmed that these cells, when stimulated optogenetically, can contract, decreasing their luminal diameter and reduce blood flow, but with slower kinetics than similar stimulation of mural cells on upstream pial and precapillary arterioles. importantly, this vasoconstriction was associated with a decrease in red blood cell velocity and flux in the same capillaries, showing that the induced levels of constriction were sufficient to alter blood flow, and in fact led to significant “stalling” of rbc flow. interestingly, “blebs” were seen in up to 30% of the pericytes. the authors hypothesized that these blebs reflected disruption of the actin cytoskeleton due to induced supra-physiological contraction. this slow vasoconstriction was inhibited by the clinically used vasodilator fasudil, a rho-kinase inhibitor that blocks contractile machinery and promotes vasodilation. during optogenetic stimulation of capillary pericytes, fasudil attenuated relative and absolute capillary constriction in a dose-dependent manner and also led to a dose-dependent alleviation of persistent flow stalls. this has important translational relevance as this drug is used to treat cerebral vasospasm after subarachnoid hemorrhage in japan, and could have a role in preventing the no reflow phenomenon in ischemic stroke after endovascular interventions. capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and slower to dilate towards baseline following optogenetically induced vasoconstriction compared to pre-capillary arterioles. optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux selectively in capillaries lacking pericyte contact. these data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain. this could have major implications for other disorders related to cerebral hypoperfusion. figure 5. the skull bone marrow immune profile changes with aging. the composition of myeloid cell populations in the skull bone marrow changes with aging with significantly higher relative frequencies of neutrophils in aged skull (top). skull bone marrow lymphocyte compartment contains significantly higher cd8+ t lymphocytes and activated cd11b high b lymphocytes in naïve aged mice when compared with young skull (bottom). reprinted from honarpisheh et al. (13). conclusion many exciting and novel papers were published in 2021. the compendium of papers highlighted demonstrate our growing understanding of vascular malformations, neuro-immune communication, and vascular function in aging and disease. one important caveat to consider is that many of these diseases predominately affect older individuals, such as stroke and amyloid related diseases. as the field moves forward it will be important to consider the age of the animals used, and also confirming that these pathways are involved in human disease using clinical samples to ensure translational relevance. for example, recent work in our own laboratory has shown that the skull immune compartment changes dramatically with age (figure 5). age-related changes in both peripheral and cns immunity will be an important avenue of exploration in the future. acknowledgements i would like to thank felix moruno manchon, phd, juneyoung lee, phd, pedram honarpisheh, phd, aki urayama, phd, sophie ren, phd, alex choi, md, and eunhee kim, phd, for their assistance in selection of these manuscripts. ldm is supported by the national institute of health (nih) with grants from the ninds (r37 ns096493, r01 ns108779) and nia (r01 ns103592). references nikolaev si, fish je, radovanovic i. somatic activating kras mutations in arteriovenous malformations of the brain. n engl j med. 2018 apr 19;378(16):1561-1562. https://doi.org/10.1056/nejmc1802190 al-olabi l, polubothu s, dowsett k, andrews ka, stadnik p, joseph ap, knox r, pittman a, clark g, baird w, bulstrode n, glover m, gordon k, hargrave d, huson sm, jacques ts, james g, kondolf h, kangesu h, keppler-noreuil km, khan a, lindhurst mj, lipson m, mansour a, o’hara j, mahon c, mosica a, moss a, murthy a, ong j, parker ve, rivière j-p, sapp jc, sebire nj, shah r, sivakumar b, thomas a, virasami a, waelchli r, zeng z, biesecker lg, barnacle a, topf m, semple rk, patton ee, kinsler va. mosaic ras/mapk variants cause sporadic vascular malformations which respond to targeted therapy. j clin invest. 2018 nov 1;128(11):5185. https://doi.org/10.1172/jci124649 park es, kim s, huang s, yoo jy, körbelin j, lee tj, kaur b, dash pk, chen pr, kim e. selective endothelial hyperactivation of oncogenic kras induces brain arteriovenous malformations in mice. ann neurol. 2021 may 89(5):926-941. https://doi.org/10.1002/ana.26059 ren aa, snellings da, su ys, hong cc, castro m, tang at, detter mr, hobson n, girard r, romanos s, lightle r, moore t, shenkar r, benavides c, beaman mm, müller-fielitz h, chen m, mericko p, yang j, sung dc, lawton mt, ruppert jm, schwaninger m, körbelin j, potente m, awad ia, marchuk da, kahn ml. pik3ca and ccm mutations fuel cavernomas through a cancer-like mechanism. nature. 2021 jun 594(7862):271-276. https://doi.org/10.1038/s41586-021-03562-8 shi l, sun z, su w, xu f, xie d, zhang q, dai x, iyer k, hitchens tk, foley lm, li s, stolz db, chen k, ding y, thomson aw, leak rk, chen j, hu x. treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke. immunity. 2021 jul 13;54(7):1527-1542. https://doi.org/10.1016/j.immuni.2021.04.022 brioschi s, wang wl, peng v, wang m, shchukina i, greenberg zj, bando jk, jaeger n, czepielewski rs, swain a, mogilenko da, beatty wl, bayguinov p, fitzpatrick jaj, schuettpelz lg, fronick cc, smirnov i, kipnis j, shapiro vs, wu gf, gilfillan s, cella m, artyomov mn, kleinstein sh, colonna m. heterogeneity of meningeal b cells reveals a lymphopoietic niche at the cns borders. science. 2021 jul 23;373(6553). https://doi.org/10.1126/science.abf9277 schafflick d, wolbert j, heming m, thomas c, hartlehnert m, börsch al, ricci a, martín-salamanca s, li x, lu in, pawlak m, minnerup j, strecker jk, seidenbecher t, meuth sg, hidalgo a, liesz a, wiendl h, meyer zu horste g. single-cell profiling of cns border compartment leukocytes reveals that b cells and their progenitors reside in non-diseased meninges. nat neurosci. 2021 sep 24(9):1225-1234. https://doi.org/10.1038/s41593-021-00880-y cugurra a, mamuladze t, rustenhoven j, dykstra t, beroshvili g, greenberg zj, baker w, papadopoulos z, drieu a, blackburn s, kanamori m, brioschi s, herz j, schuettpelz lg, colonna m, smirnov i, kipnis j. skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and cns parenchyma. science. 2021 jul 23;373(6553). https://doi.org/10.1126/science.abf7844 d'errico p, ziegler-waldkirch s, aires v, hoffmann p, mezö c, erny d, monasor ls, liebscher s, ravi vm, joseph k, schnell o, kierdorf k, staszewski o, tahirovic s, prinz m, meyer-luehmann m. microglia contribute to the propagation of aβ into unaffected brain tissue. nat neurosci. 2022 jan 25(1):20-25. https://doi.org/10.1038/s41593-021-00951-0 perosa v, oltmer j, munting lp, freeze wm, auger ca, scherlek aa, van der kouwe aj, iglesias je, atzeni a, bacskai bj, viswanathan a, frosch mp, greenberg sm, van veluw sj. perivascular space dilation is associated with vascular amyloid-β accumulation in the overlying cortex. acta neuropathol. 2022 mar 143(3):331-348. https://doi.org/10.1007/s00401-021-02393-1 mojiri a, walther bk, jiang c, matrone g, holgate r, xu q, morales e, wang g, gu j, wang r, cooke jp. telomerase therapy reverses vascular senescence and extends lifespan in progeria mice. eur heart j. 2021 nov 7;42(42):4352-4369. https://doi.org/10.1093/eurheartj/ehab547 hartmann da, berthiaume aa, grant ri, harrill sa, koski t, tieu t, mcdowell kp, faino av, kelly al, shih ay. brain capillary pericytes exert a substantial but slow influence on blood flow. nat neurosci. 2021 may 24(5):633-645. https://doi.org/10.1038/s41593-020-00793-2 honarpisheh p, bryan rm, mccullough ld. aging microbiota-gut-brain axis in stroke risk and outcome. circ res. 2022 apr 15;130(8):1112-1144. https://doi.org/10.1161/circresaha.122.319983 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. soluble brain homogenates from diverse human and mouse sources preferentially seed diffuse aβ plaque pathology when injected into newborn mouse hosts feel free to add comments by clicking these icons on the sidebar free neuropathology 3:9 (2022) original paper soluble brain homogenates from diverse human and mouse sources preferentially seed diffuse aβ plaque pathology when injected into newborn mouse hosts brenda d. moore1,2,3, yona levites1,2,3, guilian xu1,2,3, hailey hampton1,2, munir f. adamo1,2, cara l. croft1,2,3, hunter s. futch1,2, corey moran1,2, susan fromholt1,2, christopher janus1,2,3, stefan prokop2,3,4,5, dennis dickson6, jada lewis1,2,3, benoit i. giasson1,2,3, todd e. golde1,2,3,7, david r. borchelt1,2,3 1 department of neuroscience, college of medicine, university of florida, gainesville, fl 32610, usa 2 center for translational research in neurodegenerative disease, college of medicine, university of florida, gainesville, fl 32610, usa 3 mcknight brain institute, college of medicine, university of florida, gainesville, fl 32610, usa 4 department of pathology, university of florida, gainesville, fl 32610, usa 5 fixel institute for neurological diseases, university of florida, gainesville, fl 32610, usa 6 department of neuroscience, mayo clinic, jacksonville, fl, usa 7 department of neurology, college of medicine, university of florida, gainesville fl 32610, usa corresponding author: brenda d. moore · 1275 center dr · po box 100159, 352-273-7868 · gainesville, fl 32610 · usa brendar@ufl.edu submitted: 18 january 2022 accepted: 28 february 2022 copyedited by: henry robbert published: 23 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3766 additional resources and electronic supplementary material: supplementary material keywords: amyloid-β, tau, α-synuclein, seeding, alzheimer’s disease, lewy body dementia abstract background: seeding of pathology related to alzheimer’s disease (ad) and lewy body disease (lbd) by tissue homogenates or purified protein aggregates in various model systems has revealed prion-like properties of these disorders. typically, these homogenates are injected into adult mice stereotaxically. injection of brain lysates into newborn mice represents an alternative approach of delivering seeds that could direct the evolution of amyloid-β (aβ) pathology co-mixed with either tau or α-synuclein (αsyn) pathology in susceptible mouse models. methods: homogenates of human pre-frontal cortex were injected into the lateral ventricles of newborn (p0) mice expressing a mutant humanized amyloid precursor protein (app), human p301l tau, human wild type αsyn, or combinations thereof. the homogenates were prepared from ad and ad/lbd cases displaying variable degrees of aβ pathology and co-existing tau and αsyn deposits. behavioral assessments of app transgenic mice injected with ad brain lysates were conducted. for comparison, homogenates of aged app transgenic mice that preferentially exhibit diffuse or cored deposits were similarly injected into the brains of newborn app mice. results: we observed that lysates from the brains with ad (aβ+, tau+), ad/lbd (aβ+, tau+, αsyn+), or pathological aging (aβ+, tau-, αsyn-) efficiently seeded diffuse aβ deposits. moderate seeding of cerebral amyloid angiopathy (caa) was also observed. no animal of any genotype developed discernable tau or αsyn pathology. performance in fear-conditioning cognitive tasks was not significantly altered in app transgenic animals injected with ad brain lysates compared to nontransgenic controls. homogenates prepared from aged app transgenic mice with diffuse aβ deposits induced similar deposits in app host mice; whereas homogenates from app mice with cored deposits induced similar cored deposits, albeit at a lower level. conclusions: these findings are consistent with the idea that diffuse aβ pathology, which is a common feature of human ad, ad/lbd, and pa brains, may arise from a distinct strain of misfolded aβ that is highly transmissible to newborn transgenic app mice. seeding of tau or αsyn comorbidities was inefficient in the models we used, indicating that additional methodological refinement will be needed to efficiently seed ad or ad/lbd mixed pathologies by injecting newborn mice. abbreviations ad alzheimer’s disease, lbd lewy body disease, ad/lbd alzheimer’s disease/lewy body disease, aβ amyloid-β, αsyn α-synuclein, app amyloid precursor protein, pa pathological aging, gfap glial fibrillary acidic protein, mapt microtubule-associated protein tau, ndc nondemented controls, wt wild type, caa cerebral amyloid angiopathy, elisa enzyme linked immunosorbent assay, sds sodium dodecyl sulfate, fa formic acid, thio-s thioflavin s. background the amyloid cascade hypothesis posits that deposition of the aβ peptide is an initiating factor in alzheimer’s disease (ad) with downstream effects of increased inflammatory response, altered neuronal homeostasis, altered tau phosphorylation leading to tangle formation, neuronal dysfunction, and cognitive decline (1, 2). the interaction between aβ and tau pathology has been modeled in transgenic animals that co-express human amyloid precursor proteins (app) with mutations associated with familial ad (fad) and wild-type or mutant human tau (3-12). multiple studies report that the presence of aβ pathology exacerbates the severity of tau pathology, leading to increased tau phosphorylation or increased levels of neurofibrillary tangles (3, 6, 8, 10, 11). interactions between aβ and tau pathology have also been modeled in mice by utilizing the prion-like capacity of aβ and tau aggregates to seed accelerated ad pathology. aβ containing seeds, derived from human ad and pathological aging (pa) brain lysates, have been shown to accelerate the time-course and severity of aβ deposition in amyloid precursor protein (app) transgenic models as well as impact the morphology of the seeded deposits (for review see (13)). seeding preparations can consist of human brain homogenates, app transgenic mouse brain homogenates, enriched preparations derived from such homogenates and synthetic aβ fibrils (13). in most of these published studies, seeds are injected into adult app mice that are a few months away from developing aβ pathology on their own and in such cases the inherent pathological tendencies of the app host model can contribute to the pathological outcome. partially purified tau aggregates from ad brains were found to seed enhanced neuritic tau pathology in the 5xfad mouse model and the appnl-g-f/human mapt double knock-in model (14); however, neither of these models produced pathology resembling neurofibrillary tangles. in appswe/ps1de9 and appnl-f/nl-f mice crossed to humanized htau mice, the presence of aβ pathology did not produce significant enhancement of tau seeding (15). these latter studies illustrate the need to refine seeding approaches towards producing models that reproduce a broader spectrum of ad-related pathology. recent studies have begun to use seeding approaches to create models of mixed ad or lewy body disease (lbd) pathology. in parkinson’s disease and lbd, the presence of aβ plaques is associated with cortical αsyn pathology (16, 17). similar to studies of aβ/tau interactions, investigations of aβ and αsyn interaction in transgenic mice have suggested a possible synergistic relationship (18, 19). in seeding models involving the injection of αsyn preformed fibrils, aβ deposits accelerated αsyn pathogenesis and spread throughout the brain of 5xfad mice (20) and in bigenic mice co-expressing appswe/ps1de9 and wt αsyn (21) . one of the goals of our study was to further explore the potential to use seeding as a means to generate novel mouse models of mixed ad and lbd pathology. a subset of cognitively normal aged individuals exhibit substantial aβ pathology at autopsy (22). these individuals, which we use the designation of pathologic aging (pa), may represent prodromal, presymptomatic ad (23-26). the amyloid pathology of pa brains has typically been described as diffuse in nature whereas ad brains contain both diffuse and compact/neuritic deposits of aβ (26-28). cognitively normal individuals with pa generally have very low levels of tau pathology (26). rigorous biochemical examination of aβ from the pre-frontal cortex of pa and ad brain revealed extensive overlap in aβ levels, peptide profiles, solubility, and sds-stable oligomeric assemblies (25). thus, pa brains provide a source of human brain-derived aβ seeds that can be compared to human ad brain preparations that contain both aβ and tau seeds. nearly all of the published studies involving injection of aβ, tau, or αsyn seeds into the brains of transgenic mice have followed a protocol of injecting the seeds stereotaxically into the brains of young adult mice. in this study, we have examined the efficacy of intracerebroventricular injection of homogenates prepared from ad, pa, and ad/lbd donors into newborn app, app/tau, or app/αsyn host mice. in prior studies, we had established that injection of recombinant adeno-associated virus into newborn mice was an effective approach to obtaining widespread dispersion of injected virus (29, 30). we therefore hypothesized that misfolded seeds of aβ, tau, or αsyn might similarly be more widely dispersed to more effectively seed pathology. in fact, we observed efficient seeding of αsyn pathology in newborn mice, expressing a53t human αsyn (m83 line), by injecting brain lysates from patients with multisystem atrophy (31). here we performed brain lysate injection studies at p0 in the lateral ventricles of host transgenic mice that co-express humanized mutant app with either mutant human tau-p301l or wild-type human αsyn (m20 line). importantly, the recipient transgenic mice used in the current studies develop pathology at late ages or not at all; enabling sensitive evaluation of potential seeding. we observed that injecting newborn app, app/tau, or app/αsyn mice with homogenates from diverse pathological specimens, including pa brains, produced robust diffuse aβ deposition. though the human homogenates used contained coexisting αsyn and tau seeds, we were not able to directly induce lbd or tau pathology in recipient models tested here. we compared the data from human lysates to lysates prepared from transgenic mouse donors finding again that diffuse aβ deposits were preferentially seeded when these preparations were injected in newborn mice. app mice seeded with ad brain lysates, exhibiting diffuse aβ pathology, also showed no significant impairments in a contextual fear paradigm of cognitive performance. our findings indicate that the injection of human brain homogenates from ad and ad/lbd donors into newborn app mice preferentially induces diffuse aβ pathology. interestingly, pa brains induce the same pathology implying that ad and ad/lbd brains contain amyloid seeding entities that are shared with pa brains. methods description of ad, pa and ndc cases for this study we utilized a subset of the human brain samples that our group previously analyzed (25). tissue sections and frozen pre-frontal cortex (ad = 2, pa = 4, and ndc = 2) were obtained from the mayo clinic brain bank with informed consent, in accordance with the mayo clinic institutional review board, using previously described acquisition and diagnostic analyses (26, 32, 33). samples were initially cryo-pulverized to allow for multiple studies. we previously analyzed the aβ peptide profile, quantity, and solubility in these brain samples (25). in this study, we analyzed two brains from ad patients (both aged 84), four pa brains from subjects (age range = 78 to 83) without clinical evidence of dementia, and two brains with rare or no ad lesions from elderly individuals without clinical evidence of a neurological illness (ages 75 and 78). in addition, human brain samples were also obtained from the university of florida neuromedicine human brain tissue bank following institutional regulations and previously described classification (34-36). we analyzed tissue sections and frozen pre-frontal cortex from two ad patients (ages 82 and 86), two cases with ad/lbd (ages 81 and 83), and two control individuals without pathology (ages 52 and 82). table 1 summarizes the cases studied in this report. table 1. list of human brain samples used in this study. pathological diagnosis (pathdx), lewy body disease (lbd) classification, amyloid plaque score (thal phase), braak stage, consortium to establish a registry for alzheimer’s disease (cerad) protocol, 2% sds and 70% formic acid (fa) solubilized aβ40 and 42 (25), cerebral amyloid angiopathy (caa), apolipoprotein e (apoe) genotype, gender, and age are reported. ad, alzheimer’s disease; pa, pathological aging; ndc, non-demented controls; oa, optic atrophy; tr, transynaptic degeneration; gba, gross brain atrophy; x, below detection levels; nd, not determined, f, female; m, male. transgenic mice the transgenic mice used in this study have been described previously and are summarized in table 2. the prp.appsi mice express mouse app-695 cdna with a humanized aβ sequence and fad mutations (appswe/ind) using the moprp.xho vector, which was co-injected with a vector to express egfp in skin (first described in (37)). the itau-p301l mice express human 0n4r tau-p301l using a tet-regulated vector that includes a minimal cmv promoter (38). tau levels were assessed by western blotting. pbs lysates of 2.5-6 month old p301l mice were heated at 95°c for five minutes in the presence of denaturing sds sample buffer, were separated on a 4-12% bis-tris gel (bio-rad, hercules, ca) in 3-(n-morpholino)propanesulfonic acid (mops) running buffer (bio-rad, hercules, ca) and transferred onto pvdf membrane. the membrane was blocked in casein blocking buffer and incubated overnight at 4°c with primary antibodies to tau, tau-13 (anti-human tau2-18; covance) and 3026 (rabbit polyclonal antibody raised against full length 0n3r human tau and also reacts with 0n4r human tau (39, 40)), and anti-β-actin (sigma-aldrich, st. louis, mo) before incubation with secondary antibody, fluorophore conjugated alexafluor 680 anti-mouse igg (thermo fisher scientific, waltham, ma). protein bands were detected and quantified using the multiplex odyssey infrared imaging system (bio-rad, hercules, ca). table 2. mouse strains used in this study. caa, cerebral amyloid angiopathy; na, not applicable. the m20 mice express wild type αsyn under the control of the prion promoter and do not develop αsyn pathology (41). prp.huaβ/ps1 mice express human appswe and human ps1de9. prp.moaβ/ps1 mice express mouse appswe mutation and human ps1de9, and tet.moaβ mice expresses inducible mouse appswe (37). these mice were bred in-house. prp.appsi and m20 were maintained on hybrid c57bl6/c3h backgrounds, following a breeding scheme in which transgene-positive males were bred to nontransgenic f1 b6/c3 female mice. the itau-p301l mice we used had been backcrossed to the fvb/ncr mouse strain for more than 10 generations. appsi/tau-p301l and appsi/αsyn mice were generated by intercrossing mice that where hemizygous for the respective transgenes. prp.appsi mice were genotyped by visualizing gfp expression, which is possible because these mice were co-injected with a transgene that expresses gfp in the skin integrated next to app transgene. prp.appsi/itau-p301l mice were genotyped for app by illumination and visualization with special filter goggles (bls ltd, budapest, hungary) and for tau by pcr of tail dna. itau-p301l and m20 mice were genotyped by pcr of tail dna. prp.appsi mice destined for behavioral studies were backcrossed one generation onto c57bl6. all animals were housed 1-5 per cage with unlimited access to food and water with a 14-hour light and 10-hour dark cycle. all experiments involving mice were approved by the university of florida institutional animal care and use committee (iacuc) and conducted in accordance with nih guidelines. human brain lysates human and mouse brain lysates were prepared as previously described (42). briefly, frozen tissue was homogenized at 10% (w/v) in sterile pbs, subjected to vortex and sonication (3 x 5 sec) and then centrifuged 3000 x g for 5 min at 4°c. lysates were immediately aliquoted and stored at -80°c until needed. as previously described (25), the aβ levels and solubility in the lysates was assessed by western blotting. briefly, lysates were heated at 50°c for three minutes in the presence of denaturing sample buffer, were separated on a 4-12% bis-tris gel (bio-rad, hercules, ca) in 1x2-(n-morpholino)ethanesulfonic acid (mes) running buffer (bio-rad, hercules, ca) and transferred onto nitrocellulose membrane (bio-rad, hercules, ca). the membrane was boiled in pbs for 5 min, blocked in starting block (thermo fisher scientific [formerly thermo scientific], waltham, ma) and incubated overnight at 4°c with primary antibody ab5 (human aβ1-16 specific; t.e. golde) before incubation with the secondary antibody, fluorophore conjugated alexa fluor 680 anti-mouse igg (thermo fisher scientific, waltham, ma). protein bands were detected using the multiplex odyssey infrared imaging system (bio-rad, hercules, ca). brain seeding with neonatal cerebral ventricle injections injections of human brain lysates and lysates from a 27mo prp.appsi mouse, a 25mo prp.huaβ/ps1 mouse, a 24mo tet.moaβ mouse, and a 24mo prp.moaβ/ps1 mouse were performed as described previously with recombinant adeno-associated virus (43). briefly, p0 pups were cryoanesthetized and 2 ul of human or mouse brain lysates were bilaterally injected into the cerebral ventricle using a 10 ul hamilton syringe with a 30-inch needle (hamilton company, reno, nv). pups were placed on a heating pad for recovery and returned to their home cage. cohorts of mice were euthanized at 6, 9, 12, and 18 months for analysis. both male and female mice were analyzed (see tables s1 and s2 for description of cohorts). mouse brain tissue collection mice were anesthetized with isoflurane and perfused transcardially with 20 ml of cold pbs. the brains were cut sagittally through the midline, and one hemibrain was drop-fixed in 4% paraformaldehyde in pbs (ph 7.5) for ~48 hrs at 4°c followed by processing and paraffin embedding. the other hemibrain was snap frozen with isopentane on dry-ice and then stored at -80⁰c until it was thawed and homogenized in preparation for elisa measurements of aβ peptide levels. histology and immunochemistry paraffin sections (5 μm) were used for all the histology and immunochemistry studies. campbell-switzer silver (44) and thio-s (37) tissue staining methods were performed as previously described. immunochemistry followed standard protocols described previously (37). to assess amyloid pathology, embedded sections were immunohistochemically stained with a biotinylated pan-aβ antibody ab5 (1:500; t.e.g.) and developed using vectastain elite abc kit (vector laboratories, burlingame, ca) followed with 3,3’-diaminobenzidine (dab) substrate (vector laboratories, burlingame, ca). to evaluate tau pathology, embedded sections were immunohistochemically stained with 7f2 (1:10,000; ben giasson (39)), cp27 (1:1000; peter davies), at8 (1:1000; thermofisher scientific), and mc1 (1:1000; peter davies). to assess αsyn pathology, sections were stained with 9c10 (1:1000; ben giasson) (31). after development by 3,3’-diaminobenzidine (dab) (vector laboratories, burlingame, ca) substrate and counterstaining with hematoxylin, the slides were coverslipped and images were taken using an olympus bx60 microscope or scanned by aperio® xt system (leica biosystems, buffalo gove, il, usa) or zeiss microscope (carl zeiss, oberkochen, germany) and analyzed using the zen 2.6 program (carl zeiss, oberkochen, germany). blinded observers then reviewed the images and scored the burden of aβ pathology based on the following criteria: “+++” = heavy aβ burden with too many deposits to count. “++” = abundant pathology with >30 deposits per section. “+” = consistent pathology with > 5 deposits per section. “+/-” = inconsistent pathology with <3 deposits per section. brain amyloid extraction human brain samples were originally analyzed by moore (25). briefly, frozen pre-frontal cortex tissue was cryo-pulverized in liquid nitrogen and then sequentially extracted with tris-buffered saline (tbs), radioimmunoprecipitation buffer (ripa), 2% sodium dodecyl sulfate (sds), and 70% formic acid (fa) containing protease inhibitor cocktail (roche, indianapolis, in, usa). after harvesting the mouse brain, the left hemisphere was flash-frozen in isopentane. the frozen cortex was sequentially extracted with protease inhibitor cocktail (roche) containing tris-buffered saline, ripa buffer, 2% sds, and 70% formic acid (fa) as described previously at a concentration of 150 mg/ml (25). elisa aβ levels from the 2% sds and 70% fa extracted samples were quantified by sandwich elisa as described previously (25). total aβ was captured with mab ab9 and detected by hrp-conjugated mab 4g8 (human aβ17-24; covance, princeton, nj, usa); aβ1-40 was captured with monoclonal antibody (mab) ab9 (human aβ1-16; t.e. golde) and detected by horseradish peroxidase (hrp)-conjugated mab 13.1.1 (human aβ35-40 specific; t.e. golde); aβ1-42 was captured with mab 2.1.3 (human aβ35-42 specific; t.e. golde) and detected by hrp-conjugated mab ab9. elisa results were analyzed using softmax pro software (molecular devices). contextual fear conditioning transgenic prp.appsi and nontransgenic littermates, seeded with ad cases 1 and 2, and ndc case 7, were aged to 12 months and subjected to contextual fear conditioning as previously described (45, 46). briefly, in the contextual fear conditioning test, mice learn the association between the training chamber, which represents an initially neutral conditional stimulus (cs) and an aversive, brief electric foot-shock, unconditional stimulus (us), which takes place in the training context. we have previously established that 0.45 ma electric current elicits robust avoidance response and results in strong freezing response after 2 cs-us pairings (45). during the training session, mice were allowed to explore the training chamber for 120 sec, with a 2 sec foot shock immediately following a 30 sec tone (80 db) (day 1; see fig. 4a). the mice recover for 60 sec and then receive another 2 sec foot shock following a 30 sec tone. the final post-us period is 60 sec. for the context test session, on day 3, mice were exposed to the same training context and the freezing presentation during the 300 sec was recorded by an image analysis system (freezeframe, actimetrics). on day 4, mice experienced the tone session, and were placed in a modified chamber (altered inserts and smell), during the first 180 sec the mice were allowed to explore the new environment. during the second 180 sec period the tone was delivered and the percent freezing was recorded. no shock was applied during context or tone session on days 3 and 4. results characterization of ad/lbd, ad, pa, and ndc cases in this study, we selected a subset of ad, pa, and ndc donors that were previously analyzed (25) for examination of seeding activity in app, app/tau, and tau transgenic mice (table 1, ad 1 and 2, pa 1, 2, 3, 4, and ndc 1 and 2). all 8 of these donors exhibited some degree of lewy body pathology (lbd). two of the donors selected for the study were sub-categorized with cerebral amyloid angiopathy (caa); pa 3 had a high abundance of vascular amyloid with a caa score of 2+-3+ while ad 1 had moderate levels of vascular aβ deposition with a score of 1+ (table 1). as expected, the two ad brains had significant accumulation of tau, indicated by braak staging (5.5 and 6) (table 1). the pa and ndc brains had lower abundance of tau than the ad brains, but were similar to each other (2, 2.5, 3, 2, and 2, 3, respectively) (table 1). in this set of cases, all also exhibited incidental, diffuse, αsyn pathology. to these 8, we added 6 additional donors that included two ad (no lbd), two with lewy-body variant ad (ad/lbd), and 2 additional controls that were free of all types of pathology (table 1, ad 3 and 4, ad/lbd 1 and 2, and ndc 3 and 4). these brains were used in seeding app, app/α-synuclein (αsyn), and αsyn mice so that we would be able to compare ad and ndc that lacked αsyn pathology to ad/lbd brains with high levels of αsyn pathology. to characterize the pathology in the ad and pa brains used to seed the app/tau, app, and tau mice, sections were stained with a pan-aβ antibody (fig. s1a), thioflavin s (thio-s) (fig. s1b), and with a phospho-specific tau antibody (fig. s1c). we observed widespread aβ deposition of both diffuse and compact amyloid in both ad and pa (fig. s1a, b), with a subset showing striking caa. pa 3 had numerous, thio-s positive, vascular amyloid deposits, consistent with the assessment of caa as 2+-3+ (fig. s1a, b). similarly, we observed thio-s positive amyloid staining surrounding several vessels within ad 1, consistent with a caa score of 1+ (fig. s1a, b). ad 2 contained several cored thio-s positive deposits while the compact deposits in pa 1, 2, and 4 had little to no thio-s staining. as expected, ad 1 and 2 contained both substantial phosphorylated tau in form of dystrophic neurites, neuropil threads and neuronal inclusions while all four of the pa were negative for tau deposits (fig. s1c). ndc 1 and 2 were negative for aβ and tau deposits by both immunostaining and thio-s staining. cerebral injection of brain lysates into newborn app/tau and app mice results in widespread, robust amyloid deposition to gain a better understanding of the type of aβ pathology that unseeded prp.appsi mice produce, we harvested breeder mice at various ages to assess phenotypic variation (fig. s2a and b) (37). at 12 months of age, when deposition begins to occur in the brains of prp.appsi mice, any given section through the cortex and hippocampus may exhibit 1 or 2 diffuse tufted deposits and/or cored deposits (score +/-). at this age, aβ deposition also begins to appear in the meninges surrounding the cerebellum (fig. s2a). approximately 50% of prp.appsi mice at 11-13 months of age exhibit no aβ pathology or show only meningeal deposition in the cerebellum (fig. s2a). to concurrently examine whether tau pathology could be augmented by seeding from these lysates, the prp.appsi mice were crossed to a line of mice that express human tau p301l (itau-p301l) (table 2) (38, 47). the itau-p301l mice express mutant human tau at levels 2-3 fold higher than nontransgenic mice (fig. s3). to compare the relative ability of homogenates prepared from ad and pa cases to seed aβ deposition in these mice, lysates from the pre-frontal cortex of the ad, pa, and ndc were injected into the cerebral ventricles of newborn mice at p0 (31, 42, 43). we confirmed that the lysates contained aβ by immunoblotting and elisa (fig. s1d and table 1). aβ was detectable by immunoblot in each of the lysates used for injection (fig. s1d), and enzyme-linked immunosorbent assay (elisa) measurements confirmed high levels of sds-soluble and formic acid (fa) soluble aβ42 in each brain lysate (table 1). we hypothesized that injection of these lysates in p0 mice would result in widespread dispersion of the aβ or tau seeds, and potentially offer the best chance to extensively alter the type of pathology that would be induced by seeding. to assess amyloid pathology, hemibrains of seeded mice were stained with a pan-aβ antibody and thio-s. in mice injected with either ad or pa lysates, we observed significant induction of aβ deposition, comprised primarily of diffuse deposits throughout the cortex and hippocampus, with some vascular deposition in the pia surrounding the cortex and within the hippocampal fissure (fig. 1b, c). mice injected with homogenates from ndc 1 and 2, and mice that were not injected, had little aβ pathology by 12 months of age (fig. 1b, c). examples of cases with the most severe pathology are shown in figure 1, while images of animals with the least pathology are shown in figure s4. in all cases, however, the induced aβ pathology exhibited a diffuse, thio-s negative morphology (fig. s5). the levels of induced aβ pathology in mice that co-expressed appsi and tau-p301l were similar to that of mice that expressed only appsi (fig. 1b versus c, s4a versus b). mice that were transgenic for only tau-p301l, or were non-transgenic, showed no evidence of aβ deposition (data not shown). to compare the data across all of the mice examined, aβ pathology was qualitatively scored by three independent observers (fig. 1d, e). at 12 months, only pa 4 was scored as having a level of aβ pathology that approached mice injected with the ad cases; both of which exhibited widespread, diffuse pathology (fig. 1b, c). aβ pathology in mice seeded by pa 1, 2, and 3 was scored as less abundant than in mice seeded by the ad cases (fig. 1d, e). we confirmed the high seeding activity of ad 1 and 2, and pa 4 by assessing aβ pathology at 9 months post-injection. although the severity of pathology varied, multiple animals injected with each of these lysates exhibited aβ deposition (fig. s6a). notably, dilution of the brain lysates by 10-fold prior to injection greatly diminished seeding activity (fig. s6b). to further examine the relative seeding activity of pa lysates, we injected newborn prp.appsi mice with each of the 4 pa lysates and ndc 1, and analyzed pathology at 18 months post-injection. although the mice injected with the pa lysates appeared to have more severe aβ deposition, we also observed some significant aβ pathology in animals injected with the ndc lysate (fig. s7). whether this outcome was due to some small amount of aβ seed in the ndc 1 will require further study. collectively, these studies demonstrated that ad and pa brains contain misfolded forms of aβ that preferentially seed diffuse aβ pathology. fig. 1 cerebral injection of brain lysate at p0 induces widespread, robust amyloid deposition. overall schematic of experiments (a) images of prp.appsi/tau-p301l (b) and prp.appsi (c) mice injected with ad, pa, or ndc lysate at p0. brain sections (hemibrain) stained with biotinylated anti-aβ mab ab5 (anti-aβ 1-16) and counterstained with hematoxylin. cases with the most abundant amyloid pathology are shown. scale bar: 100 μm. qualitative analysis of amyloid deposition in seeded prp.appsi/tau-p301l (d) and prp.appsi (e) mice. three observers independently scored representative slides from each seeded mouse. the amyloid pathology in each mouse was categorized on a scale from (-) as no pathology to (+++) as abundant pathology. each point represents a rating score by an observer (3 observations per animal). the number of seeded mice for each scale of amyloid burden by the three observers is reported. a subset of mice injected with ndc lysate were rated as + based on 3-5 plaques per section, and thus we set this rating as baseline (marked by a dash line). only the two ad cases and pa 4 consistently scored above +. the number of prp.appsi/tau-p301l or prp.appsi mice injected with each lysate, respectively, were as follows: ad 1: n=4, n=3; ad 2: n=4, n=3; pa 1: n=3, n=3; pa 2: n=3, n=5; pa 3: n=5, n=4; pa 4: n=2, n=5; ndc 1: n=3, n=3; ndc 2: n=5, n=5; uninjected: n=3, n=3. two of the donor cases were identified as having caa pathology, ad 1 and pa 3, and were scored 1+ and 2+-3+, respectively (table 1). to determine if caa pathology was seeded in the recipient mice, we stained sections with thio-s and searched for evidence of vascular amyloid. in mice seeded with pa 3, we observed thio-s positive, vascular pathology in 6 of the 7 recipient mice; however, the incidence of this pathology was limited (fig. s8). although ad 1 also contained caa pathology, we did not observe thio-s positive, vascular pathology in mice seeded by this homogenate. these findings suggest that it may be possible to selectively increase caa using seeding, but enhancing such pathology may require purification of cerebral vessels before preparation of the seeds. we biochemically confirmed our histological data by analyzing aβ levels from sequentially extracted brain lysates using c-terminal specific antibodies in sandwich elisas to measure aβ40 and aβ42 specifically. in our aβ elisas the detection limit was approximately 0.04 pmol/g. in the brains of all seeded mice, the levels of aβ40, both sds-soluble and fa, were 10-100-fold lower than that of aβ42 (fig. 2a, b). consistent with the presence of abundant diffuse aβ pathology, we detected elevated levels of aβ42 in sds-soluble fractions from mice seeded with ad 1 and 2, and pa 4 (fig. 2c). somewhat surprisingly, the levels of aβ42 in the fa-soluble fractions were similar to that of the sds fraction despite the absence of cored aβ deposits (fig. 2c and d). notably, there was considerable variation in the levels of aβ in these seeded animals, which rendered relatively few indications of statistical differences (fig. 2e-h). for aβ40 measures, the only instance in which the seeded mice had levels that were higher than uninjected controls, or mice injected with ndc lysate, were mice seeded by ad 1 (fig. 2e, f). for measurements of sds-soluble aβ42, the brains of mice seeded with ad 1, 2, and pa 4 were the only examples in which the levels were statistically higher than the levels in mice seeded with ndc 1 and 2, or uninjected mice (fig. 2g). in the fa-soluble fractions, only the ad lysates possessed higher levels of aβ42 than the brains of mice seeded with the two ndc lysates or uninjected mice (fig. 2h). collectively, these findings demonstrated that both ad and pa brains have the potential to seed diffuse aβ pathology, with the ad brains appearing to be slightly more potent. fig. 2 biochemical analysis of sequentially extracted aβ42 and aβ40 levels by end-specific sandwich elisa. prp.appsi/tau-p301l and prp.appsi mice were seeded by ad, pa, or ndc lysate at p0 and aged 12 months. (a, e) 2% sds-extracted aβ40, (b, f) 70% formic acid aβ40, (c, g) 2% sds-extracted aβ42, and (d, h) 70% formic acid aβ42. data plotted as scatter dot plot of prp.appsi/tau-p301l (square) and prp.appsi (circle) ± standard error of the mean. the number of prp.appsi/tau-p301l or prp.appsi mice injected with each lysate, respectively, were as follows: ad 1: n=5, n=4; ad 2: n=6, n=5; pa 1: n=5, n=4; pa 2: n=4, n=5; pa 3: n=5, n=4; pa 4: n=3, n=5; ndc 1: n=5, n=4; ndc 2: n=5, n=5; uninjected: n=5, n=3. aβ42 and aβ40 levels were quantified with corresponding one-way anova with multiple comparisons test (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001). to determine whether the injection of these lysates into the brains of newborn prp.appsi/itau-p301l mice also induced tau pathology, we stained the brains of the seeded animals with antibodies specific for phosphorylated tau (at8) and misfolded tau (mc1). immunostaining with the antibody cp27, which is specific for human tau, confirmed the presence of human p301l tau in the bigenic mice, but we observed no obvious reactivity with at8 or mc1 antibodies (fig. s9). to determine if the induction of amyloid would secondarily induce αsyn pathology, we examined the seeding activity of individuals with ad and lbd pathology. to characterize the pathology of these ad, ad/lbd, and ndc brains, we stained sections with a pan-aβ antibody (fig. 3a), thio-s (fig. 3b), αsyn antibody (fig. 3c), and a phospho-specific tau antibody (fig. 3d). both ad 3 and 4 contained numerous amyloid deposits that were compact and thio-s positive (fig. 3a, b), had widespread tau positive inclusions (fig. 3d), and lacked αsyn pathology (fig. 3c). ad 3 showed striking thio-s positive amyloid staining surrounding vessels, consistent with a caa score of 3+ (fig. 3b, table 1). we also observed caa staining with ad/lbd 1 (fig. 3b). the ad/lbd cases showed modest aβ pathology with some αsyn pathology and sparse tau deposition (fig. 3a, c, d). ndc 3 contained some amyloid pathology, but both lacked αsyn and tau pathology (fig. 3). fig. 3 characterization of ad, ad/lbd, and control cases. representative image from the cortex of 2 ad cases, 2 ad/lbd cases, and 2 ndc cases stained with (a) biotinylated anti-aβ mab 33.1.1 (anti-aβ 1-16). scale bar: 100 μm, (b) thio-s. scale bar: 50 μm, (c) anti-αsyn mab 9c10 (anti αsyn 2-21). arrowheads depict lewy bodies. scale bar: 100 μm, and anti-ptau mab 7f2 (pthr205). scale bar: 150 μm. prp.appsi/m20 and prp.appsi mice injected with ad brains developed widespread, robust aβ pathology by 12 months post-injection (fig. 4a and b). aβ deposition was primarily diffuse and thio-s negative (fig. s10). there was no difference in aβ seeding capacity between prp.appsi/m20 and prp.appsi mice. interestingly, ad/lbd 1 and 2 also promoted deposition of aβ in mice expressing appsi with and without αsyn (fig. 4), indicating that the ad/lbd brains contained considerable levels of aβ seeds despite a much lower burden of aβ pathology (fig. 3). none of the injected animals developed appreciable αsyn pathology (data not shown). although two of the donor cases contained significant caa pathology, caa was not seeded in the recipient mice (fig. s10). fig. 4 cerebral injection of ad and ad/lbd brain lysates results in widespread amyloid deposition and caa. overall schematic of the experiment (a). newborn p0 mice were injected with ad, ad/lbd, and ndc brain lysates and aged 12 months. representative brain sections (hippocampus, cortex, and meninges) of (b) prp.appsi/line m20 and (c) prp.appsi mice stained with biotinylated anti-aβ mab ab5 (anti-aβ 1-16) and counterstained with hematoxylin. n=4-8. scale bar: hippocampus: 250 μm, cortex and meninges: 50 μm. seeding diffuse aβ deposition does not impair performance in a fear-conditioning cognitive task to assess whether the diffuse aβ pathology seeded by ad brain produces cognitive deficits, an additional cohort of prp.appsi mice were seeded with ad 1 and 2, or ndc 1, and then were aged 12 months before behavioral testing in a fear-conditioning paradigm. control groups were mice that were not injected with brain lysate and nontransgenic littermate mice that were either injected with the same lysates or uninjected. the performance of the animals in the contextual fear memory test was conducted twice. in the first round of testing, the performance of the seeded mice was not statistically different from that of the control uninjected mice (fig. 5a). overall, in this first round of testing, the percentage of time animals that exhibited freezing behavior was relatively low. in the second round of testing, we expected to reinforce memory of the adverse cue (context or tone) and we observed that re-tested mice showed increased levels of freezing behavior (fig. 5b); however, there was still no statistically significant difference in the performance of the seeded mice relative to any control. these findings indicate that the diffuse aβ pathology seeded by these homogenates of human ad brain does not produce a meaningful impairment in the systems involved in fear-conditioning memory tasks. fig. 5 diffuse amyloid seeding does not cause cognitive impairment in fear-conditioning. prp.appsi mice seeded by ad and ndc lysates, were aged 12 months and subjected to contextual fear conditioning. mean percentage freezing ± standard error of the mean exhibited by prp.appsi and nontransgenic (ntg) littermates injected with ad cases 1 or 2 and ndc case 7. uninjected mice were the control group. (a) context and tone paradigm. (b) a subset of mice were re-tested the following week, context and tone test. n = 6-8/tg, n = 12-16/ntg mice per group. assessment of aβ seeding efficiency in appsi mice to examine the relative seeding efficiency of different types of aβ pathology in prp.appsi mice, we used an approach in which homogenates from the brains of 4 different lines of aged app and app/ps1 mice were used to seed accelerated aβ deposition (described in table 2). at these advanced ages, each of the mice used to produce inoculum had substantial aβ pathology (fig. s11). following the strategy used above, we injected the homogenates containing aβ seeds into newborn mice on neonatal day 0, which were then aged to 9, 12, or 15 months before euthanasia and neuropathological analysis by campbell switzer silver stain and thio-s staining. the type of pathology noted and the relative abundance score are noted at the bottom of each column of images in figure 6. in all of the prp.appsi mice injected with brain homogenate seeds from mice that primarily exhibited cored plaques, we observed an obvious shift in the type of aβ deposits in recipient mice to match the pathology found in the donor mice used for seeding (fig. 6). self-seeding of newborn prp.appsi mice with brain homogenates from aged prp.appsi mice produced diffuse deposits (fig. 6), which was the predominant form of aβ pathology in the prp.appsi animal used to generate the seeding homogenate (fig. s11). similarly, the predominant pathology in the ~25 month old tet.moaβ animal used to prepare seeding homogenate was diffuse aβ deposits (fig. s11)(48), and brain homogenates from this animal efficiently seeded the deposition of human aβ in the prp.appsi mice to produce diffuse deposits (fig. 6). prp.appsi mice seeded with homogenates from donors that had primarily cored deposits had lower aβ burden scores, but importantly we observed a shift in the neuropathologic features of the recipient mice to a much higher incidence of cored deposits (fig. 6). interestingly, the morphology of deposits in the seeded prp.appsi mice was not quite identical to the source prp.huaβ/ps1 mice in that the seeded deposits were smaller and appeared to be more compact than the donor line. these small dense deposits were thio-s positive (fig. 6). similar small dense core deposits that were thio-s positive were observed in the cortex of prp.appsi mice injected with brain homogenates from the prp.moaβ/ps1 mice (fig. 6). collectively, these findings demonstrate the relative ease with which diffuse aβ pathology can be induced in prp.appsi mice. fig. 6 comparison of seeding activity between transgenic mice that exhibit diffuse versus cored aβ pathology. we selected brains from four sources that exhibit either diffuse or cored neuritic aβ deposits (see fig. s11). newborn prp.appsi mice were injected with homogenate from each of the four sources identified at the top of the figure. compared to uninjected mice, prp.appsi mice injected with brain homogenates from any source showed an induction of aβ pathology. inoculum from aged prp.appsi and tet.moaβ mice, which primarily exhibit diffuse aβ pathology (37, 48), robustly seeded diffuse pathology in the injected prp.appsi mice (severity of pathology and number of animals indicated at the bottom of the figure). inoculum from aged prp.huaβ/ps1 (a.k.a. appswe/ps1de9) and prp.moaβ/ps1 mice, which primarily exhibit cored neuritic deposits (37, 48), induced the deposition of a limited number of cored aβ deposits in injected prp.appsi mice with little or no diffuse deposits. d = diffuse aβ pathology. m = mixed diffuse and cored deposits. c = cored, neuritic deposits. discussion we investigated whether injection of neonatal app, app/tau, or app/αsyn mice with ad or ad/lbd brain homogenates could be used as a paradigm to generate mice that model the mixed pathology associated with each disease. despite the widespread induction of diffuse aβ deposition by injection of four different ad brain lysates, and the presence of tau seeds in these lysates, tau pathology was not induced. for comparison, we also injected pa brain lysates, finding a similar induction of diffuse aβ deposition without induction of tau pathology. similarly, injection of ad/lbd brain lysates induced diffuse aβ deposition, but αsyn pathology was not induced. in all cases, the induced aβ pathology exhibited a diffuse, thio-s negative, morphology. prp.appsi mice injected with ad brain lysates that develop diffuse aβ pathology showed no significant impairment in a fear-conditioning cognitive task. our findings suggest that seeding the brains of neonatal transgenic mice with ad, ad/lbd, and pa brain homogenates can efficiently induce diffuse aβ deposition but neither ad nor ad/lbd brains seeded concurrent tau or αsyn pathology. the most common route of administering aβ seeding preparations is by stereotaxic injection into the hippocampus of adult app transgenic mice (reviewed in (13)). in host mice that develop amyloid pathology at relatively young ages, the induced pathology created by seeding may be localized to the site of injection and overlying cortex (13); whereas, in models that do not develop deposits until late in life, the induced pathology may be more wide-spread (reviewed in (13)). the prp.appsi host we used in the present study develops pathology on its own between 12-14 months of age. in testing the approach of injecting aβ seeds into newborn mice from this model, we aspired to attain a widespread distribution of seeding material and induce aβ, tau, or αsyn pathology throughout the brain. this approach replicates previous studies where p0 injection of adeno-associated virus resulted in widespread distribution in the brain (49). injection of newborn hamsters with scrapie prions has been shown to accelerate onset of prion disease (50). additionally, we have observed that intracerebral injection of newborn a53t αsyn mice with brain lysates from multiple system atrophy donors induces αsyn pathology and motor impairment (31), and that intraspinal injection of newborn mice expressing mutant superoxide dismutase (sod1) can accelerate the onset of paralysis and pathology (51, 52). thus, in performing newborn injections our goal was to initiate pathological cascades as early as possible in order to determine whether there may be distinct synergies between aβ and tau, or aβ and αsyn, which can be elaborated by seeding. we recognize that there are a large number of potential mouse models that could have been used for these studies. the app mouse model we used for these studies was selected because prp.appsi mice do not inherently develop aβ pathology until 12-14 months of age and because this model can exhibit a full spectrum of aβ pathology including cored-neuritic, diffuse, and vascular deposition (see supplemental fig. s2). the tau model we chose to use expresses human p301l tau at relatively low levels and does not develop tau pathology on its own. when paired with mice that express the tetracycline transactivator in the rtg4510 model these develop a robust tau pathology (38). in paradigms in which tau expression in rtg4510 mice is induced early and then suppressed by doxycycline, the low level of “leaky” tau expression in this model is sufficient to sustain neurofibrillary tangle pathology (38). in our paradigm, we asked whether neonatal seeding of this tau model could induce a sustained pathology in the same way that early expression of the transgene at high levels produced sustained pathology. alternatively, we were interested to determine whether we could detect any synergy between concurrent aβ pathology and tau seeding. the αsyn model we chose to use expresses human wt αsyn at levels that do not cause pathology (41). wt αsyn (m20) mice can be seeded when injected with high levels of purified αsyn fibrils, but are not easily seeded by human brain homogenates (31). our goal in choosing the m20 wt αsyn mice was to develop a model of human αsyn pathology and assess whether concurrent aβ pathology could synergize αsyn seeding. our method of seed preparation followed commonly used protocols where we injected a soluble fraction; frontal cortex homogenized in pbs (10% w/v) followed by sonication and low speed centrifugation, as previously described (42, 53, 54). by this method, we expected sonication to fragment all types of seeds with a mixture of seeds remaining in suspension after low-speed centrifugation. as we have observed here, the most common outcome of aβ seeding with similar preparations is induction of diffuse aβ pathology (reviewed in (13)). in order to efficiently seed mixed pathology, it may be necessary to optimize preparations of each type of seed independently. interestingly, injection of the supernatant and pellet of fractionated app23 brain homogenate resulted in morphologically different aβ deposits, with the supernatant fraction seeding diffuse, congo red negative aβ and the pellet seeding deposits similar to the total lysate, a mixture of diffuse and punctate, congo red positive deposits (55). several studies have shown induction of tau pathology after injection of synthetic tau fibrils, brain extract from mutant p301s tau mouse, or human ad, corticobasal degeneration, and progressive supranuclear palsy brain lysates (56-62). in these studies, the human brain lysates were enriched for tau seeds by sequential fractionation with the addition of sucrose and/or sarkosyl, creating fractions that contain potent tau seeds. similarly, injection of the fractionated homogenate from brain lysates of individuals with lbd resulted in the induction of αsyn, indicating that with enrichment it is possible to seed αsyn pathology directly from human brain (20, 63, 64). because one of our goals was to determine whether we could detect synergy between aβ and tau or αsyn pathology, we chose to avoid enriching for any particular type of seeding activity. the absence of tau or αsyn pathology in our seeded models indicates that the type of aβ deposition we generated did not synergize to induce, or exacerbate, tau or αsyn pathology. we demonstrated that the aβ seeding activity of brain lysates from pa cases was similar to ad and ad/lbd cases. these findings agree with a previous study that reported brain homogenates from a human pa case seeded diffuse aβ deposition in tg2576 app mice (65). although mice seeded by pa brains scored as having less amyloid positivity at 12 months post-injection than mice injected with ad brain lysates, the difference between pa and ad brain seeding activity was less evident by 18 months post-injection. our study was not powered or designed to determine whether homogenates from ad and pa brains have quantitative differences in seeding activity. all three of the pa cases tested were able to seed amyloid pathology, supporting the hypothesis that the aβ pathology in pa is similar to ad (25). interestingly, several of the brain lysates we used were from tissues that exhibited relatively high levels of caa pathology (see table 1). pa 3 and ad 3 were both scored as having the highest levels of caa, with significant caa in ad/lbd 1 and 2. in mice injected with pa 3, we observed modest seeding of parenchymal caa pathology in the host mice but in all other mice the only obvious vascular deposition was meningeal. augmented caa pathology has been observed in app23 mice, which develop caa, by intraperitoneal injection of app23 lysates (66). caa pathology was also induced in crnd8 mice by injection of synthetic aβ42 oligomers, generated in the presence of anionic micelles composed of fatty acids (67). recent studies have documented early onset cerebrovascular aβ pathology in individuals receiving dura mater transplants and cadaveric pituitary-derived growth hormone, suggesting that caa may be transmitted iatrogenically (68, 69). collectively, these results suggest that caa could be a distinct conformer of aβ that may be independently propagated. not all aβ conformers seeded as proficiently as vascular aβ. tissues from ad and ad/lbd brains that were used to prepare these lysates contained both diffuse and compact aβ deposits, with significant levels of formic acid extractable aβ. yet, even in mice aged to 18 months of age, diffuse aβ deposits were the dominant form of pathology. these results mirror similar studies in which injection of human brain homogenates resulted in induction of diffuse aβ pathology, with relatively few thio-s or congophilic compact deposits (49, 65, 70, 71). the lack of conversion of the diffuse deposits to cored plaques indicates that these types of amyloid deposition are not freely interchangeable, supporting the idea that each type of aβ pathology arises from different amyloid strains with distinct seeding capabilities. this conclusion is also supported by our studies of aβ seeding in which transgenic mice were used as the seed source. newborn prp.appsi mice injected with transgenic mouse brain homogenates prepared from mice that exhibit high levels of diffuse aβ pathology produced a robust induction of diffuse aβ deposition by 12 months post-injection. by contrast, brain homogenates prepared from mice that predominantly produce cored, thio-s positive, aβ deposits induced minimal aβ deposition by 12 months. these results are consistent with previous studies with app/ps1 and app23 mice. injection of appps1 mice that typically develop compact, punctate plaques, with app23 seeds results in a mixture of diffuse, filamentous aβ as well as compact plaques (42, 72). injection of app23 mice that typically develop mixed pathology, both cored and diffuse plaques, with appps1 homogenate results in plaques that are more diffuse than the punctate deposits seen in appps1 mice injected with appps1 homogenate (42, 72). thus, although it is possible to seed cored deposits in app mice (42, 55, 73-75), it appears that diffuse aβ pathology is more easily seeded. together these results suggest that aβ plaques differ in their morphology, seeding ability, and impact on cognitive function. current amyloid therapies aim to reduce amyloid deposition without much consideration of the type of plaque that is being targeted. reduction of diffuse amyloid may at first appear to be beneficial, since it appears to be the dominate seeding strain, however, it may not be the most pathological strain. cognitive behavior studies of the seeded appsi mice reported here, and of a non-seeded bri-aβ42 model that also shows primarily diffuse aβ pathology (76), failed to associate diffuse amyloid with reduced performance in a fear-conditioning memory task. if diffuse aβ pathology is less damaging, then therapies that only reduce this pathology may not produce therapeutic benefit in ad. conclusions in conclusion, we assessed the type of pathology induced when neonatal app transgenic mice were injected with brain lysates prepared from ad, ad/lbd, and pa brains, finding robust and widespread induction of aβ pathology. the induced aβ pathology was diffuse, which has been reported in other seeding studies where adult animals were the recipients of injections (13). our results are consistent with earlier reports in finding that diffuse aβ is easily seeded by crude homogenates of human brain. the diffuse aβ pathology induced in these models was not accompanied by secondary tau or αsyn pathology in bigenic mice co-expressing app/tau or app/asyn. seeded mice with diffuse aβ pathology were not impaired in a fear-conditioning memory task. the seeding approach described here may be useful in producing mice that model aspects of non-ad pathological aging, providing a useful comparison model to mice that exhibit pathological features more similar to ad and ad/lbd pathology. ethics approval human brain tissue was obtained from the mayo clinic brain bank and university of florida neuromedicine human brain tissue bank with informed consent following institutional regulations. all animal procedures were approved by the university of florida institutional animal care and use committee. consent for publication not applicable. availability of data and materials all data generated and analyzed during this study are included in this manuscript. competing interests the authors declare that they have no competing interests. funding this work was supported by funding from the national institute of health; grant numbers r21ag046125, r21ag055113, u54ns110435, p50ag047266, p30ag066506. authors’ contributions study concept and design: bdm, yl, jl, big, drb. pathological analysis: sp and dd. acquisition of data: bdm, yl, gx, hh, mfa, clc, hsf, cm, sf, sp, dd. statistical analysis: bdm and drb. analysis and interpretation of the data: bdm, yl, gx, cj, jl, big, teg, drb. drafting of the manuscript: bdm and drb. acquisition of funding: jl, big, teg, drb. all authors read and approved the final manuscript. acknowledgements the authors would like to thank the patients and their families that donated tissue. we thank dr. peter davies (feinstein institute for medical research, manhasset, ny, usa) posthumously for antibodies to tau. conflict of interest teg is a co-founder of lacerta therapeutics. references 1. hardy ja, higgins ga. alzheimer's disease: the amyloid cascade hypothesis. science. 1992;256(5054):184-5. 2. selkoe dj, hardy j. the amyloid hypothesis of alzheimer's disease at 25 years. embo mol med. 2016;8(6):595-608. 3. lewis j, dickson dw, lin wl, chisholm l, corral a, jones g, et al. enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and app. science. 2001;293(5534):1487-91. 4. oddo s, caccamo a, shepherd jd, murphy mp, golde te, kayed r, et al. triple-transgenic model of alzheimer's disease with plaques and tangles: intracellular abeta and synaptic dysfunction. neuron. 2003;39(3):409-21. 5. paulson jb, ramsden m, forster c, sherman ma, mcgowan e, ashe kh. amyloid plaque and neurofibrillary tangle pathology in a regulatable mouse model of alzheimer's disease. am j pathol. 2008;173(3):762-72. 6. guo q, li h, cole al, hur jy, li y, zheng h. modeling alzheimer's disease in mouse without mutant protein overexpression: cooperative and independent effects of abeta and tau. plos one. 2013;8(11):e80706. 7. angulo sl, orman r, neymotin sa, liu l, buitrago l, cepeda-prado e, et al. tau and amyloid-related pathologies in the entorhinal cortex have divergent effects in the hippocampal circuit. neurobiol dis. 2017;108:261-76. 8. bennett re, devos sl, dujardin s, corjuc b, gor r, gonzalez j, et al. enhanced tau aggregation in the presence of amyloid beta. am j pathol. 2017;187(7):1601-12. 9. rodriguez ga, barrett gm, duff ke, hussaini sa. chemogenetic attenuation of neuronal activity in the entorhinal cortex reduces abeta and tau pathology in the hippocampus. plos biol. 2020;18(8):e3000851. 10. saul a, sprenger f, bayer ta, wirths o. accelerated tau pathology with synaptic and neuronal loss in a novel triple transgenic mouse model of alzheimer's disease. neurobiol aging. 2013;34(11):2564-73. 11. xu g, ulm bs, howard j, fromholt se, lu q, lee bb, et al. tapping into the potential of inducible tau/app transgenic mice. neuropathol appl neurobiol. 2022;in press. 12. koller ej, ibanez kr, vo q, mcfarland kn, gonzalez de la cruz e, zobel l, et al. combinatorial model of amyloid beta and tau reveals synergy between amyloid deposits and tangle formation. neuropathol appl neurobiol. 2021;48(2):e12779. 13. ulm bs, borchelt dr, moore bd. remodeling alzheimer-amyloidosis models by seeding. mol neurodegener. 2021;16(1):8. 14. vergara c, houben s, suain v, yilmaz z, de decker r, vanden dries v, et al. amyloid-beta pathology enhances pathological fibrillary tau seeding induced by alzheimer phf in vivo. acta neuropathol. 2019;137(3):397-412. 15. nies sh, takahashi h, herber cs, huttner a, chase a, strittmatter sm. spreading of alzheimer tau seeds is enhanced by aging and template matching with limited impact of amyloid-beta. j biol chem. 2021;297(4):101159. 16. hansen l, salmon d, galasko d, masliah e, katzman r, deteresa r, et al. the lewy body variant of alzheimer's disease: a clinical and pathologic entity. neurology. 1990;40(1):1-8. 17. perry rh, irving d, blessed g, fairbairn a, perry ek. senile dementia of lewy body type. a clinically and neuropathologically distinct form of lewy body dementia in the elderly. j neurol sci. 1990;95(2):119-39. 18. clinton lk, blurton-jones m, myczek k, trojanowski jq, laferla fm. synergistic interactions between abeta, tau, and alpha-synuclein: acceleration of neuropathology and cognitive decline. j neurosci. 2010;30(21):7281-9. 19. masliah e, rockenstein e, veinbergs i, sagara y, mallory m, hashimoto m, et al. beta-amyloid peptides enhance alpha-synuclein accumulation and neuronal deficits in a transgenic mouse model linking alzheimer's disease and parkinson's disease. proc natl acad sci u s a. 2001;98(21):12245-50. 20. bassil f, brown hj, pattabhiraman s, iwasyk je, maghames cm, meymand es, et al. amyloid-beta (abeta) plaques promote seeding and spreading of alpha-synuclein and tau in a mouse model of lewy body disorders with abeta pathology. neuron. 2020;105(2):260-75 e6. 21. lloyd gm, dhillon js, gorion km, riffe c, fromholt se, xia y, et al. collusion of alpha-synuclein and abeta aggravating co-morbidities in a novel prion-type mouse model. mol neurodegener. 2021;16(1):63. 22. yamada m, tsukagoshi h, otomo e, hayakawa m. systemic amyloid deposition in old age and dementia of alzheimer type: the relationship of brain amyloid to other amyloid. acta neuropathol. 1988;77(2):136-41. 23. golde te, schneider ls, koo eh. anti-abeta therapeutics in alzheimer's disease: the need for a paradigm shift. neuron. 2011;69(2):203-13. 24. jack cr, jr., lowe vj, weigand sd, wiste hj, senjem ml, knopman ds, et al. serial pib and mri in normal, mild cognitive impairment and alzheimer's disease: implications for sequence of pathological events in alzheimer's disease. brain. 2009;132(pt 5):1355-65. 25. moore bd, chakrabarty p, levites y, kukar tl, baine am, moroni t, et al. overlapping profiles of abeta peptides in the alzheimer's disease and pathological aging brains. alzheimers res ther. 2012;4(3):18. 26. dickson dw, crystal ha, mattiace la, masur dm, blau ad, davies p, et al. identification of normal and pathological aging in prospectively studied nondemented elderly humans. neurobiol aging. 1992;13(1):179-89. 27. dickson dw, crystal ha, bevona c, honer w, vincent i, davies p. correlations of synaptic and pathological markers with cognition of the elderly. neurobiol aging. 1995;16(3):285-98; discussion 98-304. 28. golde te, dickson d, hutton m. filling the gaps in the abeta cascade hypothesis of alzheimer's disease. curr alzheimer res. 2006;3(5):421-30. 29. ayers ji, fromholt s, sinyavskaya o, siemienski z, rosario am, li a, et al. widespread and efficient transduction of spinal cord and brain following neonatal aav injection and potential disease modifying effect in als mice. mol ther. 2015;23(1):53-62. 30. kim jy, grunke sd, levites y, golde te, jankowsky jl. intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. j vis exp. 2014(91):51863. 31. dhillon js, trejo-lopez ja, riffe c, levites y, sacino an, borchelt dr, et al. comparative analyses of the in vivo induction and transmission of alpha-synuclein pathology in transgenic mice by msa brain lysate and recombinant alpha-synuclein fibrils. acta neuropathol commun. 2019;7(1):80. 32. saido tc, kawashima s, tani e, yokota m. upand down-regulation of calpain inhibitor polypeptide, calpastatin, in postischemic hippocampus. neurosci lett. 1997;227(2):75-8. 33. schmidt ml, didario ag, otvos l, jr., hoshi n, kant ja, lee vm, et al. plaque-associated neuronal proteins: a recurrent motif in neuritic amyloid deposits throughout diverse cortical areas of the alzheimer's disease brain. exp neurol. 1994;130(2):311-22. 34. mckeith ig, boeve bf, dickson dw, halliday g, taylor jp, weintraub d, et al. diagnosis and management of dementia with lewy bodies: fourth consensus report of the dlb consortium. neurology. 2017;89(1):88-100. 35. montine tj, phelps ch, beach tg, bigio eh, cairns nj, dickson dw, et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol. 2012;123(1):1-11. 36. charidimou a, martinez-ramirez s, shoamanesh a, oliveira-filho j, frosch m, vashkevich a, et al. cerebral amyloid angiopathy with and without hemorrhage: evidence for different disease phenotypes. neurology. 2015;84(12):1206-12. 37. xu g, ran y, fromholt se, fu c, yachnis at, golde te, et al. murine abeta over-production produces diffuse and compact alzheimer-type amyloid deposits. acta neuropathol commun. 2015;3:72. 38. santacruz k, lewis j, spires t, paulson j, kotilinek l, ingelsson m, et al. tau suppression in a neurodegenerative mouse model improves memory function. science. 2005;309(5733):476-81. 39. strang kh, goodwin ms, riffe c, moore bd, chakrabarty p, levites y, et al. generation and characterization of new monoclonal antibodies targeting the phf1 and at8 epitopes on human tau. acta neuropathol commun. 2017;5(1):58. 40. xia y, bell bm, giasson bi. tau k321/k353 pseudoacetylation within kxgs motifs regulates tau-microtubule interactions and inhibits aggregation. sci rep. 2021;11(1):17069. 41. giasson bi, duda je, quinn sm, zhang b, trojanowski jq, lee vm. neuronal alpha-synucleinopathy with severe movement disorder in mice expressing a53t human alpha-synuclein. neuron. 2002;34(4):521-33. 42. meyer-luehmann m, coomaraswamy j, bolmont t, kaeser s, schaefer c, kilger e, et al. exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. science. 2006;313(5794):1781-4. 43. chakrabarty p, rosario a, cruz p, siemienski z, ceballos-diaz c, crosby k, et al. capsid serotype and timing of injection determines aav transduction in the neonatal mice brain. plos one. 2013;8(6):e67680. 44. vallet pg, guntern r, hof pr, golaz j, delacourte a, robakis nk, et al. a comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in alzheimer's disease. acta neuropathol. 1992;83(2):170-8. 45. hanna a, iremonger k, das p, dickson d, golde t, janus c. age-related increase in amyloid plaque burden is associated with impairment in conditioned fear memory in crnd8 mouse model of amyloidosis. alzheimers res ther. 2012;4(3):21. 46. chakrabarty p, hudson vj, iii, sacino an, brooks mm, d'alton s, lewis j, et al. inefficient induction and spread of seeded tau pathology in p301l mouse model of tauopathy suggests inherent physiological barriers to transmission. acta neuropathol. 2015;130(2):303-5. 47. lewis j, mcgowan e, rockwood j, melrose h, nacharaju p, van slegtenhorst m, et al. neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (p301l) tau protein. nat genet. 2000;25(4):402-5. 48. xu g, fromholt se, chakrabarty p, zhu f, liu x, pace mc, et al. diversity in abeta deposit morphology and secondary proteome insolubility across models of alzheimer-type amyloidosis. acta neuropathol commun. 2020;8(1):43. 49. walker lc, callahan mj, bian f, durham ra, roher ae, lipinski wj. exogenous induction of cerebral beta-amyloidosis in betaapp-transgenic mice. peptides. 2002;23(7):1241-7. 50. mckinley mp, dearmond sj, torchia m, mobley wc, prusiner sb. acceleration of scrapie in neonatal syrian hamsters. neurology. 1989;39(10):1319-24. 51. ayers ji, fromholt s, koch m, debosier a, mcmahon b, xu g, et al. experimental transmissibility of mutant sod1 motor neuron disease. acta neuropathol. 2014;128(6):791-803. 52. bidhendi ee, bergh j, zetterstrom p, andersen pm, marklund sl, brannstrom t. two superoxide dismutase prion strains transmit amyotrophic lateral sclerosis-like disease. j clin invest. 2016;126(6):2249-53. 53. eisele ys, bolmont t, heikenwalder m, langer f, jacobson lh, yan zx, et al. induction of cerebral beta-amyloidosis: intracerebral versus systemic abeta inoculation. proc natl acad sci u s a. 2009;106(31):12926-31. 54. ziegler-waldkirch s, d'errico p, sauer jf, erny d, savanthrapadian s, loreth d, et al. seed-induced abeta deposition is modulated by microglia under environmental enrichment in a mouse model of alzheimer's disease. embo j. 2018;37(2):167-82. 55. langer f, eisele ys, fritschi sk, staufenbiel m, walker lc, jucker m. soluble abeta seeds are potent inducers of cerebral beta-amyloid deposition. j neurosci. 2011;31(41):14488-95. 56. peeraer e, bottelbergs a, van kolen k, stancu ic, vasconcelos b, mahieu m, et al. intracerebral injection of preformed synthetic tau fibrils initiates widespread tauopathy and neuronal loss in the brains of tau transgenic mice. neurobiol dis. 2015;73:83-95. 57. clavaguera f, bolmont t, crowther ra, abramowski d, frank s, probst a, et al. transmission and spreading of tauopathy in transgenic mouse brain. nat cell biol. 2009;11(7):909-13. 58. guo jl, narasimhan s, changolkar l, he z, stieber a, zhang b, et al. unique pathological tau conformers from alzheimer's brains transmit tau pathology in nontransgenic mice. j exp med. 2016;213(12):2635-54. 59. guo jl, lee vm. neurofibrillary tangle-like tau pathology induced by synthetic tau fibrils in primary neurons over-expressing mutant tau. febs lett. 2013;587(6):717-23. 60. boluda s, iba m, zhang b, raible km, lee vm, trojanowski jq. differential induction and spread of tau pathology in young ps19 tau transgenic mice following intracerebral injections of pathological tau from alzheimer's disease or corticobasal degeneration brains. acta neuropathol. 2015;129(2):221-37. 61. narasimhan s, guo jl, changolkar l, stieber a, mcbride jd, silva lv, et al. pathological tau strains from human brains recapitulate the diversity of tauopathies in nontransgenic mouse brain. j neurosci. 2017;37(47):11406-23. 62. clavaguera f, akatsu h, fraser g, crowther ra, frank s, hench j, et al. brain homogenates from human tauopathies induce tau inclusions in mouse brain. proc natl acad sci u s a. 2013;110(23):9535-40. 63. masuda-suzukake m, nonaka t, hosokawa m, oikawa t, arai t, akiyama h, et al. prion-like spreading of pathological alpha-synuclein in brain. brain. 2013;136(pt 4):1128-38. 64. recasens a, dehay b, bove j, carballo-carbajal i, dovero s, perez-villalba a, et al. lewy body extracts from parkinson disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys. ann neurol. 2014;75(3):351-62. 65. kane md, lipinski wj, callahan mj, bian f, durham ra, schwarz rd, et al. evidence for seeding of beta -amyloid by intracerebral infusion of alzheimer brain extracts in beta -amyloid precursor protein-transgenic mice. j neurosci. 2000;20(10):3606-11. 66. eisele ys, obermuller u, heilbronner g, baumann f, kaeser sa, wolburg h, et al. peripherally applied abeta-containing inoculates induce cerebral beta-amyloidosis. science. 2010;330(6006):980-2. 67. saha j, dean dn, dhakal s, stockmal ka, morgan se, dillon kd, et al. biophysical characteristics of lipid-induced abeta oligomers correlate to distinctive phenotypes in transgenic mice. faseb j. 2021;35(2):e21318. 68. preusser m, strobel t, gelpi e, eiler m, broessner g, schmutzhard e, et al. alzheimer-type neuropathology in a 28 year old patient with iatrogenic creutzfeldt-jakob disease after dural grafting. j neurol neurosurg psychiatry. 2006;77(3):413-6. 69. jaunmuktane z, mead s, ellis m, wadsworth jd, nicoll aj, kenny j, et al. evidence for human transmission of amyloid-beta pathology and cerebral amyloid angiopathy. nature. 2015;525(7568):247-50. 70. morales r, duran-aniotz c, castilla j, estrada ld, soto c. de novo induction of amyloid-beta deposition in vivo. mol psychiatry. 2012;17(12):1347-53. 71. fritschi sk, langer f, kaeser sa, maia lf, portelius e, pinotsi d, et al. highly potent soluble amyloid-beta seeds in human alzheimer brain but not cerebrospinal fluid. brain. 2014;137(pt 11):2909-15. 72. heilbronner g, eisele ys, langer f, kaeser sa, novotny r, nagarathinam a, et al. seeded strain-like transmission of beta-amyloid morphotypes in app transgenic mice. embo rep. 2013;14(11):1017-22. 73. eisele ys, fritschi sk, hamaguchi t, obermuller u, fuger p, skodras a, et al. multiple factors contribute to the peripheral induction of cerebral beta-amyloidosis. j neurosci. 2014;34(31):10264-73. 74. fritschi sk, cintron a, ye l, mahler j, buhler a, baumann f, et al. abeta seeds resist inactivation by formaldehyde. acta neuropathol. 2014;128(4):477-84. 75. ye l, rasmussen j, kaeser sa, marzesco am, obermuller u, mahler j, et al. abeta seeding potency peaks in the early stages of cerebral beta-amyloidosis. embo rep. 2017;18(9):1536-44. 76. kim j, chakrabarty p, hanna a, march a, dickson dw, borchelt dr, et al. normal cognition in transgenic bri2-abeta mice. mol neurodegener. 2013;8:15. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. lessons learned from a career in neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 2:27 (2021) reflections lessons learned from a career in neuropathology roy o. weller faculty of medicine, southampton university, united kingdom address for correspondence: roy o. weller, bsc md phd frcpath, emeritus professor of neuropathology · clinical neurosciences · ld66, mail point 806, south academic block, level d · southampton university hospital southampton so16 6yd · united kingdom rcn@soton.ac.uk submitted: 09 october 2021 accepted: accepted: 12 october 2021 published: 25 october 2021 https://doi.org/10.17879/freeneuropathology-2021-3634 additional resources and electronic supplementary material: supplementary material keywords: clinical neuropathology, research, students, lymphatic drainage of the brain, alzheimer’s disease, cerebral amyloid angiopathy, personal reflections, family introduction writing an autobiography was probably the last thing on my mind when i was asked to add mine to a growing list of personal accounts published in free neuropathology. it was no easy task to summarise my life in what i hope will be an interesting account for my readers. but, in the end, it has been rewarding for me at least to state what i think have been the important events, ideas and attitudes that have shaped my life and career in neuropathology and to recognise some of the many people who have taught, influenced, helped me and offered their friendship over many years. as for the lessons learned, i hope they will become apparent as the account proceeds. early years i was born in a south-eastern suburb of london a year before the outbreak of the second world war. our house was two miles from the river thames and woolwich arsenal but, despite this vulnerable position, our home suffered no damage from bombing. it did mean, however, that there were many periods when we spent nights in an air-raid shelter in the garden. i was too young to appreciate the deprivations of wartime england and the terrible time that my parents endured. the first school that i attended was partially destroyed by a v2 rocket and my classmates and i transferred to another school that had already been half destroyed by incendiary bombs. despite this, all i can remember is a very happy childhood and education. at the age of 11 years, i transferred to a grammar school in the centre of london, close to tower bridge. founded in 1588, st olave’s and st saviour’s school that i attended had moved to a beautiful early victorian building in 1840 to make room for the construction of london bridge station. the school is now in kent and the original 1840 building is a 5-star hotel. i received a classical education with some concentration on languages; english, latin, french, greek and german have all been of great value to me, not only in medicine but also when i travelled widely in europe as an undergraduate and during my postgraduate career. science was also strong in my school and this enabled me to enter guy’s hospital medical school in london as a medical student in 1956. my motivation for studying medicine was mainly the attraction of the basic sciences, particularly biochemistry, but i soon became enthralled with patient contact and clinical medicine. during my undergraduate studies i spent a year, gaining a bachelor of science degree in anatomy under excellent tuition from roger warwick and peter williams who were both editors of gray’s anatomy. i visited many of the medical schools in london for lectures and demonstrations during this year which gave me a clear insight into academic medicine and was probably a major factor in determining my postgraduate career in neuropathology. early postgraduate career having completed my medical education, i took a teaching post in anatomy at guy’s and a research post with professor john cavanagh (figure 1) to work on peripheral neuropathies, most especially experimental diphtheritic neuropathy in chickens (much to the amusement of my friends). my objective was to determine the ultrastructural sequences involved in the initial stages of segmental demyelination. john was an ideal supervisor at the beginning of my research career as he would present me with a problem and leave me to solve it, only giving advice when asked; john also taught me the basis of the scientific method and how to view earlier findings in my field with scepticism. i was introduced to the technical aspects and analysis of transmission electron microscopy (tem) that was still a relatively new technique. the skills i learnt at that time have served me throughout my career. in my first paper in 1965 1, i showed that, prior to breakdown of the myelin sheath in segmental demyelination, acid phosphatase-containing lysosomes gathered in cytoplasmic pockets on the inner aspect of the myelin sheath. this gave some indication of how the myelin was removed without damaging the axon that it encompassed. figure 1. john b cavanagh 1997. my second post at guy’s was with professor colin adams, who was a lipid histochemist of world renown. he had a gift for adapting techniques to identify individual lipids, particularly in atherosclerosis and in multiple sclerosis. the major task for my phd was to define the ultrastructure of intracellular lipid accumulations in atherosclerosis. this was no easy matter as the lipids are soluble in the organic solvents used in preparation for tem. by a combination of polarised light microscopy, negative staining and routine tem (figure 2), i identified the multi-lamellated structure of the liquid crystalline lipid droplets that appeared to be the template for esterification of the cholesterol that they contain 2,3. it was during this period, that i started my education in clinical neuropathology with henry urich at the london hospital. henry’s teaching was direct and very suitable for me in the early stages of my career. figure 2. intracellular lipid droplets in atherosclerosis. top left and then clockwise: tem showing removal of lipid from cells during preparation. birefringent liquid crystalline droplets of cholesterol and phospholipids isolated from an atherosclerotic lesion. similar droplets examined as negative stained tem showing lamellated structure 2,3. us-nih-fellowship at the albert einstein college of medicine in new york having gained my ph.d. in 1967, it was time for a change, so my wife, francine, and our two children, adrienne, aged three and timothy aged two years embarked on a boat for the usa. i had gained an us-national institutes of health (us-nih) postdoctoral fellowship in neuropathology with dr robert terry at the albert einstein college of medicine in new york. it was a very different but highly stimulating environment. bob terry was very generous with his time and ideas and this led to numerous interesting discussions about pathology of the nervous system. i learned much about clinical neuropathology from bob and his colleagues, especially kinuko suzuki. for my research, i investigated the effects of acute hydrocephalus upon the brains of adult rabbits, infant dogs and in biopsies from very young children. my main teacher and collaborator in this project was henryk wisniewski who was an excellent animal surgeon and experimenter. henryk devised a technique for producing hydrocephalus by injecting highly viscous, but non-inflammatory, silicon oil through a lumbar intrathecal catheter into the posterior fossa of adult rabbits and young dogs to compress the fourth ventricle and impede the drainage of csf. i examined the brains in large-area 1 μm resin sections and by tem. by sequential examination of the olfactory bulbs in adult rabbits and the walls of the lateral ventricles in infant dogs, we showed that as the ventricles dilate in the early stages of hydrocephalus, the ependyma ruptures and the flow of csf into the periventricular white matter results in extensive csf oedema with a mild degree of axonal degeneration. as hydrocephalus progresses, the csf oedema subsides and the periventricular white matter displays extensive reactive astrocytosis, and axonal degeneration 4,5. a similar sequence of changes was revealed in biopsies from very young children with acute hydrocephalus 6. these observations suggested that treating children with shunts in the early stages of hydrocephalus would preserve axons and improve prognoses. our findings were confirmed several years later when ct scans clearly revealed the presence of periventricular oedema in the acute stages of hydrocephalus. the sequence of changes of ependymal rupture, increasing csf oedema and destruction of periventricular white matter is well demonstrated in a congenital model of hydrocephalus in mice that i bred later when i returned to guy’s (figure 3). figure 3. progressive hydrocephalus in the mouse showing (a) normal mouse brain. (b) early stages of hydrocephalus with csf oedema of the white matter. (c) severe oedema with destruction of the white matter and preservation of grey matter. (d) sem showing almost total destruction of the cerebral white matter and rupture of the ependyma 7. my family and i spent a most rewarding year in new york. we lived in an apartment block in the bronx very near the einstein. as the months progressed we adapted to the very low temperatures in the winter in new york and the high temperatures and humidity in the summer. our children became bilingual through contact with their friends at nursery school; they would speak with a new york accent and vocabulary between themselves and their friends but spoke english when communicating with francine and me. we explored manhattan widely and travelled to a variety of cities in the us to appreciate the diversity of the country. as we finally left new york in september 1968, we travelled by greyhound bus to a conference in new orleans before flying home – quite an adventure! i have very pleasant memories of the team of people with whom i worked at the einstein (figure 4). through meetings of the american association neuropathologists (aanp), i remained in contact with bob terry for many years. similarly, i retained contacts with henryk wisniewski, who had a very successful research career in demyelinating diseases and in alzheimer’s disease. figure 4. neuropathology group at the albert einstein college of medicine 1968. first row, left to right: drs kinuko suzuki, mariene lenger, krystyna wisniewska, kytja voeller and anne johnson. second row, left to right: drs roy weller, henryk wisniewski, robert d. terry, john andrews, jan leestma and ivan herzog. third row, left to right: drs steven shayvitz, michael shelanski, richard snyder, john prineas and carlos araoz. i have kinuko-suzuki to thank for my much of my training in diagnostic neuropathology and for her friendship that lasts to the present day. of the younger members of the team, michael shelanski became professor of pathology at columbia university in new york, john prineas returned to australia as a neurologist in sydney to continue his research on multiple sclerosis. jan leestma developed a very successful career in forensic neuropathology in chicago; we are still in contact. after the photograph in figure 4 was taken, cedric raine and peter spencer joined bob terry’s department. cedric developed a very successful career in multiple sclerosis research. peter spencer moved into toxicology and together with herb shaumburg published a large multi-author book on clinical and experimental neurotoxicology to which my colleague in southampton, john mitchell, and i contributed a chapter on a fascinating mexican segmental demyelinating neuropathy due to ingestion of fruit and seeds of the buckthorn plant (karwinskia humboltiana) 8. it was rumoured that mexican women would administer small amounts of the fruit or seeds to their husbands so that that they would feel weak and stay at home rather than spending the evenings drinking with their friends. return to england in the fall of 1968, my family and i returned to england and i was appointed lecturer in pathology at guy’s hospital medical school. i continued my close connections with neuropathology by working in peter daniel’s department at the institute of psychiatry in london. my joint appointment allowed me to learn much from peter and from his colleagues. i also broadened my knowledge of clinical diagnostic pathology in general by engaging in the analysis of renal biopsies in collaboration with david turner, a fellow pathologist, and renal physicians, stewart cameron and chisholm ogg. this was a very fruitful time as correlation of the ultrastructural changes in renal biopsies with clinical syndromes was still in its early stages. together with barry nester, i published mid-20th-century diagnoses for three post-mortem kidneys that had been preserved by richard bright in the 1820-30s 9. for me, this was a valuable time for establishing contacts in london and elsewhere in the uk and for considering my ultimate career. during the 1970s i started to regularly attend conferences abroad, mainly in europe. this was essential for my continuing education and for maintaining contact with colleagues. i also benefitted by visiting many interesting countries and cities and meeting a great variety of different people. in 1971, i was fortunate to spend a short period in berlin with jorge cervós navarro that not only greatly increased my experience in neuropathology, but also allowed me to appreciate life in east berlin, then in the soviet bloc, and to visit the famous pergamon museum and its marvellous exhibits from ancient greece. it was through jorge that i was introduced to the german neuropathology society. at this time, trips to interesting nearby attractions were a feature of the german conferences. following a meeting in heidelberg, for example, we visited bad dürkheim and the dürkheimer wurstmarkt (sausage market but in reality the world’s largest wine festival) (figure 5). i retained contact with jorge for many years and together we wrote a book on the “pathology of peripheral nerves” that was published in 1978. figure 5. bad dürkheim (germany), 1971, fotoschiessen. as our colleague in the centre hit the target, the photo was taken with jorge cervós navarro (left) and roy weller (right). the text book – “pathology of peripheral nerves” (lower right) – was one product of my collaboration with jorge. university of southampton school of medicine in early 1972, the opportunity arose to join the new medical school that had been established in 1971 in the university of southampton on the southern coast of the uk. it was a combined national health service (nhs) and university post for which the job description was brief and to the point: “provide a diagnostic service for neuropathology for the wessex regional neurological centre, teach neuropathology and perform research”. following a thorough assessment of the post and the environment in southampton, i applied and was appointed to start in january 1973. i have never regretted the move to southampton. my family and i moved to live in winchester, just north of southampton; an ancient roman city and mediaeval capital of england, one hour by train from london and 40 min by car from heathrow. clinical neuropathology service the 30 years that i worked in southampton were delightful and i greatly appreciated the friendship of my colleagues. my relationships with clinical and university colleagues worked through collaboration and mutual respect for each others’ areas of expertise. the two neurosurgeons, jason brice and john garfield, who were working in the wessex neurological centre (there are now 14 neurosurgeons) when i arrived and the three neurologists, stanley graveson, peter robinson and lee illis (now 21 neurologists) had very professional attitudes. their aim was clearly to provide an excellent clinical service that required a high quality and rapid diagnostic neuropathology service from me, together with weekly multidisciplinary team meetings to discuss individual patients. i had soon realised that my clinical colleagues had many challenges and that my role was to provide them with all the assistance that i could. as we built up an atmosphere of mutual collaboration my life in southampton remained stable and pleasurable. neuropathology in southampton had a defined laboratory with its own staff but it was part of a larger department of histopathology/cell pathology with prof dennis wright at its head. this arrangement had many advantages, not least the wide range of expertise and facilities. regular clinical pathology and research meetings allowed the sharing of ideas and information about new developments. social activities were enthusiastically pursued which helped with cohesion of the department; there was also a constant circulation of pathology trainees from the uk and other countries through neuropathology providing a very interesting international environment. there were some disadvantages, such as competition for space and staff but the advantages heavily outweighed the disadvantages. from the time that i arrived in southampton, i was responsible for neuropathological services for the wessex region of some 3 million people. richard goodbody, who had long been involved in brain tumour diagnoses and in analysing post-mortem brains, very helpfully continued as part-time in this field. i covered brain, nerve and muscle biopsies, ophthalmic biopsies together with post-mortem brains and teaching sessions for trainee pathology staff, clinicians, medical students and nurses. my workload was very high and i was careful to prioritise so that i could cover all fields for which i was employed. i soon acquired a very extensive experience of diagnostic neuropathology and felt able to compile textbooks on neuropathological subjects (see later section) during the 1970s, there were rapid advances in clinical neurology, neurosurgery and neuroradiology with the introduction of ct scanning and mri that had a huge influence on diagnostic neuropathology. tumours and other lesions in the brain and spinal cord were well visualised, but instead of reducing the workload in neuropathology, the number of biopsies steadily increased. there were significant advances in the technical aspects of neuropathology. immunocytochemistry, for both frozen and paraffin sections, and later genetic techniques were incorporated into the diagnostic pathology and neuropathology services. ray hunt and barbara davis led the technical team for neuropathology and together with phillip steart, jean buontempo and judy mepham were always very anxious and able to advance the scope and quality of the service. trainees, fellows and staffing in clinical neuropathology in addition to trainee pathologists and neurosurgeons from southampton rotating through neuropathology, a number of fellows from other institutions joined me over the years. ricardo campora came in the 1970s from seville and worked with me for a year before returning to spain to practice neuropathology. ricardo is extremely able and became professor of pathology and head of department in seville. i am still frequently in touch with ricardo and his wife conchita. for a few years i taught a neuropathology course in seville and received tremendous hospitality there; we also meet on occasion in london. laura chavez from mexico city worked with me as did markus tolnay from basel and jim lowe from nottingham; all have had successful careers in neuropathology and we remain in contact. in the 1980s john grant came to work with me as a trainee in neuropathology. this changed my life as it gave me much more flexibility in how i spent my time. john was always very reliable and a great inspiration; when he left southampton john gained experience in zürich before embarking on a very successful career as a consultant pathologist in cambridge, where he has been head of department for a number of years. patrick gallagher was a consultant cardiac pathologist who also joined the neuropathology diagnostic team which again was of great benefit to the service. david ellison arrived as a trainee in neuropathology in the early 1990s and soon made significant progress in research into the genetics of brain tumours. david became a consultant neuropathologist in southampton before gaining a professorship in newcastle and then moving to memphis, tennessee, to pursue his world-renowned career in the pathology and genetics of childhood brain tumours. soon after david ellison left southampton, james nicoll came from glasgow; he brought tremendous expertise in dementia research, particularly in the roles of microglia. james has made a considerable contribution to the study of post-mortem brains in patients following immunotherapy for alzheimer’s disease. i retired in 2003 and left clinical neuropathology in southampton in the extremely capable hands of james nicoll. after my retirement the clinical neuropathology service was augmented by two very competent consultants, mark walker and mark fabian. research in this account, i take a personal view of how my research progressed in southampton and i include the names of some, but by no means all, of the many people involved. serendipity and the views of my colleagues worldwide played a major role in how my research progressed. i am indebted to the students and postdocs who contributed so much to our research programme and stimulated its progress. a number of themes initially emerged from my research including structural changes in peripheral neuropathies and myopathies, regeneration in nerves and muscle and detailed analyses of brains with growth hormone-related creutzfeldt-jakob disease. postdoctoral fellows, lawrie haynes and gary caine used tumour biopsy material to investigate the effects of pharmacological agents on tissue culture preparations of gliomas. with the introduction of immunocytochemistry for paraffin sections developed by dennis wright and his colleagues in our department, we were able to investigate the uptake of proteins by normal and neoplastic astrocytes. major research themes four major interconnected themes dominated most of my research in southampton and led to advances and revision of scientific concepts. (i) the structure of the leptomeninges and their relationships to the brain. (ii) the detailed pathways for lymphatic drainage of the brain. (iii) the relationship between age-related failure of lymphatic drainage and the accumulation of amyloid-β (aβ) in artery walls in cerebral amyloid angiopathy (caa) and in the brain in alzheimer’s disease. the latter advances were mainly through the work of roxana carare, who following completion of her phd formed her own research group that included cheryl hawkes and many others (figure 6). roxana’s work progressed to identify potential therapies for alzheimer’s disease and caa and to early clinical trials. roxana is now professor of clinical anatomy in southampton. (iv) through a very fruitful and lasting collaboration with britta engelhardt in bern, switzerland, we also investigated the relationship between lymphatic drainage of the brain and neuroimmunological diseases with relevance to disorders such as multiple sclerosis. a list of over 200 peer-reviewed papers that my colleagues and i published can be obtained from pubmed. i will only cite a few of those papers in the following text. figure 6. roxana carare and her group. students and technical staff with cheryl hawkes (right front) next to roy weller and roxana carare. why do research? for me, the main reasons for engaging in research are to satisfy my curiosity and to take the opportunity to present unique data to colleagues at scientific conferences and in scientific journals. i had been exposed to research during my bsc course at medical school and in my early postgraduate years working with john cavanagh and colin adams. bob terry encouraged research in all his fellows and led by example. by the time i arrived in southampton, research was well ingrained within me. southampton has been, for me, an ideal environment for research through excellent facilities and multiple disciplines within the university available for consultation and collaboration. with a heavy diagnostic workload in biopsies and post-mortem brains, i was exposed to many unsolved problems in human neurobiology and neuropathology that provided a stimulus for hypothesis-driven experimental research. another important stimulus for research was the requirement that each medical student in southampton perform a research project from october to may in the fourth year of the course. this meant that a constant stream of two or three highly intelligent, hard-working enthusiastic 22 year old students worked in neuropathology each year with their individual projects but collaborating closely with bsc and phd students, post-docs and research fellows. my strategy was to begin a research theme with straight-forward observational projects for the medical students that generated hypotheses to drive experimental research for the phd students and postdoctoral fellows. many students presented papers at british neuropathological society (bns) meetings and published papers in peer-reviewed scientific journals. all groups benefitted their future careers by acquiring skills in spoken and written communication. how did i benefit from students and others involved in research projects? the students educated me by their observations, the papers that they read and from the knowledge that they had gained in their recent medical education. retaining an open mind was very important: each student was given a problem to solve but advised not to read the previous literature until they had results and could form their own opinions. there were numerous occasions upon which students burst into my office crying “the literature is wrong!” in this way they built up a healthy scepticism for previous findings. i advised students not to believe anything they read and not to believe anything that i told them until they had verified it for themselves. structure of the human leptomeninges and their relationship to the brain and blood vessels although my interest in fluid balance in the brain started with my work on hydrocephalus in new york, it was reawakened in southampton by student projects using scanning electron microscopy (sem) to examine human leptomeninges. margaret upton 11 identified the intricate pattern of channels by which csf passes through arachnoid granulations to the blood in venous sinuses. margaret hutchings made a startling discovery in 1986 using sem on the surface of human brain by showing that the pia mater is reflected from the surface of the brain on to arteries and veins in the subarachnoid space thus separating csf in the subarachnoid space from the brain 10 (figure 7). this exploded the longstanding concept that there was a direct communication between csf and brain. margaret also showed that erythrocytes in subarachnoid haemorrhage do not penetrate the pia mater to enter the brain. we later confirmed that solutes and macrophages do pass through the pia mater that forms a continuous sheet on the surface of well-fixed normal brain as shown by ruth alcalado 12. no pores in the pia have been identified by tem and the holes seen in sem images are probably post mortem or fixation artefact. figure 7. sem of the meningeal surface of the human cerebral cortex showing the arachnoid mater (ar) and branches of an artery (a) spreading over the surface of the pia mater (p). the circle depicts the point at which a branch of the artery enters the cortex and how the pia mater is reflected on to the artery thus separating the subarachnoid space from the brain. (b) is an enlarged view of the reflected pia mater (p). artefactual holes are seen in the pia in this post-mortem specimen. arrow (lower left) identifies a filiform trabecula crossing the subarachnoid space 10. in the 1980s, en-tan zhang came from beijing to work with me in southampton. en-tan is a medically qualified anatomist and he made a major contribution to establishing the relationships between the leptomeninges and arteries as they enter the brain. using well fixed human biopsy brain material, en-tan showed that a single layer of pia mater coats arteries as they enter the surface of the cerebral cortex and that there is no “periarterial space” between the glia limitans and the artery wall 13. astrocytes in the glia limitans, pia mater, smooth muscle cells and endothelium together with the intervening layers of basement membrane form a compact structure, with no perivascular space (figure 8). the long-described virchow-robin spaces do not exist in the cortex and if present they are mainly due to swelling of astrocyte processes from poor fixation as emphasised by milton brightman in the 1960s. en-tan’s observations completely changed our perception of the relationship between csf and the brain. subsequently, roxana carare’s group showed that tracers from the csf enter the brain, not along perivascular spaces (as there are none) but along basement membranes between the glia limitans and the single layer of pia mater that surrounds each artery as it enters the brain 14. basement membranes between smooth muscle cells in the tunica media were later shown by roxana carare to be the pathways for flow of fluid and solutes out of the brain. due to the absence of periarterial spaces, there are no periarterial pathways for the entry of inflammatory cells from the csf into the brain. periarterial spaces do form in other the parts of the brain, especially in the basal ganglia and around arteries in the white matter. at both of these sites, arteries are surrounded by two layers of pia mater and the periarterial space develops between them 15,16. figure 8. tem of a well-fixed human cortical artery showing part of the wall and surrounding brain. the layers of glia limitans, pia mater, smooth muscle cells and endothelium adjacent to the lumen are all compacted with no preriaterial space (see also 13). lymphatic drainage of the brain there are two extracellular fluids associated with the brain and spinal cord: (i) csf in the ventricles and subarachnoid spaces and (ii) interstitial fluid (isf) in the extracellular spaces of the brain and spinal cord. pathways for lymphatic drainage of csf are almost completely separate from lymphatic drainage of isf from the brain. lymphatic drainage of csf shinya kida, a neurosurgeon from japan, joined us in southampton in the early 1990s and embarked upon a search for the drainage pathways for csf in the rat with a medical student andreas pantasis. lymphatic drainage of csf to cervical lymph nodes had been demonstrated by schwalbe in 1869, and bradbury and cserr showed in the 1980s that lymphatic drainage of csf occurred through the cribriform plate of the ethmoid bone but the details were unclear. shinya and andreas 17 demonstrated direct drainage of indian ink tracer from the subarachnoid space along distinct vessels that pass through the cribriform plate alongside olfactory nerves into the nasal submucosa en route to cervical lymph nodes. such channels were also shown to exist in post mortem humans brains by the injection of myodil tracer into the csf 18 and by reconstruction from serial sections of the human cribriform plate and nasal mucosa by effie djuanda in her fourth year medical student project 19. other channels for drainage of csf identified in these studies included dural lymphatics and the sheaths of cranial nerves 17,20. drainage of csf by the nasal route has been demonstrated in humans using pet scanning 20. the balance between lymphatic drainage of csf to cervical or lumbar lymph nodes and drainage through arachnoid granulations has yet to be determined. the small size and paucity of arachnoid villi in the rat suggests that they are quantitatively less important for the drainage of csf in rodents than lymphatic drainage. in humans, arachnoid granulations do not develop until the age of two years which suggests that lymphatic drainage may also be an important route for the drainage of csf in humans. arachnoid granulations may compensate for variations in csf pressure that occur with change of body position; this speculation still requires direct verification. lymphatic drainage of interstitial fluid (isf) and solutes from the brain there are no conventional lymphatic vessels in the brain or spinal cord. in the 1980s helen cserr and her colleagues 21 injected minute amounts of radioactive human serum albumin as a tracer into the basal ganglia of the rat brain, and showed that the tracer drained to cervical lymph nodes along the walls of cerebral arteries. only 10-15% of tracer leaked into the csf emphasising that the pathways for lymphatic drainage for csf from the subarachnoid spaces are separate from drainage of isf from the brain itself. en-tan zhang attempted to define the drainage pathways for isf in more detail in the late 1980s by injecting indian ink particles directly into the basal ganglia of the rat brain. the ink particles tracked along the outside of arteries, were taken up by perivascular macrophages and remained in situ 22. this technique did not identify the drainage pathways for isf but did identify a pathway that was later used for convection-enhanced delivery (ced) of drugs directly into the brain 23. the results from en-tan’s work led to discussions with alex roher from phoenix, arizona, who suggested that cerebral amyloid angiopathy (caa) may be related to periarterial lymphatic drainage of the brain. we subsequently published a joint paper in american journal of pathology in 1998 with the title “cerebral amyloid angiopathy: amyloid β accumulates in putative interstitial fluid drainage pathways in alzheimer’s disease“ 24. breakthrough a major breakthrough in our research on lymphatic drainage of the brain came from the work of roxana carare in her phd project that i supervised jointly with hugh perry. roxana injected formalin-fixable fluorescent dextrans or soluble amyloid-β (aβ) into mouse brains and examined the drainage pathways by confocal microscopy. in her paper, “solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroimmunology” 25, roxana defined the pathways for the lymphatic drainage of fluid and solutes from brain tissue that we later termed the “intramural peri-arterial drainage (ipad) pathways” 14. following the injection of minute volumes (0.5 μl) of tracer into the caudate nucleus of the mouse brain, tracer spread through the extracellular spaces of the brain and within 5 mins had entered the lymphatic drainage pathway (ipad) in the basement membranes of capillaries and basement membranes between smooth muscle cells in the tunica media of cerebral arteries. the drainage pathway (ipad) extended into leptomeningeal arteries. no tracer was seen around veins in normal animals following intracerebral injections although animals with age-related impairment of ipad did show some tracer around veins 26. confusion considerable confusion in the scientific literature has resulted from the injection of excessive volumes of tracer into the mouse brain that results in leakage of tracer into the csf giving the false impression that lymphatic drainage of the brain occurs via the csf. furthermore, injecting excessive amounts of tracer into the brains of mice and not examining the animals until 1 hour later, after ipad is complete, result in pooling of tracer around veins and has given the erroneous impression that lymphatic drainage of the brain is along the walls of veins. supporting evidence for ipad in the human brain caa in mice and humans provides convincing supporting evidence that ipad is the route for lymphatic drainage of fluid and solutes from the brain parenchyma. (i) the pattern of distribution of aβ in capillary and artery basement membranes in caa exactly mirrors the distribution of aβ and other tracers in ipad pathways in the animal experiments 27. (ii) caa occurs in transgenic mice in which there is excessive production of aβ in neurons; in these mice the origin of the aβ is definitely from cells in the brain 28. age-related failure of elimination of aβ by ipad is associated with caa and alzheimer’s disease by combining experimental studies in the mouse with observations of human caa and alzheimer’s disease, the following main conclusions have been reached as summarised in 29. ipad is impaired by age-related changes in the walls of arteries and by caa 26. both theoretical models and experimental studies suggest that the motive force for ipad is derived from waves of smooth muscle cell contraction (vasomotion) that pass along the walls of cerebral arteries in the opposite direction to blood flow, but in the same direction as ipad 29,30. age and the presence of apolipoprotein e ε4 (apoe4) is associated with the impaired ipad 31 and this correlates with age and apoe4 as major risk factors for alzheimer’s disease and caa in humans. caa also affects capillaries; painstaking cutting of hundreds of serial transverse sections of arteries in regions of capillary caa by hong yeen yow (bsc student) suggest that capillary caa is associated with thrombotic occlusion of penetrating cortical arteries 19. age-related impairment of ipad appears to be associated with loss of homeostasis in the brain as reflected by accumulation of fluid in white matter hyperintensities on mri and the failure of elimination of aβ with age and in alzheimer’s disease. dilatation of periarterial spaces in the white matter is associated with caa and probably reflects failure of ipad. translational neuroscience: therapeutic strategies for caa and alzheimer’s disease: facilitating ipad for the past 20 years there have been many trials of immunotherapy for alzheimer’s disease but with little success in reducing cognitive decline in the treated patients. studies of post mortem brains following aβ immunotherapy have shown that although aβ plaques are removed from the cerebral cortex both arterial and capillary caa are increased in severity 32,33. it does appear that age-related failure of ipad is a rate-limiting step in aβ immunotherapy and this emphasises the importance of facilitating ipad in the treatment of caa and alzheimer’s disease. potential new therapeutic strategies could act via ipad by modulating vasomotion as the postulated motive force for ipad. this may be accomplished by stimulating contraction of vascular smooth muscle cells directly or through their nerve supplies. another therapeutic possibility is the use of chaperone molecules to enhance the clearance of aβ along the basement membranes that form the ipad pathways 29. lymphatic drainage of the brain and neuroimmunology the brain is an immunologically privileged site 34 as shown by experiments in which skin allografts implanted in the brain survive for longer periods than allografts in other organs. rapid rejection of brain allographs does occur, however, when similar allografts are implanted into the animal’s skin. the leptomeninges and csf spaces do not show the same immunological privilege as brain tissue 34. one of the major differences is that csf drains directly into lymphatic vessels that allow fluid, antigens and antigen presenting cells to drain directly from the csf to lymph nodes. ipad pathways are too narrow to allow the traffic of apcs from the brain to lymph nodes 25. this may be a major factor in immunological privilege of the brain. cervical lymph nodes have a major role in immunological reactions in the brain as shown by marian phillips, dong sun (ph.d. students), jonathan lake and michelle needham (bsc medical students) and by jon laman and his group 35-37. our first task was to enhance experimental autoimmune encephalomyelitis (eae), in the cerebral hemispheres of rats so that accurate measurements of inflammation could be made. in most models of eae following injection of antigen into the footpads, inflammation is concentrated in the spinal cord. however, shinya kida had previously shown that a wound to the surface of the rat cerebral cortex in the form of a cryolesion resulted in activation of perivascular macrophages throughout the cerebral hemispheres. thus we showed that a cryolesion on the surface of the cortex in rats, 7 days post injection (dpi) of eae antigens into the foot pad of the rat resulted in enhanced inflammation in the cerebral hemispheres at day 15 dpi. removal of cervical lymph nodes on day 7 dpi resulted in a 50% reduction in eae-related inflammation at day 15 dpi. these results suggest that cervical lymph nodes play a key role in cerebral eae 35. furthermore, adoptive transfer of lymphocytes from animals with cryolesion eae resulted in a predominance of eae lesions in the cerebral hemispheres in naive recipients rather than spinal cord eae 36. our involvement in neuroimmunology brought us into contact with britta engelhardt in bern and her excellent studies on the receptor-mediated entry of lymphocytes into the brain through post-capillary venules. we are still in collaboration 34. figure 9. summary of pathways for the flow of fluid and solutes into and out of the brain. (a) csf flows into the brain along basement membranes shared by pia mater and glia limitans on outer surfaces of cortical arteries as they penetrate the brain. (b) interstitial fluid (isf) and solutes such as aβ flow out of the brain to lymph nodes along ipad pathways. (c) details of the pathway by which csf flows into the cerebral cortex along pial-glial basement membranes and then enters the brain to flow out along ipad. reproduced from 29. original design by roy weller and graphic by artfact graphics. award of the gertrud-reemtsma prize for international translational neuroscience 2020 figure 10. the prize winners of the international prize for translational neuroscience 2020: left to right: roy weller, maiken nedergaard, and mathias jucker. i was greatly honoured by the award of the international prize for translational neuroscience by the council of the gertrud reemstma foundation, administered by the max planck society. the prize had been the k. j. zülch-prize until 2019 with many famous and worthy winners from 1990. i was awarded the prize together with maiken nedergaard and mathias jucker (figure 10). my award was for the southampton group’s “ground-breaking scientific discoveries in the field of lymphatic drainage of the brain and its relationship to the aetiology of alzheimer’s disease”. of course the prize was awarded as much of the work performed by the many medical students, ph.d. students and postdocs who worked with me in southampton. roxana carare, cheryl hawkes, en-tan zhang, shinya kida and dong sun deserve special mention. nevertheless, i felt it was a great honour for our group. due to covid-19 i could not attend the award ceremony on september 10th 2020 in cologne, germany, in person so i presented my lecture on zoom. management roles throughout most of my career in southampton i was involved in management in the nhs, southampton university, and in regional, national and international organisations. it was part of my role to assist with the organisation of the institutions to which i belonged. furthermore i was rewarded by meeting many interesting people while performing management tasks. within two years of my arrival in southampton, i became chairman of the whole of pathology; this was in a very active period when all pathology departments were moving into a newly-built pathology block. at that time, in the mid-1970s, there were virtually no management courses and i learnt the necessary skills by observing others, how they chaired meetings, coordinated events and managed people so that the whole system ran smoothly and within budget. soon after my promotion to professor of neuropathology in 1978, i became deputy dean of medicine, which included, among other very interesting tasks, chairing the committee for selection of medical students; this gave me insight into the great variety of skills exhibited by the applicants to medical school. following my four-year term as deputy dean, i was appointed chairman of the university library committee. not only did this coincide with the delivery to the library of the papers of the duke of wellington, but it was also the time that the library acquired the papers of lord louis mountbatten. the papers were markedly different in their condition. the wellington papers were written with indian ink on parchment such that they could be cleaned with large india rubber balls in sinks of cold water. the mountbatten papers, on the other hand, were the fourth or fifth typed copy and required urgent transcription. for some 20 years i chaired various regional and national committees that ensured high quality training of pathologists locally in wessex and nationally through the royal college of pathologists. this was the time during which neuropathology became an independent discipline within the royal college of pathologists and training and examinations were formalised. the establishment of the european confederation of neuropathological society (eurocns) also allowed a european program of education and qualifications in neuropathology to be established. british neuropathological society the british neuropathological society (bns) plays valuable roles in neuropathology in the uk, both in monitoring the quality of clinical neuropathology and in encouraging research. the annual bns scientific meeting is always very interesting and informative. in 2000, the bns organised an international neuropathology society congress in birmingham, uk, with some 700 delegates. i chaired the committee that organised the scientific programme and david graham was president. due to the hard work and diligence of my many colleagues, the congress was a great success. i was elected president of the bns for 2001-2002. major figures such as hume adams and david graham in glasgow, james ironside and jeanne bell in edinburg, raj kalaria in newcastle, james lowe in nottingham, seth love in bristol, peter lantos in london and many others played very significant roles in organising the bns such that the bns conferences attracted significant numbers of neuropathologist from europe, usa and other countries. editor of neuropathology and applied neurobiology in 1975, john cavanagh founded the journal neuropathology and applied neurobiology (nan), that was adopted by the bns as the society journal. i followed john as editor in chief of nan between 1988 and 1998. this was a most rewarding time for me, organising the publication of research articles and review articles from an international body of authors. i learned much about the organisation of scientific journals and gradually nan started to make a profit that has benefited the bns financially. john had organised nan with the original publishers, blackwell, so that the profits were shared between the publisher and the bns. elizabeth whelan was my main contact with the publisher and our relationship was excellent throughout my time as editor, largely due to elizabeth’s skill and dedication. james lowe followed me as editor in 1999 and there has been a succession of very successful editors since. the impact factor of nan has increased steadily from 3.9 in 2014 to 7.09 in 2019. tom jacques is the present editor in chief and nan continues to play a significant role in international neuropathology. editorial boards i have been a member of editorial boards of a number of journals but the ones i remember in particular are the boards of brain pathology, acta neuropatholgica and more recently, free neuropathology. werner paulus made a particular impression on me as editor of acta neuropatholgica; his very inspired decisions on which papers to accept for the journal ensured that the impact factor for acta neuropatholgica rose to 18. werner is the founding editor in chief of free neuropathology. books published 1978-2013 i decided to harness the clinical material associated with the diagnostic service to publish a number of text books; this was partly as a self-education exercise. some books i wrote alone or with another single author or i acted as editor as well as a contributor. the rewards for my efforts were that writing and editing consolidated my knowledge of neuropathology and built lasting friendships with multiple authors who showed great tolerance for my editorial changes. my first was “pathology of peripheral nerves” by r o weller and j cervós-navarro, published by butterworths, london in 1978 and my second resulted from a course that we ran in southampton: “clinical neuropathology.” a multi-author book edited by r o weller, m swash, dl mclellan and c l scholtz, published by springer verlag, berlin in 1983. the japanese edition of “clinical neuropathology” was translated by t mientani and published by nishimura co ltd in 1983. “a colour atlas of neuropathology” by r o weller, published by harvey miller and oxford university press in 1984 and i contributed to “mcalpine’s multiple sclerosis”, by w b matthews, e d acheson, j r bachelor and r o weller. edited by w. b. matthews, published by churchill livingstone, edinburgh in 1985. as part of “systemic pathology”, third edition: general editor, w st c symmers. i edited a multi-author “volume 4: nervous system, muscle and eyes”, published by churchill livingstone, edinburgh in 1990. in 2002 i contributed to “diagnostic pathology of nervous system tumours”, by j w ironside, t h moss, d n louis, j s lowe and r o weller, published by churchill livingstone, edinburgh. i was appointed director of the book series sponsored by the international society of neuropathology (isn), publishing “neurodegeneration. the molecular pathology of dementia and movement disorders”, second edition. edited by d w dickson and r o weller. series director, r. o. weller. published by wiley-blackwell, chichester, uk in 2011. as “neurodegeneración”, the book was published in spanish by editorial medica panamericana, madrid and buenos aires. the second book in the isn series was “muscle disease: pathology and genetics”, second edition. edited by h h goebel, c a sewry and r o weller, series director, r. o. weller. published by wiley-blackwell, chichester, in 2013. i also contributed chapters to a number of books including: gray’s anatomy, greenfield’s neuropathology, clinical and experimental neurotoxicology and histology for pathologists. international conferences and visits to other neuropathology units attending conferences was essential for my continuing education and for maintaining contact with colleagues. i also benefitted from visiting many interesting countries and cities throughout the world and meeting a great variety of different people. i regularly participated in the annual meetings of the british, french and german neuropathology societies and occasionally, the scandinavian and italian neuropathology society meetings. i developed strong friendships with many members of those societies; francoise gray, jacqeline mikol and homa adle-biassette to name but a few. a particular favourite conference of mine was the meeting of the swiss society of neuropathology in st. moritz every two years. not only was the meeting held amidst beautiful scenery, the train journey was spectacular and there was ample opportunity for skiing and social interaction for discussing scientific data. i made many friends at these meetings. in addition to european meetings, i attended many conferences in the usa, including annual meetings of the american association of neuropathology. other conferences to which i contributed include those in india, australia and brazil. photographs of many of the friends that i made at these international meetings appear in an excellent autobiography by sam ludwin 38. in 1988 paul kleihues kindly invited me to work for four months in zürich. there was a most interesting group of young neuropathologists working with paul at the time, otmar wiestler, andreas von deimling and adriano aguzzi all of whom subsequently developed very successful careers and with whom i developed lasting friendships. while working for six weeks in harare in zimbabwe in 1991, i was involved mainly in teaching with occasional and most stimulating visits by car to other parts of the country. martin lewis was there at the time; he was an english pathologist working in florida and we made a long tour around zimbabwe in a 1968 mercedes car that he had borrowed. martin had extensive experience of africa that is outlined in his book “the call of africa”. particularly rewarding were my visits to beijing after my retirement at the invitation of wei wei zhang. quite apart from her tremendous hospitality, wei wei and her husband accompanied me on very interesting trips to other chinese cities to meet many researchers and clinicians and to see the sights. i shall ever be indebted to professor zhang for introducing me to so much chinese culture and cuisine. post-retirement when i was about to retire, i realised that leaving what seemed like a lifetime in neuropathology would be very difficult. through the kindness of my colleagues, i took gradual retirement over 4-5 years moving to part-time involvement in clinical diagnostic neuropathology. i also remain involved on a consultancy basis with professor roxana carare’s group who, following her ph.d., developed a very successful research programme in southampton with substantial grant income. i watched with great interest and some advice to the development of the ipad work and i was involved in the discussion during the preparation of papers for publication. this was very helpful in my disengagement from neuropathology. in addition, i was asked to be chairman of the biomedical grants advisory board for the alzheimer’s society from 2013 to 2018 which kept me in touch with developments in research into alzheimer’s disease. living in winchester gave me the opportunity to become a guide in the cathedral. this 900-year-old building and its contents are a mine of information regarding british history in the last 2000 years. i edited the winchester cathedral record that published research articles concerning the cathedral for four years and i have been actively involved in researching the cathedral for my own benefit and the benefits of my fellow guides and visitors. family if it had not been for the support that i have received from my family, my career in medicine and neuropathology would not have been possible. my loving parents greatly encouraged me in my education and my wife, francine, has offered her full support to me and to our moves around the uk and abroad. throughout our 60 years of happy marriage, i have admired francine’s independence and i am indebted to her for expanding my knowledge and involvement in the arts, especially music and literature. we have both sung in choirs for almost the whole of our married life and continue to do so with great enjoyment. our two children, adrienne and timothy have been a huge joy to us as have their spouses and our four grandchildren. acknowledgments i would like to thank professor roxana carare for all her help in the final editing and submission of the paper to free neuropathology. references weller ro. diphtheritic neuropathy in the chicken: an electron-microscope study. j pathol bacteriol 1965; 89: 591-8. weller ro. cytochemistry of lipids in atherosclerosis. j pathol bacteriol 1967; 94(1): 171-82. weller ro, clark ra, oswald wb. stages in the formation and metabolism in intracellular lipid droplets in atherosclerosis. an electron microscopical and biochemical study. j atheroscler res 1968; 8(2): 249-63. weller ro, wisniewski h. histological and ultrastructural changes with experimental hydrocephalus in adult rabbits. brain 1969; 92(4): 819-28. weller ro, wisniewski h, shulman k, terry rd. experimental hydrocephalus in young dogs: histological and ultrastructural study of the brain tissue damage. j neuropathol exp neurol 1971; 30(4): 613-26. weller ro, shulman k. infantile hydrocephalus: clinical, histological, and ultrastructural study of brain damage. j neurosurg 1972; 36(3): 255-65. weller ro. pathology of cerebrospinal fluid and interstitial fluid of the cns: significance for alzheimer disease, prion disorders and multiple sclerosis. j neuropathol exp neurol 1998; 57(10): 885-94. mitchell j, weller ro, evans h, arai i, daves gd, jr. buckthorn neuropathy: effects of intraneural injection of karwinskia humboldtiana toxins. neuropathol appl neurobiol 1978; 4(2): 85-97. weller ro, nester b. histological reassessment of three kidneys originally described by richard bright in 1827-36. br med j 1972; 2(816): 761-3. hutchings m, weller ro. anatomical relationships of the pia mater to cerebral blood vessels in man. j neurosurg 1986; 65(3): 316-25. upton ml, weller ro. the morphology of cerebrospinal fluid drainage pathways in human arachnoid granulations. j neurosurg 1985; 63(6): 867-75. alcolado r, weller ro, parrish ep, garrod d. the cranial arachnoid and pia mater in man: anatomical and ultrastructural observations. neuropathol appl neurobiol 1988; 14(1): 1-17. zhang et, inman cb, weller ro. interrelationships of the pia mater and the perivascular (virchow-robin) spaces in the human cerebrum. j anat 1990; 170: 111-23. albargothy nj, johnston da, macgregor-sharp m, et al. convective influx/glymphatic system: tracers injected into the csf enter and leave the brain along separate periarterial basement membrane pathways. acta neuropathol 2018; 136(1): 139-52. doi: 10.1007/s00401-018-1862-7. macgregor sharp m, bulters d, brandner s, et al. the fine anatomy of the perivascular compartment in the human brain: relevance to dilated perivascular spaces in cerebral amyloid angiopathy. neuropathol appl neurobiol 2019; 45: 305-8. pollock h, hutchings m, weller ro, zhang et. perivascular spaces in the basal ganglia of the human brain: their relationship to lacunes. j anat 1997; 191(pt 3): 337-46. kida s, pantazis a, weller ro. csf drains directly from the subarachnoid space into nasal lymphatics in the rat. anatomy, histology and immunological significance. neuropathol appl neurobiol 1993; 19(6): 480-8. johnston m, zakharov a, papaiconomou c, salmasi g, armstrong d. evidence of connections between cerebrospinal fluid and nasal lymphatic vessels in humans, non-human primates and other mammalian species. cerebrospinal fluid research 2004; 1: 2-15. weller ro, djuanda e, yow hy, carare ro. lymphatic drainage of the brain and the pathophysiology of neurological disease. acta neuropathol 2009; 117(1): 1-14. de leon mj, li y, okamura n, et al. cerebrospinal fluid clearance in alzheimer disease measured with dynamic pet. j nucl med 2017; 58(9): 1471-6. szentistvanyi i, patlak cs, ellis ra, cserr hf. drainage of interstitial fluid from different regions of rat brain. american journal of physiology 1984; 246: f835-44. zhang et, richards hk, kida s, weller ro. directional and compartmentalised drainage of interstitial fluid and cerebrospinal fluid from the rat brain. acta neuropathol 1992; 83(3): 233-9. barua nu, bienemann as, hesketh s, et al. intrastriatal convection-enhanced delivery results in widespread perivascular distribution in a pre-clinical model. fluids barriers cns 2012; 9: 2. weller ro, massey a, newman ta, hutchings m, kuo ym, roher ae. cerebral amyloid angiopathy: amyloid beta accumulates in putative interstitial fluid drainage pathways in alzheimer’s disease. am j pathol 1998; 153(3): 725-33. carare ro, bernardes-silva m, page am, nicoll ja, perry vh, weller ro. solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroimmunology. neuropathol appl neurobiol 2008; 34(2): 131-44. hawkes ca, hartig w, kacza j, et al. perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy. acta neuropathol 2011; 121(4): 431-43. carare ro, hawkes ca, jeffrey m, kalaria rn, weller ro. review: cerebral amyloid angiopathy, prion angiopathy, cadasil and the spectrum of protein elimination failure angiopathies (pefa) in neurodegenerative disease with a focus on therapy. neuropathol appl neurobiol 2013; 39(6): 593-611. herzig mc, van nostrand we, jucker m. mechanism of cerebral beta-amyloid angiopathy: murine and cellular models. brain pathol 2006; 16: 40-54. carare r, aldea r, agarwal n, et al. clearance of interstitial fluid (isf) and csf (clic) group—part of vascular professional interest area (pia). cerebrovascular disease and the failure of elimination of amyloid-β from the brain and retina with age and alzheimer’s disease-opportunities for therapy. alzheimer’s dement 2020; 12: e12053. aldea r, weller ro, wilcock dm, carare ro, richardson g. cerebrovascular smooth muscle cells as the drivers of intramural periarterial drainage of the brain. front aging neurosci 2019; 11: 1. hawkes ca, sullivan pm, hands s, weller ro, nicoll ja, carare ro. disruption of arterial perivascular drainage of amyloid-beta from the brains of mice expressing the human apoe ε 4 allele. plos one 2012; 7(7): e41636. nicoll ja, wilkinson d, holmes c, steart p, markham h, weller ro. neuropathology of human alzheimer disease after immunization with amyloid-beta peptide: a case report. nat med 2003; 9(4): 448-52. boche d, nicoll j, weller r. immunotherapy for alzheimer’s disease and other dementias. curr opin neurol 2005; 18(6): 720-5. engelhardt b, vajkoczy p, weller ro. the movers and shapers in immune privilege of the cns. nat immunol 2017; 18(2): 123-31. phillips mj, needham m, weller ro. role of cervical lymph nodes in autoimmune encephalomyelitis in the lewis rat. j pathol 1997; 182(4): 457-64. lake j, weller ro, phillips mj, needham m. lymphocyte targeting of the brain in adoptive transfer cryolesion-eae. j pathol 1999; 187(2): 259-65. van zwam m, huizinga r, heijmans n, et al. surgical excision of cns-draining lymph nodes reduces relapse severity in chronic-relapsing experimental autoimmune encephalomyelitis. j pathol 2009; 217(4): 543-51. ludwin s. autobiography series: in search of knowledge and joy: my life as a neuropathologist. j neuropathol exp neurol 2018; 77(2): 162–75. 🎉 🎂 celebrating professor roy weller: 🎊 🥂 83 years old, over 5 decades of contributions to neuropathology prof johannes attems consultant neuropathologist, newcastle university and editor, acta neuropathologica the first time i met professor roy weller was at the british neuropathological society winter meeting in 2001; in the break after the session where i had presented, he the famous professor approached me the unknown junior doctor from vienna to discuss my talk! it was a nearly 20 minute discussion about the pathogenesis of cerebral amyloid angiopathy and professor weller listened to what i had to say and even produced some drawings illustrating the perivascular clearance. this first meeting with professor weller was hugely inspiring and motivating and certainly had a big impact on my decision to continue with research into neuropathology and i still think often back to this day in 2001! naturally many more stimulating discussions between us followed (on all topics, professional and private) and i am looking forward to many more discussions in the future. very many thanks for all your support roy! prof roxana carare clinical neurosciences, university of southampton roy weller has catalyzed and energized the research into lymphatic drainage of the brain, inspiring and allowing us to pursue this with translational value for cerebral amyloid angiopathy and alzheimer’s disease. he has taught me and many others the scientific method and testing hypotheses based on a solid overview of past and present observations on the human brain. the research group that i lead is a result of his continuous efforts to help with the direction of research, interpreting results, analytical, critical and forward-thinking discussions. we are grateful for his attention to detail and educating us to be researchers of high integrity and a deep quest for advancing science. prof mony de leon weill cornell medicine director, brain health imaging institute i am delighted to celebrate professor roy weller’s 80th birthday by reflecting on his contributions to pathology and to radiology. however, using just a few words to do this, owing to his extensive and influential scholarly history, is a remarkably difficult but a highly refreshing task. taking advantage of a personal and arbitrary starting point, my first encounter with roy’s work was on his work in dementia and hydrocephalus. weller, a longtime developer of animal models of hydrocephalus and an advocate for innovative dementia treatment options, would write in the j neurol neurosurg psychiatry 1989, “when confronted with a patient with dementia, gait dyspraxia, and incontinence, we suggest that it is more realistic to look for a remediable hydrocephalic component to the illness than to consider that the patient must have either alzheimer’s disease, multi-infarct dementia, or normal pressure hydrocephalus alone.” in the late 1970s i was a gerontology student studying alzheimer disease (ad) and in 1980 i received an nyu-brookhaven national lab (bnl) neuroimaging post-doctoral fellowship. with the emergence of human imaging studies during the late 1970s, my mentor and colleague neuroradiologist ajax george and i were awarded our first nih-nia funded studies of ct atrophy in dementia and in 1980, collaboration with alfred wolf and joanna fowler at bnl, nih-ninds funded fdg-pet studies of ad began. at the time, it was commonly believed that imaging had little to contribute to the ad diagnosis due to background aging effects. one real-world test of our ad diagnostic imaging work was to evaluate if our measurements differentiated between ad and hydrocephalus. we quantified ct scans for regional ventriculomegaly, evaluated compressed sulci and csf pooling, we published and post mortem validated a ct scale for periventricular white matter pathology, and tested whether the hippocampus, a site of early ad pathology, was preserved in hydrocephalus. we ran fdg-pet scans before and after shunting looking for metabolic recovery, and collected biopsy samples during shunting for evidence for ad. this work led to another point of connection between my work and roy weller’s, interactions with two pathologists henry wisniewski and robert terry (both deceased) who both worked with roy and who would become my valued friends and advisors. today, the radiological diagnosis is an essential part of the differential dementia examination, and multiple avenues of imaging research, guided by roy’s insights, continue. one such insight, and clearly for me, roy’s most important contribution was for his anatomico-physiological studies examining the normal and pathological clearance of csf and isf from the brain. these studies, which continue today, are now led at the university of london in southampton by professor roxana carare, a former weller mentee. historically, these studies revealed the anatomical pathways for isf drainage along basement membranes surrounding smooth muscle cells of cerebral arteries and along capillaries, and provided evidence that impaired interstitial flow contributed to parenchymal and vascular amyloid accumulations as well as being negatively impacted by amyloid accumulations. this mechanistic message was heard by many others, including the radiological community where currently pet and mri imaging studies are underway today translating the weller and carare observations of a direct link between impaired vascular biology and ad and most recently other neurological diseases defined by misfolded proteinopathies. thus, we wish roy a happy birthday and applaud him and his colleagues for their continued efforts to treat dementia by providing pathology grounded data and testable hypotheses. prof margaret esiri consultant neuropathologist, john radcliffe hospital, oxford i first encountered roy at my first bns meeting in (probably) 1971 or 1972. he came over as a calm, confident deliverer of a presentation – i’ve forgotten what it was on. i think he may have been at guy’s at the time. i was very impressed! i have gone on being impressed with roy’s very significant contributions to neuropathology ever since. long may they continue! happy birthday, and many more to come, roy! prof masafumi ihara head of the cardiovascular centre, osaka, japan it is my great pleasure to contribute to an article celebrating prof. weller's 80th birthday. prof. weller is an internationally recognized neuropathologist and well-known to have established the important concept of perivascular drainage pathway (now called ipad) as an abeta clearance system. i had been inspired by the concept and had a great opportunity to meet him in the xviith international congress of neuropathology in salzburg (icn 2010). i remember i had a good discussion with him in front of a poster of his team. i visited his and prof. carare’s laboratory in southampton with three of my phd students in 2012 and have been collaborating with the southampton team. our collaboration led to my investigator-led clinical trial of a vasoactive drug for mci patients to determine whether ipad can be facilitated clinically. dr. satoshi saito, my former phd student, joined the southampton team in 2018, came back to japan in 2020 after learning a lot not only from prof. carare but also from prof. weller, and is now working as a talented physician scientist in my department. thus, prof. weller academically guided us as an established neuropathologist but not only that, he very kindly acted as a tour guide of winchester for me a few years ago! prof. weller has continued impacting my research team for over 10 years with his plentiful neuropathological knowledge and we would like to send our heartfelt congratulations to prof. weller. prof raj kalaria neurovascular pathologist, newcastle university and chairman, international society for vascular behavioral and cognitive disorders (vascog) i had first heard about roy from helen cserr, many moons ago! besides the ‘art’ of neuropathology, roy’s profound writings on drainage pathways of the brain intrigued me. in the late 1980s when i was still at case western reserve university training in vascular neuropathology, i had communicated with roy about a paper in neuropathology and applied neurobiology. at the time, roy was editor-in-chief of the journal. few years later, i met roy face-to-face when i was interviewed for a job in southampton with the hope that the family and i could quickly return to england. i did not get the post but i thought it was such a privilege and an honour to meet the pleasant and humble professor weller! i had also thought somehow i should still keep in touch, especially that i developed a strong interest in cerebrovascular mechanisms and vascular pathology. i was absorbed in mechanisms of cerebral amyloid angiopathy (caa) although at the time i searched for explanations on cystatin c caa, which causes profound intracerebral haemorrhages. cystatin c is enriched in the brain and csf but it was also found in the cervical lymph glands of icelandic patients. using the cystatin c reasoning, i was determined to disprove the giants selkoe and wisniewski, who were both proposing at the time that cerebral aβ amyloidosis was initiated by a protein(s) from the circulation (even papers in nature) akin to the classical pathways discussed by glenner and others how amyloidosis is caused in end organs. large nih funds were even spent on studies how aβ could be transported from the blood-to-brain rather than searching for routes how it leaves the brain. all above was proven to be inaccurate and i was glad that roy’s seminal work glued it altogether suggesting that aβ is not only produced in the brain but ‘pouring’ out of it along perivascular pathways as one of the main routes. roy and later roxana beautifully demonstrated the mechanisms involved in caa and how proteins including aβ, prions, cystatin c drain out formulating the ipad hypothesis. this is also of importance because we have used the tonsils (lymphatic nodes) to test individuals who might have prions and infected with new variant cjd. to show, i understood the ‘drains’ of the brain, i was proud to give a talk on weller’s hypothesis of caa at the isn conference in turin in 2004. even though, i did not end up in southampton, i am very fond of roy and he has been an excellent mentor, advisor, collaborator and a good friend with whom one could relax, have a drink or two in the pub and discuss the ‘nuts and bolts’ of vascular neuropathology rather than daily mundane matters. over the years, it has been one of the most exciting journeys with roy. our regular interactions at the bns meetings, vascog and isn conferences including the one roy organized in 2000 in birmingham were refreshing, delightful and enjoyable. having dinners at such gatherings and other times is something i always looked forward to and will cherish. thank you roy and congratulations on reaching 80, true to what the scriptures say ‘the span of our life is 70 years or 80 if one is especially strong…’ (psalms 90:10). prof dietmar thal consultant neuropathologist, leuven, belgium roy weller: a pioneering neuropathologist. the first time i heard a talk given by roy weller, he was explaining the contribution of perivascular clearance on several brain disorders. this was 1994. perivascular clearance mechanisms became of clear importance for amyloid pathology in alzheimer’s disease. here, prof. weller had a strong impact on my thinking about how cerebral amyloid angiopathy develops in ad, namely as a result of perivascular clearance. for me, this could also explain interactions between cerebral small vessel disease and hypertension with ad by impairing the perivascular clearance. prof. weller’s concept of perivascular clearance is not only the basis of our understanding of cerebral amyloid angiopathy pathogenesis but had even a bigger impact on the understanding of fluid and protein drainage of the brain in general. prof hugh perry emeritus professor, university of southampton it was my pleasure to meet and get to know roy weller when i moved to southampton in the late 1990s. roy was a generous and welcoming colleague and as a basic scientist, mostly involved in pre-clinical models, he was there to remind us of the need to be aware of the human perspective. he had a great breadth of knowledge in neuropathology and also an understanding of the history behind many key observations in neuropathology. his research into how interstitial fluid carrying proteins from the brain drained from the brain was one of his passions. this is, of course, a very important component of understanding interactions between the immune system and the central nervous system. as the rapidly growing field of neuroimmunology advances so the research that roy championed will remain relevant and new discoveries will amplify the importance of his favoured research topic. prof stephen wharton consultant neuropathologist, sheffield institute of translational neuroscience, and ex-editor, neuropathology and applied neurobiology i first came across roy in the 1980s. i was presenting some (rather incomplete) results from my intercalated bsc project at a meeting of the anatomical society in southampton. i received a kind and supportive question from an audience member and was told, “that’s roy weller”. later, when i began training in neuropathology i used his textbook on nervous system, muscle and eyes, part of the comprehensive symmers series and enjoyed its clarity and approachability. roy of course was editor of neuropathology and applied neurobiology for ten years or so, and i had the opportunity to work with him more closely when i subsequently became editor after jim lowe. roy agreed to remain on the editorial board where he was always an enthusiastic contributor with good ideas to move the journal forward. he was also a great source of advice, both scientific and for those tricky situations that sometimes arise with papers. since then i have continued to enjoy conversations about neuropathology at various meetings. i have always enjoyed these stimulating conversations and i, like many others, have appreciated his kindness and advice over many years. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurotrauma: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:26 (2022) review neurotrauma: 2022 update david s. priemer1-3, daniel p. perl1,2 1 the department of defense/uniformed services university brain tissue repository, bethesda, md, usa 2 department of pathology, f. edward hébert school of medicine, uniformed services university, bethesda, md, usa 3 henry m. jackson foundation for the advancement of military medicine, inc., bethesda, md, usa corresponding author: daniel p. perl · department of pathology · f. edward hébert school of medicine · uniformed services university (usu) · 4301 jones bridge rd · bethesda, md 20814 · usa daniel.perl@usuhs.edu submitted: 24 october 2022 accepted: 01 november 2022 copyedited by: shino magaki published: 15 november 2022 https://doi.org/10.17879/freeneuropathology-2022-4495 keywords: traumatic brain injury, chronic traumatic encephalopathy, tau, interface astroglial scarring, diffuse axonal injury, multidimensional mri abstract the year 2021 was highlighted by many notable advancements in the field of neurotrauma and associated neuropathology. after a thorough review of the new literature, we call attention to what we feel are among the most impactful studies and publications. in brief, 2021 was marked by published consensus papers related to the diagnosis of chronic traumatic encephalopathy (cte) and its clinical counterpart, traumatic encephalopathy syndrome. there was also progress toward our understanding of the impact of traumatic brain injury (tbi) on the general population, and how strongly cte pathology may, or may not, commonly underlie long term clinical sequelae following tbi. next, a critical new study has identified that acetylated tau protein, which has been found to be increased in the brains of alzheimer’s disease and cte patients, can be induced by tbi, is neurotoxic, and that its reduction via already-existent therapeutics is neuroprotective. there are also several important updates that pertain to military and blast tbi, particularly as they pertain to establishing causality of interface astroglial scarring. in addition, and for the first time, a specific signature for diffuse axonal injury has been identified in ex vivo tissues using multidimensional magnetic resonance imaging, providing promise for the clinical diagnosis of this lesion. finally, several important radiologic studies from 2021 have highlighted long-standing structural reductions in a number of brain regions following both mild and severe tbi, emphasizing the need for neuropathologic correlation. we end by highlighting an editorial piece discussing how tbi is portrayed in entertainment media and how this impacts public perception of tbi and its consequences. introduction through the emergence of new viral variants, and the waxing and waning of health measures taken by both governments and private industry, the covid-19 pandemic remained an obstacle to ongoing research work related to neurotrauma in the year 2021. however, a review of the published literature in 2021 reveals that interest and advancement has not been slowed, but rather appears to have increased. for example, a simple search for the term ‘traumatic brain injury’ (tbi) on pubmed reveals 5,387 results for the year 2021, which is the most of any year on record. correspondingly, a number of important papers appeared that are of considerable impact to our neuropathology-oriented readership. here, we present our admittedly biased list of highlight papers that were published in the year 2021, with commentary on the information they provided and why we chose to include them in this year’s listing. we are quick to note that a few of these papers have involved our own participation; we do not apologize for these choices, as we think others tasked with the judgement of important contributions to the field of neurotrauma would have similarly considered them. updates to neuropathological criteria for chronic traumatic encephalopathy (cte) and its proposed clinical syndrome the light that is the study of cte burned as brightly as ever in 2021, and perhaps is best represented by the publication of the highly awaited conclusions of the second national institute of neurological disorders and stroke (ninds)/ national institute of biomedical imaging and bioengineering (nibib) consensus meeting to define neuropathological criteria for cte.1 five years in the making, this paper details the results of the reconvening of the ninds/nibib consensus panel in the year 2016 to refine the diagnostic criteria for cte that were set after the first panel meeting in 2015. through the evaluation of 27 cases of different tauopathies, the panel of neuropathologists was able to identify and discern cte according to the original diagnostic criteria, with high rates of agreement while both blinded and unblinded to gross neuropathological findings and clinical histories. therefore, the panel agreed to largely uphold the original definition of the pathognomonic lesion of cte, but with some refinement that can be appreciated in the comparison below: definitions of the pathognomonic lesion for a minimal diagnosis of cte: first ninds/nibib consensus meeting (2015)2: “p-tau aggregates in neurons, astrocytes, and cell processes around small vessels in an irregular pattern at the depths of the cortical sulci.” second ninds/nibib consensus meeting (2016, published 2021): “p-tau aggregates in neurons, with or without glial tau in thorn-shaped astrocytes, at the depth of a cortical sulcus around a small blood vessel, in deeper cortical layers not restricted to subpial and superficial region of the sulcus” as can be seen, the updated definition for the pathognomonic lesion maintains that a diagnostic cte lesion must be at a cortical sulcal depth and that it must display perivascular neurofibrillary changes, but now more strongly emphasizes neuronal involvement as necessary, and details that p-tau pathology cannot be limited to the superficial aspects of the cortex. these refinements were made in the hopes that they will lead to more reliable delineation between cte and other entities, such as age-related tau astrogliopathy (artag) (see figure 1). figure 1. comparison of chronic traumatic encephalopathy (cte) and sulcal tau astrogliopathy. a and b (black bars are 900µm and 200µm, respectively): pathognomonic cte lesion with tau aggregates in neurons (and glia) around small blood vessels at the depth of a sulcus, with tau pathology not restricted to superficial cortical layers. c and d (black bars are 600µm and 200µm, respectively): in contrast, glial tau aggregates in a sulcal depth without perivascular predilection and limited to superficial cortical layers is not diagnostic for cte, but is rather most fitting of tau astrogliopathy. in addition to a refinement of cte minimal diagnostic criteria, the second ninds/nibib consensus panel on cte introduced a working protocol/workflow for the neuropathological evaluation of a brain for cte, which it hopes to be of service to neuropathologists in the community. further, the panel ambitiously proposed the first consensus-based scheme for staging the severity of cte pathology as either “low cte” or “high cte” according to a checklist of criteria. on the clinical side, the year 2021 also saw the publication of the details of the first ninds consensus workshop to define the diagnostic criteria for traumatic encephalopathy syndrome (tes),3 the clinical syndrome which is intended to correspond to cte neuropathology. the workshop was intended to improve upon the first proposed diagnostic criteria for tes that were published in 2014.4 as part of the workshop, a multispecialty panel of experts in tbi and its sequelae reviewed all published cases of neuropathologically confirmed cte, and where possible, carefully assessed predictive validity of clinical features in relation to cte pathology in 298 cases, and used a modified delphi method to agree upon a restructured, stepwise process for the clinical diagnosis of tes. in short, a diagnosis of tes foremost requires a substantial history of repetitive head impacts and cognitive and/or neurobehavioral dysregulation (core clinical features) with a progressive course unexplained by another disorder. supportive clinical features for tes which the panel concluded had insufficient predictive value to be included as core clinical features include delayed onset of symptoms, motor symptoms (e.g. parkinsonism), and relatively non-specific psychiatric features (e.g. anxiety, depression, paranoia). beyond this, the panel also agreed upon provisional criteria (for research purposes) for determining the level of certainty of cte pathology in a clinical case based on meeting tes criteria, varying levels of impact tbi history (particularly long-standing contact sports history), presence or absence of supportive clinical features as described above, and severity of clinical dementia. there is no evidence, as yet, supporting the predictive value of these clinical criteria based on subsequent neuropathology evaluation. time will judge the success of the neuropathological diagnostic criteria and staging scheme for cte, and also of the new proposed clinical criteria for tes. almost certainly, new issues will arise. however, the continued pursuits of consensus agreement in the face of rapidly evolving, and not uncommonly controversial, data are a promising sign for the future. additional consensus meetings to evaluate newly emerging data will clearly be needed. cte in the community crucial and yet unanswered questions with respect to cte concern its frequency in the general community, and its impact on members of the general public with a history of tbi (concerns largely raised by frightening media accounts). in the almost two decades since the resurgence of tbi and cte in the eyes of the public and the scientific community because of its discovery in american football players and other contact sport athletes, collection of materials and data by a number of research centers is starting to bear fruit. with the use of the national health and nutrition examination surveys (nhnes), whose participants are selected to be representative of the civilian general population, and who undergo a rather thorough interview process, physical examination, and blood and urine collection, schneider and colleagues reported a large scale analysis of 7,390 participants over the age of 40 years to determine a prevalence estimate of prior head injury with loss of consciousness (loc) and associated disability.5 of the 7,390 participants, 944 had a history of self-reported head injury with loc. of those 944 with this degree of head injury, 47.4% were noted to be living with a disability in at least one domain of functioning (e.g. activities of daily living, work limitation, memory and confusion limitation, etc.), which was significantly higher (p<0.001) than those without a history of head injury. extrapolating the data to a prevalence estimate for the general public, the study estimated that there are 11.4 million individuals above the age of 40 with a history of head injury and loc and who suffer disability in at least one domain of functioning. it should be pointed out that this figure is more than twice the current prevalence estimated for alzheimer’s disease. as neuropathologists we naturally wonder what pathologies could underlie this staggering new public health data, and perhaps many of us would suspect cte. however, evidence that has been published from a community cohort in the last year seems to suggest otherwise. in a study entitled “the delayed neuropathological consequences of traumatic brain injury in a community-based sample” published in frontiers in neurology,6 authors postupna et al. reported the neuropathological findings of 532 brains from deceased elderly individuals (average age at death: 87 years) consecutively donated to the adult changes in thought study, which itself is focused on aging and dementia in community-dwelling individuals. one hundred seven of these cases had a history of at least one remote head injury associated with loc (most participants sustained their first tbi with loc at less than 25 years of age). of the 532 cases, only 3 (0.6%) brains had diagnostic cte lesions. further, and more surprisingly, none of these 3 cases were among the 107 subjects with a history of tbi and associated loc. while it may be inappropriate to draw conclusions about the general population from this particular study, when viewed in juxtaposition with the aforementioned nhnes study, it appears that disability relating to one or even a few past instances of tbi with loc in the general community (as opposed to cohorts selected for repetitive neurotrauma, such as contact sports athletes) is not widely attributable to cte, and that different mechanisms are likely involved. future study is necessary. reducing acetylated tau is neuroprotective following brain injury the literature has long established that tbi is associated with increased risk of developing clinical alzheimer’s disease and dementia in general, albeit with extremely limited neuropathologic correlation studies.7,8 nonetheless, this suggests that tbi may potentiate or exacerbate neurodegenerative disease and therefore that neurodegenerative proteins may be a therapeutic target for the prevention of long-term cognitive sequelae of tbi. however, precise pathophysiologic links between tbi and neurodegeneration have proven rather elusive. using studies that pointed to acetylated tau as increased in cte, alzheimer’s disease, and other tauopathies as a basis for their research, shin et al. conducted a hallmark study to determine whether tbi induces acetylated tau and thus establish a potential link between tbi and neurodegenerative disease, to establish that acetylated tau is neurotoxic, and to investigate whether inhibition of tau acetylation could be neuroprotective after tbi.9 the authors used a multimodal mouse model for tbi, and showed that acetylated tau protein (acetylated at positions k263 and k270, corresponding to k274 and k281 in humans) was rapidly induced by tbi in both the mouse cerebral cortex and hippocampus, selectively in neurons, and that this was dose-dependent. further, they demonstrated that acetylated tau levels not only rose acutely in injured mice, but remained elevated for months following injury. in the same study, the authors additionally analyzed human frontal cortex specimens of elderly individuals and demonstrated that acetylated tau accumulation was significantly higher in alzheimer’s disease cases when compared to controls, and amongst the alzheimer’s disease cases acetylated tau levels were significantly higher in those who had a remote history of tbi(s) versus those who did not. in a separate component of the study, the authors sought to determine if acetylated tau was directly neurotoxic. they conducted both in vitro and in vivo genetic studies on cultured human neuroblasts exposed to acetylated tau and transgenic mice with mutations that mimic tau acetylation at k263 and k270, respectively. in the cultured human cell lines the authors found that tau acetylation specifically increased neuronal cell death, and in the transgenic mice there was a significant degree of axonal degeneration particularly in the cerebral cortex and hippocampus compared to controls after one year of life. in a translation of their work to therapeutics, the authors experimented with 3 agents that are established inhibitors of processes that promote tau acetylation or promotors of tau deacetylation: cgp3466b omigapil (gapdh nitrosylation inhibitor), salsalate (non-steroidal anti-inflammatory drug and p300/cbp acetyltransferase inhibitor), and aminopropyl carbazole p7c3-a20 (nampt activator that increases preservation of nad+). administration of all three agents blocked tau acetylation, protected against axonal degeneration, and provided significant protection from neurocognitive deficits following tbi in mice. as if the above data were not enough, the authors finally also demonstrated that acetylated tau levels in mouse plasma were elevated following tbi, establishing the potential for acetylated tau to serve as a blood biomarker for neurodegenerative risk following tbi, and noted that the aforementioned neuroprotective therapies decreased concentrations of acetylated tau in plasma following tbi. collectively this laudable study, published in cell, establishes a potential relationship between tbi and neurodegeneration in the form of acetylated tau, demonstrates that acetylated tau is neurotoxic, and provides a basis for the assessment for and protection from neurodegenerative risk after tbi. updates from the battlefield published 100 years after frederick mott’s seminal reporting of the gross neuropathologic features of acute blast exposure in the brains of world war i military personnel who had died from high explosives,10 a new entity was posited to represent a long-term neuropathological consequence of blast exposure in 2016: interface astroglial scarring (ias).11 ias describes a pattern of glial scarring, visible by glial fibrillary acidic protein (gfap) immunohistochemistry, at brain interfaces (e.g. subpial glial plate, around penetrating cortical vessels, grey-white matter junctions, and structures lining the ventricles). at the time of publication, it was suggested that injury at brain interfaces was compatible with the biophysics of blast waves passing through tissues, though this was presented without experimental data in the brain. as expected, this newly described entity was met with criticism,12 much of which was valid and could not be addressed without more study. the year 2021 brought two important publications in support of ias as a novel entity that is caused by blast exposure. in the journal of neuropathology and experimental neurology, schwerin and colleagues reported the results of a ferret model of blast exposure and provided the first animal correlate of blast-induced ias.13 ferrets were chosen because, unlike lissencephalic rodent brains with relatively limited translational capability, the brain of a ferret is more similar to humans in that it is gyrencephalic, has a high white-to-grey matter ratio and a well delineated grey-white matter junction, and has a ventrally positioned hippocampus (see figure 2). in the study, ferrets were anesthetized and exposed to compressed air shock waves, mimicking exposure to a primary blast wave, and sacrificed after survival periods of 1, 4, or 12 weeks. immunohistochemistry for gfap, particularly beginning at 4 weeks survival and in multiply exposed ferrets (four blast exposures compared to one), showed striking astrocyte immunoreactivity precisely at brain interfaces, including the subpial plate, grey-white matter junctions, and surrounding penetrating vessels, thereby reproducing the pattern of ias that was published in human blast cases. further, in demonstrating that blast exposure produces astrogliosis in a similar distribution to that seen in human blast cases, the study transversely also provides evidence that ferrets may serve as a translatable animal model for human tbi. figure 2. comparison of ferret brain with human and other animal species. though relatively small, ferret brains show striking structural similarities with that of primate and human brains, particularly relative to the brains of rodents which themselves are lissencephalic, lack clear grey-white matter interfaces, and have substantially less white matter. the biophysics of a blast wave producing stresses at brain interfaces has also now been supported using an artificial head model. in a study entitled “localizing clinical patterns of blast traumatic brain injury through computational modeling and simulation” miller et al. describe a human head model – simplified but nonetheless complete with distinct skull, cerebrospinal fluid, white and grey matter forming gyri and sulci geometrically similar to human brains, vasculature, and ventricles – which was blast-loaded at three different overpressures and from three different directions (front blast, side blast, and a more complex but true-to-life “wall blast” in which a reflecting surface was introduced for the blast wave to bounce back at the head).14 what the authors found was that strain from blast exposure within the model was most significant in perivascular regions, the subpial plate, and the periventricular regions, i.e. an interface pattern of mechanical stress from the blast wave. simply put, the investigators demonstrated, in an idealized human head model, support for the hypothesis that tbi from blast injury primarily concerns structural interfaces in the brain. further study of different forms of tbi, especially impact tbi, using this model will be critical to additional understanding and establishing its utility in the study of human tbi. a radiologic signature for diffuse axonal injury in what may come as a surprise to some neuropathologists who are quite familiar with this entity, diffuse axonal injury (dai) was purely a pathologic diagnosis without a sensitive or specific radiologic correlate prior to the year 2021. dai is essentially invisible to conventional computed tomography (ct) and magnetic resonance imagining (mri) scans. in a landmark study, authors benjamini et al. applied multidimensional mri to ex vivo samples of human brain with and without neuropathologically confirmed dai (10 cases in total).15 multidimensional mri is an emerging imaging technique that encodes multiple contrasts (e.g. t1, t2) together to provide a “multidimensional” distribution of these components combined with artificial intelligence to allow for enhanced separation of different biological elements within a heterogeneous tissue sample. using this new technique, the authors were able to identify a unique mri signature that allowed them to produce radiologic findings in tissue that precisely mirrored the distribution of app immunohistochemistry on sections cut from the same samples, and further allowed them to blindly differentiate all of the dai and non-dai cases. though this study was performed on ex vivo tissues and utilized preclinical mri technology, the authors have nonetheless provided a bedrock for the potential future clinical detection of dai and more subtle axonal injury in living patients. with further improvements of the multidimensional mri modality, studies scanning whole brains and brains in vivo, and advancements in clinical mri system technology, this imaging breakthrough may soon redefine the clinical assessment of tbi. long-term structural changes in the brain following tbi with clinical correlation there were a number of imaging studies published in 2021 that assessed the volume and/or integrity of a variety of brain structures in the subacute and chronic stages in mild tbi patients. churchill et al. describe reductions of cingulate gyrus blood flow, particularly in the posterior cingulate chronically (1 year after return-to-play) in adult contact sport athletes with a history of concussion, along with increased mean diffusivity in the corpus callosum (cc) (toward the splenium), as compared to control subjects, indicating potential long-term effects of mild tbi on these midline brain structures.16 in a related study, wang et al. conducted diffusion tensor imaging and functional mri on 42 mild tbi patients and 42 matched controls.17 in the tbi patients, they found evidence of structural impairment in the cc which expanded from the anterior-to-midbody of the cc in acute/subacute phases following tbi into both more anterior and posterior regions of the cc in chronic phase (6-12 months), with corresponding evidence of impairment in interhemispheric connectivity. further, these findings correlated to reduced executive function parameters on clinical testing of the patients. finally, authors meier et al. assessed the hippocampus via mri in a group of 106 collegiate athletes.18 they report that hippocampal volume was reduced in athletes with a history of concussion(s), in comparison to those without, and further that hippocampal volume was inversely correlated with the number of previous concussions. this was observed along with a greater association of various neuropsychiatric symptoms. as it pertains to severe tbi, authors tomaiulo et al. describe the long-term mri findings in a group of 25 patients with a history of severe non-missile tbi but without large focal lesions taken at one year and nine years following head injury. they report significant volume reduction in both grey matter (frontotemporal region, crests of gyri, amygdala, hippocampus, basal ganglia, and thalamus) and white matter (cc, fornix, parasagittal white matter, cerebral peduncles) at one year following injury.19 at nine years, there were no significant increases in grey matter reduction, but white matter reduction continued particularly in the posterior body of the cc, and in the white matter under-surface of several cerebral lobes. the above studies, all image-based, are alarming and should serve as a call to action for neuropathologists interested in studying tbi. the need to pathologically characterize and corroborate the growing myriad of long-term structural changes that occur in the brain from tbi as suggested by radiologic evaluation is obvious. in closing as you have read, there were several important developments in the field of neurotrauma in the year 2021. given the sheer volume of publications produced in the field over this time, this article could easily have continued on toward a tome’s length, and admittedly it was quite difficult to refrain from discussing more. however, we hope that you as the reader have enjoyed this summation of the literature that we chose to highlight. to conclude this writing, we would like to bring to attention a poignant short article entitled “the banality of head injury in the punisher”, written by daniel m. donaldson et al. and published in lancet neurology in 2021.20 in this article, the authors examined 26 episodes of the actionand violence-packed tv series the punisher, which focuses on an antihero from the marvel comic universe who employs vigilante, and usually lethal, methods to fight crime. in their assessment, the authors identified 125 instances of head trauma inflicted on characters in the show, including 12 inflicted on the punisher himself, and determined the severity of the tbi events (as best it could be achieved) by the glasgow coma scale. by their assessment, 62% of the head injuries depicted in the show were severe, with an initial glasgow coma scale of 3 in most cases. however, in almost all individuals who survived these severe tbi events, neurologic symptoms following the initial neurological impairment were virtually nonexistent (i.e. no abnormality in mental status after return of consciousness, no lasting neurocognitive deficits, etc.). indeed, all 12 tbi events suffered by the punisher, even when severe, resulted in no neurologic deficits following a return of consciousness. further, significant or long-term structural damage to the face/skull was not appreciated in all but 1 case, even in circumstances where there was substantial disfigurement of other parts of the body from the same general traumatic events. of course, this phenomenon is far from unique to the punisher, as consumers of action tv series and movies are well aware. instead, the authors’ analysis of this one tv show highlights how inaccurately violence, particularly tbi, is presented in media productions and thus to the public as a whole. the authors suggest that the portrayal of the severity (or lack thereof) of tbi in media may augment our perception of tbi and expected outcomes from tbi in real life, and we agree with this supposition. what can be done about this is uncertain, but as neuropathologists and tbi researchers we are uniquely suited to have a seat at the table. disclaimer the information/content, conclusions, and/or opinions expressed herein do not necessarily represent the official position or policy of, nor should any official endorsement be inferred on the part of, uniformed services university, the department of defense, the us veterans administration, the u.s. government or the henry m. jackson foundation for the advancement of military medicine, inc. conflicts of interest the authors do not have any conflict of interest to declare. references 1. bieniek kf, cairns nj, crary jf, et al. (tbi/cte research group). the second ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. j neuropathol exp neurol. 2021;80(3):210-219. https://doi.org/10.1093/jnen/nlab001 2. mckee ac, cairns nj, dickson dw, et al. the first ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. acta neuropathol. 2016;131(1):75-86. https://doi.org/10.1007/s00401-015-1515-z 3. katz di, bernick c, dodick dw, et al. national institute of neurological disorders and stroke consensus diagnostic criteria for traumatic encephalopathy syndrome. neurology. 2021;96(18):848-863. https://doi.org/10.1212/wnl.0000000000011850 4. montenigro ph, baugh cm, daneshvar dh, et al. clinical subtypes of chronic traumatic encephalopathy: literature review and proposed research diagnostic criteria for traumatic encephalopathy syndrome. alzheimers res ther. 2014;6(5):68. https://doi.org/10.1186/s13195-014-0068-z 5. schneider alc, wang d, gottesman rf, selvin e. prevalence of disability associated with head injury with loss of consciousness in adults in the united states: a population-based study. neurology. 2021;97(2):e124-e135. https://doi.org/10.1212/wnl.0000000000012148 6. postupna n, rose se, gibbons le, et al. the delayed neuropathological consequences of traumatic brain injury in a community-based sample. front neurol. 2021;12:624696. https://doi.org/10.3389/fneur.2021.624696 7. li y, li y, li x, et al. head injury as a risk factor for dementia and alzheimer's disease: a systematic review and meta-analysis of 32 observational studies. plos one. 2017;12(1):e0169650. https://doi.org/10.1371/journal.pone.0169650 8. shively s, scher ai, perl dp, diaz-arrastia r. dementia resulting from traumatic brain injury: what is the pathology? arch neurol. 2012;69(10):1245-1251. https://doi.org/10.1001/archneurol.2011.3747 9. shin mk, vázquez-rosa e, koh y, et al. reducing acetylated tau is neuroprotective in brain injury. cell. 2021;184(10):2715-2732.e23. https://doi.org/10.1016/j.cell.2021.03.032 10. mott fw. the effects of high explosives upon the central nervous system. lecture i. lancet. 1916;187(4824):331–338. https://doi.org/10.1016/s0140-6736(00)52963-8 11. shively sb, horkayne-szakaly i, jones rv, kelly jp, armstrong rc, perl dp. characterisation of interface astroglial scarring in the human brain after blast exposure: a post-mortem case series. lancet neurol. 2016;15(9):944-953. https://doi.org/10.1016/s1474-4422(16)30057-6 12. hoge cw, wolf j, williamson d. astroglial scarring after blast exposure: unproven causality. lancet neurol. 2017;16(1):26. https://doi.org/10.1016/s1474-4422(16)30342-8 13. schwerin sc, chatterjee m, hutchinson eb, et al. expression of gfap and tau following blast exposure in the cerebral cortex of ferrets. j neuropathol exp neurol. 2021;80(2):112-128. https://doi.org/10.1093/jnen/nlaa157 14. miller st, cooper cf, elsbernd p, kerwin j, mejia-alvarez r, willis am. localizing clinical patterns of blast traumatic brain injury through computational modeling and simulation. front neurol. 2021;12:547655. https://doi.org/10.3389/fneur.2021.547655 15. benjamini d, iacono d, komlosh me, perl dp, brody dl, basser pj. diffuse axonal injury has a characteristic multidimensional mri signature in the human brain. brain. 2021;144(3):800-816. https://doi.org/10.1093/brain/awaa447 16. churchill nw, hutchison mg, graham sj, schweizer ta. acute and chronic effects of multiple concussions on midline brain structures. neurology. 2021;97(12):e1170-e1181. https://doi.org/10.1212/wnl.0000000000012580 17. wang z, zhang m, sun c, et al. single mild traumatic brain injury deteriorates progressive interhemispheric functional and structural connectivity. j neurotrauma. 2021;38(4):464-473. https://doi.org/10.1089/neu.2018.6196 18. meier tb, españa ly, kirk aj, et al. association of previous concussion with hippocampal volume and symptoms in collegiate-aged athletes. j neurotrauma. 2021;38(10):1358-1367. https://doi.org/10.1089/neu.2020.7143 19. tomaiuolo f, cerasa a, lerch jp, et al. brain neurodegeneration in the chronic stage of the survivors from severe non-missile traumatic brain injury: a voxel-based morphometry within-group at one versus nine years from a head injury. j neurotrauma. 2021;38(2):283-290. https://doi.org/10.1089/neu.2020.7203 20. donaldson dm, holtmann h, nitschke j, et al. the banality of head injury in the punisher. lancet neurol. 2021;20(7):509. https://doi.org/10.1016/s1474-4422(21)00160-5 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. survey of neuroanatomic sampling and staining procedures in alzheimer disease research center brain banks feel free to add comments by clicking these icons on the sidebar free neuropathology 4:6 (2023) original paper survey of neuroanatomic sampling and staining procedures in alzheimer disease research center brain banks juan c. vizcarra1, andrew f. teich2, brittany n. dugger3, david a. gutman4, and the alzheimer’s disease research center digital pathology working group5 department of biomedical engineering, emory university & georgia institute of technology, atlanta, usa department of pathology and cell biology, department of neurology, the taub institute for research on alzheimer’s disease and the aging brain, columbia university, new york, new york, usa department of pathology and laboratory medicine, university of california-davis, sacramento, california, usa department of neuropathology, emory university, atlanta, georgia, usa members of the group and their affiliations can be found in supplementary material 2. corresponding authors: brittany n. dugger · department of pathology and laboratory medicine · university of california, davis · research iii, third floor, suite 3400a · 4645 2nd ave. · sacramento, ca, 95817 · usa, bndugger@ucdavis.edu david a. gutman · department of pathology and laboratory medicine · emory university · 1364 clifton rd · atlanta, ga, 30322 · usa, dgutman@emory.edu additional resources and electronic supplementary material: supplementary material 1 supplementary material 2 supplementary document github repository: https://github.com/gutman-lab/adrc-np-survey-2023 submitted: 25 february 2023 accepted: 28 march 2023 copyedited by: georg haase and cinthya aguero published: 13 april 2023 https://doi.org/10.17879/freeneuropathology-2023-4696 keywords: neuropathology, adrcs, brain banks, tissue repository, biobank abstract the collection of post-mortem brain tissue has been a core function of the alzheimer disease research center’s (adrcs) network located within the united states since its inception. individual brain banks and centers follow detailed protocols to record, store, and manage complex datasets that include clinical data, demographics, and when post-mortem tissue is available, a detailed neuropathological assessment. since each institution often has specific research foci, there can be variability in tissue collection and processing workflows. while published guidelines exist for select diseases, such as those put forth by the national institute on aging and alzheimer association (nia-aa), it is of importance to denote the current practices across institutions. to this end a survey was developed and sent to united states based brain bank leaders, collecting data on brain region sampling, including anatomic landmarks used, staining (including antibodies used), as well as whole-slide-image scanning hardware. we distributed this survey to 40 brain banks and obtained a response rate of 95% (38 / 40). most brain banks followed guidelines defined by the nia-aa, having h&e staining in all recommended regions and targeted region-based amyloid beta, tau, and alpha-synuclein immunohistochemical staining. however, sampling consistency varied related to key anatomic landmarks/locations in select regions, such as the striatum, periventricular white matter, and parietal cortex. this study highlights the diversity and similarities amongst brain banks and discusses considerations when amalgamating data/samples across multiple centers. this survey aids in establishing benchmarks to enhance dialogues on divergent workflows in a feasible way. introduction brain banks are a fundamental resource to facilitate scientific research to better understand the complex biology of the human brain. human tissues are a critical component in understanding a wide range of disorders that affect human health, including alzheimer disease, parkinson’s disease, and chronic traumatic encephalopathy (cte), as well as understanding the effects of normal aging.1,2 within the alzheimer disease and related disorders (adrd) community, the national institute of health (nih) has supported many brain-banking efforts, including alzheimer disease research centers (adrcs).3–5 many brain banks have been collecting tissue for decades and have developed specific protocols on sectioning, staining, and preserving samples typically driven by a specific research area or focus. for example, in a research repository focused on amyotrophic lateral sclerosis (als), the spinal cord may be routinely included in the sampling schematic, while this may not be routine in one focused on alzheimer disease (ad).6,7 a key mandate of adrcs is to aggregate and share the data from research participants, including clinical data, neuropsychological assessments, patient demographics, and when available, neuropathology (np) assessments as well as tissue samples.8 the np data provides the ground truth diagnosis, based on a review of a specific set of stained brain region slides, using standardized semi-quantitative rating scales developed over the past several decades. for ad these include the consortium to establish a registry for alzheimer's disease (cerad) score, braak neurofibrillary tangle stage, thal amyloid phase, and others.9–13 guidelines regarding adrd diagnosis have been previously published and updated as research progresses.14–17 following initial autopsies, a typical protocol will involve the dissection of brain tissue and placement into small cassettes (∼30x20x5 mm, but this can vary) to be paraffinized, sectioned, and then stained either histochemically or immunohistochemically for select markers. however, numerous variations exist, such as large format free floating sections that may be 40-80 µm thick. the specific staining protocols, sample preparations including slice thickness, antibodies or other reagents used for processing, and the anatomic regions surveyed are just a few parameters that can vary from center to center, bank to bank, or even case to case.18,14 furthermore, with the advent and more widespread availability of whole slide image (wsi) technology, it is now feasible to digitize neuropathology datasets at high resolution. these datasets are more easily shared than physical slides and there is a need to understand how best to harmonize workflows. data harmonization efforts will require the creation of a standardized data dictionary in order to facilitate data sharing in the spirit of the fair guidelines (findability, accessibility, interoperability, and reusability).19 given potential variability in staining, sectioning, blocking parameters, and naming conventions, we created, distributed, and evaluated a survey that assessed the current landscape across research institutes to identify common practices in adrd brain banks (supplementary document). materials and methods the authors, along with input from the adrc digital pathology working group, produced and disseminated a survey to brain bank leaders in the spring / summer of 2022, see supplementary table s1 for list of submitted surveys and supplementary material 2 for names of members of the adrc group and their affiliations. survey questions focused on obtaining data for the immunohistochemistry (ihc) procedures used, sampling of brain hemispheres, wsi scanners available, antibody usage, and brain region staining and sampling procedures. for anatomic areas, select landmarks were surveyed to identify heterogeneity between sampling procedures among adrcs (for the complete survey, see supplementary material 1). the survey questions were developed using the jotform.com platform (jotform inc, san francisco, ca). a url link to the survey was emailed to 39 past and current adrc neuropathology core leaders and one non-adrc brain bank leader. participation was voluntary. responses contained no personally identifiable information and results were anonymized. survey responses were compiled in the fall of 2022. survey entries were exported into an excel spreadsheet file for analysis. all analyses were done using open-source python libraries through custom code found in the github repository, including the resulting excel file: https://github.com/gutman-lab/adrc-np-survey-2023. categorical data were presented as frequencies and percentages. most questions in the survey had multi-select answers that were not mutually exclusive (multiple answers possible for each question) or were free text-answers. questions regarding the counter staining-process for ihc were yes/no questions. results the survey requests were sent out to 39 past and present adrc np core leaders, plus one additional brain bank leader not affiliated with an adrc. the survey obtained a response rate of 95% (38/40). most surveys were completed by the center’s np leader or co-leader (33/38). the survey collected center information regarding the ihc procedure used, sampling of brain hemispheres, wsi scanners available, antibody usage, and brain region staining and sampling procedures. ihc processing and counterstaining were similar across centers, with most respondents stating they use hematoxylin counterstain with diaminobenzidine (dab) as the chromogen (brown) without enhancement (30/38). other responses included using neutral red with dab as chromogen with nickel enhancement (3/38) and only dab with nickel enhancement (1/38). four participants specified only using hematoxylin counterstain but neither dab as chromogen with or without nickel enhancement, presumably meaning no implementation of ihc, an alternative approach, or a misinterpretation of the survey question. four of the 38 respondents did not specify the brain region they sampled. of the remaining 34, most denoted sampling the brain's left hemisphere (32/34), 13 sampled both hemispheres, and two sampled only the right hemisphere. answers to this question were not mutually exclusive and included sampling the brain's left, right, or both hemispheres. average section thickness varied considerably across centers, with the most common section size being 5 µm (12/38), followed by 8 µm (7/38), 4 µm (6/38), and 6 µm (5/38). some centers (3/38) sample within a range of sizes, i.e., 5 7 µm. the thinnest section size sampled was 2 µm and the thickest was 80 µm. most centers have a single expert or neuropathologist do all the blocking (23/37), with the remaining utilizing a group of individuals that routinely perform the blocking (14/37); responses were free text answers. the type of wsi scanner available was also surveyed for all institutions. the most common type of scanner denoted was the aperio / leica (25/38). other scanners included olympus, zeiss, huron, philips, hamamatsu nanozoomer, keyence, and 3d histotech. most respondents had access to only a single type of scanner (29/38), while four had two scanner types available. five respondents stated they had no access to a wsi scanner. these data are similar to previously published results.20 with respect to ihc, most respondents utilize the at8 antibody when staining for tau (23/37, antibody information missing for one center) or the phf1 antibody (14/37). some respondents stated using more than one antibody variant for tau staining, with two centers using three, and two centers using two antibodies. other tau antibodies used include cp13 and rd3/rd4. amyloid beta (aβ) staining showed more antibodies used by respondents, with 12 different antibodies in current use. 4g8 (13/37) and the 6e10 (9/37) antibodies are the most common. only one respondent stated using multiple antibodies for aβ staining (10d5, 4g8, and 6e10). for alpha-synuclein (ɑsyn) and tdp-43, respondents specified phospho-specific or non-phospho-specific antibody use. most respondents use a phospho-specific antibody for tdp-43 (29/37). for ɑsyn, about half of the respondents use each type of antibody (phospho-specific: 18, non-phospho specific: 17), and two respondents specified alternative approaches (lb509 emd millipore & millipore #ab5038p). additionally, one respondent used both phospho and non-phospho-specific antibodies. nineteen brain regions were surveyed to assess the stains applied, anatomical landmarks included, and sampling methodology. the survey identified a set of highly consistent combinations of stains and regions across centers/banks. for example, the h&e stain was denoted to be used by all respondents. however, there was a set of six regions (central gyri, periventricular white matter, anterior hippocampus, olfactory bulb, posterior cingulate gyrus, and temporal pole) that were not universally stained with h&e. other stains, like ɑsyn, were more targeted, with frequent use in the midbrain, amygdala, and anterior cingulate gyrus (n≥30) and moderate use for the frontal gyri, posterior hippocampus, temporal lobe, medulla, olfactory bulb, parietal gyri, and the pons (n≥13) (figure 1). figure 1. heatmap showing the number of centers using specific stains for different sampled brain regions. the vertical axis displays the 19 brain regions surveyed and the horizontal axis displays the major stains surveyed. the number in the heatmap signifies the number of centers that use the stain for that region. ɑ-syn: alpha-synuclein, aβ: amyloid-beta, h&e: hematoxylin & eosin. as anatomic regions can be vast and vary in mediolateral, superior/inferior, or rostrocaudal aspects, we also surveyed specific anatomic landmarks within select sampled brain regions. for all landmark questions, the responses were not mutually exclusive. the inclusion of specific anatomic landmarks varied considerably across respondents. for example, for the frontal gyri region, most respondents consistently sample the middle frontal gyrus (32/37 centers that collect the region), but only a few respondents stated that they also sample the inferior frontal gyrus (8/37). for the cerebellum, nearly all respondents stated they sampled the dentate nucleus (36/37), but less than one-third of respondents sampled the vermis (11/37). similarly, for the thalamus and subthalamic nuclei region, not all respondents included the subthalamic nuclei (30/37). other landmarks in this region were sampled by a subset of brain banks (figure 2 shows responses for select regions, additional results are included in supplementary figure s1). figure 2. bar plots showing the anatomical landmarks sampled in four brain regions. the dashed line represents the total number of submitted surveys (38). the dotted line represents the number of respondents that answered the question (varies per region). the n value in the vertical label corresponds to the value of the dotted line. all answers were non-mutually exclusive. mt: mammilla-thalamic, ac: anterior commissure, additional region landmark responses can be found in supplementary figure s1. for most regions, the majority of respondents stated sampling after coronal slicing (>80%) as opposed to before. for the cerebellum region, the survey asked if the sampling was done with longitudinal, transverse, or coronal slicing. most centers use longitudinal sectioning when sampling the cerebellum (21/38), with a subset using transverse (n=6) or coronal (n=5) sectioning, or some other approach (n=5) (figure 3). additional questions not reported are those regarding the antibody vendor, the select set of regions and the number of gyri and sulci targeted during sampling (survey answers can be found at https://github.com/gutman-lab/adrc-np-survey-2023). these questions were free text answers, and while we attempted to glean data from these, there was too high a level of variability to draw conclusions. figure 3. for 18 out of the 19 regions (olfactory bulb excluded), the number of centers that sample the region via different sectioning approaches is shown using stacked bar plots. for 17 regions, the survey asked if the centers sampled the region after or before coronal slicing. for the cerebellum region, the options were: longitudinal, transversal, and coronal slicing. the numbers show the value of the stacked bar. the label in each bar is the brain region. discussion overall, we received survey responses from 95% of sites, providing detailed information regarding neuropathology (np) protocols in select brain banks within the united states. across respondents, the most consistently sampled regions include the frontal gyri, visual cortex, midbrain, posterior hippocampus, and striatum (37/38 centers). the least sampled regions include the posterior cingulate gyrus and the temporal pole (18/38). sampled regions were denoted to be stained with h&e most often, concerning ihc, ɑsyn staining was most frequently conducted on the midbrain & amygdala (>32 centers), and aβ staining was most frequently used in striatum, frontal gyri, cerebellum, and posterior hippocampus (>29 centers). there was variability for tissue thickness, and the specific antibodies/epitopes for tau, ɑsyn, and aβ. the use of specific landmarks to aid in localizing sample regions showed a much higher degree of heterogeneity (figure 2). brain regions can be relatively large, and assigning generic terms or talairach/mni space or brodmann area may not be sufficient for optimal consistency in sampling.21,22 for denoting the presence/absence of a particular neuropathologic feature, a level of precise anatomic location within a nucleus may or may not be necessary, especially when assessing overall diagnoses. however, depending on what nuclear subregions are examined, the distribution of aggregate proteins can vary, for example in the amygdala, and these may alter specific correlations.23–25 there is also variability in the performance of staining procedures, and how effectively they may reveal pathologies, as some have reported for ɑsyn ihc.26 additional studies, including those from the brainnet europe consortium and from persons within the adrc network, have aided in understanding intra-rater reliability, accuracy to clinical diagnoses, and validation of methods in multi-institutional cohorts.10–13, 27–30 depending on the specific scientific question, understanding these additional details may be important.14,15 given the recent advancements in digital pathology, to examining other communities that have converted to a digital format, such as the radiology community, can be advantageous. within the radiology imaging community, standardization of imaging protocols has been achieved across centers, driven in part by the alzheimer’s disease neuroimaging initiative (adni), now starting its fourth iteration.31,32 despite significant differences in mri scanners across individual institutions, these efforts have facilitated the development of comparable imaging acquisition protocols across centers. the inherent digital nature of mri datasets has facilitated not only the sharing of the original imaging sequences but also allows the sharing of standardized quantitative measures of brain structure and pathology. for example, mri volumes are often run through a freesurfer pipeline that produces volumetric information on individual brain structures, cortical thickness, and white matter/gray matter volume, among other detailed statistics.33–36 while data sharing is also a key focus of the adrc network, sharing the physical autopsy slide sets between research centers may often be impractical. there is a great expense in properly packing and shipping large sets of glass slides between centers, although individual sections or blocks can reasonably be shared at a limited scale. the increased availability of whole slide imaging (wsi) platforms is enhancing the ability to share high-resolution digital pathology images.37,38 these, in turn, can be utilized alongside machine learning/artificial intelligence (ml/al) workflows to tackle questions that are otherwise difficult to solve by conventional or traditional approaches. for example, early work in this domain has shown that computational workflows can identify and quantify neuropathological hallmarks of the disease, such as aβ plaques, in a scalable manner.39–43 however, before similar studies can be replicated at scale across a large, diverse collection of images, it is imperative to begin developing a standardized data model to capture pre-analytic variables, as these have been denoted to alter machine learning algorithm outputs.42,44 ml models are notorious for making mistakes in unpredictable ways when being subject to novel images (i.e., different from images used during model training).45–48 thus, it is to be expected that models trained on images from 5 µm sections would not have similar results when assessing images from 80 µm sections. similarly, the different staining or sampling approaches between adrcs might produce images that vary slightly or significantly when viewed through an ml workflow. understanding the data is equally important as developing the computational approach, and the results of this survey will help in understanding the depth of heterogeneity in the adrc’s collective database. conflicts of interest dr. gutman has done consulting work with histowiz inc llc. acknowledgements we immensely thank the adrc digital pathology working group, dr. jonathan d. glass, dr. marla gearing, dr. katherine l. lucot, and kevin nzenkue for their helpful suggestions and feedback on this project. funding this work was supported in part by grants from the national institute on aging (nia) of the national institutes of health (nih) under award numbers p30-ag066511-03s2, nih u01 ag061357-04s1, p30ag072972, r01ag052132, r01ag056519, r01ag062517 and p30ag066462. references 1. carlos, a. f. et al. from brain collections to modern brain banks: a historical perspective. alzheimers. dement. 5, 52–60 (2019). 2. beach, t. g. alzheimer’s disease and the ‘valley of death’: not enough guidance from human brain tissue? j. alzheimers. dis. 33 suppl 1, s219–33 (2013). 3. yong, w. h., dry, s. m. & shabihkhani, m. a practical approach to clinical and research biobanking. methods mol. biol. 1180, 137–162 (2014). 4. alzheimer’s disease research centers. national institute on aging. https://www.nia.nih.gov/health/alzheimers-disease-research-centers. 5. beekly, d. l. et al. the national alzheimer’s coordinating center (nacc) database: an alzheimer disease database. alzheimer dis. assoc. disord. 18, 270–277 (2004). 6. pantoni, l. et al. postmortem examination of vascular lesions in cognitive impairment: a survey among neuropathological services. stroke 37, 1005–1009 (2006). 7. alafuzoff, i. et al. the need to unify neuropathological assessments of vascular alterations in the ageing brain: multicentre survey by the brainnet europe consortium. exp. gerontol. 47, 825–833 (2012). 8. rfa-ag-24-001: alzheimer’s disease research centers (p30 clinical trial not allowed). https://grants.nih.gov/grants/guide/rfa-files/rfa-ag-24-001.html. 9. mirra, s. s. et al. the consortium to establish a registry for alzheimer’s disease (cerad). part ii. standardization of the neuropathologic assessment of alzheimer's disease. neurology 41, 479–486 (1991). 10. braak, h. & braak, e. neuropathological stageing of alzheimer-related changes. acta neuropathol. 82, 239–259 (1991). 11. thal, d. r., rüb, u., orantes, m. & braak, h. phases of aβ-deposition in the human brain and its relevance for the development of ad. neurology (2002). 12. attems, j. et al. neuropathological consensus criteria for the evaluation of lewy pathology in post-mortem brains: a multi-centre study. acta neuropathol. 141, 159–172 (2021). 13. newell, k. l., hyman, b. t., growdon, j. h. & hedley-whyte, e. t. application of the national institute on aging (nia)-reagan institute criteria for the neuropathological diagnosis of alzheimer disease. j. neuropathol. exp. neurol. 58, 1147–1155 (1999). 14. montine, t. j. et al. national institute on aging-alzheimer’s association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol. 123, 1–11 (2012). 15. hyman, b. t. et al. national institute on aging-alzheimer’s association guidelines for the neuropathologic assessment of alzheimer's disease. alzheimers. dement. 8, 1–13 (2012). 16. lucot, k. l. et al. assessment of current practices across alzheimer’s disease research centers biorepositories. biopreserv. biobank. (2022) doi:10.1089/bio.2022.0022. 17. shakir, m. n. & dugger, b. n. advances in deep neuropathological phenotyping of alzheimer disease: past, present, and future. j. neuropathol. exp. neurol. 81, 2–15 (2022). 18. king, a., bodi, i. & troakes, c. the neuropathological diagnosis of alzheimer’s disease-the challenges of pathological mimics and concomitant pathology. brain sci 10, (2020). 19. wilkinson, m. d. et al. the fair guiding principles for scientific data management and stewardship. sci data 3, 160018 (2016). 20. scalco, r. et al. the status of digital pathology and associated infrastructure within alzheimer’s disease centers. j. neuropathol. exp. neurol. (2023) doi:10.1093/jnen/nlac127. 21. dervin, j. co-planar stereotaxic atlas of the human brain 3-dimensional proportional system: an approach to cerebral imaging 1988j. talairich and p. tournoux mark rayport georg thieme verlag. stuttgart, new york 3 13 711 701 1 price dm 268. pp. 122. illustrations 130. the journal of laryngology & otology 104, 72–72 (1990). 22. evans, a. c., janke, a. l., collins, d. l. & baillet, s. brain templates and atlases. neuroimage 62, 911–922 (2012). 23. nelson, p. t. et al. the amygdala as a locus of pathologic misfolding in neurodegenerative diseases. j. neuropathol. exp. neurol. 77, 2–20 (2018). 24. braak, h. et al. amygdala pathology in parkinson’s disease. acta neuropathol. 88, 493–500 (1994). 25. cykowski, m. d. et al. patterns of amygdala region pathology in late-nc: subtypes that differ with regard to tdp-43 histopathology, genetic risk factors, and comorbid pathologies. acta neuropathol. 143, 531–545 (2022). 26. beach, t. g. et al. evaluation of alpha-synuclein immunohistochemical methods used by invited experts. acta neuropathol. 116, 277–288 (2008). 27. mirra, s. s. the cerad neuropathology protocol and consensus recommendations for the postmortem diagnosis of alzheimer’s disease: a commentary. neurobiol. aging 18, s91–s94 (1997). 28. del tredici, k. & braak, h. to stage, or not to stage. curr. opin. neurobiol. 61, 10–22 (2020). 29. beach, t. g., monsell, s. e., phillips, l. e. & kukull, w. accuracy of the clinical diagnosis of alzheimer disease at national institute on aging alzheimer disease centers, 2005–2010. j. neuropathol. exp. neurol. 71, 266–273 (2012). 30. montine, t. j. et al. multisite assessment of nia-aa guidelines for the neuropathologic evaluation of alzheimer’s disease. alzheimers. dement. 12, 164–169 (2016). 31. weiner, m. w. et al. impact of the alzheimer’s disease neuroimaging initiative, 2004 to 2014. alzheimers. dement. 11, 865–884 (2015). 32. weber, c. j. et al. the worldwide alzheimer’s disease neuroimaging initiative: adni-3 updates and global perspectives. alzheimers. dement. 7, e12226 (2021). 33. saygin, z. m. et al. high-resolution magnetic resonance imaging reveals nuclei of the human amygdala: manual segmentation to automatic atlas. neuroimage 155, 370–382 (2017). 34. hedges, e. p. et al. reliability of structural mri measurements: the effects of scan session, head tilt, inter-scan interval, acquisition sequence, freesurfer version and processing stream. neuroimage 246, 118751 (2022). 35. mulder, e. r. et al. hippocampal volume change measurement: quantitative assessment of the reproducibility of expert manual outlining and the automated methods freesurfer and first. neuroimage 92, 169–181 (2014). 36. fischl, b. freesurfer. neuroimage 62, 774–781 (2012). 37. kumar, n., gupta, r. & gupta, s. whole slide imaging (wsi) in pathology: current perspectives and future directions. j. digit. imaging 33, 1034–1040 (2020). 38. niazi, m. k. k., parwani, a. v. & gurcan, m. n. digital pathology and artificial intelligence. lancet oncol. 20, e253–e261 (2019). 39. wong, d. r. et al. deep learning from multiple experts improves identification of amyloid neuropathologies. biorxiv 2021.03.12.435050 (2021) doi:10.1101/2021.03.12.435050. 40. vizcarra, j. c. et al. validation of machine learning models to detect amyloid pathologies across institutions. acta neuropathol commun 8, 59 (2020). 41. tang, z. et al. interpretable classification of alzheimer’s disease pathologies with a convolutional neural network pipeline. nat. commun. 10, 2173 (2019). 42. oliveira, l. c. et al. preanalytic variable effects on segmentation and quantification machine learning algorithms for amyloid-β analyses on digitized human brain slides. j. neuropathol. exp. neurol. (2023) doi:10.1093/jnen/nlac132. 43. signaevsky, m. et al. artificial intelligence in neuropathology: deep learning-based assessment of tauopathy. lab. invest. (2019) doi:10.1038/s41374-019-0202-4. 44. jones, a. d. et al. impact of pre-analytical variables on deep learning accuracy in histopathology. histopathology 75, 39–53 (2019). 45. finlayson, s. g. et al. adversarial attacks on medical machine learning. science 363, 1287–1289 (2019). 46. eykholt, k. et al. robust physical-world attacks on deep learning models. arxiv [cs.cr] (2017). 47. cisse, m., adi, y., neverova, n. & keshet, j. houdini: fooling deep structured prediction models. arxiv [stat.ml] (2017). 48. szegedy, c. et al. intriguing properties of neural networks. arxiv [cs.cv] (2013). copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurooncology: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:4 (2022) review neurooncology: 2022 update pieter wesseling1,2, jacob s. rozowsky2 1 department of pathology, amsterdam university medical centers/vumc, brain tumor center amsterdam, de boelelaan 1117, 1081hv amsterdam, the netherlands 2 laboratory for childhood cancer pathology, princess máxima center for pediatric oncology, heidelberglaan 25, 3584 cs utrecht, the netherlands corresponding author: pieter wesseling · department of pathology · amsterdam university medical centers/vumc · de boelelaan 1117 . 1081 hv amsterdam · the netherlands p.wesseling@amsterdamumc.nl submitted: 14 february 2022 accepted: 23 february 2022 copyedited by: henry robbert published: 24 february 2022 https://doi.org/10.17879/freeneuropathology-2022-3804 keywords: brain tumor, neuropathology, molecular diagnostics, glioblastoma, medulloblastoma abstract this ‘neurooncology: 2022 update’ presents topics that were selected by the authors as top ten discoveries published in 2021 in the broader field of neurooncological pathology. this time, the spectrum of topics includes: papers with a direct impact on daily diagnostic practice of cns tumors in general and with information on how to improve grading of meningiomas; studies shedding new light on the oncogenesis of gliomas (in particular ‘optic gliomas’ and h3-mutant gliomas); several ‘multi-omic’ investigations unraveling the intra-tumoral heterogeneity of especially glioblastomas further; a study indicating the potential of ‘repurposing’ prozac® for the treatment of glioblastomas; liquid biopsy using csf for assessment of residual medulloblastoma. in the last part of this review some other papers are mentioned that didn’t make it to this (quite subjective) top ten list. introduction for the third year in a row, the first author of this paper was invited to contribute a review on the top ten discoveries in neurooncology published in the previous year. rather than checking over 10,000 papers on tumors of the central nervous system (cns) published in 2021 (as was done for the previous review), he now restricted himself to screening the higher-ranked neurooncology and neuropathology journals and the top-journals in oncology and science with a broader scope. furthermore, he teamed up with the last author, a young fulbright scholar from the usa that he supervises for a period of 9 months in the netherlands. the last author mainly used a twitter®-based approach for identifying promising candidates for our list of top ten discoveries in 2021. interestingly, there was quite some overlap between the results of these different search strategies (but more research is needed for sorting out if twitter® is a reliable source of information in this respect, also because it appears that in other areas of life the information shared by tweets can cause chaos rather than order). the final list of topics selected by the authors as very interesting and/or very important for the broader field of neurooncological pathology this time looks like this: who 2021 classification of cns tumors 1 glioma driver mutations in the normal human brain 2 neuronal activity-dependent initiation of nf1-mutant ‘optic gliomas’ 3 oncogenic role of h3 mutations in histogenesis context 4,5 epigenomic insights into glioma cell differentiation and plasticity 6,7 proteogenomic and metabolomic characterization of glioblastomas 8 genomic and immunologic heterogeneity of gliomas and brain metastases 9 therapeutic interference with glioblastoma cell membranes using prozac® 10 liquid biopsy assessment of residual medulloblastoma 11 improved ‘histomolecular’ grading of meningiomas 12-14 of note, all the papers just mentioned became officially available in 2021, but the printed version may have lagged somewhat behind. also, and like in previous years, one has to realize that there is of course quite a subjective component in the selection process that resulted in this list. for example, the strong interest of the last author in computational biology aspects of cns tumors may well have played a role in the selection of topics 5 and 6 especially. to make up to some degree for this rather subjectively composed bouquet of papers, in the discussion section of this review some papers/topics are mentioned that didn’t make it to this top ten list but that certainly are interesting as well. hopefully, this review thereby again helps to appreciate the wealth of (sometimes mind-boggling) information provided by the papers that were used as building blocks for the present manuscript. topic 1: who 2021 classification of cns tumors 1 publication of a new who cns tumor classification may in itself not be perceived as a major scientific achievement. however, quite some of the changes implemented in the fifth edition of the who classification of tumors of the central nervous system (who cns5 classification) are the result of very good science, followed by (hopefully) ‘smart translation’ of the findings into a new classification, which definitely has a major impact on clinical neurooncology. since november 2021, the who cns5 classification is available online via https://tumourclassification.iarc.who.int, and the printed ‘blue book’ version can now be purchased as well (figure 1). the review by louis dn et al. published in neuro-oncology provides a very informative summary of major general changes in the who cns5 classification and of specific changes in the different taxonomic categories 1. figure 1. covers of the who cns tumor blue books throughout the years.in these blue books, the actual who cns tumor classification is the list of tumors that are recognized as distinct tumor types (see e.g. table 1 for the 5th edition); the rest of the information in these books concerns explanation of the pathological (nowadays histological and molecular) characteristics of these tumors, and the most salient information on their clinical and radiological context. molecular characteristics were for the first time introduced as defining criteria in the revised 4th edition, especially for diffuse gliomas in adults and for some embryonal tumors. in the 5th edition, molecular features are introduced as essential diagnostic criteria for many more tumors. in line with recommendations of the cimpact-now consortium 15, salient changes in this new classification are the separation of (groups of) pediatric-type low-grade and pediatric-type high-grade diffuse gliomas from adult-type diffuse gliomas, further refinement of the classification of ependymal tumors, and the addition of a few newly recognized tumors in the category of cns embryonal tumors. table 1 provides an overview of these changes in the who cns5 classification (with # and * indicating the newly introduced and the provisionally accepted tumor types, respectively). another important change is that grading of neoplasms is performed in the who cns5 classification within (rather than across) tumor types, with arabic (instead of roman) numerals now used for the different grades. thus, in the who cns5 classification there is only one entry for e.g. astrocytoma, idh-mutant, and one for meningioma (rather than separate entries for e.g. anaplastic astrocytoma, idh-mutant, or for atypical meningioma). also, glioblastoma, idh-mutant should now be diagnosed as astrocytoma, idh-mutant, cns who grade 4. table 1. who classification of primary cns tumors, 5th edition (2021).major changes in the who cns5 classification are the separation of (low-grade and high-grade) pediatric-type diffuse gliomas from adult-type diffuse gliomas; the recognition of several new ependymal, embryonal and other tumor types; assigning of cns who grades within tumor types; coining the most malignant idh-mutant diffuse astrocytic tumor as astrocytoma, idh-mutant, cns who grade 4; and changing the name of diffuse midline glioma (dmg), h3 k27m-mutant into dmg, h3k27-altered. see summary of louis dn et al. 1 or, even better, the (digital version of the) ‘blue book’ for much more information of these changes. in red & capitals & italics: overarching categories of tumors; in bold and italics: groups of tumor types, and under these groups (with a few exceptions) the individual tumor types; # = newly defined tumor type compared to the 2016 edition; * = provisional tumor type; @ = tumor with revised nomenclature or revised placement. click here to view a large version of this table. furthermore, the presence of tert promoter mutation, egfr amplification and/or the combination of gain of complete chromosome 7 and loss of complete chromosome 10 can now be used for diagnosing an adult-type, histologically lower grade, idh-wildtype diffuse glioma as glioblastoma, idh-wildtype (cns who grade 4), and the presence of homozygous cdkn2a/b loss to diagnose a histologically lower grade, idh-mutant diffuse astrocytoma as cns who grade 4. for yet other tumors the name or their placement in the classification was changed as well (@ in table 1). for example, h3k27m-mutant after diffuse midline glioma (dmg) was changed into h3k27-altered because there are h3-wildtype dmgs especially in children that do show loss of nuclear h3k27me3 staining and with a similar prognosis as dmgs, h3k27m-mutant. also, the name rela fusion-positive for a subset of supratentorial ependymoma was changed into zfta fusion-positive, as zfta (‘zinc finger translocation associated’, the new name for c11orf95) appears to be the more frequent fusion partner in these tumors (most frequently showing fusion with rela). compared to the revised fourth edition, the who cns5 tumor classification certainly is an improvement. however, as discussed in somewhat more detail in the last part of this review, this new classification brings several challenges as well, e.g., related to the (lack of) availability of molecular diagnostic tools, finding the optimal therapeutic management for newly defined tumor types, and the fact that a more refined taxonomy of cns tumors makes it more difficult to perform studies on a large number of patients. topic 2: glioma driver mutations in the normal human brain 2 cancer is a disease characterized by the accumulation of genomic aberrations that confer a proliferative advantage. also, somatic mutations and copy number variations have been described to accrue with age in (seemingly) normal tissues 16. however, relatively little is known about this phenomenon in the normal brain. ganz j and maury ea et al. (with lee ea and walsh ca as corresponding authors) published a paper in cancer discovery 2 in which they used targeted gene sequencing on cerebral gray and white matter samples of 110 individuals to investigate the presence of oncogenic variants in normal brain tissue. the 121-gene panel was selected for genes implicated in different diseases and previously characterized oncogenic drivers. the authors hypothesized that somatic mutations would accumulate in brain tissue over time and (as neurons have a low proliferation capacity) would be enriched in cerebral white matter. the authors identified 35 variants present in normal brain tissue: 12 of the variants were in known proto-oncogenes or tumor suppressor genes, and 6 were known driver mutations of gliomas (idh1, ptpn11, pten, nf1, apc, mtor). the authors validated the variant allele frequencies (vafs) from their gene panel with ultra-deep ion torrent sequencing. the most frequently found mutation was idh1 r132h. in one patient, two adjacent white matter samples had vastly different variant allele frequencies (vafs) of idh1 r132h (5% vs 0.9%). because a 5% vaf is high for a localized oncogenic mutation, the authors suggest that in this tissue, the mutation should be considered as a clonal event that conferred cells with a proliferative advantage. using single-nucleus rnaseq, they found that the idh1 r132h mutation was enriched in glial cells. interestingly (and thus in contrast to what has previously been described for other organs), in this study the accumulation of oncogenic mutations in brain tissue did not positively correlate with age. in fact, all of the patients with identified somatic variants were under 30. furthermore, in this study oncogenic mutations were found to be present in 5.4% of the non-diseased human brains, while the incidence of primary brain tumors in the population is much lower. these results thus raise some intriguing next questions, such as: what are the mechanisms for postnatal elimination of the mutant clones? what is the ‘tipping point’ that eventually causes brain tumor formation? maybe the acquisition of a second hit (such as tp53 and/or atrx mutation for idh-mutant astrocytomas, and 1p/19q codeletion for idh-mutant oligodendrogliomas) 17? and do maybe these findings help to explain the occurrence of ‘dual genotype’ oligoastrocytomas with histologically distinct astrocytic and oligodendroglial areas 18? topic 3: neuronal activity-dependent initiation of nf1-mutant ‘optic gliomas’ 3 over the past few years, we have begun to appreciate the unique role of neurons for glioma growth. for example, two papers that were already summarized in topic 7 of the ‘top ten discoveries in 2019’ review in this journal demonstrated the presence of bona fide neuron-glioma synapses in high-grade gliomas, with electrochemical-induced depolarization of the glioma membrane promoting proliferation of the tumor cells 19,20. these discoveries have boosted the interdisciplinary field of ‘cancer neuroscience’ 21. therapies that target the neuron-glioma interface, especially through neuroligin-3 signaling (nlgn3), are now under investigation as a possible treatment for these malignant gliomas 22,23. in 2021, pan y et al. (with monje m and gutmann dh as corresponding authors) published a study in nature on the impact of neuronal activity in the optic nerve on the formation and growth of ‘optic gliomas’ in mice with nf1 mutation 3. the authors started with an authenticated genetically engineered mouse model which mimics the neurofibromatosis type 1 (nf1) childhood predisposition syndrome: a germline nf1 mutation (nf1+/-) combined with an acquired somatic nf1 mutation in neural progenitor cells (nf1-/-). when these mice are 9 weeks of age, gliomas form along the optic nerve, resembling what is seen in children with nf1 (and in the vocabulary of the who cns tumor classification then generally concern pilocytic astrocytomas). by stimulating the retinal ganglion cells, the authors found an increase in optic nerve volume and proliferation rate, proving that optic nerve activity can increase optic glioma growth. next, the authors investigated the effect of visual experience on the initiation and growth of these optic gliomas. rearing mice in the dark from 6 weeks of age (before optic glioma formation) prevented the formation of tumors. additionally, compared with mice raised in a regular light cycle, dark-rearing mice rescued retinal ganglion cell death and prevented optic nerve damage. pan y et al. further characterized the role of nlgn3, a synaptic adhesion molecule, in these models and found that it operates under a similar mechanism as in high-grade gliomas: adam10 sheddase cleaves and releases nlgn3 into in the tumor microenvironment, which stimulates proliferation of optic glioma cells. when the authors introduced a brain penetrant inhibitor of adam10 in their model, no tumor formed in the optic nerve. these insights raise many interesting follow up questions. for example, would limiting light exposure—e.g., with light-blocking glasses—and/or interference with nlgn3 during certain developmental periods in children help to prevent optic glioma formation? hopefully, these findings will sooner or later provide new opportunities for the therapeutic management of children with nf1. topic 4: oncogenic role of h3 mutations in histogenesis context 4,5 mutations affecting histone h3 are a defining feature in a particular subset of high-grade diffuse gliomas in children and suggest an epigenetic driver of cancer. the two more common mutations, h3.3 p.k28m (k27m) and h3.3 p.g35r/v (g34r/v), are mutually exclusive and typically lead to tumor formation in different cns locations (h3 k27m in midline structures, and h3 g34r/v in the cerebral hemisphere). two recent studies aimed to discern the oncogenic mechanisms of these h3.3 mutations using stem cell models of brain development and gliomagenesis. haag d et al. (with wernig m and pfister sm as senior authors) published a study in cancer cell in which they investigated the effects of the h3 k27m mutation, which co-occurs in diffuse midline gliomas (dmgs) with a mutation in tp53 in 77% of cases 4. previous single cell rnaseq studies of dmgs, h3 k27m-mutant revealed that these tumors may originate from an oligodendrocyte progenitor cell (opc) 24. haag et al. inserted a k27m mutation in neural stem cell (nsc) and opc lines, which increased proliferation in these cell lines. interestingly, only nsc lines with k27m and knock-out of tp53 formed malignant tumors in mouse xenografts that resembled the pathology of dmgs. as this finding is inconsistent with prior reports of the cell-of-origin, the authors hypothesized that the k27m mutation could cause nsc to adopt an opc-like transcriptional profile. to this end, the authors used chromatin immunoprecipitation sequencing (chip-seq) to describe the interactions between mutant h3.3 and dna. they found h3.3 enrichment in bivalent chromatin domains which maintains the expression genes necessary for pluripotency and stem cell identity. this can explain how k27m-mutant nscs can mimic opc transcriptional programs and could drive these tumors. bressan rb et al. (with pollard sm as lead contact) described the oncogenic effects of the g34r mutation in nsc lines derived from the forebrain and hindbrain in a paper in cell stem cell 5. forebrain nsc lines with only the g34r mutation had low tumorigenic capacities. however, combination of knockout of tp53 and amplification of pdgfra in these cells resulted in increased proliferation and the formation of malignant tumors when xenografted in mice. the authors then sought to describe the epigenetic effects of the h3 g34r mutation. unlike the k27m mutation, which causes wide-spread epigenetic and transcriptional changes, the authors found that the g34r mutation reduces binding with zmynd11, a tumor suppressor that alters the elongation and splicing of highly expressed genes. in the context of brain tumors, this results in higher expression of genes that regulate forebrain development and locks nscs into a proliferative and non-differentiated state. together, these papers very nicely show how pre-clinical stem cell modeling can untangle the mechanisms of cancer initiation in general, and of the oncogenesis of h3.3 mutant tumors in particular. topic 5: epigenomic insights into glioma cell differentiation and plasticity 6,7 single-cell (sc) rnaseq studies of diffuse gliomas have revealed unprecedented insights into their intra-tumoral heterogeneity, wherein glioma cells from the same tumor exist in different transcriptionally defined cell states 25-27. many of these cell states mirror the neurodevelopmental hierarchy, and it has been suggested that tumor cells can transition between more or less differentiated states. such a plasticity may well contribute to resistance to therapy. as gene regulation and dna methylation (dname) can largely control these cell states, back-to-back papers published in nature genetics by johnson kc and anderson kj et al. (with verhaak rgw as senior author) 6 and chaligne r, gaiti f, silverbush d, and schiffman js et al. (supervised by suvá ml and landau da) 7 measured dname and rna expression at the single-cell level of idh-wildtype glioblastomas in adults (idh-wt gbms) and in idh-mutant (idh-mt) diffuse gliomas. using scdname, both articles report on how glioma cells from the same patient exist in different methylation states, while cells from different patients can exist in the same states. these studies re-affirm that diffuse gliomas, especially gbms, have high intra-tumoral heterogeneity, with malignant cells adopting different epigenotypes. obviously, the bulk methylation assays that are frequently used to classify (glial) brain tumors fail to account for this level of intra-tumoral heterogeneity that is uncovered by such single-cell analyses. both articles took different approaches to describe the epigenomic landscape that gives rise to the differentiation hierarchies in gliomas. chaligne et al. report that, compared to idh-mt gliomas, polycomb complexes in idh-wt gbms are more hypermethylated in stem-like cells than in differentiated cells. this suggests a role for dname in maintaining a stem-like phenotype of gbm cells, thereby contributing to their treatment resistance and progression. however, this was not seen in idh-mt gliomas, suggesting a different role of dname in maintaining their stem-like cell states. furthermore, the authors constructed lineage trees using scdname and found that cells within each clade had the same chromosomal aberrations. this highlights how sub-clonal mutations are related with the epigenotype of glioma cells. interestingly, when the authors inferred glioma cell state (npc, opc, ac, mes) based on scrnaseq for idh-wt gbms, they found that cells within the same lineage adopted different transcriptional states. however, the reverse was found for idh-mt glioma, where closely related cells obtained similar cell states. altogether, these results confirm that idh-wt gbms have higher cell plasticity than idh-mt glioma, and dname contributes to the glioma stem-like identity. johnson et al. also investigated the contribution of ‘dname disorder’ (i.e., aberrant methylation which allows cells to adapt to diverse methylation states) for maintaining the stem-like identity in gliomas. dname disorder could allow for the glioma cells to overcome environmental stressors and contribute to resistance to treatment. this hypothesis was tested in vitro by exposing two glioma cell lines to hypoxic conditions and irradiation. the authors found that under such environmental stressors, dname disorder increases with time, potentially allowing the cells to adopt and retain their stem-like identities. topic 6: proteogenomic and metabolomic characterization of glioblastoma 8 wang l-b et al. (with the clinical proteomic tumor analysis consortium) published a study which integrated whole exome/whole genome sequencing, bulk and single-nucleus rna sequencing (rnaseq), dna methylation, proteome, phospho-proteome, acetylome, lipidome and metabolome datasets for 100 treatment-naïve glioblastomas. landmark studies previously already comprehensively characterized the genome and transcriptome of glioblastoma (see e.g. 28). a deep ‘multi-omic’ approach, like wang l-b et al. undertook, provides very interesting further insights into functional and potentially targetable differences between glioblastoma subtypes. based on their findings, especially with regard to gene expression, and protein and phosphoprotein abundances, wang l-b et al. defined three ‘multi-omic subtypes’ which closely resemble tcga (the cancer genome atlas) glioma subtypes: the proneural-like subtype was enriched for neurotransmission and synaptic vesicle gene-sets; the mesenchymal-like subtype was enriched for immune system activation, phagocytosis, and glycolysis; finally, the classical-like subtype was enriched for chromatin modification, dna repair, and mrna splicing. the authors also defined immune-based subtypes based on gene-set enrichment analysis of rnaseq data. subtypes ranged from low-enrichment of all immune cells (especially classical and proneural-like glioblastomas) to enrichment of lymphocytes, microglia, and macrophages (mesenchymal-like glioblastomas). the authors integrated these observations with single nucleus rnaseq of 18 samples spanning all multi-omic and immune subtypes, and characterized expression programs found in the neoplastic, stromal, and immune cells. interestingly, this rnaseq analysis revealed upregulation of epithelial-mesenchymal transition related genes in both glioma and immune cells of mesenchymal-like glioblastomas. these differences could be observed on the protein-level as well. last but not least, the authors also noticed subtype-specific enrichment of lipids and metabolites. for example, they noticed idh mutation status-associated differences in glycolysis-related metabolites. not surprisingly, idh-mutant gliomas showed increased levels of 2-hg, as well as decreased glutamate and serine. phosphoproteomic data highlighted therapeutic opportunities for treating glioblastoma, with plcg1 and ptpn11 as signaling hubs for receptor tyrosine kinases. importantly, all the data published by wang et al. is open access and can thus be used as a resource for future studies of glioblastomas. indeed, such a rich dataset adds depth to the previous knowledge from genomic and transcriptomic investigations of glioblastomas and can hopefully pave the way for more effective, personalized treatments for these tumors. topic 7: genomic and immunologic heterogeneity of gliomas and brain metastases 9 immunotherapies for primary and secondary brain tumors are a ‘hot topic’ in the field of neurooncology (see for example also topic 9 of the ‘top ten discoveries of the year 2019’ review in this journal). in 2021, schaettler mo and richters mm et al. (with griffith m and dunn gp as supervising authors) published a study in cancer discovery investigating intra-tumoral heterogeneity of somatic variants, neo-antigens, and infiltrating t-cells in gliomas as well as in brain metastases. for this study, the authors collected 93 spatially separated tumor samples from 30 patients. these samples were subjected to whole exome, rna and t-cell receptor sequencing (wes, rnaseq, tcr-seq). interestingly, while brain metastases had considerably more somatic variants per tumor than gliomas, these variants were mostly clonal, so spanning all tumor samples of a patient. when analyzing the sub-clonal variants of gliomas, the authors noted that sequencing only one region of the tumor missed 40% of variants that were obtained when sequencing three regions. additionally, they found that 9/16 gliomas in this study showed characteristics of multiple transcriptional subtypes (classical, mesenchymal, neural and/or proneural). integrating wes and rnaseq data allowed the authors to predict hla class i and ii neo-antigens expressed in the tumors. they found that the neo-antigens in brain metastases were more often clonal. this could have implications for the development of personalized immunotherapies, as clonal neoantigens would be better targets. using rnaseq data, the authors also described the immune composition of the tumors. brain metastases had robust infiltration of monocyte-derived macrophages, while gliomas were more enriched for microglia gene signatures. finally, the results of tcr-seq analysis revealed that spatially distinct samples of brain metastases had more similar t-cell clones than those of gliomas. altogether, these findings highlight the considerable intra-tumoral heterogeneity in somatic variants, transcriptome, and neo-antigens of gliomas. this can be expected to pose significant challenges for developing effective immunotherapies. comparatively, brain metastases are more homogenous tumors, which could partially explain the clinical benefits of immune checkpoint inhibition when treating patients with those latter tumors. topic 8: therapeutic interference with glioblastoma cell membranes using prozac® 10 so far, little progress has been made with improving the prognosis for patients with idh-wildtype glioblastomas (idh-wt gbms). gbms are known to have an altered lipid composition, with molecular alterations being linked to differential plasma membrane remodeling 29. for example, gbms are frequently driven by the amplification of egfr on extra-chromosomal dna (ecdna). furthermore, variants in the extracellular egfr domain (especially egfrviii) cause constitutive activation 30,31. these signaling molecules are organized into lipid rafts on the plasma membrane. in a study of bi j et al. published in cell reports (with mischel ps as lead contact), the authors identified that gbms are dependent on sphingomyelin phosphodiesterase 1 (smpd1), an enzyme that converts sphingomyelin to ceramide and alters the composition of the plasma membrane. in the tcga (the cancer genome atlas) cohort of gbm samples, increased expression of smpd1 was associated with shorter overall survival, suggesting that targeting this pathway could be a therapeutic strategy for gbm. interestingly, fluoxetine (sold under the brand name prozac®; a brain-penetrant, fda-approved selective serotonin reuptake inhibitor (ssri) used to treat multiple psychiatric disorders) inhibits smpd1 activity. in vitro experiments of gbm patient-derived cell lines treated with fluoxetine was found to result in an increase in lysosomal stress and dose-dependent cell death. also, temozolomide showed synergistic effects with fluoxetine. these results were confirmed in in vivo studies of gbm orthotopic xenografts, wherein 6 of 8 mice receiving both temozolomide and fluoxetine showed no tumor recurrence after 5 months of treatment. over-expression of smpd1 in vitro and in vivo reversed the effects of fluoxetine. next, the authors examined electronic medical records from 180 million americans who were diagnosed with gbm between 2003 and 2017. the patients with gbm who, for psychiatric reasons, were also treated with fluoxetine (prozac®) during the disease course of their brain tumor had significantly longer overall survival. this result was not found for gbm patients treated with other ssris. in conclusion, the results of the study of bi et al. indicate that fluoxetine/prozac® can maybe be ‘re-purposed’ and integrated with standard of care of patients diagnosed with gbm in order to help improve their survival. obviously, controlled clinical trials are necessary to substantiate this idea further. also, it would be interesting to learn more about other compounds that might be good candidates for therapeutic interference with (the composition of) gbm cell membranes. topic 9: liquid biopsy assessment of residual medulloblastoma 11 a current problem in the management of children with medulloblastoma is assessing residual disease during treatment and predicting which patients relapse locally or through dissemination to the leptomeninges (which, unfortunately, frequently leads to death) 32. currently, mri and cytology assessment of the cerebrospinal fluid (csf) are used to detect relapse. however, these diagnostic modalities are generally only useful once the tumor has progressed substantially. medulloblastomas typically have few driver mutations and are characterized by high levels of chromosomal instability (copy number variants). in a study from 2020, escudero et al. found that csf-derived cell-free dna (cfdna) recapitulated molecular characteristics of the initial medulloblastomas, including the oncogenic drivers 33. cfdna from the csf could also be used to classify medulloblastomas into the four molecular sub-groups. following up on these findings, liu api, smith ks, and kumar r et al. (with gajjar a, robinson gw and northcott pa as senior authors) published a paper in cancer cell evaluating the utility of cfdna to assess measurable residual disease (mrd) for guiding the therapeutic management of medulloblastoma patients 11. they performed low-coverage whole genome sequencing on cfdna from 476 csf samples, representing 123 patients, and derived cnvs as a biomarker for mrd. of the 105 csf samples collected at baseline, the authors detected cnvs in 64% of the samples. cnvs were not detected in cfdna from csf of patients without oncological diseases that were used as control. using multivariate regression, they found that detection of baseline samples was associated with tumor location and subgroup, wherein detection was lowest for tumors centered within cerebellar hemispheres or vermis, and for the shh subgroup. additionally, the detection of mrd in samples post-radiotherapy, mid-chemotherapy, or at the end of therapy was associated with worse progression free survival (pfs), while baseline cnv detection was not prognostic for pfs. the authors did find a high correlation between cnvs detected at baseline in the csf and in the primary tumor, confirming the results from escudero et al. comparing serial samples of cfdna, 75% of patients had a more ‘unstable’ genome with a loss of chromosome 10q at recurrence. mrd was detected for all patients with persistent disease recurrence, demonstrating the high accuracy for this assay. of note, of the patients with medulloblastoma recurrence, mrd was detected by csf cfdna analysis at least 3 months before appearing on mri or based on cytology analysis. this study demonstrates that detection of cnvs from csf-derived cfdna may allow for a more sensitive evaluation of disease progression in children with medulloblastoma than the currently available tools. the authors therefore advocate for incorporating such cfdna analysis of liquid biopsies to inform management of these aggressive cancers. topic 10: improved (histo)molecular grading of meningiomas 12-14 until recently, the diagnosis who grade ii meningiomas was based on the presence of increased mitotic activity, brain invasion, presence/absence of three or more of the following features: high cellularity, small cells with a high nucleus: cytoplasm ratio, prominent nucleoli, patternless/sheet-like growth, and foci of necrosis. furthermore, particular histological phenotypes were used to assign a who grade ii (clear cell, chordoid) or who grade iii (rhabdoid, papillary) to meningiomas, while very high mitotic activity and/or overtly malignant cytomorphology were considered as sufficient for the diagnosis of malignant/anaplastic meningioma (who grade iii) as well. however, the prognostic meaning of this histology-based grading system was suboptimal, and it became increasingly clear that molecular data can be used to refine grading of these tumors. indeed, in the who cns5 classification, presence of tert promoter mutation and/or homozygous cdkn2a and/or cdkn2b deletion are now listed as criteria for meningioma, cns who grade 3. furthermore, in this new classification, the rhabdoid and papillary phenotypes are no longer considered as cns who grade 3 based on their histological phenotype alone 1,34. nassiri f et al. (with aldape k and zadeh g as supervising authors) published a paper in nature providing a wealth of matched multidimensional bulk and single-cell molecular and clinical data on a large cohort of meningiomas, enriched for the higher-grade tumors according to who 2016 criteria 12. unsupervised sample-wise clustering of gene-level somatic copy-number alterations (cnas), dna methylome, and transcriptome data in isolation revealed six stable subgroups for each datatype with clinically relevant and significant differences in outcome. additional (second-order) clustering revealed four stable molecular groups (mg1–mg4) without a clear one-to-one relationship between molecular group and who grade. classification by molecular groups was independently associated with recurrence-free survival as assessed by multivariable cox regression, even after accounting for known prognostic clinical factors. mg3 and mg4 tumors (carrying the most unfavorable outcomes) were found to be high-aneuploidy tumors with losses in chromosomes 22q, 1p, 6q, 14, and 18. in addition, mg4 meningiomas showed a gain of chromosome 1q and a loss of chromosome 10. such findings have the potential to supersede existing molecular and clinically used classifications and grading schemes. indeed, two studies published in 2021 show that prognostication for patients with meningioma can be improved by taking particular copy number variations (cnvs) into account (figure 2). driver j et al. (corresponding authors bi wl and santagata s) published a paper in neuro-oncology evaluating whether the use of chromosomal copy-number data provided more accurate prediction of time to recurrence for patients with meningioma than the traditional who grades 14. their discovery cohort consisted of 527 patients diagnosed with meningioma, and two independent cohorts of 172 meningioma patients were used for validation of the findings. based on mitotic count and the presence/absence of the loss of (arms of) particular chromosomes (1p, 3p, 4, 6, 10, 14q, 18, 19), and of homozygous deletion of cdkn2a the authors developed a scheme with three integrated grades (1-3). this grading approach was found to more accurately identify meningioma patients at risk for recurrence than the traditional who grading system. figure 2. assessment of chromosomal copy number aberrations for improved grading of meningiomas.both in the study by driver j et al. 14 and by maas sln et al. 13 the presence/absence of particular chromosomal losses is used for assessment of prognosis of meningiomas. in the ‘driver approach’, this is combined with information on cdkn2a/b status and number of mitoses, while in the ‘maas approach’ the grade as traditionally assigned based on histological features and the suggested methylation family using the meningioma classifier (benign – intermediate – malignant) are taken into account as well. importantly, next to methylation array-based grading system for meningiomas, the study of maas et al. presents some alternatives for a more stepwise approach for grading of meningiomas, taking into account that particular histological phenotypes (angiomatous, psammomatous, secretory) are strongly associated with cns who grade 1 behavior, while presence of tert promoter mutation and/or cdkn2a/b loss indicate high-risk tumors. maas sln, stichel d, hielscher t, sievers p et al. (supervisors von deimling a and sahm f) published a study in the journal clinical oncology in which dna methylation profiling and copy-number information were generated for 3,031 meningiomas of 2,868 patients and mutation data for 858 samples 13. both cnvand methylation family-based subgrouping independently resulted in increased prediction accuracy of risk of recurrence compared with who grading. prediction power for outcome was assessed in a retrospective cohort of 514 patients, and validated on a retrospective cohort of 184 as well as on a prospective cohort of 287 cases. combining different risk stratification approaches into an integrated molecular-morphologic score resulted in substantial further increase in accuracy. again (like in the study of driver et al.), the integrated scores were found to separate tumors more precisely for risk of progression, especially so at the diagnostically challenging interface of cns who grade 1 and grade 2 tumors. discussion in 2021 again an amazing amount of information with relevance for neurooncological pathology has been published. hopefully, this review helps readers keep up with what’s new in this respect. like who tumor classifications in general, and as already stated in the section on topic 1, the who cns5 classification represents work in progress with room for further improvements. obviously, this new classification brings further challenges as well. for example, for more cns tumors it is now impossible to reach a state-of-the-art ‘histomolecular’ diagnosis in case molecular tools for assessment of essential diagnostic characteristics (or immunohistochemistry for reliable surrogate markers, see below) are not available. in those situations, adding nos (not otherwise specified) to the histology-based diagnosis is the way to go 35. furthermore, designing the optimal therapeutic management for newly defined tumor types is challenging. and while a more precise classification facilitates enrollment of more homogeneous populations of patients in clinical studies, the higher granularity of cns tumor taxonomy makes it more difficult to perform studies on a large number of patients for particular tumor types. still, one would like to think that patients suffering from a cns tumor are better served by a more precise diagnosis because this allows for a better estimation of prognosis and, hopefully sooner than later, for a more tailored and effective therapeutic approach. immunohistochemistry for surrogate markers can indeed provide a way for making a bona fide, ‘histomolecular’ who cns5 diagnosis. some examples of stains that are already often used in clinical practice are immunohistochemistry for mutant proteins (idh1 r132h, h3 k27m, h3 g34v/r, braf v600e), for loss of staining for normal proteins in the tumor cell nuclei (atrx, ini1, brg1, h3k27me3), and for abnormal location or intensity of a protein in the tumor cell nuclei (e.g. stat6, p53). the information provided by the studies of driver et al. and of maas et al. (see topic 10) shows that the care for patients with meningioma can be improved by assessment of particular molecular characteristics for the grading of these tumors 13,14. at the same time, there may be an opportunity for immunohistochemistry here as well: for example, loss of h3k27me3 staining of tumor cell nuclei in meningioma has been reported as an additional tool for identification of meningiomas with higher risk of recurrence 36,37. furthermore, in the study of nassiri f et al. (see topic 10), particular proteins were found to be highly enriched in the different molecular groups (mg1: s100b; mg2: scgn; mg3: acadl; mg4: mcm2) 12. further validation of such immunohistochemical approaches and comparison with the results as presented by driver et al. and by maas et al. is needed. also, acknowledging that the molecular underpinnings in meningiomas diagnosed in kids are distinct from those in adults 38, pediatric meningiomas may require an adapted grading system. while cns tumors can now be much more precisely characterized than a few decades ago, the translation of this increased knowledge into more effective treatments is seriously lagging behind. quite some of the topics discussed in this review concern studies that further elucidate the pathobiology of particular cns tumors. hopefully, such knowledge can be exploited for more effective therapies as well, e.g., by targeting of epigenetic regulation of neural or oligodendrocyte precursor states/cells (topic 4), by remodeling the epigenome of glioma cells in order to alter the differentiation hierarchy of gliomas (topic 5), by exploiting the new information obtained by multi-omic investigations for improved targeting of the pathways involved in glioblastomas (topic 6), and/or by improving strategies for selection of patients for targeted (immune)therapies based on immunophenotyping studies and information on the spatial distribution of genomic alterations of gliomas/glioblastomas (topic 7). in a study of > 10,000 cancer patients across 20 different cancer types, transcriptomic analysis allowed for the identification of four distinct tumor-microenvironment subtypes which correlated with patient response to immunotherapy. by integrating transcriptomic and genomic data, a global tumor portrait can be designed, describing the tumor framework, mutational load, immune composition, anti-tumor immunity, and immunosuppressive escape mechanisms, guiding therapeutic decision-making 39. the finding that visual experience and neuronal activity is required for the formation and growth of optic gliomas may provide new targets for therapeutic interference for these tumors (topic 3), and maybe some ‘old drugs’ can indeed be re-purposed in order to improve the outcome for patients with glioblastoma, e.g. because they interfere with the cell membranes of the tumor cells (topic 8). interestingly, a recent (‘seed & soil’) study on the metastatic potential of 500 human cancer cell lines spanning 21 types of solid tumor, breast cancer cells capable of metastasizing to the brain were found to have an altered lipid metabolism, perturbation of which indeed resulting in curbed development of brain metastasis 40. as discussed under topic 9, a liquid biopsy approach using detection of cnvs in csf-derived cfdna may allow for improved recognition of disease progression in children with medulloblastoma. however, in a very recent study of 258 pediatric brain tumors ‘across all histopathologies’, cnvs were detected in only 20% of csf-derived cfdna, and the fact that no genomic aberrations could be detected in liquid biopsies from patients with low-grade gliomas may indicate that its utility is more promising for patients with aggressive brain tumors 41. papers published in 2021 that the authors considered as (very) interesting, but that didn’t make it to the top ten list include: publications presenting potential new tumor types (e.g., supratentorial neuroepithelial tumors, plagl1 fusion-positive 42 and histologically polyphenotypic neuroepithelial tumors, patz1 fusion-positive 43); the report of detailed molecular analysis of (plus clinical variables in) a series of 191 medulloblastomas in adults 44; a study reporting that germline variants in the e-cadherin gene cdh1 are (in addition to non-cns tumors, especially of the stomach and breast) associated with increased risk of neuroepithelial tumors/oligodendrogliomas, idh-mutant, 1p/19q-codeleted 45; papers reporting the accuracy of different molecular tests for assessment 1p/19q status 46, and for mgmt promoter methylation status 47,48. also, for those with a keen interest in neuroimmunooncology it may be good to know that dural sinuses appear to act as a ‘neuroimmune interface’ where brain antigens are surveyed, and that may show ageand disease-related dysfunction 50, and that bone marrow niches in the skull and vertebral column act as myeloid cell reservoirs for the meninges and cns parenchyma 51. acknowledging that for multiple decades histological slides formed the basis for the diagnosis of (cns) tumors, one can argue that for a long time clinical (neuro)pathologists have acted as masters of ‘thin slicing’ (i.e., making quick inferences about characteristics of a tumor using a limited amount of tissue). nowadays, however, for the pathological diagnosis of a rapidly increasing number of cns tumors assessment of particular molecular characteristics is required as well. and in the near future, spatial and/or multi-omics analysis of these tumors may also be required. one can expect that by that time, diagnostic support tools will become available, such as tools for artificial intelligence (ai)-guided analysis of digitized, histological slides 52, and visualization tools for integration of the multi-omic profiling data and for guidance of therapeutic decision-making 39 (figure 3). while such a transition from thin slicing towards augmented reality indicates that the future of neurooncological pathology is bright, it wouldn’t mean too much unless these developments indeed can help to drastically improve the prognosis of patients that suffer from a cns tumor. figure 3. neurooncological pathology: from thin slicing to augmented realityas histological slides formed the basis for the diagnosis of (cns) tumors for multiple decades, one can argue that for a long time clinical (neuro)pathologists have acted as masters of ‘thin slicing’. this term was initially used especially in psychology and philosophy for a situation in which one makes very quick inferences about the state, characteristics or details of an individual or situation with minimal amounts of information. in the context of tumor pathology, it could literally mean the ability to make quick inferences about characteristics of a tumor using very thin slices of tissue. nowadays, however, for an increasing number of cns tumors, molecular characterization is necessary as well. in the near future, spatial and/or multi-omics analysis of cns tumors may be required for an optimal assessment of the diagnosis (including prognosis) and for prediction of the best therapeutic management. one can expect that by that time, diagnostic (ai-guided and visual) support tools have become available that guide the neurooncological pathologist from a more bounded rationality into an augmented reality. meanwhile, it is important to realize that the judgement of an experienced (neuro)pathologist based on thin-slicing/histological slides will remain invaluable for an optimal diagnosis and indeed in quite some situations (e.g., tumors that can readily be recognized based on an h&e stained section +/some additional immunohistochemical stains, and in case of problems with representativeness or a differential diagnosis with non-neoplastic lesions) can even be more accurate than judgements based on much more, non-histological information. references louis dn, perry a, wesseling p, et al: the 2021 who classification of tumors of the central nervous system: a summary. neuro oncol 23:1231-1251, 2021 ganz j, maury ea, becerra b, et al: rates and patterns of clonal oncogenic mutations in the normal human brain. cancer discov 12:172-185, 2022 pan y, hysinger jd, barron t, et al: nf1 mutation drives neuronal activity-dependent initiation of optic glioma. nature 594:277-282, 2021 haag d, mack n, benites goncalves da silva p, et al: h3.3-k27m drives neural stem cell-specific gliomagenesis in a human ipsc-derived model. cancer cell 39:407-422 e13, 2021 bressan rb, southgate b, ferguson km, et al: regional identity of human neural stem cells determines oncogenic responses to histone h3.3 mutants. cell stem cell 28:877-893 e9, 2021 johnson kc, anderson kj, courtois et, et al: single-cell multimodal glioma analyses identify epigenetic regulators of cellular plasticity and environmental stress response. nat genet 53:1456-1468, 2021 chaligne r, gaiti f, silverbush d, et al: epigenetic encoding, heritability and plasticity of glioma transcriptional cell states. nat genet 53:1469-1479, 2021 wang lb, karpova a, gritsenko ma, et al: proteogenomic and metabolomic characterization of human glioblastoma. cancer cell 39:509-528 e20, 2021 schaettler mo, richters mm, wang az, et al: characterization of the genomic and immunologic diversity of malignant brain tumors through multisector analysis. cancer discov 12:154-171, 2022 bi j, khan a, tang j, et al: targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug. cell rep 37:109957, 2021 liu apy, smith ks, kumar r, et al: serial assessment of measurable residual disease in medulloblastoma liquid biopsies. cancer cell 39:1519-1530 e4, 2021 nassiri f, liu j, patil v, et al: a clinically applicable integrative molecular classification of meningiomas. nature 597:119-125, 2021 maas sln, stichel d, hielscher t, et al: integrated molecular-morphologic meningioma classification: a multicenter retrospective analysis, retrospectively and prospectively validated. j clin oncol 39:3839-3852, 2021 driver j, hoffman se, tavakol s, et al: a molecularly integrated grade for meningioma. neuro oncol, 2021 louis dn, wesseling p, aldape k, et al: cimpact-now update 6: new entity and diagnostic principle recommendations of the cimpact-utrecht meeting on future cns tumor classification and grading. brain pathol 30:844-856, 2020 martincorena i: somatic mutation and clonal expansions in human tissues. genome med 11:35, 2019 barthel fp, wesseling p, verhaak rgw: reconstructing the molecular life history of gliomas. acta neuropathol 135:649-670, 2018 barresi v, lionti s, valori l, et al: dual-genotype diffuse low-grade glioma: is it really time to abandon oligoastrocytoma as a distinct entity? j neuropathol exp neurol 76:342-346, 2017 venkatesh hs, morishita w, geraghty ac, et al: electrical and synaptic integration of glioma into neural circuits. nature 573:539-545, 2019 venkataramani v, tanev di, strahle c, et al: glutamatergic synaptic input to glioma cells drives brain tumour progression. nature 573:532-538, 2019 monje m, borniger jc, d'silva nj, et al: roadmap for the emerging field of cancer neuroscience. cell 181:219-222, 2020 venkatesh hs, johung tb, caretti v, et al: neuronal activity promotes glioma growth through neuroligin-3 secretion. cell 161:803-16, 2015 venkatesh hs, tam lt, woo pj, et al: targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma. nature 549:533-537, 2017 filbin mg, tirosh i, hovestadt v, et al: developmental and oncogenic programs in h3k27m gliomas dissected by single-cell rna-seq. science 360:331-335, 2018 tirosh i, venteicher as, hebert c, et al: single-cell rna-seq supports a developmental hierarchy in human oligodendroglioma. nature 539:309-313, 2016 venteicher as, tirosh i, hebert c, et al: decoupling genetics, lineages, and microenvironment in idh-mutant gliomas by single-cell rna-seq. science 355, 2017 neftel c, laffy j, filbin mg, et al: an integrative model of cellular states, plasticity, and genetics for glioblastoma. cell 178:835-849 e21, 2019 brennan cw, verhaak rg, mckenna a, et al: the somatic genomic landscape of glioblastoma. cell 155:462-477, 2013 bi j, ichu ta, zanca c, et al: oncogene amplification in growth factor signaling pathways renders cancers dependent on membrane lipid remodeling. cell metab 30:525-538 e8, 2019 lemmon ma, schlessinger j, ferguson km: the egfr family: not so prototypical receptor tyrosine kinases. cold spring harb perspect biol 6:a020768, 2014 nathanson da, gini b, mottahedeh j, et al: targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant egfr dna. science 343:72-76, 2014 hill rm, plasschaert sla, timmermann b, et al: relapsed medulloblastoma in pre-irradiated patients: current practice for diagnostics and treatment. cancers (basel) 14, 2021 escudero l, llort a, arias a, et al: circulating tumour dna from the cerebrospinal fluid allows the characterisation and monitoring of medulloblastoma. nat commun 11:5376, 2020 goldbrunner r, stavrinou p, jenkinson md, et al: eano guideline on the diagnosis and management of meningiomas. neuro oncol 23:1821-1834, 2021 louis dn, wesseling p, paulus w, et al: cimpact-now update 1: not otherwise specified (nos) and not elsewhere classified (nec). acta neuropathol 135:481-484, 2018 behling f, fodi c, gepfner-tuma i, et al: h3k27me3 loss indicates an increased risk of recurrence in the tubingen meningioma cohort. neuro oncol 23:1273-1281, 2021 nassiri f, wang jz, singh o, et al: loss of h3k27me3 in meningiomas. neuro oncol 23:1282-1291, 2021 kirches e, sahm f, korshunov a, et al: molecular profiling of pediatric meningiomas shows tumor characteristics distinct from adult meningiomas. acta neuropathol 142:873-886, 2021 bagaev a, kotlov n, nomie k, et al: conserved pan-cancer microenvironment subtypes predict response to immunotherapy. cancer cell 39:845-865 e7, 2021 jin x, demere z, nair k, et al: a metastasis map of human cancer cell lines. nature 588:331-336, 2020 pages m, rotem d, gydush g, et al: liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free dna in peripheral blood, csf, and urine. neuro oncol, 2022 sievers p, henneken sc, blume c, et al: recurrent fusions in plagl1 define a distinct subset of pediatric-type supratentorial neuroepithelial tumors. acta neuropathol 142:827-839, 2021 alhalabi kt, stichel d, sievers p, et al: patz1 fusions define a novel molecularly distinct neuroepithelial tumor entity with a broad histological spectrum. acta neuropathol 142:841-857, 2021 coltin h, sundaresan l, smith ks, et al: subgroup and subtype-specific outcomes in adult medulloblastoma. acta neuropathol 142:859-871, 2021 forster a, brand f, banan r, et al: rare germline variants in the e-cadherin gene cdh1 are associated with the risk of brain tumors of neuroepithelial and epithelial origin. acta neuropathol 142:191-210, 2021 brandner s, mcaleenan a, jones he, et al: diagnostic accuracy of 1p/19q codeletion tests in oligodendroglioma: a comprehensive meta-analysis based on a cochrane systematic review. neuropathol appl neurobiol, 2021 mcaleenan a, kelly c, spiga f, et al: prognostic value of test(s) for o6-methylguanine-dna methyltransferase (mgmt) promoter methylation for predicting overall survival in people with glioblastoma treated with temozolomide. cochrane database syst rev 3:cd013316, 2021 brandner s, mcaleenan a, kelly c, et al: mgmt promoter methylation testing to predict overall survival in people with glioblastoma treated with temozolomide: a comprehensive meta-analysis based on a cochrane systematic review. neuro oncol 23:1457-1469, 2021 carlson jc, cantu gutierrez m, lozzi b, et al: identification of diverse tumor endothelial cell populations in malignant glioma. neuro oncol 23:932-944, 2021 rustenhoven j, drieu a, mamuladze t, et al: functional characterization of the dural sinuses as a neuroimmune interface. cell 184:1000-1016 e27, 2021 cugurra a, mamuladze t, rustenhoven j, et al: skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and cns parenchyma. science 373, 2021 jin l, shi f, chun q, et al: artificial intelligence neuropathologist for glioma classification using deep learning on hematoxylin and eosin stained slide images and molecular markers. neuro oncol 23:44-52, 2021 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 64th meeting of the french society of neuropathology meeting abstracts , december 2nd. 2022 feel free to add comments by clicking these icons on the sidebar free neuropathology 4:5 (2023) meeting abstracts 64th meeting of the french society of neuropathology meeting abstracts december 2nd, 2022 centre hospitalier sainte-anne (paris) amphithéâtre de l'institut de psychiatrie et de neurosciences the french society of neuropathology was created in 1989, succeeding the french club of neuropathology set up in 1965.   submitted: 22 march 2023 accepted: 23 march 2023 published: 04 april 2023 https://doi.org/10.17879/freeneuropathology-2023-4776 keywords: french society of neuropathology, sfnp, meeting abstracts, 64th meeting dec. 2022   neuro-oncology   free neuropathol 4:5:2-3 clinico-pathological and epigenetic heterogeneity of diffuse gliomas with fgfr3:tacc3 fusion alice métais1,2, arnault tauziède-espariat1,2, jeremy garcia3, romain appay3,4, emmanuelle uro-coste5, david meyronet6,7,8, claude-alain maurage9, fanny vandenbos10, valérie rigaud11, dan christian chiforeanu12, johan pallud13,2, yann suhan senova14, raphaël saffroy15, carole colin4, myriam edjlali16,17, pascale varlet1,2, dominique figarella-branger3,4 and contributors of the biopathology renoclip-loc network18 ghu psychiatrie et neurosciences, site sainte-anne, service de neuropathologie, paris, france institut de psychiatrie et neurosciences de paris (ipnp), umr_s1266, inserm, université de paris, equipe ima-brain (imaging biomarkers for brain development and disorders), paris, france aphm, chu timone, service d'anatomie pathologique et de neuropathologie, marseille, france aix-marseille univ, cnrs, inp, inst neurophysiopathol, marseille, france department of pathology, toulouse university hospital, toulouse, france groupe hospitalier est, département de neuropathologie, hospices civils de lyon, bron, france claude bernard university lyon 1, lyon, france department of cancer cell plasticity – inserm u1052 cancer research center of lyon, lyon, france department of pathology, lille university hospital, lille, france department of neuropathology, hôpital pasteur, nice, france department of pathology, gui de chauliac hospital, montpellier university medical center, montpellier, france service d'anatomie et cytologie pathologiques, pontchaillou university hospital, rennes, france department of neurosurgery, ghu paris psychiatrie et neurosciences, paris, france departments of neurosurgery and psychiatry, assistance publique-hôpitaux de paris (aphp) groupe henri-mondor albert-chenevier, créteil, france department of biochemistry and oncogenetic, aphp, paul-brousse hospital, villejuif, france department of radiology, aphp, hôpitaux raymond-poincaré & ambroise paré, dmu smart imaging, gh université paris-saclay, u 1179 uvsq/paris-saclay, paris, france laboratoire d'imagerie biomédicale multimodale (biomaps), université paris-saclay, cea, cnrs, inserm, service hospitalier frédéric joliot, orsay, france biopathology reno-clip-loc network: a. rousseau (angers), c. godfraind (clermont-ferrand), g. gauchotte (nancy), k. mokhtari and f. bielle (paris), f. escande (lille), f. fina (marseille) gliomas with fgfr3::tacc3 fusion mainly occur in adults, display pathological features of glioblastomas (gb) and are usually classified as glioblastoma, idh-wildtype. however, cases demonstrating pathological features of low-grade glioma (lgg) lead to difficulties in classification and clinical management. we report a series of 8 gb and 14 lgg with fgfr3::tacc3 fusion. tert promoter mutation was recorded in all gb and 6/14 lgg. among the 7 cases with a methylation score >0.9 in the classifier (v12.5), 2 were classified as gb, 4 as ganglioglioma (gg) and 1 as dysembryoplastic neuroepithelial tumor (dnet). t-sne analysis and unsupervised hierarchical clustering showed epigenetic heterogeneity among fgfr3::tacc3 fused gliomas. relevant factors associated with a better prognosis were age <40, fgfr3(ex17)::tacc3(ex10) fusion type and lack of tert promoter mutation. among gliomas with fgfr3::tacc3 fusion, age, tert promoter mutation, pathological features, dna-methylation profiling and fusion subtype are of interest to determine patients’ risk.   free neuropathol 4:5:4 chromothripsis, one major genetic instability factor in glioblastoma, is rare in idh-mutant gliomas baptiste sourty1,2, laëtitia basset1,2, emmanuel garcion2, audrey rousseau1,2 département de pathologie, chu angers, 4 rue larrey, 49933 angers, france univ angers, nantes université, inserm, cnrs, crci2na, sfr icat, f-49000 angers, france introduction: chromothripsis (ct) and whole-genome duplication (wgd) lead to massive chromosomal alterations that characterize genomic instability. those events are well described in glioblastomas, but scarcely in idh-mutant gliomas. their better prognosis may be related to their genomic stability compared to glioblastomas. methods: pangenomic profiles of 301 gliomas were analyzed by snp array (196 glioblastomas; 105 idh-mutant gliomas). tumor ploidy and ct events were assessed through manual screening and bioinformatics. results: thirty-seven glioblastomas (18.8%) displayed ct versus 5 idh-mutant gliomas (4.8%, p = 0.0007) (all high-grade astrocytomas). wgd was detected in 18 glioblastomas (9.2%) and 9 idh-mutant gliomas of any subtype and grade (8.6%), preceding 75% of chromosomal losses. conclusion: ct is rare in idh-mutant gliomas compared to glioblastomas, contributing to the genomic stability of oligodendrogliomas and grade 2 astrocytomas. ct occurrence in high-grade astrocytomas may underlie aggressive biological behavior. wgd occurs early, as much in idh-mutant gliomas as in glioblastomas.   free neuropathol 4:5:5-6 molecular and clinical diversity in primary central nervous system lymphoma i. hernández-verdin1, e. kirasic1, k. wienand2, k. mokhtari1,3, s. eimer4, h. loiseau5, a. rousseau6, j. paillassa7, g. ahle8, f. lerintiu9, e. uro-coste10, l. oberic11, d. figarella-branger12, o. chinot13, g. gauchotte14, l. taillandier15, j-p. marolleau16, m. polivka17, c. adam18, r. ursu19, a. schmitt20, n. barillot1, l. nichelli21, f. lozano-sánchez22, m-j. ibañez-juliá23, m. peyre1,24, b. mathon1,24, y. abada22, f. charlotte25, f. davi26, c. stewart27, a. de reyniès28, s. choquet25, c. soussain29, c. houillier22, b. chapuy2, k. hoang-xuan1,22, a. alentorn1,22* institut du cerveau-paris brain institute-icm, inserm, sorbonne université, cnrs, f-75013 paris, france department of hematology and medical oncology, university medical center göttingen, 37075 göttingen, germany and department of hematology, oncology and cancer immunology, campus benjamin franklin, charité universitätsmedizin berlin, corporate member of freie universität berlin and humboldt-universität zu berlin, 12203, berlin, germany department of neuropathology, groupe hospitalier pitié salpêtrière, aphp, f-75013, paris, france department of pathology, chu de bordeaux, hôpital pellegrin, 33076, bordeaux, france department of neurosurgery, bordeaux university hospital center, pellegrin hospital, place amélie raba-léon, 33076, bordeaux and ea 7435 imotion, university of bordeaux, bordeaux, france departement of pathology, pbh, chu angers, 4, rue larrey, 49933 angers cedex 9, france and crcina, université de nantes université d'angers, angers, france department of hematology, chu angers, 4, rue larrey, 49933 angers cedex 9, france department of neurology, hôpitaux civils de colmar, colmar, france department of neuropathology, hôpitaux civils de colmar, 68000 strasbourg, france department of pathology, chu de toulouse, iuc-oncopole, 31300 toulouse and inserm u1037, cancer research center of toulouse (crct), 31100 toulouse, université toulouse iii paul sabatier, 31062 toulouse, france department of hematology, iuc toulouse oncopole, toulouse, france neuropathology department university hospital timone, aix marseille university, marseille and inst neurophysiopathol, cnrs, inp, aix-marseille university, marseille, france department of neuro-oncology, chu timone, aphm, marseille, france and inst neurophysiopathol, cnrs, inp, aix-marseille university, marseille, france department of biopathology, chru nancy, chru/icl, bâtiment bbb, rue du morvan, 54511, vandoeuvre-lès-nancy and department of legal medicine, chru nancy, vandoeuvre-lès-nancy and inserm u1256, university of lorraine, vandoeuvre-lès-nancy and centre de ressources biologiques, bb-0033-00035, chru, nancy, france department of neuro-oncology, chru-nancy, université de lorraine, nancy, france department of hematology, chu amiens-picardie, amiens, france department of anatomopathology, lariboisière hospital, assistance publique hopitaux de paris, university of paris, paris, france pathology department, bicêtre university hospital, public hospital network of paris, le kremlin bicêtre, france department of neurology, université de paris, ap-hp, hôpital saint louis, 75010, paris, france department of hematology, institut bergonié hospital, bordeaux, france department of neuroradiology, sorbonne université, assistance publique-hôpitaux de paris, groupe hospitalier pitié-salpêtrière-charles foix, paris, france department of neurology-2, sorbonne université, assistance publique-hôpitaux de paris, groupe hospitalier pitié-salpêtrière-charles foix, paris, france department of neurology, perpignan hospital, perpignan, france department of neurosurgery, sorbonne université, assistance publique-hôpitaux de paris, groupe hospitalier pitié-salpêtrière-charles foix, paris, france department of pathology, aphp, hôpital pitié-salpêtrière and sorbonne university, paris, france department of hematology, aphp, hôpital pitié-salpêtrière and sorbonne university, paris, france broad institute of mit and harvard, cambridge, ma 02142, usa inserm umr_s1138 centre de recherche des cordeliers université pierre et marie curie et université paris descartes, paris, france hematology unit, institut curie, 92210 saint-cloud, france primary central nervous system lymphoma (pcnsl) is a distinct extranodal lymphoma presenting with limited stage disease but variable response rates to treatment despite homogenous pathological presentation. the likely underlying molecular heterogeneity and its clinical impact is poorly understood. we performed a comprehensive multi-omic analysis (whole-exome sequencing, rna-seq, and methyl-seq) in a discovery cohort of 147 immunocompetent pcnsls and a validation cohort of 93 pcnsls. these data were integrated and correlated with the clinico-radiological characteristics and outcomes of the patients, allowing us to identify four significant clusters within pcnsl with shared causative biologic factors of disease and outcome. we found evidence of the microenvironment playing a key role where in two clusters was associated with hypermethylation and in one with high proliferation and polycomb repressive complex 2 activity. meningeal infiltration was associated with a group enriched for hist1h1e mutations. functional analysis on proposed targets supports potential precision-medicine strategies in these pcnsl subtypes.   mini-symposium in memoriam pr charles duyckaerts pr danielle seilhean   free neuropathol 4:5:7 the role of environmental factors on sporadic creutzfeldt-jakob disease mortality: evidence from an age-period-cohort analysis angéline denouel, msc1, jean-philippe brandel, md1,2, danielle seilhean, md, phd1, jean-louis laplanche, phd3,4, alexis elbaz, md, phd5†, stéphane haik, md, phd1,2† cnrs umr 7225, inserm u1127, paris brain institute, sorbonne universités, paris, france ap-hp, centre national de référence des maladies de creutzfeldt-jakob, groupe hospitalier pitié-salpêtrière, paris, france département de biochimie et biologie moléculaire, hôpitaux lariboisière-fernand widal, paris, france inserm, umr 1144, “optimisation thérapeutique en neuropsychopharmacologie”, paris, france université paris-saclay, uvsq, univ. paris-sud, gustave roussy, inserm, u1018, team « exposome, heredity, cancer, and health », cesp, 94807, villejuif, france † these authors share senior authorship sporadic creutzfeldt-jakob disease (scjd) is the most common form of human prion diseases. its origin is still unknown and the role of exogenous factors remains possible. we aimed to study scjd mortality from data collected over 25-years (1992-2016) of active surveillance in france using an age-period-cohort (apc) model in order to better understand scjd origin. our study revealed that several factors influence mortality of scjd over time. indeed, apc analyses highlighted processes linked to aging through an age effect, improvement of surveillance system through a period effect, and, unexpectedly, showed a cohort effect supporting the role of unknown environmental risk factors in disease occurrence. in addition, an age-dependent gender effect was shown with a shift in men-to-women mortality ratio at the age peak. this approach was performed for all scjd cases and for patients associated with the most frequent strain of scjd (i.e. the m1 strain) with similar results.   free neuropathol 4:5:8 astrocytic permeability disorder in spheroid leukoencephalopathy with csf1r mutation teresa dot gomara1, sabrina leclère2,3, isabelle plu1,2,3, susana boluda casas1,2,3, danielle seilhean1,2,3 department of neuropathology, pitié-salpêtrière hospital, aphp, sorbonne university, paris, france neuroceb brainbank, france cnrs umr7225, inserm u1127, paris brain institute, sorbonne university, france adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (alsp) is a rare, progressive neurological disease associated with mutations in csf1r (colony-stimulating factor-1 receptor) gene, known to control the production, differentiation and function of macrophages. we analyzed a series of seven cases of alsp with regards to microglial, astrocytic and axonal markers. we observed a progressive loss of microglia associated with a change in the shape and size of remaining cells. the distribution of astrocytic aquaporin-4 (aqp4) was significantly different in alsp compared to controls. in the relatively spared subcortical regions, an abundance of ramified iba1+ microglia was noticed as well as a strong expression of astrocytic aqp4. in contrast, in the most severely affected regions these markers were extinguished. these arguments provide evidence for the toxic role of microglia and disorders of water flow between cells as factors in the progression of lesions in the disease.   free neuropathol 4:5:9 neuropathological differences between down syndrome and familial alzheimer’s disease with app duplication: role of endothelial cells in cerebral amyloid angiopathy a. kasri1, s. boluda1, l. valay1, m. danjou1, v. montecalvo1, c. duyckaerts1, l. stimmer1, e. gkanatsiou2, g. brinkmalm2,3, y. vermeiren4,5, s. e. pape6, p. p. de deyn4,5, m. irmler7,8, j. becker7,8, h. zetterberg2,9, a. strydom6, m.-c. potier1 paris brain institute, icm, cnrs umr7225 inserm u1127 – upmc, paris, france institute of neuroscience and physiology, the sahlgrenska academy at the university of gothenburg, gothenburg, sweden sahlgrenska university hospital, clinical neurochemistry laboratory, mölndal, sweden department of biomedical sciences, neurochemistry and behavior, institute born-bunge, university of antwerp, antwerp, belgium department of neurology and alzheimer center, university of groningen, university medical center groningen (umcg), groningen, netherlands institute of psychology and neuroscience, king’s college london, 16 de crespigny park, london, united kingdom helmholtz zentrum münchen, neuherberg, germany technical university munich, tum school of life sciences, freising department of molecular neuroscience, ucl institute of neurology, queen square, london, united kingdom while amyloid plaques are common in all ad cases, caa is mainly found in familial ad with duplications of the app gene (dup-app), down syndrome (ds), and specific app mutations. mechanisms leading to these differences are not yet understood. we investigated the diversity of neuropathological phenotypes in sporadic ad (sad), dup-app, app mutations, ds with or without dementia (d), and control cases. in addition, we analysed endothelial cells derived from ipsc lines (ipsc-d-ecs) of patients to model the vessel wall forming the blood-brain barrier. aβ deposits in the parenchyma were numerous in sad, ds-d and app mutations, but less abundant in dup-app sections (12 sad, 7 dup-app, 3 ds, 10 ds-d and 9 app mutations cases). conversely, aβ deposits in the blood vessels (arteries and arterioles) were prominent in dup-app, less abundant in ds-d and scarce in sad and app mutations. only dup-app cases showed aβ deposits in the capillaries. despite striking differences in aβ pathologies, all cases with dementia had high tau pathology. ipsc-d-ecs secreted substantial amounts of aβ peptides. we identified changes in the morphology and tight junctions of ipsc-d-ecs with dup-app as well as specific gene expression dysregulations, suggesting intrinsic remodelling of ecs of the blood-brain barrier in dup-app. our study reveals new pathophysiological mechanisms involved in specific aβ production and deposition in the blood vessel wall of patients carrying dup-app involving ec. differences between dup-app and ds suggest the presence in the ds populations of protective factors against caa.   free neuropathol 4:5:10 genesis and plasticity of the als concept in research: what function for what history? anne fenoy1, danielle seilhean2, claire crignon3 umr 8011 snd, initiative humanités biomédicales, sorbonne université, paris, france inserm u1127, cnrs u7225, sorbonne université, institut du cerveau, paris, france & ap-hp, hôpital pitié-salpêtrière, département de neuropathologie, paris, france umr 7117, archives poincaré, université de lorraine, nancy, france the identification of als by jean-martin charcot in 1873 appears as a founding moment in the history of neuropathology and neurology. through the anatomical-clinical method, charcot was able to provide a first description of the main mechanisms of the disease by combining clinical and neuropathological observations. the history of knowledge of als is often reduced to the history of charcot, whose work is commemorated, notably by the use of the eponym "charcot's disease". reducing the story to a hagiographic approach can lead to a misguided view of how als research works and how it has evolved over time. a combined philosophical, historical, and observational field approach allows us to understand the evolution of the concepts and models developed. the comparison of representations questions the evolution of practices and models and their interpretation, from the original case study to the models, up to the use of animal models.   free neuropathol 4:5:11 neurofilament accumulations in amyotrophic lateral sclerosis patients’ motor neurons impair axonal initial segment integrity cynthia lefebvre-omar1, elise liu1, carine dalle1, boris lamotte d'incamps2, stéphanie bigou1, clément daube1, marc davenne1, noémie robil3, coline jost-mousseau1, françois salachas1,4, lucette lacomblez1,4, danielle seilhean1,5, christian s. lobsiger1, stéphanie millecamps1, séverine boillée1, delphine bohl1 sorbonne université, institut du cerveau – paris brain institute – icm, inserm, cnrs ap-hp, hôpital de la pitié-salpêtrière, paris, france université de paris, saints-pères paris institut des neurosciences, cnrs, paris, france genosplice technology, paris, france département de neurologie, centre de référence sla ile de france, assistance publique hôpitaux de paris (ap-hp), sorbonne université, hôpital de la pitié-salpêtrière, paris, france département de neuropathologie, assistance publique hôpitaux de paris (ap-hp), sorbonne université, hôpital de la pitié-salpêtrière, paris, france neurofilament (nf) levels in patient’ fluids have emerged as the prime biomarker of amyotrophic lateral sclerosis (als) disease progression, while nf accumulation in mns of patients is one of the oldest pathological hallmarks. however, the way how nf accumulations contribute to mn degeneration remains unknown. to assess nf accumulations and study the impact on mns, we compared mns derived from induced pluripotent stem cells (ipsc) of als patients carrying different mutations. our results show that the integrity of the mn axonal initial segment (ais), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of phosphorylated nf-m/h accumulations in mns. our results expand the understanding of how nf accumulation could dysregulate components of the axonal cytoskeleton and disrupt mn homeostasis. thus, preserving ais integrity could open new therapeutic opportunities for als.   free neuropathol 4:5:12 design of a customizable relational database to study clinic pathological correlations in autopsied series isabelle journe-mallet1, julien gouju1, frederique etcharry-bouyx2, valerie chauvire2, virginie guillet-pichon2,3,4, christophe verny3,4, franck letournel1, philippe codron1,4,5,6 laboratoire de neurobiologie et neuropathologie, centre hospitalier universitaire d’angers, angers, france centre mémoire de ressource et de recherche, centre hospitalier universitaire d’angers, angers, france centre de référence des maladies neurogénétiques, centre hospitalier universitaire d’angers, angers, france univ angers, inserm, cnrs, mitovasc, sfr icat, angers, france centre de référence des maladies neuromusculaires aoc, centre hospitalier universitaire d’angers, angers, france centre de ressources et de compétences sur la sla, centre hospitalier universitaire d'angers, angers, france the analysis of clinicopathological correlations in autopsy series remains a central method to improve our understanding of neurodegenerative diseases. however, this approach requires a wealth of information to be relevant, and the use of standard spreadsheet software to collect and manage such volume of data is hardly suitable. to overcome this constraint, we used an open-source database management systems (dbms) to design a customizable neuropathology form with 456 data entry fields. this approach allowed us to optimize the collection of clinical and pathological data from our brain collection, with an average filling time of about 10 minutes per patient. next, we could easily retrieve information from the generated database (22,885 data points) with multiple and conditional queries to study clinicopathological correlations and to quickly identify cases for both diagnosis and research purpose. the large amount of generated data in clinicopathological studies should encourage a more systematic use of dbms. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodevelopmental disorders: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:8 (2022) review neurodevelopmental disorders: 2022 update miguel sabariego-navarro1, álvaro fernández-blanco1, cesar sierra1, mara dierssen1,2,3,4 1 center for genomic regulation, the barcelona institute for science and technology, barcelona, spain 2 neurosciences research program, hospital del mar medical research institute, barcelona, spain 3 university pompeu fabra, barcelona, spain 4 biomedical research networking center for rare diseases (ciberer), barcelona, spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona · spain mara.dierssen@crg.eu submitted: 09 february 2022 accepted: 08 march 2022 copyedited by: christian thomas published: 21 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3801 keywords: mitovesicles, repeatome, trnas methylation, chromatin remodeler, autism spectrum disorder, aicardi-goutières syndrome abstract with a prevalence of 2-4% of the worldwide population, neurodevelopmental disorders (ndds) comprise a heterogeneous group of disorders associated with neurodevelopmental dysfunction, including intellectual disability (id), autism spectrum disorder (asd), down syndrome (ds) and attention-deficit/hyperactivity disorder (adhd) among others. however, due to their heterogeneity and overlapping clinical features, ndds such as asd are often misdiagnosed, while for others with more distinct symptoms, such as rett syndrome or ds, the mechanisms underlying their pathogenesis remain elusive. last year, important steps in the mechanistic understanding of several ndds have been achieved. new preclinical models demonstrated causality between pak3 mutations and disorders associated with social deficiencies. arid1b mutations have been linked to neuroectoderm specification in coffin-siris syndrome and dna damage was established as an important pathologic mechanism in aicardi-goutières syndrome. moreover, alterations in basic molecular processes including translation and histone acetylation have been established as major traits in the pathology of x-linked id and rett syndrome, revealing new pathogenetic mechanisms. last year, advances in bioinformatics have begun to shed light on the human repeatome, a largely unexplored part of our genome, and how alterations in these sequences have a central role in asd. the role of mitochondria in neuropathology was clarified last year with the discovery of previously unknown vesicles derived from mitochondria with a putative role in ds. an interesting discovery in the field of basic neurodevelopment showed that during postnatal brain development, changes in genome architecture and transcriptional dynamics progress independently of sensory experience. finally, our neurocentric views of ndds are changing as new players such as astrocytes are revealed to be crucial in neuropathology. the role of astrocytes has been clarified for some pathologies such as asd and ds, linking well-known genetic mutations to impaired astrocyte function. abbreviations adar1 double-stranded rna-specific adenosine deaminase, ags aicardi-goutières syndrome, asd autism spectrum disorder, atp adenosine triphosphate, cgas gmp-amp synthase, cko conditional knockout, dip-c diploid chromatin conformation capture, dna deoxyribonucleic acid, ds down syndrome, ev extracellular vesicles, gaba gamma-aminobutyric acid, γh2ax: h2a histone family member x, hipsc human induced pluripotent stem cells, iba1 ionized calcium-binding adaptor molecule 1, id intellectual disability, ifn interferon, ip3r2 2 inositol 1,4,5-trisphosphate receptors, lc-ms liquid chromatography-mass spectrometry, mecp2 methyl cpg binding protein 2, ndd neurodevelopmental disorder, rna ribonucleic acid, rnaseh2 ribonuclease h2, nmda n-methyl-d-aspartic acid or n-methyl-d-aspartate, nova1 neuro-oncological ventral antigen 1,   psd95 postsynaptic density 95, rtt rett syndrome, rps4y1 40s ribosomal protein s4, tads topologically associated domains, trs tandem-repeat sequence, trex1 three prime repair exonuclease 1, trna transfer rnas, xlid x-linked intellectual disability. introduction single-term search in the pubmed database with the search term “neurodevelopmental disorders” from january 1st to december 31st 2021 retrieved more than 10,000 papers. in this review, we selected the 10 most interesting discoveries in the field during the last year. instead of centering our efforts around papers published in the highest impact factor journals, we decided to select the discoveries that open new research avenues and that can be expected to have substantial implications in the study of neurodevelopmental disorders paving the way to a change of paradigms. the major steps made during 2021 comprise very different topics, from the discovery of new shared molecular mechanisms involved in several neurodevelopmental syndromes such as aicardi-goutières syndrome or coffin-siris to disentangling the complexity of autism spectrum disorders by the identification of new players at the network, on the cellular and the molecular levels. advancement in modeling tools holds the promise to not only increase our understanding of the origin of neurodevelopmental disorders, including their evolutionary aspects, but also to start proving causality. in the last year, the role of several underexplored biological underpinnings such as trnas methylation or mitochondrial-derived extracellular vesicles were clarified in neurodevelopmental disorders, opening new perspectives for diagnosis and treatment. finally, new tools appeared to study human neurodevelopmental disorders, partially solving some of the associated problems with the use of animal models. the final list of papers and topics of 2021, selected as very interesting and/or important for the field of neurodevelopmental disorders, comprises: reconsidering the molecular neuropathological basis of aicardi-goutières syndrome (ags). understanding cortical neurodevelopment from evolution. new 3d in vitro models to unveil neuropathological mechanisms of human neurodevelopmental disorders. new insights and tools to explore the genome architecture and transcriptional dynamics in the mammalian brain development. new cellular mechanisms and new tools to establish causality in asd. new mechanism in asd pathology: astrocytes, atp and gabaergic signaling. disentangling the role of repeatome in asd. key switch between chromatin remodeler baf subunits explains coffin-siris syndrome neuropathology. trnas methylation and intellectual disability. mitovesicles: a new player in the neuropathology of neurodevelopmental disorders. reconsidering the molecular neuropathological basis of aicardi-goutières syndrome (ags) aicardi-goutières syndrome (ags) is a rare neurodevelopmental disorder. the main neuropathological signs are progressive microcephaly associated with basal ganglia and white matter calcifications, leukodystrophy, cerebral atrophy, and variable increase of lymphocyte count in the cerebrospinal fluid. it is caused by recessive mutations in nine known genes identified until now, namely trex1, rnaseh2a, rnaseh2b, rnaseh2c, samhd1, adar1, mda5, lsm11 and rnu7-1 that result in upregulation of type i interferon (ifn) signaling and is accompanied by neuroinflammation [1]. increased type i ifn activity is detected in cerebrospinal fluid and serum, and increased expression of ifn-stimulated gene transcripts in peripheral blood. moreover, a positive correlation between the level of cerebrospinal fluid ifn activity and the degree of motor and intellectual disability has been reported [2]. astrocytes are the main cell type that produce ifn-α in the brain and magnetic resonance image studies in patients suffering ags show calcifications in the white matter and white matter signal changes in the frontotemporal regions with associated astrocytosis [3]. interestingly, ags-related genes are critical in nucleic acid metabolism function [4, 5], and involved in the removal of redundant endogenous or exogenous dna, rna, or dna/rna hybrids. it is assumed that failure to remove intracellular remnants of nucleic acids leads to stimulation of the innate and adaptive immune responses, so that type i interferonopathies would represent a failure of selfversus non-self-discrimination, as these essential antiviral systems can also be triggered by host dna and rna. the ribonuclease h2 (rnaseh2), linked to ags, is a genome surveillance factor, which has the role of constantly inspecting dna integrity and removing ribonucleotides that incorporate by error into replicating dna [2]. however, the role of ifn hyperactivity and rnaseh2 mutations and their possible functional interaction on ags neuropathology is poorly understood. although the neurotoxic potential of type i ifn was highlighted by studies that recapitulated the neuropathological features of ags in transgenic mice chronically producing ifn-α from astrocytes [6], available genetic ags mouse models fail to show type i ifn response and inflammation in the brain [7, 8]. now, aditi et al. have provided evidence that dna damage-dependent signaling rather than type i ifn signaling underlies neurodegeneration, in this class of interferonopathy. they generated a murine model of neural rnaseh2b inactivation (rnaseh2bnes-cre) [9]. these mice showed smaller cerebellar volume and reduced granule cell and interneuron number. the authors found that reduced cerebellar volume was explained by the loss of proliferative neurons as a consequence of dna damage-induced cell death. they found increased dna double-strand breaks in cells using the h2a histone family member x (γh2ax) marker. the authors used rna-sequencing to compare cerebellar tissue of rnaseh2bnes-cre and controls. they found that genes involved in type i ifn-α response pathway were particularly enriched in rnaseh2bnes-cre cerebellar cells indicating that rnaseh2 deficiency leads to an increased type i ifn response. consistent with the upregulation of neuroinflammatory genes in rnaseh2bnes-cre cerebellum, they also found astroand microglial genes such as glial fibrillary acidic protein (gfap) and ionized calcium-binding adaptor molecule 1 (iba1) were particularly upregulated in rnaseh2bnes-cre cerebellum. these data suggest an ongoing astrocytosis and microglial activation that are hallmarks of neuroinflammation. remarkably, inactivation of tumor protein p53, which induces apoptosis in damaged cells, rescued dna damage-induced cell death and cerebellar atrophy but led to accumulation of cytoplasmic dna damage that resulted in the development of medulloblastoma. the authors also demonstrated that neurodegeneration of rnaseh2bnes-cre mice was not driven by type i ifn signaling since the deletion of cyclic gmp–amp synthase (cgas), a factor that is required for type i ifn activation, led to similar phenotypes in rnaseh2bnes-cre mice. all these data together provide new levels of understanding about ags neuropathology and suggest that neuroinflammation is not the main cause of ags phenotypes, but is a consequence derived from increased dna damage due to rnaseh2b mutations. in this regard, those individuals with additional mutations in dna repair genes might be more prone to present exaggerated immune responses as a consequence of increased dna damage. this discovery constitutes a paradigm-shift in interferonopathies and changes the assumptions about the genetic and molecular causes of ags. reconsidering the leading cause of ags neuropathology can help not only to disentangle the complex neuropathology of this neurodevelopmental disorder, but also to specifically design strategies that would help to ameliorate the symptoms associated with dna damage-driven neuroinflammatory response in ags. understanding cortical neurodevelopment from evolution genetic disease is a necessary product of evolution. in fact, evolutionary pressures have established the risk for many neuropsychiatric or neurodevelopmental diseases. nearly all genetic variants that influence disease risk have human-specific origins. fundamental biological systems, such as dna replication, transcription and translation, evolved very early in the history of life, to give rise to cellular life, but also created the potential for disease. among those, alternative splicing is a form of genetic regulation that enables the production of multiple proteins from a single gene. differences in splicing arose rapidly during a recent evolutionary transition and appear to contribute to adaptation but also to brain disorder. it is known that the transcriptional machinery is often mutated in neurodevelopmental disorders [10]. the brain displays the most complex pattern of alternative splicing of the body [10], with neuron-specific alternative exons such as dna-binding proteins and histone modifying enzymes that regulate evolutionarily conserved transcription. given that alternative splicing is necessary for the proper cortical development [11, 12], a better understanding of how mutations in genes associated with alternative splicing would help to unravel the enormous complexity of several ndds. one interesting example is the neuro-oncological ventral antigen 1 (nova1), which encodes a rna-binding protein that acts as a brain-specific splicing factor. it regulates neuronal alternative splicing of genes responsible for synapse formation in the developing nervous system [13]. increasing evidence has implicated nova1 in numerous pathological processes and neurodevelopmental disorders [14, 15]. mutations in nova1 have been associated with impairment in the development of the motor system [16] and severe neurodevelopmental delays [17]. last year, a comprehensive analysis of genetic variation between modern humans and neanderthals performed by trujillo et al. detected a specific nova1 genetic variation [18]. even though the genomes of humans and neanderthals are very similar, this archaic nova1 version could help to explain how nova1 variants can differentially regulate alternative splicing during human brain development. to study the functional relevance of nova1 mutations, authors used crispr-cas9 genome-editing technology to introduce the archaic variant into the genome of human induced pluripotent stem cells (hipscs). hipscs carrying the archaic mutation (nova1ar/ar) and control hipscs (nova1hu/hu) were used to generate cortical organoids to investigate the impact of the nova1ar/ar variant on the human brain. trujillo and colleagues found that nova1ar/ar cortical organoids were smaller than nova1hu/hu, a phenotype that was explained by a reduced number of rosettes and a higher number of apoptotic cells in nova1ar/ar along with a reduced number of proliferating cells compared to the nova1hu/hu organoids. the authors also found several genes differentially expressed in nova1ar/ar such as rps4y1 and nnat, which are involved in neuronal differentiation, tdgf1, involved in cell migration and proliferation, or pax6 and lhx5, transcription factors associated with cell differentiation during brain development. trujillo and colleagues investigated the gene-splicing variants of nova1ar/ar and nova1hu/hu using single-nuclei rna-seq and identified differences in alternative splicing events affecting 122 genes. remarkably, most of the differentially spliced genes in nova1ar/ar organoids are involved in synaptogenesis and neuronal connectivity. more in detail, nova1ar/ar cells had reduced levels of preand postsynaptic markers such as psd95 and nmda, closely related with activity-dependent plasticity. finally, they evaluated how these synaptic changes could impact neuronal network activity using a multielectrode array that measures several electrophysiological properties. nova1ar/ar cells showed increased number of bursts, lower synchrony and increased variability compared with nova1hu/hu organoids (fig. 1). overall, their results suggest that the archaic nova1 variant causes changes in alternative splicing of genes involved in brain development, proliferation and synaptic plasticity that might explain how mutations of the nova1 gene can contribute to different alterations in ndds. specifically, nova1 would negatively impact neuronal proliferation and survival, but also synaptic connectivity as a result of the reduced expression of proteins important for activity-dependent plasticity. this new study helps disentangle the evolutionary origins of ndds, also pointing to relevant biological processes. fig. 1. schematic representation of how the archaic nova1 variant affects cellular, molecular and neuronal network activity in cortical organoids. a) nova1 archaic version was introduced in ipscs derived from humans. nova1hu/hu and nova1ar/ar hipscs were differentiated into cortical organoids and their cellular, molecular and physiology were studied. b) size of nova1ar/ar organoids was smaller than nova1ar/ar organoids due to decreased neuronal proliferation and increased apoptosis. single-nuclei rna-seq revealed reduced levels of synaptic proteins such as psd95. the study of electrophysiological properties by means of a multielectrode array revealed an increased burst activity in nova1ar/ar cells along with higher variability and reduced levels of synchrony when compared to nova1hu/hu. new 3d in vitro models to unveil neuropathological mechanisms of human neurodevelopmental disorders until recently, the study of neurodevelopmental disorders (ndds) has mainly relied on in vivo animal models to characterize the changes occurring during early brain development. however, the establishment of in vitro models of brain development derived from hipscs represents an unprecedented opportunity to study key brain developmental processes, such as neuronal cell morphogenesis, migration and connectivity in human tissue. although this field is still in its infancy, an increasing number of studies demonstrate its potential to shed light on the mechanisms underlying ndds. this potential is illustrated by recent advances in the study of rett syndrome (rtt), a ndd characterized by cognitive, speech, behavioral and motor problems, that starts in the first year of life and that predominantly affects females. rett syndrome is a rare and complex x-linked neurodevelopmental disorder associated with mutations in methyl cpg binding protein 2 (mecp2) that cause its loss of function. mecp2 is an epigenetic reader that binds to methylated dna to promote transcriptional repression. male homozygous mecp2-/y mice are the most frequently used rtt model due to the early development of severe phenotypes, but even though the use of these models has significantly contributed to the understanding of the mechanisms underlying rtt, their clinical relevance might be limited. in fact, their face validity is constrained as male mice are used to model a disease mainly affecting females, and female patients have a milder phenotype than the one observed in mice. to overcome these limitations, several methodologies have been established to model rtt with hipscs from patients by developing 2d-based neuronal differentiation protocols. more recently, the generation of 3d human brain organoids, a more complex approach that better recapitulates human neurodevelopment in vitro, has been established. these culture platforms are increasingly contributing to a better understanding of the rtt etiology. the tremendous potential of such methodologies is illustrated by the recent work of samarasinghe et al. [19] who showed that brain organoids derived from induced pluripotent stem cells from individuals with rtt reproduce patterns of electrical brain activity that resembled seizures, a hallmark of the condition. using calcium sensor imaging and extracellular recording approaches, they demonstrated highly abnormal and epileptiform-like activity, accompanied by transcriptomic differences revealed by single-cell analyses. interestingly, trujillo et al. [20] used an innovative pipeline to model rtt neurodevelopment demonstrating that neurons differentiated from human mecp2-ko hipscs show altered expression of synapse-relevant genes, synaptic morphology, and decreased calcium and network activities compared to control neurons. an in silico neural network simulation affirmed that synaptic structural parameters link with neural network activity, supporting the approach of rescuing synaptic structure and increasing activity. they differentiated mecp2-ko hipsc into in vitro models of increasing complexity. first, 2d monolayer neuronal cultures revealed an impaired neurotransmission. the same hipsc were then used to produce a novel system of 3d brain neurospheres derived from a 50/50 mixture of healthy and mecp2-ko hipsc to better mimic the mosaicism observed in female patients, a phenomenon produced by random x-inactivation. this new model will be of great utility to investigate rtt and other x-linked genetic conditions. as a last step, complex 3d mecp2-ko cortical organoids were also produced. interestingly, this pipeline revealed important features of rtt pathology. the researchers found that rtt organoids displayed smaller diameters and both mecp-ko neuronal cultures and organoids showed that synaptic and neurotransmitter pathophysiology principally concentrated in glutamatergic and cholinergic dysregulation. furthermore, nefiracetam, a cholinergic, gabaergic, and glutamatergic agonist, and pha 543613, a α7-nachr agonist, had a rescuing effect supporting glutamatergic and cholinergic dysregulation. new insights and tools to explore the genome architecture and transcriptional dynamics in the mammalian brain development neurodevelopmental processes rely on the orchestration of finely tuned gene expression programs, which depend on the regulation of chromatin structure. over the last few years, mutations in genes encoding factors that can modify chromatin regulation and nuclear architecture have emerged as a frequent cause of ndds. post mortem studies on brains from patients with different ndds found chromatin and other epigenetic alterations linked with abnormalities in neural development. this is the case of opitz-kaveggia and fryns-lujan syndromes, caused by mutations in med12 [21], and cornelia de lange syndrome, which results from mutations in nipbl or in the cohesin subunits coding genes smc1 and smc3 [22]. mutations in the ctcf gene are also associated with intellectual disability [23, 24]. on the other hand, our knowledge of the critical role of the genome’s 3d organization in gene regulation is steadily increasing. this is important since the expression of a given gene in a specific time and cellular type is known to depend on its location within the nucleus and on its contacts with other loci, forming topologically associated domains (tads). however, although previous studies have contributed to characterize the transcriptome and 3d genome in the brain, they failed to answer this question mainly due to the use of bulk chromatin conformation capture techniques, which can only assess averaged maps of the 3d genome. this constitutes an important limitation in a highly heterogeneous tissue such as the brain. now, tan et al. [25] have overcome this limitation thanks to the use of a new diploid chromatin conformation capture (dip-c) method [26], a chromatin conformation capture method that allows to distinguish between the two haplotypes of each chromosome, which allowed them to create a 3d genome atlas of the developing mouse forebrain, including ~2,000 single-cell contact maps and ~800 3d structures from two brain regions and 6 time points. in parallel, they also produced a single-cell transcriptomic atlas to shed light on the “structure-function” relationship question during neurodevelopment. importantly, this approach revealed a major post-natal transcriptomic and 3d structure reorganization. in other words, the gene expression and chromatin architecture profiles of postmitotic neurons continue to mature during postnatal development. at the 3d genome architecture level, the radial positioning (preference for the nuclear periphery or nuclear core) of genes was the most prominent difference between neonatal and adult neurons. a similar phenomenon was previously thought to be unique for certain gene clusters, such as olfactory receptors (or), in olfactory receptor neurons [27] development. importantly, during developmental stages, ors genes move to the nuclear interior, detaching from the nuclear laminin, thereby allowing their activation, to ensure that each olfactory receptor neuron expresses one, and only one, or, a phenomenon called the “one neuron-one receptor expression” [27]. these results, however, show that this behavior might represent a generalizable principle of neuron development not restricted to olfactory receptor neurons. strikingly, tan et al. also showed that these transformations of the 3d genome are independent of sensory experience, suggesting that the 3d transformation is genetically predetermined. altogether, these results provide new tools and a conceptual framework to shed light on the molecular mechanisms governing neurodevelopmental processes at an unprecedented resolution. new cellular mechanisms and new tools to establish causality in asd throughout the last years, advances in the field of genetics have led to the discovery of hundreds of genes that contribute to serious deficits in communication, social cognition, and behavior of autism spectrum disorder (asd). however, these genes only account for 10–20% of cases, and similar pathogenic variants may lead to very different levels of affectation. in fact, understanding causal relationships between genes and phenotypes remains a challenge. recent evidence suggests that dysfunction of rho family guanosine triphosphatases (rho-gtpases) contributes substantially to the pathogenesis of ndds [28, 29], and as many as 20 genes encoding rho gtpase regulators and effectors are listed as asd risk genes [30, 31]. those genes are promising candidates to explain asd neuropathology. rho gtpase signaling is essential for cellular signaling and cytoskeleton dynamics [32, 33]. one interesting example is the p21-activated kinase 3 (pak3), which is a downstream component of the rho-gtpase cascade. genetic studies in humans revealed that mutations of pak3 are associated with moderate to severe intellectual disability (id) and with synaptic alterations in post mortem human tissues [34-36]. animal models with pak3 mutations (mpak3) also recapitulate these phenotypes and have shown that pak3 is important for recognition memory [37, 38]. in fact, pak activity, in combination with rac (a rho-gtpase), has been suggested to be responsible for the autistic cellular and clinical phenotypes in mouse models of asd, and may be related to synaptic plasticity alterations [39]. however, a causal link between these mutations and autistic phenotypes has not yet been directly demonstrated. one possible problem is the lack of spatial-temporal resolution of the investigations. for example, a gene mutation may have specific neuropathological impact on a subpopulation of cells, which cannot be selectively targeted. this is the case with memories, which are believed to be stored in a subpopulation of neurons that get activated and modify their functional properties during learning. those are called engram cells [40, 41], and might be specifically affected by pak3 mutations. for this reason, zhou et al. designed a tetracycline-inducible system (tta/teto) under the control of cfos, to express a mutant pak3 form tagged with green fluorescent protein (mpak3-gfp) specifically in neurons that were activated during social interaction [42]. cfos is an immediate early gene associated with neuronal activity and synaptic plasticity and is often used to identify engram cells [40, 41, 43]. the authors showed that disrupting pak3 signaling by the expression of mpak3 in cfos-expressing neurons during social interactions in fact was sufficient to impair social memory. contrarily, rescuing mpak3 levels in the same animals restored social memory deficits. the fact that pak3 disruption in just a subset of cfos positive cells is sufficient to produce social memory impairment underscores the critical importance of increasing the resolution of the techniques we use. in this regard, an increasing number of studies are using single-cell omics for resolving the cell-specific impact of specific mutations, and single-cell rna seq data are available from post mortem neurotypical human brain regions. even though the mechanisms by which mpak3 was altering social memory engrams were not covered in this study, to date this is the first experimental demonstration that establishes the necessity of pak-dependent molecular changes in engram cells underlying social memory pathology. certainly, this cell-specific system can be used to establish molecular causality of memory deficits in different mouse models of ndds given the ability to fine-tune the expression of particular genes that could be potentially implicated in the neuropathology of these disorders in an inducible manner. increasing the resolution and selectivity of our experimental approaches will certainly uncover new neuropathological mechanisms and possibly help to understand the actual contribution of risk genes to the pathogenesis of autism and other ndds. new mechanisms in asd pathology: astrocytes, atp and gabaergic signaling the research in asd has traditionally focused on neuronal dysfunction, as many asd genes encode for proteins that have a role in synaptic function, thus making the views of the pathological features of asd mainly “neurocentric”. however, selectively focusing on neuronal mechanisms has proven to be limited, and thus, alternative biological analyses may help to reveal previously unknown cellular and molecular mechanisms. astrocytes are newly identified players in several brain pathologies, and could play an important role in asd. in fact, rna sequencing revealed a close association between asd and the genes related to glial cell activation, immune and inflammatory categories [44]. studies in post mortem brain tissue from donors affected by asd have shown alterations in the expression of astrocyte markers, and ipscs derived from asd patients present alterations in astrocytic development [45, 46]. one recent preprint shows that astrocytes from asd patients cause autistic phenotypes when engrafted in mouse pups, establishing an important milestone in the asd field [47]. however, whether astrocytes alterations are the cause or the consequence of asd and how this glial cell type contributes to asd pathology remains unclear. interestingly, de novo mutations in type 2 inositol 1,4,5-trisphosphate receptors (ip3r2) gene have been identified in asd patients [48]. this receptor mediates the activation of astrocytes by increasing cytoplasmic calcium signaling [49], and previous studies in ip3r2 knockout (ip3r2 ko) mice showed selective dysfunction of astrocytes but not neurons leading to social behavior impairment, as revealed by increased repetitive behaviors and impaired social approach [50]. wang et al. [51] now show that astrocyte calcium signaling dysfunction can result in asd-like behavior in mice. the authors generated a conditional knockout (cko) mouse for ip3r2 using an astrocyte-specific promoter, aldh1l1, that induces the ip3r2 knockout during adulthood. this astrocyte specific ip3r2 ko in adult mice was sufficient to produce autism-related behaviors, suggesting that these phenotypes are not just a consequence of altered neurodevelopment. to further investigate the mechanism underlying social behavior alterations and astrocyte dysfunction in ip3r2 mutant mice, they analyzed the levels of different gliotransmitters. they found a strong reduction in atp levels, a known gliotransmitter, in ipr32 cko mice. this reduction in atp concentrations was only found in astrocytes but not in neurons isolated from ipr32 cko mice, indicating astrocytes-specific alterations. using chemogenetic techniques and an atp sensor under an astrocytic promoter, they demonstrated that atp release was significantly reduced in ip3r2 cko and that atp administration led to a total rescue of social interaction. this indicates the need of atp release by astrocytes for proper social interaction. this finding establishes a new pathogenic mechanism: lower release of atp after astrocyte activation leads to an increase of gabaergic inhibitory activity. however, the pathogenetic mechanisms are certainly more complicated, as shown by other recent studies reporting that astrocyte dysfunction could lead to an altered microglial state [52], and lead to increased neuroinflammatory processes. in conclusion, astrocyte activity, mediated by calcium signaling pathways, plays a pivotal role in asd pathology. disentangling the role of repeatome in asd approximately half of the human genome consists of repetitive dna sequences, and is called the repeatome. repetitive dna consists of more than one million tandem repeats, sections of dna in which a sequence is replicated many times in tandems, interspersed repeats and copy number variants, which biology remains under-studied and poorly annotated, being considered as “junk dna” compared to the protein coding dna [53]. however, more than 50 diseases such as huntington disease, fragile x, various ataxias and a major subset of amyotrophic lateral sclerosis and frontotemporal dementia cases are caused by an expansion of a tandem-repeat sequence (trs), showing the importance of this non-coding dna in some pathologies [54]. repeat-associated non-atg translation (ran translation) of toxic peptides may contribute to the pathogenesis of those disorders and emerging evidence suggests that trs can also regulate gene expression in healthy individuals [54]. nonetheless, the role of trs in polygenic pathologies such as asd remains largely unexplored. even though changes in the number of copies of large segments of dna have been implicated in asd pathology [55], the capacity to analyze alterations in trs from a genome-wide point of view was, until now, unachievable. mitra et al. have now developed a new bioinformatic tool capable of analyzing trs in people with asd and their family members [56]. this new bioinformatic tool that the authors named monstr, allowed the researchers to discover that trs expansions are more common in people with asd than in their unaffected siblings. the expansions occurred in genes involved in brain development such as foxp1. some genes previously associated with asd, e.g. pdcd1 or kcnb1 present some tandem-repeat mutations, increasing the possibility that these repeats are involved in asd pathology. in a recent paper, trost et al. [57] also analyzed the role of trs in asd but with a different approach, not taking into account the appearance of de novo mutations. this study used three times more genomes as compared to the analysis of mitra et al., thus substantially increasing the statistical power. although the studies identified different mutations, probably due to the differences in their methodological approach, they were more complementary than controversial. interestingly, the study from trost et al. relates several trs expansions with particular clinical features like cognitive disturbances. the findings open a new research line that could contribute to disentangle the contribution of trs to other neurodevelopmental disorders and pathologies. however, the most critical question now is how these alterations in trs generate disturbances in neurodevelopmental processes, accounting for the multifactorial nature of asd with the environment playing a pivotal role, thus opening the question of how the environment interacts with these alterations in trs. key switch between chromatin remodeler baf subunits explains coffin-siris syndrome neuropathology the brain exhibits an extraordinarily precise morphogenesis that leads to the correct cell types and proportions at the appropriate sites. during the neurodevelopmental processes that lead to the formation of the central nervous system, cell fate is acquired by the decline in neural stem cell self-renewal and amplification potential to give rise to fate-committed progenitors that will ultimately undergo differentiation into neurons (neurogenic) or glial cells (gliogenic) [58]. the establishment of these cell fates requires the orchestration of timely regulated gene expression programs which, in turn, depend upon epigenetic and chromatin regulators. atp-dependent chromatin remodeling factors have been described to be vigorous regulators of chromatin state and dna accessibility, both of which have an important impact on the coordination of gene expression. interestingly, a substantial proportion of ndd associated genes are involved in chromatin and/or transcriptional regulation including the broad family of atp-dependent chromatin remodelers including the multi-subunit brg1/brm associated factor (baf) chromatin modifier family, also known as switch/sucrose non-fermentable (mswi/snf) chromatin remodeling complex. a dna-binding domain targets the swi/snf complex to specific genes and facilitates dna access for transcription factors. these complexes play a central role in different developmental processes. all described canonical configurations of baf require the presence of a subunit containing an at-rich dna binding domain (arid). namely, arid1b and its paralog arid1a encode for the two largest, mutually exclusive, subunits of the complex: arid1a and arid1b. interestingly, the arid1b gene, but not arid1a, is among the most commonly mutated genes in asd and in syndromic and non-syndromic ids [59]. mutations observed include deletions and truncations due to de novo nonsense or frameshift mutations as well as translocations leading to arid1b haploinsufficiency in the individuals with intellectual disability. furthermore, de novo haploinsufficient mutations in this gene are found in ~75% of coffin-siris syndrome patients, a neurodevelopmental condition characterized by learning disability and craniofacial features. despite the biochemical knowledge on their mechanisms of action, the roles of chromatin remodeling factors during brain development have not been identified. furthermore, the brain defects that arise as a consequence of changes in chromatin structure, dynamics and function remain largely unexplored. paglaroli et al. [60] have studied the molecular role of arid1b in cell fate commitment during neurodevelopment. using arid1b+/– coffin-siris patient-derived hipscs, they described for the first time that a switching mechanism between arid1b containing baf (arid1b-baf) and arid1a-baf is required for lineage specification and for exiting the pluripotent state. in pscs, pluripotency is conveyed via binding of an arid1a-containing baf to pluripotency-associated enhancers of the sox2 and nanog networks. in order to perform transition to a multipotent state, arid1b-baf is transiently activated to replace arid1a-baf at the sox2/nanog enhancers and elicits their repression (see fig. 2). the haploinsufficient mutation of arid1b in coffin-siris syndrome impairs the switch from arid1a-baf to arid1b-baf, thus maintaining arid1a-baf at pluripotency enhancers. this leads to aberrant binding of sox2 that interferes with the gene expression patterns required for neurodevelopment. altogether, these alterations result in an impaired cranial neural crest differentiation that accounts for the phenotypes observed in coffin-siris patients. these findings provide the molecular mechanisms underlying not only coffin-siris syndrome but also in some non-syndromic ids and asd in which arid1b is frequently mutated [59]. fig. 2. schematic representation of arid1a-baf switch to arid1b-baf during psc to multipotent state transition (top). in arid1b+/mutations, arid1a-baf is maintained at pluripotent enhancers, leading to an impaired neurodevelopment (bottom). trnas methylation and intellectual disability transfer rnas (trna) are small and highly abundant rna molecules in the cell, representing around 10% of the total cell’s rna pool [61]. trna help decode a messenger rna (mrna) sequence into a protein, coupling transcriptional and translational processes. trnas are highly structured and contain a high number of post-translational modifications such as methylation and acylation. these modifications contribute to the stability and integrity of trnas, to the codon-anticodon interaction, which contributes to translation efficiency and to the rna quality control and regulation of trna localization in the cell. neuronal function requires high translation efficiency due to the high translation rate and alterations in translation are now considered a pivotal pathogenic mechanism in several neurologic disorders, especially in ndds [62]. one of the most common trna modifications is methylation, with more than 30 methylated nucleotides found at different positions in trnas. trnas methylation is fundamental for the normal function of different organs, and the disruption of this process is associated with cancer, neurodegeneration and intellectual disability (id) and neurological phenotypes are often the primary manifestation of mutations affecting the trna regulome [63, 64]. trna methyltransferase genes such as trmt10a, trmt1, ftsj1, elp1, wdr4 and nsun2 have been linked to different forms of id [65-67]. the human ftsj1rna 2’-o-methyltransferase 1 (ftsj1) gene is located on the x chromosome and several mutations in this gene have been associated with the development of x-linked intellectual disability (xlid) in several families [68]. nagayoshi et al. have analyzed why the loss of ftsj1 and its methyltransferase activity would lead to the development of id despite its ubiquitous expression [69]. using ftsj1 ko mice and xlid patient-derived cells, they observed a reduction in 2’o-methylated nucleotides in 11 species of trna including trnaphe, a ftsj1 substrate. among all trna species, trnaphe is the most reduced in ftsj1 ko mouse brain compared to the subtle reduction of the other trnas. trna-sequencing demonstrated an accumulation of fragments of trnaphe in the ftsj1 ko mouse brains. these findings revealed that the absence of the ftsj1 methylation leads to a cleavage in specific positions, and, ultimately, to perturbed decoding at phenylalanine (phe) codons. the authors hypothesized that alterations in phe codon translation leads to dysfunction in the translation of fundamental proteins for neuron function as they found dysregulation in genes involved in synapse organization and cell projection, including wnt7b and ctnnb1 as two interesting candidates related to the wnt signaling pathway [70]. ftsj1 ko showed an increase of thin dendritic spines indicating immaturity of synapses, and a decrease in the length and width of the postsynaptic density indicating alterations in connectivity. finally, the authors demonstrate that ftsj1 ko mice show altered ltp and ltd, and behavioral impairment with increased anxiety, slower spatial learning and poor associative learning. these results establish for the first time how alterations in the normal physiology of trna methyltransferases lead to aberrant translation, altered cell biology, impaired electrophysiological properties and, ultimately, behavioral deterioration. mitovesicles: a new player in the neuropathology of neurodevelopmental disorders mitochondria, the organelles classically seen as the powerhouse of the cell, also play a role in a plethora of crucial cellular functions, from regulation of calcium homeostasis, initiation of apoptosis, thermoregulation, red-ox balance, to epigenetic regulation by means of methylation, acetylation, or phosphorylation. genes encoding mitochondrial proteins are increasingly associated with a wide variety of ndds such as epileptic encephalopathy, id, or asd and recent reports show that proteins associated with intellectual disability are linked to mitochondrial function. interestingly, mitochondrial components can be mediators for intraand inter-cellular communication under physiological and pathological conditions, being transferred through extracellular vesicles (evs). the study of evs, defined as heterogeneous nanoscale vesicles secreted into the extracellular space, is a growing field in biomedicine and particularly in neuroscience due to its potential as a biomarker. microvesicles and exosomes are the main types of ev, with subtle differences in composition [71]. in a recent study, the ability of evs to transfer mitochondrial components was examined in fmr1 knockout mice, a model of fxs [72]. the authors found reduced mitochondrial components in mitochondrial fractions from cortical tissues and astrocytes of fmr1 ko mouse brains, and were able to monitor mitochondrial dysfunction in evs. in fact, mitochondrial components are the least studied components in evs [73]. during years, there was a controversy about the nature of this secreted mitochondrial material with evidences suggesting the secretion of exosomes packed with mitochondrial material or the secretion of whole mitochondria, leading to exchange of these organelles between cells [74-76]. however, the lack of standardized methodologies to accurately separate these different components impeded the proper study of the biological mechanisms behind the different subtypes of evs [77]. d’acunzo et al. now developed a new method based on density gradients to achieve high resolution isolation of different ev subpopulations. this new method led to the finding of a new class of previously unknown, mitochondria-derived evs [78]. first, by using a high-resolution step gradient, the authors purified different ev fractions from mouse brains and demonstrated that the content and composition of evs was different between the isolated fractions. using cryogenic electron microscopy, they identified three brain ev subtypes based on protein content, morphology, electron density and size. then, they analyzed the protein composition of these different fractions, and identified a new class of vesicles with high abundance in mitochondrial markers, “mitovesicles”, of mitochondrial origin. using liquid chromatography-mass spectrometry (lc-ms) on the fraction enriched with mitovesicles, the authors provide data about their protein composition, with 279 mitochondria-specific proteins of 673 proteins identified in this fraction. alterations in mitochondria, from the fetal to the adult stage, are a pathological signature of patients with down syndrome (ds) [79]. the authors hypothesized that the newly described mitovesicles were aberrant in ds. in a trisomic mouse model of ds, ts[rb(12.17)]2cje (ts2), the authors found alterations in mitochondrial proteins in ev fractions and a higher number of mitovesicles in the parenchyma. they replicated these results in humans using post mortem brains of individuals with ds. although the pathogenic role of these alterations in number and in protein composition of mitovesicles remains unknown, ds cells are deficient in the elimination of mitochondria, a process called mitophagy, and thus, the excess of mitovesicles could contribute to the removal of damaged mitochondria, working as a homeostatic process to reduce cellular stress. another possibility is that mitovesicles may activate microglia through acting as proinflammatory agents, which would explain the over-activated microglia detected in several ds mouse models [80]. this study describes and characterizes for the first time a new type of ev derived from mitochondria and also describes a potential role in an ndd such as ds. discussion the complexity and heterogeneity of the myriad of ndds makes it difficult to decipher the ultimate mechanisms of ndd neuropathology. accumulating evidence suggests that their multidimensional nature can be explained by the interaction of numerous genetic and environmental factors. luckily, we are now facing exciting times in which we can study neurodevelopmental disorder from a holistic perspective using advanced omics and cell engineering techniques that are increasing the temporo-spatial resolution of our mechanistic understanding. techniques such as single cell omics or spatial transcriptomics and proteomics help us to unravel the enormous diversity and complexity of both cell composition and cell type-specific molecular signatures throughout the brain. the number of publications associating new cellular players such as the astrocytes with brain alterations have increased exponentially in the last three decades suggesting an important role of this cell type in the onset and progression of several ndds. every year, selected discoveries open new avenues to investigate ndds from completely unexplored perspectives and serve as building blocks to better comprehend the enormous complexity of ndds, hopefully leading to better diagnostics and new therapeutic avenues. acknowledgements the lab of md is supported by the secretaria d’universitats i recerca del departament d’economia i coneixement de la generalitat de catalunya (grups consolidats 2017 sgr 926). we also acknowledge the support of the agencia estatal de investigación (pid2019-110755rb-i00/aei / 10.13039/501100011033), h2020 sc1 go-ds21-848077 and 101057454-psych-strata, jerôme lejeune foundation #2002, nih (grant number: 1r01eb 028159-01), fundació la marató-tv3 (#2016/20-30), ministerio de ciencia innovación y universidades (rtc2019-007230-1 and rtc2019-007329-1). cs received an fi grant (2020fi_b2 00143) from the agència de gestió d’ajuts universitaris i de recerca (agaur) de la generalitat de catalunya; ms received an fpu fellowship (fpu19/04789) and afb received an fpi-so fellowship (pre2018-084504) of the ministerio de universidades, spain. we acknowledge support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa and the cerca programme /generalitat de catalunya. the ciber of rare diseases is an initiative of the isciii. references crow, y.j. and n. manel, aicardi-goutieres syndrome and the type i interferonopathies. nat rev immunol, 2015. 15(7): p. 429-40. crow, y.j., et al., characterization of human disease phenotypes associated with mutations in trex1, rnaseh2a, rnaseh2b, rnaseh2c, samhd1, adar, and ifih1. am j med genet a, 2015. 167a(2): p. 296-312. cuadrado, e., et al., chronic exposure of astrocytes to interferon-alpha reveals molecular changes related to aicardi-goutieres syndrome. brain, 2013. 136(pt 1): p. 245-58. rice, g.i., et al., mutations involved in aicardi-goutieres syndrome implicate samhd1 as regulator of the innate immune response. nat genet, 2009. 41(7): p. 829-32. lee-kirsch, m.a., the type i interferonopathies. annu rev med, 2017. 68: p. 297-315. campbell, i.l., et al., structural and functional neuropathology in transgenic mice with cns expression of ifn-alpha. brain res, 1999. 835(1): p. 46-61. pokatayev, v., et al., rnase h2 catalytic core aicardi-goutieres syndrome-related mutant invokes cgas-sting innate immune-sensing pathway in mice. j exp med, 2016. 213(3): p. 329-36. mackenzie, k.j., et al., ribonuclease h2 mutations induce a cgas/sting-dependent innate immune response. embo j, 2016. 35(8): p. 831-44. aditi, s.m.d., et al., genome instability independent of type i interferon signaling drives neuropathology caused by impaired ribonucleotide excision repair. neuron, 2021. 109(24): p. 3962-3979 e6. porter, r.s., et al., neuron-specific alternative splicing of transcriptional machineries: implications for neurodevelopmental disorders. mol cell neurosci, 2018. 87: p. 35-45. su, c.h., et al., alternative splicing in neurogenesis and brain development. front mol biosci, 2018. 5: p. 12. weyn-vanhentenryck, s.m., et al., precise temporal regulation of alternative splicing during neural development. nat commun, 2018. 9(1): p. 2189. meldolesi, j., alternative splicing by nova factors: from gene expression to cell physiology and pathology. int j mol sci, 2020. 21(11): p. 3941. xin, y., et al., neuro-oncological ventral antigen 1 (nova1): implications in neurological diseases and cancers. cell prolif, 2017. 50(4): p. e12348. parikshak, n.n., et al., genome-wide changes in lncrna, splicing, and regional gene expression patterns in autism. nature, 2016. 540(7633): p. 423-7. jensen, k.b., et al., nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. neuron, 2000. 25(2): p. 359-71. ruggiu, m., et al., rescuing z+ agrin splicing in nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing. proc natl acad sci u s a, 2009. 106(9): p. 3513-8. trujillo, c.a., et al., reintroduction of the archaic variant of nova1 in cortical organoids alters neurodevelopment. science, 2021. 371(6530). samarasinghe, r.a., et al., identification of neural oscillations and epileptiform changes in human brain organoids. nat neurosci, 2021. 24(10): p. 1488-1500. trujillo, c.a., et al., pharmacological reversal of synaptic and network pathology in human mecp2-ko neurons and cortical organoids. embo mol med, 2021. 13(1): p. e12523. philibert, r.a. and a. madan, role of med12 in transcription and human behavior. pharmacogenomics, 2007. 8(8): p. 909-16. remeseiro, s., et al., reduction of nipbl impairs cohesin loading locally and affects transcription but not cohesion-dependent functions in a mouse model of cornelia de lange syndrome. biochim biophys acta, 2013. 1832(12): p. 2097-102. gregor, a., et al., de novo mutations in the genome organizer ctcf cause intellectual disability. am j hum genet, 2013. 93(1): p. 124-31. jaitner, c., et al., satb2 determines mirna expression and long-term memory in the adult central nervous system. elife, 2016. 5: p. e17361. tan, l., et al., changes in genome architecture and transcriptional dynamics progress independently of sensory experience during post-natal brain development. cell, 2021. 184(3): p. 741-758 e17. tan, l., et al., three-dimensional genome structures of single diploid human cells. science, 2018. 361(6405): p. 924-8. winick-ng, w., et al., cell-type specialization is encoded by specific chromatin topologies. nature, 2021. 599(7886): p. 684-91. reichova, a., et al., abnormalities in interactions of rho gtpases with scaffolding proteins contribute to neurodevelopmental disorders. j neurosci res, 2018. 96(5): p. 781-8. scala, m., et al., pathophysiological mechanisms in neurodevelopmental disorders caused by rac gtpases dysregulation: what's behind neuro-racopathies. cells, 2021. 10(12): p. 3395. zeidan-chulia, f., et al., exploring the multifactorial nature of autism through computational systems biology: calcium and the rho gtpase rac1 under the spotlight. neuromolecular med, 2013. 15(2): p. 364-83. guo, d., et al., rho gtpase regulators and effectors in autism spectrum disorders: animal models and insights for therapeutics. cells, 2020. 9(4): p. 835. sit, s.t. and e. manser, rho gtpases and their role in organizing the actin cytoskeleton. j cell sci, 2011. 124(pt 5): p. 679-83. hall, a. and c.d. nobes, rho gtpases: molecular switches that control the organization and dynamics of the actin cytoskeleton. philos trans r soc lond b biol sci, 2000. 355(1399): p. 965-70. allen, k.m., et al., pak3 mutation in nonsyndromic x-linked mental retardation. nat genet, 1998. 20(1): p. 25-30. bienvenu, t., et al., missense mutation in pak3, r67c, causes x-linked nonspecific mental retardation. am j med genet, 2000. 93(4): p. 294-8. magini, p., et al., a mutation in pak3 with a dual molecular effect deregulates the ras/mapk pathway and drives an x-linked syndromic phenotype. hum mol genet, 2014. 23(13): p. 3607-17. hayashi, m.l., et al., altered cortical synaptic morphology and impaired memory consolidation in forebrainspecific dominant-negative pak transgenic mice. neuron, 2004. 42(5): p. 773-87. castillon, c., et al., the intellectual disability pak3 r67c mutation impacts cognitive functions and adult hippocampal neurogenesis. hum mol genet, 2020. 29(12): p. 1950-68. dong, t., et al., inability to activate rac1-dependent forgetting contributes to behavioral inflexibility in mutants of multiple autism-risk genes. proc natl acad sci u s a, 2016. 113(27): p. 7644-9. ryan, t.j., et al., memory. engram cells retain memory under retrograde amnesia. science, 2015. 348(6238): p. 1007-13. ramirez, s., et al., creating a false memory in the hippocampus. science, 2013. 341(6144): p. 387-91. zhou, s. and z. jia, disruption of pak3 signaling in social interaction induced cfos positive cells impairs social recognition memory. cells, 2021. 10(11): p. 3010. reijmers, l.g., et al., localization of a stable neural correlate of associative memory. science, 2007. 317(5842): p. 1230-3. voineagu, i., et al., transcriptomic analysis of autistic brain reveals convergent molecular pathology. nature, 2011. 474(7351): p. 380-4. edmonson, c., m.n. ziats, and o.m. rennert, altered glial marker expression in autistic post-mortem prefrontal cortex and cerebellum. mol autism, 2014. 5(1): p. 3. russo, f.b., et al., modeling the interplay between neurons and astrocytes in autism using human induced pluripotent stem cells. biol psychiatry, 2018. 83(7): p. 569-78. allen, m., et al., astrocytes derived from asd patients alter behavior and destabilize neuronal activity through aberrant ca2+ signaling. biorxiv, 2021.. gilman, s.r., et al., rare de novo variants associated with autism implicate a large functional network of genes involved in formation and function of synapses. neuron, 2011. 70(5): p. 898-907. srinivasan, r., et al., ca(2+) signaling in astrocytes from ip3r2(-/-) mice in brain slices and during startle responses in vivo. nat neurosci, 2015. 18(5): p. 708-17. petravicz, j., et al., loss of ip3 receptor-dependent ca2+ increases in hippocampal astrocytes does not affect baseline ca1 pyramidal neuron synaptic activity. j neurosci, 2008. 28(19): p. 4967-73. wang, q., et al., impaired calcium signaling in astrocytes modulates autism spectrum disorder-like behaviors in mice. nat commun, 2021. 12(1): p. 3321. traetta, m.e., et al., long-lasting changes in glial cells isolated from rats subjected to the valproic acid model of autism spectrum disorder. front pharmacol, 2021. 12: p. 707859. hannan, a.j., tandem repeats and repeatomes: delving deeper into the 'dark matter' of genomes. ebiomedicine, 2018. 31: p. 3-4. hannan, a.j., tandem repeats mediating genetic plasticity in health and disease. nat rev genet, 2018. 19(5): p. 286-98. schaaf, c.p., et al., a framework for an evidence-based gene list relevant to autism spectrum disorder. nat rev genet, 2020. 21(6): p. 367-76. mitra, i., et al., patterns of de novo tandem repeat mutations and their role in autism. nature, 2021. 589(7841): p. 246-50. trost, b., et al., genome-wide detection of tandem dna repeats that are expanded in autism. nature, 2020. 586(7827): p. 80-6. sokpor, g., et al., chromatin remodeling baf (swi/snf) complexes in neural development and disorders. front mol neurosci, 2017. 10: p. 243. hoyer, j., et al., haploinsufficiency of arid1b, a member of the swi/snf-a chromatin-remodeling complex, is a frequent cause of intellectual disability. am j hum genet, 2012. 90(3): p. 565-72. pagliaroli, l., et al., inability to switch from arid1a-baf to arid1b-baf impairs exit from pluripotency and commitment towards neural crest formation in arid1b-related neurodevelopmental disorders. nat commun, 2021. 12(1): p. 6469. jurkin, j., et al., the mammalian trna ligase complex mediates splicing of xbp1 mrna and controls antibody secretion in plasma cells. embo j, 2014. 33(24): p. 2922-36. parenti, i., et al., neurodevelopmental disorders: from genetics to functional pathways. trends neurosci, 2020. 43(8): p. 608-21. swinehart, w.e. and j.e. jackman, diversity in mechanism and function of trna methyltransferases. rna biol, 2015. 12(4): p. 398-411. abedini, s.s., et al., trna methyltransferase defects and intellectual disability. arch iran med, 2018. 21(10): p. 478-85. yew, t.w., et al., trna methyltransferase homologue gene trmt10a mutation in young adult-onset diabetes with intellectual disability, microcephaly and epilepsy. diabet med, 2016. 33(9): p. e21-5. zhang, k., et al., an intellectual disability-associated missense variant in trmt1 impairs trna modification and reconstitution of enzymatic activity. hum mutat, 2020. 41(3): p. 600-7. abbasi-moheb, l., et al., mutations in nsun2 cause autosomal-recessive intellectual disability. am j hum genet, 2012. 90(5): p. 847-55. dai, l., et al., positive association of the ftsj1 gene polymorphisms with nonsyndromic x-linked mental retardation in young chinese male subjects. j hum genet, 2008. 53(7): p. 592-7. nagayoshi, y., et al., loss of ftsj1 perturbs codon-specific translation efficiency in the brain and is associated with x-linked intellectual disability. sci adv, 2021. 7(13): p. eabf3072. brafman, d. and willert, k., wnt/beta-catenin signaling during early vertebrate neural development. dev neurobiol, 2017. 77(11): p. 1239-59. stahl, a.l., et al., exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases. pediatr nephrol, 2019. 34(1): p. 11-30. ha, b.g., et al., depletion of mitochondrial components from extracellular vesicles secreted from astrocytes in a mouse model of fragile x syndrome. int j mol sci, 2021. 22(1): p. 410. zhang, b., et al., stimulated human mast cells secrete mitochondrial components that have autocrine and paracrine inflammatory actions. plos one, 2012. 7(12): p. e49767. guescini, m., et al., astrocytes and glioblastoma cells release exosomes carrying mtdna. j neural transm (vienna), 2010. 117(1): p. 1-4. phinney, d.g., et al., mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle micrornas. nat commun, 2015. 6: p. 8472. hayakawa, k., et al., transfer of mitochondria from astrocytes to neurons after stroke. nature, 2016. 535(7613): p. 551-5. jeppesen, d.k., et al., reassessment of exosome composition. cell, 2019. 177(2): p. 428-45 e18. d'acunzo, p., et al., mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in down syndrome. sci adv, 2021. 7(7): p. eabe5085. izzo, a., et al., mitochondrial dysfunction in down syndrome: molecular mechanisms and therapeutic targets. mol med, 2018. 24(1): p. 2. pinto, b., et al., rescuing over-activated microglia restores cognitive performance in juvenile animals of the dp(16) mouse model of down syndrome. neuron, 2020. 108(5): p. 887-904 e12. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology and epilepsy surgery: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:12 (2022) review neuropathology and epilepsy surgery: 2022 update ingmar blümcke1 1 department of neuropathology, university hospital erlangen, germany corresponding author: ingmar blümcke · department of neuropathology · university hospital erlangen · schwabachanlage 6 · erlangen · germany bluemcke@uk-erlangen.de submitted: 17 march 2022 accepted: 25 april 2022 copyedited by: vanessa goodwill published: 03 may 2022 https://doi.org/10.17879/freeneuropathology-2022-3813 keywords: brain, seizure, malformation, brain tumor, molecular diagnostics abstract the impact of a precise histopathology diagnosis and molecular workup for surgical patient management remains a controversial issue in epileptology with a lack of diagnostic agreement as root cause. very recent advances in genotype-phenotype characterization of epilepsy-associated developmental brain lesions, including the first diagnostically useful dna methylation studies, opened new avenues and will help to finally resolve these issues. a series of most recent articles were decisively selected by the author to exemplify the areas of improvement in neuropathology and epilepsy surgery. these topics include the progress in genotype-phenotype association studies of focal cortical dysplasia (fcd) leading to the discovery of new molecularly defined entities, i.e. mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (moghe), slc35a2 altered. these studies also triggered the first update of the international fcd consensus classification scheme from 2011, which will hopefully support diagnostic agreement in clinical practice and research. the dilemma of new tumor entities proposed by the 5th edition of the who classification primarily associated with early seizure onset yet not well introduced to the epileptology community will also be discussed in the light of emerging experimental evidence when transfecting the developing murine brain with the single most important genetic alteration for both carcinoand epileptogenesis, i.e. braf v600e. introduction the impact of a precise histopathology diagnosis and molecular workup for patient management remains a controversial issue in epileptology 1, i.e., the decision making for surgical treatment when a patient with focal epilepsy is considered drug-resistant and prediction of postsurgical seizure freedom should be based on the underlying cause. this discussion continues despite large enough patient series suggesting a positive prediction of scientifically defined brain lesions for long-term postsurgical seizure control for more than 5 years2. a root cause of these inconsistencies is likely due to lack of diagnostic agreement across histopathology laboratories around the world, a fact widely recognized in the scientific literature3-7. the introduction of molecular diagnostics has helped to overcome this issue in the field of neurooncology with the best renowned examples of abandoning mixed oligo-astrocytomas and introducing molecularly defined and clinically/therapeutically relevant tumor subtypes. literature published in 2021 about the topic of neuropathology and epilepsy surgery elicited new hope for epilepsy centers around the world to better connect with molecular diagnostics and integrated genotype-phenotype pathology diagnosis, which will finally move the field toward personalized medicine and targeted treatment options. the following chapters will address ten topics of interest to the author based on papers published in 2020-2022 and with reference to other older topic-related key data. topic 1: the molecular phenotype of human epilepsies at the single cell level topic 2: brain somatic mutations in cortical malformations (mcd) topic 3: dna-methylation classifier for malformations of cortical development topic 4: new clinico-pathologically and genetically defined entities: moghe and fcd1a topic 5: toward a first update of the international consensus classification of fcd topic 6: news from low-grade epilepsy-associated brain tumors (leat): human studies topic 7: news from leat: animal models topic 8. what is new about hippocampal sclerosis: human herpes virus infection and the impact of inflammatory infiltrates on neuronal cell loss topic 9. the fine structure of the epileptogenic neocortex and white matter topic 10: what is still missing to further the advancement of neuropathology and epilepsy surgery? topic 1: the molecular phenotype of human epilepsies at the single cell level the inception of single cell biology has completely revolutionized the study of neurological diseases and has the potential to answer many of the yet unresolved questions in the neurosciences8. the advancement in molecular-genetic barcoding technology (figure 1) at the single cell level including single-cell genomics and transcriptomics finally reached the realm of epileptology. human surgical tissue samples of clinically well-characterized patients have ever been a valuable resource for research, although vast differences in the patients’ genetic background, clinical histories and medical treatment pose major challenges to comprehensive data analysis. focusing on single cell genomics, transcriptomics, and finally also epigenomics in focal epilepsy research, will circumvent the averaging artifact when studying bulk brain tissue and will reveal the kind of granularity that is needed for investigating the consequences of network alterations at the single cell level8. figure 1: single-cell genomic applications using single cell combinatorial indexing or droplet based cell isolation with barcoding 8 single cell combinatorial indexing (sci) and droplet-based cell isolation are the most popular barcoding strategies used by most high-throughput single cell genomic applications. sci uniquely tags the nucleic acid molecules in each cell through serial mixing, splitting, and barcoding. the higher the number of barcoding steps, the higher the number of cells that can be uniquely tagged in each experiment. droplet-based techniques rely on physical isolation of individual cells and engineered barcoded beads in nanoliter droplets, which limits their scalability but they produce less noisy results. with permission from the author 8. whereas single cell genomics have already been applied using human epilepsy surgery brain samples9, pfisterer and colleagues described in 2020 for the first time single nucleus transcriptomics in human temporal lobe epilepsy and non-epileptic subjects10. they deciphered dysfunctional neuronal subtypes in the seizure active cortical region and found that the largest transcriptomic changes occurred in distinct subtypes of principal neurons, as classified by the layer-specific marker genes cux2, rorb, themis, and fezf2, and gabaergic interneurons, as classified by the marker genes pvalb, sst, vip, and id2. as a result, fezf2 positive neurons of layers 5 and 6 and cux2 neurons of layers 2 and 3 were more affected than other subtypes of the same families. similarly, parvalbumin and somatostatin interneurons showed more of an epileptogenic signature than other gabaergic interneurons. furthermore, the subtypes with the largest epilepsy-related transcriptomic changes belong to same circuits. in addition, glutamate signaling exhibited a strong dysregulation in epilepsy, as indexed by layer-specific transcriptional changes in multiple glutamate receptor genes and upregulation of genes coding for ampa receptor auxiliary subunits10. overall, these data confirmed neuron-specific molecular phenotypes in the epileptic neocortex, which may become novel targets for anti-epileptic precision medicine in the near future. over the past 5 years dna-methylation has gained significant interest and momentum in neuropathology. the development of dna-methylation based classifier for epilepsy-associated brain malformations will be discussed further below (see topic 3 for further reading). dna methylation has not yet been addressed at the single cell level in human neurosurgical specimens though. in the mouse brain, however, a first comprehensive assessment of the epigenomes of mouse brain cell types was published in 202111. the researchers used single-nucleus dna methylation sequencing from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas and identified 161 cell clusters with distinct spatial locations and projection targets11. furthermore, they constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. these features revealed the repetitive usage of regulators in excitatory and inhibitory cells to determine cellular subtypes. furthermore, an artificial neural network model was developed to predict neuron cell-type identity and their spatial brain localization. the creation of a comprehensive dna methylation-based atlas will establish the epigenetic basis for neuronal diversity and spatial organization throughout the mammalian brain, being also instrumental when developing a human methylome brain atlas in the near future, including epilepsy surgery brain samples from various disorders, e.g. cortical malformations and hippocampal sclerosis. topic 2: brain somatic mutations in cortical malformations fifty years ago, in 1971, taylor and colleagues coined the term focal cortical dysplasia (fcd) for peculiar lesions causing drug resistant epilepsy12, which is now the most common cause for epilepsy surgery in children13. the study of resected human fcd tissue using advanced genomic technologies (figure 1) has led to remarkable advances in understanding the genetic basis of fcd (figure 2)14. mechanistic parallels have emerged between these malformative lesions and low-grade and epilepsy-associated developmental brain tumors (leat). two comprehensive review articles published in 2021 and 2022 recapitulated the avenue from recognition of somatic variants in genes related to cell growth, i.e. the mtor pathway in fcd type 2, which were acquired in neuronal progenitors during neurodevelopment14,15. the timing of the genetic event and the specific gene involved during neurodevelopment is likely to drive the nature and size of the lesion, and is maybe also related to the lobar localization. the proposed two-hit mechanism in patients presenting primarily with germline mutations, however, e.g., depdc5, has been scientifically approved but remains often difficult to detect with standard technologies. along these lines, lee and coworkers newly reported pathogenic brain-specific somatic ras homolog enriched in brain (rheb) variants in three patients with cortical malformations16. interestingly, the somatic variant load directly correlated with the size of the malformation and upregulated mtor activity was confirmed in dysplastic tissues. laser capture microdissection showed enrichment of rheb variants in dysmorphic neurons and balloon cells, i.e. fcd type 2b. these findings added rheb as an additional mtor pathway gene to the diagnostically relevant gene panel for mcd. that the extent of dysplastic brain directly correlated with the somatic variant load suggested that cortical malformations with cytopathological features, i.e. fcd type 2, represent a disease continuum from a regionally localized dysplasia to lobar or full blown hemimegalencephaly. figure 2: “one brain – many genomes” 17 somatic variants are spontaneously acquired during neurodevelopment. all the somatic variants in a progenitor cell are passed down to its daughter cells. the number of cells carrying a specific variant is an indirect marker for the developmental time point at which it was generated. with permission from the author 8. brain mosaicism, however, can only be detected in tissue obtained from autopsy or brain biopsy. liquid biopsy using cell-free dna derived from cerebrospinal fluid (csf) could yet be another source for genetic testing, as successfully proven in malignant brain tumors. in 2021, ye and coworker published a proof of principle study demonstrating that csf liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable18. first, they sequenced csf cell-free dna (cfdna) in 28 patients with focal epilepsy and 28 controls (using droplet digital polymerase chain reaction (pcr)). they detected somatic mutations in three patients, i.e., the lis1 p.lys64* variant at 9.4% frequency in one patient with subcortical band heterotopia; the tsc1 p.phe581his*6 variant at 7.8% frequency in another patient with fcd and thirdly the braf p.val600glu variant at 3.2% frequency in a patient with ganglioglioma. they also determined that cfdna was brain-derived by using whole-genome bisulfite sequencing and demonstrating an enrichment of brain-specific dna methylation patterns. in a second parallel study and publication, same results were obtained from csf collected during epilepsy surgery19. somatic variants were detected in cfdna from 3 epileptic patients with known somatic mutations previously identified in brain tissue (out of 12 patients included in the study). both proof-of-principle studies provide evidence that brain mosaicism can be detected in the csf-derived cfdna and open future avenues for detecting the mutant allele driving epilepsy in csf. topic 3: dna-methylation classifier for malformations of cortical development the histopathological work-up of epilepsy surgery specimens is often based only on routine hematoxylin and eosin (h&e) staining with its many difficulties and pitfalls in reliable identification of anatomical landmarks and cortical layering. this applies particularly to samples not resected anatomically en bloc, or when compromised by peri-operative artefacts or diagnostic procedures such as intracranial electroencephalography (eeg) recordings, laser ablation or thermo-coagulation. it has been a long-standing effort in histopathology, therefore, to support the microscopic diagnoses with objectifiable diagnostic measures. indeed, the same issue applied to the scenario in neuro-oncology before the discovery and introduction of predictive and prognostic genetic markers for the differential diagnosis of high-grade glioma and embryonal brain tumors, which helped to develop a reliable and widely used open-access dna methylation-based brain tumor classifier20. jabari and coworkers presented such a first dna methylation classifier for cortical malformations in 2021, which opens the avenue for an objective genotype-phenotype approach in epilepsy surgery using routinely processed (archival) formalin-fixed and paraffin embedded (ffpe) tissue samples21. this work included a series of 308 histopathologically defined samples to cover the broad spectrum of mcd, including most common fcd subtypes, as well as polymicrogyria, hemimegalencephaly, tuberous sclerosis complex, mild malformations of cortical development (mmcd), and moghe specimens as well as non-mcd epilepsy samples and non-epilepsy post-mortem controls. the possibility to apply such a dna methylation classification for non-neoplastic lesions has been questioned and is an ongoing challenge due to the often low content of abnormal cells and admixture with architecturally normal-appearing cortical areas. the possibility to microscopically dissect an area of the lesion with the most cell dense abnormality from ffpe tissue blocks was an important foundation, therefore, to the success of this project. the study did also follow a classifier approach different from the well-established heidelberg classifier for brain tumors. in fact, their paper disclosed a graph in the supplemental material showing how all of their mcd samples would assemble with the heidelberg classifier (figure 3). this highlights the challenge but also the benefit of advanced bioinformatics for developing a dna methylation classifier. it also highlights the need for a disease-specific pipeline addressing structural brain lesions in epilepsy and potential confounders. as an example, seizures themselves alter the dna methylation map as demonstrated in a recent in vitro culture model “epilepsy in a dish”22. the variable seizure burden in a given patient, the affected brain region (i.e. grey and/or white matter), and the surgical tissue sample (e.g., representing the lesion or its periphery), present an ongoing issue when using the dna methylation methodology for human brain specimens. assessing as many potential confounders in the new pipeline published by jabari et al. helped to separate the histopathologically labelled groups into well distinguishable entities e.g., taking into account the different referral centres, seizure onset, disease duration before surgery, cellular heterogeneity, batch effects, and sex. finally, a test cohort of 43 independent surgical samples from different epilepsy centres was used to test the precision of the dna methylation-based mcd classifier. amongst this cohort were 19 samples previously reviewed by an international consensus expert panel (will be discussed further below). all samples from the test cohort were accurately assigned to their histopathologically agreed disease classes by the algorithm. this report provides a first dna methylation-based mcd classification scheme suitable across major histopathological disease entities observed in epilepsy surgery. there is still a lot of work to do, however. the impact of increasingly recognized brain somatic mutations in fcd and other mcd entities (see chapters above), were not yet addressed in this study and need further attention. figure 3: dna methylation-based classifier using a new deep learning algorithm adapted for epilepsy surgery brain samples (on the left) compared to the heidelberg brain tumor classifier (on the right) on the left: umap uniform manifold approximation and projection of the decision boundaries of the trained deep learning algorithm showing the distribution of 308 datasets (taken from 21; this is an open access article distributed under the terms of the creative commons cc by license). colors correspond to the following coding ctrl – post-mortem control, ncx – neocortex, wm – white matter; fcd – focal cortical dysplasia type 1, 2 and 3 according to ilae classification scheme; moghe mild malformation with oligodendroglial hyperplasia in epilepsy, pmg polymicrogyria, hme hemimegalencephaly, tle – temporal lobe epilepsy without cortical dysplasia, tsc tuberous sclerosis complex, mmcd mild malformations of cortical development. on the right: t-sne – “t-distributed stochastic neighbour embedding” of the brain tumor classifier from heidelberg of the same samples shown on the left. topic 4: new clinico-pathologically and genetically defined entities: moghe and fcd1a in 2021, we witnessed the exciting rise of a new, genetically defined cortical malformation associated with early onset focal epilepsy. the term “mild malformation with oligodendroglial hyperplasia in epilepsy” (moghe) was already coined in 201723 but received only little attention by the histo-/neuropathology community. when epileptologists described its mri fingerprint in 201924, many more reports became available cumulating in 2021 as a distinct clinico-pathological moghe pattern in children with early seizure onset and mostly affecting the frontal lobe25-28. two other papers in 2021 almost simultaneously assigned brain somatic mutations in the udp-galactose transporter slc35a2 gene to moghe6,29. the slc35a2 mutation was first discovered in a cohort of focal epileptic brain lesions in 2018, replicated in 2019 and originally assigned to either mmcd or fcd ilae type 1 entities30,31. notwithstanding, both of these differential histopathology diagnoses are difficult and poorly defined. bonduelle and coworker specifically searched for slc35a2 mutations in histopathologically confirmed moghe and detected pathogenic mutations in 9 out of 20 new cases included in their series (45%). the frequency of somatic variants were usually above 10%, ranging from 1.4% to 52%. droplet digital pcr of microdissected cells from one moghe case confirmed variant enrichment in abnormally clustering oligodendroglial cells and heterotopic neurons, suggesting that the mutation targeted neuroglial progenitors during brain development. these data provide compelling evidence for a consistent genotype-phenotype correlation in moghe with slc35a2 mosaicism as a diagnostic and probably also prognostic marker, as more than 60% of moghe patients benefit from gross total resection of the lesion25. the slc35a2 gene opens another avenue for targeted treatment in mcd and is outside of the well-recognized mtor pathway associated with fcd ilae type 2 lesions15. a recent report showed a clinical improvement in response to oral d‐galactose supplementation in several slc35a2-congenital disorder of glycosylation (cdg) patients, caused by monoallelic pathogenic variants in slc35a2 on chromosome xp11.23, resulting in reduction of seizure frequency 32. evaluating this therapeutic approach in patients with brain mosaic mutations of slc35a2 and moghe, and for whom surgery is not an option or failed to reduce seizures due to the large extent of the lesion, could offer a personalized treatment strategy. yet, there is no targeted animal model of slc35a2 brain mosaicism to clarify the precise pathogenicity. it also remains to be shown how a primary defect in glycosylation in neuroglial progenitors leads to the histopathological features described in moghe, i.e. patchy oligodendroglial hyperplasia and heterotopic neurons in the white matter (figure 4). figure 4: simplified representation of pathophysiology and neuropathology findings in slc35a2-related moghe cases on the left: slc35a2 loss-of-function variants cause a defect in protein/sphingolipid glycosylation in the cell. on the right: loss of slc35a2 function leads to a moghe phenotype, with clusters of increased density of olig2-positive cells in the white matter and deep cortical layers. l1-l6: layers 1-6; wm: white matter; figure taken from 29, an open access article distributed under the terms of the creative commons cc by license. in 2021, another comprehensive study described the clinico-pathological and molecular presentation of fcd ilae type 1a33. holthausen identified a cohort of 19 young children with drug-resistance from seizure onset and severe eeg abnormalities almost always emphasized in the posterior (temporo-parieto-occipital) quadrant. the lack of focal neurological deficits but severe cognitive impairment was another hallmark of holthausen’s disease. important for the diagnostic work-up is the mri signature presenting as subtle hypoplasia of the epileptogenic area with focus on the white matter. abundance of cortical microcolumns, best visible by immunohistochemical stainings using neun antibodies and detected in all 19 cases assigned holthausen’s disease to fcd ilae type 1a (figure 5), molecularly confirmed by dna methylation analysis. these defining features coined the provisional term “multilobar unilateral hypoplasia with severe epilepsy in children (muhsec) and is referred to holthausen’s disease herein. to the best of my knowledge, this case series is a first comprehensive electro-clinical and anatomo-pathological description of a pure fcd ilae type 1 cohort in the contemporary peer reviewed literature. the three fcd1 subtypes have been almost hypothetically defined by the ilae classification scheme as architectural abnormality of the 6-layered neocortex without frank signs of cytopathology, i.e., dysmorphic neurons or balloon cells. up to date, there is no clear description available for either fcd 1b (horizontal dyslamination) or fcd 1c (horizontal and vertical dyslamination), although the latter is often associated with developmental vascular lesions, e.g., meningeal angiomatosis of sturge-weber34, or perinatal stroke, and assigned to fcd 3c or 3d, respectively (figure 5)6,35. there was always concern that different patterns of cortical dyslamination are caused by different aetiologies and pathophysiological mechanisms, which will have implications for the diagnostic work-up as well as for the therapeutic management36. yet, we have to await further studies until the issue of fcd type 1 subtypes and their clinical relevance for patient management and treatment options will be finally resolved. figure 5: fcd subtypes of the ilae classification scheme of 2011 as visualized with neun immunohistochemistry upper panel with the pial surface always presented on top. neun immunohistochemistry with hematoxylin counterstain. scale bar on upper left = 250 μm, applies to all images of upper panel. scale bar on lower panel = 2500 μm. fcd1a: 2-year old girl with right temporo-parieto-occipital seizures. the histograph reveals abundance of microcolumns. this is even more visible on the low-power magnification of the same patient in the lower panel with arrows indicating several large regions with microcolumns and heterotopic neurons in the white matter. fcd 2b: 5-year old boy with left frontal lobe epilepsy. note the large (dysmorphic) neurons in an otherwise unlayered neocortex. fcd 3a: 28-year old male patient with left-sided temporal lobe epilepsy and hippocampal sclerosis (not shown). note the pathognomonic narrowing of cortical layer ii and depletion of pyramidal neurons in layer iii. fcd3c: 20-year old male patient with sturge-weber syndrome. note the meningeal angiomatosis on top and the abundant microcolumnar arrangement of neurons in the neocortex. adjacent cortical areas also revealed disorganization of cortical layers in a horizontal direction (although not visible by this magnification). fcd3d: 14 year old girl with seizures and intrauterine stroke. note the architectural abnormalities in cortical layers ii-v. topic 5: toward a first update of the international consensus classification of fcd fcd are the most common malformations of cortical development recognized in epilepsy surgery case series2,13. clinical diagnosis is usually based on the presurgical evaluation including highor ultra-high-resolution mri37, but should be always confirmed histopathologically from surgically resected brain samples. hence, the agreement of microscopic diagnoses remains ever challenging, which has been documented many times in the scientific literature3-7. in 2021, a new approach was launched by a consortium nominated by the ilae task force of fcd. they iteratively reviewed a consecutive cases series of 22 patients from a single center in the united states and with each patient being clinically (presurgically) suspicious for any type of fcd6. in addition, five independent laboratories performed genetic testing with fcd-specific gene panel sequencing from tissue and blood samples of the very same 22 patients. four rounds of histopathology agreement were performed amongst 20 internationally renowned colleagues from 16 countries. the group started with a series of 196 h&e stains made available through an open access digital slide review platform. notably, agreement was very low (kappa value = 0.16), and all reviewers were also asked to propose useful immunohistochemical stains from sections with the most prominent histopathologic changes. antibodies directed against the neun, map2 and non-phosphorylated neurofilament epitopes were requested most often and can thus be considered as most valuable for the histopathology work-up of epilepsy surgery specimens6. notwithstanding, the panel of recommended immunostainings cover many more epitopes, which may become necessary in the differential diagnosis of difficult-to-classify epileptogenic brain lesions38. this panel has been recently extended including mutation-specific antibodies directed against brafv600e and idh1r132h, as well as the ps6 epitope to visualize an activated mtor pathway (najm et al. the ilae fcd classification update 2022, in revision). with the addition of requested immunohistochemical stains the agreement increased to a kappa-value of 0.35 in the second round. during the third round, all previous answers were anonymously disclosed using the delphi consensus method. mri and histographs showing specific features of each lesion were also disclosed for the consensus discussion. this increased the diagnostic agreement to a kappa value of 0.5. in the final round, the results from genetic testing were available, i.e., pathogenic mtor, depdc5, akt3, nprl3 and slc35a2 mutations in 7 cases. the final agreement based on all available information was substantial with a kappa value of 0.686. this study will define the path towards an integrated genotype-phenotype diagnosis in fcd and the authors proposed a number of amendments to be recognized in an update of the 2011 ilae classification scheme (table 1). most importantly, the authors suggested to include two new entities, which were predominantly related to the white matter and already described in the scientific literature, but not yet well recognized in the neuropathology work-up of epilepsy surgery specimens. the moghe entity has been already discussed above and the association with pathogenic slc35a2 brain somatic mutations was confirmed in the presented case series. yet there is no hotspot mutation nor any immunohistochemical surrogate marker available for slc35a2. the other entity is that of mmcd with abundance of heterotopic neurons in the white matter as a defining hallmark. this lesion type will surely remain a matter of concern and discussion, as few heterotopic neurons are also present in the normal white matter of the non-epileptic cortex, as well as in several other epileptogenic disease conditions, first and foremost in fcd ilae type 1a (holthausen’s disease) and moghe. in addition, the term mmcd should be used only when any other pathology finding was ruled out, including any other principal lesion such as hippocampal sclerosis, developmental brain tumors or glial scars acquired early during life. the authors further suggested to include a panel of immunohistochemical markers to confirm the diagnosis of fcd or when no lesion can be microscopically defined6. table 1: proposed amendments to the ilae classification scheme of fcd fcd, focal cortical dysplasia, hs, hippocampal sclerosis, mmcd, mild malformation of cortical development, moghe, mmcd with oligodendroglial hyperplasia in epilepsy; * mmcd: not associated with any other principal lesion, such as hippocampal sclerosis, brain tumor, or vascular malformation; for reference values see39-42. ** no definite fcd on histopathology: a descriptive report is recommended to highlight anatomical ambiguities (if applicable). table modified from 6, an open access article distributed under the terms of the creative commons cc by license. the proposed amendments to the classification scheme presented in table 1 were taken from the discussed publication6, whereas the actual ilae classification update is still under peer review and expected to be published in due course of 2022 (najm et al. the ilae fcd classification update 2022, in revision). this fcd classification update will propose for the first time a multi-layered integration of all available diagnostic information, including histopathology (layer 1), genetics (layer 2) and imaging (layer 3), in order to achieve a final diagnosis. this approach may also compensate for the lack of accuracy or agreement shown for each diagnostic layer on its own, due to the aforementioned issues and challenges in the histopathology assessment6. it will then be the task of the treating physician to assemble all these data to a final genotype-phenotype diagnosis. a likely scenario to foster and promote this final diagnosis would be a post-surgical patient management conference, including the epileptologists, neurosurgeon, neuropathologist, neuroradiologist and neurogeneticist. the proposition to add the level of genetic testing to a comprehensive, reliable and integrative genotype-phenotype diagnosis represents an important step towards precision medicine in the realm of epileptology, as has been successfully implemented in neuro-oncology. genetic testing should ideally be performed from dna extracted from a microscopically assessed surgical brain tissue and paired blood sample, to facilitate the identification of brain mosaicism that represent post-zygotic mutational events. whether or not such genetic testing should become mandatory in clinical practise or should be applied foremost in scientific research will be a matter of further discussion and will always depend on available resources in each epilepsy (surgery) centre. the possibility to detect brain somatic mutations in cell-free dna obtained from cerebrospinal fluid using targeted droplet digital pcr was discovered in 2021 and described by two papers18,19. these findings offer the opportunity to establish a genetic diagnosis even before surgery, or in patients not eligible for surgery, and to guide the implementation of targeted therapies into the arena of epileptology. topic 6: news from low-grade epilepsy-associated brain tumors (leat): human studies the fifth edition of the who classification of brain tumors is now available online and in print43. this 5th edition introduced six new entities pertinent to the arena of epileptology (out of 22 overall new tumor types, 27%), namely the many variants and subtypes of glio-neuronal tumors and low-grade gliomas, i.e., paediatric type diffuse low-grade gliomas, mapk pathway-altered; diffuse astrocytoma, myb or mybl-altered; polymorphous low-grade neuroepithelial tumor of the young; diffuse glioneuronal tumor with oligodendroglioma-features and nuclear clusters; myxoid glioneuronal tumor; and multinodular and vacuolating neuronal tumor. their molecular-pathologic features have been scientifically defined but the epileptological disease condition with early seizure onset and drug-resistant epilepsy as clinical hallmarks and their long-term seizure outcome following gross surgical resection were rarely addressed at a comprehensive level. as a result, most of the new tumor entities are not yet introduced to the clinical arena of epileptologists and a careful review of published histopathology images in the new who classification suggests that these new entities mimic well-established tumors of the leat family (leat – low-grade and epilepsy-associated tumors)43. one example is that of diffuse astrocytoma, mybor mybl1-altered, which was first described in 2004 as isomorphic astrocytoma in a series of 19 patients with long-term and drug-resistant epilepsy44,45 or the polymorphous low-grade neuroepithelial tumor of the young (plnty), which shares many structural (i.e., glio-neuronal) and molecular (i.e., cd34, braf v600e) similarities with gangliogliomas (gg)43,46. a recently published genotype-phenotype study from beijing, china addressing 30 patients with gg confirmed the presence of braf v600e in the majority of their gg cases (77%) when submitted to targeted next-generation sequencing using a panel of 131 genes47. there was no low-grade paediatric type diffuse glioma, plnty, nor any other of the new subtypes specified above present in their series. along these lines, a group of french neuropathologists have asked the question whether the staggering number of new leat entities make a difference in the clinical management of epilepsy48. they focused on a subgroup of 72 leat cases, predominantly presenting with an oligodendroglial phenotype, and tried to classify them according to the 2021 who classification scheme. based on rna-sequencing, multiplexed digital pcr, dna methylation analysis and histopathology review they identified only two major clusters of biological significance, with cluster 1 being enriched with dysembryoplastic neuroepithelial tumors (dnt) at histology, belonging to the lgg-dnt methylation class with cd34 negativity and fgrf1 alterations; while cluster 2 was classified histopathologically as gg, belonging to the lgg-gg methylation class, with braf v600e mutation and cd34 positivity. the same conclusion was already drawn in 2019 by an international leat review panel of 25 experts from 18 countries when studying their series of 30 leat7. the current situation questions, therefore, the clinico-pathological relevance of the various types recognized by the who within the spectrum of leat48. it will remain a continuous debate and negotiation to bring together all experts, including histopathologists, epileptologists and neuro-oncologists, to define a successful strategy for best patient management. these tumors rarely progress into malignancy but offer the opportunity for long-term seizure freedom when neurosurgically resected following a comprehensive epileptology work-up2. this will require new measures and parameters for the definition of leat when addressing the specific needs of patients with early onset drug-resistant epilepsy. topic 7: news from leat: animal models braf v600e is the most common genetic driver in leat and is now variably assigned to several tumor entities listed in the 2021 who classification update (see above). notwithstanding, none of these entities share an easy to classify histopathology phenotype and the failure to establish and agree upon the diagnosis across countries and laboratories is well known from published literature49. intuitively, one would expect that a gene driver mutation such as braf v600e would establish a single diagnostic entity rather than being implicated in separate neuroepithelial tumors, i.e., paediatric type diffuse low-grade gliomas, mapk pathway-altered; plnty; pleomorphic xanthoastrocytoma; gg, dnt, or multinodular and vacuolating neuronal tumor. when braf v600e is experimentally introduced in utero into the developing murine brain by intraventricular electroporation, the resulting phenotype was consistently described as biphasic gg by koh et al. in 201850. the resulting lesion also showed intrinsic epileptogenic properties in the neuronal cell lineage, whereas tumorigenic properties were attributed to the proliferation active glial cell lineage. rna sequencing analysis of patients’ brain tissues with the mutation revealed that braf v600e-induced epileptogenesis is mediated by re1-silencing transcription factor (rest), which is a regulator of ion channels and neurotransmitter receptors associated with epilepsy50. moreover, they found that seizures were treatable in mice with the fda-approved braf v600e inhibitor vemurafenib, as well as various genetic inhibitors of rest. accordingly, this study provides direct evidence of a braf somatic mutation contributing to the intrinsic epileptogenicity in paediatric brain tumors and suggests that braf and rest could be treatment targets for intractable epilepsy. cases-cunillera and co-worker have used the same technique of in utero electroporation to extend these studies towards braf v600e mutations with doubleand triple-hits51. in their most recent animal model, aberrant braf expression in murine neural progenitors generated benign glio-neuronal neoplasms of the gg phenotype only in concert with active mtor-signaling, i.e. by phosphorylated akt (pakt). additional somatic trp53-loss generated anaplastic gg, a grading scale and terminology not supported anymore by the current who classification scheme. functionally, only braf/pakt altered tumors showed substantial neuronal activity which spread to the adjacent neocortical tissue, which was interpreted as correlate of increased epileptogenicity51. in both animal studies, however, cd34 immunoreactivity was an immunohistochemical surrogate of the experimentally induced gg50,51. such experimental leat models, which reproduce the variable genetic and histopathologic tumor phenotypes, will be important tools to further our knowledge and finally assess targeted therapies. topic 8. what is new about hippocampal sclerosis?: human herpes virus infection and the impact of inflammatory infiltrates on neuronal cell loss hippocampal sclerosis (hs) remains the single most common cause of focal epilepsy amenable to epilepsy surgery13. its etiology remains, however, to be further clarified with most researchers probably agreeing with the statement that hs is an end stage resulting from multiple etiologies. this may also be reflected by the different histopathologic patterns of segmental neuronal cell loss in hs being recognized in the ilae consensus classification scheme of 201352. however, inflammation remains a major topic of interest in the pathogenesis of mesial temporal lobe epilepsy (mtle) with or without hs. i will briefly discuss three publications highlighting this topic. tröscher and colleagues analyzed the presence of t cells in various mtle subgroups to answer the question of how much inflammation is present and whether the presence of t cells is associated with seizures and/or neuronal cell loss in the hippocampus53. their detailed histopathological investigation of the involvement of t cells in various mtle subgroups, i.e., gangliogliomas, febrile seizures, postinfectious encephalitis and rasmussen encephalitis w/o hs, suggested that t cell numbers correlated with the degree of neuronal cell loss rather than seizure frequency or disease duration. their quantification also showed that t cell numbers were significantly elevated in all mtle groups compared to healthy post-mortem controls. however, cd3+ as well as cd8+ t cell numbers varied among their mtle subgroups and nearly all mtle groups revealed elevated numbers of t cells years after the precipitating injury. hippocampal infection with human herpes virus 6 (hhv-6) has been demonstrated in patients with mtle already in 200354 and confirmed thereafter in several scientific publications. it can be envisioned, therefore, as one key etiological cause of mtle with hs. this knowledge gains further momentum from the publication of theodore and coworkers published in 2021, detecting hhv-6a and hhv-6b strains in fresh human tissue samples obtained from 87 patients with drug-resistant epilepsy submitted to epilepsy surgery55. twenty-nine of their 54 patients with hs (54%), six of 23 with fcd (26%), and one of three with a history of encephalitis (33%) were positive for hhv-6. this contrasts the overall low percentage of hhv-6 dna detection in only 6% of human brain samples with non-neurologic illness56. a febrile seizure history was not significantly associated with hhv-6 detection. however, patients with hhv-6 positive hs had significantly lower age at seizure onset than those with other pathologies. also, there was a trend for hhv-6 positive patients to have higher binding of [11c]pbr28 in positron emission tomography (pet), the latter being a marker for reactive astrocytes and activated microglia, suggesting an inflammatory pathomechanism. indeed, these findings reinforce the potential role and impact for hhv-6 in the etiology of mtle with hs. however, the same group of researchers published a neuroimaging approach in 2020 to address the effects of hhv-6 on hippocampal volume in patients with hippocampal sclerosis57. they used mri post-processing to segment cortical structures and to obtain an asymmetry index between hippocampal volumes ipsilateral and contralateral to the seizure focus when comparing between hhv-6 positive and negative patients. in this study, however, hhv-6 negative patients had significantly greater asymmetry and lower total hippocampal volume ipsilateral to the seizure focus compared to hhv-6 positive patients. disease duration and age of onset did not affect these results. their data suggest that hhv-6 still play a role in mtle but with hhv-6 having a less severe effect on hippocampal damage. this controversy will be ongoing, however, and in need of more studies to clarify the issue. topic 9. the fine structure of the epileptogenic neocortex and white matter gray-white matter blurring of the anterior temporal lobe (gwmb) is a common neuroimaging finding in patients with hs58. it is often assigned to fcd, i.e., fcd ilae type 1, although systematic correlation studies of in vivo and ex vivo mri with histopathology showed disturbed axonal myelination of the affected white matter as an underlying structural correlate rather than any signature of cortical (neuronal) malformation58. whether or not such white matter alterations were due to secondary axonal degeneration, e.g., neuronal cell loss in the ipsilateral hippocampus, has yet to be clarified but was already suggested as severity aggravates with disease duration58. this topic was once again addressed in a study by demrath and co-worker in 2021, when studying mri-histopathology correlations in twenty patients with unilateral temporal lobe epilepsy, gwmb and hs59. anterior temporal lobe white matter t1 relaxation times and diffusion measures were analyzed on the side of hs, on the side contralateral to hs, and in ten normal controls. resected brain tissue was further evaluated at the ultrastructural level from three patients without gwmb and four patients with gwmb addressing axon density and diameter, the relation of the axon diameter to the total fiber diameter, and the thickness of the myelin sheath59. as a result, hs with gwmb of the anterior temporal lobe was related to prolonged t1 relaxation and axonal loss. a less pronounced reduction in axonal fraction was also found on imaging in gwmb-negative temporal poles compared to normal controls. contralateral values did not differ significantly between patients and normal controls. reduced axonal density and axonal diameter were histopathologically confirmed in samples with gwmb compared to temporal poles without gwmb. the authors concluded that gwmb should be considered as an imaging correlate for disturbed axonal maturation that can be quantified with advanced diffusion imaging. in my view, however, the data does not provide true evidence for “disturbed axonal maturation” in the sense of a maldevelopmental feature. similar findings may occur as secondary retrograde damage and/or repair of the axonal compartment as suggested previously58. in 2021, another study addressed the fine microstructure of the epileptic neocortex using capricious golgi-impregnations to further analyze synaptic networks of glutamatergic and gabaergic axon terminals60. alterations in dendritic morphology and spine loss have been reported both in epilepsy animal models and in human brain tissues from patients with epilepsy61. however, it is still unclear whether these dendritic abnormalities relate to the cause of epilepsy or are generated by seizure recurrence. rossini and coworker investigated these fine neuronal structures in cortical specimens from 28 patients with different neuropathologically defined etiologies, i.e., fcd type 1a and type 2, and non-lesional neocortex obtained from tle with hs. autoptic brain tissues were used for comparison. three-dimensional reconstructions of golgi-impregnated neurons revealed severe dendritic reshaping and spine alteration in the core region of fcd type 2. dysmorphic neurons showed increased dendritic complexity, reduction of dendritic spines and occasional filopodia-like protrusions emerging from the soma (figure 6). surprisingly, the intermingled normal-looking pyramidal neurons also showed severe spine loss and simplified dendritic arborization. no changes were observed outside the dysplasia in perilesional tissue or in neocortical tissue obtained from the other patient groups. these data confirmed a rather normal appearing fine morphological aspect of neurons and dendritic spines in the epileptogenic neocortex, with the exception of type 2 dysplastic lesions and argue against the concept that long-lasting epilepsy will produce per se any dendritic pathology. figure 6: filopodia-like protrusions in dysmorphic neurons of fcd 2b low power microphotographs of golgi-impregnated dysmorphic neuron from a fcd 2b on the left, showing the presence of numerous short filopodia-like protrusions emerging from the soma. higher magnification on right. modified from 60 with permission from the author. the missing topic 10: what is still missing to further the advancement of neuropathology and epilepsy surgery? we are already facing another major advancement in neuropathology, which will soon be recognized as the era of digital neuropathology. artificial intelligence (ai)-based disease classifier and ever growing digital slide suites will substitute the routine work-up and may be able to also predict essential molecular features of the underlying disease condition to inform precision medicine treatment. these topics have not yet been publicized in peer reviewed journals with the exception of a deep learning-based algorithm to differentiate fcd type 2 from cortical tuber on routine h&e-stained glass slides in 202062. ai-based algorithms may even be able to replicate histochemical and immunohistochemical stainings from unstained slides without actually performing the staining procedures63,64. this potential is yet to be fully explored, including within the field of neuropathology and epilepsy surgery, and we will await upcoming publications addressing this fascinating matter as it may well reshape our next future in neuropathology. conclusion during the past decade, there have been considerable advances in understanding the genetic and morphogenic processes underlying cortical malformations and developmental brain tumors15. focal brain malformations result from somatic (post-zygotic) variants in several genes related to the mtor pathway in focal cortical dysplasia type 2, akt3, depdc5, mtor, nprl2/3, pik3ca, rheb, tsc1/2 and others, or in the galactose transporter gene slc35a2 in moghe, which were acquired early during cortical development. the timing of the genetic event, the specific gene involved and the targeted precursor cell population will determine the nature and size of the lesion, whether developmental malformation or a brain tumor. there is also emerging evidence of epigenetic processes underlying a ‘molecular memory’ in epileptogenesis22, which also helped to establish a diagnostic dna methylation classification scheme21. this knowledge will finally accumulate into a better understanding of why and how patients with these lesions have epilepsy and to move toward precision medicine in patients with drug-resistant focal epilepsy. these topics will be continuously addressed and discussed also at the annual ilae summer school of neuropathology and epilepsy surgery (https://www.ilae.org/congresses). please address your application to bluemcke[at]uk-erlangen.de references jehi l, braun k. does etiology really matter for epilepsy surgery outcome? brain pathol. 2021 jul;31:e12965. lamberink hj, otte wm, blümcke i, braun kpj. seizure outcome and use of antiepileptic drugs after epilepsy surgery according to histopathological diagnosis: a retrospective multicentre cohort study. lancet neurol. 2020 sep;19:748-757. chamberlain wa, cohen ml, gyure ka, kleinschmidt-demasters bk, perry a, powell sz, et al. interobserver and intraobserver reproducibility in focal cortical dysplasia (malformations of cortical development). epilepsia. 2009 dec;50:2593-2598. coras r, de boer oj, armstrong d, becker a, jacques ts, miyata h, et al. good interobserver and intraobserver agreement in the evaluation of the new ilae classification of focal cortical dysplasias. epilepsia. 2012 aug;53:1341-1348. blumcke i, aronica e, becker a, capper d, coras r, honavar m, et al. low-grade epilepsy-associated neuroepithelial tumours the 2016 who classification. nat rev neurol. 2016 dec;12:732-740. blumcke i, coras r, busch rm, morita-sherman m, lal d, prayson r, et al. toward a better definition of focal cortical dysplasia: an iterative histopathological and genetic agreement trial. epilepsia. 2021 jun;62:1416-1428. blumcke i, coras r, wefers ak, capper d, aronica e, becker a, et al. challenges in the histopathological classification of ganglioglioma and dnt: microscopic agreement studies and a preliminary genotype-phenotype analysis. neuropathol appl neurobiol. 2019 oct;45:95-107. khoshkhoo s, lal d, walsh ca. application of single cell genomics to focal epilepsies: a call to action brain pathol. 2021 jul;31:e12958. d'gama am, woodworth mb, hossain aa, bizzotto s, hatem ne, lacoursiere cm, et al. somatic mutations activating the mtor pathway in dorsal telencephalic progenitors cause a continuum of cortical dysplasias. cell rep. 2017 dec;21:3754-3766. pfisterer u, petukhov v, demharter s, meichsner j, thompson jj, batiuk my, et al. author correction: identification of epilepsy-associated neuronal subtypes and gene expression underlying epileptogenesis. nat commun. 2020 nov;11:5988. liu h, zhou j, tian w, luo c, bartlett a, aldridge a, et al. dna methylation atlas of the mouse brain at single-cell resolution nature. 2021 oct;598:120-128. taylor dc, falconer ma, bruton cj, corsellis ja. focal dysplasia of the cerebral cortex in epilepsy. j neurol neurosurg psychiatry. 1971;34:369-387. blümcke i, spreafico r, haaker g, coras r, kobow k, bien cg, et al. histopathological findings in brain tissue obtained during epilepsy surgery. n engl j med. 2017 oct;377:1648-1656. lee ws, baldassari s, stephenson sem, lockhart pj, baulac s, leventer rj. cortical dysplasia and the mtor pathway: how the study of human brain tissue has led to insights into epileptogenesis. int j mol sci. 2022 jan;23. blumcke i, budday s, poduri a, lal d, kobow k, baulac s. neocortical development and epilepsy: insights from focal cortical dysplasia and brain tumours. lancet neurol. 2021 nov;20:943-955. lee ws, baldassari s, chipaux m, adle-biassette h, stephenson sem, maixner w, et al. gradient of brain mosaic rheb variants causes a continuum of cortical dysplasia. ann clin transl neurol. 2021 jan 12. evrony gd. one brain, many genomes science. 2016;354:557-558. ye z, chatterton z, pflueger j, damiano ja, mcquillan l, harvey as, et al. cerebrospinal fluid liquid biopsy for detecting somatic mosaicism in brain. brain communications. 2021;3. kim s, baldassari s, sim ns, chipaux m, dorfmuller g, kim ds, et al. detection of brain somatic mutations in cerebrospinal fluid from refractory epilepsy patients. ann neurol. 2021 jun;89:1248-1252. capper d, jones dtw, sill m, hovestadt v, schrimpf d, sturm d, et al. dna methylation-based classification of central nervous system tumours. nature. 2018 mar;555:469-474. jabari s, kobow k, pieper t, hartlieb t, kudernatsch m, polster t, et al. dna methylation-based classification of malformations of cortical development in the human brain. acta neuropathologica. 2021 nov. jablonski j, hoffmann l, blumcke i, fejtova a, uebe s, ekici ab, et al. experimental epileptogenesis in a cell culture model of primary neurons from rat brain: a temporal multi-scale study. cells. 2021 nov;10. schurr j, coras r, rossler k, pieper t, kudernatsch m, holthausen h, et al. mild malformation of cortical development with oligodendroglial hyperplasia in frontal lobe epilepsy: a new clinico-pathological entity. brain pathol. 2017 jan;27:26-35. hartlieb t, winkler p, coras r, pieper t, holthausen h, blümcke i, et al. age-related mr characteristics in mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (moghe). epilepsy behav. 2019 feb;91:68-74. gaballa a, woermann fg, cloppenborg t, kalbhenn t, blumcke i, bien cg, et al. clinical characteristics and postoperative seizure outcome in patients with mild malformation of cortical development and oligodendroglial hyperplasia. epilepsia. 2021 dec;62:2920-2931. seetharam r, nooraine j, mhatre r, ramachandran j, iyer rb, mahadevan a. mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (moghe): a widespread disease with an apparently focal epilepsy. epileptic disord. 2021 apr;23:407-411. mendes coelho vc, morita-sherman m, yasuda cl, alvim mmk, amorim bj, tedeschi h, et al. magnetic resonance imaging findings and clinical characteristics in mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy in a predominantly adult cohort. epilepsia. 2021 jun;62:1429-1441. di giacomo r, deleo f, garbelli r, marucci g, del sole a, dominese a, et al. mild malformation of cortical development with oligodendroglial hyperplasia (moghe): neurophysiological fingerprints of a new pathological entity. clin neurophysiol. 2021 jan;132:154-156. bonduelle t, hartlieb t, baldassari s, sim ns, kim sh, kang hc, et al. frequent slc35a2 brain mosaicism in mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (moghe). acta neuropathol commun. 2021 jan;9:3. sim ns, seo y, lim js, kim wk, son h, kim hd, et al. brain somatic mutations in slc35a2 cause intractable epilepsy with aberrant n-glycosylation. neurol genet. 2018 dec;4:e294. baldassari s, ribierre t, marsan e, adle-biassette h, ferrand-sorbets s, bulteau c, et al. dissecting the genetic basis of focal cortical dysplasia: a large cohort study. acta neuropathol. 2019 dec;138:885-900. witters p, tahata s, barone r, ounap k, salvarinova r, gronborg s, et al. clinical and biochemical improvement with galactose supplementation in slc35a2-cdg. genet med. 2020 jun;22:1102-1107. holthausen h, coras r, tang y, bai l, wang i, pieper t, et al. multilobar unilateral hypoplasia with emphasis on the posterior quadrant and severe epilepsy in children with fcd ilae type 1a. epilepsia. 2022 jan;63:42-60. miyata h, kuwashige h, hori t, kubota y, pieper t, coras r, et al. variable histopathology features of neuronal dyslamination in the cerebral neocortex adjacent to epilepsy-associated vascular malformations suggest complex pathogenesis of focal cortical dysplasia ilae type iiic. brain pathol. 2022 jan:e13052. blumcke i, cendes f, miyata h, thom m, aronica e, najm i. toward a refined genotype-phenotype classification scheme for the international consensus classification of focal cortical dysplasia. brain pathol. 2021 jul;31:e12956. najm im, sarnat hb, blümcke i. the international consensus classification of focal cortical dysplasia a critical update 2018. neuropathol appl neurobiol. 2018 feb;44:18-31. wang i, oh s, blümcke i, coras r, krishnan b, kim s, et al. value of 7t mri and post-processing in patients with nonlesional 3t mri undergoing epilepsy presurgical evaluation. epilepsia. 2020 sep;61:2509-2520. blumcke i, aronica e, miyata h, sarnat hb, thom m, roessler k, et al. international recommendation for a comprehensive neuropathologic workup of epilepsy surgery brain tissue: a consensus task force report from the ilae commission on diagnostic methods. epilepsia. 2016 feb;57:348-358. rojiani am, emery ja, anderson kj, massey jk. distribution of heterotopic neurons in normal hemispheric white matter: a morphometric analysis. j neuropathol exp neurol. 1996;55:178-183. muhlebner a, coras r, kobow k, feucht m, czech t, stefan h, et al. neuropathologic measurements in focal cortical dysplasias: validation of the ilae 2011 classification system and diagnostic implications for mri. acta neuropathol. 2012 feb;123:259-272. thom m, sisodiya s, harkness w, scaravilli f. microdysgenesis in temporal lobe epilepsy. a quantitative and immunohistochemical study of white matter neurones. brain. 2001 nov;124:2299-2309. emery ja, roper sn, rojiani am. white matter neuronal heterotopia in temporal lobe epilepsy: a morphometric and immunohistochemical study j neuropathol exp neurol. 1997 dec;56:1276-1282. louis dn, perry a, wesseling p, brat dj, cree ia, figarella-branger d, et al. the 2021 who classification of tumors of the central nervous system: a summary. neuro oncol. 2021 aug;23:1231-1251. wefers ak, stichel d, schrimpf d, coras r, pages m, tauziede-espariat a, et al. isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of mybl1 or myb and a benign disease course. acta neuropathol. 2020 jan;139:193-209. blumcke i, luyken c, urbach h, schramm j, wiestler od. an isomorphic subtype of long-term epilepsy-associated astrocytomas associated with benign prognosis. acta neuropathol. 2004 may;107:381-388. huse jt, snuderl m, jones dt, brathwaite cd, altman n, lavi e, et al. polymorphous low-grade neuroepithelial tumor of the young (plnty): an epileptogenic neoplasm with oligodendroglioma-like components, aberrant cd34 expression, and genetic alterations involving the map kinase pathway. acta neuropathol. 2016 nov;133:417-429. wang y, wang l, blumcke i, zhang w, fu y, shan y, et al. integrated genotype-phenotype analysis of long-term epilepsy-associated ganglioglioma. brain pathol. 2021 aug:e13011. metais a, appay r, pages m, gallardo c, silva k, siegfried a, et al. low-grade epilepsy-associated neuroepithelial tumours with a prominent oligodendroglioma-like component: the diagnostic challenges. neuropathol appl neurobiol. 2022 feb;48:e12769. slegers rj, blumcke i. low-grade developmental and epilepsy associated brain tumors: a critical update 2020. acta neuropathol commun. 2020 mar;8:27. koh hy, kim sh, jang j, kim h, han s, lim js, et al. braf somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors nat med. 2018 nov;24:1662-1668. cases-cunillera s, van loo kmj, pitsch j, quatraccioni a, sivalingam s, salomoni p, et al. heterogeneity and excitability of braf v600e-induced tumors is determined by akt/mtor-signaling state and trp53-loss. neuro oncol. 2021 dec. blumcke i, thom m, aronica e, armstrong dd, bartolomei f, bernasconi a, et al. international consensus classification of hippocampal sclerosis in temporal lobe epilepsy: a task force report from the ilae commission on diagnostic methods epilepsia. 2013 jul;54:1315-1329. troscher ar, sakaraki e, mair km, kock u, racz a, borger v, et al. t cell numbers correlate with neuronal loss rather than with seizure activity in medial temporal lobe epilepsy. epilepsia. 2021 jun;62:1343-1353. donati d, akhyani n, fogdell-hahn a, cermelli c, cassiani-ingoni r, vortmeyer a, et al. detection of human herpesvirus-6 in mesial temporal lobe epilepsy surgical brain resections. neurology. 2003 nov;61:1405-1411. theodore wh, leibovitch e, billioux bj, inati sk, zaghloul k, heiss j, et al. human herpesvirus 6 and epilepsy. epilepsia open. 2021 dec;6:777-780. lin ct, leibovitch ec, almira-suarez mi, jacobson s. human herpesvirus multiplex ddpcr detection in brain tissue from lowand high-grade astrocytoma cases and controls infect agent cancer. 2016;11:32. akinsoji eo, leibovitch e, billioux bj, abath neto ol, ray-chaudhury a, inati sk, et al. hhv-6 and hippocampal volume in patients with mesial temporal sclerosis. ann clin transl neurol. 2020 sep;7:1674-1680. garbelli r, milesi g, medici v, villani f, didato g, deleo f, et al. blurring in patients with temporal lobe epilepsy: clinical, high-field imaging and ultrastructural study. brain. 2012 aug;135:2337-2349. demerath t, donkels c, reisert m, heers m, rau a, schroter n, et al. gray-white matter blurring of the temporal pole associated with hippocampal sclerosis: a microstructural study involving 3 t mri and ultrastructural histopathology. cereb cortex. 2021 sep. rossini l, de santis d, mauceri rr, tesoriero c, bentivoglio m, maderna e, et al. dendritic pathology, spine loss and synaptic reorganization in human cortex from epilepsy patients. brain. 2021 feb;144:251-265. blumcke i, suter b, behle k, kuhn r, schramm j, elger ce, et al. loss of hilar mossy cells in ammon's horn sclerosis. epilepsia. 2000;41:s174-s180. kubach j, muhlebner-fahrngruber a, soylemezoglu f, miyata h, niehusmann p, honavar m, et al. same same but different: a web-based deep learning application revealed classifying features for the histopathologic distinction of cortical malformations. epilepsia. 2020 mar;61:421-432. rivenson y, wang h, wei z, de haan k, zhang y, wu y, et al. virtual histological staining of unlabelled tissue-autofluorescence images via deep learning. nat biomed eng. 2019 jun;3:466-477. de haan k, zhang y, zuckerman je, liu t, sisk ae, diaz mfp, et al. deep learning-based transformation of h&e stained tissues into special stains. nat commun. 2021 aug;12:4884. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 65th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 2:22 (2021) meeting abstracts 65th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts september 1–3, 2021 submitted: 23 august 2021 accepted: 23 august 2021 published: 25 august 2021   dear participants, dear colleagues, it is our great pleasure to welcome you to the 65th annual meeting of the german society of neuropathology and neuroanatomy the brain and nerve microenvironment in health and disease which will be held as a virtual meeting from september 1–3, 2021. the meeting will bring together basic and clinical researchers, physicians as well as junior scientists and phd students from different disciplines of basic and clinical neuroscience. we will have outstanding lectures by and with some of the most renowned international experts in the field of neuro-oncology, neuroinflammation, neurodegeneration and muscle and nerve diseases and look forward to exciting scientific discussions. there will also be a special and timely section on the effects of covid-19 on the central and peripheral nervous system. the three days will offer exciting insights into different areas of basic and clinical neuroscience. we have also encouraged early career scientists to present their scientific findings in short talks and poster presentations. we are therefore particularly thankful that the abstracts of the meeting, which in their sum provide the best overview of the high scientific standing of the field, will be published in free neuropathology. finally, we would like to thank you all for your active contribution to this conference in these difficult times. we also thank all supporters for their financial help. we wish you a stimulating and exciting conference. yours sincerely, prof. dr. med. till acker conference chair (justus liebig university giessen) pd. dr. med. anne schänzer dr. med. hildegard dohmen https://doi.org/10.17879/freeneuropathology-2021-3500 keywords: german society for neuropathology and neuroanatomy, dgnn, meeting abstracts, 65th meeting sep. 2021 contents neuroinflammation ni-01 ni-02 ni-03 neurooncology adult i (from mechanisms to translation) noa-01 noa-02 noa-03 noa-04 noa-05 noa-06 noa-07 muscle & nerve diseases mnd-01 mnd-02 neurodegeneration / covid-19 and neuropathology nd-01 nd-02 nd-03 neurooncology pediatrics nop-01 nop-02 postersession i – neuroinflammation pi-01 pi-02 pi-03 postersession ii – neurooncology pii-01 pii-02 pii-03 poster session iii – muscle & nerve piii-01 piii-02 piii-03 piii-04 poster session iv – neurodegeneration piv-01 piv-02 piv-03 piv-04 piv-05 piv-06     neuroinflammation ni-01 free neuropathol 2:22:4 microbiota-derived acetate enables the metabolic fitness of the cns innate immune system during health and disease d. erny1 1 institut für neuropathologie, neurozentrum, freiburg i. br., germany introduction: as tissue macrophages of the central nervous system (cns) microglia constitute the pivotal immune cells of this organ. microglial features are strongly dependent on environmental cues such as commensal microbiota. gut bacteria are known to continuously modulate microglia maturation and function by the production of short-chain fatty acids (scfa). however, the precise mechanism of this crosstalk is unknown. objectives & results: here we determined that the immature phenotype of microglia from germ-free (gf) mice is epigenetically imprinted by h3k4me3 and h3k9ac on metabolic genes associated with substantial functional alterations including increased mitochondrial mass and specific respiratory chain dysfunctions. we identified acetate as the essential microbiome-derived scfa driving microglia maturation and regulating the homeostatic metabolic state, and further showed that it is able to modulate microglial phagocytosis and disease progression during neurodegeneration. conclusion: these findings indicate that acetate is an essential bacteria-derived molecule driving metabolic pathways and functions of microglia during health and perturbation.   ni-02 free neuropathol 2:22:5 multiple sclerosis immunopathological patterns and their mri correlates i. metz1, r. gavrilova2, s. weigand3, y. guo2, n. zalewski2, m. gloth1, o. tobin2, h. lassmann4, j. tillema2, b. erickson2, j. parisi5, b. popescu6, s. becker7, f. könig8, j. frischer9, w. brück1, c. lucchinetti2 1 university medical center göttingen, georg-august-university, institute of neuropathology, göttingen, germany 2 mayo clinic, department of neurology, rochester, ny, united states 3 mayo clinic, department of health sciences research, rochester, ny, united states 4 medical university vienna, department of neuroimmunology, center for brain research, vienna, austria 5 mayo clinic, department of laboratory medicine and pathology, rochester, ny, united states 6 university of saskatchewan, department of anatomy, physiology and pharmacology, and cameco ms neuroscience research center, saskatchewan, canada 7 university medical center göttingen, department of palliative medicine, göttingen, germany 8 klinikum kassel, institute of pathology, kassel, germany 9 medical university vienna, department of neurosurgery, vienna, austria introduction: early active multiple sclerosis lesions can be classified into three main immunopathological patterns of active demyelination (patterns i-iii) by histology. these patterns are inter-individually heterogeneous but intra-individually stable. in pattern i and ii, a t-celland macrophage-associated demyelination is suggested, with pattern ii only showing signs of a humoral immune response. pattern iii is characterized by inflammatory lesions with an oligodendrocyte degeneration. patterns suggest pathogenic heterogeneity, and we postulated that they have distinct mri correlates that may serve as biomarkers. objectives: to analyze the mri correlates of multiple sclerosis immunopathological patterns. patients and methods: we evaluated in an international collaborative retrospective cohort study the mri lesion characteristics of 789 pre-biopsy and follow-up mris in relation to their histopathologically classified immunpathological patterns (n=161 subjects) and lesion edge features (n=112). results: a strong association of a ring-like enhancement and a hypointense t2-weighted rim (t2w rim) with pattern i and ii, but not pattern iii, was observed. only a fraction of pattern iii patients showed a ring-like enhancement, and this was always atypical. ring-like enhancement and t2w rims colocalized, and ring-like enhancement showed a strong association with macrophage rims as shown by histology. a strong concordance of mri lesion characteristics, meaning that different lesions showed the same features, was found, indicating lesion homogeneity within individual patients. conclusion: we provide robust evidence that mri characteristics reflect specific histological features of ms immunopatterns and that ring-like enhancement and t2w hypointense rims might serve as a valuable non-invasive biomarker to differentiate pathological patterns of demyelination.   ni-03 free neuropathol 2:22:7 microvascular vulnerability to cytokine toxicity is a critical initiating factor in cerebral interferonopathies b. viengkhou1, e. hayashida1, s. mcglasson2, k. emelianova2, y. crow2, a. pagenstecher3, d. hunt2, m. hofer1 1 the university of sydney, charles perkins centre, sydney, australia 2 the university of edinburgh, edinburgh, united kingdom 3 university of marburg, dept. neuropathology, marburg, germany cerebral interferonopathies are a group of devastating diseases characterised by overproduction of interferon-alpha (ifn-α), leading to diffuse brain damage. importantly, the critical cellular mediators of ifn-α are unknown. here we use a mouse model of brain-targeted ifn-α production to identify endothelial cells as the major mediator of ifn-α neurotoxicity. specifically, we have created a single cell atlas of the "cerebral interferome" using single cell rna sequencing of brain cells of wild type and ifn-α transgenic mice. by linking this dataset with neuropathological studies of cerebral interferon overproduction, we identified a role for cerebral endothelial cells in the initiation of brain disease. we then confirmed a critical role for endothelial interferon signalling through targeted deletion of the type i interferon receptor ifnar on endothelial cells of the transgenic mice. remarkably, this intervention led to near-complete rescue of the complex multicellular disease phenotype. importantly, we show that diffuse aspects of interferonopathic brain disease such as calcification and neuronal loss arise as a downstream consequence of cytokine-induced microvascular disease. in conclusion, our data identify damage to the cerebral microvasculature as the critical first step in interferon neurotoxicity, and pinpoint ifnar signalling within endothelial cells as a key site for potential therapeutic intervention.   neurooncology adult i (from mechanisms to translation) noa-01 free neuropathol 2:22:8 molecular mechanism of therapy resistance in malignant melanoma brain metastasis e. schumann1, r. koll1, j. onken2, k. jürchott3, t. redmer4, j. radke1 1 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 2 charité – universitätsmedizin berlin, department of neurosurgery, berlin, germany 3 charité – universitätsmedizin berlin, institute of medical immunology, berlin, germany 4 university of veterinary medicine vienna, department of medical biochemistry, vienna, austria introduction: malignant melanoma (mm) has the highest potential to disseminate to the cns. about 45% of patients suffer from brain metastasis, which likely proceed continuously during the course of disease. genetically and molecularly distinct subclones lead to tumor heterogeneity, therapy resistance and poor prognosis. previous studies suggested increased metastatic spread to and within the brain during braf inhibitor (brafi) therapy, which is caused by upregulation of a subset of molecular drivers that control migratory and invasion such as the nerve growth factor receptor cd271/ngfr. objectives: to investigate the molecular features of migration and invasion of patient derived cell lines from mm brain metastases (bm) that were therapy-responsive or therapy-resistant to brafi, radiotherapy and immune checkpoint inhibitors. patients & methods: we isolated and cultured patient derived cell lines from mm bm (n = 9) and dna-sequencing (n = 5) and transcriptome analyses (n = 2) of cell lines and concordant tumor (n = 2). the incucyte® live-cell analysis was used to perform high throughput scratch wound assays with patient derived cell lines, which were genetically modified to overexpress or downregulate the expression of ngfr. results: transcriptome profiling of brafi resistant mm bm revealed that the invasive potential increased during disease progression, which was accompanied by upregulation of ngfr expression. this phenotype was preserved in patient derived cells lines, which demonstrated significantly higher potential of two-dimensional in vitro migration (90% vs. 76% after 100 hours). furthermore, cd271 knockdown was associated with loss-of-expression of several genes involved in migration and invasion such as erbb3, tcf19 and baalc. conclusion: brain metastases are the major cause of death in metastasized mm. our study provides a longitudinal perspective on the progression of brain metastasis and their mechanisms leading to therapy resistance.   noa-02 free neuropathol 2:22:9 molecular characterization of sporadic endolymphatic sac tumours and comparison to von hippel-lindau disease-related tumours l. schweizer1, f. thierfelder1, c. thomas2, p. soschinski2, h. y. kim1, r. jödicke1, n. woltering2, a. förster1, d. teichmann1, c. siewert1, k. klein1, s. schmid1, m. nunninger3, u. w. thomale4, j. onken5, h. mühleisen6, j. schittenhelm7, m. tatagiba8, a. von deimling9, d. e. reuss9, d. a. solomon10, f. l. heppner1, a. koch1, c. hartmann11, o. staszewski12, d. capper1 1 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 2 university hospital münster, department of neuropathology, münster, germany 3 charité – universitätsmedizin berlin, department of radiology, berlin, germany 4 charité – universitätsmedizin berlin, department of neurosurgery, division pediatric neurosurgery, berlin, germany 5 charité – universitätsmedizin berlin, department of neurosurgery, berlin, germany 6 synlab labor für pathologie mutlangen, mutlangen, germany 7 institute of pathology and neuropathology, university of tübingen, department of neuropathology, tübingen, germany 8 university hospital tübingen, department of neurosurgery, tübingen, germany 9 heidelberg university hospital, department of neuropathology, institute of pathology, heidelberg, germany 10 university of california, division of neuropathology, department of pathology, san francisco, ca, united states 11 hannover medical school (mhh), department of neuropathology, hannover, germany 12 faculty of medicine, university of freiburg, institute of neuropathology, freiburg i. br., germany introduction. endolymphatic sac tumours (elsts) are low-grade tumours arising from the endolymphatic sac in the temporal bone. elsts can be locally aggressive and may even expand into the posterior fossa representing a rare differential diagnosis for tumours of the cerebellopontine angle. although inactivation of the von hippel-lindau gene (vhl) on chromosome 3p25 is considered to be the major cause of hereditary endolymphatic sac tumours (elsts), the genetic background of sporadic elst is largely unknown. objectives. the aim of this study was to determine the prevalence of vhl alterations in sporadic elsts and compare their characteristics to vhl-disease-related tumours. materials and methods. we analyzed 11 sporadic and 11 vhl-disease-related elsts by targeted sequencing and dna methylation analysis. results. vhl mutations and small deletions detected by targeted deep sequencing were identified in 9/11 sporadic elsts (82%). no other cancer-related genetic pathway was altered except for tert promoter mutations in two sporadic and one vhl-disease-related elsts (15%). loss of heterozygosity of chromosome 3 was found in 6/10 (60%) vhl-disease-related and 10/11 (91%) sporadic elsts resulting in biallelic vhl inactivation in 8/10 (73%) sporadic elsts. dna methylation profiling did not reveal differences between sporadic and vhl-disease-related elsts, but reliably distinguished elst from morphological mimics of the cerebellopontine angle. vhl patients were significantly younger at disease onset compared to sporadic elsts (29 vs. 52 years, p < 0.0001, fisher's exact test). vhl-disease status was not associated with an increased risk of recurrence, but the presence of clear cells was found to be associated with shorter progression-free survival (p = 0.0002, log-rank test). conclusion. biallelic inactivation of vhl is the main mechanism underlying elsts, but unknown mechanisms beyond vhl may rarely be involved in the pathogenesis of sporadic elsts. fig. 1 fig. 2   noa-03 free neuropathol 2:22:12 establishment and characterization of a meningioma cell model with ragc knockout r. diaz peregrino1, j. kahr1, n. waldt1, e. kirches1, c. mawrin1 1 otto-von-guericke universität magdeburg, institut für neuropathologie, magdeburg, germany amino acid sensors are essential for cell growth and carcinogenesis via the mtorc1 pathway. iomm lee cells are suitable to study the meningioma pathogenesis, and amino acid sensors like ragc can be investigated in these cells. the aims of this work are (i) generate a meningioma cell line with ragc deficiency; (ii) determine its morphology; (iii) characterize it in vitro using cell viability assay, trypan blue cell counting assay, colony formation assay, and brdu assay; (iv) and measure its motion. ragc knockout was achieved through crispr-cas9 system and corroborated by western blot. the area and circularity of iomm ragcko and iomm scr (scramble control) cells were determined by phalloidin staining. in vitro assays at 24 and 48 hours were performed to scr and ragcko cells cultured in normal and leucine-depleted medium. wound healing assay, velocity, displacement, acceleration, and percentage of moving cells were measured in both cell lines seeded in the standard and leucine-free medium. cell area was similar between cell genotypes, but ragcko cells were more elongated than scr cells. based on cell genotype comparison, ragcko cells were more metabolically active and displayed more cell proliferation than scr cells. centered on media comparison, cell numbers and cell proliferation decayed in both cell lines when cultivating them in the leucine-free medium. only ragcko cells had a reduction in atp production under leucine withdrawal. finally, ragcko cells exhibited higher motility than scr cells judged by the motion parameters. moreover, the motility of ragcko cells was resistant towards leucine depletion and even increased in comparison to scr cells. ragcko cells displayed increased proliferation and atp production in leucine-supplied scenarios and accelerated migration under leucine-deprived conditions. changes in akt and pp70s6 signaling may be involved in both phenomena. furthermore, the morphological modifications of ragcko cells might enhance their motion.   noa-04 free neuropathol 2:22:13 the role of 2-oxoglutarate homeostasis in tumour invasion and metastasis in glioblastoma a. alserw1, n. bögürcü-seidel1, s. seidel2, s. gräf1, s. zukunft3, i. fleming3, s. günther4, m. looso4, a. németh1, b. garvalov1,5, t. acker1 1 justus-liebig-university gießen, institute for neuropathology, gießen, germany 2 institute of cell biology and neuroscience and buchmann institute for molecular life sciences (bmls), goethe university, frankfurt a. m., germany 3 goethe university frankfurt, institute for vascular signalling, frankfurt a. m., germany 4 max planck institute for heart and lung research, bioinformatics core unit (m.l., p.g.), bad nauheim, germany 5 european center for angioscience (ecas), department of microvascular biology and pathobiology, mannheim, germany introduction: metabolic reprogramming has been associated with tumour invasion and metastasis. epithelial/proneural-to-mesenchymal transition (emt/prmt) is a key process behind cancer cell dissemination, which is regulated by several transcription factors, including snail. 2-oxoglutarate (2-og) is a metabolite whose intracellular level depends on the activity of isocitrate dehydrogenase 1 (idh1). the metabolite acts as a co-substrate for 2-og-dependent dioxygenases (2-ogdds) which regulate different cellular activities. while mutant-idh-induced, 2-ogdd-dependent tumorigenic mechanisms have been described in detail, it is less well understood how wildtype idh and 2-og levels control emt and invasion. objectives: we aim to define the homeostatic function of idh1 and 2-og in the regulation of emt and tumour progression primarily in glioblastoma, as well as in breast and lung cancer models. materials and methods: boyden chamber, western blot, if, rt-qpcr, rnaseq and metabolite ms assays are employed to determine the role of idh1 and 2-og levels on emt regulators and cell invasion. idh1 knockdown and several functional assays are used to further explore the underlying mechanisms. results: we observed that tgfβ; an established emt inducer results in idh1 downregulation, with a concomitant increase in snail expression and invasion. strikingly, 2-og supplementation reverts tgfβ induced snail at the mrna and protein level as well as cellular invasion in vitro. in line with a functional role of idh1 in emt, idh1deficiency promotes invasion and metastasis and enhances a cancer stem cell phenotype. importantly, we identify hif and mtor signalling as two crucial mechanistic pathways involved in snail regulation by 2-og. conclusion: collectively, our findings emphasize the interplay between metabolism and metastasis and reveal novel mechanistic insights into the role of wildtype idh and intracellular 2-og levels in the metabolic control of tumor invasion and emt/prmt.   noa-05 free neuropathol 2:22:14 infratentorial idh mutant astrocytoma is a distinct subtype r. banan1, d. stichel1, a. bleck2, b. hong3, u. lehmann4, a. suwala1, a. reinhardt1, d. schrimpf1, r. buslei5, c. stadelmann-nessler6, k. ehlert7, m. prinz8, t. acker9, j. schittenhelm10, d. kaul11, l. schweizer12, d. capper12, p. n. harter13, n. etminan14, d. t. w. jones15, s. m. pfister16, c. herold mende17, w. wick18, f. sahm1, a. von deimling1, c. hartmann2, d. e. reuss1 1 heidelberg university hospital, department of neuropathology, institute of pathology, heidelberg, germany 2 hannover medical school (mhh), department of neuropathology, institute of pathology, hannover, germany 3 hannover medical school (mhh), department of neurosurgery, hannover, germany 4 hannover medical school (mhh), institute of pathology, hannover, germany 5 klinikum bamberg, institute of pathology, bamberg, germany 6 university medical center göttingen, institute of neuropathology, göttingen, germany 7 university of greifswald, department of pediatric oncology and hematology, greifswald, germany 8 university of freiburg, institute of neuropathology, freiburg i. br., germany 9 institute of neuropathology, university of gießen, gießen, germany 10 university hospital tübingen, department of neuropathology, tübingen, germany 11 charité – universitätsmedizin berlin, department of radiation oncology and radiotherapy, berlin, germany 12 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 13 goethe university frankfurt, institute of neurology (edinger institute), frankfurt a. m., germany 14 university hospital mannheim, university of heidelberg, department of neurosurgery, mannheim, germany 15 german cancer research center (dkfz), pediatric glioma research group, heidelberg, germany 16 university hospital heidelberg, department of pediatric oncology and hematology, heidelberg, germany 17 university hospital heidelberg, department of neurosurgery, division of experimental neurosurgery, heidelberg, germany 18 university of heidelberg medical center, neurology clinic, heidelberg, germany introduction: diffuse idh-mutant astrocytic tumors are rarely diagnosed in infratentorial structures. only few studies have reported single cases of infratentorial diffuse gliomas harboring idh mutations, while suffering small case numbers. objectives: in this multi-institutional study, we aimed to analyze infratentorial idh-mutant astrocytomas in a larger series with respect to clinical and molecular parameters. patients and methods: tumor samples of patients with diffuse infratentorial astrocytomas from different german neuropathology departments were investigated using immunohistochemistry, pyrosequencing, genome-wide dna methylation analysis and ngs panel sequencing. overall survival data were assessed using kaplan–meier method. results: we identified a series of 43 infratentorial idh-mutant astrocytomas from 42 patients. about 80% of idh mutations were of non-idh1-r132h variants which are rare in supratentorial astrocytomas. most frequently, idh1-r132c/g and idh2-r172s/g mutations were present. moreover, atrx-loss and mgmt promoter methylation typically associated with idh mutations in supratentorial astrocytomas, were significantly less frequent in the infratentorial compartment. panel sequencing revealed two samples with idh1-r132c/h3f3a-k27m co-mutations. methylation analysis and copy number profiling provided further evidence for a molecular distinctiveness of these tumors. clinical outcome of patients with infratentorial idh-mutant astrocytomas was significantly better than in patients with diffuse midline gliomas, h3k27m-mutant (p < 0.005) and poorer than in those with supratentorial idh-mutant astrocytomas (p = 0.028). conclusion: the presented data highlight the very existence and distinctiveness of infratentorial idh-mutant astrocytomas implying that molecular testing is critical for detection of these tumors, since many of these tumors cannot be identified by immunohistochemistry applied for the mutated idh1-r132h protein or loss of atrx.   noa-06 free neuropathol 2:22:16 dna methylation profiling and molecular grading of stereotaxic brain tumor biopsies k. filipski1,2,3, j. hench4, m. armbrust1, p. s. zeiner2,3,5, t. i. hartung1, e. steidl6, j. quick-weller7, t. fenton8, j. p. steinbach2,3,5, m. czabanka7, d. capper9,10, s. frank4, k. h. plate1,2,3, m. t. forster7, p. n. harter1,2,3 1 university hospital frankfurt, neurological institute (edinger institute), frankfurt a. m., germany 2 german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany 3 frankfurt cancer institute (fci), frankfurt a. m., germany 4 basel university hospital, department of neuropathology, institute of pathology, basel, switzerland 5 university hospital frankfurt, dr. senckenberg institute of neurooncology, frankfurt a. m., germany 6 university hospital frankfurt, institute of neuroradiology, frankfurt a. m., germany 7 university hospital frankfurt, department of neurosurgery, frankfurt a. m., germany 8 university of kent, school of bioscience, kent, united kingdom 9 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 10 german cancer consortium (dktk), partner site berlin, german cancer research center (dkfz), heidelberg, germany introduction: dna methylation profiling of cns tumors emerges as a powerful tool to support and refine histological diagnoses. tumor entity-specific dna methylation signatures allow for alignment with methylation classes and copy number profiling (cnp), the latter being of particular interest in terms of molecular grading regarding the upcoming who classification. when gross tumor resection is precluded, stereotaxic biopsies might not be representative of the bulk tumor, underlining the need for objective molecular diagnostic parameters. objective: here we investigated the diagnostic potential and accuracy of dna methylation profiling and molecular grading on small stereotaxic brain tumor biopsies. materials & methods: ffpe samples of 237 patients were subjected to dna methylation analysis including the heidelberg brain tumor classifier, cnp and tumor deconvolution using methylcibersort. results: median dna concentration was 6.26 ng/µl. 90.5% of stx samples (n=232) allocated to previously defined dna methylation classes, with 61.6% reaching classifier scores of > 0.84. combined gain of chromosome 7 and loss of chromosome 10 or egfr amplification were detectable in 61% and 38% of idh wildtype glioma (n=156), respectively. 64.5% of histological lower grade, idh wildtype gliomas (n=31) were eligible for upgrading to molecular glioblastoma. 58% of idh mutant gliomas (n=31) presented with 1p/19q codeletion and 23% with homozygous deletion of cdkn2a/b. recursive partitioning pointed at a minimum of 29.42% cancer cells for a 0.83 probability to reach a matching calibrated score in glioblastoma, idh wildtype. a dna input threshold of 3.53 ng/µl yielded matching dna methylation classes with a probability of 0.69. conclusion: our findings demonstrate broad applicability of dna methylation profiling and molecular grading to challengingly small, ill-defined brain tumor specimens with a special impact on grading of histologically lower grade, non-idh mutant diffuse gliomas.   noa-07 free neuropathol 2:22:18 prediction of meningioma methylation classes using ai-assisted digital histopathology j. sehring1, h. dohmen1, m. manke1, d. amsel1, a. németh1, a. mukhopadhyay2, t. acker1 1 justus-liebig-university gießen, institute of neuropathology, gießen, germany 2 technical university darmstadt, medical and environmental computing, darmstadt, germany introduction: current studies suggest that dna methylation-based molecular classification of meningioma has a higher predicting power for tumor recurrence than the current histology-based who classification. however dna methylation profiling is costly, time-consuming and not widely available. therefore, an ai-histology-based prediction of the six dna methylation classes would be very beneficial and could additionally complement the molecular classification. deep neural networks and their visual decoding power offer a promising tool to address this problem. objectives: our goal is to predict meningioma methylation classes using ai-assisted digital histopathology. materials and methods: h&e stained histological slides were digitized with a hamamatsu nanozoomer s360 to obtain whole slide images(wsi). multiple image patches per patient were extracted and encoded into feature space using a pre-trained resnet50. those features were aggregated in a multiple instance learning setting using an attention network to predict the methylation class. results: we have collected wsi data of 167 patients so far. most specimens belong to the most frequently diagnosed methylation subgroups, namely benign-1 (n=36), benign-2 (n=55) and intermediate-a (n=60). three separate two-class classifications (benign-1 vs. benign-2, benign-1 vs. intermediate-a and benign-2 vs. intermediate-a) are currently under development. patients were allocated into training, validation and test sets (0.7/0.1/0.2) and we evaluated the patient-wise balanced accuracy on the test set in a 5-fold cross-validation. in addition, the attention weights for the image patches of a wsi were visualized for interpretability. conclusion: we employ a framework for the prediction of methylation classes based on histological features. visualization is used for an enhanced interpretability of the black-box network. this ai-framework may support, complement, and accelerate meningioma classification in the future.   muscle & nerve diseases mnd-01 free neuropathol 2:22:19 in the spotlight: nk cell receptors in inclusion body myositis c. nelke1, m. pawlitzki2, t. ruck1 1 university hospital duesseldorf, deparment for neurology, neurology, düsseldorf, germany 2 university hospital münster, department of neurology with intitute of translational neurology, münster, germany introduction: inclusion body myositis (ibm) is the most common inflammatory idiopathic myopathy (iim) of old age. as of yet, effective treatment approaches are lacking. a deeper immunological understanding is necessary to overcome this roadblock. as natural killer (nk) cells orchestrate the extent of inflammatory responses by cytolytic properties in iim capable of inducing bystander self-tissue damage, we aimed to characterize nk cell patterns in ibm. objectives: to determine nk cell patterns in ibm and identify drivers of nk cell activation. patients & methods: we included 20 ibm patients and 20 age and sex matched non diseased controls (ndc). peripheral blood was collected and peripheral blood mononuclear cells (pbmc) were isolated by density gradient centrifugation. differences between groups were analyzed using unpaired student"s t test or mann–whitney-u test as appropriate. anti-cn-1a-antibodies were detected in patient serum using the euroimmun immunoassay. results: the activated cd56dim population was reduced in peripheral blood of ibm patients as compared to controls. comparing surface expression as measured by mean fluorescence intensity, we observed a significant increase of nkg2a and nkg2d levels on the cd56dim nk cell subset in ibm patients as compared to ndc. other surface receptors remained unchanged. next, we were interested whether the antibody status of ibm patients affects expression of surface receptors. consequently, comparison of anti-cn-1a positive and anti-cn-1a negative patients revealed that nkg2a level do not differ, while increased nkg2d levels were associated with anti-cn-1a-ab positivity. conclusion: here, we report distinct nk cell patterns in ibm patients. activated cd56dim nk cells are reduced in peripheral blood as compared to ndc. nkg2a and nkg2d levels are increased on cd56dim nk cells. lastly, increased nkg2d levels were associated with anti-cn-1-ab serostatus. fig. 1 fig. 2   mnd-02 free neuropathol 2:22:21 mutations in bves can lead to early onset of cardiac symptoms without clinical signs of muscle dystrophy in children and adolescents a. gangfuß1, a. hentschel2, m. gonzales3, a. schönecker4, a. töpf5, a. sickmann2, a. nishimura6, u. schara-schmidt1, a. hahn7, a. roos1, a. schänzer6 1 university duisburg-essen, department of pediatric neurology, centre for neuromuscular disorders, centre for translational neuroand behavioral sciences, essen, germany 2 leibniz-institut für analytische wissenschaften isas e.v., dortmund, germany 3 justus-liebig-university gießen, pediatric heart center, gießen, germany 4 university duisburg-essen, department of pediatric cardiology, essen, germany 5 newcastle university and newcastle hospitals nhs foundation trust, the john walton muscular dystrophy research centre, translational and clinical research institute, newcastle upon tyne, united kingdom 6 newcastle university and newcastle hospitals nhs foundation trust, the john walton muscular dystrophy research centre, translational and clinical research institute, newcastle upon tyne, united kingdom 7 justus-liebig-university gießen, department of child neurology, gießen, germany introduction: limb-girdle muscular dystrophies occur in children and adults and may be associated with a high variability of the clinical phenotype. in some subtypes, cardiac involvement is the first and thus key symptom. recently, blood vessel endothelial substance (bves), a gene encoding popeye domain containing protein 1 (popdc1), has been implicated as a causative for limb-girdle dystrophy type r25 (lgmdr25), associated with cardiac arrhythmia and variable skeletal muscle involvement. until today, only 10 adult patients with this rare autosomal recessive lgmd have been described. objectives: with this study, we aim to broaden the phenotypic spectrum and improve the pathophysiological understanding of lgmdr25. materials & methods: hence, we describe the clinical phenotype of four affected children of two families with homozygous variants (c.457>t; p.gln153ter and c.578t>g, p.ile193ser) in blood vessel endothelial substance (bves). additionally, we performed detailed muscle biopsy analysis from two affected patients including immunofluorescence, electron microscopic and proteomic studies. results: highly elevated creatine kinase and cardiac involvement are present in all patients. muscle weakness was not present in any of the patients, considering the oldest being 19 years old. however, histological and ultrastructural analysis revealed a myopathy. immunohistochemistry showed an absence of sarcolemnal expression of bves confirming the diagnosis. additional proteomic analyses were indicative for an impairment of the calcium balance and thus a potential influence on the contractile apparatus. conclusion: to conclude, lgmdr25 should be considered in children with cardiac symptoms and highly elevated creatine kinase, even in the absence of muscle weakness. a muscle biopsy is helpful to confirm the diagnosis and to increase the understanding of the disease pathology.   neurodegeneration / covid-19 and neuropathology nd-01 free neuropathol 2:22:23 combining postmortem single cell analysis with an induced pluripotent stem cell model to study dysregulated pathways in frontotemporal dementia o. al dalahmah1, r. kühn1, t. nguyen1, m. deture2, m. siegelin1, d. dickson2, j. p. vonsattel1, j. goldman1, p. canoll1, g. hargus1 1 columbia university, department of pathology and cell biology, new york, ny, united states 2 mayo clinic, jacksonville, fl, united states introduction: frontotemporal dementia (ftd) is a group of early-onset dementias leading to an impairment of behavior, language and cognition. ftd can be caused by mutations in the mapt gene encoding the microtubule-associated protein tau resulting in pronounced atrophy of the frontal and temporal lobes, basal ganglia and brain stem areas. objectives: currently, the underlying mechanisms of neurodegeneration are not clearly understood and curative options do not exit. materials & methods: here, we performed single nucleus rna sequencing (snrna-seq) on postmortem brain tissue from ftd patients carrying the maptn279k mutation and from healthy control individuals to identify dysregulated pathways in patient cells at single cell resolution. results: we found significant changes in pathways related to cell metabolism and neuroinflammation in patient neurons that we tested further in a stem cell model of ftd using patientand control individual-derived induced pluripotent stem cells (ipscs). patient ipsc-derived neurons with the maptn279k mutation demonstrated tau pathology, an impairment of neurite outgrowth and an increased but reversible oxidative stress response. ftd neurons also showed altered metabolic profiles with an increased basal mitochondrial respiration and increased atp production indicating an increased energy demand in these cells. interestingly, ftd neurons also had a significant effect on the survival of host neurons and on glial cell responses when transplanted into the brains of immunocompromised mice indicating a potential immunomodulatory role of patient neurons in vivo. conclusion: these findings demonstrate that a combinatorial approach applying snrna-seq on patient brain tissue and a dynamic ipsc model comprises a powerful tool to identify disease phenotypes in neural cells at risk in ftd. such stem cell model could also be used as a cellular platform for high-throughput drug screening assays to identify potential therapeutic targets in ftd.   nd-02 free neuropathol 2:22:24 deep learning assisted quantitative assessment of histopathological markers of alzheimer"s disease and cerebral amyloid angiopathy v. perosa1,2, a. scherleck3, m. kozberg1, l. smith4, t. westerling-bui4, c. a. auger5, s. vasylechko6, s. m. greenberg1, s. j. van veluw1,5 1 massachusetts general hospital, 1j. philip kistler stroke research center, boston, ma, united states 2 otto-von-guericke university, department of neurology, magdeburg, germany 3 rush university medical center, 3rush alzheimer disease center, chicago, il, united states 4 aiforia inc, cambridge, ma, united states 5 massachusetts general hospital, massgeneral institute for neurodegenerative disease, charlestown, ma, united states 6 boston children's hospital, computational radiology laboratory, boston, ma, united states introduction: traditionally, analysis of neuropathological markers in neurodegenerative diseases has relied on visual assessments of stained sections. resulting semiquantitative scores often vary between individual raters and research centers, limiting statistical approaches. to overcome these issues, we have developed six deep learning-based models, that identify some of the most characteristic markers of alzheimer"s disease (ad) and cerebral amyloid angiopathy (caa). methods: deep learning-based models were trained to differentially detect parenchymal amyloid β (aβ)-plaques, vascular aβ-deposition, iron and calcium deposition, reactive astrocytes, microglia, as well as fibrin extravasation. the models were trained on digitized histopathological slides from brains of patients with ad and caa, using a workflow that allows neuropathology experts to train convolutional neural networks (cnns) on a cloud-based graphical interface. results: validation of all models indicated a very good to excellent performance compared to three independent expert human raters. furthermore, the aβ and iron models were consistent with previously acquired semiquantitative scores in the same dataset and allowed the use of more complex statistical approaches. discussion: the presented workflow is easy for researchers with pathological expertise to implement and is customizable for additional histopathological markers. the implementation of deep learning-assisted analyses of histopathological slides is likely to promote standardization of the assessment of neuropathological markers across research centers, which will allow specific pathophysiological questions in neurodegenerative disease to be addressed in a harmonized way and on a larger scale.   nd-03 free neuropathol 2:22:25 exercise ameliorates alzheimers pathology across generations a. herring1, y. münster1, n. kurapati1, r. khadzhiev1, k. keyvani1 1 university duisburg-essen, institute of neuropathology, essen, germany introduction: an active lifestyle reduces the risk of dementia and cognitive decline in alzheimer"s diseases (ad) patients. animal studies have shown that environmental enrichment increases the cognitive reserve and interferes with the pathomechanisms of ad over a very extended period of ontogenesis, from in utero stimulation via maternal running during pregnancy until late exercise in a full-blown stage disease. objectives: here we asked whether protective effects of exercise against ad can be inherited across several generations and if so, what epigenetic modifications enable transgenerational transmission. materials & methods: in the founder generation (f0), tgcrnd8 mice (disease onset around postnatal day 90, p90) and wildtype littermates had either access to voluntary wheel running for six months, starting at p30, or were housed without exercise (control). from p90, either exercised mice were mated or mice that experienced a sedentary life style. the offspring was solely housed under control conditions and crossed until the third progenitor generation. the progeny (f1-f3) was phenotyped for memory and explorative behaviour as well as amyloid beta (aβ) pathology in an advanced stage disease (p210). to detect epigenetic transmitters, we determined the cerebral expression profile of micrornas involved in aβ metabolism. results: we here show that great-grandparental running reduced aβ plaque burden, enhanced cerebral levels of the aβ efflux transporter mdr1 and further increased recognition memory and explorative behaviour in transgenic mice until the f3 generation. further, the cerebral expression of micrornas that promote aβ pathology (mirnas 342-3p, 16-5p, 9-5p) was strongly reduced following ancestral exercise. conclusion: we conclude that protective effects of exercise can propagate across generations. ongoing experiments aim to decode further epigenetic modifications that enable transgenerational inheritance.   neurooncology pediatrics nop-01 free neuropathol 2:22:26 co-activation of sonic hedgehog and wnt signaling in murine retinal precursor cells drives ocular lesions resembling intraocular medulloepithelioma m. dottermusch1,2, p. sumislawski1, j. krevet3,4, h. voß5, m. middelkamp1, h. bartsch6, k. sotlar6,7, a. korshunov8, m. glatzel1, u. schüller1,4,9, j. neumann1,2 1 university medical center hamburg-eppendorf, institute of neuropathology, hamburg, germany 2 university medical center hamburg-eppendorf, center for molecular neurobiology (zmnh), hamburg, germany 3 ludwig-maximilians-university munich, center for neuropathology, münchen, germany 4 research institute children’s cancer center hamburg, hamburg, germany 5 university medical center hamburg-eppendorf, institute of clinical chemistry and laboratory medicine, hamburg, germany 6 ludwig-maximilians-university munich, department of pathology, münchen, germany 7 paracelsus medical university, department of pathology, salzburg, austria 8 german cancer consortium (dktk), german cancer research center (dkfz), clinical cooperation unit neuropathology (g380), heidelberg, germany 9 university medical center hamburg-eppendorf, department of pediatric hematology and oncology, hamburg, germany introduction: intraocular medulloepithelioma (io-mepl) is a rare embryonal ocular neoplasm, which prevalently occurs in young children and is commonly treated by enucleation of the eye. io-mepls share common histomorphological features with variants of cns embryonal tumors with multilayered rosettes (etmr), referred to as intracranial medulloepitheliomas. while sonic hedgehog (shh) and wnt signaling pathways are crucial for etmr pathogenesis, the impact of these pathways on human io-mepl development is unclear. objectives: our objective in this study was to explore the relevance of shh and wnt signaling in human io-mepls and demonstrate the effect of simultaneous shh and wnt activation in mouse retinal precursor cells. materials & methods: using nanostring technology, gene expression data was obtained from ffpe tumor samples of 8 human io-mepls, as well as 16 intracranial embryonal tumors comprising etmrs, shh-medulloblastomas (mbs), wnt-mbs and group 4-mbs. in order to unravel the function of shh and wnt signaling for io-mepl pathogenesis in vivo in a time point and cell specific manner, the tamoxifen inducible creert2-lox system was utilized to activate shh and wnt signaling in sox2or raxexpressing retinal precursor cells. results: io-mepls and etmrs displayed similar gene expression patterns and significant simultaneous overrepresentation of both shh and wnt target genes. co-activation of both pathways in early sox2or raxexpressing precursor cells resulted in infiltrative ocular lesions that displayed extraocular expansion. histomorphological, immunohistochemical and molecular features of these lesions revealed strong concordance with those of human io-mepls. conclusion: we report a relevant role of wnt and shh signaling in io-mepl and report a mouse model for this rare disease, setting the foundation for a targeted therapeutic approach with the aim of more commonly feasible eye salvage in affected patients.   nop-02 free neuropathol 2:22:28 characterisation of spinal diffuse midline gliomas l. stegat1, u. schüller1,2,3, a. wefers1 1 university medical center hamburg-eppendorf, institute of neuropathology, hamburg, germany 2 university medical center hamburg-eppendorf, department of pediatric hematology and oncology, hamburg, germany 3 research institute children’s cancer center hamburg, hamburg, germany introduction: diffuse midline gliomas (dmgs) are malignant gliomas that grow in midline structures of the central nervous system. due to their aggressive and diffuse growth and a two-year survival rate of less than 10%, dmgs are graded as who grade iv. depending on the localization, median age of patients is about 11-20 years. genetically, tumors are defined by a k27m-mutation in one of the highly homologous genes encoding histone protein h3. while some studies state that the global dna methylation pattern is correlated with the mutated histone gene, others suggest that it differs between dmgs of different localisations. objectives: since dmgs most frequently occur in pons and thalamus, comparatively little is known about spinal dmgs. therefore, we histologically, molecularly and clinically characterised spinal dmgs and analysed in which aspects they differ from dmgs of other localizations as this may have diagnostical and/or clinical implications. materials & methods: our cohort currently consists of 17 spinal dmgs plus dmgs of different localisations. histological and immunohistochemical analysis as well as molecular analyses (dna methylation, dna panel sequencing) were done from ffpe tissue. clinical data were analysed with prism. results: spinal dmgs were histologically very heterogeneous, both with regard to different areas of single tumors as well as when comparing different tumors. preliminary data suggest that there may be some differences to dmgs of different localisations, e.g. with regard to cell differentiation. first t-sne analyses of dna methylation data did not indicate a separation of spinal dmgs from those of different localisations. 10/11 spinal dmgs sequenced so far were h3f3a k27m-mutant, while one tumor had a hist1h3b k27m mutation. mean age was 22 years. conclusion: first analyses suggest slight histological differences of spinal dmgs compared to those of other localisations. further histological, molecular and clinical analyses are ongoing.   postersession i – neuroinflammation pi-01 free neuropathol 2:22:29 neurofilament light chains in serum are a biomarker for the acute axonal damage in ms lesions: a histological-serological correlative study a. s. beutler1, n. kruse1, l. stork1, m. gloth1, c. stadelmann-nessler1, w. brück1, i. metz1 1 university medical center göttingen, georg-august-university, institute of neuropathology, göttingen, germany introduction: multiple sclerosis (ms) is an inflammatory demyelinating disease of the cns, associated with a variable axonal damage. the axonal damage is responsible for the irreversible clinical disability, and within the cns no relevant neuroaxonal regeneration occurs. thus, in ms patients it is of utmost importance to monitor and hinder the axonal damage from the earliest stages on. a promising biomarker are neurofilament light chains measured in serum (snfl). however, snfl do not provide any information regarding the localization of the neuroaxonal damage, and multiple factors influence snfl levels. objectives: to perform a histopathological-serological correlative study to show the pathological correlate of elevated snfl levels in ms patients. patients and methods: 106 ms subjects with biopsy tissue and blood sample available were characterized for their acute axonal damage with the anti-app staining and the chronic axonal damage with the bielschowsky silver staining. results were correlated with the neurofilament light chain levels in serum measured with the single molecular array (simoa) technique. results: high snfl levels were present in biopsied ms patients (median 59 pg/ml) compared to healthy controls (19 pg/ml), and levels were influenced by the age of patients, relapses prior blood sampling as well as the time interval after biopsy. correlating histological parameters with snfl, multiple regression analyses showed that the acute axonal damage within early active ms lesions significantly contributed to the snfl (p<0.01), but not the axonal density. importantly, snfl correlated with the clinical disability at biopsy as well as at last follow-up. conclusion: snfl mirror the acute axonal damage in early ms lesions as shown by histology, and correlate with the clinical disability of patients. they are thus a promising biomarker to monitor the axonal damage ex vivo in ms patients. this study was sponsored by novartis pharma gmbh.   pi-02 free neuropathol 2:22:30 mgmt-promoter methylation in non-neoplastic diseases of the central nervous system s. teuber-hanselmann1, k. worm2, a. junker1 1 universitätsklinikum essen, institut für neuropathologie, essen, germany 2 universitätsklinikum essen, institut für pathologie, essen, germany introduction: o6-methylguanine-dna-methyltransferase (mgmt) plays a pivotal role in cellular defense by repairing alkylated dna. hypermethylation of the mgmt promoter region results in gene silencing, an effect seen in various tumors, like gliomas. mgmt hypermethylation was postulated to be attributed to cancers or to malignant transformation of precancerous lesions and that consequently it could be used as a tumor biomarker. but while mgmt hypermethylation was also detected in various peripheral inflammatory diseases, it has not yet been investigated in non-neoplastic cns diseases. objectives: to investigate mgmt promoter methylation levels in various non-neoplastic cns diseases. methods: mgmt promoter methylation and expression levels of dna demethylases tet1 and tet2 were investigated via pyrosequencing and immunohistochemistry on human autopsy or biopsy ffpe tissue samples with pml (10), ms (28), toxoplasmosis (6), cmv (1), hsv1 (1) or hiv (2) infection, mycotic encephalitis (4), brain abscesses (3), central pontine / extrapontine myelinolysis (cpm/epm) (8), wallerian degeneration (3) and controls without cns pathologies (8). results: slight mgmt hypermethylation was seen in non-neoplastic cns diseases, all of which were associated with damaged myelin sheaths, i.e. inflammatory and demyelinating (ms and pml), as well as non-inflammatory metabolic or degenerative cns diseases (cpm/epm, wallerian degeneration), while there were no associations with infectious non-demyelinating diseases or distinct pathogens. reduced expression levels of tet1 could possibly be the cause for mgmt hypermethylation. conclusion: mgmt hypermethylation is not restricted to neoplastic or infectious diseases but occurs in chronic cns diseases that lead to damage of the myelin sheath in various ways. while this gives new insights into epigenetic and pathophysiological processes involved in de-/remyelination, it also reduces the specificity of mgmt hypermethylation as a tumor biomarker.   pi-03 free neuropathol 2:22:31 chronic retrotransposon expression in mouse glial cells is associated with spatial learning deficits without an overt inflammatory response r. sankowski1, g. monaco1, m. prinz1 1 universitätsklinikum freiburg, institut für neuropathologie, freiburg i. br., germany introduction: retrotransposons are mobile genomic elements comprising approximately 40 percent of the human and mouse genomes. activation of retrotransposons has been associated with an inflammatory response in the brain and peripheral tissues. we have previously shown that chronic retrotransposon activation in b cell deficient mice was associated with astrogliosis and spatial learning deficits. objective: we were wondering about cell-type dependent retrotransposon expression in the b cell deficient mouse model of constitutive retrotransposon activation. materials & methods: here, we have utilised single-nucleus and single-cell rna-sequencing to profile the expression of retrotransposons in hippocampi and cortices of b cell deficient mice. results: we could identify increased expression of long-terminal repeat and non-long terminal repeat retrotransposons in microglia and astrocytes compared to other cell types. surprisingly, these expression changes were not associated with an increased expression of genes encoding inflammatory mediators. we confirmed these results using bulk rna sequencing. conclusion: our findings underscore the ability of brain tissues to attenuate an inflammatory response despite continuing presence of an immunogenic agent with direct therapeutic implications for unresolving chronic inflammatory conditions.   postersession ii – neurooncology pii-01 free neuropathol 2:22:32 dna methylation-based classification of sinonasal tumors p. jurmeister1, s. glöß2, r. roller3, s. schmid2, r. fritz3, a. thieme2, c. friedrich3, i. hoffmann3, m. leitheiser3, p. mertins4, f. klauschen1, d. capper2 1 ludwig-maximilians-university munich, institute of pathology, münchen, germany 2 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 3 charité – universitätsmedizin berlin, institute of pathology, berlin, germany 4 max-delbrück-centrum für molekulare medizin, berlin, germany introduction: the histopathological diagnosis of sinonasal tumors is challenging as it encompasses a heterogenous spectrum of diverse differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (snucs). objectives: in this study, we aimed to develop a dna methylation-based classification algorithm for sinonasal tumors and to elucidate the molecular background of snucs. materials & methods: we established a cohort of dna methylation profiles from 374 sinonasal tumor specimens, covering 17 tumor entities and normal control tissue. furthermore, we performed dna sequencing and mass spectrometry-based proteomics of selected cases. results: a machine learning algorithm was able to reliably classifiy sinonasal tumors based on their dna methylation profiles with clinical-grade accuracy. we further showed that tumors with snuc morphology can be assigned to four molecular classes defined by distinct epigenetic, mutational and proteomic profiles. this included two classes with clear neuroendocrine differentiation in proteomics analysis which were characterized by either idh2 mutations or swi/snf chromatin remodeling complex (smarca4/arid1a) mutations and overall favorable clinical course. the third group comprised highly aggressive tumors, driven by smarcb1-deficiency. the fourth snuc class contained tumors that actually represented previously misclassified adenoid-cystic carcinomas, which was supported by recurrent myb translocations and the resemeblance of these specimens with serous cell of submucosal glands in proteomics analysis. conclusions: our dna methylation-based machine learning algorithm could greatly improve the diagnostics of challenging sinonasal tumors. furthermore, we provide solid proof that sinonasal tumors are not as undifferentiated as their current terminology implies, but rather represent distinct molecular groups with at least two classes showing clear neuroendocrine differentiation. fig. 1   pii-02 free neuropathol 2:22:34 deep learning aided mitotic figure count in human meningiomas based on the open-source software sliderunner s. jabari1, c. bertram2, r. coras1, a. maier3, i. blümcke1, m. aubreville4 1 university hospitals erlangen, institute for neuropathology, erlangen, germany 2 veterinärmedizinische universität wien, institut für pathologie, vienna, austria 3 lehrstuhl für mustererkennung, fau, erlangen, germany 4 image understanding and medical application of artificial intelligence, ingolstadt, germany introduction: the world health organization (who) classification of brain tumors describes three grades of meningioma: who i-iii. a major factor that distinguishes the classes is the number of mitotic figures. detecting and counting mitotic figures reliably in meningioma is, therefore, an essential part of the diagnostic workup. objectives: however, detecting mitotic figures quickly, reliably, and comprehensively in a tissue section is a major challenge. for this reason, we developed a technical assistance system that detects mitotic figures on whole-slide-images and visualizes these as a heat map showing the mitotic density in almost real-time. patients & methods: meningioma samples were retrospectively collected from the dept. of neuropathology erlangen, germany. all samples were rereviewed by an expert neuropathologist and subsequently digitized using the hamamatsu s60 digital slide scanner. the slides have been completely annotated for mitotic figures and we provide secondary annotations for mitotic figure look-alikes. additionally to a blinded two-expert manual annotation, we developed an algorithm-aided dataset, where potentially missed mitotic figures were detected by a deep neural network and subsequently assessed by a human expert. we used a pretrained deep-neuronal network approach (retinanet) to validate our data set. finally, a plugin for sliderunner, an open-source software, was built for convenience enabling real-time identification of mitotic figures in virtual slides. results: out of 69 whole slide images of the complete data set, 46 were randomly assigned to the training set and 23 to be the test set. a preliminary evaluation yielded an f1 score of 0.659. conclusion: error-prone and tedious mitotic figure counts and region of interest identification may be assisted in the future with the help of modern deep learning algorithms integrated into a user-friendly software suite.   pii-03 free neuropathol 2:22:35 intracranial ependymoma in adults, a methylome-based analysis m. träger1, l. schweizer2,3, p. vajkoczy4, k. fukuoka5, k. ichimura5, u. schüller6, l. dührsen7, m. müther8, w. paulus9, c. thomas9, j. schittenhelm10, f. eckert11, k. m. niyazi12, d. f. fleischmann12, m. dorostkar13, p. feyer14, s. a. may15, d. moskopp16, h. badakhshi17, s. wecker17, c. radke18, j. walter19, d. capper2,3, d. kaul1,3 1 charité – universitätsmedizin berlin, department of radiation oncology, berlin, germany 2 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 3 german cancer consortium (dktk), partner site berlin, german cancer research center (dkfz), heidelberg, germany 4 charité – universitätsmedizin berlin, department of neurosurgery, berlin, germany 5 national cancer center research institute, division of brain tumor translational research, tokyo, japan 6 university medical center hamburg-eppendorf, institute of neuropathology, hamburg, germany 7 university medical center hamburg-eppendorf, department of neurosurgery, hamburg, germany 8 university hospital münster, department of neurosurgery, münster, germany 9 university hospital münster, institute of neuropathology, münster, germany 10 university hospital tübingen, department of neuropathology, tübingen, germany 11 university hospital tübingen, department of radiation oncology, tübingen, germany 12 university hospital, lmu munich, department of radiation oncology, münchen, germany 13 ludwig-maximilians-university munich, center for neuropathology, münchen, germany 14 vivantes hospital neukölln, berlin, germany 15 klinikum chemnitz, department of neurosurgery, chemnitz, germany 16 vivantes klinikum im friedrichshain, department of neurosurgery, berlin, germany 17 ernst von bergmann medical center, academic teaching hospital of humboldt university berlin (charité), department of clinical radiation oncology, potsdam, germany 18 ernst von bergmann medical center, academic teaching hospital of humboldt university berlin (charité), department of pathology, potsdam, germany 19 medical center saarbruecken, department of neurosurgery, saarbrücken, germany introduction: recently dna methylation profiling became widely accepted as a method to identify biologically distinct subgroups of ependymoma. however, due to the rarity of these tumors in adults, previous studies have failed to achieve a sufficient number of patients to characterize the landscape of dna methylation profiles, their clinical characteristics and prognosis in this age group. objectives: we investigated dna methylation profiles of adult intracranial ependymoma collected from 12 different centers and sought to correlate molecular subgroups to clinical characteristics and outcome. materials and methods: tumors from 157 adult patients histologically classified as who grade ii or iii intracranial ependymoma between 1990 and 2020 were subjected to dna methylation profiling using the illumina human methylation 450k or epic bead chip platform. profiles were matched with the dkfz brain tumor classifier and detected molecular subgroups were correlated with overall survival (os) and progression free-survival (pfs). results: in 68.8% (108/157) of cases, the suggested methylation class confirmed diagnosis of an ependymal tumor with the additional assignment of a molecular subgroup (49 pfb, 36 se, 17 rela, 3 pfa, 2 spinal epn and1 myxopapillary epn) whereas 5.7% (9/157) were not compatible with the initial diagnosis of ependymoma. the remaining 25.5% (40/157) did not reach a calibrated score ≥0.9 and were considered not classifiable. median follow-up was 54 months. five and 10-year pfs rates were 54.1% and 40.6% for se, 65.2% and 37.1% for pf-b, 49% and 49% for rela tumors. the 5and 10-year os rates were 70.1% and 49.1%for se, 95.4% and 85.5% for pf-b and 100% and 75% for rela tumors. conclusion: this cohort of adult intracranial ependymoma comprises various molecular subgroups and even different tumor entities. most patients with ependymal tumors confirmed by dna methylation profiling show a favorable prognosis. however, factors that affect survival need to be determined.   poster session iii – muscle & nerve piii-01 free neuropathol 2:22:37 imaging of extended tissue areas by automated large-scale electron microscopy c. dittmayer1, h. h. goebel1,2, f. l. heppner1, w. stenzel1 1 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 2 johannes-guttenberg universität, department of neuropathology, mainz, germany introduction: traditional imaging of sections in diagnostic or research settings using transmission electron microscopy (tem) is limited by manual acquisition procedures. thus, analysis of samples is restricted to images of few preselected regions that may be non-representative and lack correlation to their microanatomical context. modern scanning electron microscopes allow unbiased digitization of entire large sections and improved analysis, but performance is limited by preparation artifacts. objectives: we aimed at an improved preparation of sections for large-scale electron microscopy (em) and at a pipeline for data processing and analysis. material & methods: we achieved wrinkle-free collection of sections by a combined glow-discharge and ethanol-based smoothening procedure and reduced filming and staining artifacts. a scanning electron microscope with a scanning transmission electron microscopy detector was used for pre-irradiation and imaging. imaging parameters were varied to achieve fast imaging and adequate image quality. we used trakem2 and nip2 for stitching of image tiles to generate coherent bigtif datasets, thus allowing in-depth analysis via qupath. results: our preparation workflow allowed to reliably prepare samples with virtual absence of limiting artifacts, such wrinkles and stain precipitates. up to 12 entire sections were automatically digitized in about 6 days using 7.3 nm pixel size and 1.0 µs dwell time. our data processing pipeline allowed batch generation of bigtif files with minimal operator involvement. conclusion: we propose a highly reliable workflow for preparation, imaging and data processing of samples with superior quality to enable routine large-scale em in diagnostic and research settings. we demonstrate the high pragmatic value of our workflow with data of different published and unpublished projects in the field of muscle pathology and ultrastructural characterization of sars-cov-2 in autopsy tissues.   piii-02 free neuropathol 2:22:38 nanostring technology distinguishes anti-tif-1γ+ from anti-mi-2+ dermatomyositis patients c. preuße1,2, p. eede1, l. heinzeling3,4, k. freitag1,5, r. koll1,6, w. froehlich3, u. schneider7, y. allenbach8, o. benveniste8, a. schänzer9, h. h. goebel1, w. stenzel1, j. radke1,6 1 charité – universitätsmedizin berlin, neuropathology, berlin, germany 2 münster university hospital, neurology with institute for translational neurology, münster, germany 3 university hospital of erlangen, dermatology, erlangen, germany 4 lmu klinikum, dermatology, münchen, germany 5 german center for neurodegenerative diseases (dzne) within the helmholtz association, berlin, germany 6 german cancer consortium (dktk), german cancer research center (dkfz), berlin, germany 7 charité – universitätsmedizin berlin, rheumatology, berlin, germany 8 pitié-salpêtrière university hospital, internal medicine and clinical immunology, paris, france 9 justus-liebig-university gießen, neuropathology, gießen, germany introduction: dermatomyositis (dm) is a systemic idiopathic inflammatory disease affecting skeletal muscle and skin, clinically characterized by symmetrical proximal muscle weakness and typical skin lesions. recently, myositis-specific autoantibodies (msa) became of utmost importance because they strongly correlate with distinct clinical manifestations and prognosis. antibodies against transcription intermediary factor 1γ (tif-1γ) are frequently associated with increased risk of malignancy, a specific cutaneous phenotype and limited response to therapy in adult dm patients. anti-mi-2 autoantibodies, in contrast, are typically associated with classic dm rashes, prominent skeletal muscle weakness, better therapeutic response and prognosis, and less frequently with cancer. the correlation of cancer and mi-2 autoantibodies, however, remains debated. objectives: to further investigate the both clinically distinct dm subgroups and visualize differences or similarities on gene and protein level. patients & methods: we analyzed 30 dm patients (n=15 mi2+ and n=15 tif-1γ+) and 8 non-disease controls (ndc) by nanostring, qpcr, and immunohistochmistry. results: both anti-tif-1γ+ and anti-mi-2+ patients" skeletal muscle biopsies revealed strong dysregulation of immune response-related genes compared to ndcs. in line with previous reports, these included well-known type 1 ifn-inducible genes; however, we also identified subgroup-specific differences. conclusion: our results help to stratify both subgroups and predict, which dm patients require an intensified diagnostic procedure and might have a poorer outcome. additionally, we demonstrate that the nanostring technology can be used as a very sensitive method to clearly differentiate two clinically distinct dm subgroups, namely mi2+ and tif-1γ+ dermatomyositis. potentially, this could also have implications for the therapeutic approach.   piii-03 free neuropathol 2:22:40 immunomodulatory function of gm-csf in idiopathic inflammatory myopathies d. cengiz1,2, t. müntefering2, j. schubert1, c. preuße3, w. stenzel3, s. meuth2, t. ruck2 1 clinic for neurology with institute for translational neurology, university hospital, münster, germany 2 heinrich-heine university düsseldorf, department of neurology, medical faculty, düsseldorf, germany 3 charité universitätsmedizin, department of neuropathology, berlin, germany introduction: idiopathic inflammatory myopathies (iims) are characterized by chronic inflammation of the muscle, resulting in muscle weakness and pain. pathogenesis is driven by the interaction of skeletal muscle, muscle endothelial and immune cells. cytokines play a central role in controlling these interactions. gm-csf is a cytokine, which is significantly involved in the development of autoimmune diseases and is currently being investigated in clinical trials for therapy. objectives: the role of gm-csf in the inflammatory processes of iims is largely unknown. therefore, the aim of this work is to investigate its immunomodulatory function. materials & methods: murine skeletal muscle and endothelial cells were stimulated with gm-csf and other proand anti-inflammatory cytokines. to analyze the impact of these conditions, flow cytometric analysis was performed. furthermore, the immunological relevance of gm-csf on immune cell migration and adhesion were investigated. the expression of gm-csf was also evaluated in human muscle biopsies of iim patients. results: stimulation with proand anti-inflammatory cytokines regulate the expression of gm-csf and the gm-csf receptor in muscle and endothelial cells. gm-csf led to increased expression of costimulatory molecules on skeletal muscle cells, as well as decreased expression of adhesion molecules on endothelial cells. stimulation also resulted in increased immune cell migration across muscle endothelial barriers. in human muscle biopsies of iim patients, histological analysis revealed high expression of gm-csf. conclusion: the gm-csf signaling pathway affects the cellular interactions of muscle, endothelial, and immune cells in the skeletal muscle. these findings could contribute to elucidate autoimmune processes in iims and identify new therapeutic targets.   piii-04 free neuropathol 2:22:41 effect of vasoactive therapy in patients with systemic sclerosis on dermal small nerve fibers, langerhans-cells and vessel density. l. bajors1, f. höcketstaller2, m. köhm2,3, s. seidel4, r. wolf5, a. roth1, u. drott2,6, a. schänzer1 1 justus-liebig-university gießen, institute of neuropathology, gießen, germany 2 goethe university frankfurt, department of rheumatology, frankfurt a. m., germany 3 goethe university frankfurt, fraunhofer project group translational medicine and pharmacology, ime, frankfurt a. m., germany 4 goethe university frankfurt, institute of cell biology and neuroscience and buchmann institute for molecular life sciences (bmls), frankfurt a. m., germany 5 philipps university marburg, department of dermatology and allergology, marburg, germany 6 median klinik schlangenbad, department of rheumatology, wiesbaden, germany introduction: systemic sclerosis (ssc) is an orphan immune-mediated disease affecting multiple organ systems. most commonly, clinical symptoms such as skin fibrosis and digital ulceration occur early in disease course. here, vasculopathy is an underlying aetiology. additional, small nerve fibers (snf) might be affected in in ssc patients and contribute to disease progression. material and methods: 21 ssc patients received intravenous medication iloprost for 10 days (95% females; 56,1±11,9 years). 7 healthy subjects served as controls (71% f; 51,6±18,3 y). skin biopsies were taken from the volar forearm before and 3 months after treatment. epidermal nerve density (end), langerhans cells (lc) and vessel density (vd) were analysed at immunofluorescence-stained sections using a zeiss axioscanner and imagej software. the skin pathology was categorized with a histological skin score. results: the ssc group showed no significant difference of end and lc density (13,09±4,26 snf/mm; 259,8±121,5 lc/mm²) compared to controls (10,8±3,11 sfn/mm, p=0,198; 335,4±124,86 lc/mm², p=0,091) or after treatment (12,71±5,82 snf/mm, p=0,49; 305,4±98,5 lc/mm², p=0,098). the vd was significantly increased in the ssc group compared to controls (3,01±1,10µm²/100µm²; 2,18±1,12µm²/100µm²; p=0,046). in the ssc group, the vd was significantly decreased after treatment (2,51±0,78µm²/100µm²; p=0,044). the histological skin score showed overall low fibrotic alterations in both groups. conclusion: in our study, ssc patients showed a higher vd compared to controls and vd was affected during short-term iloprost-therapy. megacapillaries is a key feature of ssc and the results should be correlated with results of capillary microscopy. end and lc were not altered during treatment in ssc patients. maybe these findings depend on the low skin score showed in the patients. to investigate further correlations, the results will be correlated with clinical parameters.   poster session iv – neurodegeneration piv-01 free neuropathol 2:22:42 the interplay between neuroimmune activation and blood brain barrier dysfunction in alzheimer's disease m. armbrust1, a. dominguez-belloso1, r. figueiredo2, k. gneiding1, s. guérit1, s. thom1, p. winter2, k. h. plate1, p. n. harter1, k. devraj1, s. liebner1 1 goethe university hospital, neurological institute, edinger institute, frankfurt a. m., germany 2 genxpro gmbh, frankfurt a. m., germany introduction: alzheimer's disease (ad) is characterized by amyloid-β aggregation, hyperphosphorylated tau protein, neuroimmune activation, neuronal loss and blood brain barrier (bbb) dysfunction. the contribution of the latter to disease genesis and progression is poorly understood. objectives: to investigate wnt/β-catenin and bbb-specific alterations in ad. materials and methods: transgenic mice harbouring triple-mutated (swdi) human app were used for 3'-rna-sequencing (massive analysis of cdna ends: mace) of isolated mouse brain microvessels (mbmvs) and of facs-sorted cells of the neurovascular unit (endothelial cells (ecs), mural cells, astrocytes and microglia (mg)). an activation of the wnt/β-catenin pathway in ecs was conducted in a cre-loxp-based β-catenin gain-of-function (gof)-ad mouse model. tracer permeability assays, immunohistochemistry and immunofluorescence analyses as well as behavourial tests were performed in ad and ad-gof mice. results: gene ontology analysis revealed a downregulation of wnt/β-catenin signalling in mbmvs and facs-sorted ecs of ad mice. dkk2, encoding dickkopf wnt pathway inhibitor 2, was upregulated in mbmvs and mg. in human ad brain tissue, dkk2 was detected in mg. additionally, an increase in bbb permeability correlating with cognitive decline and a decrease of vascular lymphoid enhancer-binding factor 1 expression, a target and key player in wnt/β-catenin signalling, was observed in ad mice. a presymptomatic activation of the wnt/β-catenin pathway in ecs of gof-ad mice partially prevented cognitive decline. conclusion: dkk2 upregulation in microvessel-associated mg could contribute to the wnt/β-catenin pathway repression observed at the bbb in ad. together with the prevention of cognitive decline after presymptomatic activation of the wnt/β-catenin pathway, our findings indicate a potential link between neuroimmune activation and bbb dysfunction and propose the wnt/β-catenin pathway as a potential therapeutic target.   piv-02 a deep learning approach for monitoring parietal-dominant alzheimer's disease in world trade center responders at midlife not available for publication   piv-03 free neuropathol 2:22:44 amyloid-β dimers (aβ-s8c) are antiprions inhibiting seeded nucleation in vivo e. van gerresheim1, a. müller-schiffmann1, s. schäble2, c. korth1 1 heinrich-heine university düsseldorf, neuropathology, düsseldorf, germany 2 heinrich-heine university düsseldorf, experimental psychology, düsseldorf, germany introduction: alzheimer"s disease (ad) is a neurologic disorder, were amyloid-beta (aβ) aggregation causes massive neuronal death. aβ expansion can follow prion-like replication from aβ seeds. with the current animal models, based on the overexpression of aβ, used to study the aβ cascade and prion-like expansion, it is impossible to distinguish between exogenous de novo seeding and accelerated spontaneous plaque development. so far, seeding of aβ in wild type animals has not been successful. objectives: here we studied the effects of aβ-s8c dimers on aβ prion spreading and induced behavioral changes. materials and methods: tgdimer mice, expressing human app751 (swe, k670 n/ m671l, s679c), only develop aβ-s8c dimers and do not develop aβ plaques, but do associate to existing plaques, were crossed with gfap-luc mice, enabling bioluminescence (bli) monitoring of aβ aggregation-associated astrogliosis. mice were intra-hippocampal inoculated with aβ aggregates. wild type and app23 mice, aβ aggregate inoculated, were used as controls. cognition was probed in the observer-free paradigm, the cognitionwall, for reward-dependent learning and burrowing as species-specific behavior. results: we observed no aβ aggregation-associated astrogliosis or impaired cognition in the tgdimer mice inoculated with aβ aggregate. for the positive control, the app23 mice, an increase in bli signal started at 11 months of age and at 18 months of age, app23 mice showed impaired discrimination learning in the cognitionwall. conclusions: aβ-s8c dimers inhibit aβ aggregate spreading and are thus anti-prions. they may have a regulatory role for aggregate spreading suggesting that aβ prion effects are actively regulated rather than a passive process. the difference in time of start of aβ aggregate spreading and impaired discrimination learning in app23 mice suggests that behavioral consequences are disparate from initial neuropathological signs.   piv-04 free neuropathol 2:22:45 a decline in peroxisomal numerical abundance occurs late during alzheimer´s disease progression and is particulary found in the hippocampus e. semikasev1, b. ahlemeyer1, t. acker2, a. schänzer2, e. baumgart-vogt1 1 institute of anatomy and cell biology, medical cell biology, jl university, gießen, germany 2 institute of neuropathology, jl university, gießen, germany peroxisomal biogenesis disorders are inherited metabolic diseases caused by mutations in the pex genes. in severe forms, patients have profound defects in the brain demonstrating the importance of this organelle for neuronal survival and function. additionally, dysfunction of the peroxisomal metabolism, e.g. plasmalogen synthesis or the degradation of very long-chained fatty acids, has to been linked to neurodegenerative diseases such as alzheimer´s disease (ad). to study the role of peroxisomes in ad, we analyzed the density of this organelle in the perikarya of pyramidal cells in the hippocampus (cornu ammonis, subiculum and the entorhinal cortex layer iii) as well as neocortical regions (frontal, temporal, parietal und occipital) of brain from patients with different neuropathological stages. we used double immunofluorescence staining techniques to detect pex14 (as marker for the total peroxisome numerical abundance) and β-amlyoid plaques or neurofibrillary tangles (nfts)s in different brain regions. human brains were genderand age-matched and classified into 5 patient groups based to the abc-score. all 42 brain samples were from the institute of neuropathology in gießen. quantification of peroxisome number per area (density) was performed by imagej free software. with ongoing stages of ad, we measured a progressive decline in the density of peroxisomes in pyramidal cells of all hippocampal and neocortical areas. the effect was even more pronounced when we included known ad risk factors such as vascular angiopathies. interestingly, the decrease in the density of peroxisomes at late stages in ad was more evident in patients ≤80 years old than in patients >81 years old. however, we did not find a correlation between the intraneuronal peroxisome density and the amount of β-amyloid and nfts. our data suggest that peroxisomes may play a protective role in ad and their decline in numerical density – especially in the hippocampus – occurs late during ad progression.   piv-05 free neuropathol 2:22:46 its not all about covid19 incidental finding of hereditary neuroferritinopathy in a 78-year-old woman diagnosed with covid-19 associated pneumonia v. umathum1,2, d. amsel1, a. weber3, c. becker4, c. kasan4, a. may5, t. acker1, a. schänzer1 1 justus-liebig-university gießen, institute of neuropathology, gießen, germany 2 bundeswehrkrankenhaus ulm, institute of pathology and molecular pathology, ulm, germany 3 justus-liebig-university gießen, institute of human genetics, gießen, germany 4 institute of pathology, cytology and molecular pathology, wetzlar, germany 5 agaplesion evangelisches krankenhaus, department of pulmonology, gießen, germany introduction: neuroferritinopathy (nf) is an autosomal-dominant movement disorder due to mutations in the l-ferritin gene (ftl) on chromosome 19q13.33. nf belongs to neurodegeneration with brain iron accumulation (nbia) disorders, a group of hereditary neurodegenerative diseases with a prevalence of 1/1.000.000. patients with nf present clinically with progressive extrapyramidal movement symptoms with variable phenotype. histopathologically, nf is characterized by iron deposits and formation of ferritin inclusion bodies (ibs). clinical history: a 78-year-old woman was hospitalized with a severe covid-19 pneumonia and after three months died from covid19-associated pneumonia. during her 3-month clinical course she showed neurological symptoms of delirium and restlessness. cranial mri was not performed. neuropathological findings: brain weight was 1172 g. no abnormalities were seen in cross sections. histological examination revealed frequent atypical iron accumulation throughout the brain particularly in the basal ganglia. the nuclear spherical iron inclusions were localized in neurons, microglia and oligodendrocytes stained for prussian blue and immunohistochemically with antibodies against anti-ferritin light chain. electron microscopy showed fragmented nuclei with inclusions and chromatin accumulation toward the membrane consistent with ibs. moderate microcystic vacuolization and partial loss of myelination of nerve fibres were evident. discussion and conclusion:131 cases of nf have been described so far in the literature with 80% in france and the uk and 20% throughout the world. movement disorders or cognitive impairments were not reported in our patient, maybe due to a lack of full penetrance that occurs at the age of 60, in general. in our patient, the brain pathology is suspicious for a nf. most of the patients harbour an adenine insertion at position 460-461 of the ftl-gene. whole exome sequencing from brain tissue is in progress to confirm the diagnosis.   piv-06 free neuropathol 2:22:47 role of purinergic receptor p2y12 in alzheimer's disease r. maruccia1,2, a. a. gabr1, m. j. pietrowski1, b. a. tambe1, a. halle1,2 1 deutsches zentrum für neurodegenerative erkrankungen, bonn, germany 2 universitätsklinikum bonn, institut für neuropathologie, bonn, germany microglia have recently moved into prime focus of ad research due to the discovery of risk loci and genes that are linked to immune mechanisms, some of them highly expressed in microglia. however, immune signaling pathways in microglia that modulate ad progression, for example by removing aβ plaques, have remained poorly understood. the purinergic receptor p2y12 is activated by extracellular atp and adp and is highly expressed in platelets and microglia. p2y12 is crucial for several microglial functions such as chemotaxis, blood-brain barrier closure, synaptic plasticity, interaction with neuronal somata and phagocytosis. thus, a compromised microglial phagocytic removal of aβ and impaired plaque barrier formation upon p2y12 deletion may affect ad progression. here we analyze 3d-reconstructed confocal images of brain tissue from ad models to assess the role of p2y12 in the progression of aβ pathology, microglia-plaque interaction and inter-cellular interaction between microglial cells under neurodegenerative conditions. our preliminary findings suggest a region-dependent modulation of microglial coverage and co-localization with plaques in models with p2y12 deletion and therefore that this receptor may affect microglial interaction with aβ plaques. future studies investigating plaque load changes and the interplay between microglia, astrocytes and neurons will be needed to better understand inter-cellular pathways that contribute to p2y12-related effects in ad pathology. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. intraoperative confocal laser endomicroscopy for brain tumors potential and challenges from a neuropathological perspective feel free to add comments by clicking these icons on the sidebar free neuropathology 3:24 (2022) opinion pieces intraoperative confocal laser endomicroscopy for brain tumors potential and challenges from a neuropathological perspective theoni maragkou1, karl quint2, bianca pollo3, ekkehard hewer4 1 institute of pathology, university of bern, bern, switzerland 2 quint healthcare, fürth, germany 3 unit of neuropathology, fondazione irccs istituto neurologico carlo besta, milano, italy 4 institute of pathology, lausanne university hospital and university of lausanne, lausanne, switzerland corresponding author: ekkehard hewer · institut universitaire de pathologie · bugnon 25 · ch-1011 lausanne · switzerland ekkehard.hewer@chuv.ch submitted: 16 august 2022 accepted: 21 october 2022 copyedited by: georg haase published: 23 november 2022 https://doi.org/10.17879/freeneuropathology-2022-4369 keywords: confocal laser endomicroscopy, intraoperative consultation, digital biopsies, brain tumors, resection margin, in vivo microscopy abstract confocal laser endomicroscopy (cle) represents a new non-invasive in vivo imaging technique that holds considerable promise in neurosurgery and neuropathology. cle is based on the principle of optical sectioning which uses pinholes placed in the light path to selectively image photons of a specific focal plane by filtering out photons above and below the focal plane. potential indications of cle in neurosurgery and neuropathology include intraoperative tumor diagnosis and staging as well as assessment of tumor resection margins notably in the case of diffusely infiltrating gliomas. cle-based tumor analysis in near-real time may also have a significant impact on future tumor resection strategies. we here discuss the technical features of cle, its potential for wide-field imaging, its role in comparison to established histological techniques for intraoperative tumor assessment and its position in digital pathology and telepathology. based on our group’s experience with a commercially available confocal laser endomicroscope (zeiss convivo), we critically address the current state of intraoperative cle in brain tumor surgery, the applicability of classical histological criteria and the strategies required to further improve the diagnostic accuracy of cle. we finally discuss how a widespread use of cle in neurosurgery may modify the role of neuropathologists in intraoperative consultation, generating both new opportunities and new challenges. introduction intraoperative consultation in the form of frozen section analysis and/or intraoperative cytology is an integral part of neuropathology. its relative importance compared to other diagnostic activities is arguably higher in neuropathology than in most other domains of pathology. this is due to a variety of reasons, including (a) the high percentage of brain tumor surgeries among all surgical procedures, (b) the absence of preoperative biopsy in most cases meaning that intraoperative consultation is usually the first opportunity to obtain a tissue-based diagnosis, (c) the large variety of tumor types with often overlapping imaging features and (d) the difficulty to distinguish tumoral tissue macroscopically from non-tumoral tissue. nevertheless, intraoperative consultation has not benefited from any major improvements for several decades and remains currently afflicted by the drawbacks of established histological and cytological methods. these comprise tissue freezing leading to morphological artifacts, tissue consumption, delays in the transfer of tissue samples from the operating room to the laboratory and difficulties in cell and tissue processing. in vivo microscopy based on various techniques such as confocal laser endomicroscopy (cle) or optical coherence tomography has been used for a long time in ophthalmology and has become available for clinical routine for different indications such as gastrointestinal diseases (1), pulmonary mucosal lesions or urological disorders. our opinion piece is based on our own experience with a commercially available zeiss convivo confocal laser endomicroscope in both preclinical and clinical settings. we here first review technical aspects of cle and then describe the characteristics of the first clinical-grade cle device available for neurosurgical applications. finally we discuss potential advantages and limitations of in vivo cle as well as its impact on diagnostic practice in surgical neuropathology. with this contribution to the scientific literature, we aim to inform potential future users, to stimulate discussion and research on cle, and to discuss the potential benefit of cle in neurosurgery and neuropathology. limitations of histological and cytological methods in neuropathology and neurosurgery traditionally, intraoperative diagnosis in neurosurgery has relied on standard pathological techniques such as frozen sections and intraoperative cytology, which are followed by analysis of formalin-fixed, paraffin-embedded (ffpe) tissue for definite diagnosis. while these latter techniques are still being considered the gold standard, some of their drawbacks are prolonged time for sampling, transferring, processing and interpreting the samples, artifacts and sampling errors, all which limit their applicability for rapid interpretation of tissue samples. in addition, standard pathological techniques involve a multitude of different processes and specialized personnel. in the case of ffpe tissue, they involve tissue fixation, dehydration, rinsing, embedding in hot paraffin, cooling, freezing in preparation for cutting, microtome cutting, transferring to glass slides, and drying. in routine hematoxylin and eosin (h&e) staining, several more steps are being performed: dewaxing, dehydration, hematoxylin staining, differentiation in mild acid, bluing, eosin staining, dehydration, clearing and cover-slipping, with various washing and alcohol incubation steps in between. all these procedures can take up to one full day for basic staining, and several more days for advanced subsequent investigations such as immunohistochemistry and molecular analyses (2–4). for digital pathology, the final slides need to be scanned, processed, analyzed and archived. confocal laser endomicroscopy in neurosurgery the disadvantages of traditional histologic and cytologic techniques are increasingly being overcome by newer technologies. confocal laser endomicroscopy (cle) allows in vivo noninvasive histological imaging using optical sectioning. by placing pinholes in the light path, optical sectioning enables the microscope to selectively image a specific focal depth by mechanically filtering most out-of-focus photons above and below the focal plane. this allows in situ visualization of tissue with higher contrast compared to conventional wide-field microscopy, eliminating out-of-focus background and scattered light. in addition, endomicroscopy allows for three-dimensional image reconstruction and greater variety of imaging parameters. most importantly, this technology enables real-time histopathological analysis with increased diagnostic yield without the traditional histopathological process of excision, fixation and staining. only recently this new technological advancement has been applied to neurosurgery, by using a wide range of fluorescent dyes as contrast enhancers. a single clinical-grade device for cle, the zeiss convivo, is hitherto commercially available with clearance by the food and drug administration (fda) of the united states of america and ce marking in the european union. routine clinical experience with the cle device is currently being investigated in several clinical trials. preclinical research indicates that the cle device could improve tumor visualization at the tumor margin and speed up intraoperative diagnosis. furthermore, the cle device is designed to be used directly in the operating room and to allow telepathology, thus bringing the neuropathologist even closer to the neurosurgeon. confocal laser endomicroscopy beyond neurosurgery standard histology and confocal laser endomicroscopy are not interchangeable nor can one method fully replace the other. tissue samples can be analyzed by standard histology and subsequent elaborated investigations. both techniques are time-consuming which can become limiting in the case of frozen sections. by contrast, confocal laser microscopy of tissues yields almost instantaneous information but is not amenable to traditional subsequent analyses that require a resected physical sample. it might, however benefit from new analytical methods such as artificial intelligence. such methods are being increasingly employed in radiology and termed radiomics or radiogenomics. unlike the situation in neurosurgery and neuropathology, cle has already been adopted by multiple medical specialties including gastroenterology, pulmonology otorhinolaryngology and urology since early the 2000s. it is therefore worthwhile to analyze how cle has been integrated in the workflows of these medical specialties (table 1). in gastroenterology for instance, cle has proven its clinical utility for the diagnosis of esophageal premalignant lesions and neoplasms such as barrett’s esophagus with low-grade and high-grade dysplasia (5,6) and esophageal cancer (7). the usefulness of cle in the detection of esophageal, stomach and colon cancer has moved beyond early proof of concept to large clinical trials (8–14). table 1: selected clinical in vivo studies reporting interobserver agreement for cle imaging and consistency of cle-based diagnosis with histopathology in various precancerous lesions and cancer entities n.r.: not reported, cle: confocal laser endomicroscopy, wlc: white light cystoscopy, fna: sodium fluorescein as for all new methods, interpretation criteria for cle have to be developed, validated and adopted by the medical community. while interpretation guidelines for cle images are in their infancy in neuropathology, various interpretation criteria and classification systems have been developed and validated in urology and gastroenterology owing to the earlier adoption of cle in these areas. these diagnostic systems range from simple criteria such as the presence of dark aggregates of cells as the cle criterion for malignant mucinous lesions of the pancreas (15) to elaborate morphological criteria and diagnostic scores such as the ones published for dysplasia for barrett’s esophagus (16,17), oral squamous cell carcinomas (18) and bladder cancer (19) and others. these diagnostic systems use features of tissue and cellular architecture found in cle images to distinguish normal from malignant tissue, or to distinguish between different types of lesions. scoring methods assign positive and negative scores to specific tissue or cell features. if the score is beyond a predetermined threshold, there is a very high likelihood of malignancy (18). similar to all diagnostic procedures requiring human interpretation, cle is also affected by the pathologist’s subjectivity. in medical disciplines that require interpretation of diagnostic images, such as pathology and radiology, interobserver studies are commonly done to ensure that results are reproducible. diagnostic methods showing higher levels of agreement between observers are deemed more reliable. while this aspect has not yet been fully investigated for brain tumors, reports on interobserver agreement exist in various tumor entities (table 1). confocal laser endomicroscopy in the context of digital pathology and telepathology implementation of cle will not happen in a void, but in the context of existing workflows and current developments. first, cle integrates well into the current movement towards digital pathology. a fundamental challenge of digital pathology is digital microscopy, i.e. the adoption of digital images. this made workflows more complex in almost all applications as glass slides cannot be eliminated but remain the source of the ultimate digital image. this contrasts with the situation in digital radiology where workflows were simplified by the elimination of films. from this perspective, cle as well as other techniques for in vivo microscopy offers the advantage of being intrinsically digital which has a number of positive consequences, including the absence of tissue consumption, the lack of a delay for scanning glass slides and the possibility of teleconsultation. in the conceptual framework of telepathology (table 2), cle is most similar to dynamic telemicroscopy since it is potentially very interactive with the possibility of real-time discussion of findings with the neurosurgeon who holds the cle probe and needs to identify areas of interest, and with a second person who operates the cle device. table 2: paradigms for telepathology and their respective advantages and limitations confocal laser endomicroscopy in the context of intraoperative consultation: strengths and limitations traditionally, frozen section and/or cytological preparations are used in variable combination for intraoperative diagnosis in most centers. both techniques have specific strengths and limitations. while frozen sections provide more information on the tissue architectural level and are more similar to histological slides, cytological preparations provide better nuclear morphological details and are not susceptible to freezing artifacts. given that intraoperative cytology requires only minimal technical equipment, it is performed on-site in some centers which eliminates transfer to the pathology laboratory and accelerates processing. it requires however the presence of neuropathologists on-site or the presence of equipment for telepathology. cle will likely result in faster turnaround times even than on-site cytology while being intrinsically prone to teleconsultation. this may turn out particularly advantageous given the limited availability of neuropathologists in many places. the implementation of cle in clinical routine will however require both neurosurgeons and neuropathologists to adopt a novel conceptual approach. table 3: time requirement for intraoperative microscopic diagnosis using cle and competing techniques the ultimate clinical utility of cle may very well differ in terms of specific indications. when neuropathologists are required to assess sample adequacy for diagnosis and ancillary diagnostic techniques, the highest possible diagnostic accuracy probably obtained with frozen section and cytology will arguably outweigh any advantage in turnaround time or non-invasiveness of cle. conversely, cle may turn out as the method of choice for the identification of residual tumor cell infiltration in the periphery of resection cavities, because its speed and non-invasiveness allow for much more extensive sampling than tissue biopsies. cle thus has the potential to change current paradigms for maximal safe resection of brain tumors. a hybrid approach combining cle and tissue biopsies may be envisioned for intraoperative diagnosis. here, cle would provide a faster response, while frozen section or cytology would serve as confirmatory techniques. table 4: expected potential of cle to supplement or replace frozen sections any novel technique can potentially add something to the existing armamentarium, either by adding precision as historically seen with gene sequencing or methylome analysis or by providing information even if limited more rapidly than existing techniques. current diagnostic practice in neuropathology is characterized by a whole set of different diagnostic modalities. in terms of turnaround times, any technique that is slower than existing ones may be of interest if it adds, either alone or as a complement to other techniques, diagnostic precision. hematoxylin-eosin-stained slides of ffpe tissue will typically be available within one day, immunohistochemical stainings may require an additional working day. based on this, patients may be informed about their diagnosis before they leave hospital. molecular analyses often require around one week which is sufficient for discussion in a multidisciplinary tumor conference before starting adjuvant therapy. frozen sections and cytology are sufficiently fast for intraoperative consultation, which makes a somewhat lower precision than that of histological slides acceptable. their turnaround times are however typically too long for extensive repetitive sampling. therefore, cle may prove clinically highly useful by allowing repeated analysis or analysis of multiple sites, even if its accuracy were ultimately found to be somewhat lower than that of frozen sections (figure 1). figure 1: schematic illustration of turnaround time versus diagnostic precision. an ideal technique would fit in the upper left corner, i.e. offer the greatest possible accuracy with the shortest possible turnaround. nevertheless, any new technique that is either more precise or faster than available techniques is expected to improve the diagnostic armamentarium. in the case of cle, the turnaround time in order of one to few minutes allows for multiple repetitive analyses, which are impractical with frozen sections due to the time constraints for their preparation. for a given question, cle may provide significant benefit by providing fast results even if its precision were lower than that of frozen sections. general aspects of interpretation correlation between confocal laser endomicroscopy and standard histology in vivo confocal endomicroscopy produces a novel representation of tissue structures that neuropathologists are well accustomed to interpret (figure 2). as for any new technique, it will be crucial to learn to what extent previously defined morphological criteria can be applied and to identify the differences as compared to traditional techniques. our experience with cle images matches h&e slides for different types of lesions. the cellular structures most consistently identifiable in confocal laser endomicroscopy are cell nuclei due to their fainter fluorescence as compared to adjacent tissue. nuclear size, nuclear size variation as well nuclear spatial distribution are generally well visualized by cle. on static images, red blood cells are occasionally difficult to distinguish from neural nuclei as they produce a similar type of negative image due to their low fluorescence. we have found their uniform size and round shape useful for their identification. figure 2: example of morphologic patterns in glioblastoma in h&e slides (a, b, c) and in cle (d), illustrating how similar morphological findings of the same lesions can be visualized with both imaging techniques. red circles indicate giant cells, green circles indicate necrotic areas. correlation of cle-based morphology and standard histology in clinical routine will likely be an important basis of continuous training for all neuropathologists, allowing them to refine diagnostic criteria. it should be kept in mind, however, that any sole morphological analysis of cns tumors has significant limitations in the age of a molecular tumor classification. in particular, tumor types defined by molecular alterations cannot be diagnosed with certitude by histology or by any other morphological technique including cle. cle may face similar limitations regarding lesions that are considered high-grade solely due to molecular features. neither of these limitations, however, fundamentally thwart the clinical utility of intraoperative cle. three clinical trials are currently investigating cle in neurosurgery and neuropathology: a 200 patient multicenter trial in germany (invivo, nct04597801), a 200 patient trial in berne, switzerland (clebt, nct04280952) and a trial in milano, italy (besta institute review board, verbal n. 72/2020) focusing on the concordance between cle and definitive histopathological analysis. the results of these trials will clarify the potential clinical benefit of cle in neurosurgery and neuropathology and define the role of cle in the neurosurgical armamentarium. development and refinement of diagnostic criteria for cle cle is a promising method which achieves in vivo imaging during neurosurgery at high magnification and in real-time without tissue manipulation. cle is expected to help to distinguish more easily between healthy and neoplastic tissue, thus addressing a major challenge in brain tumor surgery. by optimizing tumor resection and preventing tumor relapse, cle is expected to offer a significant advantage to patients. in order to achieve these goals, the neuropathologist and/or the neurosurgeon will require basic knowledge of the cytoarchitecture of different tumor types and subtypes imaged by cle. in addition, entity-specific interpretation criteria and tumor classification guidelines are needed, similar to the previous situation in urology and gastroenterology. here, we would first like to discuss and present the general aspects of interpretation of cle images, based on the thorough examination of thousands of cle images and the corresponding histology from different brain tumor samples, and before establishing definite diagnostic criteria for each tumor type and subtype. in general, our cle images revealed features similar to already known tissue architecturea and morphology. the cle images correlated well with the corresponding histology (figure 2). unique aspects of individual cells and other surrounding tumor tissue elements were observed while the cle probe acquired images throughout its focal-depth range. cle images of brain tumors demonstrated high-contrast background with features of hypercellularity, atypic nuclei, microvascular proliferation, necrosis, and/or abnormal cell clustering and grouping (figures 3, 6, 7). by contrast, cle images of normal brain tissue showed a rather quiet, dark and normocellular background (figure 3e). erythrocytes were visualized as abundant, round to oval shaped cells that appeared to be much smaller than tumor cells. these features were already sufficient to allow a quick intraoperative diagnosis and to classify the tissue as normal or pathologic, independent of the exact tumor type and subtype, supporting the intraoperative decision making. figure 3: examples of morphologic patterns in various tumors on in vivo cle images recorded in comparison with microscopically matched h&e slides from the same region of interest. pleomorphic cells and hypercellularity in a case of glioblastoma multiforme (gbm) (a-b), a case of anaplastic oligodendroglioma (c-d), a case of high-grade glioma (hgg) showing a region of normal brain tissue and a highlighted vessel (arrow in h&e slide, bright streak in cle image) adjacent to the neoplastic tissue (e-f), and a case of anaplastic ependymoma (g-h). possible use cases for cle as mentioned previously, cle images showed the presence or absence of typical histomorphological characteristics of high-grade gliomas (figures 2 and 3) as observed on routine histological examination such as microvascular proliferation and/or necrosis in addition to hypercellularity and pleomorphism. distinguishing between a low-grade glioma (cns who grade 1/2) and a high-grade glioma (cns who grade 3/4) is important, because the therapeutic options differ: simple resection in low-grade tumors versus additional radiation and chemotherapy in high-grade tumors. an intraoperative statement regarding the malignant potential of the tumor is often asked, and can help the neurosurgeon decide if further resection should be undertaken or not. however, making a distinction between a low-grade glioma cns who grade 2 and a high-grade glioma cns who grade 3 can be very demanding, since the differentiation between these tumors mostly relies on the presence of mitoses, which are hitherto difficult to identify on cle images. these limitations should however not affect the integration of molecular data into the tumor grading. furthermore, cle is able to access the center of a brain tumor which may be useful for rapid tumor recognition and confirmation. last but not least, cle bears the big advantage to evaluate the borders of the tumor, recognizes abnormal histological features, and optimizes surgical resection in marginal regions if necessary. due to the diffuse growth pattern of most glial tumors, it is often challenging during surgery to distinguish neoplastic tissue from normal or reactive brain tissue without special or enhanced imaging techniques, often resulting in incomplete tumor resection. here, virtual biopsies of gliomas at the tumor margins using cle are of particular interest, because they allow an immediate histological assessment. in such cases, a specific pattern of cellular structures showing delineation of nuclei appearing darker than cytoplasm can often be noticed. moreover, small clusters of tumor cells or individual tumor cells within brain tissue can sometimes be identified, and a tumor border can often be delineated. figure 4: representation of the field of view of the zeiss convivo device superimposed over a low-power histological image of a glioblastoma. it should be pointed out that the field of view of cle images is small, approximately 475 μm x 267 μm in the case of the zeiss convivo (figure 4). using cle to decide whether or not to extend tumor removal can thus be challenging. thus, when cle provides an intraoperative preliminary diagnosis based on high magnification images, and a low-power histological image is not available yet, attention must be paid to avoid a potential pitfall for misleading diagnosis, as the entire lesion cannot be overviewed in a single cle image yet. consequently, the focus should be on detecting abnormal histoarchitectural features in such cases. due to the intrinsically digital nature of cle images, image stitching techniques (figure 5) can compensate the small field of view of current generation cle devices. by scanning the tissue surface of the region of interest, cle can generate a digital map in near real-time. here, the cle probe tip constantly acquires images which are digitally stitched together as they are acquired. the resulting digital map then covers a much greater area and allows a better overview of the region of interest than the individual cle images. figure 5: experimental image processing (a,b) using stitching techniques. serial images with overlapping content can be stitched together and result in a greater field of view. illustrative example of two (a) and three (b) serial images of vasculature with overlapping image content. the images were extracted from an image series recorded over several seconds during which the cle probe was moved along the tissue surface. tilting of the probe tip or tissue compression during image series acquisition can result in perspective distortion, which has been corrected for in these examples. composite of 45 serial images of tumor tissue (c, d): the cle probe was maneuvered along the tissue surface while serial images were acquired. the images were aligned based on overlapping content (c), which results in a much greater field of view compared to the individual images. because the images are stacked onto each other, the borders of the individual images are clearly recognizable, as the images on top of the stack overlay the images further below. further image processing methods, such as blending, can create a seamless tissue map and allow assessment of the tissue in its continuity (d). for clinical purposes it has to be made sure that such methods do not introduce diagnostically relevant artifacts. confocal laser endomicroscopy of specific tumor types in our experience, cle of metastatic tumor lesions presented mostly high-contrast, clear and good quality images with histomorphological features that were highly diagnostic of the tumor type (figure 6). a sharp delineation between the neoplastic and healthy brain tissue as well as a grouped growth pattern of tumor cells displaying a high nuclear-to-cytoplasmic ratio, sometimes with a detectable prominent nucleolus, were easily recognizable in cle images. figure 6: comparative examples of metastases in in vivo and ex vivo cle images and h&e slides. shown are a metastasis of a poorly differentiated carcinoma (a-b) and reactive changes (gliosis and discrete lymphocytic inflammatory infiltrate) at the border of the same lesion (c-d), metastatic adenocarcinoma (e-f) and metastasis of ductal breast carcinoma (g-h, h&e of frozen section). for illustrative purposes, both in vivo and ex vivo cle images are included showing that morphology is represented consistently independent of the acquisition modality (a, c, and e: ex vivo; g: in vivo). imaging of meningiomas often revealed moderate hypercellular tissue with relatively monomorphic tumor cells and patterns characteristic of a subtype, such as a fibrous background, whorls or microcystic changes (figure 7). psammoma bodies were always easily identified when present in the tumor sample. meningiomas of cns who grade 2 and 3 seemed to have increased cellular atypia than cns who grade 1 tumors, although this distinction was mostly hard to draw. figure 7: comparative examples of tumor morphology in ex vivo cle images and matched h&e slides in two distinct cases of meningioma (a-b and c-d) and microcystic meningioma (e-f). while in vivo imaging is the preferred usage scenario of cle, ex vivo imaging is useful for training purposes. it enables recording images over a longer period than what is practicable during surgery. it can also help to point out specific tissue characteristics that might otherwise be more time-consuming to achieve intraoperatively. in the same manner, schwannomas exhibited a fascicular architecture composed of spindled cells with elongated cytoplasmic processes, which were easy to recognize on cle images. features such as antoni a and antoni b patterns or verocay bodies were not always detected, even when present in the matched histological samples. another important aspect to consider when evaluating cle images as a neuropathologist, is the quality of the images obtained by the neurosurgeon. in the beginning, lack of experience in handling the cle probe can cause difficulties in obtaining adequate images for diagnosis. indeed, the area of interest can be obscured by hand instability or by acquisition of images with blood or movement artifacts. at the beginning of the learning curve, we found that ex vivo cle imaging was very useful for training purposes. such ex vivo images can be easily obtained, allow careful selection of the imaging window and artifact-free depiction of the features of interest. when both the neurosurgeon and the neuropathologist gain experience with the cle probe, high quality in vivo images are then guaranteed. potential impact of cle on the role of neuropathologists the future integration of cle in clinical routine may have a significant impact on the role of neuropathologists in the interdisciplinary team and on neuropathologists’ workload. on one hand, the intrinsic suitability of cle for telepathology may facilitate distribution of the workload onto a larger team of neuropathologists possibly working at different hospital sites. this may also increase efficiency since the neuropathologists would no longer need to move to the frozen section laboratory or to stay there between subsequent intraoperative consultations. furthermore, closer interaction due to increased and real-time intraoperative consultation may enhance the visibility of neuropathologists to clinical colleagues and thereby contribute favorably to the role of neuropathologists in the interdisciplinary team. on the other hand, a possible increase in intraoperative consultations may increase the workload of neuropathologists and their limited workforce. some neuropathologists may be sceptic about cle since cle gives neurosurgeons immediate access to a tool of microscopic analysis. there could be fears that the neurosurgeons may be tempted to interpret cle images on their own, which ultimately might be seen to render neuropathologists superfluous. we are convinced, however, that such fears would not be warranted and that neuropathologists have any reason to be confident in the importance of their morphological competences. when neurosurgeons became more and more comfortable in interpreting cle images, this will undoubtedly enhance the quality of the interdisciplinary collaboration and of the entire diagnostic process. challenges in the clinical implementation of cle further challenges to be considered for cle are economic aspects. clinical implementation of cle technology may be initially restricted to prosperous countries able to afford the cost of the novel technology. on a global scale, many people do not have access to adequate neurosurgical care for brain tumors. we deem it easier to implement in vivo cle in an existing setting of micro-neurosurgery, than to build up infrastructure for frozen section and train staff from scratch in neuropathology. in this scenario, an added benefit would be the easier cle technology dissemination of knowledge with, e.g. via joint expert teleconsultations from the operating room, as compared to standard slide-based histology. in many healthcare systems, billing in neuropathology is closely linked to technical analyses of tissues, which means that adequate reimbursement for reading of cle images may be difficult to obtain. furthermore, specific training will have to be developed and implemented in order to permit widespread implementation of cle. additionally, meticulous quality control procedures assessing diagnostic accuracy and turnaround times will be necessary. finally, the respective roles, strengths and limitations of frozen section, intraoperative cytology and cle depending on the clinical context will have to be clarified in order to allow this entire armamentarium of diagnostic modalities to unfold its full potential. conclusion and outlook cle has a significant potential as an additional tool in intraoperative consultation for cns lesions. the main strengths of cle relate to its speed, the possibility to analyze tissue without prior resection, and its intrinsic suitability for telepathology. the commercial availability of a first clinical-grade device will facilitate future widespread application. data obtained through previous experimental use of cle will provide a basis for interpretation in clinical use cases. further refinement of diagnostic criteria and of diagnostic accuracy in routine settings will be paramount. furthermore, cle offers unique perspectives for intraoperative diagnosis at the tissue level, thereby enhancing the role of neuropathologists in the interdisciplinary care team. ultimately, cle promises to complement the intraoperative diagnostic armamentarium in neuropathology and neurosurgery and thereby to contribute favorably to the clinical management of cns tumors. conflicts of interest the departments of neurosurgery in milan and bern receive fees from carl zeiss meditec ag, oberkochen, germany, for lectures at international congresses and support for research using the zeiss convivo device. dr. karl quint receives fees for coordinating the community of researchers and clinicians working with the zeiss convivo device. prof. ekkehard hewer and dr. maragkou have received honoraria from carl zeiss meditec ag. carl zeiss meditec ag has not played any role in analysis or interpretation of the data and has not been involved in the preparation of the manuscript. references 1. goetz m. confocal laser endomicroscopy, current indications and future perspectives in gastrointestinal diseases. endoscopia (2012) 24:67–74. 2. sadeghipour a, babaheidarian p. making formalin-fixed, paraffin embedded blocks. methods mol biol clifton nj (2019) 1897:253–268. https://doi.org/10.1007/978-1-4939-8935-5_22 3. sy j, ang l-c. microtomy: cutting formalin-fixed, paraffin-embedded sections. methods mol biol clifton nj (2019) 1897:269–278. https://doi.org/10.1007/978-1-4939-8935-5_23 4. canene-adams k. preparation of formalin-fixed paraffin-embedded tissue for immunohistochemistry. methods enzymol (2013) 533:225–233. https://doi.org/10.1016/b978-0-12-420067-8.00015-5 5. pilonis nd, januszewicz w, di pietro m. confocal laser endomicroscopy in gastro-intestinal endoscopy: technical aspects and clinical applications. transl gastroenterol hepatol (2022) 7:7. https://doi.org/10.21037/tgh.2020.04.02 6. di pietro m, bertani h, oʼdonovan m, santos p, alastal h, phillips r, ortiz-fernández-sordo j, iacucci m, modolell i, reggiani bonetti l, et al. development and validation of confocal endomicroscopy diagnostic criteria for low-grade dysplasia in barrett’s esophagus. clin transl gastroenterol (2019) 10:e00014. https://doi.org/10.14309/ctg.0000000000000014 7. xiong y-q, ma s-j, zhou j-h, zhong x-s, chen q. a meta-analysis of confocal laser endomicroscopy for the detection of neoplasia in patients with barrett’s esophagus. j gastroenterol hepatol (2016) 31:1102–1110. https://doi.org/10.1111/jgh.13267 8. meining a, bajbouj m, schmid rm. confocal fluorescence microscopy for detection of gastric angiodysplasia. endoscopy (2007) 39:e145–e145. https://doi.org/10.1055/s-2007-966109 9. kiesslich r, goetz m, neurath mf. confocal laser endomicroscopy for gastrointestinal diseases. gastrointest endosc clin n am (2008) 18:451–466, viii. https://doi.org/10.1016/j.giec.2008.03.002 10. kiesslich r, burg j, vieth m, gnaendiger j, enders m, delaney p, polglase a, mclaren w, janell d, thomas s, et al. confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. gastroenterology (2004) 127:706–713. https://doi.org/10.1053/j.gastro.2004.06.050 11. hsiung p-l, hsiung p-l, hardy j, friedland s, soetikno r, du cb, wu ap, sahbaie p, crawford jm, lowe aw, et al. detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. nat med (2008) 14:454–458. https://doi.org/10.1038/nm1692 12. kakeji y, yamaguchi s, yoshida d, tanoue k, ueda m, masunari a, utsunomiya t, imamura m, honda h, maehara y, et al. development and assessment of morphologic criteria for diagnosing gastric cancer using confocal endomicroscopy: an ex vivo and in vivo study. endoscopy (2006) 38:886–890. https://doi.org/10.1055/s-2006-944735 13. meining a, wallace mb. endoscopic imaging of angiogenesis in vivo. gastroenterology (2008) 134:915–918. https://doi.org/10.1053/j.gastro.2008.02.049 14. wang td, friedland s, sahbaie p, soetikno r, hsiung p-l, liu jtc, crawford jm, contag ch. functional imaging of colonic mucosa with a fibered confocal microscope for real time in vivo pathology. clin gastroenterol hepatol off clin pract j am gastroenterol assoc (2007) 5:1300–1305. https://doi.org/10.1016/j.cgh.2007.07.013 15. feng y, chang x, zhao y, wu d, meng z, wu x, guo t, jiang q, zhang s, wang q, et al. a new needle-based confocal laser endomicroscopy pattern of malignant pancreatic mucinous cystic lesions (with video). endosc ultrasound (2020) 10:200–206. https://doi.org/10.4103/eus.eus_35_20 16. gaddam s, mathur sc, singh m, arora j, wani sb, gupta n, overhiser a, rastogi a, singh v, desai n, et al. novel probe-based confocal laser endomicroscopy criteria and interobserver agreement for the detection of dysplasia in barrett’s esophagus. am j gastroenterol (2011) 106:1961–1969. https://doi.org/10.1038/ajg.2011.294 17. tofteland n, singh m, gaddam s, wani sb, gupta n, rastogi a, bansal a, kanakadandi v, mcgregor dh, ulusarac o, et al. evaluation of the updated confocal laser endomicroscopy criteria for barrett’s esophagus among gastrointestinal pathologists. dis esophagus off j int soc dis esophagus (2014) 27:623–629. https://doi.org/10.1111/dote.12121 18. oetter n, knipfer c, rohde m, von wilmowsky c, maier a, brunner k, adler w, neukam f-w, neumann h, stelzle f. development and validation of a classification and scoring system for the diagnosis of oral squamous cell carcinomas through confocal laser endomicroscopy. j transl med (2016) 14:159. https://doi.org/10.1186/s12967-016-0919-4 19. wu j, wang y-c, dai b, ye d-w, zhu y-p. optical biopsy of bladder cancer using confocal laser endomicroscopy. int urol nephrol (2019) 51:1473–1479. https://doi.org/10.1007/s11255-019-02197-z 20. chang tc, liu j-j, hsiao st, pan y, mach ke, leppert jt, mckenney jk, rouse rv, liao jc. interobserver agreement of confocal laser endomicroscopy for bladder cancer. j endourol (2013) 27:598–603. https://doi.org/10.1089/end.2012.0549 21. liem eiml, freund je, savci-heijink cd, de la rosette jjmch, kamphuis gm, baard j, liao jc, van leeuwen tg, de reijke tm, de bruin dm. validation of confocal laser endomicroscopy features of bladder cancer: the next step towards real-time histologic grading. eur urol focus (2020) 6:81–87. https://doi.org/10.1016/j.euf.2018.07.012 22. breda a, territo a, guttilla a, sanguedolce f, manfredi m, quaresima l, gaya jm, algaba f, palou j, villavicencio h. correlation between confocal laser endomicroscopy (cellvizio®) and histological grading of upper tract urothelial carcinoma: a step forward for a better selection of patients suitable for conservative management. eur urol focus (2018) 4:954–959. https://doi.org/10.1016/j.euf.2017.05.008 23. wu j, wang y-c, dai b, ye d-w, zhu y-p. optical biopsy of bladder cancer using confocal laser endomicroscopy. int urol nephrol (2019) 51:1473–1479. https://doi.org/10.1007/s11255-019-02197-z 24. kiesslich r, gossner l, goetz m, dahlmann a, vieth m, stolte m, hoffman a, jung m, nafe b, galle pr, et al. in vivo histology of barrett’s esophagus and associated neoplasia by confocal laser endomicroscopy. clin gastroenterol hepatol off clin pract j am gastroenterol assoc (2006) 4:979–987. https://doi.org/10.1016/j.cgh.2006.05.010 25. vennelaganti s, vennalaganti p, mathur s, singh s, jamal m, kanakadandi v, rai t, hall m, gupta n, nutalapati v, et al. validation of probe-based confocal laser endomicroscopy (pcle) criteria for diagnosing colon polyp histology. j clin gastroenterol (2018) 52:812–816. https://doi.org/10.1097/mcg.0000000000000927 26. sievert m, mantsopoulos k, mueller sk, eckstein m, rupp r, aubreville m, stelzle f, oetter n, maier a, iro h, et al. systematic interpretation of confocal laser endomicroscopy: larynx and pharynx confocal imaging score. acta otorhinolaryngol ital organo uff della soc ital otorinolaringol e chir cerv-facc (2022) 42:26–33. https://doi.org/10.14639/0392-100x-n1643 27. abramov i, dru ab, belykh e, park mt, bardonova l, preul mc. redosing of fluorescein sodium improves image interpretation during intraoperative ex vivo confocal laser endomicroscopy of brain tumors. front oncol (2021) 11:668661. https://doi.org/10.3389/fonc.2021.668661 28. acerbi f, pollo b, de laurentis c, restelli f, falco j, vetrano ig, broggi m, schiariti m, tramacere i, ferroli p, et al. ex vivo fluorescein-assisted confocal laser endomicroscopy (convivo® system) in patients with glioblastoma: results from a prospective study. front oncol (2020) 10:606574. https://doi.org/10.3389/fonc.2020.606574 29. belykh e, zhao x, ngo b, farhadi ds, byvaltsev va, eschbacher jm, nakaji p, preul mc. intraoperative confocal laser endomicroscopy ex vivo examination of tissue microstructure during fluorescence-guided brain tumor surgery. front oncol (2020) 10:599250. https://doi.org/10.3389/fonc.2020.599250 30. abramov i, park mt, belykh e, dru ab, xu y, gooldy tc, scherschinski l, farber sh, little as, porter rw, et al. intraoperative confocal laser endomicroscopy: prospective in vivo feasibility study of a clinical-grade system for brain tumors. j neurosurg (2022) 1:1–11. https://doi.org/10.3171/2022.5.jns2282 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. my pathway to a career in neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 3:10 (2022) reflections my pathway to a career in neuropathology clive harper university of sydney and royal prince alfred hospital corresponding author: clive harper · rpa neuropathology · 94 mallett st · camperdown, nsw 2050 · australia clive.harper@sydney.edu.au submitted: 09 march 2022 accepted: 10 march 2022 copyedited by: henry robbert published: 12 april 2022 https://doi.org/10.17879/freeneuropathology-2022-3809 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, reflections, alcohol brain damage, thiamine deficiency, brain banking and premortem donor program, international collaborations i grew up in balgowlah heights in sydney. my father was a physicist at csiro but was also involved with the national standards commission. in 1970 he was appointed executive member of the metric conversion board that was established to oversee the metric conversion process in australia. it has been said that the reason that this proceeded so smoothly over the next ten years was due to his remarkable management skills. he was awarded an australian honour (ao) for this contribution. i attended the local primary school and, for my secondary education, the manly boys high school. i won a commonwealth scholarship to sydney university and commenced dentistry in 1961. after the successful completion of my first year i applied to transfer from dentistry to medicine and was accepted. i completed my medical degree in 1966 and began working as a resident medical officer at the royal north shore hospital (rnsh) in 1967. after two years i commenced another five years of post-graduate training in pathology. the course included separate years in histopathology, microbiology, haematology and biochemistry. towards the end of this pathology training all i could think of was travelling overseas. i think that my father’s travels and connections with overseas friends had caused my ‘travel bug’. i had no idea what i wanted to do or where i wanted to go. figure 1: clive sits at his microscope in neuropathology at royal perth hospital in 1984. however, at a dinner party one evening i met a pathologist from glasgow who mentioned that one of his colleagues was looking for a locum pathologist. i immediately wrote (no emails or faxes in those days) and waited patiently to hear from professor hume adams, head of the department of neuropathology at the university of glasgow/southern general hospital. i remember how excited i was when i received a letter from him offering me a job as lecturer in neuropathology in glasgow, beginning september 1972. hume even organised accommodation for us and it was all go. i was married with two children aged five and two. at that time australia was actively repatriating mentally disturbed immigrants. the government always sent a doctor along with the patients. i requested such a position and was invited to meet stephan in ryde psychiatric hospital. he was to return to zagreb in yugoslavia (now croatia). he was only about 21 years old and the nurses said that he could not speak english. a government car picked me up in the morning and we drove to the hospital to pick up stephan. then on to mascot to take the qantas flight to rome and then zagreb. i had bought my onward ticket to london. jann and the kids travelled on the same flight, direct to london. stephan and i were seated in a separate zone with no one close by and i had my medical case with drugs and syringes to keep stephan quiet, if necessary. as we took off stephan turned to me and said, “why don’t we have a drink”. he had been foxing about his english the whole time just to get a free trip home. i was able to wander about and keep an eye on the kids. stephan and i had to change planes in rome while my wife and kids went straight on to london. when we arrived in zagreb stephan’s family were there to meet him. he had lost a lot of weight, grown a beard and his hair was very long so they did not recognise him! immigration immediately picked up on this and i was dragged off by security and interrogated as to his true identity. in those days yugoslavia was totally communist and everyone seemed to be carrying guns. i was terrified and the australian embassy official who was meant to meet us had not turned up (surprise, surprise!). finally, stephan’s sister and uncle arrived and they immediately hugged him and everything settled down. i was very pleased to get back on the next flight and get out of my first communist country. figure 2: professor hume adams and clive enjoy a moment in neuropathology at the southern general hospital (university of glasgow) in 1973. arrival in glasgow, scotland hume adams had organised accommodation for us and we were able to move straight into our new home in white inch, about three miles from the southern general hospital. it was a 2-story tenement with three bedrooms and was very comfortable but pretty cold, even for summer. the owner even offered to babysit for us – what a bonus. we organised a local school for our son dal and i went to the hospital to meet my new colleagues. i found hume to be a delightful chap, about 45 years old. the department seemed to be ‘buzzing’. the hospital was a new innovative ‘one-stop shop’ for the neurosciences. it combined neurology, neurosurgery, neuroradiology and neuropathology in the one building and the clinical groups met every week to ensure the smooth running of the institute. getting back to work was a bit of a challenge. i had not done any anatomical pathology for three years so was pretty rusty. i had been training in biochemistry, microbiology and haematology. moreover, neurosciences had always been a bit lost on me so it was a sharp learning curve! fortunately, hume believed in a long training/apprentice period and so i spent a lot of time observing the techniques of brain dissection. my main role was doing the autopsies. we moved about a lot each week working in different hospitals all over glasgow and in some of the major country hospitals. soon after i started work the hospital/institute had an official opening. on the first evening, there was a huge formal dinner at the university refectory. the invited guest for the official opening was wilder penfield, director of the montreal neurosciences institute in canada. his neurosurgical fame related to in-vivo electrical stimulation of the brain to identify specific regions that control motor function. he would buzz the brain with electrodes during his operations and watch to see which muscle contracted! sounds primitive but this work was at the forefront of neuroscience at the time. i found his speech inspiring and felt an almost ‘religious conversion’. this really was the beginning of my major interest in neuropathology. a few weeks after we arrived in glasgow, hume had a dinner party and we met a number of other pathologists. several became famous pathologists in later life – alistair cochrane and roddy mcsween. there was also an ophthalmological pathologist who asked me to be involved in a research project involving the electronmicroscopic study of optic nerves from a group of baboons who had been subjected to ‘toxic amblyopia’. this was my first real taste of neuropathological research and probably set the direction of my future career. hume’s hospitality was extraordinary. in the first four months he had invited us to his home twice, took us to a dinner party and a wine tasting and paid for all of this – so different to the australian work scene. holiday in europe may 1973 in may 1973 the weather was warming and my parents arrived to visit. i organised to take my annual holidays and we all drove in our camper to europe. i had taken the opportunity of organising several job interviews in europe. hume had asked me to stay in glasgow but the weather drove us all mad and we were keen to experience life in a ‘foreign’ country and to master a second language. i had done a lot of school french and, during our time in glasgow, i had done private french lessons with a friend. he said “i don’t need any payment, just give me as much whisky as i want during the lessons”! it cost a fortune but my french did improve. i had interviews for jobs in neuropathology in paris and in geneva and lausanne in switzerland. i was offered all three jobs and chose lausanne. we moved in september 1973. hume had a farewell party for us and i was very sad to leave. i still consider hume to be my mentor. we remained friends for life and visited him in 2012 in glasgow. this was 40 years after we first met. he was then 84 years old. as most of you know hume passed away in 2020 aged 90 years. lausanne, switzerland 1973-74 we drove down through london, crossed the channel and drove on to lausanne. it took about two weeks to find an apartment. it was a six-story block in the hills behind lausanne, overlooking the town and across the lake to the alps. the neuropathology laboratory was beside the hôpital cantonal de lausanne. it was in an office block and we were housed in the basement. there was a nice café on the ground floor and we often went upstairs at morning tea for a coffee and ‘pomme de vie’ liqueur. there were five other medical staff in neuropathology – professor rabinowicz (swiss), dr. alphonse probst (swiss), deraux (german), and two young portuguese trainees (cousins, manuel and juan sanches). it was an excellent setup with lots of technical staff and lots of specialised equipment (em included). the department specialised in developmental neuropathology with lots of quantitative analyses. quantitation later became my speciality in the alcohol cases we studied in perth and sydney. one of my responsibilities was to do most of the autopsies. the other trainees did not have autopsy experience. the department provided a service for the whole canton (state) so i was often sent off with a technician to do autopsies in country towns, usually high up in the mountains. in wintertime i sometimes packed my skis in the van and, after finishing work, would go for a ski. the french language came slowly. i was challenged after six weeks by having to present a case report to an evening conference. i practiced for ages and did a reasonable job until question time. i had no idea what they were asking in their rapid french. future directions should i try to take a clinical job next year? neurology is the obvious choice if i wish to proceed in neuropathology. the idea of a university appointment is still in the back of my mind. teaching would give me a lot of satisfaction but i must remember what a small fraction of teaching occurs in neuropathology – hours per year. the idea of having to do a phd or md is not very appealing. perhaps these things will become clearer in time? despite all of this self-analysis i have not made any decisions. if only i could do clinical medicine without having to work on nights and weekends – that would be hard to take again! i have been reading my old swiss diaries and i constantly refer to my lack of direction and confusion about my career pathway. correspondence with the clinical superintendent in my previous sydney hospital (rnsh peter williamson) led me to believe that i should not return to australia until i had done at least one year in the usa. one week later i wrote in my diary: “here i am with a letter half written to apply for a job as assistant neuropathologist in perth. it is not really like going home so i don’t have the anxieties re arriving home dissatisfied with lack of travel”. i am still waiting for a reply from the cornell medical school in new york but can’t really see it matching up to the perth job with byron kakulas. in fact, as we walked around the lausanne lakefront on sunday, looking at the boats on the lake, we began talking about getting a boat in western australia. the idea was very appealing. anyway, it is just as well that the perth job came up because the new york job did not come through. i guess i should set my sights on my career for a while and try to achieve something really great by the time i am 35-40 years. a professorship would suit me fine but a lot of water under the bridge before then! it’s the idea of publishing papers that really upsets me – they are just not my scene. no one reads them so what’s the point? i wish i could develop some original thought pattern. currently, i am fully occupied trying to learn the basics of neuropathology!” whilst working in lausanne i attended neuropathology conferences in innsbruck, austria and in budapest, hungary (international society of neuropathology). at the latter conference i met byron kakalus from perth, australia. we had never met and he seemed delighted to meet another australian training in neuropathology. he immediately invited me to join his department in perth after i finished working in lausanne. i told him that i was waiting to hear about a position in boston but he said that the perth position was only available if i came immediately. since the family had already planned a three-month trip in italy, yugoslavia and greece after leaving lausanne we compromised and i arranged to commence in perth after this holiday. perth 1975 we flew into perth from switzerland via the uk and were met by my new boss, professor byron kakulas. i started work at the royal perth hospital immediately. byron had set up the biggest and best neuropathology department in australia. it was big even by international standards. he had trained in boston at the massachusetts general hospital with two world famous neurologists/neuropathologists, raymond adams and pearson richardson. there were excellent facilities and large numbers of secretarial and technical staff. there were even two registrars and several supplementary overseas trainees. the registrars, peter blumbergs and tony tannenberg and i worked closely together for several years until they took up senior neuropathology positions in adelaide and brisbane, respectively. they are good friends to this day and we often laugh about that first week and the tough time that they gave me! i really loved my work – a mixture of clinical neuropathology, teaching and small clinical research projects. byron loved having international visitors and we ran an annual neuropathology course for trainee registrars as well as undergraduates. i met henry urich (london university), raymond adams, pearson richardson (mass general) again and many others. there was an excellent clinical program for all of the neuroscience groups in perth. the group met weekly, on a rotation basis, in the four main hospitals (royal perth, fremantle, sir charles gardiner and the children’s hospital). this created a harmonious working relationship between neurology, neurosurgery, neuroradiology and neuropathology. figure 3: staff and visitors to neuropathology at royal perth hospital in about 1981. byron kakulas (front row left) , had a wide group of international colleagues including the famous neurologist/neuropathologist raymond adams, bullard professor of neuropathology at harvard medical school and chief of neurology at massachusetts general hospital. professor adams is sitting next to byron. one of the enjoyable and challenging weekly clinical programs was the clinico-pathological conference. we (the neuropathologists) would prepare a case as an unknown and ask one of the neurologists to discuss the case. some of these cases were so good that i decided to write them up and publish them in the medical journal of australia (mja) following the example in the nejm . this was a great way to start getting publications together. i negotiated directly with the editor of the medical journal of australia and sent him two cases each year (see below): harper cg. progressive difficulty in walking and speech. med j aust 1977, 2:370-3. harper cg. recurrent subarachnoid haemorrhage and obesity. med j aust 1977, 2:865-7. harper cg. multisystem febrile illness. med j aust 1978, 1:205-8. the department had a huge workload of cases, mostly from the department of forensic medicine (coroners). every forensic autopsy brain was sent to us for examination. in addition, we did many of the autopsies for royal perth hospital and did the brain examinations on all of the cases. the total number of brains examined each year was more than 1000. each week we had a ‘brain review’. the most interesting cases were selected and demonstrated to pathology, neurology and neurosurgery trainees. some medical students also attended and it was run as a question and answer session. i replicated these sessions when i moved to sydney. as time went by i became aware that some additional training in neuropathology in the usa would enhance my career. i asked byron what he would recommend and he helped me obtain a fellowship (paid by us grant money) in philadelphia with a greek/american colleague, nicholas gonatas. i applied for leave without pay for 12 months and the hospital supported my application and even paid for my travel. a locum was appointed to my position victor ojeda was from south america but had been working in new zealand. travel to philadelphia via europe – 1977-78 it was blizzarding when we arrived in new york. we stayed in a hotel that night and then took the amtrak train to philadelphia. we were met at penn station by my father’s friend, bill zeiter. he and his wife had offered us accommodation until we found a house. the hospital work was very stimulating. i was responsible for some of the brain biopsy reporting with the head of department, nick gonatas and several other trainees – tomahiko mizutani and stavros balloyannis. they were both neurologists from japan and greece, respectively and were in the usa to get some neuropathology experience. we have kept in touch from time to time. the three of us were responsible for the hospital autopsies and for preparing the clinico-pathological cases each week, as we did in perth. we were working with and meeting many famous neurologists, neurosurgeons and neuropathologists every week. there were regular meetings and conferences and nick gonatas travelled constantly in the usa and to europe. it was very stimulating. nick was the elected president of the american neuropathology association which meant that we had even more interaction with american and international neuropathologists. by chance, there was also a link with my old glasgow colleagues. david graham (i was his locum in glasgow while he worked in philadelphia) had been working with the neurosurgeons in philadelphia on an animal model (monkeys) of head injuries. each of the fellows was allocated a research project and our main laboratory supervisor was nick’s wife, jackie. she had meticulous laboratory habits and trained us well. we were working with animal models of neurotoxicity using toxic ricin. we were studying the effects of toxic ricin on animal nervous tissues using electron microscopy and immunohistochemistry. i was able to publish three papers in the first year. even more importantly, the personal interactions in the department were invaluable to my research education and intellectual development. gonatas nk, harper cg, mizutani t, gonatas j. superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde transport. j histochem cytochem 1979, 27:723-34. harper cg, gonatas jo, steiber a, gonatas nk. in vivo uptake of wheat germ agglutinin horseradish peroxidase conjugates into neuronal gerl and lysosomes. brain res 1980, 188:465-72. harper cg, gonatas jo, mizutani t, gonatas nk. retrograde transport and effects of toxic ricin in the autonomic nervous system. lab investigations 1980, 42:396-404. my first significant clinico-pathological manuscript was published while i was in the usa in 1979. the work had been done in perth using forensic autopsy material. this was really the beginning of my lifetime interest in the primary and secondary effects of alcohol on the human brain. wernicke’s encephalopathy is caused by thiamine (vitamin b1) deficiency and is very common in alcoholics. harper cg. wernicke’s encephalopathy a more common disease than realised. j neurol neurosurg psychiatry 1979, 42:226-31. by chance, the editor of this journal was prof. ian simpson from glasgow where i worked in 1973. i do not like to admit it but i think that this helped significantly in getting the paper published. i was not just a name without a face! this was the beginning of an important realisation that interpersonal relationships are critical to one’s career. i would go so far as to say that it was one of the main factors that contributed to the success of my career. during spring and summer our family travelled almost every weekend in our camper van. we had medical friends in washington dc (colin and helen masters), connecticut (michael and heather greenaway) and in boston (chris and stephanie burke). colin was working with dr. carlton gajdusek, who discovered the cause of prion disorders like kuru and creutzfeldt-jacob disease. he was awarded the nobel prize for this work. both hume adams and henry urich visited and stayed with us in philadelphia. figure 4: staff from the departments of neuropathology and neurology at the hospital of the university of pennsylvania. nick gonatas, head of neuropathology, is on the left of the front row next to clive. after finishing my 18 months work in philadelphia, we took a well-earned break of three months and went camping in mexico and guatemala. en route, i attended the annual conference of the american association of neuropathology (aanp) in kansas city. this was where i first met jim powers. he was working at einstein, ny, with bob terry and cedric reine. jim and i have been friends ever since. in 2013 jim and i arranged to meet with bob terry in california. therese and i had invited jim and cedric raine to travel with us through arizona in our hired rv. we were all taking bob terry out to dinner in the rv. we stopped to pick up another person, dik horoupian. when he stepped into the rv bob nearly fell over – they had worked together for many years. jim had coordinated all these arrangements without anyone knowing. at the aanp conference in 1985 jim introduced me to paul garren. paul came to work with me in sydney as assistant neuropathologist in 1986 for one year. jim has now retired as neuropathologist from rochester, new york, and is living in north carolina. in 2013 my wife and i travelled with jim powers and cedric raine in a hired rv (motorhome) through arizona – we had a ball! figure 5: special get together in 2013 in san diego to meet robert (bob) terry. left to right: cedric raine. bob terry, dik horoupian, and jim powers. royal perth hospital 1980 i returned to perth in 1980 full of enthusiasm for research, teaching and clinical work. the weekly program and thursday ‘brain cut’ demonstration was always popular with the hospital clinicians and trainees. i was keen to bring the western australian neuroscientists together and, along with serge bajada, decided to start an ’annual neurosciences colloquium’. i organised a venue (perth railway station – where the indian pacific arrives) and funding from several pharmaceutical companies. we invited an interstate guest and called for papers. the meeting was a great success and we ran it for about five years. it generated a number of collaborations between groups like veterinary science and medicine in perth. i did some work with the vet school at murdock university on ‘staggers’, a nutritional disease of sheep and cattle that damages the brain (rye grass toxicity). many famous visitors came to visit our department. byron had worked in boston during the 1960s and had many international connections. they included raymond adams who i had already met because he held a position at l’hôpital cantonal in lausanne, switzerland where i worked in 1973. he was a very modest man. he wrote the classic text on wernicke-korsakoff disease (caused by vitamin b1 deficiency). i studied the prevalence of this disease in perth and australia in the 1970s and 1980s and our data was used by the federal department of health to mandate the addition of vitamin b1 (thiamine) to bread to help improve everyone’s b1 levels in australia. harper cg. the incidence of wernicke’s encephalopathy in australia a neuropathological study of 131 cases. j neurol neurosurg psychiatry 1983, 46:593-8. raymond adams worked closely with another delightful fellow – pearson richardson. sir john walton, senior neurologist at queens square in london also visited the department. i was delighted when my mentor from glasgow, hume adams decided to visit perth. it was fabulous to catch up with hume and eileen again. my time in the usa had given me a real taste for research and i now had a feel for how to move ahead. i decided to begin an active research program and to apply for research funds to support the work. we had a very close working relationship with the department of forensic medicine in perth (coroners) and studied most of the brains from the coroner’s autopsies (about 3,000 per year). this was a unique source of cases and allowed us to carry out high quality epidemiologic studies. john hilton was the head of department and we became good friends. later, i encouraged him to move to sydney to join the university of sydney and he became head of department of forensic medicine in glebe. my research began with the simple aim of establishing whether or not people who drank alcohol had smaller brains than those who were nonor social drinkers. this work was published in two articles and was the beginning of my life-long thematic research on the effects of alcohol on the brain: harper cg, blumbergs pc. brain weights in alcoholics. j neurol neurosurg psychiatry 1982, 45:383-40. harper cg. neuropathology of brain damage caused by alcohol. med j aust 1982, 2:277-82. the neurosciences in perth was very strong and a new initiative was started in the early 1980s – ‘the australian brain foundation’. this was an australia wide foundation but western australia was one of the leading states and raised most of the start-up funds. i was the medical director of the state foundation for a period. later, through the foundation, i put together a proposal to the wa lotteries commission for funds to purchase an electronic counting machine for brain nerve cells in autopsy material. in june 1982 i did a reconnaissance trip around the world to check out different systems for automated cell counting. next, i flew to new york to meet ralph holloway, an anthropologist at columbia university. he was interested in our work on aboriginal brains. ralph and i worked together for a number of years and published several papers. this was the beginning of a whole new area of research for our group. figure 6: the local media were entranced with our research and came up with some clever headlines. the staff at royal perth hospital by 1984 i was developing an active research program and was looking for honours and phd students. i put out a leaflet to the science and medical students inviting them to apply for an honours year at the hospital and had an application from jillian kril. i think she was 17 years old at the time! jill is now professor of neuropathology at the university of sydney. she completed her honours year on the project ‘brain shrinkage in alcoholics’. we made foam casts of the inside of the skulls of hundreds of autopsy cases so that we could estimate the intracranial volume and compare it with the brain volume (estimated by archimedes principle). this enabled us to calculate how much the brain had shrunk during life. the catchphrase was: “too much drink and your brain will shrink”. the media loved the work and we had lots of publicity. several important manuscripts arose from this research in perth. harper cg, kril jj, backhouse a. measuring brain atrophy. neurology 1986, 36:1147 harper c, kril j, daly j. are we drinking our neurones away? brit med j 1987, 294:534-6. in june 1985 i visited my first ‘brain bank’ in boston – the beginning of another important part of my professional life. our decision to move back to sydney at the end of 1985 and leave our idyllic lifestyle in perth was not an easy one. my principal motivator was professor jim mcleod, head of neurology at royal prince alfred hospital (rpah). neuropathology in sydney was pretty weak compared to the strength of all of the fabulous clinical and experimental neurosciences. it was quite a task to convince my family that this was the right thing to do. personally, i was very anxious that i was moving beyond my level of competence and thought that i might not cope with the challenge. however, jim mcleod convinced me that ‘all of the stars were aligned’ and promised his personal help and advice. one of the main players was the dean of medicine, professor richard guy. he was a senior neurosurgeon and was still working at rpah. in those days the dean of medicine was able to handle both the clinical and administrative jobs. another important player was john allsop, a senior neurologist and chairman of the rpah board. in retrospect, it was the best thing i could ever have done. i was getting into a ‘semi-retirement’ mode in perth at the age of 42 years – far too young. a major challenge was just what i needed. since that time, i have not met anyone who has made a similar major change in direction in their professional career who has regretted his/her decision and i encourage others to do the same – go for it!! i reflected back to a statement i wrote while i was in switzerland in 1974: “i guess i should set my sights on my career for a while and try to achieve something really great by the time i am 35-40 years. a professorship would suit me fine but a lot of water under the bridge before then!” mind you, it was not all that straightforward. the position was advertised and then interviews were arranged in sydney. i think i was the only applicant but, even so, it was a harrowing experience. there were 10 on the interview panel including the head of medicine, professor bickerton blackburn. i remember him asking, “what is all this nerve cell counting about, is that real research? ” i had known prof. blackburn as a medical student and he always seemed totally unapproachable – my interview did not change this opinion. ironically, in 1994, i rented prof. blackburn’s apartment in paris for 6 weeks when i was working with jean-jacques hauw at l’hôpital salpetriere. the blackburn family were a medical force to behold. sir charles blackburn, the father of bickerton blackburn, was a pioneer of clinical research at the university of sydney. in the 1960s he forged strong links between researchers and clinical practitioners at rpah and within the faculty of medicine. he was responsible for establishing key academic and research posts and for appointing highly competent staff like john allsop and jim mcleod to fill these positions. my wife and i flew to sydney in november 1985 to look for houses before we moved from perth. she had already decided that we would live on the northern beaches. after about three days of house hunting we walked into the house in bayview and we both said: this is it! there were lots of farewell parties but the final party was held in the qantas lounge at the airport on the 7th december 1985. my parents were delighted that we were finally returning to sydney after 13 years away. dad wrote me a letter congratulating me – i am still touched when i read it today. dad was not an ‘expressive’ person! i never remember him telling me that he loved me. while still working in perth we (myself and the university design team) had put together plans for the new neuropathology laboratories $250,000 had been donated by the ramaciotti foundation. i based the plans on the neuropathology laboratories in perth that worked very well. i discussed my appointment with roger pamphlett who was about to head off to london for more neuropathology training and he said he would love to come to work with me as senior lecturer in neuropathology once he completed his training and examinations. it all seemed to be falling into place. i also asked jillian kril if she would be interested in coming to the new department in sydney. she was keen to do a phd and i was able to offer her a job as a research assistant. she joined our group in 1986. building works on our labs did not commence until i had been in sydney for many months. i was given tremendous support by my colleagues at the university department of pathology. david cameron was the head of department but sue dorsch, a dynamic immunologist/pathologist also played a key role. by choice, sue later became involved in university administration and finally became the deputy vice chancellor. for many years she actively supported the development of neuropathology. there were many professorial staff who liked the old style of university life. you taught a few students and played about with a bit of ‘hobby’ research. some disappeared for months at a time without explanation and there was little that the administration seemed to be able to do! finally, the laboratories were finished. professor guy officiated at the formal opening of the neuropathology labs on 26th november 1986. the minister for health attended as well as many dignitaries from the university and rpah. the nsw minister for health, the hon. peter anderson, gave his welcoming address and declared the neuropathology laboratories open. when i commenced work at the university, i was invited by the vice chancellor to a luncheon. he held these lunches regularly to meet his new senior staff. it was delightful to meet professor ward. he was very receptive to the needs of our new department and, in no time, i had significant additional funding. today, one is lucky to ever meet the vice chancellor in your entire career! at the luncheon i sat next to the head of telecom. he asked me if i was familiar with faxes. i had to declare my ignorance but, after he described the efficiency of faxes, i returned to my office and called the university telecommunications office. i explained the situation and said that i would like an additional telephone connection in my office for a fax machine. the director said, “who do you think you are – what if every professor calls and asks for an additional phone connection?” i made some enquiries and found out that i could simply buy a ‘line splitter’ and plug it into my existing phone outlet. i could then use the second line for my new fax machine! one of the delightful things about coming “home to sydney” was that there were staff in the university, hospital and state government who were fellow university medical students. when i called to make an appointment with the new south wales (nsw) dept. of health i found that i would be talking with sue morey. she and i had been good friends at uni and, over the next couple of years, she opened doors and provided support that would otherwise have been impossible to achieve. its ‘who you know not what you know’! here, the vice chancellor is opening our new-look pathology museum. my relationships with my pathology colleagues in rpah were far less cordial. the head of the department wanted me to go onto the regular general pathology reporting roster for skin, breasts, bowels etc. i explained that my training was exclusively in neuropathology. after some tense discussions it was agreed that i would take on the responsibility of the hospital autopsies and i did not have to go onto the general roster. frankly, it would have been dangerous and the risk of errors in diagnosis would have been significant. there were several of the hospital pathologists who i knew very well – peter russell and stan mccarthy. they were very supportive. many years later peter became head of the department and things ran much more smoothly. i had studied with stan when we did our pathology training. i also played against him in the inter-hospital rugby – these were ferocious games. we used to train 2-3 times each week and then play a ‘round robin’ against all of the teaching hospitals attached to the university of sydney. i had to stop playing in about 1970 when i broke two lateral spines on my lumbar vertebrae in one of the games. our coach was max elliott, an ex-wallaby representative for australia and respiratory physician. the history of the department of pathology at the university of sydney in october 1850 australia's first institution of tertiary education was established in sydney. the faculty of medicine at sydney university formally came into being on 13 june 1856 and thus is the oldest faculty of medicine in australia and new zealand. provision for teaching of pathology was made in 1883 after funds were made available in 1882 for the establishment of a chair of anatomy and physiology, and for lectureships in subjects including pathology. a significant event in the history of the department of pathology was the opening of the neuropathology unit in 1986. the establishment of this new facility resulted from the combined efforts of the nsw department of health, through the royal prince alfred hospital and the university of sydney. the refurbishment of the laboratories was made possible by generous funding to the university of sydney from the nathan roberts and phyllis henderson bequests and the ramaciotti foundation. the neuropathology unit also has responsibilities for medical undergraduate and postgraduate teaching, together with instruction groups which include nurses, physiotherapists and speech pathologists. international travel and research staff i had told sydney university and rpah before i accepted my appointment that i had already planned a big round-the-world scientific trip in september 1986. they had agreed that this would be ok. the hospitals funded three weeks of conference travel for staff specialists annually. first i flew to london where i had arranged to meet up with roger pamphlett and to meet the staff at queens square. roger had worked with us in neuropathology in perth for a year of his post-graduate neurology training. he enjoyed the neuropathology so much that he decided to make it his career. when i was invited to take the sydney chair in 1985 we made a pact that he would come and join me when he returned from queens square, london. we had a fabulous working relationship for 24 years at usyd. i next visited cambridge and gave a lecture at the maudsley hospital. i met peter lantos and prof. lishmann who were very interested in our research. peter lantos invited me to his “london club” for lunch. we remained friends for many years. international fellows jörg klekamp, a german medical student visited me when i was in perth in 1980. he came as part of an optional clinical term in his german medical curriculum. this was the beginning of a long working collaboration with the hannover medical school. his mentor, prof. kretschmann, had been doing volumetric and morphometric studies for many years and had established normal numbers during brain development. i visited hannover in 1982 and 1984 for this collaboration. one of the high points of the 1984 visit was a night of jazz in an underground cellar – i got so involved that i almost missed my early morning flight! jörg and his partner aggie met me again in london in 1985 to discuss if they could come to sydney to work for a year or so (they stayed three years). they wanted to come to sydney to do more work on brain volumes in aboriginal cases. prof. kretschmann organised a fellowship to help support their visit to sydney. jörg and aggie arrived in sydney in may 1986. they were a lovely couple and worked incredibly hard on their project (comparative neuroanatomy between caucasian and aboriginal brains). we published four manuscripts together. sadly, the work was seen as ‘racist’ by some international colleagues and my abstract submitted to the american neuropathology association (aanp) meeting was rejected. this was my only rejection (of a paper) in my career! klekamp j, riedel a, harper c, kretschmann hj. morphometric study on the postnatal growth of non-cortical brain regions in australian aborigines and caucasians. brain res 1989 riedel a, klekamp j, harper c, kretschmann hj. morphometric study on the postnatal growth of the cerebellum of australian aborigines and caucasians. brain res 1989 klekamp j, riedel a, harper c, kretschmann hj. quantitative changes during the postnatal maturation of the human visual cortex. j neurol sci 1991 riedel a, klekamp j, harper c, kretschmann hj. morphometric study on the postnatal growth of the hippocampus in australian aborigines and caucasians. brain res 1991 the interesting results of this research were picked up in the 1990s and were used in a film entitled ‘the difference’, a bbc documentary, produced and directed by mike smith (1999). this film highlighted the difference between aboriginal and caucasian children in their abilities to use either visual or auditory memory. the aboriginal children did extraordinarily well when they were challenged using visual memory compared with the caucasian kids. the opposite was seen when the children used auditory memory. this supported our research findings that aboriginal brains have larger, and presumably better organised visual cortices than caucasians. the implications of these scientific findings for educating aboriginal children are self-evident. jörg is now professor of neurosurgery in germany and we still keep in touch. it always creates a great sense of satisfaction to see students and colleagues who you have trained and worked with succeed in their chosen professional pathways. media presentations on alcohol our biomedical scientific alcohol story had a great attraction for the media and, over the years, i think i became quite adept at dealing with interviews. below are some of the projects that we were involved with. i was invited back to perth in 1987 by the wa department of health to launch a ‘healthy drinking program.’ in 1992 i ‘starred’ in a documentary made by the abc. i had a dreadful time trying to remember my lines and finally the director said “just ad-lib please clive”. i am now far more respectful of actors! alcohol and brain damage – produced by barbara edwards, dept. of health, wa 1987 choose your poison! documentary by abc 1988 film on addiction produced by robyn davidson – “this time next time”, 1992 research and international friends international interest in our research really began to take off after i visited peter carlen in toronto, canada in 1989. peter worked at the addiction research foundation. i had an introduction through an aussie (jim rankin) who had worked with peter. he was a leader in ‘reversibility of alcohol related brain damage’ and was very interested in our quantitative work on the autopsy cases. partially reversible cerebral atrophy and functional improvement in recently abstinent alcoholics. carlen pl, wilkinson da, wortzman g, holgate r. can j neurol sci. 1984 a fascinating area of research. commenting on a previous editorial by clive g. harper. carlen pl. br j addict. 1988 peter opened the door on international alcohol research for me. peter invited me to the next combined conference for the international society for biomedical research on alcohol (isbra) and the research society of alcoholism (rsa). there are very close ties between the international (isbra) and the us societies (rsa) for alcohol research. this was really the beginning of our research becoming recognised internationally. for the rest of my career the rsa and isbra conferences became the main focus of my international conference travel. previously, i had been attending pathological, neuropathological and neurological conferences but the more specialised meetings were much more relevant. two isbra conferences have been held in australia – 1994 and 2006. i was heavily involved in the organisation of both of these meetings. the first rsa meeting that i attended was on marco island in florida. i was welcomed into the society by a group with whom i just clicked. they included john littleton and susan baron, david lovinger, dennis twombly, rubin and kathy gonzales, fulton crews, and the society ceo, deb sharp. most of this group were basic neuroscientists so they welcomed someone with a clinical background. it was a revelation – so many people working on alcohol related organ damage. deb sharp ran/runs the conferences brilliantly and was always able to help and support our australian research group. deb encouraged therese and i to attend the rsa conference in denver, colorado in 2017 even though we had retired. one of the more senior rsa members was oscar parsons, a clinical psychologist. oscar recognised the importance of the structural brain changes we had described in alcoholic cases. we really clicked at the dinner dance each year because he also loved to dance. oscar appreciated my unorthodox style – not everyone does! i also met most of the more senior members who played an important role in my subsequent research and in helping me obtain grants from niaaa (national institute for alcohol addiction and alcoholism) in the usa (director tk lee). i met matz berglund, a psychiatrist from malmö, sweden. he was keen for us to study the autopsy brains of alcoholics from sweden. he suggested that we could transfer the brains to the new south wales tissue resource centre (trc). every person in sweden has a centralised medical record that includes data on general health, eating and drinking habits. this information is invaluable when correlated with the harvested brains. i visited matz in malmö in 1989 to discuss this possibility. i recall being in the hotel room when cnn announced the huge earthquake that had struck san francisco. i watched in horror as they showed the motorways, bridges and buildings that had collapsed. unfortunately, the swedish government would not let the brains leave the country so that our collaboration was cut short. there was a lot of serious science at these conferences and a lot of wonderful collaborations were developed. for many years we worked closely with edie sullivan and dolf pfefferbaum at the sri in palo alto, california. they invited us to their labs at sri in palo alto for six weeks to complete a study using mri on formalin fixed brains. we had intended to camp in a friend’s vw camper but there were no camping areas close by. edie booked us into a downtown motel called ‘the mermaid inn’. it became obvious on the first night with all the movement about the corridors and banging of doors that this was a brothel. edie was horrified at her mistake and she invited us to stay in their beautiful home. most recently we stayed at edie and kevin’s home in palo alto when we were in the usa in 2017. in 1992 i attended the isbra conference in bristol, in the uk. i met another australian who was big in the alcohol field john saunders. he and i ran two subsequent isbra conferences in australia (gold coast in 1994 and sydney in 2006). i also met many of the key european alcohol research workers like karl mann from heidelberg. figure 7: professors edie sullivan (left) and dolf pfefferbaum (right) from stanford university joined my wife (therese garrick) and me at the isbra conference dinner in sydney in 2006. neuropathology training and courses i saw training of staff in neuropathology as one of our critical roles. this included not just pathologists but also neurologists, neurosurgeons and radiologists. as a result, i began negotiations soon after i arrived in sydney with these medical groups. senior neurology and neurosurgery staff realised the enormous benefits of adding neuropathology to their trainee programs. we soon had regular rotating appointments with trainees from neurology, neurosurgery and pathology. two of the rotating neurosurgical registrars are still good friends – david mcdowell, a senior neurosurgeon in canberra and charlie teo, a well-known neurosurgeon in sydney. charlie was awarded the prestigious australian of the year award in 2016. one of my pathology rotating registrars was michael buckland. he now holds my position as head of neuropathology at royal prince alfred hospital. one of his main interest is the molecular biology of brain tumours. michael has also been instrumental in starting a brain bank for sports head injuries. the australian sports brain bank is located at the brain and mind centre and is partnered with the concussion legacy foundation in the usa. in addition, i decided to start an annual postgraduate neuropathology training course. this was a huge undertaking. i was able to convince my australian neuropathology colleagues (and friends) to contribute to this enterprise. initially, the main support group were peter blumbergs (adelaide), tony tannenberg (brisbane) and colin masters, catriona mclean and michael gonzales from melbourne. my own staff were also involved – roger pamphlett, michael rodriguez, roger stankovic, therese garrick, steve kumjew, donna sheedy and jillian kril. we limited the number of students to about 20 initially because each student needed a microscope and set of histological slides. there was a big emphasis on practical aspects of neuropathology and each student dissected a brain. the first year was a huge amount of work for our technicians but, after that, it became easier. the program was a huge success and, over the years, there were others who visited and contributed including our mate jim powers from rochester, new york. the students paid to attend so travel, accommodation and social event costs were covered for our teaching staff. each january they would fly into sydney for the four-day course over a weekend. we had a lot of fun together and i still receive thanks from many of those clinicians who attended. we even had international participants from new zealand and jordan. figure 8: michael buckland, then registrar in neuropathology, attended and presented at the 2001 american association of neuropathologists. it was about this time (1993) that i met therese garrick. she had been appointed to a research position with the biological schizophrenia research team (bsrt). her role was to attend the department of forensic medicine each morning to review the cases of the day to see if any were suitable for inclusion in our new brain bank (nsw tissue resource centre). on her first day she was told by stan catts (director of the bsrt) “meet professor harper at the forensic institute and he will show you around”. the meeting went something like: “hello professor harper, my name is therese garrick”. i responded with “hello therese (incorrect pronunciation) please call me clive”. she responded with the correct pronunciation of her first name and i repeated it incorrectly again. i am not sure why i did not get it but, to this day, i still pronounce it incorrectly! this was the beginning of a wonderful friendship/relationship and we have been married for 20 years. jim powers flew out from rochester to join us for the celebrations in 2000. figure 9: official dinner at the 2001 american association of neuropathologists. from the left, gavin dixon, michael buckland, maria sarris, therese garrick and clive harper. we were all working in neuropathology at the university of sydney. fern yang was another of my favourite staff members. she and her husband migrated to australia in 1990 and both had medical degrees from china. these degrees could not be registered in australia so they decided to do master’s/phd degrees at the university of sydney. fern came to work with me in neuropathology and her husband found a position in physiology. she had a delightful buoyant personality and was a pleasure to work with. after completing her master’s degree she became our muscle/nerve biopsy technician. sadly, in about 2000 she developed a cancer of the bile duct and died after a 4-year battle. jordan and syria – april 1992 bashar anabtawi, a jordanian/palestinian pathologist had come to our annual postgraduate neuropathology course in sydney in 1990. he wanted to develop his skills in muscle and nerve pathology. we got on very well and, as he left sydney he asked me if i would be prepared to come to jordan to help him develop a nerve and muscle laboratory for the middle east. at the time, most of the biopsies from jordan were sent to london. i jumped at the opportunity. bashar was good friends with the australian ambassador in jordan and the next thing we knew we were invited to stay at the consulate in jordan – what an opportunity. this adventure was amazing. apart from the work we visited damascus, and the carved city and temples in petra. in addition, we visited pella, with its ancient greek ruins. we climbed mount sinai to see the sun rise in the morning and then, in the afternoon, went for a swim in the ‘dead sea’. the ambassador was bob bowker – we are still friends with bob and his wife jenny. we share our love of camping around australia and own identical camping rigs (active campers). working in paris 1993 in 1993 i was invited to work in paris for six weeks with jean-jacques hauw at the hôpital universitaire pitié salpêtrière. my aim was to review all of their cases of the wernicke korsakoff syndrome and determine the prevalence of that disease in their paris forensic population. we published the data in 1995. harper c, fornes p, duyckaerts c, lecomte d, hauw j-j. an international perspective on the prevalence of the wernicke-korsakoff syndrome. metabolic brain disease, 1995. nisad in 1994 i negotiated to work with the nisad (neuroscience institute for schizophrenia and allied disorders) group of researchers and to take on some new staff to help with the brain bank. i went to forensic medicine each morning looking for donor cases for the brain bank. if there were any cases that had a history of schizophrenia or alcoholism that might be suitable for the brain bank the nisad staff called the relatives of the deceased to ask if they would consider donating their loved ones brain to the brain bank – a tough job! figure 10: students at our annual postgraduate neuropathology course at the university of sydney studying the histopathology slides for the course in 2004. research grants the neuropathology lab was going gangbusters with good financial support from the nh&mrc and nisad and various other smaller funding bodies. in 1990 i had given a lecture in washington, dc, to the niaaa. in discussions with the director, enoch gordis, he asked if i had ever considered submitting a grant application to the niaaa to support the australian brain bank. when dr. glenda halliday joined our research group we put together a research grant application to niaaa in the usa. the niaaa is one subsection within the huge us national institutes of health (nih). we were successful with this application in 1993 and were awarded $750,000. this allowed us to appoint several new staff members for the brain bank and therese garrick was reappointed as clinical manager. as part of the grant i was expected to travel to the usa each year to attend the research society of alcoholism. i formed many wonderful working relationships and friendships with members of the rsa over the next 20 years. the grant was a great honour – only one or two other australian research groups received grants from the nih. the funding has continued to this day (2020). figure 11: lecturers at the annual postgraduate neuropathology course at the university of sydney in 2004. from the left: jillian kril (sydney), clive harper (sydney), judith fryer (sydney), renata kalnins (melbourne), roger pamphlett (sydney), tony tannenberg (brisbane) and peter blumbergs (adelaide). ‘using our brains’ – a new initiative at the university of sydney in the early 2000s we realised that we needed to obtain more ‘normal brains’ to allow our brain bank research to prosper. we came up with the idea of asking the public to consider donating their brains while they were alive. here is the documentation of the huge public program that we launched in 2002. i was blessed by having a young friend, james pegum, assist with the six-month lead-up and launch in the nsw parliament house. he ably assisted with all of the subsequent media activity. i think that i was on every sydney television and radio station during the launch week. one of the main reasons for the success of this initiative was that gough whitlam agreed to become a donor and help launch the program. james went on to a career in it and is now a part-owner of privia. he is married to ria and they are living in orange. they have two boys, william and charlie and we see each other regularly. figure 12: official launch of the ‘using our brains’ donor program in the new south wales state parliament house in 2002. clive harper is talking with retired prime minister gough whitlam (centre) who volunteered as our first donor. sri lanka – 2002, 2007 and 2008 throughout the 2000s forensic medicine at glebe had been inviting postgraduate pathology students from sri lanka to sydney for an additional one year’s training. this was supported strongly by the university of colombo. in fact, it was a requirement that each student spend one year in another country to complete their training in pathology. i spoke to john hilton (director of forensic medicine) and he put me in touch with the coordinator of the colombo program. subsequently, a number of students came and worked with us in pathology at rpah. the first student was geethika jayaweera we are still good friends. geethika is now one of the most senior pathologists in the sri lankan army. there were many other trainees who visited sydney for 12 months and this helped to build strong links between our countries. as a result of this program i was invited to visit colombo as their external examiner in postgraduate anatomical pathology in 2002, 2006 and 2007. on each occasion, after we had completed the examinations with the postgraduate students, we spent two weeks travelling around their beautiful country. back in colombo we met up with geethika and her family and went to the local cricket club. we met arjuna ranatunga, the captain of the sri lanka team when they won the 1996 cricket world cup. he was nicknamed captain cool and is regarded as the pioneer who helped to lift sri lankan cricket from underdog status. after retirement, he worked in many posts of sri lankan cricket administration and then entered politics in 2005. ranatunga became the cabinet minister of transport and civil aviation. he was very charming. the sri lankan cricketers have a soft spot for australia as shane warne raised funds to rebuild the galle cricket ground after the 2004 tsunami. muscat, oman – 2008 and 2009 in 2008 i was invited to run a neuropathology course in muscat, oman. it ran very smoothly and was well accepted. following this, the dean of medicine at the sultan qaboos university approached me to ask if i would be interested in coming to work at the university/hospital in the future. i was very attracted to the idea and thought that it might fit in well with our plans for retirement. i negotiated with my staff and the university and rpah and changed my role to part-time. i was responsible for doing the neuropathology reports for the brain bank cases a 20 hour per week appointment. the pathology sides of the cases could be sent to me anywhere in the world – including oman. our neuropathology team was now 26 people. our five months term in 2009 in oman was fantastic. we enjoyed the enthusiasm of our students and the atmosphere of both the hospital and university. we lived on campus but took the opportunity of exploring oman at every opportunity in our borrowed tent and sleeping bags. on many occasions, as we camped on the beaches and in the deserts, we were offered food and drinks by passing locals. the omani’s hospitality was amazing. this was the perfect ‘exit plan’ and we moved into retirement smoothly and easily. i have no regrets about retiring and have enjoyed every moment. one of the main activities has been travel and catching up with our world-wide network of friends from both our work and social lives. we are also doing volunteer work with a local toy recycle group, catering to the nsw rural fire services and on our extended travels, working with blazeaid. they are another volunteer group who help aussie farmers after fires and floods to refence their properties. awards royal prince alfred research foundation medal for excellence in research i was encouraged to put my name ‘in the ring’ for the prestigious royal prince alfred (rpa) research foundation medal for excellence in research. i was one of two doctors selected by the committee and we were invited to present our research in lecture format so that the committee could select the winner. my younger son, rye, was able to come along –this was the first time he has heard me speak in public. i won the rpa foundation medal for excellence in research in 2006. this was the press release: “rpa medal winner identifies approaches to repair alcohol related brain damage. professor clive harper has won the prestigious rpa foundation medal for excellence in research for his ground-breaking research into alcohol-induced brain damage.” figure 13: farewell luncheon with all of the examiners at the university of colombo after completion of the examinations for postgraduate anatomical pathology in 2002. australia day honours award in the following year (2007) i was delighted to be included in the australia day honours list and was awarded an am (member of the order of australia). this is the citation: professor clive gordon harper department of pathology, university of sydney nsw 2006 for service to medicine in the field of neuroscience, particularly research into the neuropathological consequences of alcohol-related brain damage, and through contributions to public health policy. the presentation of awards was at nsw parliament house. professor marie bashir was the governor general and officiated. i had met her professionally when she was the clinical director of mental health services for the central sydney area health service. she was very charming and told me that she had been following my career and the development of the brain bank. the henri begleiter excellence in research award in 2009 i received another award that i am very proud of: the henri begleiter excellence in research award. nominations for this award are accepted from research society of alcoholism members. recipients of this award receive a plaque and cash award at the annual meeting. this award is given to an individual demonstrating innovation or creativity and excellence in their research and/or someone whose work has a major impact on the field. recipients of this award: ting-kai li (2007), adolf pfefferbaum (2008), clive harper (2009), jan hoek (2010), marlene oscar berman (2011), linda spear (2012), kathleen grant (2013), george fein (2014), john krystal (2015), bernice porjesz (2016), raymond anton (2017), sarah jo nixon (2018). the award was made at the san diego rsa conference by the president, peter monti. my personal philosophy has always been to find the best staff to work with regardless of whether or not they are smarter than you … most have been smarter! always be available to staff and to look for opportunities to help staff in their personal development, such as encouraging attendance at conferences, even for junior staff. always be on the lookout for new opportunities and do not be afraid of the extra work. go the extra yard and don’t be afraid to ask the hard questions. be totally honest and up front with staff and colleagues. i have rarely been disappointed by my staff and consider that i have been blessed in my career. i was awarded emeritus status by the university of sydney and give occasional consultative advice to my colleagues. in our retirement my wife and i have joined a number of volunteer groups including blazeaid. the peninsular senior citizens toy recycle group and the local rural fire service (catering division). blazeaid is particularly challenging. it is a volunteer-based organisation that works with families and individuals in rural australia after natural disasters such as fires and floods. working alongside the rural families, we help to rebuild fences and other structures that have been damaged or destroyed (https://blazeaid.com.au). in our spare time we swim, cycle, sail and travel in our 4x4 camper rig. last year we were on the road, around australia, for seven months! after covid we hope to travel overseas again to meet up with our international colleagues and friends. figure 14: camping in 2021 on magnetic island in queensland with the local koala. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodegeneration: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:13 (2022) review neurodegeneration: 2022 update john f. crary neuropathology brain bank & research core, department of pathology, nash family department of neuroscience, department of artificial intelligence & human health, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary, md-phd · friedman brain institute · ronald m. loeb center for alzheimer’s disease · icahn school of medicine at mountsinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 18 april 2022 accepted: 05 may 2022 copyedited by: georg haase published: 10 may 2022 https://doi.org/10.17879/freeneuropathology-2022-3866 keywords: neurodegeneration, neuropathology, aging, microglia, alzheimer disease, tauopathy, huntington’s disease, α-synucleinopathy, tdp-43 proteinopathy, traumatic brain injury abstract here, we review a collection of recent manuscripts and research trends on the neuropathology of neurodegeneration that are considered by the author to be among the potentially most impactful. to the greatest extent possible, we chose to focus on histopathological studies that are most relevant to experimental and diagnostic neuropathology. while there has been an abundance of important recent discoveries and developments in neurodegenerative disease research, there was a deliberate effort here to provide balance to prevent disease categories and experimental approaches from overshadowing the others. the result is a diverse series of outstanding studies, together showing the landscape of progress across neurodegenerative disorders. one is a stereological study examining dystrophic microglia in aging. we highlight the first large genetic study of primary age-related tauopathy, showing convergence and divergence from classical alzheimer’s disease. there were further advances in the neuropathological criteria and staging of chronic traumatic encephalopathy. links suggesting a causal role for tmem106b in tdp-43 proteinopathy emerged. attempts to subtype alzheimer’s disease on the molecular level were made. evidence for a role for the vegf family in cognitive impairment was advanced. comparison of gene expression profiles from myeloid cells in peripheral blood and brain tissues from parkinson’s disease patients revealed pathways that may lead to new mechanistic insights and biomarkers. a large autopsy series identified an increased frequency of central nervous system developmental malformations in huntington’s disease. a robust and reliable system for assessing lewy body pathology was proposed. finally, we continue to be plagued by the covid-19 pandemic, with lingering concerns of a long-term link with neurodegeneration. 1. neurodegeneration-specific alterations in microglial morphology as the data from genetic, cellular and animal studies accumulates, the importance of microglial and neuroimmune function continues to grow. microglia take on a number of different morphologies, including ramified, hypertrophic and dystrophic, that are demonstrable using various histopathological techniques. the importance of the dystrophic morphology is currently unclear and has been implicated in disease and age-related cellular senescence. unfortunately, there has been a dearth of rigorous histopathological human studies looking at them across the aging spectrum. critically, our understanding of dystrophic microglia has been hindered by a lack of well-designed and implemented quantitative studies of these various microglial morphological patterns across the human lifespan and in disease. shahhidehpour et al. performed a stereological study, published in the neurobiology of aging, specifically investigating these microglial morphologies in human post-mortem brain tissues across the lifespan (shahidehpour et al., 2021). the authors set out to test the hypothesis that age causes microglial dystrophy. abnormalities in microglial function may be a strong contributor to age-related neurodegenerative disease, with cellular senescence being a critical hallmark of aging. the strength of the study was the rigorous stereological counts of microglial morphologies including hypertrophic and dystrophic types alongside total, across a collection of brains ranging in age in years from teenage to nineties (figure 1). surprisingly, while an age-associated increase in the total number of dystrophic microglia was found, this was not significant when compared to the total number of microglia which also increased with age. in contrast, the total number of dystrophic microglia was greater in subjects with a number of neurodegenerative diseases, including alzheimer's disease, dementia with lewy bodies, and limbic predominant age-related tdp-43 encephalopathy. the authors also provided evidence for a role of altered iron homeostasis in these dystrophic microglia using ferritin light chain as a marker of intracellular iron indicating a possible mechanism. this result pinpoints microglia dystrophy as a critical disease-associated signature in neurodegeneration. figure 1. examples of ramified (a, b), hypertrophic (c, d) and dystrophic microglia (e, f) reproduced with permission from shahidehpour et al. (shahidehpour et al., 2021). 2. pathogenetic study in part clarifies genetic overlap with ad and identifies jade1 in 2014, a group of neuropathologists, led by the author of this article, defined criteria for a new category of neurodegenerative change, termed primary age-related tauopathy (part) (crary et al., 2014). subsequently, there have been many clinical and neuropathological studies that have explored the contours and implications of this designation, both challenging and supporting its scientific validity as an independent age-associated neurodegenerative category. while the terminology has been broadly adopted, part remains controversial and poorly understood. one of the biggest gaps in knowledge is the extent to which part represents a distinct pathological process or shares mechanistic underpinnings with ad and perhaps the other tauopathies. this was addressed with the first genome-wide association study (gwas) in primary age-related tauopathy published online in november 2021 in acta neuropathologica (farrell et al., 2022). given that part is a ubiquitous neuropathological feature in essentially all aged human brains, farrell et al. conducted a case-only quantitative trait analysis using braak neurofibrillary stage as an endophenotype. while this was a relatively small gwas (n=647), it was noted that most similar studies rely on clinical phenotypes, which are noisy relative to autopsy-derived endophenotypes, such as the braak neurofibrillary tangle (nft) stage used in this study. looking at candidate genes implicated in alzheimer’s disease and tauopathies revealed some associations, inducing with the mapt h1 haplotype and other candidate loci, including slc24a4, ms4a6a, hs3st1 and eif2ak3. as with previous studies, there was no association between part and the most important and common risk allele for sporadic alzheimer’s disease, apoe. the study also identified a novel locus on chromosome 4 that met genome-wide significance. given that the locus contains a number of genes, the investigators used a computational approach, including examination of a single-cell tangle-containing rna-seq dataset, to nominate gene for apoptosis and differentiation in epithelia 1 (jade1) as the best candidate in the locus. the jade1 protein has some interesting neuropathological features, co-localizing by immunohistochemistry with nft in all tauopathies except pick disease, the prototype tauopathy with three microtubule-binding domain repeats (3r). this was intriguing given that jade1 protein biochemically interacts with only tau isoforms that contain 4 microtubule-binding domain repeats (4r). finally, the jade1 homolog in drosophila, rhinoceros (rno), was shown to be a potent regulator of cell death in the fly eye and brain. together, these findings suggest that jade1 might be a component of a pathway that shared by many tauopathies. 3. neuropathological criteria for chronic traumatic encephalopathy: consensus update as awareness and scientific interest in chronic traumatic encephalopathy (cte) continues to expand, the neuropathological criteria continue to be a focus area. concussions are extremely common, especially in military populations and contact sports athletes, and the long-term neurological sequelae remain under-investigated. previously, the nih convened a consensus panel of neuropathologists to establish preliminary consensus criteria for the neuropathological diagnosis of cte (mckee et al., 2016). this landmark study has been highly influential, but questions persisted. for example, it was unclear as to what is the minimum requirement for a cte diagnosis. further, it was important to know whether cte could be reliably differentiated from age-related tauopathies like primary age-related tauopathy (part) and aging-related tau astrogliopathy (artag). further, a four-tiered staging system has been proposed but has not been independently validated (mckee et al., 2013). to address this, the nih convened a second consensus panel meeting with eight neuropathologists expert in neurodegenerative tauopathies (bieniek et al., 2021). the panel blindly reviewed 27 cases (17 cte and 10 other tauopathies), applying the 2016 cte criteria. first, there was substantial agreement in the diagnosis across raters which validated the robustness of the approach. next, the panel turned to staging and developed a simplified and practical tool for the assessment of the cte stage (figure 2). the tool involves assessment of the 11 brain regions proposed by the first consensus meeting with a minimum threshold as a single pathognomonic lesion (with or without glial tangles). a point system was devised encompassing the numerous features of cte with a cutoff of five, dichotomizing “low" and “high" cte neuropathologic change. this simplified system had excellent correlation with the cte stage as previously described (mckee et al., 2013). numerous challenges and controversies in the field remain, but this represents a milestone in establishing rigor and reproducibility in cte autopsy studies and provides a foundation for further advances in the field. figure 2. flowchart for diagnosis of cte reproduced with permission from bieniek et al. (bieniek et al., 2021). 4. a causal role for tmem106b in tdp-43 pathology inclusions containing the tar dna-binding protein 43 (tdp-43) are the hallmark neuropathological feature of a number of neurodegenerative diseases with divergent neuroanatomical vulnerability and symptomatology including amyotrophic lateral sclerosis (als), frontotemporal lobar degeneration (ftld-tdp), and limbic age-related tdp-43 encephalopathy (late). the underlying reasons for these divergent pathoanatomical signatures are unclear and numerous factors likely contribute. while mutations in the tdp-43 gene are only rarely associated with tdp-43 proteinopathy, genetic variation in other genes including tmem106b and c9orf72 are much more frequent in this context. to explore this, mao et al. performed a rigorous pathoanatomical study published in acta neuropathologica examining the influence of genetic variation in tmem106b and c9orf72 on the burden and distribution of tdp-43 proteinopathy in 899 brains from the university of pennsylvania center for neurodegenerative disease research (cndr) brain bank, including cases with als, ftld-tdp, alzheimer’s disease and lewy body disease (mao et al., 2021). tmem106b risk alleles for ftld-tdp were demonstrated to modify tdp-43 burden in als (but curiously not in ftld-tdp). c9orf72 expansion was associated with tdp-43 burden in both als and ftld-tdp. the interaction between tdp-43 and tmem106b was confirmed in a cellular model. these data are intriguing because they suggest a causal role for tmem106b in some forms of tdp-43 pathology (figure 3). figure 3. schematic showing the effects of tmem106b on tdp-43 proteinopathy reproduced with permission from mao et al. (mao et al., 2021). 5. defining molecular subtypes for alzheimer’s disease according to a widely held dogma, alzheimer’s disease arises secondarily from amyloid-beta toxicity that in turn leads to neurofibrillary degeneration and cell death. however, there is increasing evidence that ad is more heterogeneous than recognized, manifesting not only in the ad mimics such as limbic age-related tdp-43 encephalopathy and comorbid dementias such as lewy body disease and cerebrovascular disease) but also within the group of subjects that meet the neuropathological criteria for ad. neuropathological subtypes of ad have been proposed (janocko et al., 2012; murray et al., 2011) and gene expression studies in ad have been previously performed, but defining a molecularly defined subtype has never been attempted. armed with a large rna-seq dataset of over 1543 transcriptomes derived from four brain regions from two independent brain bank series, neff et al. set out to define molecular subtypes of ad (neff et al., 2021). to accomplish this, they leveraged a novel clustering method which uncovered five stable subtypes in three classes: a, b1/2 and c1/2 (figure 4). one of these subtypes termed class c (“typical") which represented about one third of all cases, displayed expression changes similar to those previously reported in alzheimer’s disease (blalock et al., 2011) with increased immune response and decreased synaptic signaling. the class c cases also showed strong up-regulation of pathways involving amyloid-beta clearance, fiber formation and scavenger receptor activity. in contrast, expression changes in class a (“atypical") were generally opposite to class c (blalock) changes. class b (“intermediate") did not show changes in either direction. tau-related pathways were strongly upregulated in both a and b1/2 subtypes. certain pathway changes were subtype-specific illustrated by the up-regulation of protein degradation-related genes (e.g., ubiquitination) in class a or the up-regulation of organic acid-related genes in class b. of note, the different classes were associated with distinct clinical and neuropathological phenotypes. class c1 for instance showed a more frequent association with increased amyloid plaque burden than the tau-related class a and also had an increased apoe ε4 allele frequency whereas class a displayed increased mapt pathway activation and decreased nft association, thus potentially representing a resilient subtype. many questions remain, especially how these subtypes align with previously proposed ad subtypes. nevertheless, this study provides evidence that ad is likely not a single entity and paves the way towards additional studies addressing this critical barrier. figure 4. molecular subtypes of alzheimer’s disease defined by gene expression changes. changes in the mean expression of gene pathways are indicated for ad subtypes a, b1, b2, c1 and c2 in comparison to normal control samples. data from a previous study msigdb are indicated on the right. changes in gene expression are grouped by major areas of biological activity. reproduced with permission from neff et al. (neff et al., 2021). 6. cerebrovascular disease: vegf gene family expression in cognitive aging and alzheimer’s neuropathology vascular endothelial growth factor (vegf) is an angiogenic factor induced by hypoxia that plays an important role in angiogenesis. the vegf gene family has several members which interact with a complex set of receptors and co-receptors which trigger (figure 5) various signaling pathways highly relevant to neurodegeneration such as neurogenesis, neuroprotection, apoptosis, cell proliferation, and inflammation. likewise, previous studies have linked vegf signalling to various neurodegenerative and cerebrovascular diseases. figure 5. illustration of the vegf family of growth factors and their receptors. asterisks indicate genes that are differentially expressed between ad and controls. reproduced with permission from mahoney et al. (mahoney et al., 2021). a focused study published in molecular psychiatry by mahoney et al. found intriguing direct evidence that changes in vegf signaling are closely associated with the development of ad neuropathology and the progression of cognitive impairment (mahoney et al., 2021). the authors leveraged the religious orders study (ros) and the rush memory and aging project (map), using global composited cognitive scores from 17 neuropsychological tests and autopsy measurements of amyloid plaque and tau burden from a total of 531 subjects with normal cognition, mild cognitive impairment (mci) and ad-type dementia. the authors found that expression in the frontal cortex of four key components of vegf signaling, vegfb, flt4, flt1 and pgf, were associated with accelerated cognitive decline. all four of these genes were also associated with increased tau and amyloid burden. additional work is required to elucidate exactly how vegf signaling relates to ad neuropathology and cerebrovascular disease. this study, which requires additional confirmation and replication with molecular studies and cellular/animal modeling, spotlights the potential importance of vegf in dementia. 7. monocytes and microglia in parkinson’s disease evidence is accumulating that the immune system plays a critical role in parkinson’s disease (pd). investigating the pathogenic events occuring in critical immune cells especially in the pd brain remains of great interest. myeloid cells and microglia have been understudied. genetic studies have identified a large number of risk loci that alter the expression of nearby genes in easily accessible peripheral monocytes and there is enrichment of gene sets expressed in microglia. there are many functions of myeloid cells, including clearing of debris and maintaining brain homeostasis, that are highly relevant to pd pathogenesis. further, alpha-synuclein can promote microglial release of neurotoxic factors potentially leading to death of dopaminergic neurons. intriguingly, the possibility that alpha-synuclein pathology might be initiated in the enteric nervous system further highlights the potential importance of peripheral myeloid cells to influence pathogenesis. how myeloid cells are involved in pd is unclear and the extent to which specific immune cell types, in the periphery or in the central nervous system, participate remains a critical knowledge gap. in a paper published in nature aging by navarro et al., an international team of researchers conducted a study looking at monocytes in peripheral blood as well as monocytes and microglia in post mortem brains from pd patients (navarro et al., 2021). the authors sorted cd14+ peripheral monocytes and microglia from the blood and brains and performed a large-scale unbiased single cell transcriptomic study. the strength of this study are the direct comparisons between gene expression in the peripheral blood with the brain as most studies in pd have focused on dopaminergic neurons, fibroblasts, or whole blood. they identified genes and co-expression networks dysregulated in pd myeloid cells. they identified genes that are involved in mitochondrial and proteasomal function that are highly expressed in a proinflammatory intermediate subpopulation. they identified 17 variants that influence mrna expression and pd risk. this enabled the authors to show that there are actually opposite mitochondrial transcriptomic profiles between microglia and macrophages (down regulated) versus peripheral monocytes (upregulated). these results provide further support for a prominent role for immune dysfunction in the pathogenesis of pd. 8. neurodevelopmental alterations in huntington’s disease huntington’s disease (hd) is the prototype hyperkinetic movement disorder caused by polyq repeat expansion in the huntingtin gene (htt). while the neuropathology of hd has been extensively studied, accumulating evidence from cellular, animal and human studies pointed to the existence of neurodevelopmental abnormalities in hd. of note, barnat et al. have reported defects in neuronal migration in the brains of mutant htt-fetuses and linked them to alterations in the interkinetic nuclear migration of progenitor cells (barnat et al, science 2020). neuropathological evidence in adult human hd has however been lacking. in a paper published in acta neuropathologica, hickman et al. provided the first neuropathological evidence that malformations are present in adult human hd (hickman et al., 2021) through systematic evaluation of two large brain collections: a discovery cohort from the new york brain bank (nybb) at columbia university and a validation cohort from the harvard brain tissue resource center (hbtrc), total n=3918. of 130 hd brains examined from the nybb, eight (6.2%) had at least one malformation compared to 12 (0.75%) in the 1600 non-hd brains. periventricular nodular heterotopias (pnh) were the most frequent malformation (figure 6), confirming abnormalities in neuronal migration in hd. intriguingly, the neurons in the pnh contained p62-positive aggregates indicative of ongoing proteinopathy. other cerebral malformations included hamartomas, cerebellar nodular heterotopias, and one multinodular vacuolating tumor of the cerebellum (mvnt). the frequency of hd-associated cerebral malformations was also significantly increased in the hbtrc cohort but at a lower frequency than in the nybb cohort, which was likely explained by differences in sampling and assessment protocols. the asymmetric nature of the cerebral malformations in hd suggested that they were secondary to somatic mutation. their higher frequency in women may be related to sexual dimorphism. the possibility that hd brains contain additional developmental or neurodevelopmental abnormalities merits further investigation. nevertheless, this study provides concrete evidence that developmental abnormalities occur in human brains with hd and provide the foundation for a new avenue into studying htt-related mechanisms. figure 6. a periventricular nodular heterotopia (pnh) in a 41-year-old woman with hd. gross (a) and low power (lh&e stain) images (b) of a subependymal heterotopia containing non-dysmorphic neurons (c) that are immunopositive for htt and p62 (d, arrows). reproduced with permission from hickman et al. (hickman et al., 2021). 9. criteria for lewy body pathology: approaching consensus the spectrum of lewy body disease (lbd) pathology includes parkinson’s disease (pd), dementia with lewy bodies (dlb) and pd with mild cognitive impairment or dementia. currently, a neuropathological diagnosis of lewy body disease can be rendered using any of four different proposed systems (adler et al., 2019; braak et al., 2003; leverenz et al., 2008; mckeith et al., 2005). all use semi-quantitative assessment of the neuroanatomical distribution of lewy pathology in neurites and cell bodies, but each has specific strengths and weaknesses. each system suffers from issues related to interrater reliability and/or the inability to classify certain cases with lewy pathology (lp), for example cases with olfactory bulb-only or amygdala-only disease. this stems from the heterogenous pathoanatomical distribution of lp. in 2021, new neuropathological consensus criteria that aim to address the limitations of these systems, termed the lp consensus criteria (lpc), were developed and published in acta neuropathologica in order to improve interrater reliability and to unequivocally classify all possible cases with lp (attems et al., 2021). to improve inter-rater reliability, the authors implemented a dichotomized scoring system, with lp scored as either present or absent. the diagnostic categories were: olfactory-only, amygdala-predominant, brainstem, limbic, and neocortical lp. the 16 raters from 13 centers applied the lpc and each of the four existing systems to 34 cases with lp from the newcastle brain tissue resource and the university of pennsylvania brain bank. interrater reliability was good for the lpc, being comparable to the mckeith and leverenz system (krippendorff’s alpha ≈ 0.6), and considerably better than for braak and adler (beach). the lpc system was as applicable as the beach system and able to unequivocally classify essentially all cases, which was not the case for the leverenz, mckeith and braak systems which failed in 10%, 25% and nearly 30% of cases respectively. given the reliability and applicability of the lpc system, we expect that it will be adopted broadly in the post-mortem evaluation of cases with lewy body pathology. 10. long-covid intersecting with neurodegeneration during the covid-19 pandemic, a number of neuropathological studies addressed the changes in the human brain from patients succumbing to acute sars-cov-2 infection. microthrombi and strokes were among the most common findings together with indirect inflammatory and autoimmune changes but encephalitis was rare. after the peak of the pandemic, roughly one third of patients emerged with a clinical syndrom often termed post-acute sequelae of sars-cov-2 infection (pasc) and commonly referred to as “long-covid". cardiac and pulmonary symptoms are prominent in pasc but there are also many alarming non-specific neurological and psychiatric symptoms including fatigue, cognitive complaints (“brain fog"), sensorimotor defects, headaches, insomnia, depression, and post-traumatic stress disorder (taquet et al., 2021). a meta analysis confirmed decreased cognitive function seven months following covid-19 infection (crivelli et al., 2022). pasc has drawn attention in the field of neurodegeneration given the possibility that infection with the virus may trigger a long-term neurodegenerative process (lennon, 2020). the mechanisms whereby pasc may be linked to neurodegeneration remained however elusive. many investigators have raised the questions as to whether sars-cov-2 can cause neurodegeneration through its neurotropism or otherwise (krey et al., 2021). some investigators have proposed that sars-cov-2 enters the brain and may activate an ad-like program (shen et al., 2022). the ace2 covid-19 receptor was indeed shown to be upregulated in the hippocampal formation of ad patients (zhao et al., 2021), which may explain why medial temporal lobe structures might be vulnerable, potentially contributing to some post-infection memory impairment and neurodegeneration (ritchie et al., 2020). notably, signaling pathways leading to tau hyperphosphorylation in ad including gsk3β may be activated in post-mortem covid-19 brains (reiken et al., 2022). adand covid-19 signaling pathways may converge at the level of the nrlp3 inflammasome through dysregulation of amyloid clearance by microglia (cama et al., 2021, p. 3). alternatively, sars-cov-2 infection may worsen ad for various reasons (villa et al., 2022). while this constitutes perhaps the most urgent and potentially important research trend at this time, it remains to be seen whether there will be sustainable momentum. acknowledgement dr. crary receives research funding from the nih (r01ag054008, r01ns095252, r01ag062348, r01ns086736, u54ns115266, u54ns115322). we further acknowledge the rainwater charitable trust/tau consortium, david and elsie werber, alexander saint amand scholar award and karen strauss cook research scholar award. references adler, c. h., beach, t. g., zhang, n., shill, h. a., driver-dunckley, e., caviness, j. n., mehta, s. h., sabbagh, m. n., serrano, g. e., sue, l. i., belden, c. m., powell, j., jacobson, s. a., zamrini, e., shprecher, d., davis, k. j., dugger, b. n., & hentz, j. g. (2019). unified staging system for lewy body disorders: clinicopathologic correlations and comparison to braak staging. journal of neuropathology and experimental neurology, 78(10), 891–899. https://doi.org/10.1093/jnen/nlz080 attems, j., toledo, j. b., walker, l., gelpi, e., gentleman, s., halliday, g., hortobagyi, t., jellinger, k., kovacs, g. g., lee, e. b., love, s., mcaleese, k. e., nelson, p. t., neumann, m., parkkinen, l., polvikoski, t., sikorska, b., smith, c., grinberg, l. t., … mckeith, i. g. (2021). neuropathological consensus criteria for the evaluation of lewy pathology in post-mortem brains: a multi-centre study. acta neuropathologica, 141(2), 159–172. https://doi.org/10.1007/s00401-020-02255-2 barnat, m., capizzi, m., aparicio, e., boluda, s., wennagel, d., kacher, r., kassem, r., lenoir, s., agasse, f., braz, b.y., liu, j.-p., ighil, j., tessier, a., zeitlin, s.o., duyckaerts, c., dommergues, m., durr, a., humbert, s., (2020). huntington's disease alters human neurodevelopment. science, 369(6505), 787-793. https://doi.org/10.1126/science.aax3338 bieniek, k. f., cairns, n. j., crary, j. f., dickson, d. w., folkerth, r. d., keene, c. d., litvan, i., perl, d. p., stein, t. d., vonsattel, j.-p., stewart, w., dams-o’connor, k., gordon, w. a., tripodis, y., alvarez, v. e., mez, j., alosco, m. l., & mckee, a. c. (2021). the second ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. journal of neuropathology and experimental neurology, 80(3), 210–219. https://doi.org/10.1093/jnen/nlab001 blalock, e. m., buechel, h. m., popovic, j., geddes, j. w., & landfield, p. w. (2011). microarray analyses of laser-captured hippocampus reveal distinct gray and white matter signatures associated with incipient alzheimer’s disease. journal of chemical neuroanatomy, 42(2), 118–126. https://doi.org/10.1016/j.jchemneu.2011.06.007 braak, h., del tredici, k., rüb, u., de vos, r. a. i., jansen steur, e. n. h., & braak, e. (2003). staging of brain pathology related to sporadic parkinson’s disease. neurobiology of aging, 24(2), 197–211. https://doi.org/10.1016/s0197-4580(02)00065-9 cama, v. f., marín-prida, j., acosta-rivero, n., acosta, e. f., díaz, l. o., casadesús, a. v., fernández-marrero, b., gilva-rodríguez, n., cremata-garcía, d., cervantes-llanos, m., piniella-matamoros, b., sánchez, d., del rosario-cruz, l., borrajero, i., díaz, a., gonzález, y., pentón-arias, e., montero-gonzález, t., guillen-nieto, g., & pentón-rol, g. (2021). the microglial nlrp3 inflammasome is involved in human sars-cov-2 cerebral pathogenicity: a report of three post-mortem cases. journal of neuroimmunology, 361, 577728. https://doi.org/10.1016/j.jneuroim.2021.577728 crary, j. f., trojanowski, j. q., schneider, j. a., abisambra, j. f., abner, e. l., alafuzoff, i., arnold, s. e., attems, j., beach, t. g., bigio, e. h., cairns, n. j., dickson, d. w., gearing, m., grinberg, l. t., hof, p. r., hyman, b. t., jellinger, k., jicha, g. a., kovacs, g. g., … nelson, p. t. (2014). primary age-related tauopathy (part): a common pathology associated with human aging. acta neuropathologica, 128(6), 755–766. https://doi.org/10.1007/s00401-014-1349-0 crivelli, l., palmer, k., calandri, i., guekht, a., beghi, e., carroll, w., frontera, j., garcía-azorín, d., westenberg, e., winkler, a. s., mangialasche, f., allegri, r. f., & kivipelto, m. (2022). changes in cognitive functioning after covid-19: a systematic review and meta-analysis. alzheimer’s & dementia: the journal of the alzheimer’s association. https://doi.org/10.1002/alz.12644 farrell, k., kim, s., han, n., iida, m. a., gonzalez, e. m., otero-garcia, m., walker, j. m., richardson, t. e., renton, a. e., andrews, s. j., fulton-howard, b., humphrey, j., vialle, r. a., bowles, k. r., de paiva lopes, k., whitney, k., dangoor, d. k., walsh, h., marcora, e., … crary, j. f. (2022). genome-wide association study and functional validation implicates jade1 in tauopathy. acta neuropathologica, 143(1), 33–53. https://doi.org/10.1007/s00401-021-02379-z hickman, r. a., faust, p. l., rosenblum, m. k., marder, k., mehler, m. f., & vonsattel, j. p. (2021). developmental malformations in huntington disease: neuropathologic evidence of focal neuronal migration defects in a subset of adult brains. acta neuropathologica, 141(3), 399–413. https://doi.org/10.1007/s00401-021-02269-4 janocko, n. j., brodersen, k. a., soto-ortolaza, a. i., ross, o. a., liesinger, a. m., duara, r., graff-radford, n. r., dickson, d. w., & murray, m. e. (2012). neuropathologically defined subtypes of alzheimer’s disease differ significantly from neurofibrillary tangle-predominant dementia. acta neuropathologica, 124(5), 681–692. https://doi.org/10.1007/s00401-012-1044-y krey, l., huber, m. k., höglinger, g. u., & wegner, f. (2021). can sars-cov-2 infection lead to neurodegeneration and parkinson’s disease? brain sciences, 11(12), 1654. https://doi.org/10.3390/brainsci11121654 lennon, j. c. (2020). neurologic and immunologic complications of covid-19: potential long-term risk factors for alzheimer’s disease. journal of alzheimer’s disease reports, 4(1), 217–221. https://doi.org/10.3233/adr-200190 leverenz, j. b., hamilton, r., tsuang, d. w., schantz, a., vavrek, d., larson, e. b., kukull, w. a., lopez, o., galasko, d., masliah, e., kaye, j., woltjer, r., clark, c., trojanowski, j. q., & montine, t. j. (2008). empiric refinement of the pathologic assessment of lewy-related pathology in the dementia patient. brain pathology (zurich, switzerland), 18(2), 220–224. https://doi.org/10.1111/j.1750-3639.2007.00117.x mahoney, e. r., dumitrescu, l., moore, a. m., cambronero, f. e., de jager, p. l., koran, m. e. i., petyuk, v. a., robinson, r. a. s., goyal, s., schneider, j. a., bennett, d. a., jefferson, a. l., & hohman, t. j. (2021). brain expression of the vascular endothelial growth factor gene family in cognitive aging and alzheimer’s disease. molecular psychiatry, 26(3), 888–896. https://doi.org/10.1038/s41380-019-0458-5 mao, f., robinson, j. l., unger, t., posavi, m., amado, d. a., elman, l., grossman, m., wolk, d. a., lee, e. b., van deerlin, v. m., porta, s., lee, v. m. y., trojanowski, j. q., & chen-plotkin, a. s. (2021). tmem106b modifies tdp-43 pathology in human als brain and cell-based models of tdp-43 proteinopathy. acta neuropathologica, 142(4), 629–642. https://doi.org/10.1007/s00401-021-02330-2 mckee, a. c., cairns, n. j., dickson, d. w., folkerth, r. d., keene, c. d., litvan, i., perl, d. p., stein, t. d., vonsattel, j.-p., stewart, w., tripodis, y., crary, j. f., bieniek, k. f., dams-o’connor, k., alvarez, v. e., gordon, w. a., & tbi/cte group. (2016). the first ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. acta neuropathologica, 131(1), 75–86. https://doi.org/10.1007/s00401-015-1515-z mckee, a. c., stern, r. a., nowinski, c. j., stein, t. d., alvarez, v. e., daneshvar, d. h., lee, h.-s., wojtowicz, s. m., hall, g., baugh, c. m., riley, d. o., kubilus, c. a., cormier, k. a., jacobs, m. a., martin, b. r., abraham, c. r., ikezu, t., reichard, r. r., wolozin, b. l., … cantu, r. c. (2013). the spectrum of disease in chronic traumatic encephalopathy. brain: a journal of neurology, 136(pt 1), 43–64. https://doi.org/10.1093/brain/aws307 mckeith, i. g., dickson, d. w., lowe, j., emre, m., o’brien, j. t., feldman, h., cummings, j., duda, j. e., lippa, c., perry, e. k., aarsland, d., arai, h., ballard, c. g., boeve, b., burn, d. j., costa, d., del ser, t., dubois, b., galasko, d., … consortium on dlb. (2005). diagnosis and management of dementia with lewy bodies: third report of the dlb consortium. neurology, 65(12), 1863–1872. https://doi.org/10.1212/01.wnl.0000187889.17253.b1 murray, m. e., graff-radford, n. r., ross, o. a., petersen, r. c., duara, r., & dickson, d. w. (2011). neuropathologically defined subtypes of alzheimer’s disease with distinct clinical characteristics: a retrospective study. the lancet. neurology, 10(9), 785–796. https://doi.org/10.1016/s1474-4422(11)70156-9 navarro, e., udine, e., de paiva lopes, k., parks, m., riboldi, g., schilder, b. m., humphrey, j., snijders, g. j. l., vialle, r. a., zhuang, m., sikder, t., argyrou, c., allan, a., chao, m. j., farrell, k., henderson, b., simon, s., raymond, d., elango, s., … raj, t. (2021). dysregulation of mitochondrial and proteolysosomal genes in parkinson’s disease myeloid cells. nature aging, 1(9), 850–863. https://doi.org/10.1038/s43587-021-00110-x neff, r. a., wang, m., vatansever, s., guo, l., ming, c., wang, q., wang, e., horgusluoglu-moloch, e., song, w.-m., li, a., castranio, e. l., tcw, j., ho, l., goate, a., fossati, v., noggle, s., gandy, s., ehrlich, m. e., katsel, p., … zhang, b. (2021). molecular subtyping of alzheimer’s disease using rna sequencing data reveals novel mechanisms and targets. science advances, 7(2), eabb5398. https://doi.org/10.1126/sciadv.abb5398 reiken, s., sittenfeld, l., dridi, h., liu, y., liu, x., & marks, a. r. (2022). alzheimer’s-like signaling in brains of covid-19 patients. alzheimer’s & dementia: the journal of the alzheimer’s association. https://doi.org/10.1002/alz.12558 ritchie, k., chan, d., & watermeyer, t. (2020). the cognitive consequences of the covid-19 epidemic: collateral damage? brain communications, 2(2), fcaa069. https://doi.org/10.1093/braincomms/fcaa069 shahidehpour, r. k., higdon, r. e., crawford, n. g., neltner, j. h., ighodaro, e. t., patel, e., price, d., nelson, p. t., & bachstetter, a. d. (2021). dystrophic microglia are associated with neurodegenerative disease and not healthy aging in the human brain. neurobiology of aging, 99, 19–27. https://doi.org/10.1016/j.neurobiolaging.2020.12.003 shen, w.-b., logue, j., yang, p., baracco, l., elahi, m., reece, e. a., wang, b., li, l., blanchard, t. g., han, z., frieman, m. b., rissman, r. a., & yang, p. (2022). sars-cov-2 invades cognitive centers of the brain and induces alzheimer’s-like neuropathology. biorxiv: the preprint server for biology, 2022.01.31.478476. https://doi.org/10.1101/2022.01.31.478476 taquet, m., geddes, j. r., husain, m., luciano, s., & harrison, p. j. (2021). 6-month neurological and psychiatric outcomes in 236 379 survivors of covid-19: a retrospective cohort study using electronic health records. the lancet. psychiatry, 8(5), 416–427. https://doi.org/10.1016/s2215-0366(21)00084-5 villa, c., rivellini, e., lavitrano, m., & combi, r. (2022). can sars-cov-2 infection exacerbate alzheimer’s disease? an overview of shared risk factors and pathogenetic mechanisms. journal of personalized medicine, 12(1), 29. https://doi.org/10.3390/jpm12010029 zhao, y., li, w., & lukiw, w. (2021). ubiquity of the sars-cov-2 receptor ace2 and upregulation in limbic regions of alzheimer’s disease brain. folia neuropathologica, 59(3), 232–238. https://doi.org/10.5114/fn.2021.109495 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. post-mortem neuropathologic examination of a 6-case series of car t-cell treated patients feel free to add comments by clicking these icons on the sidebar free neuropathology 3:23 (2022) original paper post-mortem neuropathologic examination of a 6-case series of car t-cell treated patients nuria vidal-robau1*, gabriela caballero1*, ivan archilla1, andrea ladino2, sara fernández2, valentín ortiz-maldonado3, montserrat rovira3, marta gómez-hernando3, julio delgado3, maría suárez-lledó3, carlos fernández de larrea3,8, olga balagué1, gerard frigola1, abel muñoz1, estrella ortiz1, teresa ribalta1, miguel j. martinez5,6, maria angeles-marcos5, marta español-rego7, azucena gonzález7, daniel benitez-ribas7, eugenia martinez-hernandez4, pedro castro2,8, iban aldecoa1,9 1 pathology department, biomedical diagnostic centre (cdb), hospital clinic of barcelona – university of barcelona, barcelona, spain 2 medical intensive care unit, hospital clinic of barcelona – university of barcelona, barcelona, spain 3 haematology department, hospital clinic of barcelona – university of barcelona, barcelona, spain 4 neurology department, hospital clinic of barcelona – university of barcelona, barcelona, spain 5 microbiology department, biomedical diagnostic centre (cdb), hospital clinic of barcelona – university of barcelona, barcelona, spain 6 barcelona institute for global health (isglobal), hospital clinic of barcelona – university of barcelona, barcelona, spain 7 immunology department, biomedical diagnostic centre (cdb), hospital clinic of barcelona – university of barcelona, barcelona, spain 8 august pi sunyer biomedical research institute (idibaps) – university of barcelona, barcelona, spain 9 neurological tissue bank, biobank of hospital clinic of barcelona – idibaps, barcelona, spain   * these authors contributed equally corresponding author: iban aldecoa ansorregui · pathology department. 3rd stair, 5th floor · hospital clinic of barcelona · st. villaroel 170 · 08036 barcelona · spain ialdecoa@clinic.cat submitted: 04 august 2022 accepted: 20 october 2022 copyedited by: alessia sciortino published: 27 october 2022 https://doi.org/10.17879/freeneuropathology-2022-4365 keywords: hematologic malignancies, chimeric antigen receptor (car) t-cell, cytokine release syndrome (crs), neurotoxicity, neuropathology, immunohistochemical stains abstract introduction: chimeric antigen receptor (car) t-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. adverse events are common, and neurotoxicity is one of the most important. however, the physiopathology is unknown and neuropathologic information is scarce. materials and methods: post-mortem examination of 6 brains from patients that underwent car t-cell therapy from 2017 to 2022. in all cases, polymerase chain reaction (pcr) in paraffin blocks for the detection of car t cells was performed. results: two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. the neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly cd8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. microbiological studies were negative for neurotropic viruses, and pcr failed to detect car t -cells. another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. the remaining 4 cases only showed a mild patchy gliosis and microglial activation, and car t cells were detected by pcr only in one of them. conclusions: in this series of patients that died after car t-cell therapy, we predominantly found non-specific or minimal neuropathological changes. car t-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings. introduction chimeric antigen receptor (car) t-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. normal and malignant b-cells express the cd19 protein in the cell membrane. lymphodepletion followed by transfer of autologous t-cells that have been genetically modified to express a car targeting cd19 have been effective in clinical trials, producing remission of refractory or relapsed acute lymphoblastic leukemia (all), chronic lymphocytic leukemia and non-hodgkin’s lymphoma (nhl)1–5. recently, bcma targeted cars have been used to treat multiple myeloma6. among toxic effects expected after lymphodepletion and anti-cd19 car t-cell infusion, cytokine release syndrome (crs) and neurotoxicity are the most relevant but, unlike crs, the physiopathology of neurological events is less known and neuropathologic information is scarce7. clinical presentation develops mostly as non-specific brain dysfunction without focal signs,most of them being mild and transient. nevertheless, focal symptoms such as seizures and fatal cerebral oedema have been described. neurotoxicity can occur in the context of severe crs, where disruption of the blood-brain barrier (bbb) by cytokine production and subsequent endothelial activation leads to an increase in proinflammatory cytokine levels in cerebrospinal fluid (csf)7–10. however, neurotoxicity is also associated with expansion and activation of car t-cells that lead to a direct parenchymal car t-cell infiltration. in addition to crs following car t-cell infusion, factors that favor car t-cell proliferation in blood, such as the addition of fludarabine to cyclophosphamide lymphodepletion chemotherapy9, are also associated with an increased risk of neurotoxicity. fludarabine is a antineoplastic agent that added to the conditioning regimens improves the expansion and persistence of car t-cells, as well as duration of remission and disease-free survival2,4,5. its contribution to neurotoxicity may be driven by different mechanisms, and is yet to be elucidated. however, fludarabine-associated neurotoxicity has a later onset, and cerebral oedema is not usually reported11. reports of neuropathological findings after car t-cell therapy are scarce, independent of the presence or absence of neurological symptoms prior to death. we only found 4 cases described in the literature, which showed variable and non-specific neuropathological changes7–10. here, we report detailed neuropathological findings of 6 patients who died after car t-cell therapy12. materials and methods six brains from patients that underwent car t-cell therapy from 2017 to 2022 in a tertiary university hospital were examined. we selected all cases with car t-cell therapy from requested medical autopsies at the pathology department during this period. patients received ari-0001 (anti-cd19) and ari-0002h (anti-bcma, for patients with multiple myeloma) based car t treatment. for ari-0001, the planned target cell dose varied depending on the patient's disease: typically, 1 × 106 ari-0001 cells/kg for patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia; and 5 × 106 ari-0001 cells/kg for non-hodgkin lymphoma patients. full details of ari-0001 and ari-0002h cell production and a phenotypic characterization of these cells can be found elsewhere6,12–14. clinical data was retrospectively retrieved from the electronic medical records. formalin-fixed and paraffin-embedded tissue sections from the frontal cortex, visual cortex, cingulate, hippocampus, amygdala, thalamus, basal ganglia, white matter, midbrain, pons, medulla oblongata, spinal cord and cerebellum that had been stained with hematoxylin-eosin (he) were histologically assessed using light microscopy. in some of the cases the study was extended with immunohistochemistry (ihc). ihc was performed on 5-µm formalin-fixed, paraffin-embedded (ffpe) sections using the roche benchmark ultra platform with usual antigen retrieval protocols. the primary antibodies used were cd3 (2gv6, roche), cd20 (l26, roche), cd4 (sp35, roche), cd68 (kp-1, roche), glial fibrillary acidic protein (gfap) (ep672y, roche), terminal deoxynucleotidyl transferase (tdt) (roche), beta amyloid (de2b4, roche), tau (at8, thermo-fisher scientific), neurofilament (2f11, roche), herpes simplex virus (hsv)-1 (10a3, roche), hsv2 (dbm15.69, sanbio), human herpes virus (hhv)-6 (a & b, gp 60/110, millipore), hhv-8 (13b10, roche), cytomegalovirus (cmv) (8b1.2, 1g5.2, and 2d4.2, roche), epstein-barr virus (ebv) latent membrane protein (lmp)-1 (cs1-4, roche), epstein-barr virus (ebv)-encoded small rnas (eber) (roche), and polyomavirus (mrq-4, roche). ihc studies were performed in the initial diagnostic workup when clinical suspicion or neuropathological evidence of any pathologic process was observed. in addition, a systematic retrospective analysis was performed: cd3 and cd20 were screened on hippocampal and midbrain sections in all cases, on spinal cord sections in all cases except case 1, on frontal cortex in all cases except cases 2 and 3, and on basal ganglia only in cases 4 and 6. gfap ihc was performed on frontal sections in all cases, on basal ganglia in all cases except case 3, on hippocampus in all cases except case 1, and on midbrain only in case 4. polymerase chain reaction (pcr) in paraffin blocks was performed in case 5 for the detection of hhv-6, west nile virus, adenovirus, enterovirus, measles, mumps and lymphocytic choriomeningitis. samples of all cases from hippocampus, midbrain, and spinal cord (only in cases 2 to 6) were used for car t-cell detection by pcr. five micrometer sections were obtained from paraffin blocks, and dna was extracted using qia amp dna ffpe tissue kit (qiagen, hilden, germany). real-time pcr was used to measure number of copies/cell. genomic dna was amplified as previously described13. amplification reactions were performed in triplicate. an eight-point standard curve was generated using a sequence close to the cdkn1a gene (genebank: z86995) as comparator. the test was calibrated to detect a number of copies ranging from 5 to 106 per 500 ng of genomic dna. results see table 1 for a summary of the cases here presented and the other cases reported in the literature, and figure 1 for the comparison of gliosis and t-cell infiltrates of the cases. table 1. clinical data and histopathologic findings of our cases and previously described cases (modified from7) m: male; f: female; n/a: not applicable. ffpe: formalin fixed paraffin embedded. figure 1. figures co to f1: gfap immunohistochemistry of hippocampus (upper third ca1 sector -right sideand subiculum – left side-) and parahippocampal area (lower third, transenthorinal cortex) of a control (co) and cases 2 (b1), 3 (c1), 4 (d1), 5 (e1) and 6 (f1). the control was a 52-year-old male who died due to an acute bilateral adrenal hemorrhage with extensive clasmatodendrosis (insets, parahippocampal white matter, 400x). the cases showed variable gliosis, predominantly mild, higher in case 5 (e1) who had an encephalitis, and case 4 (d1) who died with an extensive acute pneumonia. both had mild and isolated clasmatodendritic changes in white matter (see insets). row 3 and 4 show t-cell infiltrates (cd3+) in parahippocampal cortex (left half) and substantia nigra pars compacta (right half), in cases 1 (a2), 2 (b2), 3 (c2), 4 (d2), 5 (e2) and 6 (f2). only case 5 (e2) presented marked infiltrates, while other cases has only scattered to isolated perivascular t-cells. case 2 (b2), which was positive for cart pcr in brain tissue, did not have significantly higher t-cell infiltrates (figures co, b1 to f1 20x scale bar 500 µm; insets 400x, scale bar 20 µm. figures a2 to f2 200x, scale bar 50 µm). case 1 a 44-year-old woman with acute b-cell lymphoblastic leukemia presented to an outside hospital with central nervous system (cns) involvement and a paravertebral mass (d8-d10) without spinal compression. at disease onset, she had lymph node involvement and leukocytosis and was refractory to five lines of treatment. at her first consultation in our center she did not present any neurological symptoms. basal brain magnetic resonance imaging (mri) showed a nonspecific lacunar lesion in the left frontal white matter and discrete homogeneous dural enhancement in cerebral convexity; the cerebrospinal fluid (csf) study was normal. after the conditioning with cyclophosphamide and fludarabine, she received car-cd19 treatment. three days after the infusion she developed fever, pulmonary interstitial infiltrates, and pleural effusion. the radiology was suggestive of non-cardiogenic oedema, with isolated colonies of staphylococcus haemolyticus in respiratory samples culture. she received antimicrobial therapy, depletive treatment, pleural drainage, and non-invasive mechanical ventilation, as well as tocilizumab, as a crs could not be ruled out. her clinical status improved, although she had persistent fever with negative microbiological culture. fourteen days after the infusion she progressed with fever, hemodynamic instability and multi organ failure, as well as car t-cells amplification in peripheral blood. with the diagnosis of crs, a second dose of tocilizumab was administered with only partial improvement. steroids were added, but the patient developed confusion, suggesting neurotoxicity. the electroencephalogram (eeg) showed generalized slowing, compatible with encephalopathy, and the brain mri showed two non-specific signal abnormalities/foci of signal alteration without mass effect or contrast uptake, lateral to the left ventricular atrium and in the right temporo-occipital area adjacent to the ventricular occipital horn. flow cytometry (fc) of the csf revealed the presence of mature t lymphocytes and isolated blasts, and the microbiological csf study was weakly positive for hhv-6. nineteen days after the infusion she presented rapidly progressive respiratory and hemodynamic deterioration requiring intubation and vasoactive treatment. the tracheal aspirate was positive for stenotrophomona maltophila and the diagnostic of septic shock due to nosocomial pneumonia was established. antibiotic treatment was escalated. however, she progressed with pulmonary hemorrhage and died 23 days after the infusion. neuropathologic gross examination revealed a 1280 g fixed brain with diffuse petechiae in the dura mater. coronal sections showed slight discoloration of right temporal white matter. histology showed mild arteriosclerosis and arteriolar hyalinosis, minimal lymphocytic perivascular infiltrate (t-cell predominant) and occasional intravascular infiltration by atypical cells with tdt positivity (figure 2), confirming the presence of leukemic cells, without remarkable parenchymal involvement. car t-cell pcr in the brain was negative and there were no signs suggestive of encephalitis associated to viral infection. full body autopsy demonstrated massive dissemination of the lymphoproliferative process affecting the lungs, liver, spleen, bone marrow, lymph nodes and mammary gland. the cause of death was attributed to disease progression. figure 2. remarkable histological features: case 1 presented scant intravascular infiltration of the leukemia (a), which was positive for tdt (a, inset). case 5 showed an encephalomyelitis, with predominant involvement of limbic areas (b, entorhinal cortex), basal ganglia (c, hypothalamus), brainstem (d, substantia nigra) and spinal cord (e, lumbar spinal cord). t-cell lymphocytic infiltrates were both cd8+ and cd4+, with predominance of the former (f and g, cd8+ and cd4+ infiltrates in lumbar spinal cord, which did not show marked tropism for motor neurons (arrowheads)) (a 400x, scale bar 20 µm, inset 600x, scale bar 20 µm; b to g, 200x, scale bar 50 µm). case 2 a 19-year-old woman presented with primary mediastinal diffuse large b-cell lymphoma with infiltration of soft tissues. she had a protracted course with persistent mediastinal disease, gastric relapse, superior vena cava syndrome and pericardial and pleural malignant effusions. she received multiple therapy lines including autologous stem cell transplant, local radiotherapy, and several chemotherapy lines comprising burkimab protocol and ibrutinib, before considering car t treatment. her initial mri showed mild global cerebral atrophy without other remarkable findings. on admission she was hypotensive and tachycardic and positron emission tomography (pet) – computed tomography (ct) scan showed pericardial infiltration in addition to supra and infradiaphragmatic lymphadenopathies, with soft tissue, muscle, bone, and pleural multifocal enhancement. the presence of bone marrow aplasia delayed the conditioning with fludarabine-cyclophosphamide. respiratory distress increased after conditioning along with the pleural effusion, and she was admitted to the intensive care unit (icu) to receive car-cd19 infusions. the following 48 hours, her respiratory status worsened, adding hemodynamic instability and fever. a nosocomial pneumonia was diagnosed by x-ray and ultrasound examination, without microbiological isolation, and she received antibiotics and fluconazole. despite treatment, her status worsened, requiring invasive mechanical ventilation and vasoactive treatment. subsequently, she presented superior vena cava syndrome and worsening of pericardial effusion with progression of hemodynamic instability. although she received corticosteroids and rituximab aiming to diminish the tumoral mass, she progressed to organic failure, and died 33 days after car t infusion. neurological symptoms were not detected in the course of the disease, although it was difficult to examine the patient due to sedation administered during invasive mechanical ventilation. neuropathologic gross examination was unavailable. histology revealed basal ganglia mild gliosis, and occasional perivascular hemosiderin deposits in parenchymal blood vessels of the white matter and basal ganglia. immunohistochemical studies showed scarce perivascular cd3+ t-cells with minimal intraparenchymal extension in the midbrain tegmentum, as well as in perithalamic white matter and cervical spinal cord. t-cells were not detected in the hippocampus, and there were almost no cd20+ b-cells. pcr for the detection of car t-cells in paraffin blocks of midbrain and spinal cord was positive, while it was negative in the hippocampus. full body autopsy revealed a large mediastinal mass with pericardial extension causing severe constrictive pericarditis, as well as extension to cervical region, diaphragm, rib wall and skin, and multiorgan tumor implants. no clear histological signs of acute neutrophilic pneumonia were found, although the immunosuppressive and neutropenic status may have had hindered the presence of histological signs. the cause of death was attributed to disease progression. case 3 a 19-year-old man with acute b-cell lymphoblastic leukemia was referred to our center one year after disease onset. he had relapsed disease despite induction protocol and six consolidation treatment cycles, as well as one cycle of inotuzumab-ozogamicine. he received cyclophosphamide and corticosteroid bolus as bridge therapy before the infusion of car-cd19. his initial brain mri showed multiple bilateral cortical and subcortical frontoparietal signal abnormalities suggesting chronic hemorrhages from multiple cerebral cavernomatosis, but no contraindication for the car t therapy was found. after conditioning with fludarabine and cyclophosphamide, antibiotics were started due to fever and isolation of enterococcus faecalis in urine culture. twelve hours after the first car t-cell infusion, he presented fever and hemodynamic instability refractory to fluids. he was transferred to the intensive care unit (icu) with the diagnosis of crs grade 3. tocilizumab, high doses of methylprednisolone and siltuximab, as well as support treatment with several vasopressors, mechanical ventilation, and empirical treatment with antifungal and antibacterial agents were administered. however, he developed refractory multiorgan failure, with negative microbiological analyses, and he died five days after car t-cell infusion. no neurological symptoms were noted, and he was clinically diagnosed as fatal crs (grade 5). neuropathologic gross examination revealed a 1255 g fixed brain. coronal sections showed mild congestive appearance of left amygdala, putamen, front-temporo-parieto-occipital white matter, and pontine nuclei, as well as mild greyish discoloration of white matter, midbrain tegmentum and pons. the brainstem showed moderate substantia nigra depigmentation. histology confirmed mild white matter oedema with mild gliosis, mainly in basal ganglia (ventral thalamus) and midbrain tegmentum/periaqueductal grey matter. blue dark neurons were observed in cortical areas, predominantly in layers ii and iii, suggestive of incipient mild hypoxic-ischemic changes. histology also revealed mild congestion and minimal arteriosclerosis of parenchymal blood vessels with occasional perivascular histiocytes with hematic component, without perivascular tissue damage. no evidence of venous dilations or vascular malformations were observed; the changes reported by mri study were attributed to hemodynamic fluctuations in life rather than to the presence of established vascular malformations. pcr for the detection of car t-cells in paraffin blocks was negative. full body autopsy revealed no significant findings. the cause of death was attributed to crs after car t-cell infusion. case 4 a 63-year-old male patient with a grade 3a, stage iv follicular lymphoma with abdominal extranodal and mesenteric involvement that had been treated with a first line of r-chop and had reached partial response. following maintenance with rituximab for 2 years, he remained stable one more year, until progression of the abdominal mass, new gastric infiltration, and pet hypermetabolism in the esophagus. after a third line with obinotuzumab and bendamustine, a failed attempt of autologous stem cell aphaeresis, and maintenance with obinutuzumab, a fourth line with idelalisib was started. however, the abdominal disease progressed with involvement of peripancreatic, renal and iliac lymph nodes, the patient was not eligible to allogeneic stem cell transplant, and he received car-cd19 therapy with prior fludarabine and cyclophosphamide conditioning. after the third day of consecutive infusion, the patient developed fever with mild hypotension that was diagnosed as grade 2 crs and had a good response to tocilizumab. he had an episode of toxic epidermolytic necrolysis (diagnosed by skin biopsy) with severe extensive rash and oral lesions. he also presented with severe aplasia, as well as a septic shock due to urinary tract infection that was treated in the icu. after initial improvement, he presented a pulmonary hemorrhage with colonization by candida glabrata, and developed kidney failure, hyperkaliemia, tracheobronchitis, more episodes of septic shock due to bacteremia of intestinal and skin origin (bacteroides fragilis, pseudomona aeruginosa, enterococcus faecium), and persistent candidemia as well as cmv reactivation. due to persistent skin lesions, an alternative diagnosis of car t-cell associated dermatosis was suspected. immunosuppression was restarted, improving the skin lesions, but it had to be stopped due to bone marrow aplasia and worsening neutropenia. of note, the refractory follicular lymphoma showed morpho-metabolic decrease in post car t infusion ct and pet scans, attributed to the presence of circulating car t-cells. eventually, the last episode of septic shock and ventilator associated pneumonia were refractory, which led to death after 4 months in the icu at 5 months of car t-cell therapy. he died without evidence of neurological symptoms in the course of the disease, and a brain computerized tomography (ct) done 45 days after the car t infusions showed no significant findings. neuropathologic gross examination showed a 1240 g fixed brain. coronal sections showed mild congestive changes in caudate, putamen, thalamus, and parieto-occipital white matter. histology revealed cortical gliosis in frontal and parahippocampal cortices, in possible relation to acute hypoxic-ischemic injury, as well as mild scattered foci of gliosis with minimal clasmatodendrosis in frontal and parahippocampal subcortical white matter, highlighted with gfap immunohistochemistry (see figure 1). mild parenchymal blood vessel arteriosclerosis and arteriolar hyalinosis, with occasional hemosiderin deposits, and isolated perivascular rarefaction in deep vessels was observed. histology also showed isolated diffuse cortical amyloid beta deposits, and minimal amygdala neurofibrillary pathology. pcr for the detection of car t-cells in paraffin blocks was negative. full body autopsy excluded residual neoplasia and evidenced a pneumonic process in context of the septic condition. the final cause of death was a respiratory failure. case 5 a 28-year-old man with b-cell acute lymphoblastic leukemia with disease progression with orbital infiltration. after diagnosis, induction, and consolidation treatment, he received haploidentical stem cell transplant (sct), which was previously conditioned with thiotepa, fludarabine, busulfan and prophylaxis of graft versus host disease (gvhd) with tacrolimus and cyclophosphamide. he developed hemorrhagic cystitis, cmv reactivation, escherichia coli orchitis and tacrolimus nephrotoxicity as complications, but never gvhd. he relapsed one year after sct with testicular infiltration, which required blinatumomab rescue treatment and lymphocyte donor infusion plus intrathecal prophylaxis, but ultimately led to radiotherapy and posterior bilateral orchiectomy. two years after diagnosis, he had progressive disease and was planned for car-cd19 therapy. his initial brain mri showed few nonspecific subcortical white matter hyperintense signal abnormalities, and tumor infiltration of the right orbit. after lymphodepletion with fludarabine and cyclophosphamide, he received a first car t-cell infusion with good tolerance, no signs of crs and good response observing non-neoplastic b-cells. due to loss of car t-cells in peripheral blood, he received two reinfusions in a 10-month period. the second was followed by an early loss of car t-cells due to antibodies, and the third infusion was made after plasma exchange to remove the antibodies. after the third infusion, he presented with fever without a clear origin that was treated with antibiotics, with low clinical suspicion of crs due to the absence of analytical alterations. almost one month after this last infusion, he presented with confusion, bradypsychia and generalized tremor. repeated, a brain mri showed no significant changes, and persistent tumor infiltration of the right orbit. eeg study showed diffuse slowing, and there were not significant findings in the csf study. the neurological symptoms were refractory, and he died 43 days after the third car t-cell infusion. neuropathologic gross examination showed a 1385 g fixed brain. coronal sections showed softening in both putamen. histology revealed diffuse gliosis predominant in basal ganglia, hippocampus, substantia nigra and brainstem. the most relevant finding was interstitial and perivascular lymphocytic infiltrates, together with histiocytic infiltrates, which were severe in anterior horns and grey matter of spinal cord, and to a lesser extent in the midbrain and hippocampus (figure 2). immunohistochemical studies revealed that lymphocytic infiltrates were cd3+ t-cells, predominantly cd8+, with practically no b-cells being observed, and were accompanied by severe cd68+ microglial activation with foamy histiocytes. neurofilament staining showed occasional phosphorylated somas as well as isolated axonal spheroids in cortical and hippocampal white matter, without axonal loss in spinal cord tracts. gfap staining showed mild and focal clasmatodendritic changes in the white matter. all the findings above were concordant with the diagnosis of a diffuse lymphocytic encephalomyelitis predominant in brainstem, spinal cord and limbic system, with milder disease in cortex and basal ganglia, and relative sparing of cerebellum. no viral inclusions were observed, and virus detection stains for hsv-1, hsv-2, hhv-6, hhv-8, cmv, ebv and polyomavirus, and pcr for hhv-6, west nile virus, adenovirus, enterovirus, measles, mumps and lymphocytic choriomeningitis were negative. pcr in paraffin blocks for the detection of car t-cells was negative. full body autopsy revealed extensive pulmonary hemorrhage with no apparent signs of infection and foci of extramedullary hematopoiesis in the liver. after all, the cause of death was attributed to an unspecified encephalomyelitis, suggesting primarily an infectious etiology but not ruling out immune causes. case 6 a 71-year-old woman with multiple myeloma iga kappa r-iss ii, diagnosed from a scalp extramedullary plasmacytoma. cytogenetic showed high risk (17p deletion) and it relapsed after four therapy lines. she received fludarabine and cyclophosphamide conditioning after bridging therapy. after one week of the first car-bcma infusion, she was admitted to the icu due to a grade 2 crs with fever and hypotension that were successfully treated with one dose of tocilizumab and corticosteroids. ten days later, she presented episodes of hypotension without fever or alterations in chest and brain ct or echocardiography. in the differential diagnosis, neurotoxicity related to treatment was considered, corticosteroid dose was increased, and antibiotics were added. she worsened with agitation, tachypnoea, and respiratory failure; and progressed to refractory multiorgan failure with ischemic hepatitis, renal failure, and hemorrhagic complications in the context of thrombocytopenia. a macrophagic activation syndrome was suspected due to ldh and ferritin increase, and treatment was attempted with corticosteroids, anakinra, siltuximab and cyclophosphamide. amid this situation, she presented a blood culture positive for escherichia coli without detectable septic focus. despite all measures, she remained in multiorgan failure with acidosis and hyperlactacidemia, and she died 19 days after car t-cell therapy start. additional neurotoxicity signs were not evident. neuropathologic gross examination showed a 1045 g fixed brain with mild hippocampal and amygdalar atrophy. neuropathologic evaluation revealed mild arteriosclerosis (with focal mural calcification in globus pallidus arteries) and diffuse minimal gliosis. histology revealed scattered perivascular t-cells (cd3+) in basal nuclei, frontal cortex, and hippocampus. minimal microglial activation highlighted with cd68 accompanied these findings. she also had a definite primary age related tauopathy or part (braak stage i/vi, thal 0) and mild periamygdaline age related tau astrogliopathy or artag. pcr for the detection of car t-cells in paraffin blocks was negative. the most remarkable systemic finding was a submassive acute hepatic necrosis. the prominent center lobular location, the lack of cytopathic changes and negative immunohistochemical stains for hsv-1, hsv-2, hvv-8, cmv, ebv lmp1 and eber oriented to a hemodynamic–hypovolemic etiology. there were additional acute hemorrhagic foci, like in myocardium and intestines, but the lack of tissue ischemia or inflammatory reaction surrounding them suggested they were acute premortem ischemic changes. remarkably, there was hypercellular bone marrow with t-cell expansion and hemophagocytosis. immunohistochemistry for cd68 in different organs showed moderate histiocytic activation in the spleen and mild activation in the heart, liver, intestine and brain. in the latter, it did not seem to induce secondary tissue alterations, hence its impact may had been limited. a lithic focus in the cranium was constituted mainly by fibrotic reactive tissue without evidence of plasma cell disease. after all, the cause of death was attributed to submassive liver necrosis of probable ischemic cause. discussion here we present the neuropathological findings of 6 deceased patients, 3 women and 3 men, who received a cd19(5) and bcma(1) targeted car t-cell therapy. three of them had a b-cell acute lymphoblastic leukemia, one a follicular lymphoma 3a grade, one a primary mediastinal diffuse large b-cell lymphoma, and the other a multiple myeloma with plasmocytoma. only 2 of them presented explicit neurological symptoms in the course of the disease. brain gross examination did not show significant alterations in any of the patients. histological findings in one of the patients with neurological symptoms were mild arteriosclerosis and arteriolar hyalinosis, minimal lymphocytic perivascular infiltrate (mainly cd5+) and leukemic cells, in line with the systemic progression of the hematologic malignancy as the cause of death. the histological examination of the other patient with neurological symptoms showed a severe perivascular and interstitial lymphocytic infiltration, predominantly cd8+, admixed with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, along with a diffuse gliosis of basal ganglia, hippocampus, and brainstem. microbiologic studies were negative for neurotropic viruses, and pcr failed to detect car t-cells. finally, the cause of death was attributed to a lymphocytic encephalomyelitis of unknown etiology, infectious versus toxic, without being able to demonstrate viral infection nor car t-cells in brain tissue. one of the cases without neurological signs showed mild clasmatodendrosis in context of terminal hypoxic-ischemic damage. the rest showed a mild patchy gliosis and microglial activation, with no other remarkable histologic changes. car t-cells were detected by pcr only in one of them, without histological particularities. reports of neuropathological findings after car t-cell therapy are scarce. neuropathological findings in autopsies of different types of patients vary, but there are common changes suggesting astrocyte dysfunction, blood-brain barrier disruption with endothelial damage and activation, and a subsequent increase in vascular permeability. we found four cases described in the literature, all of them with neurological involvement that led to death. the autopsy of a patient who died from fulminant cerebral oedema showed signs of blood-brain-barrier disruption, clasmatodendrosis and microglial activation8. the findings in the autopsy of another patient who died from progressive neurologic deterioration were diffuse gliosis with severe neuronal loss and degeneration of white matter, dense infiltration of macrophages with numerous microglial cells, as well as a cd8+ infiltrate. it is worth noting that before the hematologic malignancy, he had the diagnosis of optic atrophy, which could alter the findings10. the autopsy findings of another 2 patients were vascular in nature, with multifocal microhemorrhages, perivascular infiltration of cd8+ cells, intravascular von willebrand factor (vwf) binding, and platelet microthrombi, along with more severe vascular findings, such as karyorrhexis and fibrinoid vessel wall necrosis. reactive microglia was also noted, the percentage of t-cells in the parenchyma mirrored the percentage of those cells in the csf9. it is worth highlighting that the clinical setting of patients undergoing car t-cell therapy makes it difficult to determine the cause of the observed neurological symptoms. while crs is widely studied, neurological toxicity itself remains less understood. neurological damage due to the underlying hematological disease, opportunistic infections in a setting of severe immunosuppression, and pharmacological toxicity of accompanying chemotherapy drugs are conditions that should be taken into consideration when evaluating the possible effects of car t-cell therapy. fludarabine, a drug used for the conditioning with cyclophosphamide in all our cases, seems to promote a discernible form of neurotoxicity, with unspecific but distinctive gliosis and white matter involvement11. basic neuropathologic assessment allows reliably detecting or discarding malignant cell infiltration in the central nervous system. it also enables to demonstrate the presence of inflammation with or without infectious agents in neurological tissue, although their absence does not completely rule out infection as in our fifth case. alterations of the bbb cannot be reliably studied in he alone and specially without a prior technical background. nevertheless, without clear evidence of brain weight gain, oedema, and white matter changes, nor minimal vascular damage and perivascular cellular reactions, bbb disruption may be at best limited in cases without neurological involvement in life. on the other hand, astrocyte injury can be highlighted with ihc showing gfap aberrant beading and fragmentation, a phenomenon called clasmatodendrosis, that relates to blood barrier dysfunction8,15. we reexamined our cases with gfap, and only saw scattered foci of clasmatodendrosis in 2 cases, one with hypoxic-ischemic changes in the context of nosocomial pneumonia and one with encephalomyelitis. clasmatodendrosis has been related to acute metabolic disturbances in astrocytes (hypoxia, ischemia, hypoglycemia…)15, and as such may not be pathognomonic of crs brain pathology or fludarabine neurotoxicity, for example, especially when is mild and focal and may have alternative causes. the relative lack of clasmatodendrosis as well as overall gliosis in most of our cases would support the notion that bbb dysfunction is not a systematic feature in car t-cell treated critical patients when they do not have specific and severe neurological symptomatology. we present 6 patients who died after car t-cell therapy, 2 of whom displayed neurologic clinical manifestations with only one showing significant neuropathological findings. this case had an encephalomyelitis of immune-toxic versus infectious origin, being the findings in the rest of the patients unspecific and minimal, with mild chronic vascular disease and changes suggestive of terminal hypoxic-ischemic etiology. interestingly, the encephalomyelitis changes could not be related to the presence of car t-cells in brain tissue, while we were able to demonstrate car t-cells in brain tissue of one patient without relevant histological changes nor neurological symptoms. in conclusion, patients that die after car t-cell therapy show non-specific or minimal neuropathological changes, especially when they lack evident neurological symptoms and these do not contribute in patient death. car t-cell related direct toxicity may not be the only cause for neurological symptoms, and autopsy may detect other pathological processes, such as disease progression. car t-cell therapy is a new therapeutic approach that needs further research to elucidate its potential adverse effects and the underlying pathological mechanisms behind them. conflicts of interest the authors have no conflicts of interest that relate to the content of this article. acknowledgments we are thankful to the pathology technicians in the immunohistochemistry and molecular sections of the pathology department for the performed studies. funding the authors have not received specific funding for this study. cfl thanks the spanish institute of health carlos iii (projects pi19/00669 and ici19/00025; co-funded by the european union). references 1. kochenderfer, j. n. et al. chemotherapy-refractory diffuse large b-cell lymphoma and indolent b-cell malignancies can be effectively treated with autologous t cells expressing an anti-cd19 chimeric antigen receptor. j. clin. oncol. 33, 540–549 (2015). pmid: 25154820. https://doi.org/10.1200/jco.2014.56.2025 2. turtle, c. j. et al. cd19 car-t cells of defined cd4+:cd8+ composition in adult b cell all patients. j. clin. invest. 126, 2123–2138 (2016). pmid: 27111235. pmcid: pmc4887159. https://doi.org/10.1172/jci85309 3. maude, s. l. et al. chimeric antigen receptor t cells for sustained remissions in leukemia. n. engl. j. med. 371, 1507–1517 (2014). pmid: 25317870. pmcid: pmc4267531. https://doi.org/10.1056/nejmoa1407222 4. turtle, c. j., riddell, s. r. & maloney, d. g. cd19-targeted chimeric antigen receptor-modified t-cell immunotherapy for b-cell malignancies. clin. pharmacol. ther. 100, 252–258 (2016). pmid: 27170467. https://doi.org/10.1002/cpt.392 5. turtle, c. j. et al. immunotherapy of non-hodgkin’s lymphoma with a defined ratio of cd8+ and cd4+ cd19-specific chimeric antigen receptor-modified t cells. sci. transl. med. 8, 355ra116 (2016). pmid: 27605551. pmcid: pmc5045301. https://doi.org/10.1126/scitranslmed.aaf8621 6. oliver-caldes, a. et al. first report of cart treatment in al amyloidosis and relapsed/refractory multiple myeloma. j. immunother. cancer 9, e003783 (2021). pmid: 34876408. pmcid: pmc8655576. https://doi.org/10.1136/jitc-2021-003783 7. hunter, b. d. & jacobson, c. a. car t-cell associated neurotoxicity: mechanisms, clinicopathologic correlates, and future directions. j. natl. cancer inst. 111, 646–654 (2019). pmid: 30753567. https://doi.org/10.1093/jnci/djz017 8. torre, m. et al. neuropathology of a case with fatal car t-cell-associated cerebral edema. j. neuropathol. exp. neurol. 77, 877–882 (2018). pmid: 30060228. https://doi.org/10.1093/jnen/nly064 9. gust, j. et al. endothelial activation and blood–brain barrier disruption in neurotoxicity after adoptive immunotherapy with cd19 car-t cells. cancer discov. 7, 1404–1419 (2017). pmcid: pmc5718945. https://doi.org/10.1158/2159-8290.cd-17-0698 10. schuster, s. j. et al. chimeric antigen receptor t cells in refractory b-cell lymphomas. n. engl. j. med. 377, 2545–2554 (2017). pmid: 29226764. pmcid: pmc5788566. https://doi.org/10.1056/nejmoa1708566 11. lowe, k. l. et al. fludarabine and neurotoxicity in engineered t-cell therapy. gene therapy. 25, 176–191 (2018). pmid: 29789639. https://doi.org/10.1038/s41434-018-0019-6 12. ortíz-maldonado, v. et al. cart19-be-01: a multicenter trial of ari-0001 cell therapy in patients with cd19+ relapsed/refractory malignancies. mol. ther. 29, 636–644 (2021). pmid: 33010231. pmcid: pmc7854276. https://doi.org/10.1016/j.ymthe.2020.09.027 13. castella, m. et al. development of a novel anti-cd19 chimeric antigen receptor: a paradigm for an affordable car t cell production at academic institutions. mol. ther. methods clin. dev. 12, 134–144 (2019). pmid: 30623002. pmcid: pmc6319086. https://doi.org/10.1016/j.omtm.2018.11.010 14. castella, m. et al. point-of-care car t-cell production (ari-0001) using a closed semi-automatic bioreactor: experience from an academic phase i clinical trial. front. immunol. 11, 482 (2020). pmid: 32528460. pmcid: pmc7259426. https://doi.org/10.3389/fimmu.2020.00482 15. balaban, d., miyawaki, e. k., bhattacharyya, s. & torre, m. the phenomenon of clasmatodendrosis. heliyon 7, e07605 (2021). pmid: 34368479. pmcid: pmc8326353. https://doi.org/10.1016/j.heliyon.2021.e07605 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 62nd annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) feel free to add comments by clicking these icons on the sidebar free neuropathology 3:25 (2022) meeting abstracts 62nd annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts october 13–15, 2022 saskatoon, sk submitted: 31 october 2022 accepted: 01 november 2022 published: 16 november 2022   the canadian association of neuropathologist – association canadienne des neuropathologistes (canp-acnp) held their 62nd annual meeting at the delta bessborough in saskatoon, sk from october 13th to 15th, 2022, under the leadership of dr. robert hammond, president of the canp-acnp, dr. peter schutz, secretary treasurer of the canp-acnp, and with technical support from canp administrator, colleen fifield. the academic program comprised 15 scientific abstracts, 9 unknown cases, a mini-symposium on competence based medical education in neuropathology, and the presidential symposium on multiple sclerosis and immune mediated demyelinating disease. digital pathology images from the 9 unknown cases are available for viewing online (www.canp.ca). the unknown case sessions were moderated by dr. andrew gao. the presidential symposium 2022 on multiple sclerosis and immune mediated demyelinating disease featured the gordon mathieson lecture given by dr. g. r. wayne moore entitled demyelination, multiple sclerosis, and mri and the david robertson lecture given by dr. michael levin entitled multiple sclerosis and future therapies. the program was completed by three invited presentations with dr. e. ann yeh presenting on pediatric multiple sclerosis and immune mediated demyelination, dr. tanja kuhlmann presenting on neuropathology of ms and stem cells and dr. pamela kanellis presenting on outlook of patients and public on ms research and treatment in canada. the mary tom award for best clinical science presentation by a trainee went to dr. christopher newell (supervisor dr. j. joseph), and the morrison h. finlayson award for best basic science presentation by a trainee was won by dr. erin stephenson (supervisor dr. v.w. yong). the following abstracts were presented at the 62nd annual meeting of the canadian association of neuropathologists – association candienne des neuropathologistes (canp-acnp) in october 2022.   https://doi.org/10.17879/freeneuropathology-2022-4496 keywords: canadian association of neuropathologists, canp, meeting abstracts, 62nd meeting oct. 2022 contents abstract 1 effects of prenatal cocaine exposure on the human developing brain. a neuropathological study. abstract 2: utilizing neurodevelopmental time windows of hypoxic-ischemic pathology to infer brain maturity in patients with congenital cardiothoracic defects abstract 3: spinal nerve root blood in pediatric autopsy cases is not necessarily a marker of trauma at that site abstract 4: multifocal necrotizing leukoencephalopathy post-covid-19 infection: expanding the “neuro-covid” spectrum abstract 5: the international disseminated pediatric low grade glioma consortium: project goals and preliminary results abstract 6: glioblastoma, idh-wildtype with fgfr3-tacc3 fusion has unusual histologic, molecular and clinicoradiologic characteristics abstract 7: infiltrative pattern in pediatric ganglioglioma abstract 8: clinical implementation of methylation profiling: the sickkids experience abstract 9: cerebral amyloidoma: pathological findings from diagnostic biopsy to autopsy abstract 10: identification and validation of immunohistochemical antibodies against transcriptomically distinct human hippocampal astrocyte subtypes abstract 11: cadherin-23 is essential for the normal organisation of cerebellar mossy fibre synapses abstract 12: the extracellular matrix transcriptome following demyelination abstract 13: nodding syndrome: characterizing the newest tau proteinopathy in africa abstract 14: mri-pathological correlations show differential patterns of ex vivo texture in als abstract 15: lymphoid and plasma-cell infiltrates with amorphous eosinophilic material deposition in the leptomeninges: a challenging case     abstract 1 free neuropathol 3:25:4 effects of prenatal cocaine exposure on the human developing brain. a neuropathological study. catherine fallet-bianco1, mubina jovanovic1, dorothée bouron-dal soglio1 1department of pathology, hôpital sainte-justine-université de montreal, montreal, qc, canada recent studies report that cocaine use among pregnant women continues to be a public health concern. experimental studies have shown that prenatal cocaine exposure is responsible for disturbances affecting neurogenesis, neuronal migration and neurotransmitter systems. however, human-based studies are impacted by confounding factors: multi-drug use, lack of neuropathological studies. we describe the neuropathological phenotype observed in two fetuses after prenatal exposure to cocaine. in a 34-year-old woman using cocaine for several years, ultrasonography at 24 gestational weeks revealed a severe microcephaly with a severe global disorganization of brain development. in the second case, a 19-year-old mother using cocaine, ultrasonography at 20 weeks showed a severe microcephaly with bilateral schizencephaly resulting from multiple hemorrhages. a vasoconstriction of maternal-fetal arteries is, classically, the mechanism considered responsible for the disturbed brain development. a vascular disruption is probably the cause of the lesions in the second case. the changes in the first case are similar to those generated in experimental models and confirm that cocaine has direct toxic effects on the neurogenesis, gliogenesis and neuronal migration in the developing brain. the wide spectrum of brain lesions depends on multiple factors; the most important is probably the moment of exposure in gestation, as well as dose, frequency of cocaine use, and genetic susceptibility.   abstract 2 free neuropathol 3:25:5 utilizing neurodevelopmental time windows of hypoxic-ischemic pathology to infer brain maturity in patients with congenital cardiothoracic defects karina chornenka1, christopher dunham2 1 department of pathology and laboratory medicine, university of british columbia, vancouver, bc, canada 2 division of anatomical pathology, children’s and women’s health centre of bc, vancouver, bc, canada neuroradiologic investigations have demonstrated that cerebral development is delayed by 2-4 weeks in infants suffering from congenital heart defects and congenital diaphragmatic hernia (chd/cdh). these estimates are based upon application of the “total maturation score” (tms) system that evaluates brain development using magnetic resonance imaging (mri) by assessing myelination, cortical gyration, insular development, t1 white matter signal intensity and the involution of the germinal matrix (n engl j med 2007;357: 1928-1938, j pediatr surg 2012;47:453-461). these infants often require surgical correction of their malformations shortly after birth, but unfortunately some do not survive. of those coming to autopsy, it is not uncommon to encounter acute hypoxic ischemic injury (hii). we examined three individuals born at term with chd/cdh, ranging in age from 3 to 5 months, who died shortly after surgery and displayed evidence of neuropathology that included acute hii characterized by periventricular leukomalacia (pvl) and pontosubicular necrosis (psn). pvl and psn are typically encountered in the context of prematurity and occur during specific developmental time windows (i.e., pvl: 24-32 weeks gestational age; psn: 20 weeks gestational age – 2 months postnatal) (acta neuropathol 1995;90:7-10, acta neuropathol 2005;110:563-578). given the ages of the affected individuals herein, we suggest that the presence of premature-type neuropathology in the form of acute hii could be used to support the hat infants with chd/cdh incur delayed brain development. moreover, based on our observations we propose that the delay in cerebral development could be longer than previous estimates using mri.   abstract 3 free neuropathol 3:25:6 spinal nerve root blood in pediatric autopsy cases is not necessarily a marker of trauma at that site marc r. del bigio1 1 department of pathology, university of manitoba and diagnostic services manitoba, winnipeg, mb, canada increasingly, the forensic examination of suspected child abuse cases includes dissection of the entire cervical spine; hemorrhage along nerve roots is postulated to indicate forces exerted by shaking. seventy pediatric spinal cords encased in dura mater were examined (including 12 en bloc cervical spines); age 0-58 months. the mechanism of deaths were: suspected abusive trauma 35; accident 13; undetermined or natural without head/neck trauma 12; forceps injury at birth 3; birth related intracranial hemorrhage 7. prominent spinal subarachnoid hemorrhage was present in 30 cases; hemorrhage was detected along spinal nerve roots at the cervical level in 14/30 and at the lumbosacral level in 8/30 in the absence of definite injury to the spinal column. two cases with definite evidence of bone/ligamentous injury to the spine and the 2 forceps injury cases had extensive epidural hemorrhage ± subarachnoid hemorrhage extending the entire length of the cord. anatomical studies in humans, tracer studies, and experimental hemorrhage show direct communication of the cranial and spinal subarachnoid and subdural spaces with extension to the dorsal root ganglia. i conclude that the majority of spinal nerve root hemorrhages in pediatric trauma cases are simply markers of subarachnoid or subdural hemorrhage elsewhere. additional work is needed to determine if en bloc spine dissection, which is invasive and time consuming, adds significant information to the autopsy.   abstract 4 free neuropathol 3:25:7 multifocal necrotizing leukoencephalopathy post-covid-19 infection: expanding the “neuro-covid” spectrum jacob a. houpt1, lee cyn ang1,2, qi zhang1,2 1 department of pathology and laboratory medicine, london health sciences centre, london, ontario, canada 2 department of clinical neurological sciences, london health sciences centre, london, ontario, canada multifocal necrotizing leukoencephalopathy (mnl), also known as disseminated necrotizing leukoencephalopathy (dnl), is a rare pathological phenomenon defined by numerous discrete foci of necrosis with predilection for ventral pontine white matter. while exact etiology and pathogenic mechanisms have yet to be elucidated, mnl is most often reported on post-mortem examination in the context of immunocompromised (ex: receiving chemotherapy or with untreated hiv-aids) or critically ill patients (ex: metastatic cancer, sepsis). coronavirus disease 2019 (covid-19) is a viral respiratory illness with variable severity depending on host immune response and comorbid conditions. in its most serious cases, covid-19 can elicit both harmfully insufficient and excessive immune responses. we report on a case of covid-19 pneumonia which progressed to a clinical impression of sepsis, declining respiratory status, and death in an unvaccinated, previously well patient in which there were findings consistent with mnl throughout the midbrain and basis pontis. there was no encephalitis, microthrombi, infarcts, or other previously reported neuropathological findings. this index case was again compared and contrasted against four additional mnl cases examined at the london health sciences centre. the neuropathological findings are examined and described for these 5 cases, all with clinical scenarios involving systemic critical illness and/or immune system compromise and with similar ventral pontine focal necrosis, myelin loss, and limited reactive glial response. though numerous radiological studies have suggested a disseminated necrotizing leukoencephalopathic process underlying covid-19-associated encephalopathy, this case represents the first to offer pathological correlation supported by autopsy confirmation of mnl in a patient with covid-19 infection.   abstract 5 free neuropathol 3:25:8 the international disseminated pediatric low grade glioma consortium: project goals and preliminary results adrian levine1,4, julie bennett2, joseline haizel-cobbina3, liana nobre2, michael dewan3, uri tabori2, cynthia hawkins1 1 department of pediatric laboratory medicine, hospital for sick children, toronto, on, canada 2 division of neuro-oncology, hospital for sick children, toronto, on, canada 3 department of neurosurgery, vanderbilt university medical center, nashville, tn, usa 4 clinician investigator program, university of british columbia, bc, canada although most pediatric lgg (plgg) have excellent long-term survival, there is a subset of cases that disseminate (dplgg) and have very poor outcomes. it is unknown why these tumors behave in such an aggressive way. we believe that biological mechanisms, distinct from those present in non-disseminated lgg, underlie this metastatic ability, but so far there has been no in-depth investigation of this. to improve our understanding of this rare patient population, we have created a new consortium to compile dlgg cases across from across the world. we are collecting comprehensive clinical information, including the tumor presentation and pattern of dissemination, surgical outcomes and subsequent treatment courses, and quality of life outcomes. cases with available tissue will be tested with our glioma ngs panel for the most common mutations and fusions, and methylation array for copy number and classification. our data thus far on the first 53 cases demonstrates the dplgg has much worse prognosis than the overall plgg population. virtually all dplgg progress at 5 years, compared to a quarter of other plgg, and the disseminated cases are approximately 5-times more likely to die at 10 years. this can affect children of all ages and does not carry a gender predilection. we observe three patterns of dissemination – 40% of patients present with a localized mass and have secondary dissemination, 35% with disseminated tumor and a clear dominant mass, and 25% with disseminated disease without a dominant mass. the most common molecular alteration is braf fusions.   abstract 6 free neuropathol 3:25:9 glioblastoma, idh-wildtype with fgfr3-tacc3 fusion has unusual histologic, molecular and clinicoradiologic characteristics namita sinha1, sherry krawitz1, jai j.s. shankar2, saranya kakumanu3, ken aldape4, marc r.del bigio1 1 department of pathology, university of manitoba, winnipeg, mb, canada 2 department of radiology, university of manitoba, winnipeg, mb, canada 3 cancer care manitoba, university of manitoba, winnipeg, mb, canada 4 laboratory of pathology, national cancer institute, nih, bethesda, maryland, usa according to 2021 world health organization (who) classification, term ‘glioblastoma’ is reserved for cns who grade 4 astrocytic neoplasm that is idh-wildtype. a small subset of glioblastomas may have unusual clinical, histological and molecular profiles that may need appropriate molecular work-up for definitive diagnosis, and some of them may be reported as high-grade glioma, not otherwise specified, if complete molecular work up is lacking. we present two cases: a 48-year-old female with imaging revealing right occipital solid, cystic, mass demonstrating nodular calcification; and a 53-year-old male with imaging revealing multifocal enhancing mass with extensive calcification in the right parietal, occipital and temporal regions. the cerebral tumors in both patients were relatively circumscribed high-grade glioma composed of gfap immunoreactive monomorphic ovoid cells, with endothelial proliferation, predominantly infarct-type necrosis, and abundant calcospherites. both tumors showed occasional mitosis with ki-67 of ~ 5-10%. mass array analysis showed no idh, h3f3a, braf v600e mutation in these two cases and revealed tert promoter mutation in the second case. because of unusual histology, methylation profiling was pursued in these two cases that matched to glioblastoma. interestingly, ngs study showed fgfr3-tacc3 fusion in both the cases. follow-up imaging showed tumor progression in these two patients, with recurrence at 9 months in the second patient. both the patients are clinically well at 7 months and 15 months of follow-up. a few case reports are published with similar histologic and molecular findings. our cases contribute to expanding histologic, molecular and clinical spectrum of fgfr3-tacc3 fused glioblastoma.   abstract 7 free neuropathol 3:25:10 infiltrative pattern in pediatric ganglioglioma murad alturkustani1 1 department of pathology, faculty of medicine, king abdulaziz university, jeddah, saudi arabia ganglioglioma is a well-circumscribed low-grade glioneuronal tumor with a broad morphological spectrum. diffuse glioneuronal tumors are used to describe cases with infiltrative growth. molecular studies of some of these cases were consistent with ganglioglioma. this work aims to clarify the growth patterns in ganglioglioma. the available slides and clinical and molecular information for ganglioglioma cases under the open pediatric brain tumor atlas from the children’s brain tumor network database were reviewed to confirm the integrated diagnosis and to evaluate the growth patterns in these cases. infiltration is defined as the presence of neoplastic cells among the nonneoplastic parenchyma. the diagnosis of ganglioglioma was confirmed in 16 of 46 cases (nine females and seven males; age ranges from eight months – 19 years with a mean of 9.9 years). the infiltrative pattern was identified in 5 cases as the predominant pattern and in another 5 cases combined with a circumscribed nodule, while only 6 cases had predominant circumscribed growth. this work confirms the presence of an infiltrative/diffuse variant of ganglioglioma. awareness of this variant should help with infiltrative tumors, as the differential includes diffuse glioma, which is usually idh wild type in this population.   abstract 8 free neuropathol 3:25:11 clinical implementation of methylation profiling: the sickkids experience adrian levine1,2, michelle ku1, scott ryall1, cynthia hawkins1 1 department of pediatric laboratory medicine, hospital for sick children, toronto, on, canada 2 clinician investigator program, university of british columbia, bc, canada over the past decade, methylation profiling and machine learning based classification has had a major impact on the diagnostic framework for cns tumors, however there remains inconsistent and limited access to clinical testing for this analysis. at sickkids, we have implemented a clinically validated methylation assay using the illumina epic platform for the heidelberg cns tumor classifier, copy number profiling, and mgmt promoter methylation status. we have now run 136 samples for clinical use (plus additional samples for validation), which have been classified through both v11 and v12.5 of the heidelberg classifier. many external samples were not received with a histologic diagnosis, those that do have a diagnosis include 34 low grade gliomas (lgg), 19 high grade gliomas (hgg), 10 ependymomas, 4 pineal tumors, and 4 medulloblastoma/embryonal tumor. overall, only 34 samples (25%) classified with confidence score >0.85 on the v11 classifier and 77 samples (57%) on v12.5. the medulloblastomas all classified with high confidence scores, as did 7/10 ependymomas, and 2/3 papillary tumors of the pineal region. 9/19 hggs classified with high confidence – most of which were methylation-defined subtypes of idh-mutant and idh-wildtype astrocytomas or glioblastomas, with unclear clinical significance. notably, though 2 of the cases that were referred as hgg were in fact other tumor types, one being an atrt, and the other an ependymoma with zfta-fusion. the classifier had particular challenge with lggs, of which only 17 (50%) classified with high confidence, including one that was erroneously called “control tissue, reactive tumor microenvironment”.   abstract 9 free neuropathol 3:25:12 cerebral amyloidoma: pathological findings from diagnostic biopsy to autopsy shervin pejhan1, adrian budhram2, joseph megyesi2, lee cyn ang1,2, robert hammond1,2, qi zhang1,2 1 department of pathology & lab medicine, university of western ontario, on, canada 2 department of clinical neurological sciences, university of western ontario, on, canada amyloidoma is a rare condition defined by the aggregation of amyloid proteins forming a mass-like lesion. while brain-restricted amyloidomas have been reported, their course and pathogenesis are poorly understood. we are reporting a case of multifocal brain amyloidoma in a 71-year-old man, which is significant for its protracted clinical course and progression of neurological symptoms. a pair of stereotactic biopsies revealed the underlying pathology. the pathological findings were unusual with a dual picture of perivascular lymphoplasmacytic infiltration in the frontal area, accompanied by amyloid deposits in occipital white matter. proteomic analysis of the amyloid protein by mass spectrometry was also curious as it characterised both lambda light chain and amyloid beta-protein without evidence of monoclonality. further assessments found no involvement of other organ systems. with the progression of symptoms and lack of definitive treatment, the patient underwent palliative whole-brain irradiation, with no improvement, and passed away shortly thereafter. assessment of post-mortem brain tissue showed massive amyloid deposition in the periventricular white matter with sparse perivascular lymphocytes and fewer plasma cells than the biopsy samples. special staining, immunohistochemistry, electron microscopy, and mass spectrometry confirmed multifocal cerebral amyloidoma, lambda light chain type. the collection of the biopsy and autopsy findings may be the result of lesions observed at different stages of progression, treatment effects, or a not-previously described pattern of brain amyloidoma. studying this case is beneficial to gaining insight into the pathobiology of this rare condition and to inform future studies and therapeutic interventions.   abstract 10 free neuropathol 3:25:13 identification and validation of immunohistochemical antibodies against transcriptomically distinct human hippocampal astrocyte subtypes brenden joseph1,2, kaitlin sullivan3, mark s. cembrowski3, veronica hirsch-reinshagen1,2 1 department of pathology & laboratory medicine, university of british columbia, vancouver, bc, canada 2 division of neuropathology, vancouver general hospital, vancouver, bc, canada 3 department of cellular and physiological sciences, djavad mowafaghian centre for brain health, university of british columbia, vancouver, bc, canada our understanding of astrocyte function has steadily evolved from an early conceptualization as simplistic support cells to a current appreciation for their diverse and critical physiological roles. there is emerging evidence for the existence of distinct astrocyte subtypes with differential genomic expression, spatial localization, morphology, and physiological function. however, little is known about their specialized functions or the role of specific subtypes in human disease. a lack of established subtype-specific immunohistochemical markers represents a significant barrier to further investigation. to address this limitation, we aim to identify transcriptomically distinct human astrocyte subtypes and validate immunohistochemical markers for these. to identify distinct transcriptomic subtypes of human astrocytes, we used single cell nuclear rna sequencing of 11,204 astrocytes in human hippocampal surgical samples from 5 patients with intractable mesiotemporal epilepsy. canonical correlation and seurat cluster analysis identified 6 distinct clusters of astrocytes with unique transcriptomes and revealed several candidate marker genes unique to each cluster. we are currently validating c8orf34 and nrg4 as subtype specific markers for cluster 5, which was the most transcriptomically unique cluster. the study’s current and future directions include the selection of commercially available antibodies against candidate marker gene products and validation of these subtype-specific antibodies using doubleand triple-label immunofluorescence microscopy with other established markers for various cns cell types. establishing validated antibodies will lead to the discovery of subtype-specific involvement of astrocytes in human neurological and psychiatric conditions and advancements in our understanding of human astrocyte physiology.   abstract 11 free neuropathol 3:25:14 cadherin-23 is essential for the normal organisation of cerebellar mossy fibre synapses madison t. gray1,2,3, julie l. lefebvre2,3 1 department of pathology and laboratory medicine, western university, london, on, canada 2 neuroscience and mental health, hospital for sick children, toronto, on, canada 3 department of molecular genetics, faculty of medicine, university of toronto, toronto, on, canada to understand the physiological basis of neurological disease, one must first identify which neurons are connected and how they form those connections. here, we identify a novel cell adhesion molecule signature in golgi cells, an inhibitory interneuron in the granular layer of the cerebellar cortex. golgi cells receive excitatory input from both precerebellar mossy fibres and cerebellar granule cells, while providing inhibition to those same granule cells in a feedback loop. we show that both golgi cells and their mossy fibre partners express cadherin 23 (cdh23) – a pattern conserved in evolution. using explant cultures in vitro and ectopic viral expression in vivo, we demonstrate that cdh23 is sufficient to induce the formation of synapses by mossy fibres. the known in vivo roles of cdh23 depend on binding protocadherin 15 (pcdh15) in trans. however, here we show that pcdh15 is not expressed in mossy fibre neurons, golgi cells, or their cerebellar targets. a cdh23 mutant incapable of binding pcdh15 nonetheless induces mossy fibre synapses in vitro and in vivo, confirming that the synaptic organiser function of cdh23 is independent of pcdh15 binding. finally, analysis of a cdh23-null mouse reveals that cdh23 is not necessary for anatomical synapse formation but mossy fibres. however, loss of cdh23 results in enlarged, sparse mossy fibre terminals suggesting a role for cdh23 in mossy fibre bouton development.   abstract 12 free neuropathol 3:25:15 the extracellular matrix transcriptome following demyelination erin l. stephenson1, samira ghorbanigazar2, charlotte d’mello2, rajiv william jain2, v. wee yong2 1 department of pathology and laboratory medicine, university of calgary, calgary, ab, canada 2 hotchkiss brain institute, university of calgary, calgary, ab, canada the extracellular matrix (ecm) of the central nervous system (cns) is an interconnected network of proteins and sugars. the ecm has critical roles not only in homeostasis, but ecm remodeling in neurological diseases impacts both injury and repair. multiple sclerosis (ms) is a chronic inflammatory and degenerative disease of the cns. here, we evaluated ecm changes in ms lesions compared to controls using databases generated in-house through spatial rna-sequencing, and through a public resource of single-nucleus rna sequencing. our results found widespread changes in ecm molecules and their interacting proteins, including alterations to proteoglycans and glycoproteins within inactive and active ms lesions. some highly upregulated members, including serglycin and sparc-related proteins, have not previously been investigated and their role on ms lesion evolution and disease course remains unknown. our results emphasize that there are profound changes to the ecm following demyelination.   abstract 13 free neuropathol 3:25:16 nodding syndrome: characterizing the newest tau proteinopathy in africa kenneth g. kodja1,2,3, sylvester onzivua4, david clutterbuck2, david l. kitara5, amanda fong2, michael s. pollanen1,2,3 1 department of pathobiology and laboratory medicine, university of toronto, toronto, on, canada 2 ontario forensic pathology service, toronto, on, canada 3 tanz centre for research in neurodegenerative diseases, university of toronto, toronto, on, canada 4 department of pathology, college of health sciences, makerere university, kampala, uganda 5 department of surgery, gulu university, gulu, uganda nodding syndrome (ns) is an acquired tau proteinopathy plaguing the remote rural communities in eastern sub-saharan africa. it has been proposed that infection with the nematode parasite onchocerca volvulus triggers an autoimmune attack against the human protein leiomodin-1. this theory is dependent on the constitutive neuronal expression of leiomodin-1. we tested this hypothesis by exploring the histologic distribution of this protein in the normal human brain. subsequent immunostaining of cerebellar tissue and c. elegans (a proxy for onchocerca volvulus) using an antibody recognizing the n-terminus of leiomodin-1 was conducted. our study failed to identify the presence of leiomodin-1 immunoreactivity in neurons or glia. our results also suggest the possibility of leiomodin-1 antibody cross-reactivity between human purkinje cell membranes and the body wall of c. elegans; a finding explained by the homology between leiomodin-1 and tropomodulin. this outcome does not support the hypothesis of autoimmunity involving onchocerca volvulus and leiomodin-1. we also investigate the signature laminar distribution of cortical tau pathology in ns. through morphometric analysis, our current study, in agreement with the literature, suggests that primary tauopathies (the likes of progressive supranuclear palsy, corticobasal degeneration, pick’s disease and globular glial tauopathy) are associated with severe lower layer (iv-vi) pathologic burden, independent of clinical symptomology. interestingly, we found predominant involvement of the upper layers (i-iii) in ns; a laminar profile similar to the amyotrophic lateral sclerosis and parkinsonism-dementia complex of guam. the present study lays the foundation for future work investigating potentially unique mechanisms of propagation and neurodegeneration in ns.   abstract 14 free neuropathol 3:25:17 mri-pathological correlations show differential patterns of ex vivo texture in als avyarthana dey1,2, collin luk2, abdullah ishaque1,2, peter seres3, dennell krebs2, yee-hong yang4, julia keith5,6, sumit das1,7, ekaterina olkhov-mitsel5, sanjay kalra1,2 1 neuroscience and mental health institute, university of alberta, edmonton, ab, canada 2 division of neurology, faculty of medicine and dentistry, university of alberta, edmonton, ab, canada 3 peter s. allen mri center, university of alberta, edmonton, ab, canada 4 faculty of science – computing science, university of alberta, edmonton, ab, canada 5 laboratory medicine and molecular diagnostics, sunnybrook health sciences centre, toronto, on, canada 6 department of laboratory medicine & pathobiology, university of toronto, toronto, on, canada 7 department of laboratory medicine & pathology, faculty of medicine and dentistry, university of alberta, edmonton, ab, canada in vivo magnetic resonance imaging (mri) has provided a reliable biomarker for amyotrophic lateral sclerosis (als). however, lack of pathological specificity of these biomarkers impedes their integration in clinical investigations. to address this lack of specificity and establish their relationship with disease pathology, the aim of the current study was to examine the associations between ex vivo neuroimaging and histopathological features. we hypothesized that histopathology of the cervical spinal cord (csc) and precentral gyrus (pcg) shows associations with texture of the posterior limb of internal capsule (plic). formalin-fixed brains of 13 als patients underwent excision of the pcg, plic, and csc. t1-weighted mri was performed for pcg and plic from which texture features – namely autocorrelation (autoc), energy (energ), inverse difference normalized (indnc) – were extracted. quantitative histological assessments were performed using qupath to assess the density of axons in the corticospinal tract on nf immunolabeling, and extent of macrophage infiltration of the corticospinal tract on cd68 immunolabeling in the csc. qualitative assessments were performed to assess superficial spongiosis and presence of tdp-43 inclusions in the pcg. pearson’s-r correlational analysis was performed between texture and histological features. statistical significance was set at p < 0.05. a negative correlation was observed between plic autoc and presence of tdp-43 inclusions (r = -0.652, p = 0.022) as well as superficial spongiosis (r = -0.585, p = 0.046) in the pcg. a positive correlation was observed between plic energ and cd68 density (r = 0.714, p = 0.009). the pcg and csc demonstrate patterns of histopathological changes associated with plic texture features in als.   abstract 15 free neuropathol 3:25:18 lymphoid and plasma-cell infiltrates with amorphous eosinophilic material deposition in the leptomeninges: a challenging case gianluca lopez1,2, karam han1, shino d. magaki1, sophie x. song3, noriko salamon4, kanwarpal s. kahlon5, inna keselman6, ausaf a. bari7, harry v. vinters1 1 section of neuropathology, department of pathology and laboratory medicine, ronald reagan ucla medical center and david geffen school of medicine, los angeles, ca, usa 2 university of milan, milan, italy 3 section of hematopathology, department of pathology and laboratory medicine, ronald reagan ucla medical center and david geffen school of medicine, los angeles, ca, usa 4 department of radiology, david geffen school of medicine, university of california, los angeles, los angeles, ca, usa 5 division of hematology-oncology, david geffen school of medicine, university of california, los angeles, los angeles, ca, usa 6 department of neurology, david geffen school of medicine, university of california, los angeles, los angeles, ca, usa 7 department of neurosurgery, david geffen school of medicine, los angeles, ca, usa a 65-year-old female presented with recurrent seizures. she had a history of intractable epilepsy after a motor vehicle accident, which was treated several decades ago with left anteromedial temporal lobectomy and hippocampectomy; hippocampal sclerosis was noted at the time. at the time of her last hospital presentation, she underwent magnetic resonance imaging, which showed a leptomeningeal enhancement in the left parieto-occipital lobe. she underwent a biopsy for diagnostic purposes, which showed a diffuse lymphoid infiltrate predominantly involving the leptomeninges, with extension into the virchow-robin spaces and with deposits of amorphous eosinophilic material. the lymphoid population comprised small atypical lymphocytes, scattered plasma cells, and plasmacytoid lymphocytes. with immunohistochemistry, the lymphoid infiltrate was positive for cd20, cd19, and bcl2; a subset of cells were positive for cd138 and mum1; lambda light chain restriction was noted on rna in situ hybridization. negative stains included cd3, cd5, cd10, cd21, cd23, and bcl6. the ki-67 proliferative index was estimated at 5%. on congo red stain, the amorphous material showed green birefringence under polarized light. myd88 mutation was not detected. the patient had no evidence of systemic amyloidosis, monoclonal igm gammopathy, urinary bence jones protein, or bone marrow or other organ sites involvement. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. multiple sclerosis: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:3 (2023) review multiple sclerosis: 2023 update tanja kuhlmann1,2, jack antel2 1 institute of neuropathology, university hospital münster, 48149 münster, germany 2 neuroimmunology unit, montreal neurological institute, mcgill university, 3801 québec, canada corresponding author: tanja kuhlmann · institute of neuropathology · university hospital münster · pottkamp 2 · 48149 münster · germany tanja.kuhlmann@ukmuenster.de submitted: 02 february 2023 accepted: 01 march 2023 copyedited by: monica miranda published: 09 march 2023 https://doi.org/10.17879/freeneuropathology-2023-4675 keywords: multiple sclerosis, ebv, remyelination, slowly expanding lesions, spatial transcriptomics abstract multiple sclerosis (ms) is the most frequent inflammatory and demyelinating disease of the central nervous system (cns). significant progress has been made during recent years in preventing relapses by using systemic immunomodulatory or immunosuppressive therapies. however, the limited effectiveness of such therapies for controlling the progressive disease course indicates there is a continuous disease progression independent of relapse activity which may start very early during the disease course. dissecting the underlying mechanisms and developing therapies for preventing or stopping this disease progression represent, currently, the biggest challenges in the field of ms. here, we summarize publications of 2022 which provide insight into susceptibility to ms, the basis of disease progression and features of relatively recently recognized distinct forms of inflammatory/demyelinating disorders of the cns, such as myelin oligodendrocyte glycoprotein antibody-associated disease (mogad). introduction multiple sclerosis (ms) is the most frequent inflammatory demyelinating disease of the central nervous system (cns) and a leading cause for permanent neurological disability in young adults. the variability in disease course amongst cases is well known. significant progress has been made during recent years in using systemic immune-system directed therapies to prevent relapses that characterize the initial disease course. however, the limited effectiveness of such therapies for the later-recognized progressive disease course indicates there is a continuous disease progression independent of relapse activity which may start very early during this condition 1,2. we select articles published in 2022 that provide insight into susceptibility to ms, the basis of disease progression, and features of relatively recently recognized distinct forms of inflammatory/demyelinating disorders of the cns (figure 1). ms has a strong hereditary component, but also environmental factors play an important role, as shown in studies of monozygotic twins discordant for the disease. several articles published in 2022 support that epstein-barr virus (ebv), which has long been implicated in ms, plays an important role in the development of ms. this raises the tantalizing possibility that vaccination against ebv may eliminate ms. another topic, which continues to be heavily discussed in the ms field, is the impact of the microbiome, not only in the gut but also in the lung, on ms disease course. potential mechanisms driving disease progression include persistent focal inflammation resulting in slowly expanding lesions, meningeal inflammation causing cortical demyelination and neuronal injury, and inflammation induced diffuse changes in white matter (wm) as well as loss of compensatory mechanism, such as remyelination and brain plasticity 3. we summarize several pathological and imaging studies, which aim to dissect the underlying mechanisms driving disease progression. however, these mechanisms most likely vary over time, adding another layer of complexity for the successful development of new treatment strategies to prevent or stop disease progression. furthermore, individual patient specific factors may modulate ms pathology significantly. this is supported by results from a single nuclei rna sequencing (snrnaseq) study of human ms tissue samples which reveals that the variability in gene expression between patients is higher than between lesions 4. based on findings from animal studies, it is assumed that promotion of remyelination can prevent disease progression. however, the evidence that remyelination in ms is neuroprotective is relatively sparse. in 2022, an imaging study provides first evidence that lack of remyelination in ms is associated with increased brain atrophy, a maker of neurodegeneration 5. figure 1: insights into disease course of ms. we selected articles published in 2022 providing insights into the susceptibility to ms, potential modulators of the disease course and new technologies to monitor the disease or to dissect underlying pathways. finally, we discuss the most recent findings in myelin oligodendrocyte glycoprotein antibody-associated disease (mogad), a relatively recently newly defined inflammatory and demyelinating disease recognized by the presence of anti-myelin oligodendrocyte glycoprotein (mog) antibodies in the serum. (i) ms susceptibility and disease course – population based studies role of ebv in susceptibility to ms epidemiologic based studies in 2022 bolstered the evidence for ebv infection being a necessary albeit, not sufficient trigger, for development of ms. bjornevik et al. examined the records of a cohort of more than 10 million active us military personnel in which 801 of 955 cases that developed ms could be evaluated with regard to ebv serology 6. all but one were serologically positive at the time of diagnosis. although 35 had initially been serologically negative, 34 converted prior to diagnosis. seroconversion rate was 57% in the non-ms cohort. no enhanced conversion rate was found for other viruses; cytomegalovirus (cmv) conversion may have been protective. in a retrospective cohort study of 2,576,011 individuals born in denmark between 1971 and 2018, rostgaard et al. found that having a sibling 0–2 years younger reduces the probability of ms by 25% compared to having no siblings. furthermore, having a second sibling 6–8 years younger reduces this probability by 30% 7. they concluded that having younger siblings reduces the risk of infectious mononucleosis and the risk of ms in a mirror-like manner, suggesting that the reduction in ms risk results from earlier primary ebv infection. the basis whereby ebv infection links to disease susceptibility and/or disease course remains under investigation. ebv is known to infect b cells and these cells have been detected in ms lesions in some studies, but still require unequivocal confirmation. b cells are implicated in producing factors that contribute to underlying tissue injury. support for the postulate that ebv specific humoral and/or cellular immune mediators contribute to the disease pathology is supported by the findings by lanz et al., regarding cross-reactivity between ebv and neural tissue components 8. they demonstrated high affinity molecular mimicry between the ebv transcription factor epstein–barr nuclear antigen 1 (ebna1) and the cns protein glialcam, as well as presence of cross reactive antibodies in the cerebrospinal fluid (csf) of ms patients. interestingly, these cross-reactive antibodies bound to rodent oligodendrocytes and astrocytes, glia limitans and, likely, perivascular glial cells in the hippocampus and brainstem, a pattern reminiscent of the expression pattern of aquaporin-4 (aqp4). anti-aqp4 antibodies are the hallmark of neuromyelitis optica (nmo), another inflammatory demyelinating disease with pathological features distinct from ms 9,10. further support for the potential relevance of ebv specific humoral and/or cellular immune mediators in ms pathogenesis is the study by schneider-hohendorf et al. conducted on ms disease discordant monozygotic twins, showing a broader ebv-specific t cell receptor (tcr) repertoire in the affected one of the pair 11. these combined population and laboratory based studies raise the issue of potential benefit of ebv directed vaccines in the very young population. however, one need consider the potential risk of such vaccination, including inducing a neural-target directed cross reactive immune response and persistent modulation of overall immune-regulatory mechanisms which may have protective purposes. microbiome impacts on disease susceptibility and course in ms having identified that an array of environmental factors contribute to the susceptibility and course of ms, the challenge remains to understand the underlying mechanisms for such effects. further research is needed to find out how these factors can act via modulating the systemic immune system to acquire pro-inflammatory properties. to dissect the influence of genetic predisposition versus environmental factors on shaping the immune system, ingelfinger et al. applied a combination of antibody-based and single-cell technologies to define the peripheral immune signatures of 61 monozygotic (mz) twin pairs discordant for ms (+ healthy mz twin and dizygotic twin pairs) 12. they could discern both genetically (cd25 expression by naïve helper t cells) and environmentally (helper t cells with a dysregulated cd25-il-2 axis) determined features of an ms-associated immune signature. the microbiome serves as a major interaction of a host with the environment. the gut is long recognized as a major resident niche for the immune system, being a major site of host immune interaction with the enormous population of resident microbes. the results of the international multiple sclerosis microbiome study (imsms), derived from an analysis of the gut microbiome of 576 ms patients (36% untreated) and genetically unrelated household healthy controls (1,152 total subjects), indicated that the phytate degradation pathway was over-represented in untreated ms, while pyruvate-producing carbohydrate metabolism pathways were significantly reduced 13. microbiome composition, function, and derived metabolites were all modified in response to disease-modifying treatments. as pointed out by hosang et al., the lung (airway) also serves as a niche for immune cells supporting their long-term survival and maturation into migration-competent effector t cells 14. smoking is a recognized risk factor for the development of ms. hosang et al. demonstrated the potential for the interaction of this niche with the environment to influence the susceptibility to autoimmune neurologic disease, using the experimental autoimmune encephalomyelitis (eae) model. treating animals with antibiotics (neomycin) shifted the microbiota towards lipopolysaccharide (lps)-enriched phyla without an effect on the gut microbiome. the underlying mechanism of effect implicated was that lps would usually induce a type 1 interferon response in microglia and reduce immune activation within the cns. however, lps is also used to induce a pro-inflammatory response in human microglia 15. the above studies indicate the potential for the microbiome in multiple niches to impact on ms throughout its disease course. microbiome influences on adaptive immune cells would impact on their initial activation and migration into the cns as part of new lesion formation. modulation of the endogenous glia cells populations (astrocytes, microglia) by products derived from the microbiome would impact on the extent to which these glial cells participate in tissue injury and repair processes throughout the ms disease course. progression independent of relapse activity the natural history of multiple sclerosis has been considered in terms of relapsing and progressive categories; the latter could occur in those with previous clinical relapses (secondary progressive (sp)) or not (primary progressive (pp)). specific criteria have been defined for diagnosis of either relapsing or pp forms of the disease that now include paraclinical measures (csf, imaging) as well as clinical features 16. progression needs not be sustained in either sp or pp forms resulting in use of the term “confirmed progression” (over 3-6 months) in clinical trials. more recent focus is whether progressive forms are associated with ongoing disease activity, as defined by imaging or clinical activity, introducing the term progression independent of relapse activity (pira) 1,2,17. this designation has acquired increased significance in that regulatory approval for agents in sp ms has been denied for cases lacking of documented activity. lublin et al. used the novartis-oxford multiple sclerosis (no.ms) data pool, spanning all multiple sclerosis phenotypes and paediatric multiple sclerosis, to evaluate ∼200,000 expanded disability status scale (edss) transitions from >27,000 patients with ≤15 years follow-up 18. their conclusions were that although relapses contribute to the accumulation of disability, primarily early in multiple sclerosis, pira started early in the disease process, occurred in all phenotypes and became the principal driver of disability accumulation in the progressive phase of the disease. tur et al. could show that pira, after a first demyelinating event, was not uncommon and suggested an unfavorable long-term prognosis, especially if it occurs early in the disease course 19. focal inflammation as driver of disease progression the observation that disease progression is driven by mechanisms other than peripherally-driven acute focal inflammatory lesions, raise the issue of the pathogenic mechanisms responsible for progression. this has become a hot topic for both histopathologic and serial neuroimaging studies, with a challenge of how to reconcile these two approaches. both have implicated pathogenic processes in the white matter, cortical grey, periventricular, and spinal cord regions of the cns. as regards focal white matter lesions, beynon et al. examined chronic lesion activity (cla) ( assessed by t1-hypointense lesion volume increase within baseline t2-non-enhancing lesions identified as either slowly expanding (sels) or not slowly expanding (non-sels)) in a 108 week phase 3 clinical trial of natalizumab (ascend) in sp ms patients 20. they found that cla in both sels and non-sels was greater in patients with spms and confirmed disability progression than in those with no progression. sel prevalence was lower in the absence of acute lesion activity (ala) (5% vs. 19% in subjects with ala), while cla remained associated with disability progression. in view of the association of ala with sel prevalence, the authors suggest that the effect of natalizumab on cla may be related to its effect on ala and the suppression of ala in mixed active/inactive lesions. further studies are required to understand whether and to which extent the established immunosuppressive and immunomodulatory ms treatments target sel (with or without paramagnetic rims). (ii) ms susceptibility and disease course – tissue based studies meningeal inflammation correlates with lesion activity the meninges consists of the dura mater and the leptomeninges comprising pia mater and arachnoid. during recent years, a number of papers have elucidated the relationship between leptomeningeal inflammation and subpial cortical lesions 21-23. subpial lesions are more frequently found in close proximity to meningeal infiltrates and, subpial demyelination is associated with neuronal, oligodendroglial and astrocytic injury and loss; cortical pathology is now considered as a driver of disease progression 22. subpial lesions are characterized by reduced extent of grey matter (gm) inflammation compared to white matter lesions; therefore, the question occurs whether additional mechanisms may contribute to the development of these lesions. an obvious possibility would be that soluble factors secreted by the inflammatory cells contribute to subpial demyelination and cellular injury. earlier pathological studies could not identify a correlation between meningeal inflammation and number of wm lesions 23,24. ahmed et al. addressed the question whether meningeal infiltrates correlate with wm lesion activity 25. they analyzed the density of meningeal lymphocytes and their topographical association with grey (cortical) and white matter lesions in 27 patients with progressive ms and nine controls. numbers of t and b cells per unit length of meninges were significantly increased in ms compared to controls and number of meningeal lymphocytes correlated with extent of subpial demyelination, confirming earlier studies 22,23,24,26. patients with high meningeal lymphocytic counts had more cortical lesions compared to patients with low lymphocytic counts, but no difference in the percentage of subpial, leukocortical or intracortical lesions. importantly, patients with a high density of meningeal lymphocytic infiltrates had a higher proportion of active and mixed lesions and a lower percentage of inactive or remyelinated lesions than patients with a low density of meningeal infiltrates. interestingly, no correlation between meningeal myeloid infiltrates and subpial demyelination or wm lesion activity was detected. the authors speculated that the correlation of the extent of meningeal inflammation and the presence of higher proportions of active and mixed lesions may represent two independent pathogenic mechanisms which reflect an overall higher disease activity. in line with this observation are studies which demonstrated a correlation between numbers of leptomeningeal b cells and perivascular t and b cells in wm or brain stem lesions 27,28. cellular trafficking to and from the meninges the bi-directional communication between the systemic and cns compartments is a central process contributing to the disease course of multiple sclerosis. to be considered are the connections whereby systemic immune constituents can access the cns and how materials released within the cns are cleared from this compartment and transferred back to the systemic compartment. in context of immune-brain interactions, entry via the blood-brain barrier (bbb) has been the classic pathway. blocking immune cell passage with adhesion molecule directed inhibitors or antibodies is an effective therapy in the eae model and translates into clinical therapy (natalizumab is an approved therapy for relapsing ms). a further pathway involves blood-csf trafficking that underlies immune seeding within the meninges. the observed pathologic changes in ms in the subpial grey matter and periventricular regions support the impact of immune constituents from the csf-meninges acting on the brain parenchyma. drieu et al., taking advantage of the capacity to pharmacologically or genetically delete selective cell populations in mice, showed that populations of perivascular and leptomeningeal macrophages, referred to as parenchymal border macrophages (pbms), regulate arterial motion that drives csf flow 29. the cns 'waste clearance' system is defined as the glymphatic system. carotenuto et al. used an array of different magnetic resonance imaging (mri) techniques to identify dysfunction of the glymphatic system function in multiple sclerosis and found an association with clinical disability, disease course, demyelination and neurodegeneration 30. transport of neural tissue breakdown products, specifically myelin constituents, back to regional lymph nodes has previously been documented by showing their presence in cervical lymph nodes of ms cases 31.the detection of lymphatic dural vessels, which connect csf and draining cervical lymph nodes, raises the question of the relevance of these vessels and dural inflammation for autoimmune disease such as multiple sclerosis 32. merlini et al. have studied the functional role of dural and leptomeningeal inflammation in eae using intravital microscopy to address this question 32. they could show that the leptomeninges were highly inflamed in acute and chronic eae, whereas the dura was only minimal affected. t cells adhered more weakly, antigen presentation was less efficient and activation of autoreactive t cells was lower in the dura compared to the leptomeninges suggesting that the dura plays only a minor role in autoimmune inflammation. these observations were matched by findings in ms brains. histological studies analyzing the extent of inflammation in leptomeninges compared to the dura observed significant t cell infiltrates only in the leptomeninges, but not in the dura. diffuse white mater inflammation and it consequences for the myelin-axon unit the pathogenic mechanisms driving disease progression independent of new lesions are, so far, only incompletely understood. potential mechanisms include the expansion of focal lesions, as described above, but also diffuse changes in normal appearing white matter in ms. in their study, van den bosch et al. elucidate the consequences of diffuse microglia activation and low-level t cell infiltration for axon-myelin units in the normal appearing white matter (nawm), using high-resolution immunohistochemistry (ihc) and transmission electron microscopy (tem) 33. they compared the ultrastructural characteristics of the axon-myelin unit in non-lesional optic nerves from 8 people with ms and 8 controls. in the optic nerves of ms patients, they observed, as expected, more activated and phagocytic microglia and more t cells than in controls. they analyzed the structure of the nodes of ranvier and found an elongation of paranodes and juxtaparanodes, as well as an increased overlap between paranodal and juxtaparanodal regions, confirming the results of earlier studies 34,35. when analyzing the structure of the myelin sheath, they found a decrease in the g-ratio compared to controls, suggesting a thicker myelin sheath in ms optic nerves. however, more detailed analyses demonstrated a loss of myelin compaction as the underlying cause for the decreased g-ratio. tem analyses revealed a higher percentage of axons containing mitochondria in patients with ms, which was confirmed by high-resolution ihc. the overlap of paranodes and juxtaparanodes, less compacted myelin and mitochondrial frequency in axons correlated with the number of activated microglia. these findings suggest that activated microglia contribute to nodal and myelin disorganization in nawm in ms patients, which may result in a higher axonal energy demand and increased mitochondrial numbers to compensate it. further studies are required to determine whether the same changes occur throughout the cns in patients with ms, when these changes start and whether these pathological changes correlate with pira. (iii) ms susceptibility and disease course new technologies in vivo imaging to assess consequences of demyelination and presence of re-myelination in ms emerging concepts suggest that a combination of persisting focal and diffuse inflammation within the cns and a gradual failure of compensatory mechanisms, including remyelination and brain plasticity, result in disease progression. in demyelinating animal models, remyelination reduces axonal as well as neuronal damage and is associated with clinical recovery 36-38. therefore, promotion of remyelination appears to be a promising new treatment approach in ms to prevent disease progression. however, despite increasing knowledge regarding the molecular mechanisms regulating deand remyelination in animal models, the development of new remyelination promoting drugs is still a challenge. to date, clinical trials testing experimentally identified drugs have had limited success. the failure of these clinical trials might be at least partly explained by our still limited knowledge about the natural course and clinical effects of remyelination in ms patients. tonietto et al. combined voxel based positron emission tomography (pet) and magnetization transfer mri (mtr) approaches to study the influence of lesion location on myelin repair and the correlation between remyelination and grey matter atrophy 5. at baseline, the probability of demyelinated voxels was highest close to the ventricle and, increasing distance from the ventricle was associated with an increased myelin content in lesions. they also reported that the probability of a demyelinated voxel to remyelinate in the follow-up period correlated with the distance from the ventricle. these findings are in line with histopathological studies, observing a lower extent of remyelination in periventricular compared to subcortical lesions 39,40. interestingly, tonietto et al. also observed that failure of remyelination was associated with lower thalamic volume, an imaging marker of neurodegeneration 5. additionally, they found a weak association between periventricular remyelination failure and regional cortical atrophy depending on the number of cortex-derived tracks passing through periventricular white matter. these finding suggest that lack of periventricular remyelination contributes to cortical damage in ms. however, the small sample size and the relatively short follow-up are limitations of these findings. further studies are required to determine the relationship between remyelination (failure) and brain atrophy. arnold et al. reported that siponimod therapy (expnd trial) improved magnetic transfer ratios (mtr), a measure of myelination within new lesions and across grey and white matter 41. this was associated with reduced loss of cortical grey matter, thalamic, and total brain volume, again suggesting a beneficial effect of remyelination for brain atrophy. as mentioned, the challenge remains to distinguish therapeutic effects acting via impact on inflammation from neuroprotective and repair promoting effects. to date, no therapy has received regulatory approval based entirely on the latter. a challenge remains how to use advances in imaging (and other para-clinical measures (electrophysiology, biomarkers)) to rapidly predict meaningful efficacy of therapies aimed at the latter, as so successfully done for immune-therapies for relapsing ms. new imaging techniques may help to differentiate repair and injury mechanisms. rahmandazeh et al. combined cross-sectional, longitudinal and post-mortem histopathology-imaging validation studies to evaluate, for the first time, the usefulness of quantitative susceptibility mapping (qsm) for the identification of different ms lesion types, including remyelinated lesions 42. qsm quantifies the distribution of magnetic susceptibility in tissues and is sensitive to myelin content and iron accumulation in the brain 43,44. due to its ability to detect iron, it has been used in the past for the detection of paramagnetic rim lesions (prls), which histopathologically, at least partly, correspond to mixed active/inactive lesions (= chronic active lesions). in this cross-sectional study, the authors analyzed 1,621 lesions and classified them in the qsm maps as either isointense (29 %), hypointense (4 %), hyperintense (52 %), lesions with a hypointense rim (1 %) and prls (13 %). the qsm histopathological correlation study revealed that iso/hypointense lesions mostly corresponded to remyelinated lesions, hyperintense lesions to inactive lesions and prls mostly to mixed active/inactive lesions. however, one caveat is that hypo-intensity in qsm indicates complete remyelination in the absence of ongoing inflammation; therefore, it appears less suitable for the detection of ongoing remyelination during the first five to six months after lesion formation 45. further studies comparing different imaging technologies with histopathological findings are required to evaluate the advantages and limitations of these approaches in the follow-up of remyelination in people with ms. novel molecular technologies to dissect ms pathogenesis in human tissue sections a number of studies so far have analyzed human ms tissue samples by snrnaseq 46-48. however, these studies included only a relatively small number of tissue samples from few patients. macnair et al. analyzed in their not yet peer-reviewed study the largest cohort of ms and control tissue samples by snrnaseq 4. they analysed 740,000 snrnaseq profiles from 165 samples including wm lesions (n = 62), normal appearing wm (n = 17), gm lesions (n = 39) and normal appearing gm (n = 16) from 55 ms patients, as well as, 15 wm and 16 gm tissue samples from 28 controls. they reached a median sequencing depth of 4,194 nuclei/sample, 3,154 reads/nucleus and 1,702 genes per nucleus. unsurprisingly, the authors observed highly cell-type specific changes in gene expression in wm and gm lesions in ms. interestingly, cell-type specific changes were largely shared across lesions, and patient identity was a much stronger driver of variability in cell type-specific expression for many genes than lesion type. nevertheless, since the composition of ms lesions is heterogeneous and the authors did not dissect specific lesion areas, for example the rim of mixed lesions, more specific signatures may have been overlooked. the authors observed an enrichment of ms risk genes differentially expressed in immune cells, but also in neurons, astrocytes and oligodendrocytes, supporting the notion that not only immune cells but also neural cells play a role for ms disease risk and progression. they also found an enrichment of different oligodendroglial subpopulations in lesions and nawm in ms compared to control wm; however, again donor id was a stronger driver of variability than lesion type. they identified three different oligodendroglial patterns. the first pattern was similar to controls, the second was characterized by a high proportion of an oligodendroglial subtype upregulating cellular stress genes and, the third pattern showed high proportions of early oligodendrocytes and reduced levels of more mature oligodendrocytes, suggestive of an arrest of oligodendroglial maturation or differentiation. these data suggest that patients may respond differently to potential remyelination promoting drugs. the challenge for the future will be to develop measure to identify patients who will especially benefit from remyelination promoting drugs. one limitation of snrnaseq is the lack of spatial resolution. this is of special interest for ms lesions, which may be spatially heterogeneous with respect to extent and composition of inflammation (such as mixed lesions) or remyelination (for example, partially remyelinated lesions). spatial transcriptomics allows transcriptomic analyses in a spatially highly defined manner. kaufmann et al. combined this technology with high sensitivity proteomics, published single cell-rna sequencing data and in vivo perturbation model data to dissect underlying disease mechanisms of progressive ms and to identify new drug targets 49. they focussed their analysis on cortical grey matter from 13 progressive ms patients. there was a heterogenous distribution of neurodegeneration within the tissue and across patients, and pseudotemporal analyses allowed the identification of early neurodegenerative pathways in cortical ms tissue. interestingly, these pathways were cell-type specific; however their regulation was highly connected between cell types suggesting multicellular pathological networks driving neurodegeneration. kaufmann et al. confirmed earlier studies describing significant synaptic loss in cortical ms lesions and normal appearing grey matter (nagm) 50. they also found an inverse correlation between genes regulating synaptic plasticity and maintenance and genes regulating inflammation and tissues remodelling expressed by astrocytes and innate immune cells. interestingly, their data suggested that chronic inflammation only in combination with local failure of neurotrophic support result in full extent of multicomponent neurodegeneration. one caveat might be that the authors used pseudotemporal biostatistical approaches to identify early neurodegenerative events. it would be desirable to validate these results in tissue samples from ms patients with ongoing cortical demyelination; but these lesions are rarely available in post-mortem tissue collections. using complex and elegant biostatistical analyses including in vivo cns disease model data and their own spatial transcriptomic and proteomic results they identified and prioritized cns enriched receptors as well as new pharmacological drug targets. (iv) other demyelinating diseases multiple sclerosis remains a diagnosis based on carefully considered clinical and mri based criteria plus csf findings of immunoglobulin (ig) synthesis and oligoclonal bands. although considered as an autoimmune disorder, no specific antigen-directed immune response is yet incorporated into the diagnostic criteria. however, over the last years, antigen specific immune responses have been identified in patients who would previously have been included within the ms umbrella. the first of these was nmo featuring aqp4directed antibodies. consensus clinical syndromes and mri findings related to optic nerve, spinal cord, area postrema, other brainstem, diencephalic, or cerebral presentations are now recognized 51. more recent has been recognition of monophasic or recurrent syndromes linked to presence of anti-mog antibody. these expanding syndromes now include optic neuritis (on), transverse myelitis (tm), encephalitis, and seizures, as well as aseptic meningitis and peripheral nervous system demyelination (reviewed in 52). mogad can be presented across the age spectrum. a specific question of interest addressed by gaudioso et al. is the frequency of mog-igg and aqp4-igg among patients with pediatric-onset multiple sclerosis (poms) 53. in their review of 493 cases, mog-igg was positive in 30 cases (6%) and zero controls; none were nmo-antibody positive. twenty-five of the 30 positive patients were judged to have mogad clinical criteria, 5 maintained a diagnosis of multiple sclerosis (ms). only 44% had serum epstein-barr virus (ebv) positivity and only 20% had cerebrospinal fluid oligoclonal bands. these distinct “ms-related” disorders are receiving increasing attention with regard to optimal treatment paradigms, both for acute episodes and for prevention of recurrences. cerebral cortical encephalitis (cce) is a recently recognized phenotype of mogad. it is characterized by cortical t2-fluid-attenuated inversion recovery hyperintensity on mri 54. valencia-sanchez et al. presented the clinical, imaging and pathological features of 19 cce patients, identified by screening their cohort of 285 mogad patients 55. the majority of cce occurred in patients with childhood onset of mogad and many patients presented with epilepsy. pathology details were available in two patients. in one patient inactive perivascular demyelinated lesions in cortex and subcortical white matter were observed. the second biopsy was characterized by marked meningeal inflammation, extensive macrophage and microglia activation in the meninges as well as cortex and extensive subpial demyelination. the cd4/cd8 ratio was variable in these two cases and varied between 2.4 and 0.7. few b cells aggregates were found in the meninges of one patient; terminal complement activation products were absent. extensive subpial demyelination has been described to be specific for multiple sclerosis 56. this recent report, together with earlier mogad case reports demonstrate that subpial demyelination can also be found in other inflammatory demyelinating diseases and raises speculation regarding the role of humoral immune constituents and soluble inflammatory mediators as contributors 57,58. a further recently addressed issue is whether sars-cov-2 vaccination programs could result in increased rates of mogad. francis et al reported a total of 25 cases referred to specialized mogad centers in the uk after the introduction of the covid vaccination program; their conclusion was “these observations might support a causative role of the chadox1s vaccine in inflammatory cns disease and particularly mogad” 59. summary the development of successful systemic immunomodulatory or anti-inflammatory treatment options for ms within the last two decades resulted in the discovery of progression independent of relapse activity. to dissect the underlying mechanisms driving this progression represents currently the biggest challenge in the field of ms. no animal model for the progressive disease phase of ms exists. therefore, clinically well characterized human tissue collections and modern technologies, such as sc/snrna sequencing and spatial transcriptomics will be essential to dissect the cellular and molecular mechanisms driving disease progression and to detect potential biomarkers. clinical, imaging and other para-clinical studies are required to further validate candidate pathways and potential biomarkers. here, we summarized some studies which started to disentangle the underlying pathways for disease progression and may serve as blueprint for future studies. references 1. kappos, l. et al. contribution of relapse independent progression vs relapse associated worsening to overall confirmed disability accumulation in typical relapsing multiple sclerosis in a pooled analysis of 2 randomized clinical trials. jama neurol 77, 1132 1140, https://doi.org/10.1001/jamaneurol.2020.1568 (2020). 2. university of california et al. silent progression in disease activity free relapsing multiple sclerosis. ann neurol 85, 653 666, https://doi.org/10.1002/ana.25463 (2019). 3. kuhlmann, t. et al. multiple sclerosis progression: time for a new mechanism driven framework. lancet neurol 22, 78 88, https://doi.org/10.1016/s1474 4422(22)00289 7 (2023). 4. macnair, w. et al. single nuclei rnaseq stratifies multiple sclerosis patients into three distinct white matter glia responses. biorxiv, https://doi.org/10.1101/2022.04.06.487263 (2022). 5. tonietto, m. et al. periventricular remyelination failure in multiple sclerosis: a substrate for neurodegeneration. brain 146, 182 194, https://doi.org/10.1093/brain/awac334 (2023). 6. bjornevik, k. et al. longitudinal analysis reveals high prevalence of epstein barr virus associated with multiple sclerosis. science 375, 296 301, https://doi.org/10.1126/science.abj8222 (2022). 7. rostgaard, k., nielsen, n. m., melbye, m., frisch, m. & hjalgrim, h. siblings reduce multiple sclerosis risk by preventing delayed primary epstein barr virus infection. brain, https://doi.org/10.1093/brain/awac401 (2022). 8. lanz, t. v. et al. clonally expanded b cells in multiple sclerosis bind ebv ebna1 and glialcam. nature 603, 321 327, https://doi.org/10.1038/s41586 022 04432 7 (2022). 9. hoftberger, r. & lassmann, h. immune mediated disorders. handb clin neurol 145, 285 299, https://doi.org/10.1016/b978 0 12 802395 2.00020 1 (2017). 10. wingerchuk, d. m. & lucchinetti, c. f. neuromyelitis optica spectrum disorder. n engl j med 387, 631 639, https://doi.org/10.1056/nejmra1904655 (2022). 11. schneider hohendorf, t. et al. broader epstein barr virus specific t cell receptor repertoire in patients with multiple sclerosis. j exp med 219, https://doi.org/10.1084/jem.20220650 (2022). 12. ingelfinger, f. et al. twin study reveals non heritable immune perturbations in multiple sclerosis. nature 603, 152 158, https://doi.org/10.1038/s41586 022 04419 4 (2022). 13. imsm consortium. gut microbiome of multiple sclerosis patients and paired household healthy controls reveal associations with disease risk and course. cell 185, 3467 3486 e3416, https://doi.org/10.1016/j.cell.2022.08.021 (2022). 14. hosang, l. et al. the lung microbiome regulates brain autoimmunity. nature 603, 138 144, https://doi.org/10.1038/s41586 022 04427 4 (2022). 15. dorion, m. f. et al. systematic comparison of culture media uncovers phenotypic shift of primary human microglia defined by reduced reliance to csf1r signaling. glia, https://doi.org/10.1002/glia.24338 (2023). 16. thompson, a. j. et al. diagnosis of multiple sclerosis: 2017 revisions of the mcdonald criteria. lancet neurol 17, 162 173, https://doi.org/10.1016/s1474 4422(17)30470 2 (2018). 17. lublin, f. d. et al. defining the clinical course of multiple sclerosis: the 2013 revisions. neurology 83, 278 286, https://doi.org/10.1212/wnl.0000000000000560 (2014). 18. lublin, f. d. et al. how patients with multiple sclerosis acquire disability. brain 145, 3147 3161, https://doi.org/10.1093/brain/awac016 (2022). 19. tur, c. et al. association of early progression independent of relapse activity with long term disability after a first demyelinating event in multiple sclerosis. jama neurol, https://doi.org/10.1001/jamaneurol.2022.4655 (2022). 20. beynon, v. et al. chronic lesion activity and disability progression in secondary progressive multiple sclerosis. bmj neurol open 4, e000240, https://doi.org/10.1136/bmjno 2021 000240 (2022). 21. bevan, r. j. et al. meningeal inflammation and cortical demyelination in acute multiple sclerosis. ann neurol 84, 829 842, https://doi.org/10.1002/ana.25365 (2018). 22. magliozzi, r. et al. a gradient of neuronal loss and meningeal inflammation in multiple sclerosis. ann. neurol 68, 477 493, https://doi.org/10.1002/ana.22230 (2010). 23. magliozzi, r. et al. meningeal b cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology. brain 130, 1089 1104, https://doi.org/10.1093/brain/awm038 (2007). 24. howell, o. w. et al. meningeal inflammation is widespread and linked to cortical pathology in multiple sclerosis. brain 134, 2755 2771, https://doi.org/10.1093/brain/awr182 (2011). 25. ahmed, s. m. et al. accumulation of meningeal lymphocytes correlates with white matter lesion activity in progressive multiple sclerosis. jci insight 7, https://doi.org/10.1172/jci.insight.151683 (2022). 26. howell, o. w. et al. extensive grey matter pathology in the cerebellum in multiple sclerosis is linked to inflammation in the subarachnoid space. neuropathol appl neurobiol 41, 798 813, https://doi.org/10.1111/nan.12199 (2015). 27. fransen, n. l. et al. absence of b cells in brainstem and white matter lesions associates with less severe disease and absence of oligoclonal bands in ms. neurol neuroimmunol neuroinflamm 8, https://doi.org/10.1212/nxi.0000000000000955 (2021). 28. reali, c. et al. b cell rich meningeal inflammation associates with increased spinal cord pathology in multiple sclerosis. brain pathol 30, 779 793, https://doi.org/10.1111/bpa.12841 (2020). 29. drieu, a. et al. parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid. nature 611, 585 593, https://doi.org/10.1038/s41586 022 05397 3 (2022). 30. carotenuto, a. et al. glymphatic system impairment in multiple sclerosis: relation with brain damage and disability. brain 145, 2785 2795, https://doi.org/10.1093/brain/awab454 (2022). 31. fabriek, b. o. et al. in vivo detection of myelin proteins in cervical lymph nodes of ms patients using ultrasound guided fine needle aspiration cytology. j neuroimmunol 161, 190 194, https://doi.org/10.1016/j.jneuroim.2004.12.018 (2005). 32. merlini, a. et al. distinct roles of the meningeal layers in cns autoimmunity. nat neurosci 25, 887 899, https://doi.org/10.1038/s41593 022 01108 3 (2022). 33. van den bosch, a. m. r. et al. ultrastructural axon myelin unit alterations in multiple sclerosis correlate with inflammation. ann neurol, https://doi.org/10.1002/ana.26585 (2022). 34. wolswijk, g. & balesar, r. changes in the expression and localization of the paranodal protein caspr on axons in chronic multiple sclerosis. brain 126, 1638 1649, https://doi.org/10.1093/brain/awg151 (2003). 35. gallego delgado, p. et al. neuroinflammation in the normal appearing white matter (nawm) of the multiple sclerosis brain causes abnormalities at the nodes of ranvier. plos biol 18, e3001008, https://doi.org/10.1371/journal.pbio.3001008 (2020). 36. irvine, k. a. & blakemore, w. f. remyelination protects axons from demyelination associated axon degeneration. brain 131, 1464 1477, https://doi.org/10.1093/brain/awn080 (2008). 37. duncan, i. d., brower, a., kondo, y., curlee, j. f., jr. & schultz, r. d. extensive remyelination of the cns leads to functional recovery. proc. natl. acad. sci. u. s. a 106, 6832 6836, https://doi.org/10.1073/pnas.0812500106 (2009). 38. sarrazin, n. et al. failed remyelination of the nonhuman primate optic nerve leads to axon degeneration, retinal damages, and visual dysfunction. proc natl acad sci u s a 119, e2115973119, https://doi.org/10.1073/pnas.2115973119 (2022). 39. patrikios, p. et al. remyelination is extensive in a subset of multiple sclerosis patients. brain 129, 3165 3172, https://doi.org/10.1093/brain/awl217 (2006). 40. goldschmidt, t., antel, j., konig, f. b., brück, w. & kuhlmann, t. remyelination capacity of the ms brain decreases with disease chronicity. neurology 72, 1914 1921, https://doi.org/10.1212/wnl.0b013e3181a8260a (2009). 41. arnold, d. l. et al. effect of siponimod on magnetic resonance imaging measures of neurodegeneration and myelination in secondary progressive multiple sclerosis: gray matter atrophy and magnetization transfer ratio analyses from the expand phase 3 trial. mult scler 28, 1526 1540, https://doi.org/10.1177/13524585221076717 (2022). 42. rahmanzadeh, r. et al. a new advanced mri biomarker for remyelinated lesions in multiple sclerosis. ann neurol 92, 486 502, https://doi.org/10.1002/ana.26441 (2022). 43. absinta, m. et al. persistent 7 tesla phase rim predicts poor outcome in new multiple sclerosis patient lesions. j clin invest 126, 2597 2609, https://doi.org/10.1172/jci86198 (2016). 44. chen, w. et al. quantitative susceptibility mapping of multiple sclerosis lesions at various ages. radiology 271, 183 192, https://doi.org/10.1148/radiol.13130353 (2014). 45. chen, j. t., collins, d. l., atkins, h. l., freedman, m. s. & arnold, d. l. magnetization transfer ratio evolution with demyelination and remyelination in multiple sclerosis lesions. ann. neurol 63, 254 262, https://doi.org/10.1002/ana.21302 (2008). 46. jakel, s. et al. altered human oligodendrocyte heterogeneity in multiple sclerosis. nature 566, 543 547, https://doi.org/10.1038/s41586 019 0903 2 (2019). 47. schirmer, l. et al. neuronal vulnerability and multilineage diversity in multiple sclerosis. nature 573, 75 82, https://doi.org/10.1038/s41586 019 1404 z (2019). 48. absinta, m. et al. a lymphocyte microglia astrocyte axis in chronic active multiple sclerosis. nature 597, 709 714, https://doi.org/10.1038/s41586 021 03892 7 (2021). 49. kaufmann, m. et al. identification of early neurodegenerative pathways in progressive multiple sclerosis. nat neurosci 25, 944 955, https://doi.org/10.1038/s41593 022 01097 3 (2022). 50. jurgens, t. et al. reconstruction of single cortical projection neurons reveals primary spine loss in multiple sclerosis. brain 139, 39 46, https://doi.org/10.1093/brain/awv353 (2016). 51. wingerchuk, d. m. et al. international consensus diagnostic criteria for neuromyelitis optica spectrum disorders. neurology 85, 177 189,https://doi.org/10.1212/wnl.0000000000001729 (2015). 52. marignier, r. et al. myelin oligodendrocyte glycoprotein antibody associated disease. lancet neurol 20, 762 772, https://doi.org/10.1016/s1474 4422(21)00218 0 (2021). 53. gaudioso, c. m. et al. mog and aqp4 antibodies among children with multiple sclerosis and controls. ann neurol, https://doi.org/10.1002/ana.26502 (2022). 54. ogawa, r. et al. mog antibody positive, benign, unilateral, cerebral cortical encephalitis with epilepsy. neurol neuroimmunol neuroinflamm 4, e322, https://doi.org/10.1212/nxi.0000000000000322 (2017). 55. valencia sanchez, c. et al. cerebral cortical encephalitis in myelin oligodendrocyte glycoprotein antibody associated disease. ann neurol, https://doi.org/10.1002/ana.26549 (2022). 56. junker, a. et al. extensive subpial cortical demyelination is specific to multiple sclerosis. brain pathol 30, 641 652, https://doi.org/10.1111/bpa.12813 (2020). 57. hoftberger, r. et al. the pathology of central nervous system inflammatory demyelinating disease accompanying myelin oligodendrocyte glycoprotein autoantibody. acta neuropathol 139, 875 892, https://doi.org/10.1007/s00401 020 02132 y (2020). 58. takai, y. et al. myelin oligodendrocyte glycoprotein antibody associated disease: an immunopathological study. brain 143, 1431 1446, https://doi.org/10.1093/brain/awaa102 (2020). 59. francis, a. g. et al. acute inflammatory diseases of the central nervous system after sars cov 2 vaccination. neurol neuroimmunol neuroinflamm 10, https://doi.org/10.1212/nxi.0000000000200063 (2023). copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. from amaurotic idiocy to biochemically defined lipid storage diseases: the first identification of gm1-gangliosidosis feel free to add comments by clicking these icons on the sidebar free neuropathology 4:12 (2023) flashback from amaurotic idiocy to biochemically defined lipid storage diseases: the first identification of gm1-gangliosidosis burkhard s. kasper1, christian thomas2, anne albers2, ekkehard m. kasper3, konrad sandhoff4 friedrich-alexander-universität erlangen-nuremberg, university hospital, dept. neurology; erlangen, germany institute of neuropathology, university hospital münster, münster, germany division of neurosurgery, hamilton health sciences, mcmaster university faculty of health sciences, hamilton, on, canada limes institute, membrane biology & biochemistry unit, bonn university, bonn, germany corresponding author: burkhard s. kasper · epilepsy center · department of neurology · friedrich-alexander university erlangen-nuremberg · schwabachanlage 6 · 91054 erlangen · germany burkhard.kasper@uk-erlangen.de additional resources and electronic supplementary material: supplementary material submitted: 22 may 2023 accepted: 15 july 2023 copyedited by: jeffrey nirschl published: 08 august 2023 https://doi.org/10.17879/freeneuropathology-2023-4845 keywords: lipid storage disease, gm1-gangliosidosis, amaurotic idiocy, tay-sachs-disease, beta-galactosidase original paper: on a biochemically special form of infantile amaurotic idiocy. jatzkewitz h., sandhoff k., biochim. biophys. acta 1963; 70; 354-356. see supplement 1. abstract on february 23rd 1936, a boy-child (“kn”) died in an asylum near munich after years of severe congenital disease, which had profoundly impaired his development leading to inability to walk, talk and see as well as to severe epilepsy. while a diagnosis of “little’s disease” was made during life, his postmortem brain investigation at munich neuropathology (“deutsche forschungsanstalt für psychiatrie”) revealed the diagnosis of “amaurotic idiocy” (ai). ai, as exemplified by tay-sachs-disease (tsd), back then was not yet understood as a specific inborn error of metabolism encompassing several disease entities. many neuropathological studies were performed on ai, but the underlying processes could only be revealed by new scientific techniques such as biochemical analysis of nervous tissue, deciphering ai as nervous system lipid storage diseases, e.g. gm2-gangliosidosis. in 1963, sandhoff & jatzkewitz published an article on a “biochemically special form of ai” reporting striking differences when comparing their biochemical observations of hallmark features of tsd to tissue composition in a single case: the boy kn. this was the first description of “gm1-gangliosidosis”, later understood as resulting from genetically determined deficiency in beta-galactosidase. here we present illustrative materials from this historic patient, including selected diagnostic slides from the case “kn” in virtual microscopy, original records and other illustrative material available. finally, we present results from genetic analysis performed on archived tissue proving beta-galactosidase-gene mutation, verifying the 1963 interpretation as correct. this synopsis shall give a first-hand impression of this milestone finding in neuropathology. introduction in history of science, there has always been and will be discussion, to whom to ascribe to the first description of a disease. addressing a specific publication as the first description can turn out quite differently, depending on whether or not one is focusing on a disease’s clinical manifestations, its tissue pathology, pathophysiological mechanism or underlying genetic cause. further complicating matters, similar findings are sometimes reported independently or even simultaneously by separate researchers at different places. as thinking and knowledge about human disease evolve over time, which include terminology and classifications, it could well be that we end up with more than one author credited for the first description of a particular disease. individual patients and the details behind groundbreaking findings, however, are rarely appreciated in detail, albeit some of them have become linked to (neuro-)history at least by their initials, such as “h.m.” [1]. concerning the “lipidoses”, exemplified by tay-sachs disease (tsd), syndromal appearance of certain symptoms in children had been well observed and reported as early as at the end of the 19th century by warren tay [55] and bernard sachs [36] establishing “tay-sachs-disease” as an eponym [6], as well as “amaurotic family idiocy” (afi) as another common term [35]. the rather dramatic clinical presentation in tsd/afi and comparable diseases, such as niemann-pick’s, was accompanied by striking pathologic findings post-mortem, especially in the cns, where neurons were found enlarged and ballooned, obviously filled by aberrant material [25]. the first pathoanatomic description of tsd appears to have been given by sachs himself in 1887 [36]. in the 20th century tsd/afi brains were studied extensively both histopathologically and later also ultrastructurally (e.g. [8], [9], [10], [38], [55], [56]) laying the foundation for a concept of lysosomal lipid storage disease (review see [11], [25], [27], [58]). cns tissue pathology in tsd and other storage diseases appeared rather uniform, resulting from accumulation of some storage material strikingly altering neuronal morphology, a finding early appreciated in cns histopathology ([12], [57], [46-48]). afi subtypes for quite a long time were separated on clinical grounds only by predominating manifestation age. specific techniques allowing a more precise analysis, deciphering the underlying molecular pathobiology and establishing a clear understanding of different disease forms, were not available. this changed in the mid-to-late 1930s, when klenk coined the term “gangliosides” to describe characteristic acid glycolipid components of neuronal cells isolated from tsd brains [19-21]. it took more than 20 years from then to identify the first ganglioside structure g1 [24], now called gm1, and to apply better techniques such as thin-layer chromatography allowing for separation and characterization of the different ganglioside components in diseased versus normal brain tissues in the 1960s. further important breakthrough discoveries were achieved in the understanding of nervous system within a few years ([14], [15], [24], [34], [43], [53]) deciphering the “gangliosidoses”, including classic tsd as gm2-gangliosidosis. research activities and knowledge gain then exploded, leading to one of the most astonishing advances in understanding of lipid storage-related cns-disease (for review see: [2], [16]), which for decades before had remained enigmatic and summed up under different terms of afi, “amaurotic idiocy” (ai) or “infantile amaurotic idiocy” (iai), a hot topic in early neuropathology. up to today, many aspects of lipid storage related disease mechanisms have been elucidated. this includes many of the enzymes involved, their functional dynamics in health and disease, their structure(s), co-factors and substrates including cellular pathways & regulation, the role of the lysosomal compartment, the regulating genes and their mutational spectrum (for review see [2], [16], [22], [28], [42]). after decades characterized by restriction to symptom-oriented diagnosis and, at best, post mortem validation in a few selected cases, we are now able to recognize these diseases intra vitam by surrogate biomarkers and/or molecular testing. it is a fascinating journey to follow the historic lines of these developments. here we present and re-appreciate a key patient, “kn”, including his post mortem tissues as well as the understanding derived from analyzing his tissue probes biochemically: this boy died 1936 in munich from a fatal course of iai at the age of 7¾. by diligent biochemical analysis of this patient’s brain tissue, performed after more than 25 years of formalin-fixation, jatzkewitz & sandhoff revealed a “biochemically special form of infantile amaurotic idiocy” [14] later to be named “gm1-gangliosidosis” [51]. this can be acknowledged as the first definite characterization of this disease [16, 28], eliciting that tsd/ai/iai was a spectrum of different diseases rather than a uniform entity. what exactly did the authors describe in this study? jatzkewitz & sandhoff presented a surprising result of a thin-layer-chromatographic lipid compound analysis performed on brain tissue from an “ai”-patient, for this single case (patient “kn”) out of an ai-series clearly displayed an unusual “aberrant” pattern on chromatography when compared to chromatography bands of other “ai-tsd”-materials and normal brain (see figure 1). kn’s analysis did not show the accumulation of “tay-sachs-ganglioside” (=gm2 ganglioside) but showed accumulation of the major normal “ganglioside g1” according to kuhn & wiegandt [24], i.e. ganglioside a according to klenk and gm1-ganglioside according to svennerholm. why is this a milestone paper? the paper’s key finding summarized on less than three pages convincingly demonstrates that a special and biologically different variant of tsd-like disease had been found, which turned out to be correct. in contrast to classic tsd showing accumulation of gm2-ganglioside and its sialic acid free residue ga2, here a different metabolic block had to be suggested since other substances were stored, identified as “ganglioside gm1” and its sialic acid free residue [14] [41], which turned out to be correct [2]. the distinct identity of these storage compounds was proven by chemical, biochemical and enzymatic procedures [43]. while several papers and textbooks refer to a different study as the one having identified gm1 as the storage compound in this type of disease [30], this clearly came later ([16], [28]). at this stage, all the knowledge to be revealed later about metabolic chains and enzymes involved had not yet been known [44]. figure 1. original chromatography analysis by sandhoff: “kn” = lane 7. formalin-fixed control = lane 5 (photography by k. sandhoff, gangliosides labelled; original legend). see full article by jatzkewitz & sandhoff 1963 [14] in supplement 1 (illustrating the metabolic path).thin-layer chromatogram of lipid extracts of brain tissue from two usual infantile cases and one from one special form of late-infantile amaurotic idiocy (jatzkewitz and sandhoff 1963) adsorbent, 400-µm-thick layer of kieselgel g, merck; solvent system, propanol-conc. ammonia-water (6:2:1); height of the solvent front, 15cm; detection, anisaldehyde, sulfuric acid in acetic acid (reagent of kägi-miescher). 1, 20µg of neuraminic acid-free residue of ganglioside tay-sachs (b’, now named ga2, rf 0.33); 2, 250µg of total lipid extract of the brain cortex of a case of iai (fresh tissue); 3, 20µg of ganglioside tay-sachs (a’, gm2, rf 0.37); 4, 250µg of total lipid extract of the brain cortex of a case of iai, preserved for formalin for 26 years; 5, 250µg of total lipid extract of normal brain cortex, preserved in formalin for 26 years; 6, 20µg of ganglioside a (gm1, rf 0.30); 7, 250µg of total lipid extract of the brain cortex of a special form of late iai, preserved in formalin for 26 years; 8, 20µg of neuraminic acid-free residue of ganglioside a (ga1, rf 0.26). [author’s addendum: rf means “retention factor”]. what exactly was new based on the knowledge back then? up to this 1963 study’s result, ai and tsd was perceived as an entity with varying clinical courses and was classified according to manifestation age. classic microscopic histomorphological analysis was not able to clearly differentiate between various entities, and therefore could not resolve the ai-enigma, maybe except separation of the “neuronal ceroid-lipofuscinoses” (ncl) as a separate group in ai-like disease ([46-48] [57]). of note, ncl as a term was coined later ([50],[62]). the biology and biochemical nature of these diseases were largely unknown. what turned out as being correct or incorrect since then? some authorities were convinced that essentially everything was already known about tsd up to 1960. as late as 1957, seitelberger had stated that “…it has been…proven, that the cellular metabolic disturbance constituting the individual forms [of tsd] are mainly the same” [49]. when k. sandhoff following his analysis of a randomly selected first ai-case searched for further brain specimens in munich neuropathology archives, he was confronted by the institute’s head g. peters, who told him boldly: “…if you aim to know about tay-sachs-disease, read my book and you will know everything” (sandhoff, personal memory). this was the starting point. newly developed biochemical analytic methods opened new windows: it turned out that diverse lipid compounds represent essential building blocks of nervous tissue, that their metabolism is highly sophisticated biochemically and can be altered causing various specific diseases (for review see [45], [2]). concerning the “biochemically special form” of ai as identified in 1963, the postulated metabolic block in ganglioside a degradation later was identified and characterized as impairment of specific enzyme activities, mostly beta-galactosidase ([2], [16], [44]). at what stage of the author´s career was the paper published? k. sandhoff was a young student at the very beginnings of his career as a researcher (figure 2). figure 2: konrad sandhoff 1962, during the time of his thesis (photo shared by k sandhoff). this study was his debut publication. having finished studying chemistry in 1962, he turned towards lipid compound analysis from nervous tissue by joining the department of neurochemistry headed by horst jatzkewitz at munich max-planck institute for psychiatry, encouraged and supported by 1964 nobel laureate prof. fedor lynen. after failed attempts to isolate presumed sialic-acid-free gangliosides (“asialo-gangliosides”) from cattle brain, his attention turned towards new targets for investigation: rather by chance, in 1962 he decided to choose a first human brain sample in the lab labelled “ai” [44]. in this very first analysis, using chromatographic techniques, he was able to reveal two bands, which he named “a” and “b” (later identified as gm2-ganglioside and its corresponding asialo-residue ga2). in order to validate his finding, he widened his analysis to include tissue probes from other 12 brains with ai/tsd picked from the archives at munich neuropathology, “kn” being one of them ([43], [44]). how was the paper received over time? issues were raised whether the results presented were rather a methodological artifact caused by long-term formalin fixation, although this was largely ruled out by the experiments presented, since matching long-term fixed control samples had been included ([14], see figure 1). in the realm of anglo-american work this type of lipidosis became rather known as norman-landing-disease or landing’s disease for after an early case reported by norman [29] landing published a clinical report in 1964 ([26], [37], which was followed by identification of gm1-storage [30] and galactosidase deficiency [31]. in one early review, o’brien questioned, whether the patient from the jatzkewitz-sandhoff-paper had the disease at all [30]. the jatzkewitz-sandhoff paper [14] prompted further group-activities supported by national and international colleagues, who agreed to provide selected tissues probes incl. fresh and fresh-frozen samples from “ai” patients for further analysis by sandhoff. several researchers knew each other personally and also cooperated, such as sandhoff and o’brien or sandhoff and h. moser/boston (sandhoff, personal communication, [39]). such cooperation rapidly expanded the experience and led to description of further lipid storage disease variants, especially with respect to the gm2-gangliosidoses, including the hexosaminidase-0-variant later known as “sandhoff’s disease” ([23], [33], [40]). beyond this, further variants were described, including cases with storage but normal enzyme levels, indicating the presence of co-factors [44]. what happened to the original histological slides and patient documentation until today? kn’s original slides were found within the historical archive of the max planck institute for psychiatry in munich by registrar c. dücker after intense search by bs kasper had revealed enough detail in order to clearly identify them correctly, for there was no specific finding aid. the crucial informations were patient initials (“kn”) and age (“7¾“), as well as the institutional archive case number (55/36) listed in the sandhoff doctoral thesis only ([43], see supplement 2). an earlier paper on ai from munich [8], which had recruited its cases from the munich neuropathology collection also, served as cross reference, since it happened to list respective overlapping information including patient initials and age (see supplement 3). thus, tissue samples of “case 55/36” could be identified in two case boxes labelled “ai” at mpi archives (see supplement 4). luckily, the mpi archive stored one single additional document relating to “kn”, a clinical report giving some more details about the patient child, including full name, date of birth, date of death and the name of the caring medical institution prior to his death (see supplement 5). this finally allowed to identify further clinical records preserved within another munich archive, i.e. the archbishop of munich & freising’s archive (see supplement 6); for reasons of clerical law and tradition, this archive keeps documents much longer than done by average public hospitals. the munich psychiatric hospital’s archive did no longer store any documents (explored by p. rauh, see acknowledgement). what is known about the patient behind the paper? the patient designated “kn” in the respective publications ([8], [14], [15]) was a boy named johann knott, born april 11th 1928. early on, he lived with a foster mother in munich, but soon was put under guardianship by munich city administration. this way he came into an asylum in schönbrunn near munich early due to his severe disability labelled “little’s disease”. asylum notes describe the kid as unable to talk, unable to walk, suffering from epilepsy and being “very poor”. he was referred to the munich university hospital (“nervenklinik”) in june 1932, where he stayed until april 1933. at admission he was described as apathic and without reaction to external stimuli. he was noted to show impaired swallowing and an aberrant breathing pattern. physical and neurological examination was reported with brisk reflexes, abnormal muscular tone, but ocular fundi were reported as normal. the boy had multiple epileptic seizures every day described as tonic without further detail. affected by pneumonia, he died at schönbrunn february 23rd 1936 (see supplements 5-7). it is important to note, that kn’s death and postmortem investigation in no way is related to the nazi euthanasia program (confirmation with support by p. rauh; see acknowledgement, please read supplement 10). the role of german neuropathology as well as clinical medicine during the ns-regime is at the focus of ongoing research (see supplement 10). what can be seen on the original slides? at the historical archive of the munich max-planck-institute for psychiatry, 194 microscopic slides were found stored together with the original diagnostic report (see supplements 4 & 9) and the clinical note mentioned above (supplement 5). the slides encompass a systematic selection of cns sites including specimens of the spinal cord, selected brain stem levels such as medulla and pons, cerebellum and various cortical samples quite differently stained. stains according to the original report and (inconstant) slide labelling included bielschowsky-, nissl-, holzer-, mallory-, heidenhain-, “fat-” and “pikrofuchsin”-stains. while a significant number of slides were no longer useable due to severe aging and bleaching, several slides were preserved quite well. the reader is invited to explore a selection of well-preserved slides here (see figure 3 and slide-links). it is interesting to compare these examples to the original drawings drawn by van gieson, provided in bernard sachs’s 1887 publication [35] on “amaurotic idiocy” (see figure 4, and supplement 8). figure 3. original historical slides from selected cns sites of patient “kn”. specimen were well preserved stained mallory, an additional neocortical specimen underwent some “fat-stain”, not otherwise specified (scanned with zeiss axioscan.z1). the cerebral slides (lower row: neocortex and hippocampal region) illustrate the severe cortical pathology characterized by shrinking of the cortical band with profound loss of neurons. residual neurons show the typical swollen, ballooned morphology of a cns lipidosis. when going into detail on large magnifications, the neuronal pathology is seen well in brainstem also, esp. in the medulla/olive specimen (upper row, middle). within the hippocampus proper there seems to be profound loss of pyramidal neurons, which could represent hippocampal sclerosis (this specimen was not available in other stains). clicking into the respective picture will lead you to the full virtual slide. figure 4. original drawings by i. van gieson (1866-1913), from sachs’s publication 1887 [36] (courtesy of the national library of medicine). see supplement 8. have the original materials been used for modern molecular approaches and what was the result? to the best of our knowledge, the original materials of the patient johann knott presented here so far were not used for any modern molecular analysis later on, although materials participated in one subsequent ganglioside quantification analysis [15]. for this re-visitation, we performed a genetic analysis using a selected fraction of kn’s archived material using only sections no longer usable for microscopy due to aging effects. this procedure was covered by a positive vote of the ethics commission at munich university/lmu (nr. 22-1021): materials & methods: formalin-fixed paraffin-embedded (ffpe) cerebellar sections from the historical archive of the max planck institute for psychiatry in munich were used as follows. genomic dna was extracted from three stained sections. the slides were incubated in xylene (bergchemie j. c. bröcking) for 14 days to enable lifting of the cover slip. tissue was overlayed with incubation buffer containing proteinase k (maxwell ffpe plus dna kit, promega), scraped from the surface using a scalpel and resuspended for transfer into a 1.5 ml reaction tube to perform dna extraction using the maxwell ffpe plus dna kit (promega). dna was quantified using the quantifluor one dsdna system (promega) and 20 ng were subjected to the infinium hd ffpe restore protocol (illumina, zymo). finally, 1 µl of the restored gdna was inspected by genomic dna screentape assay (agilent) to determine dna integrity number (din) and concentration (figure 5a). although dna integrity was low (1.0), the exome 2.0 kit (twist bioscience) could be successfully applied for library preparation and sequencing was performed on a novaseq 6000 device. after base calling and adapter trimming, alignment to the human reference genome (homo_sapiens.grch37.75_complete) and variant calling were performed using dragen v07.021.624.3.10.10. variants were annotated using the variant effect predictor (v100) for functional annotations, pathogenicity scores, population allele frequencies and to determine the effect of called variants on genes, transcripts, and protein sequence. variants were manually inspected with the integrative genomics viewer (igv). results are illustrated in figure 5. figure 5. (a) dna fragment analysis. fragment analysis of gdna isolated from formalin-fixed paraffin-embedded tissue shows high dna degradation. the figure shows dna fragment sizing and quantification using genomic dna screentape assay. (b) igv screenshots showing representative images of the two heterozygous mutations: glb1 (nm_000404.4): c.400g>a; p.g134r (top) and glb1 (nm_000404.4): c.931g>a; p.g311r (bottom). both mutations have been described in patients with gm1 gangliosidosis [28]. epilogue amaurotic idiocy (ai), as exemplified by tay-sachs-disease (tsd), had remained a fatal, enigmatic condition for a long period of time. ai’s impressive cellular pathology, affecting neuronal cells throughout the nervous system quite specifically, was recognized as early as 1887 ([36] supplement 8). after separating the tissue pathology later to be known as ceroid-lipofuscinosis [62; 46-48] classic neuromorphology for decades had not much to contribute to the differentiation of ai, for its cns pathology appeared rather uniform [25]. significant advances in this field were largely driven by biochemical insight into composition and distribution of central nervous compounds and by applying newly emerging techniques between 1920 and 1960, exemplified by the works of klenk [19-21], kuhn & wiegandt [24], svennerholm [53] and sandhoff ([42], [45]). sandhoff’s original observation [14] revisited here opened the door to the realization that tsd-like ai was not a homogenous entity, but a spectrum of neurometabolic diseases [2]. triggered by detailed analysis of johann knott (kn) and subsequent findings the field expanded and evolved rapidly ([2], [16], [44], [45]) leading to in depth characterization of the different forms of lipid storage disease, including enzymes & genes involved. thus, gm1-gangliosidosis today can be summarized as a rare autosomally inherited lipid catabolism defect caused by deficiency of beta-galactosidase, encoded by the gene glb1 on chromosome 3, leading to lysosomal storage of ganglioside gm1 and its sialic-free residue ga1 ([2], [16], [28]] encompassing various clinical forms ([3], [16], [52]). the term “ai” has long been abandoned and is now replaced by more specific terms representing our refined understanding. acknowledgements we thank: c. dücker / historical archive registrar at mpi psychiatry munich for generous cooperation e. binder / head of mpi psychiatry munich for kindly supporting this project p. rauh / institute of history and ethics of medicine, tu munich, for fruitful discussion, sharing his knowledge about munich archives, and help with clarifying the historical context of the patient dr rauh is co-head of the research project entitled "brain research at institutes of the kaiser wilhelm society in the context of nazi injustices: brain specimens in institutes of the max planck society and identification of victims" (tu munich, university vienna, oxford brookes university, leopoldina) r. egensperger, h. leithäuser / lmu neuropathology munich for excellent support with digital scanning d. heuss / neurology at erlangen university, for support with his “flying microscope” g. treffler / munich from the munich-freising archbishop‘s archive c. döbereiner / erlangen university library for great help with many remote references supplementary material suppl. 1 jatzkewitz & sandhoff (1963) [14]; re-use permitted by elsevier, license no 5582300877941 suppl. 2 key information retrieved from the thesis by k sandhoff (1965) suppl. 3 full text: ai paper by escola 1961 [8] also incl. “kn” suppl. 4 slide boxes at mpi archive munich suppl. 5 clinical note about “kn” from historical mpi archive / munich suppl. 6 patient “kn” death certificate from the archbishop’s archive / munich suppl. 7 short autopsy report from schönbrunn suppl. 8 full-text: sachs b (1887) incl. illustrations by van gieson [36] suppl. 9 original histopathology report from historical mpi archive / munich suppl. 10 about the context of johann knott’s death in 1936 slide links spinal cord, mallory https://omero-imaging.uni-muenster.de/webclient/img_detail/314443 brainstem: medulla, mallory https://omero-imaging.uni-muenster.de/webclient/img_detail/314446 brainstem: pons, mallory https://omero-imaging.uni-muenster.de/webclient/img_detail/314449 neocortex, mallory https://omero-imaging.uni-muenster.de/webclient/img_detail/314452 neocortex, ‘fat-stain’ https://omero-imaging.uni-muenster.de/webclient/img_detail/314455 hippocampal region, mallory https://omero-imaging.uni-muenster.de/webclient/img_detail/314458 references 1. annese j, schenker-ahmed nm, bartsch h, maechler p, sheh c, thomas n, kayano j, ghatan a, bresler n, frosch mp, klaming r, corkin s. postmortem examination of patient h.m.'s brain based on histological sectioning and digital 3d reconstruction. nat commun. 2014; 5: 3122. https://doi.org/10.1038/ncomms4122 2. breiden b, sandhoff k. lysosomal glycosphingolipid storage disease. ann rev. biochem 2019; 88: 461 485. https://doi.org/10.1146/annurev-biochem-013118-111518 3. caciotti a, garman sc, rivera colon y, procopio e, catarzi s, et al. 2011. gm1 gangliosidosis and morquio b disease: an update on genetic alterations and clinical findings. biochim. biophys. acta 1812:782–90. https://doi.org/10.1016/j.bbadis.2011.03.018 4 czech h. forschen ohne skrupel. die wissenschaftliche verwertung von opfern der psychiatriemorde in wien. in: eberhard g, neugebauer w; von der zwangssterilisierung zur e.rmordung. zur geschichte der ns euthanasie in wien teil 2; 143 163; wien/köln/weimar, böhlau 2002. 5. czech h, weindling p, druml c. from scientific exploitation to individual memorialization: evolving attitudes towards research on nazi victims' bodies. bioethics 2021; 35 (6): 508 517. https://doi.org/10.1111/bioe.12860 6. desnick rj, kaback mm (ed) tay sachs disease, academic press, london sand diego 2001. 7. diamond ef. reflections on the 50th anniversary of the nuremberg doctor’s trials. the linacre quarterly 1997; 64 (2): 17 20. https://doi.org/10.1080/20508549.1999.11878376 8. escola j. [über die prozessausbreitung der amaurotischen idiotie im zentralnervensystem in verschiedenen lebensaltern und besonderheiten der spätform gegenüber der pigmentatrophie]. arch.f. psychiatrie u. zeitschrift f. d. ges. neurologie 1961; 202: 95 112. https://doi.org/10.1007/bf00342813 9. escola pico j. über die feinstruktur der speichersubstanzen bei der infantilen form der familiären amaurotischen idiotie. acta neuropathol 1964; 3: 289 294. https://doi.org/10.1007/bf00684405 10. escola pico j. über die ultrastruktur der speichersubstanzen bei spätfällen von familiärer amaurotischer idiotie. acta neuropathol 1964; 3: 309 318. https://doi.org/10.1007/bf00691839 11. gravel ra, kaback mm, proia rl, sandhoff k et al. (2001) the gm2 gangliosidoses. in: scriver cr, beaudet al, sly ws, valle d (eds) the metabolic & molecular bases of inherited disease, 8th edn. mcgraw hill, new york, pp 3827–3876. 12. haymaker w, neuburger kt, hurteau ww (ed). amaurotic family idiocy (slide 52). in: atlas of neuropathology prepared at the army medical museum, office of the surgeon general, u.s. army, 1944. http://resource.nlm.nih.gov/41220590r 13. hudson l. from small beginnings: the euthanasia of children with disabilities in nazi germany. j pediatr child health 2011; 47: 508 511. https://doi.org/10.1111/j.1440-1754.2010.01977.x 14. jatzkewitz h, sandhoff k. on a biochemically special form of infantile amaurotic idiocy. biochim. biophys. acta 1963; 70; 354 356. https://doi.org/10.1016/0006-3002(63)90764-9 15. jatzkewitz h, pilz h, sandhoff k. the quantitative determination of gangliosides and their derivatives in different forms of amaurotic idiocy. j neurochemistry 1965; 12: 135 144. https://doi.org/10.1111/j.1471-4159.1965.tb06749.x 16. johnson wg. galactosidase deficiency: gm1 gangliosidosis, morqio b disease, and galactosialidosis. in: rosenberg’s molecular and genetic basis of neurological and psychiatric disease, volume 1, 6th edition (ed. rosenberg rn and pascual jm), academic pres, london san diego 2020 17. kipfelsberger t. die konfrontation der associationsanstalt schönbrunn mit den nationalsozialistischen „euthanasie“ maßnahmen. diss. med. tu münchen, 2017 18. kinzelbach a, neuner s, hohendorf g, buschmann m, rauh p. between institutional routine, hereditary health policy, and “ns euthanasia” killings. the dissection laboratory (prosector position) at the german research institute for psychiatry (1926 1962). medizinhist j 2022; 57: 332 362. https://doi.org/10.25162/mhj-2022-0012 19. klenk e. die fettstoffe des gehirns bei amaurotischer idiotie und niemann pick’scher krankheit. ber ges physiol 1937; 96: 659 660. 20. klenk e. niemann pick’sche krankheit und amaurotische idiotie. hoppe seyler’s z physiol chem 1939; 262: 128 143. 21. klenk e. über die ganglioside, eine neue gruppe von zuckerhaltigen gehirnlipoiden. hoppe seyler’s z. physiol. chem. 1942; 273:76–86 22. kolter t. ganglioside biochemistry. international scholarly research network. isrn biochemistry2012, article id 506160, 36 pages; https://doi.org/10.5402/2012/506160 23. krivit w, desnick rj, lee j, moller f, sweeley cc, snyder pd, sharp hl. generalized accumulation of neutral glycoshpingolipids with gm2 ganglioside accumulation in the brain – sandhoff’s disease (variant of tay sachs disease). am j med 1972; 52: 763 770. https://doi.org/10.1016/0002-9343(72)90082-4 24. kuhn r, wiegandt h. die konstitution der ganglio n tetraose und des gangliosids g1. chem. ber. 1963; 96: 866 880. 25. lake bd. lysosomal enzyme deficiencies. in: greenfield’s neuropathology, 4th edition, editors adams jh, corsellis jan, duchen lw. edward arnold, london 1984, 491 572. 26. landing bh, silverman fn, craig jm, jacoby md, lahey me, chadwick dl. familial neurovisceral lipidosis. am j dis child 1964; 108: 503 22. pmid: 14209687. 27. lyon g, kolodny eh, pastores gm. neurology of hereditary metabolic diseases of children, 3rd edition. mc graw hill, new york 2006 28. nicoli er, annunziata i, d’azzo a, platt fm, tifft cj, stepien km. gm1 gangliosidosis – a mini review. frontiers in genetics 2021; 12. article 734878. https://doi.org/10.3389/fgene.2021.734878 29. norman rm, urich h, tingey am, goodbody ra. tay sachs disease with visceral involvement and its relation to niemann pick disease. j pathol bacteriol 1959; 78: 409 421. https://doi.org/10.1002/path.1700780208 30. o’brien js, stern mb, landing bh, o’brien jk, donnel gn. generalized gangliosidosis: another inborn error of ganglioside metabolism? am j dis child. 1965; 109: 338 346. pmid: 14261015. 31. okada s, o’brien js. generalized gangliosidosis: beta galactosidase deficiency. science; 1968; 160: 1002 1004. https://doi.org/10.1126/science.160.3831.1002 32. peiffer j. assessing the neuropathological research carried out on victims of the “euthanasia” programme. medizinhist j 1999; 34: 339 355. pmid: 10783589. 33. pilz h, müller d, sandhoff k, ter meulen v. tay sachssche krankheit mit hexosaminidase defekt. deutsch med wschr 1968; 39: 1833 1839 and 1843 1845. https://doi.org/10.1055/s-0028-1110836 34. pilz h, sandhoff k, jatzkewitz h. a disorder of ganglioside metabolism with storage of ceramide lactoside, monosialo ceramide lactoside and tay sachs ganglioside in the brain. j neurochemistry 1966; 13: 1273 1282. https://doi.org/10.1111/j.1471-4159.1966.tb04290.x 35. sachs b. a family form of idiocy. generally fatal and associated with early blindness (amaurotic family idiocy) new york medical journal, may 30, vol. 64 1896: 1 22. http://resource.nlm.nih.gov/101501959 36. sachs b. on arrested cerebral development with special reference to its cortical pathology. j of nervous and mental disease, vol xiv 1887 37. sacrez r, juif jg, gigonnet jm, gruner je: la maladie de landing, ou idiote amaurotique infantile precoce avec gangliosidose generalisee, pediatrie 1967: 22: 143. pmid: 4973280. 38. samuels s, korey sr, gonatas j, terry rd, weiss m. studies in tay sachs disease. iv. membranous cytoplasmic bodies. j neuropath exp neurol 1963; 22:81–97. https://doi.org/10.1097/00005072-196301000-00005 39. sandhoff k, telephone conversations with bs kasper on 22.3.2022 (2h), 27.9.2022 (1h), 23.12.2022 (1h) 40. sandhoff k, andreae u, jatzkewitz h. deficient hexosaminidase activity in an exceptional case of tay sachs disease with additional storage of kidney globoside in visceral organs. life sciences 1968; 7: 283 288. https://doi.org/10.1016/0024-3205(68)90024-6 41. sandhoff k. variation of beta n acetylhexosaminidase pattern in tay sachs disease. febs letters 1969; 4 (4): 351 354. https://doi.org/10.1016/0014-5793(69)80274-7 42. sandhoff k, harzer k. gangliosides and gangliosidoses: principles of molecular and metabolic pathogenesis. j neurosci 2013; 33 (25): 10195 10208. https://doi.org/10.1523/jneurosci.0822-13.2013 43. sandhoff k. die amaurotische idiotie des menschen als störung des glycosphingolipoidstoffwechsels. thesis, munich university (1965) dissertation [german] 44. sandhoff k. the gm2 gangliosidoses and the elucidation of the beta hexosaminidase system. advances in genetics 2001; 44: 67 91. https://doi.org/10.1016/s0065 2660(01)44072 7 45. sandhoff k. my journey into the world of sphingolipids and sphingolipidoses. proc jpn acad ser b phys biol sci 2012; 88: 554 582. https://doi.org/10.2183/pjab.88.554 46. spielmeyer w. über familiäre amaurotische idiotien. neurol. centralblatt 1905; 24: 620 621 47. spielmeyer w. über eine besondere form von familiärer amaurotischer idiotie. neurol centralblatt 1906; 25: 51 55 48. spielmeyer w. klinische und anatomische untersuchungen über eine besondere form von familiärer amaurotischer idiotie. histol und histopathol 1908 (2): 193 251 & tafeln xxii xxiii 49. seitelberger f, vogel g, stepan h. spätinfantile amaurotische idiotie. archiv für psychiatrie und zeitschrift f. d. ges. neurologie 1957; 196: 54 190. https://doi.org/10.1007/bf00354507 important note: in this publication by seitelberger et al, according to evidence published by czech 2002 [4], brain specimen from euthanasia victims from the spiegelgrund institution were integrated [4]. 50. simonati a, williams re. neuronal ceroid lipofuscinosis: the multifaceted approach to the clinical issues, an overview. front neurol. 2022 mar 11;13: 811686. https://doi.org/10.3389/fneur.2022.811686 51. suzuki k, chen gc: brain ceramide hexosides in tay sachs disease and generalized gangliosidosis (gm1 gangliosidosis), j. lipid res. 8: 105 113, 1967. pmid: 14564716. 52. suzuki y, oshima a, nanba e. 2001. β galactosisdase deficiency (β galactosidosis): gm1 gangliosidosis and morquio b disease. in: the metabolic and molecular bases of inherited diseases, ed. cr scriver, al beaudet, ws sly, d valle, pp. 3775 3809. new york: mcgraw hill 53. svennerholm l. the chemical structure of normal human brain and tay sachs gangliosides. biochem. biophys research communications 1962; 9 (5): 436 441. https://doi.org/10.1016/0006-291x(62)90030-x 54. tay w. symmetrical changes in the region of the yellow spot in each eye of an infant. transactions of the ophthalmological society u.k 1881; 1: 55–57. https://doi.org/10.1001/archneur.1969.00480070114014 55. terry rd and korey sr membranous cytoplasmic granules in infantile amaurotic idiocy. nature 1960; 188, 1000 1002. https://doi.org/10.1038/1881000a0 56. terry rd, weiss m. studies in tay sachs disease ii. ultrastructure of the cerebrum. j neuropathol exp neurol 1963; 22: 18 55. https://doi.org/10.1097/00005072-196301000-00003 57. vogt h. über familiäre amaurotische idiotie und verwandte krankheitsbilder. monatsschrift für psychiatrie und neurologie 1905; 18: 161 171, 310 357. 58. walkley su. cellular pathology of lysosomal storage disease. brain pathology 1998; 8: 175 193. https://doi.org/10.1111/j.1750-3639.1998.tb00144.x 59. weindling p, hohendorf g, hüntelmann ac, kindel j, kinzelbach a, loewenau a, neuner s, palacz ma, zingler m, czech h. the problematic legacy of victim specimens from the nazi era: identifying the persons behind the specimens at the max planck institutes for brain research and of psychiatry. j hist neurosci. 2021 oct 18:1 22. https://doi.org/10.1080/0964704x.2021.1959185 60. zeidman la. hans jacob and brain research on hamburg “euthanasia” victims “awaiting further brains!”. neurology 2017 ;88: 1089–1094. https://doi.org/10.1212/wnl.0000000000003712 61. zeidman la. brain science under the swastika. ethical violations, resistance, and victimization of neuroscientists in nazi europe. oxford university press, oxford 2020. 62. zeman w, dyken p. neuronal ceroid lipofuscinosis (batten’s disease): relationship to amaurotic family idiocy? pediatrics 1969; 44: 570-583. pmid: 5346636. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuromuscular disease: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:5 (2022) review neuromuscular disease: 2022 update marta margeta1 1 department of pathology, university of california, san francisco, ca, usa address for correspondence: marta margeta · ucsf pathology, box 0511 · 513 parnassus ave., hsw-514 · san francisco, ca 94143 · usa marta.margeta@ucsf.edu submitted: 16 february 2022 accepted: 02 march 2022 copyedited by: henry robbert published: 04 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3805 keywords: covid-19, guillain-barré syndrome, mlip, vwa1, optic neuropathy, autophagy, peripheral neuropathy, systemic sclerosis, microvessels, idiopathic inflammatory myopathy, gene expression profiling, pompe disease abstract this review highlights ten important advances in the neuromuscular disease field that were reported in 2021. as with prior updates in this article series, the overarching topics include (i) advances in understanding of fundamental neuromuscular biology; (ii) new / emerging diseases; (iii) advances in understanding of disease etiology and pathogenesis; (iii) diagnostic advances; and (iv) therapeutic advances. within this general framework, the individual disease entities that are discussed in more detail include neuromuscular complications of covid-19 (another look at the topic first covered in the 2021 review), autosomal recessive myopathy caused by mlip mutations, autosomal recessive neuromuscular disease caused by vwa1 mutations, leber’s hereditary optic neuropathy, myopathies with autophagic defects, trna synthetase-associated charcot-marie-tooth disease, systemic sclerosis-associated myopathy, humoral immune endoneurial microvasculopathy, and late-onset pompe disease. in addition, the review highlights a few other advances (including new insights into mechanisms of muscle and nerve regeneration and the use of gene expression profiling to better characterize different subtypes of immune-mediated myopathies) that will be of significant interest for clinicians and researchers who specialize in neuromuscular disease. abbreviations ars aminoacyl-trna synthetase, ass anti-synthetase syndrome, assm anti-synthetase syndrome-associated myositis, avm autophagic vacuolar myopathy, casa chaperone-assisted selective autophagy, covid-19 coronavirus disease 2019, ck creatine kinase, cmt charcot-marie-tooth disease, dm dermatomyositis, ert enzyme replacement therapy, gaa acid α-glucosidase, gbs guillain-barré syndrome, hiem humoral immune endoneurial microvasculopathy, hmgcr 3-hydroxy-3-methylglutaryl-coa reductase, iim idiopathic inflammatory myopathy, imnm immune-mediated necrotizing myopathy, isr integrated stress response, lhon leber’s hereditary optic neuropathy, lopd late-onset pompe disease, m6p mannose-6-phosphate, mct1 monocarboxylate transporter 1, mlip muscular a-type lamin-interacting protein, mmcp minimal myositis with capillary pathology, msa myositis specific antibody, msp multiple system proteinopathy, musc muscle stem cells, mxa myxovirus (influenza virus) resistance 1, interferon-inducible protein p78, nampta nicotinamide phosphoribosyl transferase a, nmd neuromuscular disease, sibm sporadic inclusion body myositis, ssc systemic sclerosis, vwa1 von willebrand factor a containing 1 protein.   in this annual update, i will briefly describe ten neuromuscular field advances from last year that i consider to be most important and/or interesting; as in the earlier updates (margeta, 2020b, 2021), these advances will be grouped into different “discovery clusters” and listed in no particular order. advances in fundamental neuromuscular biology with implications for neuromuscular disease 1. revisiting the role of macrophages in nerve and muscle regeneration in the last year’s review (margeta, 2021), i highlighted the 2020 discovery of a crosstalk between macrophages and satellite cells in skeletal muscle repair. excitingly, two studies published in 2021 extended that work by further dissecting the molecular mechanisms by which macrophages promote regeneration not only in skeletal muscle (ratnayake et al., 2021) but also in peripheral nerves (jha et al., 2021). together with other recent work in the field of regenerative medicine, these two studies provide evidence that macrophages – which are readily detectable within necrotic muscle fibers (fig. 1a-b) and adjacent to degenerating peripheral nerve axons (fig. 1c-d) – play a major role in the pns repair, extending well beyond the long-recognized phagocytic clearance of necrotic cellular debris at injury sites. figure 1. macrophages home on degenerating muscle and nerve fibers. a representative field from an h&e-stained muscle section (a) shows muscle fibers in varying stages of necrosis and repair, starting with early coagulative necrosis, progressing through myophagocytosis, and ending with regeneration mediated by macrophage-activated satellite cells; in this case, the fiber injury was immune-mediated, but similar changes are seen in all necrotizing myopathies regardless of the underlying etiology. the cd68 stain of the same area (b) highlights macrophages within necrotic muscle fiber segments (myophagocytosis). a representative field from an h&e-stained peripheral nerve section (c) shows a marked loss of large myelinated axons and abundant digestion chambers (fragments of degenerating axons) in a case of acute axonal neuropathy. the cd68 stain of the same microscopic field (d) highlights endoneurial macrophages associated with degenerating axon fragments. the stains were performed on formalin-fixed, paraffin-embedded tissue; scale bar, 50 μm. ratnayake and colleagues used sophisticated genetic and pharmacologic approaches to delineate the macrophage-stem cell interactions that are required for muscle wound repair in both zebrafish and mice (ratnayake et al., 2021). using the zebrafish laser-ablation injury model, they showed that a subset of tissue-resident macrophages that are recruited to the wound following injury remained at the injury site, creating a “dwelling” macrophage population that had spherical morphology (in contrast to transient macrophages, which had a stellate appearance); these dwelling macrophages formed intimate connections with muscle stem cells (musc) that were reminiscent of dendritic cell-t cell immunological synapses, forming a transient pro-proliferative stem cell niche that persisted until musc underwent cell division. based on their gene expression profiles, dwelling macrophages were shown to be a heterogenous cell population; however, the authors identified a single “mature” subpopulation (defined by expression of markers associated with the anti-inflammatory macrophage phenotype, such as arginase 2 and matrix metalloproteinase-9) that mediated muscle repair by stimulating musc proliferation. to promote musc mitogenesis, these mature dwelling macrophages (which constitute ~70% of all dwelling macrophages) secreted nicotinamide phosphoribosyl transferase a (nampta), which bound to the chemokine receptor ccr5 expressed by musc; interestingly, this mitogenic effect of nampta was independent of its enzymatic activity. the zebrafish findings were replicated in mice using the volumetric muscle loss model, which simulates irrecoverable muscle injury that occurs following trauma, tumor excision, or infarction (quarta et al., 2017; sicari et al., 2012): delivery of human recombinant nampt (the human analogue of zebrafish nampta) into the excisional muscle defect using a fibrin hydrogel led to a complete restoration of muscle architecture, with an increase in the number of proliferating satellite cells and centrally nucleated, regenerating muscle fibers relative to the control condition (fibrin hydrogel without nampt). while these results are very promising from the tissue engineering standpoint, it remains to be seen whether a similar approach can be used to promote muscle fiber regeneration in muscle dystrophies and other muscle diseases that show a significant loss of muscle volume. the study by jha and colleagues (jha et al., 2021) focused on the role of macrophage metabolism in the peripheral nerve repair following mechanical injury, and their nerve regeneration findings in many ways echo the muscle regeneration findings made by ratnayake et al. using the murine sciatic nerve crush model of wallerian degeneration, jha et al. showed that the activity of monocarboxylate transporter 1 (mct1, encoded by slc16a1 gene) in macrophages, but not in dorsal root ganglion neurons, schwann cells, or perineurial cells, plays a key role in axon regeneration. selective deletion of mct1 from macrophages did not affect macrophage recruitment to the portion of the nerve distal to the site of injury; instead, it impaired the ability of recruited macrophages to phagocytose axon and myelin debris. in addition, mct1 deletion led to a decrease in the expression of macrophage transcription factor atf3, thereby attenuating macrophage reprogramming into a pro-regenerative, anti-inflammatory phenotype characterized by high expression of arginase 1 and chitinase-like 3. excitingly, the authors also showed that i.v. treatment with bone marrow-derived wt macrophages fully restored peripheral nerve regeneration in mice with macrophage-selective mct1 deletion, while macrophage-specific mct1 upregulation led to improved nerve regeneration. taken together, these findings suggest that adoptive transfer of patient-derived macrophages engineered to upregulate their mct1 activity could be used to treat severe peripheral nerve injuries. however, this study has one very significant caveat: while both anti-regenerative and pro-regenerative effects of various experimental manipulations could be demonstrated on the tissue level (by quantifying regenerating axon clusters, relative thickness of axon myelin sheaths, and the number and distribution of neuromuscular junctions in the gastrocnemius muscle), they were not accompanied by parallel measures of behavioral recovery. one possible explanation for this discrepancy is that behavioral recovery actually preceded tissue recovery in this mouse model, suggesting that alternate recovery / behavioral compensation pathways are important, at least in rodents; nonetheless, this animal model discrepancy needs to be resolved before exploring the efficacy of macrophage-based therapies for human axonal neuropathies. newly defined / emerging neuromuscular diseases 2. neuromuscular complications of covid-19: what have we learned after one more year of the pandemic? covid-19, the novel infectious disease caused by sars-cov-2, primarily targets the respiratory system but can also affect many other tissues and organs, including the pns. in the last year’s review (margeta, 2021), i summarized what was known about the neuromuscular complications of covid-19 at the end of 2020; given the preliminary nature of those investigations, i decided to revisit this topic in the current review, providing an update on what has been learned since then. one area of intense interest within the neuromuscular field has been the possible link between covid-19 and guillain-barré syndrome (gbs), an acute immune-mediated polyneuropathy characterized by ascending weakness, mild-moderate sensory abnormalities, and pain. most gbs cases are triggered by a bacterial or viral infection that precedes neurologic symptoms by 1-3 weeks; the strongest association is seen with campylobacter jejuni bacterium (~100 gbs cases / 100 000 c. jejuni infections) and with zika virus (~50 gbs cases / 100 000 zika virus infections). while small early studies from italy and spain suggested that gbs could also be triggered by a preceding sars-cov-2 infection (filosto et al., 2021; fragiel et al., 2021), no such link was found in a more comprehensive epidemiologic study performed in the uk (keddie et al., 2021) [for a more detailed discussion of the 2020 data, see the last year’s review (margeta, 2021)]. discrepancies between these initial studies prompted animated discussion among the gbs experts, but also led to additional investigations undertaken by several different groups; taken as a whole, this new evidence provides further support for a lack of a strong link between covid-19 and gbs. for example, the data originally reported by keddie at al. reflected the first uk covid-19 surge (in march and april 2020); extension of the same study until the end of 2020 showed essentially the same findings, with a large increase in the number of covid-19 cases in the fall of 2020 that was accompanied by a decrease, rather than an increase, in the number of gbs cases during the same time period [(lunn et al., 2021) and fig. 2]. moreover, as reported by the same group of investigators in a very recently published medrχiv preprint (keh et al., 2022), this disassociation between covid-19 and gbs numbers has persisted into 2021, with a clear uptick in gbs cases observed in march and april of 2021 (secondary to covid-19 vaccination; further discussed below), but no similar increase was seen in august and september of 2021, during the delta variant-associated covid-19 surge. (no data is yet available for the most recent covid-19 surge caused by the omicron variant of sars-cov-2.) a similar lack of association between covid-19 and gbs was observed by a team of investigators in singapore (umapathi et al., 2021). in contrast, a weak association between sars-cov-2 infection and gbs (1.5 excess gbs cases per 100 000 sars-cov-2-positive tests) was reported by a different group of uk investigators (patone et al., 2021); however, that study was based on the hospital coding data, which are not always entirely accurate, and included gbs cases that were diagnosed on the same day as covid-19 (suggesting a coincidental rather than causative relationship). finally, no excess gbs cases were observed by researchers of the international gbs outcome study consortium, which includes gbs experts from china and japan in addition to several european countries. while this study was not designed for systematic gbs surveillance, a significant increase in the gbs case incidence should have led to an observable increase in the study inclusion rate; instead, the inclusion rate in the first 5 months of 2020 was comparable to inclusion rates in 2017, 2018 and 2019 (luijten et al., 2021). taken together, the preponderance of data available to date suggests that a preceding sars-cov-2 infection is not a major risk factor for development of gbs; however, it remains possible that rare gbs cases could be triggered by infection with this virus. figure 2. daily uk infections with covid-19 by pcr (blue bars) and monthly cases of gbs. 2020 = red dotted line, 2019 = green dashed line, secondary y-axis. the graphs demonstrate no visible increase in gbs in the last quarter of 2020 with a rise in case numbers between 30% and 1000% more than in march/april. note significant alterations in testing occurred in the uk in april 2020 resulting in the subsequent enhanced detection of most symptomatic cases. hospital admissions were 30% higher on 1 january 2021 than in april 2020. sources: covid cases (left axis, blue bars) https://coronavirus.data.gov.uk/details/cases; nhse national immunoglobulin database courtesy mdsas, manchester, uk. [with publisher’s permission, this figure and its legend are reproduced from (lunn et al., 2021).] in contrast to a lack of definite association between gbs and covid-19, there is a clear link between gbs and the first dose of the chadox1 ncov-19 (astrazeneca) adenoviral covid-19 vaccine, with an excess risk of ~0.4-0.6 gbs cases for each 100 000 administered vaccine doses (keh et al., 2022; patone et al., 2021). interestingly, a similar excess gbs risk was seen in the us during the 1976-77 flu vaccination campaign, with 0.5-0.6 gbs cases observed for each 100 000 doses of the “swine flu” vaccine given that year; the risk attributable to later influenza vaccines has been significantly lower (~0.1 gbs cases for each 100 000 vaccines), and no increase in the gbs risk has been observed with the mrna-based covid vaccines, bnt162b2/tozinameran (pfizer) and mrna-1273 (moderna). given that all these covid-19 vaccines target the spike protein of sars-cov-2, the differences among them suggest that the excess gbs risk is attributable to the adenoviral vector rather than the sars-cov-2 spike protein antigen; supporting this hypothesis, a warning about a link between the adenovirus-based ad26.cov2.s (j&j) covid-19 vaccine and gbs was announced by the us food and drug administration in july 2021 (https://fda.gov/news-events/press-announcements/coronavirus-covid-19-update-july-13-2021), although no formal scientific study about this association has been published to date. what about covid-19-associated myopathy? although initial studies were extremely limited, they suggested that weakness and high creatine kinase (ck) levels seen in a significant subset of covid-19 patients were immune-mediated rather than due to direct viral infection of skeletal muscle [reviewed in (margeta, 2021)]. based on the two large autopsy case-control studies published since then (aschman et al., 2021; suh et al., 2021), this initial impression was essentially correct. aschman and colleagues examined deltoid and quadriceps muscles from 43 patients with covid-19 and compared them to the same muscles sampled from 11 severely ill patients negative for sars-cov-2. they found that ~60% of patients who died with severe covid-19 showed evidence of immune-mediated muscle pathology including some combination of mild-severe inflammation (mostly consisting of cd8+ t cells and macrophages), degenerating muscle fibers, and mhc-i and mhc-ii upregulation in muscle fiber sarcolemma (the latter mostly seen in chronic cases). three samples showed perifascicular upregulation of mhc-i and mhc-ii; however, no sarcoplasmic upregulation of dermatomyositis (dm) marker mxa [myxovirus (influenza virus) resistance 1, interferon-inducible protein p78; also known as mx1] was seen in any of the specimens. (interestingly, some covid-19 samples showed increased mxa expression in the endomysial capillaries, possibly reflecting a nonspecific response to systemic viral infection.) in addition, a few covid-19 samples showed evidence of small and medium-vessel angiitis. although sars-cov-2 rna was detectable by pcr in some muscle homogenates, immunohistochemistry against sars-cov-2 spike protein was negative in all cases and no viral particles were demonstrated on electron microscopy (aschman et al., 2021). very similar findings were reported by suh et al., who studied psoas muscles from 35 patients who died following covid-19 infection and 10 severely ill patients who were covid-19-negative. in addition to diffuse type 2 fiber atrophy (which was present in almost all cases and controls), these authors found evidence of immune-mediated muscle pathology in ~70% of covid-19 cases, with necrotizing myopathy in 9 patients, t celland macrophage-mediated myositis in 8 patients, and isolated diffuse or multifocal mhc-i upregulation in additional 8 patients. like aschman et al., suh et al. observed perifascicular pattern of mhc-i upregulation without concurrent sarcolemmal mxa upregulation in one covid-19 case, while mxa expression restricted to endomysial capillaries was seen in 8 covid-19 cases. immunohistochemistry against sars-cov-2 nucleocapsid protein was negative in all 35 covid-19 cases. in contrast to the findings in these autopsy studies, direct viral infection of muscle fibers was recently documented by both immunohistochemistry and electron microscopy in muscle biopsies from two of three covid-19 patients with critical illness myopathy and persistent sars-cov-2 positivity on pcr testing (dodig et al., 2022). it is currently unclear what accounts for the discrepancy between these three studies; one possibility is that the difference reflects the sample type used (muscle biopsy vs. autopsy), although that does not seem likely given (1) that sars-cov-2 viral infection can be demonstrated by both immunohistochemistry and electron microscopy in the lungs of autopsied covid-19 patients and (2) that no sars-cov-2 was definitively detected in other muscle biopsies from covid-19 patients reported to date. more likely, this difference can be attributed to the effects of a long-standing and persistent sars-cov-2 infection, which was documented in the patients described by dodig et al. but is not a common feature of covid-19. importantly, and regardless of the specific reason for the rarity of this finding, the study by dodig and coauthors demonstrates that direct sars-cov-2 infection of skeletal muscle can occur, and raises the possibility that such infection is present in the early stages of covid-19 in many patients, acting as a trigger for the subsequent immune-mediated muscle pathology that develops in some instances. 3. autosomal recessive myopathy caused by truncating mutations of mlip (muscular a-type lamin-interacting protein) unexplained persistent elevation of serum ck levels points to a chronic myopathic process that is likely genetic in origin, but identifying the specific underlying etiology can be challenging if no other symptoms and signs are present to narrow the differential diagnosis. when chronic hyperckemia is associated with exercise intolerance and exercise-induced rhabdomyolysis, the underlying problem is usually metabolic; if, on the other hand, chronic ck elevation is accompanied by muscle weakness and wasting, the patient likely has a muscular dystrophy. two interesting papers published in 2021 (lopes abath neto et al., 2021; salzer-sheelo et al., 2021) uncovered a novel autosomal recessive myopathy that is caused by loss-of-function mutations in mlip (muscular a-type lamin-interacting protein; also known as muscular lmna-interacting protein) and is clinically characterized by persistent basal hyperckemia. among the twelve affected individuals identified to date, eight developed symptoms in childhood (with symptom onset between 8 months and 7 years of age); their clinical findings included exertional myalgia, episodes of rhabdomyolysis (occasionally exercise-induced, but without clear trigger in most cases), and/or mild progressive proximal muscle weakness in addition to chronically elevated ck levels. the remaining four individuals were identified based on genetic screening of the subject cohort enrolled in the university of maryland study of old order amish; based on the evaluation of banked serum specimens (available for 3 of 4 individuals), those patients, who currently range from 24 to 67 years in age, also have chronic hyperckemia, but no other known neuromuscular symptoms. muscle biopsies were performed for 7 of 8 clinically affected patients and showed a wide spectrum of histopathologic findings, ranging from mild nonspecific features to necrotizing and – less frequently – dystrophic changes. thus, it is not yet entirely clear whether to classify this new disease as a metabolic myopathy or a muscular dystrophy; while muscular dystrophy is more likely, the final classification will require careful clinical follow-up and repeat muscle biopsies of the known symptomatic individuals (who currently range from 5-19 years of age), as well as identification of additional patients by genetic testing of individuals with otherwise unexplained chronic hyperckemia. mlip is a ubiquitously expressed, alternatively spliced protein (23-57 kda) that directly interacts with lamins a/c (two intermediate filaments, both derived from the lmna gene, that are the main constituents of the nuclear envelope); its expression is highest in the heart, skeletal muscle, and brain, but its function remains poorly understood (ahmady et al., 2011). in a murine model, mlip deletion in the heart led to metabolic abnormalities, abnormal adaptation to stress, and accelerated progression from compensated cardiac hypertrophy to decompensated heart failure (cattin et al., 2015; huang et al., 2015). in contrast, mlip deletion in murine skeletal muscle led to increased central nucleation but no obvious neuromuscular phenotype (liu et al., 2020). in humans, variants in the mlip genetic locus [a single coding missense variant (nm_138569, c.475g>a, p.val159ile) and several noncoding variants] have been shown to increase the risk of dilated cardiomyopathy (esslinger et al., 2017); however, prior to studies by lopes abath neto et al. and salzer-sheelo et al., it was not known whether mlip mutations can directly cause human disease. twelve affected individuals from 8 unrelated families that are described in these two studies all carry either homozygous or heterozygous biallelic truncating mutations of mlip gene, with mutation hotspots in exons 4 and exons 9; while the molecular consequences of these mutations still need to be fully elucidated, initial work suggests that they lead to nonsense-mediated mrna decay and presumed loss of protein expression [although the latter is likely specific for individual splice variants and has therefore been difficult to establish (lopes abath neto et al., 2021)]. interestingly, 1 of 8 affected children and all 4 affected adults with biallelic mlip mutations showed evidence of mild cardiac involvement, raising the possibility that mlip deficiency causes cardiac abnormalities in humans as well as mice; this is particularly intriguing given the broad spectrum of human disease associated with lmna mutations, which includes cardiomyopathy in addition to muscular dystrophy, peripheral neuropathy, lipodystrophy, and premature aging (ho and hegele, 2019). future work will be required to fully define the nature and scope of skeletal and cardiac muscle phenotypes caused by mlip deficiency and to elucidate whether mlip interaction with lamins a/c plays an important role in the pathogenesis of this new human disease. 4. autosomal recessive neuromuscular disease caused by mutations in vwa1 (von willebrand factor a containing 1 protein) in the past, a disease was typically defined based on its unique clinicopathologic features, with the underlying etiology and pathogenesis established later. in the era of comprehensive genetic testing, this traditional sequence is being inverted: new genetic diseases are identified and defined based on their etiology, and future work is required to fully define their clinicopathologic features. as with the mlip-associated recessive myopathy described in discovery #2, two different research groups (deschauer et al., 2021; pagnamenta et al., 2021) used a genetic approach to identify a novel autosomal recessive neuromyopathy caused by mutations in vwa1 (von willebrand factor a containing 1 protein), also known as warp (von willebrand factor a domain-related protein). deschauer et al. started their study in a more “conventional” manner, by performing exome sequencing of 10 individuals with presumed genetic neuromuscular disease but without genetic diagnosis; 2 of these 10 patients turned out to carry biallelic truncating mutations in vwa1, a gene not previously associated with human disease but with high expression in peripheral nerve and skeletal muscle tissues. follow-up analyses of the patients’ families and additional “unsolved” patient cohorts identified 13 additional individuals with biallelic vwa1 mutations; 5 of 6 identified mutations are predicted to result in protein truncation, and all 6 are predicted to result in a loss of protein function. pagnamanta et al. used a slightly different approach: they identified vwa1 as a potentially interesting gene based on its high expression in the pns and searched the data from the 100k genome project (turnbull et al., 2018) for individuals carrying biallelic vwa1 variants. this analysis ultimately yielded 10 unrelated patients that fit the inclusion criteria, with 7 additional individuals identified through follow-up studies of other patient cohorts. interestingly, 15 of these 17 patients (from 14 of 15 separate families) carry the same truncating vwa1 mutation (10-bp insertion, nm_022834, c.62_71dup; pgly25argfster74) in either homozygous or heterozygous state; this mutation was also identified in 3 of 6 families described by deschauer et al., and is estimated by pagnamanta et al. to have arisen in europe ~8000 years ago. interestingly, the same 10-bp tandem repeat is deleted (rather than duplicated) in other affected individuals, suggesting that it is prone to de novo alterations. what is the clinicopathologic phenotype of the vwa1-associated neuromuscular disease? the affected individuals typically develop slowly progressive non-length dependent axonal motor neuropathy that presents with distal weakness and foot deformities, often in early childhood; however, proximal weakness with scapular winging and mildly elevated ck level is sometimes also present, and it is not entirely clear whether this myopathic involvement represents a separate phenotype of vwa1 deficiency, or whether myopathic changes are secondary to the primary neuropathic process. muscle biopsies were evaluated for a subset of cases (7 of 32 patients across both studies had biopsies that were available for review), showing a similarly complex picture: well-developed chronic neurogenic changes were accompanied by some myopathic features (including fiber size variation, internal nucleation, endomysial fibrosis, fiber lobulation and/or whirling, and mitochondrial alterations) but no basement membrane abnormalities. vwa1 is a 445 aa extracellular matrix protein that contains the n-terminal von willebrand factor a domain followed by two fibronectin type iii repeats; it is expressed in the cartilage and in the basement membranes of peripheral nerves (endoneurium), skeletal muscle (endomysium), and the cns vasculature, where it interacts with collagen vi and perlecan (two proteins that are also associated with genetic nmd) (allen et al., 2009). vwa1-null mice show a delayed response to painful stimuli and impairment of fine motor coordination; histologically, their peripheral nerves show aberrant fusion of schwann cell basement membranes, while their skeletal muscles and other vwa1-expressing tissues show no obvious abnormalities (allen et al., 2009). knockdown of zebrafish vwa1 gene (zebrafish vwa1 analogue) leads to dose-dependent abnormalities of motor neuron development, with reduced axon branching of primary motor neurons, axon truncation of secondary motor neurons, and reduced number of neuromuscular junctions, as well as some muscle fiber disorganization (pagnamenta et al., 2021). taking together the human and animal model data, it is clear that vwa1-deficiency affects motor neuron axons and leads to neuropathic changes in the affected musculature; however, it remains to be established whether these abnormalities represent a developmental or degenerative disease, and whether there is a separate but concurrent myopathic phenotype. in addition, it needs to be elucidated how the subtle basement membrane abnormalities lead to these neuromuscular phenotypes. advances in understanding of etiology and pathogenesis of neuromuscular diseases 5. impaired repair of mitochondrial respiratory chain complex 1 causes autosomal recessive variant of leber’s hereditary optic neuropathy leber’s hereditary optic neuropathy (lhon), first defined as a clinical entity in 1871, is a degenerative disease of retinal ganglion cells and their axons that presents with subacute bilateral painless loss of central vision. most lhon cases are maternally inherited; three point mutations in mitochondrial dna (mtdna), each of which alters the coding sequence of a different subunit of mitochondrial respiratory chain complex i, account for ~90% of the maternally inherited cases, with other mtdna mutations accounting for additional ~5%. however, approximately 5% of lhon patients carries no pathogenic mtdna mutations; in their groundbreaking work published last year, stenton et al. showed that many of these previously unresolved lhon cases carry biallelic missense mutations in the gene encoding dnajc30 chaperone (stenton et al., 2021). among 33 individuals with dnajc30-mutated, autosomal recessive form of lhon (arlhon) characterized by stenton and colleagues, 29 carry the same missense mutation (nm-032317, c152a>g, ptyr51cys; estimated to have arisen in eastern europe ~85 generations / ~2100 years ago); 2 additional missense dnajc30 mutations were identified in the remaining 4 patients. clinically, arlhon cases are indistinguishable from mtlhon cases, although arlhon has a significantly earlier age of onset (19.9+/-7.9 vs. 30.7+/-15.0 years). like mtlhon, arlhon is characterized by incomplete disease penetrance, with some homozygous mutation carriers not expressing the clinical phenotype. interestingly, mutation penetrance is sex-dependent in both mitochondrial and autosomal recessive forms of lhon, with disease prevalence 5-10 times higher in male compared to female mutation carriers; both incomplete penetrance and male predominance are unusual for an autosomal recessive disease, and the mechanism(s) underlying these phenomena are yet to be elucidated. importantly, arlhon patients have a better response to treatment with idebenone [a coenzyme q10 analogue that bypasses complex i and is currently approved for lhon treatment in europe (amore et al., 2021)] than mtlhon patients; this difference in the treatment response highlights the clinical significance of early molecular diagnosis of arlhon cases. notably, these genetic and clinical findings were replicated by another very recent study (kieninger et al., 2022), which also identified two additional dnajc30 pathogenic variants. how do dnajc30 mutations cause lhon? all three initially identified variants are located in the j domain, a conserved portion of dnajc30 with homology to the heat shock family of proteins. muscle biopsies from arlhon patients showed a small but significant defect in complex i activity (but no change in complex iv and complex v activities), and this complex i-specific defect was recapitulated in patient-derived fibroblast cell lines and dnajc30-knockout hek293 cells. interestingly, the functional defect of complex i was accompanied by a small but consistent increase (rather than decrease) in the expression level of complex i subunits, with no corresponding increase in the level of mrnas that encode these subunits. the increase in the complex i subunit expression level was accompanied by a decrease in the turnover of subunits that comprise the n-module, which normally have the highest turnover rate due to a high degree of oxidative damage (szczepanowska et al., 2020). taken together, these data indicate that dnajc30 maintains structure and function of complex i by facilitating exchange of oxidatively damaged subunits comprising its n-module; impairment of this repair mechanism leads to accumulation of damaged type i complexes, functional complex i defect, and lhon clinical phenotype. interestingly, dnajc30 was also shown to interact with several subunits of complex v / atp-synthase (tebbenkamp et al., 2018); however, clinical significance of that finding is unclear given that muscle biopsies from patients with dnajc30 mutations show no changes in complex v abundance and no functional complex v deficits. it is worth noting that complex i dysfunction is thought to play an important role in the pathogenesis of parkinson disease, cancer, and diabetes; a long-term clinical follow up of arlhon patients will be necessary to determine whether they are at increased risk for these common age-associated disorders. 6. autophagy defects in skeletal myopathies: the etiologic spectrum widens degradative macroautophagy (hereafter referred to as autophagy) is a conserved catabolic pathway required for cellular adaptation to nutrient deprivation and for degradation of damaged intracellular organelles and insoluble protein aggregates. autophagy is a highly regulated cellular process that involves multiple steps (starting with autophagy induction and ending with degradation of sequestered cargo by lysosomal hydrolases), disruption of each can lead to human disease [reviewed in detail in (margeta, 2020a)]. from the muscle pathology perspective, the most recognizable autophagy defect involves inhibition of the bulk autophagic flux, which results in the autophagic vacuolar myopathy (avm) phenotype; however, defects of autophagy induction and cargo sequestration can also result in muscle disease (fig. 3). several new diseases with autophagic impairment were identified in 2021, expanding the spectrum of etiologies that cause skeletal myopathy through dysregulation of autophagy; interestingly, however, all of them map on one of the four previously identified pathogenetic pathways illustrated in fig. 3. figure 3. the role of autophagic defects in the pathogenesis of skeletal myopathies. in myopathies with autophagy induction defects, the accumulation of damaged cellular organelles activates signaling cascades that contribute to fiber atrophy and apoptosis; however, there is no accumulation of autophagosomes. in contrast, autophagic vacuolar myopathies are caused by defects in the general autophagic flux that lead to a massive accumulation of autophagosomes and undigested cargo, ultimately resulting in a reduction of the mechanical force generated during muscle contraction (i.e., muscle weakness) and a failure of muscle regeneration. finally, cargo recognition defects lead to two different, defect-specific disease phenotypes: a granulophagy defect results in rimmed vacuole formation and inclusion body myopathy, while a casa defect causes myofibrillar myopathy by impairing z-disc stability. the electron micrographs illustrate (a) subsarcolemmal accumulation of damaged mitochondria that show marked variation in their size and shape; (b) an autophagic vacuole with an accumulation of undigested, electron-dense autophagic cargo; (c) a rimmed vacuole with a rim of autophagic material and a center that consists of aggregated proteins; and (d) myofibrillar disorganization with streaming of the z-disc material. [this figure and its legend are reproduced from (margeta, 2020a) with permission from the annual review of pathology: mechanisms of disease, volume 15 © 2020 by annual reviews, http://www.annualreviews.org/.] when there is a block of autophagy induction, autophagosomes do not normally form and therefore cannot significantly accumulate; however, there is accumulation of damaged and degenerating intracellular organelles, which ultimately leads to fiber atrophy and apoptosis. the autophagy induction defect plays an important role in the pathogenesis of at least some muscular dystrophies and congenital myopathies; however, the complete block of autophagy induction is embryonically or perinatally lethal in animal models, and no human disease has previously been attributed to mutations in one of the core autophagy genes (margeta, 2020a). that changed in 2021, when collier at al. identified five independent families with autosomal recessive mutations in atg7 (a core autophagy-related protein that is required for lipidation of lc3, the key step in induction of degradative autophagy); the affected individuals have a complex developmental disorder that commonly includes cerebellar hypoplasia, corpus callosum abnormalities, facial dysmorphism, and mild proximal myopathy (collier et al., 2021). muscle biopsies were performed on a few affected individuals and showed mild myopathic changes mimicking those seen in the autophagy-deficient murine muscle, with moderate mitochondrial abnormalities, lipofuscin accumulation, subsarcolemmal accumulation of autophagic cargo receptor p62/sqstm1 (encoded by sqstm1 gene), and diminished levels of lc3-ii (the lipidated, autophagosome-bound form of lc3); while some autophagosomes were present, there was no accumulation of autophagic vacuoles. intriguingly, another potential myopathy with impaired autophagy induction was reported last year by napolitano and co-authors (napolitano et al., 2021): while these authors classified their patient 6 as having an avm, his muscle biopsy showed high p62 and low lc3-ii (as would be expected with the autophagy initiation defect) rather than high levels of both p62 and lc3-ii (as would be expected in a true avm caused by the autophagic flux defect). interestingly, that individual was found to carry a potentially deleterious variant in tbc1d5 gene, which has not previously been associated with human disease but was shown to regulate autophagy initiation (popovic and dikic, 2014). specific classification of tbc1d5-associated myopathy aside, napolitano et al. showed that the avm pathology mimicking late-onset pompe disease (lopd) can be associated with mutations in genes not previously associated with this clinicopathologic phenotype (such as titin, dysferlin, plectin, and perilipin 3, among others); this highlights the importance of genetic diagnosis for lopd, which can be treated by enzyme replacement therapy (ert; further discussed in advance #10) – a treatment that is unlikely to be effective if the underlying molecular defect is not acid α-glucosidase (gaa) deficiency. cargo recognition defects, which result in selective (rather than global) autophagy dysfunction, can also cause muscle disease: impairment of granulophagy (autophagy of stress granules, which are ribonucleoprotein foci that form under cellular stress and, if persistent, need to be degraded via autophagy) leads to an inclusion body myopathy phenotype, while impairment of chaperone-assisted selective autophagy (casa), which is critical for the z-disc maintenance, results in myofibrillar myopathy (fig. 3). each of these phenotypes has been associated with mutations in many different genes; in 2021, two new genes were added to this ever-growing list. in the first study, leoni et al. showed that autosomal dominant missense variants in annexin a11, a 505 aa calcium-dependent phospholipid-binding protein, lead to hereditary inclusion body myopathy, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration (leoni et al., 2021); given that other mutations in anxa11 (annexin a11-encoding gene) were previously shown to cause amyotrophic lateral sclerosis and frontotemporal degeneration (zhang et al., 2018), anxa11 is now the sixth gene known to cause multiple system proteinopathy (msp type 6). the other 5 genes associated with the msp phenotype encode either rna-binding proteins that form stress granules (hnrnpa2b1, hnrnpa1, and matr3 genes) or autophagy-associated regulatory proteins (vcp and sqstm1 genes); while the precise function of annexin 11 still needs to be elucidated, it most likely falls in the latter category given that other annexin a proteins play a role in autophagy regulation (xi et al., 2020). interestingly, rimmed vacuoles in msp-6 patients are positive for annexin 11 in addition to tdp-43, while rimmed vacuoles in sporadic inclusion body myositis (sibm) and other inclusion body myopathies are annexin 11-negative (leoni et al., 2021). in the second study, johari and colleagues showed that autosomal dominant missense mutations in smpx (small muscle protein x-linked) cause distal myopathy with protein inclusions (johari et al., 2021). smpx is a small (88 aa), proline-rich protein that is highly expressed in slow muscle fibers and cardiomyocytes, where it localizes to costameres and intermyofibrillar spaces; its function is currently unknown, but loss-of-function smpx mutations cause non-syndromic hearing loss without muscle weakness. muscle biopsies from patients with smpx-related distal myopathy, who do not have hearing loss, showed lc3-positive rimmed vacuoles, abundant smpx-and p62-positive sarcoplasmic inclusions, as well as smpx-negative inclusions that were positive for αb-crystallin and myotilin in addition to casa chaperones bag3 and hspb8; however, there was no significant myofibrillar disarray on electron microscopy. thus, it is not yet clear whether this new disease should be classified as a rimmed vacuolar (inclusion body) myopathy or as a myofibrillar myopathy; while a lot of additional work is needed to fully elucidate its pathogenesis, initial experiments suggest that smpx may affect stress granule dynamics (johari et al., 2021). thus, it will be interesting to see whether smpx mutations can also cause other diseases in the msp spectrum, either alone or in combination with mutations in other msp-associated genes. 7. activation of the integrated stress response plays a key role in the pathogenesis of trna synthetase-associated subtypes of axonal charcot-marie-tooth disease non-syndromic inherited sensory-motor peripheral neuropathies, also known as charcot-marie-tooth (cmt) diseases, are broadly classified into demyelinating, axonal, and intermediate subtypes, each of which is associated with a large number of causative genes and with more than one mode of inheritance (stavrou et al., 2021; zhang et al., 2021). within the axonal cmt category, an interesting and relatively recently recognized disease subset is caused by mutations in aminoacyl-trna synthetases (arss), which are ubiquitously expressed enzymes that charge amino acids onto their cognate trnas during protein synthesis. of 20 arss, 5 have been associated with autosomal dominant forms of axonal cmt disease [yars (tyrosyl-rs; cmtdic), gars (glycyl-rs; cmt2d), aars (alanyl-rs; cmt2n), hars (histidyl-rs; cmt2w), and mars (methionyl-rs; cmt2u)], while two (wars / tryptophanyl-rs and kars / lysyl-rs) have been associated with hereditary motor neuropathy type 9 (hmn9) and autosomal recessive intermediate cmt disease cmtrib, respectively. (of note, gars mutations can cause hmn5a in addition to cmt2d, while hars mutations can cause hmn2 in addition to cmt2w.) not much was known about pathogenesis of ars-associated forms of cmt until last year, when two fascinating studies were published back-to-back in science, together providing important mechanistic insights and therapeutic promise for this subcategory of inherited neuropathies (spaulding et al., 2021; zuko et al., 2021). spaulding et al. used three different mouse models of gars-cmt (cmt2d) and one mouse model of yars-cmt (cmtdic) to investigate molecular pathways that are affected by mutant gene expression (spaulding et al., 2021). in all four investigated mouse models, they found that the integrated stress response (isr) was selectively activated in alpha motor neurons and a subset of sensory neurons; the isr activation preceded neurologic symptom onset, was associated with increased expression of genes regulated by transcription factor atf4, and required sensor kinase gcn2. [the isr is a conserved cellular signaling pathway that alters gene expression program in response to various cellular stressors, including protein homeostasis defects, nutrient deprivation, viral infection, and oxidative stress (costa-mattioli and walter, 2020). these stresses are sensed by four specialized kinases (perk, gcn2, pkr and hri), activation of which ultimately leads to phosphorylation of the eukaryotic translation initiation factor eif2 and general reduction in protein synthesis along with an increase in translation of specific mrnas, such as atf4. if these adaptive responses fail to mitigate cellular stress, the isr triggers programmed cell death.] genetic deletion of gcn2 completely rescued the neuropathy phenotype in the gars mutant mice, with beneficial effects of deletion observed behaviorally, physiologically (as improved sciatic nerve conduction velocity), and pathologically (as ameliorated axon loss, increased axon diameter, and decreased muscle denervation). while only one of three gars-cmt mouse models was tested in these genetic studies, similar results were observed in a different gars-cmt model following pharmacologic inhibition of gcn2, with treatment that was initiated at disease onset (2 weeks of age); interestingly, the effect of pharmacologic gcn2 inhibition was greater in male than female mice, but the reason for this sex difference is currently unclear. in a parallel study, zuko et al. investigated mechanisms underlying inhibition of global protein synthesis that was previously documented in 6 different gars-cmt and yars-cmt mouse lines and could not be rescued by overexpression of wt gars or yars. while manipulation of translation initiation and upstream regulatory pathways had no significant effect, overexpression of trnagly rescued both the protein synthesis deficit and neuropathy phenotype in two drosophila models of gars-cmt. these findings were replicated in two mouse models of gars-cmt, with trnagly overexpression resulting in an essentially complete behavioral, physiologic, and pathologic rescue of peripheral neuropathy (zuko et al., 2021). in addition, zuko and colleagues showed that inhibition of global protein synthesis in this mouse model was due to trnagly sequestration by mutant gars, which led to ribosome stalling at gly codons; as a result, the neuropathy in gars-cmt mice was worsened by concurrent deletion of the ribosome rescue factor gtpbp2, which by itself does not lead to nerve damage. finally, connecting the dots between the two studies, zuko et al. showed that trnagly overexpression abrogated activation of the isr in the gars-cmt mouse model, indicating that depletion of the normal trnagly pool and consequent ribosome stalling are the upstream signals that lead to the isr activation. taken together, these two studies suggest that ars mutations cause autosomal dominant axonal cmt disease through a toxic gain-of-function mechanism: by binding more tightly to their cognate trnas, they deplete the free trna pool, ultimately leading to ribosome stalling, aberrant isr activation, and axonal degeneration. despite the very comprehensive nature of this work, additional questions need to be answered. for example, it remains to be shown whether the same (or similar) molecular mechanism plays a role in the pathogenesis of the aars-, hars-, and mars-associated cmt, and in axonal cmt more broadly. in addition, it is not clear which downstream effect of the isr activation (protein synthesis inhibition or the atf4 pathway activation) leads to axonal degeneration. nonetheless, and very excitingly, this work opens a new direction for the cmt treatment development: manipulation of the isr is emerging as a promising therapeutic avenue for several age-associated diseases, including neurodegeneration and cancer (costa-mattioli and walter, 2020), and at least some subtypes of axonal cmt can now be added to that list. [as an aside, ars enzymes also play an important role in anti-synthetase syndrome (ass), where they are targeted by disease-causing autoantibodies (histidyl-rs is targeted by anti-jo-1 antibodies, alanyl-rs by anti-pl12 antibodies, glycyl-rs by anti-ej antibodies, lysyl-rs by anti-sc antibodies, and tyrosyl-rs by anti-yrs antibodies; witt et al., 2016). ass and cmt disease have entirely different clinical manifestations, so the underlying pathogenetic mechanisms are likely also different despite this common link; while there is some evidence that protein translation is affected in ass – see advance #9 – it remains to be established whether the isr activation and/or protein synthesis inhibition play a role in the ass pathogenesis.] advances in neuromuscular disease diagnostics 8. microvascular pathology in immune-mediated neuromuscular disease major vascular pathology (such as vasculitis or amyloid vasculopathy) has long been recognized as an important cause of neuromuscular disease, and vessel evaluation on routine stains is a standard component of diagnostic neuromuscular pathology workup. the role of microvascular pathology is less well understood, but some microvascular abnormalities [such as deposition of c5b9 (complement membrane attack complex) in the endomysial capillaries] are included in the diagnostic criteria for several idiopathic inflammatory myopathies (iims) including dm, anti-synthetase syndrome-associated myositis (assm), and immune-mediated necrotizing myopathy (imnm). two studies published last year (siegert et al., 2021; trikamji and pestronk, 2021) expanded the spectrum of microvascular abnormalities seen in immune-mediated neuromuscular disorders, highlighting that careful evaluation of endomysial and endoneurial microvessels (arterioles and capillaries) can increase the diagnostic yield of muscle and nerve biopsies. systemic sclerosis (ssc) is a systemic autoimmune disease characterized by fibrosis of skin and internal organs, vasculopathy, and evidence of immune system dysregulation; skeletal muscle involvement is variable, with some patients developing a superimposed specific iim or less specific “overlap myositis”, while others (~20%) have a poorly understood muscle disease specific to ssc. to better define the latter disease entity, siegert et al. performed detailed retrospective evaluation of muscle biopsies from 18 ssc patients with muscle symptoms (derived from a larger cohort of 367 ssc patients) using routine light microscopy as well as semi-quantitative large scale electron microscopy [described as advance #9 in last year’s review (margeta, 2021)]. two thirds of evaluated ssc biopsies (12 of 18) showed a novel morphologic pattern (which the authors termed “minimal myositis with capillary pathology”; mmcp) characterized by sparse endomysial t cells, rare necrotic and regenerating muscle fibers, diffuse mhc-i and focal mhc-ii upregulation, and enlarged capillaries with prominent pericytes but no capillary c5b9 deposition; ultrastructurally, mmcp cases showed well-developed but mild capillary pathology, including thickening and reduplication of basement membranes, reactive endothelial changes, and capillary ensheathment by pericyte processes (fig. 4). similar but more severe capillary alterations were seen in other 6 ssc muscle biopsies, which also showed additional capillary abnormalities (such as tubuloreticular inclusions) and more prominent inflammatory changes; therefore, capillary pathology is a uniform feature of mmcp cases but is not pathognomonic for this condition, and must be interpreted in the context of other muscle biopsy findings. interestingly, mmcp ssc cases were clinically less severe than non-mmcp ssc cases, with higher frequency of localized (versus diffuse) skin sclerosis, less prominent internal organ involvement, and lower ck levels; however, these muscle abnormalities developed early in the disease course, with mean disease duration of 3 years at the time of biopsy (<1 year in 6 of 12 cases). figure 4. ultrastructural characterization of skeletal muscle biopsies with histological minimal myositis with capillary pathology (mmcp) phenotype. entire ultrathin sections were recorded by large-scale digitization at 7.3 nm pixel size; digitally magnified regions of interests (roi) of patients 3 (a, d, g–l), 8 (b, m), 6 (e), 2 (f), 18 (n; severe necrotizing myositis with capillary pathology and fibrosis), 1 (o) and non-diseased control (c). a markedly thickened capillaries can be clearly identified at low magnification (arrows). b capillary with pronounced thickening and reduplication (arrow) of the basement membrane (bm) as well as ensheathment by pericyte processes (white asterisk), mildly activated endothelium (black asterisk), lumen (lu), adjacent muscle fiber (mf). c healthy capillary with thin bm (arrow): note that no reduplication is apparent, endothelium with no signs of activation (asterisk), lumen with erythrocyte (er). different types of bm (bm) thickening were detected (d–f): distinct reduplication (d; arrows), endothelium (asterisk), fibroblast (fi); fuzzy appearance with less pronounced reduplication (e): note that the bm (arrow) directly underneath the endothelium (asterisk) is clearly visible, fibrous long spacing collagen (arrowhead); homogeneous thickened bm (f), capillary lumen (lu), neighboring muscle fiber (mf) with basal lamina (white arrow). different types of endothelial activation (g–i): increased size and number of endothelial cells (g; asterisk, lu lumen), note the granular appearance of the cytoplasm, probably mostly due to ribosomes; prominent endothelial membrane organelles (h; black asterisk, er erythrocyte in lumen), note the prominent and mostly small pericyte processes demonstrating mild ensheathment (white asterisks); degraded capillary (i), showing some remaining membrane structures, probably of endothelial or pericyte origin (arrowheads), almost empty bm “sack” with mild reduplication (arrows). [this figure and its legend were adopted from figure 3 in (siegert et al., 2021); this use is permitted under the creative commons attribution 4.0 international license.] while at least some aspects of microvascular pathology are routinely evaluated in muscle biopsies, the same is not true for nerve biopsies; the 2021 study by trikamji and pestronk suggests that this approach to nerve biopsy processing should probably change. these authors have retrospectively analyzed a cohort of 16 patients with a motor-sensory axonal polyneuropathy and complement deposition in endoneurial microvessels, detected by c5b9 immunohistochemistry performed on nerve cryosections. this patient cohort showed unique clinicopathologic features that could reflect a new / previously unrecognized autoimmune neuropathy, which the authors termed “humoral immune endoneurial microvasculopathy” (hiem). clinically, hiem patients showed progressive asymmetric distal weakness that was more prominent in lower extremities; there was no evidence of a systemic inflammatory process. pathologically, there was a patchy / asymmetric axon loss that preferentially affected myelinated axons, with rare digestion chambers and rare regenerating axon clusters but no significant inflammation; strong c5b9 staining was seen surrounding the endothelium in all or nearly all endoneurial microvessels, and was also detectable in endomysial capillaries in the majority of paired muscle biopsies (trikamji and pestronk, 2021). a potentially confounding factor is that similar capillary changes were previously documented in nerve and muscle biopsies from diabetic patients (yell et al., 2018); 56% of the patients in the hiem cohort also had diabetes, and the mean age of onset was lower in diabetic than non-diabetic patients. however, and very compellingly, 15 of 16 hiem patients showed a measurable strength improvement following a short course of corticosteroid therapy; this would not be expected if the observed nerve pathology was mainly due to chronic (and likely irreversible) diabetic neuropathy. while very intriguing, these findings need to be replicated in other patient cohorts, with corticosteroid treatment effectiveness confirmed by double-blind clinical trials. in addition, both nerve and muscle biopsies from hiem patients should be evaluated by electron microscopy: it is possible that ultrastructural evaluation will yield additional useful clues, either from pathogenetic or diagnostic perspective. nonetheless, trikamji and pestronk’s findings suggest that a portion of each nerve biopsy should be frozen for specialized immunohistochemical studies, which is currently not a standard operating protocol in the majority of neuromuscular pathology laboratories: in addition to enabling identification of potentially treatable hiem patients, such processing would also facilitate detection of immunoglobulin deposits that are important for accurate classification of amyloid and vasculitic neuropathies. 9. gene expression profiling in idiopathicinflammatory myopathies based on the current clinicoseropathologic criteria, iims are divided into four distinct subcategories [dm, assm, imnm, and sibm; for a brief overview of the diagnostic criteria and other recent advances in the iim field, see the 2020 update in this article series (margeta, 2020b)]. an interesting study published last year (amici et al., 2021) has used a gene expression profiling approach to take a deeper look into these disorders; while the current data is mainly interesting from the standpoint of disease pathogenesis, this study also provides a glimpse into the use of transcriptomic profiles for disease diagnostics. the authors used rna-sequencing data from 119 iim muscle biopsies [39 dm cases, 18 assm cases, 49 imnm cases, and 13 sibm cases; all assm cases were positive for anti-jo-1 antibodies, while the dm and imnm groups were heterogenous with respect to the patient’s serologic status, with 5-40 cases per myositis-specific antibody (msa) subgroup] and 20 normal muscle biopsies to generate transcriptome maps and identify gene expression modules that are dynamically regulated in iims. some of the results were not surprising; for example, dm biopsies showed activation of the type 1 interferon-inducible gene module, which was not significantly upregulated in other iim subtypes, while sibm cases were unique in showing consistent overexpression of t cell genes. other findings, however, were novel and somewhat surprising. for example, two gene modules – the stress response acute phase response module and the neutrophil degranulation module – were upregulated across all iim subtypes (including imnm cases, which generally lack significant inflammation), as was the cytoskeleton remodeling module, which includes tgf-β and could be related to endomysial fibrosis seen in many long-standing iim cases; in contrast, the large muscle function, redox metabolism, and ubiquitination gene module was downregulated across all iim subtypes. expression changes in some gene modules were more specific and characteristic for individual iim subtypes: for example, the previously mentioned upregulation of the type 1 interferon-induced gene module in dm cases was strongly correlated with downregulation of the titin transcript, while the assm cases showed upregulation of the rna binding, splicing, and translation genes. in addition to studying gene expression changes across different iim subtypes, the authors of this study converted individual trascriptomic profiles into images suitable for computational analysis by a convolutional neural network. unsupervised hierarchical clustering by neural network-derived features led to an interesting hierarchy of different patient subgroups, with the severe dm cases clustering closer to the assm cases and severe imnm cases than to the mild dm and imnm cases; one severe anti-mi-2 dm case was even misclassified within the assm group. this finding is congruent with the experience on a diagnostic muscle pathology service, where one can occasionally see a significant histopathologic overlap between anti-mi-2 dm, assm, and severe imnm (fig. 5). interestingly, in the group of 119 iim cases studied by amici at al., disease severity did not correlate with a specific msa subtype; instead, both mild and severe cases were seen in each msa-defined subgroup. if validated by future studies and found to correlate with the response to therapy and/or patient outcomes, this finding suggests that transcriptomic analysis might be able to extract clinically useful information not captured by serotype / disease subtype, and that gene expression profiling therefore may at some point become a routine component of diagnostic muscle biopsy work-up. excitingly, the authors of this study have shared the source code and model weights on their website (https://github.com/mendillolab), and this tool can be used by other researchers who perform muscle transcriptomics to further test and improve their algorithm. figure 5. histologic overlap between assm (a-c), anti-mi-2 dm (d-f), and a severe case of anti-hmgcr imnm (g-i). a typical assm case shows perifascicular fiber atrophy and necrosis (a) along with marked perimysial abnormalities [edema, fragmentation, macrophage-rich inflammation (best visualized on acid phosphatase stain; b) and a linear pattern of alkaline phosphatase positivity (c)]. anti-mi-2-associated dm often leads to a more severe muscle involvement than other dm subtypes and can histologically mimic assm; in this example (d-f), there was a prominent perimysial involvement including macrophage-rich inflammation (e) and strong alkaline phosphatase staining (f). markers of type 1 interferon response (such as mxa) can help differentiate dm from assm, but in the ucsf experience mxa upregulation is often weak in anti-mi-2 dm cases. typical imnm cases show little to no inflammation and are generally easily distinguished from other autoimmune myopathies; however, severe cases (such as the one shown in g-i) can be diagnostically challenging. this anti-hmgcr imnm case showed abundant macrophage-rich inflammatory infiltrate in the perimysium (h) along with well-developed perimysial alkaline phosphatase positivity (i); the only clue to the correct diagnosis was dense punctate p62 staining of muscle fibers (inset in g), which is not typically seen in either dm or assm. representative cryosection images are shown for all three cases; scale bars, 100 μm. advances in neuromuscular disease treatment 10. clinical trials: late-onset pompe disease revisited pompe disease is an autosomal recessive metabolic disorder caused by loss-of-function mutations in gaa, the lysosomal glycogen-catabolizing enzyme; depending on the level of residual gaa activity, the disease can present in infancy, childhood, or adulthood. while early onset pompe disease is a systemic disorder that affects multiple organs systems, lopd primarily affects skeletal musculature and is characterized by progressive weakness of the axial, limb-girdle, and respiratory muscles; pathologically, vacuolated muscle fibers show an accumulation of membrane-bound as well as free glycogen and a build-up of autophagic debris (fig. 6), so this disease is currently classified as an avm (fig. 6). ert with alglucosidase alfa, recombinant form of human gaa, was approved in 2006, constituting a breakthrough in pompe disease management; however, most lopd patients on this therapy experience a short-term improvement followed by progressive decline. the failure of ert in lopd is thought to primarily reflect inefficient delivery of alglucosidase alfa to skeletal muscle lysosomes: efficient lysosomal targeting is based on binding of mannose 6-phosphate (m6p)-glycans and bis-m6p-glycans within the recombinant protein to the cation-independent m6p receptor in the target tissue, and only a minor fraction of originally developed recombinant gaa contains bis-m6p ligands. figure 6. muscle biopsy findings in lopd. a representative h&e-stained cryosection (a) shows largely unremarkable skeletal muscle with a single highly vacuolated muscle fiber in this microscopic field (black arrowhead). the same microscopic field from a pas (periodic acid-schiff)-stained cryosection (b) shows two vacuolated muscle fibers with mild glycogen accumulation (black arrowheads); the less vacuolated fiber at the bottom of the image shows no obvious changes on the corresponding h&e-stained section (a). electron microscopy shows an increase in the lysosomal (membrane-bound) glycogen (white arrowheads in c) as well as accumulation of free sarcoplasmic glycogen accompanied by electron dense autophagic debris (d). the focal nature of these pathologic findings is typical for lopd, although the degree of muscle fiber involvement varies from case to case. scale bars: a and b, 60 μm; c and d, 2 μm. to try circumventing this problem, two new versions of recombinant gaa were developed and tested against the current standard of care (alglucosidase alfa), with the results of both clinical trials published last year in lancet neurology (diaz-manera et al., 2021; schoser et al., 2021). to generate the first investigational drug, avalglucosidase alfa, synthetic bis-m6p-glycane-containing oligosaccharides were chemically conjugated to recombinant human gaa; the safety and efficacy of this drug was evaluated by the comet study, a phase 3, randomized, double-blind clinical trial involving 55 sites in 20 countries (diaz-manera et al., 2021). the second investigational drug, cipaglucosidase alfa, is a novel recombinant human gaa with a high bis-m6p glycan content that is co-administered with a pharmacologic chaperone (miglustat) to increase its bioavailability; the safety and efficacy of this drug combination was evaluated by the propel study, also a phase 3, randomized, double-blind clinical trial that involved 62 sites in 24 countries (schoser et al., 2021). the comet trial enrolled 100 ert-naïve lopd patients who were randomized into equally sized avalglucosidase alfa and alglucosidase alfa groups; the propel trial, on the other hand, had a parallel group design (28 ert-naïve participants and 95 participants previously treated with alglucosidase alfa), with two thirds of participants in each subgroup receiving cipaglucosidase alfa + miglustat and one third receiving alglucosidase alfa + placebo. while the details of the study design differed slightly between the two trials, they had similar duration (49 weeks for comet and 52 weeks for propel) and their outcomes were quite similar: (1) the safety of both investigational drugs was high, with no new safety signals reported; (2) neither investigational drug was inferior to alglucosidase alfa, the current standard of care; and (3) while there was a trend toward increased efficacy for both investigational drugs, neither reached a statistically significant primary end-point. given the very promising results of prior preclinical (mouse model) studies and initial phase 1/2 trials (meena et al., 2020; puertollano and raben, 2021), these results are somewhat disappointing. intriguingly, however, the cipaglucosidase alfa + miglustat treatment was significantly more effective than the alglucosidase alfa + placebo treatment in the ert-experienced subgroup of the propel study, which is arguably most representative of the segment of the lopd patient population with the greatest need for new and more effective lopd therapies. both trials have now moved into their last phase (open label extended treatment period), the aim of which is to assess the long-term safety and efficacy of these new drugs; it will be interesting to see whether the difference in the efficacy profile between either investigational drug and alglucosidase alfa becomes greater over time or whether they will show a similar loss of efficacy over time. in addition, it will be interesting to directly compare the two new lopd drugs and determine whether one of them is superior to the other with respect to the size of initial improvement, effect duration, or both. most importantly, however, the modest results of these large and expensive clinical trials indicate that the cure for lopd remains elusive and highlight the need for a novel, ideally more effective and durable, lopd treatment approach going forward. disclosure statement the author receives research support from astellas gene therapies (formerly known as audentes therapeutics, inc) as a member of the muscle biopsy review committee for the aspiro (nct03199469) and fortis (nct04174105) clinical trials, which are evaluating the safety and efficacy of gene transfer therapy for x-linked myotubular myopathy (aspiro) and lopd (fortis). acknowledgements i am grateful to drs. nigel g. laing, gina ravenscroft, and benedikt schoser for helpful input during the conceptualization stage of this review. in addition, i would like to thank ms. christine lin for assistance with figure preparation. references ahmady, e., deeke, s.a., rabaa, s., kouri, l., kenney, l., stewart, a.f., and burgon, p.g. (2011). identification of a novel muscle a-type lamin-interacting protein (mlip). j biol chem 286, 19702-19713. allen, j.m., zamurs, l., brachvogel, b., schlotzer-schrehardt, u., hansen, u., lamande, s.r., rowley, l., fitzgerald, j., and bateman, j.f. (2009). mice lacking the extracellular matrix protein warp develop normally but have compromised peripheral nerve structure and function. j biol chem 284, 12020-12030. amici, d.r., pinal-fernandez, i., christopher-stine, l., mammen, a.l., and mendillo, m.l. (2021). a network of core and subtype-specific gene expression programs in myositis. acta neuropathol 142, 887-898. amore, g., romagnoli, m., carbonelli, m., barboni, p., carelli, v., and la morgia, c. (2021). therapeutic options in hereditary optic neuropathies. drugs 81, 57-86. aschman, t., schneider, j., greuel, s., meinhardt, j., streit, s., goebel, h.h., buttnerova, i., elezkurtaj, s., scheibe, f., radke, j., et al. (2021). association between sars-cov-2 infection and immune-mediated myopathy in patients who have died. jama neurol 78, 948-960. cattin, m.e., wang, j., weldrick, j.j., roeske, c.l., mak, e., thorn, s.l., dasilva, j.n., wang, y., lusis, a.j., and burgon, p.g. (2015). deletion of mlip (muscle-enriched a-type lamin-interacting protein) leads to cardiac hyperactivation of akt/mammalian target of rapamycin (mtor) and impaired cardiac adaptation. j biol chem 290, 26699-26714. collier, j.j., guissart, c., olahova, m., sasorith, s., piron-prunier, f., suomi, f., zhang, d., martinez-lopez, n., leboucq, n., bahr, a., et al. (2021). developmental consequences of defective atg7-mediated autophagy in humans. n engl j med 384, 2406-2417. costa-mattioli, m., and walter, p. (2020). the integrated stress response: from mechanism to disease. science 368, eaat5314. deschauer, m., hengel, h., rupprich, k., kreiss, m., schlotter-weigel, b., grimmel, m., admard, j., schneider, i., alhaddad, b., gazou, a., et al. (2021). bi-allelic truncating mutations in vwa1 cause neuromyopathy. brain 144, 574-583. diaz-manera, j., kishnani, p.s., kushlaf, h., ladha, s., mozaffar, t., straub, v., toscano, a., van der ploeg, a.t., berger, k.i., clemens, p.r., et al. (2021). safety and efficacy of avalglucosidase alfa versus alglucosidase alfa in patients with late-onset pompe disease (comet): a phase 3, randomised, multicentre trial. lancet neurol 20, 1012-1026. dodig, d., tarnopolsky, m.a., margeta, m., gordon, k., fritzler, m.j., and lu, j.q. (2022). covid-19-associated critical illness myopathy with direct viral effects. ann neurol, epub ahead of print. esslinger, u., garnier, s., korniat, a., proust, c., kararigas, g., muller-nurasyid, m., empana, j.p., morley, m.p., perret, c., stark, k., et al. (2017). exome-wide association study reveals novel susceptibility genes to sporadic dilated cardiomyopathy. plos one 12, e0172995. filosto, m., cotti piccinelli, s., gazzina, s., foresti, c., frigeni, b., servalli, m.c., sessa, m., cosentino, g., marchioni, e., ravaglia, s., et al. (2021). guillain-barre syndrome and covid-19: an observational multicentre study from two italian hotspot regions. j neurol neurosurg psychiatry 92, 751-756. fragiel, m., miro, o., llorens, p., jimenez, s., pinera, p., burillo, g., martin, a., martin-sanchez, f.j., garcia-lamberechts, e.j., jacob, j., et al. (2021). incidence, clinical, risk factors and outcomes of guillain-barre in covid-19. ann neurol 89, 598-603. ho, r., and hegele, r.a. (2019). complex effects of laminopathy mutations on nuclear structure and function. clin genet 95, 199-209. huang, z.p., kataoka, m., chen, j., wu, g., ding, j., nie, m., lin, z., liu, j., hu, x., ma, l., et al. (2015). cardiomyocyte-enriched protein cip protects against pathophysiological stresses and regulates cardiac homeostasis. j clin invest 125, 4122-4134. jha, m.k., passero, j.v., rawat, a., ament, x.h., yang, f., vidensky, s., collins, s.l., horton, m.r., hoke, a., rutter, g.a., et al. (2021). macrophage monocarboxylate transporter 1 promotes peripheral nerve regeneration after injury in mice. j clin invest 131, e141964. johari, m., sarparanta, j., vihola, a., jonson, p.h., savarese, m., jokela, m., torella, a., piluso, g., said, e., vella, n., et al. (2021). missense mutations in small muscle protein x-linked (smpx) cause distal myopathy with protein inclusions. acta neuropathol 142, 375-393. keddie, s., pakpoor, j., mousele, c., pipis, m., machado, p.m., foster, m., record, c.j., keh, r.y.s., fehmi, j., paterson, r.w., et al. (2021). epidemiological and cohort study finds no association between covid-19 and guillain-barre syndrome. brain 144, 682-693. keh, r.y.s., scanlon, s., datta-nemdharry, p., donegan, k., cavanagh, s., foster, m., skelland, d., palmer, j., machado, p.m., keddie, s., et al. (2022). covid-19 vaccination and guillain-barré syndrome: analyses using the national immunoglobulin database. medrxiv, 2021.2012.2014.21267418. kieninger, s., xiao, t., weisschuh, n., kohl, s., ruther, k., kroisel, p.m., brockmann, t., knappe, s., kellner, u., lagreze, w., et al. (2022). dnajc30 disease-causing gene variants in a large central european cohort of patients with suspected leber's hereditary optic neuropathy and optic atrophy. j med genet. leoni, t.b., gonzalez-salazar, c., rezende, t.j.r., hernandez, a.l.c., mattos, a.h.b., coimbra neto, a.r., da graca, f.f., goncalves, j.p.n., martinez, a.r.m., taniguti, l., et al. (2021). a novel multisystem proteinopathy caused by a missense anxa11 variant. ann neurol 90, 239-252. liu, j., huang, z.p., nie, m., wang, g., silva, w.j., yang, q., freire, p.p., hu, x., chen, h., deng, z., et al. (2020). regulation of myonuclear positioning and muscle function by the skeletal muscle-specific cip protein. proc natl acad sci u s a 117, 19254-19265. lopes abath neto, o., medne, l., donkervoort, s., rodriguez-garcia, m.e., bolduc, v., hu, y., guadagnin, e., foley, a.r., brandsema, j.f., glanzman, a.m., et al. (2021). mlip causes recessive myopathy with rhabdomyolysis, myalgia and baseline elevated serum creatine kinase. brain 144, 2722-2731. luijten, l.w.g., leonhard, s.e., van der eijk, a.a., doets, a.y., appeltshauser, l., arends, s., attarian, s., benedetti, l., briani, c., casasnovas, c., et al. (2021). guillain-barre syndrome after sars-cov-2 infection in an international prospective cohort study. brain 144, 3392-3404. lunn, m.p., carr, a.c., keddie, s., pakpoor, j., pipis, m., and willison, h.j. (2021). reply: guillain-barre syndrome, sars-cov-2 and molecular mimicry and ongoing challenges in unravelling the association between covid-19 and guillain-barre syndrome and unclear association between covid-19 and guillain-barre syndrome and currently available data regarding the potential association between covid-19 and guillain-barre syndrome. brain 144, e47. margeta, m. (2020a). autophagy defects in skeletal myopathies. annu rev pathol 15, 261-285. margeta, m. (2020b). top ten discoveries of the year: neuromuscular disease. free neuropathol 1, 4. margeta, m. (2021). neuromuscular disease: 2021 update. free neuropathol 2, 3. meena, n.k., ralston, e., raben, n., and puertollano, r. (2020). enzyme replacement therapy can reverse pathogenic cascade in pompe disease. mol ther methods clin dev 18, 199-214. napolitano, f., terracciano, c., bruno, g., de blasiis, p., lombardi, l., gialluisi, a., gianfrancesco, f., de giovanni, d., tummolo, a., di iorio, g., et al. (2021). novel autophagic vacuolar myopathies: phenotype and genotype features. neuropathol appl neurobiol 47, 664-678. pagnamenta, a.t., kaiyrzhanov, r., zou, y., da'as, s.i., maroofian, r., donkervoort, s., dominik, n., lauffer, m., ferla, m.p., orioli, a., et al. (2021). an ancestral 10-bp repeat expansion in vwa1 causes recessive hereditary motor neuropathy. brain 144, 584-600. patone, m., handunnetthi, l., saatci, d., pan, j., katikireddi, s.v., razvi, s., hunt, d., mei, x.w., dixon, s., zaccardi, f., et al. (2021). neurological complications after first dose of covid-19 vaccines and sars-cov-2 infection. nat med 27, 2144-2153. popovic, d., and dikic, i. (2014). tbc1d5 and the ap2 complex regulate atg9 trafficking and initiation of autophagy. embo rep 15, 392-401. puertollano, r., and raben, n. (2021). new therapies for pompe disease: are we closer to a cure? lancet neurol 20, 973-975. quarta, m., cromie, m., chacon, r., blonigan, j., garcia, v., akimenko, i., hamer, m., paine, p., stok, m., shrager, j.b., et al. (2017). bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss. nat commun 8, 15613. ratnayake, d., nguyen, p.d., rossello, f.j., wimmer, v.c., tan, j.l., galvis, l.a., julier, z., wood, a.j., boudier, t., isiaku, a.i., et al. (2021). macrophages provide a transient muscle stem cell niche via nampt secretion. nature 591, 281-287. salzer-sheelo, l., fellner, a., orenstein, n., bazak, l., lev-el halabi, n., daue, m., smirin-yosef, p., van hout, c.v., fellig, y., ruhrman-shahar, n., et al. (2021). biallelic truncating variants in the muscular a-type lamin-interacting protein (mlip) gene cause myopathy with hyperckemia. eur j neurol, epub ahead of print. schoser, b., roberts, m., byrne, b.j., sitaraman, s., jiang, h., laforet, p., toscano, a., castelli, j., diaz-manera, j., goldman, m., et al. (2021). safety and efficacy of cipaglucosidase alfa plus miglustat versus alglucosidase alfa plus placebo in late-onset pompe disease (propel): an international, randomised, double-blind, parallel-group, phase 3 trial. lancet neurol 20, 1027-1037. sicari, b.m., agrawal, v., siu, b.f., medberry, c.j., dearth, c.l., turner, n.j., and badylak, s.f. (2012). a murine model of volumetric muscle loss and a regenerative medicine approach for tissue replacement. tissue eng part a 18, 1941-1948. siegert, e., uruha, a., goebel, h.h., preusse, c., casteleyn, v., kleefeld, f., alten, r., burmester, g.r., schneider, u., hoppner, j., et al. (2021). systemic sclerosis-associated myositis features minimal inflammation and characteristic capillary pathology. acta neuropathol 141, 917-927. spaulding, e.l., hines, t.j., bais, p., tadenev, a.l.d., schneider, r., jewett, d., pattavina, b., pratt, s.l., morelli, k.h., stum, m.g., et al. (2021). the integrated stress response contributes to trna synthetase-associated peripheral neuropathy. science 373, 1156-1161. stavrou, m., sargiannidou, i., christofi, t., and kleopa, k.a. (2021). genetic mechanisms of peripheral nerve disease. neurosci lett 742, 135357. stenton, s.l., sheremet, n.l., catarino, c.b., andreeva, n.a., assouline, z., barboni, p., barel, o., berutti, r., bychkov, i., caporali, l., et al. (2021). impaired complex i repair causes recessive leber’s hereditary optic neuropathy. j clin invest 131, e138267. suh, j., mukerji, s.s., collens, s.i., padera, r.f., jr., pinkus, g.s., amato, a.a., and solomon, i.h. (2021). skeletal muscle and peripheral nerve histopathology in covid-19. neurology 97, e849-e858. szczepanowska, k., senft, k., heidler, j., herholz, m., kukat, a., hohne, m.n., hofsetz, e., becker, c., kaspar, s., giese, h., et al. (2020). a salvage pathway maintains highly functional respiratory complex i. nat commun 11, 1643. tebbenkamp, a.t.n., varela, l., choi, j., paredes, m.i., giani, a.m., song, j.e., sestan-pesa, m., franjic, d., sousa, a.m.m., liu, z.w., et al. (2018). the 7q11.23 protein dnajc30 interacts with atp synthase and links mitochondria to brain development. cell 175, 1088-1104 e1023. trikamji, b., and pestronk, a. (2021). treatable, motor-sensory, axonal neuropathies with c5b-9 complement on endoneurial microvessels. muscle nerve 63, 506-515. turnbull, c., scott, r.h., thomas, e., jones, l., murugaesu, n., pretty, f.b., halai, d., baple, e., craig, c., hamblin, a., et al. (2018). the 100 000 genomes project: bringing whole genome sequencing to the nhs. bmj 361, k1687. umapathi, t., er, b., koh, j.s., goh, y.h., and chua, l. (2021). guillain-barre syndrome decreases in singapore during the covid-19 pandemic. j peripher nerv syst 26, 235-236. witt, l.j., curran, j.j., and strek, m.e. (2016). the diagnosis and treatment of antisynthetase syndrome. clin pulm med 23, 218-226. xi, y., ju, r., and wang, y. (2020). roles of annexin a protein family in autophagy regulation and therapy. biomed pharmacother 130, 110591. yell, p.c., burns, d.k., dittmar, e.g., white, c.l., 3rd, and cai, c. (2018). diffuse microvascular c5b-9 deposition is a common feature in muscle and nerve biopsies from diabetic patients. acta neuropathol commun 6, 11. zhang, h., zhou, z.w., and sun, l. (2021). aminoacyl-trna synthetases in charcot-marie-tooth disease: a gain or a loss? j neurochem 157, 351-369. zhang, k., liu, q., liu, k., shen, d., tai, h., shu, s., ding, q., fu, h., liu, s., wang, z., et al. (2018). anxa11 mutations prevail in chinese als patients with and without cognitive dementia. neurol genet 4, e237. zuko, a., mallik, m., thompson, r., spaulding, e.l., wienand, a.r., been, m., tadenev, a.l.d., van bakel, n., sijlmans, c., santos, l.a., et al. (2021). trna overexpression rescues peripheral neuropathy caused by mutations in trna synthetase. science 373, 1161-1166. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. ex situ perfusion fixation for brain banking: a technical report feel free to add comments by clicking these icons on the sidebar free neuropathology 3:22 (2022) original paper ex situ perfusion fixation for brain banking: a technical report andrew t. mckenzie1,2,3,4, emma woodoff-leith1,2,3, diana dangoor1,2,3, alessandra cervera1,2,3, hadley walsh ressler1,2,3, kristen whitney1,2,3, kristen dams-o’connor5,6, zhuhao wu1,7,8, elizabeth m. c. hillman9, alan c. seifert10, john f. crary1,2,3 1 department of neuroscience, icahn school of medicine at mount sinai, new york, new york, usa 2 friedman brain institute, departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa 3 neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa 4 department of psychiatry, icahn school of medicine at mount sinai, new york, new york, usa 5 department of rehabilitation and human performance, icahn school of medicine at mount sinai, new york, new york, usa 6 department of neurology, icahn school of medicine at mount sinai, new york, new york, usa 7 department of cell, developmental, and regenerative biology, icahn school of medicine at mount sinai, new york, new york, usa 8 black family stem cell institute, icahn school of medicine at mount sinai, new york, new york, usa 9 laboratory for functional optical imaging, departments of biomedical engineering and radiology and the mortimer b. zuckerman mind brain behavior institute, columbia university, new york, new york, usa 10 biomedical engineering and imaging institute, department of diagnostic, molecular and interventional radiology, and graduate school of biomedical sciences, icahn school of medicine at mount sinai, new york, new york, usa corresponding author: john f. crary · department of pathology · icahn school of medicine at mount sinai · icahn building 9th floor, room 20a · 1425 madison avenue · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 15 august 2022 accepted: 20 september 2022 copyedited by: georg haase published: 28 september 2022 https://doi.org/10.17879/freeneuropathology-2022-4368 keywords: brain banking; perfusion fixation; perfusion pressure; postmortem interval; tissue morphology; rnascope; ex vivo neuroimaging abstract perfusion fixation is a well-established technique in animal research to improve preservation quality in the study of many tissues, including the brain. there is a growing interest in using perfusion to fix postmortem human brain tissue to achieve the highest fidelity preservation for downstream high-resolution morphomolecular brain mapping studies. numerous practical barriers arise when applying perfusion fixation in brain banking settings, including the large mass of the organ, degradation of vascular integrity and patency prior to the start of the procedure, and differing investigator goals sometimes necessitating part of the brain to be frozen. as a result, there is a critical need to establish a perfusion fixation procedure in brain banking that is flexible and scalable. this technical report describes our approach to developing an ex situ perfusion fixation protocol. we discuss the challenges encountered and lessons learned while implementing this procedure. routine morphological staining and rna in situ hybridization data show that the perfused brains have well-preserved tissue cytoarchitecture and intact biomolecular signal. however, it remains uncertain whether this procedure leads to improved histology quality compared to immersion fixation. additionally, ex vivo magnetic resonance imaging (mri) data suggest that the perfusion fixation protocol may introduce imaging artifacts in the form of air bubbles in the vasculature. we conclude with further research directions to investigate the use of perfusion fixation as a rigorous and reproducible alternative to immersion fixation for the preparation of postmortem human brains. introduction a detailed examination of the brain in various settings, including educational, clinical, and research domains, is critically dependent upon the quality of brain preservation. human tissue-based research, which plays a significant role in studying brain diseases, relies on the brain banking process to sufficiently preserve the postmortem brain. for example, in the study of brain disorders such as alzheimer’s disease, it is essential to have well-maintained histoarchitecture and biomolecular structure to reliably distinguish the features of brain donors with and without the disorder (lucassen et al., 1997). the goal of the initial preservation procedure is to interrupt postmortem degradative processes in a way that prepares the brain for long-term storage, while minimizing damage resulting from the preservation process itself. with access to intact, well-preserved human brain tissue, investigators can conduct a diverse set of next-generation brain mapping studies, including high-resolution ex vivo neuroimaging (pallebage-gamarallage et al., 2018), connectomics studies using high-throughput serial section electron microscopy (shapson-coe et al., 2021), and volumetric 3d histologic imaging of the brain’s cellularand molecular-level organization (patel et al., 2022). all existing brain preservation procedures face trade-offs in the extent and type of tissue preservation achieved and the consequent degree of information loss. in non-human animal studies, perfusion fixation is widely considered to be the state of the art for achieving the highest-quality morphologic preservation of the whole brain (bodian, 1936; karlsson and schultz, 1965; mcfadden et al., 2019). in human tissue brain banking, immersion fixation is the most common preservation method employed. a key conundrum in immersion procedures is that the outer regions of the brain are liable to be over-fixed and the inner regions of the brain are liable to be under-fixed, both of which have the potential to limit optimal and consistent antigen preservation. while sectioning tissue prior to immersion may allow fixative to penetrate inner brain regions earlier, this technique can damage tissue morphology (adickes et al., 1997). perfusion fixation is a potential solution for this challenge and may more consistently preserve tissue morphology and antigenicity than immersion fixation. the perfusion technique may allow for a shorter fixation period, without requiring pre-sectioning, by penetrating inner brain regions more quickly. perfusion fixation is also expected to limit tissue autolysis, a postmortem change that inevitably occurs in the inner brain regions before the immersion fixative can reach those areas (beach et al., 1987; mcfadden et al., 2019). perfusion can either be performed via an intrathoracic approach in the case of smaller animals or via an intracarotid approach in the case of larger animals such as pigs or elephants (manger et al., 2009; musigazi et al., 2018). the two classes of methods to perform perfusion fixation are ex situ methods, where the brain is extracted from the skull prior to perfusion, and in situ methods, where the brain remains inside the skull during perfusion (mcfadden et al., 2019) (figure 1). in situ approaches often involve cannulation of one or both carotid arteries, with or without also cannulating the vertebral arteries. ex situ approaches often involve cannulation of the internal carotid arteries and the vertebrobasilar artery system. figure 1. two major approaches for performing perfusion fixation example approaches of human brain perfusion fixation using an in situ (brain inside of the skull); a) or ex situ (brain removed from the skull); b) approach. while only an ex situ perfusion protocol is described in this report, we include an illustration of the in situ method as well to demonstrate this alternative approach. also, while the use of surgical sutures is an alternative approach that is illustrated here, in our protocol, hemostats are used to clamp cannulae in place. arrows represent the direction of perfusate flow during perfusion. scm = sternocleidomastoid muscle. illustrations by jill k. gregory, certified medical illustrator, fellow of the association of medical illustrators. the purpose of this technical report is to describe our use of perfusion fixation for human brain banking as a quality improvement project. we used an ex situ approach, which can be more easily integrated into the autopsy suite workflow and which is compatible with the anatomical donation of the brain alone, as opposed to a full-body donation. our aim is to describe our experience in implementing this procedure to provide pertinent information for the research community. there have been several previous descriptions of ex situ approaches to perfusion fixation in brain banking described in the literature (adickes et al., 1997; beach et al., 1987; de oliveira et al., 2012; grinberg et al., 2008; halliday et al., 1988; insausti et al., 1995; waldvogel et al., 2006; welikovitch et al., 2018). we recognize that there are likely many other approaches. we also suspect that perfusion fixation methods in brain banking will likely evolve in the future as more researchers engage with the procedure. materials and methods patient samples all specimens were obtained and de-identified at the icahn school of medicine at mount sinai in accordance with its policies, regulations, and institutional review board recommendations. the use of the perfusion fixation protocol was developed as part of an ongoing quality improvement project in the neuropathology brain bank & research core. the goal of the project is to see whether it is possible to decrease fixation times and thereby mitigate antigenicity loss of formalin-sensitive antigens in intact brains. the specimens were a convenience sample of brains processed at the neuropathology brain bank & research core, with no specific exclusion criteria. perfusion fixation methods we used the same perfusion circuit setup throughout the development of the protocol, with or without an in-line pressure sensor. details of the procedure, including the use of different reagents, were iterated upon over the course of the study. in our perfusion circuit, fluid in a beaker, which consists of either washout or fixative solution, is pumped through silicone tubing via a peristaltic pump with a variable pump drive speed, which mediates the flow rate (see table 1 for details of equipment used). downstream of the pump, there is a pulse dampener that acts to prevent significant pulsations in fluid flow. the next component in the circuit is the pressure sensor, which connects to a digital pressure monitor. finally, there is a y connector that splits the circuit into two circuits, each of which connects to a catheter that can be used to cannulate one blood vessel. following cannulation, the cannulae are clamped in place with hemostats in all cases. if it is preferred to perfuse through only one of the two catheters at a time, the perfusion tubing following the y connector for the other catheter can be clamped with a hemostat to stop the flow through that component of the circuit. all perfusion procedures are performed at room temperature. † catheter bevels are filed to have blunt ends prior to use in perfusion. we performed this procedure on two types of specimens, termed either ‘whole brain’ or ‘hemibrain’. the term hemibrain refers to one of the two cerebral hemispheres, which is obtained after a hemi-sectioning procedure designed to retain enough of the circle of willis for adequate vascular access. three hemi-brains (cases 1-3) and two whole brains (cases 4-5) were subjected to perfustion fixation. in case 1, the hindbrain was not removed prior to the perfusion procedure; however, in all other cases, the hindbrain was detached following transection through the upper midbrain to vascular access. thus, whole brain specimens have both cerebral hemispheres, but some brainstem structures removed. there were a couple of differences in how perfusion fixation was performed for hemibrain and whole brain specimens. first, for hemibrains, a plastic container with muslin cloth was used to suspend the specimen without immersing it in fixative, allowing the perfusate to drain into the bottom of the container during the procedure. for whole brains, there was a focus on volumetric analysis with ex vivo neuroimaging. as a result, for the whole brain cases, the specimen was suspended in a formalin bath, because suspending the brain in fluid was expected to less shape deformation during the procedure. second, for whole brain perfusions, fixation was accelerated after the initial procedure by injecting fixative into ventricular spaces in the brain using a 50 ml syringe. we employed this additional fixation step to mimic how fixative more readily diffuses into the ventricular system for hemibrains, assuming that the septum pellucidum does not act as a barrier to diffusion, which is expected to be the case following brain hemisection. following perfusion, the brains were fixed via additional immersion in 10% (w/v) neutral buffered (sodium phosphate) formalin for approximately two weeks. in the perfusion fixation literature, this subsequent immersion step is referred to as postfixation (mcfadden et al., 2019). the two whole brain specimens were imaged with mri as described below. then the brains were dissected coronally into slabs 3-5 mm thick in the anterior to posterior direction. brains were systematically sampled for neuropathologic postmortem diagnosis and placed into cassettes for processing. the samples were embedded in paraffin and 5-7 μm thick sections were cut serially on a microtome, mounted on glass slides, deparaffinized, and stained with hematoxylin and eosin (h&e) or luxol fast blue counterstained with h&e for microscopic examination. the slides for cases 1, 2, and 3 were viewed and photographed using a nikon eclipse ci microscope. the slides for cases 4 and 5 were imaged using a philips ultra fast scanner. rna in situ hybridization rna in situ hybridization was performed using the fully automated rnascope 2.5 lsx reagent kit-red (advanced cell diagnostics, catalog #322750) according to the manufacturer’s instructions for formalin-fixed paraffin-embedded (ffpe) brain tissue. 5 µm ffpe tissue sections prepared from perfusion fixation brain tissue were baked for 1 hour at 70° c, and the rnascope assay was run on the leica bond rx automated slide stainer platform (leica biosystems). target retrieval was performed for 20 minutes at 95° c followed by 15 minutes protease iii treatment at 40° c and amplification and detection steps according to the manufactures protocol. a probe for the housekeeping gene ubiquitin c (hs-ubc, catalog # 312028) was used to assess mrna detection. slides were visualized using a nikon eclipse brightfield microscope with a nikon ds-fi3 camera and nis elements software. ex vivo whole brain mri imaging following perfusion, both of the two ex vivo whole brain specimens were placed in a custom-built imaging container (boonstra et al., 2021), immersed in fluorinert (3m, saint paul, mn), and exposed to vacuum for 15 minutes to dislodge air bubbles (stram et al., 2022). the container was sealed, and the specimen was imaged using a 7 tesla whole-body mri scanner (magnetom 7t as, siemens, erlangen, germany) equipped with a 32-channel radiofrequency head coil (nova medical, wilmington, ma). after acquisition of localizer images and main magnetic field shimming by 3d field mapping, the specimens were imaged with a 3d multi-echo gradient-echo pulse sequence with the following parameters: repetition time = 38 ms, echo times = 5, 10, 15, 20, 25, 30 ms, flip angle = 18°, resolution = 0.38 mm isotropic, in-plane acceleration by grappa (griswold et al., 2002) with acceleration factor r=2, readout bandwidth = 260 hz/px, bipolar readouts, non-selective excitation. individual echo images were co-registered by fsl flirt (jenkinson et al., 2002; jenkinson and smith, 2001), and combined into a single image by root-sum-of-squares of individual echoes. results implementation of the perfusion fixation protocol we report our deployment of the ex situ perfusion fixation procedure on five brains in the form of individual case summaries. in all instances, we used a perfusion circuit with the same core design and evaluated different variations of the procedure. for each case, we describe operational parameters, case-specific factors of the procedure, and metrics recorded regarding the quality of the perfusion fixation for that case (table 2). * semicolons denote vessels cannulated separately and sequentially. † volume perfused approximate. ‡ flow rate is mediated by pump drive speed. with our circuit, 50 rotations per minute (rpm) corresponds to approximately 38 ml/min and 60 rpm to 50 ml/min. § not available. case 1. the perfusion fixation procedure was performed with a hemibrain specimen from a 90+-year-old man with a clinical diagnosis of sepsis who had died of pneumonia. the postmortem interval (pmi) was 52 hours prior to the procedure. grossly, the blood vessels were assessed as having moderate atherosclerosis. the internal carotid and posterior cerebral arteries were cannulated with a 21g catheter. the vessels were first perfused with 0.5 l of phosphate buffered saline (pbs) with sodium azide (0.01% wt/vol) as a washout solution for 15 minutes. during this washout process, the blood was not found to substantially clear on visual inspection of the surface of the brain. the vessels were next perfused with 1.5 l of fixative solution, consisting of 10% (w/v) formalin and 1% (w/v) glutaraldehyde. qualitatively, at the end of the fixation procedure, palpation revealed that areas near the perfusate entry were found to be stiffer than other areas of the brain, suggestive of rapid fixation in those areas. case 2. this procedure was performed with a hemibrain specimen from a 66-year-old woman with a clinical diagnosis of sepsis who had died of cardiac arrest. the pmi was 44 hours. for most of the pmi, the brain was stored at refrigerator temperature (4° c). the anterior carotid artery and middle cerebral artery were cannulated with 19g catheters. with this specimen, it was more difficult to cannulate the vessels as only partial remnants of the original vessels remained after brain removal. the vessels were first perfused with 0.5 l of washout solution at a pump speed of 50 rpm, consisting of pbs with mannitol (4.5% wt/vol) and sodium nitrite (1% wt/vol). mannitol was employed to assist with opening of the blood brain barrier, while sodium nitrite was used to assist with vasodilation (ikeda et al., 2003; palay et al., 1962). during this washout process, the apparent clearance of blood from some blood vessels was visualized. the vessels were then perfused with about 1 l of fixative, consisting of 10% formalin and 1% glutaraldehyde, at a pump speed of 60 rpm. physical stiffening of the hemibrain post perfusion was not appreciated, possibly suggesting inadequate perfusion in this case, although we did not use precise measurements of stiffness. case 3. the perfusion fixation procedure was performed with a hemibrain specimen from a 77-year-old man with a diagnosis of diffuse lewy body disease who had died of pneumonia. the pmi was 12 hours. grossly, the blood vessels were assessed as having mild atherosclerosis. the middle cerebral artery and posterior cerebral artery were cannulated with 19g catheters. to streamline the procedure, washout solution and glutaraldehyde were omitted. the vessels were perfused with 2 l of 10% (w/v) formalin at a pump speed of 60 rpm for 30-40 minutes. at the end of the procedure, the anterolateral aspect of the perfused hemibrain had significant swelling. the reason for the swelling was unclear, but the shorter pmi of the specimen in case 3 may have led to a lower resistance to perfusate flow than in cases 1 and 2, leading to a higher effective perfusion pressure, potentially resulting in blood brain barrier disruption (schwarzmaier et al., 2022). case 4. this perfusion fixation procedure was performed on a whole brain specimen from a 90+-year-old woman who was positive for covid-19 at the time of death. the pmi was 24 hours. the blood vessels were cannulated with 23g catheters in three different ways. no washout solution was perfused. we first cannulated the bilateral internal carotid arteries and perfused approximately 250 ml of 10% formalin at an initial pump speed of 50 rpm that was subsequently raised to 60 rpm. during this process, it was possible to visualize blood being removed from an artery in the right frontal cortex. the inferior frontal gyrus and inferior temporal gyrus appeared to stiffen. however, there was a concern that the bilateral anterior frontal poles were becoming slightly edematous, although the gross effects of this could not be clearly seen post-perfusion. additionally, at the beginning of the perfusion, the pressure monitor was at 1.5 psi (77.6 mmhg), and it increased to approximately 2.1 psi (108.6 mmhg), suggestive of a possible blockage or buildup of fluid. as a result, the perfusion through the bilateral internal carotid arteries was stopped. we next cannulated and perfused two other blood vessels to assess whether the same swelling phenomenon would occur. after dissection to remove the cerebellum, it was possible to visualize the circle of willis, which was intact. we cannulated and perfused through the anterior cerebral arteries bilaterally at a pump speed of 50 rpm. arteries in the right hemisphere temporal cortex were visualized being drained of blood. however, there was a concern that the vessels may have become clogged because an unintentional pressure increase was detected in the pressure sensor. we then switched to perfusing through the basilar artery at a pump speed of 50 rpm. after a few minutes of perfusion through the basilar artery, the fixative appeared to be accumulating near the circle of willis rather than penetrating through the blood vessels into the deeper parts of the brain, so this perfusion was stopped. leakage from the superior cerebellar arteries, which are severed upon removal of the cerebellum, may have contributed to the observed accumulation of fixative around the circle of willis. for injection of fixative, we suspended the whole brain by tying a string around the circle of willis and placing the specimen into a container filled with formalin. to access the third ventricle, we directly inserted a syringe through the floor of the third ventricle at the posterior aspect of the tuber cinereum and injected 25 ml of 10% formalin. we then inserted a syringe into the cerebral aqueduct and injected 25 ml of 10% formalin there. the brain was then placed on a rocker at refrigerator temperature for postfixation. the gross anatomic condition of this brain shows that the brain grossly appears more pale, suggestive of less remaining blood in vessels, over the course of the preservation process (figure 2). figure 2. serial gross images of a donated human brain during ex-situ perfusion the gross anatomic condition of the brain (case 4) is shown via a high-resolution photograph after extraction from the skull but prior to perfusion (a), following perfusion fixation and further dissection of the circle of willis but prior to immersion fixation (b), and following both perfusion fixation and post-perfusion immersion fixation (c). note that the cerebellum has been removed from the brain specimen between steps (a) and (b). additionally, the frontal pole has been removed at step (c). case 5. the perfusion fixation procedure was performed on a whole brain specimen from a 64-year-old woman with a clinical diagnosis of metastatic adenocarcinoma. the pmi was 7 hours. the blood vessels were cannulated with 19g catheters in three different ways. we first cannulated the bilateral middle cerebral arteries, then we cannulated one of the anterior cerebral arteries, and then we cannulated one of the vertebral arteries leading into the basilar artery. after each of these vessel cannulations, we perfused 10% formalin for 5-10 minutes, with no washout solution. the posterior communicating arteries were small and there was no evidence of intact collateral circulation through the circle of willis. when perfusing through each of the blood vessels systems in this case, there was clearing of blood appreciated in the proximal areas of the tissue next to the cannulated blood vessel. manual palpation revealed stiffening of areas of brain tissue distal to the cannulated vessels. the pressure monitoring showed a range of 1.5 psi (77.6 mmhg) to 2 psi (103.4 mmhg) when perfusing through two blood vessels. higher pressures were found, even up to 3 psi (155.1 mmhg), when perfusing through just one blood vessel. at one point, we also noticed an air bubble to be present in the perfusion tubing, although it is unclear what impact this had on the perfusion quality. after perfusion, we used a syringe to perform injection fixation of the brain. fixative was introduced into the third ventricle, the fourth ventricle via a superior approach through the anterior corpus callosum, and each of the lateral ventricles after sectioning through the midbrain and cerebellum. for each injection, we used 25-50 ml of 10% formalin. we infer that the injection was made into the ventricles rather than into the brain parenchyma because a partial flow of fluid could be seen coming out of the perforation in the third ventricle during injection of fixative into the lateral ventricles. additionally, there was no evidence upon sectioning that the striatum was disrupted, as might have been expected from parenchymal injection. however, because we could not directly visualize the injection into the ventricles, we cannot completely exclude that some of the fixative may have directly entered the brain parenchyma. histology and neuroimaging data histology data from the perfused brains showed that the expected tissue morphology and cell shape were present, which was similar to typical findings from immersion-fixed brains (figure 3). there were no clear alterations due to the edema observed in the perfused brains. figure 3. routinely histology stainings of tissue sections from perfused brains representative histology sections were luxol fast blue, hematoxylin and eosin (lh&e)-stained in case 1 (a-c) or hematoxylin and eosin (h&e)-stained from case 2 (d), case 3 (e), case 4 (f-g), and case 5 (h-i). sections were derived from the striatum (a-b), corpus callosum (c), temporal cortex (d), white matter of the temporal pole (e), substantia nigra (f), hippocampus – dentate gyrus (g), mid-frontal cortex (h), and striatum (i). tissue morphology shows expected findings, suggesting that the perfusion process did not adversely affect tissue morphology. red blood cells were observed in some vessels, indicating absence of complete perfusion. all scale bars 100 μm except for sub-figures d (1000 μm), g (50 μm), and h (200 μm). to assess the distribution of single rna molecules we performed rna in situ hybridization analysis in one of the perfused brains (figure 4). specifically, we utilized the rnascope in situ hybridization (ish) assay, which is a commercial rna ish technology that detects individual mrna molecules on a single-cell level, in the superior frontal region of case 5 (wang et al., 2012). we observed a strong and robust ubiquitin c signal in all cell types including neurons, glia and blood vessels, suggesting that mrna species can be detected in perfused brains (figure 4), as expected from previous results using the rnascope in immersion-fixed human brain tissue (baleriola et al., 2014). figure 4. robust detection of ubiquitin c rna by in situ hybridization in a perfusion-fixed brain representative low (a) and high (b) magnification images of the rnascope assay in the superior frontal region of case five. strong and robust signal of ubiquitin c mrna is detected in all cell types including neurons (black arrow), glia (black arrowhead), and blood vessels (red arrow). scale bars 1 mm (a) and 50 µm (b). we also performed ex vivo mri scans following perfusion of the two whole brains. while pure immersion-based diffusion of formalin has the potential to create an artifact on ex vivo mri scans corresponding to the fixation front, in the mri scans of perfusion-fixed brains there were no prominent rim artifacts (i.e. variations in signal intensity in superficial versus deep regions) related to inhomogeneous fixation (nazemorroaya et al., 2022). however, the perfused brains contained numerous small hypointensities seen on multi-echo gradient-echo with root-sum-of-squares echo recombination images, which are not present in comparative immersion-fixed brains scanned with the same protocol (figure 5). these hypointensities are consistent with susceptibility-induced signal loss due to small air bubbles distributed in the vasculature. therefore, one of the potential downsides of the current perfusion protocol may be the inadvertent introduction of air bubbles into the tissue. these hypointensities are less likely to be related to perivascular spaces, which typically appear as hyperintensities on these mri contrasts. however, the effect of these small hypointensities on histological features is uncertain and we have not detected any parenchymal air bubble artifacts in the preliminary histology data reviewed so far. figure 5. perfusion-fixed brains contain numerous small hypointensities on mri scans multi-echo gradient-echo with root-sum-of-squares echo recombination images of a comparison immersion-fixed brain (a) as well as two perfusion-fixed whole brains: case 4 (b) and case 5 (c). numerous small hypointensities are visualized in the two perfusion-fixed brains but not in the immersion-fixed brain imaged with the same protocol. discussion in its simplest form, perfusion fixation can be thought of as an extension of immersion fixation, except that fixative solution extravasates from the blood vessels rather than penetrates from outside brain surfaces. this thereby increases the surface area by which fixatives can penetrate the tissue by orders of magnitude and homogenizes its distribution. however, introducing additional variables causes other potential alterations to the perfused brains that might contribute to artifacts or batch effects in comparison to immersion-fixed brains. glutaraldehyde, for instance, is not typically used in brain banking and there was a theoretical concern that it might affect the results of downstream immunolabelling assays, introducing the risk of batch effect (mrini et al., 1995). for this reason, we removed glutaraldehyde from the fixative solution. in further cases, we also removed the washout solution from the protocol. because formaldehyde takes a significant time to fix tissue, it is unclear if a washout process is necessary to prevent fixation of debris within blood vessels when perfusing with formaldehyde, while using a washout solution adds to procedural complexity and creates a possible batch effect. in our view, the value of perfusing washout solution prior to perfusing fixative remains an open question, with both potential upsides and downsides (mcfadden et al., 2019; tao-cheng et al., 2007). a limitation of this technical report is that the recorded outcome metrics were largely qualitative. because edema was often seen and red blood cells were observed in some vessels, we expect that perfusion fixation was incomplete. however, no quantitative analysis has been performed to determine whether there is a change in autolytic artifacts, blood vessel shape, or other histologic features in the perfused brains compared to brains preserved with the standard immersion protocol (frigon et al., 2022). as a result, we were unable to quantify the extent of partial perfusion fixation. in future studies, both operational and histologic perfusion fixation quality outcome metrics should be measured more quantitatively. for example, the use of an indentation device that measures both force and displacement could precisely measure tissue stiffness (elkin et al., 2011). additionally, the use of video monitoring during the perfusion fixation procedure could help to precisely record and quantify any changes in brain pigmentation or volume. more rigorous outcome metrics would also help to monitor and make any adjustments necessary during the procedure and to optimize the procedure for future cases. one major challenge in ex situ brain perfusion is the presence of air bubbles. consistent with this, mri images from our perfusion-fixed brains showed hypointensities that may correspond to small air bubbles generated or distributed in the vasculature during the perfusion procedure. because air bubbles will accumulate in sulci, ventricles, and blood vessels as soon as the brain is extracted from the skull, it is an immense challenge to completely avoid air bubbles during ex situ perfusion fixation (bolliger et al., 2018). air bubbles can also form in the perfusion circuit, which may introduce an unnecessary impediment to perfusion flow. when air bubbles are present in blood vessels, they can theoretically obstruct the flow of fixative. for future iterations of the procedure, we are currently working on a strategy using drip chambers in the perfusion circuit to decrease the potential for air bubbles to be perfused into the blood vessels. keeping the tubing of the perfusion circle at a vertical angle may also help bubbles to rise to the top rather than continuing through the perfusion circuit. when a catheter is placed into a blood vessel for cannulation, there is a need to secure it in place, or it will move due to the perfusate flow. here, we employed hemostats to secure the catheters. with this approach, the length of vessel required for cannulation is limited by the diameter of the hemostat. with the use of our mosquito hemostats, approximately 1 cm of blood vessel is required to adequately secure the cannula. the presence of atherosclerosis did not appear to affect the ability to seal the cannulated vessels. however, hemostats are unwieldy and can obstruct the field of vision. an alternative approach is to use surgical sutures; however, this requires significant skill and takes more time. we are currently evaluating alternative approaches to address the problem of securing catheters in the blood vessels. it is also important to note the necessity of clamping arteries that are downstream of the cannulated artery and that are not wished to be perfused. the failure to clamp severed superior cerebellar arteries may have contributed to the fixative accumulation that occurred during perfusion through the basilar artery in case 4. the two main methods for driving fluid in perfusion fixation are gravity-based systems and peristaltic pumps. with gravity, a constant pressure is provided with variable flow rate, whereas with a peristaltic pump, a consistent oscillating flow rate is provided irrespective of resistance (scouten et al., 2006). by monitoring the in-line pressure in our peristaltic pump-based perfusion circuit, we can detect whether there is a build-up of resistance to flow, as indicated by an increase in pressure. for a given cannula size, it is possible to control the pressure by varying the pump speed. therefore, if a pressure build-up is detected, we can either decrease the pump speed and/or cannulate other arteries (schwarzmaier et al., 2022; scouten et al., 2006). because the perfusion circuit will have a higher resistance when perfusing through one blood vessel as opposed to multiple, lower flow rates may need to be used when perfusing through only one vessel, to avoid raising the perfusion pressure to excessive values, as was seen in case 5. the ideal pressure value for perfusion fixation, or even the one that best simulates brain-specific physiologic perfusion pressure, is unclear. at lower perfusion pressures, the perfusion may be incomplete and hence fail to sufficiently extravasate into the tissue; while at higher perfusion pressures, the blood brain barrier could be disrupted or blood vessels could rupture (saliani et al., 2017; schwarzmaier et al., 2022). currently, we target an in-line perfusion pressure in the range of 70-100 mm hg based on theoretical expectations. further investigation is necessary to query the optimal perfusion pressure for the ex situ human brain. we speculate that the tissue swelling that we sometimes detected may have resulted from an overly high perfusion pressure (schwarzmaier et al., 2022). if tissue swelling is detected, suggesting increased resistance to flow, then an alternative approach may be to decrease the perfusion pressure and to perfuse for a longer period of time. related to perfusion pressure is the issue of postmortem interval. the postmortem intervals in our sample varied considerably, from 7 hours to 52 hours, which likely contributed to differences in tissue or blood vessel integrity between cases. we hypothesize that factors contributing to resistance to flow are likely to accumulate in brains that have undergone a more severe agonal state or longer postmortem interval prior to the procedure. in those cases, a higher perfusion pressure may be necessary to achieve sufficient perfusate flow throughout the brain. determining the optimal perfusion pressure range in brain banking and identifying ways to modify the perfusion pressure during the procedure are important areas for further research. in addition to uncertainties regarding perfusion pressure, the duration of perfusion fixation to provide adequate tissue fixation is also unclear. in rodent studies, as few as 5 minutes of perfusion fixation at optimal pressure has been found to be sufficient for proper tissue preservation (schwarzmaier et al., 2022). in brain banking studies, however, reported times of perfusion fixation have varied widely, from 5 minutes to 2 hours, with 15-30 minutes being a commonly reported time range (mcfadden et al., 2019). because of the potential for inadequate perfusate flow in human brains, methods that allow for precise monitoring during the perfusion procedure, such as ultrasound or mri, may help investigators to tailor the perfusion time for each brain, albeit at higher cost (vrselja et al., 2019). taken together, the amount of time required for adequate perfusion fixation and postfixation in brain banking that preserves tissue morphology while minimizing alterations of antigenicity is an important open research question. conclusion in our standard immersion fixation protocol, the whole brain or hemibrain is immersed in formalin for two to four weeks prior to further sectioning and storage procedures. based on previous data and biophysical principles, there are theoretical reasons to expect that preservation using the perfusion fixation protocol may yield improved histology quality (mcfadden et al., 2019). however, we do not have any systematic outcome data comparing our perfusion procedure to an immersion procedure. we also encountered several challenges in implementing the perfusion fixation procedure, including the intermittent occurrence of tissue swelling and the potential for introducing air bubbles into the vasculature. the added value to preservation quality using a perfusion fixation approach may depend in part on the agonal phase and the postmortem interval, during which thrombi may accumulate and blood vessels undergo autolysis, thereby restricting perfusate flow (hansma et al., 2015). further research and development are needed to improve the rigor and reproducibility of perfusion fixation methods and to assess the value of the procedure in improving preservation quality. acknowledgements the authors would like to thank helmut heinsen, henry waldvogel, kátia cristina de oliveira, maglóczky zsófia, ricardo insausti, and thomas beach for helpful advice in setting up the perfusion fixation system. we gratefully acknowledge the following funding sources: nih grants rf1mh128969, r01ag062348, rf1ns115268, r01ag054008, r01ns095252, r01ag060961, r01ns086736, an alzheimer’s disease research center (adrc) developmental project funding award to a.t.m. (p30ag066514), the rainwater charitable foundation, and an alexander saint-amand fellowship to j.f.c. abbreviations pbs: phosphate buffered saline; pmi: postmortem interval; mri: magnetic resonance imaging; rpm: rotations per minute. author contributions a.t.m., k.d.-o.c., a.s., z.w., e.m.c.h., and j.f.c. contributed to the original conception of the study; a.t.m., e.w.-l., a.c., h.w., and j.f.c. performed perfusion fixation studies; e.w.-l. and d.d. performed histology studies; k.w. performed rna in situ hybridization studies; a.s. performed mri imaging and data analysis; a.t.m. and j.f.c. wrote the original draft of the manuscript. all authors read and approved the manuscript. conflict of interest the authors declare that they have no competing financial interests. data availability all histology and mri data presented is available to the academic community upon request. references adickes, e.d., folkerth, r.d., sims, k.l., 1997. use of perfusion fixation for improved neuropathologic examination. arch. pathol. lab. med. 121, 1199–1206. baleriola, j., walker, c.a., jean, y.y., crary, j.f., troy, c.m., nagy, p.l., hengst, u., 2014. axonally synthesized atf4 transmits a neurodegenerative signal across brain regions. cell 158, 1159–1172. https://doi.org/10.1016/j.cell.2014.07.001 beach, t.g., tago, h., nagai, t., kimura, h., mcgeer, p.l., mcgeer, e.g., 1987. perfusion-fixation of the human brain for immunohistochemistry: comparison with immersion-fixation. j. neurosci. methods 19, 183–192. https://doi.org/10.1016/s0165-0270(87)80001-8 bodian, d., 1936. a new method for staining nerve fibers and nerve endings in mounted paraffin sections. anat. rec. 65, 89–97. https://doi.org/10.1002/ar.1090650110 bolliger, s.a., tomasin, d., heimer, j., richter, h., thali, m.j., gascho, d., 2018. rapid and reliable detection of previous freezing of cerebral tissue by computed tomography and magnetic resonance imaging. forensic sci. med. pathol. 14, 85–94. https://doi.org/10.1007/s12024-018-9955-0 boonstra, j.t., michielse, s., roebroeck, a., temel, y., jahanshahi, a., 2021. dedicated container for postmortem human brain ultra-high field magnetic resonance imaging. neuroimage 235, 118010. https://doi.org/10.1016/j.neuroimage.2021.118010 de oliveira, k.c., nery, f.g., ferreti, r.e.l., lima, m.c., cappi, c., machado-lima, a., polichiso, l., carreira, l.l., ávila, c., alho, a.t.d.l., brentani, h.p., miguel, e.c., heinsen, h., jacob-filho, w., pasqualucci, c.a., lafer, b., grinberg, l.t., 2012. brazilian psychiatric brain bank: a new contribution tool to network studies. cell tissue bank. 13, 315–326. https://doi.org/10.1007/s10561-011-9258-0 elkin, b.s., ilankova, a., morrison, b., 2011. dynamic, regional mechanical properties of the porcine brain: indentation in the coronal plane. j. biomech. eng. 133, 071009. https://doi.org/10.1115/1.4004494 frigon, è.-m., dadar, m., boire, d., maranzano, j., 2022. antigenicity is preserved with fixative solutions used in human gross anatomy: a mice brain immunohistochemistry study. https://doi.org/10.1101/2022.05.06.490908 grinberg, l.t., amaro, e., teipel, s., dos santos, d.d., pasqualucci, c.a., leite, r.e.p., camargo, c.r., gonçalves, j.a., sanches, a.g., santana, m., ferretti, r.e.l., jacob-filho, w., nitrini, r., heinsen, h., brazilian aging brain study group, 2008. assessment of factors that confound mri and neuropathological correlation of human postmortem brain tissue. cell tissue bank. 9, 195–203. https://doi.org/10.1007/s10561-008-9080-5 griswold, m.a., jakob, p.m., heidemann, r.m., nittka, m., jellus, v., wang, j., kiefer, b., haase, a., 2002. generalized autocalibrating partially parallel acquisitions (grappa). magn. reson. med. 47, 1202–1210. https://doi.org/10.1002/mrm.10171 halliday, g.m., li, y.w., joh, t.h., cotton, r.g., howe, p.r., geffen, l.b., blessing, w.w., 1988. distribution of substance p-like immunoreactive neurons in the human medulla oblongata: co-localization with monoamine-synthesizing neurons. synapse 2, 353–370. https://doi.org/10.1002/syn.890020403 hansma, p., powers, s., diaz, f., li, w., 2015. agonal thrombi at autopsy. am. j. forensic med. pathol. 36, 141–144. https://doi.org/10.1097/paf.0000000000000162 ikeda, m., nagashima, t., bhattacharjee, a.k., kondoh, t., kohmura, e., tamaki, n., 2003. quantitative analysis of hyperosmotic and hypothermic blood-brain barrier opening. acta neurochir. suppl. 86, 559–563. https://doi.org/10.1007/978-3-7091-0651-8_114 insausti, r., tuñón, t., sobreviela, t., insausti, a.m., gonzalo, l.m., 1995. the human entorhinal cortex: a cytoarchitectonic analysis. j. comp. neurol. 355, 171–198. https://doi.org/10.1002/cne.903550203 jenkinson, m., bannister, p., brady, m., smith, s., 2002. improved optimization for the robust and accurate linear registration and motion correction of brain images. neuroimage 17, 825–841. https://doi.org/10.1016/s1053-8119(02)91132-8 jenkinson, m., smith, s., 2001. a global optimisation method for robust affine registration of brain images. med. image anal. 5, 143–156. https://doi.org/10.1016/s1361-8415(01)00036-6 karlsson, u., schultz, r.l., 1965. fixation of the central nervous system for electron microscopy by aldehyde perfusion. 1. preservation with aldehyde perfusates versus direct perfusion with osmium tetroxide with special reference to membranes and the extracellular space. j. ultrastruct. res. 12, 160–186. https://doi.org/10.1016/s0022-5320(65)80014-4 lucassen, p.j., chung, w.c., kamphorst, w., swaab, d.f., 1997. dna damage distribution in the human brain as shown by in situ end labeling; area-specific differences in aging and alzheimer disease in the absence of apoptotic morphology. j. neuropathol. exp. neurol. https://doi.org/10.1097/00005072-199708000-00007 manger, p.r., pillay, p., maseko, b.c., bhagwandin, a., gravett, n., moon, d.-j., jillani, n., hemingway, j., 2009. acquisition of brains from the african elephant (loxodonta africana): perfusion-fixation and dissection. j. neurosci. methods 179, 16–21. https://doi.org/10.1016/j.jneumeth.2009.01.001 mcfadden, w.c., walsh, h., richter, f., soudant, c., bryce, c.h., hof, p.r., fowkes, m., crary, j.f., mckenzie, a.t., 2019. perfusion fixation in brain banking: a systematic review. acta neuropathol. commun. 7, 146. https://doi.org/10.1186/s40478-019-0799-y mrini, a., moukhles, h., jacomy, h., bosler, o., doucet, g., 1995. efficient immunodetection of various protein antigens in glutaraldehyde-fixed brain tissue. j. histochem. cytochem. off. j. histochem. soc. 43, 1285–1291. https://doi.org/10.1177/43.12.8537644 musigazi, g.u., de vleeschauwer, s., sciot, r., verbeken, e., depreitere, b., 2018. brain perfusion fixation in male pigs using a safer closed system. lab. anim. 52, 413–417. https://doi.org/10.1177/0023677217752747 nazemorroaya, a., aghaeifar, a., shiozawa, t., hirt, b., schulz, h., scheffler, k., hagberg, g.e., 2022. developing formalin-based fixative agents for post mortem brain mri at 9.4 t. magn. reson. med. 87, 2481–2494. https://doi.org/10.1002/mrm.29122 palay, s.l., mcgee-russell, s.m., gordon, s., grillo, m.a., 1962. fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide. j. cell biol. 12, 385–410. https://doi.org/10.1083/jcb.12.2.385 pallebage-gamarallage, m., foxley, s., menke, r.a.l., huszar, i.n., jenkinson, m., tendler, b.c., wang, c., jbabdi, s., turner, m.r., miller, k.l., ansorge, o., 2018. dissecting the pathobiology of altered mri signal in amyotrophic lateral sclerosis: a post mortem whole brain sampling strategy for the integration of ultra-high-field mri and quantitative neuropathology. bmc neurosci. 19, 11. https://doi.org/10.1186/s12868-018-0416-1 patel, k.b., liang, w., casper, m.j., voleti, v., li, w., yagielski, a.j., zhao, h.t., perez campos, c., lee, g.s., liu, j.m., philipone, e., yoon, a.j., olive, k.p., coley, s.m., hillman, e.m.c., 2022. high-speed light-sheet microscopy for the in-situ acquisition of volumetric histological images of living tissue. nat. biomed. eng. 6, 569–583. https://doi.org/10.1038/s41551-022-00849-7 saliani, a., perraud, b., duval, t., stikov, n., rossignol, s., cohen-adad, j., 2017. axon and myelin morphology in animal and human spinal cord. front. neuroanat. 11. https://doi.org/10.3389/fnana.2017.00129 schwarzmaier, s.m., knarr, m.r.o., hu, s., ertürk, a., hellal, f., plesnila, n., 2022. perfusion pressure determines vascular integrity and histomorphological quality following perfusion fixation of the brain. j. neurosci. methods 372, 109493. https://doi.org/10.1016/j.jneumeth.2022.109493 scouten, c.w., o’connor, r., cunningham, m., 2006. perfusion fixation of research animals. microsc. today 14, 26–33. https://doi.org/10.1017/s1551929500057631 shapson-coe, a., januszewski, m., berger, d.r., pope, a., wu, y., blakely, t., schalek, r.l., li, p.h., wang, s., maitin-shepard, j., karlupia, n., dorkenwald, s., sjostedt, e., leavitt, l., lee, d., bailey, l., fitzmaurice, a., kar, r., field, b., wu, h., wagner-carena, j., aley, d., lau, j., lin, z., wei, d., pfister, h., peleg, a., jain, v., lichtman, j.w., 2021. a connectomic study of a petascale fragment of human cerebral cortex. https://doi.org/10.1101/2021.05.29.446289 stram, m.n., seifert, a.c., cortes, e., akyatan, a., woodoff-leith, e., borukhov, v., tetlow, a., alyemni, d., greenberg, m., gupta, a., krausert, a., mecca, l., rodriguez, s., stahl-herz, j., guzman, m.a., delman, b., crary, j.f., dams-o’connor, k., folkerth, r.d., 2022. neuropathology of pediatric sars-cov-2 infection in the forensic setting: novel application of ex vivo imaging in analysis of brain microvasculature. front. neurol. 13, 894565. https://doi.org/10.3389/fneur.2022.894565 tao-cheng, j.-h., gallant, p.e., brightman, m.w., dosemeci, a., reese, t.s., 2007. structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. j. comp. neurol. 501, 731–740. https://doi.org/10.1002/cne.21276 vrselja, z., daniele, s.g., silbereis, j., talpo, f., morozov, y.m., sousa, a.m.m., tanaka, b.s., skarica, m., pletikos, m., kaur, n., zhuang, z.w., liu, z., alkawadri, r., sinusas, a.j., latham, s.r., waxman, s.g., sestan, n., 2019. restoration of brain circulation and cellular functions hours post-mortem. nature 568, 336–343. https://doi.org/10.1038/s41586-019-1099-1 waldvogel, h.j., curtis, m.a., baer, k., rees, m.i., faull, r.l.m., 2006. immunohistochemical staining of post-mortem adult human brain sections. nat. protoc. 1, 2719–2732. https://doi.org/10.1038/nprot.2006.354 wang, f., flanagan, j., su, n., wang, l.-c., bui, s., nielson, a., wu, x., vo, h.-t., ma, x.-j., luo, y., 2012. rnascope: a novel in situ rna analysis platform for formalin-fixed, paraffin-embedded tissues. j. mol. diagn. jmd 14, 22–29. https://doi.org/10.1016/j.jmoldx.2011.08.002 welikovitch, l.a., do carmo, s., maglóczky, z., szocsics, p., lőke, j., freund, t., cuello, a.c., 2018. evidence of intraneuronal aβ accumulation preceding tau pathology in the entorhinal cortex. acta neuropathol. (berl.) 136, 901–917. https://doi.org/10.1007/s00401-018-1922-z copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodevelopmental disorders: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:6 (2021) review neurodevelopmental disorders: 2021 update alfonsa zamora-moratalla1, maría martínez de lagrán1, mara dierssen1,2,3 1 centre for genomic regulation (crg), the barcelona institute of science and technology, dr. aiguader 88, barcelona 08003, spain 2 universitat pompeu fabra (upf), barcelona, spain 3 centro de investigación biomédica en red de enfermedades raras (ciberer), spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona · spain mara.dierssen@crg.eu submitted: 23 february 2021 accepted: 12 march 2021 copyedited by: cathryn cadwell published: 24 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3268 keywords: autism spectrum disorder, asd, neurodegenerative disorders, next generation sequencing, microbiome, preterm birth abstract one of the current challenges in the field of neurodevelopmental disorders (ndds) is still to determine their underlying aetiology and risk factors. ndds comprise a diverse group of disorders primarily related to neurodevelopmental dysfunction including autism spectrum disorder (asd), developmental delay, intellectual disability (id), and attention-deficit/hyperactivity disorder (adhd) that may present with a certain degree of cognitive dysfunction and high prevalence of neuropsychiatric outcomes. last year, advances in human genomics have begun to shed light on the genetic architecture of these disorders and large-scale sequencing studies are starting to reveal mechanisms that range from unique genomic dna methylation patterns (i.e. “episignatures”) to highly polygenic conditions. in addition, the contribution of de novo somatic mutations to neurodevelopmental diseases is being recognized. however, progressing from genetic findings to underlying neuropathological mechanisms has proved challenging, due to the increased resolution of the molecular and genetic assays. advancement in modelling tools is likely to improve our understanding of the origin of neurodevelopmental disorders and provide insight into their developmental mechanisms. also, combined in vivo editing of multiple genes and single-cell rna-sequencing (scrna-seq) are bringing us into a new era of understanding the molecular neuropathology of ndds. abbreviations adhd attention-deficit/hyperactivity disorder, art assisted reproductive technology, asd -autism spectrum disorder, atac-seq assay for transposase-accessible chromatin using sequencing, cas9 crispr-associated protein 9, cb cingulum bundle, chip-seq chromatin immunoprecipitation sequencing, cnvs copy-number variants, crispr clustered regularly interspaced short palindromic repeats, degs differentially expressed genes, dlpfc -dorsolateral prefrontal cortex, eeg electroencephalographic, e/i excitation and inhibition, encode encyclopedia of dna elements, ept extremely preterm, ewas epigenome-wide association studies, flhs floating-harbor syndrome, geo gene expression omnibus, gf germ-free, go gene ontology, gw gestational week, hd huntington disease, hnpcs human neural progenitor cells, htt huntingtin, id intellectual disability, ipscs induced pluripotent stem cells, iq intelligence quotient, lgd likely gene-disruptive, lincrna long intergenic noncoding rna, mipscs miniature inhibitory postsynaptic currents, mhtt mutant htt, mri magnetic resonance imaging, ndds neurodevelopmental disorders, nfl neurofilament light protein, nlgs neuroligins, npcs neural progenitor cells, nrt nucleus reticularis thalami, nrxs neurexins, pmds phelan-mcdermid syndrome, pscs pluripotent stem cells, prehd premanifest hd gene carriers, prdd-seq parallel rna and dna analysis after deep sequencing, ptms post-translational modifications, rem rapid eye movement, rna-seq rna-sequencing, scrna-seq single-cell rna-seq, rsts1 rubinstein-taybi syndrome 1, sipscs spontaneous inhibitory postsynaptic currents, spf specific pathogen free, swds spike-wave discharges, vz ventricular zone, wes whole-exome sequencing, wgs whole-genome sequencing introduction one of the current challenges in the field of neurodevelopmental disorders (ndds) is still to determine their underlying aetiology and risk factors. ndds comprise a diverse group of disorders primarily related to neurodevelopmental dysfunction including autism spectrum disorder (asd), developmental delay, intellectual disability (id), and attention-deficit/hyperactivity disorder (adhd) that may present with a certain degree of cognitive dysfunction and high prevalence of neuropsychiatric outcomes. last year, advances in human genomics have begun to shed light on the genetic architecture of these disorders and large-scale sequencing studies are starting to reveal mechanisms that range from unique genomic dna methylation patterns (i.e. “epi-signatures”) to highly polygenic conditions. in addition, the contribution of de novo somatic mutations to neurodevelopmental diseases is being recognized. however, progressing from genetic findings to underlying neuropathological mechanisms has proved challenging, due to the increased resolution of the molecular and genetic assays. advancement in modelling tools is likely to improve our understanding of the origin of neurodevelopmental disorders and provide insight into their developmental mechanisms. also, combined in vivo editing of multiple genes and single-cell rna-sequencing (scrna-seq) are bringing us into a new era of understanding the molecular neuropathology of ndds. 1. progress in the use of big data for the understanding the neuropathology of neurodevelopmental disorders most nnds have a major inherited component which compromises correct brain development; however, the evaluation of the genetic causes of ndds remains challenging because of genetic and phenotypic heterogeneity. one well-known example is asd, which has been associated with mutations in a wide range of genes, but relatively few genes or loci are identified in sufficient cases to prove statistical significance at the genome-wide level. such rare genetic developmental diseases are a challenge for diagnosis, as patients with the same genetic defect present with varying degrees of symptoms and phenotypes (haghshenas et al., 2020), that can be due to molecular interactions between their associated genes, as in the case of floating-harbor syndrome (flhs) and rubinstein-taybi syndrome 1 (rsts1). in the last years, the fast pace of development of next-generation sequencing technologies, such as gene panels, whole-exome sequencing (wes), and whole-genome sequencing (wgs) technologies have enhanced our ability to diagnose the ndds. of these, wes achieves a diagnostic rate of 30–53% for ndds and wgs improves the diagnostic rates even more (42-62%). the use of high-throughput allows broader range of variant detection, including noncoding and regulatory regions, and the discovery of novel disease-associated genes, has shed light on the genetic, genomic and epigenomic key players of ndds and will also help in the diagnosis and stratification of the population with those disorders. changes in gene expression are also widely studied to characterize various human diseases and successfully used to predict molecular and cellular processes in complex neurodevelopmental diseases. high-throughput expression profiling has become routine, and as a consequence, a vast amount of public rna-sequencing (rna-seq) datasets has been generated and is available from online repositories, such as gene expression omnibus (geo; barrett et al., 2013). up to the second quarter of 2019, geo hosted more than 112,000 data series comprising more than 3,000,000 samples. this massive amount of biological data brings great opportunity for generating prominent biological hypotheses and is widely used to shed light into neuropathological mechanisms. however, these large datasets were produced for diverse purposes, are sometimes difficult to interpret, and are not friendly to large-scale data integration. therefore, increasing sophistication in the statistical methods and well-organized resources are required to enable efficient and extensive integrated analysis. one interesting example is the work of rahman et al., who performed a meta-analysis using two publicly available rna-seq studies from asd cerebral cortex (rahman et al., 2020). they performed integrative rna-seq gene expression profiling in cortex to identify transcriptional gene signatures altered in 15 asd subjects compared to 15 controls. the study revealed core signatures of differentially expressed genes (degs) associated with asd, including already known markers of asd and novel hub genes. the authors detected 235 unique degs, not identified by the individual studies, supporting the increased statistical power of the meta-analysis approach (rau et al., 2014; walker et al., 2008). seven of these degs (pak1, dnah17, dock8, dapp1, pcdhac2, erbin, and slc7a7), were previously reported to be deferentially expressed in asd. gene ontology (go) and pathways analysis was then used as a tool for identifying molecular pathways enriched by the degs. in their meta-analysis, rahman et al. showed altered osteoclast differentiation, tnf signaling pathway, and complement and coagulation cascade pathways in asd, revealing new previously unidentified genes. moreover, topological analysis of protein–protein interaction of the asd brain cortex revealed proteomics hub gene signatures. however, although meta-analysis is a powerful tool, it is also controversial due to the heterogeneity in terms of platform (different microarray and sequencing techniques), and sources (from peripheral blood to induced pluripotent stem cells [ipscs] or other tissues). jensen et al. (jensen et al., 2020), in an interesting study, added yet another level of complexity: the existence of comorbidities between ndds. this is an important step, given the high degree of co-occurrence of autism with id that may blur the de novo variants and copy-number variants (cnvs) identified in autistic individuals. the study analyzed 2290 individuals from the simons simplex collection for de novo likely gene-disruptive (lgd) variants and cnvs, and determined their relevance regarding intelligence quotient (iq) and social responsiveness scale measures. by analyzing pathogenic de novo genetic variants in individuals with autism who had either id or normal cognitive function, they determined whether genes associated with autism also contribute towards id comorbidity. their study showed that pathogenic de novo variants disrupting autism-associated genes contribute towards autism and id comorbidity so that gene discoveries in autism are biased towards genes that also contribute towards comorbid id (jensen et al., 2020). in contrast, individuals with high-functioning autism are less probable to carry de novo lgd variants in candidate autism genes and tend to present with disruption of genes with less functional relevance towards neurodevelopment. these results highlight the relevance of dissecting phenotypic heterogeneity in family-based sequencing studies of complex diseases. another recent example is the identification of a non-syndromic asd subtype characterized by dyslipidemia using massive multimodal data triangulation from wes, neurodevelopmental expression patterns, electronic health records and healthcare claims (luo et al., 2020). 2. dna episignatures genes associated with asd are enriched for pathways affecting neuronal homeostasis and embryonic development. however, no single genetic variant has been found that accounts for more than 1% of disease liability. this may be in part due to the fact that environmental factors are also known to contribute to asd risk, especially during the prenatal period. epigenetic mechanisms including dna methylation, histone post-translational modifications (ptms), noncoding rna, and chromatin architecture have been proposed to account for the sex bias, gene-environment interactions, and developmental origins of asd aetiology (ciernia & lasalle, 2016). epigenome-wide association studies (ewas) have identified asd-associated locus-specific differential methylation of genes involved in synaptic transmission or microglia, and disease-associated epigenetic signatures (nardone et al., 2014). ewas in post-mortem brains have identified one of the most novel and promising areas of research of ndds, the identification of dna methylation called "episignatures". those are defined as the cumulative dna methylation patterns occurring at multiple cpg dinucleotides across the genome. in the last years, an expanding number of genetic syndromes have been shown to have unique genomic dna methylation patterns. the first clinical genome-wide dna methylation assay, “episign,” used genome-wide dna methylation analysis for the screening of 14 syndromes known to harbor episignatures. this first study showed that dna methylation patterns are stable and specific to certain syndromes, and occur consistently across all of the individuals affected with the same syndrome (aref-eshghi et al., 2018). this has been confirmed recently by the identification of 34 disease-specific episignatures mapping onto 42 genetic syndromes, thus increasing the number of conditions that can effectively be diagnosed through dna methylation testing (aref-eshghi et al., 2020). the authors examined emerging patterns of overlap, and similarities and hierarchical relationships across episignatures. aref-eshghi et al. implemented a uniform approach for mapping dna methylation signatures in numerous syndromes to enable unbiased comparisons. this allowed identification of their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. through mass screening of a large cohort of subjects with developmental delays and congenital anomalies, they demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases. an interesting study has taken a slightly different approach. mordaunt et al. using whole-genome bisulfite sequencing identified a distinct dna methylation signature over regulatory regions and genes relevant to early fetal neurodevelopment in the cord blood from newborns later diagnosed with asd (mordaunt et al., 2020). the development of the forebrain, including the assembly of the expanded human cerebral cortex, linked to the distinctively human features affected in ndds, is a lengthy process that involves diversification and expansion of neural progenitors, generation and positioning of layer-specific glutamatergic neurons, cellular migration of gabaergic neurons, and formation and maturation of glial cells. disruption of these cellular events by either genetic or environmental factors can lead to neurodevelopmental disease, including asd and id. these complex cellular processes require highly synchronized regulatory activity underlying these events, which, if perturbed, can cause disease. the authors had previously used whole-genome bisulfite sequencing in placenta samples and identified differential methylation of genes associated with asd (zhu et al., 2019). now, they obtained umbilical cord blood samples from asd and typically developing subjects from two high-familial risk prospective cohorts (i.e., cohorts following child’s early development of younger siblings of a child already diagnosed with asd through) in order to identify an epigenomic signature of asd at birth. their findings suggest that epigenetic dysregulation in asd may originate during early prenatal development in a sex-specific manner and converge on brain-relevant genes to disrupt neurodevelopment. although the study has some limitations as the selection of high-familial risk cohorts and the limitation in statistical power, it opens a new framework for the prognosis and diagnosis of ndds. 3. chromatin dynamics in neurodevelopment as mentioned above, epigenetic gene regulation plays a crucial role in controlling developmental transitions and cell differentiation, and is widely hypothesized to partly mediate risk for ndds such as asd or schizophrenia. therefore, tracking epigenetic changes in specific forebrain cell lineages over long time periods, has the potential to unravel the molecular programs that underlie cell specification in the human cerebral cortex and, by temporally mapping disease risk onto these changes, to identify cell types and periods of increased disease susceptibility. in the last years, chromatin accessibility has emerged as an accurate proxy for the cellular regulatory potential. chromatin encodes epigenetic information in the form of post-translational histone modifications and accessibility to dna binding factors (allis & jenuwein, 2016). chromatin regulation is affected by genetic alterations causative of ndds (de rubeis et al., 2014; pinto et al., 2014; lasalle, 2013), suggesting the existence of two classes of ndds. the first class is produced by mutations in chromatin regulators, as occurs in rett syndrome (schmidt et al., 2020). importantly, those may target several convergent molecular axes, with genes either belonging to the same class (e.g. lysine demethylases) or operating in the same molecular pathway (e.g. polycomb-mediated chromatin regulation). the second class comprises those ndds caused by environmentally-induced epigenetic dysfunction. interestingly, accessible chromatin regions exhibit a high heritability enrichment, indicating that sequence conservation can further refine functional risk genetic variants for disorders with a strong neurodevelopmental component, such as schizophrenia (bryois et al., 2018). efforts to define the transcriptomic and epigenomic landscapes of the developing human forebrain included multilevel analyses with characterization of spatiotemporal gene expression in the cortex (pollen et al., 2015; nowakowski et al., 2018), the molecular signature of cortical progenitors (johnson et al., 2015; pollen et al., 2015), and epigenetics of early brain development (amiri et al., 2018). now, gorkin and partners of the encyclopaedia of dna elements (encode) project, have launched an atlas of chromatin development (gorkin et al., 2020), a genomic resource profiling epigenomics of mammalian development. they initially used a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states to systematically examine the state and accessibility of chromatin in the developing mouse fetus. to map chromatin states, the authors performed chromatin immunoprecipitation with sequencing (chip–seq) for a set of eight histone modifications that can distinguish between functional elements and activity levels. methods like chromatin immunoprecipitation and reduced representation bisulfite sequencing allow the investigation of epigenetic modifications on a genome-wide scale. however, one of the potential limitations of these methods is that you need to already have an idea about what epigenetic mechanisms are at play. thus, the authors complemented this technique with assay for transposase-accessible chromatin using sequencing (atac–seq; buenrostro et al., 2013), optimized for use on frozen tissues, to assay chromatin accessibility. atac-seq identifies accessible dna regions by probing open chromatin with hyperactive mutant tn5 transposase (picelli et al., 2014) that inserts sequencing adapters into open regions of the genome. the authors systematically mapped chromatin state and accessibility across 72 distinct tissue-stages of mouse development, and carried out integrative analyses incorporating additional epigenomic and transcriptomic data sets from the same tissue-stages. importantly, their analysis allowed them to integrate chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. as such, they could identify target genes and demonstrate tissue-specific enrichments of variants associated with disease in humans. approximately 1–4% of the genome differed in chromatin state between tissues at the same stage, and 0.03–3% differed between adjacent stages of the same tissue. this resource will help to map genetic risk for disease and to shed light into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders. data from this and all phases of encode are publicly available through the encode portal (https://www.encodeproject.org). although encouraging, data on mouse development do not completely mimic human forebrain development, which is, to a large extent, inaccessible for cellular-level study. the lack of availability of primary brain tissue samples and the limitations of conventional in vitro cellular models have precluded a detailed mechanistic understanding of corticogenesis in disease states. using long-term three-dimensional (3d) directed differentiation of human pluripotent stem cells (pscs) into dorsal and ventral forebrain domains as well as primary brain tissue samples, a recently published work (trevino et al., 2020) found that organoids intrinsically undergo chromatin state transitions that are closely related to human forebrain development. trevino et al., also applied atac-seq in combination with rna-seq to map the epigenetic and gene expression signatures of neuronal and glial cell lineages over 20 months in vitro. the authors identified epigenetic alterations putatively driven by specific transcription factors and discovered a dynamic period of chromatin remodeling during human cortical neurogenesis identifying key transcription factors that may coordinate over time to drive these changes. this approach may bring new insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders. 4. sequencing perturbed cortex development the growing number of genetic disruptions identified in human genetic studies far exceeds the ability to study their functions, or perform rigorous genotype-phenotype correlations, which may vary substantially across different cell types and states. until now, genetic screens have systematically analyzed individual gene function in mammalian cells or in vivo in knockout models, analyzing each perturbation separately. also, screens have been performed in a pooled format, measuring, for example, cell autonomous phenotypes, such as growth, drug resistance, or marker expression. both screening methods are time and labor intensive and do not allow the study of genetic interactions. comprehensive analysis of genetic interactions has been performed in yeast between pairs of genes (costanzo et al., 2016). in mammals, only small sets of pre-selected pairs have been assessed for cell viability (bassik et al., 2013) or morphology (laufer et al., 2013), but very few studies have examined higher order interactions or coupled those with a high content scalable readout. the newest addition to the genomic arsenal is single-cell clustered regularly interspaced short palindromic repeats (crispr) screening techniques, independently termed perturb-seq, crisp-seq, or crop-seq, that combine pooled crispr screening with scrna-seq to allow functional crispr screening in single-cells. crispr are dna sequences found in the genomes of prokaryotic organisms such as bacteria and archaea, which are used to detect and destroy dna from similar bacteriophages during subsequent infections. cas9 (or "crispr-associated protein 9") is an enzyme that uses crispr as a guide to recognize and cleave specific strands of dna. cas9 enzymes together with crispr sequences form the basis of the crispr-cas9 technology, used for gene editing. crispr targeting in vivo, especially in mammals, can be difficult and time consuming when attempting to determine the effects of more than a single gene. however, such studies may be required to identify pathological gene variants with effects in specific cells along a developmental trajectory. pool gene targeting followed by single-cell rna-sequencing of perturbed cells in the brain is a powerful methodology to reveal traits of individual cells in heterogeneous populations such as those in brain tissue (dixit et al., 2016). perturbations with single-cell sequencing readouts with increased throughput and enhanced resolution offer the possibility to explore the dynamics of transcription factors in development and genes whose expressions are differential between cell states or across the different areas of a tissue. perturb-seq was developed in 2016 (dixit et al., 2016), involving crispr/cas9 to perform multi-locus gene perturbation with massive parallel scrna-seq. as cellular behaviors typically depend on coordinated expression of many genes and translated proteins, unbiased sequencing methods can extract genome-wide profiles from single cells without prior knowledge. jin et al. (2020) have now used perturb-seq to explore the effects of in vivo genetic disruptions of risk genes of asd or ndds across diverse cells in the developing mouse cortex combined with single-cell transcriptome sequencing. they evaluated 35 asd de novo loss-of-function risk genes in multiple mouse embryos, using crispr-cas9 to introduce frameshift mutations in pools of these risk genes. this allowed them to investigate how diverse mutations affect cell types and states in the developing organism. this method identified networks of gene expression in neuronal and glial cells that suggest new functions in asd-related genes. using weighted gene correlation network analysis, they identified 14 covarying gene modules representing transcriptional programs in different types of cortical cells that affect common biological processes across multiple cell types and/or represent cell type–specific features. perturbations in nine of those asd/ndd genes had significant effects across cortical projection neurons, cortical inhibitory neurons, astrocytes, and oligodendrocytes. one example is ankyrin, which interacts with ion channels in excitatory neurons and stabilizes gabaergic synapses. ank2 mutants show misregulation of intracellular calcium homeostasis and calcium channel expression in excitatory neurons, and ectopic connectivity, but now perturb-seq data could identify additional roles of ank2 in those interneurons co-expressing the ndnf gene. oligodendrocytes and astrocytes were also affected by multiple risk gene perturbations. for example, chd8 modulates oligodendrocyte differentiation and maturation, by directly interacting with oligodendrocyte maturation genes. as highlighted in a comment on this work (treutlein & camp, 2020) in vivo perturb-seq can reveal neuronal and glial effects of sets of asd/ndd risk genes associated with autism. 5. study of lineage diversification in the developing neocortex as shown above, asd susceptibility genes are strongly interconnected and many act as genetic regulators of neurodevelopment of the cerebral cortex. however, the core underlying neuropathologies cannot be fully elucidated without understanding the cellular architecture of the human cortex, underlying its susceptibility to disease. the cerebral cortex is a complex structure formed by a wide repertoire of neural cells shaping its unique configuration. in only two areas of the adult mouse neocortex, single-cell transcriptomics has already identified at least 55 excitatory and 60 inhibitory neuron types (ecker et al., 2017; tasic et al., 2016) and a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected in the middle temporal gyrus of human cortex (hodge et al., 2019). such tremendous cellular complexity of mature neurons is especially intriguing, given that those come from a limited number of progenitors. this apparent paradox leads to one of the most crucial questions in neurodevelopment: how the myriad of neuronal varieties located in the neocortex can arise from a reduced number of progenitor types. to solve this question, researchers are using next-generation single cell functional genomic techniques, which enable comprehensive and unbiased characterization of the progenitor populations from the early fetal stage to neuronal maturation. to elucidate the lineage relationships among different cell types during brain development, last year huang and colleagues (huang et al., 2020) developed a novel method in which parallel rna and dna analysis after deep sequencing (prdd-seq) allows simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation in post-mortem human cerebral cortex. this innovative approach revealed some conserved human cell lineage patterns, including that inhibitory and excitatory neurons diverge early in humans, and that excitatory neurons form following a similar “inside-out” order as seen in animal models. their work shows that at least some human neural progenitor cells (hnpcs) demonstrate restricted cell type output and that excitatory and inhibitory neurons are generated from distinct progenitor regions, supporting a model initially established in mice. the authors estimate that the number of progenitor cells that generate the excitatory neurons within a cortical column is ~10. by contrast, inhibitory neurons show complex, subtype-specific patterns of neurogenesis, and not all of them are conserved relative to mouse. using single-cell rna-sequencing and in vivo validation, li et al. (2020) have now revealed previously unrecognized neural stem and progenitor cell diversity within the fetal mouse and human neocortex, including multiple types of radial glia and intermediate progenitors. this novel cellular diversity catalogue they describe is characterized by mixed transcriptional profiles and not so much by morphological classes, which may indicate unique functional or state-dependent transcriptional profiles. in addition, most of the previously unrevealed types of basal progenitors have a human counterpart, indicating again the inter-species conservation of neurodevelopmental processes. li et al. postulate that transcriptional priming, a phenomenon whereby mrna for proteins that will be expressed in progeny is present (but not translated) in the parent cell, underlies the diversification of a subset of ventricular radial glial cells in developing mouse and human brain (li et al., 2020). transcriptional priming of radial glial cells would generate specific types of basal progenitors and neurons, being thus a driver of precursor and lineage diversity. however, those studies still elude the temporal scale in which progenitor populations at the early foetal stage go through neuronal maturation. another recent study has also systematically profiled single-cell transcriptome of the four cortical lobes and the pons. however, in this study they included the temporal dimension by analyzing transcriptional landscapes from human embryo to mid gestation, covering more than 5 months of critical developmental stages (fan et al., 2020). this strategy allowed accurate temporal and spatial resolution of developmental processes. as expected, the authors observed marked distinctions between cerebral cortex and the pons, in both molecular regulations and developmental patterns. the pons, an evolutionarily ancient structure, develops earlier showing abundant interneurons at early embryonic stages whereas in the cerebral cortex interneurons start to appear beginning at the early mid-fetal stage, suggesting a delayed development of neurons in the cerebral cortex. besides, molecular and electrophysiological results pointed out an asynchronous cell development in different regions of the cortex, with maturation occurring earlier in the rostral regions than in the caudal ones, and regional differences on the lateral side of the developing cortex appeared more conspicuous during the neuron maturation stage in the frontal lobe. the authors emphasize the important role of long intergenic noncoding rna (lincrna) in regional and cell type maintenance. these temporal differences in neuronal maturation could be essential for proper neural network construction and be beyond the establishment of certain neurodevelopmental disorders. one limitation of this study, however, is that post-mortem samples are collected at different gestational ages only allow a raw estimation of the temporal evolution of cell lineages but not a longitudinal tracing of the progenitors. a tool that tracks single-cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. this is exactly what el nachef et al. (el-nachef et al., 2020) have now developed: a novel tool to track in vitro dynamic behaviors at the single-cell level based on the rainbow mouse models. they developed a stable cre-inducible rainbow reporter hpscline that provides up to 18 unique membrane-targeted fluorescent barcodes. because dna recombination is permanent the color barcode of a cell is inherited by daughter cells during cell division, enabling determination of the parental origin of cellular progeny and tracking them longitudinally by live-cell imaging. using this technique, the authors have tracked neural progenitor cells (npcs) over a month of cortical neuron differentiation into 3d cultures. npcs in the monolayer culture had self-organized into radially aligned cells forming a ventricular zone (vz) in these aggregates. the finding that monolayer cultures generate 3d cortical structures suggests that the differentiation protocol robustly recapitulates in vivo signaling where the planar neural plate gives rise to the neural tube and its derivatives through morphogenesis. in addition, longitudinal live imaging enabled determination of the timing of key neurogenesis events, such as vz formation and neurite outgrowth. these results strengthen the use of organoids derived from human pscs for the study of neurodevelopmental processes. understanding cellular mechanisms that lead to dysregulated npc expansion might help the understanding of neurodevelopmental disorders characterized by microcephaly or megacephaly. 6. microbiome: the hidden player behind neurodevelopmental disorders all the works presented above have revealed a remarkably complex molecular neuropathology involving a myriad of genetic architectures and regulatory elements, including unexpected peripheral players, such as the microbiota, in ndds. the human intestine harbors trillions of microbial cells which form a symbiotic relationship with the host and play a vital role in both health and disease (huttenhower et al., 2012). the importance of microbiota to human health is suggested by the observation that dysbiotic shifts in microbial communities have been associated with a number of human diseases, including obesity, inflammatory bowel disorders, or gastrointestinal cancer (schulz et al., 2014; flintoft, 2012; morgan et al., 2012; qin et al., 2012). recent findings also suggest that gut dysbiosis can be involved in the development of a variety of neuropsychiatric disorders, such as asd, depression and schizophrenia (grochowska et al., 2018). however, the high interindividual diversity due to genetics, age, diet, and health condition poses severe limitation to the study of asd gut microbiome (lloyd-price, 2016), the results being highly dependent on the cohort studied. the composition of intestinal microbiome affects health from the prenatal period throughout adulthood, and should be thought of as an organ system with important effects on child development (ronan et al., 2021). in fact, gut microbiota plays a major role in the bidirectional communication between the gastrointestinal tract and the central nervous system and there is increasing evidence of an important role of the gut microbiota in brain development and behavior (hoban et al., 2016; vuong et al., 2017; warner, 2019). to date, most of our understanding of the interaction between the microbiota and the brain has come from rodent models, in which the gut microbiota is linked to brain signaling mechanisms and affective phenotypes, such as anxiety or depression-like behavior. animal studies have clearly demonstrated effects of the gut microbiota on brain gene expression profiles and neurochemical metabolism impacting behavior and performance. interestingly, transfer of fecal gut microbiota from humans with depression into rodents appears to induce animal behaviors that are hypothesized to indicate depression-like states (kelly et al., 2016). germ-free (gf) animals also show clear behavioral differences compared to conventionally raised mice, or mice raised free of specific disease-causing organisms (specific pathogen free; spf) (bäckhed et al., 2007), including social interaction disturbances similar to those of asd; stress-related and anxiety-related behaviors; learning and memory deficits; and impaired motor control (vuong et al., 2017). some of the biochemical changes resulting from germ-free status are irreversible, even after colonization of the animals with normal gut microbiota later in life. other abnormalities, such as anxiety-related behavior, can be ameliorated after reconstitution of the gut microbiota (clarke et al., 2013). dysbiosis has been associated with pediatric diseases, including autism, adhd, asthma, and allergies. however, although several studies of shotgun-based metagenome analysis have been reported for asd gut microbiome (laue et al, 2020; wang et al, 2019), the high interindividual diversity, influenced by a wide range of factors such as genetics, age, diet, and health conditions (lloyd-price et al., 2016) impedes the correct identification of disease-associated microbiome features with stochastic false positives or negatives and the findings are highly dependent on the samples collected (surana & kasper, 2017). to overcome this problem, a team of researchers in china proposed a new analytical strategy, a quasi-paired cohort analysis that successfully allowed them to identify a new gut microbe deficiency in asd (zhang et al., 2020) children. in their paper, zhang et al. applied this novel strategy to a metagenomic study of the asd microbiome. they collected stool samples from 39 children diagnosed with asd, and 40 ageand sex-matched neurotypical children of similar metabolic backgrounds, referring to the profile of metabolic pathways inferred from the metagenomic data. this approach allowed them to transform the original cohort into a paired cohort, thus controlling for individual diversity and increasing statistical power. each of the stool samples were subjected to metagenomic sequencing and the team focused most specifically on 18 microbial species that have previously been linked to asd. using the quasi-paired cohort analytical strategy, the authors identified significant deficiencies in detoxifying enzymes and pathways, strongly correlated with biomarkers of mitochondrial dysfunction in asd children. then they applied diagnostic models based on these detoxifying enzymes, and could accurately distinguish asd individuals from controls of their cohort. in fact, the dysfunction score inferred from the model was proportionally increased with the clinical rating scores of asd. these results were further proofed in an independent cohort of 65 asd children. these results suggest a gut microbiome impact on detoxification in asd, and a potential role of impaired intestinal microbial detoxification in toxin accumulation and mitochondrial dysfunction, a core component of asd pathogenesis (castora, 2019). this is a very interesting finding as toxin exposure is one of the main etiological factors of asd (von ehrenstein et al., 2019). the impaired detoxification capability of the intestinal microbiome would reveal a new mechanism explaining this increased vulnerability to toxicant exposure of patients with asd. moreover, microbial detoxification capabilities could thus be a new therapeutic target for patients with asd. emerging evidence suggests that the microbiota’s influence on the central nervous system seems likely to be established as traits early in life, and potentially lead to later adverse mental health outcomes (walker et al., 2008). in fact, the gut microbiome development evolves throughout the lifespan from a composition that is simpler and more unstable during development to a highly diverse and stable composition in the adult (fouhy et al., 2012). even transient changes in gut microbial composition, induced by various intrinsic and extrinsic factors, may drive enduring shifts in the microbiota composition when the gut microbiota has not fully matured or is generally unstable, such as during early life. in fact, the vulnerability of microbiota composition during adolescence may explain why this period shows increasing incidence of psychiatric illnesses, including anxiety and mood disorders, psychosis, eating disorders, personality disorders and substance abuse (borre et al., 2014). the pathophysiology of these disorders has been commonly interpreted as arising from aberrations of maturational changes that normally occur in the adolescent brain. now, a recent study (lach et al., 2020) by lach et al. has revealed that even transient microbiota depletion has long-lasting effects on microbiota composition and leads to increased anxiety-like behavior. the study was performed in mice exposed to antibiotic treatment for 3 weeks to achieve microbiota depletion during the adolescent period or in the adulthood. this transient microbiota depletion had long-lasting effects on microbiota composition but only when antibiotics were administered in adolescent mice, in which it was associated with a significantly increased anxiety-like behavior. to further understand what pathways of communication between the gut and the brain are responsible for such changes at this time period, the authors analyzed gene expression in the amygdala and again they showed that it was more severely affected in mice treated with antibiotics during adolescence. this highlights the vulnerability of the gut microbiota during the adolescent period, influencing gene expression and behavior even in adulthood. this is especially important in the actual covid-19 crisis (fore et al., 2020; headey et al., 2020), which poses severe risks to the nutritional status of young children and adolescents, especially in lowand middle-income countries, as diet is a potentially modifiable determinant of the functionality of gut microbiome throughout life (ghosh et al., 2020; jebeile et al., 2019; liu et al., 2019; sonnenburg & sonnenburg, 2019). it is thus important not to neglect the later consequences of this crisis, forcing millions of families to rely on nutrient-poor alternatives that may not only lead to later obesity and diabetes, but also to a mental health pandemic (pan et al., 2021). 7. neuroligin in neurodevelopmental disorders alterations in synapse formation and function are a hallmark of neurodevelopmental and neuropsychiatric disorders. synapses are formed by a complex process that is controlled by synaptic organizer molecules, including trans-synaptic adhesion molecules and secreted factors (ferrer-ferrer & dityatev, 2018; yuzaki, 2018). a key component of these organizational protein complexes are the synaptic adhesion proteins called neuroligins (nlgs), a family of post-synaptic cell-adhesion proteins which act trans-synaptically with neurexins (nrxs) another group of pre-synaptic cell-adhesion molecules. the nlgs and nrxs were one of the first pairs of synaptic adhesion molecules to be characterized, and their mutation was quickly shown to be associated with autism, and later with schizophrenia (jamain et al., 2003; südhof, 2008; sun et al., 2011). synaptic organizational protein complexes play central roles both in orchestrating synapse formation and in defining the functional properties of synaptic transmission that together shape the flow of information through neuronal networks. however, although a myriad of evidence supports the involvement of genetic alterations in neuroligin genes, including point mutations, missense mutations and internal deletions in patients with asd, their precise pathogenetic mechanism in ndds still remains elusive. there are five members of the nlg family in humans and other primates: nlg1, 2, 3, 4x, and 4y. mutations in nrx1, nlg3 and nlg4 genes are linked with asd and other neurodevelopmental disorders (parente et al., 2017; etherton et al., 2011; sun et al., 2011; südhof, 2008; dean & dresbach, 2006; lisé & el-husseini, 2006). several nlgn2 variants have been identified in individuals with schizophrenia, autism, and anxiety disorder (parente et al., 2017). one longstanding hypothesis in the field is that asd arises from a disruption of the neuronal network activity due to perturbation of the synaptic excitation and inhibition (e/i) balance. the nlgs family regulates the balance between excitation and inhibition activity in the brain circuit by being able to modify the excitatory (nlgn4) / inhibitory (nlgn2) balance. in particular, the nlg2 subtype is highly expressed at inhibitory postsynaptic structures, and it is required to establish appropriate inhibitory synapse function during development, but also appears to play a role in synapse maintenance in adulthood (liang et al., 2015; troyano-rodriguez et al., 2019). as such, nlg2 is a potent regulator of the e/i balance: knockdown of nlg2 reduces gaba receptors and gaba-mediated synaptic transmission, resulting in increased neuronal excitability (liang et al., 2015) while overexpression of nlg2 in transgenic mice leads to motor discoordination, social impairment and abnormal electroencephalographic (eeg) activities, characteristics shared by asd and epilepsy (hines et al., 2008). this is interesting since high prevalence of epileptiform activity emerges as a common pathophysiological hallmark of autism (subota et al., 2017; horváth et al., 2016; plioplys et al., 2007). nlg2 might be thus a common player explaining the comorbidity, as asd and epilepsy frequently coexist in the same individual, suggesting a common neurodevelopmental basis for these disorders. however, even though genome-wide association studies recently allowed for the identification of a substantial number of genes involved in asd and epilepsy, there was no direct evidence to support the specific involvement of nlg2 in epileptic seizures. now, in a recent paper, cao et al. (cao et al., 2020) demonstrate that loss of nlg2 produces seizures and behavioral deficits that are associated with gabaergic transmission in thalamic neurons. they systematically performed cortical eeg recordings in freely moving nlg1, nlg2, and nlg3 knockout mice and their wild-type littermates in awake, rapid eye movement (rem) sleep, and non-rem sleep states, simultaneously monitoring their motor activity by electromyographic recordings. interestingly, only nlg2 knockout mice exhibited abnormal 5-8hz spontaneous spike-wave discharges (swds), an activity pattern that was correlated with behavioral arrest episodes characteristic of absence seizures. supporting their hypothesis that those arrest episodes were reflecting absence, these were ameliorated by the anti-absence seizure drug ethosuximide. this might result from the markedly reduced gabaergic and glycinergic inhibitory synaptic transmission, as assessed in the form of miniature or spontaneous inhibitory postsynaptic currents (mipscs or sipscs) in nlg2 knockout mice. in fact, restoring gabaergic transmission by optogenetic activation in a projection from the thalamic reticular nucleus to the ventrobasal thalamus also partially normalized these phenotypes, indicating that they may result from loss of feedforward inhibition in the circuit, the only region, together with the central amygdala, reported to date to have near-normal inhibitory synaptic transmission in the absence of nlg2 (babaev et al., 2016). the same rescuing effect was achieved by postsynaptic nlg2 expression in the thalamic neurons and optogenetic activation of the nucleus reticularis thalami (nrt) thalamic pathway was able to partially rescue gabaergic transmission, swds, and behavior arrests in nlg2 knockout mice. previous results have indicated that the imbalance in the excitation/inhibition tone in the thalamocortical circuitry is linked with the swds, absence seizures and other forms of epileptic behaviors (fogerson & huguenard, 2016; maheshwari & noebels, 2014; paz et al., 2011). the results of cao and colleagues now provide strong evidence that nlgn2-mediated gabaergic transmission is a powerful regulator of thalamocortical network activities related to absence seizures and may provide a molecular mechanism underlying high rates of comorbidity of asds with increased epileptic seizures, providing a link between asd phenotype and absence seizures through a nlg2-mediated inhibition at the nrt-thalamic synapse. 8. are neurodegeneration diseases programmed from development? since some years ago, accumulating evidence is revealing similarities and frequent complex interactions between neurodevelopmental and neurodegenerative disorders. neurodevelopmental conditions can manifest as lifelong weaknesses in cognitive functions and a number of works suggest that certain neurodevelopmental disorders may entail greater risk for specific neurodegenerative disorders. an interesting example is huntington disease (hd) a neurodegenerative disease characterized by dyskinesia, progressive involuntary movements, behavioral and psychiatric disturbances, and dementia. these symptoms are accompanied by widespread neurodegeneration that likely starts in the striatum (nopoulos, 2016) but also involves fronto-striatal circuitry malfunction. it is prevalent in adult individuals, with a mean age at onset of ~45 years (langbehn et al., 2010) and initiates with motor disabilities, such as involuntary movements. hd is caused by mutation of the huntingtin (htt) gene, as a result of cag trinucleotide repeat expansion with repeat length ranging from 10 to 35 in the normal population. repeat lengths between 36 and 39 cause hd at reduced penetrance, and when expanded to 40 or more repeats, mutant htt (mhtt) causes hd at full penetrance. despite symptoms of hd manifesting in adulthood, the aberrant htt protein is present much earlier in persons carrying the disease-causing mutation. in fact, htt is involved in brain development, axonal transport, synaptic function and cell survival and mhtt impairs npc division, neuronal migration and maturation (saudou & humbert, 2016). mutation of htt in early life is enough to induce hd features in adult mice and shows that there is a developmental component in the disease (arteaga-bracho et al., 2016; molero et al., 2016) and hd mice present in fact thinner cortices (godin et al., 2010). there are a number of lines of evidence that show cognitive function starts to decline and functional deficits appear around 15 years before clinical onset (lee et al., 2012; paulsen et al., 2008; tabrizi et al., 2020). an interesting approach was taken by the kids-hd study, which includes a unique cohort of children and adolescents who are at risk for adult-onset hd (lee et al., 2017). a recent study on this cohort using neuroimaging data of children and adolescent carriers of mhtt showed that developmental trajectories in the striatum and globus pallidus were markedly different between gene-expanded and gene-non-expanded individuals (van der plas et al., 2019), a pattern that was exaggerated with cag expansion length > 50. the striking difference in developmental patterns suggests that pathogenesis of hd begins with abnormal brain development, where children who carry the gene expansion exhibit different trajectories of brain growth than those who did not inherit the expansion. now, scahill et al. (2020) have showed that clinical biomarkers of hd neurodegeneration such as cerebrospinal fluid neurofilament light protein (nfl) are already significantly elevated as early as 24 years from predicted onset of clinical symptoms in a cohort of premanifest hd gene carriers (prehd) young adults, in which motor, cognitive, and psychiatric function are preserved,. a curious finding of tereshchenko et al. (tereshchenko et al. 2020) also indicates that measures of growth are abnormal in child and adolescent carriers of mhtt decades before hd onset. specifically, gene expanded males were taller than gene non-expanded males in adolescents of similar weight. furthermore, barnat et al. (barnat et al. 2020) and difiglia (difiglia 2020), have gone even earlier, and studied human brain cortex of hd mutation carrier fetuses and healthy controls at gestational week 13 (gw13) and of a hd knock-in mouse model at embryonic day 13.5 (e13.5), which correspond to gw13. in this phase of development, progenitor cells in the vz are extending processes towards the apical and basal surfaces of the neuroepithelial wall. the authors found, both in human fetuses and in mouse embryos, abnormalities in the developing cortex, including mislocalization of mhtt and junctional complex proteins, defects in npc polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. in normal development, npcs in the vz reach out to both the apical and basal surfaces of the neuroepithelial wall, and their cellular nuclei shuttle back and forth as the cell cycle progresses. with the aberrant mhtt, epithelial junctions are disrupted, epithelial polarity is disturbed, and the cell cycle favors premature neuronal differentiation. these data are in good agreement with previous experimental data that htt regulates cellular adhesion, polarity, and epithelial organization (godin et al., 2010). in summary, alterations in neurodegenerative genes or proteins during developmental stages might program neurodegeneration disorders in adulthood. however, given their neurodevelopmental effects, it is unknown why these disorders do not emerge until adult stages. 9. shank-3: the missing link the wide variety of ndds that present with motor symptoms is well established. in recent years, there has been an increased interest in the motor deficits of asd in order to find a target to diagnose the disease as early as possible in young children. however, the landscape of motor deficits is quite broad and may involve brain, peripheral nervous system or neuromuscular junction. asd patients may present with motor-related abnormalities such as delayed motor development (ozonoff et al., 2008), impairment of gross and fine motor functions (provost et al, 2007) and deficits in basic motor control (jansiewicz et al., 2006). some patients may have trouble with coordinating movements between the left and right side of the body, low muscle tone or balance. one possible candidate to explain part of these symptoms is shank3. haploinsufficiency of the shank3 gene represents one of the most common single-gene mutations in asd and has been associated with neural circuit alterations in several brain areas (holder and quach, 2016; leblond et al., 2014; betancur et al., 2013). shank3 is a structural protein accumulated in postsynaptic densities, which contributes to excitatory synapse function and plays various roles in development (wang et al., 2014). moreover, the shank3 gene is the main contributing factor in phelan-mcdermid syndrome (pmds) that is generally characterized by neonatal skeletal muscle hypotonia despite normal growth, severely delayed speech, moderate to profound developmental delay, and mild dysmorphic features (sarasua et al., 2014; kolevzon et al., 2014; soorya et al., 2013). hypotonia is critical for diagnosis because it is one of the earliest clinical hallmarks of pmds. indeed lutz et al. (lutz et al., 2020) now reported the first experimental evidence that directly links the mutation of shank3 with peripheral muscle atrophy. they investigated the role of shank3 on motor unit development using a combination of patient-derived human ipscs, shank3δ11 (−/−) mice, and pmds muscle biopsies from patients. the authors show that hypotonia in shank3 deficiency might be caused by dysfunction in all elements of the voluntary motor system: motoneurons, neuromuscular junctions and striated muscle fibers. these findings provide a new perspective on the function of shank3, beyond the central nervous system, and suggests a future treatment strategy for shank3-associated asd. furthermore, recent evidence shows that somatosensory neurons in shank3-mutated mice are dysfunctional and contribute to tactile phenotypes (orefice et al., 2019). therefore, impaired proprioception and altered somatosensory feedback to the central nervous system of patients with pmds and asd may worsen symptoms and induce a delay in motor development. abnormal functional communication between brain areas has recently arisen as a feasible biomarker for asds (emerson et al., 2017; lewis et al., 2013). interestingly, zhou et al. (2019) showed evidence of atypical behavior and altered brain circuits in shank3-mutant macaques. the authors used crispr-cas9-mediated gene-editing technology to genetically engineer non-human primate models (macacafascicularis and their offspring), which might better approximate the behavioral and neural phenotypes of asds than do rodent models. magnetic resonance imaging (mri) in shank3 mutants revealed altered local and global connectivity patterns among brain areas that were indicative of circuit abnormalities. this was paralleled by social and learning impairments, sleep disturbances, repetitive behaviours and motor deficits. the works of lutz et al. (2020) and zhou et al. (2019), together, make it clear that shank3 protein is affecting the physiological functioning of the motor system from the cerebral cortex to the nerve endings of the body's extremities. in addition to these findings, using multiparametric mri analysis by diffusion tensor imaging and volumetry, it has recently been shown that the lack of shank3 induces mainly changes in white matter (jesse et al., 2019). in shank3-deficient patients with pmds the authors found a significant pattern of white matter alterations in the long association fiber tracts. white matter alterations were also observed in heterozygous isoform-specific shank3 knockout mice, with a minimal change in gray matter. the delayed achievement of motor milestones seen in pmds may be associated with white matter changes in the sensorimotor, cortico-striatal and cortico-spinal tracts. these abnormalities during development could affect myelin formation and connections between different brain areas, altering the flow of information in brain networks, and leading to the motor disturbances and asd associated behaviors observed in shank3 mutant mice (jamarillo et al., 2020). interestingly, early genetic restoration of shank3 improves the behavioral deficits (figure 1). asd shows a very complex pathology associated with shank3 mutations that induce alterations in functional and structural motor network organization within the central and periphery nervous system. further investigation using different animal models will be needed to move these findings from basic research to the clinic and ultimately in order to find new therapeutic strategies at early ages. figure 1: possible mechanisms associated with shank3 mutation. mutant shank3 induces deficits in the central and peripheral nervous system. early genetic restoration of shank3 is able to prevent the beginning of the asd-like behavioral deficits. possible mechanisms associated with shank3 rescue are depicted. 10. the effects of being too early prematurity, gestational age, or weight at birth have been identified as a risk factor for ndds, including those distinguished by autistic features. brain injury is more severe in extremely preterm (ept) newborns (those born with less than 25-30 weeks gestational age). their survival has increased remarkably in the last decades, making even more obvious the consequences of underdevelopment. data from 70’s indicated that survival was less than 20%, while recent data in the united kingdom reports that 35% of the babies born at 22 weeks, 38% born at 23 weeks, and 60% born at 24 weeks, survive (mactier et al., 2020). among the babies who survive, the risk of complications or ongoing disability increases with decreased gestational age. among the possible complications are anxiety, asd, cerebral palsy, epilepsy and a significant number of cases of cognitive impairment is detected during childhood. however, the majority of survivors do not have a severe, life-limiting disability (marret et al., 2013). thus, to what extend ept children have long-term deficits at different neurological levels needs to be explored. last year, erdei et al. reported that ept children had higher rates of cognitive impairment relative to their term-born peers, with odds rate of any impairment ranging from 3.7 at the age of two, to 5.0 at age 6 (erdei et al., 2020) however, most children born ept experience mild (27.2%) rather than severe (10.7%) cognitive impairment at 12 years. also, a recent study from the epicure team that followed a cohort of 5,391 infants born ept in the united kingdom and ireland from 1995, found that 60% of young adults (19-year-old) who were ept are impaired in at least one neuropsychological area, often cognition and visuomotor abilities (o’reilly et al., 2020). the cohort continues to present lower scores compared to term-born peers in general cognitive functioning (iq), visuomotor skills, as well as in prospective memory and aspects of language and executive functions. interestingly, the damage seems progressive, as the proportion of ept participants with intellectual impairment (iq <70) increased by 6.7% between 11 and 19 years of age. for a small proportion of ept individuals, intellectual impairment may only become apparent later in adolescence or adulthood when cognitive demands become more complex. the heterogeneity in the onset and severity of the neuropsychological and cognitive outcomes among the ept individuals and how to predict neurodevelopment impairments is one of the unsolved issues in the field. from the neurobiological point of view, the ept prenatal period concurs with the final stages of neurogenesis in the human telencephalon, neuronal migration, differentiation and maturation, and the very early stages of cortical myelination, conferring to the brain high susceptibility. therefore, interruption of the normal fetal environment during the development process is expected to have an impact in the brain conformation and connectivity. many neuropathological hallmarks of brain injury are found in preterm born infants (burkitt et al., 2019; yum et al., 2019; strunk et al., 2014) including neuroinflammation, oligodendrocyte maturation arrest and hypomyelination, axonopathy, reduced fractional anisotropy and cortical volume as determined by mri, and many of these differences persist into adolescence and adulthood. the neuroimaging studies revealed alterations in white and grey matter microstructure, impaired cortical folding and disturbances to regional brain growth (hinojosa-rodríguez et al., 2017). a recent study comparing brain connectivity using resting state functional mri in very premature infants and age-matched healthy fetuses with structurally normal brain mris, revealed stronger connectivity in sensory input and stress-related areas in premature infants suggesting that extra-uterine environment exposure alters the development of select neural networks even in the absence of structural brain injury (de asis-cruz et al., 2020). brain structures whose maturation is highly dependent on external stimuli during the postnatal period are more susceptible to being affected due to the housing conditions, poverty of maternal care and associated pathologies along with the ept condition. this is the case of the thalamocortical pathway whose maturation is subjected to critical developmental windows during the neonatal period (kostoví & judaš, 2002). the thalamus is a relevant structure as it acts as a hub for integration of cortical activity which regulates cortical power across a range of frequencies (malekmohammadi et al., 2015; fitzgerald et al., 2013). recently, the relation between thalamic myelination level and its neurophysiological functioning at rest in preterm infants has been explored (nunes et al., 2020). this study revealed that the thalamic volume reduction is associated with atypical frequency-specific neurophysiological power in preterm birth. moreover, thalamic intensity is associated with negative neurocognitive outcomes. neurocognitive difficulties were associated with increased frontal and occipital delta, and occipital theta, as well as decreased power mostly involving frontal and occipital beta activity. these data together suggest that environmental exposure in preterm newborns might play a crucial role in the outcome of neurophysiological outcomes, being relevant to the development of standardized protocols of medical care during the earlier postnatal life of ept individuals. other studies are focusing more on the analysis of the spatial microstructure alterations and not so much on long-range connections, with the aim of predicting the outcomes based on the heterogeneity among ept individuals. one latest example is the imaging study of the cingulum bundle (cb), an anterior-to-posterior white matter tract connecting prefrontal areas with the insula, amygdala and hippocampal and parahippocampal cortices that has been associated with complex attention processes (i.e., shifting and divided attention [schulte et al., 2008]). the maturation of the cb can be compromised in ept since myelination begins postnatally and continues until the first 2 years of life. brenner et al. have shown in a longitudinal study in preterm infants at 5 years of age that white matter microstructure is altered specifically in the right anterior cb and correlates with the emergence of psychiatric symptoms (brenner et al., 2021). this is interesting as dysfunction of anterior cb has been associated with internalizing disorders, autism, and attention-deficit/hyperactivity disorder. based on the existing knowledge of the precise spatial and temporal mapping of cortical gene expression during the human fetal period, ball et al. (ball et al., 2020) have linked molecular mechanisms using gene expression patterns to non-invasive measures of cortical development by mri in early life (ball et al., 2020). the authors demonstrated that non-invasive imaging of the cortical structure in the neonatal brain is sensitive to differential spatiotemporal patterns of gene expression during gestation. specifically, regional variation in cortical morphometry and microstructure reflects differences in developmental maturity and tissue composition across cortical areas. in addition, they found that interruption to this developmental programming by preterm birth is associated with significant cortical alterations that appear to reflect the selective vulnerability of developing cortical glial populations. nowadays, mainly due to late maternal age, assisted reproductive technology (art) is widely used as a solution for specific infertility problems. however, although the association between the use of art and preterm birth and associated neurodevelopmental risks has been explored in several studies (liu et al., 2017) the result is still inconclusive and has mainly been explored for asd. to analyze the existent of risk of neurodevelopmental disorders among children who are born from this technology, djuwantono and colleagues (djuwantono et al., 2020) published a meta-analysis aimed at quantifying the relative risks of cerebral palsy, intellectual disability, asd, and behavioral problems in children born from different art methods. their study indicates that children conceived by art are at higher risk of acquiring cerebral palsy. cerebral palsy has been associated with preterm birth and low body weight, features also common with art. remarkably, the intracytoplasmic sperm injection technique increased risks of intellectual disability and asd (catford et al., 2018). this technique is mainly recommended in cases of sperm immobility and poor-quality oocytes, associated with higher rates of dna fragmentation as are also advanced maternal age, unexplained infertility, or cryopreservation (inaba et al., 2016). this might be related to neurodevelopmental disorders, as some studies correlate infantile autism with elevated degree of dna damage (porokhovnik et al., 2016). sperm dna damage might be associated with reduced numbers and proportion of trophectoderm cells, promote embryo arrest and induce the activation of apoptotic machinery, all associated with higher prevalence of neurodevelopmental disorders (sedó et al., 2017). colophon the combination of new sequencing techniques and more efficient gene editing tools, with machine learning and deep learning techniques is revealing unexpected complexities in the cellular and molecular neuropathology of ndds. also, revisiting older concepts with new perspectives is changing the perspectives of how development may influence brain function in the long term. we are certainly entering in a new and exciting era of the genomic neuropathology, that combined with classical techniques will help in understanding the rules behind pathogenesis of disturbed brain development. acknowledgements the lab of md is supported by the secretaria d’universitats i recerca del departament d’economia i coneixement de la generalitat de catalunya (grups consolidats 2017 sgr 926). we also acknowledge the support of the agencia estatal de investigación (pid2019-110755rb-i00/aei / 10.13039/501100011033), the european union's horizon 2020 research and innovation programme under grant agreement no 848077, jerôme lejeune foundation, nih (grant number: 1r01eb 028159-01), marató tv3 (#2016/20-30), jpnd heroes project. we acknowledge support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa and the cerca programme / generalitat de catalunya. the ciber of rare diseases (ciberer) is an initiative of the isciii. references allis, c. d., & jenuwein, t. (2016). the molecular hallmarks of epigenetic control. nature reviews genetics, 17(8), 487–500. https://doi.org/10.1038/nrg.2016.59 amiri, a., coppola1, g., roychowdhury, t., liu f., pochareddy, s., shin y., safi a., song, l., zhu, y., sousa, a. m. m., gerstein, m., crawford, g. e., sestan, n., abyzov a., vaccarino, f. m. (2018). transcriptome and epigenome landscape of human cortical development modeled in organoids. science, 362(6240), eaat6720. https://doi.org/10.1126/science.aat6720 aref-eshghi, e., kerkhof, j., pedro, v. p., barat-houari, m., ruiz-pallares, n., andrau, j. c., lacombe, d., van-gils, j., fergelot, p., dubourg, c., cormier-daire, v., rondeau, s., lecoquierre, f., saugier-veber, p., nicolas, g., lesca, g., chatron, n., sanlaville, d., vitobello, a., … sadikovic, b. (2020). evaluation of dna methylation episignatures for diagnosis and phenotype correlations in 42 mendelian neurodevelopmental disorders. american journal of human genetics, 106(3), 356–370. https://doi.org/10.1016/j.ajhg.2020.01.019 aref-eshghi, e., rodenhiser, d. i., schenkel, l. c., lin, h., skinner, c., ainsworth, p., paré, g., hood, r. l., bulman, d. e., kernohan, k. d., boycott, k. m., campeau, p. m., schwartz, c., & sadikovic, b. (2018). genomic dna methylation signatures enable concurrent diagnosis and clinical genetic variant classification in neurodevelopmental syndromes. american journal of human genetics, 102(1), 156–174. https://doi.org/10.1016/j.ajhg.2017.12.008 arteaga-bracho, e. e., gulinello, m., winchester, m. l., pichamoorthy, n., petronglo, j. r., zambrano, a. d., inocencio, j., de jesus, c. d., louie, j. o., gokhan, s., mehler, m. f., & molero, a. e. (2016). postnatal and adult consequences of loss of huntingtin during development: implications for huntington’s disease. neurobiology of disease, 96, 144–155. https://doi.org/10.1016/j.nbd.2016.09.006 babaev, o., botta, p., meyer, e., müller, c., ehrenreich, h., brose, n., lüthi, a., & krueger-burg, d. (2016). neuroligin 2 deletion alters inhibitory synapse function and anxiety-associated neuronal activation in the amygdala. neuropharmacology, 100, 56–65. https://doi.org/10.1016/j.neuropharm.2015.06.016 bäckhed, f., manchester, j. k., semenkovich, c. f., & gordon, j. i. (2007). mechanisms underlying the resistance to diet-induced obesity in germ-free mice. proceedings of the national academy of sciences of the united states of america, 104(3), 979–984. https://doi.org/10.1073/pnas.0605374104 ball, g., seidlitz, j., o’muircheartaigh, j., dimitrova, r., fenchel, d., makropoulos, a., christiaens, d., schuh, a., passerat-palmbach, j., hutter, j., cordero-grande, l., hughes, e., price, a., hajnal, j. v., rueckert, d., robinson, e. c., & edwards, a. d. (2020). cortical morphology at birth reflects spatiotemporal patterns of gene expression in the fetal human brain. plos biology, 18(11), e3000976. https://doi.org/10.1371/journal.pbio.3000976 barnat, m., capizzi, m., aparicio, e., boluda, s., wennagel, d., kacher, r., kassem, r., lenoir, s., agasse, f., bra, b. y., liu, j. p., ighil, j., tessier, a., zeitli, s. o., duyckaerts, c., dommergues, m., durr, a., & humbert, s. (2020). huntington’s disease alters human neurodevelopment. science, 369(6505), 787–793. https://doi.org/10.1126/science.aax3338 barrett, t., wilhite, s. e., ledoux, p., evangelista, c., kim, i. f., tomashevsky, m., marshall, k. a., phillippy, k. h., sherman, p. m., holko, m., yefanov, a., lee, h., zhang, n., robertson, c. l., serova, n., davis, s., & soboleva, a. (2013). ncbi geo: archive for functional genomics data sets update. nucleic acids research, 41(d1), d991–d995. https://doi.org/10.1093/nar/gks1193 bassik, m. c., kampmann, m., lebbink, r. j., wang, s., hein, m. y., poser, i., weibezahn, j., horlbeck, m. a., chen, s., mann, m., hyman, a. a., leproust, e. m., mcmanus, m. t., & weissman, j. s. (2013). a systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. cell, 152(4), 909–922. https://doi.org/10.1016/j.cell.2013.01.030 betancur, c., & buxbaum, j. d. (2013). shank3 haploinsufficiency: a “common” but underdiagnosed highly penetrant monogenic cause of autism spectrum disorders. molecular autism, 4(1). https://doi.org/10.1186/2040-2392-4-17 borre, y. e., o’keeffe, g. w., clarke, g., stanton, c., dinan, t. g., & cryan, j. f. (2014). microbiota and neurodevelopmental windows: implications for brain disorders. trends in molecular medicine, 20(9), 509–518. https://doi.org/10.1016/j.molmed.2014.05.002 brenner, r. g., smyser, c. d., lean, r. e., kenley, j. k., smyser, t. a., cyr, p. e. p., shimony, j. s., barch, d. m., & rogers, c. e. (2021). microstructure of the dorsal anterior cingulum bundle in very preterm neonates predicts the preterm behavioral phenotype at 5 years of age. biological psychiatry, 89(5), 433–442. https://doi.org/10.1016/j.biopsych.2020.06.015 bryois, j., garrett, m. e., song, l., safi, a., giusti-rodriguez, p., johnson, g. d., shieh, a. w., buil, a., fullard, j. f., roussos, p., sklar, p., akbarian, s., haroutunian, v., stockmeier, c. a., wray, g. a., white, k. p., liu, c., reddy, t. e., ashley-koch, a., … crawford, g. e. (2018). evaluation of chromatin accessibility in prefrontal cortex of individuals with schizophrenia. nature communications, 9(1). https://doi.org/10.1038/s41467-018-05379-y buenrostro, j. d., giresi, p. g., zaba, l. c., chang, h. y., & greenleaf, w. j. (2013). transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, dna-binding proteins and nucleosome position. nature methods, 10(12), 1213–1218. https://doi.org/10.1038/nmeth.2688 burkitt, k., kang, o., jyoti, r., mohamed, a. l., & chaudhari, t. (2019). comparison of cranial ultrasound and mri for detecting brain injury in extremely preterm infants and correlation with neurological outcomes at 1 and 3 years. european journal of pediatrics, 178(7), 1053–1061. https://doi.org/10.1007/s00431-019-03388-7 cao, f., liu, j. j., zhou, s., cortez, m. a., snead, o. c., han, j., & jia, z. (2020). neuroligin 2 regulates absence seizures and behavioral arrests through gabaergic transmission within the thalamocortical circuitry. nature communications, 11(1), 3744. https://doi.org/10.1038/s41467-020-17560-3 castora, f. j. (2019). mitochondrial function and abnormalities implicated in the pathogenesis of asd. progress in neuro-psychopharmacology and biological psychiatry, 92, 83–108. https://doi.org/10.1016/j.pnpbp.2018.12.015 catford, s. r., mclachlan, r. i., o’bryan, m. k., & halliday, j. l. (2018). long-term follow-up of icsi-conceived offspring compared with spontaneously conceived offspring: a systematic review of health outcomes beyond the neonatal period. andrology, 6(5), 635–653. https://doi.org/10.1111/andr.12526 ciernia, a. v., & lasalle, j. (2016). the landscape of dna methylation amid a perfect storm of autism aetiologies. nature reviews neuroscience, 17(7), 411–423. https://doi.org/10.1038/nrn.2016.41 clarke, g., grenham, s., scully, p., fitzgerald, p., moloney, r. d., shanahan, f., dinan, t. g., & cryan, j. f. (2013). the microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner. molecular psychiatry, 18(6), 666–673. https://doi.org/10.1038/mp.2012.77 costanzo, m., vandersluis, b., koch, e. n., baryshnikova, a., pons, c., tan, g., wang, w., usaj, m., hanchard, j., lee, s. d., pelechano, v., styles, e. b., billmann, m., van leeuwen, j., van dyk, n., lin, z. y., kuzmin, e., nelson, j., piotrowski, j. s., … boone, c. (2016). a global genetic interaction network maps a wiring diagram of cellular function. science, 353(6306), aaf1420. https://doi.org/10.1126/science.aaf1420 de asis-cruz, j., kapse, k., basu, s. k., said, m., murnick, j., chang, t., du plessis, a., imaging, b., science, d., & haven, n. (2020). molecular identity of human outer radial glia during cortical development. neuroimage, 219, 1–25. https://doi.org/10.1016/j.neuroimage.2020.117043 de rubeis, s., he, x., goldberg, a. p., poultney, c. s., samocha, k., cicek, a. e., kou, y., liu, l., fromer, m., walker, s., singh, t., klei, l., kosmicki, j., fu, s. c., aleksic, b., biscaldi, m., bolton, p. f., brownfeld, j. m., cai, j., … buxbaum, j. d. (2014). synaptic, transcriptional and chromatin genes disrupted in autism. nature, 515(7526), 209–215. https://doi.org/10.1038/nature13772 dean, c., & dresbach, t. (2006). neuroligins and neurexins: linking cell adhesion, synapse formation and cognitive function. trends in neurosciences, 29(1), 21–29. https://doi.org/10.1016/j.tins.2005.11.003 difiglia, m. (2020). an early start to huntington’s disease. science, 369(6505), 771–772. https://doi.org/10.1126/science.abd6215 dixit, a., parnas, o., li, b., chen, j., fulco, c. p., jerby-arnon, l., marjanovic, n. d., dionne, d., burks, t., raychowdhury, r., adamson, b., norman, t. m., lander, e. s., weissman, j. s., friedman, n., & regev, a. (2016). perturb-seq: dissecting molecular circuits with scalable single-cell rna profiling of pooled genetic screens. cell, 167(7), 1853-1866.e17. https://doi.org/10.1016/j.cell.2016.11.038 djuwantono, t., aviani, j. k., permadi, w., achmad, t. h., & halim, d. (2020). risk of neurodevelopmental disorders in children born from different art treatments: a systematic review and meta-analysis. journal of neurodevelopmental disorders, 12(1), 1–13. https://doi.org/10.1186/s11689-020-09347-w ecker, j. r., geschwind, d. h., kriegstein, a. r., ngai, j., osten, p., polioudakis, d., regev, a., sestan, n., wickersham, i. r., & zeng, h. (2017). the brain initiative cell census consortium: lessons learned toward generating a comprehensive brain cell atlas. neuron, 96(3), 542–557. https://doi.org/10.1016/j.neuron.2017.10.007 el-nachef, d., shi, k., beussman, k. m., martinez, r., regier, m. c., everett, g. w., murry, c. e., stevens, k. r., young, j. e., sniadecki, n. j., & davis, j. (2020). a rainbow reporter tracks single cells and reveals heterogeneous cellular dynamics among pluripotent stem cells and their differentiated derivatives. stem cell reports, 15(1), 226–241. https://doi.org/10.1016/j.stemcr.2020.06.005 emerson, r. w., adams, c., nishino, t., hazlett, h. c., wolff, j. j., zwaigenbaum, l., constantino, j. n., shen, m. d., swanson, m. r., elison, j. t., kandala, s., estes, a. m., botteron, k. n., collins, l., dager, s. r., evans, a. c., gerig, g., gu, h., mckinstry, r. c., … piven, j. (2017). functional neuroimaging of high-risk 6-month-old infants predicts a diagnosis of autism at 24 months of age. science translational medicine, 9(393). https://doi.org/10.1126/scitranslmed.aag2882 erdei, c., austin, n. c., cherkerzian, s., morris, a. r., & woodward, l. j. (2020). predicting school-aged cognitive impairment in children born very preterm. pediatrics, 145(4), e20191982. https://doi.org/10.1542/peds.2019-1982 etherton, m., földy, c., sharma, m., tabuchi, k., liu, x., shamloo, m., malenka, r. c., & südhof, t. c. (2011). autism-linked neuroligin-3 r451c mutation differentially alters hippocampal and cortical synaptic function. proceedings of the national academy of sciences of the united states of america, 108(33), 13764–13769. https://doi.org/10.1073/pnas.1111093108 fan, x., fu, y., zhou, x., sun, l., yang, m., wang, m., chen, r., wu, q., yong, j., dong, j., wen, l., qiao, j., wang, x., & tang, f. (2020). single-cell transcriptome analysis reveals cell lineage specification in temporal-spatial patterns in human cortical development. science advances, 6(34), eaaz2978. https://doi.org/10.1126/sciadv.aaz2978 ferrer-ferrer, m., & dityatev, a. (2018). shaping synapses by the neural extracellular matrix. frontiers in neuroanatomy, 12(40). https://doi.org/10.3389/fnana.2018.00040 fitzgerald, t. h. b., valentin, a., selway, r., & richardson, m. p. (2013). cross-frequency coupling within and between the human thalamus and neocortex. frontiers in neuroengineering, 7(84). https://doi.org/10.3389/fnhum.2013.00084 flintoft, l. (2012). disease genomics: associations go metagenome-wide. nature reviews genetics, 13(11), 756–757. https://doi.org/10.1038/nrg3347 fogerson, p. m., & huguenard, j. r. (2016). tapping the brakes: cellular and synaptic mechanisms that regulate thalamic oscillations. neuron, 92(4), 687–704. https://doi.org/10.1016/j.neuron.2016.10.024 fore, h. h., dongyu, q., beasley, d. m., & ghebreyesus, t. a. (2020). child malnutrition and covid-19: the time to act is now. the lancet, 396(10250), 517–518). https://doi.org/10.1016/s0140-6736(20)31648-2 fouhy, f., ross, r. p., fitzgerald, g., stanton, c., & cotter, p. d. (2012). composition of the early intestinal microbiota:knowledge, knowledge gaps and the use of high-throughput sequencing to address these gaps. gut microbes, 3(3), 203–220. https://doi.org/10.4161/gmic.20169 ghosh, t. s., rampelli, s., jeffery, i. b., santoro, a., neto, m., capri, m., giampieri, e., jennings, a., candela, m., turroni, s., zoetendal, e. g., hermes, g. d. a., elodie, c., meunier, n., brugere, c. m., pujos-guillot, e., berendsen, a. m., de groot, l. c. p. g. m., feskins, e. j. m., … o’toole, p. w. (2020). mediterranean diet intervention alters the gut microbiome in older people reducing frailty and improving health status: the nu-age 1-year dietary intervention across five european countries. gut, 69(7), 1218–1228. https://doi.org/10.1136/gutjnl-2019-319654 godin, j. d., colombo, k., molina-calavita, m., keryer, g., zala, d., charrin, b. e. c., dietrich, p., volvert, m. l., guillemot, f., dragatsis, i., bellaiche, y., saudou, f., nguyen, l., & humbert, s. (2010). huntingtin is required for mitotic spindle orientation and mammalian neurogenesis. neuron, 67(3), 392–406. https://doi.org/10.1016/j.neuron.2010.06.027 gorkin, d. u., barozzi, i., zhao, y., zhang, y., huang, h., lee, a. y., li, b., chiou, j., wildberg, a., ding, b., zhang, b., wang, m., strattan, j. s., davidson, j. m., qiu, y., afzal, v., akiyama, j. a., plajzer-frick, i., novak, c. s., … ren, b. (2020). an atlas of dynamic chromatin landscapes in mouse fetal development. nature, 583(7818), 744–751. https://doi.org/10.1038/s41586-020-2093-3 grochowska, m., wojnar, m., & radkowski, m. (2018). the gut microbiota in neuropsychiatric disorders. acta neurobiologiae experimentalis, 78(2), 69–81. https://doi.org/10.21307/ane‑2018‑008 haghshenas, s., bhai, p., aref-eshghi, e., & sadikovic, b. (2020). diagnostic utility of genome-wide dna methylation analysis in mendelian neurodevelopmental disorders. international journal of molecular sciences, 21(23), 1–14. https://doi.org/10.3390/ijms21239303 headey, d., heidkamp, r., osendarp, s., ruel, m., scott, n., black, r., shekar, m., bouis, h., flory, a., haddad, l., & walker, n. (2020). impacts of covid-19 on childhood malnutrition and nutrition-related mortality. the lancet, 396(10250), 519–521. https://doi.org/10.1016/s0140-6736(20)31647-0 hines, r. m., wu, l., hines, d. j., steenland, h., mansour, s., dahlhaus, r., singaraja, r. r., cao, x., sammler, e., hormuzdi, s. g., zhuo, m., & el-husseini, a. (2008). synaptic imbalance, stereotypies, and impaired social interactions in mice with altered neuroligin 2 expression. journal of neuroscience, 28(24), 6055–6067. https://doi.org/10.1523/jneurosci.0032-08.2008 hinojosa-rodríguez, m., harmony, t., carrillo-prado, c., van horn, j. d., irimia, a., torgerson, c., & jacokes, z. (2017). clinical neuroimaging in the preterm infant: diagnosis and prognosis. neuroimage: clinical, 16, 355–368. https://doi.org/10.1016/j.nicl.2017.08.015 hoban, a. e., moloney, r. d., golubeva, a. v., mcvey neufeld, k. a., o’sullivan, o., patterson, e., stanton, c., dinan, t. g., clarke, g., & cryan, j. f. (2016). behavioural and neurochemical consequences of chronic gut microbiota depletion during adulthood in the rat. neuroscience, 339, 463–477. https://doi.org/10.1016/j.neuroscience.2016.10.003 hodge, r. d., bakken, t. e., miller, j. a., smith, k. a., barkan, e. r., graybuck, l. t., close, j. l., long, b., johansen, n., penn, o., yao, z., eggermont, j., höllt, t., levi, b. p., shehata, s. i., aevermann, b., beller, a., bertagnolli, d., brouner, k., … lein, e. s. (2019). conserved cell types with divergent features in human versus mouse cortex. nature, 573(7772), 61–68. https://doi.org/10.1038/s41586-019-1506-7 holder, j. l., & quach, m. m. (2016). the spectrum of epilepsy and electroencephalographic abnormalities due to shank3 loss-of-function mutations. epilepsia, 57(10), 1651–1659. https://doi.org/10.1111/epi.13506 horváth, a., szcs, a., barcs, g., noebels, j. l., & kamondi, a. (2016). epileptic seizures in alzheimer disease. alzheimer disease and associated disorders, 30(2), 186–192. https://doi.org/10.1097/wad.0000000000000134 huang, a. y., li, p., rodin, r. e., kim, s. n., dou, y., kenny, c. j., akula, s. k., hodge, r. d., bakken, t. e., miller, j. a., lein, e. s., park, p. j., lee, e. a., & walsh, c. a. (2020). parallel rna and dna analysis after deep sequencing (prdd-seq) reveals cell type-specific lineage patterns in human brain. proceedings of the national academy of sciences of the united states of america, 117(25), 13886–13895. https://doi.org/10.1073/pnas.2006163117 huttenhower, c., gevers, d., knight, r., abubucker, s., badger, j. h., chinwalla, a. t., creasy, h. h., earl, a. m., fitzgerald, m. g., fulton, r. s., giglio, m. g., hallsworth-pepin, k., lobos, e. a., madupu, r., magrini, v., martin, j. c., mitreva, m., muzny, d. m., sodergren, e. j., … white, o. (2012). structure, function and diversity of the healthy human microbiome. nature, 486(7402), 207–214. https://doi.org/10.1038/nature11234 inaba, y., miyashita, s., somfai, t., geshi, m., matoba, s., dochi, o., & nagai, t. (2016). cryopreservation method affects dna fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts. cryobiology, 72(2), 86–92. https://doi.org/10.1016/j.cryobiol.2016.03.006 jamain, s., quach, h., betancur, c., råstam, m., colineaux, c., gillberg, c., soderstrom, h., giros, b., leboyer, m., gillberg, c., bourgeron, t., nydén, a., philippe, a., cohen, d., chabane, n., mouren-siméoni, m. c., brice, a., sponheim, e., spurkland, i., … van maldergem, l. (2003). mutations of the x-linked genes encoding neuroligins nlgn3 and nlgn4 are associated with autism. nature genetics, 34(1), 27–29. https://doi.org/10.1038/ng1136 jansiewicz, e. m., goldberg, m. c., newschaffer, c. j., denckla, m. b., landa, r., & mostofsky, s. h. (2006). motor signs distinguish children with high functioning autism and asperger’s syndrome from controls. journal of autism and developmental disorders, 36(5), 613–621. https://doi.org/10.1007/s10803-006-0109-y jaramillo tc, xuan z, reimers jm, escamilla co, liu s, powell cm (2020) early restoration of shank3 expression in shank3 knock-out mice prevents core asd-like behavioral phenotypes. eneuro. 7(3), eneuro.0332-19.2020. https://doi: 10.1523/eneuro.0332-19.2020 jebeile, h., gow, m. l., baur, l. a., garnett, s. p., paxton, s. j., & lister, n. b. (2019). association of pediatric obesity treatment, including a dietary component, with change in depression and anxiety: a systematic review and meta-analysis. jama pediatrics, 173(11), e192841. https://doi.org/10.1001/jamapediatrics.2019.2841 jensen, m., smolen, c., & girirajan, s. (2020). gene discoveries in autism are biased towards comorbidity with intellectual disability. journal of medical genetics, 57(9), 647–652. https://doi.org/10.1136/jmedgenet-2019-106476 jesse s, müller hp, schoen m, asoglu h, bockmann j, huppertz hj, rasche v, ludolph ac, boeckers tm, kassubek j (2019) severe white matter damage in shank3 deficiency: a human and translational study. ann clintranslneurol 7(1), 46-58. https://doi: 10.1002/acn3.50959 jin, x., simmons, s. k., guo, a., shetty, a. s., ko, m., nguyen, l., jokhi, v., robinson, e., oyler, p., curry, n., deangeli, g., lodato, s., levin, j. z., regev, a., zhang, f., & arlotta, p. (2020). in vivo perturb-seq reveals neuronal and glial abnormalities associated with autism risk genes. science, 370(6520), eaaz6063. https://doi.org/10.1126/science.aaz6063 johnson mb, wang pp, atabay kd, murphy ea, doan rn, hecht jl, walsh ca. (2015) single-cell analysis reveals transcriptional heterogeneity of neural progenitors in human cortex. nat neurosci. 18(5), 637-46. https://doi: 10.1038/nn.3980 kelly, j. r., borre, y., o’ brien, c., patterson, e., el aidy, s., deane, j., kennedy, p. j., beers, s., scott, k., moloney, g., hoban, a. e., scott, l., fitzgerald, p., ross, p., stanton, c., clarke, g., cryan, j. f., & dinan, t. g. (2016). transferring the blues: depression-associated gut microbiota induces neurobehavioural changes in the rat. journal of psychiatric research, 82, 109–118. https://doi.org/10.1016/j.jpsychires.2016.07.019 kolevzon, a., angarita, b., bush, l., wang, a. t., frank, y., yang, a., rapaport, r., saland, j., srivastava, s., farrell, c., edelmann, l. j., & buxbaum, j. d. (2014). phelan-mcdermid syndrome: a review of the literature and practice parameters for medical assessment and monitoring. journal of neurodevelopmental disorders, 6(1). springer new york llc. https://doi.org/10.1186/1866-1955-6-39 kostoví, i., & judaš, m. (2002). correlation between the sequential ingrowth of afferents and transient patterns of cortical lamination in preterm infants. anatomical record, 267(1), 1–6. https://doi.org/10.1002/ar.10069 lach, g., fülling, c., bastiaanssen, t. f. s., fouhy, f., donovan, a. n. o., ventura-silva, a. p., stanton, c., dinan, t. g., & cryan, j. f. (2020). enduring neurobehavioral effects induced by microbiota depletion during the adolescent period. translational psychiatry, 10, 382. https://doi.org/10.1038/s41398-020-01073-0 langbehn, d. r., hayden, m. r., paulsen, j. s., johnson, h., aylward, e., biglan, k., kieburtz, k., oakes, d., shoulson, i., guttman, m., landwehrmeyer, b. g., nance, m., ross, c., & stout, j. (2010). cag-repeat length and the age of onset in huntington disease (hd): a review and validation study of statistical approaches. american journal of medical genetics, part b: neuropsychiatric genetics, 153(2), 397–408. https://doi.org/10.1002/ajmg.b.30992 lasalle, j. (2013). autism genes keep turning up chromatin. oa autism, 1(2), 14. https://doi.org/10.13172/2052-7810-1-2-610 laue he., korrick, sa., baker er., karagas mr., madan jc. (2020). prospective associations of the infant gut microbiome and microbial function with social behaviors related to autism at age 3 years. scientific reports, 10, 15515. https://doi.org/10.1038/s41598-020-72386-9 laufer, c., fischer, b., billmann, m., huber, w., & boutros, m. (2013). mapping genetic interactions in human cancer cells with rnai and multiparametric phenotyping. nature methods, 10(5), 427–431. https://doi.org/10.1038/nmeth.2436 leblond, c. s., nava, c., polge, a., gauthier, j., huguet, g., lumbroso, s., giuliano, f., stordeur, c., depienne, c., mouzat, k., pinto, d., howe, j., lemière, n., durand, c. m., guibert, j., ey, e., toro, r., peyre, h., mathieu, a., … bourgeron, t. (2014). meta-analysis of shank mutations in autism spectrum disorders: a gradient of severity in cognitive impairments. plos genetics, 10(9). https://doi.org/10.1371/journal.pgen.1004580 lee, j. k., ding, y., conrad, a. l., cattaneo, e., epping, e., mathews, k., gonzalez-alegre, p., cahill, l., magnotta, v., schlaggar, b. l., perlmutter, j. s., kim, r. e. y., dawson, j. d., & nopoulos, p. (2017). sex-specific effects of the huntington gene on normal neurodevelopment. journal of neuroscience research, 95(1–2), 398–408. https://doi.org/10.1002/jnr.23980 lee, j. k., mathews, k., schlaggar, b., perlmutter, j., paulsen, j. s., epping, e., burmeister, l., & nopoulos, p. (2012). measures of growth in children at risk for huntington disease. neurology, 79(7), 668–674. https://doi.org/10.1212/wnl.0b013e3182648b65 lewis, j. d., theilmann, r. j., townsend, j., & evans, a. c. (2013). network efficiency in autism spectrum disorder and its relation to brain overgrowth. frontiers in human neuroscience, 7(845). https://doi.org/10.3389/fnhum.2013.00845 li, z., tyler, w. a., zeldich, e., baró, g. s., okamoto, m., gao, t., li, m., sestan, n., & haydar, t. f. (2020). transcriptional priming as a conserved mechanism of lineage diversification in the developing mouse and human neocortex. science advances, 6(45), eabd2068. https://doi.org/10.1126/sciadv.abd2068 liang, j., xu, w., hsu, y. t., yee, a. x., chen, l., & südhof, t. c. (2015). conditional neuroligin-2 knockout in adult medial prefrontal cortex links chronic changes in synaptic inhibition to cognitive impairments. molecular psychiatry, 20(7), 850–859. https://doi.org/10.1038/mp.2015.31 lisé, m. f., & el-husseini, a. (2006). the neuroligin and neurexin families: from structure to function at the synapse. cellular and molecular life sciences, 63(16), 1833–1849. https://doi.org/10.1007/s00018-006-6061-3 liu, l., gao, j., he, x., cai, y., wang, l., & fan, x. (2017). association between assisted reproductive technology and the risk of autism spectrum disorders in the offspring: a meta-analysis. scientific reports, 7, 46207. https://doi.org/10.1038/srep46207 liu, r. t., walsh, r. f. l., & sheehan, a. e. (2019). prebiotics and probiotics for depression and anxiety: a systematic review and meta-analysis of controlled clinical trials. neuroscience and biobehavioral reviews(102), 13–23. https://doi.org/10.1016/j.neubiorev.2019.03.023 lloyd-price, j., abu-ali, g., & huttenhower, c. (2016). the healthy human microbiome. genome medicine, 8, 51. https://doi.org/10.1186/s13073-016-0307-y luo, y., eran, a., palmer, n., avillach, p., levy-moonshine, a., szolovits, p., & kohane, i. s. (2020). a multidimensional precision medicine approach identifies an autism subtype characterized by dyslipidemia. nature medicine, 26(9), 1375–1379. https://doi.org/10.1038/s41591-020-1007-0 lutz, a. k., pfaender, s., incearap, b., ioannidis, v., ottonelli, i., föhr, k. j., cammerer, j., zoller, m., higelin, j., giona, f., stetter, m., stoecker, n., alami, n. o., schön, m., orth, m., liebau, s., barbi, g., grabrucker, a. m., delorme, r., … boeckers, t. m. (2020). autism-associated shank3 mutations impair maturation of neuromuscular junctions and striated muscles. science translational medicine, 12(547). https://doi.org/10.1126/scitranslmed.aaz3267 mactier, h., bates, s. e., johnston, t., lee-davey, c., marlow, n., mulley, k., smith, l. k., to, m., & wilkinson, d. (2020). perinatal management of extreme preterm birth before 27 weeks of gestation: a framework for practice. archives of disease in childhood: fetal and neonatal edition, 105(3), f232–f239. https://doi.org/10.1136/archdischild-2019-318402 maheshwari, a., & noebels, j. l. (2014). monogenic models of absence epilepsy. windows into the complex balance between inhibition and excitation in thalamocortical microcircuits. progress in brain research, 213(c), 223–252. https://doi.org/10.1016/b978-0-444-63326-2.00012-0 malekmohammadi, m., elias, w. j., & pouratian, n. (2015). human thalamus regulates cortical activity via spatially specific and structurally constrained phase-amplitude coupling. cerebral cortex, 25(6), 1618–1628. https://doi.org/10.1093/cercor/bht358 marret, s., marchand-martin, l., picaud, j. c., hascoët, j. m., arnaud, c., rozé, j. c., truffert, p., larroque, b., kaminski, m., & ancel, p. y. (2013). brain injury in very preterm children and neurosensory and cognitive disabilities during childhood: the epipage cohort study. plos one, 8(5), 1–9. https://doi.org/10.1371/journal.pone.0062683 molero, a. e., arteaga-bracho, e. e., chen, c. h., gulinello, m., winchester, m. l., pichamoorthy, n., gokhan, s., khodakhah, k., & mehler, m. f. (2016). selective expression of mutant huntingtin during development recapitulates characteristic features of huntington’s disease. proceedings of the national academy of sciences of the united states of america, 113(20), 5736–5741. https://doi.org/10.1073/pnas.1603871113 mordaunt, c. e., jianu, j. m., laufer, b. i., zhu, y., hwang, h., dunaway, k. w., bakulski, k. m., feinberg, j. i., volk, h. e., lyall, k., croen, l. a., newschaffer, c. j., ozonoff, s., hertz-picciotto, i., fallin, m. d., schmidt, r. j., & lasalle, j. m. (2020). cord blood dna methylome in newborns later diagnosed with autism spectrum disorder reflects early dysregulation of neurodevelopmental and x-linked genes. genome medicine, 12(1), 1–25. https://doi.org/10.1186/s13073-020-00785-8 morgan, x. c., tickle, t. l., sokol, h., gevers, d., devaney, k. l., ward, d. v., reyes, j. a., shah, s. a., leleiko, n., snapper, s. b., bousvaros, a., korzenik, j., sands, b. e., xavier, r. j., & huttenhower, c. (2012). dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. genome biology, 13(9), r79. https://doi.org/10.1186/gb-2012-13-9-r79 nardone, s., sharan sams, d., reuveni, e., getselter, d., oron, o., karpuj, m., & elliott, e. (2014). dna methylation analysis of the autistic brain reveals multiple dysregulated biological pathways. translational psychiatry, 4(9), e433. https://doi.org/10.1038/tp.2014.70 nopoulos, p. c. (2016). huntington disease: a single-gene degenerative disorder of the striatum. dialogues in clinical neuroscience, 18(1), 91–98. https://doi.org/10.31887/dcns.2016.18.1/pnopoulos nowakowski, t. j., bhaduri, a., pollen, a. a., alvarado, b., mostajo-radji, m. a., lullo, e. di, haeussler, m., liu, s. j., velmeshev, d., ounadjela, j. r., shuga, j., wang, x., lim, d. a., west, j. a., leyrat, a. a., james, w., & kriegstein, a. r. (2018). spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex. science, 358(6368), 1318–1323. https://doi.org/10.1126/science.aap8809 nunes, a. s., kozhemiako, n., hutcheon, e., chau, c., ribary, u., grunau, r. e., & doesburg, s. m. (2020). atypical neuromagnetic resting activity associated with thalamic volume and cognitive outcome in very preterm children. neuroimage: clinical, 27, 102275. https://doi.org/10.1016/j.nicl.2020.102275 orefice, l. l., mosko, j. r., morency, d. t., wells, m. f., tasnim, a., mozeika, s. m., ye, m., chirila, a. m., emanuel, a. j., rankin, g., fame, r. m., lehtinen, m. k., feng, g., & ginty, d. d. (2019). targeting peripheral somatosensory neurons to improve tactile-related phenotypes in asd models. cell, 178(4), 867-886.e24. https://doi.org/10.1016/j.cell.2019.07.024 o’reilly, h., johnson, s., ni, y., wolke, d., & marlow, n. (2020). neuropsychological outcomes at 19 years of age following extremely preterm birth. pediatrics, 145(2), e20192087. https://doi.org/10.1542/peds.2019-2087 ozonoff, s., young, g. s., goldring, s., greiss-hess, l., herrera, a. m., steele, j., macari, s., hepburn, s., & rogers, s. j. (2008). gross motor development, movement abnormalities, and early identification of autism. journal of autism and developmental disorders, 38(4), 644–656. https://doi.org/10.1007/s10803-007-0430-0 pan, k. y., kok, a. a. l., eikelenboom, m., horsfall, m., jörg, f., luteijn, r. a., rhebergen, d., oppen, p. van, giltay, e. j., & penninx, b. w. j. h. (2021). the mental health impact of the covid-19 pandemic on people with and without depressive, anxiety, or obsessive-compulsive disorders: a longitudinal study of three dutch case-control cohorts. the lancet psychiatry, 8(2), 121–129. https://doi.org/10.1016/s2215-0366(20)30491-0 parente, d. j., garriga, c., baskin, b., douglas, g., cho, m. t., araujo, g. c., & shinawi, m. (2017). neuroligin 2 nonsense variant associated with anxiety, autism, intellectual disability, hyperphagia, and obesity. american journal of medical genetics, part a, 173(1), 213–216. https://doi.org/10.1002/ajmg.a.37977 paulsen, j. s., langbehn, d. r., stout, j. c., aylward, e., ross, c. a., nance, m., guttman, m., johnson, s., macdonald, m., beglinger, l. j., duff, k., kayson, e., biglan, k., shoulson, i., oakes, d., & hayden, m. (2008). detection of huntington’s disease decades before diagnosis: the predict-hd study. journal of neurology, neurosurgery and psychiatry, 79(8), 874–880. https://doi.org/10.1136/jnnp.2007.128728 paz, j. t., bryant, a. s., peng, k., fenno, l., yizhar, o., frankel, w. n., deisseroth, k., & huguenard, j. r. (2011). a new mode of corticothalamic transmission revealed in the gria4 -/model of absence epilepsy. nature neuroscience, 14(9), 1167–1175. https://doi.org/10.1038/nn.2896 picelli, s., björklund, a. k., reinius, b., sagasser, s., winberg, g., & sandberg, r. (2014). tn5 transposase and tagmentation procedures for massively scaled sequencing projects. genome research, 24(12), 2033–2040. https://doi.org/10.1101/gr.177881.114 pinto, d., delaby, e., merico, d., barbosa, m., merikangas, a., klei, l., thiruvahindrapuram, b., xu, x., ziman, r., wang, z., vorstman, j. a. s., thompson, a., regan, r., pilorge, m., pellecchia, g., pagnamenta, a. t., oliveira, b., marshall, c. r., magalhaes, t. r., … scherer, s. w. (2014). convergence of genes and cellular pathways dysregulated in autism spectrum disorders. american journal of human genetics, 94(5), 677–694. https://doi.org/10.1016/j.ajhg.2014.03.018 plioplys s, dunn dw, caplan r. (2017) 10-year research update review: psychiatric problems in children with epilepsy. j am acad child adolesc psychiatry 46(11), 1389-402. https://doi:10.1097/chi.0b013e31815597fc pollen, a. a., nowakowski, t. j., chen, j., retallack, h., sandoval-espinosa, c., nicholas, c. r., shuga, j., liu, s. j., oldham, c., diaz, a., lim, d. a., leyrat, a. a., west, j. a., & kreigstein, a. r. (2015). molecular identity of human outer radial glia during cortical development. cell 163(1), 55–67. https://doi.org/10.1016/j.cell.2015.09.004 porokhovnik, l. n., kostyuk, s. v., ershova, e. s., stukalov, s. m., veiko, n. n., korovina, n. y., gorbachevskaya, n. l., sorokin, a. b., & lyapunova, n. a. (2016). the maternal effect in infantile autism: elevated dna damage degree in patients & their mothers. biomeditsinskaya khimiya, 62(4), 466–470. https://doi.org/10.18097/pbmc20166204466 provost, b., heimerl, s., & lopez, b. r. (2007). levels of gross and fine motor development in young children with autism spectrum disorder. physical and occupational therapy in pediatrics, 27(3), 21–36. https://doi.org/10.1300/j006v27n03_03 qin, j., li, y., cai, z., li, s., zhu, j., zhang, f., liang, s., zhang, w., guan, y., shen, d., peng, y., zhang, d., jie, z., wu, w., qin, y., xue, w., li, j., han, l., lu, d., … wang, j. (2012). a metagenome-wide association study of gut microbiota in type 2 diabetes. nature, 490(7418), 55–60. https://doi.org/10.1038/nature11450 rahman, m. r., petralia, m. c., ciurleo, r., bramanti, a., fagone, p., shahjaman, m., wu, l., sun, y., turanli, b., arga, k. y., islam, m. r., islam, t., & nicoletti, f. (2020). comprehensive analysis of rna-seq gene expression profiling of brain transcriptomes reveals novel genes, regulators, and pathways in autism spectrum disorder. brain sciences, 10(10), 747. https://doi.org/10.3390/brainsci10100747 rau, a., marot, g., & jaffrézic, f. (2014). differential meta-analysis of rna-seq data from multiple studies. bmc bioinformatics, 15(1), 91. https://doi.org/10.1186/1471-2105-15-91 ronan, v., yeasin, r., & claud, e. c. (2021). childhood development and the microbiome—the intestinal microbiota in maintenance of health and development of disease during childhood development. gastroenterology, 160(2), 495–506. https://doi.org/10.1053/j.gastro.2020.08.065 sarasua, s. m., boccuto, l., sharp, j. l., dwivedi, a., chen, c. f., rollins, j. d., rogers, r. c., phelan, k., & dupont, b. r. (2014). clinical and genomic evaluation of 201 patients with phelan-mcdermid syndrome. human genetics, 133(7), 847–859. https://doi.org/10.1007/s00439-014-1423-7 saudou, f., & humbert, s. (2016). the biology of huntingtin. neuron, 89(5), 910–926). https://doi.org/10.1016/j.neuron.2016.02.003 scahill, r. i., zeun, p., osborne-crowley, k., johnson, e. b., gregory, s., parker, c., lowe, j., nair, a., o’callaghan, c., langley, c., papoutsi, m., mccolgan, p., estevez-fraga, c., fayer, k., wellington, h., rodrigues, f. b., byrne, l. m., heselgrave, a., hyare, h., … tabrizi, s. j. (2020). biological and clinical characteristics of gene carriers far from predicted onset in the huntington’s disease young adult study (hd-yas): a cross-sectional analysis. the lancet neurology, 19(6), 502–512. https://doi.org/10.1016/s1474-4422(20)30143-5 schmidt, a., zhang, h., & cardoso, m. c. (2020). mecp2 and chromatin compartmentalization. cells, 9(4), 878. https://doi.org/10.3390/cells9040878 schulte, t., müller-oehring, e. m., javitz, h., pfefferbaum, a., & sullivan, e. v. (2008). callosal compromise differentially affects conflict processing and attentional allocation in alcoholism, hiv, and their comorbidity. brain imaging and behavior, 2(1), 27–38. https://doi.org/10.1007/s11682-007-9014-z schulz, m. d., atay, ç., heringer, j., romrig, f. k., schwitalla, s., aydin, b., ziegler, p. k., varga, j., reindl, w., pommerenke, c., salinas-riester, g., böck, a., alpert, c., blaut, m., polson, s. c., brandl, l., kirchner, t., greten, f. r., polson, s. w., & arkan, m. c. (2014). high-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity. nature, 514(7253), 508–512. https://doi.org/10.1038/nature13398 sedó, c. a., bilinski, m., lorenzi, d., uriondo, h., noblía, f., longobucco, v., lagar, e. v., & nodar, f. (2017). effect of sperm dna fragmentation on embryo development: clinical and biological aspects. jornal brasileiro de reproducao assistida, 21(4), 343–350. https://doi.org/10.5935/1518-0557.20170061 sonnenburg, e. d., & sonnenburg, j. l. (2019). the ancestral and industrialized gut microbiota and implications for human health. nature reviews microbiology, 17(6), 383–390. https://doi.org/10.1038/s41579-019-0191-8 soorya, l., kolevzon, a., zweifach, j., lim, t., dobry, y., schwartz, l., frank, y., wang, a. t., cai, g., parkhomenko, e., halpern, d., grodberg, d., angarita, b., willner, j. p., yang, a., canitano, r., chaplin, w., betancur, c., & buxbaum, j. d. (2013). prospective investigation of autism and genotype-phenotype correlations in 22q13 deletion syndrome and shank3 deficiency. molecular autism, 4(1). https://doi.org/10.1186/2040-2392-4-18 strunk, t., inder, t., wang, x., burgner, d., mallard, c., & levy, o. (2014). infection-induced inflammation and cerebral injury in preterm infants. the lancet infectious diseases, 14(8), 751–762. https://doi.org/10.1016/s1473-3099(14)70710-8 subota, a., pham, t., jetté, n., sauro, k., lorenzetti, d., & holroyd-leduc, j. (2017). the association between dementia and epilepsy: a systematic review and meta-analysis. epilepsia, 58(6), 962–972. https://doi.org/10.1111/epi.13744 südhof, t. c. (2008). neuroligins and neurexins link synaptic function to cognitive disease. nature, 455(7215), 903–911. https://doi.org/10.1038/nature07456 sun, c., cheng, m. c., qin, r., liao, d. l., chen, t. t., koong, f. j., chen, g., & chen, c. h. (2011). identification and functional characterization of rare mutations of the neuroligin-2 gene (nlgn2) associated with schizophrenia. human molecular genetics, 20(15), 3042–3051. https://doi.org/10.1093/hmg/ddr208 surana, n. k., & kasper, d. l. (2017). moving beyond microbiome-wide associations to causal microbe identification. nature, 552(7684), 244–247. https://doi.org/10.1038/nature25019 tabrizi, s. j., flower, m. d., ross, c. a., & wild, e. j. (2020). huntington disease: new insights into molecular pathogenesis and therapeutic opportunities. nature reviews neurology, 16(10), 529–546. https://doi.org/10.1038/s41582-020-0389-4 tasic, b., menon, v., nguyen, t. n., kim, t. k., jarsky, t., yao, z., levi, b., gray, l. t., sorensen, s. a., dolbeare, t., bertagnolli, d., goldy, j., shapovalova, n., parry, s., lee, c., smith, k., bernard, a., madisen, l., sunkin, s. m., … zeng, h. (2016). adult mouse cortical cell taxonomy revealed by single cell transcriptomics. nature neuroscience, 19(2), 335–346. https://doi.org/10.1038/nn.4216 tereshchenko, a., van der plas, e., mathews, k. d., epping, e., conrad, a. l., langbehn, d. r., & nopoulos, p. (2020). developmental trajectory of height, weight, and bmi in children and adolescents at risk for huntington’s disease: effect of mhtt on growth. journal of huntington’s disease, 9(3), 245–251. https://doi.org/10.3233/jhd-200407 treutlein, b., & camp, j. g. (2020). sequencing perturbed cortex development. science, 370(6520), 1038–1039. https://doi.org/10.1126/science.abf3661 trevino, a. e., sinnott-armstrong, n., andersen, j., yoon, s. j., huber, n., pritchard, j. k., chang, h. y., greenleaf, w. j., & pașca, s. p. (2020). chromatin accessibility dynamics in a model of human forebrain development. science, 367(6476), eaay1645. https://doi.org/10.1126/science.aay1645 troyano-rodriguez, e., wirsig-wiechmann, c. r., & ahmad, m. (2019). neuroligin-2 determines inhibitory synaptic transmission in the lateral septum to optimize stress-induced neuronal activation and avoidance behavior. biological psychiatry, 85(12), 1046–1055. https://doi.org/10.1016/j.biopsych.2019.01.022 van der plas, e., langbehn, d. r., conrad, a. l., koscik, t. r., tereshchenko, a., epping, e. a., magnotta, v. a., & nopoulos, p. c. (2019). abnormal brain development in child and adolescent carriers of mutant huntingtin. neurology, 93(10), e1021–e1030. https://doi.org/10.1212/wnl.0000000000008066 von ehrenstein, o. s., ling, c., cui, x., cockburn, m., park, a. s., yu, f., wu, j., & ritz, b. (2019). prenatal and infant exposure to ambient pesticides and autism spectrum disorder in children: population based case-control study. the bmj, 364, i962. https://doi.org/10.1136/bmj.l962 vuong, h. e., yano, j. m., fung, t. c., & hsiao, e. y. (2017). the microbiome and host behavior. annual review of neuroscience, 40, 21–49. https://doi.org/10.1146/annurev-neuro-072116-031347 walker, e., hernandez, a. v., & kattan, m. w. (2008). meta-analysis: its strengths and limitations. cleveland clinic journal of medicine, 75(6), 431–439. https://doi.org/10.3949/ccjm.75.6.431 wang, m., wan, j., rong, h., he, f., wang, h., zhou, j., cai, c., wang, y., xu, r., yin, z., zhou, w. (2019) alterations in gut glutamate metabolism associated with changes in gut microbiota composition in children with autism spectrum disorder. msystems, 4(1), e00321-18. https://doi.org/10.1128/msystems.00321-18 wang, x., xu, q., bey, a. l., lee, y., & jiang, y. h. (2014). transcriptional and functional complexity of shank3 provides a molecular framework to understand the phenotypic heterogeneity of shank3 causing autism and shank3 mutant mice. molecular autism, 5(1), 30. https://doi.org/10.1186/2040-2392-5-30 warner, b. b. (2019). the contribution of the gut microbiome to neurodevelopment and neuropsychiatric disorders. pediatric research, 85(2), 216–224. https://doi.org/10.1038/s41390-018-0191-9 yum, s. k., im, s. a., seo, y. m., & sung, i. k. (2019). enlarged subarachnoid space on cranial ultrasound in preterm infants: neurodevelopmental implication. scientific reports, 9(1), 19072. https://doi.org/10.1038/s41598-019-55604-x yuzaki, m. (2018). two classes of secreted synaptic organizers in the central nervous system. annual review of physiology, 80, 243–262. https://doi.org/10.1146/annurev-physiol-021317-121322 zhang, m., chu, y., meng, q., ding, r., shi, x., wang, z., he, y., zhang, j., liu, j., zhang, j., yu, j., kang, y., & wang, j. (2020). a quasi-paired cohort strategy reveals the impaired detoxifying function of microbes in the gut of autistic children. science advances, 6(43), eaba3760. https://doi.org/10.1126/sciadv.aba3760 zhou, y., sharma, j., ke, q., landman, r., yuan, j., chen, h., hayden, d. s., fisher, j. w., jiang, m., menegas, w., aida, t., yan, t., zou, y., xu, d., parmar, s., hyman, j. b., fanucci-kiss, a., meisner, o., wang, d., … yang, s. (2019). atypical behaviour and connectivity in shank3-mutant macaques. nature, 570(7761), 326–331. https://doi.org/10.1038/s41586-019-1278-0 zhu, y., mordaunt, c. e., yasui, d. h., marathe, r., coulson, r. l., dunaway, k. w., jianu, j. m., walker, c. k., ozonoff, s., hertz-picciotto, i., schmidt, r. j., & lasalle, j. m. (2019). placental dna methylation levels at cyp2e1 and irs2 are associated with child outcome in a prospective autism study. human molecular genetics, 28(16), 2659–2674. https://doi.org/10.1093/hmg/ddz084 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 50 years of surgical pathology / 36 years of neuropathology. my way / a personal journey feel free to add comments by clicking these icons on the sidebar free neuropathology 2:28 (2021) reflections 50 years of surgical pathology / 36 years of neuropathology my way / a personal journey sverre j. mørk university of bergen, clinical institute 1, jonas lies vei 91b, 5021 bergen, norway corresponding author: sverre j. mørk · medvitno · skeielia 62 · jonas lies vei 91b · 5239 bergen · norway mork@uib.no submitted: 13 october 2021 accepted: 19 october 2021 copyedited by: henry robbert published: 08 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3639 keywords: neuropathology, reflections, autobiography i: the start 1 drøbak i was born in drøbak (norway) in 1942. drøbak is a village-like small town at the eastern shoreline of the oslofjord (the photo at the top, from the 1960s, shows our dark house dead centre). i grew up here, where everyone ‘knew’ each other. my neighbourhood is depicted in the oil painting from the spring of 1945 (the artist, anton thoresen, lived in the red house). in these safe surroundings, we played cowboys and indians and hide and seek. 1 drøbak 2 giessen 3 oslo 4 kongsberg 5 larsnes 6 ørland air base 7 meråker i always wanted to be a physician. after 12 years of effortless schooling, my final grades lacked a few points for studying medicine in oslo or bergen, the only two medical faculties in norway at that time. in the 1960s and 70s, 50% of norwegian physicians studied medicine abroad. germany, austria, switzerland, great britain, and ireland were among the most popular countries. i applied to and was accepted by universities in scotland, austria, and germany. i did not fancy studying in big cities like berlin, hamburg, munich, or vienna, with all their social diversions (or ‘opportunities’). so, among the actual medical faculties, i chose the one that looked ‘safest’ to me. 2 giessen the medical school of the justus liebig university in (hessen, deutschland) presented a bed-side-teaching concept where the 11 half-year semesters were organised in a school-like schedule (i.e., less freedom of choice for composing your curriculum). the fact that only a few students were accepted every year (‘…kleine zahl seiner medizinstudenten…’) was a perfect fit for me. low numbers of consemesters and transparency (‘who is missing today? almost all on board!’) are two ways to harness young and drifting spirits. steady surveillance by my peers turned out to be a helpful remedy. so, on october 30th 1961, i listened to the first lecture in german. after 3 weeks, i lowered the language barrier enough to understand most of what i heard. although generally restless, i stayed at the justus liebig university from october 1961 until august 1967. i kept the same room (#19) in the new dormitory ‘landgraf ludwig haus’ from autumn 1961 until i left for norway in august 1967. the drawing (45x15cm) by my little brother erling hung above my bed; it has been with me ever since. it depicts the cycle of life and i have been through six of the depicted stages. the right figure fits me best now. sverre, sabine, hans, and peter minutes after the last examination, 1967, and 50 years later (2017). the 12th semester was a tough stretch of examinations, all oral, 19 in all. we were examined in groups of four (the same four students during the whole ‘staatsexamen’). we all did well. here, in the summer of 1967, we were a relieved and happy quartet standing in front of my 11m2 studio after the very last examination. 3 oslo back in oslo, half a year of additional studies (‘tilleggskurs’) were required to pass the last hurdle to be an authorised physician. i was granted preliminary permission to practice medicine for the duration of this course. for the rest of 1967, i worked at the acute psychiatric ward for men of the oslo city hospital, which added to my mental health. i successfully completed ‘national examinations’ in forensic psychiatry, forensic medicine, hygiene, social medicine, and prescription theory, and was awarded my diploma on december 12th 1967. it states that with my staatsexamen from giessen, with the additional course included, i was granted the norsk medisinsk embedseksamen. no crystal ball could tell that four years later i would face many examinations every day executing diagnostic anatomical pathology (histopathology). i learned to see and register what the microscope shows you, internalizing what you see, processing these images into a conclusion, and revealing a correct and crisp diagnosis. 4 kongsberg on january 1st 1968, i started 1.5 years of internship (six months of surgery, six months of internal medicine, lastly half a year of general practice). there was an official raffle for a number that would give you your place in the queue for choosing ‘your’ hospital as well as the country district where you’d spend the last stretch before being certified to practice medicine. i drew a low number, and decided to stay in the south of norway, where i picked the community hospital in kongsberg. there i met and worked together with another intern, tore böhmer halvorsen, who three years later was to be decisive in my way into pathology. i had family and friends in and around drøbak on the east shore of the oslofjord. kongsberg was located inland on the west side of the fjord. during 1968, i’d travel those one-hundred-and-twenty kilometres countless times, through nights and early mornings, probably driving too fast. the interns and other young doctors lived in small townhouses close to the clinic. i had memorable times as a bachelor: socialising, playing bridge, driving on ice-covered lakes, skiing, dancing, sailing, and soccer. 5 larsnes the final six months of my internship took place within the boundaries of the district sande, north of stad, the roughest coastal stretch of norway. my assignment was to assist the county doctor’s general practice. from monday to friday, the local bus unloaded patients at 8:30 in front of the district doctor’s office in larsnes. at the very start most ‘customers’ wanted the attention of the ‘old’ doctor. with some luck in handling, i was quickly accepted by the inhabitants, giving the older doctor and me equal workload. in this large district with several islands and no bridges i had many demanding visits during the winter and spring, going out to islands in bad weather. but in june of 1969, military service in the norwegian airforce called. 6 ørland air base students of medicine, theology, and dentistry could absolve part of the 15 months of mandatory military service during their studies. i finished my recruit training (‘boot camp’) in between semesters. in the summer of 1969, i did a six-week officers course followed by one year as a flight surgeon in a care-of-the-flyer program at ørland air base west of trondheim. to fill this assignment, it was important to get an impression, a real feeling, of how it is to be a fighter pilot. some of the enlisted doctors in the air force did get one single flight with a fighter jet. airtime for the northrop f-5 freedom fighter was precious. all non-fighter pilots within the air base would give ‘anything’ to be taking off in a fighter jet. i was lucky to be granted this experience five times for a total of 6 hours and 25 minutes. i flew tactical missions, night flight, interceptions, and granted control of the jet for some minutes, pretending that the top of the clouds was solid ground! exciting and unforgettable! i met my wife kari during this period and made many life-long friendships. 7 meråker after i completed my obligatory military service, i had not yet decided what kind of a physician i wanted to be. a friend in the military persuaded me to spend the next year in rural meråker (approx. 3,000 inhabitants). the single country doctor was leaving to specialise in psychiatry and had no successor. following my friend’s call, i filled the vacancy for 12 months (1970-1971). it was quite a challenge for a green and inexperienced 28-year-old physician. i still remember my existential thoughts passing the local church that had a huge and grassy graveyard with only a few stones in a corner… now (photo as of july 2021) the trees have multiplied and grown; still there is abundant free space. decades ago, the people in rural areas of norway were not accustomed to unnecessarily bothering the doctor. calls during late evenings and nights occurred, on average, once a month. it was like a fairy-tale for rural doctors today. my medical practice went rather smoothly. when more complicated situations occurred (e.g., marriage counselling), i referred my patients to other competent persons or institutions. generally, childbirths are among the most exciting things in life but i was terrified to being called to a difficult one. thankfully, for me and the meråker community, we were lucky enough to have a brilliant country nurse who acted as a midwife when necessary. her soothing expertise was not called for during my tenure but nurse (söster) inger’s familiarity with generations of inhabitants was indispensable for my practice of medicine there. one story has stayed with me to this day: an old man was found in a river on top of his volvo van. he had fallen asleep on his way to his swedish home, and then the local river flooded after heavy rainfall. the roaring river swept his van away and took it 100 meters downstream, where it snagged on large rocks. a farmer saw the incident and fetched me and other neighbours. we dropped a small boat in the river and secured it with an exceptionally long rope and then made our way to the driver. i was in the back of the boat to catch him. a journalist happened to pass by and shot the telling photo (from the newspaper adresseavisen): my colleague and friend tore b. halvorsen (from internship in kongsberg) had started pathology training in trondheim, the third largest norwegian city. trondheim was located 1.5 hours from meråker. he tried to convince me to start working in the pathology department with him. we had many arguments back and forth, but he won the day he said, ‘the workforce is young, enthusiastic, and easy-going’. i realised that whatever i ended up doing, basic knowledge in general and special pathology would be a benefit. after one year of being the only md for 3,500 persons, it was tempting to enter the safe harbour of a hospital. during my last months in meråker, i had already sent two work applications to hospitals in hamar (internal medicine) and arendal (general surgery). these were specialties miles apart thematically and geographically. the idea was to have some time before choosing the eventual ‘right' specialty. none of my applications were even confirmed received. as the saying goes, ‘one bird in the hand is better than ten on the roof’. i accepted the ‘offer’ from the pathology department in trondheim. late letters of acceptance from both clinics arrived, but too late. i had already promised to join my friend in trondheim. we (kari, baby pia and i) left meråker without having contributed to an increase in the number of tombstones. luckily. ii: the start of my surgical pathology trondheim 1971-1974 sentralsykehuset i sør-trøndela ‘aller anfang ist schwer’. my backbone in anatomical/surgical pathology was formed in the practice of a rather large community hospital. during the years in trondheim, i was influenced by two colleagues, who had entered the pathology field some 8 to 12 months earlier than me. they were experienced fellows compared to the ‘rookie’. my colleagues (tore b. halvorsen and arne ødegaard) managed to keep me ‘downstairs’ doing autopsies for half-a-year. i was told that was the customary entrance to anatomical pathology. starting out with post-mortem examinations should give you ample time (no hurry) to get a grip on the microscopy of tissues and changes therein. thus, my friends (tore and arne, see the next photo) had it easy, cuddled with biopsies, while i, lifting the burdens of autopsies off their shoulders, strived to ‘see’ and to ‘read’ what microscopy offered me. i longed for the real thing to experience the task of being the doctor’s doctor in real time. that is what surgical pathology is about. in goethe’s words: ‘vor die therapie haben die götter die diagnose gestellt’. but i was doing the essential work of diagnostic pathology: establishing borders between normal appearances, physiological changes within the boundaries of normality, and initial changes signalling disease within tissues. after only autopsies for month after month, i longed to address pathology in real time – biopsies. the chief physician of the pathology lab in trondheim was painstakingly meticulous. his goal was not to miss anything in an autopsy or biopsy. he wanted minimal aberrations from the ‘normal’ to be written down on the final ‘list of diagnosis’ on the front page of the autopsy report. i recall 20 or more items on the final list. we’d discuss the individual order of findings with minor, or even no clinical significance to give it its proper position on the list. i still use some of his elegant, descriptive phrases which characterise the microscopic picture in a few, well-chosen words. an awkward situation in the autopsy room: the ‘whole’ lung department came for a demonstration. we had interesting findings and good discussions. in the end, there was something in the air… they complained about the long time it took to get autopsy reports. i protested, knowing that we were pretty prompt. later the same day, a doctor from the lung department informed me that one of the secretaries had a drawer where she puts reports related to ‘dead patients’ without telling anyone. my second ‘boss’ had been on a leave-of-absence as a surgical pathologist in uganda. he was quick to recognise the important players in a tissue section and was effective in his diagnostic service. nor did he dwell on details of no or minimal clinical significance. he was an efficient surgical pathologist. i learned from both, as i did from all the continued education courses in the following years. in 1973 i attended a tumour biology course in uppsala, sweden. the main lecturer was professor jan pontén who headed the institute of pathology at uppsala university. this course became a key experience for me. pontén had a unique presence; he was part of what he presented be it cells, tissues, neoplasia (‘tumours’, new growths, cancers). his research led to insights into growth capacities and proliferation dynamics in normal and neoplastic cells in cultures (in vitro) and in tissues (in vivo). the course involved exciting presentations on glioma tissue culture, multicellular spheroids, contact inhibition, growth capacity and proliferation dynamics. bengt westermark’s lectures also left a lasting impact. he presented many things that were new to me in my second year of diagnosing common surgical biopsies. for me, he added a third and a fourth dimension – he revealed the sheer complexity of the still-life lying in front of your eyes on the glass slide. you would have to imagine what is just a few microns below and above the actual tissue level. you have to think of the 4th dimension: the time aspect, how the present lesion could have evolved and what lies ahead (prognostication). the equation of ‘positive’ (proliferation/cell divisions/mitoses/mitotic figures) and ‘negative’ elements (cell death/apoptosis/apoptotic figures/necroses) defines the biological aggressiveness of a neoplastic tumour. location and eventual spread add to the equation. the tumour biology course contributed to my interpretation ability but it also instilled in me the importance of trying to understand the biology and natural history of human cancers. since there were no neurosurgeons north of oslo, the ‘university hospital’ trondheim planned to start a neurosurgery unit at the hospital where i worked. the plan materialised in the mid-1970s with the appointment as rolf ringkjöb as the first neurosurgeon in trondheim. to be prepared for the challenges this would bring, i applied for a junior position in the neuropathology section, department of pathology, rikshospitalet (the national hospital of norway) in oslo. the main task was to get accustomed to biopsy material from the peripheral, and central nervous system, the eye and orbit, as well as from skeletal muscle. the plan was to return to trondheim to serve the neurosurgery department there. iii: the start of my neuropathology rikshospitalet (the national hospital of norway) 1974-1976 rikshospitalet (rh) was a hospital where special, rare, and difficult cases found their way. the neurosurgical department in oslo served the whole country (about 3.500.000 individuals in the 1970s). the need for a set of criteria for histopathological diagnoses grew in the 1960s. we needed a necessary foundation for relevance of international cooperation in epidemiology and clinicopathological studies on cancer incidence and survival. the first ‘blue book’ was: kreyberg l: histological typing of lung tumors. geneva, world health organization, 1967. the pathology department at the national hospital (rh) was influenced by the prominent pathologist leiv kreyberg (1896-1984). he headed the institute of general and experimental pathology from 1938-1964. professor kreyberg’s scientific contributions were many. he left permanent footprints on carcinogenesis research, in typing and classification of lung cancer as well as diagnostic histopathology with emphasis on tumour biology. olav hilmar iversen (1922-1997) headed the merged pathology department from 1964 to 1990. he recruited ‘aspiring scientists’ to focus on skin cancer (chalones), cell proliferation and growth control. one of them, ole didrik laerum was to become a major influencer in my personal and scientific journey. my mentor to be, ole didrik laerum, had done his phd thesis on metabolic events related to cell population kinetics during early stages of mouse skin carcinogenesis. laerum was inspired to follow a scientific career, and joined manfred rajewsky’s group at the max planck institute of virus research in tübingen, germany, in 1971 as a postdoc. around that time n-nitroso compounds were known to induce brain tumours in many different animal species. the rat turned out to be the animal of choice for experimental tumour production. laerum and rajewsky focused on the induction of brain tumours in the offspring of bdix-rats after administration of ethyl-nitrosourea (enu) at the 18th day of gestation, a standardised system developed by ivancovic, druckrey and others. carcinogenesis was simultaneously followed in cell culture (in vitro) as well as in enu-treated rats (in vivo). aagot christie löken (1911-2007), led the neuropathology section from 1952 until 1978. the official painting of her indicates the gentle intelligent person she was. to get into this new (for me) exciting field, works by percival bailey & harvey cushing, hans joachim scherer, klaus joachim zülch, lucien j rubinstein, for example, were mandatory reading. not least, the works of the neuropathologist scherer deserve a note. he published in german during the late 1930s and 1940s (also in english). scherer performed enlightening histological studies on the development of malignant brain tumours in humans. he described how tumour cells invade the adjacent brain tissue by following existing anatomical structures and taking shape after them (secondary structures of scherer). this was in distinction to the primary arrangement of the cells in the tumour itself. and this was just one of his many pioneering assessments. my neuro-oncological literature studies were educational and helpful, and i’ve returned to these books throughout my career. from the fall of 1974, the section of neuropathology was to be my workplace for the next two years. here i was to meet the spectrum of primary brain tumours, the challenge of cell culture, and the excitement of experimental brain tumour research. this photo of a brain section with grey and white matter and a brain tumour with a bleed (hemorrhage) was taken by norbert wey from a case i examined with a young colleague, jens pahnke, in 2002. (department of pathology, university hospital zurich, switzerland) early on, dr. löken asked me if i would be interested in working up some special brain tumour cases for eventual publication. of course, i said ‘yes’. she suggested to focus on a group of neoplastic brain lesions with a varied connective tissue-like arrangement mixed with a common primary pattern of a malignant brain tumour. this new growth (tumour) is called gliosarcoma, a tumour pattern that needed clarification. as a newcomer to this intricate field (neuropathology/neuro-oncology), the first thing i had to do was to find out what kind of pattern warranted the use of this wording/diagnosis. microscopically, i saw biphasic growth with one clear cut, intrinsic, malignant brain tumour, and another component with aggressive mesenchymal features. evidently, this last tissue component could present in many forms. long story short, my brain and i needed to start out with a tumour that had a more delineated histology. new to brain tumours, ependymoma seemed to be a relatively clearer defined entity. therefore, i proposed to go through the national hospital’s files to collect all tumours with this diagnosis. it turned out to be 148 cases of ependymomas of the brain and spinal cord in the archives from 1953 to 1974.this number reflects occurrence in the whole population of norway during that period. after revisions and exclusion of plexus papillomas and subependymomas, we started a follow-up study of 101 cases. more on this project later (vide infra, part 2). the mds of the pathology department met every morning at 8:00. i was early for my first morning meeting. i placed myself at the long table. rather quickly i realised that my table position was wrong and unwanted. i repositioned, back to the wall (‘junge burschen, zweite reihe’) and fell in line. time and again visiting pathologists and scientists ‘misplaced’ themselves, finding a professor standing tall alongside his/her assigned chair. writing this, i recall another feeling: the anxiety fuming around the more scientifically oriented leading pathologists when, on rare occasions, a frozen section from a tumour-suspect breast lesion arrived under the microscope. my backbone (i felt like having one at the time!) in anatomical/surgical pathology was formed in the practice of a rather large community hospital. thus, i felt confident evaluating the more common surgical biopsies. my self-centered ‘expertise’ was not often called for. our section was run by two technicians and two mds. i was in the starting block and enjoyed the challenges. we had a small frozen section area in front of the operating theatre on the 7th floor of the old block. when we were called upon, we had to leave our new high-rise and go into the main building which had a pater noster (see illustration) for staff only. a fun ride, especially when daring to go over the top. photo credit: l. weber, 2014 my first frozen section: i have a habit of examining the slide before reading clinical info. i thought (and still think) that swiftness is a way of signalling proficiency. i quickly gave my interpretation over the intercom: ‘meningioma’. my name said nothing to the neurosurgeon, nor did my diagnosis impress the operator, who called out that he was in the middle of the pituitary gland. he said that there was no way the lesion was a meningioma. probably followed by a few more words not fit to print. the next few days, i checked through all files and found close to ten cases where ‘meningioma’ was called on frozen and changed to adenoma after viewing the permanent sections. a comfort of sorts. the work with the special brain tumour, ependymoma, stayed with me for a couple of years. at that time, the norwegian population was ethnically consistent. we had one of the oldest cancer registers in the world, kreftregisteret (est. 1951), with complete, mandatory reports of cancer cases from all pathology departments in norway. this registry could be linked up to the mortality register of the ‘statistics norway’ (statistisk sentralbyrå, est. 1876). thus, we could obtain a follow-up rate of 100%. having such a setting, we wanted to establish the frequency of ependymomas in a well-defined, homogeneous population, and determine survival times after brain surgery. the final stages of the manuscript had a special and fortunate course. karin (blikstad) miller, a colleague of mine, knew that i worked on a brain tumour project, and also that i had little experience in such an undertaking. she ‘offered me’ her husband saying that he loved to help aspiring authors with all sides of a manuscript. after retirement in 1968, ashton miller (1908-1992) moved to oslo with his wife, karin. he had been president of the section of urology of the royal society of medicine in england. the result of his help with language, disposition, and corrections led to our shot at a high-ranking journal for eventual publication. i chose cancer, a top cancer journal (of the american cancer society) at that time. miller’s ingenious help with corrections and suggestions proved invaluable. it turned out to be a bull’s-eye (see below). juan rosai asked for and got permission to use one figure (a kaplan-meyer plot showing survival related to location of the tumour) in ackerman’s surgical pathology (juan rosai / sixth edition, 1981). six years later lucien j rubinstein ‘confessed’ to me that he was one of the reviewers. he had advised the editor to ‘accept it as is’. ependymoma paper: manuscript mailed form bergen, november 8th, 1976 to the editorial office of cancer, washington, d.c. acknowledged as received, november 12th, 1976. letter of acceptance, december 3rd, 1976. accepted directly, unchanged after 3 weeks. published, cancer 40:907-915, 1977. ole didrik laerum returned to oslo in 1972, where he continued in vitro and in vitro research on human and experimental brain tumours. he had brought with him a breeding pair of bdix-rats, a gift from druckrey personally. in the summer of 1973, he got a call for an academic position at the gade institute, haukeland university hospital, in bergen. the call was furnished with means to continue and expand his research facilities. he accepted the call and moved to bergen with his family and a pair of rats! from the summer of 1974, he embarked on a unique academic and scientific career in bergen. in 1976, a flow cytometer (an advanced cell sorter based on laser measurements) enforced the research landscape at gade’s. he successfully guided 44 candidates to phds since then. in addition, laerum served as rector of the university of bergen, 1990-1995. i took over parts of laerum’s cell culture duties in oslo. from 1975 on, i was repeatedly asked to join laerum in bergen. going there would probably secure an academic career for me. so… iv: the gade institute, department of pathology, haukeland university hospital, bergen, norway gade’s institute was originally named ‘dr. med. f.g. gades pathological anatomical laboratory’. fredrik georg gade (1855-1933) was the son of a wealthy merchant in bergen. he was a pathologist who defended his thesis ‘about pathological anatomical changes in the tissues of neurotrophic origin’ in 1900. gade donated nok 150.000 (inherited from his father) to build a house exclusively for pathology (which included bacteriology at that time). the gade institute was inaugurated on march 15th 1912. on friday october 1st 1976, i started on another stretch of my way as a consultant in surgical pathology and neuropathology at the gade institute in bergen. that same autumn, i was board certified in the specialty of pathology. going to bergen, i brought three years of anatomical/surgical pathology with two years of neuropathology, added as a subspecialty to complete the fiveyear minimum of specialty training for being a certified pathologist in norway. this duality, surgical and neuropathology, stayed with me from then on. the beginning was demanding, with lots to learn, in many ways and in many fields. ernest glück was the oldest and the most experienced surgical pathologist in bergen at that time. he took me under his wing and enabled me to become a doctor’s doctor at the haukeland university hospital. he had an open door, always willing to help when i felt insecure diagnostically. on a double-headed microscope with him, my suggestions were corrected or affirmed. it took almost half a year before i was joined by my family, and we could start settling in. however, in my scientific life, i settled in immediately with the group laerum had started to build at the gade institute. the long presence of the hanseatic league merchants in the city of bergen (1350-1750) has contributed to the unique bergen dialect. bergeners have a saying: ‘i’m not from norway, i’m from bergen’. the wet climate is another well-known fact. many jokes about the climate in bergen are ‘floating’ around you probably have heard at least one? neurosurgeons, neurologists, ophthalmologists, and ear-nose-throat doctors in bergen were pleased to have a ‘fresh neuropathologist’ aboard. recent biopsies and brain autopsies were presented in the presence of neuroradiologists, neurologists, neurosurgeons, and others, with interest in the actual case. my colleagues interacted for the best of the person in focus (also called patient). in other words, these were clinicopathological conferences in the same form as at the rh in oslo (all over, for that matter). except that i always used gloves. i recall that dr. löken sometimes demonstrated bare-handed. a young and enthusiastic neurologist, harald nyland, contacted me early on. he tried to persuade me to take specimens from fresh autopsy brains. ☹ that was against my neuropathological workbook. to find and eventually confirm a suspected pathology in the central nervous system, you should first secure the brain, eventually with the spinal cord, trough fixation in 10% formalin (4% formaldehyde solution) for at least 2 weeks. harald insisted upon the necessity of using unfixed brain tissue in the search for immune-active cells in the plaques, typically seen by the naked eye (grossly/macroscopically) in this brain disorder. this research resulted in significant findings (to be presented further down the road). however, since immunohistochemistry emerged to be effective on formalin-fixed tissue, the standard fixation method was used on most post-mortem brain examinations. a thorough study of post-mortem brains is still fundamental to understand neurological diseases. gade’s staff around 1980, ‘none mentioned, none forgotten’. exception: professor emeritus erik waaler, in front, who discovered the rheumatoid factor in 1939. he went to bergen and opened the medical faculty here in 1947. an open mind a touch of subjectivity is inherent in histopathological diagnoses. a reality is evidenced through inter-observer as well as intra-observer variance in histopathological assessments. since i am easily influenced, i long ago settled into a routine of ignoring the specimen’s clinical, anamnestic data. my policy is to grab the object glass, glance at the slide on its way to the position between light source and objectives. to look through the microscope before reading at the clinical data (age, gender, actual problem et cetera) is my way to lower bias in reading the histology. that is, before i acknowledge the clinical problem, and the eventually suggested, tentative diagnosis (tx). starting every biopsy assessment with an open mind, trying to decide the source of the material (organ?), then a quick glance for changes, has worked well for me. it is not a time-consuming ordeal, one second, sometimes a few, does it. the trick is to convert the evaluation (reading) of every biopsy specimen into an internal competition. this habit makes the daily workload fascinating to handle. i’ve not had a single day of boredom in the company of my microscope and a carefully produced tissue section. i’d say that this habit (modus operandi) has fed alertness and positivity. in my office i had two computer screens in front of me. one hooked up to the pathology department and the other connected to the servers of the university for surfing the web to keep updated. in this way i could confer with the newest data without having to shut down the one with sensitive patient information. as mentioned before, the bdix rat was an asset that laerum used to the fullest extent for the years to come. the breeding of the bdix rats was in the hand of animal technician tore-jacob raa. he kept breeding 35 generations! the +40-year-old photo shows us injecting tumour cells intraperitoneally: during the 1970s, marc mareel and leo de ridder at the university of ghent, belgium, developed organotypic methods for the study of invasiveness by malignant tumour cells in vitro. chick heart tissue was found to be well suited for invasion studies. in bergen, laerum cooperated with the group in ghent, and i was naturally involved in these activities. this cooperation led to my thesis: ‘characterization of premalignant and malignant cells from the nervous system’, which i defended publicly at the university in bergen, may 1983. v: international activities the idea to start a society of neuropathology in the nordic countries originated chiefly from erna christensen and edith reske-nielsen in denmark, aagot christie löken in norway, and patrick sourander in sweden. ansgar torvik, oslo and yngve olsson, uppsala were involved as well. the scandinavian society of neuropathology was to be a forum for persons interested in clinical and experimental neuropathology. the founding meeting was held at rigshospitalet, copenhagen, denmark on january 23rd 1965. erna christensen and patrick sourander were among those few who re-formed the international committee of neuropathology into the international society of neuropathology in copenhagen in 1967. sourander was the grandfather of modern neuropathology in scandinavia. the scandinavian society had joint meetings with british, german, italian and other nation’s neuropathological societies. these were friendly and inspiring conferences with social events. several nordic neuropathologists were members of other nations’ neuropathological societies. the winter and summer meetings of the british neuropathological society (bns) were especially popular among scandinavians, i would say. in the bns summer meeting 1979 in oxford i presented ‘ploidy of human intracranial neoplasms studied by flow cytometry’. i did not follow up ploidy and brain tumours, since i did not feel that this would have an impact on prognostication of an individual person’s brain tumour. (two roads) the first large and overwhelmingly impressive meeting on my way was that of the viiith international congress of neuropathology, washington, d.c., usa, september 24-29th 1978. i still have the program and the list of participants, as well as the abstracts of that meeting. this event was special for americans too, since it represented the first international congress of neuropathology to be held in the united states. the earlier congresses are listed here: i 1952 rome ii 1955 london iii 1957 brussels iv 1961 munich v 1965 zurich vi 1970 paris vii 1974 budapest viii 1978 washington, d.c. kenneth m earle was the president and henry de forest webster the secretary general of that congress. i have good memories of both these gentlemen. the same applies to officers of the aanp from that meeting: asao hirano, john j kepes, richard l davies, and michael n hart. from the congress, one memorable recollection: a big audience in a dark, huge room with a large picture projected onto the wall. there was a tiny, fuzzy structure barely visible, in the middle. this was stanley prusiner’s first image of a scrapie associated particle. if body language and unclear murmurs were any indication, the audience was not convinced that there was a specific structure to be seen in the image. in the program leaflet, i ticked off a platform presentation: ‘gfa and intermediate filament protein in glioma cells’, given by anders paetau of helsinki university, helsinki, finland. it must have been my first acquaintance with this household molecule to be. the 1980s in 1980, i returned to washington, d.c., to spend three weeks at the armed forces institute of pathology (afip) in bethesda, maryland, just outside the city limits. the afip had made life easier for the world’s surgical pathologist through their soft-covered fascicles (‘a set of books being published in instalments as separate pamphlets’), which presented the tissue patterns of tumours. these inexpensive atlases of tumor pathology standardised nomenclature, classification, and diagnostic, histological criteria of human tumours of all primary sites. i got permission to visit the departments of neuropathology and ophthalmic pathology. after a quick photo session i had an id card, and then the huge concrete building (‘atom bomb secure’, they said) was free to me. my plan for the first week was to see as much eye tumours as possible in the unit of ophthalmic pathology, headed by lorenz e zimmerman. on monday morning, ‘second in command’, ramon l font welcomed us (around 10 us and international eye aficionados) with actual, current cases of diseases of the eye and orbit. he really grilled us for diagnostic suggestions. originally from cuba, he spoke with an accent. i recall him correcting the eye doctor (from mexico) in front of me, who had answered ‘leukemia’; ramon font with a prompt correction: ‘leukemia, leukemia’. the doctor in front of me uttered in his cupped hand, ‘at least i speak two languages’. zimmerman had finished editing the blue book on eye and orbital tumors. the manuscript was being printed. he was so kind to give me access to the background material, most importantly, a box with glass slides. the content was identical to the material sent to leading ophthalmic pathologists worldwide. the rest of the week i was completely occupied by ‘reading’ the different lesions through the microscope. with the box came the diagnoses offered by every one of the cooperating international ophthalmic pathologists (more than 10 of them). i kept my eyes (see ‘an open mind’ above) away from the judgment of the international expertise. i worked the microscope in a relaxed mood (‘no rush’) and wrote down my assessment of every case. only then would i check the diagnoses of the specialists from the international editorial board. the head of department of ophthalmology in bergen, professor torstein berthelsen (1923-2008) did contribute financially to my study tour to the afip. the week ended with a feeling of having the stamina to adequately serve the eye doctors of western norway. the last two weeks of my way of the afip experience turned out to be great as well. i was the only ‘free player’ there. i was fortunate to have the personal attention of the neuropathology staff, especially gary clark and james henry, who helped in picking interesting cases to study during my stay. vernon armbrustmacher supplied me with muscle biopsies. i recall memorable lunches with kenneth earle (chief of neuropathology) and leslie h sobin (editor of innumerable books on histological classification of tumours, and poetry (see example in box)). despite all the studies we try some tumours our efforts defy; we know not their name nor what is their game so we say they are unclassified l h sobin i made life-long friends at the afip. the afip experience added substantially to my brain’s library of diseases. afip is history now. wikipedia: in a blow to the us’s pathology community, the armed forces institute of pathology will close its doors sept. 15 (2011), the victim of government cost-cutting initiatives. the washington, d.c.-based facility, which holds around 95 million tissue samples in its repository and employs platoons of ‘renowned scientific consultants’, has been a ‘global resource for disease diagnosis and analysis’ for nearly 150 years. returning to scandinavia in 1980 on the flight back to scandinavia, i was seated next to a young japanese who told me of his years in palo alto at stanford university which was the highlight of his time in the us. his positive tale about palo alto/stanford planted a seed in me. wasn’t the eminent diagnostician and renowned author of the brain tumour ‘bible’ situated in stanford? he was. the unrivalled textbook on brain tumour pathology used since the 1960s was pathology of tumours of the nervous system (published by edward arnold). this book was co-authored by dorothy s russel and lucien j rubinstein. it was first published in 1959. in total, five editions of this critically acclaimed, highly appraised book have been published. after defending my thesis in may 1983 i felt ready for a change. i planned to join lucien j rubinstein for a one-year sabbatical. i wrote a letter (september 20th 1980) introducing myself, asking for a possible stay under his guidance. professor rubinstein’s response came some weeks thereafter. he was pleased with my ‘interest in coming to work with us’ and he detailed interesting projects they were pursuing. he asked for three letters of reference, ‘possibly one from dr. löken, whom i know of course quite well’. the letter also stated: ‘we are planning to move our neuropathological operation from stanford university to the university of virginia in charlottesville’. this was a surprising statement; i had planned for both: stanford and rubinstein. the latter had to be the preference, of course. so, instead of sunny california, virginia would be my state of residence, if i got the opportunity to team up with ljr. opportunity knocked, and a few years later, i went through the door. (see viii) ix international congress of neuropathology, vienna, austria september 5-10th 1982 wednesday, september 8th: at 16:30 i presented ‘in situ characterization of t lymphocyte subpopulations in multiple sclerosis (ms) brains’. at 16:45 ute traugott presented ‘localization of t and b cells in multiple sclerosis (ms) plaques’. these presentations were awaited with interest, not least due to the take-home message of traugott and raine’s abstract: ‘shown here for the first time in the human brain’. the chairpersons (ingrid allen and ellsworth c alvord) decided that the two last oral presentations of the afternoon were to be discussed together at the end. the discussion i was nervous and cannot recollect much about the discussion. i recall that the meeting room was half dark, with thick curtains hiding the sun outside. one person stood up, asking me if i ‘believed’ those cells i presented really were lymphocytes. i ended up saying, in the affirmative, that ‘i believe they are’. then he pointed at me, looked around in the audience and called out (screamed, in my ears/brain): ‘he’s a believer…, he’s a believer!’. this aggressive comment hit me in the chest, like a heart attack. after the session closed, i was told that this person was known to act rudely/impolitely in discussions. he, a virologist, from the us west coast, was mikkel gammelsten (name changed to old norse by me). i still remember the few neuropathologists who patted my back afterwards. but this unpleasant occurrence is still with me. to cure ‘it’, i reset my focus to the message we presented in 1982. the cedric raine group penned it for us: ‘t cells, shown here for the first time in the human cns’. we could not have written it better ourselves. now it pleases me to look back on that afternoon session in vienna 40 years ago and re-establish the fact that our group from haukeland university hospital (harald nyland, department of neurology), roald matre (immunologist, broegelmann research laboratory, and i) were the ‘first’ to show evidence that t-cells were at the site of ms lesions. we even gave an estimate of the ratio (5:1) between helper and suppressor lymphocytes, as well as presenting cd8 positive cytotoxic t-cells in the parenchyma. our findings were published in a letter to the editor under the heading ‘t lymphocyte subpopulations in multiple sclerosis lesions’ in december 1982 (new england journal of medicine 307:1643-1644, 1982) and those of traugott and raine in january 1983. this fact seems forgotten in most references to this topic. the results presented in vienna 1982 have been repeatedly confirmed during later years. in 1986 the scandinavian neuropathological society hosted the xth international congress of neuropathology. this meeting took place in stockholm, sweden with patrick sourander as president of the congress. it was a huge success, scientifically as well as financially, not least through the economic efficacy of yngve olsson. to my knowledge, the number of participants (1,300) have never been surpassed by later international and european neuropathology congresses. in 2002 our society hosted the euro-cns meeting in helsinki, finland, with matti haltia and hannu kalimo as chief organisers. the 12th european congress of neuropathology was organised by bjarne winther kristensen, denmark from may 31st – june 3rd 2021. our society also organises the northern lights neuroscience symposia. vi: northern lights neuroscience symposia during the preparation for the 1986 event in stockholm, the idea came up to start a high-quality series of neuroscience symposia. i took the challenge of organizing the first such meeting. my cooperation with neurologist harald nyland had focused on multiple sclerosis for some years, and it was convenient to select themes related to this disease. the leader gerd hagen of the norwegian ms society provided financial support so we could invite active researchers from any country to give the newest research in their field of expertise. all the 17 internationally acknowledged scientists complied with our invitation to contribute to the first northern lights neuroscience symposium. the scientific focus was ‘myelin and demyelination’. one of the invited, bruce d trapp, would turn out to be a valuable research partner for the next 30 years! bergen is the hometown of the composer edvard grieg. no rain and extraordinary nice weather, cloudless blue skies, and up to 25oc daily for the meeting (may 20-23rd 1987), which coincided with the opening of the bergen international music festival. this is a yearly main norwegian cultural event existing since 1953. a fine combination of science and culture. kuo-chun ma (shanghai) with yngve olsson (uppsala). most participants came from europe, many from north america and a few from asia and africa. some of us still remember dr. kuo-chun ma from shanghai. he had been taken from the dinner table and imprisoned for eight years by the gang of four. he had learned english well enough that he could serve as an interpreter. that was reason enough for imprisonment. he was one of the first mds allowed to travel out of china. kari skullerud and yngve olsson could tell you more about the astonishing dr. ma, a highly intelligent and flexible personality. probably they will do so here in free neuropathology. sam ludwin (kingston) and hans lassmann (vienna) enjoy luncheon at hotel norge, bergen (first nlns, may 1987). there have been numerous nlns since then. to mention a few: the one in 1995 (the vith nlns on inflammatory muscle disorders) was an adventurous and highly praised event. the location was svalbard/spitsbergen and kari skullerud (neuropathologist, oslo) had the idea and organised the symposium. ellsworth c ‘buster’ alvord (1923-2010) and wife nancy (left) took part in our first nlns. he was an outstanding neuropathologist and scientist from seattle (university of washington). numerous attendants attested to the quality of all aspects of this very first nlns gathering with a focus on myelin and demyelination. see reactions tagged for the facsimile. harald nyland and i organised another symposium on multiple sclerosis in 1996. we used a painting by the artist nicolai astrup (1880-1928) as an eye catcher. in english the title of the oil painting is ‘midsummer eve bonfire’. for us it symbolises the various aspects of demyelinating lesions. multifocal fires of different age and intensity, some burned out, others with flames high! by the way, nicolai astrup was introduced for the first time in the usa just this august 2021. the venue: the clark art institute, williamstown, ma. the boston globe writes: ‘the best artist you’ve never heard of’ and adds: ‘the wall street journal and the new yorker, no less, are cheerleaders’. vii: international activities continue the ixth nlns in reykjavik, 1998, was organised by my good friend gudmundur georgsson. he is standing on the left. in front peter stubbe-teglbjerg (aalborg) with wife, center mara popovic (ljubljana) and marie bojsen-möller (aarhus), then henning laursen (copenhagen), inger mazanti (southampton), and henrik schröder (odense). american association of neuropathologists inc. and other activities i have attended many annual meetings of this society, and met so many people who left an impression on me. the list of all those whom i feel a special bond with would be too long. but i must mention here two persons: one a neuropathologist and the second the spouse of a neuropathologist. i met bernd walter scheithauer in june 1984 at my first annual meeting of the american association of neuropathologists. during a lunchbreak i saw him having a beer at the pool bar in the holiday inn at the embarcadero, san diego, ca. when i think of him, i recall a quote from the tv show mad men: ‘when a man goes into a room, he brings his whole life with him’. i joined him and swapped stories interchanged with information about our similar ‘upbringing’ in europe. in later meetings, i noticed that bernd was augmenting newcomers, disregarded their origin. he trained in the department of pathology at stanford university school of medicine during the last half of the 1970s. there he was influenced by rubinstein who was the director of neuropathology at stanford at that time. from 1979 bernd worked in the department of laboratory medicine and pathology at the mayo clinic in rochester, minnesota. his neuro-oncological expertise was undisputed, and he enjoyed displaying it. he welcomed many norwegian pathologists to stay with him at the mayo clinic in rochester, minnesota. we met many times on both sides of the atlantic, presenting cases at workshops etc. in prague, toronto, zurich, and at other venues. both of us, among others, were searching for histological features that could predict survival of persons with oligodendrogliomas. at the 1984 meeting i presented preliminary data on oligodendroglioma. the full work ‘oligodendroglioma. histologic evaluation and prognosis’ was published in 1986. in a review titled ‘oligodendroglioma: diagnosis and prognosis’ (seminars in diagnostic pathology 4:352-261, 1987) janet m bruner (chief neuropathologist, m. d. anderson hospital and tumor institute, houston, texas) notes: ‘detailed clinical analysis in two large series of cases,2,3 with similar attention to the relation of prognosis and histologic features in two companion studies4,5 have provided concrete and apparently valid criteria for grading’. grading of oligodendrogliomas was an issue of interest not only for neuropathologists. bernd was used to the so-called st. anne-mayo grading system, which was primarily for astrocytomas. nuclear atypia, mitosis, endothelial proliferation, and necrosis as the morphologic criteria to assign a grade, 1-4, according to presence of cero to all four criteria. the norwegian experience was that microcystic change, necrosis, and cell density were the only histologic features of prognostic significance. it was surprising that nuclear atypia, mitosis, and endothelial proliferation did not display effect on survival. it was a fertile soil for discussions and further studies (more on this topic under ‘1990’ later). he opened his home to me, and i am happy to say that he also stayed in our home in norway a couple of times. bernd died in 2011. he is very much missed. since san diego (1984), i have visited arlington, atlanta, boston, cleveland, denver, minneapolis, new orleans, orlando, pittsburgh, portland, st. louis, salt lake city, san francisco, seattle, and washington, d.c., but have not done much tourism, unfortunately. usually, it is airport hotel airport like for most readers of free neuropathology, i’m sure. annual meetings usually occurred in the first half of june, starting on thursday, and ending early afternoon on saturday. when your home base reimburses attendance fees, with paid leave of absence included, you are reluctant to leave a meeting. but i could get in some exploring by having a stayover to sunday (ostensibly to save on airfare). i will never forget one such sunday: june 11th 2000, in atlanta. it was the day of pentecost. i went for a walk around noon. a woman in a glorious white dress, came towards me down the sidewalk. to my surprise, i recognised a lady i had met once or twice in charlottesville 12 years before. she beamed, telling me about her experience in an episcopal church just minutes ago. after entering the packed church, she stood out. she was called by the chaplain to present herself, and then experienced open arms. she ended up singing and swaying with the whole vibrant parish. she had planned this months ago when she realised that the meeting her husband was going to also coincided with the celebration of pentecost in the deep south. later we got acquainted with each other well. working in zurich, i spent many a weekend enjoying the hospitality of elisabeth and elias perentes who both worked at novartis in basel and lived just over the border to france. in 2003, a lorry ran a red traffic light, crashed into two cars, and killed both drivers, one of whom was elisabeth driving home after work. i will never forget the flamboyant elisabeth perentes on that hot sunday in atlanta. viii: lucien julien rubinstein, uva, charlottesville university of virginia, charlottesville the brilliant thomas jefferson (1743-1826) was born in shadwell, just outside charlottesville, virginia. he was one of the ‘founding fathers’ and finalised the wordings of the ‘constitution of the united states of america’. jefferson planned a new university close to his home monticello in albemarle county, virginia. he was the architect and designer of the campus with its special features like the rotunda, colonnade and the serpentine wall, qualities that make the university of virginia stand out even today. after defending my thesis in may 1983, i was ready to join lucien j rubinstein for a one-year sabbatical leave of absence from haukeland university hospital in bergen. late june 1983 lucien met me at the little charlottesville-albemarle airport outside of charlottesville, virginia. leaving the airplane, i was struck by the burning sun and the warm, moist air (40oc). in other words, my first experience with steaming hot virginian weather. the air was ‘dead’, not even a little breeze gave relief. after a few welcoming words, he told me that when you live in america, the first thing you should do is to buy a car. mobility is a must here, he stressed. in the parking lot, lucien’s open mustang was a pleasant surprise. i recall his gray hair in the wind speeding down route 29 south to the small town (approx. 25,000 inhabitants). he took me to my temporary quarters. i was to live in a huge white barn, from another century with barren rooms, high ceilings and worn-out furniture. in most rooms, there were slowly rotating ceiling fans. i did not notice any students. it was hotter inside than out, and ‘summer empty’. as we looked around, i think my facial expression and body movements signalled reluctance to be ‘dumped’ there. after some minutes of silent disbelief, it must have dawned upon lucien that this residence was not appropriate for a 41-year-old cold-blooded norwegian visiting associate professor of the honourable uva. from norway, i had tried to find a flat or townhouse in charlottesville or outskirts (albemarle county) without success. it would be easier when being there in person, i thought. it took some weeks before i found a fitting place for my family (wife kari and our children, pia, 12, ine, 11, and erik, 2 years). in the intervening time, i was offered lodging in ljr’s home, a one-stock redbrick house on the hillside close to the famous resort boar’s head inn. the adjacent, even posher, farmington country club had been their first choice, but ljr’s application for membership was declined for unknown reasons. the interior of lucien’s and wife mary m herman’s home was elegant and even cosy. i was offered a nice bedroom with a queen-size bed, soft and comfortable. the room had a feminine flavour. it took a while before i realised that i occupied mary’s bedroom… i felt welcome and even comfortable in mary and lucien’s house the two weeks of my stay in ednam drive. mostly i was offered a quick breakfast with lucien. he worked on a new edition of pathology of the nervous system, usually from early morning, not seldom in his morning-robe. therefore, he often came in late. taking lucien’s advice, i took my first test drive, which proved to be an unpleasant encounter. raised on cars with manual gearshift, my initial attempt to shift led to an immediate clash with the windshield. without prolonging my essay, i will skip all the details about problems with old and big american cars, only to say that i needed assistance to find where to fill the gas tank. one of my first days in charlottesville, i was introduced to lennart heimer, a neuroanatomist/physiologist, originally from gothenburg. uva was evidently proud to have him in their midst. heimer spoke english with a heavy swedish accent, which didn’t seem to hurt him in academia. i felt relieved to continue with my own scandinavian drawl. neuropathology had offices, labs and special rooms for cell/tissue culture, electron microscopy (ultrastructure), and biochemistry as a special section of the department of pathology. it was situated within the old colonial redbrick building with ‘medical school’ above the entrance. an antique, thin, squeaky, and almost worn-out door can be seen above. exiting it, you entered a small lawn close to a cosy, little street, called an avenue (university avenue). although not broad, wide, or lined by trees, ‘avenue’ makes sense (avenir – access to) since it leads to the original uva campus. university avenue had some nice small restaurants, coffee shops, fast-food spots, and bookstores. i remembered the site of the very first lunch break lucien invited me to. this picture, taken through the front screen of my rental car, shows the red front of the smallest joint in the avenue. we had sloppy joes (without the bun). my assigned office space was shared with two neuropathology fellows, estelle t may and josé m bonnin. we had weekly lab meetings, were we talked projects, progress, assigned workplaces and equipment. my first lab meeting was rather special. a disagreement about glass pipettes used by sozos was awkward to listen to. they were taken from mary’s side of the lab bench! i uttered something about spoiling time in discussing pettiness. my voicing did not fall in good soil, as the saying goes in norwegian. i was registered as a difficult new boy on the block, a notion further enabled a few weeks later. the summer of 1983 was very warm with record highs. on my lunch break each day, it was a shock to leave the cool air-conditioned interior entering a sauna with clothes on (never did that, by the way). now and then we (estelle, josé and i) had our burger king whopper on the grass close to the serpentine wall surrounding the original thomas jefferson’s uva. our office door had a little window, on the inside of which i put a little black and white version of edvard munch’s ‘the scream’. it was facing the hallway and hung there long enough to annoy passers-by. someone tacked up a message telling me to remove the image. it was an intuitive or subconscious cry from me that i see clearly now. amazing grace comes to my mind: ‘was blind, but now i see’. i recently wondered if my fellow fellows felt the same unease at the time. they most probably did. as the time went by, i felt more relaxed, especially during the weekends. this ‘saturday in the office’ posture was more casual than necessary. note the small microscope compared to the ‘monsters’ of today. family life in charlottesville expanded just a few days after kari, pia, ine and erik’s arrival in the us on august 11th 1983. we rented a townhouse in ‘four seasons’ just outside the charlottesville city limits. after a few days, we found out about a lake not too far away. at chris green lake we enjoyed cooling off in the hot and humid august with swimming and making ‘bombs’ (pictured here). on the first day in the water, a passing young swimmer asked our girls if they were from norway. her mother was. the young girl came up to us on the beach with the telephone number to her home. one phone call and we quickly got anchored to people that we still have as friends, close to 40 years later. other social activities: the head of neurological surgery (john a jane) was exceptionally gracious. i was able to attend two of his garden parties. ‘christmas in june’ was a yearly event hosted by ‘dr. and mrs. john a. jane’. it was a fantastic party, which people looked forward to for weeks. in a corner of their huge lawn, a dixieland band entertained with feel-good vibrations. there was plenty of food and beverages. some persons even followed the suggested theme of the party for ‘optional masquerade and/or masks.’ the christmas party in june was an extraordinary successful socialising event. i was introduced to many interesting people. i recall: ‘you have to meet lennart heimer, he is from sweden, and has recently published a neuroanatomy textbook.’ i had not heard heimer’s name before. his ‘the human brain and spinal cord: functional neuroanatomy and dissection guide’ had just been published in 1983. the reviews were excellent and so was the book, which i promptly acquired. lennart’s wife was norwegian, and there were further scandinavian ladies married to american scientist and businessmen. they met once a month, and kari was wholeheartedly taken in. in our four seasons neighbourhood we connected to neighbours with children from abroad, like peru, israel, finland, and canada. after a harsh start, our two girls thrived in the public jack jouett middle school. they were considered just very shy americans. after those first awkward weeks, things turned around. neuropathology continued lucien’s office was relatively modest in size, soft-coloured interior, decorated in beige and brown. a two-headed microscope was the first thing you saw when entering this sacred place. you did not want rush in there. i felt tension in the air from the start. (was it only me?) however, i found the weekly review of the recently arrived consultation cases (‘ccs’) most educational. brain tumour cases came from all over the world. the fellows and i were assigned cases for presentation at the review meeting, and i greatly valued the scholarly discussions related to these rare and mostly difficult cases. the significance of patterns and elements of the microscopic changes were deliberated in extenso. back at our desks, we dictated microscopic descriptions and diagnoses. the front page, for the ease of the submitter to see, gave a clear conclusion/diagnosis. this was followed by a very wordy description to ensure that every possibility was taken into consideration. rubinstein’s wordings in letters to editors (and others) were polite, starting and ending with ‘thank you for’ with interspersed ‘...would you be so kind as to…’, and ‘…please find...’, a custom i have adopted which has suited me well. another matter was his quest for excellence in microphotography with many retakes and increases of magnification (‘go to a higher mag!’). he typically stressed the fact that you are writing for educated and interested readers, so you do not have to explain everything. trust that your audience is ‘level’. i recall a special incidence: at the double microscope, he rubbed his face and moaned over the quality of the tissue section we were looking at. a long silence, and then ‘who ordered the lab to do this (immunostaining, my comment) gfap?’ i did. more moaning, then: ‘technicians must respect the persons who give them orders’, meaning that he himself had to order immunohistochemistry if he wanted the result to be ‘perfect’. in other words, i was not respected. a deep sadness hit me, i felt both bad and sad. ‘they’re like animals’ he said. i thought i didn’t hear right! lucien was unique in his position as a world authority on brain tumours with a more than complex personality, not without ‘musicality’ and a sort of sweetness and vulnerability. in january of 1990, lucien j rubinstein died at 65. the eulogy, written by scott r vandenberg, bernd w scheithauer, and darrell d bigner, has a passage that i would like to share with you. ‘lucien rubinstein had a remarkable dramatic presence. not many people could fail to remember the first encounter with him. all were affected by his charming manner, subtle and, at times mischievous wit, or lancinating wrath’. my wife would easily have stayed in charlottesville for another year or two. i, however, longed for doing neuropathology and diagnostic surgical pathology back home. (two roads) lucien had a special liking for embryonal tumours, so i’ll close the uva experience with three images. ix: the 1980s continued during the year as a visiting associate professor at uva, i continued work on my oligodendroglioma project, which i had started years earlier. through recommendation by kari skullerud (neuropathologist, the national hospital of norway, oslo), we (the norwegian cancer society) hired a young, inexperienced person, arnhild tollefsrud. her intelligent suggestions and hard work included correct registration of hundreds of data points from 208 persons with a oligodendroglioma diagnosis. her insightful work provided sound foundation for three manuscripts. the first, ‘oligodendroglioma. histologic evaluation and prognosis’, was sent to the journal of neuropathology and experimental neurology (jnen). it turned out to be quite an ordeal to get through the review process of the jnen, with many back-and-forth letters between me and the powerful editor-in-chief, john mossy. some sentences from our correspondence might be of interest. i wrote: ‘our contribution is one for (and by) neuropathologists and surgical pathologists, and not for experimental neurologists. we feel that our manuscript reveals the materials, methods, and results in a way that compares favourably with other contributions in this field. we therefore hope you will accept it in its present form. for example, to give a more ‘precise definition’ of oligodendroglioma than used in authoritative textbooks and in the who classification seems beyond our competence and scope’. i asked mossy to return the manuscript to us, and politely added: ‘i do not regret sending it to you, as your comments and criticism have improved the article’. but then it was accepted for publication. janet m bruner (houston) found an error in figure 7 of our jnen oligo-article. we were impressed by her ingenuity and gave a reply in a letter to the editor (journal of neuropathology and experimental neurology 45, 1986). ellsworth c alvord suggested further avenues of studying histological correlation of oligodendroglioma with prediction of survival. (two roads) the two other ‘oligo-manuscripts’ were accepted and published by the journal of neurosurgery after a thorough review. high on life, after getting an extremely nice comment from the editor-in-chief, william f collins, i sent in a suggestion for the cover page of the issue in which our article would appear. i got no feedback. oh well… after publication of the oligodendroglioma papers in jn and jnen, many neuropathologists and neurosurgeons reacted in a positive manner. i gave ‘oligo-talks’ in europe and in the states. following one in in memphis, tennessee, the neuropathologist, f curtis dohan jr, wanted to discuss some interesting cases with me. entering the microscopy room, i was met by a 12-headed microscope loaded with 10 residents. ten (!)and all were residents of the largest neurosurgical department in the southern us. the cases ‘curt’ had sought out for discussion were certainly not straightforward. i remember that we had lengthy discussions before concluding. thinking back, i must say that the quality of questions and comments from those mainly neurosurgical residents surpassed anything i had experienced before. the 1990s two key events in my way occurred in 1990. in january i received an invitation from paul kleihues (zurich/lyon) to participate in a brain tumour meeting in zurich. the invitation had a questionnaire attached, where you answered, ‘by circling the answers which correspond best to your current opinion on classification and grading’. at the end of march 1990 paul called together a who working group to discuss histological typing of tumours of the central nervous system. the main purpose was to get consensus on the nomenclature to be used in the next version of the iacc brain tumour classification. although the meeting took place more than 30 years ago, you can recognise the expertise of the participants by reading some of the last names in the official photo. scott vandenberg sat in for lucien rubinstein who died in january 1990. there were slide-viewing sessions, short presentations on specific problems followed by panel discussions, and occasional decisions by voting. for me, a highly educational and excellent experience. one evening, peter burger, bernd scheithauer, roy weller and i discussed criteria for grading oligodendroglioma among other things. bernd and i agreed to take this further. we wanted to do an ‘oligo study’ (heading of a letter from bernd to me, july 9th, 1990) comparing different grading systems. i had the impression that ‘my’ system would be part of the study, but something went wrong. i am not sure what it was. the study was now ‘oligodendroglioma grading project’. in the summer of 1993 i had fulfilled my contribution to the somewhat changed study/project. caterina gianinni compiled the input from six neuropathologists and six surgical pathologists and was the first author of ‘oligodendrogliomas: reproducibility and prognostic value of histologic diagnosis and grading’ (journal of neuropathology and experimental neurology 60(3):248-62, 2001). the second event came after the xith international congress of neuropathology, kyoto, japan 1990, which was my first and only visit to asia. i had a gap of two or three days between the end of the congress and an ms meeting in the same city. i was completely on my own; no one understood what i said. i did not interact with anyone for days. at least it felt so. (writing this, i reflect that feeling down and being depressed is not the same.) that aside, for the second time in life, i felt so down that i considered it depression. i avoided entering the balcony of my hotel room… on the way back home, i bought a cd at the airport in singapore. it was the misspelling on the cover that hit me: beethoven’s third symphony (‘erotica’)! back in norway, beethoven helped me over my down period/depression. from the first day, i played his ‘eroica’ very loudly (very, very) driving to and from work. it helped very much. i did not miss a day’s work. the ‘third’ is a dynamic source of mental energy. x: department of neurosciences, lerner research institute, cleveland clinic, cleveland, ohio the first time i met bruce trapp was in vienna, 1982. he was in company with my friend henning laursen (neuropathologist at rigshospitalet, copenhagen). they knew each other from the time they spent as postdocs at the national institutes of health, bethesda, maryland (laboratory of neuropathology and neuroanatomical sciences, national institute of neurological and communicative disorders and stroke). after short introductory remarks, henning suggested that we come together in a ‘heuriger’ the same evening. ‘heuriger’ is a word for austrian wine taverns serving this year’s wine (heuer: this year) that they produce themselves. they serve their wines in gardens, and welcome everyone warmly; just what i needed following the infamous afternoon session. outside on bleachers, a couple of americans and scandinavians had a jovial and laid-back time in a pleasant atmosphere. since then, bruce trapp and my way have crossed many times. in one aanp annual meeting, bruce asked me if i knew any ‘bright norwegians’ (!) who could be recruited to join him at the department of neurology, johns hopkins school of medicine, baltimore, maryland. i said yes. back in bergen, a young md, lars bö, took the challenge and met with bruce trapp and the chairman of the neurology department, john ‘jack’ griffin, to check out the ‘premises’ in 1990. lars and his wife, elisabeth, went to baltimore in 1991, supported by the norwegian research council. he developed into an open-minded, innovative, and rational researcher. bruce trapp was, and still is, a well-known myelin researcher. his ultrastructural studies on myelin are of exceptional quality and won him the ‘weil award for best paper on experimental neuropathology’ presented at the aanp annual meeting 1986. so, when harald nyland and i prepared for a meeting on myelin and demyelination, bruce trapp was one of the 17 international experts we invited to bergen. in the 1990s the lerner research institute at the cleveland clinic inc. (cleveland, oh) wanted to add a department of neuroscience to its stock of nine research departments. bruce trapp applied and was offered the position of director of the newly established unit. he started developing neuroscience at ccf in 1994, and lars fit well into the structure that bruce had in mind. we, wife kari and son erik (born 1980) moved to join the ‘trapp lab’ for a one-year-sabbatical in 1997/98. meeting the trapp family was an enjoyable event. after a short while we were treated and felt like family. our families are closely connected still. i could write pages about caring carol, the twins brian & dan the man, sara, david, and bruce. i hope it will be done by others. the collaboration of neuroscience in cleveland and neuropathology in bergen developed in a persistently friendly and inspiring atmosphere. i learned the basics of preparing and immunostaining free-floating 30μm thick sections for confocal microscopy by ansi chang. she was, and still is, an extremely skilled and intelligent person who i am glad i met and with whom i still keep in touch. she instructed me to be an approved user of the leica aristoplan laser scanning microscope. through confocal microscopy and computer-based, three-dimensional reconstructions, we studied pathological changes in multiple sclerosis brains. many hours were spent on the confocal microscope looking for clues. the attraction of the ‘collapsed’ stacks of the laser-scanned immune-stained 30μm thick sections from the frozen specimen of brain tissue was evident in images. the image is stained for myelin and microglia. it was a good feeling to know that you had removed the cns at post-mortem, fixated, selected areas to study, cut, and stored tissue wet or in paraffin blocks. i brought some of the tissue to the us in my hand luggage. the co-operation between cleveland and bergen resulted in many publications (the first results were published in 1994, and the last paper last year in 2020. one, in the new england journal of medicine had a special impact since our findings initiated an editorial (‘demyelinating diseases ― new pathological insights, new therapeutic targets’) in the same journal. our results were presented on national tv and, in february of 1998, reached the front page of the new york times. in other words, the old knowledge that ms lesions affect grey matter of the brain as well as white matter came in ‘new wrappings’ and got some great attention. i had long since noticed that the characteristic lesions in ms brains did not spare the grey matter. so i got the idea to sample grey and white matter from preselected areas disregarding macroscopic changes/lesions. we wanted to see what the eyes did not see; therefore, we searched for a new way to examine ms brains. we did not sample macroscopic lesion. rather we stuck to sampling brain material strictly from preselected areas. we weren’t looking for macroscopically evident ms lesion; instead, we took tissue probes from predetermined areas. (i was experienced in cutting formalin fixed brain in the frontal plane coronal sectioning). a section through the anterior commissure turned out to be a fine landmark for getting similar brain areas from different individuals with ms. from this section we sampled the superior temporal, the cingular gyrus, and parietal tissue. we revealed the extent and pattern of demyelination in the cerebral cortices and underlying white matter of 20 ms brains. tissue from the same preselected brain areas from people without neurological disease served as controls. our findings were published in the jnen in 2003 and undermined the amount and importance of grey matter (cortical) changes in the brains of ms patients. lars bö defended his phd dissertation ‘multiple sclerosis: immunopathological studies of inflammatory central nervous system demyelination’ at the university of bergen, in 1998. the five peer-reviewed articles of his dissertation have, as of may 2021, been cited 5,639 times. space was precious in the energetic and crowded neuroscience floor. so, when the newly ‘added’ neuropathologist/researcher susan staugaitis arrived in cleveland, i had to ‘give up’ my office to her. she was split between diagnostic service for pathology and research in neuroscience. susan was intelligent, sharp, and well informed. she was an outspoken and confident contributor in many aanp and other scientific meetings. we had lot of engaging discussions. susan sadly had a glioblastoma diagnosed in 2014, a fact that she was extraordinarily open about. in 2017, i sent her a christmas greeting with this photo from the library room. drs susan staugaitis (✝) and ansi chang were treasured colleagues of mine in the trapp lab. susan: ‘it goes without saying that i am still alive. i never believed i would be around this long. i am doing my best to stay as strong and safe as possible as i continue to live alone in my home. it helps when we can look at things from multiple perspectives and find the part of the glass that is half full’. she died in august 2018. xi: other activities medical university of lübeck in the year 1360 the hanseatic league (the german guild of merchants) created an overseas office in bergen. for almost 400 years, they dominated commerce and social life here. hanseatic influence is still very much evident in surnames, buildings, and the wharf (‘german wharf’ or ‘tyskebryggen’, after wwii best just called ‘bryggen’) in the centre part of the city. in the 1970s, a ‘hansa-related’ idea was born at an international forensic science meeting where professors otto pribilla from lübeck and johan chr. giertsen from bergen happened to meet. they found it timely to try to establish an official contact between the two hanseatic cities through their respective universities. a general agreement of cooperation was officially acknowledged by both parties in 1976. the aim was and is to foster better contact through student exchange, postgraduate education, and research within the fields of medicine and natural sciences. i had the honour of taking giertsen’s place as coordinator of this cooperation from 1993 to 2011; most of the time together with the surgeon professor jan fredrik halvorsen. we enjoyed interactions with many team players in lübeck. manfred oehmichen, eberhard schwinger, and michael seyfahrt were among the proactive professors, to name a few. the main burden of this successful treaty was carried by the administrators peter mühlhausen (oberamtsrat) in lübeck and johannes (johs) teigland (faculty director), as well as torill knag in bergen. in 1995 the general agreement of cooperation between the universities was extended without limitation. universitäts spital zürich 2002 in 2002, i spent an interesting year at the universitäts spital zürich. i was asked to take care of the diagnostic neuropathology for a year or so. phillip u heitz was the director of the institute of pathology and molecular pathology, with its highly qualified medical and technical staff. i especially remember the productive, pleasant, and friendly atmosphere within the department. social skills were ‘comme il faut’, highlighted by free ski weekends in davos. lifts, lodging, and food were paid for by the department! the institute of neuropathology was completely focused on prion research, enigmatically led by adriano aguzzi. this meant they were continually searching for a clinically oriented neuropathologist to take care of the diagnostic routine for a longer or shorter period. i went for one year (december 2001march 2003). i intended to combine diagnostic ‘neurosurgical’ work with microglia research but that did not materialise. i did, however, learn a lot about prions and psychology, and witnessed the immense effort of postdocs to satisfy their manager during the weekly progress report (and in-between). i connected with many of the optimistic, eager, dedicated, young scientist during my period there. 12 years later, one of them, jens pahnke, became professor of neuropathology at the oslo university hospital. his track record has been and is superb. i appreciated working with the fine technical staff of the institute of neuropathology. this photo shows that we had a good time in between skiing, needless to mention afterwards! miscellaneous major changes have increased the effectivity of doing diagnostic pathology and research over the years. i’ll just mention a few elements that made my way easier. one general problem that gummed up the works all over was the backlog created by secretaries typewriting manuscripts. if you had a few minor changes, pages or the whole report had to be typed anew. from 1973 the ibm ‘golf ball’ typewriter with an internal correction feature reduced the backlog for typing and retyping manuscripts. another problem was that all manuscripts had to be submitted with original photos to be accepted for review. there were strict instructions for authors, especially regarding illustrations, and you had to pay for coloured illustrations. it seemed that every journal had different rules. the invention of the letraset’s dry rub-down instant lettering made it easier to put arrows on figures. they were large, half transparent sheets with letters, stars, arrows and more. letraset was ‘expensive’ and popular; you had to ask around to find the right sheet. it was not uncommon to find the tokens you needed for your illustration ‘scratched out’. it took time to order new sheets. letraset was very popular for years before desktop publishing took over. to find and check out correct references, libraries had meter after meter with index medicus occupying their shelfs. they received new, heavy books annually. you had to carry one to your desk, turn the pages, carry it back to the shelf, get the next one, and so on. during the 1990s, the introduction of the internet and world-wide-web (www) accelerated the shift from printed to online information, especially with the start of pubmed. also in the 1990s, powerpoint transformed the process of preparing and giving presentations. the development of immunohistochemistry (ihc) opened possibilities in characterizing tissue, cells, and cell products. at first, you were lucky if you knew someone who had produced an antibody and was willing to share it. now the production of antibodies is a huge industry providing opportunities for research and industrial applications. (order online and receive your antibody in a couple of weeks!) for me, it was great experience to effectively pinpoint the inborn superintendent of the microenvironment of the normal brain, the microglia (mg)! they are almost invisible in conventional staining (he). the importance of mgs cannot be overrated. they are everywhere in the cns, acting as sensors (housekeepers) of the local milieu. they are constantly working, switching between quiet monitoring physiologic activity and active efforts to keep changes from doing harm. tissue micro arrays (tmas) became popular in the 2000s. small cores of tissue (for example tumour material) could be cut from a ‘donor’ paraffin block and, one by one, placed in an ordered way in columns and rows to the recipient block. as large number of samples could be examined in a single cut from one recipient paraffin block, thus creating an exceptionally cost-effective and timesaving research tool. the digitizing of specimen slides with computer-assisted technology has led to digital pathology as a powerful and expanding line of work for easing many aspects of diagnostic pathology. special scanning machines convert the tissue specimen on the glass slides to a seamless image on your computer screen. this allows for a wealth of possibilities related to histological assessment (diagnosis), research and teaching. i have worked as a digital pathologist for many years now. my first experience was in costa del sol (spain) in 2015. there i performed microscopic evaluation and diagnoses on surgical biopsies in between rounds of golf and trips to the beach. digital pathology allows for quicker measurements, easier comparisons, straightforward sharing of information, and effortless transfer of digitised images for consultation and second opinions. this home-office type of diagnostic, digital pathology is perfect in this sars-cov-2/covid-19 pandemic. polymerase chain reaction (pcr) needs no introduction, but i will tell the following historical anecdote: as a postdoc at the institute for enzyme research, university of wisconsin 1968-1970, the norwegian kjell kleppe (bergen based) found ways to copy larger amounts of dna from a limited quantity (studies on polynucleotides. xcvi. repair replications of short synthetic dna’s as catalyzed by dna polymerases. journal of molecular biology 56(2):341-361, 1971). kary b. mullis used more heat resistant enzymes in the reactions and was given the nobel prize in chemistry for 1993 ‘for his invention of the polymerase chain reaction’. neither the nobel committee nor the laureate mentioned the pioneering work of kjell kleppe. the importance of kleppe’s work has been emphasised recently (see jonathan d kaunitz in digestive diseases and sciences 60(8):2230-2231, 2015; https://en.wikipedia.org/wiki/pcr). haukeland university hospital, bergen, norway the norwegian cancer registry contains complete, mandatory reports of all cancer cases in the country. since the registry can be linked to the mortality register of ‘statistics norway’, a follow-up rate of 100% is obtainable. in cooperation with fröydis langmark (director of the norwegian cancer registry) and arild helseth, we performed population-based epidemiological studies on intracranial gliomas, primary intraspinal neoplasms, and meningioma. we also analysed the occurrence of multiple primary neoplasms in people with meningioma. results were published in 1988 and 1989. in 1989, are helseth defended his thesis ‘a population-based survey of neoplasms of the central nervous system in norway’ in bergen, for the phd degree. another fruitful collaboration was that with christos d katsetos, the neuropathologist of the departments of pathology and laboratory medicine at hahneman university and st. christopher’s hospital for children hospital, philadelphia. we knew each other from mutual uva experience (both, at different times, disciples of lucien j rubinstein). this cooperation led to his successful public defence of ‘neurobiology of class iiib-tubulin isotype: differential cellular expression in human development and neoplasia’ for the phd degree at the university of bergen in 2002. paul kleihues (zurich/lyon/zurich) and roy weller (southampton) were the evaluators of and opponents in the public discussion of the dissertation at the haukeland university hospital, bergen. (sadly, christos passed away in 2017.) in the central block of haukeland university hospital, forensic pathology is located next to labs and offices of neuropathology. we have had much to do with each other. years of oil-related professional diving in the north sea has claimed many lives. likewise, amateur diving is another diving activity that sometimes can be fatal. forensic post-mortem examinations of such cases always gave rise to examinations of brains and often the spinal cord too. surprisingly, there is very little damage to be found even in long-time professional saturation divers. one should think that the pressure and the stress of such activity takes its toll. we were surprised, after examining 8 to 64 (!) tissue blocks from the spinal cord of twenty divers with immunohistochemistry, to conclude that the cords did not reveal tissue changes. while i am happy that the study was published in the journal undersea & hyperbaric medicine, i now think that i perhaps should have published this thorough report somewhere else with wider reach. norwegian ms registry & biobank research related to brain tumours, and ms have been closest to my heart. in cooperation between the norwegian multiple sclerosis competence centre and the norwegian ms registry & biobank, we have organised cns material for research purposes. we have stored clinical data, paraffin blocks, stained, and unstained slides from more than 40 persons with ms. in many cases bioengineers brynhild haugen and laila vårdal embedded whole coronal sections from ms brains into paraffin. they are cut by a large sliding microtome (not many of them around) into 20μ sections, mounted on glass slides in glycerol, and cover slipped for microscopic analysis. the manoeuvring via water bath to glass is technically challenging, as is the staining process. the images (above and below) show how it can be done! regarding ms, there is so much to wonder about. looking through the microscope on ihc stained lesions, i still haven’t been able to grasp who or what is the wrongdoer? angiocentricity of lesions is for me not a general feature. staring at different highlighted antigens from the same lesion gives me an uneasy feeling that the key is there, you just cannot add up the clues to unlock the enigma. i think of the philosopher henri bergson: ‘the eyes only see what the mind is prepared to comprehend’. above, a thin (20μ) coronal brain sections immunostained for myelin (dark brown). it shows bilaterally irregular, and sharply bordered, large, demyelinated areas adjacent to lateral ventricles. look closely and you will notice the amount and variation of the widespread myelin deficits in the cortex. to me it looks as if the players in demyelinated foci have their own agenda! to the left, a single reactive astrocyte dominates the field with scattered myelin rests. (two intact internodes?). to the right you see a tiny myelin debris in a cortical (autofluorescence) field with total loss of myelin and a solitary microglia cell (watch dog) with multiple tiny cell processes, which do not signal activation. what happened to the myelin? one can wonder… then i consider: what about the bioactive substances that are present in the same spot, untagged? this slightly tilted 3d presentation shows a very active cortical ms lesion; the most active picture i have seen. microglia is in red, and myelin is in green. the yellow colour signals co-localisation. the morphology is clearly that of acutely activated microglia engulfing myelin. think about this image as a point in time in a video, with a dynamic remodelling of the cytoplasm, with extensions and retractions in an undulating mode. it goes from slim microglia via this morphology to full-blown macrophages. hopefully methods of the future will start to bring us nearer to the ultimate goal: to unravel the mystery of multiple sclerosis. above a conventionally cut section where demyelination (black=myelin in subcortical white matter) affects the full thickness of the cortex can be seen. the extremely sharp border between affected and normal brain should say something about how the wrongdoer moves in the microenvironment. it is a well-known fact that the movement of cerebrospinal fluid (csf, oedema) differs greatly within the cns. the restriction of fluid movement is much higher in grey matter than in white matter. the complexities of the multi-layered and compact grey matter lead to slower drive of substances here. our (bö et al.) findings presented in jnen in 2003, showed purely cortical subpial demyelination in an extent not demonstrated earlier. this indicates that the csf transports myelin-harming substances which lead to demise of myelin in ms. (fluid transport through the ependyma of the ventricular walls is unrestricted; this being so, white matter oedema drains directly into the csf.) i have a gut feeling considering initial events in ms. recent studies have revealed prominent clonal expansion of cd8 positive t-cells within the cns during ms (journal of immunology 206(1), 2021). this is just one example that has confirmed our findings, first reported in vienna in 1982. the photograph shows my closest co-workers and friends from before 2000 to 2012 and beyond: the bioengineers edith fick, laila vårdal, and brynhild haugen. many are those who have helped me along my way. in bergen, i have had expert assistance on different occasions by anne marie austerheim, karin eliassen, gerd lillian halseth, nina holmelid, randi lavik, karen böhm nilssen, bendik nordanger, tore-jacob raa, laila vårdal, and grethe waaler. i left diagnostic neuropathology after turning 70 in 2012. i felt like i was one of the last surviving members of the he only (+ some ihcs) era of neuropathologists squeezing the most out of basic, classic, cost-efficient, affordable, and fast histopathology. after april 2012, i continued as a consultant in general surgical pathology (at 30% of full time) through 2019. that was the end of my affiliation to the haukeland university hospital. however, i am still engaged in digital diagnostics. i have spent my pathology career believing in short, relevant descriptions, quick responses, and up-to-date diagnoses. i am content as an emeritus professor sharing a 15 m2 office with two other emeriti. one of them is ole didrik laerum, the one who got me to bergen in the first place. each of us have a desk, one microscope, and one computer. rarely are all three of us present at the same time, so the space is adequate. i hope to accomplish a few more things yet. the last years have come and gone like snow in april. in the last decade, my beloved wife, kari, who has been a source of love and order for our close family, started to lose us and herself. she was diagnosed with dementia. she was so full of life, a loving mother, and a talented artist. her care enabled my career and family life, so it was difficult when the tables were turned, and i found myself caregiving for her. my close-knit family has helped me greatly in coping with this challenging time, for which i am endlessly thankful. she has transitioned to a care home, but i visit her frequently, and she frequently comes back to herself. her face often lights up when she sees a frequent visitor, whom she sometimes recognises as ‘sverre’. i have told myself, ‘de-mentia is not a-mentia’, and for kari, that is true. she has retained her use of language. on one recent visit, she exclaimed, ‘there are more people here!’ ‘just me’, i said. ‘well’, kari said. ‘i am here too!’ for the last 50 years, she has been a wonderful companion and remains one now, even as we come to the end. the challenge i was given by the free neuropathology has helped me to concentrate on times past. the time spent has been valuable. i have by now wound up most items that i had put aside. private letters, reports, material related to international co-operation, journals, books, folders with obituaries, reprints, and photographs. i thank werner paulus and tibor hortobágyi for this opportunity to illuminate my existence. brian trapp helped me with language corrections and inputs. a mild acceptance of how things have turned out, the ‘you could have done better’ have been overtaken by the perception of the facts of my way. all the way, robert frost’s poem (the atlantic monthly, 1915) has been with me. the road not taken by robert frost two roads diverged in a yellow wood, and sorry i could not travel both and be one traveler, long i stood and looked down one as far as i could to where it bent in the undergrowth; then took the other, just as fair, and having perhaps the better claim, because it was grassy and wanted wear; though as for that the passing there had worn them really about the same. and both that morning equally lay in leaves no step had trodden black. oh, i kept the first for another day! yet knowing how way leads on to way, i doubted if i should ever come back. i shall be telling this with a sigh somewhere ages and ages hence: two roads diverged in a wood, and i--i took the one less traveled by, and that has made all the difference. with my friend, tore, who led me into the road taken 50 years ago copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuromuscular disease: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:2 (2023) review neuromuscular disease: 2023 update marta margeta department of pathology, university of california, san francisco, ca, usa corresponding author: marta margeta· ucsf pathology, box 0511 · 513 parnassus ave., hsw-514 · san francisco, ca 94143 · usa marta.margeta@ucsf.edu submitted: 07 february 2023 accepted: 22 february 2023 copyedited by: biswa ramani published: 27 february 2023 https://doi.org/10.17879/freeneuropathology-2023-4682 keywords: long covid, guillain-barré syndrome, wallerian degeneration, nodes of ranvier, muscle regeneration, chaperonopathy, target fiber, inclusion body myositis, facioscapulohumeral dystrophy, myotonic dystrophy, amyotrophic lateral sclerosis abstract this review highlights ten important advances in the neuromuscular disease field that were reported in 2022. as with prior updates in this article series, the overarching topics include (i) advances in understanding of fundamental neuromuscular biology; (ii) new / emerging diseases; (iii) advances in understanding of disease etiology and pathogenesis; (iv) diagnostic advances; and (v) therapeutic advances. within this general framework, the individual disease entities that are discussed in more detail include neuromuscular complications of covid-19 (another look at the topic first covered in the 2021 and 2022 reviews), dnajb4-associated myopathy, nmnat2-deficient hereditary axonal neuropathy, guillain-barré syndrome, sporadic inclusion body myositis, and amyotrophic lateral sclerosis. in addition, the review highlights a few other advances (including new insights into mechanisms of fiber maturation during muscle regeneration and fiber rebuilding following reinnervation, improved genetic testing methods for facioscapulohumeral and myotonic muscular dystrophies, and the use of sarm1 inhibitors to block wallerian degeneration) that will be of significant interest for clinicians and researchers who specialize in neuromuscular disease. abbreviations ad autosomal dominant, adpr adenosine diphosphate ribose, aidp acute inflammatory demyelinating polyneuropathy, als amyotrophic lateral sclerosis, aman acute motor axonal neuropathy, amsan acute multifocal sensorimotor neuropathy, ar autosomal recessive, cnm centronuclear myopathy, cox cytochrome c oxidase, covid-19 coronavirus disease 2019, dm dystrophia myotonica (myotonic dystrophy), emg electromyography, fshd facioscapulohumeral dystrophy, gbs guillain-barré syndrome, iefnd intraep-idermal nerve fiber density, ivig intravenous immunoglobulin, lmn lower motor neuron, mac membrane attack complex, mfn2 mitofusin 2, mhc-i major histocompatibility complex i, mnd motor neuron disease, myhc myosin heavy chain, ncs nerve conduction studies, nor node(s) of ranvier, pasc post-acute sequalae of sars-cov-2 infection, pm-mito polymyositis with mitochondrial abnormalities, sc schwann cell, sfn small fiber neuropathy, sibm sporadic inclusion body myositis, str short tandem repeat, xlmtm x-linked myotubular myopathy in this annual update, i will briefly describe ten neuromuscular field advances from 2022 that i consider to be most important and/or interesting; as in the prior updates (margeta, 2020, 2021, 2022), these advances will be grouped into different “discovery clusters” and listed in no particular order. advances in fundamental neuromuscular biology with implications for neuromuscular disease 1. regulation of the neonatal-adult maturation checkpoint during skeletal muscle regeneration in the past two reviews (margeta, 2021, 2022), i described the crosstalk between macrophages and satellite cells (muscle stem cells) that initiates skeletal muscle repair. in this review, the focus will be on a later stage of myofiber regeneration, which recapitulates muscle development and involves myofiber growth and maturation from a fetal to the adult state. in mammals, there are seven distinct isoforms of myosin heavy chain (myhc; the major contractile component of thick filaments) – two isoforms that are normally expressed only during development [embryonic (myhc-emb / mhc3) and neonatal (myhc-neo / mhc8)] and four isoforms that are expressed in mature muscle fibers [one slow isoform (mhc-slow / mhc7; type 1 fibers) and three fast isoforms: myhc-2a / mhc2 (type 2a fibers), myhc-2x / mhc1 (type 2x fibers) and myhc-2b / mhc4 (type 2b fibers)] (schiaffino et al., 2015). during muscle repair, regenerated myofibers mature over ~14 days by recapitulating developmental sequence of myhc expression: newly formed fibers first express myhc-emb, followed by myhc-neo, and finally one of the adult myhc isoforms. [this aspect of the regeneration process is diagnostically useful: in adult muscle, immunohistochemistry for developmental myhc isoforms can be used to detect regenerating fibers [(sewry et al., 2021) and fig. 1.]. figure 1. regenerating muscle fibers express developmental myosin heavy chain isoforms, myhc-emb (mhc3) and/or myhc-neo (mhc8). dual immunohistochemical stain for myhc-emb (red) and myhc-neo (brown), here performed on cryosections, can be used to detect regenerating fibers at different stages of maturation. (a) in normal adult skeletal muscle, there is no staining for either myhc-emb or myhc-neo. (b) a case of immune-mediated necrotizing myopathy (imnm) shows frequent regenerating fibers, most of which are in the early stages of maturation and express only myhc-emb (an example is highlighted by the green arrow). a few regenerating fibers are in the late stages of maturation and express only myhc-neo (an example is highlighted by the orange arrow), while several regenerating fibers are transitioning from an early to a late maturation stage and co-express both myhc-emb and myhc-neo (dark orange sarcoplasmic staining; an example is highlighted by the black arrow). (c) a case of sporadic inclusion body myositis (sibm) shows abundant regenerating fibers spanning the entire spectrum of maturation. (d) in dermatomyositis (dm), both early and late regenerating fibers are mainly found in the perifascicular areas. scale bars: a and d, 200 µm; b and c, 50 µm. wang and co-authors have now elucidated molecular and cellular mechanisms that regulate the neonatal / adult transition checkpoint, a key step in this maturation sequence (wang et al., 2022). using a myotoxin-induced model of skeletal muscle injury, they showed that expression of mitofusin 2 (mfn2; a mitochondrial membrane protein that initiates mitochondrial fusion and is induced in activated satellite cells) is required for maturation of newly formed myofibers: in mfn2-deficient mice, myotoxic muscle injury led to formation of early (centrally nucleated, mhc3-expressing) regenerating fibers, but these fibers were smaller than regenerating myofibers in wild-type (wt) mice and ultimately arrested their maturation at the neonatal (mhc8-expressing) developmental stage. this maturation defect was not due to a metabolic abnormality in satellite cells; rather, mfn2 deletion led to nuclear translocation of transcription factor nfatc2 (nuclear factor of activated t cells 2) and increased expression of hif1α (hypoxia-induced factor 1α), ultimately resulting in the lack of repressive methylation marks at the mhc8 genetic locus, which are required for the neonatal / adult fate transition. in wt animals, expression of hif1α protein was induced following muscle injury, but its level returned to the preinjury baseline within 5 days; in mfn2-null animals, hif1α levels remained elevated 14 days post-injury. importantly, sustained hif1α signaling was both necessary and sufficient for the arrest of regenerating myofibers at the neonatal stage: treatment of mfn2-/mice with px-478 (a chemical compound that reduces hif1α levels) for 14 days, starting at 14 days after muscle injury, enabled mfn2-/regenerating myofibers to escape the maturation arrest, adopt the adult fate, and grow closer to a normal adult fiber size. the same rescue could be achieved through genetic deletion of hif1α or through inhibition of h3k27 demethylases; conversely, the effect of mfn2 deletion could be replicated by genetic manipulation of vhl (von-hippel lindau tumor suppressor) signaling, which leads to hif1α stabilization independent of mfn2 signaling. finally, the authors used an ischemic model of muscle injury to show that in wt mice, repressive methylation marks at the mhc8 locus started appearing on day 9 (coincidental with reperfusion), followed by fiber growth and transition to the adult fate on day 10, and complete downregulation of mhc8 expression by day 12. in contrast, myofiber maturation was significantly accelerated in animals lacking hif1α, with completely differentiated fibers lacking any mhc8 expression present on day 8. taken together, these data indicate that mfn2/hif1α signaling regulates myofiber fate specification through the epigenetic control of myhc-neo / mhc8 expression, and that this checkpoint enables synchronous development of all skeletal muscle tissue elements. however, it should be noted that all experiments in this study were done on the mouse tibialis anterior muscle, which is largely composed of type 2 fibers; thus, it remains to be shown whether a similar maturation checkpoint also affects type 1 fibers. what is the relevance of this regulatory pathway for human disease? while that question needs to be investigated in much greater detail in the future, initial experiments performed by wang and co-authors offer some tantalizing clues. because maturation-arrested regenerating fibers are reminiscent of atrophic, centrally nucleated fibers seen in severe centronuclear myopathies (cnms), they evaluated biopsies from infantile patients with cnm 1 (caused by mutations in dynamin 2) and cnm 2 / x-linked myotubular myopathy (xlmtm, caused by mutations in myotubularin 1); in both diseases, they detected myofibers strongly positive for mhc8 as well as nuclear nfatc2 and sarcoplasmic carbonic anhydrase 3 (a hif1α target). while mhc3+ and mhc8+ positive fibers can be seen in any myopathy that involves significant fiber necrosis (fig. 1), they are generally not very numerous in congenital myopathies, raising the possibility that the mfn2/hif1α-controlled maturation checkpoint is specifically dysregulated in cnms. interestingly, a different 2022 study demonstrated that epigenetic alterations are a conserved feature of xlmtm and that histone deacetylase inhibition is a promising therapeutic strategy for this severe muscle disease (volpatti et al., 2022). while altered muscle development is one of the epigenetically modified pathways uncovered by volpatti et al., it remains to be shown whether changes in the methylation of the mhc8 genetic locus are part of the xlmtm epigenetic disease signature. interestingly, autosomal dominant mutations in mfn2 cause axonal charcot-marie-tooth disease type 2a (cmt 2a) but are not known to produce a significant myopathic phenotype. it is possible that cmt 2a patients exhibit atypical muscle development and/or aberrant response to muscle injury that contributes to their weakness but has been masked by their peripheral neuropathy; that needs to be explored by future studies. in addition, it needs to be investigated whether pharmaceutical treatments that turn off this maturation checkpoint can accelerate muscle repair following injury and/or prove beneficial for any muscle disease characterized by significant fiber necrosis and regeneration. newly defined / emerging neuromuscular diseases 2. neuromuscular complications of covid-19: post-acute sequelae of sars-cov-2 infection (“long covid”) syndrome covid-19, the novel infectious disease caused by sars-cov-2, primarily targets the respiratory system but also affects many other tissues and organs, including the pns. in the past two reviews (margeta, 2021, 2022), i discussed the emerging understanding of acute neuromuscular complications of covid-19; in this review, i will summarize what is currently known about neuromuscular involvement in the post-acute sequelae of sars-cov-2 infection (pasc) syndrome (also known as “post-covid conditions” or “long covid”). the cdc (centers for disease control and prevention, a branch of the united states government) defines pasc as continuation of covid-19 symptoms – or the emergence of new symptoms – four weeks or longer after initial sars-cov-2 infection; given this broad definition and the continuously changing spectrum of viral variants, the true prevalence of pasc is not known, but the current estimates range from 5-30%. while pasc can include a wide array of symptoms, some of the most common and particularly debilitating concerns (including fatigue / exertional intolerance, dysautonomia, and sensory symptoms such as paresthesias and numbness) suggest that the pns is affected. two small retrospective studies published in 2022 explored whether autonomic and sensory symptoms experienced by pasc patients can be attributed to small fiber neuropathy (sfn). the first study included 13 patients (8 women and 5 men) who had acute covid-19 infection in march-april 2020 and developed paresthesias 0-2 months later; the patients were evaluated by electromyography / nerve conduction studies (emg/ncs) and two punch skin biopsies (from distal leg and proximal thigh) to determine intraepidermal nerve fiber density (iefnd) (abrams et al., 2022). of these 13 patients, only one had severe covid, and none had a history of prior neurologic symptoms or evidence of large fiber polyneuropathy on emg/ncs. seven of 13 patients also reported orthostatic discomfort; four of these seven patients underwent autonomic function testing, with abnormal findings reported in three. reduced iefnd was seen in 46% patients (6 of 13; all female), all of whom had clinical findings consistent with sfn (mainly a decreased pinprick sensation in distal legs); the biopsy findings were also more severe distally. in contrast, most patients with normal biopsy results showed no objective sensory abnormalities on neurologic exam. the second study enrolled 17 patients (11 women and 6 men) who developed covid-19 between february 2020 and january 2021 and presented with new neuropathy symptoms 0.1 to 14.9 months following covid onset; again, only 1 of these 17 patients had a severe covid infection (oaklander et al., 2022). in this cohort, 16.7% of electrodiagnostic studies showed abnormal findings, while 62.5% of lower leg skin biopsies and 50% of upper thigh biopsies showed reduced iefnd. importantly, in both studies the clinicopathologic evaluation was performed with a significant delay due to pandemic-related disruptions in health care delivery; thus, it is possible that partial or complete small fiber recovery occurred in some patients in the interval between the symptom onset and assessment, leading to false-negative test results. both studies should be considered preliminary given their small size and confounding by referral bias; however, the reported findings are very similar and – if conformed in larger cohorts – suggest that the pasc-associated peripheral neuropathy reflects small nerve fiber injury. the underlying disease mechanisms also remain to be determined; however, a delay between covid infection and neurologic symptom onset in all but one of these 30 pa-tients, along with therapeutic effectiveness of the repeated ivig treatment in 5 patients who received it, suggest that the pasc-associated sfn is due to post-infectious dysregulation of the immune system. persistent fatigue is experienced by 50-70% of pasc patients, making it one of the most common pasc symptoms. however, it has been challenging to determine whether this relatively subjective and nonspecific clinical symptom is caused by functional response of muscle tissue to circulating inflammatory mediators, microcirculatory abnormalities, or an underlying skeletal myopathy. to start addressing this question, hejbøl and colleagues evaluated 16 patients (13 women and 3 men) with chronic post-covid fatigue; none of the patients had been severely ill with covid or hospitalized in intensive care unit, and clinicopathologic evaluation for myopathy (which included quantitative emg and skeletal muscle biopsy) occurred 5-14 months following acute infection (hejbøl et al., 2022). among these 16 patients with pasc fatigue, 69% also had paresthesias, but none had abnormal deep tendon reflexes or altered vibratory sensation, and ncs were normal in all 16. in addition to the fatigue, 81% of study patients had myalgia, 50% objective muscle weakness, and 75% myopathic findings on emg; serologic studies were generally unremarkable, except for one patient with a marginally elevated creatine kinase level and another with elevated tif1-γ antibody titer but normal creatine kinase level. remarkably, muscle biopsies from all 16 patients showed histologic and/or ultrastructural muscle fiber pathology; the findings were variable, but included prominent nucleoli and non-selective fiber atrophy (suggestive of fiber regeneration), basal lamina reduplication, myofibrillar disorganization, and mitochondrial abnormalities (fig. 2). in addition, inflammation and/or mhc-i upregulation was present in 62% of biopsies, while capillary abnormalities (including basal lamina reduplication and capillary loss/degeneration) were detected in 75% of biopsies. finally, one patient showed a loss of non-myelinated, small caliber axons in the intramuscular nerve twigs. the spectrum of findings seen in these specimens is very broad and does not point to a specific disease mechanism; however, both mitochondrial and capillary pathology could provide an explanation for fatigue, while basal lamina reduplication could be due to increased tgfβ (transforming growth factor β) levels, which often accompany chronic low-grade inflammation. like the two sfn studies described above, this study has several important limitations, including small cohort size, lack of patients from later stages of the pandemic (who were infected by sars-cov-2 variants other than the ancestral strain), and lack of a control group (i.e., patients with covid-19 who did not develop fatigue or other pasc symptoms); nonetheless, the findings suggest that muscle biopsy is the most sensitive test for diagnosis of the pasc-associated myopathy. figure 2. myopathy as a cause of fatigue in long-term post-covid-19 symptoms: histopathological changes in muscle fibers. (a, b) muscle fiber atrophy. (a) atrophic fiber and (b) folds in the basal lamina, both in patient 9. (c–f) muscle fiber damage indicated by basal lamina proliferation. (c) basal lamina duplication (arrow) in patient 16. (d, e) multiple myofiber basal lamina (d) and (e) higher magnification showing continuity between the different layers (arrows) in patient 14. (f) from patient 6, showing aggregation of nuclei, and multiple layers of myofiber basal lamina (arrow). (g–i) myofibrillary disorganization: (g) streaming of z-bands in patient 14, (h) disorganized myofibrils in patient 13, and (i) myofibril disorganization and a cytoplasmic body in patient 1. (j–l) mitochondrial changes: (j) a cytochrome c oxidase-negative fiber (blue) and (k) subsarcolemmal accumulation of structurally abnormal mitochondria from patient 8, and (l) subsarcolemmal accumulation of ultrastructural normal-appearing mitochondria in patient 9. scale bars: k = 500 nm; c, e, h, and l = 1 μm; a, b, d, f, g, and i = 2 μm; j = 100 μm. [this figure and its legend are reproduced without modifications from (hejbøl et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by-nc-nd international license.] collectively, these three studies provide initial evidence that neuromuscular symptoms commonly seen in pasc patients are associated with – and likely attributable to – structural abnormalities of peripheral nerves and skeletal muscles; however, these preliminary findings should be replicated in larger and temporally more homogenous cohorts of pasc patients. furthermore, additional work is required to establish whether similar nerve and muscle abnormalities are detectable in patients with myalgic encephalomyelitis / chronic fatigue syndrome, which shares many clinical features with the pasc syndrome. 3. dnajb4-associated myopathy: a new chaperonopathy molecular chaperones play a key role in the maintenance of protein homeostasis by facilitating folding of newly synthetized proteins and degradation of misfolded or damaged / unfolded proteins; the latter role is particularly critical in skeletal muscle, where mechanical stress leads to constant damage to sarcomeric proteins and where misfolded protein aggregates cannot be diluted through cell division. as a result, although chaperones and co-chaperones are ubiquitously expressed proteins, skeletal myopathy is generally the main (and often only) clinical manifestation of many chaperonopathies. dnaj/hsp40 co-chaperones determine the specificity of the chaperone/client interaction and are highly evolutionarily conserved from yeast to humans; mutations in several dnaj co-chaperones from the b family (dnajb2, dnajb5 and dnajb6) cause neuromuscular disease, but prior to 2022 no human disease has been linked to dnajb4. dnajb6 is a close homolog of dnajb4; it localizes to the z-disc and, together with hspb8 and bag3, plays a critical role in the z-disc maintenance via chaperone-assisted selective autophagy. autosomal dominant mutations in dnajb6 lead to two distinct clinical phenotypes, limb-girdle muscular dystrophy d1 and distal myopathy, both of which are histologically characterized by myofibrillar disorganization, protein aggregates, and rimmed vacuoles (sarparanta et al., 2020). last year, two separate research groups showed that mutations in dnajb4 cause a similar but distinct skeletal myopathy that is inherited in either autosomal recessive (ar; weihl et al., 2023) or autosomal dominant (ad; inoue et al., 2023) manner. weihl and co-authors used a reverse genetic approach to identify four patients from three different families who carry autosomal recessive, loss-of-function mutations in dnajb4 that either severely abrogate protein expression (c.856a>t; p.lys286ter and c.785t>c; p.leu262ser) or lead to expression of a non-functional protein (c.74g>a; p.arg25gln). clinically, these patients presented in the first decades of life with spinal rigidity and severe diaphragmatic weakness that led to early-onset respiratory failure. in contrast, inoue and colleagues used a more traditional forward genetic approach: they identified a single family with an autosomal dominant, slowly progressive myopathy characterized by onset in the 3rd to 5th decade, asymmetric thumb and grip weakness, symmetric distal weakness, respiratory failure, scoliosis, and ultimately loss of ambulation, and showed that a heterozygous missense variant of dnajb4 (c.270t>a; p.phe90leu) segregated with this clinical phenotype. muscle pathology was similar in both ad and ar forms of dnajb4 disease, with fiber size variation, rubbed out fiber centers on oxidative stains, rimmed vacuoles in atrophic fibers, and very large central inclusions / protein aggregates (up to 30-40 sarcomeres in length) in non-atrophic fibers that stained red on modified gomori trichrome, had an amorphous “woolly” or granulofilamentous appearance on em, and were immunopositive positive for p62, hsp70, desmin, myotilin, filamin c, and (in the ad form of disease) for dnajb4. ultrastructurally, there was also widespread degeneration of sarcomeres, but z-discs were relatively spared, with no definite z-band widening or streaming. interestingly, inoue and co-authors showed that this pathology is more prominent in type 1 muscle fibers, providing explanation for the preferential clinical involvement of muscles that are type 1 fiber-predominant (soleus, diaphragm, paraspinals, as well as thenar and hypothenar muscles). impressively, both groups generated model mice that recapitulated key aspects of human dnajb4 disease, including its unique myopathologic features and preferential involvement of type 1 muscle fibers; this was true both for knockout dnajb4-/mice (which model ar human disease; created independently by both groups) and for dnajb4f90l/+ knock-in mice (which model ad human disease; created by inoue et al.), suggesting that the f90l dnajb4 mutation has a dominant negative effect. of course, a lot remains to be discovered about this new disease. for example, it remains to be established why type 1 muscle fibers are more vulnerable to dnajb4 deficiency than type 2 fibers. in addition, it is not entirely clear why there are significant clinicopathologic differences between dnajb6 and dnajb4 diseases given the large degree of structural homology between these two proteins. one clue that has emerged from the work of inoue et al. is a subtle difference in the subcellular localization of these two proteins: while dnajb4 is localized to the close vicinity of the z-disc, dnajb6 directly colocalizes with this structure. future studies will be necessary to delineate the functional differences between these two co-chaperones, particularly with respect to their roles in chaperone-assisted selective autophagy, and in formation and/or degradation of rna stress granules. 4. nmnat2-deficient, sarm1-dependent hereditary axonal neuropathy (“sarmopathy”) wallerian degeneration is a highly regulated, active process of axon destruction; it is typically seen following acute mechanical or ischemic injury, but can also be caused by toxins, nutritional deficiencies, immune-mediated injury (further discussed in advance #5), and genetic alterations. regardless of the underlying etiology, wallerian degeneration is triggered by nad+ hydrolase sarm1 (sterile alpha and toll/interleukin-1 receptor motif-containing 1), the activity of which is regulated by the axonal nmn/nad+ ratio (osterloh et al., 2012; figley et al., 2021). under normal conditions, the activity of sarm1 (which is often described as “the central executioner of a cell-autonomous axon degeneration program”) is kept in check by a highly labile nad+-generating enzyme nmnat2 (nicotinamide mononucleotide adenylyltransferase 2); nmnat2 is trafficked from the soma into the axon, where it catalyzes synthesis of nad+ using nmn and atp as substrates (conforti et al., 2014). axonal injury disrupts axon transport of nmnat2, resulting in the buildup of nmn and depletion of nad+ distal to the injury site; this increases the nmn/nad+ ratio and activates sarm1, further depleting nad+ levels and causing metabolic crisis that ultimately leads to axon fragmentation. not surprisingly, deletion of nmnat2 in mouse models leads to perinatal lethality; however, mice lacking both nmnat2 and sarm1 are viable and protected against axonal degeneration. in humans, nmnat2 deficiency is very rare; however, homozygous r232q nmnat2 variant (which causes severe reduction in nmnat2 activity) has been linked to fetal akinesia deformation sequence (lukacs et al., 2019), while homozygous t94m nmnat2 variant (which reduces nmnat2 thermal stability) causes mild sensorimotor axonal polyneuropathy with erythromelalgia (huppke et al., 2019). (erythromelalgia is a syndrome that consists of sporadic attacks of burning pain, redness, and swelling, typically affecting the lower extremities; it is most often caused by gain-of-function mutations in scn9a, which encodes sodium channel nav1.7.) in their tour-de-force study published last year, dingwall and coauthors expanded the spectrum of human nmnat2-associated disease by showing that heterozygous nmnat2 loss-of-function mutations cause severe hereditary axonal neuropathy through a sarm1-dependent, macrophage-mediated non-cell autonomous pathway; consequently, the authors have termed this new disease “sarmopathy” (dingwall et al., 2022). the two affected brothers described by dingwall et al. share a very unique clinical phenotype that includes (i) repeated episodes of acute multifocal sensorimotor axonal neuropathy (amsan) that mimic guillain-barré syndrome (gbs), are typically triggered by acute infection, and include severe pain, erythromelalgia, flaccid quadriparesis, and respiratory failure; and (ii) chronic axonal motor neuropathy that progresses between these acute episodes and leads to distal weakness, scoliosis, and wheelchair dependence in the third decade of life. the patients carry compound heterozygous nmnat2 variants (the previously identified pathogenic variant c.695g>a; p.arg232gln / r232q and a novel variant c.292g>a; p.val98met / v98m); each of these variants was inherited from one of the patients’ asymptomatic parents and was shown to markedly reduce nmnat2 enzymatic activity. to further study this new hereditary neuropathy, the authors generated nmnat2v98m/r232q model mice. like the two patients, these model mice developed an age-dependent, motor-predominant axonal polyneuropathy that led to severe hindlimb wasting by 9-12 months of age and was characterized by a severe loss of axons in mixed and motor nerves (fig. 3a-b); in contrast, sensory nerves were largely spared (fig. 3c), as were motor neuron cell bodies in the ventral horn of the spinal cord. unlike the two patients, the model mice did not develop amsan episodes or hypersensitivity to pain, possibly because they were housed in a pathogen-free animal facility and thereby spared from acute infections. as expected, the axonal neuropathy phenotype in model mice was rescued by simultaneous deletion of sarm1 (fig. 3d-f), demonstrating that sarm1 activation was necessary for axon degeneration; a similar, but slightly less effective rescue was achieved by transgenic expression of a dominant negative variant of sarm1. more surprisingly, the sarm1 effect in this mouse model was not entirely cell-autonomous; instead, it was at least in part mediated by macrophage activation, and could therefore be blocked by macrophage depletion (via monoclonal antibody against colony stimulating factor 1 receptor). importantly, macrophage depletion was effective not only when treatment was started at birth, but also when it was started at 4 months of age (by which point mice already developed significant neuropathy); this suggests that blocking ongoing axonal degeneration can tip the balance toward axonal regeneration and functional recovery. figure 3. nmnat2 variants cause progressive axon loss in mice in a sarm1-dependent manner. (a–c) representative images of sciatic (a), femoral (b), and sural (c) nerves in 9–12-month-old nmnat2v98m/r232q (n = 9) or wt (n = 5) mice. percent axonal area/total nerve area are indicated to the right (n = 4–11 mice per age cohort, per genotype). (d–f) representative images of sciatic (d), femoral (e), and sural (f) nerves in 9–12-month-old nmnat2v98m/r232q; sarm1-ko mice. percent axonal area/total nerve area is calculated below each corresponding nerve (n = 3–11 mice per age cohort, per genotype). statistical significance was determined by 2-way anova with multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. scale bars, 50 μm. [this figure and its legend were adopted from figures 3 (a-c) and 5 (d-f) in (dingwall et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by international license.] as always, many questions remain to be answered. in particular, it is not clear why acute attacks in human patients involve both sensory and motor nerves, while chronic neuropathy predominantly affects motor nerves in humans as well as mice. [preferential involvement of motor axons in the mouse model can be attributed to differences in macrophage activation between sensory and motor nerves; however, given that increased sarm1 activity is observed in both nerve types, it is not clear what leads to these nerve-specific differences in macrophage activation. it is also possible that motor neurons are particularly vulnerable to sarm1 activation; in agreement with that possibility, hypermorphic sarm1 variants were found to be enriched in patients with amyotrophic lateral sclerosis (als); (gilley et al., 2021; bloom et al., 2022)]. moreover, it is not understood why nmnat2 deficiency primarily affects peripheral nerves in both mice and humans when sarm1 acts as the central regulator of wallerian degeneration in both cns and pns; a plausible explanation that needs experimental confirmation is that longer length of pns axons makes them more vulnerable to nmnat2 deficiency. finally, the prodegenerative effect of macrophage activation in the nmnat2v98m/r232q mouse model was entirely unexpected, not only because it was previously thought that sarm1 acts in an entirely cell-autonomous manner, but also because of a large body of prior research that demonstrated a key role of macrophages in axon regeneration [as discussed in advance #1 of my last update (margeta, 2022)]. these discrepancies suggest that macrophage effects on axonal degeneration are complex and context-dependent, and raise the possibility that macrophage depletion therapies could be either beneficial or detrimental, depending on the specific clinical scenario. one intriguing possibility, which needs to be addressed by future studies, is that macrophage activation promotes degeneration of damaged but still viable axons in chronic axonal neuropathies, while promoting axonal regeneration following severe insults (such as nerve trauma or ischemia) that result in catastrophic axonal injury. advances in understanding of etiology and pathogenesis of neuromuscular diseases 5. axonal injury in guillain-barré syndrome starts at the nodes of ranvier gbs is an autoimmune disorder of the peripheral nerves and spinal roots that is typically triggered by a preceding infection or vaccination; nerve injury is the result of autoantibody-induced complement fixation and macrophage activation. while gbs is traditionally considered to be a demyelinating disease (also known as acute inflammatory demyelinating polyneuropathy / aidp), it can also manifest as a primary acute motor or sensorimotor axonal neuropathy (aman / amsan). bystander axonal injury can also develop in the aidp variant of gbs, secondary to schwann cell (sc) injury. regardless of the disease variant, the long-term severity of gbs is ultimately determined by the extent of axon loss, which is less reversible than the myelin loss; however, the mechanisms that underlie the primary and secondary axon injury in gbs have been difficult to dissect because the key autoantibody targets (such as gm1 gangliosides) are often expressed by both neurons and scs. to circumvent this challenge, a research group in scotland created model mice that selectively express gm1 ganglioside on either sc or neuronal membranes; the peripheral nerves of these mice were then exposed to monoclonal anti-gm1 antibodies and normal human serum (as a source of complement) to trigger acute or subacute nerve injury, either ex vivo (in the triangularis sterni nerve-muscle preparations) or in vivo (mcgonigal et al., 2022). in both mouse models, early injury [following a short (4 h) treatment with anti-gm1 antibodies and complement] occurred at the nodes of ranvier (nor); this is concordant with the neuropathologic studies of patients with gbs, which demonstrated nodal autoantibody and complement deposition (hafer-macko et al., 1996a; hafer-macko et al., 1996b) along with alterations of the nodal ultrastructure (griffin et al., 1996; vallat et al., 2020). in neuronal gbs model mice (which express gm1 selectively in neurons), formation of the membrane attack complex (mac) pores in the nodal axolemma led to calpain-mediated cleavage of adaptor protein ankyrin-g, dispersion and disappearance of the nodal sodium channel clusters, and conduction failure; however, there were no structural changes in the paranodal sc loops and no disruption of the cell adhesion complexes that form “transverse bands” at the axo-glial interface. in glial gbs model mice (which express gm1 selectively in scs), injury started with the mac-induced distortion of the paranodal sc loops and calpain-mediated cleavage of the sc scaffolding protein ankyrin-b, leading to disruption of transverse bands, dispersion of the sodium channel clusters, and conduction failure (fig. 4). thus, the ultimate functional consequence – acute conduction failure – was the same in both mouse models, although the underlying disease mechanisms and associated structural changes were different. acute conduction failure – which is seen in many human gbs patients in addition to these experimental disease models – is often temporary and reversible; however, it can also lead to subsequent axonal transection / wallerian degeneration. indeed, extended (20 h) treatment with anti-gm1 antibodies and complement in vivo led to a loss of the distal motor axon integrity in glial mice, indicative of secondary / bystander axonal degeneration due to nodal sc injury. (the authors have yet to perform similar extended treatment studies in their neuronal mice.) taken together, these results point to nor as the site of initial injury in both neuronal/axonal and glial/demyelinating forms of gbs. [interestingly, nor is also the main site of injury in autoimmune nodopathies, which are caused by antibodies that directly target glial or neuronal cell-adhesion molecules expressed in the nodal membranes; clinically, autoimmune nodopathies show significant overlap with gbs, but are typically more severe and resistant to ivig immunomodulatory therapy (martin-aguilar et al., 2022).] figure 4. ultrastructural evaluation of diaphragms from in vivo gbs models. neuronal and glial mice were dosed i.p. with 50 mg/kg anti-gm1 ab followed 16 hours later with 30 μl/g normal human serum (nhs) (injury) or nhs only (control). (a) a normal paranode from glial control tissue. this image is also representative of the neuronal control nor (not shown). (b) higher magnification of boxed region from a shows tight junctions (large arrowhead) between the paranodal loops, and transverse bands (tbs, small arrowheads) at the axo-glial junction between the axon and paranodal loops. (c) injured glial nors show severely disrupted paranodal loop organization compared with control. (d) magnification of boxed area from c, shows tbs are present between the paranodal loops and axon at the juxtaparanodal-proximal paranode (above black line); however, they are absent at the node-proximal border (above white line, right of asterisk). (e) injured neuronal nors show no architectural disruption. (f) neuronal control motor nerve terminal (mnt) displays normal architecture and contains synaptic vesicles (black arrows). (g) disturbance to the injured neuronal mnt includes an absence of neurofilament, synaptic vesicles, and the formation of dense or vacuolated mitochondria (white arrows). results are representative of analysis from 8–10 nors per mouse (n = 3/genotype/treatment). [this figure and its legend were adopted from figure 6 in (mcgonigal et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by international license.] what is the relevance of these findings for human gbs? since gm1 gangliosides (and other ganglioside antigens) are expressed by neurons as well as scs, both of these mechanisms are likely in play in human disease, and which mechanism dominates in any given gbs patient likely depends on a combination of factors, including antibody titer / specificity and intrinsic host factors that shape disease vulnerability. moreover, these findings have potential diagnostic implications: taken together with the 2020 ultrastructural study of peripheral nerves from patients with gbs and other nodo-paranodopathies (vallat et al., 2020), which showed ultrastructural nodal pathology similar to ultrastructural abnormalities seen in glial gbs mice (fig. 4), they suggest that electron microscopic evaluation of nor could help delineate immune-mediated from ischemic and other causes of axonal injury, and should therefore be included in the nerve biopsy workup whenever an obvious cause of axon degeneration (such as vasculitis) is not detected in a nerve biopsy. 6. target formation is indicative of muscle fiber reinnervation target fibers are one of the classic diagnostic features of a neurogenic change secondary to lower motor neuron (lmn) injury. targets are most commonly seen in type 1 fibers, are best visualized on oxidative stains, and in the cross-sectional view consist of several distinct concentric layers / zones – typically a central pale area devoid of mitochondrial activity and reminiscent of a core, an intermediate zone that shows a decreased but not entirely absent mitochondrial activity, and an outer zone that resembles the rest of the myofiber and often shows an increase in mitochondrial activity (fig. 5a-b). ultrastructurally, the central zone contains the z-disc material and is devoid of glycogen and mitochondria, the intermediate zone shows disorganized myofilaments with mild z-line abnormalities, while the outer zone contains ultrastructurally normal myofilaments. similar formations that have a less distinctively layered appearance are called “targetoid” and can be more difficult to distinguish from cores if they are not accompanied by true targets and/or other diagnostic features of neurogenic change. however, targets are not present in all muscle biopsies with neuropathic changes, and can be seen both in small angulated and normally sized fibers. as a result, it has been unclear whether target formation reflects denervation [as originally proposed (engel, 1961)] or reinnervation [as suggested by subsequent animal model studies (de reuck et al., 1977)]. a recent proteomic study done by the kley lab sheds a new light on this old question, providing compelling evidence that target formation reflects myofibril assembly that occurs following reinnervation (krause et al., 2022). figure 5. target fibers. (a) nadh-tr stain shows two type 1 fibers with central target formations; on myosin heavy chain immunohistochemistry, these fibers were positive for myhc-slow (mhc7; not shown). the same microscopic field, captured on consecutive cryosections, is shown in all main figure panels; target formations are highlighted by arrowheads. (b) the multilayered appearance of target formations (arrowheads), with the central pale zone, dark mitochondria-rich outer rim, and an intermediate zone in between, is more obvious on sdh histochemistry. (c) desmin immunohistochemistry highlights the inner and intermediate zones of target formations, where new myofibrils are assembled; in some cases, desmin staining is most prominent in the intermediate zone (main panel), while in others it is equally prominent in both zones (inset). (orange line, inner zone boundary; green line, intermediate zone boundary; black line, outer zone boundary.) (d). lc3 immunohistochemistry highlights autophagosomes, a key component of the quality-control machinery responsible for degradation of misassembled or damaged myofibrils; in some target formations, lc3 staining is most prominent in the central zone (main panel), while in others it is most prominent in the intermediate zone (inset). different patterns of target formation staining observed with desmin and lc3 immunostains could reflect different stages in myofibril assembly following fiber reinnervation. scale bar, 50 µm. to investigate the molecular composition of targets, krause and co-authors employed a non-biased, hypothesis-free proteomic strategy: they used 20 muscle biopsies from patients with neurogenic muscle atrophy to micro-dissect targets and analogously sized portions of uninvolved type 1 fibers, and then compared the protein composition of both samples via label-free mass spectroscopy. out of 1026 proteins identified in these samples, 55 proteins were overrepresented and 40 proteins underrepresented in targets compared to the control regions of type 1 fibers. unsurprisingly, the underrepresented proteins were mainly mitochondrial proteins. the overrepresented proteins were more interesting and could be classified into four main groups based on their subcellular localization and/or function: (i) z-disc and actin dynamics (15 proteins, including filamin c and desmin), (ii) myosins and myosin-associated proteins (10), (iii) protein biosynthesis (10, including ribosomal proteins), and (iv) molecular chaperones (5, including alpha(b)-crystallin and bag3); the remaining 15 proteins were classified as “other.” the key mass spectroscopy findings were then validated by immunofluorescent staining and showed zonal pattern of expression, with filamin c, other z-disc proteins, and chaperones in the central zone, desmin mainly in the intermediate zone (fig. 5c), and myosins in both zones. the model that emerged from these proteomic findings suggests that the central and intermediate zones are the sites of new myofibril synthesis; premyofibrils are formed in the central zone, and then assembled and linked with the existing myofibrils in the intermediate zone. chaperones and other components of the machinery involved in chaperone-assisted selective autophagy (such as autophagosomal protein lc3; fig. 5d) are enriched in both zones, providing a quality-control mechanism that ensures that nascent myofibrils are either properly built and assembled or immediately degraded. while this study represents a major advance in our understanding of the reinnervation-induced myofiber rebuilding mechanisms, some aspects of this process still remain to be elucidated. for example, it is not clear why targets are mainly seen in type 1 fibers. does the same process occur in a less structured / morphologically less distinctive way in type 2 fibers, or are reinnervated type 2 fibers rebuilt through entirely different molecular mechanisms? furthermore, it remains to be established how the motor neuron axon and muscle fiber interact to orchestrate this highly organized process of new myofibril formation. 7. sporadic inclusion body myositis revisited: what comes first, inflammation or myodegeneration? sporadic inclusion body myositis (sibm) primarily affects people older than 50 and has distinctive clinicopathologic features that differentiate it from other idiopathic inflammatory myopathies; clinically, there is predominant involvement of quadriceps and finger flexor muscles, while pathologically, there is endomysial inflammation accompanied by diffuse mhc-i upregulation and invasion of intact muscle fibers by cytotoxic cd8+ t lymphocytes and macrophages, along with mitochondrial abnormalities, chronic myopathic features resembling muscular dystrophy, rimmed vacuoles, and p62and tdp-43-positive protein aggregates. however, pathogenesis of sibm remains poorly understood. in particular, the role of inflammation has remained controversial: although sibm muscle biopsies typically show prominent inflammatory infiltrates, the disease does not respond to immunosuppression (or any other therapy) and generally leads to wheelchair dependence ~15 years following the diagnosis. several important studies published in 2019 [and discussed in the first paper in this review series (margeta, 2020)] suggested that the lack of response to immunomodulatory therapy is due to involvement of terminally differentiated effector memory t cells (so-called temra cells), which are positive for klrg1 (killer cell lectin-like receptor g1; a marker of t cell exhaustion) and resistant to steroid-induced apoptosis (greenberg et al., 2019; knauss et al., 2019). several important follow-up studies were published in 2022, prompting me to revisit this important topic in the current review. our understanding of sibm pathogenesis has been limited by the lack of a disease model that replicates both inflammatory and myodegenerative features of this disease. to circumvent this barrier, britson and colleagues developed a xenograft model of sibm, in which human muscle biopsy tissue is transplanted into immunodeficient mice; following transplantation, transacted mature muscle fibers degenerate and are replaced by newly formed fibers generated from satellite cells that were harnessed along with the graft (britson et al., 2022). remarkably, in sibm xenografts – but not in the xenografts generated from relatively normal muscle biopsies or from biopsies showing other types of myositis – regenerated myofibers showed typical features of sibm, including mhc-i positivity, rimmed vacuoles, loss of nuclear tdp-43 expression, and mitochondrial abnormalities (cox deficiency). in addition, sibm xenografts contained donor-derived oligoclonal cd8+klrg1+ temra cells that were enriched compared to the source sibm biopsies. finally, ~50% of sibm xenografts (but only 3% of control xenografts) showed expression of cryptic exons in tdp-43-regulated mrnas, indicative of functional tdp-43 deficiency. (tdp-43 is an rna-binding protein that maintains the health of the transcriptome by repressing incorporation of non-conserved cryptic exons during pre-mrna splicing; cryptic exon expression in the tdp-43-regulated, muscle-specific mrna transcripts was 84% sensitive and 99% specific marker of sibm in the cohort of 119 myositis patients enrolled in this study). having developed the xenograft sibm model, britson et al. used to it to evaluate whether treatment with monoclonal anti-cd3 antibody okt3 would improve various aspects of sibm pathology. the okt3 treatment, which almost completely depleted t cells and significantly reduced the number of klrg1-positive cells in sibm xenografts after 4 months of weekly infusions, led to a significant decrease in the extent of mhc-i upregulation; however, it had no significant effect on myodegenerative sibm features (cox-negative fibers, rimmed vacuoles, and p62-positive protein aggregates). in addition, there was no change in the aberrant expression of cryptic exons. these results quite definitively demonstrate that myodegenerative pathology is intrinsic to sibm muscle and can persist independent of sustained inflammation or circulating factors, providing an additional explanation for refractoriness of sibm to standard immunosuppressive treatments, but also diminishing hope that therapies that directly target klrg1-positive temra cells (which are already in development) will significantly benefit sibm patients. however, it is less clear what these findings mean for our understanding of sibm pathogenesis: while the authors of this study favor a model in which tdp-43 dysfunction is an early event in the disease progression that then drives aberrant mrna splicing, neoantigen expression, and secondary inflammation, it is equally possible that chronic inflammation within the human xenograft donor prior to the biopsy (and/or within the early xenograft, prior to the okt treatment) leads to genetic and/or epigenetic alterations in satellite cells, which then recapitulate tdp-43 dysfunction and myodegenerative changes in newly formed muscle fibers. additional work will be required to distinguish between these two models of sibm pathogenesis; in particular, it should be established whether aberrant expression of tdp-43-regulated cryptic exons is also present in hereditary inclusion body myopathies, which show sibm-like myodegenerative features but lack inflammation. interestingly, the results of another 2022 paper provide support for inflammation as an early event in sibm disease progression (kleefeld et al., 2022). in their cross-sectional study, kleefeld and colleagues examined the similarities and differences between polymyositis with mitochondrial pathology (pm-mito) and sibm. in contrast to sibm, which has a distinctive clinical phenotype, pm-mito is not a clinically well-defined entity; pathologically, however, it shows some sibm-like features (lymphocytic endomysial inflammation, muscle fiber invasion by inflammatory cells, and mitochondrial pathology / cox-deficient fibers) but lacks rimmed vacuoles, chronic myopathic changes, and p62or tdp-43-positive protein aggregates (blume et al., 1997; hiniker et al., 2013). although most patients with pm-mito over time progress to sibm, at least partial response to immunosuppression has been observed in a subset of cases (levine and pestronk, 1998; winkler et al., 2021), raising the possibility that pm-mito and sibm exist on a spectrum, with pm-mito an early – and potentially treatable – stage of sibm. the findings reported by kleefeld et al. support this model: they found an essentially identical inflammatory molecular signature in pm-mito and sibm biopsies, with greater activation of type ii than type i interferon response and expression of cryptic exons in both sets of cases (although frequency of cryptic exon expression was greater in sibm than pm-mito biopsies). in contrast to these similarities, klrg1 and gbp6 (guanylate binding protein family member 6) genes were differentially expressed, with both klrg1 and gbp6 mrna upregulation detectable only in sibm biopsies. in agreement with this finding, gbp6-positive macrophages, gbp6-expressing myofibers, and klrg1-positive t cells were significantly more abundant in sibm than pm-mito samples. overall, most differences between pm-mito and sibm biopsies were quantitative rather than qualitative, with a gradient of severity observed even within the pm-mito group (which could be histologically divided into “mild”, “typical”, and “pre-ibm” subgroups). importantly, this progression of severity was also observed in serial biopsies from two individuals, the first showing pm-mito and the second (5-7 years later) sibm. based on these findings, kleefeld and colleagues recommended that pm-mito should be renamed early sibm and that both disorders together should be classified as “sibm-spectrum disease” (sibm-sd). in addition, they suggested that the difference in klrg1 expression between early and late sibm stages reflected disease duration, with emergence of exhausted t cells a consequence of prolonged inflammation / antigenic stimulation and a harbinger of transition into a therapy-resistant end stage of sibm-sd. in agreement with that hypothesis, another recent study used “deep immunophenotyping” of blood t and nk cells from sibm patients to show that increased cd8+ t cell differentiation correlated with sibm duration (goyal et al., 2022). among many questions that still need to be answered is the “status” of polymyositis without mitochondrial abnormalities, which is histologically identical to pm-mito except for the absence of ragged red and cox-negative fibers (hiniker et al., 2013). it is not currently known whether polymyositis is a separate disease, or an even earlier stage of sibm-sd that can be effectively treated by standard immunosuppressive therapy and is therefore less likely to progress to a chronic inflammation-driven, treatment-resistant end stage. given that inflammation can lead to secondary mitochondrial abnormalities in other inflammatory myopathies (hedberg-oldfors et al., 2022), it is possible that emergence of cox-negative fibers in pm-mito represents the first step on the pathway towards treatment resistance in the setting of persistent chronic inflammation. advances in neuromuscular disease diagnostics 8. improved genetic testing techniques for facioscapulohumeral and myotonic muscular dystrophies despite widespread availability and relatively low cost of advanced genetic testing techniques (such as syndrome-specific multigene panels and whole exome sequencing), 50-75% of presumed genetic neuromuscular disease cases currently remain undiagnosed (cummings et al., 2017). the underlying reasons for these diagnostic challenges are complex and varied, but include the inability of these sequencing techniques to detect structural rearrangements and copy number variants. fortunately, two new genetic testing strategies developed in 2022 will alleviate some of these challenges, simplifying the diagnosis of two common muscular dystrophies (erdmann et al., 2022; stevanovski et al., 2022). facioscapulohumeral muscular dystrophy (fshd) is the third most common muscular dystrophy; it has a fairly unique clinical presentation, with the facial and shoulder girdle weakness that progresses to involve distal legs and the pelvic girdle. however, the age of onset is highly variable (from infancy to late adulthood) and the phenotype can also vary, making the diagnosis challenging. histopathologic features of fshd are relatively unique (fig. 6), but not sufficiently specific to enable definitive diagnosis; hence, directed genetic testing is generally required at some point of diagnostic work-up. molecularly, fshd is caused by aberrant expression of dux4 (double homeobox 4) protein in skeletal muscle, which has myotoxic effects. dux4 is a transcription factor that regulates expression of genes important for preand post-implantation development, but is epigenetically silenced in most adult tissues except in thymus and testis, where it has an unknown function (mocciaro et al., 2021). in fshd, there is a loss of dux4 repression, but that derepression occurs through mechanisms that differ between two disease subtypes, fshd1 and fshd2. in fshd1 (~95% of cases), dux4 derepression is caused by contraction of the d4z4 macrosatellite repeat array in the subtelomeric region of chromosome 4q35 to <12 repeating units; in fshd2 (~5% of cases), there is global hypomethylation of the d4z4 array due to mutations in one of the several genes that encode proteins required for epigenetic suppression. (a permissive haplotype 4qa or 4qal, which provides a polya signal for dux4 mrna, is required for phenotypic disease expression in either disease subtype.) currently, genetic diagnosis of fshd requires (i) confirmation of the permissive haplotype, (ii) determination of the d4z4 repeat length, typically via southern blotting (which requires a large amount of high molecular weight dna), and (iii) sequencing of epigenetic suppressor genes if the d4z4 repeat length is found to be within normal limits. the new, much more streamlined approach for fshd testing developed by erdmann et al. is based on methylation profiling: following confirmation of the permissive haplotype, a high-throughput methylation profile analysis is used to determine the global methylation level of the entire d4z4 repeat array as well as the regional methylation of its most distal repeat unit; individuals with isolated distal hypomethylation have fshd1, while individuals with both global and distal hypomethylation have fshd2 (erdmann et al., 2022). not only does this new diagnostic approach require much smaller quantity of dna and less laboratory effort, it also detects cases that are missed with traditional methods (for example, patients with complex chromosomal rearrangements that cannot be detected by southern blotting) or cases that are in the gray zone between fshd1 and fshd2 (which represent the two ends of the epigenetic disease continuum). in addition, erdmann et al. have shown that the d4z4 repeat methylation profile is a biomarker of fshd severity: in their cohort of 148 patients, the distal methylation level was better correlated with clinical findings (including the age of onset) than the d4z4 repeat length. finally, this study provides definitive evidence that fshd pathogenesis is ultimately driven by epigenetic rather than genetic mechanisms. figure 6. histologic findings in facioscapulohumeral dystrophy (fshd). (a) a representative h&e-stained section shows fiber size variation, endomysial fibrosis, and perimysial inflammation that focally extends into the endomysium. (b) moderately frequent lobulated fibers (arrowheads) are best seen on nadh-tr histochemistry. (c) mhc-i is diffusely upregulated in muscle fibers. (d) cd31 immunohistochemistry highlights enlarged endomysial capillaries. based on the biopsy findings, the patient underwent genetic testing for fshd that showed pathogenic d4z4 repeat contraction (8 repeats) detected on a 4qa permissive haplotype, diagnostic of fshd1. all stains were performed on cryosections; scale bar, 50 µm. myotonic dystrophy (dystrophia myotonica; dm) is the most common muscular dystrophy in adults and the second most common muscular dystrophy overall. as with fshd, there are two distinct dm subtypes, dm1 (which accounts for >95% of cases) and dm2 (which is much rarer). although their clinical features and causative genes differ, both dm1 and dm2 are short tandem repeat (str) expansion diseases: dm1 is caused by expansion of the ctg repeat in the 3’ untranslated region of dmpk gene, while dm2 is caused by expansion of the cctg repeat in intron 1 of cnbp/znf9 gene. interestingly, the mechanism of myotonia is the same in both dm subtypes: mutant rnas sequester rna-binding and processing proteins, leading to incorrect splicing of clc-1 chloride channel (which is required for the maintenance of normal resting membrane potential). histopathology of dm1 and dm2 is strikingly similar, with very high internal nucleation and frequent subsarcolemmal nuclear aggregates in the setting of usual dystrophic changes (fig. 7); therefore, the definitive diagnosis requires genetic testing. although small str expansions (< 300 bp) can be detected by conventional pcr, genetic diagnosis of most str expansion disorders (which in addition to dm1 and dm2 include huntington’s disease, fragile x syndrome, hereditary cerebellar ataxias, c9orf72-related als / frontotemporal degeneration, and other neurologic diseases) currently relies on southern blotting and/or tandem repeat-primed pcr, and therefore faces many challenges already described for fshd in addition to requiring separate probes and/or primers for each str. these challenges are either solved or bypassed by the new genetic strategy developed by stevanovski et al., which relies on programmable targeted long-read nanopore sequencing to genotype all known neuropathogenic strs in parallel, using a single molecular assay (stevanovski et al., 2022). one particularly appealing feature of the platform used for this assay is that it permits flexible inclusion of targets, so that a specific assay used for any given patient can be individually tailored based on the clinician’s input and/or the patient’s preferences; another is that sequencing can be combined with methylation profiling, providing epigenetic as well as genetic information in a single step. as a result, this new genetic testing approach will likely advance basic research in addition to patient care: much remains to be learned about the fundamental biology of the str expansion disorders and the key variables that distinguish normal and disease-causing alleles, and the availability of a relatively inexpensive high-resolution molecular str assay should accelerate research progress in all these areas. figure 7. histologic findings in myotonic dystrophy (dm). (a) a representative h&e-stained formalin-fixed, paraffin embedded section shows fiber size variation, endomysial fibrosis with fatty infiltration, frequent subsarcolemmal nuclear aggregates, and nuclei running in chains along the longitudinal fiber axis (arrowhead). (b) an h&e-stained cryosection shows similar findings, with prominent subsarcolemmal nuclear aggregates and a marked increase in internal nuclei. (c) an acid phosphatase-stained cryosection shows a fiber with prominent sarcoplasmic acid phosphatase-positive granules (asterisk). based on the biopsy findings, the patient underwent genetic testing for myotonic dystrophy that showed >15600 repeats in one allele of cnbp/znf9 gene, diagnostic of dm2. (histologic findings in dm1 are very similar.) scale bars: a and b, 100 µm; c, 50 µm. 9. accumulation of phosphorylated tdp-43 in motor nerves and intramuscular nerve twigs is a diagnostic marker of amyotrophic lateral sclerosis als is a neurodegenerative disease that typically affects both upper and lower motor neurons and is the most common cause of motor neuron disease (mnd) in adults; pathologically, it is characterized by selective motor neuron loss that is accompanied by aggregates of phosphorylated tdp-43 (ptdp-43) in the remaining motor neurons and glia. during life, als diagnosis is based on the clinical criteria; however, there is a significant phenotypic heterogeneity among als patients, and up to 20% of patients with an lmn-predominant form of als are misdiagnosed prior to death. based on the recent work from two separate research groups in italy and japan, immunohistochemical detection of ptdp-43 aggregates in motor nerve biopsies (riva et al., 2022) and/or motor nerve twigs in muscle biopsies (kurashige et al., 2022) may be a useful tool for closure of that diagnostic gap. riva and colleagues performed a retrospective cohort study to assess whether tdp-43 accumulation in motor nerve biopsies can serve as a useful biomarker of als. they retrospectively reviewed the clinical course of 113 patients who underwent a diagnostic biopsy of the anterior motor branch of the obturator nerve, typically for evaluation of a diagnostically confusing lmn syndrome, during the 25-year period between 1994 and 2019. of these 113 patients, 102 had sufficient clinical data to be included in the study; 71 (69.6%) were ultimately diagnosed with als. in the first part of this study, the authors performed blinded histopathology review of original slides for all 102 study participants; 61 biopsies were diagnosed as pathologic mnd, 16 were diagnosed as pathologic motor neuropathy, while 25 were non-diagnostic. excluding the cases with pathologic diagnosis of motor neuropathy (which had 100% sensitivity and 100% specificity for the ultimate diagnosis of motor neuropathy), the specificity of pathologic mnd pattern for the final diagnosis of als in that patient cohort was 66.6%, while its sensitivity was 78.9%. (interestingly, the degree of axon loss in als cases was predictive of the overall and post-biopsy survival length.) in the second part of the study, immunohistochemistry with polyclonal anti-tdp-43 and anti-p(s409/410)tdp-43 antibodies was performed on biopsies from 80 patients for which sufficient archival tissue was available; in this subcohort, 57 patients (71.3%) were ultimately diagnosed with als. accumulation of tdp-43 in myelinated axons and sc cytoplasm was more commonly detected in als patients than in non-als patients, but this difference was statistically significant only when anti-ptdp-43 antibodies were used; overall, the presence of ptdp-43 aggregates had the specificity of 65.2% and sensitivity of 98.2% for the final diagnosis of als. interestingly, some of the patients with a false-positive ptdp-43 signal in their motor nerve biopsies were ultimately diagnosed with inclusion body myositis or idiopathic motor neuropathy. while this finding represents a limitation from the diagnostic perspective, it highlights the mechanistic connections between these disorders: multisystem proteinopathies, which clinically manifest as one or more of these diseases, have been etiologically linked to impaired biology of tdp-43-containing rna stress granules (korb et al., 2021). interestingly, axonal ptdp-43 aggregates were detected in 11 of 11 als cases with normal (and therefore nondiagnostic) motor nerve biopsies, suggesting that ptdp-43 accumulation is an early event in the disease progression that precedes lmn degeneration and axon loss. one limitation of the current study is that the authors did not evaluate whether specificity and sensitivity of the motor nerve biopsy in the als diagnosis could be improved by combining the results of standard histopathologic analysis with ptdp-43 immunohistochemistry; this important question should be assessed in future studies from this or other research groups. kurashige et al. approached the same question in a slightly different way, by focusing on ptdp-43 accumulation in the intramuscular motor nerve twigs (kurashige et al., 2022). in the first part of their investigation, these authors performed an autopsy case-control study that included 10 patients with autopsy-confirmed sporadic als and 12 control, non-als patients; for each case, muscle samples were obtained from the tongue, diaphragm, biceps brachii, and rectus femoris. the second part of the project was a retrospective cohort study, which started by screening 450 patients who underwent a diagnostic muscle biopsy at the authors’ institutions between 2004 and 2019; after all patients with a myopathy and patients with pathogenic variants in the als-associated genes were excluded, the study cohort consisted of 114 patients, 71 of whom had biopsies that included intramuscular nerve twigs. all study samples were stained with both polyclonal and monoclonal anti-p(s409/410)tdp-43 antibodies, which gave identical results. in autopsy cases, axonal ptdp-43-positive aggregates (but no fus-, p62-, or ubiquitin-positive aggregates) were detected in all als patients but not in any of the controls (fig. 8); ~50% of nerve twigs were involved in any given case, with no significant differences among the four sampled muscle groups. in the biopsy portion of the study, 33 of 71 cases with intramuscular nerve twigs (46.5%) showed axonal ptdp-43-positive aggregates; all these patients were ultimately diagnosed with als (9 with the lmn-predominant form), while none of 38 ptdp-43-negative patients received the als diagnosis, and were instead ultimately diagnosed with other mnds or neuropathies. (interestingly, only 4 of the 114 patients in the muscle biopsy cohort were clinically suspected of having als prior to the biopsy.) figure 8. axonal ptdp-43–positive accumulations in intramuscular nerve bundles of skeletal muscle in the postmortem case-control study. (a and d) h&e-staining in axons of intramuscular nerve bundles showed no differences between patients with amyotrophic lateral sclerosis (als) and those without als except for the density of intramuscular nerves (original magnification: a, ×40; d, ×60). (b) immunohistochemical analysis with mouse monoclonal ptdp-43–positive accumulations in intramuscular nerve bundles (arrowheads) of patients with spontaneous als (sals) (original magnification, ×40). (e) immunostaining with mouse monoclonal antibody against ptdp-43 in patients with non-als diseases did not reveal any abnormalities in intramuscular nerve bundles (original magnification, ×40). (c) immunohistochemical analysis with rabbit polyclonal ptdp-43 antibody also revealed ptdp-43–positive accumulations in intramuscular nerve bundles (arrowheads) of patients with sals (original magnification, ×60). (f) immunostaining with rabbit polyclonal antibody against ptdp-43 in patients with non-als diseases did not reveal any abnormalities in intramuscular nerve bundles (original magnification, ×40). [this figure and its legend were adopted from figure 2 in (kurashige et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by international license.] taken together, these two studies provide compelling evidence that immunohistochemical detection of ptdp-43-positive protein aggregates in motor axons is a useful diagnostic marker of als. while the results of both studies need to be replicated by other researchers, these findings suggest that muscle biopsy evaluation is a particularly promising diagnostic approach: not only are muscle biopsies easier to do and have lower morbidity than motor nerve biopsies, they also showed higher overall specificity and sensitivity than nerve biopsies (perhaps because potentially confounding myopathic biopsies could easily be excluded). of course, the utility of muscle biopsies for als diagnosis is limited by the lack of intramuscular nerve twigs in many specimens (~40% in this study cohort); obtaining larger tissue samples and performing serial level sections will probably help decrease the number of inadequate biopsies if this evaluation becomes a standard component of the als diagnostic work-up. advances in neuromuscular disease treatment 10. sarm1 inhibitors block wallerian degeneration in preclinical models of axonal neuropathy as discussed in advance #4, sarm1 is the central regulator of intrinsic axon destruction program / wallerian degeneration; deletion of sarm1 in mice prevents axonal degeneration in both cns (for example, in mouse models of traumatic brain injury) and pns (in mouse models of axotomy and chemotherapy-induced peripheral neuropathy). the central role of sarm1 in wallerian degeneration raised the possibility that sarm1 blockade would benefit patients with a broad spectrum of neurologic disorders, leading to a major effort by multiple different research groups (both in academia and biotechnology companies) to develop small molecule inhibitors of sarm1. initial proof-of-principle studies with catalytic site sarm1 inhibitors were published in 2021 (bosanac et al., 2021; hughes et al., 2021), with more comprehensive preclinical studies of catalytic inhibitors (bratkowski et al., 2022; shi et al., 2022) and a discovery of a new class of allosteric sarm1 inhibitors (feldman et al., 2022) reported in 2022. sarm1 is a multi-domain enzyme that functions as an oligomer; it contains an auto-inhibitory n-terminal armadillo repeat (arm) domain, repeat sterile alpha motif (sam) domains that are required for dimerization / oligomerization, and a catalytic c-terminal domain that includes the toll/interleukin receptor (tir) motif. the tir domain contains the nad+ hydrolase activity that catabolizes nad+ into nicotinamide, adenosine diphosphate ribose (adpr), and cyclic adpr (which is the second messenger that triggers the wallerian degeneration pathway). under normal conditions, nad+ is bound to the arm domain, resulting in autoinhibition of the sarm1 activity; when the nmn/nad+ ratio increases (typically due to a decreased production of nad+ by nmnat2), nmn binds to the arm domain instead of nad+, resulting in a conformational change that leads to oligomerization of the tir domain and relief of sarm 1 autoinhibition (feldman et al., 2022). several groups have developed non-competitive, adduct-forming small molecule inhibitors of the sarm1 nad+ hydrolase site, with most comprehensive characterization to date reported by bratkowski and colleagues. these non-competitive sarm1 inhibitors function as pseudo-substrates / pro-drugs that intercept nad+ hydrolysis and form inhibitor-adpr adducts, which act as highly potent inhibitors of the catalytic site, with slow on and off rates and the dissociation constant in the low nanomolar range. while all studies have shown that these catalytic site inhibitors prevent axonal degeneration in vitro, bratkowski et al. went beyond cellular axon injury models: they used preclinical mouse models of acute peripheral nerve injury (a sciatic nerve transection model and a model of vincristine-induced peripheral neuropathy) to demonstrate that these drugs are both safe and effective in vivo, with efficacy approaching the efficacy of genetic sarm1 deletion. specifically, treatment with nb-3 (the compound with chemical properties most suitable for in vivo use) suppressed an increase in the plasma level of neurofilament light chain (a biomarker of axonal injury) and led to preservation of distal nerve fiber integrity in both acute nerve injury models; in addition, it attenuated toxin-induced allodynia / neuropathic pain in the vincristine model. (the transection model cannot be used for functional studies because connections between the cns and periphery are completely disrupted.) critically, the nb-3 treatment was effective not only when it was administered prophylactically but also when it was given several hours after injury, a treatment paradigm that is more relevant for potential clinical use. in a separate line of the sarm1 inhibitor research, feldman and colleagues used a chemical proteomic approach to discover an entirely novel class of sarm1 inhibitors: these electrophilic compounds (tryptoline acrylamides) target cysteine c311 in the autoregulatory arm domain, resulting in allosteric inhibition of sarm1 enzymatic activity (feldman et al., 2022). thus far, these new compounds were evaluated only in the cellular models of axon degeneration, where their efficacy was similar to the efficacy of catalytic site inhibitors; it remains to be determined whether they will be similarly safe and effective in vivo. regardless of the in vivo effectiveness of these specific compounds, this study provides initial evidence that sarm1 can be pharmacologically inhibited through allosteric as well as enzymatic mechanisms; because different classes of sarm1 inhibitors are likely to have different side effects and different optimal clinical uses, this finding is both important and exciting. where is this research headed? first, there should be additional preclinical testing of all compounds discovered to date using models of chronic axonal injury (including the nmnat2 deficiency model described in advance #4) that more closely resemble clinical scenarios where sarm1 inhibitors will be used. [in the setting of catastrophic acute axonal injury, degeneration of the distal segment of the injured axon creates a cellular and molecular environment that promotes effective axon regeneration (simon and watkins, 2018; li et al., 2023); therefore, inhibiting sarm1 activation in human patients who experienced traumatic or ischemic nerve injury would probably not be beneficial, and might even be harmful.] second, there should be a continued effort to discover additional sarm1 inhibitors that belong to different chemical classes and/or have different mechanisms of action. finally, the best performing compounds should rapidly proceed to clinical trials. given the incredible pace of research in this field over the last few years, it will probably not take too long before axon-protective medications become available for treatment of human patients with a broad array of peripheral – and possibly central – nervous system disorders. disclosure statement the author receives research support from astellas gene therapies (formerly known as audentes therapeutics, inc) as a member of the muscle biopsy review committee for the aspiro (nct03199469) and fortis (nct04174105) clinical trials, which are evaluating the safety and efficacy of gene transfer therapy for x-linked myotubular myopathy (aspiro) and late onset pompe disease (fortis). acknowledgements i am grateful to drs. nigel g. laing, gina ravenscroft, and benedikt schoser for helpful input during the conceptualization stage of this review. in addition, i would like to thank ms. christine lin for assistance with figure preparation. references abrams, r.m.c., simpson, d.m., navis, a., jette, n., zhou, l., and shin, s.c. (2022). small fiber neuropathy associated with sars-cov-2 infection. muscle nerve 65, 440-443. https://doi.org/10.1002/mus.27458 bloom, a.j., mao, x., strickland, a., sasaki, y., milbrandt, j., and diantonio, a. (2022). constitutively active sarm1 variants that induce neuropathy are enriched in als patients. mol neurodegener 17, 1. https://doi.org/10.1186/s13024-021-00511-x blume, g., pestronk, a., frank, b., and johns, d.r. (1997). polymyositis with cytochrome oxidase negative muscle fibres. early quadriceps weakness and poor response to immunosuppressive therapy. brain 120, 39-45. https://doi.org/10.1093/brain/120.1.39 bosanac, t., hughes, r.o., engber, t., devraj, r., brearley, a., danker, k., young, k., kopatz, j., hermann, m., berthemy, a., et al. (2021). pharmacological sarm1 inhibition protects axon structure and function in paclitaxel-induced peripheral neuropathy. brain 144, 3226-3238. https://doi.org/10.1093/brain/awab184 bratkowski, m., burdett, t.c., danao, j., wang, x., mathur, p., gu, w., beckstead, j.a., talreja, s., yang, y.s., danko, g., et al. (2022). uncompetitive, adduct-forming sarm1 inhibitors are neuroprotective in preclinical models of nerve injury and disease. neuron 110, 3711-3726. https://doi.org/10.1016/j.neuron.2022.08.017 britson, k.a., ling, j.p., braunstein, k.e., montagne, j.m., kastenschmidt, j.m., wilson, a., ikenaga, c., tsao, w., pinal-fernandez, i., russell, k.a., et al. (2022). loss of tdp-43 function and rimmed vacuoles persist after t cell depletion in a xenograft model of sporadic inclusion body myositis. sci transl med 14, eabi9196. https://doi.org/10.1126/scitranslmed.abi9196 conforti, l., gilley, j., and coleman, m.p. (2014). wallerian degeneration: an emerging axon death pathway linking injury and disease. nat rev neurosci 15, 394-409. https://doi.org/10.1038/nrn3680 cummings, b.b., marshall, j.l., tukiainen, t., lek, m., donkervoort, s., foley, a.r., bolduc, v., waddell, l.b., sandaradura, s.a., o'grady, g.l., et al. (2017). improving genetic diagnosis in mendelian disease with transcriptome sequencing. sci transl med 9, eaal5209. https://doi.org/10.1126/scitranslmed.aal5209 de reuck, j., de coster, w., and vander eecken, h. (1977). the target phenomenon in rat muscle following tenotomy and neurotomy. a comparative light microscopic and histochemical study. acta neuropathol 37, 49-53. https://doi.org/10.1007/bf00684540 dingwall, c.b., strickland, a., yum, s.w., yim, a.k., zhu, j., wang, p.l., yamada, y., schmidt, r.e., sasaki, y., bloom, a.j., et al. (2022). macrophage depletion blocks congenital sarm1-dependent neuropathy. j clin invest 132, e159800. https://doi.org/10.1172/jci159800 engel, w.k. (1961). muscle target fibres, a newly recognized sign of denervation. nature 191, 389-390. https://doi.org/10.1038/191389a0 erdmann, h., scharf, f., gehling, s., benet-pages, a., jakubiczka, s., becker, k., seipelt, m., kleefeld, f., knop, k.c., prott, e.c., et al. (2022). methylation of the 4q35 d4z4 repeat defines disease status in facioscapulohumeral muscular dystrophy. brain, epub ahead of print. https://doi.org/10.1093/brain/awac336 feldman, h.c., merlini, e., guijas, c., demeester, k.e., njomen, e., kozina, e.m., yokoyama, m., vinogradova, e., reardon, h.t., melillo, b., et al. (2022). selective inhibitors of sarm1 targeting an allosteric cysteine in the autoregulatory arm domain. proc natl acad sci u s a 119, e2208457119. https://doi.org/10.1073/pnas.2208457119 figley, m.d., gu, w., nanson, j.d., shi, y., sasaki, y., cunnea, k., malde, a.k., jia, x., luo, z., saikot, f.k., et al. (2021). sarm1 is a metabolic sensor activated by an increased nmn/nad(+) ratio to trigger axon degeneration. neuron 109, 1118-1136. https://doi.org/10.1016/j.neuron.2021.02.009 gilley, j., jackson, o., pipis, m., estiar, m.a., al-chalabi, a., danzi, m.c., van eijk, k.r., goutman, s.a., harms, m.b., houlden, h., et al. (2021). enrichment of sarm1 alleles encoding variants with constitutively hyperactive nadase in patients with als and other motor nerve disorders. elife 10, e70905. https://doi.org/10.7554/elife.70905 goyal, n.a., coulis, g., duarte, j., farahat, p.k., mannaa, a.h., cauchii, j., irani, t., araujo, n., wang, l., wencel, m., et al. (2022). immunophenotyping of inclusion body myositis blood t and nk cells. neurology 98, e1374-e1383. https://doi.org/10.1212/wnl.0000000000200013 greenberg, s.a., pinkus, j.l., kong, s.w., baecher-allan, c., amato, a.a., and dorfman, d.m. (2019). highly differentiated cytotoxic t cells in inclusion body myositis. brain 142, 2590-2604. https://doi.org/10.1093/brain/awz207 griffin, j.w., li, c.y., macko, c., ho, t.w., hsieh, s.t., xue, p., wang, f.a., cornblath, d.r., mckhann, g.m., and asbury, a.k. (1996). early nodal changes in the acute motor axonal neuropathy pattern of the guillain-barre syndrome. j neurocytol 25, 33-51. https://doi.org/10.1007/bf02284784 hafer-macko, c., hsieh, s.t., li, c.y., ho, t.w., sheikh, k., cornblath, d.r., mckhann, g.m., asbury, a.k., and griffin, j.w. (1996a). acute motor axonal neuropathy: an antibody-mediated attack on axolemma. ann neurol 40, 635-644. https://doi.org/10.1002/ana.410400414 hafer-macko, c.e., sheikh, k.a., li, c.y., ho, t.w., cornblath, d.r., mckhann, g.m., asbury, a.k., and griffin, j.w. (1996b). immune attack on the schwann cell surface in acute inflammatory demyelinating polyneuropathy. ann neurol 39, 625-635. https://doi.org/10.1002/ana.410390512 hedberg-oldfors, c., lindgren, u., visuttijai, k., loof, d., roos, s., thomsen, c., and oldfors, a. (2022). respiratory chain dysfunction in perifascicular muscle fibres in patients with dermatomyositis is associated with mitochondrial dna depletion. neuropathol appl neurobiol 48, e12841. https://doi.org/10.1111/nan.12841 hejbøl, e.k., harbo, t., agergaard, j., madsen, l.b., pedersen, t.h., ostergaard, l.j., andersen, h., schroder, h.d., and tankisi, h. (2022). myopathy as a cause of fatigue in long-term post-covid-19 symptoms: evidence of skeletal muscle histopathology. eur j neurol 29, 2832-2841. https://doi.org/10.1111/ene.15435 hiniker, a., daniels, b.h., lee, h.s., and margeta, m. (2013). comparative utility of lc3, p62 and tdp-43 immunohistochemistry in differentiation of inclusion body myositis from polymyositis and related inflammatory myopathies. acta neuropathol commun 1, 29. https://doi.org/10.1186/2051-5960-1-29 hughes, r.o., bosanac, t., mao, x., engber, t.m., diantonio, a., milbrandt, j., devraj, r., and krauss, r. (2021). small molecule sarm1 inhibitors recapitulate the sarm1(-/-) phenotype and allow recovery of a metastable pool of axons fated to degenerate. cell rep 34, 108588. https://doi.org/10.1016/j.celrep.2020.108588 huppke, p., wegener, e., gilley, j., angeletti, c., kurth, i., drenth, j.p.h., stadelmann, c., barrantes-freer, a., bruck, w., thiele, h., et al. (2019). homozygous nmnat2 mutation in sisters with polyneuropathy and erythromelalgia. exp neurol 320, 112958. https://doi.org/10.1016/j.expneurol.2019.112958 inoue, m., noguchi, s., inoue, y.u., iida, a., ogawa, m., bengoechea, r., pittman, s.k., hayashi, s., watanabe, k., hosoi, y., et al. (2023). distinctive chaperonopathy in skeletal muscle associated with the dominant variant in dnajb4. acta neuropathol 145, 235-255. https://doi.org/10.1007/s00401-022-02530-4 kleefeld, f., uruha, a., schanzer, a., nishimura, a., roos, a., schneider, u., goebel, h.h., schuelke, m., hahn, k., preusse, c., et al. (2022). morphologic and molecular patterns of polymyositis with mitochondrial pathology and inclusion body myositis. neurology 99, e2212-e2222. https://doi.org/10.1212/wnl.0000000000201103 knauss, s., preusse, c., allenbach, y., leonard-louis, s., touat, m., fischer, n., radbruch, h., mothes, r., matyash, v., bohmerle, w., et al. (2019). pd1 pathway in immune-mediated myopathies: pathogenesis of dysfunctional t cells revisited. neurol neuroimmunol neuroinflamm 6, e558. https://doi.org/10.1212/nxi.0000000000000558 korb, m.k., kimonis, v.e., and mozaffar, t. (2021). multisystem proteinopathy: where myopathy and motor neuron disease converge. muscle nerve 63, 442-454. https://doi.org/10.1002/mus.27097 krause, k., eggers, b., uszkoreit, j., eulitz, s., rehmann, r., guttsches, a.k., schreiner, a., van der ven, p.f.m., furst, d.o., marcus, k., et al. (2022). target formation in muscle fibres indicates reinnervation a proteomic study in muscle samples from peripheral neuropathies. neuropathol appl neurobiol, e12853. https://doi.org/10.1111/nan.12853 kurashige, t., morino, h., murao, t., izumi, y., sugiura, t., kuraoka, k., kawakami, h., torii, t., and maruyama, h. (2022). tdp-43 accumulation within intramuscular nerve bundles of patients with amyotrophic lateral sclerosis. jama neurol 79, 693-701. https://doi.org/10.1001/jamaneurol.2022.1113 levine, t.d., and pestronk, a. (1998). inflammatory myopathy with cytochrome oxidase negative muscle fibers: methotrexate treatment. muscle nerve 21, 1724-1728. https://doi.org/10.1002/(sici)1097-4598(199812)21:123.0.co;2-2 li, x., zhang, t., li, c., xu, w., guan, y., li, x., cheng, h., chen, s., yang, b., liu, y., et al. (2023). electrical stimulation accelerates wallerian degeneration and promotes nerve regeneration after sciatic nerve injury. glia 71, 758-774. https://doi.org/10.1002/glia.24309 lukacs, m., gilley, j., zhu, y., orsomando, g., angeletti, c., liu, j., yang, x., park, j., hopkin, r.j., coleman, m.p., et al. (2019). severe biallelic loss-of-function mutations in nicotinamide mononucleotide adenylyltransferase 2 (nmnat2) in two fetuses with fetal akinesia deformation sequence. exp neurol 320, 112961. https://doi.org/10.1016/j.expneurol.2019.112961 margeta, m. (2020). top ten discoveries of the year: neuromuscular disease. free neuropathol 1, 4. https://doi.org/10.17879/freeneuropathology-2020-2627 margeta, m. (2021). neuromuscular disease: 2021 update. free neuropathol 2, 3. https://doi.org/10.17879/freeneuropathology-2021-3236 margeta, m. (2022). neuromuscular disease: 2022 update. free neuropathol 3, 5. https://doi.org/10.17879/freeneuropathology-2022-3805 martin-aguilar, l., lleixa, c., and pascual-goni, e. (2022). autoimmune nodopathies, an emerging diagnostic category. curr opin neurol 35, 579-585. https://doi.org/10.1097/wco.0000000000001107 mcgonigal, r., campbell, c.i., barrie, j.a., yao, d., cunningham, m.e., crawford, c.l., rinaldi, s., rowan, e.g., and willison, h.j. (2022). schwann cell nodal membrane disruption triggers bystander axonal degeneration in a guillain-barre syndrome mouse model. j clin invest 132, e158524. https://doi.org/10.1172/jci158524 mocciaro, e., runfola, v., ghezzi, p., pannese, m., and gabellini, d. (2021). dux4 role in normal physiology and in fshd muscular dystrophy. cells 10, 3322. https://doi.org/10.3390/cells10123322 oaklander, a.l., mills, a.j., kelley, m., toran, l.s., smith, b., dalakas, m.c., and nath, a. (2022). peripheral neuropathy evaluations of patients with prolonged long covid. neurol neuroimmunol neuroinflamm 9, e1146. https://doi.org/10.1212/nxi.0000000000001146 osterloh, j.m., yang, j., rooney, t.m., fox, a.n., adalbert, r., powell, e.h., sheehan, a.e., avery, m.a., hackett, r., logan, m.a., et al. (2012). dsarm/sarm1 is required for activation of an injury-induced axon death pathway. science 337, 481-484. https://doi.org/10.1126/science.1223899 riva, n., gentile, f., cerri, f., gallia, f., podini, p., dina, g., falzone, y.m., fazio, r., lunetta, c., calvo, a., et al. (2022). phosphorylated tdp-43 aggregates in peripheral motor nerves of patients with amyotrophic lateral sclerosis. brain 145, 276-284. https://doi.org/10.1093/brain/awab285 sarparanta, j., jonson, p.h., kawan, s., and udd, b. (2020). neuromuscular diseases due to chaperone mutations: a review and some new results. int j mol sci 21, 1409. https://doi.org/10.3390/ijms21041409 schiaffino, s., rossi, a.c., smerdu, v., leinwand, l.a., and reggiani, c. (2015). developmental myosins: expression patterns and functional significance. skelet muscle 5, 22. https://doi.org/10.1186/s13395-015-0046-6 sewry, c.a., feng, l., chambers, d., matthews, e., and phadke, r. (2021). importance of immunohistochemical evaluation of developmentally regulated myosin heavy chains in human muscle biopsies. neuromuscul disord 31, 371-384. https://doi.org/10.1016/j.nmd.2021.02.007 shi, y., kerry, p.s., nanson, j.d., bosanac, t., sasaki, y., krauss, r., saikot, f.k., adams, s.e., mosaiab, t., masic, v., et al. (2022). structural basis of sarm1 activation, substrate recognition, and inhibition by small molecules. mol cell 82, 1643-1659. https://doi.org/10.1016/j.molcel.2022.03.007 simon, d.j., and watkins, t.a. (2018). therapeutic opportunities and pitfalls in the treatment of axon degeneration. curr opin neurol 31, 693-701. https://doi.org/10.1097/wco.0000000000000621 stevanovski, i., chintalaphani, s.r., gamaarachchi, h., ferguson, j.m., pineda, s.s., scriba, c.k., tchan, m., fung, v., ng, k., cortese, a., et al. (2022). comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing. sci adv 8, eabm5386. https://doi.org/10.1126/sciadv.abm5386 vallat, j.m., magy, l., corcia, p., boulesteix, j.m., uncini, a., and mathis, s. (2020). ultrastructural lesions of nodo-paranodopathies in peripheral neuropathies. j neuropathol exp neurol 79, 247-255. https://doi.org/10.1093/jnen/nlz134 volpatti, j.r., ghahramani-seno, m.m., mansat, m., sabha, n., sarikaya, e., goodman, s.j., chater-diehl, e., celik, a., pannia, e., froment, c., et al. (2022). x-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by hdac inhibition. acta neuropathol 144, 537-563. https://doi.org/10.1007/s00401-022-02468-7 wang, x., jia, y., zhao, j., lesner, n.p., menezes, c.j., shelton, s.d., venigalla, s.s.k., xu, j., cai, c., and mishra, p. (2022). a mitofusin 2/hif1alpha axis sets a maturation checkpoint in regenerating skeletal muscle. j clin invest 132, e161638. https://doi.org/10.1172/jci161638 weihl, c.c., topf, a., bengoechea, r., duff, j., charlton, r., garcia, s.k., dominguez-gonzalez, c., alsaman, a., hernandez-lain, a., franco, l.v., et al. (2023). loss of function variants in dnajb4 cause a myopathy with early respiratory failure. acta neuropathol 145, 127-143. https://doi.org/10.1007/s00401-022-02510-8 winkler, m., von landenberg, c., kappes-horn, k., neudecker, s., kornblum, c., and reimann, j. (2021). diagnosis and clinical development of sporadic inclusion body myositis and polymyositis with mitochondrial pathology: a single-center retrospective analysis. j neuropathol exp neurol 80, 1060-1067. https://doi.org/10.1093/jnen/nlab101 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodevelopmental disorders: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:8 (2023) review neurodevelopmental disorders: 2023 update paulina carriba1,3, nicola lorenzón1, mara dierssen1,2,3 centre for genomic regulation (crg), the barcelona institute of science and technology, barcelona, spain universitat pompeu fabra (upf), barcelona, spain centro de investigación biomédica en red de enfermedades raras (ciberer), madrid, spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona spain mara.dierssen@crg.eu submitted: 28 february 2023 accepted: 30 april 2023 copyedited by: aivi nguyen published: 08 may 2023 https://doi.org/10.17879/freeneuropathology-2023-4701 keywords: centrosomes, clip: caudal late interneuron progenitor, human organoids, mcd: malformations of cortical development, stress granules, sudc: sudden unexplained death in childhood abstract several advances in the field of neurodevelopmental diseases (ndds) have been reported by 2022. of course, ndds comprise a diverse group of disorders, most of which with different aetiologies. however, owing to the development and consolidation of technological approaches, such as proteomics and rna-sequencing, and to the improvement of brain organoids along with the introduction of artificial intelligence (ai) for biodata analysis, in 2022 new aetiological mechanisms for some ndds have been proposed. here, we present hints of some of these findings. for instance, centrioles regulate neuronal migration and could be behind the aetiology of periventricular heterotopia; also, the accumulation of misfolded proteins could explain the neurological effects in covid-19 patients; and, autism spectrum disorders (asd) could be the expression of altered cortical arealization. we also cover other interesting aspects as the description of a new ndd characterized by deregulation of genes involved in stress granule (sg) assemblies, or the description of a newly discovered neural progenitor that explains the different phenotypes of tumours and cortical tubers in tuberous sclerosis complex (tsc) disease; and how it is possible to decipher the aetiology of sudden unexplained death in childhood (sudc) or improve the diagnosis of cortical malformations using formalin-fixed paraffin-embedded samples. abbreviations ad – alzheimer’s disease; adhd attention deficit/hyperactivity disorder; ai – artificial intelligence; asd autism spectrum disorder; bcs – balloon cells; ca – cornu ammonis; cd cluster of differentiation; cge caudal ganglionic eminence; clip caudal late interneuron progenitor; cns – central nervous system; dg – dentate gyrus; egfr epidermal growth factor receptor; eif2 eukaryotic initiation factor 2; fcd – focal cortical dysplasia; ffpe formalin-fixed paraffin-embedded; fs febrile seizure; gcs giant cells; go – gene ontology; hand – hiv-associated neurocognitive disorder; hco human cortical organoids; het – heterozygous; hiv – human immunodeficiency virus; id intellectual disability; ipscs induced pluripotent stem cells; ko – knockout; loh loss of heterozygosity; mcds – malformations of cortical development; mtor mammalian target of rapamycin; nsc – neural stem cells; orf – open reading frame; ph – periventricular heterotopia; prpf6 pre-mrna processing factor 6; rna-seq – rna sequencing; sars-cov-2 severe acute respiratory syndrome coronavirus 2; sgs stress granules; sudc sudden unexplained death in childhood; sudep sudden unexpected death in epilepsy; t-hco – transplanted human cortical organoids; ts – timothy syndrome; tsc tuberous sclerosis complex; wt wild-type. introduction for this new collection of the most relevant findings in neurodevelopmental disorders that appeared in 2022, our selection tried to encompass a wide range of aspects that we think could be of interest to neuropathologists. the topics chosen are: neurodevelopmental disorders and the proper space and time sequential events during brain neurodevelopment stress granule assemblies and neurodevelopmental disorders clip, a newly discovered interneuron progenitor, explains the divergent phenotype in tuberous sclerosis complex disorder improving the diagnostic of malformations of cortical development (mcds) diseases by dna methylation profile patterns deciphering the aetiology of sudden unexplained death in childhood (sudc) by proteomics transcriptomic dysregulation in asd occurs across the whole cerebral cortex and follows a regional gradient in vivo platform for the study of human neurodevelopmental disorders mitochondria participate in the neuron-glia crosstalk gaining insight on the role of hiv-1 in the cns novel mechanisms explaining covid-19 neurological anomalies along these subject matters we aim to discuss advances in different ndds, from brain malformations or classical neurodevelopmental conditions to more general aspects that a neuropathologist might face, such as paediatric neurological alterations associated with covid-19 or hiv-associated neurocognitive disorder (hand) in children. we also selected relevant findings regarding how formalin-fixed paraffin-embedded (ffpe) samples, the major form of stored brain samples, could be used for studying neurodevelopmental disorders, and how the use of artificial intelligence (ai) can improve the diagnosis of cortical malformations. some of the selected topics also provide new mechanistic insights, such as the newly discovered neural progenitor clip, which explains the divergent phenotypes in tuberous sclerosis complex (tsc) pathology. indeed, one of the most remarkable topics of 2022, which has acquired increasing relevance in recent years, is the importance of the spatial and temporal regulation of brain development. the coordinated and orchestrated series of cellular processes controlled by fine-tuned sets of genetic programs during neurodevelopment leads to immense cell diversity, with different features depending on their final fate, localisation, and properties distinctive from the cells from which they developed. those cells will be part of circuits that are adjusted, readjusted and refined by intrinsic and extrinsic signals (rubenstein and rakic, 1999; miyata et al., 2010; kwan et al., 2012; greig et al., 2013; wamsley et al., 2018; di bella et al., 2021; bonnefont and vanderhaeghen, 2021) in a precise spatial-temporal manner. within this choreographic arrangement, a single out-of-tune event in time or space may represent the inception of a neurodevelopmental pathological condition. finally, human-derived organoids continue to be a promising in vitro tool for modelling human physiological and pathological development (figure 1). in the last year, those systems gained popularity thanks to specific improvements in successfully modelling neurodevelopmental disorders, allowing the study of human neuronal function in an in vivo context. figure 1. schematic representation of some uses of human-derived organoids presented in this update. a) cerebral organoids derived from ipscs can be used for single-cell rna-sequencing (scrnaseq) to determine expression patterns for the study of mechanisms driving neurodevelopmental disorders. here presented in neurodevelopmental diseases and the proper space and time sequential events during brain neurodevelopment; clip, a newly discovered interneuron progenitor, explains the divergent phenotype in tuberous sclerosis complex disorder; and in transcriptomic dysregulation in asd occurs across the whole cerebral cortex and follows a regional gradient. also, cerebral organoids can be transplanted into a host brain circuit to study the functionality of human organoids, as introduced in the in vivo platform for the study of human neurodevelopmental diseases. b) new engineering organoids containing microglia have been developed last year to study novel mechanisms explaining covid-19 neurological anomalies. 1. neurodevelopmental diseases and the proper space and time sequential events during brain neurodevelopment although it has been suggested that out-of-tune events at specific time points or specific brain regions are crucial for understanding neurodevelopmental pathological conditions, few examples have established a concrete cellular process in which such timeand place-specific effects could be disentangled. o’neill et al. reported time-dependent dysregulation of the centrosome interactome at specific neuronal differentiation stages, which allowed studying the aetiology of neurodevelopmental diseases (o’neill et al., 2022). centrosomes, as anchor structures for the cell cytoskeleton, are involved in a number of cell functions, including mitosis and cell migration (wilsch-bräuninger and huttner, 2021; gönczy and hatzopoulos, 2019; vineethakumari and lüders, 2022; delgehyr et al., 2005; piel et al., 2000). to prove their time-dependent hypothesis, the authors derived neural stem cells (nsc) [15 days in culture], and differentiated neurons [40 days in culture] to forebrain identity, using human induced pluripotent stem cell (ipsc) lines. at these two stages, mass spectrometry (ms) of centrosome-associated proteins revealed large cell type-specificity, with around 60% of the neural centrosome proteins not being detected in the centrosome of other cell types. gene ontology (go) categorization indicated that, as expected, nsc centrosome-associated proteins are richer in proteins related to cell division, microtubule organization, and rna splicing; whereas in later stages, neuronal centrosome interactors are related to cytoskeleton and rna-interacting proteins. interestingly, the neural centrosome interactome is particularly enriched in rna-interacting proteins compared with other cell types. by overlaying the interactomes with published datasets of neurodevelopmental diseases with de novo variants (dnv) in autism spectrum disorder (asd), periventricular heterotopia (ph), intellectual disability (id), epileptic encephalopathy (ee) and polymicrogyria (pmg), the authors detected a clear disease association of the neural centrosome interactome. in asd, a pathological association was found for all datasets analysed, suggesting pan-cellular involvement of centrosome proteins in its aetiology. in ph, the authors identified the enrichment of the microtubule-anchoring pre-mrna processing factor 6 (prpf6). prpf6 is more abundant in the centrosome of nscs than of neurons. mutated prff6 recapitulated ph heterotopias in the periventricular cortex of early mouse embryos, along altered mrna splicing, that affected the centrosome associated brsk2 (brain-selective kinase 2) protein, involved in microtubule dynamic regulation and neural migration (barnes et al., 2007; kishi et al., 2005; nakanishi et al., 2019). indeed, rna dynamics play an important function during brain development (raj and blencowe, 2015). panagiotakos and pasca, in a perspective manuscript in neuron, remark how critical the moment and place of the events during brain development is for neurodevelopmental pathologies (panagiotakos and pasca, 2022). as an example, the temporal expression pattern of the voltage-gated sodium channels nav1.1, nav1.2, and nav1.3 isoforms explains developmental brain malformations. mutations in scn3a, encoding for nav1.3, which is elevated in immature progenitors and foetal brain neurons, can lead to abnormal neuronal migration and subsequent polymicrogyria (smith et al., 2018). instead, mutations in scn1a and scn2a encoding for nav1.1 and nav1.2, respectively (beckh et al., 1989; smith et al., 2018), which are elevated in postnatal neurons, are commonly related to infantile epilepsies (meisler and kearney, 2005). interestingly, these protein isoforms also display specific cell-type enrichment during brain development. parvalbumin (pv) cortical interneurons predominantly express nav1.1 channels during early life (yu et al., 2006), so that scn1a loss of function leads to postnatal epilepsy, which disappears in adulthood (favero et al., 2018). thus, it is relevant to differentiate the initial mechanism that triggers disease onset, from those contributing to chronic disease states. in fact, the individual variability of neuropathology onset or affectation may depend on the moment or place the alteration occurs. therefore, in addition to the proteomic and genetic information, understanding of neuropathology requires the understanding of cell-specific alterations, gene regulatory networks and protein interactomes and how they evolve and are regulated during the nervous system formation. 2. stress granule assemblies and neurodevelopmental disorders stress granules (sgs) are dynamic cytoplasmatic membrane-less compartments that assemble under a variety of stress conditions (anderson and kedersha, 2009; jain et al., 2016). a large number of sgs components and regulators have been described (jain et al., 2016; markmiller et al., 2018; yang et al., 2020), but the mechanistic dynamics of these assemblies are still unknown. accumulated data indicates that these cytoplasmatic compartments play important roles in the regulation of gene expression (buchan et al., 2008; arimoto et al., 2008; takahara and maeda, 2012; decker and parker, 2012; yang et al., 2020). sgs are detected where there are considerable pools of untranslated messengers and ribonucleoprotein particles (rnps) (protein-coding mrnas and non-protein-coding rnas, and rna-binding proteins) to shut down translation (guillén-boixet et al., 2020). thus, they seem critical for gene expression homeostasis (martin and ephrussi, 2009; wang et al., 2019), playing then relevant functions during brain development. last year, jia et al. reported a new ndd characterized by alterations in sg formation (jia et al., 2022). they detected disruptive variants of ubap2l, an essential regulator of sg formation (youn et al., 2018; cirillo et al., 2020), in patients with speech-language problems, id, motor delay, seizure, and with less prevalence in patients with adhd, asd, repetitive and aggressive behaviour, and anxiety, but without a defined ndd. the patients also presented morphological features such as facial dysmorphisms, visual impairment, hypotonia and hand and foot abnormalities. using skin fibroblast cell cultures from two patients, they showed reduced levels of ubap2l and fewer sgs under stressful conditions. the authors validated these observations in a cell line (hela) knockout (ko) for ubap2l. transfection of these ko cells with the ubap2l mutants detected in patients also led to a reduction in sgs formation. further experiments in ubap2l ko mice showed increased mortality in embryonic stages and reduced brain size compared with wild-type (wt) ubap2l+/+, and heterozygous (het) ubap2l+/littermates. ko mice showed anomalous neocortex lamination and reduction in neuronal progenitor proliferation possibly linked to altered sg dynamics during cortical neurogenesis. moreover, ubap2l+/animals showed impaired social novelty ability, abnormal spatial working memory, and more anxiety-like behaviour. all these data prompted the authors to analyse the enrichment of sg genes from published datasets of proteomics and high-throughput genome-wide screenings in curated ndd gene datasets, including from sfari (simons foundation autism research initiative) and ddg2p database (development disorder genotype phenotype database). they detected significant enrichment of sg genes, particularly sg core genes and rna-binding proteins. they also examined specific sg genes that could be related to ndd from previously reported de novo mutations (dnms), detecting 3410 variants in the coding regions of 843 sg genes. the statistical analysis showed enrichment of sg genes that clustered according to their network function, string database for protein-protein interaction (ppi), with some of the enriched genes that had not been previously implicated in ndds. although previous works have evidenced that stress conditions during embryonic stages increase the risk of ndds (kinney et al., 2008; babenko et al., 2015; fitzgerald et al., 2020; chui et al., 2020), this is the first study to identify alterations in sg assemblies as a common neuropathological feature of ndds with no defined aetiology. 3. clip, a newly discovered interneuron progenitor, explains the divergent phenotype in tuberous sclerosis complex disorder tuberous sclerosis complex (tsc) is a rare genetic condition that causes benign tumours in different parts of the body mainly the brain, kidneys, heart, skin, lungs and eyes. in the brain, tsc-associated lesions include subependymal tumours at the lateral ventricle and cortical dysplastic lesions, namely cortical tubers. both aberrant structures contain, among other cell types, giant cells (gcs), which are the histopathological hallmark of the disease (ruppe et al., 2014; gelot and represa, 2020; henske et al., 2016). these cells feature a large and central nucleus with peripheral chromatin and a prominent nucleolus, and nissl substance and neurofibrils in the cytoplasm (mizuguchi, 2007). the abnormally large size of gcs strongly indicates dysregulation of cell size control in tsc (mizuguchi, 2007). patients often develop tsc-associated neuropsychiatric disorders (tand) which include id, attention deficit/hyperactivity disorder (adhd), aggressiveness, difficulties with communication and social interaction (asd), epilepsy, seizures and psychiatric conditions (thiele, 2010). tsc is produced by the mutation of either tsc1 or tsc2. these two genes encode for proteins that inhibit mtor (mammalian target of rapamycin) signalling, which is the major regulator of cell growth. loss of regulation of this signalling pathway leads to abnormal cell development and differentiation. experimental data suggest that tsc is produced by a heterozygous germline mutation followed by somatic loss of heterozygosity (loh) in the other allele, due to loss-of-function mutations (crino, 2013; feliciano et al., 2011; feliciano et al., 2012). however, patient tissue analyses show that loh occurs only in tumours and not in dysplastic tubers (henske et al., 1996; chan et al., 2004; qin et al., 2010). moreover, mouse models with loh in either tsc1 or tsc2 cannot recapitulate the full spectrum of brain aberrations observed in patients. last year, eichmüller et al., solved the discrepancies owing to the discovery of a new interneuron progenitor (eichmüller et al., 2022). the authors found that cerebral organoids derived from patients with tsc2+/reproduced both histopathological features using different culture conditions; that is, brain tumours when cultured in high-nutrient medium, and dysplastic cortical tubers when cultured in low-nutrient medium. the characterization of the cellular composition by single-cell rna-sequencing (scrna-seq), along with exhaustive histological validation, allowed the authors to identify a specific interneuron progenitor population that gives rise to both the tumours and the cortical tuber lesions. comparisons with human foetal brain data revealed that this interneuron progenitor is first detected in the caudal ganglionic eminence (cge) during late mid-gestation, with manifest expansion and migration during late gestation. given their origin and embryonic stage, the authors called these interneuron progenitor cells clip, for “caudal late interneuron progenitor”. clip cells seem to be particularly sensitive to mtor levels, being disturbed upon loss of one copy of tsc1/2, which resulted in the over-proliferation of these progenitor cells. the authors determined that the tubers are generated from migrated clip interneurons while the tumours grow in the cge as a consequence of an additional aberration in the second allele, most probably produced by the over-proliferation of these clip cells and the contribution of other factors or cell types (figure 2). thus, although derived from the same altered progenitor clip, tuber cells do not show loh as a mechanism of action, while the tumour cells do show loh. figure 2. illustration depicting the mechanism described by eichmüller et al., 2022. the left shows normal development when neither of the two copies of tsc1/2 have mutations. on the right, when one copy of tsc1/2 is mutated, clip cells become sensitive to mtor levels, resulting in aberrant growth and expansion. clip neurons that migrate to the cortex develop into cortical tubers, and the remaining clip cells, through the participation of other additional alterations lose the other allele producing tumours. the manuscript shows how the same progenitor cell type diverges into two histopathological differential phenotypes. it also shows that clip cells depend on epidermal growth factor receptor (egfr) signalling, and that the inhibition of egfr regressed the organoid tumours, providing an alternative treatment therapy for this pathology. an interesting aspect of this manuscript is that the disease mechanism described is human-specific. indeed, human brain development encompasses the generation and/or expansion of cell types deriving large and gyrated cortices, which do not occur in small lissencephalic brains such as the rodent brain cortex. even postnatally there is extensive migration of interneurons from the cge into the cortex in humans (paredes et al., 2016; hansen et al., 2013; hodge et al., 2019), but not in mice (raju et al., 2018). the use of human organoids was key for this discovery. however, although human organoids are a powerful model system, this technology is still in its infancy. for example, the current lack of standardized protocols implies important variability among organoids from the same patient, which intrinsically puts the results in uncertainty. thus, further studies are necessary to validate clip cells and their functions. 4. improving the diagnostic of malformations of cortical development (mcds) diseases by dna methylation patterns malformations of cortical development (mcds) comprise various neurodevelopmental disorders that are a major cause of epilepsy (leventer et al., 1999), and medically stubborn childhood seizures (kuzniecky, 1995). mcds can be classified into three groups depending on their likely origin. in group i, derived from abnormal cell proliferation or apoptosis, there are hemi-megalencephaly, microcephaly, megalencephaly, and focal cortical dysplasia; in group ii, related to abnormal cell migration, we find tubulinopathies, lissencephalies and heterotopies; and in group iii polymicrogyria is produced by abnormal post-migrational development (desikan and barkovich, 2016). this heterogeneity of causes and phenotypic presentations with a broad range of symptomatology including cognitive deficits, id, and asd (barkovich et al., 2012; guerrini and dobyns, 2014), challenges neuropathologists in providing an accurate diagnosis and, consequently, an on-target prognosis and management of the affectation. as such, biomarkers to identify the type of mcd more precisely are a growing subject of research. however, biomarkers are available only for focal cortical dysplasia (fcd) type ii (d’gama et al., 2015; jansen et al., 2015; d’gama et al., 2017; baldassari et al., 2019) and mild malformations of cortical development with oligodendroglial hyperplasia (moghe) (schurr et al., 2017; bonduelle et al., 2021). currently, the diagnosis of mcd pathologies is based only on histopathological criteria providing, generally, an imprecise diagnosis. as an example, in the case of fcd type ii, two forms have been described fcdiia and fcdiib (blümcke et al., 2011), which differ in that only fcdiib contains balloon cells (bcs). bcs are enlarged cytoplasm cells that resemble gemistocytic astrocytes displaying multiple or convoluted nuclei without prominent nucleoli (mizuguchi, 2007). however, bcs histologically are very alike to gcs observed in tsc. fcdiib and tsc are both pathologies associated with dysregulation of the mtor pathway and display comparable histopathological features, particularly fcdiib and the cortical tubers, which suggests a closely related origin, although they are clearly different neuropathological entities (taylor et al., 1971; lee et al., 2022). in an attempt to improve the diagnosis of mcd pathologies, jabari et al. assayed a potential strategy based on dna methylation (jabari et al., 2022). dna methylation can be a reliable biomarker because it is preserved and, therefore, can be detected in archival human brains stored in ffpe (sahm et al., 2017; capper et al., 2018; wefers et al., 2020). moreover, the methylome manifests a combination of both the somatically acquired dna methylation alterations, and the molecular memory marks in response to environmental or pathogenic cues (kobow and blümcke, 2012; kobow et al., 2013; kiese et al., 2017; kobow et al., 2019; kobow et al., 2020). furthermore, dna methylation profile is widely used to classify cns tumours because of its reproducibility and sensitivity even in small samples (sahm et al., 2017; capper et al., 2018). thus, the purpose of this study was to find dna methylation patterns to accurately classify the different histopathological entities. the authors used surgical samples from patients with mcd and with a confirmed histopathological classification included 265 samples across all age groups and sex that demonstrated different pathological levels of the 12 major subtypes of mcd along with different controls. the authors performed a genome-wide dna methylation assay to correlate the dna methylation patterns with the histopathological classification. they used three different approaches: pairwise comparison, machine learning, and deep learning algorithms. the deep learning algorithm allowed for the most accurate discrimination providing a rationalized classification of the pathologies. then, they analysed the precision of the dna methylation-based mcd classification using a new cohort from different epilepsy centres. this test cohort contained 43 surgical ffpe samples, among which some previously underwent multiple rounds of histopathological evaluation from expert neuropathologists because of the difficulty of their classification. using the algorithm, the authors were able to accurately classify all samples from the test cohort. figure 3 depicts the flowchart the authors followed. thus, they demonstrate that dna methylation-based mcd classification is suitable across major histopathological entities and could be used to establish an integrated diagnostic classification scheme for mcd neuropathology. figure 3. flowchart of the process. top, generation of the algorithm by artificial intelligence (ai) of the correlation of the dna methylation profiles obtained from ffpe samples with confirmed histopathological mcds classification. bottom, validation of the algorithm to classify mcds based on dna methylation using a new set of ffpe samples. 5. deciphering the aetiology of sudden unexplained death in childhood (sudc) by proteomics sudden unexplained death in childhood (sudc) refers to the unexplainable death of children over 1 year of age. it is called unexplained because, after a complete review of the clinical history and the autopsy including toxicologic, genetic, metabolic and microbiology analyses, to cite some of the complementary studies, the cause of death is not determined. although the causes may be diverse, genetic variants are likely to be prone to sudc risk (crandal et al., 2020; halvorsen et al., 2021; harowitz et al., 2021; holm et al., 2012; narula et al., 2015). for example, among sudc cases, there is a high prevalence of individual or familial febrile seizure (fs) history (hefti et al., 2016; hesdorffer et al., 2015; mcguone et al., 2020). sudc shares some pathological similarities with sudden unexpected death in epilepsy (sudep) (devinsky et al., 2016; kinney et al., 2016), suggesting that they may share some coincident mechanisms that result in premature death. similar to sudc, the causes of sudep may also be diverse including genetic risk factors. since both pathological conditions show abnormalities in the hippocampus and cortex (ackerman et al., 2016; kinney et al., 2016; kon et al., 2020; mcguone et al., 2020), these brain regions, particularly the hippocampus, have attracted most sudc studies. however, no conclusive results are available to date (leitner et al., 2022a; roy et al., 2020). in this scenario, leitner et al., have defined differential protein abundance in several brain areas of sudc cases (19 cases), including the frontal cortex, hippocampal dentate gyrus (dg), and cornu ammonis (ca1-3). the study compared cases with and without febrile seizure history (sudc-fs and sudc-nofs) and without febrile seizure with control cases (n = 19) matched by age, sex, brain weight and post-mortem interval (leitner et al., 2022b). the authors micro-dissected the aforementioned brain regions from autopsy ffpe tissue to perform label-free quantitative proteomic analyses. the proteomic analyses revealed no differential hippocampal neuropathology between sudc-fc and sudc-nofc. instead, principal component analysis (pca) revealed a significant separation between sudc and controls in the frontal cortex, but not regarding fs history. differential protein abundance analysis showed significant differences between sudc and control cases in 660 proteins of the frontal cortex, while only 170 in the dg and 57 in ca1-3. pathway analysis revealed 238 signalling pathways in the frontal cortex, mainly involved in the activation of oxidative phosphorylation, inhibition of eif2 (eukaryotic initiation factor 2) signalling, and glutamate receptor signalling. in the dg, they mainly found pathways involving activation of the acute phase response and inhibition of reelin signalling, while in hippocampal ca1-3, the only involved signalling pathway was in the acute phase response associated with cellular stress response (liu and qian, 2014; brace et al., 2016; leitner et al., 2022b). the fact that they detected protein overlap in more than one signalling pathway supports the involvement of these signalling pathways. the authors also used weighted gene correlation network analysis (wgcna) to correlate the proteomic results with the clinical history. in the frontal cortex there were common affected enriched signaling pathways in sudc and sudep, some of which showed oppository effects in sudc and sudep (e.g., oxidative phosphorylation and eif2 signalling), while other proteomic pathways were similar(e.g., mitochondrial enzyme cox6b1). as a corollary, besides the relevant information provided, one of the most remarkable findings is that while the frontal cortex is not a studied region in sudc, it showed the most altered proteomic changes. hence, the authors put forward the relevance of the frontal cortex in this condition and the necessity of performing studies in this region to detect possible neuropathological signs associated with sudc to reduce and prevent the risk of this condition. 6. transcriptomic dysregulation in asd occurs across the whole cerebral cortex and follows a regional gradient asd is a prototypical example of neuropathology in which a definitive aetiology has not yet been determined. in the last years, comprehensive omics assessments have been conducted to determine risk genes (de la torre-ubieta et al., 2016) or differential patterns of splicing or gene isoform expression in asd (wu et al., 2016; sun et al., 2016; gandal et al., 2018). despite the heterogeneity of factors that drive to asd pathology, molecular profiling studies have found consistent patterns of transcriptomic and epigenetic dysregulation (ramaswami et al., 2020), involving upregulation of astrocytes, microglia, and neural immune genes; and downregulation of synaptic, neurite morphogenesis, and neuronal energy pathway genes accompanied by attenuation of gene-expression gradients in cortical association regions (voineagu et al., 2011; wu et al., 2016; parikshak et al., 2016; gandal et al., 2018). the work of gandal et al. explores whether these alterations are more widespread throughout the cortex and proposes that asd pathology is the physiological manifestation of altered cortical arealization (gandal et al., 2022). to support this hypothesis, they did bulk rna-sequencing (rna-seq) analysis to identify altered genes and alternative spliced gene isoforms. they used threefold more samples than previous works (voineagu et al., 2011; parikshak et al., 2016), analysing 725 post-mortem brain samples spanning 11 cortical areas from 112 individuals of both sexes and with ages ranging from 2 to 68, totalling 49 subjects with idiopathic asd and 54 neurotypical controls. they found transcriptomic changes across the cortex of asd patients with an anterior-to-posterior gradient, with the most remarkable differences in the primary visual cortex. in agreement with previous reports (walker et al., 2019), the greater differential expression detected was related to alternative splicing and differential isoform expression. thus, their findings indicate that molecular alterations, mainly in alternative splicing and isoform expression, in asd transcriptome extend beyond the associative cortex to broadly involve primary sensory regions. this may explain the altered sensory processing observed in individuals with asd. an interesting aspect is that the differences in gene expression that account for demarcating the different cortical regions, because they define the cytoarchitecture, connectivity, and function of a particular region, were attenuated in asd. this indicates that the cortical regions in asd patients are molecularly more homogeneous, and therefore, less differentiated and specialised. again, this attenuated expression followed a gradient pattern, which was more particularly affected in the posterior regions such as the primary visual cortex. the authors also determined whether the transcriptomic changes detected were reflected in the cell-specific type gene expression. by sn-rna-seq and methylation profiles, they determined a substantial differential expression profile in excitatory neuron classes and glia cells, once more, with a regional gradient more prominent in occipital and parietal cell types than in prefrontal cortex (pfc). this reiterative regional gradient may reflect a reminiscence of the buildout of the cortical cytoarchitecture, i.e., its patterning and connectivity, which depend on both cell intrinsic factors genetic and epigenetic regulatory programmes, and extrinsic signals, –such as morphogen gradients (cadwell et al., 2019). the data suggests that this process is altered in asd, indicating an early developmental alteration in cortical arealization, affecting local neuronal circuits, synaptic homeostasis, and leading to the asd manifestations. 7. in vivo platform for understanding the neuropathology of human neurodevelopmental diseases some years ago, several studies showed that it was possible to transplant human neurons into the rodent cortex that were able to establish connections with the rodent cells (espuny-camacho et al., 2013; mansour et al., 2018; real et al., 2018; linaro et al., 2019; kitahara et al., 2020; xiong et al., 2021), thus providing an in vivo platform to study human developmental disorders. although the hope was that those tools would allow uncovering circuit-level phenotypes from patient-derived cells and test therapeutic strategies, they had some problems. part of those have now been solved in the work of revah et al. (revah et al., 2022). to facilitate integration of the transplant they transplanted 3d human cortical organoids (hco) into the somatosensory cortex of immunodeficient rats, at early postnatal stage, in which corticocortical and thalamocortical innervation have not yet been completed, thus minimising the endogenous circuitry alteration (kichula and huntley, 2008). the novelty of this work is thus that they transplanted intact organoids, rather than a dissociated cell suspension; and at very early postnatal stages, rather than in adult rats. this strategy allows better synaptic and axonal integration of the human cells. hence, the transplanted hcos (t-hco) displayed more mature properties compared to not transplanted same-age hcos. the t-hco showed vascularization and the presence of microglia. snrna-seq revealed the canonical expression pattern of major cortical cell classes, even though t-hco did not present anatomical lamination. transplanted neurons showed a more mature morphological phenotype, i.e., larger somas, more dendrites, larger processes, and higher dendritic spine density, and more mature electrophysiological properties. the authors traced t-hco innervation with retrograde rabies showing integration of t-hco neurons in the rat brain's circuitry. fibre photometry, two-photon calcium imaging, electrophysiology, and sensory stimuli by deflecting the rat’s whiskers revealed the functionality of the integration, as t-hco stimulation evoked response in rat neurons indicating functional innervation. finally, by in vivo optogenetics they showed that activation of t-hco could modify the rats’ behavioural response. the authors used this system for studying timothy syndrome (ts), a severe genetic disorder caused by the mutation in the l-type voltage-sensitive calcium channel cav1.2 (ebert and greenberg, 2013). they compared the evolution of t-hco derived from control individuals and patients with ts with their non-transplanted counterparts (hco). ts t-hco neurons showed altered dendritic morphology with an extensive number of primary dendrites but with an overall reduction in the mean and total dendritic size. also, these neurons displayed increased synaptic spine density that impaired their electrophysiological properties. these phenotypes could only be detected in ts t-hco but not in non-transplanted organoids with the same differentiation stage, thus indicating that organoid transplantation allowed for better recapitulation of the disease phenotype that was elusive in non-transplanted organoids. 8. mitochondria participate in the neuron-glia crosstalk mitochondria play crucial roles in the regulation of cellular energy and metabolism; therefore, it is not surprising that this organelle is involved in all developmental stages with important functions in neuronal differentiation. for this reason, several mitochondrial disorders present an abnormal neuronal and neurological development (son and han, 2018). mitochondrial dysfunction has been widely associated with neurological and psychiatric diseases such as schizophrenia, bipolar depression, asd and rett syndrome (son and han, 2018). one example is leigh syndrome, which is caused by a mutation in mitochondrial dna, leading to dysfunctional mitochondrial complexes. the disorder usually manifests within the first year of life and leads to rapid degeneration of physical and mental abilities, ultimately leading to death within 2-3 years (murphy and craig, 1975). however, this close relationship between the mitochondria and the cns is not only applicable to neurons. in fact, it is astrocytic’ mitochondria that support neuronal development and function. in cases of neuronal damage, such as in brain ischemia, astrocytes release functional mitochondria into the extracellular medium via a mechanism that involves the activation of the cluster of differentiation 38 (cd38) and cyclic adenosine diphosphate (adp)-ribose signalling (hayakawa et al., 2016; english et al., 2020). this process facilitates neuronal recovery. at the same time, damaged neurons expel damaged mitochondria, which are in turn absorbed by the surrounding astrocytes to be recycled. in a paper published last year, gao et al. showed that the release of mitochondria might function as a signalling element between neurons and glial cells (gao et al., 2022). they also proved that, under physiological conditions, neurons release mitochondria into the extracellular medium. then, they tested different pathological conditions in vitro, such as acidosis, hydrogen peroxide or high levels of nmda (n-methyl d-aspartate) or glutamate, simulating brain ischemia. they detected a significant increase in the number of released damaged mitochondria by the neurons (gao et al., 2022), and proved that astrocytes uptake these mitochondria and trigger the rescuing response. hence, they speculate that, under stress conditions, mitochondria could serve as a “help-me” signal. if replicated, this discovery would represent a huge step forward in understanding the mechanisms behind neuronal stress response that could shed light on common neurodevelopmental/neurodegenerative disorders. 9. gaining insight on the role of hiv-1 in the cns since its discovery, the human immunodeficiency virus (hiv) has been associated with several neurological conditions. hiv-1 infection has long been known to have an impact on the nervous system, culminating in hiv-associated neurocognitive disorder (hand) (clifford and ances, 2013), as the brain, along with the bone marrow, is known to be a reservoir for the virus. an interesting aspect is how hiv-1 infection affects neurodevelopment. in the era of antiretroviral therapy (art), this question translates to how maternal infection affects offspring development. properly treated mothers give birth to uninfected children in almost 99% of cases (wedderburn et al., 2022). however, hiv-exposed children generally show a slower development of some neurological functions. in particular, a poorer expressive language and gross motor function compared to their hiv-unexposed counterparts (wedderburn et al., 2022). however, the mechanisms underlying this phaenomenon remain unclear. one hypothesis states that the virus could directly affect the development of the foetus, while other researchers think that it is the chronic systemic inflammation of the mother due to the infection what could affect the offspring. however, research has been hindered by the lack of a satisfactory model, owing to the complexity derived from the involvement of both the nervous and immune systems. for this reason, researchers have long been searching for a novel model to study this phenomenon. brain organoids, in fact, could provide an insight on neuronal development and in recent years, protocols for microglia-containing brain organoids have been developed, hence allowing for this technology to be applied to hiv research field (ormel et al., 2018). to prove so, a study in 2022 by gumbs et al. showed that the cluster of differentiation 4 (cd4) and cysteine-cysteine chemokine receptor 5 (ccr5)– expressing microglia were susceptible to hiv-1 infection in a human brain organoid model (gumbs et al., 2022). they tested this infection model on both microglia-containing organoids and organoid-derived microglia (omg), with positive results in both cases. albeit the obvious limitations of the organoid model, the results of this study represent a significant advancement in the hiv research field, as these organoids could help elucidate the mechanisms behind the effects of the virus on the brain and test novel therapies. 10. novel mechanisms explaining covid-19 neurological anomalies the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic has had a huge impact on everyone's lives, causing millions of deaths worldwide and changing perpetually our society. although the disease was initially known to be hazardous to the respiratory system, soon several comorbidities with long-lasting effects were associated with the virus. it was soon discovered that the infection could lead to several significant neurological complications (chen et al., 2022). these effects vary largely from patient to patient, depending on factors such as age, weight, health status and pre-existing conditions. however, both shortand long-lasting neurological effects have been reported in all populations, from children to adults. regarding the effects of covid-19 on children, we should distinguish between the effects due to the pandemic and those due to the direct effects of the virus. a meta-analysis study by hessami et al. highlighted that infants born or raised during the pandemic showed a higher rate of communication impairment than the pre-pandemic cohort (hessami et al., 2022). given the nature of the study and the lack of other significant neurodevelopmental impairments in the general paediatric population, we can speculate that this communication impairment was an effect of the lack of social stimuli because of the lockdown rather than the virus itself. however, the direct neurological consequences of covid-19 infection have also been reported in both children and adults. these effects range from temporary anosmia and ageusia to memory loss, meningitis, stroke, and neurodegeneration (ledford, 2022). data on the possible long-term neurological effects of sars-cov-2 coronavirus are inconclusive. several studies have found associations with alzheimer’s disease (ad) risk, cardiovascular damage, or neurological sequelae. an analysis in the uk found a slight overall reduction of grey matter in the brains of recovered individuals. also, that the virus infects brain support cells and induces inflammation similar to parkinson’s or ad, and that severe covid-19 causes detectable brain ageing (douaud et al., 2022). despite the significant amount of research conducted on and the neurotropic nature of the virus, the molecular mechanisms linking covid-19 to these neurological symptoms are still unclear. a study published in 2022 by charnley et al. showed that some viral open reading frame (orf) proteins can assemble into amyloid-like aggregates and cause neurotoxicity (charnley et al., 2022). using an algorithm, they pinpointed two short regions of orf6 and orf10 as those responsible for the assembly. they then tested the peptides corresponding to these regions and showed that they self-assembles into amyloid-like structures. finally, these peptides were tested on neuroblastoma sh-sy5y cells, proving to be neurotoxic (charnley et al., 2022). we can presume that some of the neurological consequences of a covid-19 infection not only share symptomatology with some common neurodegenerative disorders (e.g., ad), but also share an accumulated misfolding protein-driven pathogenesis. it will be now important to test these mechanisms during development. funding the lab of m.d. is supported by the departament d'universitats, recerca i societat de la informació de la generalitat de catalunya (grups consolidats 2022), the agencia estatal de investigación (pid2019-110755rb-i00/aei/10.13039/501100011033), the european union’s horizon 2020 research and innovation programme under grant agreements nº 848077 (go-ds21) and under grant agreement nº 899986 (icod). horizon europe (horizon-hlth-2021-stayhlth-01-psych-strata). this reflects only the author's view, and the european commission is not responsible for any use that may be made of the information it contains. jerôme lejeune foundation (grant number 2002), nih (grant number: 1r01eb 028159-01), marató tv3 (#2022-12-30-31-32). the crg acknowledges the support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa, and the cerca programme/generalitat de catalunya. the ciber of rare diseases (ciberer) is an initiative of isciii. references ackerman mj, andrew ta, baker am, devinsky o, downs jc, keens t, kuntz j, lin p, lear-kaul kc, reichard r, robinson da. an association of hippocampal malformations and sudden death? we need more data. forensic sci med pathol. 2016 jun;12(2):229-31. https://doi.org/10.1007/s12024-016-9765-1. epub 2016 mar 26. pmid: 27017493. anderson p, kedersha n. rna granules: post-transcriptional and epigenetic modulators of gene expression. nat rev mol cell biol. 2009 jun;10(6):430-6. https://doi.org/10.1038/nrm2694. pmid: 19461665. arimoto k, fukuda h, imajoh-ohmi s, saito h, takekawa m. formation of stress granules inhibits apoptosis by suppressing stress-responsive mapk pathways. nat cell biol. 2008 nov;10(11):1324-32. https://doi.org/10.1038/ncb1791. epub 2008 oct 5. pmid: 18836437. babenko o, kovalchuk i, metz ga. stress-induced perinatal and transgenerational epigenetic programming of brain development and mental health. neurosci biobehav rev. 2015 jan;48:70-91. https://doi.org/10.1016/j.neubiorev.2014.11.013. epub 2014 nov 24. pmid: 25464029. baldassari s, ribierre t, marsan e, adle-biassette h, ferrand-sorbets s, et al. dissecting the genetic basis of focal cortical dysplasia: a large cohort study. acta neuropathol. 2019 dec;138(6):885-900. https://doi.org/10.1007/s00401-019-02061-5. epub 2019 aug 23. pmid: 31444548; pmcid: pmc6851393. barkovich aj, guerrini r, kuzniecky ri, jackson gd, dobyns wb. a developmental and genetic classification for malformations of cortical development: update 2012. brain. 2012 may;135(pt 5):1348-69. https://doi.org/10.1093/brain/aws019. epub 2012 mar 16. pmid: 22427329; pmcid: pmc3338922. barnes ap, lilley bn, pan ya, plummer lj, powell aw, raines an, sanes jr, polleux f. lkb1 and sad kinases define a pathway required for the polarization of cortical neurons. cell. 2007 may 4;129(3):549-63. https://doi.org/10.1016/j.cell.2007.03.025. pmid: 17482548. beckh s, noda m, lübbert h, numa s. differential regulation of three sodium channel messenger rnas in the rat central nervous system during development. embo j. 1989 dec 1;8(12):3611-6. https://doi.org/10.1002/j.1460-2075.1989.tb08534.x. pmid: 2555170; pmcid: pmc402042. blümcke i, thom m, aronica e, armstrong dd, vinters hv, et al. the clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc task force of the ilae diagnostic methods commission. epilepsia. 2011 jan;52(1):158-74. https://doi.org/10.1111/j.1528-1167.2010.02777.x. epub 2010 nov 10. pmid: 21219302; pmcid: pmc3058866. bonduelle t, hartlieb t, baldassari s, sim ns, kim sh, et al. frequent slc35a2 brain mosaicism in mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (moghe). acta neuropathol commun. 2021 jan 6;9(1):3. https://doi.org/10.1186/s40478-020-01085-3. pmid: 33407896; pmcid: pmc7788938. bonnefont j, vanderhaeghen p. neuronal fate acquisition and specification: time for a change. curr opin neurobiol. 2021 feb;66:195-204. https://doi.org/10.1016/j.conb.2020.12.006. epub 2021 jan 5. pmid: 33412482; pmcid: pmc8064025. brace le, vose sc, stanya k, gathungu rm, marur vr, et al. increased oxidative phosphorylation in response to acute and chronic dna damage. npj aging mech dis. 2016 oct 13;2:16022. https://doi.org/10.1038/npjamd.2016.22. pmid: 28721274; pmcid: pmc5514997. buchan jr, muhlrad d, parker r. p bodies promote stress granule assembly in saccharomyces cerevisiae. j cell biol. 2008 nov 3;183(3):441-55. https://doi.org/10.1083/jcb.200807043. pmid: 18981231; pmcid: pmc2575786. cadwell cr, bhaduri a, mostajo-radji ma, keefe mg, nowakowski tj. development and arealization of the cerebral cortex. neuron. 2019 sep 25;103(6):980-1004. https://doi.org/10.1016/j.neuron.2019.07.009. pmid: 31557462; pmcid: pmc9245854. capper d, jones dtw, sill m, hovestadt v, schrimpf d, et al. dna methylation-based classification of central nervous system tumours. nature. 2018 mar 22;555(7697):469-474. https://doi.org/10.1038/nature26000. epub 2018 mar 14. pmid: 29539639; pmcid: pmc6093218. chan ja, zhang h, roberts ps, jozwiak s, wieslawa g, lewin-kowalik j, kotulska k, kwiatkowski dj. pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: biallelic inactivation of tsc1 or tsc2 leads to mtor activation. j neuropathol exp neurol. 2004 dec;63(12):1236-42. https://doi.org/10.1093/jnen/63.12.1236. pmid: 15624760. charnley m, islam s, bindra gk, engwirda j, ratcliffe j, zhou j, mezzenga r, hulett md, han k, berryman jt, reynolds np. neurotoxic amyloidogenic peptides in the proteome of sars-cov2: potential implications for neurological symptoms in covid-19. nat commun. 2022 jun 13;13(1):3387. https://doi.org/10.1038/s41467-022-30932-1. pmid: 35697699; pmcid: pmc9189797. chen y, yang w, chen f, cui l. covid-19 and cognitive impairment: neuroinvasive and blood‒brain barrier dysfunction. j neuroinflammation. 2022 sep 7;19(1):222. https://doi.org/10.1186/s12974-022-02579-8. pmid: 36071466; pmcid: pmc9450840. chui a, zhang q, dai q, shi sh. oxidative stress regulates progenitor behavior and cortical neurogenesis. development. 2020 mar 11;147(5):dev184150. https://doi.org/10.1242/dev.184150. pmid: 32041791; pmcid: pmc7075051. cirillo l, cieren a, barbieri s, khong a, schwager f, parker r, gotta m. ubap2l forms distinct cores that act in nucleating stress granules upstream of g3bp1. curr biol. 2020 feb 24;30(4):698-707.e6. https://doi.org/10.1016/j.cub.2019.12.020. epub 2020 jan 16. pmid: 31956030. clifford db, ances bm. hiv-associated neurocognitive disorder. lancet infect dis. 2013 nov;13(11):976-86. https://doi.org/10.1016/s1473-3099(13)70269-x. pmid: 24156898; pmcid: pmc4108270. crandall lg, lee jh, friedman d, lear k, maloney k, et al. evaluation of concordance between original death certifications and an expert panel process in the determination of sudden unexplained death in childhood. jama netw open. 2020 oct 1;3(10):e2023262. https://doi.org/10.1001/jamanetworkopen.2020.23262. pmid: 33125496; pmcid: pmc7599447. crino pb. evolving neurobiology of tuberous sclerosis complex. acta neuropathol. 2013 mar;125(3):317-32. https://doi.org/10.1007/s00401-013-1085-x. epub 2013 feb 6. pmid: 23386324. de la torre-ubieta l, won h, stein jl, geschwind dh. advancing the understanding of autism disease mechanisms through genetics. nat med. 2016 apr;22(4):345-61. https://doi.org/10.1038/nm.4071. pmid: 27050589; pmcid: pmc5072455. decker cj, parker r. p-bodies and stress granules: possible roles in the control of translation and mrna degradation. cold spring harb perspect biol. 2012 sep 1;4(9):a012286. https://doi.org/10.1101/cshperspect.a012286. pmid: 22763747; pmcid: pmc3428773. delgehyr n, sillibourne j, bornens m. microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function. j cell sci. 2005 apr 15;118(pt 8):1565-75. https://doi.org/10.1242/jcs.02302. epub 2005 mar 22. pmid: 15784680. desikan rs, barkovich aj. malformations of cortical development. ann neurol. 2016 dec;80(6):797-810. https://doi.org/10.1002/ana.24793. epub 2016 nov 11. pmid: 27862206; pmcid: pmc5177533. devinsky o, hesdorffer dc, thurman dj, lhatoo s, richerson g. sudden unexpected death in epilepsy: epidemiology, mechanisms, and prevention. lancet neurol. 2016 sep;15(10):1075-88. https://doi.org/10.1016/s1474-4422(16)30158-2. epub 2016 aug 8. pmid: 27571159. d'gama am, geng y, couto ja, martin b, boyle ea, et al. mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia. ann neurol. 2015 apr;77(4):720-5. https://doi.org/10.1002/ana.24357. epub 2015 feb 26. pmid: 25599672; pmcid: pmc4471336. d'gama am, woodworth mb, hossain aa, bizzotto s, hatem ne, et al. somatic mutations activating the mtor pathway in dorsal telencephalic progenitors cause a continuum of cortical dysplasias. cell rep. 2017 dec 26;21(13):3754-3766. https://doi.org/10.1016/j.celrep.2017.11.106. pmid: 29281825; pmcid: pmc5752134. di bella dj, habibi e, stickels rr, scalia g, brown j, et al. molecular logic of cellular diversification in the mouse cerebral cortex. nature. 2021 jul;595(7868):554-559. https://doi.org/10.1038/s41586-021-03670-5. epub 2021 jun 23. erratum in: nature. 2021 aug;596(7873):e11. pmid: 34163074; pmcid: pmc9006333. douaud g, lee s, alfaro-almagro f, arthofer c, wang c, mccarthy p, lange f, andersson jlr, griffanti l, duff e, jbabdi s, taschler b, keating p, winkler am, collins r, matthews pm, allen n, miller kl, nichols te, smith sm. sars-cov-2 is associated with changes in brain structure in uk biobank. nature. 2022 apr;604(7907):697-707. https://doi.org/10.1038/s41586-022-04569-5. epub 2022 mar 7. pmid: 35255491; pmcid: pmc9046077. ebert dh, greenberg me. activity-dependent neuronal signalling and autism spectrum disorder. nature. 2013 jan 17;493(7432):327-37. https://doi.org/10.1038/nature11860. pmid: 23325215; pmcid: pmc3576027. eichmüller ol, corsini ns, vértesy á, morassut i, scholl t, et al. amplification of human interneuron progenitors promotes brain tumors and neurological defects. science. 2022 jan 28;375(6579):eabf5546. https://doi.org/10.1126/science.abf5546. epub 2022 jan 28. pmid: 35084981; pmcid: pmc7613689. english k, shepherd a, uzor ne, trinh r, kavelaars a, heijnen cj. astrocytes rescue neuronal health after cisplatin treatment through mitochondrial transfer. acta neuropathol commun. 2020 mar 20;8(1):36. https://doi.org/10.1186/s40478-020-00897-7. pmid: 32197663; pmcid: pmc7082981. espuny-camacho i, michelsen ka, gall d, linaro d, hasche a, et al. pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo. neuron. 2013 feb 6;77(3):440-56. https://doi.org/10.1016/j.neuron.2012.12.011 . pmid: 23395372. favero m, sotuyo np, lopez e, kearney ja, goldberg em. a transient developmental window of fast-spiking interneuron dysfunction in a mouse model of dravet syndrome. j neurosci. 2018 sep 5;38(36):7912-7927. https://doi.org/10.1523/jneurosci.0193-18.2018. epub 2018 aug 13. pmid: 30104343; pmcid: pmc6125809. feliciano dm, quon jl, su t, taylor mm, bordey a. postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell tsc1 deletion. hum mol genet. 2012 feb 15;21(4):799-810. https://doi.org/10.1093/hmg/ddr511. epub 2011 nov 7. pmid: 22068588; pmcid: pmc3263992. feliciano dm, su t, lopez j, platel jc, bordey a. single-cell tsc1 knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in mice. j clin invest. 2011 apr;121(4):1596-607. https://doi.org/10.1172/jci44909. epub 2011 mar 14. pmid: 21403402; pmcid: pmc3069783. fitzgerald e, hor k, drake aj. maternal influences on fetal brain development: the role of nutrition, infection and stress, and the potential for intergenerational consequences. early hum dev. 2020 nov;150:105190. https://doi.org/10.1016/j.earlhumdev.2020.105190. epub 2020 sep 10. pmid: 32948364; pmcid: pmc7481314. gandal mj, haney jr, wamsley b, yap cx, parhami s, et al. broad transcriptomic dysregulation occurs across the cerebral cortex in asd. nature. 2022 nov;611(7936):532-539. https://doi.org/10.1038/s41586-022-05377-7. epub 2022 nov 2. pmid: 36323788; pmcid: pmc9668748. gandal mj, zhang p, hadjimichael e, walker rl, chen c, et al. transcriptome-wide isoform-level dysregulation in asd, schizophrenia, and bipolar disorder. science. 2018 dec 14;362(6420):eaat8127. https://doi.org/10.1126/science.aat8127. pmid: 30545856; pmcid: pmc6443102. gao l, liu f, hou pp, manaenko a, xiao zp, wang f, xu tl, hu q. neurons release injured mitochondria as "help-me" signaling after ischemic stroke. front aging neurosci. 2022 mar 3;14:785761. https://doi.org/10.3389/fnagi.2022.785761. pmid: 35309888; pmcid: pmc8926840. gelot ab, represa a. progression of fetal brain lesions in tuberous sclerosis complex. front neurosci. 2020 aug 21;14:899. https://doi.org/10.3389/fnins.2020.00899. pmid: 32973442; pmcid: pmc7472962. gönczy p, hatzopoulos gn. centriole assembly at a glance. j cell sci. 2019 feb 20;132(4):jcs228833. https://doi.org/10.1242/jcs.228833. pmid: 30787112. greig lc, woodworth mb, galazo mj, padmanabhan h, macklis jd. molecular logic of neocortical projection neuron specification, development and diversity. nat rev neurosci. 2013 nov;14(11):755-69. https://doi.org/10.1038/nrn3586. epub 2013 oct 9. pmid: 24105342; pmcid: pmc3876965. guerrini r, dobyns wb. malformations of cortical development: clinical features and genetic causes. lancet neurol. 2014 jul;13(7):710-26. https://doi.org/10.1016/s1474-4422(14)70040-7. epub 2014 jun 2. pmid: 24932993; pmcid: pmc5548104. guillén-boixet j, kopach a, holehouse as, wittmann s, jahnel m, et al. rna-induced conformational switching and clustering of g3bp drive stress granule assembly by condensation. cell. 2020 apr 16;181(2):346-361.e17. https://doi.org/10.1016/j.cell.2020.03.049. pmid: 32302572; pmcid: pmc7181197. gumbs sbh, berdenis van berlekom a, kübler r, schipper pj, gharu l, boks mp, ormel pr, wensing amj, de witte ld, nijhuis m. characterization of hiv-1 infection in microglia-containing human cerebral organoids. viruses. 2022 apr 16;14(4):829. https://doi.org/10.3390/v14040829. pmid: 35458559; pmcid: pmc9032670. halvorsen m, gould l, wang x, grant g, moya r, et al. de novo mutations in childhood cases of sudden unexplained death that disrupt intracellular ca2+ regulation. proc natl acad sci u s a. 2021 dec 28;118(52):e2115140118. https://doi.org/10.1073/pnas.2115140118. pmid: 34930847; pmcid: pmc8719874. hansen dv, lui jh, flandin p, yoshikawa k, rubenstein jl, alvarez-buylla a, kriegstein ar. non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences. nat neurosci. 2013 nov;16(11):1576-87. https://doi.org/10.1038/nn.3541. epub 2013 oct 6. pmid: 24097039; pmcid: pmc4191718. harowitz j, crandall l, mcguone d, devinsky o. seizure-related deaths in children: the expanding spectrum. epilepsia. 2021 mar;62(3):570-582. https://doi.org/10.1111/epi.16833. epub 2021 feb 14. pmid: 33586153; pmcid: pmc7986159. hayakawa k, esposito e, wang x, terasaki y, liu y, xing c, ji x, lo eh. transfer of mitochondria from astrocytes to neurons after stroke. nature. 2016 jul 28;535(7613):551-5. https://doi.org/10.1038/nature18928. erratum in: nature. 2016 sep 14;539(7627):123. pmid: 27466127; pmcid: pmc4968589. hefti mm, kinney hc, cryan jb, haas ea, chadwick ae, crandall la, trachtenberg fl, armstrong dd, grafe m, krous hf. sudden unexpected death in early childhood: general observations in a series of 151 cases: part 1 of the investigations of the san diego sudc research project. forensic sci med pathol. 2016 mar;12(1):4-13. https://doi.org/10.1007/s12024-015-9724-2. epub 2016 jan 19. pmid: 26782961; pmcid: pmc4752958. henske ep, jóźwiak s, kingswood jc, sampson jr, thiele ea. tuberous sclerosis complex. nat rev dis primers. 2016 may 26;2:16035. https://doi.org/10.1038/nrdp.2016.35. pmid: 27226234. henske ep, scheithauer bw, short mp, wollmann r, nahmias j, hornigold n, van slegtenhorst m, welsh ct, kwiatkowski dj. allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. am j hum genet. 1996 aug;59(2):400-6. pmid: 8755927; pmcid: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc1914733. hesdorffer dc, crandall la, friedman d, devinsky o. sudden unexplained death in childhood: a comparison of cases with and without a febrile seizure history. epilepsia. 2015 aug;56(8):1294-300. https://doi.org/10.1111/epi.13066. epub 2015 jun 29. pmid: 26120007. hessami k, norooznezhad ah, monteiro s, barrozo er, abdolmaleki as, arian se, zargarzadeh n, shekerdemian ls, aagaard km, shamshirsaz aa. covid-19 pandemic and infant neurodevelopmental impairment: a systematic review and meta-analysis. jama netw open. 2022 oct 3;5(10):e2238941. https://doi.org/10.1001/jamanetworkopen.2022.38941. pmid: 36306133; pmcid: pmc9617178. hodge rd, bakken te, miller ja, smith ka, barkan er, et al. conserved cell types with divergent features in human versus mouse cortex. nature. 2019 sep;573(7772):61-68. https://doi.org/10.1038/s41586-019-1506-7. epub 2019 aug 21. pmid: 31435019; pmcid: pmc6919571. holm ia, poduri a, crandall l, haas e, grafe mr, kinney hc, krous hf. inheritance of febrile seizures in sudden unexplained death in toddlers. pediatr neurol. 2012 apr;46(4):235-9. https://doi.org/10.1016/j.pediatrneurol.2012.02.007. pmid: 22490769; pmcid: pmc4009678. jabari s, kobow k, pieper t, hartlieb t, kudernatsch m, et al. dna methylation-based classification of malformations of cortical development in the human brain. acta neuropathol. 2022 jan;143(1):93-104. https://doi.org/10.1007/s00401-021-02386-0. epub 2021 nov 19. pmid: 34797422; pmcid: pmc8732912. jain s, wheeler jr, walters rw, agrawal a, barsic a, parker r. atpase-modulated stress granules contain a diverse proteome and substructure. cell. 2016 jan 28;164(3):487-98. https://doi.org/10.1016/j.cell.2015.12.038. epub 2016 jan 14. pmid: 26777405; pmcid: pmc4733397. jansen la, mirzaa gm, ishak ge, o'roak bj, hiatt jb, et al. pi3k/akt pathway mutations cause a spectrum of brain malformations from megalencephaly to focal cortical dysplasia. brain. 2015 jun;138(pt 6):1613-28. https://doi.org/10.1093/brain/awv045. epub 2015 feb 25. pmid: 25722288; pmcid: pmc4614119. jia x, zhang s, tan s, du b, he m, et al. de novo variants in genes regulating stress granule assembly associate with neurodevelopmental disorders. sci adv. 2022 aug 19;8(33):eabo7112. https://doi.org/10.1126/sciadv.abo7112. epub 2022 aug 17. pmid: 35977029; pmcid: pmc9385150. kichula ea, huntley gw. developmental and comparative aspects of posterior medial thalamocortical innervation of the barrel cortex in mice and rats. j comp neurol. 2008 jul 20;509(3):239-58. https://doi.org/10.1002/cne.21690. pmid: 18496871; pmcid: pmc4913357. kiese k, jablonski j, hackenbracht j, wrosch jk, groemer tw, kornhuber j, blümcke i, kobow k. epigenetic control of epilepsy target genes contributes to a cellular memory of epileptogenesis in cultured rat hippocampal neurons. acta neuropathol commun. 2017 oct 31;5(1):79. https://doi.org/10.1186/s40478-017-0485-x. pmid: 29089052; pmcid: pmc5664434. kinney dk, miller am, crowley dj, huang e, gerber e. autism prevalence following prenatal exposure to hurricanes and tropical storms in louisiana. j autism dev disord. 2008 mar;38(3):481-8. https://doi.org/10.1007/s10803-007-0414-0. epub 2007 jul 6. pmid: 17619130. kinney hc, poduri ah, cryan jb, haynes rl, teot l, et al. hippocampal formation maldevelopment and sudden unexpected death across the pediatric age spectrum. j neuropathol exp neurol. 2016 oct;75(10):981-997. https://doi.org/10.1093/jnen/nlw075. epub 2016 sep 9. pmid: 27612489; pmcid: pmc6281079. kishi m, pan ya, crump jg, sanes jr. mammalian sad kinases are required for neuronal polarization. science. 2005 feb 11;307(5711):929-32. https://doi.org/10.1126/science.1107403. pmid: 15705853. kitahara t, sakaguchi h, morizane a, kikuchi t, miyamoto s, takahashi j. axonal extensions along corticospinal tracts from transplanted human cerebral organoids. stem cell reports. 2020 aug 11;15(2):467-481. https://doi.org/10.1016/j.stemcr.2020.06.016. epub 2020 jul 16. pmid: 32679062; pmcid: pmc7419717. kobow k, blümcke i. the emerging role of dna methylation in epileptogenesis. epilepsia. 2012 dec;53 suppl 9:11-20. https://doi.org/10.1111/epi.12031. pmid: 23216575. kobow k, jabari s, pieper t, kudernatsch m, polster t, et al. mosaic trisomy of chromosome 1q in human brain tissue associates with unilateral polymicrogyria, very early-onset focal epilepsy, and severe developmental delay. acta neuropathol. 2020 dec;140(6):881-891. https://doi.org/10.1007/s00401-020-02228-5. epub 2020 sep 26. pmid: 32979071; pmcid: pmc7666281. kobow k, kaspi a, harikrishnan kn, kiese k, ziemann m, et al. deep sequencing reveals increased dna methylation in chronic rat epilepsy. acta neuropathol. 2013 nov;126(5):741-56. https://doi.org/10.1007/s00401-013-1168-8. epub 2013 sep 5. pmid: 24005891; pmcid: pmc3825532. kobow k, ziemann m, kaipananickal h, khurana i, mühlebner a, et al. genomic dna methylation distinguishes subtypes of human focal cortical dysplasia. epilepsia. 2019 jun;60(6):1091-1103. https://doi.org/10.1111/epi.14934. epub 2019 may 10. pmid: 31074842; pmcid: pmc6635741. kon fc, vázquez rz, lang a, cohen mc. hippocampal abnormalities and seizures: a 16-year single center review of sudden unexpected death in childhood, sudden unexpected death in epilepsy and sids. forensic sci med pathol. 2020 sep;16(3):423-434. https://doi.org/10.1007/s12024-020-00268-7. epub 2020 jul 25. pmid: 32712908. kuzniecky ri. mri in cerebral developmental malformations and epilepsy. magn reson imaging. 1995;13(8):1137-45. https://doi.org/10.1016/0730-725x(95)02024-n. pmid: 8750328. kwan ky, sestan n, anton es. transcriptional co-regulation of neuronal migration and laminar identity in the neocortex. development. 2012 may;139(9):1535-46. https://doi.org/10.1242/dev.069963. pmid: 22492350; pmcid: pmc3317962. ledford h. severe covid could cause markers of old age in the brain. nature. 2022 dec;612(7940):389. https://doi.org/10.1038/d41586-022-04253-8. pmid: 36471141. lee ws, baldassari s, stephenson sem, lockhart pj, baulac s, leventer rj. cortical dysplasia and the mtor pathway: how the study of human brain tissue has led to insights into epileptogenesis. int j mol sci. 2022 jan 25;23(3):1344. https://doi.org/10.3390/ijms23031344. pmid: 35163267; pmcid: pmc8835853. leitner df, mcguone d, william c, faustin a, askenazi m, et al. blinded review of hippocampal neuropathology in sudden unexplained death in childhood reveals inconsistent observations and similarities to explained paediatric deaths. neuropathol appl neurobiol. 2022 feb;48(1):e12746. https://doi.org/10.1111/nan.12746. epub 2021 jul 16. pmid: 34164845; pmcid: pmc8777468. a leitner df, william c, faustin a, askenazi m, kanshin e, et al. proteomic differences in hippocampus and cortex of sudden unexplained death in childhood. acta neuropathol. 2022 may;143(5):585-599. https://doi.org/10.1007/s00401-022-02414-7. epub 2022 mar 25. pmid: 35333953; pmcid: pmc8953962.b leventer rj, phelan em, coleman lt, kean mj, jackson gd, harvey as. clinical and imaging features of cortical malformations in childhood. neurology. 1999 sep 11;53(4):715-22. https://doi.org/10.1212/wnl.53.4.715. pmid: 10489031. linaro d, vermaercke b, iwata r, ramaswamy a, libé-philippot b, et al. xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits. neuron. 2019 dec 4;104(5):972-986.e6. https://doi.org/10.1016/j.neuron.2019.10.002. epub 2019 nov 21. pmid: 31761708; pmcid: pmc6899440. liu b, qian sb. translational reprogramming in cellular stress response. wiley interdiscip rev rna. 2014 may-jun;5(3):301-15. https://doi.org/10.1002/wrna.1212. epub 2013 dec 23. pmid: 24375939; pmcid: pmc3991730. mansour aa, gonçalves jt, bloyd cw, li h, fernandes s, quang d, johnston s, parylak sl, jin x, gage fh. an in vivo model of functional and vascularized human brain organoids. nat biotechnol. 2018 jun;36(5):432-441. https://doi.org/10.1038/nbt.4127. epub 2018 apr 16. pmid: 29658944; pmcid: pmc6331203. markmiller s, soltanieh s, server kl, mak r, jin w, et al. context-dependent and disease-specific diversity in protein interactions within stress granules. cell. 2018 jan 25;172(3):590-604.e13. https://doi.org/10.1016/j.cell.2017.12.032. pmid: 29373831; pmcid: pmc5969999. martin kc, ephrussi a. mrna localization: gene expression in the spatial dimension. cell. 2009 feb 20;136(4):719-30. https://doi.org/10.1016/j.cell.2009.01.044. pmid: 19239891; pmcid: pmc2819924. mcguone d, leitner d, william c, faustin a, leelatian n, et al. neuropathologic changes in sudden unexplained death in childhood. j neuropathol exp neurol. 2020 mar 1;79(3):336-346. https://doi.org/10.1093/jnen/nlz136. pmid: 31995186; pmcid: pmc7036658. meisler mh, kearney ja. sodium channel mutations in epilepsy and other neurological disorders. j clin invest. 2005 aug;115(8):2010-7. https://doi.org/10.1172/jci25466. pmid: 16075041; pmcid: pmc1180547. miyata t, kawaguchi d, kawaguchi a, gotoh y. mechanisms that regulate the number of neurons during mouse neocortical development. curr opin neurobiol. 2010 feb;20(1):22-8. https://doi.org/10.1016/j.conb.2010.01.001. pmid: 20138502. mizuguchi m. abnormal giant cells in the cerebral lesions of tuberous sclerosis complex. congenit anom (kyoto). 2007 mar;47(1):2-8. https://doi.org/10.1111/j.1741-4520.2006.00134.x. pmid: 17300684. murphy jv, craig l. leigh's disease: significance of the biochemical changes in brain. j neurol neurosurg psychiatry. 1975 nov;38(11):1100-3. https://doi.org/10.1136/jnnp.38.11.1100. pmid: 1206418; pmcid: pmc492163. nakanishi k, niida h, tabata h, ito t, hori y, et al. isozyme-specific role of sad-a in neuronal migration during development of cerebral cortex. cereb cortex. 2019 aug 14;29(9):3738-3751. https://doi.org/10.1093/cercor/bhy253. pmid: 30307479; pmcid: pmc7335017. narula n, tester dj, paulmichl a, maleszewski jj, ackerman mj. post-mortem whole exome sequencing with gene-specific analysis for autopsy-negative sudden unexplained death in the young: a case series. pediatr cardiol. 2015 apr;36(4):768-78. https://doi.org/10.1007/s00246-014-1082-4. epub 2014 dec 13. pmid: 25500949; pmcid: pmc4907366. o'neill ac, uzbas f, antognolli g, merino f, draganova k, et al. spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity. science. 2022 jun 17;376(6599):eabf9088. https://doi.org/10.1126/science.abf9088. epub 2022 jun 17. pmid: 35709258. ormel pr, vieira de sá r, van bodegraven ej, karst h, harschnitz o, et al. microglia innately develop within cerebral organoids. nat commun. 2018 oct 9;9(1):4167. https://doi.org/10.1038/s41467-018-06684-2. pmid: 30301888; pmcid: pmc6177485. panagiotakos g, pasca sp. a matter of space and time: emerging roles of disease-associated proteins in neural development. neuron. 2022 jan 19;110(2):195-208. https://doi.org/10.1016/j.neuron.2021.10.035. epub 2021 nov 29. pmid: 34847355; pmcid: pmc8776599. paredes mf, james d, gil-perotin s, kim h, cotter ja, et al. extensive migration of young neurons into the infant human frontal lobe. science. 2016 oct 7;354(6308):aaf7073. https://doi.org/10.1126/science.aaf7073. pmid: 27846470; pmcid: pmc5436574. parikshak nn, swarup v, belgard tg, irimia m, ramaswami g, et al. genome-wide changes in lncrna, splicing, and regional gene expression patterns in autism. nature. 2016 dec 15;540(7633):423-427. https://doi.org/10.1038/nature20612. epub 2016 dec 5. erratum in: nature. 2018 aug;560(7718):e30. pmid: 27919067; pmcid: pmc7102905. piel m, meyer p, khodjakov a, rieder cl, bornens m. the respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. j cell biol. 2000 apr 17;149(2):317-30. https://doi.org/10.1083/jcb.149.2.317. pmid: 10769025; pmcid: pmc2175166. qin w, chan ja, vinters hv, mathern gw, franz dn, taillon be, bouffard p, kwiatkowski dj. analysis of tsc cortical tubers by deep sequencing of tsc1, tsc2 and kras demonstrates that small second-hit mutations in these genes are rare events. brain pathol. 2010 nov;20(6):1096-105. https://doi.org/10.1111/j.1750-3639.2010.00416.x. epub 2010 jul 13. pmid: 20633017; pmcid: pmc2951479. raj b, blencowe bj. alternative splicing in the mammalian nervous system: recent insights into mechanisms and functional roles. neuron. 2015 jul 1;87(1):14-27. https://doi.org/10.1016/j.neuron.2015.05.004. pmid: 26139367. raju cs, spatazza j, stanco a, larimer p, sorrells sf, et al. secretagogin is expressed by developing neocortical gabaergic neurons in humans but not mice and increases neurite arbor size and complexity. cereb cortex. 2018 jun 1;28(6):1946-1958. https://doi.org/10.1093/cercor/bhx101. pmid: 28449024; pmcid: pmc6019052. ramaswami g, won h, gandal mj, haney j, wang jc, wong ccy, sun w, prabhakar s, mill j, geschwind dh. integrative genomics identifies a convergent molecular subtype that links epigenomic with transcriptomic differences in autism. nat commun. 2020 sep 25;11(1):4873. https://doi.org/10.1038/s41467-020-18526-1. pmid: 32978376; pmcid: pmc7519165. real r, peter m, trabalza a, khan s, smith ma, et al. in vivo modeling of human neuron dynamics and down syndrome. science. 2018 nov 16;362(6416):eaau1810. https://doi.org/10.1126/science.aau1810. epub 2018 oct 11. pmid: 30309905; pmcid: pmc6570619. revah o, gore f, kelley kw, andersen j, sakai n, et al. maturation and circuit integration of transplanted human cortical organoids. nature. 2022 oct;610(7931):319-326. https://doi.org/10.1038/s41586-022-05277-w. epub 2022 oct 12. pmid: 36224417; pmcid: pmc9556304. roy a, millen kj, kapur rp. hippocampal granule cell dispersion: a non-specific finding in pediatric patients with no history of seizures. acta neuropathol commun. 2020 apr 21;8(1):54. https://doi.org/10.1186/s40478-020-00928-3. pmid: 32317027; pmcid: pmc7171777. rubenstein jl, rakic p. genetic control of cortical development. cereb cortex. 1999 sep;9(6):521-3. https://doi.org/10.1093/cercor/9.6.521. pmid: 10498269. ruppe v, dilsiz p, reiss cs, carlson c, devinsky o, zagzag d, weiner hl, talos dm. developmental brain abnormalities in tuberous sclerosis complex: a comparative tissue analysis of cortical tubers and perituberal cortex. epilepsia. 2014 apr;55(4):539-50. https://doi.org/10.1111/epi.12545. epub 2014 feb 11. pmid: 24512506. sahm f, schrimpf d, stichel d, jones dtw, hielscher t, et al. dna methylation-based classification and grading system for meningioma: a multicentre, retrospective analysis. lancet oncol. 2017 may;18(5):682-694. https://doi.org/10.1016/s1470-2045(17)30155-9. epub 2017 mar 15. pmid: 28314689. schurr j, coras r, rössler k, pieper t, kudernatsch m, et al. mild malformation of cortical development with oligodendroglial hyperplasia in frontal lobe epilepsy: a new clinico-pathological entity. brain pathol. 2017 jan;27(1):26-35. https://doi.org/10.1111/bpa.12347. epub 2016 feb 22. pmid: 26748554; pmcid: pmc8029051. smith rs, kenny cj, ganesh v, jang a, borges-monroy r, et al. sodium channel scn3a (nav1.3) regulation of human cerebral cortical folding and oral motor development. neuron. 2018 sep 5;99(5):905-913.e7. https://doi.org/10.1016/j.neuron.2018.07.052. epub 2018 aug 23. pmid: 30146301; pmcid: pmc6226006. son g, han j. roles of mitochondria in neuronal development. bmb rep. 2018 nov;51(11):549-556. https://doi.org/10.5483/bmbrep.2018.51.11.226. pmid: 30269744; pmcid: pmc6283025. sun w, poschmann j, cruz-herrera del rosario r, parikshak nn, hajan hs, et al. histone acetylome-wide association study of autism spectrum disorder. cell. 2016 nov 17;167(5):1385-1397.e11. https://doi.org/10.1016/j.cell.2016.10.031. pmid: 27863250. takahara t, maeda t. transient sequestration of torc1 into stress granules during heat stress. mol cell. 2012 jul 27;47(2):242-52. https://doi.org/10.1016/j.molcel.2012.05.019. epub 2012 jun 21. pmid: 22727621. taylor dc, falconer ma, bruton cj, corsellis ja. focal dysplasia of the cerebral cortex in epilepsy. j neurol neurosurg psychiatry. 1971 aug;34(4):369-87. https://doi.org/10.1136/jnnp.34.4.369. pmid: 5096551; pmcid: pmc493805. thiele ea. managing and understanding epilepsy in tuberous sclerosis complex. epilepsia. 2010 feb;51 suppl 1:90-1. https://doi.org/10.1111/j.1528-1167.2009.02458.x. pmid: 20331728. vineethakumari c, lüders j. microtubule anchoring: attaching dynamic polymers to cellular structures. front cell dev biol. 2022 mar 3;10:867870. https://doi.org/10.3389/fcell.2022.867870. pmid: 35309944; pmcid: pmc8927778. voineagu i, wang x, johnston p, lowe jk, tian y, horvath s, mill j, cantor rm, blencowe bj, geschwind dh. transcriptomic analysis of autistic brain reveals convergent molecular pathology. nature. 2011 may 25;474(7351):380-4. https://doi.org/10.1038/nature10110. pmid: 21614001; pmcid: pmc3607626. walker rl, ramaswami g, hartl c, mancuso n, gandal mj, de la torre-ubieta l, pasaniuc b, stein jl, geschwind dh. genetic control of expression and splicing in developing human brain informs disease mechanisms. cell. 2019 oct 17;179(3):750-771.e22. https://doi.org/10.1016/j.cell.2019.09.021. erratum in: cell. 2020 apr 16;181(2):484. erratum in: cell. 2020 apr 30;181(3):745. pmid: 31626773; pmcid: pmc8963725. wamsley b, jaglin xh, favuzzi e, quattrocolo g, nigro mj, yusuf n, khodadadi-jamayran a, rudy b, fishell g. rbfox1 mediates cell-type-specific splicing in cortical interneurons. neuron. 2018 nov 21;100(4):846-859.e7. https://doi.org/10.1016/j.neuron.2018.09.026. epub 2018 oct 11. pmid: 30318414; pmcid: pmc6541232. wang r, zhang h, du j, xu j. heat resilience in embryonic zebrafish revealed using an in vivo stress granule reporter. j cell sci. 2019 oct 18;132(20):jcs234807. https://doi.org/10.1242/jcs.234807. pmid: 31558681; pmcid: pmc6826007. wedderburn cj, weldon e, bertran-cobo c, rehman am, stein dj, gibb dm, yeung s, prendergast aj, donald ka. early neurodevelopment of hiv-exposed uninfected children in the era of antiretroviral therapy: a systematic review and meta-analysis. lancet child adolesc health. 2022 jun;6(6):393-408. https://doi.org/10.1016/s2352-4642(22)00071-2. epub 2022 apr 26. pmid: 35483380; pmcid: pmc9090907. wefers ak, stichel d, schrimpf d, coras r, pages m, et al. isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of mybl1 or myb and a benign disease course. acta neuropathol. 2020 jan;139(1):193-209. https://doi.org/10.1007/s00401-019-02078-w. epub 2019 sep 28. pmid: 31563982; pmcid: pmc7477753. wilsch-bräuninger m, huttner wb. primary cilia and centrosomes in neocortex development. front neurosci. 2021 oct 21;15:755867. https://doi.org/10.3389/fnins.2021.755867. pmid: 34744618; pmcid: pmc8566538. wu ye, parikshak nn, belgard tg, geschwind dh. genome-wide, integrative analysis implicates microrna dysregulation in autism spectrum disorder. nat neurosci. 2016 nov;19(11):1463-1476. https://doi.org/10.1038/nn.4373. epub 2016 aug 29. pmid: 27571009; pmcid: pmc5841760. xiong m, tao y, gao q, feng b, yan w, et al. human stem cell-derived neurons repair circuits and restore neural function. cell stem cell. 2021 jan 7;28(1):112-126.e6. https://doi.org/10.1016/j.stem.2020.08.014. epub 2020 sep 22. pmid: 32966778; pmcid: pmc7796915. yang p, mathieu c, kolaitis rm, zhang p, messing j, et al. g3bp1 is a tunable switch that triggers phase separation to assemble stress granules. cell. 2020 apr 16;181(2):325-345.e28. https://doi.org/10.1016/j.cell.2020.03.046. pmid: 32302571; pmcid: pmc7448383. youn jy, dunham wh, hong sj, knight jdr, bashkurov m, et al. high-density proximity mapping reveals the subcellular organization of mrna-associated granules and bodies. mol cell. 2018 feb 1;69(3):517-532.e11. https://doi.org/10.1016/j.molcel.2017.12.020. epub 2018 jan 25. pmid: 29395067. yu fh, mantegazza m, westenbroek re, robbins ca, kalume f, burton ka, spain wj, mcknight gs, scheuer t, catterall wa. reduced sodium current in gabaergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. nat neurosci. 2006 sep;9(9):1142-9. https://doi.org/10.1038/nn1754. epub 2006 aug 20. erratum in: nat neurosci. 2007 jan;10(1):134. pmid: 16921370. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology of the alzheimer’s continuum: an update feel free to add comments by clicking these icons on the sidebar free neuropathology 1:32 (2020) review neuropathology of the alzheimer’s continuum: an update kurt a. jellinger institute of clinical neurobiology, vienna, austria address for correspondence: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13 · a-1150 vienna · austria kurt.jellinger@univie.ac.at submitted: 06 october 2020 accepted: 07 november 2020 copyedited by: nicole schwab published: 11 november 2020 https://doi.org/10.17879/freeneuropathology-2020-3050 keywords: alzheimer’s disease, β-amyloid, tau pathology, oligomers, amyloid angiopathy, alzheimer subtypes, regional vulnerabiliy, co-pathologies abstract alzheimer’s disease (ad), the most common form of dementia worldwide, is a mixed proteinopathy (amyloid and tau). originally defined as a clinicopathological entity, it is a heterogenous, multifactorial disorder, currently referred to as the alzheimer’s continuum. its cardinal pathological features are extracellular β-amyloid (amyloid plaques) and intraneuronal tau aggregates forming neurofibrillary tangles, which are accompanied by vascular amyloid deposits (cerebral amyloid angiopathy), synapse and neuronal loss, as well as neuroinflammation and reactive astrogliosis. in addition to “typical” ad, various subtypes with characteristic regional patterns of tau pathology have been described that show distinct clinical features, biomarker levels, and patterns of key network destructions responsible for cognitive decline. ad is frequently associated with other age-related changes including lewy and tdp-43 pathologies, hippocampal sclerosis, argyrophilic grain disease, cerebrovascular lesions, and others. these additional pathologies influence the clinical picture of ad, may accelerate disease progression, and can cause a number of challenges in our understanding of the disease including the threshold of each individual pathology to cause dementia and the possibility of underlying common etiologies. this article provides an up-to-date overview of ad neuropathology, its heterogeneity, and additional pathologies in order to explain the difficulties in the diagnosis and the failure of clinical trials in ad patients. abbreviations ad – alzheimer’s disease, adnc alzheimer’s disease neuropathological changes, ap amyloid plaque, app amyloid precursor protein, aβ β-amyloid peptide, aβo aβ oligomer, caa cerebral amyloid angiopathy, cbs corticobasal syndrome, cerad consortium to establish a registry for alzheimer disease, csf cerebrospinal fluid, cvd cerebrovascular disease, dlb dementia with lewy bodies, eoad early-onset ad, ftld frontotemporal lobar degeneration, ftld-tdp frontotemporal lobar degeneration with tdp-43, gvd granulovacuolar degeneration, hcsp-ad hippocampal sparing ad, hp-tau hyperphosphorylated tau protein, late limbic-predominant age-related tdp-43 encephalopathy, late-nc limbic-predominant age-related tdp-43 encephalopathy neuropathological change, lc locus ceruleus, load late-onset ad, lp-ad limbic-predominant ad, lppa logopenic primary progressive aphasia, ma-ad minimal-atrophy ad, mci mild cognitive impairment, mtl medial temporal lobe, nft neurofibrillary tangle, nia-aa national institute of aging/alzheimer's association, np neuritic plaque, nt neuropil thread, part primary age-related taupathy, pca posterior cortical atrophy, phf paired helical filament, sf straight filament, tdp-43 43-kda tar dna binding protein 43. 1. introduction alzheimer’s disease (ad) is the most common form of dementia, currently affecting around 50 million people worldwide. it accounts for 60-70% of dementia cases in clinical and autopsy series, but it is often associated with other confounding pathologies in the elderly. its incidence increases from 2/1.000 at age 65-74 years to 37/1.000 at age 85+ [1], and doubles every five years after age 65, with peaks in the tenth decade and slight decrease afterwards [2, 3]. the point prevalence of ad among individuals aged 60+ is 40.2/1,000 persons, the pooled annual period prevalence is 30.4/1,000, and the incidence rate is 15.8/1,000 person-years [4]. with the disproportional increase of the elderly population, the prevalence of ad will approach around 132 million worldwide and up to 16 million cases in the usa by 2050 [5, 6], ad has become a tremendous public health and socio-economic challenge of the 21st century [5]. as available treatments only target symptoms and neither slow nor reverse the progression of the disease, the development of disease-modifying therapeutic procedures is urgent [7]. ad was originally defined as a clinicopathological entity, characterized by progressive memory deficit, involvement of multiple cognitive domains, and a defining pathological substrate with deposition of amyloid-β peptide (aβ) in extracellular plaques and cerebral vasculature (cerebral amyloid angiopathy/caa), neuritic plaques defined by the presence of microtubule-associated hyperphosphorylated tau protein (hp-tau), intraneuronal aggregations of hp-tau manifesting as neurofibrillary tangles (nfts) in the cell soma, and neuropil threads (nts), which occur mainly in dendritic compartments and, to a lesser degree, in the axonal domain. these changes are accompanied by early synaptic loss [8], activated microglia [9], mitochondrial dysfunction causing energy loss [10], neuroinflammation [11], neurovascular dysfunction [12], disruption of the blood-brain barrier [13], neuronal loss and reactive astrogliosis [14]. ad, a mixed proteinopathy (amyloid, tau, tdp-43, and others), is a heterogenous disorder currently referred to as the alzheimer’s continuum [15] with several pathobiological subtypes and various co-pathologies [16]. the final definite diagnosis of ad rests with post-mortem neuropathology despite the advent of more sensitive neuroimaging and the use of reliable biomarkers [17]. even though the classical morphological features of ad have been known for many years, the recently used more sensitive immunohistochemistry techniques for aβ and hp-tau have replaced silver-staining techniques and have not only forwarded the diagnosis of ad but allowed a more scientific evaluation of the disease's pathology. for the neuropathological diagnosis of ad, the updated national institute on aging/alzheimer's association (nia/aa) 'abc' criteria are used [17]. the morphological changes involving brain regions and neuronal cell types following a stereotypical pattern [18] result from selective cellular and regional vulnerability to pathogenic factors and their progression through functionally integrated regions of the brain [19-23] as well as functional networks that result in progression of ad [24, 25]. however, ad is a heterogenous continuum with a variety of clinically and morphologically defined subtypes, currently referred to as alzheimer’s clinical syndrome [15], which presents major challenges for both diagnosis of ad, monitoring and targeting of disease progression [26]. the new definition of ad as a biologically defined spectrum, using the nia/aa framework [15], enables recognition and diagnosis of the various subtypes of ad [16]. research consensus guidelines have been proposed for the intra vitam biologically-based categorization termed 'atn', which uses combinations of in vivo biomarkers for aβ deposition (a), tau pathology (t), and neurodegeneration (n). they use cerebrospinal fluid (csf) or plasma biomarkers, pet, and functional and structural mri. the biomarker profiles and categories of the alzheimer’s spectrum referring to ad neuropathological changes (adnc) have been summarized recently [27]. 2. pathology of alzheimer’s disease 2.1. macroscopic features the ad brain often has decreased weight and at least moderate cortical atrophy most marked in the medial temporal lobes (mtls) with relative sparing the primary motor, somatosensory and visual cortices and enlargement of the lateral ventricles (ex vacuo hydrocephalus). brain atrophy often involves posterior cortical areas, most notable in precuneus and posterior cingulate gyrus in the preclinical stage of ad [28]. however, none of the macroscopic features are specific to ad, and healthy elderly people often show moderate cortical atrophy especially affecting the frontal lobes, with volume loss of the white matter [29]. medial temporal atrophy affecting amygdala and hippocampus with enlarged temporal horn is typical of ad (fig. 1). however, this is also seen in other age-related disorders such as hippocampal sclerosis [30]. figure 1. comparison between formalin-fixed brain slices of the left hemispheres (level of posterior hippocampus) of an aged nondemented individual (a) and an ad patient (b). note the marked atrophy (thinning of the gyri and deepening of the sulci) in b, in particular hippocampal atrophy (arrow in b) with widening of the inferior horn of the second ventricle (asterisk in b). photographs by courtesy of simon fraser and arthur oakley. 2.2. microscopic features the definite diagnosis of ad requires microscopic examination of multiple brain regions with semiquantitative assessment of the density of lesions and their topographical distribution. extracellular amyloid plaques (aps) and intracellular nfts that are essential for the neuropathological diagnosis, are associated with tau-positive nts, dystrophic neurites and neuritic plaques (nps), caa, reactive astrocytes and activated microglia, and neuroinflammation are present. these lesions result in loss of synapses and neurons in vulnerable regions leading to brain atrophy and the characteristic clinical picture of the disease. hirano bodies, granulovacuolar degeneration (gvd), tdp-43 deposits, and other lesions may also be present [31, 32]. 2.3. amyloid deposits aps are formed by the abnormal extracellular nonvascular accumulation and deposition of aβ peptides of varying length including those with 40 or 42 amino acids (aβ-40 and aβ-42), resulting from the sequential cleavage of the amyloid precursor protein (app) by the enzymes βand γ-secretases [33]. app, from which aβ is cleaved by endoproteolytic processing, is a large single transmembrane protein, encoded by the app gene on chromosome 21 [34, 35]. proteolytic cleavage of app develops mainly via two exclusive pathways, the amyloidogenic and the non-amyloidogenic pathway, but other alternative pathways (η-secretases, δ-secretase, etc.) have been described for the physiological processing of app [36]. the initial cut at the β-site of app is due to the β-secretase activity enzyme bace1, a transmembrane enzyme with aspartyl protease activity. clearance by β-secretase yields a slightly shorter soluble fragment (sappβ) and a correspondingly longer c-terminal fragment (ctfβ) or c99 [37]. app undergoes constitutive shedding by a protease activity called α-secretase, which appears to be a metalloprotease of the adam family. tace (adam17) is one of the α-secretase, but adam10 is more important for α-secretase activity and sappα production. adam10 is the physiologically relevant constitutive of α-secretase in primary neurons [38], as has been demonstrated in vitro and in vivo [39]. cleavage of app by α-secretase releases the soluble ectodomain of app, called sappα, and a membrane-tethered intracellular c-terminal fragment, termed ctfα of c83. the amyloidogenic (or β) cleavage of app is in direct competition with an alternative non-amyloidogenic pathway (cleavage by the α-secretase within the aβ sequence) which precludes the formation of amyloidogenic peptides and leads to soluble sappα, and has neuroprotective properties preventing aβ production [40]. however, aberrant sappα production may tilt the cells toward unregulated growth, but the underlying mechanisms are still unknown [41]. lastly, γ-secretase, a high molecular weight complex that consists of presenilin (ps1, ps2), an aspartyl membrane protease, aph-1, nicastrin and presenilin enhancer (pen2), cleaves app terminal fragments (ctfs) such as c83 and c99, releasing 3 or 4 amino acid peptides from the transmembrane fragment of app. notably, γ-secretase is active on app only following the antecedent αor β-secretase. the products of γ-secretase cleavage of c83 are a 3-kda peptide, termed p3 and an app intracellular domain (aicd), while γ-secretase cleavage of c99 yields the infamous aβ peptide and an identical aicd fragment. besides cleavage by α-, β-, and γ-secretase, other n-terminal fragments (ntfs) of app have been identified that are generated by unknown proteases [42, 43]. mounting evidence suggests that astrocytes that have increased levels of app, β-secretase (bace1), and γ-secretase play an additional role in ad by secreting significant amounts of aβ and contributing to overall aβ burden in the brain [44]. bace1 inhibition more effectively suppresses the initial process of plaque formation, rather than the subsequent phase of plaque growth, which has implications for therapeutic efficiency for the treatment of ad [45]. ad is driven by intraneuronally retained aβ produced by the ad-specific βapp-independent pathway [46]. neuronal aβ-42 is enriched in small vesicles at the presynaptic side of synapses [47]. aβ deposits contain a mixture of various isoforms. the most common are aβ-40 (under physiologic conditions around 90%), aβ-38 and aβ-42 (less than 10%). aβ-40 is produced within the trans-golgi network (tgn) whilst aβ-42 can be made in either the tgn or the endoplasmic reticulum [48]. the specific production of aβ-42 in the endoplasmic reticulum of neurons links this compartment with the generation of aβ and explains why primarily endoplasmic reticulum localized proteins such as presenilin could induce ad [49]. increased production of aβ-42 at the expense of aβ-40 is a common feature in both familial and sporadic ad [50]. the latter is believed to be more toxic than aβ-40 because of its tendency to aggregate and to form fibrils [51]. the phosphorylation of app by extracellular-regulated kinase (erk) and protein kinase c (pkc), in the proteolytic processing of app has been demonstrated to be critically modulating the generation of aβ [52]. the c-terminal app fragments (app intracellular domain) are generated by γ-secretase cleavage [53]. γ-secretase was shown to cleave near the cytoplasmic membrane boundary of app, called ε-site cleavage, as well as in the middle of the membrane domain, called γ-site cleavage, indicating that γand ε-site cleavage are regulated independently [54]. ubiquilin-1 has been shown to modulate γ-secretase-mediated ε-site cleavage and thus may play a role in regulating γ-secretase cleavage of app and other proteins [55]. further cleavage of app intracellular domain (aicd) fragments by caspase or caspase-like proteases results in additional fragments which, however, does not seem to require antecedent proteolysis of app [41]. figure 2. amyloid and neuritic plaques. a; a1. multiple diffuse amyloid plaques in the neocortex (antibody 4g8). b, b1. neuritic plaques that contain aβ and tau in distended processes (i.e. dystrophic neurites). gallyas silver stain visualizes both aggregated aβ and tau and is therefore ideal to detect neuritic plaques (ring in b, neuritic plaque; arrow in b1, dystrophic neurite; arrowhead in b1, neurofibrillary tangle). scale bars: 200 μm. from [71]. truncated aβ fragments are deposited in aps due to axonal linkage and release of app [56]. chemical imaging of evolving ap pathology in a transgenic mouse model for ad suggested initial plaque formation to be seeded by aβ-42, followed by plaque maturation upon deposition of aβ-40 as well as deposition of others [57]. due to its higher rate of fibrillization and insolubility, aβ-42 is its major component in addition to other aβ peptides [58]. a recent report demonstrated the role of hif-1alpha/lncrna bace1-as axis in the transactivator of transcription (tat)-mediated induction of astrocytic amyloidosis [59]. advanced biophysical examination of aβ derived from ad brain tissue showed polymorphic structures [60]. the terminology of aβ plaques is confusing, since a myriad of non-vascular aβ deposits have been described, but five major types can be distinguished: (a) primitive or immature plaques are spherical deposits of predominantly aβ-42 in the neuropil without a dense core and neurites; (b) diffuse plaques, usually large (50µm to several hundred µm), slightly immunoreactive and ill-limited, contain loose amyloid bundles in the neuropil without degenerating neurites and accompanying microglia (fig. 2a); (c) stellate deposits probably related to astrocytes [61]; (d) focal deposits with dense and spherical accumulations of aβ-42, surrounded by a neuritic corona containing dystrophic tau-positive neurites and astrocytic components, constituting the “cored”, “classical” or “neuritic” plaques (nps) (fig. 2b, 2b1); and finally (e) compact or burnt-out plaques with a dense core of aβ-40, absent or tau-negative, ubiquitin-positive neurites. nps have compact dense amyloid cores composed of more fibrillated forms of aβ (fig. 3). they contain tau-positive dystrophic neurites and are accompanied by synaptic loss, activated microglia and reactive astrocytes [62, 63]. there are differences in the composition of the aggregates, for example, the aβ in nps has a more varied composition with the presence of aβ 40, 42, 43, n-terminus truncated aβ and other post-transitionally modified forms [64, 65]. tau-positive nps begin early in ad, but major tau deposition follows the aβ deposition and the clustering of activated microglia [66]. recent studies unequivocally demonstrated that plaque-associated myeloid cells are derived exclusively from resident microglia [67]. in ad, microglia can eliminate aps through phagocytosis with apoe lipoprotein at an early stage of disease progression [68]. scanning transmission electron microscopy (stem) showed three types of fibrillary network structures: amorphous network, fibril bundles, and amyloid stars [69]. although diffuse non-neuritic plaques are generally present before nps, whether an individual diffuse plaque can actually transfer into an np or whether these two types develop differently, is not clear at present. a recently described type called the coarse-grained plaque, a relatively large deposit (diameter about 80 µm) characterized by multiple cores and aβ-devoid pores, is prominent in the neocortex and associated with homozygous apoeε4 status and caa. this divergent ap type is similar to caa, predominantly composed of aβ-40, and has been observed particularly in early-onset ad (eoad) [70]. figure 3. em image of amyloid core of a neuritic plaque. radiating bands of amyloid fibrils comprise the core (x). note the adjacent abnormal fibrils filled with dense bodies (arrows) and surrounding damaged myelin sheaths (x 4000). “burnt out” plaques are composed of dense cores lacking neuritic components, while the astrocytic processes penetrating the plaque core may represent a regressive stage (“remnant plaques”) [72]. “cotton wool plaques” are non-compact deposits, made of aβ-42 with sparse glial components and variable neurites but not surrounded by a neuritic corona. they can be detected with h&e staining [61]. aβ and tau each begin to aggregate in separate neuroanatomical locations and meet in the cerebral cortex in the np. this “collision” of both proteins mediated by microglia has devastating consequences in terms of neuronal loss, promoting neurodegeneration and the consequent development of cognitive decline, but this is still under investigation [73]. 2.4. distribution of amyloid deposits aps in ad brain show a typical distribution with brain areas that are connected via the “default network” typically affected early. in animal models some demonstration of “propagation” along neuronal systems has been observed [74, 75], suggesting some axonal transport of seeds that lead to extracellular deposits. most aβ deposits are located in the gray matter, while some diffuse or lake-like deposits may be seen in the subpial white matter. cortical soluble aβ protein is a neurotoxic agent [76, 77], and aβ oligomers (aβos) may trigger the early phase of the aβ seeding process, while depletion of aβos delays the aggregation process leading to a transient reduction of seed-induced aβ deposits [78]. the topography of aβ deposits depends on the stage of the disease, which leaded to several staging schemes. three stages were distinguished: stage a with amyloid deposits in the basal portions of the frontal, temporal and occipital cortex; in stage b all isocortex is involved, with primary cortices spared and the hippocampus only mildly affected; while stage c shows deposits in the whole isocortex including sensory and motor core fields [18]. others proposed five amyloid “phases” using sensitive silver staining or aβ antibodies: stage 1 or isocortical, stage 2 with additional involvement of hippocampus and entorhinal cortex, stage 3 plus striatum and diencephalic nuclei, stage 4 several brainstem nuclei and medulla oblongata, and stage 5 presenting amyloid deposits in the pons and molecular layer of the cerebellum [79, 80]. these can be reduced to three stages: 1 isocortical, 2 allocortical or limbic, and 3 subcortical. usually involved is the total isocortex, layers ii-v more than layers i and vi [18]. in advanced cases band-like diffuse aβ deposits are also seen in the subpial surface of the cortex or in the white matter close to layer vi [71]. amyloid pet-based staging of aβ pathology in vivo confirmed its progression in ad [81], and revealed higher plaque counts in entorhinal and occipital regions of typical ad, while other phenotypes showed more severe aβ deposition in frontal and parietal cortices [82]. post-mortem analysis of (18)fflutemetamol and (11)cpib pet signal showed that it is influenced by both diffuse plaques and cored plaques and, therefore, is likely a function of plaque size and density of aβ fibrils in plaques. brain regions with large volumes of diffuse plaques could yield pet retention levels comparable with lower volume/frequency of cored plaques [83]. 2.5. cerebral amyloid angiopathy aβ peptides also involve the vessel walls, as with caa, with the more soluble aβ-40 as the major constituent. 85-90% of confirmed ad cases have some degree of caa [84]. it mainly accumulates in the interstitium between the smooth cells of the tunica media. small arteries, arterioles and even capillaries in the cerebral cortex and leptomeningeal vessels are affected [85]. stage 1: vessels are affected in the isocortex, stage 2: involvement of allocortex, and stage 3: basal ganglia, thalamus, pons and medulla oblongata [86]. others distinguished four patterns [87]: type 1: aps with or without caa in the leptomeninges alone; type 2: caa in both leptomeningeal and deeper penetrating arteries (fig 1a); type 3: caa affects both precapillaries and arterioles; type 4 shows aβ deposition in and around blood vessels. genetically, type 3 (capillary subtype) is more strongly associated with the apoeε4 allele [87, 88]. two other types were distinguished: type 1 affecting capillaries, arterioles and small arteries is associated with apoeε4, whereas type 2 not involving capillaries is more likely associated with apoeε2, its most frequent form [89]. both severe caa and ad are associated with apoeε4-positive patients [88]. a more recent staging system is based on the severity of caa in a single vessel: grade 0: absence of staining, grade 1: a congophilic ring around the otherwise normal-appearing vessel, grade 2: complete replacement of the tunica media by congophilic material, grade 3: involving >50% of vessel circumference, giving a “double-barrel” appearance, and grade 4 or fibrinoid necrosis of the vessel wall with additional amyloid deposits in the surrounding neuropil (“dyshoric changes”) [88]. the parietal and occipital cortices are more vulnerable than the frontal and temporal lobe, and the leptomeningeal vessels more than the parenchymal ones [84]. aβ deposition shrinks the cerebral blood vessels by about 8% and reduces the energy supply resulting from decrease of blood flow [90]. caa can cause small infarcts in the cerebral cortex, while severe caa may lead to lobar hemorrhages in the frontal and occipital lobes and to diffuse white matter lesions (fig. 4) [91]. brain hemorrhage does not appear to be directly linked to amyloid burden in patients with caa-related intracerebral hemorrhage, because amyloid burden was similarly distributed across the brain hemispheres and no interhemispheric difference was observed for aβ burden nor for mri markers of small vessel disease [92]. caa and deep perforating arteriopathy are similar and interact with blood-brain barrier breakdown, endothelial damage, and impaired perivascular aβ drainage. both may cause ischemic lesions and intracerebral hemorrhages [93]. chronic treatment of a mouse model of ad with fungicides produced aβ fibril formation and impairment of aβ clearance through neprylisin, suggesting that fungicide residues could be a risk factor for ad via caa [94]. although several pathogenic mechanisms, including the disbalance between production and clearance of aβ creating a self-reinforcing cycle of increased vascular aβ and further caa and ad progression, have been shown, they do not explain completely the disease pathogenesis [95]. the intersection between caa and ad points to a crucial role for improving vascular function in the treatment of ad [96]. figure 4. multiple large hemorrhages in both frontal lobes (a) and occipital lobe (b). diffuse white matter destruction (c). caa in many vessels in the cerebral white matter; scale bar 70 µm (d). from [97]. 2.6. tau pathology tau protein is encoded by the mapt (microtubule-associated protein tau) gene on chromosome 17 [98], which generates a total of 6 isoforms through alternative splicing of exons 2, 3 and 10 in the cns [99]. tau protein, the main constituent of nfts, is involved in the stabilization of neurotubules that leads to the appropriate function of the neuron. its microtubule-binding regions are made of 3 or 4 repeats (3r or 4r tau), their second repeat (exon 10) being spliced in some isoforms. combined phosphorylation of ser202, thr205, and ser208 forms a unique post-translational modification configuration that promotes tau aggregation, accelerating the formation of tau filaments and eventually resulting in nft formation. tau adopts different stable conformations, consistent with the notion of 'strains' as may be seen with the concept of phenotypic diversity or with different environmental stimuli [100, 101]. truncation of tau by caspases-3 or -4 is an early event in the development of nfts [102]. the molecular mechanisms leading to the accumulation of tau are characterized by numerous translational modifications that change its conformation and structural state. recent studies indicate that the dysregulation and dislocation of splicing factor proline and glutamine rich (sfpq), the subsequent dna anomalies and aberrant dynamics of tia-1-positive stress granules in association with pathological tau may represent a critical pathway which contributes to the rapid progression of ad [103]. abberant phosphorylation and truncation make tau protein into a pathological entity; paired helical filaments (phf), the major structural constituents of nfts, exhibit a greater degree of phosphorylation than normal tau [104].tau monomers can aggregate to form oligomers and higher-order fibrils. whilst aβ can largely self assemble, tau phosphorylation is believed to be important for its aggregation [105]. phosphorylation of ser208 likely occurs at different disease stages from phosphorylation of ser202 and thr205. hp-tau accumulation causes synaptic impairment, neuronal dysfunction, and formation of nfts. tau with site-specific posttranslational modification/soluble hp-tau species impact mitochondria and facilitate neurodegeneration [106]. recent studies support the hypothesis that tau phosphorylation at ser208 strongly contributes to unique types of tau aggregates, and may be a reliable marker for the presence of mature nfts [107]. in ad, tau protein usually accumulates in the somato-dendritic and, to a lesser degree, in the axonal domains of the neuron. nfts and pretangles are due to accumulation in the soma; nts occur in dendrites, and the neuritic corona of core plaques is constituated by axonal processes filled by tau proteins (fig. 2). as major constituents of nfts and nts, they are hyperphosphorylated and aberrantly misfolded, have lost their microtubule stabilizing functions, and contribute to axonal transport deficits [105]. phfs in ad contain all 6 isoforms of tau protein including those with 3 and 4 repeats (3rand 4r-tau) in the microtubule binding domain, forming the core of phf [108]. the tau isoforms show a chronological shift: initially, early pretangles are positive only for 4r, gradually 3r is involved in mature tangles, and finally 4r is replaced by 3r in ghost tangles [109]. ultrastructurally, nfts appear as phfs, i.e., fibrils of ca. 28 nm in diameter that form pairs with a helical tridimensional conformation and a regular periodicity of 65-80 nm [110] or as helical or twisted ribbons [111]. straight filaments (sfs) show a longer crossover distance and modulations in width from 10 to 15 nm. both lesions are different from those seen in other tauopathies [112]. phfs and sfs differ in their inter-protofilament packing, and are ultrastructurally polymorph [113]. visible with cryo-em, phfs and sfs are made of two c-shaped protofilaments with a combined cross-β-β-helix structure, without variations in the filamentous structures between sporadic and inherited ad [114]. nts have an ultrastructure and immunohistochemistry similar to nfts. why, despite its axonal origin, phf tau accumulates primarily in the neuronal cell body and dendrites, is unknown. it shows in three stages: (a) pre-nfts composed of diffuse, or punctuate tau staining occur within the cytoplasm of otherwise normal-looking neurons with well-preserved neurites; or (b) mature intraneuronal nfts consist of cytoplasmic filamentous aggregates of tau displacing the nucleus toward the periphery of the soma and extending to the proximal segment of the axon. they appear as “flame-shaped tangles” in pyramidal neurons of the hippocampus (fig. 5) and layer v of association cortices and as “globose tangles” in subcortical nuclei; (c) extraneuronal “ghost” nfts in dead neurons, showing loss of their nucleus and of stainable cytoplasm [115]. total loss of functional microglia in advanced late-onset ad (load) promotes widespread intraneuronal neurofibrillary degeneration leading to brain failure [116]. neuronal tau pathology has been linked to neuronal death and cognitive decline in ad [117], while others suggested that neuronal cell loss is associated with dementia and not the presence of plaques and tangles [118]. it is generally thought that nfts impede neuronal functioning, but recent data indicate that they can be found in functionally intact neurons integrated in cortical circuits [119-121]. how hp-tau specifically mediates its toxic effects is still unknown, but oligomeric tau species, analogues to aβos, are potential toxic species besides nft tau. recent proteomic studies have identified specific proteins that interact with hp-tau, showing novel potential pathogenic mechanisms that are relevant in ad and providing insight into how hp-tau mediates its toxicity in ad [122]. figure 5. in ad, high amounts of neurofibrillary tangles and neuropil threads are seen in the hippocampus (a). ca1, ca2, and ca4 hippocampal cornu ammonis (ammon’s horn) sectors 1, 2, and 3, respectively; gr, granule cell layer of the dentate gyrus. immunohistochemistry with antibody at8. scale bar: 50 μm. from [71]. 2.7. topography and spreading pattern of tau the extent of tau pathology (nfts and nts) follows a predictable spatiotemporal progression through functionally integrated brain regions [18], which had been interpreted as a cell-to-cell spreading through prion-like propagation [123-127] or a transneuronal spread through functional networks, associated with a trigger, possibly aβ and/or neuronal network activity that could lead to progression of nft pathology [128]. since tau is expressed predominantly in neurons rather than glial cells, the detection of tau aggregates in astrocytes and oligodendroglia has given support to the concept that the release of misfolded tau from neurons (or oligodendroglia) may result in uptake into other cells [129]. microglia could potentially play a role in spreading of tau pathology [130]. transcellular progression of tau seeds has been observed in early braak stage in regions predicted to be free of hp-tau [131]. according to the original staging [18], the first nfts consistently occur in the transentorhinal (perirhinal) region (stage i) along with the entorhinal cortex, followed by the ca1 region of the hippocampua (stage ii), indicating a preclinical phase of ad which can last up to 20 years. limbic structures, such as the subiculum of the hippocampal formation are affected next (stage iii), followed by the amygdala, thalamus, and claustrum (stage iv). stages iii and iv are often correlated clinically with mild cognitive impairment (mci). in stage v, nfts spread to isocortical areas with the association areas being affected prior and more severely, followed in stage vi by the primary sensory, motor and visual areas, which is usually associated with overt dementia (fig. 6). this nft staging has been widely accepted in routine pathology and appears well correlated with the clinical status, at least in the amnestic ad. imaging in vivo tau pathology with tau-specific pet tracers identified nft pathology reflecting braak stages iv or higher. it rendered it possible to study the temporal progression of tau pathology in vivo, and, therefore, can be used as a reliable biomarker of tau pathology [132-137]. there is an inverse correlation between the accumulation of nfts and cognitive status; the spread and level of tau accumulation reflects the severity of dementia with time [61, 138-140]. the seeding activity is suggested to begin in the transentorhinal/entorhinal regions and anticipates hp-tau pathology in ad, whereas the locus ceruleus (lc) showed seeding only in later nft stages [141]. however, immunohistochemistry has detected pre-tangle material in multiple subcortical regions, especially in locus ceruleus (lc) neurons [142-144]. involvement of the subcortical nuclei, not considered in the original braak scheme, however, occurs in early stages of the disease and has important clinical consequences. the cholinergic nucleus basalis of meynert and axons of the adrenergic lc projecting neurons are affected already in braak stages 0/i, associated with severe neuronal loss, while moderate to severe deposition of tau in the lc was only seen in braak stages above iv [142]. the intralaminar nuclei of the thalamus, the pontine parabrachial region, the medullary reticular formation, the dorsal raphe nucleus, the oculomotor system, and the autonomous nuclei are also affected early and increase with disease progression [142, 145-147]. nigral pathology including hp-tau (nfts) accumulation and α-synuclein aggregates is common in elderly patients with and without ad, and may be related with extrapyramidal symptoms [148-150]. figure 6. spreading pattern of neuritic ad pathology. modified from [18]. 2.8. aβ and tau pathology chicken or egg? the causes of sporadic ad are far from being understood, while the hallmarks that distinguish ad from other neurodegenerative diseases – namely aβ plaques and nfts have been known for many years. the physiological and pathological roles of tau and aβ, and their implications for ad pathology and therapeutics have been reviewed recently [151]. many studies have linked aβ and tau and raised the possibility that protein-protein interactions are the key for both spreading and toxicity of these two abnormal proteins [152]. several models of interaction have been suggested: (1) the seeding of toxic tau is enhanced by the presence of aβ; (2) the toxicity of aβ depends of the presence of tau; (3) aβ and tau enhance each other's toxicity. modern network-based models revealed ways in which aβ and tau protein might interact with each other to enhance the propagation of ad, thus shedding light on the importance of protein clearance and protein interaction mechanisms in the development of ad pathology [153, 154] [155]. soluble oligomeric aβ is hypothesized to be a possible cause of the hyperphosphorylation of tau and the development of nfts. the presence of aps accelerates both the formation of hp-tau aggregates [156] and its interneuronal transfer [157]. the aβo hypothesis was introduced in 1998, suggesting that the brain damage leading to ad was initiated by soluble ligand-like aβos [158]. the extension of tau pathology is different from the spread of aβ deposition that is related to diffusion of soluble aβ in the extracellular space [159, 160]. quantification of adnc in formalin-fixed post-mortem human brain tissue detected high amounts of aβ in the frontal cortex and striatum, and of hp-tau in the frontal cortex and hippocampus of cases with high adnc pathology load [161]. the most recent version of the amyloid cascade hypothesis assumes ad arises from synaptic toxicity mediated by soluble aβos, leading to synaptic dysfunction and loss. age-related aggregation of aβ and its apparent downstream effects on microglia, astrocytes, and neurons, including the post-translational modification of the tau protein, seems necessary for ad symptom expression [162]. while an optimal concentration of aβ is thought to likely maintain synapses, alterations in the proteolytic processing of app may cause dyshomeostasis of aβ, increasing the levels of aβ-42, and initiating ad by setting off a chain of events that leads to the accumulation of tau and downstream neuronal cell death [163]. soluble aβos are now suggested to cause neuronal damage [76]. they are believed to insert into membranes, while others support ligand-like accumulation at particular synapses, providing a substantial molecular basis for the cause of ad [164]. recent data support the hypothesis that aβ enhances tau pathology through increased spreading of tau induced by phf in vivo [165-168], and that aβos promote tau seeding potentiating intracellular tau aggregation [169, 170]. intraneuronal aβ accumulation is suggested to precede tau pathology in the entorhinal cortex [171] and to interact with hippocampal and cortical tau pathology, while in the absence of aβ tau deposition may be insufficient for the neurodegeneration process that leads to ad [172]. many data supporting a toxic role for aβos have backed the aβo hypothesis for ad pathogenesis, but further advances in aβo structure-function studies are needed [158]. recent studies point to a role for exosomes in the spreading of toxic aβos and the associated disease progression in the ad brain [173]. however, the traditional consensus of the amyloid paradigm as a singular cause of ad has been under revision, with the accumulation of new pathobiological evidence [174]. new theories suggest that various mechanisms, including prion-like spread of aβ and tau, vasoconstrictions, growth hormone secretagogue receptor 1α (ghsr1α), and neuroinflammation, come together at a crossroad that ultimately leads to ad [11], while others suggested that extracellular aβ and tau act in parallel and upstream of app [175, 176]. however, recent findings have shown that the soluble form of app binds directly to gababr1a and modulates synaptic transmission [177], while that of aβ aggregates do not need app overexpression [178] but are performed by extracellular exosomes [173]. aβos are deposited inside synaptic terminals [179], enriched in small vesicles at the presynaptic side [47], and enhance synaptic dysfunction in ad [180]. according to others, aβ and hp-tau may develop concomitantly within synaptic terminals [181, 182] and cause abnormalities at synapses [183]. on the other hand, preclinical evidence indicates that tau pathology can progress independently of aβ accumulation and arises downstream of genetic risk factors for ad by an aberrant metabolic pathway [184]. the argument that insoluble aβ and tau deposits begin forming concomitantly in the cerebral cortex of ad brains would be consistent with the argument in favor of the pathogenic importance of tau deposition. recent quantitative studies did not find regional association between aβ-42 and insoluble tau, but a higher regional association between total aβ-42 and soluble tau phosphorylation. this provides evidence supporting the local interplay between aβ and soluble hp-tau in ad brains [185], and accumulating evidence suggests that both pathologies have synergistic effects. the complex aβ-tau interaction is important for elucidating disease pathogenesis and the design of next-generation ad therapeutical trials [153]. targeting the common epitope could be a more effective treatment strategy than targeting only aβ or tau alone [186]. mounting data suggest that the prion-like spreading of diffusible oligomers and other protein aggregates from cell to cell within the brain, probably through specific neuronal networks, may contribute to ad progression [128, 187]. app overexpression is not a prerequisite for the prion-like induction of cerebral aβ deposition that may contribute to disease progression in ad [178], and the multiple failures of previous anti-aβ drugs may suggest that in the ad brain, the accumulation of aβ could be secondary to an unknown 'initial disrupting event' [188]. processing and clearance of aβ and tau could be related to a bidirectional relationship between adnc and autophagy [189]. seeded templating and neurotoxicity are two of the most critical properties attributed to oligomers that have been documented for misfolded proteins in neurodegeneration [190]. it has been speculated that cellular prion protein (prpc) is a critical player in the interplay between aβ and tau propagation in a large group of ad cases. pre-existing hp-tau pathology interacting with prpc appears to be a prerequisite for aβ function as a hp-tau pthology acceleration via prpc [165]. toxic tau oligomers (tauos) and toxic oligomeric aβ assemblies (aos) have prionoid characteristics and are responsible for cell-to-cell spreading in the brain. both extraand intracellular aβos and tauos (not nfts and aps) may represent novel targets of ad research and therapeutic trials [191]. preventing soluble aβo formation and targeting their n-terminal residues with antibodies could be an attractive combined therapeutic approach [178]. recent studies found striking patient-to-patient heterogeneity in the hyperphosphorylated species of soluble oligomeric seed-competent tau. its seeding capacity correlates with the aggressiveness of the clinical disease, and some post-translational modification sites appeared to be associated with both seeding activity and worse clinical outcomes, suggesting that different individuals with “typical” ad have distinct biochemical features of tau that correlated with differences in the aggressiveness of clinical course [192], supporting an important causal role of tau as a driver of clinical dysfunction in ad [193]. the synergism between aβ deposition, nft neurodegeneration, and caa may be a better predictor of cognitive decline or disease progression than either pathology alone [194] (fig. 7). figure 7. staging of aβ, nft, and caa in non-demented (pre-ad) and demented ad patients. from [195]. 2.9. synaptic and neuronal loss essential neuropathological features of ad are loss of synapses and selected neuronal cells (20-40% in neocortex and 25-65% in hippocampus) as the main pathological substrate of cortical atrophy. its regional and laminar pattern parallels the distribution of nfts and has been suggested to be a better correlate of cognitive deficits than the aβ burden [139, 196]. little is known about the molecular basis of selective neuronal vulnerability in ad and the molecular pathways that lead to neurodegeneration, a key characteristic of the disease. it is the result of multiple molecular changes of interacting genes and pathways within vulnerable neurons [25]. the relationship between cellular senescence in the context of aging and ad have been reviewed recently [197]. age-related intraneuronal aggregation of aβ is colocalized with mitochondria and endosomes and less so with lysosomes and autophagosomes. understanding age-related changes in intraneuronal aβ may lead to application of countermeasures to prolong dementia-free health span [198]. the intraneuronal accumulation of aβ may involve synaptic dysfunction and the formation of aps in ad; intraneuronal aβ-42 has been reported to disrupt the normal cytoarchitecture of neurites. recent studies indicate that in ad, vulnerable-neuron-specific dysregulation of polypyrimidine tract binding protein (ptb) (ncbi gene id 5725), a regulator of alternative splicing [199], is the protein most highly correlated to tau in the principal neurons of the entorhinal cortex layer ii (ec ii). ptb could precipitate a 3r/4r tau imbalance in these neurons and explain the premature accumulation of nfts, thus explaining the vulnerability of ec ii neurons [25]. the neurotoxic effect of astrocyte-derived exosomes (ade) is evident with the overlap of ap density and c3/4 fragments (complement factors) observed in early ad [200]. although tangle-bearing neurons can be long lasting in regions where nfts occur at a presymptomatic stage, neuronal loss occurs early in the course of the symptomatic disease [201]. two mechanisms of neuronal death in ad have been discussed: one affecting tangle-bearing neurons that will lead to ghost extracellular tangles, another affecting tangle-free neurons, at least in part by apoptosis [202-204]. inflammation-induced hyperphosphorylation of tau destabilizes the microtubule-actin network and impairs axonal transport and disturbs energy metabolism in the axon, inducing further tau phosphorylation. accumulating data point to the fact that this facilitates the formation of phfs, further impairs axonal transport leading to complete blockage and axonal leakage, and induces loss of synaptic contacts promoting activation of microglia and reactive astrogliosis [56]. microglia have been shown to instigate tau pathology in diverse ways, inducing tau aggregation by proinflammatory cytokine release [205, 206], and spreading hp-tau oligomers or nfts through exosome secretion [130]. synaptic loss that is possibly driven by aβ and tau pathology has been suggested to precede neuronal loss [207]. synapses are present in aps and their total number decreases with time [61, 208]. their loss has been demonstrated ultrastructurally and immunohistochemically [209]. in late stages of ad synapse loss ranges from 10 to 60%, most severely in the frontal and mesiotemporal regions. synapse loss by activated astrocytes producing different secretomes reduce protein synthesis for synapse formation, resulting in synaptic loss found in ad [210]. there is a close relationship between aβ accumulation and synaptic loss that may provide direction for the development of potential disease-modifying treatments of ad [211]. eoad is associated with a higher burden of adnc and a higher rate of neocortical atrophy and synapse loss than the much more common and apparently sporadic load [212]. however, synaptic loss is not a unique hallmark of ad and occurs in many other brain diseases [213]. 2.10. neuroinflammation activated microglia operating as phagocytes are frequently observed around aβ plaques driving an inflammatory response, which can be activated by multiple factors in the local environment [214], in particular by the presence of aβ in the cortex, indicating a “toxic” response which corrupts neurons as collateral damage (“bystander effect”) [215]. tau-positive nps being early in ad, however, major tau deposition follows the accumulation of aβ and clustering of activated microglia. an increase in membrane attack complex formation leads to increased tau pathology and neoronal loss [216]. on the other hand, microglia may contribute to elimination of tau deposits by phagocytosis [217, 218]. different states of microglia activation, corresponding to regional activation of aβ and tau, are present simultaneously in the same brain. the clustering of activated microglia is greatest in the primary motor cortex, a region relatively spared compared to the severely affected inferior temporal cortex in ad. this suggests that microglial activation is not prominent in the early phase of ad pathophysiology [66]. recent studies in hp-tau mice demonstrated that microglia are not the agitators of tau aggregation, but different results about the involvement of microglia in tau aggregation and clearance were presented [219, 220]. thus, the functional role of microglial activation with hp-tau oligomers still remains elusive. gene-profiling technologies applied to isolated microglia have challenged the hypothesis that there is one acute-type (microglial drivers) of inflammation in the human brain causing accelerated proinflammatory damage in ad. these studies have shown that many of the microglia genes expressed in increased levels reflect a response to restore homeostasis and limit inflammatory damage [221]. on the other hand, there is an early microglia reaction to ad pathology, but a loss of healthy microglia is the prominent feature in severely affected regions of the ad brain [222]. in addition, there is a non-disease-specific response of microglia to neuronal damage, with upregulation of phagocytotic activity to remove damaged neurons and synapses by cd68 immunoreactivity of lysosomes [73]. their numbers increase on promotion to neuronal damage associated with nfts [62], which is due to enhanced production of inflammatory cytokines, such as il-21 and increase in t follicular helper cells. the strong immune response is insufficient at clearing up aβ and instead exacerbates inflammation [223]. reactive astrocytes that may react to cytokines and other agents produced by pro-inflammatory microglia, are observed around aps, though less frequently compared to microglia. reactive astroglia burden occurs later in ad and correlates mainly with tau pathology [31]. 2.11. pathology of preclinical ad amyloid and neuritic plaques and nfts occurring in non-demented elderly individuals, represent asymptomatic or preclinical ad (pre-ad), while clinical ad affects subjects with late stages of adnc. both ad and pre-ad cases often exhibit caa, which is also observed in non-ad cases, i.e., those without adnc. patients with mci do not always have adnc even though they have a risk of developing dementia in 10-12% and sometimes do not have any discernable pathology [224, 225]. the presence of nfts and caa in cases without aps, classified as non-ad, suggests that they may precede ap pathology or may present a pre-amyloid plaque stage not yet included in the current criteria for the neuropathological diagnosis of ad [226]. increased soluble/dispersible aβ in pre-ad compared to fully developed suggests that, in addition to more severe and widespread adnc, soluble aβ aggregates play a role in the conversion of pre-ad to clinical ad [31]. cognitively impaired individuals presenting with an early onset ad phenotype showed higher rates of tau pet accumulation, while among cognitively unimpaired individuals higher rates of tau accumulation were associated with faster rates of memory decline [227]. 2.12. neuropathological diagnosis of alzheimer’s disease histopathological examination of the brain has to establish that adnc are present in sufficient densities and extensions to distinguish ad from other age-related disorders [61]. because the disease affects the whole brain, it is not sufficient to make the diagnosis of ad just on one or two brain blocks; instead, multiple brain areas have to be examined and a staging protocol has to be established. the current algorithms for the pathological diagnosis of ad are based on semiquantiative assessment of aps and nfts providing reasonable interrater agreement when using standardized criteria [228]. current guidelines include (a) cut-off quantitative values for aps and tangles [17, 229]; (b) the semiquantitative assessment and age-adjustment of nps in the consortium to establish a registry for alzheimer's disease (cerad) protocol [230]; (c) topographic staging of neuritic/tau pathology [18], re-evaluated by immunohistochemistry [231]; and (d) the progress and distribution of aβ phases [79]. in order to develop a system that combines all the above pathological features, the nia/aa established a composite score comprising the extent of involvement/spread of cerebral aβ based on the progression model by the thal phases: (a), that of nfts based on the progression model of braak, (b), and the cerad score, which describes the density of neuritic amyloid plaques based on certain key locations in the neocortex, (c) (table 1). from this combination, it gave a likelihood for the degree of ad neuropathological changes in an individual case. sufficient agreement in ad diagnosis could be reached only when the lesions are considerable (braak nft stage v and vi) with 91% agreement, while for mild lesions it was poorer (for braak stage i and ii, agreement was only around 50%) [228, 232, 233]. combined braak and cerad scores in the nia-ri (national institute on aging and reagan institute) criteria that apply only to demented persons, relate dementia to adnc with high, intermediate and low likelihood [234]. they have been widely used in anglo-american neuropathology and are now replaced by the nia/aa guidelines. although the sensitivity and specificity of the nia-ri criteria has been proposed to be around 90%, only 30 to 57% of the brains of patients with the clinical diagnosis of probable ad showed “pure” adnc, thus reducing their predictive value to 38% [235]. an evaluation of the nia-ri criteria identified between 54 and 97% of ad cases with high braak or cerad stages, and eliminated between 60 and 100% of non-ad with low braak or cerad stages [232, 233]. another autopsy study reported diagnostic sensitivity ranging from 70.9 to 87.3% and specificity from 44.3 to 70.8% [236]. the recent updated nia/aa guidelines for the neuropathological assessment of ad consider adnc levels regardless of the clinical history of a given individual [17]. they include (1) the recognition that adnc may occur in the apparent absence of cognitive impairment; (2) the use of an “abc” score for adnc that incorporates histological assessment of aβ plaques (a), based on its phase assessment [79], staging of nfts, (b) based on the braak staging system [231], and scoring of nps, based on their semiquantitative assessment in at least three neocortical regions, and (c), based on cerad criteria [230]. table 2 shows how each of the three scores are transformed to state the level of adnc on a four tiered scale (non, low, intermediate, and high). the entire process of the neuropathological diagnosis of ad can be followed along the pathways shown in fig. 8. (3) more detailed approaches for assessing co-morbid conditions, such as lewy pathology, vascular brain injury, tdp-43 immunoreactive lesions, argyrophilic grain disease, and others that can complicate the pathological diagnosis and can sometimes co-exist with ad, are also considered [17]. testing of the revised nia/aa guidelines in 390 autopsy cases distinguished pure ad and non-ad dementia from non-demented cases with a sensitivity of 91% and a specificity of 99%. the sensitivity increased after exclusion of non-ad dementia cases, indicating that the revised nia/aa criteria appear practicable for distinguishing pure ad from non-ad dementia, preclinical ad, and controls [226]. the revised nia/aa guidelines for the severity score for adnc used in the ad centers program achieved an excellent agreement (κ=0.88, 95% ci 0.77-0.95), and good-to-excellent agreement for the three supporting scores [237]. figure 8. pathway of the combination of different pathological features that allows a classification of adnc according to the nia-aa guidelines. a comparative study of clinical and neuropathological diagnoses of ad in three epidemiological samples reported a sensitivity for probable ad of 93% [238]. meta-analysis of 20 (out of 1,189) studies to distinguish autopsy-verified ad from other dementias or healthy controls showed a sensitivity of 85.4% (95% ci 80.8-90%) and a specificity of 77.7% (95% ci 70.2-85.1%). values were higher for neuroimaging procedures and slightly lower for csf biomarkers, while the combination of both resulted in better results [239]. 3. pathobiological subtypes of ad recent studies showed that the neuropathology of ad is heterogenous [240-242]. the current guidelines for the neuropathological diagnosis of ad only consider the classical “plaque and tangle” phenotype but not other subtypes such as the “plaque only but without tangle formation/predominant” type with abundant amyloid, or the “little or no tau pathology” type limited to the hippocampus and abnormal hp-tau in neocortical pyramidal cells. this type, observed in 3.4-8.0% of demented subjects over age 85 years [243], frequently represents a specific type of dementia with lewy bodies (dlb)/dlb-ad [244]. the recently described “primary age-related taupathy” (part) [245], previously referred to as “nft-predominant dementia” [246], involves people over 85 years old and is associated with mild to moderate cognitive impairment [247, 248] it reveals tau pathology restricted to the mtl (braak stages 0-iv), relative absence of amyloid (thal aβ phases 0-2), total absence of nps, and rare caa [249]. the composition of nfts in part both for 3r and 4r tau isoforms is identical with those in classical ad [246], while pattern of hippocampal tau pathology differs significantly between part and ad [250, 251]. tau aggregates influence cognition and hippocampal atrophy in the absence of aβ [249]. positive correlations were reported in part between the braak nft stage and phosphorylated 43-kda tar dna-binding protein (ptdp-43) stage and density [252]. part is considered either a prodromal form or a subtype of ad [253, 254] (see table 3). mapt h1h1 genotype frequency is high in both part and limbic-predominant ad (lp-ad), and similar to typical ad, while apoeε4 is rather rare in part [255]. other genetic differences between part and ad have been described [256]. it seems that lower concentrations of aβos cause less severe tau deposition due to the fact that they can potentiate tau aggregation by promoting tau seed uptake [170]. cognitive decline in part is usually milder than in ad and correlates with tau burden. biomarkers and neuroimaging studies will be important to define part ante-mortem and to follow its natural history [257]. while the incidence of classical ad increases from the 7th to the 9th decade and later shows a mild decrease, the frequency of part increases after the age of 85 years [3]. the limbic-predominant age-related tdp-43 encephalopathy (late), a recently described disease entity mainly involving elderly people (>75 years at death), is associated with an amnestic dementia syndrome that may mimic ad [258, 259]. it shows pathogenic mechanisms of both frontotemporal lobar degeneration with tdp-43 (ftld-tdp) and ad, but there are different molecular patterns of tdp-43 pathology in various clinical phenotypes with a higher chance of ftd-like symptoms in ad + full-length tdp-43 cases [32]. recent studies indicated that in most cases, limbic-predominant age-related tdp-43 encephalopathy (late-nc) and ftld-tdp can be differentiated by applying single neuropathological criteria, e.g., the severity of cortical tdp-43 inclusions [260]. biomarkers for ante-mortem diagnosis of this syndrome are currently not available [261]. recent clinicopathological studies have enabled the identification of several pathophysiologically defined subtypes of ad. one distinguished three ad subtypes based on nft density: typical ad with balanced nft counts in the neocortex and hippocampus (75%), hippocampal sparing (hcsp), with nft counts predominantly in association cortices (11%), and limbic-predominant (lp) ad mainly involving the hippocampus (14%) [262]. these subtypes had different clinical phenotypes, with different ages at onset and rates of progression (fig. 9). patients with hippocampal sparing ad (hcsp-ad) were youngest at onset, had a higher proportion of men, and progressed more quickly than typical ad. lp-ad patients were older, more often female, and showed slower progression. age at death of the lp form was highest, while patients with hcsp-ad were youngest, indicating this type as the most aggressive. this could be related to the contribution of tdp-43 pathology, hippocampal sclerosis, and the microtubule-associated protein tau (mapt) h1h1 genotype to lp-ad, factors related to temporal lobe atrophy, older age, and slower disease progression. apoeε4 carriers more frequently had lp-ad and typical ad, whereas non-carriers more frequently presented as hcsp-ad. vascular co-pathology (ranging from 16 to 36%) was highest in the lp and lowest in the hcsp cases. typical ad had higher ap burden in occipital regions compared with lp-ad [262], while in contrast to specific tau accumulation and brain atrophy patterns among ad variants, aβ accumulation appeared rather diffuse and similarly across groups, except the ma group [263, 264]. tau pathology was closely associated with sites of neurodegeneration and brain atrophy corresponded well with nft topography and neuronal loss. [265-267]. clinical symptoms correlate with neuronal hypometabolism [262, 268, 269]. similar results were reported in a study of 933 autopsy cases of ad, all with neuritic braak stage > iv [270]. typical ad was more frequent than in the mayo series (82.5 vs 75%), while the other two subtypes were slightly less frequent. minimal-atrophy ad (ma-ad) was not included in this study. the lp-ad cases shared some morphological features with part [245], although later studies demonstrated significant pathological differences between part and lp-ad [240]. figure 9. main factors and characteristics of the four major subtypes of ad. ad: alzheimer’s disease; nft: neurofibrillary tangle; wmh: white matter hyperintensity; caa: cerebral amyloid angiopathy; eoad: early-onset alzheimer’s disease; load: late-onset alzheimer’s disease; lp-ad: limbic-predominant ad. typical ad showed greater white matter hyperintensity (wmh) burden, which may be due to wallerian degeneration induced by cortical tau pathology [271, 272], small vessel disease, or both [273-275]. tau pathology and neurodegeneration can disrupt key brain networks, which may induce memory impairment comparable to lp-ad and typical ad in the absence of overt brain atrophy of the mtl in ma-ad that shows [276-278]. distinct patterns of nft deposition in young-onset versus older-onset ad give evidence for variability in regional deposition patterns and demonstrate that different disease phenotypes have different patterns of tau pathology [279]. other atypical non-amnestic syndromes, referred to as focal ad [26, 280, 281], include logopenic primary progressive aphasia (lppa), showing higher nft density in superior temporal gyrus but thal amyloid plaques similar to amnestic ad [282-284]. the proportion of apoeε4 carriers was elevated in amnestic but not in non-amnestic forms of ad, suggesting that apoe is a selective risk factor that increases the vulnerability of memory-related medial temporal areas rather than language-related neocortices [285]. further atypical forms are posterior cortical atrophy (pca) [286], non-amnestic ad with tdp-43 pathology [287], syndromes resembling behavioral variant fronto-temporal lobe degeneration with tau pathology (bvftd-tau) [266, 288], and the corticobasal syndrome (cbs) subtype of ad that shows a higher nft density in the perirolandic cortices and greater neuronal loss in substantia nigra which may contribute to parkinsonism that uncommon in classic ad [289]. behavioral/dysexecutive ad revealed temporo-parietal-predominant atrophy. in the mayo series, pca, lppa and bvftd variants were more common in hcsp-ad than in lp and typical ad [262, 290]. accumulation of nfts and activated hypertrophic microglia associated with low neuron densities suggests that they may collectively contribute to focal neurodegeneration characteristic of primary progressive aphasia ad [291]. the pathogenic factors underlying ad subtypes are unclear and cannot be explained by aβ pathology alone, because the distribution of aβ pet retention is quite similar in all subtypes [263]. however, solid-state nuclear magnetic resonance measurements showed qualitative differences between aβ-40 and aβ-42 aggregates in the brain tissue of patients with two atypical ad clinical subtypes posterior cortical atrophy variant and a typical prolonged-duration form indicating that there are structural variations in aβ fibrils from ad clinical subtypes [60]. ma-ad, although aβ-positive, shows less tau pathology. according to recent studies, ad “subtypes” may be linked to different tau protein modifications, suggesting that ad patients may have multiple molecular drivers of an otherwise common phenotype [192]. this suggests that multiple subtypes are parts of the same ad continuum [266], which may have consequences for personalized therapeutic approaches. 4. the impact of co-pathologies ad pathology rarely occurs in isolation, while complex pathologies frequently lead to cognitive decline. the number of co-morbidities increases in the aging brain, causing mixed pathologies [3, 248, 274, 292-298]. the challenges of pathological mimics and concomitant pathologies in the neuropathologic diagnosis of ad have been critically reviewed recently [174]. the most frequent co-pathologies are cerebrovascular disease (cvd) and lewy and tdp-43 proteinopathies [31, 258, 299, 300]. in a consecutive autopsy series of 2,060 elderly demented patients and those with the clinical diagnosis of ad, adnc were present in 82.9% of all demented and in 92.8% of clinically diagnosed ad cases, but only 33.6% and 47.6%, respectively, showed pure adnc (abc 3/3/3). the others were either atypical ad forms or subtypes (including part) (7 and 6%, respectively) or exhibited additional cvd (24.3%), lewy (12.5%) or other mixed pathologies. vascular dementia in this cohort accounted for only 12.2% and 3.3%, respectively; other non-ad pathologies were present in 7.2% and 3.7% [301]. another study of demented elderly persons reported pure adnc in only 31% and multiple pathologies in 63% [302]. a review of 12 studies with 3,574 patients, irrespective of the clinical symptoms, reported adnc between 19% and 67%, lewy pathologies in 6% to 39%, vascular pathologies in 28% and 70%, tdp-43 proteinopathy in 19% to 78% [290], hippocampal sclerosis between 3% and 13%, and mixed pathologies between 8% and 70% [295]. among 447 patients with probable ad, only 3.13% showed pure adnc, 27.3% ad+cvd + other, 3% ad + cvd, 7.6% ad + other degenerative lesions, and 47% ad+cvd + other neurodegenerative lesions [293]. this list of combinations is not complete and there are other combinations with rare entities that need specific attention [174]. among 673 autopsy cases, including 320 demented, the majority showed mixed pathologies [274]. late-nc was present in 57% of ad cases and was associated with more rapid disease progression [259]. increased tdp-43 pathology in typical ad and lp-ad compared to hcsp-ad [262] was due to a strong association between hippocampal sclerosis and tdp-43, but clinical presentation seemed to be driven by morphological subtypes and not by tdp-43 pathology [290]. among 61 autopsy-proven ad cases, late-nc was present in 67.2% (ad, late-nc), however, it was not associated with an increase of the burden of early or late tau nor aβ pathology. late-nc showed a lower final mini mental state examination (mmse) score independent of tau pathology [303]. among 172 autopsy-confirmed ad cases, 19% were classified as non-amnestic, 69% of which had typical adnc, 31% were hcsp-ad, 36% tdp-43-positive, while there were no lp-ad cases [287]. in a recent study of 46 autopsy-confirmed ad cases, 63% exhibited late-nc (ad+) and a higher burden of hp-tau. this indicates a possible progression of the disease, whereas ad plus late-nc was not associated with differences in cognitive scores [304]. late-nc may also occur in isolation and has been viewed as a common brain disease in aging [258]. among 574 individuals with complete measurements of mmse and the clinical dementia rating scale sum of boxes (cdr-sb) from 39 ad centers across the usa, 63% of those given the 'gold standard' diagnosis of ad, possessed either tdp-43 proteinopathy or caa of sufficient severity to independently explain the majority of their cognitive impairment. aβ and/or tau burden, particularly in braak stages iv to vi, and small cerebral vessel disease may synergistically affect cognitive decline [305], and a significant interaction was found between braak nft stages, caa status and cognitive decline, suggesting that there is a significant interaction between tau pathology and caa on cognition within the ad clinical spectrum [306]. hence, interventions targeting caa may contribute to delay the onset of cognitive impairment, particularly in individuals with intermediate adnc [307]. this suggests that many individuals diagnosed with ad may actually suffer from a mixed dementia, and therapeutic targeting ad-related processes only may have limited efficiency in these co-morbid populations [298]. based on data from the nacc, 1,854 participants with a clinical diagnosis of ad and adnc at autopsy (confirmed ad) were studied; 204 with the clinical ad diagnosis had no adnc (ad-mimics), while 253 participants with negative clinical ad diagnosis had adnc (unidentified ad). compared to confirmed ad cases, ad-mimics (ftld-tau, hippocampal sclerosis, cerebrovascular pathology, etc.) had less severe cognitive impairment [308]. special practical considerations for the diagnosis of essential co-pathologies and their relations with ad were given recently [174]. argyrophilic grain disease (agd), a limbic-predominant 4r-tauopathy, with grain-like deposits in neuritic dendrites, oligodendroglial inclusions (“coiled bodies”), ramified astrocytes, and ballooned neurons in the amygdala, hippocampus and mtls [309], represents an age-related disorder and has been reported in up to 25% ad cases [310], and rarely occurs before the age of 75 [311]. aging-related tau astrogliopathy (artag) is defined by the presence of two types of tau-bearing astrocytes: thorn-shaped and granular/fuzzy astrocytes in the brains of old-aged individuals in different locations and anatomical regions (subependymal, subpial, perivascular, white and gray matter [312, 313]. among additional pathological changes in ad is granulovacuolar degeneration (gvd), characterized as 3-5 μm vesicles bound by a unit membrane, most frequently occurring in the pyramidal neurons of the hippocampus, usually in association with nfts. their origin and significance are unclear. despite the strong association between tau aggregation and granulovacuolar degeneration body (gvb) formation [314], intracellular aggregates of proteins other than tau can also induce gvb formation, which needs further elucidation [315]. the granule of the gvd is immunolabeled by antibodies against tubulin, ubiquitin, neurofilament, and tau [31]. they correlate with nft density, suggesting that they may be a cellular response to neuronal damage or late-stage autophagic vacuoles [316]. necrosome complex detected in gvd is associated with neuron loss in ad [317]. recent clinicopathological studies have shown that the complex cascades of the underlying pathologies in most elderly patients may lead to cognitive decline, and that the number of possible combinations due to co-morbidities increases with aging [248]. these concomitant pathologies may be harmful to individuals with low cognitive reserve such as patients with ma-ad. they can cause a number of challenges including the evaluation of the significance of each pathological entity in the manifestation of the clinical symptoms, and the threshold of each individual pathology to cause dementia [174]. total burden of comorbid pathological abnormalities, rather than any single lesion, is the most important cause of cognitive impairment, often despite clinical diagnosis of “only” ad [318]. 5. conclusions ad is a heterogeneous, multifactorial disorder, manifesting clinically and morphologically as several subtypes that have a distinct signature of network disruptions associated with their atrophy pattern and reflecting the differential spread of nft pathology and neuronal loss due to different vulnerability patterns of affected brain regions, which relates to specific molecular-functional properties of the affected neuronal systems [22, 23]. the severity of lesions corresponds to the “n” category in the new a/t/n classification for biomarkers [319]. the heterogeneity of the alzheimer’s syndrome is related to multiple pathogenic factors which induce misfolding tau, aβ, tdp-43, and other proteins, the synergetic or additive action of which results in various disease phenotypes [320]. several factors such as brain resilience may help compensate for these pathologies up to a certain level, although their relevance is still poorly understood. these problems and the increasing incidence of ad illustrate its consequences on public health and the resulting challenges for future medicine. increased sensitivity and specificity of new atn biomarker systems and more extensive clinicopathological studies in well-defined populations are needed, with post-mortem studies using the updated nia/aa criteria. the recent advent of tau pet and novel imaging and fluid-based (csf) biomarkers allows us to study the temporal progression of tau pathology in vivo [321, 322]. improving methods for disease detection and monitoring its progression may hopefully lead to the development and refinement of tau-based therapeutics. in the interest of optimizing the clinical diagnosis of ad and related disorders, neuropathological studies should use a wide range of molecular pathological methods and should evaluate multiple cns regions. an optimal and less cost-intensive strategy would be to screen specifically neurodegeneration-related proteins and to examine their cross reactions. the recent correlative work on concomitant pathologies has provide insight into the interactions of the various pathologies and their roles in causing dementing symptoms. interdisciplinary studies may improve our knowledge about the pathogenesis of the heterogeneous manifestation of ad and promote methods for its early diagnosis as the basis for further preventive and successful disease-modifying therapeutic measures. funding this research was partially funded by the society for the promotion of research in experimental neurology, vienna, austria. acknowledgments the author thanks mr. e. mitter-ferstl, phd, for secretarial and editorial work. conflicts of interest the author declares no conflict of interest. references 1. alzheimer's-association (2018) 2018 alzheimer's disease facts and figures. alzheimers dement 14:367-429 2. farfel jm, yu l, boyle pa, leurgans s, shah rc, schneider ja, bennett da (2019) alzheimer's disease frequency peaks in the tenth decade and is lower afterwards. acta neuropathol commun 7:104 3. jellinger ka, attems j (2010) prevalence of dementia disorders in the oldest-old: an autopsy study. acta neuropathol 119:421-433 4. fiest km, roberts ji, maxwell cj, hogan db, smith ee, frolkis a, cohen a, kirk a, pearson d, pringsheim t, venegas-torres a, jetté n (2016) the prevalence and incidence of dementia due to alzheimer's disease: a systematic review and meta-analysis. canad j neurol sci 43 (suppl s1):s51-s82 5. alzheimer's-association (2020) 2020 alzheimer's disease facts and figures. alzheimers dement online mar 10: doi: 10.1002/alz.12068 6. prince m, bryce r, albanese e, wimo a, ribeiro w, ferri cp (2013) the global prevalence of dementia: a systematic review and metaanalysis. alzheimers dement 9:63-75 e62 7. long jm, holtzman dm (2019) alzheimer disease: an update on pathobiology and treatment strategies. cell 179:312-339 8. ingelsson m, fukumoto h, newell kl, growdon jh, hedley-whyte et, frosch mp, albert ms, hyman bt, irizarry mc (2004) early abeta accumulation and progressive synaptic loss, gliosis, and tangle formation in ad brain. neurology 62:925-931 9. keren-shaul h, spinrad a, weiner a, matcovitch-natan o, dvir-szternfeld r, ulland tk, david e, baruch k, lara-astaiso d, toth b, itzkovitz s, colonna m, schwartz m, amit i (2017) a unique microglia type associated with restricting development of alzheimer's disease. cell 169:1276-1290 e1217 10. monzio compagnoni g, di fonzo a, corti s, comi gp, bresolin n, masliah e (2020) the role of mitochondria in neurodegenerative diseases: the lesson from alzheimer's disease and parkinson's disease. mol neurobiol 57:2959-2980 11. gallardo g, holtzman dm (2019) amyloid-beta and tau at the crossroads of alzheimer's disease. adv exp med biol 1184:187-203 12. kisler k, nelson ar, montagne a, zlokovic bv (2017) cerebral blood flow regulation and neurovascular dysfunction in alzheimer disease. nat rev neurosci 18:419-434 13. nation da, sweeney md, montagne a, sagare ap, d'orazio lm, pachicano m, sepehrband f, nelson ar, buennagel dp, harrington mg, benzinger tls, fagan am, ringman jm, schneider ls, morris jc, chui hc, law m, toga aw, zlokovic bv (2019) blood-brain barrier breakdown is an early biomarker of human cognitive dysfunction. nat med 25:270-276 14. carter sf, herholz k, rosa-neto p, pellerin l, nordberg a, zimmer er (2019) astrocyte biomarkers in alzheimer's disease. trends mol med 25:77-95 15. jack cr, jr., therneau tm, weigand sd, wiste hj, knopman ds, vemuri p, lowe vj, mielke mm, roberts ro, machulda mm, graff-radford j, jones dt, schwarz cg, gunter jl, senjem ml, rocca wa, petersen rc (2019) prevalence of biologically vs clinically defined alzheimer spectrum entities using the national institute on aging-alzheimer's association research framework. jama neurol 76:1174-1183 16. ferreira d, nordberg a, westman e (2020) biological subtypes of alzheimer disease: a systematic review and meta-analysis. neurology 94:436-448 17. montine tj, phelps ch, beach tg, bigio eh, cairns nj, dickson dw, duyckaerts c, frosch mp, masliah e, mirra ss, nelson pt, schneider ja, thal dr, trojanowski jq, vinters hv, hyman bt (2012) national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol 123:1-11 18. braak h, braak e (1991) neuropathological stageing of alzheimer-related changes. acta neuropathol 82:239-259 19. fornari s, schäfer a, jucker m, goriely a, kuhl e (2019) prion-like spreading of alzheimer's disease within the brain's connectome. j r soc interface 16:20190356 20. mattsson n, schott jm, hardy j, turner mr, zetterberg h (2016) selective vulnerability in neurodegeneration: insights from clinical variants of alzheimer's disease. j neurol neurosurg psychiatry 87:1000-1004 21. mrdjen d, fox ej, bukhari sa, montine ks, bendall sc, montine tj (2019) the basis of cellular and regional vulnerability in alzheimer's disease. acta neuropathol 138:729-749 22. grothe mj, sepulcre j, gonzalez-escamilla g, jelistratova i, schöll m, hansson o, teipel sj (2018) molecular properties underlying regional vulnerability to alzheimer's disease pathology. brain 141:2755-2771 23. wang zt, zhang c, wang yj, dong q, tan l, yu jt (2020) selective neuronal vulnerability in alzheimer's disease. ageing res rev 62:101114 24. acosta d, powell f, zhao y, raj a (2018) regional vulnerability in alzheimer's disease: the role of cell-autonomous and transneuronal processes. alzheimers dement 14:797-810 25. roussarie jp, yao v, rodriguez-rodriguez p, oughtred r, rust j, plautz z, kasturia s, albornoz c, wang w, schmidt ef, dannenfelser r, tadych a, brichta l, barnea-cramer a, heintz n, hof pr, heiman m, dolinski k, flajolet m, troyanskaya og, greengard p (2020) selective neuronal vulnerability in alzheimer's disease: a network-based analysis. neuron 107:p821-835.e812 26. warren jd, fletcher pd, golden hl (2012) the paradox of syndromic diversity in alzheimer disease. nat rev neurol 8:451-464 27. jellinger ka (2020) towards a biological definition of alzheimer disease. int j neurol neurother 7:095; doi 010.23937/22378-23001/1410095 28. zhou j, greicius md, gennatas ed, growdon me, jang jy, rabinovici gd, kramer jh, weiner m, miller bl, seeley ww (2010) divergent network connectivity changes in behavioural variant frontotemporal dementia and alzheimer's disease. brain 133:1352-1367 29. piguet o, double kl, kril jj, harasty j, macdonald v, mcritchie da, halliday gm (2009) white matter loss in healthy ageing: a postmortem analysis. neurobiol aging 30:1288-1295 30. serrano-pozo a, frosch mp, masliah e, hyman bt (2011) neuropathological alterations in alzheimer disease. cold spring harb perspect med 1:a006189 31. deture ma, dickson dw (2019) the neuropathological diagnosis of alzheimer's disease. mol neurodegener 14:32 32. tome so, vandenberghe r, ospitalieri s, van schoor e, tousseyn t, otto m, von arnim caf, thal dr (2020) distinct molecular patterns of tdp-43 pathology in alzheimer's disease: relationship with clinical phenotypes. acta neuropathol commun 8:61 33. o'brien rj, wong pc (2011) amyloid precursor protein processing and alzheimer's disease. annu rev neurosci 34:185-204 34. goldgaber d, lerman mi, mcbride ow, saffiotti u, gajdusek dc (1987) characterization and chromosomal localization of a cdna encoding brain amyloid of alzheimer's disease. science 235:877-880 35. tanzi re, gusella jf, watkins pc, bruns ga, st george-hyslop p, van keuren ml, patterson d, pagan s, kurnit dm, neve rl (1987) amyloid beta protein gene: cdna, mrna distribution, and genetic linkage near the alzheimer locus. science 235:880-884 36. coronel r, bernabeu-zornoza a, palmer c, muniz-moreno m, zambrano a, cano e, liste i (2018) role of amyloid precursor protein (app) and its derivatives in the biology and cell fate specification of neural stem cells. mol neurobiol 55:7107-7117 37. zheng h, koo eh (2011) biology and pathophysiology of the amyloid precursor protein. mol neurodegener 6:27 38. kuhn ph, wang h, dislich b, colombo a, zeitschel u, ellwart jw, kremmer e, rossner s, lichtenthaler sf (2010) adam10 is the physiologically relevant, constitutive alpha-secretase of the amyloid precursor protein in primary neurons. embo j 29:3020-3032 39. postina r, schroeder a, dewachter i, bohl j, schmitt u, kojro e, prinzen c, endres k, hiemke c, blessing m, flamez p, dequenne a, godaux e, van leuven f, fahrenholz f (2004) a disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an alzheimer disease mouse model. j clin invest 113:1456-1464 40. kojro e, fahrenholz f (2005) the non-amyloidogenic pathway: structure and function of alpha-secretases. subcell biochem 38:105-127 41. nhan hs, chiang k, koo eh (2015) the multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes. acta neuropathol 129:1-19 42. de chiara g, marcocci me, civitelli l, argnani r, piacentini r, ripoli c, manservigi r, grassi c, garaci e, palamara at (2010) app processing induced by herpes simplex virus type 1 (hsv-1) yields several app fragments in human and rat neuronal cells. plos one 5:e13989 43. portelius e, brinkmalm g, tran a, andreasson u, zetterberg h, westman-brinkmalm a, blennow k, ohrfelt a (2010) identification of novel n-terminal fragments of amyloid precursor protein in cerebrospinal fluid. exp neurol 223:351-358 44. frost gr, li ym (2017) the role of astrocytes in amyloid production and alzheimer's disease. open biol 7 45. peters f, salihoglu h, rodrigues e, herzog e, blume t, filser s, dorostkar m, shimshek dr, brose n, neumann u, herms j (2018) bace1 inhibition more effectively suppresses initiation than progression of beta-amyloid pathology. acta neuropathol 135:695-710 46. volloch v, olsen b, rits s (2020) alzheimer's disease is driven by intraneuronally retained beta-amyloid produced in the ad-specific, beta-app-independent pathway: current perspective and experimental models for tomorrow. ann integr mol med 2:90-114 47. yu y, jans dc, winblad b, tjernberg lo, schedin-weiss s (2018) neuronal abeta42 is enriched in small vesicles at the presynaptic side of synapses. life sci alliance 1:e201800028 48. greenfield jp, tsai j, gouras gk, hai b, thinakaran g, checler f, sisodia ss, greengard p, xu h (1999) endoplasmic reticulum and trans-golgi network generate distinct populations of alzheimer beta-amyloid peptides. proc natl acad sci u s a 96:742-747 49. hartmann t, bieger sc, brühl b, tienari pj, ida n, allsop d, roberts gw, masters cl, dotti cg, unsicker k, beyreuther k (1997) distinct sites of intracellular production for alzheimer's disease a beta40/42 amyloid peptides. nat med 3:1016-1020 50. hellström-lindahl e, viitanen m, marutle a (2009) comparison of abeta levels in the brain of familial and sporadic alzheimer's disease. neurochem int 55:243-252 51. walsh dm, selkoe dj (2007) a beta oligomers a decade of discovery. j neurochem 101:1172-1184 52. galvao f, jr., grokoski kc, da silva bb, lamers ml, siqueira ir (2019) the amyloid precursor protein (app) processing as a biological link between alzheimer's disease and cancer. ageing res rev 49:83-91 53. pinnix i, ghiso ja, pappolla ma, sambamurti k (2013) major carboxyl terminal fragments generated by gamma-secretase processing of the alzheimer amyloid precursor are 50 and 51 amino acids long. am j geriatr psychiatry 21:474-483 54. kametani f (2008) epsilon-secretase: reduction of amyloid precursor protein epsilon-site cleavage in alzheimer's disease. curr alzheimer res 5:165-171 55. viswanathan j, haapasalo a, kurkinen km, natunen t, mäkinen p, bertram l, soininen h, tanzi re, hiltunen m (2013) ubiquilin-1 modulates gamma-secretase-mediated epsilon-site cleavage in neuronal cells. biochemistry 52:3899-3912 56. krstic d, knuesel i (2013) deciphering the mechanism underlying late-onset alzheimer disease. nat rev neurol 9:25-34 57. michno w, wehrli p, meier sr, sehlin d, syvänen s, zetterberg h, blennow k, hanrieder j (2020) chemical imaging of evolving amyloid plaque pathology and associated abeta peptide aggregation in a transgenic mouse model of alzheimer's disease. j neurochem 152:602-616 58. masters cl, bateman r, blennow k, rowe cc, sperling ra, cummings jl (2015) alzheimer's disease. nat rev dis primers 1:15056 59. sil s, hu g, liao k, niu f, callen s, periyasamy p, fox hs, buch s (2020) hiv-1 tat-mediated astrocytic amyloidosis involves the hif-1alpha/lncrna bace1-as axis. plos biol 18:e3000660 60. qiang w, yau wm, lu jx, collinge j, tycko r (2017) structural variation in amyloid-beta fibrils from alzheimer's disease clinical subtypes. nature 541:217-221 61. duyckaerts c, delatour b, potier mc (2009) classification and basic pathology of alzheimer disease. acta neuropathol 118:5-36 62. serrano-pozo a, mielke ml, gomez-isla t, betensky ra, growdon jh, frosch mp, hyman bt (2011) reactive glia not only associates with plaques but also parallels tangles in alzheimer's disease. am j pathol 179:1373-1384 63. thal dr, capetillo-zarate e, del tredici k, braak h (2006) the development of amyloid beta protein deposits in the aged brain. sci aging knowledge environ 2006:re1 64. moro ml, phillips as, gaimster k, paul c, mudher a, nicoll jar, boche d (2018) pyroglutamate and isoaspartate modified amyloid-beta in ageing and alzheimer's disease. acta neuropathol commun 6:3 65. nicoll ja, barton e, boche d, neal jw, ferrer i, thompson p, vlachouli c, wilkinson d, bayer a, games d, seubert p, schenk d, holmes c (2006) abeta species removal after abeta42 immunization. j neuropathol exp neurol 65:1040-1048 66. paasila p, davies d, sutherland g, goldsbury c (2020) clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of abeta plaques in alzheimer’s disease. free neuropathol 1:20 doi 10.17879/freeneuropathology-12020-12845 67. reed-geaghan eg, croxford al, becher b, landreth ge (2020) plaque-associated myeloid cells derive from resident microglia in an alzheimer's disease model. j exp med 217 68. yeh fl, hansen dv, sheng m (2017) trem2, microglia, and neurodegenerative diseases. trends mol med 23:512-533 69. han s, kollmer m, markx d, claus s, walther p, fändrich m (2017) amyloid plaque structure and cell surface interactions of beta-amyloid fibrils revealed by electron tomography. sci rep 7:43577 70. boon bdc, bulk m, jonker aj, morrema thj, van den berg e, popovic m, walter j, kumar s, van der lee sj, holstege h, zhu x, van nostrand we, natté r, van der weerd l, bouwman fh, van de berg wdj, rozemuller ajm, hoozemans jjm (2020) the coarse-grained plaque: a divergent abeta plaque-type in early-onset alzheimer's disease. acta neuropathol online sep 14: doi: 10.1007/s00401-00020-02198-00408 71. attems j, jellinger ka (2013) neuropathology. in: dening t, thomas a (eds) oxford textbook of old age psychiatry (2 ed.). oxford univ. press oxford, uk, pp 87-105, doi: 110.1093/med/9780199644957.9780199644003.9780199640006 72. oide t, kinoshita t, arima k (2006) regression stage senile plaques in the natural course of alzheimer's disease. neuropathol appl neurobiol 32:539-556 73. boche d, nicoll jar (2020) understanding cause and effect in alzheimer's pathophysiology: implications for clinical trials. neuropathol appl neurobiol jul 8:https://doi.org/10.1111/nan.12642 74. harris ja, devidze n, verret l, ho k, halabisky b, thwin mt, kim d, hamto p, lo i, yu gq, palop jj, masliah e, mucke l (2010) transsynaptic progression of amyloid-beta-induced neuronal dysfunction within the entorhinal-hippocampal network. neuron 68:428-441 75. yamamoto k, tanei zi, hashimoto t, wakabayashi t, okuno h, naka y, yizhar o, fenno le, fukayama m, bito h, cirrito jr, holtzman dm, deisseroth k, iwatsubo t (2015) chronic optogenetic activation augments abeta pathology in a mouse model of alzheimer disease. cell rep 11:859-865 76. reiss ab, arain ha, stecker mm, siegart nm, kasselman lj (2018) amyloid toxicity in alzheimer's disease. rev neurosci 29:613-627 77. yu l, petyuk va, tasaki s, boyle pa, gaiteri c, schneider ja, de jager pl, bennett da (2019) association of cortical beta-amyloid protein in the absence of insoluble deposits with alzheimer disease. jama neurol 76:818-826 78. katzmarski n, ziegler-waldkirch s, scheffler n, witt c, abou-ajram c, nuscher b, prinz m, haass c, meyer-luehmann m (2020) abeta oligomers trigger and accelerate abeta seeding. brain pathol 30:36-45 79. thal dr, rub u, orantes m, braak h (2002) phases of a beta-deposition in the human brain and its relevance for the development of ad. neurology 58:1791-1800 80. walker lc (2020) aß plaques. free neuropathol 1:31, doi 10.17879/freeneuropathology-12020-13025 81. thal dr, ronisz a, tousseyn t, rijal upadhaya a, balakrishnan k, vandenberghe r, vandenbulcke m, von arnim caf, otto m, beach tg, lilja j, heurling k, chakrabarty a, ismail a, buckley c, smith apl, kumar s, farrar g, walter j (2019) different aspects of alzheimer's disease-related amyloid beta-peptide pathology and their relationship to amyloid positron emission tomography imaging and dementia. acta neuropathol commun 7:178 82. mattsson n, palmqvist s, stomrud e, vogel j, hansson o (2019) staging beta-amyloid pathology with amyloid positron emission tomography. jama neurol 76:1319-1329 83. ikonomovic md, buckley cj, abrahamson ee, kofler jk, mathis ca, klunk we, farrar g (2020) post-mortem analyses of pib and flutemetamol in diffuse and cored amyloid-beta plaques in alzheimer's disease. acta neuropathol 140:463-476 84. attems j (2005) sporadic cerebral amyloid angiopathy: pathology, clinical implications, and possible pathomechanisms. acta neuropathol 110:345-359 85. attems j, jellinger k, thal dr, van nostrand w (2011) review: sporadic cerebral amyloid angiopathy. neuropathol appl neurobiol 37:75-93 86. thal dr, griffin ws, de vos ra, ghebremedhin e (2008) cerebral amyloid angiopathy and its relationship to alzheimer's disease. acta neuropathol 115:599-609 87. allen n, robinson ac, snowden j, davidson ys, mann dm (2014) patterns of cerebral amyloid angiopathy define histopathological phenotypes in alzheimer's disease. neuropathol appl neurobiol 40:136-148 88. love s, chalmers k, ince p, esiri m, attems j, jellinger k, yamada m, mccarron m, minett t, matthews f, greenberg s, mann d, kehoe pg (2014) development, appraisal, validation and implementation of a consensus protocol for the assessment of cerebral amyloid angiopathy in post-mortem brain tissue. am j neurodegener dis 3:19-32 89. thal dr, ghebremedhin e, rub u, yamaguchi h, del tredici k, braak h (2002) two types of sporadic cerebral amyloid angiopathy. j neuropathol exp neurol 61:282-293 90. nortley r, korte n, izquierdo p, hirunpattarasilp c, mishra a, jaunmuktane z, kyrargyri v, pfeiffer t, khennouf l, madry c, gong h, richard-loendt a, huang w, saito t, saido tc, brandner s, sethi h, attwell d (2019) amyloid beta oligomers constrict human capillaries in alzheimer's disease via signaling to pericytes. science 365:eaav9518 doi 9510.1126/science.aav9518 91. attems j, jellinger ka (2014) pathologic aspects of the hemorrhagic consequences of small vessel disease on the brain. in: pantoni l, gorelick pb (eds) cerebral small vessel disease. cambridge university press cambridge, uk, pp 29-41 92. planton m, pariente j, nemmi f, albucher jf, calviere l, viguier a, olivot jm, salabert as, payoux p, peran p, raposo n (2020) interhemispheric distribution of amyloid and small vessel disease burden in cerebral amyloid angiopathy-related intracerebral hemorrhage. eur j neurol 27:1664-1671 93. schreiber s, wilisch-neumann a, schreiber f, assmann a, scheumann v, perosa v, jandke s, mawrin c, carare ro, werring dj (2020) the spectrum of age-related small vessel diseases: potential overlap and interactions of amyloid and nonamyloid vasculopathies. neuropathol appl neurobiol 46:219-239 94. lafon pa, wang y, arango-lievano m, torrent j, salvador-prince l, mansuy m, mestre-francès n, givalois l, liu j, mercader jv, jeanneteau f, desrumaux c, perrier v (2020) fungicide residues exposure and beta-amyloid aggregation in a mouse model of alzheimer's disease. environ health perspect 128:17011 95. gatti l, tinelli f, scelzo e, arioli f, di fede g, obici l, pantoni l, giaccone g, caroppo p, parati ea, bersano a (2020) understanding the pathophysiology of cerebral amyloid angiopathy. int j mol sci 21 96. greenberg sm, bacskai bj, hernandez-guillamon m, pruzin j, sperling r, van veluw sj (2020) cerebral amyloid angiopathy and alzheimer disease one peptide, two pathways. nat rev neurol 16:30-42 97. revesz t, ghiso j, lashley t, plant g, rostagno a, frangione b, holton jl (2003) cerebral amyloid angiopathies: a pathologic, biochemical, and genetic view. j neuropathol exp neurol 62:885-898 98. neve rl, harris p, kosik ks, kurnit dm, donlon ta (1986) identification of cdna clones for the human microtubule-associated protein tau and chromosomal localization of the genes for tau and microtubule-associated protein 2. brain res 387:271-280 99. guo t, noble w, hanger dp (2017) roles of tau protein in health and disease. acta neuropathol 133:665-704 100. holmes bb, diamond mi (2014) prion-like properties of tau protein: the importance of extracellular tau as a therapeutic target. j biol chem 289:19855-19861 101. kfoury n, holmes bb, jiang h, holtzman dm, diamond mi (2012) trans-cellular propagation of tau aggregation by fibrillar species. j biol chem 287:19440-19451 102. de calignon a, fox lm, pitstick r, carlson ga, bacskai bj, spires-jones tl, hyman bt (2010) caspase activation precedes and leads to tangles. nature 464:1201-1204 103. younas n, zafar s, shafiq m, noor a, siegert a, arora as, galkin a, zafar a, schmitz m, stadelmann c, andreoletti o, ferrer i, zerr i (2020) sfpq and tau: critical factors contributing to rapid progression of alzheimer's disease. acta neuropathol 140:317-339 104. mamun aa, uddin ms, mathew b, ashraf gm (2020) toxic tau: structural origins of tau aggregation in alzheimer's disease. neural regen res 15:1417-1420 105. ballatore c, lee vm, trojanowski jq (2007) tau-mediated neurodegeneration in alzheimer's disease and related disorders. nat rev neurosci 8:663-672 106. guha s, johnson gvw, nehrke k (2020) the crosstalk between pathological tau phosphorylation and mitochondrial dysfunction as a key to understanding and treating alzheimer's disease. mol neurobiol 57:5103-5120 107. xia y, prokop s, gorion km, kim jd, sorrentino za, bell bm, manaois an, chakrabarty p, davies p, giasson bi (2020) tau ser208 phosphorylation promotes aggregation and reveals neuropathologic diversity in alzheimer's disease and other tauopathies. acta neuropathol commun 8:88 108. wischik cm, novak m, thogersen hc, edwards pc, runswick mj, jakes r, walker je, milstein c, roth m, klug a (1988) isolation of a fragment of tau derived from the core of the paired helical filament of alzheimer disease. proc natl acad sci u s a 85:4506-4510 109. uchihara t (2020) neurofibrillary changes undergoing morphological and biochemical changes how does tau with the profile shift of from four repeat to three repeat spread in alzheimer brain? neuropathology online jul 22: doi: 10.1111/neup.12669 110. kidd m (1964) alzheimer's disease an electron microscopical study. brain 87:307-320 111. pollanen ms, markiewicz p, bergeron c, goh mc (1994) twisted ribbon structure of paired helical filaments revealed by atomic force microscopy. am j pathol 144:869-873 112. crowther ra (1991) straight and paired helical filaments in alzheimer disease have a common structural unit. proc natl acad sci u s a 88:2288-2292 113. fitzpatrick awp, falcon b, he s, murzin ag, murshudov g, garringer hj, crowther ra, ghetti b, goedert m, scheres shw (2017) cryo-em structures of tau filaments from alzheimer's disease. nature 547:185-190 114. falcon b, zhang w, schweighauser m, murzin ag, vidal r, garringer hj, ghetti b, scheres shw, goedert m (2018) tau filaments from multiple cases of sporadic and inherited alzheimer's disease adopt a common fold. acta neuropathol 136:699-708 115. braak e, braak h, mandelkow em (1994) a sequence of cytoskeleton changes related to the formation of neurofibrillary tangles and neuropil threads. acta neuropathol 87:554-567 116. streit wj, khoshbouei h, bechmann i (2020) dystrophic microglia in late-onset alzheimer's disease. glia 68:845-854 117. malpas cb, sharmin s, kalincik t (2020) the histopathological staging of tau, but not amyloid, corresponds to antemortem cognitive status, dementia stage, functional abilities and neuropsychiatric symptoms. int j neurosci apr 30: doi: 10.1080/00207454.00202020.01758087 118. andrade-moraes ch, oliveira-pinto av, castro-fonseca e, da silva cg, guimaraes dm, szczupak d, parente-bruno dr, carvalho lr, polichiso l, gomes bv, oliveira lm, rodriguez rd, leite re, ferretti-rebustini re, jacob-filho w, pasqualucci ca, grinberg lt, lent r (2013) cell number changes in alzheimer's disease relate to dementia, not to plaques and tangles. brain 136:3738-3752 119. kuchibhotla kv, wegmann s, kopeikina kj, hawkes j, rudinskiy n, andermann ml, spires-jones tl, bacskai bj, hyman bt (2014) neurofibrillary tangle-bearing neurons are functionally integrated in cortical circuits in vivo. proc natl acad sci u s a 111:510-514 120. santacruz k, lewis j, spires t, paulson j, kotilinek l, ingelsson m, guimaraes a, deture m, ramsden m, mcgowan e, forster c, yue m, orne j, janus c, mariash a, kuskowski m, hyman b, hutton m, ashe kh (2005) tau suppression in a neurodegenerative mouse model improves memory function. science 309:476-481 121. spires tl, orne jd, santacruz k, pitstick r, carlson ga, ashe kh, hyman bt (2006) region-specific dissociation of neuronal loss and neurofibrillary pathology in a mouse model of tauopathy. am j pathol 168:1598-1607 122. drummond e, pires g, macmurray c, askenazi m, nayak s, bourdon m, safar j, ueberheide b, wisniewski t (2020) phosphorylated tau interactome in the human alzheimer's disease brain. brain 143:2803-2817 123. braak h, del tredici k (2011) alzheimer's pathogenesis: is there neuron-to-neuron propagation? acta neuropathol 121:589-595 124. clavaguera f, duyckaerts c, haik s (2020) prion-like properties of tau assemblies. curr opin neurobiol 61:49-57 125. colin m, dujardin s, schraen-maschke s, meno-tetang g, duyckaerts c, courade jp, buee l (2020) from the prion-like propagation hypothesis to therapeutic strategies of anti-tau immunotherapy. acta neuropathol 139:3-25 126. devos sl, corjuc bt, oakley dh, nobuhara ck, bannon rn, chase a, commins c, gonzalez ja, dooley pm, frosch mp, hyman bt (2018) synaptic tau seeding precedes tau pathology in human alzheimer's disease brain. front neurosci 12:267 127. dujardin s, hyman bt (2019) tau prion-like propagation: state of the art and current challenges. adv exp med biol 1184:305-325 128. stancu ic, vasconcelos b, ris l, wang p, villers a, peeraer e, buist a, terwel d, baatsen p, oyelami t, pierrot n, casteels c, bormans g, kienlen-campard p, octave jn, moechars d, dewachter i (2015) templated misfolding of tau by prion-like seeding along neuronal connections impairs neuronal network function and associated behavioral outcomes in tau transgenic mice. acta neuropathol 129:875-894 129. narasimhan s, guo jl, changolkar l, stieber a, mcbride jd, silva lv, he z, zhang b, gathagan rj, trojanowski jq, lee vmy (2017) pathological tau strains from human brains recapitulate the diversity of tauopathies in nontransgenic mouse brain. j neurosci 37:11406-11423 130. hopp sc, lin y, oakley d, roe ad, devos sl, hanlon d, hyman bt (2018) the role of microglia in processing and spreading of bioactive tau seeds in alzheimer's disease. j neuroinflammation 15:269 131. furman jl, vaquer-alicea j, white cl, 3rd, cairns nj, nelson pt, diamond mi (2017) widespread tau seeding activity at early braak stages. acta neuropathol 133:91-100 132. jagust wj (2020) imaging tau pathology the next step. jama neurol 77:796-797 133. lowe vj, lundt es, albertson sm, przybelski sa, senjem ml, parisi je, kantarci k, boeve b, jones dt, knopman d, jack cr, jr., dickson dw, petersen rc, murray me (2019) neuroimaging correlates with neuropathologic schemes in neurodegenerative disease. alzheimers dement 15:927-939 134. marquié m, siao tick chong m, antón-fernández a, verwer ee, sáez-calveras n, meltzer ac, ramanan p, amaral ac, gonzalez j, normandin md, frosch mp, gómez-isla t (2017) [f-18]-av-1451 binding correlates with postmortem neurofibrillary tangle braak staging. acta neuropathol 134:619-628 135. ossenkoppele r, schonhaut dr, schöll m, lockhart sn, ayakta n, baker sl, o'neil jp, janabi m, lazaris a, cantwell a, vogel j, santos m, miller za, bettcher bm, vossel ka, kramer jh, gorno-tempini ml, miller bl, jagust wj, rabinovici gd (2016) tau pet patterns mirror clinical and neuroanatomical variability in alzheimer's disease. brain 139:1551-1567 136. schwarz aj, yu p, miller bb, shcherbinin s, dickson j, navitsky m, joshi ad, devous md, sr., mintun ms (2016) regional profiles of the candidate tau pet ligand 18f-av-1451 recapitulate key features of braak histopathological stages. brain 139:1539-1550 137. vogels t, leuzy a, cicognola c, ashton nj, smolek t, novak m, blennow k, zetterberg h, hromadka t, zilka n, schöll m (2020) propagation of tau pathology: integrating insights from postmortem and in vivo studies. biol psychiatry 87:808-818 138. arriagada pv, growdon jh, hedley-whyte et, hyman bt (1992) neurofibrillary tangles but not senile plaques parallel duration and severity of alzheimer's disease. neurology 42:631-639 139. nelson pt, alafuzoff i, bigio eh, bouras c, braak h, cairns nj, castellani rj, crain bj, davies p, del tredici k, duyckaerts c, frosch mp, haroutunian v, hof pr, hulette cm, hyman bt, iwatsubo t, jellinger ka, jicha ga, kovari e, kukull wa, leverenz jb, love s, mackenzie ir, mann dm, masliah e, mckee ac, montine tj, morris jc, schneider ja, sonnen ja, thal dr, trojanowski jq, troncoso jc, wisniewski t, woltjer rl, beach tg (2012) correlation of alzheimer disease neuropathologic changes with cognitive status: a review of the literature. j neuropathol exp neurol 71:362-381 140. ohm dt, fought aj, rademaker a, kim g, sridhar j, coventry c, gefen t, weintraub s, bigio e, mesulam mm, rogalski e, geula c (2020) neuropathologic basis of in vivo cortical atrophy in the aphasic variant of alzheimer's disease. brain pathol 30:332-344 141. kaufman sk, del tredici k, thomas tl, braak h, diamond mi (2018) tau seeding activity begins in the transentorhinal/entorhinal regions and anticipates phospho-tau pathology in alzheimer's disease and part. acta neuropathol 136:57-67 142. attems j, thomas a, jellinger k (2012) correlations between cortical and subcortical tau pathology. neuropathol appl neurobiol 38:582-590 143. braak h, thal dr, ghebremedhin e, del tredici k (2011) stages of the pathologic process in alzheimer disease: age categories from 1 to 100 years. j neuropathol exp neurol 70:960-969 144. grinberg lt, rüb u, ferretti re, nitrini r, farfel jm, polichiso l, gierga k, jacob-filho w, heinsen h (2009) the dorsal raphe nucleus shows phospho-tau neurofibrillary changes before the transentorhinal region in alzheimer's disease. a precocious onset? neuropathol appl neurobiol 35:406-416 145. braak h, del trecidi k (2015) neuroanatomy and pathology of sporadic alzheimer's disease. adv anat embryol cell biol 215:1-162 146. grudzien a, shaw p, weintraub s, bigio e, mash dc, mesulam mm (2007) locus coeruleus neurofibrillary degeneration in aging, mild cognitive impairment and early alzheimer's disease. neurobiol aging 28:327-335 147. rüb u, stratmann k, heinsen h, turco dd, seidel k, dunnen w, korf hw (2016) the brainstem tau cytoskeletal pathology of alzheimer's disease: a brief historical overview and description of its anatomical distribution pattern, evolutional features, pathogenetic and clinical relevance. curr alzheimer res 13:1178-1197 148. attems j, quass m, jellinger ka (2007) tau and alpha-synuclein brainstem pathology in alzheimer disease: relation with extrapyramidal signs. acta neuropathol 113:53-62 149. buchman as, shulman jm, nag s, leurgans se, arnold se, morris mc, schneider ja, bennett da (2012) nigral pathology and parkinsonian signs in elders without parkinson disease. ann neurol 71:258-266 150. burns jm, galvin je, roe cm, morris jc, mckeel dw (2005) the pathology of the substantia nigra in alzheimer disease with extrapyramidal signs. neurology 64:1397-1403 151. kent sa, spires-jones tl, durrant cs (2020) the physiological roles of tau and abeta: implications for alzheimer's disease pathology and therapeutics. acta neuropathol 140:417-447 152. kara e, marks jd, aguzzi a (2018) toxic protein spread in neurodegeneration: reality versus fantasy. trends mol med 24:1007-1020 153. busche ma, hyman bt (2020) synergy between amyloid-beta and tau in alzheimer's disease. nat neurosci 23:1183-1193 154. jucker m, walker lc (2018) propagation and spread of pathogenic protein assemblies in neurodegenerative diseases. nat neurosci 21:1341-1349 155. thompson tb, chaggar p, kuhl e, goriely a (2020) protein-protein interactions in neurodegenerative diseases: a conspiracy theory. plos comput biol 16:e1008267 156. bennett re, devos sl, dujardin s, corjuc b, gor r, gonzalez j, roe ad, frosch mp, pitstick r, carlson ga, hyman bt (2017) enhanced tau aggregation in the presence of amyloid beta. am j pathol 187:1601-1612 157. pooler am, polydoro m, maury ea, nicholls sb, reddy sm, wegmann s, william c, saqran l, cagsal-getkin o, pitstick r, beier dr, carlson ga, spires-jones tl, hyman bt (2015) amyloid accelerates tau propagation and toxicity in a model of early alzheimer's disease. acta neuropathol commun 3:14 158. cline en, bicca ma, viola kl, klein wl (2018) the amyloid-ß oligomer hypothesis: beginning of the third decade. j alzheimers dis 64:s567-s610 159. iadanza mg, jackson mp, hewitt ew, ranson na, radford se (2018) a new era for understanding amyloid structures and disease. nat rev mol cell biol 19:755-773 160. okamura n, harada r, furumoto s, arai h, yanai k, kudo y (2014) tau pet imaging in alzheimer's disease. curr neurol neurosci rep 14:500 161. keene cd, wilson am, kilgore md, bruner lt, postupna no, darvas m (2019) luminex-based quantification of alzheimer's disease neuropathologic change in formalin-fixed post-mortem human brain tissue. lab invest 99:1056-1067 162. walsh dm, selkoe dj (2020) amyloid beta-protein and beyond: the path forward in alzheimer's disease. curr opin neurobiol 61:116-124 163. selkoe dj, hardy j (2016) the amyloid hypothesis of alzheimer's disease at 25 years. embo mol med 8:595-608 164. viola kl, klein wl (2015) amyloid beta oligomers in alzheimer's disease pathogenesis, treatment, and diagnosis. acta neuropathol 129:183-206 165. gomes la, hipp sa, rijal upadhaya a, balakrishnan k, ospitalieri s, koper mj, largo-barrientos p, uytterhoeven v, reichwald j, rabe s, vandenberghe r, von arnim caf, tousseyn t, feederle r, giudici c, willem m, staufenbiel m, thal dr (2019) abeta-induced acceleration of alzheimer-related tau-pathology spreading and its association with prion protein. acta neuropathol 138:913-941 166. vergara c, houben s, suain v, yilmaz z, de decker r, vanden dries v, boom a, mansour s, leroy k, ando k, brion jp (2019) amyloid-beta pathology enhances pathological fibrillary tau seeding induced by alzheimer phf in vivo. acta neuropathol 137:397-412 167. wu hy, kuo pc, wang yt, lin ht, roe ad, wang by, han cl, hyman bt, chen yj, tai hc (2018) beta-amyloid induces pathology-related patterns of tau hyperphosphorylation at synaptic terminals. j neuropathol exp neurol 77:814-826 168. he z, guo jl, mcbride jd, narasimhan s, kim h, changolkar l, zhang b, gathagan rj, yue c, dengler c, stieber a, nitla m, coulter da, abel t, brunden kr, trojanowski jq, lee vm (2018) amyloid-ß plaques enhance alzheimer's brain tau-seeded pathologies by facilitating neuritic plaque tau aggregation. nat med 24:29-38 169. de felice fg, wu d, lambert mp, fernandez sj, velasco pt, lacor pn, bigio eh, jerecic j, acton pj, shughrue pj, chen-dodson e, kinney gg, klein wl (2008) alzheimer's disease-type neuronal tau hyperphosphorylation induced by a beta oligomers. neurobiol aging 29:1334-1347 170. shin ws, di j, cao q, li b, seidler pm, murray ka, bitan g, jiang l (2019) amyloid beta-protein oligomers promote the uptake of tau fibril seeds potentiating intracellular tau aggregation. alzheimers res ther 11:86 171. welikovitch la, do carmo s, maglóczky z, szocsics p, loke j, freund t, cuello ac (2018) evidence of intraneuronal abeta accumulation preceding tau pathology in the entorhinal cortex. acta neuropathol 136:901-917 172. wang l, benzinger tl, su y, christensen j, friedrichsen k, aldea p, mcconathy j, cairns nj, fagan am, morris jc, ances bm (2016) evaluation of tau imaging in staging alzheimer disease and revealing interactions between beta-amyloid and tauopathy. jama neurol 73:1070-1077 173. sardar sinha m, ansell-schultz a, civitelli l, hildesjö c, larsson m, lannfelt l, ingelsson m, hallbeck m (2018) alzheimer's disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. acta neuropathol 136:41-56 174. king a, bodi i, troakes c (2020) the neuropathological diagnosis of alzheimer's disease the challenges of pathological mimics and concomitant pathology. brain sci 10:479 175. gulisano w, maugeri d, baltrons ma, fà m, amato a, palmeri a, d'adamio l, grassi c, devanand dp, honig ls, puzzo d, arancio o (2018) role of amyloid-beta and tau proteins in alzheimer's disease: confuting the amyloid cascade. j alzheimers dis 64:s611-s631 176. small sa, duff k (2008) linking abeta and tau in late-onset alzheimer's disease: a dual pathway hypothesis. neuron 60:534-542 177. rice hc, de malmazet d, schreurs a, frere s, van molle i, volkov an, creemers e, vertkin i, nys j, ranaivoson fm, comoletti d, savas jn, remaut h, balschun d, wierda kd, slutsky i, farrow k, de strooper b, de wit j (2019) secreted amyloid-beta precursor protein functions as a gababr1a ligand to modulate synaptic transmission. science 363:eaao4827, doi 4810.1126/science.aao4827 178. ruiz-riquelme a, lau hhc, stuart e, goczi an, wang z, schmitt-ulms g, watts jc (2018) prion-like propagation of beta-amyloid aggregates in the absence of app overexpression. acta neuropathol commun 6:26 179. pickett ek, koffie rm, wegmann s, henstridge cm, herrmann ag, colom-cadena m, lleo a, kay kr, vaught m, soberman r, walsh dm, hyman bt, spires-jones tl (2016) non-fibrillar oligomeric amyloid-beta within synapses. j alzheimers dis 53:787-800 180. mroczko b, groblewska m, litman-zawadzka a, kornhuber j, lewczuk p (2018) amyloid ß oligomers (aßos) in alzheimer's disease. j neural transm (vienna) 125:177-191 181. koss dj, jones g, cranston a, gardner h, kanaan nm, platt b (2016) soluble pre-fibrillar tau and beta-amyloid species emerge in early human alzheimer's disease and track disease progression and cognitive decline. acta neuropathol 132:875-895 182. takahashi rh, capetillo-zarate e, lin mt, milner ta, gouras gk (2010) co-occurrence of alzheimer's disease beta-amyloid and tau pathologies at synapses. neurobiol aging 31:1145-1152 183. rajmohan r, reddy ph (2017) amyloid-beta and phosphorylated tau accumulations cause abnormalities at synapses of alzheimer's disease neurons. j alzheimers dis 57:975-999 184. van der kant r, goldstein lsb, ossenkoppele r (2020) amyloid-beta-independent regulators of tau pathology in alzheimer disease. nat rev neurosci 21:21-35 185. horie k, barthélemy nr, mallipeddi n, li y, franklin ee, perrin rj, bateman rj, sato c (2020) regional correlation of biochemical measures of amyloid and tau phosphorylation in the brain. acta neuropathol commun 8:149 186. tripathi t, khan h (2020) direct interaction between the beta-amyloid core and tau facilitates cross-seeding: a novel target for therapeutic intervention. biochemistry 59:341-342 187. catania m, di fede g (2020) one or more beta-amyloid(s)? new insights into the prion-like nature of alzheimer's disease. prog mol biol transl sci 175:213-237 188. panza f, lozupone m, dibello v, greco a, daniele a, seripa d, logroscino g, imbimbo bp (2019) are antibodies directed against amyloid-beta (abeta) oligomers the last call for the abeta hypothesis of alzheimer's disease? immunotherapy 11:3-6 189. kuang h, tan cy, tian hz, liu lh, yang mw, hong ff, yang sl (2020) exploring the bi-directional relationship between autophagy and alzheimer's disease. cns neurosci ther 26:155-166 190. jaunmuktane z, brandner s (2020) the role of prion-like mechanisms in neurodegenerative diseases. neuropathol appl neurobiol 46:522-545 191. penke b, szucs m, bogár f (2020) oligomerization and conformational change turn monomeric beta-amyloid and tau proteins toxic: their role in alzheimer's pathogenesis. molecules 25 192. dujardin s, commins c, lathuiliere a, beerepoot p, fernandes ar, kamath tv, de los santos mb, klickstein n, corjuc dl, corjuc bt, dooley pm, viode a, oakley dh, moore bd, mullin k, jean-gilles d, clark r, atchison k, moore r, chibnik lb, tanzi re, frosch mp, serrano-pozo a, elwood f, steen ja, kennedy me, hyman bt (2020) tau molecular diversity contributes to clinical heterogeneity in alzheimer's disease. nat med 26:1256-1263 193. sintini i, graff-radford j, senjem ml, schwarz cg, machulda mm, martin pr, jones dt, boeve bf, knopman ds, kantarci k, petersen rc, jack cr, lowe vj, josephs ka, whitwell jl (2020) longitudinal neuroimaging biomarkers differ across alzheimer's disease phenotypes. brain 143:2281-2294 194. pascoal ta, mathotaarachchi s, shin m, benedet al, mohades s, wang s, beaudry t, kang ms, soucy jp, labbe a, gauthier s, rosa-neto p (2017) synergistic interaction between amyloid and tau predicts the progression to dementia. alzheimers dement 13:644-653 195. thal dr, griffin ws, braak h (2008) parenchymal and vascular abeta-deposition and its effects on the degeneration of neurons and cognition in alzheimer's disease. j cell mol med 12:1848-1862 196. gomez-isla t, hollister r, west h, mui s, growdon jh, petersen rc, parisi je, hyman bt (1997) neuronal loss correlates with but exceeds neurofibrillary tangles in alzheimer's disease. ann neurol 41:17-24 197. saez-atienzar s, masliah e (2020) cellular senescence and alzheimer disease: the egg and the chicken scenario. nat rev neurosci 21:433-444 198. brewer gj, herrera ra, philipp s, sosna j, reyes-ruiz jm, glabe cg (2020) age-related intraneuronal aggregation of amyloid-beta in endosomes, mitochondria, autophagosomes, and lysosomes. j alzheimers dis 73:229-246 199. llorian m, schwartz s, clark ta, hollander d, tan ly, spellman r, gordon a, schweitzer ac, de la grange p, ast g, smith cw (2010) position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by ptb. nat struct mol biol 17:1114-1123 200. zanjani h, finch ce, kemper c, atkinson j, mckeel d, morris jc, price jl (2005) complement activation in very early alzheimer disease. alzheimer dis assoc disord 19:55-66 201. kordower jh, chu y, stebbins gt, dekosky st, cochran ej, bennett d, mufson ej (2001) loss and atrophy of layer ii entorhinal cortex neurons in elderly people with mild cognitive impairment. ann neurol 49:202-213 202. duyckaerts c (2014) alzheimer’s disease. in: kovacs gg (ed) neuropathology of neurodegenerative diseases. a practical guide. cambridge university press cambridge, pp 80-108 203. jellinger ka (2006) challenges in neuronal apoptosis. curr alzheimer res 3:377-391 204. woodhouse a, dickson tc, west ak, mclean ca, vickers jc (2006) no difference in expression of apoptosis-related proteins and apoptotic morphology in control, pathologically aged and alzheimer's disease cases. neurobiol dis 22:323-333 205. gorlovoy p, larionov s, pham tt, neumann h (2009) accumulation of tau induced in neurites by microglial proinflammatory mediators. faseb j 23:2502-2513 206. li jw, zong y, cao xp, tan l (2018) microglial priming in alzheimer's disease. ann transl med 6:176 207. forner s, baglietto-vargas d, martini ac, trujillo-estrada l, laferla fm (2017) synaptic impairment in alzheimer's disease: a dysregulated symphony. trends neurosci 40:347-357 208. scheff sw, price da (2003) synaptic pathology in alzheimer's disease: a review of ultrastructural studies. neurobiol aging 24:1029-1046 209. scheff sw, price da, schmitt fa, mufson ej (2006) hippocampal synaptic loss in early alzheimer's disease and mild cognitive impairment. neurobiol aging 27:1372-1384 210. smith hl, freeman oj, butcher aj, holmqvist s, humoud i, schätzl t, hughes dt, verity nc, swinden dp, hayes j, de weerd l, rowitch dh, franklin rjm, mallucci gr (2020) astrocyte unfolded protein response induces a specific reactivity state that causes non-cell-autonomous neuronal degeneration. neuron 105:855-866 e855 211. elliott c, rojo ai, ribe e, broadstock m, xia w, morin p, semenov m, baillie g, cuadrado a, al-shawi r, ballard cg, simons p, killick r (2018) a role for app in wnt signalling links synapse loss with beta-amyloid production. transl psychiatry 8:179 212. marshall ga, fairbanks la, tekin s, vinters hv, cummings jl (2007) early-onset alzheimer's disease is associated with greater pathologic burden. j geriatr psychiatry neurol 20:29-33 213. scheff sw, neltner jh, nelson pt (2014) is synaptic loss a unique hallmark of alzheimer's disease? biochem pharmacol 88:517-528 214. yin z, raj d, saiepour n, van dam d, brouwer n, holtman ir, eggen bjl, möller t, tamm ja, abdourahman a, hol em, kamphuis w, bayer ta, de deyn pp, boddeke e (2017) immune hyperreactivity of abeta plaque-associated microglia in alzheimer's disease. neurobiol aging 55:115-122 215. zotova e, bharambe v, cheaveau m, morgan w, holmes c, harris s, neal jw, love s, nicoll ja, boche d (2013) inflammatory components in human alzheimer's disease and after active amyloid-beta42 immunization. brain 136:2677-2696 216. britschgi m, takeda-uchimura y, rockenstein e, johns h, masliah e, wyss-coray t (2012) deficiency of terminal complement pathway inhibitor promotes neuronal tau pathology and degeneration in mice. j neuroinflammation 9:220 217. bolós m, llorens-martín m, jurado-arjona j, hernández f, rábano a, avila j (2016) direct evidence of internalization of tau by microglia in vitro and in vivo. j alzheimers dis 50:77-87 218. luo w, liu w, hu x, hanna m, caravaca a, paul sm (2015) microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody. sci rep 5:11161 219. van olst l, verhaege d, franssen m, kamermans a, roucourt b, carmans s, ytebrouck e, van der pol sma, wever d, popovic m, vandenbroucke re, sobrino t, schouten m, de vries he (2020) microglial activation arises after aggregation of phosphorylated-tau in a neuron-specific p301s tauopathy mouse model. neurobiol aging 89:89-98 220. zhu k, pieber m, han j, blomgren k, zhang xm, harris ra, lund h (2020) absence of microglia or presence of peripherally-derived macrophages does not affect tau pathology in young or old htau mice. glia 68:1466-1478 221. walker dg (2020) defining activation states of microglia in human brain tissue: an unresolved issue for alzheimer’s disease. neuroimmunol neuroinflammation 7:194-214; doi: 110.20517/22347-28659.22020.20509 222. paasila pj, davies ds, kril jj, goldsbury c, sutherland gt (2019) the relationship between the morphological subtypes of microglia and alzheimer's disease neuropathology. brain pathol 29:726-740 223. baulch je, acharya mm, agrawal s, apodaca la, monteiro c, agrawal a (2020) immune and inflammatory determinants underlying alzheimer's disease pathology. j neuroimmune pharmacol feb 22: doi: 10.1007/s11481-11020-09908-11489 224. jicha ga, parisi je, dickson dw, johnson k, cha r, ivnik rj, tangalos eg, boeve bf, knopman ds, braak h, petersen rc (2006) neuropathologic outcome of mild cognitive impairment following progression to clinical dementia. arch neurol 63:674-681 225. sperling ra, aisen ps, beckett la, bennett da, craft s, fagan am, iwatsubo t, jack cr, jr., kaye j, montine tj, park dc, reiman em, rowe cc, siemers e, stern y, yaffe k, carrillo mc, thies b, morrison-bogorad m, wagster mv, phelps ch (2011) toward defining the preclinical stages of alzheimer's disease: recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease. alzheimers dement 7:280-292 226. thal dr, von arnim c, griffin ws, yamaguchi h, mrak re, attems j, upadhaya ar (2013) pathology of clinical and preclinical alzheimer's disease. eur arch psychiatry clin neurosci 263 suppl 2:s137-145 227. jack cr, wiste hj, weigand sd, therneau tm, lowe vj, knopman ds, botha h, graff-radford j, jones dt, ferman tj, boeve bf, kantarci k, vemuri p, mielke mm, whitwell j, josephs k, schwarz cg, senjem ml, gunter jl, petersen rc (2020) predicting future rates of tau accumulation on pet. brain 143:3136-3150 228. alafuzoff i, arzberger t, al-sarraj s, bodi i, bogdanovic n, braak h, bugiani o, del-tredici k, ferrer i, gelpi e, giaccone g, graeber mb, ince p, kamphorst w, king a, korkolopoulou p, kovacs gg, larionov s, meyronet d, monoranu c, parchi p, patsouris e, roggendorf w, seilhean d, tagliavini f, stadelmann c, streichenberger n, thal dr, wharton sb, kretzschmar h (2008) staging of neurofibrillary pathology in alzheimer's disease: a study of the brainnet europe consortium. brain pathol 18:484-496 229. hyman bt, phelps ch, beach tg, bigio eh, cairns nj, carrillo mc, dickson dw, duyckaerts c, frosch mp, masliah e, mirra ss, nelson pt, schneider ja, thal dr, thies b, trojanowski jq, vinters hv, montine tj (2012) national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease. alzheimers dement 8:1-13 230. mirra ss, heyman a, mckeel d, sumi sm, crain bj, brownlee lm, vogel fs, hughes jp, van belle g, berg l (1991) the consortium to establish a registry for alzheimer's disease (cerad). part ii. standardization of the neuropathologic assessment of alzheimer's disease. neurology 41:479-486 231. braak h, alafuzoff i, arzberger t, kretzschmar h, del tredici k (2006) staging of alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. acta neuropathol 112:389-404 232. jellinger ka (2009) criteria for the neuropathological diagnosis of dementing disorders: routes out of the swamp? acta neuropathol 117:101-110 233. jellinger ka (2009) a critical evaluation of current staging of alpha-synuclein pathology in lewy body disorders. biochim biophys acta 1792:730-740 234. hyman bt, trojanowski jq (1997) consensus recommendations for the postmortem diagnosis of alzheimer disease from the national institute on aging and the reagan institute working group on diagnostic criteria for the neuropathological assessment of alzheimer disease. j neuropathol exp neurol 56:1095-1097 235. bowler jv, munoz dg, merskey h, hachinski v (1998) fallacies in the pathological confirmation of the diagnosis of alzheimer's disease. j neurol neurosurg psychiatry 64:18-24. 236. beach tg, monsell se, phillips le, kukull w (2012) accuracy of the clinical diagnosis of alzheimer disease at national institute on aging alzheimer disease centers, 2005-2010. j neuropathol exp neurol 71:266-273 237. montine tj, monsell se, beach tg, bigio eh, bu y, cairns nj, frosch m, henriksen j, kofler j, kukull wa, lee eb, nelson pt, schantz am, schneider ja, sonnen ja, trojanowski jq, vinters hv, zhou xh, hyman bt (2016) multisite assessment of nia-aa guidelines for the neuropathologic evaluation of alzheimer's disease. alzheimers dement 12:164-169 238. plassman bl, khachaturian as, townsend jj, ball mj, steffens dc, leslie ce, tschanz jt, norton mc, burke jr, welsh-bohmer ka, hulette cm, nixon rr, tyrey m, breitner jc (2006) comparison of clinical and neuropathologic diagnoses of alzheimer's disease in 3 epidemiologic samples. alzheimers dement 2:2-11 239. cure s, abrams k, belger m, dell'agnello g, happich m (2014) systematic literature review and meta-analysis of diagnostic test accuracy in alzheimer's disease and other dementia using autopsy as standard of truth. j alzheimers dis 42:169-182 240. janocko nj, brodersen ka, soto-ortolaza ai, ross oa, liesinger am, duara r, graff-radford nr, dickson dw, murray me (2012) neuropathologically defined subtypes of alzheimer's disease differ significantly from neurofibrillary tangle-predominant dementia. acta neuropathol 124:681-692 241. jellinger ka (2014) alzheimer’s disease: current clinical and neuropathologic diagnostic criteria. austin alzheimers j parkinsons dis 1:6 242. murray me, cannon a, graff-radford nr, liesinger am, rutherford nj, ross oa, duara r, carrasquillo mm, rademakers r, dickson dw (2014) differential clinicopathologic and genetic features of late-onset amnestic dementias. acta neuropathol 128:411-421 243. tiraboschi p, sabbagh mn, hansen la, salmon dp, merdes a, gamst a, masliah e, alford m, thal lj, corey-bloom j (2004) alzheimer disease without neocortical neurofibrillary tangles: "a second look". neurology 62:1141-1147 244. hansen l, salmon d, galasko d, masliah e, katzman r, deteresa r, thal l, pay mm, hofstetter r, klauber m, et al. (1990) the lewy body variant of alzheimer's disease: a clinical and pathologic entity. neurology 40:1-8. 245. crary jf, trojanowski jq, schneider ja, abisambra jf, abner el, alafuzoff i, arnold se, attems j, beach tg, bigio eh, cairns nj, dickson dw, gearing m, grinberg lt, hof pr, hyman bt, jellinger k, jicha ga, kovacs gg, knopman ds, kofler j, kukull wa, mackenzie ir, masliah e, mckee a, montine tj, murray me, neltner jh, santa-maria i, seeley ww, serrano-pozo a, shelanski ml, stein t, takao m, thal dr, toledo jb, troncoso jc, vonsattel jp, white cl, 3rd, wisniewski t, woltjer rl, yamada m, nelson pt (2014) primary age-related tauopathy (part): a common pathology associated with human aging. acta neuropathol 128:755-766 246. jellinger ka, attems j (2007) neurofibrillary tangle-predominant dementia: comparison with classical alzheimer disease. acta neuropathol 113:107-117 247. besser lm, kukull wa, teylan ma, bigio eh, cairns nj, kofler jk, montine tj, schneider ja, nelson pt (2018) the revised national alzheimer's coordinating center's neuropathology form-available data and new analyses. j neuropathol exp neurol 77:717-726 248. jellinger ka, attems j (2015) challenges of multimorbidity of the aging brain: a critical update. j neural transm (vienna) 122:505-521 249. josephs ka, murray me, tosakulwong n, whitwell jl, knopman ds, machulda mm, weigand sd, boeve bf, kantarci k, petrucelli l, lowe vj, jack cr, jr., petersen rc, parisi je, dickson dw (2017) tau aggregation influences cognition and hippocampal atrophy in the absence of beta-amyloid: a clinico-imaging-pathological study of primary age-related tauopathy (part). acta neuropathol 133:705-715 250. jellinger ka (2018) different patterns of hippocampal tau pathology in alzheimer's disease and part. acta neuropathol 136:811-813 251. zhang l, jiang y, zhu j, liang h, he x, qian j, lin h, tao y, zhu k (2020) quantitative assessment of hippocampal tau pathology in ad and part. j mol neurosci 70:1808-1811 252. zhang x, sun b, wang x, lu h, shao f, rozemuller ajm, liang h, liu c, chen j, huang m, zhu k (2019) phosphorylated tdp-43 staging of primary age-related tauopathy. neurosci bull 35:183-192 253. jellinger ka, alafuzoff i, attems j, beach tg, cairns nj, crary jf, dickson dw, hof pr, hyman bt, jack cr, jr., jicha ga, knopman ds, kovacs gg, mackenzie ir, masliah e, montine tj, nelson pt, schmitt f, schneider ja, serrano-pozo a, thal dr, toledo jb, trojanowski jq, troncoso jc, vonsattel jp, wisniewski t (2015) part, a distinct tauopathy, different from classical sporadic alzheimer disease. acta neuropathol 129:757-762 254. jellinger ka (2016) commentary on the paper "part, a distinct tauopathy, different from classical 1. sporadic alzheimer disease". j clin cell immunol 7:1000480 255. bancher c, egensperger r, kosel s, jellinger k, graeber mb (1997) low prevalence of apolipoprotein e epsilon 4 allele in the neurofibrillary tangle predominant form of senile dementia. acta neuropathol 94:403-409 256. mcmillan ct, lee eb, jefferson-george k, naj a, van deerlin vm, trojanowski jq, wolk da (2018) alzheimer's genetic risk is reduced in primary age-related tauopathy: a potential model of resistance? ann clin transl neurol 5:927-934 257. hickman ra, flowers xe, wisniewski t (2020) primary age-related tauopathy (part): addressing the spectrum of neuronal tauopathic changes in the aging brain. curr neurol neurosci rep 20:39 258. besser lm, teylan ma, nelson pt (2020) limbic predominant age-related tdp-43 encephalopathy (late): clinical and neuropathological associations. j neuropathol exp neurol 79:305-313 259. nelson pt, dickson dw, trojanowski jq, jack cr, boyle pa, arfanakis k, rademakers r, alafuzoff i, attems j, brayne c, coyle-gilchrist its, chui hc, fardo dw, flanagan me, halliday g, hokkanen srk, hunter s, jicha ga, katsumata y, kawas ch, keene cd, kovacs gg, kukull wa, levey ai, makkinejad n, montine tj, murayama s, murray me, nag s, rissman ra, seeley ww, sperling ra, white iii cl, yu l, schneider ja (2019) limbic-predominant age-related tdp-43 encephalopathy (late): consensus working group report. brain 142:1503-1527 260. robinson jl, porta s, garrett fg, zhang p, xie sx, suh e, van deerlin vm, abner el, jicha ga, barber jm, lee vm, lee eb, trojanowski jq, nelson pt (2020) limbic-predominant age-related tdp-43 encephalopathy differs from frontotemporal lobar degeneration. brain 143:2844-2857 261. teylan m, besser lm, crary jf, mock c, gauthreaux k, thomas nm, chen yc, kukull wa (2019) clinical diagnoses among individuals with primary age-related tauopathy versus alzheimer's neuropathology. lab invest 99:1049-1055 262. murray me, graff-radford nr, ross oa, petersen rc, duara r, dickson dw (2011) neuropathologically defined subtypes of alzheimer's disease with distinct clinical characteristics: a retrospective study. lancet neurol 10:785-796 263. lehmann m, ghosh pm, madison c, laforce r, jr., corbetta-rastelli c, weiner mw, greicius md, seeley ww, gorno-tempini ml, rosen hj, miller bl, jagust wj, rabinovici gd (2013) diverging patterns of amyloid deposition and hypometabolism in clinical variants of probable alzheimer's disease. brain 136:844-858 264. rabinovici gd, furst aj, alkalay a, racine ca, o'neil jp, janabi m, baker sl, agarwal n, bonasera sj, mormino ec, weiner mw, gorno-tempini ml, rosen hj, miller bl, jagust wj (2010) increased metabolic vulnerability in early-onset alzheimer's disease is not related to amyloid burden. brain 133:512-528 265. iaccarino l, tammewar g, ayakta n, baker sl, bejanin a, boxer al, gorno-tempini ml, janabi m, kramer jh, lazaris a, lockhart sn, miller bl, miller za, o'neil jp, ossenkoppele r, rosen hj, schonhaut dr, jagust wj, rabinovici gd (2017) local and distant relationships between amyloid, tau and neurodegeneration in alzheimer's disease. neuroimage clin 17:452-464 266. ossenkoppele r, pijnenburg ya, perry dc, cohn-sheehy bi, scheltens nm, vogel jw, kramer jh, van der vlies ae, la joie r, rosen hj, van der flier wm, grinberg lt, rozemuller aj, huang ej, van berckel bn, miller bl, barkhof f, jagust wj, scheltens p, seeley ww, rabinovici gd (2015) the behavioural/dysexecutive variant of alzheimer's disease: clinical, neuroimaging and pathological features. brain 138:2732-2749 267. tetzloff ka, graff-radford j, martin pr, tosakulwong n, machulda mm, duffy jr, clark hm, senjem ml, schwarz cg, spychalla aj, drubach da, jack cr, lowe vj, josephs ka, whitwell jl (2018) regional distribution, asymmetry, and clinical correlates of tau uptake on [18f]av-1451 pet in atypical alzheimer's disease. j alzheimers dis 62:1713-1724 268. hwang j, kim cm, jeon s, lee jm, hong yj, roh jh, lee jh, koh jy, na dl (2015) prediction of alzheimer's disease pathophysiology based on cortical thickness patterns. alzheimers dement (amst) 2:58-67 269. whitwell jl, dickson dw, murray me, weigand sd, tosakulwong n, senjem ml, knopman ds, boeve bf, parisi je, petersen rc, jack cr, jr., josephs ka (2012) neuroimaging correlates of pathologically defined subtypes of alzheimer's disease: a case-control study. lancet neurol 11:868-877 270. jellinger ka (2012) neuropathological subtypes of alzheimer's disease (correspondence). acta neuropathol 123:153-154 271. mcaleese ke, walker l, graham s, moya elj, johnson m, erskine d, colloby sj, dey m, martin-ruiz c, taylor jp, thomas aj, mckeith ig, de carli c, attems j (2017) parietal white matter lesions in alzheimer's disease are associated with cortical neurodegenerative pathology, but not with small vessel disease. acta neuropathol 134:459-473 272. strain jf, smith rx, beaumont h, roe cm, gordon ba, mishra s, adeyemo b, christensen jj, su y, morris jc, benzinger tls, ances bm (2018) loss of white matter integrity reflects tau accumulation in alzheimer disease defined regions. neurology 91:e313-e318 273. arfanakis k, evia am, leurgans se, cardoso lfc, kulkarni a, alqam n, lopes lf, vieira d, bennett da, schneider ja (2020) neuropathologic correlates of white matter hyperintensities in a community-based cohort of older adults. j alzheimers dis 73:333-345 274. mcaleese ke, colloby s, attems j, thomas a, francis pt (2020) mixed brain pathologies account for most dementia in the brains for dementia research cohort (abstract). neuropathol appl neurobiol 46 (suppl. 1):24 275. mcaleese ke, mohi m, graham s, baker g, walker l, decarli c, koss d, attems j (2020) the aetiology of frontal white matter lesions in alzheimer’s disease are associated with both neurodegenerative and ischemic mechanisms (abstract). neuropathol appl neurobiol 46 (suppl. 1):15 276. ferreira d, verhagen c, hernandez-cabrera ja, cavallin l, guo cj, ekman u, muehlboeck js, simmons a, barroso j, wahlund lo, westman e (2017) distinct subtypes of alzheimer's disease based on patterns of brain atrophy: longitudinal trajectories and clinical applications. sci rep 7:46263 277. ferreira d, pereira jb, volpe g, westman e (2019) subtypes of alzheimer's disease display distinct network abnormalities extending beyond their pattern of brain atrophy. front neurol 10:524 278. risacher sl, anderson wh, charil a, castelluccio pf, shcherbinin s, saykin aj, schwarz aj (2017) alzheimer disease brain atrophy subtypes are associated with cognition and rate of decline. neurology 89:2176-2186 279. lowe vj, lundt es, albertson sm, min hk, fang p, przybelski sa, senjem ml, schwarz cg, kantarci k, boeve b, jones dt, reichard rr, tranovich jf, hanna al-shaikh fs, knopman ds, jack cr, jr., dickson dw, petersen rc, murray me (2020) tau-positron emission tomography correlates with neuropathology findings. alzheimers dement 16:561-571 280. galton cj, patterson k, xuereb jh, hodges jr (2000) atypical and typical presentations of alzheimer's disease: a clinical, neuropsychological, neuroimaging and pathological study of 13 cases. brain 123 pt 3:484-498 281. lam b, masellis m, freedman m, stuss dt, black se (2013) clinical, imaging, and pathological heterogeneity of the alzheimer's disease syndrome. alzheimers res ther 5:1 282. ahmed s, de jager ca, haigh am, garrard p (2012) logopenic aphasia in alzheimer's disease: clinical variant or clinical feature? j neurol neurosurg psychiatry 83:1056-1062 283. gorno-tempini ml, brambati sm, ginex v, ogar j, dronkers nf, marcone a, perani d, garibotto v, cappa sf, miller bl (2008) the logopenic/phonological variant of primary progressive aphasia. neurology 71:1227-1234 284. spinelli eg, mandelli ml, miller za, santos-santos ma, wilson sm, agosta f, grinberg lt, huang ej, trojanowski jq, meyer m, henry ml, comi g, rabinovici g, rosen hj, filippi m, miller bl, seeley ww, gorno-tempini ml (2017) typical and atypical pathology in primary progressive aphasia variants. ann neurol 81:430-443 285. weintraub s, teylan m, rader b, chan kcg, bollenbeck m, kukull wa, coventry c, rogalski e, bigio e, mesulam mm (2020) apoe is a correlate of phenotypic heterogeneity in alzheimer disease in a national cohort. neurology 94:e607-e612 286. crutch sj, schott jm, rabinovici gd, murray m, snowden js, van der flier wm, dickerson bc, vandenberghe r, ahmed s, bak th, boeve bf, butler c, cappa sf, ceccaldi m, de souza lc, dubois b, felician o, galasko d, graff-radford j, graff-radford nr, hof pr, krolak-salmon p, lehmann m, magnin e, mendez mf, nestor pj, onyike cu, pelak vs, pijnenburg y, primativo s, rossor mn, ryan ns, scheltens p, shakespeare tj, suarez gonzalez a, tang-wai df, yong kxx, carrillo m, fox nc (2017) consensus classification of posterior cortical atrophy. alzheimers dement 13:870-884 287. sahoo a, bejanin a, murray me, tosakulwong n, weigand sd, serie am, senjem ml, machulda mm, parisi je, boeve bf, knopman ds, petersen rc, dickson dw, whitwell jl, josephs ka (2018) tdp-43 and alzheimer's disease pathologic subtype in non-amnestic alzheimer's disease dementia. j alzheimers dis 64:1227-1233 288. rascovsky k, hodges jr, knopman d, mendez mf, kramer jh, neuhaus j, van swieten jc, seelaar h, dopper eg, onyike cu, hillis ae, josephs ka, boeve bf, kertesz a, seeley ww, rankin kp, johnson jk, gorno-tempini ml, rosen h, prioleau-latham ce, lee a, kipps cm, lillo p, piguet o, rohrer jd, rossor mn, warren jd, fox nc, galasko d, salmon dp, black se, mesulam m, weintraub s, dickerson bc, diehl-schmid j, pasquier f, deramecourt v, lebert f, pijnenburg y, chow tw, manes f, grafman j, cappa sf, freedman m, grossman m, miller bl (2011) sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. brain 134:2456-2477 289. sakae n, josephs ka, litvan i, murray me, duara r, uitti rj, wszolek zk, van gerpen j, graff-radford nr, dickson dw (2019) clinicopathologic subtype of alzheimer's disease presenting as corticobasal syndrome. alzheimers dement 15:1218-1228 290. josephs ka, whitwell jl, tosakulwong n, weigand sd, murray me, liesinger am, petrucelli l, senjem ml, ivnik rj, parisi je, petersen rc, dickson dw (2015) tar dna-binding protein 43 and pathological subtype of alzheimer's disease impact clinical features. ann neurol 78:697-709 291. ohm dt, fought aj, martersteck a, coventry c, sridhar j, gefen t, weintraub s, bigio e, mesulam mm, rogalski e, geula c (2020) accumulation of neurofibrillary tangles and activated microglia is associated with lower neuron densities in the aphasic variant of alzheimer's disease. brain pathol 292. jellinger ka, attems j (2007) neuropathological evaluation of mixed dementia. j neurol sci 257:80-87 293. kapasi a, decarli c, schneider ja (2017) impact of multiple pathologies on the threshold for clinically overt dementia. acta neuropathol 134:171-186 294. power mc, mormino e, soldan a, james bd, yu l, armstrong nm, bangen kj, delano-wood l, lamar m, lim yy, nudelman k, zahodne l, gross al, mungas d, widaman kf, schneider j (2018) combined neuropathological pathways account for age-related risk of dementia. ann neurol 84:10-22 295. rahimi j, kovacs gg (2014) prevalence of mixed pathologies in the aging brain. alzheimers res ther 6:82 296. matej r, tesar a, rusina r (2019) alzheimer's disease and other neurodegenerative dementias in comorbidity: a clinical and neuropathological overview. clin biochem 73:26-31 297. robinson jl, lee eb, xie sx, rennert l, suh e, bredenberg c, caswell c, van deerlin vm, yan n, yousef a, hurtig hi, siderowf a, grossman m, mcmillan ct, miller b, duda je, irwin dj, wolk d, elman l, mccluskey l, chen-plotkin a, weintraub d, arnold se, brettschneider j, lee vm, trojanowski jq (2018) neurodegenerative disease concomitant proteinopathies are prevalent, age-related and apoe4-associated. brain 141:2181-2193 298. thomas dx, bajaj s, mcrae-mckee k, hadjichrysanthou c, anderson rm, collinge j (2020) association of tdp-43 proteinopathy, cerebral amyloid angiopathy, and lewy bodies with cognitive impairment in individuals with or without alzheimer's disease neuropathology. sci rep 10:14579 299. boyle pa, yu l, wilson rs, leurgans se, schneider ja, bennett da (2018) person-specific contribution of neuropathologies to cognitive loss in old age. ann neurol 83:74-83 300. schneider ja, arvanitakis z, bang w, bennett da (2007) mixed brain pathologies account for most dementia cases in community-dwelling older persons. neurology 69:2197-2204 301. jellinger ka (2006) clinicopathological analysis of dementia disorders in the elderly--an update. j alzheimers dis 9:61-70 302. wang bw, lu e, mackenzie ir, assaly m, jacova c, lee pe, beattie bl, hsiung gy (2012) multiple pathologies are common in alzheimer patients in clinical trials. can j neurol sci 39:592-599 303. mcaleese ke, walker l, erskine d, johnson m, koss d, thomas aj, attems j (2020) concomitant late-nc in alzheimer's disease is not associated with increased tau or amyloid-beta pathological burden. neuropathol appl neurobiol online sep 8: doi 10.1111/nan.12664 304. mcaleese ke, walker l, erskine d, attems j (2020) the impact of concomitant late-nc on hyperphosphorylated-s pathology and cognitive decline in alzheimer's disease (abstract). neuropathol appl neurobiol 46 (suppl. 1):37 305. jang h, kim hj, sim choe y, kim sj, park s, kim y, woon kim k, hyoung lyoo c, cho h, hoon ryu y, choi jy, decarli c, na dl, won seo s (2020) the impact of amyloid-beta or tau on cognitive change in the presence of severe cerebrovascular disease. j alzheimers dis 306. malek-ahmadi m, perez se, chen k, mufson ej (2020) braak stage, cerebral amyloid angiopathy, and cognitive decline in early alzheimer's disease. j alzheimers dis 74:189-197 307. robinson ac, roncaroli f, chew-graham s, davidson ys, minshull j, horan ma, payton a, pendleton n, mann dma (2020) the contribution of vascular pathology toward cognitive impairment in older individuals with intermediate braak stage tau pathology. j alzheimers dis 77:1005-1015 308. gauthreaux k, bonnett ta, besser lm, brenowitz wd, teylan m, mock c, chen yc, chan kcg, keene cd, zhou xh, kukull wa (2020) concordance of clinical alzheimer diagnosis and neuropathological features at autopsy. j neuropathol exp neurol 79:465-473 309. tolnay m, sergeant n, ghestem a, chalbot s, de vos ra, jansen steur en, probst a, delacourte a (2002) argyrophilic grain disease and alzheimer's disease are distinguished by their different distribution of tau protein isoforms. acta neuropathol 104:425-434 310. togo t, cookson n, dickson dw (2002) argyrophilic grain disease: neuropathology, frequency in a dementia brain bank and lack of relationship with apolipoprotein e. brain pathol 12:45-52 311. wurm r, klotz s, rahimi j, katzenschlager r, lindeck-pozza e, regelsberger g, danics k, kapas i, bíró za, stögmann e, gelpi e, kovacs gg (2020) argyrophilic grain disease in individuals younger than 75 years: clinical variability in an underrecognized limbic tauopathy. eur j neurol may 13:doi: 10.1111/ene.14321 312. ferrer i, garcia ma, gonzalez il, lucena dd, villalonga ar, tech mc, llorens f, garcia-esparcia p, martinez-maldonado a, mendez mf, escribano bt, bech-serra jj, sabido e, de la torre gomez c, del rio ja (2018) aging-related tau astrogliopathy (artag): not only tau phosphorylation in astrocytes. brain pathol 28:965-985 313. kovacs gg (2020) astroglia and tau: new perspectives. front aging neurosci 12:96 314. wiersma vi, van ziel am, vazquez-sanchez s, nölle a, berenjeno-correa e, bonaterra-pastra a, clavaguera f, tolnay m, musters rjp, van weering jrt, verhage m, hoozemans jjm, scheper w (2019) granulovacuolar degeneration bodies are neuron-selective lysosomal structures induced by intracellular tau pathology. acta neuropathol 138:943-970 315. wiersma vi, hoozemans jjm, scheper w (2020) untangling the origin and function of granulovacuolar degeneration bodies in neurodegenerative proteinopathies. acta neuropathol commun 8:153 316. hou x, fiesel fc, truban d, castanedes casey m, lin wl, soto ai, tacik p, rousseau lg, diehl nn, heckman mg, lorenzo-betancor o, ferrer i, arbelo jm, steele jc, farrer mj, cornejo-olivas m, torres l, mata if, graff-radford nr, wszolek zk, ross oa, murray me, dickson dw, springer w (2018) ageand disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and lewy body disease. autophagy 14:1404-1418 317. koper mj, van schoor e, ospitalieri s, vandenberghe r, vandenbulcke m, von arnim caf, tousseyn t, balusu s, de strooper b, thal dr (2020) necrosome complex detected in granulovacuolar degeneration is associated with neuronal loss in alzheimer's disease. acta neuropathol 139:463-484 318. white lr, edland sd, hemmy ls, montine ks, zarow c, sonnen ja, uyehara-lock jh, gelber rp, ross gw, petrovitch h, masaki kh, lim ko, launer lj, montine tj (2016) neuropathologic comorbidity and cognitive impairment in the nun and honolulu-asia aging studies. neurology 86:1000-1008 319. jack cr, jr., bennett da, blennow k, carrillo mc, dunn b, haeberlein sb, holtzman dm, jagust w, jessen f, karlawish j, liu e, molinuevo jl, montine t, phelps c, rankin kp, rowe cc, scheltens p, siemers e, snyder hm, sperling r (2018) nia-aa research framework: toward a biological definition of alzheimer's disease. alzheimers dement 14:535-562 320. nelson pt, trojanowski jq, abner el, al-janabi om, jicha ga, schmitt fa, smith cd, fardo dw, wang wx, kryscio rj, neltner jh, kukull wa, cykowski md, van eldik lj, ighodaro et (2016) "new old pathologies": ad, part, and cerebral age-related tdp-43 with sclerosis (carts). j neuropathol exp neurol 75:482-498 321. ricci m, cimini a, chiaravalloti a, filippi l, schillaci o (2020) positron emission tomography (pet) and neuroimaging in the personalized approach to neurodegenerative causes of dementia. int j mol sci 21:7481 322. koychev i, hofer m, friedman n (2020) correlation of alzheimer disease neuropathologic staging with amyloid and tau scintigraphic imaging biomarkers. j nucl med 61:1413-1418 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. aβ plaques feel free to add comments by clicking these icons on the sidebar free neuropathology 1:31 (2020) review aβ plaques lary c. walker department of neurology and yerkes national primate research center, emory university corresponding author: lary c. walker · department of neurology · emory university · 505 whitehead biomedical research building · 615 michael st · atlanta, ga 30322 · usa lary.walker@emory.edu submitted: 27 september 2020 accepted: 23 october 2020 copyedited by: bert m. verheijen published: 30 october 2020 https://doi.org/10.17879/freeneuropathology-2020-3025 keywords: alzheimer’s disease, amyloid, neuritic plaques, neurofibrillary tangles, senile plaques abstract aβ plaques are one of the two lesions in the brain that define the neuropathological diagnosis of alzheimer’s disease. plaques are highly diverse structures; many of them include massed, fibrillar polymers of the aβ protein referred to as aβ-amyloid, but some lack the defining features of amyloid. cellular elements in ‘classical’ plaques include abnormal neuronal processes and reactive glial cells, but these are not present in all plaques. plaques have been given various names since their discovery in 1892, including senile plaques, amyloid plaques, and neuritic plaques. however, with the identification in the 1980s of aβ as the obligatory and universal component of plaques, the term ‘aβ plaques’ has become a unifying term for these heterogeneous formations. tauopathy, the second essential lesion of the alzheimer’s disease diagnostic dyad, is downstream of aβ-proteopathy, but it is critically important for the manifestation of dementia. the etiologic link between aβ-proteopathy and tauopathy in alzheimer’s disease remains largely undefined. aβ plaques develop and propagate via the misfolding, self-assembly and spread of aβ by the prion-like mechanism of seeded protein aggregation. partially overlapping sets of risk factors and sequelae, including inflammation, genetic variations, and various environmental triggers have been linked to plaque development and idiopathic alzheimer’s disease, but no single factor has emerged as a requisite cause. the value of aβ plaques per se as therapeutic targets is uncertain; although some plaques are sites of focal gliosis and inflammation, the complexity of inflammatory biology presents challenges to glia-directed intervention. small, soluble, oligomeric assemblies of aβ are enriched in the vicinity of plaques, and these probably contribute to the toxic impact of aβ aggregation on the brain. measures designed to reduce the production or seeded self-assembly of aβ can impede the formation of aβ plaques and oligomers, along with their accompanying abnormalities; given the apparent long timecourse of the emergence, maturation and proliferation of aβ plaques in humans, such therapies are likely to be most effective when begun early in the pathogenic process, before significant damage has been done to the brain. since their discovery in the late 19th century, aβ plaques have, time and again, illuminated fundamental mechanisms driving neurodegeneration, and they should remain at the forefront of efforts to understand, and therefore treat, alzheimer’s disease. 1. aβ, amyloid, and alzheimer’s disease the most striking and yet still enigmatic pathologic features of alzheimer’s disease (ad) are lesions known for over a century as senile plaques microscopic anomalies in the parenchyma of the brain consisting of an abnormal accumulation of protein decorated by various molecules, and often including dystrophic neuronal processes and reactive glial cells (figure 1). although plaques are a frequent feature of the senescent brain and, when particularly numerous, an obligatory diagnostic marker of ad [1], the identity of the principal protein in the plaque core remained unknown until the 1980s. then, glenner and wong established a partial amino acid sequence of the protein in cerebral amyloid angiopathy (caa) from patients with ad and down syndrome [2, 3], and masters and beyreuther [4, 5] determined that the same protein is a key component of plaques. initially referred to as the β protein, a4, or β/a4, the protein now is commonly designated aβ [6]. collectively, these lesions are increasingly referred to as aβ plaques (see section 3). figure 1. 'classical' aβ (senile) plaques in the cortex of persons who had died with alzheimer's disease (ad). left, a plaque stained with the naoumenko-feigin silver method and periodic acid-schiff (pas) counterstain; an amyloid core (dark pink) is surrounded by profuse abnormal neurites (black). right, a plaque immunostained with antibody 4g8 to the aβ protein (brown) along with a nissl counterstain (blue); glial nuclei are visible in the region between the plaque core and outer corona, and within and surrounding the corona. bar = 20μm for both panels. 1.1 aβ aβ is a cleavage product of the aβ-precursor protein (app), a 695–770 amino acid, single membrane-spanning protein that is strongly expressed in the nervous system [7, 8]. aβ is generated mainly in endosomes, and its release into the extracellular space is influenced by synaptic activity [9]. to produce aβ, app is sequentially cleaved by the enzymes β-secretase [or β-amyloid cleaving enzyme (bace)] and γ-secretase [8], resulting in aβ proteins that are most often 40 or 42 amino acids in length (‘aβ40’ and ‘aβ42’), although many c-terminally and n-terminally variant and/or chemically modified aβ fragments also occur [7, 10-16]. different lengths of aβ can derive from their differential excision from app by secretases or from post-translational trimming of aβ by exopeptidases [10]. potential post-translational chemical modifications of aβ include pyroglutamylation, racemization, isomerization, oxidation, phosphorylation, n-homocysteinylation, nitration, and glycosylation [11, 17-19] (see also section 7, below). how post-translational modifications influence the process of protein aggregation in general remains poorly understood [20, 21]. aβ40 is the isoform of aβ that is most abundantly generated by neurons, but two c-terminal hydrophobic residues in aβ42 augment its tendency to self-assemble into amyloid [7, 22]. as a result, more plaques are immunoreactive for aβ42 than for aβ40 (figure 2), although the relative amounts of plaques stained for aβ40 and aβ42 vary. figure 2. adjacent cortical tissue sections from an ad patient, immunostained with antibodies r398 to aβ42 (top) and r361 to aβ40 (bottom). two of the plaques that are present in both sections are denoted by arrows. asterisks mark a blood vessel for reference. bar = 100μm. unlike plaques, cerebral aβ-amyloid angiopathy (aβ-caa) in large vessels is more consistently positive for aβ40, though aβ42 also is generally present [23]. the staining patterns of the two isoforms differ in capillary aβ-caa compared to large-vessel aβ-caa [24, 25], and in the vessel wall compared to the diffuse aβ that sometimes extends from the wall into the surrounding parenchyma (dyshoric amyloid angiopathy) [25, 26]. the mechanisms governing the ontogeny of plaques and aβ-caa also probably differ to some extent (see section 5.3). in addition to plaques and amyloid angiopathy, aβ multimerizes into a range of oligomeric species [27, 28] that can interact with cells and impair brain function [27, 29-35]. oligomers appear to be an important intermediate step in the assembly of polymeric amyloid of all types [20]. comparison of subjects expressing ad-type dementia to nondemented subjects with high aβ plaque pathology, the amount of oligomeric aβ correlates more strongly with cognitive decline than does the number of plaques per se [36]. experimental studies indicate that aβ plaques include abundant oligomers [36, 37], and that some plaques shed toxic oligomers into the surrounding parenchyma [37-39]. aβ42-oligomers have been shown to arise from secondary nucleation on aβ-amyloid fibrils during protein aggregation, directly linking them to the process of amyloidogenesis [34]. at least some aβ-oligomers are particularly potent seeds for the formation of aβ plaques [40, 41], although whether there are seed-active oligomers that differ from toxic oligomers, as has been found for prions [42], is unknown. the relationship between aβ-oligomers and the diverse plaque types [31, 33, 38] in the human brain e.g., dense-core vs diffuse also is an issue that remains incompletely defined. indeed, owing to their dynamicity and heterogeneity, the analysis of oligomers as they occur in living systems is technically challenging [20] (see also benilova et al. [43] for a critique of oligomers as toxic agents). regardless of the relative contribution of aβ-oligomers and amyloid fibrils to disease, both of these multimeric states denote the presence of an abnormal condition in the brain, i.e., the misfolding and accumulation of the aβ protein. aβ has assumed a prominent position in alzheimer research because all identified risk factors for ad increase its quantity and/or tendency to aggregate [33, 44, 45]. most notably, mutations in app and the presenilins (components of the γ-secretase complex) [22] are the only known autosomal dominant causes of ad, and a superfluous app gene due to trisomic chromosome 21 in down syndrome frequently leads to early-onset ad [35, 46, 47]. furthermore, a rare mutation that substitutes a threonine for alanine (a673t) at position 2 of aβ lowers both the production of aβ [48] and its propensity to aggregate [49]; this mutation is associated with a reduced risk of manifesting ad [48] and possibly parenchymal plaques as well [50]. contrariwise, when a valine replaces alanine at position 2 (a673v), aβ generation is increased, and the protein is more prone to aggregate, resulting in an autosomal recessive form of ad [51]. thus, there is little doubt that aβ is intimately involved in the pathogenesis of ad, although many questions remain about how plaques per se participate in the neurodegenerative process. 1.2 amyloid a persistent source of misunderstanding regarding the role of aβ in ad is the common use of the generic term ‘amyloid’ to refer to the protein aβ. in pathology, amyloid refers to ‘mainly extracellular tissue deposits of protein fibrils, recognized by certain properties, such as green-yellow birefringence after staining with congo red’ [6] (for historical considerations of amyloid, see [20, 52-55], and for more on the definition of amyloid see [20, 56, 57]. amyloid can arise from over 30 different proteins in various parts of the body in different human diseases [6, 58]. hence, ‘aβ’ the molecule and ‘amyloid’ the fibrillar mass are not synonymous. aβ refers exclusively to the protein that, when aggregated into distinctive fibrils, constitutes the specific type of amyloid that most commonly accumulates in the aging brain. the formation of amyloid involves the misfolding and self-assembly of a particular protein into filamentous structures with a distinctive cross-β architecture that is stabilized by a ‘steric zipper’ molecular motif [20, 59]. the misfolded protein has two notable characteristics that contribute to its amyloidogenicity: 1) it compels unfolded molecules of the same protein to similarly misfold by means of permissive templating [60]; and 2) the β-sheets in separate molecules hydrogen-bond to one another to form stable, filiform polymers with the β-sheets oriented perpendicular to the long axis of the polymer [59]. in this way, the misshapen proteins both corrupt and capture like proteins, which stack into protofilaments that wind together to build long, non-branching fibrils that typically range from ~6 to 13 nm in diameter [54]. these fibrils are characteristic of amyloid in general [6]. despite their shared cross-β backbone and similar appearance by conventional transmission electron microscopy, however, amyloid fibrils are polymorphic at the molecular level [20, 61-68]. although amyloid was long defined as an exclusively extracellular substance [69-71], it is now recognized to occur intracellularly as certain types of inclusion [6, 20]. the tau protein that polymerizes into neurofibrillary tangles the second mandatory pathologic hallmark of ad (figure 3) has attributes of amyloid [72]. thus, the two lesions that characterize ad pathologically plaques and tangles arise from two different proteins aβ and tau both of which can misfold and self-assemble into amyloid. figure 3. an aβ plaque (brown) alongside intracellular tau tangles (purple) in the cortex of an ad patient. combined polyclonal antibodies r398+r361 to aβ40+42 plus monoclonal antibody cp13 to hyperphosphorylated tau. bar = 20μm. despite genetic, biomarker and pathologic findings implicating aberrant aβ in the initiation of ad [9, 33, 44], tauopathy is more strongly correlated with cognitive decline than are plaques [73-78]. in the forebrain, tangles first appear in the medial temporal lobe [79, 80], but the dementia of ad is fully apparent only when tauopathy becomes severe in much of the neocortex [1, 81], a process that is facilitated by the presence of aβ pathology [9] (see also [82]). the precise nature of the mechanistic link between aβ-proteopathy and tauopathy in ad, however, remains a critical unsolved problem [45, 83, 84]. 2. the discovery and early exploration of plaques the late 19th and early 20th centuries saw a profusion of new staining methods that selectively revealed various elements in cells and tissues [85]. accordingly, the original depictions of plaques reflected what was disclosed by histologic stains and viewed with the light microscope. in 1892, paul blocq and georges marinesco at the salpêtrière hospital in paris reported microscopic ‘amas ronds’ (‘round clusters’, or ‘round heaps’) or ‘nodules de sclérose névroglique’ (‘nodules of neuroglial sclerosis’) in the brains of older epileptic patients [86].1 this report is generally considered to be the first unambiguous identification of plaques in the senescent brain [81, 87]. in 1898, emil redlich published evidence linking plaques to dementia [88]; in the brains of three elderly subjects, two of whom had died with clinically confirmed dementia, redlich described the structures as consisting of a core of uncertain substance along with surrounding astrocytes (‘spinnenzellen’) and their processes. because they resembled millet seeds under the microscope, he referred to these collective lesions as ‘miliary sclerosis’ (‘miliare sklerose’). notably, redlich also dubbed them ‘plaques’, a term that was expanded to ‘senile plaques’ by simchowicz in 1911 [89]. furthermore, redlich noted that some smaller lesions consisted of fine fibers with a cotton-like appearance [88], anticipating the use of the term ‘cotton-wool plaques’ to depict certain types of deposit today [90-94]. although alois alzheimer is often credited with instigating the burst of scientific analyses of plaques with his 1906 conference presentation in tübingen (published in 1907) [95], his report was brief, and plaques (‘miliary foci’) were only superficially mentioned.2 he did not issue his first detailed histopathologic examination of plaques until 1911 [96]. in fact, along with redlich [88], a good case can be made that oskar fischer deserves the credit for initiating the modern histopathologic analysis of dementia with a comprehensive series of reports published in 1907, 1910, and 1912 [87]. several other researchers contributed to the literature on plaques during this period, including, among others, miyake [97], lafora [98], bonfiglio [99], hübner [100], perusini [101], fuller [102], bielschowsky [103], barrett [104], simchowicz [105] and marinesco and minea [106] (see also christen [107] for a brief historical perspective on this period of research into what we now call ad). both alzheimer and fischer excelled in their analysis of plaques by implementing a silver-based staining method introduced by max bielschowsky [87] (see braak and braak [108] for a nice summary of the early development of silver stains).3 alzheimer did, however, correctly anticipate the evolution of neurology in his 1907 publication, in which he contended that the time had come to define neurologic diseases based on both their clinical and histologic characteristics [95].4 this view has a contemporary parallel in the call by an international group of experts for a biological, rather than syndromic, definition of ad [109]. furthermore, alzheimer noted in 1911 the prevailing technical inability to identify the substance in the plaque core: ‘... we have to consider the core of the plaque as an unorganized mass which emerges differently with different staining methods ... as perusini and fischer have already explained, we are not at present able to identify this mass with any of the substances known in pathological anatomy’ (translation from [110]). in addition, alzheimer highlighted the prominence of glial cells in the composition of plaques [96], a subject that has gained momentum in the 21st century, owing in part to the identification of compelling, glia-related genetic risk factors for ad [111-115] (see section 6.2). from the early 20th century on, researchers widely agreed that the main structural elements comprising plaques are abnormal neuronal processes, altered glial cells, and a central, disordered mass of unidentified material. in a 1929 review, macdonald critchley [116] wrote that the ‘modern conception of the plaque is that of a reactionary change directed against a specific metabolic process of a toxic nature’ (a description that, if we consider the material in the core to be the key toxic substance, resonates with leading 21st century concepts). many pioneering scientists attempted to explain the origin and nature of plaques based on their interpretations of static images in selectively stained tissue sections. not surprisingly, disagreement was common (see, e.g., [99], [101], [102], [117]). ferraro (1931) summarized this lack of consensus: ‘...one group of investigators favors the theory that [the plaque] originates from nerve cells, another that it originates from neuroglial elements, another from axis cylinders, and still another, from the intercellular ground substance’ [118]. soniat remarked in 1941 that ‘no less than twenty different concepts concerning their origin have appeared in the literature’ [119]. as late as 1960, liss wrote, citing three influential textbooks on pathology, that the ‘morphogenesis of senile plaques remains still an unsettled and controversial matter’ [120]. a crucial question, and a source of much of the discord among researchers, was the nature of the plaque core what does it consist of, how does it arise, what impact does it have, and what governs the proliferation of plaques in the brain? the answers to these questions would not begin to emerge for another half century. in fact, no compelling conceptual insights immediately followed the initial flurry of histopathological investigations of plaques, which, ultimately, were hampered by limitations in the available methods [119, 121]. beginning in the 1960s, theoretical and analytical advances accelerated; electron-microscopic studies showed that the mature plaque core consists of amyloid fibrils structurally similar to those in corporeal amyloidoses [122, 123], and quantitative analyses confirmed that plaque load in the brain is linked to dementia [124]. most important, however, was the molecular decipherment of aβ as the primary protein in cerebral amyloid by glenner and wong [2, 3] and masters and colleagues [4]. the genetic insights and technical tools resulting from this discovery ultimately established aβ as a critical player in the pathogenesis of ad, and the plaques that occur in normal aging and ad could, for the first time, be unified by a single, omnipresent component aberrant aβ. 1 ‘il existe de plus, disséminés dans les diverses couches de l’écorce, de petits amas ronds d’un diamètre de 60 µ environ, se distinguant du reste du tissu par une coloration beaucoup plus intense, à contours réguliers. ils apparaissent ainsi, parsemant discrètement le fond des préparations, d’une structure vaguement pointillée, ce pourquoi il est permis de considérer quelques-uns d’entre eux, au moins, comme de véritables nodules de sclérose névroglique (?).’ (question mark is in the original) 2 ‘über die ganze rinde zerstreut, besonders zahlreich in den oberen schichten, findet man miliare herdchen, welche durch einlagerung eines eigenartigen stoffes in die hirnrinde bedingt sind. er lässt sich schon ohne färbung erkennen, ist aber färbungen gegenüber sehr refractär.’ 3 many different silver stains have been developed to detect ad pathology. each method selectively reveals certain elements in the plaques, and they are sometimes considered to be less sensitive than is immunostaining with antibodies to aβ. some silver stains, however, are exquisitely sensitive even to small, diffuse aβ deposits, which have been recognized in ad tissue since the early 20th century (see, e.g., marinesco and minea [1912] [reference 106] and cowe, a. [1915] [reference 508]). note also figure 23. 4 ‘es gibt ganz zweifellos viel mehr psychische krankheiten, als sie unsere lehrbücher aufführen. in manchen solchen fällen wird dann eine spätere histologische untersuchung die besonderheit des falles feststellen lassen. dann werden wir aber auch allmählich dazu kommen, von den großen krankheitsgruppen unserer lehrbücher einzelne krankeiten (sic) klinisch abzuscheiden und jene selbst klinisch schärfer zu umgrenzen.’ 3. plaque nomenclature: the case for ‘aβ plaques’ the term ‘plaque’ (which historically has referred to a flat object such as a disk or tablet) was adopted by the medical community in the mid-to-late late 1800s to designate patch-like abnormalities such as atherosclerotic plaque or dental plaque.5 redlich [88] used the term to describe carmine-stained densities (‘intensiv gefärbten plaques’), and simchowicz [105] added the modifier ‘senile’ to denote their frequency in senescent brains, particularly in patients with senile dementia [89]. most plaques in the brain (unlike dental or atherosclerotic plaque) are not planar (one exception being the band-like subpial deposits [see figure 9]). of course, spheroidal plaques appear discoid in histologic sections, and their apparent size and composition are influenced by the plane through which they are cut (figure 4). figure 4. a neuritic aβ plaque in consecutive sections of the cortex from an ad patient; the core is evident in the left-hand image, whereas sections through the periphery (middle and right) reveal only neurites (black). serial sections may be required to unequivocally identify plaque types (a technical caveat noted by, among others, alzheimer [1911] [reference 96]). naoumenko-feigin (silver) and periodic-schiff stains. bar = 20 μm for all images. some of the designations for plaques derive from their staining characteristics. following divry’s discovery that certain plaques show amyloid-type birefringence after staining with the dye congo red [125], the terms ‘congophilic plaques’ or ‘amyloid plaques’ became common. the term ‘argyrophilic plaques’ also has been employed, owing to their detectability by various silver-based staining methods [81]. other labels such as ‘miliary plaques’, ‘drusen’,6 and ‘redlich-fischer plaques’ can be found in the earlier literature [116]. in 1972, wisniewski and terry introduced the term ‘neuritic plaques’ in recognition of the profusion of abnormal neuronal processes that invest many plaques. with the identification of the aβ protein in plaques, the term ‘aβ plaques’ is increasingly common. for the following reasons, ‘aβ plaques’ is recommended as the inclusive term that succinctly encompasses the multiplicity of these lesions under the umbrella of their shared feature aβ deposition:7 1) aβ is present in all of the plaques that are linked to ‘normal’ aging and ad, regardless of size, shape, aggregation state, location, or overall composition. 2) the term ‘senile’ is vague and arbitrary, and not all plaques occur in ‘senile’ humans. although aβ plaques become more common at older ages, they can emerge in the 4th decade of life or earlier, especially in people with some autosomal dominant forms of ad [126].8 3) plaques that are structurally similar to aβ plaques occur in other neurodegenerative disorders, yet these result from the misfolding and aggregation of different proteins. such plaque-forming proteins include the prion protein (prp) in certain spongiform encephalopathies [127, 128], the abri protein in familial british dementia [129, 130], and the adan protein in familial danish dementia [131, 132]. 4) not all aβ deposits incorporate abnormal neurites, which often are sparse or absent in diffuse plaques [133] including cotton-wool plaques [90, 93, 134] (see below). the term ‘neuritic plaques’ is suitable for the lesions that contain neurites, but these are only a subset of the entire family of aβ plaques. 5) the aβ in plaques does not always meet all of the criteria for amyloid [6] (see section 1.2). many diffuse aβ deposits in the aging brain do not show birefringence after staining with congo red. in addition, large, cotton-wool aβ plaques lacking amyloid cores are abundant in certain presenilin-1 mutant cases of autosomal dominant ad [90, 91, 93, 94, 134] and in some non-familial cases [92]. (the aβ in non-amyloid plaques from some presenilin-1 mutant cases is unusual in that it consists mostly of n-terminally truncated aβ [94], as do diffuse deposits in the cerebellum in ad [135] and down syndrome [135, 136]). the term ‘amyloid plaques’, like ‘neuritic plaques’, is appropriate for a subgroup of the lesions, but the universal constituent is aβ, whether it is in the form of amyloid or not; hence, more precise designations of plaque subtypes would be, for example, ‘aβ-amyloid plaques’ and ‘neuritic aβ plaques’. note that ‘diffuse plaques’ here refers to the fact that the aβ accumulation is ‘widely spread or scattered; not concentrated’ [137], without consideration of the nature of the aβ deposits, e.g., thread-like or punctate. ‘diffuse’ thus denotes only the characteristics of the aβ deposits, and not the dysmorphic neurites or any other component of the plaques. also, when analyzing aβ plaques histologically, it is useful to be cognizant of the plane of section, thickness of the tissue, and the limitations of a given staining protocol. plaques are 3-dimensional structures that, when large enough, are only partially captured in thin histologic sections (figure 4). furthermore, different stains recognize different components of plaques. consequently, a comprehensive assessment of plaques requires their full reconstruction and the application of suitable markers for potential components. in congruence with the trend to define ad according to its molecular underpinnings [109], defining the plaques that occur in aging and ad based on their principal proteinaceous component unambiguously distinguishes them from similar lesions in other disorders. in addition, this molecularly grounded moniker explicitly specifies the attribute that defines these plaques as unique pathologic entities: the misfolding and abnormal accumulation of the aβ protein. 5 ‘plaque (subject: medicine and health): any small patch or region of abnormal tissue within the body. see amyloid plaque, gliosis. [from french plaquer to plate, from middle dutch placken to beat metal].’ from: oxford dictionary of word origins: https://www.oxfordreference.com/view/10.1093/acref/9780199547920.001.0001/acref-9780199547920 6 note that ‘druse’ (‘geode’) differs from ‘drüse’ (with umlaut), which refers to a ‘gland’. 7 because ‘aβ’ and ‘plaque’ are both nouns, they could be connected by a hyphen (aβ-plaque). i have chosen not to include the hyphen (the ‘open form’) in order to simplify usage. in some cases (such as aβ-caa & aβ-oligomers), i have retained the hyphen for clarity. 8 w.h. mcmenemey opined in 1963: ‘...the structures first observed by blocq & marinesco (1892) and thought by them to be nodules of glial sclerosis were called by simchowicz (1910) ‘senile plaques’ an unfortunate choice of name for it has coloured our thinking for the past fifty years’ [reference 509]. 4. the anatomic distribution of aβ plaques 4.1 histology determination of the amino acid sequence of aβ [2-4] prompted the development of sensitive and specific antibodies that have facilitated the investigation of the anatomic localization, structural diversity, and biochemical composition of aβ deposits in the brain. aβ plaques become increasingly frequent as age advances [80, 138], but they are especially numerous in ad patients. the anatomic distribution of aβ plaques is variable, and it differs both among individuals and among brain regions in a given person [139-141] (figure 5). in general, association areas of the neocortex are more vulnerable and/or affected earlier than are primary motor and sensory areas [140]. aβ deposition is particularly profuse in the default mode network, an interconnected assemblage of brain regions that maintain vigorous metabolic activity when the brain is in an otherwise resting state [142]. the structure of aβ plaques is influenced in part by the architectonic characteristics of the areas in which they form [139, 143], but it is usual for several kinds of plaque to intermix within a given site (figure 6). in the neocortex, the laminar distribution of diverse aβ plaques can vary markedly [140] (figure 5). figure 5. variation in aβ deposition in adjacent cortical gyri from an ad patient. antibody 4g8, nissl counterstain. bar = 500μm. figure 6. variable morphology of aβ plaques in the cortex of an ad patient. classical dense-cored plaques with the core-space-corona pattern are in the upper left and lower right, and an irregular cloud of diffuse material is near the center, along with numerous very small patches. antibody 4g8; nissl counterstain. bar = 50μm. based on an analysis of human brains with different degrees of plaque accumulation, a spatiotemporal course of aβ plaque formation has been proposed [19, 144, 145]. there is general agreement that diffuse plaques are the earliest type to emerge, followed later by cored (amyloid) plaques [146]. according to thal and colleagues, in the first phase of the process, diffuse aβ plaques appear in the neo(iso)cortex; in the second phase, allocortex, the hippocampal formation and amygdala are affected; in the third phase, plaques arise in the basal ganglia and diencephalon; in the fourth phase they appear in the midbrain and medulla oblongata; and in the fifth phase, the pons and cerebellum are affected [19, 144, 145] (figure 7). these stages have been consolidated by serrano-pozo and colleagues [133] into an isocortical stage 1, allocortical/limbic stage 2, and subcortical stage 3. this general pattern of spread has been confirmed by a cross-sectional in vivo analysis of aβ-amyloid deposition profiles using florbetapir-pet imaging [147]. thus, in the end-stage of ad, most brain areas exhibit at least some aβ deposition. the spinal cord has been less studied; while it appears to be largely spared, plaques there have been reported in some instances [93, 148]. figure 7. the phases of aβ plaque distribution in the brain [references 19, 145]; illustration courtesy of dietmar thal, ku leuven. 4.2. in vivo imaging at the turn of the 21st century, the first imaging agents were introduced to detect amyloid in the living human brain via positron emission tomography [149, 150]. jorge barrio and colleagues introduced 2-(1-[[6-[(2-[18f]fluoroethyl)(methyl)amino]-2-naphthyl]]ethylidene) malononitrile ([18f]fddnp), which binds to both aβ-amyloid and tau tangles, and which has achieved some utility in diagnosing tauopathies [151-153]. a more aβ-selective ligand, developed by william klunk, chester mathis and colleagues, is 2-(4’-[11c]methylamino-phenyl)-6-hydroxybenzothiazole (pittsburgh compound-b [pib]) [149, 154]. derived from the chemical structure of the histologic staining agent thioflavin-t, pib crosses the blood-brain barrier and binds with high affinity and selectivity to aβ deposits in plaques and caa [154].9 pib (which is labeled with carbon-11), was followed by similar pet ligands labeled with fluorine-18 (a radiolabel with a longer half-life than carbon-11): florbetapir (amy-vid) [155, 156], florbetaben (neuraceq) [157], and flutemetamol (vizamyl) [158]. by assessing aβ-amyloid load in living subjects, these imaging agents have facilitated the differential diagnosis of ad and the longitudinal tracking of aβ accumulation. they are particularly sensitive in detecting dense-core aβ plaques, although they also bind to some extent to aβ-caa and diffuse aβ deposits [47, 159-161]. histochemical analysis of fluorescently labeled (‘clicked’) pib applied to ad tissue sections confirms the preference of pib at low concentration (100nm) for dense-core plaques [162]. interestingly, pib does not show significant high-affinity binding to aβ-amyloid deposits in aged nonhuman primates with substantial aβ burden [163], even though the amino acid sequence is identical to that of humans (see section 10). (note that binding of ligands can vary among humans; for example, a case of end-stage ad has been reported with extraordinarily high aβ load, a predominance of aβ40, and minimal high-affinity binding of pib [164]). since neither ad-like tauopathy nor dementia has been reported in nonhuman primates [165], comparative analysis of ligand binding could be useful in defining the variant molecular characteristics of aβ deposits and their relationship to disease phenotype (see sections 5.2 and 10). 9 in the 1920s, congo red was introduced as an in vivo diagnostic agent for non-cerebral amyloidosis. following intravenous injection, the rate at which congo red was cleared from the blood was thought to reflect amyloid burden in affected organs (the more amyloid to bind the dye, the more rapid its clearance from blood). for various reasons, the test never achieved widespread use (see buxbaum and linke [2012] [reference 52]). 5. the variety of aβ deposits 5.1 aβ plaques the histologic implementation of specific antibodies in the 1980s firmly established that aβ plaques in the brains of alzheimer patients comprise a remarkable variety of morphologies [143, 166-172]. several modern classification schemes have been proposed (e.g., [143, 166, 173-176]), and while there is not universal agreement on some of the terms, aβ plaques can be broadly categorized into amyloid plaques per se (with dense, congophilic cores), and a range of more loosely organized deposits of myriad sizes, shapes, densities and locations [133] (figures 5, 6, 8, 9). it is noteworthy that different genetic mutations can be associated with particular predominant plaque morphologies, as well as the presence of caa (see alzforum for a list of alzheimer-associated mutations (https://www.alzforum.org/mutations). note also that relatively few of the mutant forms of ad have been thoroughly scrutinized neuropathologically. within the general categories of plaque structure, the aβ-amyloid plaques are more or less spheroidal lesions that include ‘classical’ or ‘mature’ plaques and so-called ‘burned-out’ or ‘compact’ plaques [177, 178]. recently, a ‘coarse-grain’ plaque type with multiple small cores and a predominance of aβ40 has been described in advanced ad cases, often in association with apoe4 homozygosity and caa [179]. diffuse aβ plaques are much more numerous than are amyloid plaques in the alzheimeric brain [143] (figures 6, 8), and they span a range of compactness from vaguely aβ-immunoreactive, congo red-negative regions (e.g., ‘fleecy’ plaques [180]) to clusters of loose fibrillar material that sometimes are weakly congophilic [139, 166]. ultrastructurally, some of these diffuse deposits have been shown to include amyloid fibrils [181-183], whereas others do not [183], the latter possibly representing a pre-amyloid stage of aβ aggregation [139]. figure 8. small, often stellate aβ deposits in the cortex of an ad patient. some aβ accumulates within glial cells, most likely astrocytes (right). antibody 4g8; nissl counterstain. bars = 20μm. figure 9. band-like subpial aβ (left) in neocortical layer 1 and presubicular lake-like aβ (right) from two cases of ad. the subpial aβ can be discontinuous, confluent, or punctate. antibodies 4g8 (left) and 6e10 (right); nissl counterstain. bar = 100μm for both images. diffuse aβ plaques comprise very small, often stellate assemblies scattered about the parenchyma (figure 8), a sheet-like band of sometimes confluent, sometimes patchy material in the subpial cortex (figure 9), large ‘cotton-wool’ plaques, and very large ‘lake-like’ patches, including a distinctive cribriform deposit in the subicular complex [143, 171, 176, 184] (figure 9). abnormal neurites generally are absent or sparse in diffuse deposits [139], and this includes the cotton-wool plaques that are characteristic of some advanced ad cases [90-94, 134]. despite their abundance in the alzheimeric brain, very small diffuse deposits have received remarkably little scientific attention [175]. these probably correspond to the small (~2μm diameter) ‘sternchen’ which fischer in 1910 considered to be the first stage of plaque formation [185]. at least some of them appear to be related to astrocytes [175, 186] (figure 8), but the absence of systematic research on these ubiquitous lesions currently precludes meaningful consideration of their involvement in the proteopathic process. similarly, the immunoreactivity of some vestigial (extracellular) neurofibrillary tangles with antibodies to aβ [187-194] (figure 10) remains mechanistically undefined. figure 10. neurofibrillary tangles in the cortex of an ad patient immunostained with an antibody to aβ40. when present, this colocalization occurs mostly on extracellular ('ghost') tangles. nissl counterstain. bar = 50μm. certain types of aβ plaque are typical of the brain compartments in which they develop, e.g., in the cerebellum, basal ganglia, or different cortical regions and laminae (see [139]). in the white matter, distinctive granular accumulations of aβ [143] occur to varying degrees (figure 11). these clusters consist of fibrillar aβ lying outside of the axons, and they appear not to be associated with obvious tauopathy or other abnormalities of the axons themselves [143], although their functional significance is largely unexplored. the core-space-corona arrangement of aβ is a notable structural feature of classical aβ plaques that was noted in several early investigations (reviewed in [116, 119, 120]). these subdivisions of plaques have been given various designations in the early literature, for instance zentrum or kern, hof, ring, etc.10 in tissue that has been immunostained for aβ, classical aβ plaques have a condensed core of aβ-amyloid surrounded by an optically clear region with little aβ, and then an outer corona of more diffuse aβ [195] (see figure 1); the relatively clear intermediate space and the outer corona are occupied by neuronal and glial elements (which are considered in more detail in section 6). figure 11. aβ deposits in white matter of an ad patient comprise clusters of small puncta and filamentous bundles. left: light-micrograph of a cluster immunolabeled with antibody 4g8 (nissl counterstain). right, electron micrograph of a punctum immunolabeled with antibody 4g8 (black dots are gold particles bound to the secondary antibody). bars = 20μm (left) and 200nm (right). viewed in the electron microscope, aβ-amyloid fibrils in the plaque core are densely packed and often bundled to form a patchy matrix, and viable cellular processes there are largely absent. the more loosely organized aβ-amyloid sheaves in the space and corona interdigitate with cellular elements such as glial processes and neurites (figure 12; see also figures 18 and 20). embedded in the fibrillar meshwork of amyloid in plaques, various small, spherical particles can be seen (figure 13). the origin and significance of this material is obscure, but it could account for some of the non-aβ substances that have been detected in the cores of aβ plaques (see section 7). one possibility is that these vesicles originate from intracellular multivesicular bodies, which have been shown experimentally to be an important site of app/aβ biology [196-201]. in this regard, vesicular structures ranging from 50 to 300nm in diameter have been reported among the amyloid fibrils in a cell culture model of aβ amyloid deposition [202]. the center of the compact core in some aβ-amyloid plaques is refractory to aβ-immunostaining (figure 14), even though it is positive for the amyloid-selective dyes thioflavin-s and congo red [203]. ultrastructural analysis indicates that the material in the center of fully developed plaques often has a more granular, amorphous appearance (figure 13) than the obvious fibrils in the mantle of the core and in the periphery. classical aβ-amyloid plaques are often ascribed special relevance to neurodegeneration [1, 204], as they are much more likely to involve neuritic malformation and reactive gliosis than are the diffuse deposits [133]. in this regard, it is noteworthy that cognitively normal elderly subjects with abundant aβ plaques tend to have mostly diffuse plaques [1] with few neurites and little glial reactivity [139]. however, as noted above, there are rare cases of advanced ad in which classical plaques or dense-cored plaques are infrequent [90-93], suggesting that amyloid per se is not essential to the development of dementia. a similar situation holds for prion diseases, all of which are linked to the misfolding and self-assembly of prp [205, 206]; in some prionoses (such as gerstmann-sträussler-scheinker syndrome and new-variant creutzfeldt-jakob disease), prp-amyloid plaques can be numerous, whereas in others, little if any amyloid is present [127]. in these instances, oligomeric species of the proteins may have particular importance [20], although this has not been definitely established. figure 12. ultrastructure of fibrillar aβ in the plaque corona (left) and core (right) in an ad patient. bar = 500nm for both images. a small proportion of aβ-amyloid plaques lack the outer corona and have few or no neurites; these relatively plain structures have been thought to represent an end-stage in the evolution of plaques, and so were dubbed ‘burned out’ plaques [143, 178]. based on their apparent sequential appearance ance in the ad brain, a progression has been proposed in which plaques originate as diffuse (‘primitive’ or ‘immature’) deposits that evolve into classical (or ‘mature’) aβ plaques and then finally into burned-out plaques [143].11 while longitudinal studies in mouse models of cerebral aβ accumulation have begun to shed light on the time-course of plaque development (see section 10.2), the order of events in the human brain is still speculative [207]. figure 13. high-magnification electron micrograph of a portion of the core of an aβ-amyloid plaque in an ad patient. the fibrillarity of the material is less evident than in more peripheral zones. unidentified particles (2 are marked by arrows) of various sizes and densities are interspersed among the amyloid fibrils; these can be found both in the core and corona. bar = 200nm. figure 14. aβ plaque with an antibody-refractory central core in an ad patient. antibody 6e10; nissl counterstain. bar = 20μm. biochemical determination of the age of aβ deposits indicates that the amyloid core is older than the diffuse aβ in the corona and in diffuse plaques [208, 209]. armstrong [173] has suggested that the major plaque types mostly arise independently, rather than in an evolutionary progression. in any case, the transformation of diffuse plaques into compact amyloid might not be an inevitable occurrence; for instance, it appears that diffuse aβ deposits such as the lake-like cloud of aβ in the subicular complex (figure 9) do not progress into dense masses of amyloid, and this may be true also for cotton-wool plaques in ad cases with certain presenilin-1 mutations [90, 91, 93]. finally, it should be emphasized that the relative pathogenicity of the many different aβ plaque types in the aging human brain remains ambiguous. it is fairly certain that reactive gliosis/inflammation and the local disruption of neuronal processes in classical aβ plaques are deleterious to brain function (see section 6), but it is likely that oligomeric agents are the more directly injurious manifestation of misfolded proteins (see section 1.1). in fact, while the plaques themselves are indicative of a pathogenic molecular process, in and of themselves they may be relatively benign or even protective [210, 211], at least when inflammation and surrounding oligomers are negligible (see sections 1.1 and 6). 10 fischer [1910] [reference 185] referred to the central core as the ‘morgenstern’ (morning star), and described the structure of one type of plaque thusly: ‘auch hier ist ein zentraler morgenstern, aus dem mehr oder weniger lange büschel entspringen; der fädchenring ist ziemlich weit vom zentrum entfernt, so dass ein grosser hof entsteht, der von den strähnchen durchzogen wird’ 11 diffuse plaques have long been considered an early stage in plaque formation (see, e.g., critchley [1929] [reference 116]). 5.2 aβ strains in ad, the diverse morphological attributes of plaques might reflect, in addition to the local tissue organization, the variable truncation, folding, and molecular architecture of aβ [212, 213]. these variants are referred to as proteopathic strains, a biological concept that was adopted by the spongiform encephalopathy community to explain the alternative disease phenotypes resulting from prion infections [214, 215]. at the molecular level, the formation and propagation of aβ aggregates (as well as the proteins involved in several other proteopathies [216]), constitute a mechanism that is fundamentally similar to that of prions [217, 218] (see section 9). the capacity to spawn distinct strains is considered to be a shared property of proteins that are prone to misfolding and self-assembly [56, 59, 219]. in vitro, a given protein can create morphologically diverse amyloid fibrils under different environmental influences, for example temperature, ph, ionic strength, protein concentration [220, 221] and agitation [67, 222]. strain properties can be conveyed to newly forming amyloid fibrils; in vivo, it is thought that proteopathic strains undergo conformational selection by which the strain best suited to a given environment predominates [215, 220, 223, 224]. studies in genetically modified mouse models (which can be customized to make various types of aβ) can shed light on the factors that govern alternative plaque morphologies in the living brain [225]. the generation of aβ strains is influenced by characteristics of the aggregating aβ such as mutations, truncations and chemical modifications (see sections 1 and 7). aβ forms distinct structural strains in different subtypes of ad [226-231]. investigations of the molecular configuration of aβ fibrils in vitro have yielded insights into potential determinants of aβ strains (see, e.g., [228, 232-235]), but cryo-electron microscopic analysis of meningovascular aβ-amyloid indicates that aβ-caa fibrils formed in vivo, though polymorphic, differ in important ways from those formed in vitro [66]. a similar analytic comparison of aβ fibrils from plaques in the brain parenchyma and caa could help to explain the inconsistent co-presence of plaques and amyloid angiopathy in ad. 5.3 cerebral aβ-amyloid angiopathy (aβ-caa) several different proteins can form caa in different disorders, but aβ is the most common source of caa in the elderly [236]. aβ accumulates in the vascular wall and perivascular zone in cases of primary aβ-caa involving mutations in the gene for app [21, 237-240] and to varying degrees in nearly all cases of ad [241-244]. ad and aβ-caa share many genetic risk factors, and like aβ plaques, idiopathic aβ-caa sometimes is present in the nondemented elderly [240, 241]. caa is a significant risk factor for lobar hemorrhage [236, 245], particularly in individuals with hypertension [246]. in end-stage ad, the amount of aβ-caa varies widely, even in the presence of copious plaques [247], although the severity of aβ-caa tends to increase with increasing plaque load [21]. furthermore, in some instances, aβ-caa can emerge in the absence or near absence of aβ plaques, notably in an autosomal dominant form of aβ-caa known as hereditary cerebral hemorrhage with amyloidosis (dutch type) (hchwa-d) [239, 248, 249]. there is evidence for some diffuse parenchymal aβ deposition [250, 251] and cognitive decline [238, 252] in these cases, but the clinical phenotype probably reflects the vascular pathology more than an ad-like disorder in which plaques and tangles are abundant [253]. cognitive dysfunction [254-258] and neurodegenerative changes [259] also have been associated with idiopathic aβ-caa. in approximately 25% of end-stage ad patients, aβ-caa affecting large vessels is considered to be severe (arterioles are more often afflicted than are veins); capillary aβ-caa is less common, being severe in approximately 10% of cases [247]. in advanced aβ-caa, the amyloid often extends through the tunica adventitia and into the surrounding parenchyma, where it is pervaded by tau-immunoreactive abnormal neurites [25, 260, 261] (figure 15). for unknown reasons, in regions of the neocortex where capillary aβ-caa is focally abundant, aβ plaques are relatively scarce [25, 247, 262]. figure 15. fluorescence-immunolabeled dyshoric cerebral aβ-amyloid angiopathy (red; antibody r398) and tau-immunoreactive neurites (green; antibody cp13) in the cortex of an ad patient. bar = 50μm. in the early stages of large-vessel aβ-caa, aβ42 is more commonly present than is aβ40 [263], but in later stages aβ40 predominates [263, 264]. capillary aβ-caa, however, more often is positive for aβ42 than for aβ40 [263]. it has been suggested that the deposition of aβ in capillaries transpires by a different mechanism than that in large vessels and aβ plaques [25, 26]. quantitative spatial analysis has largely refuted the hypothesis that cerebral capillaries are the nidus of aβ plaque formation [265]. interestingly, ‘coarse-grain’ plaques, a special type of lesion (see section 5.1), are more common in cases with abundant aβ-caa, particularly capillary aβ-caa [179]. aβ-caa, like aβ-plaques, is associated with reactive gliosis and a perivascular inflammatory response [240, 260], although the presence of frank perivascular inflammation is inconsistent [25, 266]. aβ-amyloid plaques are occasionally confluent with aβ-caa (‘juxtavascular plaques’; figure 16), but the etiologic relationship between these merged lesions is uncertain. figure 16. juxtavascular aβ-plaque (arrow) in the cortex of an ad patient. antibody 4g8, nissl counterstain. bar = 20μm. various genetic, biochemical and pathophysiologic factors appear to influence how the misfolding and aggregation of the same protein aβ can lead to two different phenotypic presentations parenchymal plaques and vascular amyloid [21]. while many auxiliary molecules are present in both aβ plaques and aβ-caa, some are not shared by the two lesions [267]. thus, aβ-caa and aβ plaques likely result from at least partially distinct ontogenetic pathways [21] (in this regard, it is noteworthy that the disappearance of plaques in alzheimer patients immunized against aβ is accompanied by a [possibly transient] increase in aβ-caa, suggesting a transfer of aβ from the parenchyma to the walls of blood vessels [268]). for in depth reviews of caa, see [21, 236, 237, 240, 241, 260, 269]. 6. cellular components of aβ plaques the main cellular elements neuronal processes and glial cells in classical plaques were well-documented by pioneering investigators in the field (see [116]),12 although the nature of their involvement, and their functional relationship to the core, have been a persistent matter of speculation [207]. diffuse deposits of aβ mostly lack obvious changes in local neurons and glial cells, whereas these cells are conspicuously altered in classical aβ plaques. since classical plaques are especially numerous in most cases of late-stage ad, the associated abnormal neurites and activated glial cells probably contribute to the disturbance of brain function by the plaques [133]. 12 early descriptions of plaques included drawings that enabled the artist to clearly depict all cellular elements throughout the depth of the tissue sections in a way that photomicrography, still in its infancy, could not. the result was sometimes striking images that have been difficult to surpass in the century-plus since (see, e.g., the fine reproductions in defelipe [2010] [reference 510]). 6.1. abnormal neurites in advanced ad, many aβ plaques are decorated with an impressive profusion of dysmorphic neurites (figures 1, 4, 17). both axons and dendrites contribute neurites to plaques [207, 270]. although most swollen neurites have been reported to be axonal in origin [138, 178, 207], a quantitative analysis of plaques in humans using axonand dendrite-specific markers is needed to establish the relative involvement of these neuronal processes. tortuous, atypical neurites that are not spatially associated with plaques are fairly common in the aging brain [139], but neuritic pathology is particularly evident in many aβ-amyloid plaques. by disrupting the structure and trajectory of neuronal processes, aβ plaques are thought to interfere with the connectivity and network functionality of the brain [38]. figure 17. abnormal neurites associated with cortical aβ plaques in two ad patients. left: immunostain for neurofilament-h (antibody smi31) with a nissl counterstain; right, immunostain for a conformational epitope on tau filaments (antibody mc1). the presence of aberrant neurites that are immunoreactive for these antigens in plaques is variable. bar = 25μm (right) and 50μm (left). abnormal neurites are heterogeneous in size, shape and content. ultrastructurally, plaque-associated neurites may contain any of a number of inclusions, including, in addition to paired helical filaments, profuse mitochondria, various dense bodies, membranes, and multivesicular profiles [139, 178] (figure 18). the mitochondria appear to be in different stages of degeneration, and they have been hypothesized to be a source of the aβ-amyloid in plaques [207], as have multivesicular bodies [199, 271, 272]. the cytoskeleton is disrupted in swollen neurites [273], and studies of mouse models found that neuritic calcium (ca2+) homeostasis [274] and autophagy [275] are dysregulated in them. dickson [139] divided abnormal neurites into paired helical filament (phf)-type neurites, which are characteristic of advanced ad, and dystrophic-type neurites, which are relatively more frequent in the plaques found in aged, non-demented subjects (and in animal models, in which phfs per se are rare or absent [165]; see section 10). dickson also notes, however, that many neurites have the properties of both types, and that abnormal neurites tend to arise from axons or dendrites that just happen to be in the vicinity of the plaque [139]. this is likely to be a general rule for the presence of specific types of neurites in plaques, including those containing markers for diverse neurotransmitters (see below) and, e.g., the alpha-synuclein-positive neurites in aβ plaques that are sometimes co-morbid with synucleinopathy in lewy body disease [276]. figure 18. abnormal neurite (top) containing organelles /debris adjacent to fibrillar amyloid (bottom) in the plaque corona of a patient with ad. bar = 500nm. histochemically, lysosomal enzyme activity is pronounced in dystrophic neurites, as is histochemical reactivity for app and markers of degeneration such as chromogranin-a and ubiquitin [139]. the chemical variability of neurites may reflect, in addition to the neurons of origin, their stage of development and their response to injury or stress [277]. several early researchers, including fischer [117] and ramon y cajal (see [278]), thought that the swollen neurites in plaques represented an attempt by the neuronal processes to sprout. since then, multiple growth-promoting factors have been detected in these neurites [278, 279], and aβ deposits have been shown experimentally to induce axonal sprouting in the mouse brain [280]. considered as a whole, these observations indicate the presence of both degenerative and regenerative mechanisms in the aberrant neuronal processes that are associated with aβ plaques [133, 279]. analyses of aβ plaques in humans and aged nonhuman primates found that many different neurotransmitter systems contribute anomalous neurites to plaques [281-287], and that an individual plaque can contain neurites from multiple sources [288, 289]. these studies cast doubt on the hypothesis [290] that plaques emerge from the regression of neurites from a specific transmitter system, in particular the acetylcholinergic neurons of the basal forebrain [141]. rather, they highlight the probable role of a common catalyst (e.g., misfolded aβ and/or reactive glia) in driving neuritic dystrophy [139, 289, 291]. indeed, the influential model proposed by wisniewski and terry [178] (see also [81]) that posited neuritic abnormalities in general as the initial stage of plaque ontogeny now seems untenable, especially in light of genetic findings implicating aβ as the prime mover in the pathogenesis of ad [9, 22, 44, 45, 212]. even so, dysmorphic neurites do influence the pathologic plaque milieu [207], and it is possible that, by releasing aβ into the extracellular space, they contribute to the growth of plaques [272]. in addition, neuritic aβ plaques are generally more strongly associated with dementia than are diffuse plaques [1, 133, 204]. finally, the loss of synapses correlates strongly with the degree of dementia in ad [292-295]; synaptic pathology is especially evident in the immediate vicinity of aβ plaques (see [296, 297], possibly owing to increased oligomeric aβ in this region [297]. 6.2 glial cells of the many genetic risk factors for ad [298], two of the most potent variant genes apoe (apolipoprotein e) and trem2 (triggering receptor expressed on myeloid cells-2) are highly expressed in glial cells [115], as are several other ad-associated genes [299-301]. astrocytes and microglia are protean and interactive components of the homeostatic intrinsic immune system in the brain and spinal cord [299, 302, 303]. histologic, genetic, biochemical and physiological findings strongly implicate them in the pathogenesis of ad [111-115, 299, 303-310] (figure 19). microglia and astrocytes do not operate independently of one another, but rather jointly influence aβ processing and plaque biology [311, 312]. in the vicinity of aβ-amyloid, these glial cells together form a partially integrated ‘reactive glial net’ [313] that, while considered to be an attempt to shield nearby neurons from aβ aggregates [314], ultimately engenders a neurotoxic inflammatory microenvironment [313]. figure 19. reactive astrocytes (left; antibody to gfap) and microglia (right; antibody to iba1) in cortical aβ plaques of two ad patients. despite some overlap of the two cell types within plaques, astrocytic somata tend to be more peripherally located than are microglial somata. bar = 20μm for both panels. inflammation is both a risk factor for, and a result of, the deposition of aβ in the brain [45, 315]. the recruitment and activation of glial cells by aβ plaques has been likened to a local inflammatory reaction to a foreign body [138, 139], although glia contribute to the pathobiology of plaques in complex ways [299, 302, 305, 309, 312, 316-318]. mouse models have enabled a dynamic view of glial function and the general biology of plaques, whereas the genetic and physiologic analysis of glia in human ad is much less advanced [299]. even given the caveat that glia differ in humans compared to other species [316, 319, 320], mice have furnished unique insights into glial functionality in the living brain [304, 316, 321-324]. a growing literature underscores the ability of both microglia and astrocytes to adopt different physiologic states that influence how they contribute positively or negatively to ad (see, e.g., [299, 303, 306]). current views of glial cells thus emphasize their dual role in the pathobiology of ad: they participate in the clearance of aberrant aβ and other debris, but they also can secrete a variety of inflammationand cell-stress-related molecules [304, 325, 326]. much contemporary research seeks to define and disentangle these intricate and seemingly incompatible mechanisms. 6.2.1 microglia activated microglia are intimately associated with the fibrillar aβ in classical aβ plaques [139, 327-330] (figure 20). they occupy much of the space between the plaque core and outer corona, and their processes interdigitate with the bundles of amyloid [311, 327]. the discovery that loss of function mutations in trem2 are a strong risk factor for ad has heightened interest in the role of microglia in neurodegeneration [299, 306]. trem2 is a cell-surface immune receptor on many myeloid cells, including microglia, which exclusively express trem2 in the brain [306]. the production of trem2 is increased in ad [331], and it mediates the activation and responsiveness of microglia to aβ-amyloid plaques [332]. microglia have been thought to either phagocytose [139] or produce [311] multimeric aβ, and their functional variability makes both actions conceivable, depending on the circumstances. on the one hand, there is evidence that microglia normally impede the generation of aβ plaques; inhibition of microglial functionality in mice was found to increase plaque load [333, 334], and microglia contribute to the clearance of dense-core plaques following anti-aβ immunization therapy [335] (see also the analysis of immunized humans by nicoll and colleagues [336]). additionally, studies in mice indicate that trem2 signaling transforms homeostatic microglia into disease-associated microglia (dam), in which state they phagocytose aβ in plaques [306, 337]. impeding trem2 functionality in microglia reduces the binding of apoe to aβ-amyloid in plaques and augments the seeded propagation of aβ-amyloid [338]. (genetic knockout of trem2 also promotes the seeded aggregation and spread of tau in neuritic aβ plaques [339]). on the other hand, ultrastructural [311, 340, 341] and experimental [342] investigations have suggested that microglia can generate aβ-amyloid fibrils. in support of this hypothesis, sustained pharmacologic reduction of microglia significantly diminished aβ plaque load in a transgenic mouse model [343]. the ability of microglia to assume multiple phenotypic states underscores the complexity of their participation in the biology of aβ plaques [299, 300, 305, 344-346]; they contribute to normal brain homeostasis, but they also have injurious properties, particularly when activated [299, 300]. in mice, microglia have been found to exhibit a range of activation states, each of which involves the expression of distinct gene modules [299]. microglia become activated in the presence of aggregated aβ, and in this condition they can harm the brain both through the secretion of pro-inflammatory agents and the elimination of synapses [300]. to complicate matters further, a variety of microglial phenotypes are simultaneously present within the same brain [345], and the involvement of microglia in plaques differs as a function of age and disease stage [299]. finally, while the discovery of microglial risk factors for ad emerged from human genetic analyses [306], we know far more about microglia in rodent models than in human ad, and current evidence suggests that there are important differences that cannot be overlooked [299, 347-349]. these findings collectively highlight the challenges presented by microglia as therapeutic targets in ad. figure 20. electron micrographs of a microglial cell in an aβ-amyloid plaque of an ad patient. the white box in the image on the left denotes the region at higher magnification on the right. the fibrillar bundles of aβ interdigitate with the microglial soma. note that the microglial cytoplasm appears artifactually rarefied in this autopsy-derived tissue. bar = 500 nm (right), 2.8μm (left). 6.2.2 astrocytes in the vicinity of many aβ-amyloid plaques, astrocytes hypertrophy and increase their expression of glial fibrillary acidic protein (gfap) [316] (figure 19). the degree of astrocytic hypertrophy surrounding plaques, however, is inconsistent [317]. gfap expression is a reasonably reliable index of astrocytic reactivity, but gfap is not detectable in many healthy astrocytes, and its expression varies in different parts of the brain, in different animal species, and as a function of age.13 compared to microglia, astrocytic somata tend to localize more peripherally to the aggregated aβ in plaques [175, 302, 311, 313, 327, 330], whence their processes penetrate and to some extent encapsulate the plaques (figure 19). despite their tendency to partially segregate, astrocytes and microglia show some spatial overlap, and physical and chemical interactions between them help to define the inflammatory state of plaques [304]. activated astrocytes promote the inflammatory milieu around plaques through the generation of pro-inflammatory substances, including cytokines/chemokines, activation of the complement cascade, and reactive nitrogen and oxygen species [316]. as in the case of microglia, the role of astrocytes in neurodegeneration is complicated by their variable and sometimes paradoxical phenotypes [317]. in ad, astrocytes can both gain a toxic function and lose their normal physiologic function [316, 350, 351]. astrocytes have been shown experimentally to take up and degrade aβ [315]. they also are capable of expressing aβ [352], and astrocytes containing ample aβ are present in the alzheimeric brain [186, 316, 353, 354] (figure 8). in addition, the extent of peri-plaque reactive astrocytosis is positively correlated with cognitive status in aged subjects, and their abundance is reduced in persons expressing apolipoprotein e4, a major risk factor for ad [316]. in summary, research on microglia and astrocytes has disclosed the extraordinary malleability of these glial cells, the complexity of their involvement in plaques, and thus the attendant difficulties in targeting them therapeutically. interventions that modulate the activity of glia could either promote or hinder disease progression, depending on the state of the cells in different brain areas, their relative abundance, and the timing of therapeutic delivery in the protracted course of ad. nevertheless, the obvious importance of microglia and astrocytes in the pathobiology of ad justifies continued efforts to decipher the mechanisms by which they interact with aβ and with the other cellular components of plaques. for additional reviews of microglia and astrocytes in aging and ad, see [312, 355-357]. 13 the authors note that the findings should be interpreted cautiously in light of the pitfalls associated with histochemical methods (garwood et al. [2017] [reference 316]). this advice applies to histologic analyses in general, as methods and interpretations can vary among laboratories (e.g., alafuzoff et al. [2008] [reference 511]). 6.2.3 oligodendrocytes compared to microglia and astrocytes, oligodendrocytes have been less studied in ad [358]. their involvement in plaques has long been debated (see, e.g., the contrasting views of critchley [116] and ferraro [118]: ‘oligodendroglia apparently does not participate in the structure of plaques’ [critchley, 1929]; ‘it is certain, then, that both oligodendroglia and microglia cells are usual components of senile plaques’ [ferraro, 1931]). soniat contended that oligodendrocytes are not integral to the formation of plaques, but rather, when present, their presence is purely coincidental [119]. a recent analysis, however, has revealed oligodendrocyte progenitor cells in aβ plaques that become senescent and pro-inflammatory, in which state they are thought to augment the pathogenicity of aberrant aβ [359]. more work on oligodendrocytes in association with aβ plaques is clearly needed. 7. the broader biochemistry of aβ in plaques the number of molecules that have been linked in some way to aβ plaques is considerable (see, e.g., [139, 175, 278, 279, 360-364]), creating fertile ground for hypotheses on both the origin of plaques and the nature of ad. along with the many substances directly associated with neurons and glia, aggregated aβ itself is rich in accompanying molecules. amyloid p component is present in different types of amyloid throughout the body [6, 365], including aβ plaques [364, 366, 367]. other molecules that have been reported to directly co-localize with at least some aβ deposits include proteoglycans [6, 364, 368, 369], complement proteins [370-373], apolipoprotein e [374, 375], alpha-1 antichymotrypsin [376] and advanced glycation end products [377, 378], along with lipids, metal ions, reactive oxygen species and nucleic acids (see stewart and radford [364]). how aβ-linked substances might be involved in the pathobiology of plaques is attracting increasing attention. for instance, a study in mice found that aβ bound to nucleic acids acts as an immune signal, stimulating an antiviral response in microglia and astrocytes that instigates the complement-mediated elimination of local synapses [379]. the plaque-associated proteome can be interrogated by laser-microdissection of aβ plaques followed by mass-spectrometric analysis [380-385]. these studies have identified numerous proteins that are enriched in plaques, though whether they are directly associated with multimeric aβ or with the cellular constituents is sometimes undefined. it has been proposed that plaques mature through three biochemical stages within which the toxicity of the aggregates may differ; in stage 1, the aggregates lack both pyroglutamation at residue 3 (aβnp3e) and phosphorylation at residue 8 (pser8aβ); in stage 2, aβnp3e appears, and in stage 3, both aβnp3e and pser8aβ are present [19, 386]. post-translational chemical modifications of aβ can influence the aggregation of the protein along with the type of deposit that is formed in the brain [19, 387-391], but the mechanisms are, in many cases, still uncertain. 8. microbes and plaques the notion that microbes might participate in the genesis of plaques has been considered at least since the early 20th century.14 fischer likened mature plaques to actinomyces ‘drusen’, although he noted that they were negative for multiple bacterial stains [117]. critchley remarked in 1929 that the microbial origin hypothesis had failed to gain traction [116]. despite more recent claims that senile plaques in ad ‘are made up by spirochetes’ [392], there is still no credible evidence that aβ plaques are primarily collections of microbes or their remains. that said, there is fairly compelling evidence that certain microbial infections are risk factors for ad [393-395]. perhaps the best evidence indicates that some herpesviridae increase the probability of developing ad [394, 396, 397]. over 15 different microbes have been linked to ad by various researchers [398], but in many instances the findings are weak or contradictory (see, e.g., [399, 400]). in addition, it is important to distinguish cases of dementia in general (for which there are over 50 different causes [330]) from cases of dementia specifically due to the pathology of ad (as defined by jack and colleagues [109]). it is fair to say that no known infectious agent is universally and exclusively associated with ad [395], but it seems likely that any of several types of brain infection (including chronic infection and/or reactivation of resident microbes) can accelerate plaque formation and the pathogenesis of ad [394, 395, 401, 402]. in other words, at least in some instances the development of plaques may represent a non-specific response to various infectious organisms. aggregated aβ has antimicrobial properties [395, 403, 404], and some microbial antigens have been reported in aβ plaques [392, 405], but a systematic and comprehensive survey of microbial markers in different types of plaques and aβ-caa throughout the central nervous system has not been reported. app-transgenic mice raised in a germ-free environment develop some aβ plaques as they age, albeit fewer than mice raised in normal caging [406]. with the caveat that the mice strongly overexpress transgenic aβ, the findings suggest that infection is not required for plaque formation, but that it can trigger and/or accelerate the process. the role of infection in the causation of aβ plaques and as a risk factor for ad is an intriguing topic with potential implications for prevention and therapy, but supporting evidence for a specific role of specific microbes in pathogenesis is needed. for a critical consideration of the state of the field, see [393]. 14 i use the term ‘microbe’ here to include both conventional (living) microorganisms and viruses (but not prions). 9. the seeded induction of aβ plaque formation the prion paradigm has become the dominant mechanistic explanation for the aberrant self-assembly and propagation of misfolded proteins in the brain and elsewhere in the body [58, 205, 218, 407-409]. at the molecular level, the prion paradigm postulates that misfolded, β-sheet-rich proteins aggregate into oligomeric/polymeric assemblies that can induce protein molecules of the same type to adopt a similar conformation. in this condition, the proteins tend to stick together, with the assemblies often (but not always) amassing into amyloid deposits. in the prion diseases, misfolded prp self-assembles into highly stable multimers that are transmissible from one organism to another the first verified instance of an infectious protein particle (‘prion’) [410, 411]. human prion diseases also originate spontaneously or as a result of mutations in the gene for prp [412]. the pathological signature of the prion diseases varies considerably [127, 128], but, as in ad, the universal feature of prionopathies is the accumulation of an abnormally folded protein in this case prp in the nervous system. systematic studies in transgenic mouse models expressing human app have determined that aβ plaque formation is driven by a molecular process that is indistinguishable from the mechanism by which prions instigate disease [217, 218, 413-415] (figure 21). in this paradigm, brain extracts containing aggregated aβ are infused into the brains of susceptible mice, instigating aβ plaque development in a model-, doseand time-dependent fashion [218, 407]. analyses of seeded aggregation in experimental systems have demonstrated that aβ seeds share key properties with prions: 1) they are protein-only agents that are resistant to destruction by heat and formaldehyde; 2) they incite the formation of cerebral aβ plaques and aβ-amyloid angiopathy when introduced into the brain or into the periphery; 3) they exist in multiple sizes; and 4) they can fold into different molecular variants referred to as proteopathic strains [212, 213, 217, 218, 407] (see section 5.2). the strain-like properties of aβ deriving from different subtypes of ad can be partially transmitted to plaques via exogenous seeding in mouse models [227, 230]. figure 21. seeded aβ deposition in the hippocampal formation of a tg2576 app-transgenic mouse 5-months following unilateral injection of dilute ad brain extract into one hemisphere (left). the contralateral hippocampus in the same tissue section is on the right. antibody 4g8; bar = 100μm. these investigations highlight the prion-like seeded aggregation of aβ as the propulsive mechanism behind the formation of aβ plaques. since there is currently no evidence that ad or other cerebral proteopathies are infectious under ordinary circumstances [416, 417], it is likely that plaques ordinarily arise endogenously with the stochastic emergence, persistence and spread of aβ seeds. this process can be advanced by various environmental and endogenous risk factors that influence the likelihood that aβ will misfold and propagate in the brain [45]. under extraordinary circumstances, however, aβ plaques and aβ-caa can be instigated by exogenous aβ seeds in humans [417]. treatment of young people with growth hormone derived from cadaveric human pituitary glands, beginning in the late 1950s, resulted unexpectedly in the development of prion disease (creutzfeldt-jakob disease) many years later [418, 419]. the apparent cause was the presence of infectious prions in the preparations, probably because the large batches of pituitaries that were homogenized for extraction of growth hormone contained some glands from decedents with prion disease. researchers in england later found that both aβ plaques and aβ-caa were much more common in human growth hormone-treated subjects than in non-treated controls [420]. furthermore, aβ deposition was precipitated both in growth hormone recipients dying with [420] or without [421] creutzfeldt-jakob disease. an increase in aβ-proteopathy also has been reported in a subset of people who had received cadaveric dura mater transplants [422, 423]. the most parsimonious explanation for these findings is that some batches of therapeutic growth hormone and dura mater were tainted by aβ seeds that were present in the tissues taken from donors with ad (or incipient ad) [417]. this possibility is reinforced by the demonstrable presence of aggregated aβ in some pituitary glands [424] and dura mater [425] from ad patients. furthermore, aβ was detected in archival samples of cadaveric human growth hormone [426], and stored hormone was shown to stimulate cerebral aβ deposition when injected intracerebrally into app-transgenic mice [427]. interestingly, tauopathy was not apparent in most of these cases (even though some abnormal tau is present in alzheimeric pituitaries), and no recipients of cadaveric growth hormone or dura mater have yet been found to develop full-blown ad. whether this will happen as the subjects age further remains to be determined. 10. aβ plaques in nonhuman species 10.1 native aβ plaques naturally occurring aβ plaques and/or aβ-caa have been identified in aged animals of many species, including such diverse creatures as woodpeckers [428], bears [429-431], dogs [432-435], cats [436], camels [437] wolverines [438], and all species of nonhuman primate examined to date [165, 435, 439]. the mammalian mainstays of experimental biology rats and mice do not normally manifest plaques in old age, possibly owing to 3 amino acid differences in the n-terminal segment of aβ that render the protein less likely to aggregate [440, 441]. most research on native aβ plaques in nonhuman species has focused on primates ranging from prosimians to monkeys and apes [439], work that has yielded insights into the pathobiology of the lesions [178, 439, 442-444]. nonhuman primates express aβ with the same sequence of amino acids as in humans, and both diffuse and dense-core aβ plaques can be abundant in aged primates [439] (figure 22). some of the plaques include reactive glial cells and dysmorphic neurites [444]. mass-spectrometry has shown that post-translational modifications of aβ are similar in humans and squirrel monkeys (saimiri sciureus), and by elisa, the amount of aβ in the nonhuman primate brain sometimes exceeds that in humans with ad [163]. despite the presence of copious aberrant aβ, no nonhuman species has yet been found to exhibit the full clinicopathologic phenotype of ad as it occurs in humans [165]. specifically, a dementia-like condition has not yet been identified, and tauopathy, though often present, is generally mild, even in the presence of profuse aβ plaques. for unknown reasons, aβ-caa, especially capillary aβ-caa, is more commonly present in nonhuman primates than in humans [439, 445, 446]. although congophilic aβ plaques occur, human-like classical plaques with an aβ core, space, and outer corona (see figure 1) are rare, if they can be found at all, in prosimians and monkeys (we cannot yet rule out such lesions in great apes, as relatively few have been examined in advanced old age). surprisingly, there is little high-affinity binding of the aβ-amyloid-imaging agent pittsburgh compound-b (pib) to aβ plaques in nonhuman primates, suggesting biochemical and/or conformational differences in the protein between humans and other primates [447]. it is necessary to determine how differences in lifespan and environmental and genetic risk factors might influence the apparent species-specificity of ad and the aβ-deposition phenotype. however, current evidence suggests that, despite similarities in the sequence, expression, modification, and deposition of aβ, nonhuman primates lack the permissive connection between aβ-proteopathy and tauopathy that is critical to the occurrence of ad in humans [165]. clarifying the nature of this naturally occurring interruption of the aβ cascade in nonhuman species could reveal new pathogenic pathways for therapeutic intervention in ad. figure 22. aβ deposition (left) in the superior temporal gyrus and a neuritic plaque (right) in the hippocampal formation of two aged rhesus monkeys (macaca mulatta; 35 years and ~30 years, respectively). left: antibody 82e1 to the n-terminal segment of aβ, nissl counterstain; right: antibody 06-17 to phosphorylated neurofilaments. bars = 200μm (left) and 25μm (right). the maximum known lifespan of rhesus monkeys is 44 years (see stonebarger et al. [2020] [reference 512]). figure 23. aβ plaques in an aged (28 months) tg2576 app-transgenic mouse. diffuse deposits are black, and some dense deposits have a golden core (one in the frontal cortex is magnified at right). campbell-gallyas silver stain. bars = 1mm (left) and 50μm (right). 10.2 aβ plaques in genetically modified animals studies of naturally occurring plaques in various species have shed some light on the lesions, but there was no practical model in which plaques could be experimentally investigated until the mid-1990s. then, transgenic mice were introduced that overexpress human app with genetic mutations linked to ad [448-450]. with age, these app-transgenic mice deposit copious aβ in the brain (figure 23). they were followed by a wealth of additional models in various mouse (and later rat) strains with diverse genetic alterations, transgene expression levels, and the expression or deletion of interacting molecules [451-453]; see alzforum for a list of rodent models of ad-like pathology: https://www.alzforum.org/research-models/alzheimers-disease). not surprisingly, the sundry genetically modified animals exhibit many plaque (and caa) phenotypes. no genetically modified rodent, including those with multiple modifications, has manifested fully ad-like aβ plaques. as in nonhuman primates, the core-space-corona type of plaque is not typical of the transgenic rodent models. the plaques do, however, share several key features with those in humans; they exhibit a range of morphologies, many have bona fide amyloid cores, and they are invested by aberrant neurites and glial cells [452]. tau abnormalities occur, but human-like neurofibrillary tangles have not yet been generated in rodents. nonvertebrate transgenic animals such as fruit flies (drosophila melanogaster) [454-456] and roundworms (caenorhabditis elegans) [457, 458] have been developed to study the pathobiology of aβ. these models can be useful for analyzing molecular mechanisms and for studying the early-stage efficacy and toxicity of investigational agents, but no nonvertebrate model has yet generated aβ plaques that remotely resemble those in humans. it is difficult to overstate the impact that the introduction of genetically modified animals has had on the course of research on the mechanisms underlying plaque formation and ad. for example, transgenic rodents were used to establish the prion paradigm as the pre-eminent theory of plaque ontogeny and spread [217, 218, 407] (see section 9), they are being used to probe the role of glial cells and neuritic dystrophy in plaque pathophysiology (see section 6), and they are a vital tool in the preclinical testing of new therapeutic and diagnostic strategies [452, 459-463]. for instance, whereas the first evidence that aβ plaques and aβ-caa could be targeted by anti-aβ antibodies in the living brain came from experiments in nonhuman primates [464], genetically modified mice enabled the development of aβ-immunization as a strategy for the prevention or treatment of ad [465]. the application of longitudinal, in vivo-imaging studies in murine models has facilitated unique insights into the dynamics of aβ plaques and their cellular constituents (e.g., [323, 324, 466-469]), as well as the response of plaques and aβ-caa to therapeutic intervention [451, 452, 470]. currently, genetically modified nonhuman primates are being created with the hope that they will more completely recapitulate a human-like ad phenotype [471, 472], but no histopathologic findings have yet been reported. despite the limitation that genetically modified animals do not yet fully recapitulate ad, they will continue to play an important part in deciphering the pathobiology of aβ plaques. 11. conclusions: aβ plaques as a therapeutic objective aβ plaques are an obligatory component of the pathobiology of ad, and as such, strategies to reduce or neutralize plaques intersect with general strategies to prevent or treat ad. however, the value of plaques, in and of themselves, as therapeutic targets is uncertain. there is little question that aβ plaques, especially in their more elaborate states, are deleterious to brain tissue; they disrupt neuronal processes and synapses, they can be a source of harmful aβ-oligomers, and local glial cells create a toxic inflammatory environment. therapeutically targeting plaques thus presents both opportunity and obstacles. first, given the long, pre-symptomatic emergence and proliferation of aβ plaques (and neurofibrillary tangles) in the brain [109, 473], as well as evidence of extensive brain damage by the time dementia sets in, early prevention is likely to be the most effective strategy for subduing ad [474, 475]. the promise of prevention is underscored by the protective effects of the a673t mutation in app, which diminishes aβ production throughout life and lowers the risk of ad [48]. since the most effective preventive protocol should be initiated years, and possibly decades, before the predicted onset of dementia, testing for long-term safety and efficacy will be challenging. additionally, it is not known when, in the course of life, therapy must begin to effectively prevent or delay ad. second, it is possible that some, if not most, of the direct toxic influence of the aβ is mediated by oligomeric aβ rather than by fibrillar amyloid per se. evidence that aβ plaques can serve as a source of oligomers (section 1.1) argues that some benefit can be achieved by reducing plaque burden and thus the accompanying oligomers. it is encouraging that several of the more promising antibodies currently in clinical trials for ad show activity against oligomeric aβ [475, 476]. a recent study in mice indicates that the short-term neutralization of oligomeric aβ seeds early in life diminishes plaque formation as the animals age [477]. however, whether mitigating the production, seeding potential or toxicity of oligomeric aβ will be beneficial in humans, either as a preventive or as a treatment for discernible dementia, remains to be determined. it is also important to consider the possibility that treatments that block aβ-amyloid fibril assembly, or that disassemble plaques, might inadvertently increase the presence of toxic oligomers. third, the inability of anti-aβ immunotherapies to substantially impede dementia in symptomatic subjects, even when aβ plaques are reduced in number [336, 476, 478, 479], suggests that the dis-integration of the cerebral connectome caused by plaques and tangles is pronounced and largely irreversible once dementia commences [9, 474]. furthermore, tauopathy is an essential contributor to dementia that itself progresses by a prion-like mechanism [480, 481] that may be at least partly independent of aβ-proteopathy [336]. whether the alternative approach of lowering the inflammatory state associated with plaques will meaningfully improve behavior at this later stage of disease also has not been established. in short, once aβ-amyloid plaques and tauopathy become widespread, especially in neocortical regions [1], removing the plaques is unlikely to significantly reverse the course of dementia. even so, there is evidence that a reduction of tauopathy [336], and possibly some cognitive benefit, can be achieved in symptomatic patients by anti-aβ immunotherapy [476, 482-484]. indeed, active immunization with an1792, which targets both aβ plaques and oligomers, resulted in a long-term decrease in all components of the plaques aggregated aβ, aberrant neurites, tauopathy, and focal gliopathy along with improved indices of ‘neuronal health’ [336]. thus, notwithstanding the mostly disheartening outcome of therapeutic trials to date, current preclinical and clinical data indicate that the right anti-aβ treatment, at the right time, has a good chance of delaying or preventing ad. finally, it is imperative to remain vigilant to the impact of environmental factors and the microbiome [485, 486] on the risk of developing ad. for instance, if specific microbes are convincingly found to increase the risk of plaque formation and ad, early immunization against this infectious agent could be an effective preventive measure. it is important also to consider the possibility that interactions among genetic, microbiomic and/or environmental influences could raise disease risk well above the additive impact of individual risk factors. the search for disease-modifying therapies for ad is a broad and rapidly evolving endeavor that has been extensively reviewed (see, e.g., [9, 33, 476, 479, 487-491]. in addition to small molecules, we have entered a phase in medicine in which biologics such as antibodies [492, 493] and nucleic acid-based agents [494, 495] have unprecedented potential to treat neurodegenerative diseases. for well over a century, aβ plaques have been recognized as an important correlate of dementia in the aging brain. revealing the mechanisms by which plaques arise, proliferate, and interact with molecular and cellular elements in the nervous system will continue to yield insights into both the ontogeny and treatment of ad. 12. methods tissue samples were collected from human subjects with end-stage ad (figures 1-6, 8-20) and from aged nonhuman species with cerebral aβ deposition (figures 21-23). postmortem collection of samples by the emory university goizueta alzheimer’s disease research center brain bank was approved by the emory institutional review board. tissues from mice and monkeys were collected at emory’s yerkes national primate research center in accordance with federal and institutional guidelines for the humane care and use of experimental animals. the yerkes center is fully accredited by aaalac international. 12.1 immunohistochemistry for light-microscopy, tissue blocks were embedded, sectioned at 8-10 μm thickness, and mounted onto glass slides for staining. the following antibodies were used for immunohistochemistry: 4g8, mouse monoclonal antibody from covance (princeton, nj) raised against residues 17-24 of aβ peptide, with an epitope at residues 18-22 [496]; 6e10, mouse monoclonal antibody from covance raised against residues 1-16 of the aβ peptide, with an epitope at residues 3-8 [496]; rabbit polyclonal antibodies r361 and r398, kindly provided by dr. pankaj mehta (institute for basic research on developmental disabilities, staten island, ny), were raised against synthetic aβ32-40 and aβ33-42, respectively [497]; 82e1, mouse monoclonal antibody raised against residues 1-16 of synthetic aβ [498], from ibl (gunma, japan); cp13, mouse monoclonal antibody kindly provided by dr. peter davies (feinstein institutes for medical research, manhasset, ny), was raised against a synthetic peptide representing the region around phosphorylated serine residue 202 on the tau protein [499]; mc1, mouse monoclonal antibody, also from dr. davies, was raised against alz50-immunopurified paired helical filaments and then epitope-mapped to similar conformation-specific regions as alz50 [500]; anti-gfap, purified immunoglobulin fraction of rabbit antiserum from dako (carpinteria, ca) (catalog no. z0334), raised against gfap isolated from cow spinal cord and purified by solid-phase absorption with human and cow serum proteins; anti-iba1, rabbit polyclonal antibody from wako (osaka, japan), raised against a synthetic peptide corresponding to the c-terminus of ionized calcium-binding adapter molecule 1 (iba1), a 17-kda protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells [501, 502]; smi-31, mouse monoclonal antibody raised against a phosphorylated epitope on the neurofilament heavy subunit (nf-h) [503] from biolegend (san diego, ca); and 06-17, mouse monoclonal antibody to a phosphorylated epitope shared by the heavy and medium kda neurofilament polypeptides (generous gift of drs. ludwig and nancy sternberger, university of maryland, baltimore) [504, 505]. vectastain elite kits (vector laboratories, burlingame, ca) were used for abc-based immunodetection of antigen-antibody complexes according to the manufacturer’s instructions, with diaminobenzidine (dab) as coloring agent for images in figures 1, 2, 5, 6, 8-11, 14, 16, 17, 19, 21, & 22. in most cases, a nissl counterstain was applied after immunostaining, as noted. for dual fluorescence immunostaining (figure 15), the section was incubated in mouse monoclonal antibody cp13 (diluted in 2% normal goat serum) overnight at 4°c, rinsed, and then incubated for 90 minutes in cy2-conjugated anti-mouse secondary antibody (green; jackson labs, west grove, pa). the section was rinsed thoroughly, incubated overnight in diluted rabbit polyclonal antibody r398 at 4°c, rinsed, and placed for 90 minutes in rhodamine-red-x goat anti-rabbit secondary antibody (jackson labs). for dual immunostaining by standard transillumination light microscopy (figure 3), antibodies were sequentially applied as described above except that the polyclonal anti-aβ antibodies r398+r361 were colored with dab (brown), and the anti-tau monoclonal antibody cp13 was colored with vip (purple; vector laboratories). non-immune mouse igg or rabbit sera were used in place of the primary antibodies as negative controls. tissues shown in figures 1 (left) and 4 were stained with the naoumenko-feigin silver stain [506] followed by a periodic acid-schiff (pas) counterstain. figure 23 was stained with the campbell-gallyas silver stain [507]. light-microscopic photomicrographs were taken with a leica dmlb or dmls microscope (wetzlar, germany) and a spot flex (diagnostic instruments, sterling heights, mi) or moticam 5+ (motic, hong kong) digital camera. 12.2 electron microscopy for conventional ultrastructural analysis (figures 12, 13, 18, 20), small blocks of cortex were sub-dissected from larger, autopsy-derived tissue blocks that had been immersion-fixed in 10% neutral buffered formalin. the tissue samples were washed in phosphate buffer (0.1m, ph 7.4) and immersed in osmium tetroxide (1% in phosphate buffer) for 20 minutes. they were then rinsed in phosphate buffer and dehydrated in a graded series of ethanol and propylene oxide. uranyl acetate (1%) was added to the 70% ethanol (35 minute immersion) to improve contrast in the electron microscope. the sections were then embedded in epoxy resin (durcupan acm; fluka, ft. washington, pa) on microscope slides and heated for 48 hours at 60°c. areas of interest were selected, excised from the slide and glued onto resin blocks. ultrathin sections were cut with a leica ultracut t2 (nussloch, germany), collected onto single-slot copper grids, and stained with lead citrate. for immunogold em (figure 11, right), sections were preincubated in pbs containing 5% nonfat dry milk and then washed in tris-buffered saline (tbs)-gelatin buffer (0.02 m tris, 0.15 m nacl, 1 μl/ml fish gelatin, ph 7.6) to block nonspecific sites. sections were then incubated for 48 hours at 4°c with antibody 4g8 diluted in pbs-bsa, rinsed in tbs-gelatin, and incubated for 2 hours at room temperature in gold-conjugated goat anti-mouse fab’ fragments (dilution 1:100; nanogold [nanoprobes inc., yaphank, ny]). gold particles were silver-enhanced with the hq silver kit (nanoprobes). the tissue was then embedded and cut as described above. thin sections were examined with a zeiss em10-c electron microscope (oberkochen, germany) and digital images were captured using a dual view camera (gatan inc., pleasanton, ca). acknowledgements i gratefully acknowledge enlightening discussions with mathias jucker, dietmar thal, marla gearing, harry levine, rebecca rosen, amaryllis cintron, eric heuer, david lynn, yury chernoff, rolf warzok, silke vogelgesang, sanjeev gumber, linda cork, allan levey and james lah. hailian xiao, jean-françois paré, and jeromy dooyema provided expert technical assistance. portions of this work were supported by the metlife foundation, cart foundation, alexander von humboldt stiftung, and by national institutes of health (nih) grants p01 ag026423, p50 ag025688, r21 ns077049, and orip/od p51 od011132. references 1. nelson, p.t., et al., correlation of alzheimer disease neuropathologic changes with cognitive status: a review of the literature. j neuropathol exp neurol, 2012. 71(5): p. 362-81. 2. glenner, g.g. and c.w. wong, alzheimer’s disease and down’s syndrome: sharing of a unique cerebrovascular amyloid fibril protein. biochem biophys res commun, 1984. 122(3): p. 1131-5. 3. glenner, g.g. and c.w. wong, alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. biochem biophys res commun, 1984. 120(3): p. 885-90. 4. masters, c.l., et al., amyloid plaque core protein in alzheimer disease and down syndrome. proc natl acad sci u s a, 1985. 82(12): p. 4245-9. 5. masters, c.l. and k. beyreuther, pathways to the discovery of the abeta amyloid of alzheimer’s disease. j alzheimers dis, 2006. 9(3 suppl): p. 155-61. 6. benson, m.d., et al., amyloid nomenclature 2018: recommendations by the international society of amyloidosis (isa) nomenclature committee. amyloid, 2018. 25(4): p. 215-219. 7. selkoe, d.j., biology of β-amyloid precursor protein and the mechanism of alzheimer disease, in alzheimer disease, r.d. terry, et al., editors. 1999, lippincott, williams, and wilkins philadelphia. p. 293-310. 8. haass, c., et al., trafficking and proteolytic processing of app. cold spring harb perspect med, 2012. 2(5): p. a006270. 9. long, j.m. and d.m. holtzman, alzheimer disease: an update on pathobiology and treatment strategies. cell, 2019. 179(2): p. 312-339. 10. dunys, j., a. valverde, and f. checler, are nand c-terminally truncated abeta species key pathological triggers in alzheimer’s disease? j biol chem, 2018. 293(40): p. 15419-15428. 11. kummer, m.p. and m.t. heneka, truncated and modified amyloid-beta species. alzheimers res ther, 2014. 6(3): p. 28. 12. portelius, e., et al., mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic alzheimer’s disease. acta neuropathol, 2010. 120(2): p. 185-93. 13. portelius, e., et al., brain amyloid-beta fragment signatures in pathological ageing and alzheimer’s disease by hybrid immunoprecipitation mass spectrometry. neurodegener dis, 2015. 15(1): p. 50-7. 14. saido, t.c., et al., aminoand carboxyl-terminal heterogeneity of beta-amyloid peptides deposited in human brain. neurosci lett, 1996. 215(3): p. 173-6. 15. takami, m., et al., gamma-secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. j neurosci, 2009. 29(41): p. 13042-52. 16. tekirian, t.l., et al., n-terminal heterogeneity of parenchymal and cerebrovascular abeta deposits. j neuropathol exp neurol, 1998. 57(1): p. 76-94. 17. moro, m.l., et al., pyroglutamate and isoaspartate modified amyloid-beta in ageing and alzheimer’s disease. acta neuropathol commun, 2018. 6(1): p. 3. 18. schaffert, l.n. and w.g. carter, do post-translational modifications influence protein aggregation in neurodegenerative diseases: a systematic review. brain sci, 2020. 10(4): p. 232. 19. thal, d.r., et al., neuropathology and biochemistry of abeta and its aggregates in alzheimer’s disease. acta neuropathol, 2015. 129(2): p. 167-82. 20. ke, p.c., et al., half a century of amyloids: past, present and future. chem soc rev, 2020. 49(15): p. 5473-5509. 21. greenberg, s.m., et al., cerebral amyloid angiopathy and alzheimer disease one peptide, two pathways. nat rev neurol, 2020. 16(1): p. 30-42. 22. holtzman, d.m., j.c. morris, and a.m. goate, alzheimer’s disease: the challenge of the second century. sci transl med, 2011. 3(77): p. 77sr1. 23. roher, a.e., et al., beta-amyloid-(1-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of alzheimer disease. proc natl acad sci u s a, 1993. 90(22): p. 10836-40. 24. attems, j., f. lintner, and k.a. jellinger, amyloid beta peptide 1-42 highly correlates with capillary cerebral amyloid angiopathy and alzheimer disease pathology. acta neuropathol, 2004. 107(4): p. 283-91. 25. richard, e., et al., characteristics of dyshoric capillary cerebral amyloid angiopathy. j neuropathol exp neurol, 2010. 69(11): p. 1158-67. 26. attems, j., et al., capillary caa and perivascular abeta-deposition: two distinct features of alzheimer’s disease pathology. j neurol sci, 2010. 299(1-2): p. 155-62. 27. catalano, s.m., et al., the role of amyloid-beta derived diffusible ligands (addls) in alzheimer’s disease. curr top med chem, 2006. 6(6): p. 597-608. 28. kuo, y.m., et al., water-soluble abeta (n-40, n-42) oligomers in normal and alzheimer disease brains. j biol chem, 1996. 271(8): p. 4077-81. 29. cline, e.n., et al., the amyloid-beta oligomer hypothesis: beginning of the third decade. j alzheimers dis, 2018. 64(s1): p. s567-s610. 30. ferreira, s.t., et al., soluble amyloid-beta oligomers as synaptotoxins leading to cognitive impairment in alzheimer’s disease. front cell neurosci, 2015. 9: p. 191. 31. haass, c. and d.j. selkoe, soluble protein oligomers in neurodegeneration: lessons from the alzheimer’s amyloid beta-peptide. nat rev mol cell biol, 2007. 8(2): p. 101-12. 32. tomiyama, t. and h. shimada, app osaka mutation in familial alzheimer’s disease-its discovery, phenotypes, and mechanism of recessive inheritance. int j mol sci, 2020. 21(4): p. 1413. 33. walsh, d.m. and d.j. selkoe, amyloid beta-protein and beyond: the path forward in alzheimer’s disease. curr opin neurobiol, 2020. 61: p. 116-124. 34. flagmeier, p., et al., direct measurement of lipid membrane disruption connects kinetics and toxicity of abeta42 aggregation. nat struct mol biol, 2020. 27(10): p. 886-891. 35. chen, x.q. and w.c. mobley, alzheimer disease pathogenesis: insights from molecular and cellular biology studies of oligomeric abeta and tau species. front neurosci, 2019. 13: p. 659. 36. esparza, t.j., et al., amyloid-beta oligomerization in alzheimer dementia versus high-pathology controls. ann neurol, 2013. 73(1): p. 104-19. 37. shankar, g.m., et al., amyloid-beta protein dimers isolated directly from alzheimer’s brains impair synaptic plasticity and memory. nat med, 2008. 14(8): p. 837-42. 38. hyman, b.t., anatomical changes underlying dementia in alzheimer’s disease, in alzheimer: 100 years and beyond, m. jucker, et al., editors. 2006, springer-verlag: berlin heidelberg new york. p. 89-94. 39. koffie, r.m., et al., oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques. proc natl acad sci u s a, 2009. 106(10): p. 4012-7. 40. katzmarski, n., et al., abeta oligomers trigger and accelerate abeta seeding. brain pathol, 2020. 30(1): p. 36-45. 41. langer, f., et al., soluble abeta seeds are potent inducers of cerebral beta-amyloid deposition. j neurosci, 2011. 31(41): p. 14488-95. 42. benilova, i., et al., highly infectious prions are not directly neurotoxic. proc natl acad sci u s a, 2020. 117(38): p. 23815-23822. 43. benilova, i., e. karran, and b. de strooper, the toxic abeta oligomer and alzheimer’s disease: an emperor in need of clothes. nat neurosci, 2012. 15(3): p. 349-57. 44. hardy, j. and d.j. selkoe, the amyloid hypothesis of alzheimer’s disease: progress and problems on the road to therapeutics. science, 2002. 297(5580): p. 353-6. 45. walker, l.c., d.g. lynn, and y.o. chernoff, a standard model of alzheimer’s disease? prion, 2018. 12(5-6): p. 261-265. 46. wiseman, f.k., et al., a genetic cause of alzheimer disease: mechanistic insights from down syndrome. nat rev neurosci, 2015. 16(9): p. 564-74. 47. abrahamson, e.e., et al., neuropathological correlates of amyloid pet imaging in down syndrome. dev neurobiol, 2019. 79(7): p. 750-766. 48. jonsson, t., et al., a mutation in app protects against alzheimer’s disease and age-related cognitive decline. nature, 2012. 488(7409): p. 96-9. 49. benilova, i., et al., the alzheimer disease protective mutation a2t modulates kinetic and thermodynamic properties of amyloid-beta (abeta) aggregation. j biol chem, 2014. 289(45): p. 30977-89. 50. kero, m., et al., amyloid precursor protein (app) a673t mutation in the elderly finnish population. neurobiol aging, 2013. 34(5): p. 1518 e1-3. 51. di fede, g., et al., a recessive mutation in the app gene with dominant-negative effect on amyloidogenesis. science, 2009. 323(5920): p. 1473-7. 52. buxbaum, j.n. and r.p. linke, a molecular history of the amyloidoses. j mol biol, 2012. 421(2-3): p. 142-59. 53. puchtler, h. and f. sweat, a review of early concepts of amyloid in context with contemporary chemical literature from 1839 to 1859. j histochem cytochem, 1966. 14(2): p. 123-34. 54. sipe, j.d. and a.s. cohen, review: history of the amyloid fibril. j struct biol, 2000. 130(2-3): p. 88-98. 55. yakupova, e.i., et al., congo red and amyloids: history and relationship. biosci rep, 2019. 39(1): p. bsr20181415. 56. chiti, f. and c.m. dobson, protein misfolding, functional amyloid, and human disease. annu rev biochem, 2006. 75: p. 333-66. 57. fandrich, m., on the structural definition of amyloid fibrils and other polypeptide aggregates. cell mol life sci, 2007. 64(16): p. 2066-78. 58. westermark, g.t., et al., noncerebral amyloidoses: aspects on seeding, cross-seeding, and transmission. cold spring harb perspect med, 2018. 8(1): p. a024323. 59. eisenberg, d. and m. jucker, the amyloid state of proteins in human diseases. cell, 2012. 148(6): p. 1188-203. 60. hardy, j., expression of normal sequence pathogenic proteins for neurodegenerative disease contributes to disease risk: ‘permissive templating’ as a general mechanism underlying neurodegeneration. biochem soc trans, 2005. 33(pt 4): p. 578-81. 61. annamalai, k., et al., polymorphism of amyloid fibrils in vivo. angew chem int ed engl, 2016. 55(15): p. 4822-5. 62. eisenberg, d.s. and m.r. sawaya, structural studies of amyloid proteins at the molecular level. annu rev biochem, 2017. 86: p. 69-95. 63. fandrich, m., j. meinhardt, and n. grigorieff, structural polymorphism of alzheimer abeta and other amyloid fibrils. prion, 2009. 3(2): p. 89-93. 64. fitzpatrick, a.w.p., et al., cryo-em structures of tau filaments from alzheimer’s disease. nature, 2017. 547(7662): p. 185-190. 65. iadanza, m.g., et al., the structure of a beta2-microglobulin fibril suggests a molecular basis for its amyloid polymorphism. nat commun, 2018. 9(1): p. 4517. 66. kollmer, m., et al., cryo-em structure and polymorphism of abeta amyloid fibrils purified from alzheimer’s brain tissue. nat commun, 2019. 10(1): p. 4760. 67. petkova, a.t., et al., self-propagating, molecular-level polymorphism in alzheimer’s beta-amyloid fibrils. science, 2005. 307(5707): p. 262-5. 68. roeder, c., et al., cryo-em structure of islet amyloid polypeptide fibrils reveals similarities with amyloid-β fibrils. nat struct mol biol, 2020. 27(7): p. 660-667. 69. pepys, m.b., pathogenesis, diagnosis and treatment of systemic amyloidosis. philos trans r soc lond b biol sci, 2001. 356(1406): p. 203-10; discussion 210-1. 70. pinney, j.h. and p.n. hawkins, amyloidosis. ann clin biochem, 2012. 49(pt 3): p. 229-41. 71. bridger, j. and n.a. wright, amyloidosis, in oxford textbook of pathology, j.o.d. mcgee, p.g. isaacson, and n.a. wright, editors. 1992, oxford university press: new york. p. 406-412. 72. chen, d., et al., tau local structure shields an amyloid-forming motif and controls aggregation propensity. nat commun, 2019. 10(1): p. 2493. 73. arriagada, p.v., et al., neurofibrillary tangles but not senile plaques parallel duration and severity of alzheimer’s disease. neurology, 1992. 42(3 pt 1): p. 631-9. 74. bierer, l.m., et al., neocortical neurofibrillary tangles correlate with dementia severity in alzheimer’s disease. arch neurol, 1995. 52(1): p. 81-8. 75. crystal, h., et al., clinico-pathologic studies in dementia: nondemented subjects with pathologically confirmed alzheimer’s disease. neurology, 1988. 38(11): p. 1682-7. 76. gold, g., et al., clinical validity of a beta-protein deposition staging in brain aging and alzheimer disease. j neuropathol exp neurol, 2001. 60(10): p. 946-52. 77. nagy, z., et al., relative roles of plaques and tangles in the dementia of alzheimer’s disease: correlations using three sets of neuropathological criteria. dementia, 1995. 6(1): p. 21-31. 78. wilcock, g.k. and m.m. esiri, plaques, tangles and dementia. a quantitative study. j neurol sci, 1982. 56(2-3): p. 343-56. 79. braak, h. and e. braak, neuropathological stageing of alzheimer-related changes. acta neuropathol, 1991. 82(4): p. 239-59. 80. braak, h. and e. braak, frequency of stages of alzheimer-related lesions in different age categories. neurobiol aging, 1997. 18(4): p. 351-7. 81. tomlinson, b.e. and j.a.n. corsellis, ageing and the dementias, in greenfield’s neuropathology, j.h. adams, j.a.n. corsellis, and l.w. duchen, editors. 1984, john wiley and sons: new york. p. 951-1025. 82. he, z., et al., amyloid-beta plaques enhance alzheimer’s brain tau-seeded pathologies by facilitating neuritic plaque tau aggregation. nat med, 2018. 24(1): p. 29-38. 83. stancu, i.c., et al., models of beta-amyloid induced tau-pathology: the long and "folded" road to understand the mechanism. mol neurodegener, 2014. 9: p. 51. 84. busche, m.a. and b.t. hyman, synergy between amyloid-beta and tau in alzheimer’s disease. nat neurosci, 2020. 23(10): p. 1183-1193. 85. cook, h.c., origins of ... tinctorial methods in histology. j clin pathol, 1997. 50(9): p. 716-20. 86. blocq, p. and g. marinesco, sur les lésions et la pathogénie de l’épilepsie dite essentielle. la semaine médicale, 1892. p. 445-446. 87. goedert, m., oskar fischer and the study of dementia. brain, 2009. 132(pt 4): p. 1102-11. 88. redlich, e., ueber miliare sklerose der hirnrinde bei seniler atrophie. jahrbücher für psychiatrie und neurologie, 1898. 17: p. 208-216. 89. ohry, a. and o. buda, teofil simchowicz (1879-1957): the scientist who coined senile plaques in neuropathology. rom j morphol embryol, 2015. 56(4): p. 1545-8. 90. crook, r., et al., a variant of alzheimer’s disease with spastic paraparesis and unusual plaques due to deletion of exon 9 of presenilin 1. nat med, 1998. 4(4): p. 452-5. 91. houlden, h., et al., variant alzheimer’s disease with spastic paraparesis and cotton wool plaques is caused by ps-1 mutations that lead to exceptionally high amyloid-beta concentrations. ann neurol, 2000. 48(5): p. 806-8. 92. le, t.v., et al., cotton wool plaques in non-familial late-onset alzheimer disease. j neuropathol exp neurol, 2001. 60(11): p. 1051-61. 93. miki, t., et al., young adult-onset, very slowly progressive cognitive decline with spastic paraparesis in alzheimer’s disease with cotton wool plaques due to a novel presenilin1 g417s mutation. acta neuropathol commun, 2019. 7(1): p. 19. 94. miravalle, l., et al., amino-terminally truncated abeta peptide species are the main component of cotton wool plaques. biochemistry, 2005. 44(32): p. 10810-21. 95. alzheimer, a., über eine eigenartige erkrankung der hirnrinde. allgemeine zeitschrift für psychiatrie, 1907. 64: p. 146-148. 96. alzheimer, a., über eigenartige krankheitsfälle des späteren alters. zeitschrift für die gesamte neurologie und psychiatrie, 1911. 4: p. 356-385. 97. miyake, k., beiträge zur kenntnis der altersveränderungen der menschilchen hirnrinde. arbeiten obersteinerschen neurologische institut wien, 1906. 13: p. 212-259. 98. lafora, g.r., beitrag zur kenntnis der alzheimerschen krankheit oder präsenilen demenz mit herdsymptomen. zeitschrift für die gesamte neurologie und psychiatrie, 1911. 6(1): p. 15. 99. bonfiglio, f., di speciali reperti in un caso di probabile sifilide cerebrale. rivista sperimentale di feniatria 1908. 34: p. 196-206. 100. hübner, a.h., zur histopathologie der senilen hirnrinde. archiv für psychiatrie und nervenkrankheiten, 1909. 46(2): p. 598-609. 101. perusini, g., über klinisch und histologisch eigenartige psychische erkrankungen des höheren lebensalters, in histologische und histopathologische arbeiten über die grosshirnrinde, f. nissl and a. alzheimer, editors. 1910, fischer-verlag: jena. p. 297-351. 102. fuller, s.c., a study of the miliary plaques found in brains of the aged. american journal of insanity, 1911. 68(2): p. 147-219. 103. bielschowsky, m., zur kenntnis der alzheimerschen krankheit (präsenilen demenz mit herdsymptomen). journal für psychologie und neurologie 1911. 18: p. 1-20. 104. barrett, a.m., degeneration of intracellular neurofibrils with miliary gliosis in psychoses of the senile period. american journal of insanity, 1911. 67(3): p. 503-516. 105. simchowicz, t., histologische studien über die senile demenz, in histologische und histopathologische arbeiten über die grosshirnrinde, f. nissl and a. alzheimer, editors. 1911, fischer-verlag: jena. p. 267-444. 106. marinesco, g. and j. minea, untersuchungen über die “senilen plaques”. monatschrift für psychiatrie und neurologie, 1912. 31: p. 79-91. 107. christen, y., alois alzheimer and the myth of the pioneer, in alzheimer: 100 years and beyond, m. jucker, et al., editors. 2006, springer: berlin heidelberg new york. p. 51-55. 108. braak, h. and e. braak, neurofibrillary changes: the hallmark of alzheimer disease, in concepts of alzheimer disease: biological, clinical and cultural perspectives, p.j. whitehouse, k. mauer, and j. ballenger, editors. 2000, johns hopkins university press: baltimore. p. 53-71. 109. jack, c.r., jr., et al., nia-aa research framework: toward a biological definition of alzheimer’s disease. alzheimers dement, 2018. 14(4): p. 535-562. 110. bick, k., l. amaducci, and g. pepeu, the early story of alzheimer’s disease. 1987, philadelphia: lippincott williams and wilkins. 111. de strooper, b. and e. karran, the cellular phase of alzheimer’s disease. cell, 2016. 164(4): p. 603-15. 112. gleichman, a.j. and s.t. carmichael, glia in neurodegeneration: drivers of disease or along for the ride? neurobiol dis, 2020. 142: p. 104957. 113. heneka, m.t., et al., neuroinflammation in alzheimer’s disease. lancet neurol, 2015. 14(4): p. 388-405. 114. newcombe, e.a., et al., inflammation: the link between comorbidities, genetics, and alzheimer’s disease. j neuroinflammation, 2018. 15(1): p. 276. 115. shi, y. and d.m. holtzman, interplay between innate immunity and alzheimer disease: apoe and trem2 in the spotlight. nat rev immunol, 2018. 18(12): p. 759-772. 116. critchley, m., critical review: the nature and significance of senile plaques. journal of neurology and psychopathology, 1929. 10(38): p. 124-39. 117. fischer, o., miliare nekrosen mit drusigen wucherungen der neurofibrillen, eine regelmässige veränderung der hirnrinde bei seniler demenz. monatsschrift für psychiatrie und neurologie, 1907. 22: p. 361-372. 118. ferraro, a., the origin and formation of senile plaques. archives of neurology and psychiatry, 1931. 25(5): p. 1042-1062. 119. soniat, t.l.l., histogenesis of senile plaques. archives of neurology and psychiatry, 1941. 46(1): p. 101-114. 120. liss, l., senile brain changes, histopathology of the ganglion cells. j neuropathol exp neurol, 1960. 19: p. 559-71. 121. bouman, l., senile plaques. brain, 1934. 57(2): p. 128-142. 122. kidd, m., alzheimer’s disease--an electron microscopical study. brain, 1964. 87: p. 307-20. 123. terry, r.d., n.k. gonatas, and m. weiss, ultrastructural studies in alzheimer’s presenile dementia. am j pathol, 1964. 44: p. 269-97. 124. blessed, g., b.e. tomlinson, and m. roth, the association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects. br j psychiatry, 1968. 114(512): p. 797-811. 125. divry, p., etude histo-chimique des plaques séniles. journal belge de neurologie et de psychiatrie, 1927. 9: p. 643-657. 126. dickson, d.w., neuropathology of non-alzheimer degenerative disorders. int j clin exp pathol, 2009. 3(1): p. 1-23. 127. dearmond, s.j., et al., neuropathology of prion diseases, in prion biology and diseases, s.b. prusiner, editor. 2004, cold spring harbor laboratory press: cold spring harbor. p. 777-856. 128. dearmond, s.j. and s.b. prusiner, etiology and pathogenesis of prion diseases. am j pathol, 1995. 146(4): p. 785-811. 129. holton, j.l., et al., regional distribution of amyloid-bri deposition and its association with neurofibrillary degeneration in familial british dementia. am j pathol, 2001. 158(2): p. 515-26. 130. vidal, r., et al., a stop-codon mutation in the bri gene associated with familial british dementia. nature, 1999. 399(6738): p. 776-81. 131. holton, j.l., et al., familial danish dementia: a novel form of cerebral amyloidosis associated with deposition of both amyloid-dan and amyloid-beta. j neuropathol exp neurol, 2002. 61(3): p. 254-67. 132. vidal, r., et al., a decamer duplication in the 3’ region of the bri gene originates an amyloid peptide that is associated with dementia in a danish kindred. proc natl acad sci u s a, 2000. 97(9): p. 4920-5. 133. serrano-pozo, a., et al., neuropathological alterations in alzheimer disease. cold spring harb perspect med, 2011. 1(1): p. a006189. 134. steiner, h., et al., a pathogenic presenilin-1 deletion causes abberrant abeta 42 production in the absence of congophilic amyloid plaques. j biol chem, 2001. 276(10): p. 7233-9. 135. iwatsubo, t., et al., full-length amyloid-beta (1-42(43)) and amino-terminally modified and truncated amyloid-beta 42(43) deposit in diffuse plaques. am j pathol, 1996. 149(6): p. 1823-30. 136. lalowski, m., et al., the "nonamyloidogenic" p3 fragment (amyloid beta17-42) is a major constituent of down’s syndrome cerebellar preamyloid. j biol chem, 1996. 271(52): p. 33623-31. 137. the american heritage dictionary of the english language. 2011, houghton mifflin harcourt: boston, ma. 138. hauw, j.-j. and c. duyckaerts, alzheimer’s disease, in pathology of the aging human nervous system, s. duckett and j.c. de la torre, editors. 2001, oxford university press: new york. p. 207-263. 139. dickson, d.w., the pathogenesis of senile plaques. j neuropathol exp neurol, 1997. 56(4): p. 321-39. 140. cupidi, c., et al., neocortical variation of abeta load in fully expressed, pure alzheimer’s disease. j alzheimers dis, 2010. 19(1): p. 57-68. 141. rogers, j. and j.h. morrison, quantitative morphology and regional and laminar distributions of senile plaques in alzheimer’s disease. j neurosci, 1985. 5(10): p. 2801-8. 142. bero, a.w., et al., neuronal activity regulates the regional vulnerability to amyloid-beta deposition. nat neurosci, 2011. 14(6): p. 750-6. 143. wisniewski, h.m., et al., spectrum of morphological appearance of amyloid deposits in alzheimer’s disease. acta neuropathol, 1989. 78(4): p. 337-47. 144. thal, d.r., et al., the development of amyloid beta protein deposits in the aged brain. sci aging knowledge environ, 2006. 2006(6): p. re1. 145. thal, d.r., et al., phases of a beta-deposition in the human brain and its relevance for the development of ad. neurology, 2002. 58(12): p. 1791-800. 146. braak, h., et al., stages of the pathologic process in alzheimer disease: age categories from 1 to 100 years. j neuropathol exp neurol, 2011. 70(11): p. 960-9. 147. grothe, m.j., et al., in vivo staging of regional amyloid deposition. neurology, 2017. 89(20): p. 2031-2038. 148. ogomori, k., et al., beta-protein amyloid is widely distributed in the central nervous system of patients with alzheimer’s disease. am j pathol, 1989. 134(2): p. 243-51. 149. klunk, w.e., et al., imaging brain amyloid in alzheimer’s disease with pittsburgh compound-b. ann neurol, 2004. 55(3): p. 306-19. 150. shoghi-jadid, k., et al., localization of neurofibrillary tangles and beta-amyloid plaques in the brains of living patients with alzheimer disease. am j geriatr psychiatry, 2002. 10(1): p. 24-35. 151. barrio, j.r., et al., in vivo characterization of chronic traumatic encephalopathy using [f-18]fddnp pet brain imaging. proc natl acad sci u s a, 2015. 112(16): p. e2039-47. 152. kepe, v., et al., pet imaging of neuropathology in tauopathies: progressive supranuclear palsy. j alzheimers dis, 2013. 36(1): p. 145-53. 153. shin, j., et al., the merits of fddnp-pet imaging in alzheimer’s disease. j alzheimers dis, 2011. 26 suppl 3: p. 135-45. 154. mathis, c.a., et al., small-molecule pet tracers for imaging proteinopathies. semin nucl med, 2017. 47(5): p. 553-575. 155. clark, c.m., et al., use of florbetapir-pet for imaging beta-amyloid pathology. jama, 2011. 305(3): p. 275-83. 156. wong, d.f., et al., in vivo imaging of amyloid deposition in alzheimer disease using the radioligand 18f-av-45 (florbetapir [corrected] f 18). j nucl med, 2010. 51(6): p. 913-20. 157. sabri, o., et al., florbetaben pet imaging to detect amyloid beta plaques in alzheimer’s disease: phase 3 study. alzheimers dement, 2015. 11(8): p. 964-74. 158. vandenberghe, r., et al., 18f-flutemetamol amyloid imaging in alzheimer disease and mild cognitive impairment: a phase 2 trial. ann neurol, 2010. 68(3): p. 319-29. 159. ikonomovic, m.d., et al., post-mortem histopathology underlying beta-amyloid pet imaging following flutemetamol f 18 injection. acta neuropathol commun, 2016. 4(1): p. 130. 160. salloway, s., et al., performance of [(18)f]flutemetamol amyloid imaging against the neuritic plaque component of cerad and the current (2012) nia-aa recommendations for the neuropathologic diagnosis of alzheimer’s disease. alzheimers dement (amst), 2017. 9: p. 25-34. 161. thal, d.r., et al., different aspects of alzheimer’s disease-related amyloid beta-peptide pathology and their relationship to amyloid positron emission tomography imaging and dementia. acta neuropathol commun, 2019. 7(1): p. 178. 162. diner, i., et al., generation of clickable pittsburgh compound b for the detection and capture of beta-amyloid in alzheimer’s disease brain. bioconjug chem, 2017. 28(10): p. 2627-2637. 163. rosen, r.f., et al., comparative pathobiology of beta-amyloid and the unique susceptibility of humans to alzheimer’s disease. neurobiol aging, 2016. 44: p. 185-196. 164. rosen, r.f., et al., deficient high-affinity binding of pittsburgh compound b in a case of alzheimer’s disease. acta neuropathol, 2010. 119(2): p. 221-33. 165. walker, l.c. and m. jucker, the exceptional vulnerability of humans to alzheimer’s disease. trends mol med, 2017. 23(6): p. 534-545. 166. ikeda, s., d. allsop, and g.g. glenner, morphology and distribution of plaque and related deposits in the brains of alzheimer’s disease and control cases. an immunohistochemical study using amyloid beta-protein antibody. lab invest, 1989. 60(1): p. 113-22. 167. ikeda, s., et al., evidence of amyloid beta-protein immunoreactive early plaque lesions in down’s syndrome brains. lab invest, 1989. 61(1): p. 133-7. 168. tagliavini, f., et al., preamyloid deposits in the cerebral cortex of patients with alzheimer’s disease and nondemented individuals. neurosci lett, 1988. 93(2-3): p. 191-6. 169. tagliavini, f., et al., cerebral extracellular preamyloid deposits in alzheimer’s disease, down syndrome and nondemented elderly individuals. prog clin biol res, 1989. 317: p. 1001-5. 170. yamaguchi, h., et al., diffuse type of senile plaques in the brains of alzheimer-type dementia. acta neuropathol, 1988. 77(2): p. 113-9. 171. yamaguchi, h., et al., a variety of cerebral amyloid deposits in the brains of the alzheimer-type dementia demonstrated by beta protein immunostaining. acta neuropathol, 1988. 76(6): p. 541-9. 172. yamaguchi, h., et al., alzheimer type dementia: diffuse type of senile plaques demonstrated by beta protein immunostaining. prog clin biol res, 1989. 317: p. 467-74. 173. armstrong, r.a., beta-amyloid plaques: stages in life history or independent origin? dement geriatr cogn disord, 1998. 9(4): p. 227-38. 174. dickson, t.c. and j.c. vickers, the morphological phenotype of beta-amyloid plaques and associated neuritic changes in alzheimer’s disease. neuroscience, 2001. 105(1): p. 99-107. 175. duyckaerts, c., b. delatour, and m.c. potier, classification and basic pathology of alzheimer disease. acta neuropathol, 2009. 118(1): p. 5-36. 176. thal, d.r., et al., sequence of abeta-protein deposition in the human medial temporal lobe. j neuropathol exp neurol, 2000. 59(8): p. 733-48. 177. masters, c.l. and k. beyreuther, henryk m. wisniewski and the amyloid theory of alzheimer’s disease. j alzheimers dis, 2001. 3(1): p. 83-86. 178. wisniewski, h.m. and r.d. terry, reexamination of the pathogenesis of the senile plaque, in progress in neuropathology, h.m. zimmerman, editor. 1973, grune & stratton: new york. p. 1-26. 179. boon, b.d.c., et al., the coarse-grained plaque: a divergent abeta plaque-type in early-onset alzheimer’s disease. acta neuropathol, 2020. doi: 10.1007/s00401-020-02198-8. online ahead of print. pmid: 32926214. 180. thal, d.r., et al., fleecy amyloid deposits in the internal layers of the human entorhinal cortex are comprised of n-terminal truncated fragments of abeta. j neuropathol exp neurol, 1999. 58(2): p. 210-6. 181. davies, c.a. and d.m. mann, is the "preamyloid" of diffuse plaques in alzheimer’s disease really nonfibrillar? am j pathol, 1993. 143(6): p. 1594-605. 182. yamaguchi, h., et al., electron micrograph of diffuse plaques. initial stage of senile plaque formation in the alzheimer brain. am j pathol, 1989. 135(4): p. 593-7. 183. yamaguchi, h., et al., ultrastructure of diffuse plaques in senile dementia of the alzheimer type: comparison with primitive plaques. acta neuropathol, 1991. 82(1): p. 13-20. 184. wisniewski, h.m., et al., diffuse, lake-like amyloid-beta deposits in the parvopyramidal layer of the presubiculum in alzheimer disease. j neuropathol exp neurol, 1998. 57(7): p. 674-83. 185. fischer, o., die presbyophrene demenz, deren anatomische grundlage und klinische abgrenzung. zeitschrift für die gesamte neurologie und psychiatrie, 1910. 3: p. 371-471. 186. thal, d.r., et al., amyloid beta-protein (abeta)-containing astrocytes are located preferentially near n-terminal-truncated abeta deposits in the human entorhinal cortex. acta neuropathol, 2000. 100(6): p. 608-17. 187. allsop, d., et al., neurofibrillary tangles in some cases of dementia pugilistica share antigens with amyloid beta-protein of alzheimer’s disease. am j pathol, 1990. 136(2): p. 255-60. 188. bondareff, w., et al., molecular analysis of neurofibrillary degeneration in alzheimer’s disease. an immunohistochemical study. am j pathol, 1990. 137(3): p. 711-23. 189. hyman, b.t., et al., a4 amyloid protein immunoreactivity is present in alzheimer’s disease neurofibrillary tangles. neurosci lett, 1989. 101(3): p. 352-5. 190. sherriff, f.e., l.r. bridges, and d.s. de souza, non-alzheimer neurofibrillary tangles show beta-amyloid-like immunoreactivity. neuroreport, 1994. 5(15): p. 1897-900. 191. tabaton, m., et al., ultrastructural localization of beta-amyloid, tau, and ubiquitin epitopes in extracellular neurofibrillary tangles. proc natl acad sci u s a, 1991. 88(6): p. 2098-102. 192. yamaguchi, h., et al., secondary deposition of beta amyloid within extracellular neurofibrillary tangles in alzheimer-type dementia. am j pathol, 1991. 138(3): p. 699-705. 193. zemlan, f.p., et al., alzheimer’s paired helical filaments: amyloid precursor protein epitope mapping. brain res bull, 1994. 33(4): p. 387-92. 194. walker, l.c., et al., apolipoprotein e4 promotes the early deposition of abeta42 and then abeta40 in the elderly. acta neuropathol, 2000. 100(1): p. 36-42. 195. mott, r.t. and c.m. hulette, neuropathology of alzheimer’s disease. neuroimaging clin n am, 2005. 15(4): p. 755-65, ix. 196. edgar, j.r., et al., escrts regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-beta accumulation. j cell sci, 2015. 128(14): p. 2520-8. 197. langui, d., et al., subcellular topography of neuronal abeta peptide in appxps1 transgenic mice. am j pathol, 2004. 165(5): p. 1465-77. 198. rajendran, l., et al., alzheimer’s disease beta-amyloid peptides are released in association with exosomes. proc natl acad sci u s a, 2006. 103(30): p. 11172-7. 199. takahashi, r.h., et al., intraneuronal alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. am j pathol, 2002. 161(5): p. 1869-79. 200. willen, k., et al., abeta accumulation causes mvb enlargement and is modelled by dominant negative vps4a. mol neurodegener, 2017. 12(1): p. 61. 201. becot, a., c. volgers, and g. van niel, transmissible endosomal intoxication: a balance between exosomes and lysosomes at the basis of intercellular amyloid propagation. biomedicines, 2020. 8(8). 202. han, s., et al., amyloid plaque structure and cell surface interactions of beta-amyloid fibrils revealed by electron tomography. sci rep, 2017. 7: p. 43577. 203. schmidt, m.l., et al., chemical and immunological heterogeneity of fibrillar amyloid in plaques of alzheimer’s disease and down’s syndrome brains revealed by confocal microscopy. am j pathol, 1995. 147(2): p. 503-15. 204. nelson, p.t., h. braak, and w.r. markesbery, neuropathology and cognitive impairment in alzheimer disease: a complex but coherent relationship. j neuropathol exp neurol, 2009. 68(1): p. 1-14. 205. prusiner, s.b., an introduction to prion diseases, in prion diseases, s.b. prusiner, editor. 2017, cold spring harbor laboratory press: cold spring harbor p. 1-29. 206. will, r.g. and j.w. ironside, sporadic and infectious human prion diseases. cold spring harb perspect med, 2017. 7(1): p. a024364. 207. fiala, j.c., mechanisms of amyloid plaque pathogenesis. acta neuropathol, 2007. 114(6): p. 551-71. 208. azizeh, b.y., et al., molecular dating of senile plaques in the brains of individuals with down syndrome and in aged dogs. exp neurol, 2000. 163(1): p. 111-22. 209. fonseca, m.i., et al., the presence of isoaspartic acid in beta-amyloid plaques indicates plaque age. exp neurol, 1999. 157(2): p. 277-88. 210. lee, h.g., et al., challenging the amyloid cascade hypothesis: senile plaques and amyloid-beta as protective adaptations to alzheimer disease. ann n y acad sci, 2004. 1019: p. 1-4. 211. makin, s., the amyloid hypothesis on trial. nature, 2018. 559(7715): p. s4-s7. 212. walker, l.c., proteopathic strains and the heterogeneity of neurodegenerative diseases. annu rev genet, 2016. 50: p. 329-346. 213. lau, h.h.c., m. ingelsson, and j.c. watts, the existence of abeta strains and their potential for driving phenotypic heterogeneity in alzheimer’s disease. acta neuropathol, 2020. doi: 10.1007/s00401-020-02201-2. online ahead of print. pmid: 32743745. 214. bartz, j.c., prion strain diversity. cold spring harb perspect med, 2016. 6(12): p. a024349. 215. ghaemmaghami, s., biology and genetics of prp prion strains. cold spring harb perspect med, 2017. 7(8): p. a026922. 216. prusiner, s.b., prion diseases. 2017, cold spring harbor: cold spring harbor laboratory press. 217. jucker, m. and l.c. walker, self-propagation of pathogenic protein aggregates in neurodegenerative diseases. nature, 2013. 501(7465): p. 45-51. 218. walker, l.c. and m. jucker, neurodegenerative diseases: expanding the prion concept. annu rev neurosci, 2015. 38: p. 87-103. 219. tanaka, m., et al., the physical basis of how prion conformations determine strain phenotypes. nature, 2006. 442(7102): p. 585-9. 220. pedersen, j.s. and d.e. otzen, amyloid-a state in many guises: survival of the fittest fibril fold. protein sci, 2008. 17(1): p. 2-10. 221. yagi, h., et al., visualization and classification of amyloid beta supramolecular assemblies. biochemistry, 2007. 46(51): p. 15009-17. 222. mehta, a.k., et al., context dependence of protein misfolding and structural strains in neurodegenerative diseases. biopolymers, 2013. 100(6): p. 722-30. 223. li, j., et al., darwinian evolution of prions in cell culture. science, 2010. 327(5967): p. 869-72. 224. collinge, j. and a.r. clarke, a general model of prion strains and their pathogenicity. science, 2007. 318(5852): p. 930-6. 225. xu, g., et al., diversity in abeta deposit morphology and secondary proteome insolubility across models of alzheimer-type amyloidosis. acta neuropathol commun, 2020. 8(1): p. 43. 226. cohen, m., b. appleby, and j.g. safar, distinct prion-like strains of amyloid beta implicated in phenotypic diversity of alzheimer’s disease. prion, 2016. 10(1): p. 9-17. 227. condello, c., et al., structural heterogeneity and intersubject variability of abeta in familial and sporadic alzheimer’s disease. proc natl acad sci u s a, 2018. 115(4): p. e782-e791. 228. lu, j.x., et al., molecular structure of beta-amyloid fibrils in alzheimer’s disease brain tissue. cell, 2013. 154(6): p. 1257-68. 229. qiang, w., et al., structural variation in amyloid-beta fibrils from alzheimer’s disease clinical subtypes. nature, 2017. 541(7636): p. 217-221. 230. rasmussen, j., et al., amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of alzheimer’s disease. proc natl acad sci u s a, 2017. 114(49): p. 13018-13023. 231. watts, j.c., et al., serial propagation of distinct strains of abeta prions from alzheimer’s disease patients. proc natl acad sci u s a, 2014. 111(28): p. 10323-8. 232. fitzpatrick, a.w. and h.r. saibil, cryo-em of amyloid fibrils and cellular aggregates. curr opin struct biol, 2019. 58: p. 34-42. 233. paravastu, a.k., et al., molecular structural basis for polymorphism in alzheimer’s beta-amyloid fibrils. proc natl acad sci u s a, 2008. 105(47): p. 18349-54. 234. sachse, c., m. fandrich, and n. grigorieff, paired beta-sheet structure of an abeta(1-40) amyloid fibril revealed by electron microscopy. proc natl acad sci u s a, 2008. 105(21): p. 7462-6. 235. wang, h., et al., polymorphic abeta42 fibrils adopt similar secondary structure but differ in cross-strand side chain stacking interactions within the same beta-sheet. sci rep, 2020. 10(1): p. 5720. 236. revesz, t., et al., cerebral amyloid angiopathies: a pathologic, biochemical, and genetic view. j neuropathol exp neurol, 2003. 62(9): p. 885-98. 237. biffi, a. and s.m. greenberg, cerebral amyloid angiopathy: a systematic review. j clin neurol, 2011. 7(1): p. 1-9. 238. haan, j., et al., dementia in hereditary cerebral hemorrhage with amyloidosis-dutch type. arch neurol, 1990. 47(9): p. 965-7. 239. maat-schieman, m.l., et al., hereditary cerebral hemorrhage with amyloidosis-dutch type (hchwa-d): ii--a review of histopathological aspects. brain pathol, 1996. 6(2): p. 115-20. 240. revesz, t., et al., genetics and molecular pathogenesis of sporadic and hereditary cerebral amyloid angiopathies. acta neuropathol, 2009. 118(1): p. 115-30. 241. attems, j., sporadic cerebral amyloid angiopathy: pathology, clinical implications, and possible pathomechanisms. acta neuropathol, 2005. 110(4): p. 345-59. 242. attems, j. and k.a. jellinger, the overlap between vascular disease and alzheimer’s disease--lessons from pathology. bmc med, 2014. 12: p. 206. 243. kapasi, a. and j.a. schneider, vascular contributions to cognitive impairment, clinical alzheimer’s disease, and dementia in older persons. biochim biophys acta, 2016. 1862(5): p. 878-86. 244. vinters, h.v., emerging concepts in alzheimer’s disease. annu rev pathol, 2015. 10: p. 291-319. 245. auriel, e. and s.m. greenberg, the pathophysiology and clinical presentation of cerebral amyloid angiopathy. curr atheroscler rep, 2012. 14(4): p. 343-50. 246. olichney, j.m., et al., cerebral infarction in alzheimer’s disease is associated with severe amyloid angiopathy and hypertension. arch neurol, 1995. 52(7): p. 702-8. 247. kamara, d.m., et al., cerebral amyloid angiopathy: similarity in african-americans and caucasians with alzheimer’s disease. j alzheimers dis, 2018. 62(4): p. 1815-1826. 248. bornebroek, m., et al., hereditary cerebral hemorrhage with amyloidosis-dutch type (hchwa-d): i--a review of clinical, radiologic and genetic aspects. brain pathol, 1996. 6(2): p. 111-4. 249. kamp, j.a., et al., amyloid beta in hereditary cerebral hemorrhage with amyloidosis-dutch type. rev neurosci, 2014. 25(5): p. 641-51. 250. timmers, w.f., et al., parenchymal preamyloid and amyloid deposits in the brains of patients with hereditary cerebral hemorrhage with amyloidosis--dutch type. neurosci lett, 1990. 118(2): p. 223-6. 251. van duinen, s.g., et al., hereditary cerebral hemorrhage with amyloidosis in patients of dutch origin is related to alzheimer disease. proc natl acad sci u s a, 1987. 84(16): p. 5991-4. 252. natte, r., et al., dementia in hereditary cerebral hemorrhage with amyloidosis-dutch type is associated with cerebral amyloid angiopathy but is independent of plaques and neurofibrillary tangles. ann neurol, 2001. 50(6): p. 765-72. 253. wattendorff, a.r., et al., hereditary cerebral haemorrhage with amyloidosis, dutch type (hchwa-d): clinicopathological studies. j neurol neurosurg psychiatry, 1995. 58(6): p. 699-705. 254. case, n.f., et al., cerebral amyloid angiopathy is associated with executive dysfunction and mild cognitive impairment. stroke, 2016. 47(8): p. 2010-6. 255. pfeifer, l.a., et al., cerebral amyloid angiopathy and cognitive function: the haas autopsy study. neurology, 2002. 58(11): p. 1629-34. 256. schrag, m. and h. kirshner, neuropsychological effects of cerebral amyloid angiopathy. curr neurol neurosci rep, 2016. 16(8): p. 76. 257. thal, d.r., et al., vascular pathology in alzheimer disease: correlation of cerebral amyloid angiopathy and arteriosclerosis/lipohyalinosis with cognitive decline. j neuropathol exp neurol, 2003. 62(12): p. 1287-301. 258. xiong, l., et al., dementia incidence and predictors in cerebral amyloid angiopathy patients without intracerebral hemorrhage. j cereb blood flow metab, 2018. 38(2): p. 241-249. 259. smith, e.e., cerebral amyloid angiopathy as a cause of neurodegeneration. j neurochem, 2018. 144(5): p. 651-658. 260. cisternas, p., x. taylor, and c.a. lasagna-reeves, the amyloid-tau-neuroinflammation axis in the context of cerebral amyloid angiopathy. int j mol sci, 2019. 20(24): p. 6319. 261. grabowski, t.j., et al., novel amyloid precursor protein mutation in an iowa family with dementia and severe cerebral amyloid angiopathy. ann neurol, 2001. 49(6): p. 697-705. 262. oshima, k., et al., relative paucity of tau accumulation in the small areas with abundant abeta42-positive capillary amyloid angiopathy within a given cortical region in the brain of patients with alzheimer pathology. acta neuropathol, 2006. 111(6): p. 510-8. 263. yamaguchi, h. and m.l.c. maat-schieman, immunohistochemical analysis of amyloid beta-protein isoforms in caa, in cerebral amyloid angiopathy in alzheimer’s disease and related disorders, m.m. verbeek, r.m.w. de waal, and h.v. vinters, editors. 2000, kluwer academic publishers: dordrecht. p. 179-188. 264. roher, a.e., et al., chemical analysis of amyloid beta protein in caa, in cerebral amyloid angiopathy in alzheimer’s disease and related disorders, m.m. verbeek, r.m.w. de waal, and h.v. vinters, editors. 2000, kluwer academic publishers: dordrecht. p. 157-177. 265. kawai, m., et al., the relationship of amyloid plaques to cerebral capillaries in alzheimer’s disease. am j pathol, 1990. 137(6): p. 1435-46. 266. vinters, h.v., cerebral amyloid angiopathy. a critical review. stroke, 1987. 18(2): p. 311-24. 267. de waal, r.m.w. and m.m. verbeek, abeta-associated proteins in cerebral amyloid angiopathy, in cerebral amyloid angiopathy in alzheimer’s disease and related disorders, m.m. verbeek, r.m.w. de waal, and h.v. vinters, editors. 2000, kluwer academic publishers: dordrecht. p. 207-221. 268. boche, d., et al., consequence of abeta immunization on the vasculature of human alzheimer’s disease brain. brain, 2008. 131(pt 12): p. 3299-310. 269. yamada, m., cerebral amyloid angiopathy: emerging concepts. j stroke, 2015. 17(1): p. 17-30. 270. probst, a., et al., neuritic plaques in senile dementia of alzheimer type: a golgi analysis in the hippocampal region. brain res, 1983. 268(2): p. 249-54. 271. friedrich, r.p., et al., mechanism of amyloid plaque formation suggests an intracellular basis of abeta pathogenicity. proc natl acad sci u s a, 2010. 107(5): p. 1942-7. 272. takahashi, r.h., t. nagao, and g.k. gouras, plaque formation and the intraneuronal accumulation of beta-amyloid in alzheimer’s disease. pathol int, 2017. 67(4): p. 185-193. 273. sadleir, k.r., et al., presynaptic dystrophic neurites surrounding amyloid plaques are sites of microtubule disruption, bace1 elevation, and increased abeta generation in alzheimer’s disease. acta neuropathol, 2016. 132(2): p. 235-256. 274. kuchibhotla, k.v., et al., abeta plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks. neuron, 2008. 59(2): p. 214-25. 275. sanchez-varo, r., et al., abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young alzheimer’s mice hippocampus. acta neuropathol, 2012. 123(1): p. 53-70. 276. wirths, o., et al., lewy body variant of alzheimer’s disease: alpha-synuclein in dystrophic neurites of a beta plaques. neuroreport, 2000. 11(17): p. 3737-41. 277. dickson, t.c., et al., neurochemical diversity of dystrophic neurites in the early and late stages of alzheimer’s disease. exp neurol, 1999. 156(1): p. 100-10. 278. garcia-marin, v., p. garcia-lopez, and m. freire, cajal’s contributions to the study of alzheimer’s disease. j alzheimers dis, 2007. 12(2): p. 161-74. 279. zhan, s.s., et al., distribution of neuronal growth-promoting factors and cytoskeletal proteins in altered neurites in alzheimer’s disease and non-demented elderly. acta neuropathol, 1995. 89(4): p. 356-62. 280. phinney, a.l., et al., cerebral amyloid induces aberrant axonal sprouting and ectopic terminal formation in amyloid precursor protein transgenic mice. j neurosci, 1999. 19(19): p. 8552-9. 281. chan-palay, v., et al., distribution of altered hippocampal neurons and axons immunoreactive with antisera against neuropeptide y in alzheimer’s-type dementia. j comp neurol, 1986. 248(3): p. 376-94. 282. kitt, c.a., et al., evidence for cholinergic neurites in senile plaques. science, 1984. 226(4681): p. 1443-5. 283. kitt, c.a., et al., catecholaminergic neurites in senile plaques in prefrontal cortex of aged nonhuman primates. neuroscience, 1985. 16(3): p. 691-9. 284. kitt, c.a., et al., serotoninergic neurites in senile plaques in cingulate cortex of aged nonhuman primate. synapse, 1989. 3(1): p. 12-8. 285. morrison, j.h., et al., somatostatin immunoreactivity in neuritic plaques of alzheimer’s patients. nature, 1985. 314(6006): p. 90-2. 286. struble, r.g., et al., neuropeptidergic systems in plaques of alzheimer’s disease. j neuropathol exp neurol, 1987. 46(5): p. 567-84. 287. walker, l.c., et al., glutamic acid decarboxylase-like immunoreactive neurites in senile plaques. neurosci lett, 1985. 59(2): p. 165-9. 288. armstrong, d.m., et al., substance p and somatostatin coexist within neuritic plaques: implications for the pathogenesis of alzheimer’s disease. neuroscience, 1989. 31(3): p. 663-71. 289. walker, l.c., et al., multiple transmitter systems contribute neurites to individual senile plaques. j neuropathol exp neurol, 1988. 47(2): p. 138-44. 290. price, d.l., et al., basal forebrain cholinergic systems in alzheimer’s disease and related dementias. neuroscience commentaries, 1982. 1: p. 84-92. 291. benzing, w.c., e.j. mufson, and d.m. armstrong, alzheimer’s disease-like dystrophic neurites characteristically associated with senile plaques are not found within other neurodegenerative diseases unless amyloid beta-protein deposition is present. brain res, 1993. 606(1): p. 10-8. 292. dekosky, s.t. and s.w. scheff, synapse loss in frontal cortex biopsies in alzheimer’s disease: correlation with cognitive severity. ann neurol, 1990. 27(5): p. 457-64. 293. dekosky, s.t., s.w. scheff, and s.d. styren, structural correlates of cognition in dementia: quantification and assessment of synapse change. neurodegeneration, 1996. 5(4): p. 417-21. 294. masliah, e., et al., synaptic and neuritic alterations during the progression of alzheimer’s disease. neurosci lett, 1994. 174(1): p. 67-72. 295. terry, r.d., et al., physical basis of cognitive alterations in alzheimer’s disease: synapse loss is the major correlate of cognitive impairment. ann neurol, 1991. 30(4): p. 572-80. 296. koffie, r.m., b.t. hyman, and t.l. spires-jones, alzheimer’s disease: synapses gone cold. mol neurodegener, 2011. 6(1): p. 63. 297. spires-jones, t.l. and b.t. hyman, the intersection of amyloid beta and tau at synapses in alzheimer’s disease. neuron, 2014. 82(4): p. 756-71. 298. sims, r., m. hill, and j. williams, the multiplex model of the genetics of alzheimer’s disease. nat neurosci, 2020. 23(3): p. 311-322. 299. bohlen, c.j., et al., microglia in brain development, homeostasis, and neurodegeneration. annu rev genet, 2019. 53: p. 263-288. 300. hansen, d.v., j.e. hanson, and m. sheng, microglia in alzheimer’s disease. j cell biol, 2018. 217(2): p. 459-472. 301. neuner, s.m., j. tcw, and a.m. goate, genetic architecture of alzheimer’s disease. neurobiol dis, 2020. 143: p. 104976. 302. farfara, d., v. lifshitz, and d. frenkel, neuroprotective and neurotoxic properties of glial cells in the pathogenesis of alzheimer’s disease. j cell mol med, 2008. 12(3): p. 762-80. 303. liddelow, s.a., et al., neurotoxic reactive astrocytes are induced by activated microglia. nature, 2017. 541(7638): p. 481-487. 304. bouvier, d.s. and k.k. murai, synergistic actions of microglia and astrocytes in the progression of alzheimer’s disease. j alzheimers dis, 2015. 45(4): p. 1001-14. 305. nichols, m.r., et al., inflammatory mechanisms in neurodegeneration. j neurochem, 2019. 149(5): p. 562-581. 306. schwabe, t., k. srinivasan, and h. rhinn, shifting paradigms: the central role of microglia in alzheimer’s disease. neurobiol dis, 2020. 143: p. 104962. 307. serrano-pozo, a., et al., differential relationships of reactive astrocytes and microglia to fibrillar amyloid deposits in alzheimer disease. j neuropathol exp neurol, 2013. 72(6): p. 462-71. 308. town, t., v. nikolic, and j. tan, the microglial "activation" continuum: from innate to adaptive responses. j neuroinflammation, 2005. 2: p. 24. 309. wyss-coray, t. and j. rogers, inflammation in alzheimer disease-a brief review of the basic science and clinical literature. cold spring harb perspect med, 2012. 2(1): p. a006346. 310. johnson, e.c.b., et al., large-scale proteomic analysis of alzheimer’s disease brain and cerebrospinal fluid reveals early changes in energy metabolism associated with microglia and astrocyte activation. nat med, 2020. 26(5): p. 769-780. 311. nagele, r.g., et al., contribution of glial cells to the development of amyloid plaques in alzheimer’s disease. neurobiol aging, 2004. 25(5): p. 663-74. 312. perez-nievas, b.g. and a. serrano-pozo, deciphering the astrocyte reaction in alzheimer’s disease. front aging neurosci, 2018. 10: p. 114. 313. bouvier, d.s., et al., high resolution dissection of reactive glial nets in alzheimer’s disease. sci rep, 2016. 6: p. 24544. 314. smith, a.j., t. duan, and a.s. verkman, aquaporin-4 reduces neuropathology in a mouse model of alzheimer’s disease by remodeling peri-plaque astrocyte structure. acta neuropathol commun, 2019. 7(1): p. 74. 315. heppner, f.l., r.m. ransohoff, and b. becher, immune attack: the role of inflammation in alzheimer disease. nat rev neurosci, 2015. 16(6): p. 358-72. 316. garwood, c.j., et al., review: astrocytes in alzheimer’s disease and other age-associated dementias: a supporting player with a central role. neuropathol appl neurobiol, 2017. 43(4): p. 281-298. 317. simpson, j.e., et al., astrocyte phenotype in relation to alzheimer-type pathology in the ageing brain. neurobiol aging, 2010. 31(4): p. 578-90. 318. mandrekar-colucci, s. and g.e. landreth, microglia and inflammation in alzheimer’s disease. cns neurol disord drug targets, 2010. 9(2): p. 156-67. 319. galatro, t.f., et al., transcriptomic analysis of purified human cortical microglia reveals age-associated changes. nat neurosci, 2017. 20(8): p. 1162-1171. 320. zhou, y., et al., human and mouse single-nucleus transcriptomics reveal trem2-dependent and trem2-independent cellular responses in alzheimer’s disease. nat med, 2020. 26(1): p. 131-142. 321. baik, s.h., et al., microglia contributes to plaque growth by cell death due to uptake of amyloid beta in the brain of alzheimer’s disease mouse model. glia, 2016. 64(12): p. 2274-2290. 322. bolmont, t., et al., dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance. j neurosci, 2008. 28(16): p. 4283-92. 323. fuger, p., et al., microglia turnover with aging and in an alzheimer’s model via long-term in vivo single-cell imaging. nat neurosci, 2017. 20(10): p. 1371-1376. 324. hefendehl, j.k., et al., long-term in vivo imaging of beta-amyloid plaque appearance and growth in a mouse model of cerebral beta-amyloidosis. j neurosci, 2011. 31(2): p. 624-9. 325. ahmad, m.h., m. fatima, and a.c. mondal, influence of microglia and astrocyte activation in the neuroinflammatory pathogenesis of alzheimer’s disease: rational insights for the therapeutic approaches. j clin neurosci, 2019. 59: p. 6-11. 326. frost, g.r., l.a. jonas, and y.m. li, friend, foe or both? immune activity in alzheimer’s disease. front aging neurosci, 2019. 11: p. 337. 327. itagaki, s., et al., relationship of microglia and astrocytes to amyloid deposits of alzheimer disease. j neuroimmunol, 1989. 24(3): p. 173-82. 328. mrak, r.e., neuropathology and the neuroinflammation idea. j alzheimers dis, 2009. 18(3): p. 473-81. 329. sheng, j.g., r.e. mrak, and w.s. griffin, neuritic plaque evolution in alzheimer’s disease is accompanied by transition of activated microglia from primed to enlarged to phagocytic forms. acta neuropathol, 1997. 94(1): p. 1-5. 330. vonsattel, j.p. and e.t. hedley-whyte, dementia, in pathology of the aging human nervous system, s. duckett and j.c. de la torre, editors. 2001, oxford university press: new york. p. 156-206. 331. gratuze, m., c.e.g. leyns, and d.m. holtzman, new insights into the role of trem2 in alzheimer’s disease. mol neurodegener, 2018. 13(1): p. 66. 332. yeh, f.l., d.v. hansen, and m. sheng, trem2, microglia, and neurodegenerative diseases. trends mol med, 2017. 23(6): p. 512-533. 333. el khoury, j., et al., ccr2 deficiency impairs microglial accumulation and accelerates progression of alzheimer-like disease. nat med, 2007. 13(4): p. 432-8. 334. wyss-coray, t., et al., prominent neurodegeneration and increased plaque formation in complement-inhibited alzheimer’s mice. proc natl acad sci u s a, 2002. 99(16): p. 10837-42. 335. wilcock, d.m., et al., microglial activation facilitates abeta plaque removal following intracranial anti-abeta antibody administration. neurobiol dis, 2004. 15(1): p. 11-20. 336. nicoll, j.a.r., et al., persistent neuropathological effects 14 years following amyloid-beta immunization in alzheimer’s disease. brain, 2019. 142(7): p. 2113-2126. 337. keren-shaul, h., et al., a unique microglia type associated with restricting development of alzheimer’s disease. cell, 2017. 169(7): p. 1276-1290 e17. 338. parhizkar, s., et al., loss of trem2 function increases amyloid seeding but reduces plaque-associated apoe. nat neurosci, 2019. 22(2): p. 191-204. 339. leyns, c.e.g., et al., trem2 function impedes tau seeding in neuritic plaques. nat neurosci, 2019. 22(8): p. 1217-1222. 340. frackowiak, j., et al., ultrastructure of the microglia that phagocytose amyloid and the microglia that produce beta-amyloid fibrils. acta neuropathol, 1992. 84(3): p. 225-33. 341. wisniewski, h.m., et al., ultrastructure of the cells forming amyloid fibers in alzheimer disease and scrapie. am j med genet suppl, 1990. 7: p. 287-97. 342. stalder, m., et al., association of microglia with amyloid plaques in brains of app23 transgenic mice. am j pathol, 1999. 154(6): p. 1673-84. 343. spangenberg, e., et al., sustained microglial depletion with csf1r inhibitor impairs parenchymal plaque development in an alzheimer’s disease model. nat commun, 2019. 10(1): p. 3758. 344. hashemiaghdam, a. and m. mroczek, microglia heterogeneity and neurodegeneration: the emerging paradigm of the role of immunity in alzheimer’s disease. j neuroimmunol, 2020. 341: p. 577185. 345. paasila, p.j., et al., clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of aβ plaques in alzheimer’s disease. free neuropathology, 2020. 1(20): p. 1-18. 346. el hajj, h., et al., ultrastructural evidence of microglial heterogeneity in alzheimer’s disease amyloid pathology. j neuroinflammation, 2019. 16(1): p. 87. 347. streit, w.j., h. khoshbouei, and i. bechmann, dystrophic microglia in late-onset alzheimer’s disease. glia, 2020. 68(4): p. 845-854. 348. smith, a.m. and m. dragunow, the human side of microglia. trends neurosci, 2014. 37(3): p. 125-35. 349. geirsdottir, l., et al., cross-species single-cell analysis reveals divergence of the primate microglia program. cell, 2019. 179(7): p. 1609-1622 e16. 350. phatnani, h. and t. maniatis, astrocytes in neurodegenerative disease. cold spring harb perspect biol, 2015. 7(6): p. a020628. 351. thal, d.r., the role of astrocytes in amyloid beta-protein toxicity and clearance. exp neurol, 2012. 236(1): p. 1-5. 352. frost, g.r. and y.m. li, the role of astrocytes in amyloid production and alzheimer’s disease. open biol, 2017. 7(12): p. 170228. 353. funato, h., et al., astrocytes containing amyloid beta-protein (abeta)-positive granules are associated with abeta40-positive diffuse plaques in the aged human brain. am j pathol, 1998. 152(4): p. 983-92. 354. nagele, r.g., et al., astrocytes accumulate a beta 42 and give rise to astrocytic amyloid plaques in alzheimer disease brains. brain res, 2003. 971(2): p. 197-209. 355. habib, n., et al., disease-associated astrocytes in alzheimer’s disease and aging. nat neurosci, 2020. 23(6): p. 701-706. 356. matias, i., j. morgado, and f.c.a. gomes, astrocyte heterogeneity: impact to brain aging and disease. front aging neurosci, 2019. 11: p. 59. 357. arranz, a.m. and b. de strooper, the role of astroglia in alzheimer’s disease: pathophysiology and clinical implications. lancet neurol, 2019. 18(4): p. 406-414. 358. holtzman, d. and j. ulrich, senescent glia spell trouble in alzheimer’s disease. nat neurosci, 2019. 22(5): p. 683-684. 359. zhang, p., et al., senolytic therapy alleviates abeta-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an alzheimer’s disease model. nat neurosci, 2019. 22(5): p. 719-728. 360. atwood, c.s., et al., senile plaque composition and posttranslational modification of amyloid-beta peptide and associated proteins. peptides, 2002. 23(7): p. 1343-50. 361. friede, r.l., enzyme histochemical studies of senile plaques. journal of neuropathology and experimental neurology, 1965. 24(3): p. 477-491. 362. mcgeer, p.l., et al., pathological proteins in senile plaques. tohoku j exp med, 1994. 174(3): p. 269-77. 363. rebeck, g.w., et al., multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein e receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein. ann neurol, 1995. 37(2): p. 211-7. 364. stewart, k.l. and s.e. radford, amyloid plaques beyond abeta: a survey of the diverse modulators of amyloid aggregation. biophys rev, 2017. 9(4): p. 405-419. 365. pepys, m.b., et al., amyloid p component. a critical review. amyloid, 1997. 4(4): p. 274-295. 366. coria, f., et al., isolation and characterization of amyloid p component from alzheimer’s disease and other types of cerebral amyloidosis. lab invest, 1988. 58(4): p. 454-8. 367. kalaria, r.n. and i. grahovac, serum amyloid p immunoreactivity in hippocampal tangles, plaques and vessels: implications for leakage across the blood-brain barrier in alzheimer’s disease. brain res, 1990. 516(2): p. 349-53. 368. snow, a.d., et al., the presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in alzheimer’s disease. am j pathol, 1988. 133(3): p. 456-63. 369. snow, a.d., et al., early accumulation of heparan sulfate in neurons and in the beta-amyloid protein-containing lesions of alzheimer’s disease and down’s syndrome. am j pathol, 1990. 137(5): p. 1253-70. 370. eikelenboom, p., et al., complement activation in amyloid plaques in alzheimer’s dementia. virchows arch b cell pathol incl mol pathol, 1989. 56(4): p. 259-62. 371. eikelenboom, p. and f.c. stam, immunoglobulins and complement factors in senile plaques. an immunoperoxidase study. acta neuropathol, 1982. 57(2-3): p. 239-42. 372. ishii, t. and s. haga, immuno-electron-microscopic localization of complements in amyloid fibrils of senile plaques. acta neuropathol, 1984. 63(4): p. 296-300. 373. rogers, j., et al., complement activation by beta-amyloid in alzheimer disease. proc natl acad sci u s a, 1992. 89(21): p. 10016-20. 374. namba, y., et al., apolipoprotein e immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in alzheimer’s disease and kuru plaque amyloid in creutzfeldt-jakob disease. brain res, 1991. 541(1): p. 163-6. 375. nishiyama, e., et al., distribution of apolipoprotein e in senile plaques in brains with alzheimer’s disease: investigation with the confocal laser scan microscope. brain res, 1997. 750(1-2): p. 20-4. 376. abraham, c.r., d.j. selkoe, and h. potter, immunochemical identification of the serine protease inhibitor alpha 1-antichymotrypsin in the brain amyloid deposits of alzheimer’s disease. cell, 1988. 52(4): p. 487-501. 377. sasaki, n., et al., advanced glycation end products in alzheimer’s disease and other neurodegenerative diseases. am j pathol, 1998. 153(4): p. 1149-55. 378. smith, m.a., et al., advanced maillard reaction end products are associated with alzheimer disease pathology. proc natl acad sci u s a, 1994. 91(12): p. 5710-4. 379. roy, e.r., et al., type i interferon response drives neuroinflammation and synapse loss in alzheimer disease. j clin invest, 2020. 130(4): p. 1912-1930. 380. bastrup, j., et al., proteomic and unbiased post-translational modification profiling of amyloid plaques and surrounding tissue in a transgenic mouse model of alzheimer’s disease. j alzheimers dis, 2020. 73(1): p. 393-411. 381. liao, l., et al., proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection. j biol chem, 2004. 279(35): p. 37061-8. 382. lutz, b.m. and j. peng, deep profiling of the aggregated proteome in alzheimer’s disease: from pathology to disease mechanisms. proteomes, 2018. 6(4): p. 46. 383. xiong, f., w. ge, and c. ma, quantitative proteomics reveals distinct composition of amyloid plaques in alzheimer’s disease. alzheimers dement, 2019. 15(3): p. 429-440. 384. drummond, e., et al., proteomic differences in amyloid plaques in rapidly progressive and sporadic alzheimer’s disease. acta neuropathol, 2017. 133(6): p. 933-954. 385. nijholt, d.a., c. stingl, and t.m. luider, laser capture microdissection of fluorescently labeled amyloid plaques from alzheimer’s disease brain tissue for mass spectrometric analysis. methods mol biol, 2015. 1243: p. 165-73. 386. rijal upadhaya, a., et al., biochemical stages of amyloid-beta peptide aggregation and accumulation in the human brain and their association with symptomatic and pathologically preclinical alzheimer’s disease. brain, 2014. 137(pt 3): p. 887-903. 387. hu, z.w., et al., molecular structure of an n-terminal phosphorylated beta-amyloid fibril. proc natl acad sci u s a, 2019. 116(23): p. 11253-11258. 388. michno, w., et al., pyroglutamation of amyloid-betax-42 (abetax-42) followed by abeta1-40 deposition underlies plaque polymorphism in progressing alzheimer’s disease pathology. j biol chem, 2019. 294(17): p. 6719-6732. 389. roher, a.e., et al., structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in alzheimer’s disease. j biol chem, 1993. 268(5): p. 3072-83. 390. wildburger, n.c., et al., diversity of amyloid-beta proteoforms in the alzheimer’s disease brain. sci rep, 2017. 7(1): p. 9520. 391. wisniewski, t., j. ghiso, and b. frangione, biology of a beta amyloid in alzheimer’s disease. neurobiol dis, 1997. 4(5): p. 313-28. 392. miklossy, j., bacterial amyloid and dna are important constituents of senile plaques: further evidence of the spirochetal and biofilm nature of senile plaques. j alzheimers dis, 2016. 53(4): p. 1459-73. 393. itzhaki, r.f., et al., do infections have a role in the pathogenesis of alzheimer disease? nat rev neurol, 2020. 16(4): p. 193-197. 394. itzhaki, r.f., et al., microbes and alzheimer’s disease. j alzheimers dis, 2016. 51(4): p. 979-84. 395. moir, r.d., r. lathe, and r.e. tanzi, the antimicrobial protection hypothesis of alzheimer’s disease. alzheimers dement, 2018. 14(12): p. 1602-1614. 396. lovheim, h., et al., reactivated herpes simplex infection increases the risk of alzheimer’s disease. alzheimers dement, 2015. 11(6): p. 593-9. 397. readhead, b., et al., multiscale analysis of independent alzheimer’s cohorts finds disruption of molecular, genetic, and clinical networks by human herpesvirus. neuron, 2018. 99(1): p. 64-82 e7. 398. sochocka, m., k. zwolinska, and j. leszek, the infectious etiology of alzheimer’s disease. curr neuropharmacol, 2017. 15(7): p. 996-1009. 399. rizzo, r., controversial role of herpesviruses in alzheimer’s disease. plos pathog, 2020. 16(6): p. e1008575. 400. mawanda, f. and r. wallace, can infections cause alzheimer’s disease? epidemiol rev, 2013. 35: p. 161-80. 401. fulop, t., et al., role of microbes in the development of alzheimer’s disease: state of the art an international symposium presented at the 2017 iagg congress in san francisco. front genet, 2018. 9: p. 362. 402. fulop, t., et al., can an infection hypothesis explain the beta amyloid hypothesis of alzheimer’s disease? front aging neurosci, 2018. 10: p. 224. 403. eimer, w.a., et al., alzheimer’s disease-associated beta-amyloid is rapidly seeded by herpesviridae to protect against brain infection. neuron, 2018. 99(1): p. 56-63 e3. 404. soscia, s.j., et al., the alzheimer’s disease-associated amyloid beta-protein is an antimicrobial peptide. plos one, 2010. 5(3): p. e9505. 405. zhan, x., b. stamova, and f.r. sharp, lipopolysaccharide associates with amyloid plaques, neurons and oligodendrocytes in alzheimer’s disease brain: a review. front aging neurosci, 2018. 10: p. 42. 406. harach, t., et al., reduction of abeta amyloid pathology in appps1 transgenic mice in the absence of gut microbiota. sci rep, 2017. 7: p. 41802. 407. jucker, m. and l.c. walker, propagation and spread of pathogenic protein assemblies in neurodegenerative diseases. nat neurosci, 2018. 21(10): p. 1341-1349. 408. jaunmuktane, z. and s. brandner, invited review: the role of prion-like mechanisms in neurodegenerative diseases. neuropathol appl neurobiol, 2019. 46(6): p. 522-545. 409. ayers, j.i., n.a. paras, and s.b. prusiner, expanding spectrum of prion diseases. emerging topics in life sciences, 2020: p. 1-13. 410. prusiner, s.b., development of the prion concept, in prion biology and diseases, s.b. prusiner, editor. 2004, cold spring harbor laboratory press: cold spring harbor. p. 89-141. 411. zabel, m.d. and c. reid, a brief history of prions. pathog dis, 2015. 73(9): p. ftv087. 412. prusiner, s.b., prions. proc natl acad sci u s a, 1998. 95(23): p. 13363-83. 413. friesen, m. and m. meyer-luehmann, abeta seeding as a tool to study cerebral amyloidosis and associated pathology. front mol neurosci, 2019. 12: p. 233. 414. morales, r., k. callegari, and c. soto, prion-like features of misfolded abeta and tau aggregates. virus res, 2015. 207: p. 106-12. 415. watts, j.c. and s.b. prusiner, beta-amyloid prions and the pathobiology of alzheimer’s disease. cold spring harb perspect med, 2018. 8(5): p. a023507. 416. asher, d.m., et al., risk of transmissibility from neurodegenerative disease-associated proteins: experimental knowns and unknowns. j neuropathol exp neurol, 2020. 79(11): p. 1141-1146. 417. lauwers, e., et al., potential human transmission of amyloid β pathology: surveillance and risks. lancet neurology, 2020. 19: p. 872-878. 418. brown, p., et al., iatrogenic creutzfeldt-jakob disease, final assessment. emerg infect dis, 2012. 18(6): p. 901-7. 419. will, r.g., acquired prion disease: iatrogenic cjd, variant cjd, kuru. br med bull, 2003. 66: p. 255-65. 420. jaunmuktane, z., et al., evidence for human transmission of amyloid-beta pathology and cerebral amyloid angiopathy. nature, 2015. 525(7568): p. 247-50. 421. ritchie, d.l., et al., amyloid-beta accumulation in the cns in human growth hormone recipients in the uk. acta neuropathol, 2017. 134(2): p. 221-240. 422. frontzek, k., et al., amyloid-beta pathology and cerebral amyloid angiopathy are frequent in iatrogenic creutzfeldt-jakob disease after dural grafting. swiss med wkly, 2016. 146: p. w14287. 423. hamaguchi, t., et al., significant association of cadaveric dura mater grafting with subpial abeta deposition and meningeal amyloid angiopathy. acta neuropathol, 2016. 132(2): p. 313-315. 424. irwin, d.j., et al., evaluation of potential infectivity of alzheimer and parkinson disease proteins in recipients of cadaver-derived human growth hormone. jama neurol, 2013. 70(4): p. 462-8. 425. kovacs, g.g., et al., dura mater is a potential source of abeta seeds. acta neuropathol, 2016. 131(6): p. 911-23. 426. duyckaerts, c., et al., neuropathology of iatrogenic creutzfeldt-jakob disease and immunoassay of french cadaver-sourced growth hormone batches suggest possible transmission of tauopathy and long incubation periods for the transmission of abeta pathology. acta neuropathol, 2018. 135(2): p. 201-212. 427. purro, s.a., et al., transmission of amyloid-beta protein pathology from cadaveric pituitary growth hormone. nature, 2018. 564(7736): p. 415-419. 428. nakayama, h., et al., cerebral amyloid angiopathy in an aged great spotted woodpecker (picoides major). neurobiol aging, 1999. 20(1): p. 53-6. 429. cork, l.c., et al., neurofibrillary tangles and senile plaques in aged bears. j neuropathol exp neurol, 1988. 47(6): p. 629-41. 430. tekirian, t.l., et al., carboxy terminal of beta-amyloid deposits in aged human, canine, and polar bear brains. neurobiol aging, 1996. 17(2): p. 249-57. 431. uchida, k., et al., senile plaques and other senile changes in the brain of an aged american black bear. vet pathol, 1995. 32(4): p. 412-4. 432. cummings, b.j., et al., beta-amyloid accumulation correlates with cognitive dysfunction in the aged canine. neurobiol learn mem, 1996. 66(1): p. 11-23. 433. ishihara, t., et al., immunohistochemical and immunoelectron microscopical characterization of cerebrovascular and senile plaque amyloid in aged dogs’ brains. brain res, 1991. 548(1-2): p. 196-205. 434. uchida, k., et al., immunohistochemical analysis of constituents of senile plaques and cerebro-vascular amyloid in aged dogs. j vet med sci, 1992. 54(5): p. 1023-9. 435. zeiss, c.j., utility of spontaneous animal models of alzheimer’s disease in preclinical efficacy studies. cell tissue res, 2020. 380(2): p. 273-286. 436. nakamura, s., et al., senile plaques in very aged cats. acta neuropathol, 1996. 91(4): p. 437-9. 437. nakamura, s., et al., senile plaques in an aged two-humped (bactrian) camel (camelus bactrianus). acta neuropathol, 1995. 90(4): p. 415-8. 438. roertgen, k.e., et al., a beta-associated cerebral angiopathy and senile plaques with neurofibrillary tangles and cerebral hemorrhage in an aged wolverine (gulo gulo). neurobiol aging, 1996. 17(2): p. 243-7. 439. heuer, e., et al., nonhuman primate models of alzheimer-like cerebral proteopathy. curr pharm des, 2012. 18(8): p. 1159-69. 440. jankowsky, j.l., et al., rodent a beta modulates the solubility and distribution of amyloid deposits in transgenic mice. j biol chem, 2007. 282(31): p. 22707-20. 441. steffen, j., et al., expression of endogenous mouse app modulates beta-amyloid deposition in happ-transgenic mice. acta neuropathol commun, 2017. 5(1): p. 49. 442. price, d.l., et al., aged non-human primates: an animal model of age-associated neurodegenerative disease. brain pathol, 1991. 1(4): p. 287-96. 443. selkoe, d.j., et al., conservation of brain amyloid proteins in aged mammals and humans with alzheimer’s disease. science, 1987. 235(4791): p. 873-7. 444. walker, l.c. and l.c. cork, the neurobiology of aging in nonhuman primates, in alzheimer disease, r.d. terry, et al., editors. 1999, lippincott williams and wilkins: philadelphia. p. 233-243. 445. walker, l.c., animal models of cerebral beta-amyloid angiopathy. brain res brain res rev, 1997. 25(1): p. 70-84. 446. jakel, l., et al., animal models of cerebral amyloid angiopathy. clin sci (lond), 2017. 131(19): p. 2469-2488. 447. rosen, r.f., l.c. walker, and h. levine, 3rd, pib binding in aged primate brain: enrichment of high-affinity sites in humans with alzheimer’s disease. neurobiol aging, 2011. 32(2): p. 223-34. 448. games, d., et al., alzheimer-type neuropathology in transgenic mice overexpressing v717f beta-amyloid precursor protein. nature, 1995. 373(6514): p. 523-7. 449. hsiao, k., et al., correlative memory deficits, abeta elevation, and amyloid plaques in transgenic mice. science, 1996. 274(5284): p. 99-102. 450. sturchler-pierrat, c., et al., two amyloid precursor protein transgenic mouse models with alzheimer disease-like pathology. proc natl acad sci u s a, 1997. 94(24): p. 13287-92. 451. duyckaerts, c., m.c. potier, and b. delatour, alzheimer disease models and human neuropathology: similarities and differences. acta neuropathol, 2008. 115(1): p. 5-38. 452. jucker, m., the benefits and limitations of animal models for translational research in neurodegenerative diseases. nat med, 2010. 16(11): p. 1210-4. 453. myers, a. and p. mcgonigle, overview of transgenic mouse models for alzheimer’s disease. curr protoc neurosci, 2019. 89(1): p. e81. 454. greeve, i., et al., age-dependent neurodegeneration and alzheimer-amyloid plaque formation in transgenic drosophila. j neurosci, 2004. 24(16): p. 3899-906. 455. iijima-ando, k. and k. iijima, transgenic drosophila models of alzheimer’s disease and tauopathies. brain struct funct, 2010. 214(2-3): p. 245-62. 456. jeon, y., et al., genetic dissection of alzheimer’s disease using drosophila models. int j mol sci, 2020. 21(3): p. 884. 457. link, c.d., c. elegans models of age-associated neurodegenerative diseases: lessons from transgenic worm models of alzheimer’s disease. exp gerontol, 2006. 41(10): p. 1007-13. 458. link, c.d., et al., visualization of fibrillar amyloid deposits in living, transgenic caenorhabditis elegans animals using the sensitive amyloid dye, x-34. neurobiol aging, 2001. 22(2): p. 217-26. 459. dawson, t.m., t.e. golde, and c. lagier-tourenne, animal models of neurodegenerative diseases. nat neurosci, 2018. 21(10): p. 1370-1379. 460. gotz, j. and l.m. ittner, animal models of alzheimer’s disease and frontotemporal dementia. nat rev neurosci, 2008. 9(7): p. 532-44. 461. rockenstein, e., l. crews, and e. masliah, transgenic animal models of neurodegenerative diseases and their application to treatment development. adv drug deliv rev, 2007. 59(11): p. 1093-102. 462. gotz, j., l.g. bodea, and m. goedert, rodent models for alzheimer disease. nat rev neurosci, 2018. 19(10): p. 583-598. 463. kitazawa, m., r. medeiros, and f.m. laferla, transgenic mouse models of alzheimer disease: developing a better model as a tool for therapeutic interventions. curr pharm des, 2012. 18(8): p. 1131-47. 464. walker, l.c., et al., labeling of cerebral amyloid in vivo with a monoclonal antibody. j neuropathol exp neurol, 1994. 53(4): p. 377-83. 465. schenk, d., et al., immunization with amyloid-beta attenuates alzheimer-disease-like pathology in the pdapp mouse. nature, 1999. 400(6740): p. 173-7. 466. braakman, n., et al., longitudinal assessment of alzheimer’s beta-amyloid plaque development in transgenic mice monitored by in vivo magnetic resonance microimaging. j magn reson imaging, 2006. 24(3): p. 530-6. 467. christie, r.h., et al., growth arrest of individual senile plaques in a model of alzheimer’s disease observed by in vivo multiphoton microscopy. j neurosci, 2001. 21(3): p. 858-64. 468. dong, j., et al., multiphoton in vivo imaging of amyloid in animal models of alzheimer’s disease. neuropharmacology, 2010. 59(4-5): p. 268-75. 469. rominger, a., et al., longitudinal assessment of cerebral beta-amyloid deposition in mice overexpressing swedish mutant beta-amyloid precursor protein using 18f-florbetaben pet. j nucl med, 2013. 54(7): p. 1127-34. 470. prada, c.m., et al., antibody-mediated clearance of amyloid-beta peptide from cerebral amyloid angiopathy revealed by quantitative in vivo imaging. j neurosci, 2007. 27(8): p. 1973-80. 471. okano, h. and n. kishi, investigation of brain science and neurological/psychiatric disorders using genetically modified non-human primates. curr opin neurobiol, 2018. 50: p. 1-6. 472. seita, y., et al., generation of transgenic cynomolgus monkeys overexpressing the gene for amyloid-beta precursor protein. j alzheimers dis, 2020. 75(1): p. 45-60. 473. vermunt, l., et al., duration of preclinical, prodromal, and dementia stages of alzheimer’s disease in relation to age, sex, and apoe genotype. alzheimers dement, 2019. 15(7): p. 888-898. 474. selkoe, d.j., resolving controversies on the path to alzheimer’s therapeutics. nat med, 2011. 17(9): p. 1060-5. 475. selkoe, d.j., editorial: a is for amyloid. j prev alzheimers dis, 2020. 7(3): p. 140-141. 476. tolar, m., et al., aducanumab, gantenerumab, ban2401, and alz-801-the first wave of amyloid-targeting drugs for alzheimer’s disease with potential for near term approval. alzheimers res ther, 2020. 12(1): p. 95. 477. uhlmann, r.e., et al., acute targeting of pre-amyloid seeds in transgenic mice reduces alzheimer-like pathology later in life. nature neuroscience, 2020. (in press). 478. holmes, c., et al., long-term effects of abeta42 immunisation in alzheimer’s disease: follow-up of a randomised, placebo-controlled phase i trial. lancet, 2008. 372(9634): p. 216-23. 479. lozupone, m., et al., anti-amyloid-beta protein agents for the treatment of alzheimer’s disease: an update on emerging drugs. expert opin emerg drugs, 2020: p. 1-17. 480. clavaguera, f., c. duyckaerts, and s. haik, prion-like properties of tau assemblies. curr opin neurobiol, 2020. 61: p. 49-57. 481. holmes, b.b. and m.i. diamond, prion-like properties of tau protein: the importance of extracellular tau as a therapeutic target. j biol chem, 2014. 289(29): p. 19855-61. 482. klein, g., et al., gantenerumab reduces amyloid-beta plaques in patients with prodromal to moderate alzheimer’s disease: a pet substudy interim analysis. alzheimers res ther, 2019. 11(1): p. 101. 483. sevigny, j., et al., the antibody aducanumab reduces abeta plaques in alzheimer’s disease. nature, 2016. 537(7618): p. 50-6. 484. selkoe, d.j. and j. hardy, the amyloid hypothesis of alzheimer’s disease at 25 years. embo mol med, 2016. 8(6): p. 595-608. 485. angelucci, f., et al., antibiotics, gut microbiota, and alzheimer’s disease. j neuroinflammation, 2019. 16(1): p. 108. 486. sureda, a., et al., oral microbiota and alzheimer’s disease: do all roads lead to rome? pharmacol res, 2020. 151: p. 104582. 487. lemere, c.a., immunotherapy for alzheimer’s disease: hoops and hurdles. mol neurodegener, 2013. 8: p. 36. 488. van dyck, c.h., anti-amyloid-beta monoclonal antibodies for alzheimer’s disease: pitfalls and promise. biol psychiatry, 2018. 83(4): p. 311-319. 489. voytyuk, i., b. de strooper, and l. chavez-gutierrez, modulation of gammaand beta-secretases as early prevention against alzheimer’s disease. biol psychiatry, 2018. 83(4): p. 320-327. 490. wisniewski, t. and f. goni, immunotherapeutic approaches for alzheimer’s disease. neuron, 2015. 85(6): p. 1162-76. 491. plotkin, s.s. and n.r. cashman, passive immunotherapies targeting abeta and tau in alzheimer’s disease. neurobiol dis, 2020. 144: p. 105010. 492. kwon, s., et al., immunotherapies for aging-related neurodegenerative diseases-emerging perspectives and new targets. neurotherapeutics, 2020. p. 1-20. 493. yu, y.j. and r.j. watts, developing therapeutic antibodies for neurodegenerative disease. neurotherapeutics, 2013. 10(3): p. 459-72. 494. bennett, c.f., a.r. krainer, and d.w. cleveland, antisense oligonucleotide therapies for neurodegenerative diseases. annu rev neurosci, 2019. 42: p. 385-406. 495. chakravarthy, m., et al., nucleic acid-based theranostics for tackling alzheimer’s disease. theranostics, 2017. 7(16): p. 3933-3947. 496. kim, k.s., et al., production and characterization of monoclonal antibodies reactive to synthetic cerebrovascular amyloid peptide. neurosci res commun, 1988. 2: p. 121-130. 497. potempska, a., et al., quantification of sub-femtomole amounts of alzheimer amyloid beta peptides. amyloid, 1999. 6(1): p. 14-21. 498. horikoshi, y., et al., development of abeta terminal end-specific antibodies and sensitive elisa for abeta variant. biochem biophys res commun, 2004. 319(3): p. 733-7. 499. duff, k., et al., characterization of pathology in transgenic mice over-expressing human genomic and cdna tau transgenes. neurobiol dis, 2000. 7(2): p. 87-98. 500. jicha, g.a., et al., alz-50 and mc-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau. j neurosci res, 1997. 48(2): p. 128-32. 501. imai, y., et al., a novel gene iba1 in the major histocompatibility complex class iii region encoding an ef hand protein expressed in a monocytic lineage. biochem biophys res commun, 1996. 224(3): p. 855-62. 502. ito, d., et al., microglia-specific localisation of a novel calcium binding protein, iba1. brain res mol brain res, 1998. 57(1): p. 1-9. 503. lichtenberg-kraag, b., et al., phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with alzheimer tau. proc natl acad sci u s a, 1992. 89(12): p. 5384-8. 504. goldstein, m.e., l.a. sternberger, and n.h. sternberger, varying degrees of phosphorylation determine microheterogeneity of the heavy neurofilament polypeptide (nf-h). j neuroimmunol, 1987. 14(2): p. 135-48. 505. sternberger, l.a. and n.h. sternberger, monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. proc natl acad sci u s a, 1983. 80(19): p. 6126-30. 506. naoumenko, j. and i. feigin, a stable silver solution for axon staining in paraffin sections. j neuropathol exp neurol, 1967. 26(4): p. 669-673. 507. campbell, s.k., r.c. switzer iii, and t.l. martin, alzheimer’s plaques and tangles: a controlled and enhanced silver staining method. society for neuroscience abstracts, 1987. 13: p. 687. 508. cowe, a., der gliöse anteil der senilen plaques. zeitschrift für die gesamte neurologie und psychiatrie, 1915. 29(1): p. 92-96. 509. mcmenemey, w.h., alzheimer’s disease: problems concerning its concept and nature. acta neurologica scandinavica, 1963. 39: p. 369-380. 510. defelipe, j., cajal’s butterfiles of the soul. 2010, oxford university press: new york. 511. alafuzoff et al., inter-laboratory comparison of neuropathological assessments of beta-amyloid protein: a study of the brainnet europe consortium. acta neuropathologica, 2008. 115[5]: p. 533-546 512. stonebarger et al., amyloidosis increase is not attenuated by long-term calorie restriction or related to neuron density in the prefrontal cortex of extremely aged rhesus macaques. geroscience, 2020. doi: 10.1007/s11357-020-00259-0. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 5th asian oceanian congress of neuropathology along with the 5th annual conference of the neuropathology society of india (aocn-npsicon) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 3:14 (2022) meeting abstracts 5th asian oceanian congress of neuropathology along with the 5th annual conference of the neuropathology society of india (aocn-npsicon) hosted by: department of neuropathology, nimhans, bangalore, india meeting abstracts september 24–26, 2021 submitted: 12 may 2022 accepted: 12 may 2022 published: 19 may 2022   the 5th asian oceanian congress of neuropathology along with the 5th annual conference of the neuropathology society of india (aocn-npsicon) was held in virtual mode on september 24–26, 2021, at national institute of mental health and neurosciences (nimhans), bangalore, india, hosted by the department of neuropathology. it had 361 attendees from 20 countries from asia and oceania including india. the event brought together pathologists, clinicians and neuroscientists from all over asia and oceania with invited speakers from the usa, germany and canada. the program was very comprehensive and covered advances in the fields of neurooncology with emphasis on the upcoming who 2021 classification of cns tumors, neuromuscular disorders, epilepsy and neurodegenerative disorders through key note addresses and symposia that featured 78 distinguished international and national faculty sharing their expertise. in addition, there were case-based learning modules, opportunities for paper presentations and poster sessions for young faculty and postgraduates with several awards for young investigators, best papers and posters. a highlight of the conference was a unique debate on the hot topic of the decade: methylation-based classification of cns tumors and a panel discussion on covid-19. the participants were highly appreciative of the academic content. https://doi.org/10.17879/freeneuropathology-2021-4121 keywords: neuropathology society of india, npsi, meeting abstracts contents 1. clinicopathologic analysis of diffuse midline gliomas: a single centre study 2. low frequency of ezhip overexpression in diffuse midline gliomas, h3 wildtype 3. location specific kiaa1549::braf fusion variants in pilocytic astrocytoma 4. epithelial to mesenchymal transition (emt) in meningiomas 5. programmed death ligand 1(pd-l1) expression and tumor infiltrating immune cell subpopulations association with clinicopathological and prognostic parameters in diffuse gliomas 6. insights into molecular biology and immune micro-environment of pleomorphic xanthoastrocytoma 7. dynamics of cell-free dna in predicting response in adult diffuse glioma on chemoradiotherapy 8. regional heterogeneity in mitochondrial function in human brain ageing: implications for region-specific vulnerability to neurodegeneration 9. ryr1-related myopathies: a series of 5 patients from an indian tertiary care centre 10. utility of respiratory chain complex assays in the diagnosis of mitochondrial disorders a phenotype, histopathological and genotype correlation 11. myelination changes in white matter following severe traumatic brain injury (stbi): a neuropathological study 12. is cerebral malaria an astrocytopathy? a post mortem study of bbb dysfunction 13. does mitochondrial dysfunction play a role in pathogenesis of mesial temporal lobe epilepsy secondary to hippocampal sclerosis? 14. diffuse midline gliomas with h3k27 mutation clinicopathological correlates 15. significance of nestin and cd133 as cancer stem cell markers in diffuse gliomas and its association with idh-1 status and p53 expression 16. immunophenotypic profile of pediatric brain tumors reflecting molecular alterations 17. pdl1 expression in cns tumours 18. droplet digital pcr: a robust technique for detection of idh1 r132h mutation in formalin fixed tissue samples 19. tert promoter mutations in meningiomas: a clinicopathological correlation 20. evaluation of tert promoter mutation status in meningiomas 21. pitfalls in diagnosis of oligodendrogliomas on squash cytology 22. low prevalence of braf v600e mutations in pleomorphic xanthoastrocytoma 23. unusual primary intracranial sarcomas – ewing’s sarcoma and synovial sarcoma 24. relevance of the 4-variable risk stratification model in cns solitary fibrous tumours 25. oropharyngeal psammomatous melanotic schwannoma, non-syndromic an unusual tumour at an unusual site 26. silent corticogonadotroph adenoma (scga): a silent monster 27. molecular profiling: a key to clinical and histological enigma in an ambiguous case 28. pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease – a case report 29. anoctamin-5 muscular dystrophy: report of 2 cases with different phenotypes and genotypes from indian subcontinent 30. dysferlinopathy in a cohort of north indian patients: clinical histopathological and mutational spectrum 31. role of mitochondrial respiratory chain complexes in pathogenesis of temporal lobe epilepsy 32. gliotic and destructive brain lesions associated with drug resistant epilepsy a clinicopathological study 33. cytopathic changes of herpes zoster encephalitis in csf mimicking malignant cells 34. resolving the diagnosis of hmsn with nerve histopathology and genetics 35. understanding the er stress response to predict clinical outcome in focal cortical dysplasia patients 36. candida meningitis mimicking tuberculous meningitis in an immunocompetent patient diagnostic conundrum 37. rare case of dorsally located multiple neurenteric-cyst without spinal dysraphism – neuropathology insights and systematic review 38. efficacy of dimethyl fumarate in chronic constriction injury induced neuropathic pain in rats 39. moya moya disease: an autopsy case study 40. hematolymphoid malignancies presenting with neurological manifestations and hand-mirror cells in peripheral blood: report of two cases 41. mullerian choristoma as a cause of tethered cord syndrome: a case report in a 13-year-old worsening after the onset of menarche 42. plurihormonal pit-1-positive adenoma: a short series 43. an interesting case of extradural tumour in a pediatric patient 44. case report of rare extra axial cerebellopontine angle medulloblastoma, a meningioma mimicker: caution advised 45. potpourri of five cases of rare central nervous system tumors with review of literature 46. primary yolk sac tumor of cerebellar vermis: a case report 47. a diagnostic dilemma astroblastoma 48. meningioangiomatosis: a rare cause of refractory temporal lobe epilepsy 49. isolated cerebral rosai dorfman disease with granulomatous angiitis 50. twin tales of anaplastic ependymoma with extensive vacuolation/signet cell change and lipomatous differentiation     abstract 1 free neuropathol 3:14:5 clinicopathologic analysis of diffuse midline gliomas: a single centre study shiva soma1, megha s uppin1, arvind suman2, suchanda bhattacharjee2 1 department of pathology, nizam’s institute of medical sciences, hyderabad, india 2 department of neurosurgery, nizam’s institute of medical sciences, hyderabad, india background: diffuse midline gliomas are primary cns tumors found in midline location of brain and spinal cord. these are who grade 4 tumors with characteristic h3k27m mutation and worse prognosis. objectives: to determine clinical, pathological, radiological, surgical outcome of patients diagnosed as dmgs. materials and methods: all patients diagnosed as dmg from january 2017 to july 2020 were included following ethical approval. clinical presentation, radioimaging details were taken from medical records. histopathologic features were noted. immunohistochemistry (ihc) was performed with idh1r132h (dianova, dilution 1:200), atrx (sigma aldrich, dilution1:500), h3k27m (milipore, dilution 1:500), p53 (sigma aldrich, ready to use) using poly hrp technique on fully-automated immunostainer (x matrix, biogenex). results: study included a total 29 patients with mean age of 21±12.5years, m:f ratio of 2:1. tumor distribution was thalamus (58.6%), brain stem (27.6%), cerebellum (20.7%), dorsal spine (10.3%) and cervical spine (6.9%). in 14 patients, tumor was biopsied and in the rest stereotactic or open excision was attempted. morphology was variable in all cases. gemistocytes, low cellularity, giant cells, pxa like pattern were some of the highlights in morphology. h3k27m showed dark nuclear expression in all with loss of atrx in 21 cases. twenty-two patients were treated with radiotherapy and 21 patients died on follow up at 6 months. high survival duration was seen in patients of >18 years age (p=0.02) who received rt (p=0.05). conclusion: dmgs are aggressive tumors. ihc with surrogate molecular marker helps in diagnosis. prognosis is poor irrespective of surgery and radiotherapy.   abstract 2 free neuropathol 3:14:6 low frequency of ezhip overexpression in diffuse midline gliomas, h3 wildtype poonkodi manohar1, shilpa rao1, nandeesh bn1, yasha tc1, anita mahadevan1, vani santosh1 1 department of neuropathology, nimhans, bangalore, india background: few diffuse midline gliomas (dmgs) with h3k27me3 loss, lack h3k27m mutation. these h3 wildtype tumors have been shown to either overexpress ezhip or harbour other mutations, thus categorised as dmg, h3k27 altered. however, studies on the frequency of ezhip overexpression in dmg is sparse. objectives: the purpose of the present study was to analyse dmgs in paediatric and adult patients and study the frequency of ezhip overexpression in dmg, h3 wildtype tumors. material and methods: histological and immunohistochemical profiles of dmgs diagnosed between the years 2018 and 2020 (n=123) were reviewed and segregated into two groups; h3k27m mutant and h3 wildtype tumors. immunohistochemistry was performed on the h3k27m wildtype tumors using rabbit monoclonal antibody to ezhip(cxorf67). results: of the 123 cases, 22 were in children and 101 in adults. 20/ 22 (91%) paediatric tumors and 64/101 (63.4%) adult tumors harboured h3k27m mutation. the common location of h3k27m mutant and h3wildtype tumours was thalamus in both age groups. out of a total 39 h3wildtype tumours, only 2 showed ezhip overexpression along with loss of h3k27me3. both the tumours were located in the thalamus, with glioblastoma histology and occurred in young adults (30 and 36 years). conclusion: in our study, the majority of dmgs in paediatric age group and about two thirds in adults harboured h3k27m mutations. a very small subset of h3 wildtype tumours overexpressed ezhip. this study highlights the low frequency of ezhip overexpression in dmg h3 wildtype tumors.   abstract 3 free neuropathol 3:14:7 location specific kiaa1549::braf fusion variants in pilocytic astrocytoma sumitra sivakoti1, harsha sugur1, arivazhaghan a2, saini j3, yasha tc1, vani santosh1 1 department of neuropathology, national institute of mental health and neurosciences, nimhans, bengaluru, india 2 department of neurosurgery, national institute of mental health and neurosciences, nimhans, bengaluru, india 3 department of neuro imaging & interventional radiology, nimhans, bengaluru, india background: pilocytic astrocytomas (pa) are characterized by constitutive activation of the ras/mapk signalling pathway. the most common underlying genetic alteration is kiaa1549::braf fusion, followed by nf1, brafv600e, fgfr1 mutations, braf fusion with other proteins, ntrk family and kras mutation. objectives: to study the frequency of the most common braf genetic alterations, particularly kiaa1549::braf fusion variants and v600e mutation in pa and correlate with clinical features, and particular tumour site. material and methods: retrospective analysis of 61 pa cases was undertaken, and they were tested for kiaa1549::braf fusion and v600e mutation by qpcr and ihc respectively. whole rna from ffpe tissues was reverse transcribed to cdna. using taqman assay, kiaa1549::braf fusion testing at 16-9, 15-9 and 16-11 exons was performed by qpcr. ihc was performed on tissue microarray sections. results: braf genetic alterations were seen in majority of pa (80.3%) -kiaa1549-braf fusions accounting for 77% and v600e mutation for 3.3%. fusion at exon junction 16-9 was the most common (68%) and majority were cerebellar tumors in children less than 15years age. fusion at exon junction 16-11 (15%) was restricted to the cerebellum alone. we noted fusion at exon junction 15-9 (23%) frequently in midline location and few in cerebellum. multiple fusions (16-9, 15-9) were seen in two. brafv600e mutations (2) were restricted to supratentorial location. kiaa1549::braf fusion and brafv600e mutation were mutually exclusive. conclusion: braf alterations are common in pa, especially in children. different sites exhibit different kiaa1549::braf fusion transcripts in varying frequencies16-9 mainly in cerebellum, 15-9 mainly in midline, and 16-11 exclusively in cerebellum. infrequent supratentorial pa show brafv600e mutation. these have prognostic and possible therapeutic implications.   abstract 4 free neuropathol 3:14:8 epithelial to mesenchymal transition (emt) in meningiomas sanjay sriram1, swati mahajan1, mc sharma1, chitra sarkar1, ashish suri2, vaishali suri1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india background: epithelial-to-mesenchymal transition (emt) is a process involved in invasion and metastasis of tumors. its activation has been well documented in various malignancies and is associated with poor prognosis; however, data is limited in meningiomas. objectives: to analyse the expression of emt related proteins and transcription factors in various grades of meningiomas and to correlate with clinical parameters. materials and methods: seventy meningiomas of various histopathological subtypes and grades (who grade i=31, ii=31, iii=8) were analysed by immunohistochemistry for emt related proteins (e-cadherin, β-catenin, n-cadherin) and transcription factors (snail-1, slug). results: downregulation (loss of expression) of e-cadherin, β-catenin and upregulation of snail-1 was seen in higher frequency (82%, 72% and 69%) in who grade ii/iii meningiomas as compared to grade i meningiomas (6%, 3% and 13%) (p<0.05), thus indicating high frequency of emt pathway activation in high grade meningiomas. n-cadherin and slug expression was seen in only minority of cases. no difference in emt phenotype existed between different histomorphological subtypes of grade i meningiomas. on survival analysis patients with emt activation across all grades had shorter progression free and overall survival. further, in four paired samples analysed, emt activation was seen in all the principal tumors. conclusion: meningiomas exhibiting emt contribute to the aggressiveness and increased recurrence risk of these tumors. hence emt markers can be used for predicting the behaviour of meningiomas.   abstract 5 free neuropathol 3:14:9 programmed death ligand 1(pd-l1) expression and tumor infiltrating immune cell subpopulations association with clinicopathological and prognostic parameters in diffuse gliomas swati mahajan1, muhammed shafeeq1, mehar c sharma1, ashish suri2, vaishali suri1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india background: recent discoveries have revealed that the glioma microenvironment includes a wide variety of immune markers that play an important role in the process of tumorigenesis. objectives: we aimed to analyze the utility of immune markers in prognostic stratification and understand the potential candidature of diffuse infiltrating gliomas for immune checkpoint blockade. material and methods: one hundred gliomas (who grade ii–iv) were analyzed by immunohistochemistry for pd-l1 expressing tumor cells, tumor-infiltrating lymphocyte subsets (tils; cd4, cd8, foxp3, ctla4) and tumor-associated macrophages (tams; cd68, cd163). results: expression of pd-l1 was more frequent in adults, idh1 wild type gliomas (76%) and was significantly high in glioblastomas (gb, 74%) followed by grade iii (50%) and ii (27.5%) astrocytomas. median cd8+ tils, cd68+ and cd163+ tams density was higher among grade iv (5, 39 & 11/mm2) and grade iii (4, 35 & 10/mm2) as compared to grade ii tumors (1, 22 & 3/mm2). foxp3 and ctla-4 positive t cells were observed in minority of gbs only. three-paired cases analyzed showed upregulation of pd-l1, tams, ctla-4 and foxp3 in recurrent tumors, indicating a role of the immune markers in recurrence. further, positive correlation between pdl1 expression and cd8+ tils and tams was noted. on survival analysis, increase in pdl1 expression, tils and tams were associated with a shorter overall survival. conclusion: immune markers are frequently expressed in gliomas in a grade-dependent pattern. their analysis could aid in predicting the prognosis of patients and add potential value for immunotherapy treatments.   abstract 6 free neuropathol 3:14:10 insights into molecular biology and immune micro-environment of pleomorphic xanthoastrocytoma iman dandapath1, jyotsna singh1, swati mahajan1, prerana jha1, nidhi shukla1, rahul gupta4, amit katiyar8, vikas sharma8, sujata chaturvedi2, arvind ahuja3, meenakshi bhardwaj3, ravindra saran4, ajay garg5, mehar c sharma1, niveditha manjunath6, ashish suri6, ritu kulshreshtha7, chitra sarkar1, vaishali suri1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india 2 department of pathology, institute of human behaviour and allied sciences, new delhi, india 3 department of pathology, pgimer & dr. rml hospital, new delhi, india 4 department of pathology, gb pant institute of post graduate medical education and research, new delhi, india 5 department of neuro radiology, all india institute of medical sciences, new delhi, india 6 department of neurosurgery, all india institute of medical sciences, new delhi, india 7 department of biochemical engineering and biotechnology, indian institute of technology delhi, new delhi, india 8 ccrf, all india institute of medical sciences, new delhi, india background: pleomorphic xanthoastrocytomas (pxas, grade ii) are rare, accounting for less than 1% of astrocytomas and commonly occur in young patients. some tumours which occur or recur with malignant change are known as anaplastic (apxa, grade iii). there is limited data on their molecular characteristics and immune microenvironment. objectives: a comprehensive study highlighting the underlying molecular biology in pxas for future development of a robust and cost-effective panel of biomarkers for risk stratification and discovery of novel drug targets. methods: genome-wide expression profiling of 14 pxa and 6 apxas was performed by microarray. amongst differentially expressed genes (degs), cyclin dependent kinase 14 (cdk 14) and mitochondrial fission process 1 (mtfp 1) were validated by qrt-pcr. immune profile was analysed using immunohistochemistry for pdl1 and ctla4. results: unsupervised hierarchical clustering revealed two distinct molecular clusters (cluster 1: 10 pxa, 3 apxa and cluster 2: 4 pxa, 3 apxa), indicating molecular heterogeneity within same grade. 10 differentially upregulated and 418 downregulated genes were identified between the clusters. qrt-pcr validation of cdk 14 (upregulated in cluster 2) and mtfp 1 (upregulated in cluster 1) showed strong concordance with expression array. there was no significant difference in age, sex, immunohistochemical profile, frequency of braf mutation or cdkn2a deletion between two clusters. significantly worse progression-free survival was observed in cluster 2 (p=0.003). mrna profiling-based prediction of recurrence was efficient, independent of histological grade and of braf mutation or cdkn2a deletion. pdl1 and ctla4 expression was higher in pxa and apxas than primary glioblastomas. conclusion: study highlights distinct molecular subgroups of pxas. degs between two clusters may be used for histology independent classification, prognostication, and prospective therapeutic targets. higher up-regulation of pdl1 and ctla4 suggests candidature for immunotherapy.   abstract 7 free neuropathol 3:14:11 dynamics of cell-free dna in predicting response in adult diffuse glioma on chemoradiotherapy adil husain1,3, sridhar mishra1, rahat hadi2, nuzhat husain1 1 department of pathology, dr. ram manohar lohia institute of medical sciences, lucknow, india 2 department of radiation oncology, dr. ram manohar lohia institute of medical sciences, lucknow, india 3 department of biosciences, integral university, lucknow, india background: adult diffuse glioma (adg) is a heterogeneous primary brain tumor with a poor prognosis and treatment response. tissue biomarkers are available for diagnostic and prognostic purposes. however, obtaining tissue is invasive and has limitations. cell-free dna (cfdna) may help to meet these challenges in the management of adg. objectives: the study aimed to quantify cfdna in adg on radiotherapy/chemotherapy and mutational profiling. material and methods: the study group comprised histopathologically confirmed adg (n=50), including grade ii, iii and iv glioma, and controls (n=25). serum cfdna was extracted using charge switch gdna 1ml serum kit (invitrogen, usa) and quantified using sybr based quantitative polymerase chain reaction (qpcr). next-generation sequencing (ngs) was performed in 07 pre-operative and 05 post-operative cfdna and tissue dna on an ion personal genome machine (ion pgm) with an in-house designed ngs panel (including tp53, atrx, and idh1 and idh2). results: in patients with adg, the pre-radiotherapy cfdna level was significantly higher (median; 113.46ng/ml), (iqr; 50.73-238.71) than normal controls (median; 74.37ng/ml), (iqr; 49.67-120.00) (p=0.048). non-responders had significantly higher cfdna levels (median; 184.4ng/ml), (iqr;73.84-631.10) than responders (median; 68.12ng/ml), (iqr; 26.55-165.4)), (p=0.023). tp53 gene mutation was most common in both pre-operative and post-operative cfdna samples. conclusion: pre-radiotherapy cfdna levels are associated with survival outcomes independent of other prognostic factors. targeted ngs in pre-operative cfdna matches the results of ihc analysis with high concordance, and it may be helpful in inoperable cases or have recurred.   abstract 8 free neuropathol 3:14:12 regional heterogeneity in mitochondrial function in human brain ageing: implications for region-specific vulnerability to neurodegeneration anusha y kiran1, praseedha mol3, firdouz a bhat3, oishee chatterjee3, srinivas bharath m.m2, t.s. keshava prasad4, anita mahadevan1 1 department of neuropathology, nimhans, bangalore, india 2 department of clinical psychopharmacology and neurotoxicology, nimhans, bangalore, india 3 institute of bioinformatics, bangalore, india 4 centre for systems biology and molecular medicine, yenepoya research centre, yenepoya, india background: region specific vulnerability is implicated in neurodegenerative disorders. mitochondrial dysfunc-tion is implicated, while ageing is the greatest risk factor. it is unknown if age dependant variation in mitochon-drial function exists across brain regions. objectives: this study investigates regional differences in mitochondrial function across different neuroanatom-ical regions in healthy ageing. material and methods: five regions from post-mortem human brains (frontal cortex, cerebellum, striatum, hip-pocampus and medulla; age=080yrs) was evaluated by mitochondrial complex assays. enzyme histochemical staining and quantitative proteomics was performed in young (25±5 yrs) and old (≥65 yrs) ages. results: mitochondrial enzyme assays revealed lowered activity of complexes i & iv with age in all anatomical regions, complex ii showed increasing trend and complex iii remained unchanged. medulla revealed highest activity, followed by cerebellum. on enzyme histochemistry, nadh labelled neuronal cytoplasm of frontal, stria-tum and cerebellar glomeruli and reduced with age. sdh showed increasing intensity with age, in neurons of striatum, cerebellum and medulla whereas cox showed reduced neuronal labelling intensity in hippocampus, cerebellum and medulla, with age. mitochondrial protein expression profile in frontal, striatum and hippocampus was similar, compared to cerebellum and medulla. medulla showed highest expression energy metabolism pro-teins and antioxidants irrespective of age, followed by cerebellum. synaptic transmission & calcium transport pathways were enriched in frontal, striatum and hippocampus. conclusion: the study clearly demonstrates brain region specific mitochondrial metabolism, and redox homeo-stasis which could potentially contribute to region specific vulnerability to neurodegeneration.   abstract 9 free neuropathol 3:14:13 ryr1-related myopathies: a series of 5 patients from an indian tertiary care centre saumya sahu1, swati mahajan1, aishwarya dhall1, bandana jassal1, mohammed faruq3, vaishali suri1, rohit bhatia2, vishnu v2, mehar c sharma1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurology, all india institute of medical sciences, new delhi, india 3 csir-institute of genomics and integrative biology (csir-igib), new delhi, india background: ryr1-related myopathies (ryr1-rm) are described as a rare, clinically and histopathologically heterogeneous, and slowly progressive neuromuscular disorders. they constitute the most common class of congenital myopathies. clinical phenotypes are diverse and include king-denborough syndrome, exercise-induced rhabdomyolysis, lethal multiple pterygium syndrome, adult-onset distal myopathy, atypical periodic paralysis, mild calf-predominant myopathy, and dusty core disease. objectives: to report clinicopathological features of a group of the patients diagnosed with ryanodinopathy in a tertiary care centre from india. material and methods: a retrospective study (2019-2020) was performed on the clinical, histopathological and genetic features of all pediatric and adult patients, in whom ryr1 mutation was detected using next generation sequencing. results: five cases of ryr1-related myopathies were identified with age at onset varying from infancy to adulthood (2-39 years). a range of overlapping clinical phenotypes was noted: predominant proximal muscle involvement, facial weakness, external ophthalmoplegia, winging of scapula and mild tetraparesis associated with joint laxity. two different histopathological patterns were recognized: centronuclear (n= 2) and central core (n = 3). each case exhibited a different ryr1 mutation variant of which two novel genetic variants were revealed. further one genetic variant was reported to be associated with malignant hyperthermia susceptibility. conclusion: there is a striking clinical and genetic heterogeneity among ryr1-related myopathies with identification of two new ryr1 variants. its recognition is essential for genetic counselling and improving patient’s safety during anaesthesia to avoid episodes of hyperthermia.   abstract 10 free neuropathol 3:14:14 utility of respiratory chain complex assays in the diagnosis of mitochondrial disorders a phenotype, histopathological and genotype correlation deepha s1,3, ponmalar jn1,3, shivani sharma1,3, nagappa m2,3, govindaraj p3,5, bindu ps2,3,6, taly ab2,3, bharath mm4, gayathri n1,3 1 department of neuropathology, nimhans, bangalore, india 2 department of neurology, nimhans, bangalore, india 3 neuromuscular laboratory, department of neuropathology nimhans, bangalore, india 4 department of clinical psychopharmacology & neurotoxicology, nimhans, bangalore, india 5 centre for dna fingerprinting and diagnostics (cdfd), hyderabad, india 6 the children’s hospital at westmead clinical school, sydney medical school, the faculty of medicine and health, the university of sydney, sydney, nsw, australia background: mitochondrial respiratory chain (mrc) enzyme complex assay play a crucial role in the diagnostic workup in a patient with suspected mitochondrial disorders. determining the enzyme activities assist in defining isolated or multicomplex deficiency disorders and guide in identifying the molecular basis of the disease. objectives: to assess mitochondrial function in patients with suspected mitochondrial disorders. material and methods: this retrospective study (2014-2021) analysed a large cohort (n=1046) of cases with clinical diagnosis of mitochondrial disorders and /or other genetic and acquired skeletal muscle diseases. fresh skeletal muscle tissue was subjected to mrc enzyme complex assay by spectrophotometry as a part of routine diagnostics. results: a total of 268/1046 cases (25.6%) revealed complex deficiencies. amongst these, 208 cases [136 children (age range:10months-18years; m:f= 81:55) and 72 adults (age range:19-62 years; m:f= 39:33)] were primary mitochondrial disorders with syndromic and non-syndromic phenotypes. the syndromic phenotypes (n=65; 31.2%) include cpeo (n =15), mitochondrial myopathy (n=10), melas (n =9), leigh syndrome (n=8), progressive myoclonic epilepsy (n=11), leukodystrophy (n=4), leukoencephalopathy (n=5), encephalomyopathy (n=1), sensory ataxia neuropathy (n=1) and narp (n=1). non-syndromic presentations constituted 143 cases (68.75%). muscle histopathology findings was diagnostic in 33 (15.8%), while normal in 175 (84.13%). the deficiency included isolated complex i (n=127, 61%), isolated complex iv (n=31,14.9%), isolated complex iii (n=5, 2.4%) and multiple complexes (n= 45, 21.6%). complex i was the most common respiratory chain deficiency followed by multiple complex deficiencies, complex iv and complex iii. genetics done in over 100 cases revealed mutations across subunits of the complexes. conclusion: mrc enzyme complex assay enhanced the diagnostic yield in a large number of patients, in particular those with non-syndromic presentations as compared to conventional enzyme histochemical methods. correlation of clinical, biochemical, pathological and genetics findings will be presented.   abstract 11 free neuropathol 3:14:15 myelination changes in white matter following severe traumatic brain injury (stbi): a neuropathological study meenakshi sharma1, arulselvi subramanian2, vaishali suri3, deepak agrawal4, rajesh malhotra5, sanjeev lalwani1 1 division of forensic pathology and molecular dna, jai prakash narayan apex trauma centre, aiims, new delhi, india 2 department of laboratory medicine, jai prakash narayan apex trauma centre, aiims, new delhi, india 3 department of pathology, aiims, new delhi, india 4 department of neurosurgery, jai prakash narayan apex trauma centre, aiims, new delhi, india 5 chief, jai prakash narayan apex trauma centre, all india institute of medical sciences, new delhi, india background: white matter injury after tbi involves both axonal injury and myelin pathology that evolves throughout the post-injury time course. objectives: to examine myelination changes in post stbi patients. materials and methods: 64 post mortem brain tissues (corpus callosum), 24 non-tbi and 12 control samples from the patients who died due to stbi were collected. patients with initial gcs score ≤ 8 and age above 18 years with positive ct findings were included. routine h&e grading (3), myelin assay (lfb-pas) and ihc for myelin basic protein (mbp) microscopic changes were graded on the basis of percentage of demyelinated area. results: among stbi group, h&e grading shows moderate demyelination in 39.06% patients and normal grade in 26.5% cases, whereas lfb-pas & ihc –mbp grading, depicts severe demyelination in 29.8% patients and shows moderate demyelination in 26.5% patients. comparative to non-tbi group where 91.7% h&e grade and 83.3% lfb-pas &ihc –mbp grade shows normal grade, control cases express 100% normal myelination grades. both grading’s found to be significant in stbi compare to non-tbi and control groups. conclusion: though h&e showed subtle demyelination changes, lfb-pas & ihc –mbp grading depicted maximum changes following stbi. marked demyelination started in first week (3-10 days) after stbi with reduced demyelination after 10 days which might suggest remyelination of demyelinated areas. clinical evaluation of tbi will need to address the challenge of accurately detecting the extent and stage of myelin damage for identifying broader range of therapeutic opportunities to improve outcome after tbi.   abstract 12 free neuropathol 3:14:16 is cerebral malaria an astrocytopathy? a post mortem study of bbb dysfunction aditi goyal1, anita mahadevan1, netravathi m2, jitender saini3, satish chandra4, sk shankar1 1 department of neuropathology, nimhans, bangalore, india 2 department of neurology, nimhans, bangalore, india 3 department of neuroimaging and interventional radiology, nimhans, bangalore, india 4 department of neurology, apollo speciality hospital, bangalore, india background: pathogenesis of edema and seizures in cerebral malaria (cm) leading to mortality is unresolved. microvascular pathology with sequestration of parasitized rbcs, endothelial activation, inflammatory mediator release and disruption of blood brain barrier (bbb) is a central pathogenetic event. however, role of astrocytes, major regulators of bbb, that protect from vasogenic edema via aqp4 channels in their end feet is unexplored. objectives: to evaluate histomorphological alterations in astrocytes and their role in pathogenesis of cerebral malaria. materials and methods: clinical, demographic and neuropathological changes of six patients who succumbed to cerebral malaria were reviewed. immunohistochemistry was performed for glial (gfap, s100b) bbb (aqp4, igg) and immune (p65 (nfκb)) markers. results: patients [age range:14-40yrs; male:female=2:1; duration of illness (doi):2-21days] were arbitrarily divided into group1 (doi <10days-hyperacute) and group 2 ( >/= 10days, acute). brain showed marked cerebral edema with slate-grey discoloration. histopathologically, all showed sequestration of parasitized rbcs in microcirculation, fresh ring and/or ball haemorrhages (6), resolving haemorrhages (4), and durck granulomas (dg) (1). astrocytic alterations were striking. in hyperacute stage (group1), astrocytes were markedly stunted with attenuated processes, prominent dystrophic beading and retraction of processes, with reduced ensheathing of vessels. progressive reduction in gfap, s100b and aqp4 expression occurred with increasing doi. loss of aqp4 expression demonstrable around haemorrhages and dg, with leakage of igg from vessels in acute stage. p65 (nfkb) expression was limited to resolving haemorrhages and dg. conclusion: astrocytopathy results in bbb dysfunction in cm. understanding the pathogenetic events at microvascular interface could aid design effective therapy to prevent mortality and morbidity in cm.   abstract 13 free neuropathol 3:14:17 does mitochondrial dysfunction play a role in pathogenesis of mesial temporal lobe epilepsy secondary to hippocampal sclerosis? shwetha sd1, anita mahadevan1, srinivas bharath mm2, keshav prasad ts3, m.ravindranadh chowdary4, raghavendra k4, ajay asranna4, arivazhagan a5, malla bhaskara rao5, jitendra saini6, rose dawn bharath6, sinha s4 1 department of neuropathology, national institute of mental health and neurosciences (nimhans), bengaluru, india 2 department of clinical psychopharmacology and neurotoxicology, nimhans, bengaluru, india 3 department of centre for systems biology and molecular medicine, yenepoya research centre, mangalore, india 4 department of neurology, nimhans, bengaluru 5 department of neurosurgery, nimhans, bengaluru department of neuroimaging and interventional radiology, nimhans, bengaluru, india background: studies in animal models of temporal lobe epilepsy suggest a pathogenetic role for mitochondrial dysfunction, although validation studies in humans are scarce. objectives: we chose to evaluate the mitochondrial status in the hippocampus resected from patients with mesial temporal lobe epilepsy (mtle) through proteomic approaches. material and methods: crude mitochondrial preparations from human hippocampus samples, resected from patients with mtle, who underwent amygdalohippocampectomy (early-onset <10years of age, n=9 and late-onset >11years of age, n=9), compared with age matched normal controls (n = 9) were subjected to quantitative proteomics using high-resolution mass spectrometry (ms). ms data was validated by mitochondrial respiratory chain complex assays (ciciv). results: the ms identified 7,961 proteins among which, 190 proteins and 60 mitochondrial proteins differentially over expressed in early and late onset respectively (p<0.05). proteins associated with biological processes such as mitochondrial electron transport chain, mitochondrial translation and branched-chain amino acid catabolic process were differentially overexpressed in cases with early onset mtle, suggesting a pathogenetic role. fatty acid beta-oxidation and glutathione metabolic processes were common to both early and late onset mtle. mitochondrial respiratory complex iactivity was higher in early onset compared to late onset mtle and controls, validating the proteomics data. the activities of mitochondrial complexes ii-iv remained unaltered. conclusion: mitochondrial dysfunction in hippocampus appears to have a role in the pathogenesis of early onset mtle, in particular, mitochondrial complex i subunits. evidence for role of mitochondrial dysfunction may aid in development of novel therapeutic strategies for treatment of mtle.   abstract 14 free neuropathol 3:14:18 diffuse midline gliomas with h3k27 mutation clinicopathological correlates john abha1, chacko ag2, moorthy r2, joseph bv2, john r3, bindra m1, gowri mahasampath4, chacko g1 1 department of general pathology, christian medical college, vellore, india 2 department of neurosurgery, christian medical college, vellore, india 3 department of pediatric oncology, christian medical college, vellore, india 4 department of biostatistics, christian medical college, vellore, india background: h3k27m mutant diffuse midline gliomas are infiltrative, midline high-grade gliomas with a k27m mutation in either h3f3a or histih3b/hist1h3c and have been reported to have a poor prognosis. objectives: to assess the morphology, the h3k27m status and clinical outcomes in h3k27m positive and negative diffuse midline gliomas. material and methods: seventy-three cases of diffuse midline gliomas of who grades ii-iv from 2012 to 2020, underwent immunohistochemical evaluation using h3k27m mutation specific antibody and h3k27me3 trimethylation antibody. morphological parameters, clinical details and outcome were correlated with mutational status. results: forty four of the 73 tumours were positive for the h3k27m mutation.h3k27m mutant diffuse midline gliomas occurred more commonly in adults and in the thalamus. they corresponded most often to a who grade iii. the mib-1% was significantly higher in the mutant group of tumours. when considering the entire cohort, the h3k27m mutant group showed better overall survival and recurrence free survival compared to the wild group. however, rfs in the mutant group was worse in the supratentorial tumours compared to the infratentorial tumours and spinal cord h3k27m mutant tumours showed shorter os and rfs compared to wild type tumours. in the cohort of h3k27m mutant tumours, children showed worse os when compared to adults. conclusion: contrary to reported literature h3k27m mutant diffuse midline gliomas occurred more commonly in adults and in the thalamus. h3k27m mutant tumours had a better rfs and os than the wild-type cases. however, amongst h3k27m mutant tumours, children had worse os than adults. spinal cord h3k27m mutant tumours had a shorter rfs and os when compared to brainstem and thalamus.   abstract 15 free neuropathol 3:14:19 significance of nestin and cd133 as cancer stem cell markers in diffuse gliomas and its association with idh-1 status and p53 expression sivaranjani s1, srinivas bh1, surendra kumar verma1, gopalakrishnan ms2 1 department of pathology, jipmer, puducherry, india 2 department of neurosurgery, jipmer, puducherry, india background: based on the cancer stem cell (csc) theory, they have self-renewal, uncontrolled proliferation, multi-directional differentiation properties. we have studied cd133 and nestin, which are the most commonly used two csc markers with consistent expression in diffuse gliomas. objectives: to assess of the level of expression of csc markers; nestin and cd133 and identify the correlation among various grades of diffuse glioma, idh status and p53. material and methods: a cross-sectional retrospective study conducted in department of pathology and neurosurgery with 102 subjects on expression of cscs and correlation with that of p53 and idh1 status in adult diffuse gliomas by immunohistochemistry on ffpe sections. the scoring of expression of cd 133 and nestin was adopted from zhang et al and p53 from aruna et al. the data was further analysed. results: the diffuse gliomas were graded based on who into grade ii, iii and iv. the expression of cd133 and nestin was compared with the increasing grades of diffuse gliomas and plotted on roc curves with auc of 0.6806 and 0.6119 respectively. this expression also showed a positive correlation with the idh status of tumor. conclusion: csc markers are expressed in diffuse gliomas and have higher expression with increasing in who grade and have significant association with idh-1 mutant status. hence, it can be inferred that diffuse gliomas with the higher expression of csc markers have poorer prognosis. further, they have the potential to be used as therapeutic targets in the future.   abstract 16 free neuropathol 3:14:20 immunophenotypic profile of pediatric brain tumors reflecting molecular alterations karuna balakrishnan1, srinivas bh1, surendra kumar verma1, gopalakrishnan ms2 1 department of pathology, jipmer, puducherry, india 2 department of neurosurgery, jipmer, puducherry, india background: pediatric brain tumors are the most common solid pediatric tumors. currently, there has been a drive towards “personalised medicine” or “precision medicine,” where chemotherapy is targeted against specific driver mutations. the revised 4th edition and the yet-to-be-released 5th edition of who has considered this and included genetic information into the classification for a combined phenotypic-genotypic approach. as molecular analyses are not available in many centres in india, surrogate immunohistochemistry (ihc) markers corresponding to genetic alterations have been developed. objectives: to study the immunophenotypic profile of pediatric brain tumors corresponding to molecular alterations material and methods: this is a cross-sectional descriptive study of immunophenotyping of 51 patients with pediatric brain tumors reflecting molecular alterations on ffpe sections. immunohistochemistry for beta-catenin, gab-1, yap-1, ini-1, p53 and lin28a was done for 22 embryonal tumors. atrx, brafv600e and h3k27m was done on 27 glial tumors and idh-1 wherever necessary. brafv600e was done on 2 gangliogliomas. results: using beta-catenin, gab-1 and yap-1, 19 medulloblastomas were classified into non-wnt/shh pathway activated (16/19), shh pathway activated (3/19) and) wnt pathway activated (0/19). out of the 2 etmrs diagnosed morphologically, one showed lin28a expression, the other etmr showed ini-1 loss and hence reclassified as atrt. out of 9 pilocytic astrocytomas, 2/9 showed atrx loss, 4/9 showed brafv600e expression. h3k27m was positive in 2/2 diffuse midline gliomas. of the 3 anaplastic astrocytomas and 6 glioblastomas, 1/3 anaplastic astrocytoma was idh positive with atrx loss. hence it was reclassified as idh mutant grade 3 astrocytoma. rest all were idh negative. 1/3 anaplastic astrocytoma and 4/6 glioblastomas showed loss of atrx; hence they were assumed to be h3g34 altered diffuse hemispheric gliomas. the remaining 1/3 anaplastic astrocytoma and 2/6 glioblastomas showed retained atrx expression and hence considered diffuse pediatric high-grade glioma, h3-wildtype and idh-wildtype. conclusion: for embryonal tumors, currently, many clinical trials are underway based on this molecular classification. for example, for wnt-activated medulloblastomas, the dose of radiation has been reduced. for shh medulloblastomas, smo inhibitors have been introduced. brafv600e and h3k27m mutated tumors can also be given targeted therapies. hence, in a resource-limited setup, these surrogate ihc markers can help fine-tune the diagnosis so that appropriate treatment can be given.   abstract 17 free neuropathol 3:14:21 pdl1 expression in cns tumours divyangi paralkar1, ashwani tandon1, rekha singh2, adesh shrivastava3, neelkamal kapoor1 1 department of pathology and lab medicine, all india institute of medical sciences bhopal, india 2 department of endocrinology and metabolism, all india institute of medical sciences bhopal, india 3 department of neurosurgery, all india institute of medical sciences bhopal, india background: immune checkpoints like pdl-1 regulates tumour microenvironment. pd-1 is expressed on activated t cells while pdl-1 is expressed on antigen presenting tumor cells. pdl-1 inhibits t cell activation; decreases proliferation and cause t cell apoptosis. tumour survival is a balance between immune surveillance and cancer cell proliferation. it may be immunomodulated by pd-l1. anti pd-l1 have be emerged in the treatment for multiple cancers in advanced stage objectives: detection of pdl-1 expression in cns tumour. material and methods: cross sectional study was conducted on 99 cases of cns tumour (50 diffuse astrocytoma; 6 pilocytic astrocytoma; 7 ependymoma; 2 oligodendroglioma; 27 meningioma; 07 embryonal tumour) between january 2014 to august 2020 in department of pathology and lab medicine, all india institute of medical sciences, bhopal. results: diffuse astrocytoma total 50 cases of which 1/9 of grade ii, 1/6 of grade iii (1/1 til), 7/19 of grade iv (2/3 til) and 10/16 of recurrent glioma (4/4 til). pilocytic astrocytoma (3/6 cases with 1/1til); ependymoma 7 cases (0/ 1 supratentorial, 3/3 posterior fossa and 2/3 spinal cord (0/1 til); oligodendroglioma (0/2); meningioma 27 cases (6/18 grade i (2/3 til); 8/8 grade ii (2/2 til), 1/1 grade iii (1/1 til)); medulloblastoma 0/6 cases; atypical teratoid/rhabdoid tumour 1/1 were pdl1 positive. (43/99 with til 13/16) conclusion: 43.3% cns tumour showed pdl-1 positivity with higher percentage in high grade. 16.16 % tumour showed til with 81.25% pdl-1 expression.   abstract 18 free neuropathol 3:14:22 droplet digital pcr: a robust technique for detection of idh1 r132h mutation in formalin fixed tissue samples rituparna chakraborty1, swati mahajan1, jyotsna singh1, mehar c sharma1, chitra sarkar1, vaishali suri1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india background: mutations involving isocitrate dehydrogenase 1 (idh-1) occur in a high proportion of diffuse gliomas (≈90%), with implications on clinicopathologic diagnosis and prognosis. ihc is an easy and quick method of detecting idh1-r132h mutations, but sometimes there may be some discrepancies. traditional approaches, such as sanger sequencing is laborious and lack sensitivity due to tumor heterogeneity and low tumor purity of glioma samples. the recently developed droplet digital pcr (ddpcr) technique generates a large amount of nanoliter-sized droplets, each of which carries out a pcr reaction on one template. therefore, ddpcr provides high precision and absolute quantification of the nucleic acid target. objectives: the present study compares results of ihc with ddpcr in diffuse gliomas. material and methods: 50 diffuse infiltrating idh1 immunopositive gliomas and 25 control samples (meningiomas) were included in the study. all cases were assessed for idh1 mutations by ddpcr. a cut off criteria which includes mutant allele fraction was standardized. the detection limit was calculated using serially diluted positive mutant dna in a background of wild-type dna. results: there was 100% concordance of results between ihc and ddpcr. all control samples detected negative for idh1 mutation by ddpcr. further compared with sanger sequencing, ddpcr was less time consuming (≈3 hours) and less laborious. conclusion: ddpcr is a reliable, rapid, robust,100 % sensitive and specific method for screening the idh1 mutation. it can be used to detect even low-frequency mutation burden.   abstract 19 free neuropathol 3:14:23 tert promoter mutations in meningiomas: a clinicopathological correlation ganga kundeti1, nupur karnik1, mamta gurav1, sneha janjal1, omshree shetty1, ayushi sahay1, vijay patil2, prakash shetty3, aliasgar moiyadi3, tejpal gupta4, sridhar epari1 1 department of pathology, actrec and tata memorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india 2 department of medical oncology, actrec and tata memorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india 3 department of neurosurgery, actrec and tata 4emorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india 4 department of radiation oncology, actrec and tata memorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india background: mutations in the non-coding promoter region of tert gene has now been clearly established as one of the most dominant non-histological biomarker for aggressiveness in meningiomas. objectives: to study the pattern of tert promoter (ptert) mutations across different histological grades of meningiomas. materials and methods: diagnosed cases of meningiomas, which had been evaluated and interpretable for ptert mutations by direct target (c228 and c250) sequencing were analysed for their correlation for clinicopathological features. results: 155 cases (with age-range: 18-75 years & male to female ratio: 0.98 [males=58, females= 59]) formed the study cohort. commonest location was cerebral convexity. histologically, 63 were grade 1 (angiomatous:1; transitional:38; meningothelial: 14, fibroblastic:2 and nos:8) , of these 28 had subtle atypical features ( ≥1 and ≤ 3 atypical histological findings i.e. high cellularity, small cell change, prominent nucleoli, necrosis, mitotic activity <4/10 high power fields (hpf) and necrosis). 79 were grade 2 (clear cell:1; chordoid: 1, 77: atypical) and 13 (papillary:2; rhabdoid:1) were grade 3. 8/155 (5.2%) showed tert promoter mutation (c228t: 5, and c250t: 3). 2 (of 13; 15.4%) were grade 3, 3 (of 79; 3.8%) were grade 2 and 3 (of 63; 4.8%) were grade 1. all three ptert mutant histologically grade 1 meningiomas, showed subtle atypical features, none (n=35) of the typically grade 1 showed ptert mutations. conclusion: ptert mutations are uncommon in meningiomas and are seen only in cases with presence of histologically aggressive features.   abstract 20 free neuropathol 3:14:24 evaluation of tert promoter mutation status in meningiomas jyotsna singh1, swati mahajan1, afreen khan1, swati singh1, ashish suri2, niveditha manjunath2, mehar c sharma1, vaishali suri1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india background: meningiomas are the most common benign intracranial tumours. approximately 20% of the patients show aggressive phenotype with significant patient morbidity and mortality. tert promoter (ptert) mutations have been associated with upregulation of telomerase activity and tert mrna expression in diverse cancer types including glioblastomas and oligodendrogliomas. few studies on meningiomas have documented that presence of ptert mutations is associated with higher tumour grade, enhanced risk for recurrence and progression. objectives: to analyse the frequency of ptert mutation and assessed its prognostic significance in meningiomas. material and methods: a total of 125 cases (57 males and 68 females) of grade 1 (n=60), 2 (n=42), 3 (n=13) and control (n=10) were included in the study (2012 to 2020). they were assessed for c228t and c250t hotspot mutation in the ptert region by using sanger sequencing. samples were stratified into two groups tert mutated vs tert wild type. results: there were 111 adult and 4 paediatric cases. the mean age of the patients was 38.8 years (range 10–70 years). ptert c228t mutation was found in only 1.7% (2 of 115) meningiomas. both were grade 2 and adults. on follow up, both the patients died of tumour recurrence. conclusion: ptert mutations are infrequent in meningiomas and associated with aggressive biology. further, the predictive power of ptert status essentially allow the clinician to identify aggressive meningiomas, patients at risk for early recurrence and provides biomarker for new therapeutic interventions.   abstract 21 free neuropathol 3:14:25 pitfalls in diagnosis of oligodendrogliomas on squash cytology tista basu1, mou das1, uttara chatterjee1 1 department of pathology, institute of post graduate medical education and research, kolkata, india background: oligodendrogliomas (odg) are one of the less common gliomas. odg presents with a broad morphological spectrum and may mimic a variety of glial and non-glial neoplasms. the diagnostic peri-nuclear halo on histopathology is not seen in squash smears at all, thereby posing diagnostic difficulties in squash smears. objectives: to evaluate the diagnostic accuracy, limitations and pitfalls of intraoperative cytology in oligodendrogliomas. materials and methods: intraoperative squash smears of cns tumours stained with haematoxylin and eosin stain were evaluated followed by corresponding histopathology sections. appropriate immunostains were utilised where they were relevant. the findings were reviewed to determine the diagnostic pitfalls in twelve cases of odg from our institution in the last two years. results: amongst the 12 cases of histomorphologically proven odg, 5 were correctly diagnosed as odg grade 2 on squash smears. amongst four cases diagnosed as astrocytoma on squash, three were odg grade 2 and one was anaplastic odg (grade 3) in hpe. one case diagnosed as low grade glioma (odg) on squash cytology showed mini-gemistocytes. this was diagnosed as an anaplastic odg on hpe. two cases were incorrectly diagnosed as central neurocytoma and lymphoma respectively on squash. conclusion: the absence of properly defined intra-operative squash cytological features and sparse literature, leads to inter-observer variability during diagnosis of odgs on squash smear alone. the discerning nuclear features of an odg on squash smear are also lost with increase in grade and thus, further pose a diagnostic dilemma. mini-gemistocytes are a helpful clue for anaplasia on squash smear.   abstract 22 free neuropathol 3:14:26 low prevalence of braf v600e mutations in pleomorphic xanthoastrocytoma ranjani j1, gandham ej2, beno d1, pai r1, balakrishan r3, jasper a4, gowri m5, moorthy rk2, chacko ag2, chacko g1 1 department of pathology, christian medical college, vellore, india 2 department of neurological sciences, christian medical college, vellore, india 3 department of radiation therapy, christian medical college, vellore, india 4 department of radiology, christian medical college, vellore, india 5 department of biostastics, christian medical college, vellore, india background: the prevalence of braf v600e mutations in pleomorphic xanthoastrocytomas (pxa) and anaplastic pxas (apxa) varies from 60 to 80%. there are conflicting reports on the prognostic relevance of this mutation in pxas. objectives: to study the prevalence of braf v600e mutation in patients with pleomorphic xanthoastrocytoma (pxa) and correlate this with the outcome. materials and methods: this retrospective study included 33 patients with a diagnosis of pxa/apxa operated between 2007 and 2020. the demographic and clinico-radiological data were obtained retrospectively from the electronic database. the biopsies were reviewed and the samples were assessed for the presence of braf v600e mutation, using droplet-digital polymerase chain reaction. the histological grade and braf v600e mutational status were correlated with progression-free survival (pfs) and overall survival (os). results: there were 20 patients in the pxa group and 13 patients in the apxa group. braf v600e mutation was seen in 40 % (8/20) of the pxa and 15 % (2/13) of the apxa cases. recurrence was seen in 7/13 (55%) apxas but none in the pxa group recurred at a mean follow-up of 45months. the overall survival was significantly better in pxas compared to the apxas (p=0.02). brafv600e mutated tumours had a better os as compared to wild type tumours but this did not reach statistical significance. (p=0.364). conclusion: braf v600e mutations were seen in only 30% of pxa/apxas limiting its usefulness as a diagnostic marker. braf v600e mutant tumours had a better overall survival, however, this was not statistically significant.   abstract 23 free neuropathol 3:14:27 unusual primary intracranial sarcomas – ewing’s sarcoma and synovial sarcoma rallabandi hima bindu1, meenakshi swain1, rahul lath2, subodh raju2 1 department of histopathology, apollo hospital, hyderabad, india 2 department of neurosurgery, apollo hospital, hyderabad, india background: primary sarcomas of brain are rare – hence two cases of primary intracranial sarcomas of brain are being presented. 1. synovial sarcoma is an aggressive soft tissue sarcoma and intracranial occurrence is rare. it is characterized by a unique chromosomal translocation t(x;18) (p11.2; q11.2). 2. primary intracranial ewing sarcoma is also rare. it is characterized by chromosomal translocation t(11;22) (q24;q12) . very few cases, of primary intracranial synovial sarcoma and primary intracranial ewing’s sarcoma have been reported in the literature. objectives: to present two unusual sarcomas of brain and emphasize on the diagnostic challenges. material and methods: case reports of two primary intracranial sarcomas results: case 1 – 31 years old male on imaging was found to have left frontal sol. histopathological examination with immune stains, suggested a synovial sarcoma. fish studies done at two laboratories were negative for syt-ssx2. later the translocation was detected by rtpcr, confirming the histological diagnosis. case 2 –twenty-year male came with complaints of headache since 3 months. mri – revealed heterogeneously enhancing left tentorium based sol. histopathological examination with immune stains showed features of primitive neuroectodermal tumor. fish studies revealed ewsr 1 gene re-arrangement with the final diagnosis of ppnet. conclusion: sarcomas though rare can be seen as primary intracranial tumors, hence awareness of these entities with appropriate molecular studies is important for making the correct diagnosis.   abstract 24 free neuropathol 3:14:28 relevance of the 4-variable risk stratification model in cns solitary fibrous tumours hemanth kumar r1, poonkodi m1, moorthy rk2, chacko ag2, joseph bv2, rajesh b3, chacko g1 1 department of pathology, christian medical college, vellore, india 2 department of neurosurgery, christian medical college, vellore, india 3 department of radiation therapy, christian medical college, vellore, india background: solitary fibrous tumour/hemangiopericytomas (sft/hpc) of the cns are graded based on their morphological phenotype and mitotic activity. in contrast, sfts arising in the non-meningeal sites are prognosticated using the 4-variable risk stratification model (described in who classification of soft tissue and bone tumours, 2020) which has been observed to be an improvement over the traditional benign/malignant distinction. objectives: to determine pertinence of the 4-variable risk stratification model in meningeal sft/hpcs. materials and methods: this retrospective study identified 53 patients operated between 2014 and 2021 and diagnosed as sft/hpc on histopathology. follow up data was available for 27/53 patients. the cases were sorted into low, intermediate and high risk groups as per the 4-variable risk stratification model. the results were correlated with the follow-up data, including the presence or absence of distance metastasis. results: one tumour was diagnosed as sft, who grade i, while 6 tumours and 20 tumours were diagnosed as hpc, who grade ii and iii, respectively. all 27 tumours were immunopositive for stat6. in accordance with the 4-variable risk stratification model, 19 (70.4%) patients fell in to the low risk group, while 8 (29.6%) fell into the intermediate risk group. local recurrence was observed in 3 patients, while distant metastasis was seen in 3 patients (lung (1/3), l5-s1 spine (1/3) and submental soft tissue (1/3)). all patients with local recurrence or distant metastasis were diagnosed with hpc, who cns grade iii. among the patients with metastasis, two belonged to low risk group and one to intermediate risk group (as per the 4-variable risk stratification model). conclusion: despite the limited sample size, it is observed that the 4-variable risk stratification model validated in non-meningeal sfts did not prognosticate the meningeal counterparts appropriately. it is therefore unlikely that this model might replace the original grading described in who classification of cns tumours, 2016.   abstract 25 free neuropathol 3:14:29 oropharyngeal psammomatous melanotic schwannoma, non-syndromic an unusual tumour at an unusual site poonam elhence1, rashim sharma1, divya aggarwal1, balamurugan t1, ravindra shukla2, amit goyal3 1 department of pathology, all india institute of medical sciences, jodhpur, india 2 department of endocrinology, all india institute of medical sciences, jodhpur, india 3 department of otorhinolaryngology, all india institute of medical sciences, jodhpur, india background: psammomatous melanotic schwannoma is a rare tumour of uncertain histogenesis and indeterminate biologic behaviour with known association with carney’s syndrome. objectives: to present a rare case of an oropharyngeal psammomatous melanotic schwannoma which has no syndromic association presently. material and methods: case report of oropharyngeal sammomatous melanotic schwannoma results: a 25-year-old female presented with complaints of a swelling in her oral cavity for about ten years, gradually increasing in size and causing difficulty in swallowing. a clinical diagnosis of hemangioma was given. the swelling was excised and sent for histopathological evaluation. a circumscribed reddish to brownish black mass measuring 5x4x3.5cms was received. the cut surface showed reddish-brown to haemorrhagic areas and foci of calcification. on microscopic examination, a diagnosis of psammomatous melanotic schwannoma was given. the patient was not found to have any associated feature of carney’s syndrome on clinical examination. she had no significant personal or family history. the patient is on regular follow up and is doing well three years post-surgery. conclusion: psammomatous melanotic schwannoma is a rare tumour of uncertain biologic potential. a knowledge of this entity is helpful for correct diagnosis and appropriate patient management with long-term follow-up in view of potential malignant transformation.   abstract 26 free neuropathol 3:14:30 silent corticogonadotroph adenoma (scga): a silent monster shalini suman1, swati mahajan1, mohd sulaiman2, vaishali suri1, deepak aggrawal2, sharma mc1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india introduction: pituitary adenomas are usually benign tumors which are classified based on differentiated cell type origin. silent corticogonadotroph adenoma are rare, benign but aggressively growing biochemically silent adenoma subtype showing rare characteristics of bilineage differentiation of both corticotroph and gonadotroph. till now there is only a single study published in literature about this entity and further studies need to be done. objectives: to report a case of silent corticogonadotroph adenoma material and methods: case report of silent corticogonadotroph adenoma results: a 25year old male presented with complaints of multiple episodes of headache and vomiting since 3 weeks and bilateral loss of vision since 5-6 years. ncct brain showed large, well developed, extra-axial sellar and supra sellar predominantly solid lesion with peripheral cystic component measuring 6.3x2.7x5.3cm. histomorphological analysis showed a tumor with features of pituitary adenoma. immunohistochemistry for the hormonal profile showed diffuse positivity for acth, whereas tumor cells were negative for lh, fsh, prl, gh and tsh. in addition, the tumor cells were diffusely positive for lmwck with mib1 labelling index of 2%. hormonal profile suggested a corticotroph adenoma. however, ihc for transcription factors showed contradictory results with tumors cells positive for sf1 and gata3 while negative for tpit and pit1. integrating clinical, morphological and immunohistochemistry findings a finally diagnosis of silent corticogonadotroph adenoma was rendered. conclusion: due to the rarity of this lesion, scga may not be considered as a differentials while working up the case. the diagnosis of this subtype emphasizes increased postoperative surveillance for earlier detection of recurrences and hypopituitarism thereby reducing morbidity and improving quality of life in these patients.   abstract 27 free neuropathol 3:14:31 molecular profiling: a key to clinical and histological enigma in an ambiguous case priyanka singh1, iman dandapath1, swati mahajan1, satish verma2, ajay garg3, mehar c sharma1, vaishali suri1 1 department of pathology, all india institute of medical sciences, new delhi, 110029, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, 110029, india 3 neuroradiodiagnosis, all india institute of medical sciences, new delhi, 110029, india background: the upcoming 2021 who classification of cns tumors highlights the importance of molecular diagnostics. objectives: molecular characterization of a tumor showing ambiguous histology and clinico-radiological picture. material and methods: a 27-year-old female presented with history of headache, vomiting and vision deterioration for a month with no motor/sensory deficits. on cect, there was a well-defined lobulated, solid-cystic lesion with calcification involving anterior corpus callosum and bilateral anterior frontal lobe. craniotomy and tumor excision were done. intraoperatively, it was soft and vascular tumor extending to the left lobe. a radio-clinical diagnosis of oligodendroglioma or high-grade glioma was rendered. results: h&e-stained sections showed a tumor comprising of round monomorphic cells with clear cytoplasm forming small nests and rosettes with extensive neuropil in the background. hyalinized blood vessels, rosenthal fibres and eosinophilic granular bodies were seen. there was an occasional focus of endothelial cell proliferation. no mitotic activity or necrosis was noted. immunohistochemically, the tumor cells were positive for olig2, map2, β-tubulin while negative for gfap, neu-n, nf, cd34, ema and ebp-50. neuropil was highlighted by synaptophysin. the tumor cells were negative for idhr132h, p53 and licam and showed retained atrx expression. based on these findings, a diagnosis of glioneuronal tumor, nec, who grade 1 was suggested. molecular analysis by fish, rt-pcr and sanger sequencing exhibited mapk pathway activation. conclusion: a cryptic case with deregulation of mapk pathway is highlighted. molecular characterization is essential in such unusual cases owing to availability of targeted therapy.   abstract 28 free neuropathol 3:14:32 pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease – a case report nufina t a1, alok mohan uppar2, jitender saini3, vani santosh1 1 department of neuropathology, nimhans, bangalore, india 2 department of neurosurgery, nimhans, bangalore, india 3 department of neuroimaging and interventional radiology, nimhans, bangalore, india background: pseudotumoral hemicerebellitis is an exceptionally rare presentation in which unilateral cerebellar involvement mimics a tumour. the aetiology is diverse including post vaccination and post infection. objectives: to report a rare case of pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease material and methods: case report of pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease. results: a 11 year old girl presented with headache and vomiting for 1 month, associated with right cerebellar signs. mri revealed a t2 hyperintense contrast enhancing right cerebellar lesion with diffusion restriction, and features of tiger stripe pattern suggestive of lhermitte–duclos disease. there were no neurocutaneous markers. she underwent craniotomy with biopsy of right cerebellar tissue which revealed an irregularly expanded cerebellar folia with florid lymphoplasmacytic infiltration admixed with histiocytes in the meninges and parenchyma. this was associated with marked reduction in the granule cell layer neurons and polyfocal destruction of folial architecture. there were no granulomas, parasites, viral inclusions, or collection of dysplastic neurons or neoplastic cells. special stains for fungi, acid fast tubercle bacilli and bacteria were negative. the patient did not have recent history of fever, vaccination or drug ingestion. extensive work up for infectious aetiology in both serum and csf proved to be negative. serum and csf panel for autoimmune encephalitis, paraneoplastic neuronal and nmo–mogsd antibodies were also negative. conclusion: pseudotumoral hemicerebellitis is the close differential diagnosis for lhermitte–duclos disease radiologically. it typically has a benign course, the main management comprising supportive measures, steroids or antivirals. better understanding and awareness of this rare entity would help in accurate presurgical diagnosis and patient management.   abstract 29 free neuropathol 3:14:33 anoctamin-5 muscular dystrophy: report of 2 cases with different phenotypes and genotypes from indian subcontinent bandana jassal1, swati mahajan1, aishwarya dhall1, alvee saluja2, mohammed faruq3, vaishali suri1, roopa rajan2, mehar chand sharma1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india 2 department of neurology, all india institute of medical sciences, new delhi, india 3 csir-institute of genomics and integrative biology (csir-igib), new delhi, india background: anoctaminopathies are a group of autosomal recessive skeletal muscle disorders with various clinical phenotypes, caused byanoctamin 5 (ano5) gene mutations and the abnormal expression of ano5 protein. patients with recessive mutations in ano5 present with variable symptoms ranging from asymptomatic hyperckemia and exercise-induced myalgia to proximal and/or distal muscle weakness. objectives: we describe the clinical, pathological, and molecular findings of two unrelated patients with ano5-related muscular dystrophy. material and methods: 96 histologically identified muscular dystrophy cases were subjected to next generation sequencing using a customized panel of 54 genes (iilumina design studio). results: two patients were diagnosed with ano5-related muscular dystrophy. one patient had a pathogenic homozygous mutation of c.1406g>a in exon 14 while the other patient had a novel heterozygous mutation of c.2141c>g in exon 19 of ano5 gene. both showed two different phenotypes (limb girdle muscular dystrophy 2l and miyoshi myopathy) and histomorphological pattern. muscle biopsy of one patient in addition showed amyloid deposition in the blood vessels walls. neurologic examination was unremarkable with insignificant family history. serum creatine kinase (ck) was elevated in both. conclusion: ano5-related muscular dystrophy is a heterogeneous disease with different clinical phenotypes as well as genotypes. all muscle biopsies with unclassified muscular dystrophies should be subjected to congo-red stain to look for amyloidosis. the results of this study further suggests that screening for ano5 gene should represent an early step in the diagnostic work-up of the patients with undiagnosed muscular dystrophy and persistent asymptomatic hyperckemia even when muscle biopsy is normal.   abstract 30 free neuropathol 3:14:34 dysferlinopathy in a cohort of north indian patients: clinical histopathological and mutational spectrum aishwarya dhall1, swati mahajan1, mohammed faruq2, pankaj pathak1, uzma shamim2, neena dhiman1, bandana jassal1, vaishali suri1, rohit bhatia3, mehar chand sharma1 1 neuropathology lab, neurosciences centre, all india institute of medical sciences, new delhi, india 2 csir institute of genomics and integrative biology, new delhi, india 3 department of neurology, all india institute of medical sciences, new delhi, india background: dysferlinopathy is a group of autosomal recessive muscular dystrophy caused by mutations in the dysferlin gene (dysf). it is the second most commonly reported lgmd subtype (27%) in india after gne myopathy (31%). phenotypic variants includes miyoshi myopathy (mm), limb-girdle muscular dystrophy (lgmd2b), and other atypical phenotypes, such as the proximo-distal phenotype and distal anterior compartment myopathy. objectives: to describe the clinical, histopathological and mutational spectrum of dysferlinopathy in a cohort of patients from northern india material and methods: 96 patients (2018-19) suspected of lgmd from non-related families underwent thorough phenotypic characterization followed by muscle histopathological analysis. these cases were subjected to next generation sequencing using a customized panel of 54 genes. results: eleven patients (6 male and 5 females) were diagnosed as dysferlinopathy amounting to a prevalence of 11.4% of lgmd in north indian population. eight patients presented with proximal lgmd2b, 2 with distal mm and 1 with proximo-distal phenotype. mean age of onset and diagnosis was 24.4 years and 36 years respectively. cpk was elevated in all and ranged from 540 to 13000 u/l. histomorphological analysis showed predominant dystrophic changes with necrotic-regeneration pattern and inflammation. immunohistochemistry revealed partial to complete loss of dysferlin in all except one case. sequencing reveal 9 novel and 6 known mutations including exonic (frameshift, stop gain and snvs) and splice variants. conclusion: a high proportion of novel mutations were identified in the dysf gene thus broadening the genetic spectrum of dysferlinopathy. no genotype-phenotype correlation existed suggesting that the clinical phenotype is determined not only by dysf variants but also likely through a complex interplay of environmental, epigenetic, and genetic factors.   abstract 31 free neuropathol 3:14:35 role of mitochondrial respiratory chain complexes in pathogenesis of temporal lobe epilepsy dhanya ck1, shwetha sd1, anita mahadevan1, srinivas bharath mm2, keshav prasad ts6, m.ravindranadh chowdary3, raghavendra k3, ajay asranna, arivazhagan a4, malla bhaskara rao4, jitendra saini5, rose dawn bharath5, sinha s3 1 department of neuropathology, nimhans, bangalore, india 2 department of clinical psychopharmacology and neurotoxicology nimhans, bangalore, india 3 department of neurology, nimhans, bangalore, india 4 department of neurosurgery, nimhans, bangalore, india 5 department of neuroimaging and interventional radiology, nimhans, bangalore, india 6 department of centre for systems biology and molecular medicine, yenepoya research centre, mangalore, india background: role of mitochondrial dysfunction in seizure generation comes from frequent occurrence of epilepsy in inherited mitochondrial disorders. less is known about its role in acquired epilepsies such as temporal lobe epilepsy (tle). targeting mitochondrial oxidative stress with antioxidant treatment may prove a useful adjuvant in the management of epilepsy. objective: to determine the role of mitochondrial dysfunction in the pathophysiology of tle by assessing mitochondrial function in the temporal lobe. materials and methods: mitochondria isolated from human temporal lobe samples, resected from patients with tle who underwent anterior temporal lobectomy (early-onset <10years of age, n=9 and late-onset >11years of age, n=9), compared with age matched normal controls (n=9) subjected to assays for malate dehydrogenase, succinate dehydrogenase, mitochondrial complexes i to iv and adp/atp ratio. results: mitochondrial respiratory complex assay data analysis revealed significant increase in complex i and iii activity in early and late onset tle compared to controls. the mitochondrial complex ii activity was higher in controls compared to early and late onset tle whereas complex iv had higher activity in controls compared to early onset. the atp/adp ratio was decreased indicating reduced bioenergetics. an elevated activity of malate dehydrogenase and succinate dehydrogenase was also observed. conclusion: the specific targeting of mitochondrial oxidative stress, dysfunction, and bioenergetics may have significant role in inducing epileptogenesis with respect to respiratory chain complexes and antioxidant treatment may prove to be a useful adjuvant in the epilepsy management.   abstract 32 free neuropathol 3:14:36 gliotic and destructive brain lesions associated with drug resistant epilepsy a clinicopathological study rajalakshmi poyuran1, ramshekhar n menon2, bejoy thomas2, george c. vilanilam2, ashalatha radhakrishnan2, ajith cherian2, mathew abraham2, kesavadas c2, sanjeev v thomas2, deepti narasimhaiah1 1 department of pathology, sree chitra tirunal institute for medical sciences and technology (sctimst), trivandrum, kerala, india 2 r madhavan nayar center for comprehensive epilepsy care, sree chitra tirunal institute for medical sciences and technology (sctimst), trivandrum, kerala, india background: destructive insults during brain development result in various radiologically-defined lesions which may lead to drug resistant epilepsy (dre). their histomorphology is rarely described. objectives: to describe the histopathological features of lesions resulting from destructive brain injuries/insults with dre. materials and methods: histopathological evaluation of surgical specimens of lesions attributable to destructive brain insults over 4 years’ period. results: study included 58 cases (32 males and 26 females) with 93.1% presenting with complex partial seizures and 67.2% having early life adverse events. age of onset ranged from day 12 to 18.6 years (mean=5.6 years) with mean age at surgery of 14.7 years (2-39 years). lesions frequently involved occipital lobe (51.7%) and often had multilobar involvement. histopathological abnormalities were categorised as: (1) gliosis and atrophy (n= 25) of cortex and/or white matter showing a combination of cortical abnormalities ranging from unlayered, 4-layered and nodular architecture to complete neuronal loss. (2) exclusive layer 4 neuronal loss and gliosis (n=6) without other cortical abnormalities. (3) fcd type iiid (n=9) in which there was cortical dyslamination (microcolumnar architecture in 8 and cortical layer 2 loss in 1) adjacent to gliotic and atrophic parenchyma. (4) non-specific changes (n=18). conclusions: histomorphological changes include a combination of cortical and white matter gliosis and multiple patterns of cortical dyslamination. exclusive layer 4 neuronal loss and gliosis may represent a specific subtype. fcd type iiid often takes the form of microcolumnar architecture in the adjacent uninvolved cortex.   abstract 33 free neuropathol 3:14:37 cytopathic changes of herpes zoster encephalitis in csf mimicking malignant cells praveen bk1, ashwani tandon1, e jayashankar1, nirendra k rai2, ujjawal khurana1, dinesh p asati3 1 department of pathology and lab medicine, all india institute of medical sciences, bhopal, india 2 department of neurology, all india institute of medical sciences, bhopal, india 3 department of dermatology and venereology, all india institute of medical sciences, bhopal, india background: herpes zoster is the reactivation of varicella zoster virus which remained latent in the dorsal root ganglia after the primary infection. only 0.1%–0.2% of patients with disseminated varicella-zoster have been reported to have manifest with encephalitis, that too in severely immune-compromised individuals. we are presenting a cytological challenging case of zoster meningitis mimicking malignancy. objectives: to substantiate large worrisome cells in csf due to cytopathic change masquerading as neoplastic cells. material and methods: case presentation of herpes zoster encephalitis results: a 72-year-old male presented with chest wall swelling and csf evaluation performed for his neurological symptoms viz. fever and episodes of focal clonic seizures of right upper limb and impaired awareness. mri brain with contrast was normal. csf was evaluated for cytology and biochemical parameters, displaying worrisome large pleomorphic singly scattered cells with few lymphocytes, plasma cells and neutrophils. further clinical workup was performed to substantiate large cells. on re-examining the patient, the chest lesions were multiple, painful vesicles with surrounding erythema. on review, csf cytology shows mature and transformed lymphocytes with viral cytopathic change. nucleomegaly, cytomegaly and bi/trinucleation were noted. the enlarge nuclei shows cowdry type a nuclear inclusion. csf proteins were 57mg/dl and glucose were 63mg/dl. csf pcr was positive for varicella zoster. conclusion: large worrisome cells in csf need proper substantiation as management of neoplastic vs infective etiology are significantly different. in our case we substantiate viral etiology by several tests and treated appropriately.   abstract 34 free neuropathol 3:14:38 resolving the diagnosis of hmsn with nerve histopathology and genetics shivani sharma1, yasha tc1, madhu nagappa2, govindaraj periyasamy3, sanjib sinha2, akhilesh shroti2, ramesh siram2, parayil s bindu2, arun b taly1 1 department of neuropathology, nimhans, bengaluru, india 2 department of neurology, nimhans, bengaluru, india 3 centre for dna fingerprinting and diagnostics, hyderabad, india background: atypical presentations of hereditary motor and sensory neuropathies (hmsn) pose a great diagnostic challenge and delay diagnosis by several years. objectives: to describe the diagnostic odyssey of three patients with hmsn. material and methods: case reports of hmsn with atypical presentations results: case 1: a 71-years-old gentleman developed progressive paraesthesias and weakness of distal limbs, and imbalance while walking, from 54 years of age. examination showed distal wasting, pes cavus, global hyporeflexia, and foot drop. nerve conduction studies (ncs) showed demyelinating neuropathy. nerve biopsy showed thinly myelinated fibers, regenerating clusters, ill-formed onion-bulbs and epineurial perivascular inflammation. he was treated on lines of chronic inflammatory demyelinating polyneuropathy (cidp) with multiple courses of intravenous immunoglobulin and steroids. his deficits progressively worsened. targeted sequencing revealed a novel heterozygous missense variation in mpz (c.223g>t, p.asp75tyr). case 2: a 54-years-old gentleman manifested with progressive hearing impairment, cramps, and asymmetric weakness and wasting of lower extremities from 34 years of age. he had pes cavus, bifacial weakness, bilateral sensory neural hearing impairment, global hyporeflexia and foot drop. nerve biopsy showed hypertrophic demyelinating neuropathy. the patient was treated with plasmapheresis, steroids, cyclophosphamide and azathioprine, despite which he continued to develop progressive deficits. targeted sequencing revealed a heterozygous six-base pair deletion in mpz (c.207_212delgcccga, p.pro70_glu71del) and a heterozygous missense variation in dnmt1 (c.1018g>a, p.a340t). case 3: a 38-years-old lady presented with paresthesias, weakness and wasting of both lower limbs and poorly controlled diabetes mellitus. onion bulbs of variable sizes were seen on the nerve biopsy. she was treated on lines of cidp. genetic testing revealed pmp22 duplication. conclusion: in view of heterogeneous onset, progression and severity, the diagnosis of hmsn may be delayed. nerve biopsy coupled with genetic testing can help in avoiding misdiagnosis and inadvertent treatment.   abstract 35 free neuropathol 3:14:39 understanding the er stress response to predict clinical outcome in focal cortical dysplasia patients madamanchi kishore1, madhamanchi pradeep1,2, sita jayalakshmi3, manas panigrahi3, anuja patil3, phanithi prakash babu1 1 department of biotechnology and bioinformatics, school of life sciences, university of hyderabad, hyderabad, telangana 500046, india 2 govt. degree college for men, srikakulam district, andhra pradesh 532001, india 3 department of neurology, krishna institute of medical sciences (kims), secunderabad, telangana, india background: focal cortical dysplasia (fcd) is a significant cause of drug-resistant epilepsy (dre). complete seizure-free outcomes are not observed even after surgery in some patients, but the reasons are still unclear. the epileptic patients were found to have severe endoplasmic (er) stress that led to cellular damage and even cell death. objectives: this study focused on understanding er stress in predicting the post-surgical seizure-free outcome in fcd patients. material and methods: magnetic resonance imaging (mri) and fluoro deoxy glucose positron emission tomography (fdg-pet) tests were used to find the lesion location. after surgery, the samples were snap-frozen with liquid nitrogen and stored at -80 0c. double immunofluorescence, hematoxylin & eosin staining, western blot, thioflavin-t, and h2o2 neutralization assays were performed. the post-surgical follow-up data was observed for more than three years to determine the clinical outcome. results: 10 (34.6%) patients were females in our patient cohort, and 16 (59%) were males. 14, 8, and 4 patients were fcd i, fcd iia, and fcd iib, respectively. the follow-up study was performed for more than three years to find the post-operative seizures reoccurrence. then we categorized the patients according to international league against epilepsy (ilae) guidelines. ten (38.4%), nine (34.6%) and seven (26.9%) patients were classified into class 1 (complete seizure-free), class 2 (less frequent seizures or auras) and class 3 (frequent seizures) categories. severe er stress was observed in class 2 and 3 patients, which further caused the accumulation of reactive oxygen species (ros) and protein aggregates. in addition to this, we observed upregulation of an apoptosis initiation marker chop/gadd54 in the brain samples of ilae classes 2 and 3 patient samples. conclusion: our data suggest that fcd patients belonging to ilae class 1 showed reduced protein aggregates and limited ros had better seizure-free outcomes than the rest. understanding the er stress response severity in resected clinical samples can help predict the possibility of post-surgical seizure-free outcomes and hint at the post-surgical anti-epileptic drug (aed) therapy.   abstract 36 free neuropathol 3:14:40 candida meningitis mimicking tuberculous meningitis in an immunocompetent patient diagnostic conundrum hema av1, aditi g1, nufina ta1, nandeesh bn1, rao s1, nashi s2, nagarathna c3, saini j4, santosh v1, mahadevan a1 1 departments of neuropathology, nimhans, bengaluru, india 2 departments of neurology, nimhans, bengaluru, india 3 departments of neuromicrobiology, nimhans, bengaluru, india 4 departments of neuroimaging & interventional radiolog3, nimhans, bengaluru, india background: fungal meningitis is often associated with immunocompromise due to primary/secondary immunodeficiency, hiv, prolonged antibiotic usage, indwelling catheters, intracranial shunts etc. candida is the fourth most common cause of nosocomial infections, and the most frequent cause of systemic opportunistic fungal infections, though cns involvement is rare, representing disseminated infection in severely immunocompromised patients. objectives: we present a rare case of candida meningitis occurring in a middle aged immunocompetent host diagnosed at autopsy. material and methods: case report of candida meningitis in an immunocompetent patient. results: a 43-year-old man without co-morbidities (diabetic/hypertension, hiv seronegative), presented with a history of altered sensorium and fever for 6 day. cranial mri revealed multiple ring enhancing lesions in putamen, caudate, medial basifrontal and cerebellum. repeated csf examination showed no bacterial/fungal growth. he was managed with anti-toxoplasma and anti-tubercular regimen, along with steroids. after seven days of admission patient succumbed. autopsy revealed thick yellowish white basal exudates in interpeduncular cisterns resembling tuberculous meningitis. several hemorrhagic necrotizing lesions were seen in left medial frontal, bilateral putamen, thalamus, right insula, bilateral hippocampus and cerebellum. histopathology revealed multiple necrotizing lesions, with dense vasculitis and clusters of pseudohyphal forms area with angio-invasion closely mimicking aspergillus spp on pas and gms stains. however, gram positivity of “hyphal” forms suggested candida and cultures from csf grew candida albicans. conclusion: candida albicans is a very rare cause of chronic meningitis, unsuspected in immunocompetent hosts. an antecedent bacterial meningitis, treated with broad-spectrum high dose antibiotics, anemia are high-risk factors. although rare, this has to be considered in the differential diagnosis of all atypical meningitis even in immunocompetent individuals and serological/pcr tests for fungal etiology should be included in panel of diagnostic tests.   abstract 37 free neuropathol 3:14:41 rare case of dorsally located multiple neurenteric-cyst without spinal dysraphism – neuropathology insights and systematic review krishna kumar singh1, rakesh mishra2, ashish gupta3, neeraj dhameja1, priyanka gupta1 1 department of pathology, institute of medical science, banaras hindu university, varanasi, india 2 department of neurosurgery, institute of medical science, banaras hindu university, varanasi, india 3 department of neurosurgery, all india institute of medical sciences, bhopal, india background: neurenteric-spinal-cysts (nc) are rare. most are isolated-ventral, and intradural-extramedullary. cervical location is commonest. reports of nc which are multiple, dorsally located, intramedullary and without spinal dysraphism are extremely rare. are they different embryologically/pathologically? objectives: we present a case (less than five are reported till now) of multiple dorsally located nc without spinal dysraphism, one of which was idem and the other was intramedullary. we also present a systematic review of all such cases with focus on neuropathology, ihc, and clinic-radiological-pathological correlation. material and methods: case: a 40 years old gentleman with back pain and lower limb weakness for three years underwent gross-total-resection of d10-d11 lesion and partial-resection of conus medullaris lesion with detethering. systematic review: as per prisma 2009 literature searched in pubmed, medline, web of science, scopus, cochrane, google scholar, scielo. neuropathology, surgery, demographics, and outcome extracted. quality of studies and descriptive statistics applied. results: gross: thick glistening white capsule with cheesy material. cytopathology: occasional cell clusters with cilia. histopathology: columnar ciliated and mucinous cells, pas+. ihc: ema+, ck7+, gfap-focal+. search yielded 1,788 citations. most were male. lumbar location was common for intramedullary and complete resection was not possible. most were ema+, ck7+, and focal-+-gfap. conclusions: multiple idem and intramedullary dorsally located nc without spinal dysraphism are extremely rare. most of these lesions have characteristic histopathology and ihc findings. it is important to differentiate it from arachnoid cyst for appropriate management. these lesions are distinct from the nc with spinal-dysraphism.   abstract 38 free neuropathol 3:14:42 efficacy of dimethyl fumarate in chronic constriction injury induced neuropathic pain in rats jagjit singh1*, manisha naithani2, shalinee rao3, shailendra handu1 1 department of pharmacology, all india institute of medical sciences (aiims), rishikesh, india 2 department of biochemistry, all india institute of medical sciences (aiims), rishikesh, india 3 department of pathology, all india institute of medical sciences (aiims), rishikesh, india background: dimethyl fumarate (dmf) has shown beneficial effects in multiple sclerosis. further, dmf has demonstrated promising results in experimental models of autoimmune neuropathy and chemotherapy induced peripheral neuropathy. objectives: to study the effect and mechanism of dmf in chronic constriction injury (cci) model of neuropathic pain in rats. material and methods:30 male wistar rats were divided into 5 groups of six rats each as follows: group i: sham + carboxymethyl cellulose (cmc) 0.5%, group ii: cci + cmc 0.5%, group iii: cci +gabapentin (75 mg/kg): group iv: cci + dmf (25 mg/kg). group v: cci + dmf (50 mg/kg). animals were anesthetized before surgery. cci was induced by silk ligature, applied 2 mm apart on sciatic nerve. dmf and gabapentin were administered daily from day 0 to day 28. pain threshold was assessed using thermal hyperalgesia, mechanical allodynia and cold allodynia responses on days 0, 7, 14 and 28. animals were sacrificed on day 28, using high dose of anaesthesia, and sciatic nerve was dissected for histopathological analysis (hematoxylin & eosin and luxul fast blue). plasma proinflammatory cytokine estimation was done for il-1β, il-6 and tnf-α. levels of p38 mapk and bdnf were quantified by elisa in the dorsal horn of the spinal cord. results: a significant increase in the pain threshold parameters was observed. histopathological analysis demonstrated that dmf significantly reduced the macrophage infiltration and myelin loss in the injured nerve. a significant decrease in proinflammatory cytokines, p38 mapk and bdnf were observed in the dmf treated animals. conclusion: the present study suggested that dmf reduces cci neuropathic pain by protecting the peripheral nerves and preventing the central sensitization.   abstract 39 free neuropathol 3:14:43 moya moya disease: an autopsy case study g. supriya1, megha s uppin1, sireeshayareeda2, afshan jabeen2 1 department of pathology, nizam’s institute of medical sciences, hyderabad, india 2 department of neurology, nizam’s institute of medical sciences, hyderabad, india background: moya moya disease is a rare chronic cerebrovascular disorder characterized by progressive narrowing of distal ica and proximal components of mca and aca leading to formation of collateral vessels which resembles a 'puff of smoke' on angiography. it is an uncommon cause of stroke in adults and children. objective: to illustrate the pathologic features of moya moya disease with the help of autopsy findings. material and methods: a 31 year diabetic patient presented with slurring of speech since 20 days associated with headache, moderate, non-throbbing type. this was followed by sudden onset weakness of right upper and lower limb. examination revealed right umn facial palsy with right hemiplegia. mri brain showed infarcts in left frontal white matter region with right supraclinoid ica and right aca narrowing. patient was diagnosed as stroke with possible cns vasculitis. he was treated with iv steroids, cyclophsophamide and antibiotics. his condition did not improve. he developed pneumonia and succumbed on day 18 of admission. results: a complete body autopsy was performed after informed consent. the brain showed extensive subarachnoid hemorrhage. the sections from bilateral ica and mca showed intimal prolifetaion of smooth muscle cells with reduplication of iel, irregular undulation of the internal elastic lamina and duplication. there was no evidence of vasculitis. bilateral lobar pneumonia was seen in lungs. discussion: sporadic mmd is common in east asian countries. intracerebral or ventricular hemorrhage is the catastrophic events leading to mortality. however, sah has been described rarely. the vascular morphology of this disease is also well characterized in this autopsy study which helps to rule out fibromuscular dysplasia, atherosclerosis and vasculitis. conclusion: mmd is an uncommon cause of stroke in adults. it needs to be differentiated from moya moya syndrome which can occur due to secondary causes. subarachnoid hemorrhage is an uncommon complication.   abstract 40 free neuropathol 3:14:44 hematolymphoid malignancies presenting with neurological manifestations and hand-mirror cells in peripheral blood: report of two cases sangeetha seshagiri k, mallithavana s, indira devi b*, nandeesh b n# departments of transfusion medicine and haematology, neurosurgery* and neuropathology#, nimhans, bengaluru, india background: hematolymphoid malignancies involve myeloid and lymphoid cell lineages and affect blood, bone marrow, lymph nodes and lymphatic system. clinically they may present with features suggestive of systemic involvement. cns involvement is not uncommon. these malignancies may present with handmirror cells in peripheral blood which can be the initial findings of such malignancies. objectives: this study aims to emphasize that the presence of hand-mirror cells should prompt appropriate investigations, workup, and management. material and methods: this study includes two patients, first, an adolescent male and second, a middle-aged female patient who separately presented with back pain and bilateral lower limb weakness. t results: the peripheral blood smears of both patients revealed atypical cells, hand-mirror cells and thrombocytopenia. imaging studies showed extradural spinal lesions in both cases. flow cytometric immunophenotyping of the first patient showed features suggestive of acute myeloid leukaemia. both underwent laminectomy. the histopathological study of the laminectomy specimen of the first patient showed features of myeloid sarcoma and that of the second patient showed diffuse b cell lymphoma. conclusions: hand-mirror cells can be seen in both malignant and non-malignant conditions. however, their presence may be the initial finding in peripheral blood that indicates underlying malignancy with or without symptomatology and leucocytosis. their presence should prompt the pathologist to recommend further evaluation for the diagnosis and management of hematolymphoid malignancies.   abstract 41 free neuropathol 3:14:45 mullerian choristoma as a cause of tethered cord syndrome: a case report in a 13-year-old worsening after the onset of menarche payal nitin pandya1, raju subodh1, swain meenakshi2, lath rahul1 1 department of neurosurgery, apollo hospitals, hyderabad, india 2 department of pathology, apollo hospitals, hyderabad, india background: mullerian choristoma as a cause for tethered cord syndrome is exceedingly rare with very few cases being reported in the literature. objectives: to document a rare cause of tethered cord syndrome material and methods: case report of a 13-year-old girl with spinal dysraphism who was operated in 2013 for lipomeningomyelocele and tethered cord. results: the patient now presented to us with back pain and leg pain for 6 months duration. mri of lumbosacral spine revealed an intra medullary cystic mass in the conus medullaris associated with haemorrhage in association with the residual lipoma. intraoperatively following excision of intramural lipoma, a hemorrhagic cystic intramedullary mass was encountered and excised. histopathology of the mass revealed a uterus like structure with endometrial lining and associated structure resembling fallopian tube. a diagnosis of mullerian choristoma was made. conclusion: mullerian choristoma is a rare cause of tethered cord syndrome.   abstract 42 free neuropathol 3:14:46 plurihormonal pit-1-positive adenoma: a short series deepti narasimhaiah1, kesavadas c2, prakash nair3, rajalakshmi poyuran1 1 departments of pathology, sree chitra tirunal institute for medical sciences and technology, trivandrum, india 2 departments imaging sciences and interventional radiology, sree chitra tirunal institute for medical sciences and technology, trivandrum, india 3 departments of neurosurgery, sree chitra tirunal institute for medical sciences and technology, trivandrum, india background: plurihormonal pit-1-positive adenoma is a newly described entity in the 2017 who classification of tumors of endocrine organs with an aggressive behaviour. objectives: a histopathological study of plurihormonal pit-1-positive adenomas diagnosed at our institute in last 5 years. materials and methods: this is a retrospective study. routine haematoxylin and eosin (h & e), immunohistochemistry for pituitary hormones, pituitary transcription factors (pit-1, tpit), mib-1 and p53 were performed in all cases. results: seven cases of plurihormonal pit-1-positive adenomas were diagnosed. the patient age ranged from 10-51 years (median: 35 years) with 4 males and 3 females. four adenomas presented with acromegaly and 3 with decreased vision. all adenomas, with the exception of one were primary. four adenomas were invasive and 3 were non invasive. on immunohistochemistry, the most extensively expressed hormones were gh and prl, followed by tsh-beta and alpha-subunit. acth was negative in all, except in one adenoma. pit-1 was diffusely expressed in all adenomas and tpit was negative. the mib-1 labelling index ranged from 1%-7% (median: 3%). all patients were treated with surgery and one patient received radiotherapy for residual disease. the follow-up period was 5-24 months (median: 13 months) and all patients were alive and free of disease at last follow-up. conclusions: 1. gh, prl, tsh-beta and alpha-subunit were co-expressed in all adenomas, with one adenoma also expressing acth. 2. pit-1 was diffusely expressed in all tumors. 3. there was almost equal distribution of invasive and non-invasive adenomas and median mib-1 labelling index was 3%.   abstract 43 free neuropathol 3:14:47 an interesting case of extradural tumour in a pediatric patient kavin devani1, batuk diyora1 1 department of neurosurgery, ltmgh, sion, mumbai, india background: ewing’s sarcoma is a highly malignant bone tumour, belonging to a family of small round blue cell tumors, derived from primordial bone marrowderived mesenchymal stem cell which typically affects the pelvis and the long bones of the lower extremities. it’s primary involvement of the skull is encountered in only 1% cases. objectives: to describe a rare extradural sarcoma in paediatric patient. material and methods: case report of a pediatric case with extradural tumour. results: we present a pediatric case with rapidly growing, painless swelling over the left temporal region, not compressible and fixed to the scalp and underlying bone. patient underwent gross total resection of the tumour with adjuvant chemotherapy and radiation. he had a favourable outcome without any neurological deficit. no local or systemic recurrence was found at 12 months postoperatively. conclusion: intracranial ewing sarcoma/ppnet is a rare tumor with nonspecific clinical presentation and radiological findings. they are locally invasive. gross total excision with adjuvant chemoradiation is the mainstay of treatment.   abstract 44 free neuropathol 3:14:48 case report of rare extra axial cerebellopontine angle medulloblastoma, a meningioma mimicker: caution advised abhishek chowdhury1, poonkodi m1, aniketh shenoy2, shilpa rao1, nishanth sadashiva2, yasha tc1 1 department of neuropathology, nimhans, bangalore, india 2 department of neurosurgery, nimhans, bangalore, india background: medulloblastoma, the most common malignant paediatric brain tumour, presents as a posterior fossa intra-axial mass. it infrequently affects adults forming less than 1% of intracranial tumours. very rarely, it is found in extra-axial locations. to the best of our knowledge, less than 50 cases of extra-axial medulloblastomas have been reported in literature, majority in cerebellopontine (cp) angle, affecting mainly adults and very few with dural attachment. objectives: to present a rare case of extra-axial tentorial medulloblastoma in an adult with radiological features of a meningioma expanding the differential diagnoses of lesions with dural attachment in the cp angle materials and methods: case report of a cp angle tumour results: a 40-year-old man presented with headache, imbalance and recent onset vomiting. mri revealed an extra-axial lesion in left cp angle arising from the inferior tentorium cerebellum, with dural tail, radiologically considered as meningioma. the patient underwent excision of lesion, and dural attachment was noted intraoperatively with no cerebellar involvement. histopathology revealed a classic medulloblastoma with stromal desmoplasia, and immunoprofile consistent with shh-activated, p53 wildtype molecular subtype: beta catenin (-), gab1(+), yap1 focal (+), p53 negative, otx2(-) conclusion: medulloblastoma rarely presents in the cp angle with tentorial attachment leading to its omission in the differential diagnoses. available literature cites that wntand shh-activated subtypes are seen in extra axial medulloblastoma. this report highlights the importance of its consideration in atypical posterior fossa extra-axial lesions. strong suspicion can lead to early detection, prognostication and appropriate management.   abstract 45 free neuropathol 3:14:49 potpourri of five cases of rare central nervous system tumors with review of literature thamilselvi r1, megala c1, prem parkash a2 1 department of pathology, vinayaga mission kirupananda variyar medical college, salem, india 2 department of neurosurgery, sims chellum hospital, salem, india background: the overall annual incidence rate of all brain tumours are 7 per 100 000 population. meningioma is the most common brain tumor, accounting for about 30 percent of them. here we are presenting 5 unusual & rare cases of cns tumours. objectives: to study the clinicomorphological & radiological features of unusual central nervous tumors (cns) & its association with any systemic diseases. materials and methods: case reports of 5 cases results: 1. a 5 years old female child presented with a swelling in the frontal region. 2. 35-year-old woman was presented with leg weakness, numbness, seizures and headache for 18 months. 3. 41 years old male who presented with a 9 months history of headache, double vision & leg weakness. 4. a 50 years old male presented with headache & visual disturbances of 6 months. 5. 11 years old child was presented with bilateral nasal obstruction & discharge for one year. h/o headache for 7 months. radiological imaging was done & patients were underwent tumor resection & submitted for histopathological examination. conclusion: herewith discussing one case of langerhans cell histiocytosis (lch), one case of chordoid meningioma, one case of chordoma, one case of primary lymphoma & one case of pituitary tumor. hence, we have to correlate with clinical, radiological, histopathological & immunohistochemical markers for final diagnosis and thereby providing therapeutic implication. follow up & further treatment is needed to prevent recurrence.   abstract 46 free neuropathol 3:14:50 primary yolk sac tumor of cerebellar vermis: a case report divya aggarwal1, poonam elhence1, suryanarayanan bhaskar2, deepak jha2, vikas janu2, sarbesh tiwari3 1 department of pathology and lab medicine, aiims jodhpur, india 2 department of neurosurgery, aiims jodhpur, india 3 department diagnostic and interventional radiology, aiims jodhpur, india background: extragonadal germ cell tumors are rare and usually occur along midline of body. primary yolk sac tumor (yst) of brain is rare, however occurs usually in pineal and suprasellar regions. we present a rare case of primary cerebellar vermis yst. objectives: to report a rare case of cerebellar yolk sac tumour. material and methods: case report of biopsy proven yolk sac tumour of the cerebellum. results: a 3-year old male presented with ataxia, vomiting and decreased feeding which the parents noted around 15-20 days back. contrast-enhanced mri revealed a left cerebellar lesion involving vermis, with perilesional edema which was isointense on t1 and t2 hyperintense. intense contrast enhancement was noted. radiological possibilities suggested were medulloblastoma and atypical teratoid/rhabdoid tumor. intra-operative frozen section was sent and the smears showed markedly pleomorphic cells in small glands, papillae and dispersed singly. a diagnosis of germ cell tumor was rendered. definitive sections showed a tumor with cells arranged in a myriad of histological patterns, including reticular microcystic pattern, glands and solid areas. other classical features seen were presence of pas positive intracytoplasmic hyaline globules and numerous schiller duval bodies. a diagnosis of yolk sac tumor was rendered and the child was started on chemotherapy 2 weeks back. no mediastinal or testicular lesions were identified. conclusion: primary yst of cerebellar vermis is a rare entity. a knowledge of such entities is helpful in timely diagnosis and appropriate patient management.   abstract 47 free neuropathol 3:14:51 a diagnostic dilemma astroblastoma kuldeep singh khangarot1, surabhi tyagi1 1 department of pathology, mahatma gandhi medical college & hospital, jaipur, india background: astroblastoma is an extremely rare cns tumor, accounting for 0.45 to 2.8% of all neuroglial tumors. it was first described by bailey and cushing in 1926 and further characterized by bailey and bucy in 1930.according to published data there is slight female preponderance and bimodal age distribution with one peak between 5-10 years and other between 21-30 years. objectives: to study a rare case of astroblastoma. material and methods: after diagnosing on h&e correlation with the radiological investigation was done. subsequently immunomarker study and molecular workup by fish was done. results: on h&e differential diagnosis of i) well differentiated astroblastoma ii) papillary ependymoma iii) atypical choroid plexus papilloma /ca was given and further workup was advised. ihc marker and molecular information by fish assay confirms the diagnosis of well differentiated astroblastoma. conclusion: astroblastoma is a very rare primary brain tumor. its diagnosis is often challenging because of the astroblastic aspects that can be found in astrocytic tumors in ependymoma and in non-neuroepithelial tumors. the low incidence rate makes it difficult to conduct studies to examine tumor characteristics. the exact histogenesis of astroblastoma is controversial. patient with astroblastoma should be treated with curative intent.   abstract 48 free neuropathol 3:14:52 meningioangiomatosis: a rare cause of refractory temporal lobe epilepsy manasa gajula1, megha uppin1, sujata patnaik2, rajesh alugolu3, mudumba vijaya saradhi3 1 department of pathology, nizam’s institute of medical sciences, hyderabad, india 2 department of radiology, nizam’s institute of medical sciences, hyderabad, india 3 department of neurosurgery, nizam’s institute of medical sciences, hyderabad, india background: meningioangiomatosis is a rare meningovascular malformation or hamartomatous lesion responsible for refractory seizures. radiographic appearance can be highly variable and histopathology is necessary for confirmed diagnosis. objectives: to describe a case of meningioangiomatosis materials & methods: a 27-year male, with no co morbidities presented with seizures since 15 years. he was treated with antiepileptic agents however there was no change in seizure pattern. the frequency of seizures increased from 4-5 episodes/month to 8 -10 episodes/month. the eeg showed abnormal record with diffuse slowing of left temporal epileptiform focus. mri brain – focal fairly smooth expansion of left squamous temporal bone with slight sclerosis of inner cortical region. he underwent left temporal lobectomy and hippocampectomy results: the temporal neocortex showed a well circumscribed mass of 3x2cm with white fibrous appearance on cut section. the histopathology of the mass showed a lesion comprised of multiple vascular channels surrounded by bland spindle cells. these cells have oval to elongated nuclei with intranuclear inclusions. multiple psammoma bodies were identified. there was no atypia. the cells showed immunoexpression for ema and vimentin. hippocampus showed loss of neurons in ca1, ca3 and ca4 regions with dispersion and bilayering of granular neurons of dentate gyrus. conclusion: meningioangiomatosis is a very rare hamartomatous lesion associated with epilepsy. the preoperative consideration is important as prognosis with surgical resection is good.   abstract 49 free neuropathol 3:14:53 isolated cerebral rosai dorfman disease with granulomatous angiitis shinde sweety1, shenoy asha2 1 department of pathology, b.y.l nair hospital, mumbai, india 1 department of pathology, k.e.m hospital, mumbai, india background: rosai dorfman is a non-dendritic, non-langerhans histiocytic disorder. isolated intracranial involvement without lymphadenopathy is uncommon. only one report in brain and lung respectively documented a coexistent granulomatous angiitis. objectives: case report of an uncommon association between two rare cerebral entities. material and methods: a 30-year-old male presented with seizures, limb weakness and violent behaviour since 3 months. there was no fever, lymphadenopathy or hepatosplenomegaly. complete hemogram, esr, hepatic and renal function tests were normal. serology was negative for retrovirus and autoantibodies. on radioimaging, parietotemporal lobe showed a large mass 8.6 x 7.4 x 3.2 cm. it was hypointense on t1w1, hypointense on t2w1/ flair and heterogenously contrast enhancing. mr spectroscopy showed reversed naa/cr and increased cho/cr ratio suggestive of primary cns lymphoma, glioblastoma and tumefactive demyelination. subtotal resection was done. results: histopathology revealed sheets of mature lymphocytes, plasma cells, foamy histiocytes showing emperipolesis, touton giant cells and florid granulomatous angiitis with resultant coagulative necrosis. there was absence of eosinophils, langerhans cells or atypical lymphoid cells. the differentials included lymphomatoid granulomatosis, tuberculosis, tumefactive demyelination and wegener’s granulomatosis. tumor cells were positive for cd68 and s100, while negative for cd1a, eber and igg4. fungal and mycobacterial stains were negative. thus, rosai dorfman disease with coexistent granulomatous angiitis was diagnosed. on follow up for six months, there was no recurrence or new lesions. conclusion: only two cases in literature (cerebral and pulmonary) show granulomatous angiitis associated with emperipoletic histiocytic tumefaction. an overlap between igg4 related disease and rosai dorfman is also postulated.   abstract 50 free neuropathol 3:14:54 twin tales of anaplastic ependymoma with extensive vacuolation/signet cell change and lipomatous differentiation rashim sharma1, balamurugan thirunavukkarasu1, sudeep khera1, poonam abhay elhence1, deepak jha2, taruna yadav3 1 department of pathology and lab medicine, aiims jodhpur, india 2 department of neurosurgery, aiims jodhpur, india 3 department diagnostic and interventional radiology, aiims jodhpur, india background: ependymomas are circumscribed glial tumours with unique morphology. the diagnosis is relatively straightforward in the majority of the cases. however, there are scenarios where the morphology is obscured leading to diagnostic difficulty. we present two such cases of supratentorial anaplastic ependymoma in pediatric age group with one showing large areas of vacuolated/ signet-ring change and another showing lipomatous differentiation. objectives: to describe uncommon histological features in ependymoma material and methods: case reports of two cases with histopathological description. results: case 1 is a 9-year-old male child with a well-defined intra-axial, lobulated solid cystic lesion (4.5x3.6cm) in the right parieto-temporal region. biopsy showed monomorphic tumour cells interspersed with large areas of vacuolated cytoplasm/signet ring change in large areas posing a diagnostic challenge. these cells showed strong and diffuse perinuclear cytoplasmic dot-like positivity for ema along with diffusely positive gfap. however, the tumour recurred within 11 months showing anaplastic morphology lacking the previous features. case 2 is a 3-year-old male child with similar presentation except in this case there was lipomatous change mimicking adipocytes in large areas posing diagnostic difficulty. conclusion: lipomatous change, vacuolation and signet ring change has been reported in ependymoma in few reports. the origin of vacuolar change is still debated. some reports state it as dilation of intracytoplasmic membrane and some as metaplasia. these changes have been observed in other tumours like neurocytomas, medulloblastomas, cerebellar and spinal cord astrocytomas. there can be confusion between clear cell ependymoma and areas showing oligdendroglial proliferation as both show cytoplasmic clearing. in tumours with extensive change, search for classical areas and immunohistochemistry can facilitate the diagnosis.   copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 66th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 3:20 (2022) meeting abstracts 66th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts berlin, november 1–5, 2022 submitted: 09 august 2022 accepted: 10 august 2022 published: 10 august 2022   liebe kolleginnen und kollegen, zur 66. jahrestagung der deutschen gesellschaft für neuropathologie und neuroanatomie im rahmen der neurowoche vom 1. bis zum 5. november 2022 begrüße ich sie herzlich in berlin. die letzten jahre haben eine enorme erweiterung der analytischen methodik mit schwerpunkt auf molekularen untersuchungen gebracht. ein großer teil dieser untersuchungen wurde in unseren einrichtungen entwickelt und wird dort erbracht. in der tat hat sich die neuropathologie zu einem motor der neuroonkologischen und neurowissenschaftlichen forschung entwickelt und deutschsprachige neuropathologische institutionen haben wesentlich dazu beigetragen. ganz neue therapien bauen auf diese erkenntnisse auf. dadurch sind wir für die versorgung unserer patienten wichtiger denn jeher. deswegen sehe ich einen großen und zunehmenden bedarf dem wir neuropathologen nachkommen müssen. alle schwerpunkte unseres faches sind hiervon betroffen, die gehirntumordiagnostik, die neurodegenerativen erkrankungen, entzündung und erkrankungen der muskeln und der nerven. wir arbeiten eng mit unseren kollegen aus der neuroonkologie, neuropädiatrie, neurologie neurochirurgie und neuroradiologie zusammen. der interdisziplinäre austausch hat einen hohen stellenwert und wir freuen uns deshalb besonders, dass unsere jahrestagung in diesem jahr wieder im rahmen der neurowoche stattfindet, was die kommunikation und den wissenstransfer über die fächergrenzen beflügelt. dieses jahr wollen wir besonders die jungen neuropathologen und neuropathologinnen in den vordergrund stellen. sie sollen unser fach als lebendig und besonders zukunftsfähig erleben. von ihnen erwarten wir die dynamik, den einsatz und den ideenreichtum, der die neuropathologie in den nächsten jahren noch weiter zu einer zentralen querschnittsplattform für die neurofächer machen wird. wir haben einen kongressstrang mit wissenschaftlichen sitzungen am donnerstag, freitag und samstag ausgerichtet. sie dürfen vorträge mit jungen neuropathologischen expertinnen und experten sowie von jungen nachwuchswissenschaftlerinnen und nachwuchswissenschaftlern erwarten. ich freue mich auf lebhafte diskussionen und spannende interdisziplinäre debatten! ihr prof. dr. andreas von deimling universitätsklinikum heidelberg neuropathologie   https://doi.org/10.17879/freeneuropathology-2022-4366 keywords: german society of neuropathology and neuroanatomy, dgnn, meeting abstracts contents 1. new diagnostic methods 1.01 methylthioadenosine phosphorylase immunostaining as a surrogate marker for cdkn2a/b homozygous deletion in gliomas 1.02 artificial intelligence in morphomolecular analysis of glioblastoma 1.03 histological and molecular correlates of tspo labelling in human brain tissue 1.04 towards swift and accessible precision cns tumour diagnostics using third generation sequencing and deep transfer learning 1.05 retinal pathology as potential biomarker of symptom severity and impairment in patients with stiff person syndrome 1.06 deep learning based cerebrospinal fluid diagnostics 1.07 proteomic profiling of idh-mutant gliomas identifies hip1r/vimentin as surrogate markers for 1p/19q codeletion and enables prediction of chromosomal copy number variations 1.08 cellular digital neuropathology 2. neurooncology 2.01 using spatial transciptomics for diagnostic analysis of glioma 2.02 single cell dna amplicon sequencing reveals order of mutational acquisition in traf7 and klf4 or akt1 co-mutated meningiomas 2.03 alterations in ptpn11 and other noonan syndrome associated map-kinase signaling pathway genes accumulate in histopathologically atypical ganglioglioma with adverse postsurgical outcome 2.04 molecular refinement of pilocytic astrocytoma in adult patients 2.05 exploration of cellular origins and therapeutic targets by modeling high grade pediatric glioma of the mycn subclass in mice 2.06 the genomic and transcriptional landscape of primary central nervous system lymphoma 2.07 pmolecular mechanisms of therapy resistance in malignant melanoma brain metastasis 2.08 a peripheral nerve sheath tumor syndrome caused by postzygotic erbb2 mutations 2.09 cns-tumor patients within the impress-norway trial: first year experiences 3. neurodegeneration 3.01 cnn-supported quantification of fat compartments at abdominal mri applied to als patients 3.02 neurodegenerative iron storage disease (neuroferritinopathy) caused by a novel frameshift mutation in the ferritin heavy chain gene (fth1 c.341-342del) 3.03 the contribution of late-nc to neuron loss, granulovacuolar degeneration and dementia in alzheimer’s disease 3.04 the role of c3 inhibition in an ipsc nmj model of neuroinflammation 3.05 fast-track procedure for the neuropathological assessment of neurodegenerative diseases 3.06 neurodegeneration in hsan1 due to atl1 (gly66gln) mutation is associated with defective erprotein quality control and compromised autophagy 3.07 single-nucleus chromatin accessibility profiling in four-repeat tauopathies 3.08 application of a human stem cell transplantation model of alzheimer’s disease to examine disease-associated changes at a single cell level in vivo 4. neuroinflammation 4.01 pathological and genetic characterization of jc virus encephalopathy with an eleven-year-long disease course 4.02 reduction of oligodendrocyte populations in patients with late-onset multiple sclerosis 4.03 schwann cell remyelination is a salient feature of spinal nmo with neuroprotective potential 5. muscle / nerve 5.01 molecular profiling of skeletal muscle in infantile, juvenile and adult patients with pompe disease 5.02 expression of immune regulating proteins in skeletal muscle of different idiopathic inflammatory myopathies (iim) subtypes 5.03 long term safety and efficacy outcomes for x-linked myotubular myopathy (xlmtm) with gene replacement therapy, resamirigene bilparvovec (aspiro): preliminary results from cohort 1 in aspiro, a phase 1/2/3 study 5.04 lymphotoxin-driven chronic mucle inflammation interdepends with impaired autophagy, self-perpetuates and models inclusion body myositis in mice 5.05 novel form of congenital myopathy caused by bi-allelic mutations in uncoordinated mutant number-45 myosin chaperone b 6. free topics 6.01 deep genotype-phenotype analysis of focal cortical dysplasia type 2 differentiates between a gator-positive autophagy altered subtype 2a and mtor-positive migration deficit subtype 2b 6.02 age-dependent increase of perineuronal nets in the human hippocampus of patients with and without temporal lobe epilepsy 6.03 vakuolisierung der dura als nicht-lymphassoziierte veränderung 6.04 moghe with or without slc35a2 brain somatic mutations reveal a common phenotype of oligodendroglial regeneration and remyelination     1. new diagnostic methods 1.01 free neuropathol 3:20:5 methylthioadenosine phosphorylase immunostaining as a surrogate marker for cdkn2a/b homozygous deletion in gliomas theoni maragkou1, ekkehard hewer1,2, erik vassella1, baptiste pasquier1, stefan reinhard1, maja neuenschwander1, philippe schucht3 1 university of bern, institute of pathology, institute of pathology, bern, switzerland 2 lausanne university hospital, institute of pathology, institute of pathology, lausanne, switzerland 3 inselspital, bern university hospital, dept. of neurosurgery, dept. of neurosurgery, bern, switzerland background: homozygous deletion (hd) of the cdkn2a/b locus has emerged as an unfavorable prognostic marker in diffuse gliomas, both idh-mutant and idh-wildtype. testing for cdkn2a/b deletions can be performed by a variety of approaches, including copy number variation (cnv) analysis based on genome-wide dna methylation data, next generation sequencing (ngs) or fluorescence in-situ hybridization (fish), but questions remain regarding the accuracy of and correlation between different testing modalities. aims: in this study, we assessed the utility of s-methyl-5'-thioadenosine phosphorylase (mtap) and cellular tumor suppressor protein pl61nk4a (p16) immunostaining as surrogate markers for cdkn2a/b hd in gliomas, across different histological tumor grades and idh mutation status. question: are mtap and p16 accurate surrogate markers for cdkn2a/b hd in gliomas? methods: idh1 r132h, atrx and mtap immunohistochemistry was performed on tissue microarrays (tmas) of 301 diffuse gliomas. survival analysis was performed to assess the prognostic value of mtap. furthermore, 100 consecutive cases of gliomas were collected, in order to correlate mtap and p16 expression with the cdkn2a/b status in cnv plot of each tumor. results: mtap deficiency was associated with shortened survival in idh-mutant astrocytomas (n=75; median survival 61 vs. 137 months; p<0.0001), idh-mutant oligodendrogliomas (n=59; median survival 41 vs. 147 months; p<0.0001) and idh-wildtype gliomas (n=117; median survival 13 vs. 16 months; p=0.011). in a cohort of 100 gliomas, complete loss of mtap and p16 by immunohistochemistry was 100 % and 90 % sensitive as well as 97 % and 89 % specific for cdkn2a/b hd, respectively, as identified on cnv plot derived from genome-wide dna methylation analysis. two cases with mtap and p16 loss of expression did not demonstrate cdkn2a/b hd in cnv plot, however fish analysis confirmed the hd for cdkn2a/b. conclusions: mtap immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with cdkn2a/b status, robustness, rapid turnaround time and low-costs, while p16 immunostaining represents a good alternative for detecting cdkn2a/b hd. discovering cdkn2a/b hd through mtap and/or p16 immunohistochemistry seems to be a more accurate method than the cnv analysis derived from genome-wide dna methylation data.   1.02 free neuropathol 3:20:6 artificial intelligence in morphomolecular analysis of glioblastoma stephan balogh1, karen brengmann1, jannik sehring1, thomas kauer1, gudrun schmidt1, till acker1, daniel amsel1, hildegard dohmen1 1 institute of neuropathology, justus liebig university giessen, giessen, deutschland background: glioblastoma is the most common primary malignant brain tumor and has a poor prognosis despite existing treatment options. it is characterized by its inhomogeneous appearance and molecular heterogeneity. a detailed diagnosis is desirable, especially with regard to the emerging personalized medicine to guide treatment decisions. the increasing application of whole slide image scanners enables the digitalization of histopathological slides, collected in clinical routine diagnostics, into high-quality images that offer new possibilities for computer-aided precision diagnostics. objective: the goal of our work-in-progress project is to curate a high-resolution dataset with annotations to train an artificial intelligence to independently recognize characteristic structures of glioblastoma tissue sections, such as tumor area, vascular proliferation and necrosis. using these ai-based algorithms, we aim to gain new insights that could help refine the characterization of glioblastomas by correlating morphological information with available clinical and molecular data. methods: initially, clinical and molecular information on 200 glioblastoma patients was gathered. the corresponding hematoxylin-eosin-stained histopathological slides were then digitized using a high-throughput whole slide image scanner (hamamatsu nanozoomer s360). the annotation of key features in the images was divided into two distinct phases. prior to the actual annotation phase, two md students completed a training phase with a small batch of images (n=10) in order to get hands-on experience with the annotation software and difficult issues (qupath version 0.3.0). the ground truth was determined by an experienced neuropathologist. evaluation: the interand intraobserver variability of the two students will be evaluated with regard to the learning progress (e.g. accuracy, classification of a tissue area) and differences between the annotations of the two students, derived from subjective assessment. in the second part, correlations between morphological information (vascular proliferation and necrosis with pseudopalisading) and clinical and molecular information will be examined. these annotated slides will serve as training and test sets for in-house ai-based predictions. perspective: our data will be used as a resource for an in-house developed app that will serve as a learning solution for medical students, but also as a crowdsourcing platform for the annotation of features on small patches of whole slide images. in addition, the curated high-resolution data set will serve as input for further internally developed ai algorithms to support tumor diagnosis and therapy decisions.   1.03 free neuropathol 3:20:7 histological and molecular correlates of tspo labelling in human brain tissue lorraine weidner1,2, franziska dekorsy3, stefanie quach4, viktoria ruf5, julia lorenz1,2, peter hau2,6, jörg-christian tonn4,5, peter bartenstein3,7, matthias brendel3,7, nathalie l. albert3,7, markus j. riemenschneider1,2 1 regensburg university hospital, department of neuropathology, regensburg, deutschland 2 regensburg university hospital, wilhelm sander neuro-oncology unit, regensburg, deutschland 3 university hospital of munich, lmu munich, department of nuclear medicine, münchen, deutschland 4 university hospital of munich, lmu munich, department of neurosurgery, münchen, deutschland 5 lmu munich, center for neuropathology and prion research, münchen, deutschland 6 regensburg university hospital, department of neurology, regensburg, deutschland 7 german cancer research center (dkfz), german cancer consortium (dktk), partner site munich, heidelberg, deutschland background: tspo is frequently upregulated in neoplastically transformed tissues, including glioblastomas. this may be of use for pet imaging of brain tumors. however, due to the heterogeneity of cell populations that could contribute as tspo-pet signal source in gliomas, the imaging biomarker interpretation may be challenging. aim: we therefore dissect tspo labelling in connection with the underlying histopathological and molecular features in biopsy samples from glioma patients. question: to decipher the underlying histopathological and molecular features of tspo-pet enrichment. methods: we aim to collect a total of 75 glioma patients all characterized by mri, tspoand fet-pet. tspo protein expression and expression of cell differentiation markers are assessed immunohistochemically on consecutive sections and by multiplex stains. rna isolation has been optimized to perform rna-seq on biopsy samples and to compare regions of high and low tspo-pet signal/protein expression. to identify relevant hallmarks and go terms we use deseq2 followed by fuma and reactome as well as gsea with normalized counts. furthermore, exceeding the biopsy study we stain tissue microarrays for tspo that cover a broader spectrum of human brain pathologies as well as a spectrum of non-neoplastic tissues from different brain regions. to better understand tspo regulation, we consult data of large patient cohorts from the tcga database, perform in vitro epigenetic investigations on azaor tsa-treated patient-derived glioblastoma cell lines and analyze the tspo promoter in gliomas by direct bisulfite sequencing. results: we report the interim analysis of the glioma patients that have been included and fully histologically characterized in the biopsy study so far. our results suggest that (apart from microglia and macrophages) the glial tumor cells relevantly contribute to the overall tspo signal in these patients. rna-seq analyses comparing tspo high and low regions (both by pet and protein expression) indicate three tspo-dependent functional clusters, i.e apoptosis/dna repair, extracellular matrix organization and immune system. furthermore the tissue microrarrays show heterogeneity of tspo expression between different brain pathologies and non-neoplastic brain regions. bringing this information together with tspo-pets from respective patients/brain regions will generate a map of tspo expression in healthy and diseased brain for clinical use. finally, our epigenetic investigations suggest that a loss of tspo methylation in high-grade neoplasms may mechanistically contribute to the tspo overexpression observed in these tumors. conclusion: taken together, our approach of integrating histological, molecular and imaging data will provide unique insights into tspo-pet enrichment patterns and will help to better understand and to comprehensively describe the clinical relevance of this novel imaging biomarker.   1.04 free neuropathol 3:20:9 towards swift and accessible precision cns tumour diagnostics using third generation sequencing and deep transfer learning areeba patel1,2, helin dogan1,2, alexander wolfgang jung3, zaira seferbekova4, alexander payne5, natalie schoebe1,2, elena krause1,2, michael ritter1,2, daniel schrimpf1,2, damian stichel1,2, stefan hammelmann1,2, christina blume1,2, philipp euskirchen6, violaine goidts7, martin sill8,9, stefan pfister8,9, matthew loose5, wolfgang wick10,11, andreas von deimling1,2, david jones8,12, matthias schlesner13, moritz gerstung3,4, felix sahm1,2 1 german cancer research center, clinical cooperation unit neuropathology, heidelberg, deutschland 2 university hospital heidelberg, neuropathology, heidelberg, deutschland 3 european molecular biology laboratory, european bioinformatics institute embl-ebi, hinxton, united kingdom 4 german cancer research center, division of artificial intelligence in oncology, heidelberg, united kingdom 5 university of nottingham, deepseq, school of life sciences, nottingham, united kingdom 6 charité-universitätsmedizin berlin, neurology, berlin, deutschland 7 german cancer research center, brain tumor translational targets, heidelberg, deutschland 8 hopp children's cancer center (kitz), heidelberg, deutschland 9 german cancer research center, division of pediatric neurooncology, heidelberg, deutschland 10 german cancer research center, clinical cooperation unit neurooncology, heidelberg, deutschland 11 national center for tumor diseases, department of neurology and neurooncology program, heidelberg, deutschland 12 german cancer research center, pediatric glioma research group, heidelberg, deutschland 13 augsburg university, biomedical informatics, data mining and data analytics, augsburg, deutschland background: molecular markers are now unequivocally a requirement for integrative brain tumour diagnostics. the 2021 who classification of central nervous system (cns) tumours substantially increases the set of genes required in routine evaluation, and significantly increases the relevance of dna methylation analysis in the diagnostic process. owing to extensive setup costs and batch requirements, smaller labs and clinics might not be able to deliver molecular results for prompt clinical decisions. deep neural network architectures have been shown to predict whole genome duplications, driver gene mutations, transcriptomic associations, immune cell localisation and prognostic effects from h&e slides. third generation sequencing has enabled sequencing longer reads, shorter library preparation protocols, ability to call base modifications natively, real time analysis, and low-cost, portable devices. aims: to make precision diagnostics accessible, we introduce an integrated computational histopathology and third generation sequencing workflow for real-time cns tumour molecular diagnostics. methods: we present cns-chipa multi-task lightweight deep transfer learning model to predict key molecular alterations, methylation classification and survival from h&e stained cns tumour slides. the model provided basic information regarding the tumor type instantly. for further detail (e.g. variant of idh alteration) and subtyping, we subsequently used the predictions to formulate a custom panel for each patient. targeted sequencing and analyses were performed using rapid-cns2a custom neurooncology third generation sequencing pipeline for parallel copy-number profiling, mutational and methylation analysis that is highly flexible in target selection, requires no additional library preparation for targeting, runs efficiently on single samples, and can be initiated upon receipt of frozen sections. rapid-cns2 leverages adaptive sampling through readfish and was run using a portable minion or gridion device. results: we show that cns-chip can predict a multitude of key pathognomonic alterations (eg. idh mutation, 7 gain/10 loss, etc.) using a single model with reasonable accuracy. using a personalised panel for targeted sequencing of each sample enabled smaller target sizes, thus reducing sequencing time to an average of 24 hours. cns-chip predictions were compared to their respective rapid-cns2 results and corresponding conventional data (ngs panel sequencing and epic array analysis). we demonstrate our workflow on prospective diagnostic samples received by the department of neuropathology, university hospital heidelberg. the average turnaround time per sample was 48h. conclusions: our workflow harnessing histology-based molecular predictions to instruct targeted sequencing can be set up with low initial investment, reduces hands-on time and has the potential to facilitate reporting of integrated molecular diagnostic results in less than 48h. cns-chip combined with rapid-cns2 thus aims to make cns molecular diagnostics affordable and accessible to smaller hospitals and labs especially in lowand middle-income countries.   1.05 free neuropathol 3:20:11 retinal pathology as potential biomarker of symptom severity and impairment in patients with stiff person syndrome sabine seefried1, claudia sommer1 1 ukw, würzburg, deutschland background: stiff-person syndrome (sps) is a rare chronic autoimmune disease characterized by painful spasms and rigidity, predominantly of the axial and lower extremity muscles. autoantibodies have been reported in 80% of cases, disrupting the function of the inhibitory neurotransmitters gaba and glycine, and leading to overexcitability of the neuromuscular system and psychological comorbidities. since the retina is highly enriched with gabaergic neurons, retinal pathology may occur in sps, especially in the ganglion cell and inner plexiform layer (gcipl) of the retina, which contains most of the retinal gabaergic neurons. study aims: we aimed to detect potential abnormalities in the retina in sps patients and correlate these with symptom severity and impairment. hypotheses: we hypothesized a lower retinal thickness in sps patients in comparison to normal healthy controls and patients with diabetes and furthermore correlations between gad antibody levels and findings in retina layer thickness and correlations with severity of the disease. methods: 24 gad positive sps patients (17 female; 7 male; aged 53 ±7; 9 with diabetes) received a clinical examination and optical coherence tomography (oct) for retina layer thickness. blood was drawn for autoantibody detection. data were compared to a matched healthy cohort and a matched patients group with diabetes but no diabetic polyneuropathy. the severity of sps symptoms and impairment was assessed on the basis of the ability to walk: free walking, use of a crutch, use of two crutches, walking on the rollator or sitting in a wheelchair. results: oct showed lower retinal thickness in gcipl and lower average macular thickness (amt) in the sps patients (gcipl: 73.34±5,3; amt: 302.98 ±12,1) in comparison to healthy controls (gcipl: 76.01 ± 4,2; amt: 311.76±13,4). comparison of the 15 sps patients without diabetes with the total number of 25 sps patients showed no difference between these results. the control patients with only diabetes had no abnormalities in the oct results compared to the healthy controls, indicating that the atrophy of gcipl and aml is likely to be caused exclusively by the sps and not by diabetes. there was a positive correlation between walking ability and gcipl thickness in sps patients; higher gcipl thickness was associated with better walking ability (r² = 0,92). conclusion: this study indicates retinal involvement in sps. oct might be useful as a complementary diagnostic tool in sps, and retinal layer thickness measurements might be developed as a non-invasive biomarker for disease progression.   1.06 free neuropathol 3:20:12 deep learning based cerebrospinal fluid diagnostics leonille schweizer1,2, philipp seegerer3, hee-yeong kim4, rené saitenmacher3, amos münch1, liane barnick1, anja osterloh1, carsten dittmayer1, ruben jödicke1, debora pehl1, annekathrin reinhardt5, klemens ruprecht6, annika k wefers7, patrick harter8, ulrich schüller7, frank l heppner1, maximilian alber3, klaus-robert müller3, frederick klauschen9 1 institute of neuropathology, charité – universitätsmedizin berlin, corporate member of freie universität berlin, humboldt-universität zu berlin and berlin institute of health, berlin, berlin, deutschland 2 german cancer consortium (dktk), partner site berlin, and german cancer research center (dkfz), berlin, deutschland 3 machine-learning group, department of software engineering and theoretical computer science, technical university of berlin, berlin, deutschland 4 systems medicine of infectious disease, robert koch institute, berlin, deutschland 5 department of neuropathology, university hospital heidelberg, heidelberg, deutschland 6 department of neurology, charité university medicine berlin, berlin, deutschland 7 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, deutschland 8 neurological institute (edinger institute), goethe university, frankfurt, deutschland 9 institute of pathology, ludwig-maximilians-universität münchen, münchen, deutschland background: the analysis of cerebrospinal fluid (csf) specimens is essential for the diagnostic workup and clinical management of neurological patients and relies on differential cell typing. because blood cell cytometers are unable to identify diagnostically relevant cell types in csf samples, the current gold-standard is based on microscopic examination by specialized technicians and neuropathologists. manual differential cell count is time-consuming, labor-intensive and subjective. we therefore set out to compile a real-world csf dataset including all diagnostically relevant cell types to train a robust algorithm for cell type differentiation with the potential to solve complex diagnostic tasks. methods: we therefore developed an image analysis approach based on expert annotations of 127.455 digitized csf objects from 78 patients corresponding to 15 clinically relevant categories and trained a multiclass convolutional neural network (cnn). we applied explainable artificial intelligence (xai) methods to elucidate the most relevant image pixels for cnn predictions and compare pattern recognition to humans. we further developed a new data partitioning strategy for further machine learning projects. to assess the realistic usefulness in diagnostic practice, we validated the cnn-based approach by comparing its performance to that of seven board certified neuropathologist from different academic institutions. results: the cnn classified the 15 categories with high accuracy (mean auc 97.3%). by using xai, we could demonstrate that the cnn identified meaningful substructures in csf cells recapitulating human pattern recognition. we validated the diagnostic performance of the cnn by comparing the predictions of 511 cells selected from 12 different csf samples to seven board-certified neuropathologists blinded for clinical information. inter-rater agreement between the cnn and the ground truth was non-inferior (krippendorff’s alpha 0.79) compared to the agreement of seven human raters and the ground truth (mean krippendorff’s alpha 0.72, range 0.56-0.81). the cnn assigned the correct diagnostic label (inflammatory, hemorrhagic or neoplastic) in 10 out of 11 clinical samples compared to 7-11 out of 11 correctly labeled csfs by human raters. similar to four human raters, the cnn misclassified single highly activated b-cells as cancer cells in two samples, but indicated reduced confidence by low predicted probability vectors for the difficult cases. conclusions: our approach not only provides the basis to overcome current limitations in automated cell classification for routine diagnostics, but also demonstrates how a visual explanation framework can connect machine decision-making with cell properties and thus provide a novel versatile and quantitative method for investigating csf manifestations of various neurological diseases.   1.07 free neuropathol 3:20:14 proteomic profiling of idh-mutant gliomas identifies hip1r/vimentin as surrogate markers for 1p/19q codeletion and enables prediction of chromosomal copy number variations marius felix1, dennis friedel1, ashok kumar jayavelu2, katharina filipski3, anne-kathrin reinhard1, uwe warnken4, damian stichel1, daniel schrimpf1, andrey korshunov1, yueting wang1, tobias kessler4, nima etminan5, andreas unterberg6, christel herolod-mende6, laura heikaus7, felix sahm1, wolfgang wick4, patrick n. harter3, andreas von deimling1, david e. reuss1 1 department of neuropathology, institute of pathology, heidelberg, deutschland 2 clinical cooperation unit pediatric leukemia, german cancer research center (dkfz), heidelberg, deutschland 3 institute of neurology, edinger institute, heidelberg, deutschland 4 clinical cooperation unit neurooncology, german consortium for translational cancer research (dktk), german cancer research center (dkfz), heidelberg, deutschland 5 department of neurosurgery, mannheim, deutschland 6 department of neurosurgery, heidelberg, deutschland 7 bruker gmbh, bremen, deutschland background: idh-mutant gliomas are a common but heterogenous group of diffuse gliomas. chromosomal copy number variations (cnv) are a hallmark of many different types of cancer and 1p/19q codeletion is mandatory to differentiate “astrocytoma, idh mutant” from “oligodendroglioma, idh-mutant and 1p/19q-codeleted”. currently, loss of nuclear atrx is the only surrogate marker for an 1p/19q-wildtype status accepted by who. unmet diagnostic needs are improved surrogate markers for 1p/19q codeletion in atrx retaining gliomas and a rapid determination of cnvs in general. aims: we aimed at the identification of protein-level surrogate markers for 1p/19q codeletion suitable for immunohistochemical assays as well as proteomic signatures associated with chromosomal alterations in general. methods: we used mass-spectrometry (ms) based proteomics to analyze idh-mutant gliomas pre-characterized by dna methylation profiling. a discovery series containing 35 fresh frozen (ff) and 72 formalin fixed and paraffin embedded (ffpe) tumors were analyzed and potential biomarkers for 1p/19q codeletion were identified. a subsequent validation series consisting of 50 oligodendrogliomas and 50 astrocytomas was evaluated using immunohistochemistry to confirm potential biomarker identifications based on proteomic discoveries. furthermore, an additional validation cohort of 69 idh-mutant gliomas was stained and evaluated in a separate institution. results: proteomic data from ff and ffpe tissues were comparable. highly specific protein patters were identified, which could distinguish between oligodendroglioma and astrocytoma. oligodendrogliomas showed high hip1r and low vimentin (vim) staining intensities and astrocytomas low hip1r and high vim staining intensities. blinded evaluation of the validation cohort revealed a specificity of 100% and sensitivity 90-94% between two observers for 1p/19q prediction. combined evaluation with atrx increased sensitivity to 96%. an additional verification cohort stained and evaluated in a separate institution revealed similar prediction performances. further analyses revealed that a high proportion of the differentially regulated proteins between astrocytoma and oligodendroglioma are coded on the 1p and 19q chromosome arms. by generating virtual protein abundance means from unregulated chromosome arms, chromosomal protein ratios (cprs) were calculated, which helped predict copy number variations, showing high correlation with cnv plots from genome wide dna methylation profiles. conclusions: ms based analysis of ffpe tissue highly correlates with ff tissue, allowing in depth differential proteomic profiling. proteomics enables the discovery of new biomarkers and has great potential for the future of brain tumor diagnostics. immunohistochemistry for hip1r, vim and atrx can predict 1p/19q status with high specificity and sensitivity. cprs are a promising tool for the rapid proteome-based determination of chromosomal copy number variations.   1.08 free neuropathol 3:20:16 cellular digital neuropathology jonas franz1, christine stadelmann1 1 institut für neuropathologie, universitätsmedizin göttingen, göttingen, deutschland background: neuropathology is traditionally based on histological analysis of tissue. aside molecular pathology also the introduction of digital microscopy is changing the working environment of neuropathologists. digital pathology comes with modern algorithms for image quantification. mostly these algorithms are capable of solving sophisticated classification or segmentation problems. while classification is often applied to whole disease entities and segmentation is used to find, e.g., immunpositive areas we tried to adopt the algorithms to the traditional concept of „cellularpathologie“ as defined by r. virchow. aims: we aimed at optimizing existing image analysis algorithms and concepts specifically to the need of classification of cells on whole slide images. question: the main question was to determine a workflow which integrates on the one hand the needs of neuropathologists to steer the analysis and on the other hand to implement even advanced computer technology, e.g., with deep learning-based analyses. method: our main method focused on supervised deep learning to classify single cells in immunfluorescence stainings based on existing nuclear segmentation algorithms. result: as a result we deployed locally an image classification server for cell annotation by neuropathological experts without background in computer science in combination with an open microscopy (omero) server. manually annotated images were used to train various classification algorithms in combination with data augmentation and other techniques to optimize performance. after model selection we could reach almost human performance (>98% accuracy with <2-3% false positive rate) in an exemplary project of microglial cell classification stained by iba1. conclusion: we conclude that this concept of image analysis generalizes to various sorts of immunfluorescence stainings and might thus help to elevate the single cellular analysis to a whole tissue-based analysis with millions of cells.   2. neuroonocology 2.01 free neuropathol 3:20:17 using spatial transciptomics for diagnostic analysis of glioma michael ritter1,2, christina blume1,2, areeba patel1,2, philipp sievers1,2, helin dogan1,2, christel herold-mende3, wolfgang wick4,5,6, andreas von deimling1,2, felix sahm1,2 1 university hospital heidelberg, department of neuropathology, heidelberg, deutschland 2 german cancer research center (dkfz), german consortium for translational cancer research (dktk), clinical cooperation unit neuropathology, heidelberg, deutschland 3 university hospital heidelberg, department of neurosurgery, heidelberg, deutschland 4 german cancer research center (dkfz), german consortium for translational cancer research (dktk), clinical cooperation unit neurooncology, heidelberg, deutschland 5 university hospital heidelberg, neurology clinic, heidelberg, deutschland 6 german cancer research center (dkfz), national center for tumor diseases (nct), department of neurology and neurooncology program, heidelberg, deutschland background: the lack of specific in-situ markers of idh-wildtype glioblastoma makes it hard to distinguish between infiltrating tumour cells and reactive tissue. determination of copy number variations (cnv) of chromosomes may assist in this diagnostic challenge, but often requires a large amount of tissue, which is not always available. especially for stereotactic biopsies the available material is often quite limited. aims: in our study we wanted to test the feasibility of using spatial transcriptomics to distinguish between invading tumour cells and reactive/adjacent tissue and if it is possible to generate useful data like cnvs from small tissue fragments. methods: we applied spatial transcriptomics on 12 ffpe gbm samples indicating reactive tissue or an infiltration zone and 4 stereotactic biopsies. we determined the cnv for all samples and used a single cell dataset of infiltrating tumour cells (darmanis et al., 2017) to map the different cell types onto the tissue. results: the mapping of the tumour cells onto the sections clearly distinguished the tumour from the adjacent tissue and also allowed to further distinguish between majorly reactive tissue and tissue with a high number of infiltrating tumour cells. nevertheless, mapping on single cell level resolution requires higher resolution methods. both mapping by chr. 7 gain and chr. 10 loss and expression-based mapping produced similar results. using spatial transcriptomics on stereotactic biopsies we were able to determine the major cnvs from a 5 µm thick tissue section with a 1 mm diameter. conclusion: in summary we prove the feasibility to identify the infiltration zone and distinguish from reactive tissue using spatial transcriptomics, and we were able to derive cnv from very small tissue fractions. this can be leveraged especially if immunohistochemical stainings are not informative or too little tissue is left for the determination of cnv profiles.   2.02 free neuropathol 3:20:18 single cell dna amplicon sequencing reveals order of mutational acquisition in traf7 and klf4 or akt1 co-mutated meningiomas helin dogan1, christina blume1, areeba patel1, gerhard jungwirth2, miriam ratliff3, ralf ketter4, wolfgang wick5, christel herold-mende2, david reuss1, andreas von deimling1, felix sahm1 1 clinical cooperation unit neuropathology, german cancer consortium (dktk), german cancer research center and dept. of neuropathology, university hospital heidelberg, heidelberg, deutschland 2 dept. of neurosurgery, university hospital heidelberg, heidelberg, deutschland 3 dept. of neurosurgery, university hospital mannheim, mannheim, deutschland 4 dept. of neurosurgery, university hospital saarland, homburg, deutschland 5 dept. of neurology and neurooncology program, national center for tumor diseases, university hospital heidelberg, heidelberg, deutschland background: most meningiomas carry mutations in the tumor suppressor neurofibromatosis gene 2 (nf2) on chromosome 22q, while nf2-wildtype meningiomas account for about one third of all. in non-nf2-mutated cases, smo, polr2a, pik3ca, akt1 and klf4 mutations, the latter both typically with traf7 mutations, have been described. the combination of akt1 and klf4, respectively, with traf7 is intriguing: traf7/akt1 co-mutations are associated with meningothelial histology and basal localization, while traf7/klf4 co-mutations are highly specific for secretory meningioma without any predominant localization. since bulk molecular profiling indicates a step-wise mutational acquisition, the mutational sequence, whether the alteration in traf7 or in akt1/klf4 occurs first, has remained elusive. methods: single-cell sequencing technologies have allowed direct insight into the clonal architecture and complexity of thousands of individual cells. after evaluation of a patient with two independent meningiomas having identical somatic traf7 mutation but separate akt1/klf4 hotspot mutation, variant allele frequencies (vafs) of 62 retrospectively collected meningiomas carrying either coor single-mutations in traf7 and/or akt1 or klf4 were compared using bulk hybrid-capture panel sequencing data. additionally, a custom tumor panel comprising 392 amplicons covering 28 genes as well as the tert promoter was used along with the amplicon-based tapestri technology for single cell dna sequencing. genotype clustering analysis was finally performed to reveal the order of mutational acquisition in our cohort of traf7mut/akt1mut and traf7mut/klf4mut meningiomas (n=7). results: looking at mutational co-occurrence in bulk data, mutations assigned with higher vafs, unless explained by copy number changes, are thought to be acquired earlier than those with lower vafs. our bulk data of 28 co-mutated cases showed no significant difference in bulk-measured vafs, suggesting there was no major gap between the two time points of mutational acquisition. however, the majority of single-mutated cases (21/36) harbored mutations in traf7, while the others were either only akt1 (n=12) or klf4 (n=3) mutant. while it remains impossible to delineate clonal architecture from bulk data, our single cell data allowed grouping of cells into clonal populations. a total of 875,000 cells from 7 samples were prepared resulting in a median throughput of 2315 cells per sample and a median sequencing coverage of 105 reads per cell per amplicon. our data revealed three subclones in each sample: one wildtype clone (potentially stroma cells), one clone carrying a single mutation in traf7 (detected for 6/7 samples) and another clone harboring the co-mutations in traf7 and klf4 or akt1. conclusions: our findings strengthen the hypothesis that in traf7mut/akt1mut and traf7mut/klf4mut meningiomas, the mutation in traf7, which can occur throughout the wd40 domain of the protein, is acquired in an earlier stage than the hotspot mutation in akt1 or klf4. this study shows, that single-cell technologies on dna are useful in elucidating clonal architecture and phylogenetic trees. although single-cell dna sequencing in particular is associated with technical challenges such as false positive variant calling and allelic dropouts, high numbers of recovered cells as well as high-quality sequencing allow conclusive information on cellular zygosity and a robust analysis of mutational acquisition.   2.03 free neuropathol 3:20:20 alterations in ptpn11 and other noonan syndrome associated map-kinase signaling pathway genes accumulate in histopathologically atypical ganglioglioma with adverse postsurgical outcome lucas hoffmann1, roland coras1, katja kobow1, javier lopez-riviera2,3,4, costin leu3,4,5,6, dennis lal3,4,5,6, peter nürnberg6, christian g. bien7, thilo kalbhenn7, markus müller7, hajo hamer8, sebastian brandner9, karl rössler9,10, samir jabari1, ingmar blümcke1 1 department of neuropathology, universitätsklinikum erlangen, fau erlangen-nürnberg, partner of the european reference network (ern) epicare , erlangen, deutschland 2 department of molecular medicine, cleveland clinic lerner college of medicine, case western reserve university, cleveland, united states 3 genomic medicine institute, lerner research institute, cleveland clinic, cleveland, oh 44195, united states 4 charles shor epilepsy center, neurological institute, cleveland clinic, cleveland, united states; 5 stanley center for psychiatric research, broad institute of harvard and m.i.t, cambridge, ma 02142, united states 6 cologne center for genomics (ccg), medical faculty of the university of cologne, university hospital of cologne, cologne, germany 7 department of epileptology (krankenhaus mara), medical school, bielefeld university, bielefeld, germany 8 epilepsy center, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany, and epicare partner, erlangen, germany 9 department of neurosurgery, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany, and epicare partner, erlangen, germany 10 department of neurosurgery, medical university of vienna, vienna general hospital, vienna, austria, vienna, austria background: the ptpn11 gene is a tyrosine phosphatase non-receptor type protein linked to the map kinase signaling pathway. it was recently discovered as novel lesional epilepsy gene by large exome-wide sequencing studies. ptpn11 germline mutations have been associated with noonan syndrome, a multisystem disorder characterized by facial features, developmental delay and other organ diseases. sporadically, low-grade epilepsy-associated brain tumors (leat) also occur in noonan patients. herein, we performed a first deep phenotype-genotype analysis of low-grade developmental brain tumours with brain somatic alterations of the ptpn11 gene as compared to commonly observed leat with or without map kinase signaling pathway alterations. methods: we selected 87 leat cases recently submitted to whole exome sequencing and genotyping including 17 dysembryoplastic neuroepithelial tumours (dnt) and 70 ganglioglioma (gg). clinical data were retrieved from hospital files including postsurgical outcome (engel outcome, seizure onset, age at surgery, mri findings, location). available histopathology slides were fully digitalized for systematic microscopy analysis, including h&e and immunohistochemistry for cd34, p16, map2, neun, ki67, idh1 and p53. results: we identified a series of eight gg with ptpn11 alterations, i.e. gains in copy number variations (cnv) of the locus 12q, which showed a systematic pattern of additional cnv gains in fgfr4, rheb, nf1, kras as well as braf alterations (figure 1). histopathology pattern analysis revealed an atypical and complex glio-neuronal phenotype with subpial tumour spread and large, pleomorphic and multinuclear cellular features (figure 2). only three out of eight gg with ptpn11 alterations were free of disabling-seizures two years after surgery (engel ia outcome, 38%). this was remarkably different from our series of gg with braf alterations (n=35), gg without any genetic alteration detectable by our study paradigm (n=27) and dnt with fgfr1 alterations (n=6) with engel ia rates of 85%, 76% and 83%, respectively. conclusions: we identified a subgroup of ganglioglioma characterized by ptpn11 alterations in association with other noonan syndrome related alterations of the map kinase signaling pathway, i.e., kras, rheb, braf, and fgfr4. these tumours were further characterized by histopathological features of cellular atypia in glial and neuronal cell components as well as adverse postsurgical outcome. these features were strikingly different from other leat with defined genetic alterations in braf, e.g., v600e mutation, and fgfr1. notwithstanding, these findings need further validation as they argue for a three-tiered who grading system also for developmental, glio-neuronal tumors associated with early-onset focal epilepsy. genetic similarities to noonan syndrome and noonan syndrome associated disorders may also suggest the use of targeted treatment options against the map kinase and mtor signaling pathway. figure 1: oncoplot of ptpn11 altered leat compared to a braf-v600e mutated gg (arrow on left) and a fgfr1 altered dnt (arrow on right) figure 2: histopathology findings in a ptpn11 altered atypical ganglioglioma. a: subpial growth (arrow) with large, pleomorphic and glio-neuronal phenotype shown in b. c: abundant cd34 immunoreactivity (sp – subpial region). d: the arrow points to a bi-nucleated neuron (map2 immunohistochemistry) confirming the diagnosis of ganglioglioma.   2.04 free neuropathol 3:20:23 molecular refinement of pilocytic astrocytoma in adult patients helena bode1,2, catena kresbach1,2,3, dörthe holdhof1,2, mario m. dorostkar4,5, patrick n. harter6, jürgen hench7, stephan frank7, alicia eckhardt1,2,8, annika k. wefers3, sina neyazi1,2, david capper9,10, michael bockmayr1,2,11, ulrich schüller1,2,3 1 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, deutschland 2 research institute children’s cancer center hamburg, hamburg, deutschland 3 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, deutschland 4 center for neuropathology, ludwig-maximilians-university, munich, deutschland 5 german center for neurodegenerative diseases, munich, deutschland 6 institute of neurology (edinger institut), university hospital frankfurt, frankfurt, deutschland 7 division of neuropathology, institute of medical genetics and pathology, university of basel, basel, switzerland 8 lab of radiobiology & experimental radiation oncology, hubertus wald tumorzentrum – university cancer center hamburg, university medical center hamburg-eppendorf, hamburg, deutschland 9 german cancer consortium (dktk), partner site berlin, and german cancer research center (dkfz), heidelberg, deutschland 10 department of neuropathology, corporate member of freie universität berlin, charité, universitätsmedizin berlin and humboldt-universität zu berlin, berlin, deutschland 11 institute of pathology, corporate member of freie universität berlin, charité, universitätsmedizin berlin and humboldt-universität zu berlin, berlin, deutschland background: pilocytic astrocytomas (pa) are the most common primary central nervous system neoplasms in children. the vast majority of cases harbor kiaa1549-braf fusions and usually go along with an excellent prognosis. in contrast, pa in adult patients are rare, lack kiaa1549-braf fusions in many cases, and demonstrate a more aggressive clinical course. purpose: this project aims at characterizing adult pa regarding their molecular profile and clinical course. methods: we identified 55 cases with a histological diagnosis of pa in adulthood (≥18 years). molecular analyses of these cases included dna methylation analysis, copy number profiling, and dna sequencing for the most common mutations in the mapk-pathway. results: the mean age of our cohort was 35 years. tumors were located infratentorially (41%), supratentorially (41%), and spinally (18%). after performing global dna methylation analyses and applying the dkfz brain tumor classifier (v12.5), only 25% of these cases received a significant match to one of the reference methylation classes of pa (score ≥ 0.9). 20% matched to different entities, and 55% did not match to any brain tumor class. furthermore, only 23% of the tumors exhibited the kiaa1549-braf fusion. further analyses of tumors with a significant match to one of the three pa reference classes showed that adult patients mostly had supratentorial pa (lgg_pa_gg_st, mean age: 20 years, n=45), while children had pa in midline structures (lgg_pa_mid, mean age: 9 years, n=51) or in the posterior fossa (lgg_pa_pf, mean age: 11 years, n=159, p<0.005). among these tumors defined by dna methylation, the typical kiaa1549-braf fusion was found in 94 % of pediatric tumors and only in 45 % of tumors occurring in adults. conclusions: in summary, according to dna methylation profiling, a particularly high fraction of tumors histologically appearing as pa in adult patients do not match known reference cohorts of pa. many tumors are even reflecting other tumor entities, indicating ambiguous histological features. furthermore, even in cases that significantly match to pa regarding dna methylation, the distribution of genetic drivers differs from their pediatric counterparts.   2.05 free neuropathol 3:20:25 exploration of cellular origins and therapeutic targets by modeling high grade pediatric glioma of the mycn subclass in mice melanie schoof1,2, shweta godbole3, carolin walter4,5, matthias dottermusch3,6, thomas albert5, annika ballast5, carolin göbel1,2, sina neyazi1,2, dörthe holdhof1,2, catena kresbach1,6, gefion dorothea epplen1, mirjam blattner-johnson7,8, franziska modemann9,10, ann-kristin afflerbach1,2, alicia eckhardt1,11, vanessa thaden1, nina struve11,12, david t. w. jones7,8, kornelius kerl5, julia neumann3,6, ulrich schüller1,2,6 1 research institute children’s cancer center hamburg, hamburg, germany 2 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, deutschland 3 center for molecular neurobiology (zmnh), university medical center hamburg-eppendorf, hamburg, deutschland 4 institute of medical informatics, university of muenster, muenster, deutschland 5 department of pediatric hematology and oncology, university children’s hospital münster, muenster, deutschland 6 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, deutschland 7 hopp children's cancer center (kitz), heidelberg, deutschland 8 pediatric glioma research group, german cancer research center (dkfz), heidelberg, deutschland 9 department of oncology, hematology and bone marrow transplantation with division of pneumology, university medical center hamburg-eppendorf, hamburg, deutschland 10 mildred scheel cancer career center, university cancer center hamburg, university medical center hamburg-eppendorf, hamburg, deutschland 11 department of radiotherapy, university medical center hamburg-eppendorf, hamburg, deutschland 12 mildred scheel cancer career center hatrics4, university medical center hamburg-eppendorf, hamburg, deutschland pediatric gliomas of the mycn subclass, a recently described highly aggressive brain tumor entity, frequently carry amplifications of mycn and mutations in tp53. these tumors present with a median age of 8 years and a median overall survival of only 14 months. better treatment options are urgently needed, as the current treatment is ineffective and causes severe side effects. here, we describe the generation of a novel mouse model, which can be used for preclinical research. we bred hgfap-cre::tp53fl/fl::lsl-mycn mice, which develop large forebrain tumors with 100 % penetrance within the first 80 days of life. the murine tumors show a high similarity with human tumors in histology, gene expression, and global dna methylation pattern. single-cell gene expression analyzes of these tumors revealed a large intratumoral cell heterogeneity and, due to the similarity of the tumor cells with oligodendrocytes in different developmental stages, suggests a glial origin of these tumors. additionally, we tested the preclinical potential of our mouse model by showing sensitivity of mouse and human tumor cells to aurka inhibition in vitro. we believe that further characterization and utilization of the model will pave the way to improved treatment strategies for patients with these highly aggressive tumors.   2.06 free neuropathol 3:20:27 the genomic and transcriptional landscape of primary central nervous system lymphoma josefine radke1, naveed ishaque2, reiner siebert3, stefan wiemann4, frank heppner5 1 universität greifswald, pathologie, greifswald, deutschland 2 berlin institute of health (bih), digital health center, berlin, deutschland 3 ulm university & ulm university medical center, human genetics, ulm, deutschland 4 deutsches krebsforschungszentrum (dkfz), heidelberg, deutschland 5 charité, neuropathology, berlin, deutschland background: primary lymphomas of the central nervous system (pcnsl) are mainly diffuse large b-cell lymphomas (dlbcls) confined to the central nervous system (cns). despite extensive research, the molecular alterations leading to pcnsl have not been fully elucidated. aims: in order to provide a comprehensive description of the genomic and transcriptional landscape of pcnsl, we here performed whole-genome and transcriptome sequencing and integrative analysis of 51 lymphomas presenting in the cns, including 42 ebv-negative pcnsl, 6 secondary cns lymphomas (scnsl) and 3 ebv+ cnsl and matched controls. the results were compared to an independent validation cohort of 31 ffpe cnsl specimens (pcnsl, n = 19; scnsl, n = 9; ebv+ cnsl, n = 3) as well as 39 fl and 36 systemic dlbcl cases outside the cns. results: somatic genomic alterations in pcnsl mainly affect the jak-stat, nfkb, and b-cell receptor signaling pathways, with hallmark recurrent mutations including myd88 l265p (67%) and cd79b (63%), cdkn2a deletions (83%) and also non-coding rna genes such as malat1 (70%), neat (60%), and mir142 (80%). kataegis events, which affected 15 of 50 identified driver genes and 21 of the top 50 mutated ncrnas, played a decisive role in shaping the mutational repertoire of pcnsl. compared to systemic dlbcl, pcnsls exhibited significantly more focal deletions in 6p21 targeting the hla-d locus that encodes for mhc class ii molecules as a potential mechanism of immune evasion. mutational signatures correlating with dna replication and mitosis (sbs1, id1 and id2) were significantly enriched in pcnsl (sbs1: p = 0.0027, id1/id2: p < 1x10-4). furthermore, tert gene expression was significantly higher in pcnsl compared to abc-dlbcl (p = 0.027). although pcnsl share many genetic alterations with systemic abc-dlbcl in the same signaling pathways, transcriptome analysis clearly distinguished both into distinct molecular subtypes. ebv+ cnsl cases may be distinguished by lack of recurrent mutational hotspots apart from ig and hla-drb loci. conclusion: we show that pcnsl can be clearly distinguished from dlbcl, having distinct expression profiles, ig expression and translocation patterns, as well as specific combinations of genetic alterations.   2.07 free neuropathol 3:20:28 molecular mechanisms of therapy resistance in malignant melanoma brain metastasis elisa schumann1, randi koll2, julia onken2, karsten jürchott2, torben redmer3, josefine radke4 1 charité universitätsmedizin berlin, institut für neuropathologie, berlin, deutschland 2 charité universitätsmedizin berlin, berlin, deutschland 3 veterinärmedizinische universität wien, wien, austria 4 universität greifswald, greifswald, deutschland background: malignant melanoma (mm) is among the tumor entities with the highest potential to spread to the cns. about 45% of mm patients suffer from brain metastasis likely proceeding continuously during the course of disease. genetically and molecularly distinct subclones lead to tumor heterogeneity which is followed by therapy resistance and poor prognosis. previous studies suggested increased metastatic potential to and within the brain under braf inhibitor (brafi) therapy, which is caused by upregulation of a subset of molecular drivers controlling migratory and invasion such as the nerve growth factor receptor cd271/ngfr. aims: to gain insight into the molecular features of migration and invasion of patient derived cell lines from mm brain metastases (bm) that were therapy-responsive or therapy-resistant to brafi, radiotherapy and immune checkpoint inhibitors. methods: we generated patient derived cell lines from mm bm (n = 9) and performed dna-sequencing (n = 5) and transcriptome analyses (n = 2) of cell lines and concordant tumors (n = 2). furthermore, we used the incucyte® live-cell analysis to perform high throughput scratch wound assays with patient derived cell lines, which were genetically modified leading to overexpression or downregulation of ngfr. results: transcriptome profiling of brafi resistant mm bm revealed that the invasive potential increased during disease progression. this process was accompanied by upregulation of ngfr expression. moreover, it was preserved in patient derived cells lines, which demonstrated significantly higher potential of two-dimensional in vitro migration (90% vs. 76% after 100 hours). furthermore, cd271 knockdown was associated with loss-of-expression of several genes involved in migration and invasion. conclusions: brain metastases are the major cause of death in metastasized mm. probably, bm emerge and progress by the concerted interaction of several molecular programs that are triggered by cells of the tumor microenvironment and/or in response to therapeutic interventions. our study provides a longitudinal perspective on the progression of brain metastasis and their mechanisms leading to therapy resistance.   2.08 free neuropathol 3:20:29 a peripheral nerve sheath tumor syndrome caused by postzygotic erbb2 mutations michael ronellenfitsch1,2,3,4, isabel gugel5, dusica babovic-vuksanovic6, maximilian rauch2,7, jens schittenhelm5, martin u. schuhmann5, silvia hofer8, martina kirchner9, gerhard marquardt10, rouzbeh banan911, benedikt sauer1,3, ulrich schüller12,13,14, werner paulus15, matthias meinhardt16, tareq juratli16, albrecht stenzinger9, stefan fröhling17,18, eric legius19, andreas von deimling9,11, felix sahm9,11, joachim p. steinbach1,2,3,4, patrick harter2,3,10, victor-felix mautner12, david reuss11,20 1 dr. senckenberg institute of neurooncology, frankfurt, deutschland 2 university cancer center (uct) frankfurt, frankfurt, deutschland 3 german cancer consortium (dktk), frankfurt, deutschland 4 frankfurt cancer institute, frankfurt, deutschland 5 university hospital tübingen, tübingen, deutschland 6 mayo clinic college of medicine, rochester, united states 7 goethe university hospital, frankfurt, deutschland 8 university hospital and university of zurich, zürich, switzerland 9 heidelberg university hospital, heidelberg, deutschland 10 university hospital frankfurt, frankfurt, deutschland 11 dkfz, heidelberg, deutschland 12 university hospital hamburg-eppendorf, hamburg, deutschland 13 university medical center hamburg-eppendorf, hamburg, deutschland 14 research institute children's cancer center hamburg, hamburg, deutschland 15 university hospital münster, münster, deutschland 16 university hospital carl gustav carus, dresden, deutschland 17 nct heidelberg and dkfz, heidelberg, deutschland 18 dktk, heidelberg, deutschland 19 ku leuven and university hospital, leuven, belgium 20 universitätsklinikum heidelberg, neuropathologie, heidelberg, deutschland introduction: peripheral nerve sheath tumors (nst) are common manifestations of different tumor syndromes within the neurofibromatosis spectrum, comprised of nf1, nf2 and schwannomatosis. these are caused by inactivating germline mutations in the nf1, nf2, smarcb1 or lztr1 tumor suppressor genes respectively. neurofibromas are closely associated with nf1 and schwannomas occur in both nf2 and schwannomatosis. occurrence of neurofibroma/schwannoma hybrid tumors is reported in all these syndromes. we recently described erbb2 mutations in a significant portion of neurofibroma/schwannoma hybrid nerve sheath tumors. based on clinical criteria, these cases resembled schwannomatosis. however, the somatic genetic profile of the tumors was distinct. additionally, features untypical of schwannomatosis were present, resembling previously published descriptions of four patients with distinctive but unclassifiable clinical and pathological findings. objectives: the aim of the study was the clinicopathological and molecular characterization of erbb2-mutant peripheral nerve sheath tumors. methods: tumors were evaluated by histology. next generation sequencing (hd-panel or whole exome sequencing) was used to determine the presence of an erbb2 mutation in at least one tumor of every patient. pyro-sequencing was used to verify erbb2 mutations and to determine their presence in additional tumors from a given patient. 850k methylation profiling was used for additional characterizations. results: we identified 13 non-related patients with erbb2 mutant nst, including all 4 previously published patients with an unclassified syndrome. all but two patients were females and tumors developed slowly during adulthood. all patients had multiple nst, which were restricted to a specific anatomic region in several patients while a more widespread distribution of tumors was present in others. the histology showed quite distinctive features within the spectrum of hybrid nerve sheath tumors. strikingly, in all patients, tumors from distinct anatomic locations harbored the very same activating erbb2 mutation. the median mutant allele frequency of 12% was comparatively low (range 4%-23%) suggesting that only a subpopulation of cells harbored the mutation. no other candidate driver alteration was found by ngs panel or wes and rna-sequencing. dna methylation profiling provided evidence for a distinctive epigenetic profile of erbb2-mutant nsts. no chromosomal copy number alterations were detectable. ongoing molecular analyses will provide additional insight in the pathogenesis of erbb2-mutant nsts. conclusion: erbb2 mutations in nst do not occur as isolated somatic events in sporadic tumorigenesis or in the setting of nf1, nf2 or schwannomatosis but represent manifestations of a distinct tumor syndrome most likely caused by postzygotic erbb2-mosaicism. diagnosis of erbb2-mutant nst is of high clinical relevance due to the availability of specific erbb2 inhibitors and preliminary evidence of their effectiveness. histology is sufficiently specific for screening purposes but molecular analyses with highly sensitive methods like deep coverage ngs are mandatory for the definitive diagnosis.   2.09 free neuropathol 3:20:31 cns-tumor patients within the impress-norway trial: first year experiences pitt niehusmann1,2, hege g russnes1,3,4, katarina puco2, åsmund flobak5,6, eli sihn s. steinskog7, åse haug7, sigmund brabrand2,8, egil s. blix9,10, anne j skjulsvik5,11, ragnhild m wold12, henning leske1, hrvoje miletic13,14, petter brandal8,15, gro l. fagereng16, kjetil taskén3,17, åslaug helland3,4,8 1 oslo university hospital, department of pathology, oslo, norway 2 oslo university hospital, division for cancer medicine, oslo, norway 3 university of oslo, institute of clinical medicine, oslo, norway 4 oslo university hospital, department of cancer genetics, institute for cancer research, oslo, norway 5 norwegian university of science and technology, department of clinical and molecular medicine, trondheim, norway 6 st. olav university hospital, the cancer clinic, trondheim, norway 7 haukeland university hospital, department of oncology, bergen, norway 8 oslo university hospital, department of oncology, oslo, norway 9 uit the arctic university of norway, institute of clinical medicine, tromsø, norway 10 university hospital of north norway, department of oncology, tromsø, norway 11 st. olav university hospital, department of pathology, trondheim, norway 12 university hospital of north norway, department of pathology, tromsø, norway 13 university of bergen, department of biomedicine, bergen, norway 14 haukeland university hospital, department of pathology, bergen, norway 15 oslo university hospital, section for cancer cytogenetics, institute for cancer genetics and informatics, oslo, norway 16 oslo university hospital, institute for cancer research, oslo, norway 17 oslo university hospital, department of cancer immunology, institute for cancer research, oslo, norway background: impress-norway is a nation-wide precision medicine trial for cancer patients in norway that launched april 1st 2021. in this investigator-initiated, prospective, open-label, non-randomized combined basketand umbrella-trial, patients are enrolled into multiple parallel treatment cohorts. patients with progressive cancer disease, including primary cns-neoplasms, with no further standard therapy to offer, are eligible. all drugs available in impress-norway are regulatory approved. currently, five different pharmaceutical companies provide 16 drugs, and we are in process to acquire eight additional drugs for patients in this study. methods: comprehensive genomic profiling (gene-panel analysis of >500 genes) is performed as part of the norwegian public health care system. patients consenting to the impress-norway profiling phase contribute their clinical and molecular data for research and are screened for cell-free circulating tumor dna. patients with identified biomarkers matching available drugs are referred by the national molecular tumor board for inclusion in the impress-norway treatment phase. these patients will have extensive biobanking as well as whole genome molecular profiling of their tumors before and during treatment. in the impress-norway treatment phase, each cohort is defined by the patients’ tumor type, molecular profile of the tumor, and study drug. treatment outcome in each cohort is monitored using a simon two-stage-like ‘admissible’ monitoring plan to identify evidence of clinical activity. the primary objective in the study is clinical benefit of treatment at 16 weeks of treatment; defined as complete response, partial response, or stable disease. here, we report on patients with cns-neoplasms included in the impress-norway profiling and treatment phases. results: as of april 30th, 2022, twenty-four patients with cns-neoplasms had been included in the molecular profiling phase of impress-norway and 22 had completed evaluation at the molecular tumor board (see table 1). tumor mutation burden (tmb) in cns-tumor tissue samples ranged from 1.6-250 somatic mutations per megabase (mut/mb; median=4.7, n=23). in liquid biopsies, blood tmb ranged from 0-6 mut/mb (median=0; n=21), indicating a limited efficacy of this analysis in cns-tumor patients. in five of the 22 patients with completed evaluation, we identified biomarker, which allowed allocation to an impress-norway treatment-cohort (ratio of cns-patients with targetable biomarker was similar to the overall inclusion of patients into treatment cohorts, 67/295). one glioblastoma patient showed complete response according to rano at 39 weeks.   table 1 diagnosis treatment cohort glioblastoma, idh-wildtype (n=13) n=3 astrocytoma, idh-mutant (n=4) n=0 anaplastic meningioma (n=1) n=0 atypical meningioma (n=1) n=0 diffuse midline glioma, h3 k27-altered (n=1) n=1 high-grade astrocytoma with piloid features (n=1) n=1 myxopapillary ependymoma (n=2) n=0 supratentorial ependymoma (n=1) n=0   due to increasing test capacity, we anticipate to double the number of included cns-tumor patients within the next 6 months. whole genome sequencing data from patients included into the treatment cohorts are obtained successively. conclusion: patients with advanced cancer progressing on standard treatment are referred to treatment in impress-norway after advanced molecular diagnostics. molecular alterations indicating benefit of drugs currently available in the study are detected in a reasonable number of patients with cns-neoplasms.   3. neurodegeneration 3.01 free neuropathol 3:20:33 cnn-supported quantification of fat compartments at abdominal mri applied to als patients ina vernikouskaya1, hans-peter müller2, dominik felbel1, francesco roselli2, albert christian ludolph2, volker rasche1, jan kassubek2 1 ulm university medical center, internal medicine ii, ulm, deutschland 2 university of ulm, neurology, ulm, deutschland background: amyotrophic lateral sclerosis (als) is the most frequent adult onset neurodegenerative motor neuron disease characterized by catabolism1, and patients begin to lose weight more than 10 years before the onset of motor symptoms2. als patients have been shown to display an expanded ratio between visceral adipose tissue (vat) and subcutaneous adipose tissue (sat)3. accurate segmentation of body fat compartments from mri is, however, a challenging task due to the limited reproducibility of semi-manual delineations and artifacts. concerning organ segmentation, learning-based algorithms and especially convolutional neural networks (cnn) have been proven to outperform traditional methods in speed and reproducibility. aims: the aim of this study was to automate the discrimination of abdominal body fat compartments into sat and vat from t1-weighted mri using deep cnn and to quantify the fat ratio in patients with als as compared to the control cohort. question: may cnn-supported segmentation of body fat compartments serve for unbiased analysis of the vat/sat ratio parameter as a potential biological marker? methods: 74 als patients (age 60 ± 12, m/f 50/24) and 81 healthy subjects (56 ± 15, 42/39) underwent mri examination with multi-slice t1-weighted spin-echo sequence. all available data were split in training (50 %), validation (6 %), and test (44 %) data, based on age and bmi strata. semi-automatic segmentation of subcutaneous and visceral fat was performed with an established reference method using software package atlas4. the obtained sat/vat masks were used for training of the cnn of u-net like architecture. performance of the segmentation using cnn was evaluated in terms of dice coefficients. volumetric computation of segmented sat and vat for all test objects was performed with reference and cnn-based methods and compared by pearson correlation. vat/sat ratio was assessed. results: the dice coefficients between the cnn-supported and reference segmentations comprised 0.87 ± 0.04 for sat and 0.64 ± 0.17 for vat in the control group and 0.87 ± 0.08 for sat and 0.68 ± 0.15 for vat in the als group. a significant linear correlation between the cnn predicted and reference method with pearson coefficients 0.992 in controls and 0.977 in als patients was observed for sat, whereas lower pearson coefficients 0.653 in controls and 0.814 in als patients were obtained for vat. significant difference for the vat/sat ratio was observed when comparing als patients versus healthy subjects with the p-value of 0.002. figure 1 shows the sat and vat segmentation results in healthy controls and als patients. conclusions: the obtained results in the t1-weighted mri data in the als patient cohort could reproduce the results of a reference technique in a user-independent manner with high accuracy. cnn-supported quantification of vat/sat ratio might serve as a biological marker in als body composition assessment, potentially as a secondary read-out for clinical trials. references: 1. dupuis et al, lancet neurol. 2011 2. peter et al, eur j epidemiol. 2017 3. lindauer et al, plos one. 2013 4. müller et al, nmr biomed. 2011 figure 1: comparison of ccn-based segmentation vs. reference segmentation and vat/sat ratio plot in healthy controls and als patients.   3.02 free neuropathol 3:20:35 neurodegenerative iron storage disease (neuroferritinopathy) caused by a novel frameshift mutation in the ferritin heavy chain gene (fth1 c.341-342del) vincent umathum1,2, daniel amsel1, christina becker3, corinne kasan3, andrea may4, klaus nehmer4, andreas günther4,5, carmen selignow1, anna nishimura1, ioannis alexopoulos6, attila németh1, nadja ritschel1, axel weber7, till acker1, anne schänzer1 1 institut für neuropathologie, justus-liebig-universität, gießen, deutschland 2 institut für pathologie und molekularpathologie, bundeswehrkrankenhaus ulm, ulm, deutschland 3 institut für pathologie, zytologie und molekularpathologie mvz, wetzlar, deutschland 4 pneumologische klinik, agaplesion evangelisches krankenhaus, gießen, deutschland 5 zentrum für interstitielle und seltene lungenerkrankungen, justus-liebig-universität, gießen, deutschland 6 institute for lung health, justus-liebig-universität, gießen, deutschland 7 institut für humangentik, justus-liebig-universität, gießen, deutschland introduction: neuroferritinopathy (nf) is a rare hereditary neurodegenerative disorder associated with increased iron deposition in the brain and extracerebral organs such as the kidney, liver, skin and skeletal muscle. the ferritin complex consists of 24 subunits of ferritin light and heavy chains and converts free iron into a non-redox active storage form. clinically, the focus is on chorea-huntington-like movement disorders. so far, only mutations in the ferritin light chain gene (ftl) have been described in nf. material and methods: a 78-year-old female patient died of covid-19-associated pneumonia and was autopsied. standard staining (he, prussian blue), immunohistochemical and immunofluorescence staining as well as electron microscopic analyses were performed on paraffin-embedded formalin-fixed (ffpe) tissue of the patient from different brain regions as well as heart, lung, liver and kidney. whole slide images scans were done by hamamatsu nanozoomer s360 and evaluated morphometrically with qpath. whole-exome sequencing (wes) was done from ffpe material of the basal ganglia. results: macroscopically, no pathology was found in the brain. microscopically, numerous inclusion bodies (ib) were seen in the brain and sporadically in the liver and kidney. the ib were homogeneously sharply defined on he stains and up to 13 µm in size (normal nuclear diameter: approx. 5-6 µm), with strong fe3+ deposits in the prussian blue staining. ultrastructurally, the ib showed intranuclear, fine granular aggregates with lateralisation of the chromatin to the inner side of the membrane. immunohistochemistry for ftl and ferritin heavy chain protein (fth) showed clear nuclear expression in the ib. in contrast, the cells in the control tissue had predominantly perinuclear, cytoplasmic expression. quantitative evaluation showed increased fth expression in the patient: frontal cortex (cf): 0.2%, occipital cortex (co): 0.3%, hippocampus: 0.08%, basal ganglia (bg): 0.7%, dentate nucleus (dn): 1.7% compared to control tissue (cf: 0.06% co: 0.04% hippocampus: 0.04% bg: 0.08% dn: 0.4%. the ratio of fth expression (compared to control tissue) was highest in the bg (1:8.4), followed by the co (1:8.3) and lowest in the hippocampus (1:2.1). wes revealed a previously undescribed variant (double deletion) in the ferritin heavy chain gene (fth1 c.341-342del). wild-type sequence in ftl. discussion: the present study describes for the first time a patient with nf caused by a previously undescribed mutation in the fth1 gene with presentation of numerous ib in the brain and sporadically in extracerebral tissue. these results suggest that the function of the ferritin complex can be disturbed not only by an ftlbut also by an fth1-mutation, leading to pathological deposition of ferritin complexes. with extended analyses, it could be shown that the ib correspond to enlarged cell nuclei with intranuclear ferritin accumulations. furthermore, a high variability in distribution of the ib in different brain regions was found. summary: mutation in fth1 (c.341-342del) is associated with a rare neurodegenerative disease with increased intranuclear iron deposition mainly in the bg and dn, possibly showing a similar pathomechanism to the known ftl-mutation. if nf is clinically suspected, mutations should therefore be investigated not only in the ftl-gene but also in the fth1-gene.   3.03 free neuropathol 3:20:37 the contribution of late-nc to neuron loss, granulovacuolar degeneration and dementia in alzheimer’s disease dietmar thal1,2,3, klara gawor1,3, evelien van schoor1,3,4, sebastiaan moonen1,3,4, jolien schaeverbeke1,3,4, rik vandenberghe3,4,5, mathieu vandenbulcke3,4,6, christine a. f. von arnim7,8, marta koper1,3,4, sandra tomé1,3 1 ku-leuven, department of imaging and pathology, laboratory for neuropathology, leuven, belgium 2 uz leuven, department of pathology, leuven, belgium 3 leuven brain institute, leuven, belgium 4 ku leuven, department of neuroscience, leuven, belgium 5 uz leuven, department of neurology, leuven, belgium 6 uz leuven, department of psychiatry, leuven, belgium 7 göttingen university, department of geriatrics, göttingen, deutschland 8 ulm university, neurology, ulm, deutschland background: tdp-43 pathology in alzheimer’s disease (ad) is currently considered as a co-pathology belonging to the spectrum of limbic-predominant, age-associated tdp-43 encephalopathy (late). ad cases with tdp-43 pathology have greater medial temporal lobe atrophy and cognitive decline compared to ad cases without tdp-43. recently, we showed that the accumulation of the necrosome (executer complex of necroptosis which is a programmed from of necrosis) in granulovacuolar degeneration (gvd) in ad is associated with neuron loss. aims: here, we aim to clarify whether necroptosis in ad is related to tdp-43 pathology, i.e., late neuropathological changes (late-nc) and indicates a contribution of late-nc to the degeneration/ death of neurons in ad. by doing so, we will determine the impact of late-nc on ad-nc, neuron loss, especially necroptosis, and cognitive decline. research question: does tdp-43 pathology in late contribute to neurodegeneration via necrosome accumulation and necroptosis in ad and, if so, is this related to tau pathology. methods: we used 234 human post-mortem brains from 89 non-ad controls, 82 p-pread and 63 ad cases. we determined amyloid-β (aβ) (mtl = medial temporal lobe-based) phases, braak neurofibrillary tangle (nft) stages, late-nc stages and gvd stages, which described the anatomical spread of gvd bodies, and the frequency of gvd-affected neurons in a given region. from these, 66 cases were assessed for neuronal density in the ca1 subfield of the hippocampal formation and 186 were retrospectively assessed for clinical dementia rating (cdr) scores. in a subset of 27 cases covering 9 non-ad controls (without tdp-43 pathology), 8 symptomatic ad cases without tdp-43 pathology (adtdp-) and 10 symptomatic ad cases with tdp-43 proteinopathy (i.e.: late-nc; adtdp+), we quantified neuronal density, severity of ptdp-43, pmlkl (a component of the activated necrosome) and ptau pathology. results: we found that late-nc and braak nft stages contribute independently to gvd severity when being covariates the same model term (p < 0.001). late-nc and braak nft stages were associated with neuronal density in the hippocampus (p = 0.022 and p = 0.004, respectively). braak nft stage alone showed also an association with neuronal density (p < 0.001). partial correlation analysis corroborated these data and revealed that braak nft and late-nc stage are significantly correlated with gvd stage and cdr score. in the 27 cases used for quantitative assessments, the absence of ptdp-43 was associated with less neuronal loss in adtdpcases compared to adtdp+ (p = 0.0204). adtdp+ cases exhibited significantly more pmlkl-positive neurons, when compared to adtdpcases (p = 0.025) and non-diseased controls (p < 0.0001). consistently, adtdp+ cases also showed enhanced numbers of ptau exhibiting neurons compared to adtdp(p = 0.045) and controls (p < 0.0001). conclusions: thus, late-nc contributes independently to neuronal loss and gvd severity. further, these data highlight the impact of cytoplasmic tdp-43 aggregation on ptau pathology as well as on necroptosis activation in ad. support: fwo.   3.04 free neuropathol 3:20:39 the role of c3 inhibition in an ipsc nmj model of neuroinflam-mation scott baver1, virginia smith2, yan li1, david eyerman1, ashley robertson2, leticia lenkiu2, heather cannon2, daisy martinez2, hannah hanson2, james hesperos2 1 apellis, waltham, united states 2 hesperos, orlando, united states background: the complement cascade is a critical component of the immune system, and dysfunction of complement has been implicated in als. components of the complement cascade, including c3, are reported to be deposited on neuromuscular junctions (nmjs) of muscle biopsies in people with als. objectives: evaluate the effect of complement c3 inhibition on nmj function in response to inflammatory stimulations in a human ipsc-derived nmj model. question: while the therapeutic potential of complement system modulation has been explored via use of embryonic knockout animals in als models, its effects in clinically relevant human cell-based models are unknown. methods: nmj systems were established by plating human ipsc-derived motoneurons, skeletal myoblasts, schwann cells, microglia, and activated or inactivated thp monocytes in a compartmentalized co-culture system. activated and inactivated monocytes were plated at ratios of 4:1-1:50 (vs. skeletal myoblasts). als model systems were created using sod-1 (e100g) or tdp-43 ipsc-derived cells compared to control nmjs established from wild-type ipsc-derived cells. to determine the role of c3 on nmj function, the c3 inhibitor apl-2 (50 µg/ml) and human complement serum (0.05%) were acutely dosed for 3 hours. complement c3 expression was assessed by immunocytochemistry, and nmj number and fidelity were calculated by assessing the number of functional myotubes under indirect stimulation and the ratio of number of successful contractions to number of pulses at a given frequency, respectively. all experiments were replicated twice in triplicate. results: functional nmj systems were assessed with the addition of various ratios of activated or inactivated monocytes to skeletal muscle-side of the culture chamber. addition of activated monocytes resulted in reduced nmj number and function. in addition, while c3 expression was observed with thp-monocytes, activation of m1 macrophages increased c3 activity. human complement serum potentiated the effects of m1 macrophages; further decreasing nmj numbers and reducing nmj fidelity. acute treatment with apl-2 attenuated these effects. compared to wild-type, sod-1 and tdp-43 nmj systems reduced nmj number and fidelity. the reduction in sod-1 nmj number and fidelity was greater than that of tdp-43 nmj system. conclusions: these data demonstrate that modulating c3 with apl-2 in the presence of an inflammatory nmj environment could improve overall function of the nmj in related disease states.   3.05 free neuropathol 3:20:40 fast-track procedure for the neuropathological assessment of neurodegenerative diseases benjamin englert1,2,3, viktoria ruf1, jochen herms1,2,3 1 ludwig-maximilians university, center for neuropathology and prion research, munich, deutschland 2 german center for neurodegenerative diseases (dzne), munich, deutschland 3 munich cluster for systems neurology (synergy), munich, deutschland background: in order to exclude creutzfeldt-jakob disease we have established a fast-track assessment of all brains that we receive at the neurobiobank munich. aims and questions: we aim to evaluate the concordance between fast-track working diagnosis and the full histological work-up of the brain of patients clinically diagnosed with a neurodegenerative disease. methods: two predefined, easily accessible brain regions (frontal superior gyrus and cerebellar hemisphere) were sampled, fixed in formalin, treated with formic acid and embedded together in one paraffin block. h&e stain and six immunostains (antibodies against prion protein, α-synuclein, β-amyloid, phospho-tau, phospho-tdp43, p62) were performed. 133 cases were analysed. diseases in which a diagnosis cannot be made by analysing these two brain regions like pure amyotrophic lateral sclerosis and huntington disease have been excluded from this analysis. results: in 96.2% of cases the fast-track neuropathological diagnosis was confirmed by the conventional pathological work-up of the entire brain. only in four cases evaluation of additional brain regions was necessary to make a conclusive histopathological diagnosis. only in one case in our study (of an unusual tauopathy) the fast-track diagnosis needed to be corrected. the suspected clinical diagnosis was neuropathologically confirmed by fast-track histology in only 60.2 % of cases. in 39.8 % the clinical diagnosis was either different or ambiguous. conclusion: our study shows that histological and immunohistochemical screening of two selected brain regions (superior frontal gyrus and cerebellum) is indeed sufficient for establishing a reliable working diagnosis of patients with a neurodegenerative disease in over 96 % of cases. given the rapid workflow, a prompt response from the neuropathology to clinicians may improve the accuracy of clinical diagnosis. this procedure does not allow precise neuropathological disease staging and certain co-pathology cannot be fully appraised. diseases like pure als or hd cannot be neuropathologically verified on these two brain regions and the contribution of vascular pathology to the clinical picture cannot be precisely assessed.   3.06 free neuropathol 3:20:41 neurodegeneration in hsan1 due to atl1 (gly66gln) mutation is associated with defective erprotein quality control and compromised autophagy istvan katona1, hülya-sevcan daimagüler2, haihong guo1, priyanka tripathi1, antonio sechi3, alfred yamoah1, shelisa tey1, michael schröder1, jürgen klingelhöfer4, joachim weis1, anand goswami1 1 uniklinik rwth aachen, institut für neuropathologie, aachen, deutschland 2 uniklinik köln, kinderklinik, köln, deutschland 3 uniklinik rwth aachen, institut für zellund tumorbiologie, aachen, deutschland 4 klinikum chemnitz , klinik für neurologie, chemnitz, deutschland background: atlastin-1 (atl1) functions as a gtpase and is crucial for endoplasmic reticulum (er) shaping and er-microtubule interactions. mutations in atl1 have been reported to cause hereditary sensory and autonomic neuropathy type 1d (hsan1d) as well as hereditary spastic paraplegia 3a (spg3a). atl1 mutation have been linked to abnormal er morphology; still the molecular pathomechanism of defective er structures and their pathological consequences contributing to hsan1d and spg3a have not been investigated in detail so far. question: we asked if/how atl1 participates in autophagy process. methods: we used biochemical and immunocytochemistry approaches followed by live cell imaging in cell culture models overexpressing normal and mutant atl1 proteins. we corroborated our findings with comparative ultrastructural analysis on cell culture models and on biopsy samples. results: we observed that over-expression of mutant atl1 (gly66gln) forms protease resistant large, globular er associated aggregates in cell culture models, which further leads to er stress and structural abnormalities of er and associated compartments. interestingly while endogenous atl1 protein is degraded by ubiquitin proteasome system (ups), mutant atl1 impairs ups, induces proteotoxicity and cell death. autophagy, which activates as a compensatory mechanism, also compromises at multiple steps, probably due to deformities of er and persistent proteotoxic stress. extensive workup of skin, sural nerve and muscle biopsy material of a rare gly66gln hsan1 patient revealed prominent loss of myelinated and unmyelinated sural nerve fibres, but only minor neurogenic muscular atrophy. ultrastructural analysis on this biopsy revealed signs of altered autophagy in axons as well as prominent alterations of schwann cell nuclei/nuclear envelope. in line with this, hsan1 patient's fibroblasts showed similar defects. conclusions: overall, our results support the notion that neurodegeneration in hsan1 due to atl1 mutation is closely linked with the deformed er and associated functions including autophagy. neurons and distal axons are particularly vulnerable to such pathomechanism, thus explaining the degenerative phenotype in hsan 1 and related diseases.   3.07 free neuropathol 3:20:42 single-nucleus chromatin accessibility profiling in four-repeat tauopathies viktoria ruf1, nils briel1,2, sigrun roeber1, janina mielke1, mario m dorostkar1, otto windl1,2, thomas arzberger1,2,3, felix l. strübing1,2, jochen herms1,2,4 1 zentrum für neuropathologie und prionforschung, münchen, deutschland 2 deutsches zentrum für neurodegenerative erkrankungen, münchen, deutschland 3 klinik für psychiatrie und psychotherapie, münchen, deutschland 4 munich cluster of systems neurology (synergy), münchen, deutschland background: progressive supranuclear palsy (psp) and corticobasal degeneration (cbd) are sporadic neurodegenerative diseases characterized by aggregates of hyperphosphorylated four-repeat tau (4r-tau) in neurons, oligodendrocytes and astrocytes, where tufted astrocytes (ta) are a typical hallmark of psp, whereas astrocytic plaques (ap) are pathognomonic for cbd. the molecular mechanisms underlying tau aggregation and neurodegeneration are largely unclear. aims and questions: to characterize chromatin accessibility profiles of psp and cbd using atac-seq (assay for transposase-accessible chromatin using sequencing) to contribute to a better understanding of the underlying pathomechanisms of psp and cbd. methods: atac-seq was performed on 45,000 isolated single nuclei from the frontal cortex of 4 psp and 4 cbd patients and 5 healthy controls. after preprocessing and quality control, gene accessibility (ga), gene ontology (go) and transcription factor motif enrichment (tfme) analysis were conducted using snapatac. gchromvar was applied to map genetic risk variants to peaks and cicero/tradeseq was used for pseudotime analysis and repeated tfme analysis. results: we found that ga of tauopathy-associated genes was substantially altered in the brains of psp and cbd patients and could demonstrate that pspand ftd-associated genetic risk variants are particularly linked to astrocytic chromatin accessibility profiles. go enrichment analysis of significantly different transcription factor motifs (tfm) in astrocytes identified numerous motifs belonging to the immediate early response (ier; e.g. fos and jun family) or intracellular homeostasis and protein degradation (e.g. maf family and nfe2). moreover, pathway analysis highlighted immunological terms related to both innate and acquired immunity. pseudotime analysis of transcription factor motif enrichment (tfme) revealed for cbd a trajectory terminating in a population of cbd-derived astrocytes, whereas for psp no such disease-defined cluster was apparent. accordingly, a decrease was observed for tfs associated with early state astrocytic differentiation in cbd, while tfme of ier-related tfs considerably increased along the pseudotime axis. conclusions: our study revealed genetic dysregulation affecting neurodegenerative, neuroinflammatory and degradation processes and particularly points to a major role of astrocytes in the pathogenesis of psp and cbd. however, to understand the downstream mechanisms, further independent validation especially at the transcriptome and protein level will be necessary. github ressource: https://github.com/nes-b/snatac-seq_psp_cbd   3.08 free neuropathol 3:20:43 application of a human stem cell transplantation model of alzheimer’s disease to examine disease-associated changes at a single cell level in vivo wenhui qu1, matti lam2, aayushi mahajan3, nelson humala3, osama al dalahmah1, jason mares2, trang nguyen1, ismael santa-maria1, andrew sproul1, markus siegelin1, james goldman1, peter canoll1, vilas menon2, gunnar hargus1 1 columbia university medical center, department of pathology and cell biology, new york, united states 2 columbia university medical center, department of neurology, new york, united states 3 columbia university medical center, department of neurosurgery, new york, united states background: alzheimer’s disease (ad) is the most common type of dementia and is characterized by widespread degeneration of the central nervous system with amyloid and tau pathology leading to severe impairment of learning and memory. despite the high number of patients with ad, the molecular mechanisms leading to neurodegeneration are only partially understood and effective treatment options still do not exist. aims/questions: here, we applied a human stem cell model of ad to study disease-modifying roles of neural cells at a single cell level in vivo. methods: to this end, we differentiated induced pluripotent stem cells (ipscs) with the familial ad-associated appv717i mutation in the amyloid precursor protein as well as isogenic control cells into neural progenitor cells (npcs) and neurons. results: appv717i neurons showed reduced neurite outgrowth and demonstrated an increased susceptibility towards oxidative stress with changes in metabolic programs. we injected appv717i and control npcs into the brains of immunocompromised nsg mice resulting in neuronal grafts in both groups two months after injection with activation of microglial cells and presence of reactive astrocytes within and around the grafts. we then performed single nucleus rna sequencing (snrna-seq) on microdissected grafts to characterize gene expression and dysregulated pathways in appv717i versus control neurons as well as in astroglial cells and microglia in response to appv717i and control neurons at a single cell level. conclusions: our findings show that ipscs represent a powerful cell source to study mechanisms of disease development in vitro and in vivo. this stem cell model of ad could also be used as a cellular platform for high-throughput drug screening purposes to identify potential therapeutic targets in ad.   4. neuroinflammation 4.01 free neuropathol 3:20:44 pathological and genetic characterization of jc virus encephalopathy with an eleven-year-long disease course marco mlynek1, adriane kuttlovci1, marek jauß2, cornelia tennstedt-schenk3, lidia stork1, christine stadelmann1, imke metz1 1 institut für neuropathologie universitätsmedizin göttingen, göttingen, deutschland 2 ökumenisches hainich klinikum ggmbh, mühlhausen/thüringen, deutschland 3 institut für pathologie, mühlhausen/thüringen, deutschland the jc polyomavirus (jcpyv) typically causes progressive multifocal leukoencephalopathy (pml) in immunocompromised humans. the pathology shows demyelinated white matter lesions with infection of glial cells, but very few infected cortical pyramidal neurons. in contrast, another jcpyv-associated disease is jc virus encephalopathy (jcve), which is characterized by numerous infected neurons. while the non-coding control region of jcpyv in pml shows genetic rearrangements (so-called prototype), in jcve the archetype virus is found, which is also present in healthy individuals. we provide a detailed clinical, (histo)pathological and genetic description of a patient with jcve. we analyzed different lesion areas for viral infection and replication of glial and neuronal cells. in addition, demyelination, cell loss and axonal damage were investigated. we present a 54-year-old male patient with an exceptionally long disease course over eleven years who was diagnosed with jcve. he developed slowly progressing myoclonia, numbness of the upper and lower extremities, pharmacoresistant seizures and a general cognitive decline. mri showed a progressive atrophy of the cortex with cortical and subcortical parenchymal lesions. there was no clear evidence of immunosuppression. a variant of unclear significance in the interferon-induced with helicase c domain 1 (ifih1) gene was found. he died due to status epilepticus and pneumonia. macroscopy showed extensive cystic white matter lesions with preservation of the cortical ribbon (figure 1a). histology revealed in addition lesions with ongoing viral infection. here, the number of infected neuronal and glial cells outnumbered clearly the number of replicating cells, suggesting a limited viral replication (figure 1b). nevertheless, a pronounced decrease in oligodendrocytes was observed, while no major loss of astrocytes and neurons was found. juxtacortical lesions showed prominent axonal damage and loss. late lesions were characterized by a necrotic-cystic tissue defect located primarily at the gray-white junction and later spreading throughout the white matter. only few publications describe jcve as a new entity of jc virus pathologies characterized by a prominent infection of neurons. what distinguishes our case from others is the extraordinarily long disease course without clear evidence of immunodeficiency. a variant of unclear significance in the ifih1 gene was found. ifih1 encodes the mda5 protein (pattern-recognition-receptor), which is part of the innate immune system and is mainly responsible for the activation of the antiviral immune response such as the induction of interferon-1/-β/-α. this mutation potentially predisposed to the jcpyv infection. the slow disease progression in our patient is possibly due to relatively well-preserved immune function. the presence of a stable, archetype variant could also indicate a less productive viral variant. our detailed histological description shows that infected cells clearly outnumbered replicating cells, leading to production of virus in only single cells. despite numerous infected neurons, in the long run white matter lesions with a pronounced tissue destruction prevailed. thus, destructive white matter lesions seem to be a pivotal histopathological characteristic of jcve. figure 1: (a) cortex with juxtacortical lesion (arrow) and massive cystic changes within the white matter (wm). (b) multiple infected cortical pyramidal neurons (t-ag = early viral protein, indicating infection, neun = neuronal marker).   4.02 free neuropathol 3:20:46 reduction of oligodendrocyte populations in patients with late-onset multiple sclerosis schirin stephan1, lidia stork1, wolfgang brück1, christine stadelmann-nessler1, imke metz1 1 university medical center göttingen, institute of neuropathology, göttingen, deutschland background: over 2.8 million people worldwide suffer from multiple sclerosis (ms) a chronic inflammatory demyelinating disease of the cns. typical or normal-onset ms (noms) manifests in young adults (20-40 years old). in 3-12% of cases, the disease begins in elderly patients >50 years of age (late-onset ms or loms). such patients more often suffer from primary progressive ms, faster disease progression, and worse recovery from relapses, as well as age-related comorbidities. animal studies suggest a less efficient remyelination in elderly animals. marked remyelination, which is observed in 23-50 % of ms lesions in young adult-onset patients, could be slower and less successful in loms. a recent study showed that cells expressing bcas1 represent a subpopulation of actively myelinating oligodendrocytes, and can serve as a reliable marker of ongoing remyelination. questions: we addressed the question, if myelin regeneration in loms patients is less effective. methods: we performed histological analyses of three oligodendrocyte markers that correspond to different oligodendrocyte maturation stages (olig2 positive cells with a strong nuclear signal for oligodendrocyte precursor cells, bcas1 for myelinating oligodendrocytes, and nogoa for mature oligodendrocytes) in biopsy specimens from loms patients (n=30), and compared them with noms patients (n=25). results: the number of mature oligodendrocytes was significantly lower in the normal appearing (non-demyelinated) (p=0.02) and perilesional white matter (p<0.0001) in patients with loms compared to noms. moreover, mature oligodendrocytes in these regions showed a negative correlation with the age of patients (r=-0.5, p=0.01). there were no differences in the number of oligodendrocyte precursor cells between the two groups. also, the population of active myelinating bcas1+ oligodendrocytes was significantly lower in loms compared to noms patients in the normal-appearing white matter (p=0.03). again, the number of myelinating cells correlated negatively with the age of patients (r=-0.5, p=0.01). in both groups, the highest number of active myelinating oligodendrocytes was found in early active demyelinating lesions and here at the lesion edge. although no significant differences in the number of active myelinating cells was found in lesion areas comparing loms and noms, a trend for higher numbers of myelinating cells was observed in the center of late active lesions in noms. in general, older lesions (late active demyelinating and inactive lesions) showed lower numbers of active myelinating cells. loms patients had a significantly higher edss score (median 3.5, p=0.003) at last follow-up as compared to noms patients (median 2.0). a higher edss score was associated with a lower number of both mature and oligodendrocyte precursor cells in active ms lesions (r=-0.42, p=0.01 and r=-0.54, p=0.002 respectively). conclusions: we observed a significant reduction of active myelinating and mature oligodendrocytes in the periplaque and normal appearing white matter of loms patients. this may result in a lower remyelination within lesions. our results suggest a more efficient remyelination in noms compared to loms. importantly, we show that a higher edss at last follow-up in loms patients negatively correlates with the number of oligodendrocytes within lesions, emphasizing the importance of a loss of oligodendrocytes for clinical disability. study supported by sanofi genzyme.   4.03 free neuropathol 3:20:47 schwann cell remyelination is a salient feature of spinal nmo with neuroprotective potential carolina thomas1,2, josephine sophia owens1, parinaz yavarzadeh1, anne winkler1, christine stadelmann1 1 institut für neuropathologie universitätsmedizin göttingen, göttingen, deutschland 2 max-planck-institut für multidisziplinäre naturwissenschaften, city campus abteilung molekulare neurobiologie, göttingen, deutschland background: neuromyelitis optica (nmo) is a severe autoimmune demyelinating disorder characterized by the presence of pathogenic autoantibodies against the water channel aquaporin-4, and clinically by extensive myelitis with optic neuritis. intriguingly, despite the complete loss of astrocytes with consecutive damage and reduction of oligodendrocytes, a partial remyelination of nmo lesions has been previously described. aims: here we aimed to decipher the cellular components present in remyelinated spinal nmo lesions and to detect potential protective effects of remyelination on axonal integrity in these lesions. methods: we performed post-mortem histopathological and immunohistochemical examination of spinal lesions from a cohort of 8 patients. results: we demonstrate that schwann cells (sc) are present and are in part responsible for remyelination of nmo lesions. furthermore, we show a greater abundance of remyelination in the lesions localized along peripheral nerve entry-exit zones as well as perivascularly. in addition, no sc remyelination was observed in spinal lesions of patients suffering from multiple sclerosis. finally, axonal density was partially preserved in sc remyelinated areas. conclusions: taken together, our results indicate that scr is an exclusive feature of nmo that requires a breaching of the glia limitans with potential neuroprotective effects.   5. muscle / nerve 5.01 free neuropathol 3:20:48 molecular profiling of skeletal muscle in infantile, juvenile and adult patients with pompe disease alexander schaiter1, andreas roos2,3,4, andreas hentschel5, andreas hahn6, marek bartkuhn7,8, anne schänzer1 1 institute of neuropathology, justus liebig university, giessen, deutschland 2 department of pediatric neurology, centre for neuromuscular disorders, centre for translational neuroand behavioral sciences, university duisburg-essen, essen, deutschland 3 children's hospital of eastern ontario research institute; division of neurology, department of medicine, the ottawa hospital; and brain and mind research institute, university of ottawa, ottawa, canada 4 department of neurology, heimer institute for muscle research, university hospital bergmannsheil, ruhr-university bochum, bochum, deutschland 5 leibniz-institute für analytische wissenschaften – isas – e.v., dortmund, deutschland 6 department of child neurology, justus liebig university, giessen, deutschland 7 institute of biomedical informatics and systems medicine science unit for basic and clinical medicine, justus liebig university, giessen, deutschland 8 institute for lung health (ilh) platform for genomics and bioinformatics, justus liebig university, giessen, deutschland introduction: pompe disease is a lysosomal metabolic disease caused by mutations in the alpha 1,4-glucosidase (gaa). the gaa enzyme defect leads to accumulation of glycogen in striated muscles and a reduced muscle function. pompe disease is categorized in infantile onset (iopd) and late onset (lopd) based on gaa residual activity, genetic profile and clinical manifestation. furthermore, a juvenile onset is described, which is classified as lopd. the underlying mechanisms of pompe disease are still unknown. molecular signatures of iopd, lopd and juvenile patients are rare. the aim of this study is to identify specific differences between the subgroups of pompe disease using proteomic analysis. the first objective is to find novel protein expressions associated with the subgroups of pompe disease. the second objective is to compare the proteomic profiles of the subgroups with each other to prove the categorization by a proteomic approach. material and methods: skeletal muscle biopsies from 35 patients with pompe disease before start of enzyme replacement therapy (ert) (lopd n=22, iopd n=11, juvenile n=2) and 21 age matched controls (adult n=11, infantile n=10) were analysed using mass-spectrometry in an lfq (label-free-quantification) experiment. to identify significantly up or down regulated proteins between the groups, the raw proteomic data was further processed using maxquant, perseus and the r-programming language. principal component analysis (pca) and hierarchical clustering was used to show statistical difference between the sample groups. further, a pathway analysis of the significantly associated proteins using the kegg and the gene ontology (go) library was conducted. to validate associated pathways on another approach a gene set enrichment analysis (gsea) was calculated. to compare the proteomic profile of lopd, iopd and juvenile, the most significant proteins of each group were defined as gene-sets in gsea. results: in the iopd 13 proteins were up-regulated and 11 downregulated compared to controls. analyses of juvenile pompe showed 7 up-regulated, 37 downregulated and in lopd 19 up-regulated, 47 down-regulated proteins. the samples of lopd and juvenile shared certain protein expression e.g. fbn1, col1a2 and col3a1 were down-regulated in both groups. however, iopd and lopd showed divergent expression e.g. hk1 (phosphorylation of various hexoses and involved in innate immunity) was up-regulated in lopd and down-regulated in iopd whereas usmg5 (upregulated during skeletal muscle growth) was upregulated only in iopd. the gsea further shows a positive correlation between lopd and juvenile pompe and a negative correlation between iopd and lopd/juvenile pompe. discussion: mass-spectrometry analysis showed a significant altered regulation in skeletal muscle samples from patients with pompe disease compared to controls. interestingly most of the dysregulated proteins varied in iopd and lopd subgroup, whereas the expression profiling was similar in the juvenile patients compared to lopd. these data underline the heterogeneity in pompe disease and may indicate a divergent underlying muscle pathology in infantile patients with more immature skeletal muscle fibers compared to juvenile and adult patients.   5.02 free neuropathol 3:20:50 expression of immune regulating proteins in skeletal muscle of different idiopathic inflammatory myopathies (iim) subtypes anna nishimura1, rebecca hasseli2,3, heidrun h. krämer4, angela roth1, eva neuen-jacob5, tobias ruck6, anne schänzer1 1 institute of neuropathology, justus-liebig-university, giessen, deutschland 2 department of rheumatology and clinical immunology, campus kerckhoff, justus-liebig-university, giessen, deutschland 3 department of internal medicine ii, justus-liebig-university, giessen, deutschland 4 department of neurology, justus-liebig-university, giessen, deutschland 5 institute of neuropathology, heinrich-heine-university, düsseldorf, deutschland 6 department of neurology, medical faculty, heinrich-heine-university, düsseldorf, deutschland background: idiopathic inflammatory myopathies (iims) are autoimmune diseases classified as polymyositis (pm), dermatomyositis (dm), immune-mediated necrotizing myopathy (imnm), anti-synthetase syndrome (asys) and sporadic inclusion body myositis (sibm). adaptive and innate immune responses play a role in the pathogenesis of iim and immunomodulatory treatment is the recommended therapy. the individual regulatory mechanisms in iim subtypes might differ and a better understanding of the pathophysiology would improve the individual therapeutic approaches. with whole muscle section morphometry, we want to analyze the immune regulating proteins in certain subtypes of iim. methods: muscle biopsies from 24 adult patients with iim (average age at biopsy 54.6 years; 65.2 % female; dm n=5; imnm n=5; asys n=7; sibm n=7), neurogenic atrophy (na; n=4) and controls (hc; n=6) were included in the study. all biopsies were re-evaluated according to the common classification. the degree of pathology severity was estimated using a semiquantitative pathology score (p-score from 0 to 10). double immunofluorescence staining was performed with antibodies against mhc-1 (major histocompatibility complex 1), mhc-2, icam-1 (intercellular adhesion molecule 1) and vcam-1 (vascular cell adhesion molecule 1) and antibodies against spectrin or desmin. the sections were subsequently digitalized using a zeiss axio scan.z1 slide scanner. the coexpression was analyzed on entire sections using imagej software and quantified using the manders' coefficient (m). results: mhc-1 expression was significantly upregulated on muscle fibers of patients with asys (m=0.559), dm (m=0.609), sibm (m=0.557) and na (m=0.208) compared to hc (m=0.009). for imnm (m=0.079), the expression was significantly lower compared to asys and dm. significant upregulations of mhc-2 were found for asys (m=0.263), dm (m=0.504), sibm (m=0.336) and na (m=0.416) compared to hc (m=0.036), as well as a significant upregulation for dm compared to imnm (m=0.055). only asys samples showed a significant upregulation of icam-1 compared to hc (m=0.060), whereas in general, the expression varied strongly (asys m=0.263±0.182; dm m=0.270±0.256; sibm m=0.337±0.249; imnm m=0.335±0.295) with no significant difference between the iim subtypes. vcam-1 expression showed no significant difference between the iim subtypes. however, in comparison to hc (m=0.036) we found vcam-1 significantly upregulated in asys (m=0.200), dm (m=0.377) and sibm (m=0.371). correlating our results with the p-score, a positive correlation (r=0.770) for icam-1 expression in sibm was found. conclusion: we analyzed protein expression levels on muscle fibers in whole section analysis, which provides accurate data on larger sections. different expression patterns of mhc-1/2, icam-1 and vcam-1 were found in iim subtypes with lower expressions in imnm for mhc-1/2 and vcam-1. interestingly, in sibm icam-1 expression correlated with the pathology score. these insights might help to improve morphological diagnosis in iim subtypes and identify individual immune response patterns, which may improve the accuracy of future diagnoses.   5.03 free neuropathol 3:20:52 long term safety and efficacy outcomes for x-linked myotubular myopathy (xlmtm) with gene replacement therapy, resamirigene bilparvovec (aspiro): preliminary results from cohort 1 in aspiro, a phase 1/2/3 study astrid blaschek1, perry shieh2, nancy kuntz3, james dowling4, carsten bonnemann5, a. reghan foley5, dimah saade6, andreea seferian7, laurent servais8, neema lakshman9, cong han10, suyash prasad11, salvador rico11, westin miller9 1 klinikum der universität münchen, münchen, deutschland 2 university of california, los angeles, united states 3 ann & robert h. lurie children’s hospital of chicago, chicago, united states 4 the hospital for sick children, toronto, canada 5 national institutes of health, bethesda, united states 6 university of iowa hospitals and clinics medicine specialty clinics, iowa city, united states 7 institut de myologie, paris, france 8 mduk oxford neuromuscular centre, oxford, united kingdom 9 astellas gene therapies, california , united states 10 astellas pharma global development, northbrook, united states 11 formerly astellas gene therapies, california, united states background: xlmtm is a rare, life-threatening congenital myopathy caused by mutations in the mtm1 gene. there is no approved treatment for xlmtm. objectives: aspiro (nct03199469), a phase 1/2/3 randomized, open-label study is investigating the safety and efficacy of at132 (resamirigene bilparvovec), a single-dose gene replacement therapy for ventilatory-dependent xlmtm. question: what is the primary safety and efficacy data from asprio? methods: participants were young boys with genetically confirmed xlmtm. the primary efficacy outcome was the change in hours of daily ventilator support from baseline through week 48. the key secondary efficacy outcome was percentage of participants who achieve functionally independent sitting by week 48. we report long-term safety and key efficacy outcomes (up to 42 months [m]) for the first 6 participants dosed in aspiro, all receiving the lower-dose of 1.3 x 1014 vg/kg and compared with 15 untreated controls (including 12 participants from inceptus), as of 29jan2021. results: the mean age at dosing was 20·4m (range: 9·5-49·7m) and 19·6m (5·9-39·3m) at enrollment among dosed participants and controls, respectively. major developmental milestones achieved by all dosed participants over time is shown in figure 1. all dosed participants were ventilator dependent at first assessment; 5 (83.33%) requiring transtracheal invasive ventilation >22 hours/day and 1 (16.67%) used non-invasive ventilation of 12 hours/day. all dosed participants achieved ventilator independence, 5 remain so (mean durability 25.6m; range 18.3-36.6m) of which 4 have been decannulated. no control participants became ventilator independent or were decannulated. at baseline, 1/6 dosed participant was able to sit independently without support for 30 seconds; 5/6 participants did not have full head control and were unable to sit independently. major motor milestones were achieved in all dosed participants (figure 1); 5/6 remain independently ambulatory without assistive device (achieved mean [sd] time 21.92 [5.57]m); 4 of whom have achieved the ability to ascend stairs. 5/15 (33.3%) control participants achieved independent sitting; none achieved higher milestones. among 6 dosed in cohort 1, 4 (67%) participants experienced treatment-emergent severe adverse events; infections in 4 (67%) and respiratory/thoracic/mediastinal disorders in 1(17%). all dosed participants currently have stable liver function test values. as of january 2021, three deaths in the higher-dose cohort occurred following severe decompensated liver disease, and three deaths in the control cohort (aspiration pneumonia; cardiopulmonary failure; hepatic hemorrhage with peliosis) were observed. as of september 2021, a newly dosed participant in the lower-dose cohort experienced severe liver function test abnormalities and has died. conclusions: a rapid improvement in respiratory and motor outcomes was observed among 6 xlmtm participants dosed with at132 at 1.3 x 1014 vg/kg vs control participants; these improvements have been maintained and expanded upon over time, indicating improved strength, function, and quality of life for these dosed participants. these substantial improvements must be weighed against the occurrences of fatal serious adverse events, for which the aspiro program is on clinical hold while relevant clinical information is being gathered and reviewed. figure 1: developmental motor and respiratory milestones achieved in xlmtm patients dosed at at132 at 1.3 x 1014 vg/kg.   5.04 free neuropathol 3:20:54 lymphotoxin-driven chronic mucle inflammation interdepends with impaired autophagy, self-perpetuates and models inclusion body myositis in mice juliane bremer1, judith bauer2, jana zschüntzsch3, thomas blank4, kamil zajt1, laura anna fischer3, anna sensmeyer3, lara wiechers3, josef reichelt3, kai hofmann5, monika wolf6, corinna leuchtenberger5, priyanka tripathi1, claudia einer7, hans zischka7, adriano aguzzi6, regina reimann6, veronika kana6,8, elisabeth rushing6, marco prinz4, david liebetanz3, francesca odoardi3, joachim weis1, jens schmidt3,9, mathias heikenwälder5 1 uniklinik rwth aachen, institut für neuropathologie, aachen, deutschland 2 technische universität münchen, institut für toxikologie und umwelthygiene, münchen, deutschland 3 universitätsmedizin göttingen, klinik für neurologie, göttingen, deutschland 4 universitätsklinikum freiburg, institut für neuropathologie, freiburg, deutschland 5 dkfz heidelberg, heidelberg, deutschland 6 universitätsspital zürich, institut für neuropathologie, zürich, switzerland 7 helmholtz münchen, institut für molekulare toxikologie und pharmakologie , münchen, deutschland 8 universitätsspital zürich, klinik für neurologie, zürich, switzerland 9 universitätsklinik der medizinischen hochschule brandenburg, abteilung neurologie und schmerztherapie, rüdersdorf, deutschland background, goals and research question: inclusion body myositis (ibm) is a progressive muscular disorder characterized by muscle inflammation and degeneration including protein aggregates and altered autophagic activity. the combination of inflammatory and degenerative features has led to the assumption that this interrelationship may serve as a major driver of the disease pathology. in view of the lack of effective treatment there is an urgent need for useful model systems that reflect the presumed pathomechanisms. a reliable animal model for chronic inflammatory and degenerative features as in ibm has not been available. methods/results: here, we established mouse models with lymphotoxin expression-driven chronic inflammation of skeletal muscle and with impaired autophagy due to muscle cell specific atg5-knockout. both conditions alone induced weakness and muscular atrophy. gene expression analysis showed that, while chronic inflammation alone drives endoplasmic reticulum stress and alters autophagy/ proteostasis, autophagy disruption alone induces a pro-inflammatory state. this suggests that both, inflammation and autophagy disruption are interdependent in ibm pathogenesis. only when we genetically combined transgene-driven inflammation with autophagy impairment in mice, skeletal muscle fibers displayed characteristic molecular and neuropathological features of ibm, including protein aggregates with typical ultrastructural morphology. given that human ibm is refractory to established drugs, we aimed to mirror recent treatment failures and identify the underlying mechanisms by subjecting four months-old myositis mice to corticosteroids, anti-cd52 or anti-thy1.2 antibodies to deplete lymphocytes, or by blocking lymphotoxin beta-receptor signaling. none of these treatments was able to significantly improve muscular performance of the mice or expression profiles of molecular indicators of muscle pathology. this suggests that, once established, ibm-like pathology cannot be reverted or prevented from progression, but is a self-perpetuating condition. conclusions: in summary, the data provide unique evidence that inflammation and autophagy disruption are intertwined in ibm-like muscular pathology. it can be expected that this novel mouse model will substantially further our effort to identify better treatment modalities for ibm in the future.   5.05 free neuropathol 3:20:56 novel form of congenital myopathy caused by bi-allelic mutations in uncoordinated mutant number-45 myosin chaperone b sebahattin cirak1 1 uniklinik ulm, klinik für kinder-und jugendmedizin, sozialpädiatrisches zentrum und pädiatrische neurologie mit stoffwechsel, ulm, deutschland background: congenital myopathies (cm) form a genetically heterogeneous group of disorders, only 60% can be genetically solved. aims: discovery of novel genetically defined myopathies. question: deciphering the genomic landscape of congenital myopathies. methods: we recruited an 11-year old male of consanguineous parents, presenting with proximal weakness, gower's sign, without cardiomyopathy with a stable disease course. we performed exome sequencing and data analysis was performed with our in-house software varbank2 according to an autosomal recessive inheritance. we investigated the effect of the missense mutation by complementation assay on the zebrafish steif mutant, an unc-45b loss-of-function model. results: we have discovered and published a novel genetically defined form of cm due to a novel homozygous missense mutation in unc45b (nm_173167.2: c.2261g>a, p.arg754gln) also co-segregating in the family with three healthy siblings (dafsari et al., 2019). in our patient's muscle biopsy, core-like structures were detected mainly in the center of muscle fibers in nadh histochemistry. electron microscopy showed numerous focal core-like alterations of myofibrillar architecture with z-bands streaming. conclusions: three isoforms of unc45b are highly expressed in skeletal muscle, only one also in cardiac muscle. due to its high evolutionary conservation throughout species, a loss of unc45 results in different pathological conditions in various species: a knockdown of unc-45 resulted in dilated cardiomyopathy and a reduced muscle contractility in d. melanogaster. similarly, in unc-45b knockdown zebrafish and also in steif mutants, disrupted myofibrillogenesis associated cardiac dysfunction and paralysis was observed. injection of mutant unc-45b mrna did not rescue the steif mutant in contrast to wt mrna confirming the pathogenicity of the missense mutation.   6. free topics 6.01 free neuropathol 3:20:57 deep genotype-phenotype analysis of focal cortical dysplasia type 2 differentiates between a gator-positive autophagy altered subtype 2a and mtor-positive migration deficit subtype 2b jonas honke1, lucas hoffmann1, roland coras1, javier a. lópez-rivera2,3,4, costin leu3,4,5,6, dennis lal3,4,5,6, peter nürnberg6, tom pieper7, till hartlieb7, manfred kudernatsch7, christian g. bien8, friedrich woermann8, thomas cloppenborg8, hajo hamer9, sebastian brandner10, karl rössler11, arnd dörfler12, stéphanie baulac13, sara baldassari13, ingmar blümcke1,4 1 department of neuropathology, universitätsklinikum erlangen, fau erlangen-nürnberg, and partner of the european reference network (ern) epicare , erlangen, deutschland 1 department of molecular medicine, cleveland clinic lerner college of medicine, case western reserve university, cleveland, united states 1 genomic medicine institute, lerner research institute, cleveland clinic, cleveland, oh 44195, united states 1 charles shor epilepsy center, neurological institute, cleveland clinic, cleveland, united states 1 stanley center for psychiatric research, broad institute of harvard and m.i.t, cambridge, ma 02142, united states 1 cologne center for genomics (ccg), medical faculty of the university of cologne, university hospital of cologne, 50931 cologne, germany 1 center for pediatric neurology, neurorehabilitation, and epileptology, schoen-clinic, vogtareuth, germany 1 department of epileptology (krankenhaus mara), medical school, bielefeld university, bielefeld, germany 1 epilepsy center, universitätsklinikum erlangen, fau erlangen-nürnberg, and epicare partner, erlangen, germany 1 department of neurosurgery, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany 1 department of neurosurgery, medical university of vienna, vienna general hospital, vienna, austria 1 department of neuroradiology, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany 1 sorbonne université, institut du cerveau paris brain institute icm, inserm, cnrs, aphp, hôpital de la pitié salpêtrière, paris, france background: focal cortical dysplasia type 2 (fcd2) is the single most common cause of drug-resistant focal epilepsy in children. despite continuous progress in diagnostic methods, however, the recognition and treatment of fcd2 subtypes remain a challenging issue in clinical practice. herein, we performed a deep genotype-phenotype analysis to further elucidate the clinical-pathological and genetic presentation of fcd2. methods: patients with focal epilepsy submitted to neurosurgical treatment, histopathological confirmed diagnosis of fcd ilae type 2 and positive genetic testing were retrieved from the european epilepsy brain bank. clinical data were available from the contributing epilepsy centers. deep whole-exome sequencing with a coverage of >350x or mtor gene panel sequencing with a coverage of >1000x were obtained from fresh frozen tissue samples. histopathological analyses were performed from formalin-fixed, paraffin embedded tissue samples using he and immunohistochemistry for nf-smi32, neun, ps6, p62, and vimentin. all slides were digitalised and further analysed with qupath v.0.3.0. results: seventeen patients were identified by carrying pathogenetic variants in genes directly associated with the mtor pathway, i.e., loss-of-function in the gator1 complex (depdc5, n=7 and nprl3, n=3), or gain-of-function in the mtorc1 signalling pathway [mtor, n=7]. all patients were seizure-free after surgery with the exception of four patients carrying a depdc5 mutation. histopathological analysis revealed a fcd2a subtype in all cases with gator1 alteration, i.e., no balloon cells. in contrast, the fcd2b subtype was predominant in cases with gene variants affecting the mtorc1 signalling complex. specimens carrying mtor variants also had significantly larger dysmorphic neurons than gator1 variants [p=0.005]. in addition, five cases defined by gator1 variants showed a unique and predominantly vacuolizing phenotype (figure 1c). all cases with gator1 alterations were located in the frontal lobe and the majority was confined to the cortical ribbon not affecting the white matter (figure 1a). this pattern was reflected by subtle or negative mri findings in 9/10 patients with gator1 variants. discussion/conclusion: we describe a yet unrecognized genotype-phenotype correlation of gator1 variants with fcd2a in the frontal lobe. these lesions were histopathological further characterized by abnormally vacuolizing cells suggestive of an autophagy altered phenotype. from recently published evidence, we also hypothesize that this subtype will carry a second hit brain somatic variant in the depdc5 gene, which was, however, difficult to identify with techniques applied in our current study. in contrast, patients with fcd2b and brain somatic mtor variants showed larger lesions on mri including the white matter, suggesting compromised neural migration (figure 1b). these data may help to better understand difficult-to-diagnose and treat fcd subtypes, i.e., ilae type 2a and 2b. figure 1: genotype-phenotype analysis of fcd ilae type 2 a: 42-year-old male patient with frontal lobe epilepsy since age 5 years, histopathological confirmed fcd2a and a pathogenic depdc5 mosaicism. the arrow points to the sharp border between the cortical fcd and the normal appearing white matter (wm). ncx normal 6-layered neocortex. neurofilament smi32 immunohistochemistry. scale bar = 2.5mm (applies also to b). higher magnification in c reveals dysmorphic neurons with a vacuolizing predominant phenotype (red arrow) suggesting an autophagy altered phenotype. scale bar = 100µm (applies also to d). b: 19-year-old male patient with frontal lobe epilepsy since age 9 years, histopathological confirmed fcd2b at a bottom-of-sulcus (bos; higher magnification in d) and a pathogenic mtor mosaicism. dysmorphic neurons and balloon cells (latter not shown) were aggregating in the neocortex and white matter (arrow) suggesting a migration deficient phenotype.   6.02 free neuropathol 3:20:60 age-dependent increase of perineuronal nets in the human hippocampus of patients with and without temporal lobe epilepsy annika lehner1, lucas hoffmann1, friedrich paulsen2, hajo hamer3, katrin walther3, karl rössler4, sebastian brandner5, ingmar blümcke1 1 universitätsklinikum erlangen and fau erlangen-nürnberg, department of neuropathology, erlangen, deutschland 2 fau erlangen-nürnberg, institute of functional and clinical anatomy, erlangen, deutschland 3 universitätsklinikum erlangen and fau erlangen-nürnberg, epilepsy center, neurological institute, erlangen, deutschland 4 medical university of vienna, vienna general hospital, department of neurosurgery, vienna, austria 5 universitätsklinikum erlangen and fau erlangen-nürnberg, department of neurosurgery, erlangen, deutschland background: perineuronal nets (pnn) are a specialized extracellular matrix surrounding parvalbumin-positive gabaergic interneurons of the central nervous system, contain mainly aggrecan and lecticans, and play a role in the regulation of synaptic plasticity, brain maturation and cognitive impairment. in the human neocortex, pnn appear in their full expression at an age of eight, and alterations of pnn have been found in neurological disorders including alzheimer´s disease, schizophrenia or epilepsy. however, their role in functional maintenance of the cns as well as disease pathogenesis remains to be elucidated. aim: our aim was to histopathologically assess pnn in the hippocampus of patients with temporal lobe epilepsy (tle) compared to age-matched post-mortem control subjects. methods: formalin-fixed and paraffin-embedded neurosurgical tissue specimens of the human hippocampus were retrieved from the european epilepsy brain bank. twenty-nine patients had histopathologically confirmed hippocampal sclerosis (hs), and 11 patients suffered from tle with no hs. neuropsychology scores for cognitive tasks were retrieved from the files of the erlangen epilepsy center. hippocampus samples of 27 postmortem brains served as control (age range 3-84). pnn were visualized using antibodies directed against aggrecan (figure 1). pnn were manually recorded in the dentate gyrus, ca1, ca2, ca3, ca4 and subiculum. pnn density per mm² was then compared between different hs subtypes according to the ilae classification scheme and controls. selected cases were double immunofluorescence labeled with antibodies against parvalbumin and aggrecan (figure 1) and further analyzed on fully digitalized scans (hamamtsu nanozoomer s60) with qupath 0.3.0. results: the density of pnn increased with age in both, human controls and tle patients (figure 2). however, the density of pnn was significantly higher in all tle patients compared to age-matched controls with a non-significant increase in patients with hippocampal sclerosis compared to tle patients without hs. these alterations were also region-specific and most obvious in the subiculum, ca1 and dentate gyrus (figure 3). there was no significant correlation with cognitive impairment in our cohort of tle patients. discussion: we quantitatively described the normal distribution of pnn in the human hippocampal formation. we investigated changes with age in the number of perineuronal nets in older patients and found new evidence that epilepsy alters perineuronal nets. figure 1: triple immunofluorescence-staining at 40x magnification: a parvalbumin-positive neuron surrounded by aggrecan (left side). immunohistochemical staining of a pnn in the stratum plexiforme of the dentate gyrus (150 µm away from stratum granulosum), screenshot from qupath, 40x magnification (right side). figure 2: correlation between the age and the average number of pnn per mm² in patients with and without epilepsy figure 3: the normal distribution of pnn and alterations in epilepsy   6.03 free neuropathol 3:20:63 vakuolisierung der dura als nicht-lymphassoziierte veränderung ralf schober1, christian eisenlöffel1, max holzer2 1 klinikum st. georg, inst. f. pathologie u. tumordiagnostik, leipzig, deutschlandy 2 paul-flechsig-institut für hirnforschung, univ. leipzig, leipzig, deutschland die meningealen lymphgefäße stellen eine zwischenstation beim abtransport des liquors vom glymphatischen system des gehirns zu den zervikalen lymphknoten dar (louveau j ea 2015, aspelund a ea 2015). sie gelten als wichtiges immun-portal für das zentrale nervensystem (tavares ga u. louveau a 2021), und eine verlegung des glymphatischen systems wird pathogenetisch mit der demenz und insbesondere der alzheimer'schen erkrankung in verbindung gebracht (nedergaard m u. goldman sa 2020). histologische darstellungen der überwiegend sinus-nah in der dura gelegenen lymphkanäle beziehen sich bislang hauptsächlich auf die ratte, detailliertere untersuchungen beim menschen und insbesondere bei verschiedenen erkrankungen stehen noch aus. wir haben in 35 sektionsfällen die dura untersucht, meist im bereich des sinus sagittalis superior, teilweise zusätzlich des sinus transversus. das alter der 14 weiblichen und 21 männlichen patienten und patientinnen betrug 25-91 jahre, im durchschnitt 69 jahre. in 8 fällen war ein morbus alzheimer diagnostiziert worden, in 9 fällen alzheimer-assoziierte veränderungen, in je einem fall part und morbus parkinson. 6 fälle hatten meningitische veränderungen, 2 fälle eine meningeosis carcinomatosa, 2 fälle eine av-fistel, 3 fälle intra-/subdurale blutungen, und 3 fälle eine sinusthrombose. im histologischen bild vorherrschend und in allen außer 3 fällen nachweisbar waren jedoch nicht die lymphgefäße, sondern gruppen kleiner intraduraler vakuolen, 30-300 µ im durchmesser, meist glatt begrenzt und ohne zellbelag. bei immunhistochemischen untersuchungen in ausgewählten fällen zeigten sie keine positive reaktion auf podoplanin, cd31, cd34, s100, aβ und app. dagegen ließen sich lymphgefäße deutlich anhand ihrer positiven reaktion auf podoplanin identifizieren, sie erschienen durchweg streifenförmig-schmal und regelrecht strukturiert. die anzahl der vakuolen-gruppen und ihre dichte in der dura wurde semiquantitativ in 6 stufen von nicht vorhanden bis stark eingeteilt, und diese stufen wurden jeweils den falldaten gegenübergestellt. eine korrelation mit bestimmten erkrankungen ergab sich nicht, lediglich eine partielle korrelation mit dem alter. deutlich vermehrt waren vakuolen bei gleichzeitigem nachweis einer größeren anzahl fibrosierter pacchionischer granulationen vorhanden. zusammenfassend stellt eine vakuolisierung der dura im sinusdach-bereich eine relativ häufige veränderung dar, nicht im zusammenhang mit dem lymphsystem stehend sowie offenbar nicht krankheits-korreliert und am ehesten degenerativer natur. die befunde bestätigen und ergänzen kurze angaben der älteren literatur, wo zunächst von grüppchen großblasiger zellen gesprochen wird (schaffer j 1933), dann aber von altersveränderungen mit hohlraumbildungen durch aufquellung und zerfall von bindegewebsfasern (schaltenbrand g u. dorn e 1955).   6.04 free neuropathol 3:20:64 moghe with or without slc35a2 brain somatic mutations reveal a common phenotype of oligodendroglial regeneration and remyelination simon geffers1, lucas hoffmann1, roland coras1, katja kobow1, javier lopez-riviera2,3,4, costin leu3,4,5,6, dennis lal3,4,5,6, peter nürnberg6, stephanie bulac7, till hartlieb8,9, tom pieper8, manfred kudernatsch8,10, christian bien11, hajo hamer12, karl rössler13,14, sebastian brandtner14, christine stadelmann15, ingmar blümcke1 1 department of neuropathology, university hospital erlangen, erlangen, deutschland 2 department of molecular medicine, cleveland clinic lerner college of medicine, cleveland, united states 3 genomic medicine institute, lerner research center, cleveland clinic, cleveland, united states 4 charles shor epilepsy center, neurological institute, cleveland clinic, cleveland, united states 5 stanley center for psychiatric research, broad institute of harvard and m.i.t, cambridge, united states 6 cologne center for genomics (ccg), medical faculty of the university of cologne, university hospital of cologne, cologne, deutschland 7 sorbonne université, institut du cerveau , paris brain institute, paris, france 8 center for pediatric neurology, neurorehabilitation and epileptology, schoen klinik vogtareuth, vogtareuth, deutschland 9 research institute rehabilitation, transition, palliation, pmu salzburg, salzburg, austria 10 center for neurosurgery, epilepsy surgery, spine surgery and scoliosis, schoen klinik vogtareuth, vogtareuth, deutschland 11 department of epileptology (krankenhaus mara), bielefeld university, bielefeld, deutschland 12 epilepsy center, university hospital erlangen, fau erlangen nürnberg, erlangen, deutschland 13 department of neurosurgery, university hospital erlangen, fau erlangen nürnberg, erlangen, deutschland 14 department of neurosurgery, medical university of vienna, vienna general hospital, vienna, austria 15 institute of neuropathology, university medical center göttingen, göttingen, deutschland mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (moghe) is a recently identified disease entity associated with early onset frontal lobe epilepsy in the vast majority of reported cases. brain somatic mutations in the udp-galactose transporter slc35a2 gene were identified in about 50% of published moghe cases as underlying pathogenetic variant. herein, we performed a phenotype-genotype analysis of moghe with slc35a2 mutations compared to moghe without slc25a2 mutations in order to assess a potentially different phenotype-genotype relationship at the microscopy and clinical level. this may also help us to further untangle the yet unknown aetiological spectrum of the moghe disease. we retrieved 39 surgical brain specimens histopathologically characterized as moghe from the archives of the european epilepsy brain bank, 19 with and 20 without brain somatic slc35a2 variants and compared them with 20 age-matched controls, i.e., fcd 2a (n=10) and non-epileptic post-mortem specimens of the frontal neocortex (n=10). semi-quantitative analysis using a panel of immunohistochemical markers addressing oligodendroglial lineage (olig2), maturation (breast cancer amplifying sequence 1; bcas1), proliferation (ki67), and myelination (cnpase, nissl-lfb) were performed. the clinical presentation was not significantly different between both study cohorts (moghe with or without slc35a2 mutations), e.g., median seizure onset was at 1.2 and 1.8 years, respectively. patients were operated at 6.3 and 7.5 years, respectively, and 89% of lesions were localized in the frontal lobe. however, patients with slc35a2 mutated moghe presented with more infantile spasms (61% vs. 25%). patchy areas of increased oligodendroglial cellularity and decreased myelination were matching each other and were present in both groups (figure 1). in addition, regenerative oligodendrocytes immunoreactive for bcas1 were visible in both moghe groups and both groups had higher numbers of bcas-positive oligodendrocytes compared to our age-matched control cohort (figure 2). interestingly, moghe patients operated at an older age (>17 years; n=5) showed lower bcas and oligodendroglial cell numbers which were similar to those observed in our post-mortem control cohort. myelination deficits were also less visible in moghe patients operated at an older age (figure 3). in conclusion, our results revealed a similar genotype-phenotype correlation of moghe w/o slc35a2 brain somatic mutations. these results further suggest a similar oligodendroglial regeneration and remyelination capacity of early-onset moghe subtypes, and that the same udp-galactose transport pathway is likely to play a role in moghe with slc35a2 wildtype urging for a more systematic approach deciphering the pathogenic aetiology of the moghe disease spectrum. figure 1: patchy regions with increased oligodendroglial cell density (heatmap analysis in a, b, e) revealed a matching overlay with patchy areas of hypomyelination (f). moghe with slc35a2 mutations in a, c-f; b – moghe without slc35a2 mutation. figure 2: all moghe samples revealed a higher number of bcas1-immunoreactive oligodendrocytes at early ages, which normalize with further maturation. figure 3: all moghe samples revealed a higher degree of hypomyelination (nissl-luxol-fast-blue, lfb) at early ages, which normalize with further maturation. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. from research to diagnostic application of raman spectroscopy in neurosciences: past and perspectives feel free to add comments by clicking these icons on the sidebar free neuropathology 3:19 (2022) review from research to diagnostic application of raman spectroscopy in neurosciences: past and perspectives gilbert georg klamminger1,2,3, katrin b. m. frauenknecht2,3, michel mittelbronn2,3,4,5,6,7, felix b. kleine borgmann2,3,1,5 1 saarland university medical center and faculty of medicine, homburg, germany 2 national center of pathology (ncp), laboratoire national de santé (lns), dudelange, luxembourg 3 luxembourg center of neuropathology (lcnp), dudelange, luxembourg 4 luxembourg centre of systems biomedicine (lcsb), university of luxembourg (ul), esch-sur-alzette, luxembourg 5 department of cancer research (docr), luxembourg institute of health (lih), luxembourg, luxembourg 6 department of life sciences and medicine (dlsm), university of luxembourg, esch-sur-alzette, luxembourg 7 faculty of science, technology and medicine (fstm), university of luxembourg, esch-sur-alzette, luxembourg corresponding author: felix b. kleine-borgmann · department of oncology (donc) · 84, val fleuri; l-1526 luxembourg · luxembourg felix.kleineborgmann@lih.lu submitted: 31 may 2022 accepted: 17 july 2022 copyedit and layout by: jerry lou published: 05 august 2022 https://doi.org/10.17879/freeneuropathology-2022-4210 keywords: raman spectroscopy, neurooncology, neurodegeneration, neurosurgery, neuropathology, machine learning abstract in recent years, raman spectroscopy has been more and more frequently applied to address research questions in neuroscience. as a non-destructive technique based on inelastic scattering of photons, it can be used for a wide spectrum of applications including neurooncological tumor diagnostics or analysis of misfolded protein aggregates involved in neurodegenerative diseases. progress in the technical development of this method allows for an increasingly detailed analysis of biological samples and may therefore open new fields of applications. the goal of our review is to provide an introduction into raman scattering, its practical usage and also commonly associated pitfalls. furthermore, intraoperative assessment of tumor recurrence using raman based histology images as well as the search for non-invasive ways of diagnosis in neurodegenerative diseases are discussed. some of the applications mentioned here may serve as a basis and possibly set the course for a future use of the technique in clinical practice. covering a broad range of content, this overview can serve not only as a quick and accessible reference tool but also provide more in-depth information on a specific subtopic of interest. introduction as one special method of various vibrational spectroscopic techniques, raman spectroscopy (rs) has been an integral part in neuroscience research for some time now, be it in neuro-oncology for tumor classification1 or for the biochemical description of various protein aggregates in neurodegenerative diseases2. currently it is making its way towards a clinical implementation3. looking at the numerous advantages of rs, the reasons for an increased use in research are obvious: it enables fast and user-friendly (easy to apply) analysis for the purpose of tissue identification (e.g., identification of different brain regions in three mice strains4) by observed changes in the vibrational level of the underlying biochemical and molecular composition. compared to other advanced molecular techniques, reproducible results can be obtained with few requirements regarding sample preparation. the insensitivity to water molecules predestines the technology for its use in a biomedical context. to date, the vast majority of studies using raman spectroscopy examine unprocessed native, or frozen tissue/cells few publications make use of formalin-fixed or paraffin-embedded (ffpe) tissue because raman measurements remain challenging due to the strong contribution of paraffin wax to spectral intensity, thin specimens, and a disruption of the molecular integrity, which is related to the preceding fixation process. the long-term archivability and the large number of available samples, however, suggest use of rs ffpe tissue in pathology is desirable, e.g., for the analysis of tumor heterogeneity, or identification of very small tumor fragments, which could escape diagnostic high throughput of histology samples. the following review and perspective paper is divided into three parts: a) the basics of rs and the most common forms of its application in medical research are presented, b) the use of rs in selected neuroscience disciplines is accentuated with the aim to present different research questions – but even more importantly – the most interesting findings discovered with the help of rs, c) a future outlook for potential application of rs in research but also in the daily clinical work is provided. at this point, the minireview by payne et al.5 needs to be mentioned; it describes in a clear way not only applications of rs in neuroscience, but also sets a special focus on the technical aspects and benefits of advanced spectroscopy-based techniques depending on the particular use case. by contrast, the following work places a special emphasis on topics that will inevitably become relevant to the practicing spectroscopist at some point, such as varying tissue sample requirements in different clinical settings (surgery department/pathology department) or common data processing methods, to name a few. whenever it serves expedient the attentive reader shall be referred to additional more in-depth reading. search for relevant literature a literature search (the search terms “raman”, “raman spectroscopy” were each combined alternately with the terms “brain”, “neuro”, “neuroscience”, “brain tumor”, “tumor”, “neurooncology”, “glioma”, “neurodegeneration”, “neurodegenerative disease”, “alzheimer’s disease”, “parkinson’s disease”, “huntington”, “amyotrophic lateral sclerosis”, “prion disease“, ”multiple sclerosis”, “myelin”, “demyelination”, “stroke” “brain ischemia”, “brain injury”, “muscular diseases”, “brain infections”, “meningitis”, “psychiatry”) was performed, and online databases pubmed central® and google scholar® were browsed for relevant reviews and original articles; other types of literature, such as congress papers, letters, comments e.g., were excluded. after search results were identified, they were hand-screened for eligibility (inclusion criteria: employment of rs on brain/peripheral nervous/muscle tissue, rs on extracellular components/cells of the nervous/muscular system, or rs in relation to neurological/oncological/psychological disorders; exclusion criteria: use of vibrational spectroscopic techniques other than rs) based on title/abstract. within the responsibility of the authors, the final selection of literature was conducted based on the article full text. finally, associated bibliographies of selected publications were searched for additional relevant sources that semantically met the search criteria. only english language literature was considered – even though japanese research groups describe an employment of raman spectroscopy in rat brains, and human brains / brain tumors as early as the 90s6–9. although references to historical developments are pointed out whenever a contemplation of the historical context seemed valuable special focus is set on literature of the years 2021 and 2022, reflecting ongoing research projects/groups such as spectroscopical examination of microglial changes due to sars-cov-2 exposure10 using rs in neuroscience. principle of raman scattering and general spectrometer set up the raman effect is the process of inelastic scattering of photons; this effect was first described in 1928 by c.v. raman, who examined the characteristics of scattered photons when applying a light source on different liquids11,12. for his discovery, the indian physicist won the nobel prize in 193013, but despite the discovery of the raman effect in the first half of the 21st century, it took until late 1960s before it was first used in a biomedical context14–17. the interaction of incident light with a molecule leads to changes in the vibrational state, so that the molecule falls into an excited virtual vibrational state. when returning to the ground state, the largest amount of the incident photons is elastically scattered, which means that the energy of the scattered photon is the same as that of the incident photon (=rayleigh scattering). only a minor part of the scattered light experiences a change in its energy compared to the incident light; in fact when the molecule ends up on a different state in comparison to the ground state, the photon is inelastically scattered. depending on the interaction between the molecule and the photon, inelastically scattered light can have a higher energy (anti-stokes effect) or a lower energy (stokes effect) than the incident light, whereas in practical application mainly stokes scatter is attributed to a resulting raman signal, due to its higher intensity.18 see figure 1 for a visualization of the vibrational states transitions. figure 1. occurring optical phenomena when irradiating a biological sample with a photon source (laser). left: vibrational states (v0, v1, v2) involved in rayleigh and raman scattering. in case of elastic scattering (raleigh scattering), incoming photons temporarily change the vibrational state of a molecule after this excitation, the molecule returns back to the initial vibrational state (v0). in the case of stokes raman scattering, a molecule gains energy due to the excitation process and finally ends up in a higher vibrational state (it rises from v0 to v1) – the scattered photon has lower energy than the incident light. in anti-stokes scattering the molecule ends up on a lower vibrational state after excitation compared to the ground state (it falls from v1 to v0) – therefore, the scattered photon gains energy. right: in contrast, the phenomenon of fluorescence occurs when a molecule absorbs light and thus is temporarily transferred to a higher electronic state (v’0, v’1, v’2). in order to be raman-active as a molecule, i.e., to emit inelastic raman scattering, a change in polarizability is required this already shows a difference to a related and often confused spectroscopic technique, infrared spectroscopy, in which an absorbed photon leads to a change in the dipole moment19. another phenomenon, also based on absorption and often observable as a disruptive factor in raman measurements due to its stronger signal is fluorescence; here the molecule, excited by energy of absorbed photons, leaves the ground electronic state and is transferred to a higher electronic state as soon as it returns to the ground state, energy is re-emitted as fluorescence light20. the interaction of photons with their target molecules resulting in an inelastic raman scattering with a distinct energy difference reflects specific chemical bonds and constitutions. this spectral fingerprint can indicate the identity of the target molecule. a spectrum can therefore be defined as a representation of the intensity values (based on the degree of change in polarizability) and the differing frequencies (raman shift) in a function18,20. the x-axis displays the raman shift in the unit wavenumber cm-1, thereby the wavenumber is reciprocal to the wavelength and thus directly proportional to photon energy19. the conventional experimental application of the process using the pure raman effect is so the called spontaneous raman scattering (sprs). additionally, there are several derivative methods allowing, for example, scattering with enhanced signal intensity or reduced background noise, thus lending themselves to different applications such as raman imaging (e.g., by coherent raman spectroscopy). table 1 gives an overview of the technical background and advantages of commonly used variants of rs in neuroscience. for a more detailed insight into the theoretical aspects of rs the interested reader may refer to cialla-may et al.21, who provides a comprehensive overview in the book “micro-raman spectroscopy: theory and application” by popp et al.22. additionally, hu et al.23, shi et al.24 and evans et al.25 give a good overview about stimulated raman spectroscopy (srs) and coherent anti-stokes raman scattering (cars); zheng et al.26 wrote an instructive review about surface-enhanced raman scattering (sers). table 1. summary of commonly used raman techniques, their physical background and the associated advantages and disadvantages. the exact structure of a raman spectrometer differs depending on the manufacturer and the technology used. only general components and their function are discussed below; additional components such as an additional laser or a special raman substrate are commonly required in spectrometer setups of advanced raman techniques (table 1). with a focus lens, emitted photons of a laser source are focused on the sample, and after interaction with the sample both the elastic and the inelastic scattered photons are collected by a collecting lens. the reflected and elastically scattered light is then separated from the remaining light, typically by a dichroic mirror. a prism or diffraction grating spatially separates the light according to wavelength, leading it to a detection system a photo paper was employed in the classical setup either simultaneously on a charge-coupled device (ccd) or through a monochromator on a photomultiplier tube (pmt) (figure 2). figure 2. schematic and simplified representation of a raman spectrometer set up. as excitation source, typically lasers, is used where the manner of photon generation as well as the wavelength differ. commonly employed excitation wavelengths within the biomedical field are 532nm, 785nm, 830nm, or 1064nm for practical application specific effects on the tissue type of interest as well as potentially induced background signals must be considered individually and adapted according to the experimental setup27. most employed lasers nowadays are diode lasers; with the advantage of portability and favorable energy efficiency, they have replaced the gas-based lasers (helium neon laser, argon-ion laser) that were often used in the past. the type of proton emission can be divided into continuous-wave lasers and pulsed lasers; the former being more common in sprs and the latter being necessary in srs18,28. it is necessary to bundle photons both in the suitable focus on the sample (focus lens) and to collect the scattered photons (collecting lens) after interaction with the sample. next, raleigh scattered photons are filtered by a dichroic mirror and separated according to their wavelength using a diffraction grating; depending on the sampling aperture (exit slits/pinholes) within the setup, a certain number of photons are detected in a final step by the sensitive detection system. while the classical “scanning spectrometer” employs a rotatable grid concentrating the photons on a narrow exit slit and a photomultiplier tube behind detecting rs, modern set ups usually use a ccd detector. this multichannel way of photon detection (a multichannel array chip consisting of several pixels) allows for simultaneous registration and display of all photons, i.e., the whole raman spectra18,28,29. regular wavelength calibration (process of transferring pixel hits on the ccd detector to distinct displayed wavenumbers) is recommended to receive reproducible spectra over the entire duration of the experiment28. raman spectra can be employed in various ways. in addition to the possibility of using them as raw spectra primarily for the identification of biochemical components of a sample, methods called raman microscopy/imaging use the assignment of colors to raman bands (only a limited number of wavenumbers is acquired or analyzed)30 over a scanned sample to generate contrast. when extended to focusing through the depth of the sample, three-dimensional raman images can be built16. raman microscopy/imaging techniques31,32 and computational image generation algorithms have been advanced to generate raman images of various brain pathologies, e.g., gliomas, stroke and demyelination25 or to image metabolism in the brain33–35. using this approach of data visualization, it is possible to obtain a similar look to traditional h&e-stained slides on unstained specimen, which enables histopathological diagnosis36. in raman mapping, the whole raman spectrum for each point of the desired area of the specimen is acquired (either point by point or with an excitation laser forming a line on the sample and measuring simultaneously); using computational analysis afterwards, a visualization of differences in the spectral properties of data points is achieved30. peak assignment raman peaks may occur at first sight in various forms with different characteristics. in addition to certain single peaks that appear narrow and can be assigned to exactly one corresponding functional group, an additive effect of several adjacent raman active molecules in the sample can also result in broad peaks. furthermore, the presence of several contributing components, and thus neighbor dependent changes in the vibrational mode in one specimen, may affect the actual peak in comparison to an isolated measurement20. the application of rs in the biomedical context often pays special attention of the regions within the wavenumbers 400-2000cm-1 and 2700-3500cm-1. these regions, often referred to as "biological fingerprint regions" in the literature, are characterized by a high proportion of raman peaks arriving from functional groups of a typical biological specimen28. an introduction to the use of rs for identification of different molecular functional groups can be found in pezzotti et al.37 (rs and cell biology) , czamara et al.38 (rs and lipids), rygula et al.39 (rs and proteins) and wiercigroch et al.40 (rs and carbohydrates). by using rs on biomolecules such as proteins, it is not only possible to identify molecular functional groups i.e., differentiate between different amino acids/proteins, but also spatial confirmations can be detected since the raman signal is influenced by aromatic/non-aromatic side chains and the backbone of a protein. distinct vibrations result in certain amide bands (amide band a, b, i-vii)41; for example carbonyl stretching modes, n–h bending or c–n stretching results in the widely used amid i (1600-1690 cm−1), amid ii (1480-1580 cm-1) and amid iii peaks (1230-1300 cm−1). they allow further examination of the peptide secondary structure. in larger unordered protein measurements a precise peak attribution may not be possible due its large number of contributors18,39,42–44. lipids are ubiquitous in biological specimen, as they form the membranes of cells and organelles. depending on the literature, spectral properties resulting mainly from the hydrocarbon chain and partly from the polar head group can be assigned to the regions 1050-1200cm-1 (c-c stretching), 1250-1300cm-1 and 1400-1500cm-1 (ch2, ch3 group activity) or also to the regions below 600cm-1 and between 1000-1150 cm-1 (opposite motion of carbon atoms of the hydrocarbon chain). consistently, an area within the high wavenumber region 2700-3500cm-1 (sometimes solely the range between 2800-3100cm-1 is considered in the literature) is reported and attributed in a large part to stretching of c-h groups. in-depth analyzes of peak intensity and distribution in the high wavenumber region allow conclusions to be drawn about the saturation status of fatty acids and the aliphatic/aromatic components of steroids18,38,45,46. an interesting contribution at this point may come from krafft et al.45, who in 2005 measured and characterized twelve brain lipids and further related occurring peaks to their functional groups and pezzotti et al.47, who employed rs to visualize single (phospho-)lipids in neuronal cells. carbohydrates and underlying c-c and c-h structures give rise to bonds in various areas within the raman spectrum18. for a long time, minor attention was paid to the investigation of carbohydrates. although specific peak assignment is possible, in comparison to protein and lipids it remains less specific40. about 30 raman peaks of nucleotides, distributed over several areas within the spectrum, are mostly attributed due to purine/pyrimidine ring modes and phosphate groups (especially peaks next to 800cm-1 and 1100cm-1). they are useful for characterization of inter alia dna, trna, and nucleic acid-protein complexes18,48. spectroscopic examination not only allows for examination of these specific functional groups enumerated above, but also to display their interactions, such as protein-protein / protein-lipid interaction. their changes in spectral property under different conditions can also be measured17. on that note, lee et al.49 have even managed to use srs as a tool in neurophysiology when examining the spectral properties of neuronal membrane potential. although specific raman peaks have been described for various molecules50–56, one should be cautious when actually assigning peaks to one's own sample. while peaks may be characteristic for a certain biochemical compound, they can also arise from different sources; viz they are not specific. in order to correctly assign peaks / detect them within a spectrum, it is essential to reduce potential confounders within the sample or the experimental set-up pre/post-experimentally. a potential way to assign distinct peaks with high evidence is direct observation: targeted manipulation of a sample can help to confirm the source of a peak. the vibrational spectroscopic experimental setup rs is a fast, non-destructive, user friendly, and easy to apply on tool providing molecular information with minimal sample preparation requirements in a reproducible manner. however, a routine use of rs-based tools in neuroscience has not yet been established. regardless of the numerous advantages certain limitations have to be considered not only pre-experimentally, but also during implementation of an experiment and afterwards when visualizing and processing the obtained data. the occurrence of the physically related phenomenon of (auto-)fluorescence (photons of the pump beam are absorbed by molecules of the sample which are raised to another energy level when returning to the basic energy level a photon is emitted, see also figure 1) is regularly observed and the expected intensity in this case is well above the intensity of the raman signals. to reduce wavelengthdependent autofluorescence, a distinct wavelength of the excitation source can be selected, or sers can be used57,58. although in contrast to other sophisticated laboratory techniques (e.g., genetic/epigenetic testing) there are less requirements for a correctly prepared raman sample. a few things need to be considered in order to avoid the occurrence of spectral background noise and spectral contamination: samples must be placed on a robust raman substrate so that the selected measuring point and the focus remain stable. depending on the experimental question as well as the expected background noise and the costs, various raman substrates are available. in addition to gold or aluminum-coated glass slides (as a function of the excitation wavelength glass alone exhibits a strong and broad fluorescence background signal in the “biological fingerprint region”), special slides (low-e slides, caf2 slides, quartz slides) can be considered28. these are characterized by a low spectral background or single peak attribution. fullwood et al.59 and kerr et al.60 examined the effect of substrate choice for spectral histopathology in more detail. it has been shown that caf2 slides (exclusive peak at 321cm-1 or 322cm-1 respectively, depending on the literature)61 have the least influence on the spectral background in comparison to low-e slides and spectrosil slides. the single background peak can either be ignored due to its irrelevant occurrence out of the important range of biological components within the raman spectrum, or can be subtracted via computational analysis afterwards. as a low-cost alternative aluminum foil can be used, which itself does not generate any significant background noise62–64. furthermore, the sample condition (most commonly native/frozen or formalin-fixed) needs to be considered pre-experimentally. although fresh tissue samples allow for a straightforward attribution of raman peaks to underlying biochemical components, they must be processed and analyzed within a certain time window and cannot be stored for a longer period of time. when working with fresh tissue, dehydration and associated denaturation of functional biochemical groups need to be prevented e.g., by keeping the specimen hydrated19,65. as an alternative, raman measurements of frozen biological samples allow longer storage and at the same time still give an insight in the biochemical composition of the biological sample. nevertheless, it should be noted that a reduction in certain peak intensities and significant alteration of raman signal in comparison to native tissue were described when using frozen sections66,67. the handling of formalin-fixed, methanol-fixed, or ffpe samples is routine during the pathological workflow; even though samples allow long archivability and are broadly available, this way of fixation damages the biological raman spectrum to a certain degree since the tissue undergoes an aggressive chemical procedure68–72. both formalin and methanol fixation reproducibly alter spectral tissue properties and affect raman bands assigned to lipids, proteins, and nucleic acids73. despite formalin-induced biochemical changes such as formation of cross-links in the structure of the amino acids, spectroscopic assessment and classification of formalin-fixed biological tissue is possible66; in contrast, methanol-fixation was reported to potentially hamper the detection of tissue malignancy72,74. the prominent spectrum of bound paraffin wax is reflected in certain points at 1063, 1133, 1296 and 1441cm-1, which make a manual or digital dewaxing process necessary and require a careful interpretation of the obtained spectra75. several conditions (aggressive chemical processing, required choice of special substrate and the fineness of the tissue) hamper spectroscopic examination when employing rs on ffpe tissue in the pathology department, although spatial orientation on the sample and proper identification of certain areas are a potential advantage. in the literature different approaches used rs on processed tissue; in any case they all face similar difficulties. huang et al.68 described the effects of formalin fixation on rs of cancerous human bronchial tissue, whereas draux et al.71 described the influence of formalin and air drying on single cancer cells and attributed spectral changes to affection of nucleic acids and proteins. even though not only a loss of the original chemical composition but also potential contamination due to the process of formalin-fixation in murine brain tissue was determined by hackett et al.76, several studies proposed formalin fixation as a sufficient and favorable method for subsequent spectroscopic diagnostic77,78. as a proof of concept, stefanakis et al.79 demonstrated the feasibility of vibrational spectroscopy on formalin-fixed malignant brain tissue. employing vibrational spectroscopy on ffpe tissue, an effect on the lipid content due to the dewaxing process was reported; nevertheless, raman bands related to cellular and extracellular proteins were successfully measured80. gaifulina and colleagues81 examined large intestine ffpe tissue from rats and analyzed biochemical signals obtained with label-free rs in the processed tissue. other groups examined ffpe tissue of rectal cancer to predict radiotherapy response82, to map/analyze cervical tissue83,84, or employed rs on healthy and malignant breast85–88/ovarian89/prostatic90 tissue in various fixation states. for a good overview on the influence of tissue processing on biological raman spectra the reader may refer to the work from faoláin et al.66. during spectroscopic examination, background noise due to a nearby photon source (e.g., room light) should be considered and reduced by performing the raman measurement in a darkened area or with dimmed operating room light91–94. additional methods of spectra quality control during intraoperative measurement have also been proposed95,96. by ensuring that the laser settings (wavelength and power, duration of acquisition) are optimized for the examined sample, the best signal-to-noise ratio can be determined, and thermal tissue decomposition can be prevented. this form of sample destruction can be detected by a burned area where the former focus area of the laser is located, as well as by the presence of an additional carbon band at approx. 1500cm-1 in the raman spectrum28. data processing and computational analysis after the measurement, the large amount of data97 should be sorted and stored in a structured manner (data annotation) to address the research question properly. it is good practice to start the data processing with an initial visualization of the data. in this way clear deviations from an expected result such as strong contamination or cosmic ray artifacts (randomly occurring electromagnetic radiation) and hot pixels (overresponse of a pixel on the ccd detector to an incoming photon) can be recognized and corrected28,98,99. for a more detailed reading on potential anomalies and artifacts that may occur, see bowie et al.100. during data preprocessing, a baseline correction can be applied to the data to minimalize residual background signal and autofluorescence101,102; a common way to model and subtract the background noise to obtain the intrinsic sample spectrum103,104. additionally, a common way to further reduce the noise in the data is a smoothing technique, such as savitzky-golay filtering28,105,106. both of the above-mentioned methods must not be used without proper caution as there is always the risk of producing artifacts, as well as equalizing significant data points. in order to correct confounders that result from the experiment setup itself (e.g., slightly different dryness or thickness of the specimens) data normalization methods, such as min-max normalization or z-normalization, usually precede the actual data analysis107. specialized spectroscopy software are commercially available and enable even the inexperienced spectroscopist to use the acquired data in a structured and comprehensive manner108. due to the large amount of data, several data reduction methods are used for quick explorative purposes, above all pca (principal component analysis) is widely employed. this unsupervised clustering technique can be used to determine principal components in a big data set, which explains a significant part of the variance and reduces noise41,109.< in the last step of computational analysis, classification algorithms and machine learning techniques110,111 are commonly used to classify the spectral data either according to pre-experimental defined groups (supervised clustering) or according to new groups based on similarities in spectral properties (unsupervised clustering)112. a widely used technique in unsupervised clustering is hierarchical cluster analysis (hca), in which the data is transferred to a higher-dimensional space, cluster in a certain proximity to one another based on similar properties. a number of cluster variables can be specified individually, which forms the selected number of similar clusters103. unsupervised clustering is beneficial for exploratory research questions since no prior knowledge of possible group properties is required28. common methods used for supervised clustering are trees/random forest classifications (several decision trees in a row) or support vector machines (search for a hyperplane to distinguish between classes)91. the groups determined a priori are referred to as "classes" and the gold standard histopathology often serves as ground truth. in general, the algorithm is trained with a training data set and tested with an external validation data set afterwards. to avoid overfitting (capability of good differentiation only on the specific training data set) a validation of performance e.g., k-fold cross validation or holdout validation is performed, and metrices of algorithm performance (e.g., sensitivity, specificity, f1-score, accuracy, auroc/aupr value) are calculated afterwards based on its output113. ralbovsky and colleagues provided an overview of machine learning algorithms and their functions in raman based cancer detection112. rs in neurooncology with a growing number of publications in the last years (zhang et al.114 and banerjee et al.115 described a change in spectroscopic properties of glioma cells in comparison to astrocytes already in the mid-2000s), the neuro-oncological field is one of the largest areas of research on rs, in which the therapeutical balancing act between maximum resection of normal-brain-resembling tumorous residues and minimal surgical disruption of healthy brain functions proves particularly difficult. on the subject of rs in (neuro)oncology reviews by auner et al.20 and hollon et al.116 give a comprehensive introduction to the respective topic; for further reading on implications and current progress of rs in oncology see also santos et al.117. at first sight, use of this spectroscopic technique mainly apply to two main research focuses: on the one hand a spectroscopic detection of malignancy118,119 which in a next steps allows precise, accurate diagnosis of the tumor entity intraoperatively without having to wait for further traditional tissue processing (pathological diagnosis on frozen sections)120,121, and on the other hand real time surgery guidance i.e., live feedback intraoperatively122,123 aiming for maximal tumor resection124–126. both topics merge and evolve at a certain point; this may result in new research questions, e.g., when aiming to determine tumor infiltration zone / resection margin or when aiming for detection of tumor genetics on various states of tumor tissue. moreover, also basic research questions in oncology can be addressed with this vibrational spectroscopic technique e.g., monitoring lipotoxicity in glioblastoma cells127, observing cell response of u251 glioblastoma cells after induced apoptosis128, examining the glycosylation pattern of proteins in medulloblastoma129, or observation of redox state of mitochondrial cytochromes130, just to name a few. most research groups use sprs20 as an easy to apply, label free method. more advanced raman techniques in neurooncology131 are used predominantly in animal models132–134 – where surface enhanced resonant raman spectroscopy (serrs) detection of tumor margins135 has shown prognostic benefits136, or cars was employed for detection of different human brain tumors in a mouse model137. rs for detection of tumor group, genetic alteration and histomorphology rs can distinguish between grey and white matter and (partly) other brain regions such as cerebellum, striatum, basal forebrain both macroscopically and on cellular resolution4,138–146,147. interestingly, one analysis of the mouse brain using sers revealed a different spectral fingerprint and thus also different biochemical composition between left and right hemisphere148. spectroscopically feasible discrimination between glioma tissue and brain tissue was reported in several studies3,149–153 as well as between dura mater and meningioma, which was demonstrated to be based in part on peaks corresponding to collagen and on the higher lipid content within tumorous tissue154–156. beside these binary classification models, several studies showed the potential of rs aiming for a multiclass classification to differentiate various tumor entities within one classifier119,157–166 or to determine the primary site of metastasis167,168. using raman mapping/imaging for brain tumor visualization116,169, even special morphological features of tumors (e.g., necrosis in glioblastoma, cell density or individual cell nuclei) could be identified170–172. even though areas of tumor necrosis are typically characterized by an increased presence of proteins such as phenylalanine (around 1032cm-1, among others) as well as cholesterol esters (1739cm-1)171,173, one group proposed two distinct spectral properties within the necrosis of glioblastoma cells: “highly necrotic”, showing an increase in plasma proteins and “peri-necrotic”, exhibiting a higher lipid content174. the histopathological heterogeneity of tumor tissue samples was addressed in fresh and frozen brain sections, although possible confusion between different tumor components (i.e., tumor hemorrhage and necrosis) is described36,173. the genomic heterogeneity in glioblastoma has also been successfully addressed175. other approaches make use of an alternative advanced raman technique named stimulated raman histology176–179 (srh), where distinct wavenumbers are used for image acquisition and virtual h&e-like images are generated after computational processing. with this approach in combination with deep convolutional neural networks, amongst others hollon et al. assessed (pediatric180) brain tumors intraoperatively1,181,182. in the scope of this imaging approach, also a traditional pathological diagnosis based on digital raman histology slides seems feasible183–185. rs could be used to identify brain edema186, tumor recurrence187 or tumor margins188–194 but also tumor infiltration zones.195,196 in general, infiltrative glioma cells showed significant spectral differences in the regions of phenylalanine and amide iii (around 1030cm-1 and 1230-1300cm−1), as well as the region assigned to c-c stretching lipids and nucleotides (around 1050-1100cm-1) – just to list a few wavenumbers of interest exemplarily197. ji et al.196 report the cellularity within a sample as well as the density of axons and the ratio of lipid and protein contents as the basis for the difference in spectral properties. even single tumor cells198 were detectable using rs, something alternative imaging methods struggle with. rs was also applied to observe glioblastoma tumor evolution199, to determine the molecular subtype of glioblastoma200, and to give insight in glioma biochemistry201. rs was shown to be superior in differentiation of brain tumor and glioblastoma in comparison to 5-ala-induced fluorescence202,203, and capable to detect idh mutations in gliomas – inter alia changes in the spectral protein profile are consistently reported in case of idh mutation204–206. it also showed diagnostic value in tumor discrimination when measuring small extracellular vesicles207, or potential when tracking/detecting metabolic changes208–210 in brain tumors/cancer cells, as well as drug delivery mechanisms211 and post-therapeutic changes212 in glioblastoma cells. spectroscopic classification of different grades of brain tumors is possible213. zhou et al.214 distinguished between different who grades of gliomas using raman bands of tryptophane (around 1588cm−1, among others) and carotenoids (1008cm−1, 1157cm−1, 1521cm−1, 2320cm−1, and 2667cm−1) as well as the peak intensity ratio between proteins and lipids in the high wavenumber region (2934cm−1/2885cm−1). the group of morais et al.215 and lilo et al.216 differentiated between different grades of meningiomas. zhang et al.217 associated an intensity ratio in the high wavenumber region with different meningioma grades. while gliomas/neuroepithelial tumors and meningiomas have been described218 and morpho-chemically analyzed219,220 extensively221, some work also exist on neuroblastomas. one group differentiated between different neural crest-derived tumors in fresh and frozen tissue222,223, and ricciardi et al.224 used rs to examine changes in the biochemistry of neuroblastoma cells after exposure to radiation. medulloblastomas225, biopsies of the pituitary gland209,226, seeds of retinoblastomas227, and carcinoma metastases228 have been spectroscopically studied as well. early, intraoperative, and neuropathological diagnostics using rs perioperative ex vivo tissue assessments allow for direct and early treatment decision, e.g., when examining smear brain tumor samples94 or discriminating between primary cns (central nervous system) lymphoma and glioblastoma based on biopsies229. rs can also be applied intraoperatively (in vivo) recently even in dogs230 using a hand-held probe for tumor classification231–237, where a real-time auditory feedback mechanism has been proposed to guide the neurosurgeon238. transcranial rs, leaving the skull intact, has been proposed and demonstrated in a mouse model239. using optical spectroscopy applied on ffpe tissue, devpura et al.240 and gajjar et al.159 examined a possible application of rs to various brain tumors already in 2012/2013. shortly after, fulwood et al.241 distinguished between glioblastoma, metastases and normal brain using immersion rs on ffpe samples. livermore et al. 204 have been able to carry out the above-mentioned analysis of the idh mutation detection in glioblastoma tumors also on ffpe tissue. different histological areas can be distinguished in glioblastoma in ffpe tissue, with a sound separability between the peritumoral area and the area of necrosis242. to enable early and non-invasive cancer diagnosis, some approaches aim for identification of meningioma243 and glioma244 patients based on serum samples and resulting spectroscopic behavior. using rs as an additive technique, le reste et al.245 combine spectroscopic data and transcriptomic data for machine learning analyses on glioblastoma subtypes and related clinical outcomes. rs in neurodegenerative diseases misfolded proteins and aggregates in various diseases246–248, e.g., alzheimer's (tau and amyloid), parkinson's (alpha-synuclein), huntington's (polyglutamine), are in general accessible to vibrational spectroscopic techniques249. usage of these techniques ranges from tracking and characterization of misfolded proteins41, to potential new diagnostic methods250,251, especially in biofluids252–254. studies on the pathological hallmarks of neurodegenerative diseases used a variety of rs techniques; most frequently employed techniques are sers, ters (tip-enhanced raman spectroscopy), as well as duvrr255,256 (deep uv resonance raman), where a wavelength in the range of uv (200nm) is used as excitation source which results in an increased intensity. another common technique named roa (raman optical activity) makes use of the principle that a chiral molecule scatters left and right handed polarized photons at different intensities and so is particularly useful to analyze protein aggregates257,258. furthermore, also ir (infrared)-spectroscopy and related/modified vibrational methods are common, and a combination of techniques could lead to an increased diagnostic ability and gain of knowledge2,259–262. several ways of increasing the detectability of a sample via rs have gained popularity in the neurodegenerative field. bringing in a labelled isotope into the backbone of a peptide shifts certain amid bands and enables a demarcation from the existing amide bands emanating from the unlabeled proteins in the sample, although an overlap of raman peaks of interests may occur263,264. another similar approach integrates external probes such as unnatural amino acids with vibrational potential into the sample, which can afterwards be traced by specific raman peaks, often in the range between 1900-2900cm-1 where the interference with other peaks of the specimen is minor264–266. for further reading, devitt et al.2 provides a detailed insight into the use of rs in the field of neurodegenerative diseases. around 20 years ago conventional rs was already capable of distinguishing between ad brain tissue and healthy control brain tissue (in 2022 machine learning algorithms are useful to do the same267) and to determine the presence of amyloid-beta-sheets in senile plaques268–270. shortly after, raman signals of the hippocampus of ad rats were proposed to aid diagnosis of ad271. kurouski et al.44 give an overview of the application of rs in the course of plaque formation and structure; wilkosz et al.41 provide a comprehensive list of wavenumbers associated with protein aggregation. detailed examinations of the (secondary)-structure of beta-amyloid in various experimental set ups have been carried out using duvrr272–275 or roa44. cunha et al.276 used a combination of raman techniques for amyloid plaque characterization. sers has been used to identify tau protein and (soluble) amyloid beta277,278, and to detect amyloid-beta1-40 monomers and amyloid-beta1-40 fibrils in solution279 as well as in brain tissue280. aβ40 and aβ42281 were shown to be distinguishable. ters was used to characterize natural aβ1-42 fibrils and identify toxic oligomeric forms282,283. rs was capable of visualizing amyloid in ad brains post mortem and of displaying neuritic plaques and neurofibrillary tangles284 – even though the latter findings were questioned and measurement of lipofuscin granulates instead of plaques was proposed285 raman imaging also determined the presence of hemoproteins in senile plaques286 and allowed for reconstruction of the evolution process of different types of amyloid beta plaques287. based on rs measurements, ad-associated astrogliosis288 and lipid deposits in vicinity of fibrillary plaques were identified and further morphologically described289. beside the identification of amyloid beta290–292, for example in the surrounding of neuronal spines293, raman imaging294,295 has been used to compare the concentration of aβ in hippocampal regions and eye lens tissue296 and to determine cholesteroland sphingomyelin-rich structures surrounding amyloid plaques, thought to represent dystrophic neurites297. another research group used cars to determine a higher content of lipid, collagen and amyloid fibers in alzheimer-affected brain samples298. searching for biomarkers as an early diagnostic tool in ad299–302, human tears303, saliva,304 cerebrospinal fluid305 (different states of amyloid beta confirmations could be detected in cerebrospinal fluid already in 2008306), retinal imaging307 and blood samples308–318 have been evaluated for a potential diagnosis of ad using spectral differences arriving from platelets319 or the concentration of the neurotransmitters glutamate (glu) and γ-aminobutyric acid (gaba)320. in the course of this approach, it has been shown that cortical cataract may not be a sufficient predictor of ad296. the detection of neurotransmitters using rs has been shown and further analyzed, by ardini et al.321, lee et al322, moody et al.323–325 (i.e. rs for detection of neurotransmitters through the skull), cao et al.326 / zhou et al.327 (neurotransmitter detection in serum), ciubuc et al.328 (rs for dopamine detection and analysis), silwal et al.329 (dopamine and dopamine transporter interaction), manciu et al.330 (dopamine – serotonin interaction) and shi et al.331 (quantification of norepinephrine). in addition, rs is also suitable to examine the interaction of beta-amyloid with metal ions332–337. interestingly, detection of tau335–338 and insulin342–345 has so far been studied to a lesser extent; ozone exposure as a known risk factor has been found to lead to spectroscopically measurable changes of the hippocampus in a rat model346. in parkinson’s disease (pd), a main focus of the application of rs is the characterization of the secondary structure of alpha-synuclein338,347–349 as well as the identification of alpha-synuclein aggregations, feasible not only in the brain but also in the gut350. mensch et al.351 used roa to examine the spectral properties of α-synuclein during transition to its secondary structure. another group spectroscopically characterized the striatal extracellular matrix in a pd mouse model352. since early loss of dopaminergic neurons is an early change in patients with pd, different approaches aim for detection of dopamine353–355, e.g., in striatum of mice356, or in blood samples of patients with antipsychotic drug-induced parkinsonism357. other efforts to establish early diagnostic tests for pd, such as examination of erythrocytes and blood coagulation in pd patients358, were carried out e.g., by carlomagno et al.359 using saliva of pd patients and schipper et al.360 who combined rs and nirs (near infrared spectroscopy) to distinguish between blood samples of pd patients and a control group through different spectroscopic properties correlated with oxidative stress. mammadova et al.361 used rs in a pd mouse model to detect pathological retinal changes as a method to distinguish between healthy and diseased samples. analyzing peripheral nervous tissue in als mice and autopsies of patients suffering from als, tian et al.362 showed that raman imaging was capable of visualizing and detecting early pathological changes. different approaches distinguish between altered lipids and proteoglycans in spinal cord tissue of als mice and healthy controls363, or test the prognostic value of sers in als patients364. in addition to the many approaches to diagnose ad and pd patients by rs, others focus on als as well. for diagnostic purposes, zhang et al.365 used sers on plasma samples to distinguish between als patients and a healthy control group; morasso et al.366 proposed vibrational spectroscopy and extracellular vesicles as a potential biomarker and another research group spectroscopically examined saliva from als, pd, and ad patients, showing differences in the spectral properties of each group367. in the context of huntington disease (hd), rs has been used for quantification and visualization of aggregated polyglutamine368 and for the assessment of its structure369,370. huefner et al.371 found significant changes in the spectra related to disease progression, as well as differences corresponding to genotype and gender in serum samples of hd patients and healthy controls. in another approach, membrane composition of hd-affected and control peripheral fibroblasts were separatable using rs, suggesting that cell membrane damage may serve as future diagnostic biomarker372. rs has also been used for research on prion diseases373–378; one research group employed the method to examine the diagnostic value when analyzing blood samples of sheep to detect the altering from of prpc to prpsc379. spectroscopic examination of myelin composition in the cns and in peripheral nerve tissue rs proves useful to gain a deeper understanding of the molecular myelin composition; pezzotti et al.380 examined the physical chemistry of cocultured neuronal and schwann cells. in addition, rs may be advantageous to detect pathological processes of demyelinating diseases in the cns or in peripheral nerve tissue. carmona et al.381 studied the spectroscopic hallmarks of lipid chains in myelin membranes as well as the secondary structure of associated proteolipid proteins (plp). some publications report the possibility of detecting myelin in vivo using raman microscopy382,383; huang et al.384 described different compositions of myelin structures, whereas wang et al.385 used cars microscopy to detect not only myelin but also axons, the node of ranvier, and the schmidt-lanterman incisure. fu et al.386 visualized fiber tracts in mice brain by imaging the myelin along the axons. in 2021 lucas et al.387 used cars to determine myelination deficits in a fragile-x-syndrome mouse model. out of pure academic interest the publication of poulen et al.388, in which raman scattering on spinal cord myelin distinguishes between three different species (human, mouse, lemur), shall be mentioned at this point. few raman experiments deal with multiple sclerosis (ms)389; the process of myelin degradation can be addressed with rs not only quantitatively390 but also qualitatively. to tackle alterations in the biochemical compositions in human brains postmortem, poon et al.391–393 measured various pathologic features and showed that even normal appearing white matter next to ms lesions included spectroscopically measurable changes. imitola et al.394 correlate the presence of microglia (on a side note: even the activation of microglia is traceable using rs395) and axonal injury/demyelination using cars microscopy. fu et al.396 applied the same method to examine different time points of experimental autoimmune encephalomyelitis in mice and gasecka et al.397 used cars to detect induced autoimmune demyelination in spinal cord of mice. another approach was carried out by the team of alba-arbalat et al.398; they detected spectral changes of defined molecules in the retina (even an in vivo use of rs applied on human retina is in line with laser safety regulations399) associated not only with different phases of ms, but also age-related in healthy patients. raman-based research of myelin composition and pathology is not limited to ms, it also extends to the study of demyelination and its biochemical changes in peripheral nerve tissue400 even pathological401 and age related402 changes. using different raman techniques the remyelination process in the spinal cord of rats after iatrogenic induced demyelination403, as well as remyelination in rat sciatic nerve404, and biochemical changes during nerve injury405,406 can be tracked. another approach used cars imaging to interpret the interaction of different macrophages (resident and recruited) after wallerian degeneration407. upcoming novel fields for rs from stroke to muscular diseases to psychiatry rs has been applied in combination with infrared spectroscopy and atomic force microscopy to characterize different types of thrombi in ischemic stroke408 or to characterize atherosclerotic plaques409,410. changes in fibrin concentration in a blood clot after zonal thrombolysis with urokinase411, or the metabolic regulation of artery tone412 were examined. other research groups investigated spectroscopic changes in the hippocampus due to cerebral ischemia-reperfusion413, or spectroscopic changes in the amount of cu+ and cu2+ ions in brain ischemia414. russo et al.415 used raman traceable cytochrome c to investigate effects of insulin on the hippocampus after transient ischemic brain conditions, yamazoe et al.416 used a self-developed raman approach to detect areas of an ischemic core area. the group of caine et al.417 used a combination of imaging techniques, amongst others raman imaging, to track biochemical changes in the peri-infarct zone after induced stroke in a mouse model. as an alternative way of infarction diagnostic, fan et al.418 proposed tear rs in combination with machine learning tools as a non-invasive technique. in context of brain hemorrhages, raman imaging has been used to detect microvessels and induced hemorrhage419, as well as to track the oxygen flow in brain vessels420. furthermore, rs was employed as a method in rat brains with striatal hemorrhages to evaluate the biochemical composition after rehabilitation treatment421. employing sers, the subarachnoid hemorrhage biomarker glial fibrillary acid protein can be detected422. sers can also be used to assess complications post subarachnoid hemorrhage, like vasospasm and hydrocephalus423. in tissue conditions of brain or spine injury, rs was applied to tissue of rat models424–426 and on retinae of mice after traumatic brain injury427. biochemical changes in affected areas arising from hem or divergent levels of cholesterol were discovered428 and compared to mri scans429. rs was capable of detecting injured motor cortex areas where certain spectroscopic properties were associated with cell death430. employment of sers-based methods allow for detection of neuron-specific enolase (nse), n-acetylasparate or s-100β in blood samples as biomarkers for brain injury431–435; aiming for intraoperative assessment of molecular changes one group developed a device for intracranial spectroscopy within brain injury436. changes in the biochemical and cellular composition of rat brain after gamma radiation have been addressed by kočović et al.437. for a further reading the reader may refer to stevens et al.438, who has recently reviewed the current deployments of raman spectroscopy in traumatic brain injury in a detailed way. even muscular diseases are accessible to rs: niedieker et al.439 used cars imaging to visualize morphological hallmarks such as glycogen storage and internalized nuclei in various muscular diseases; alix et al.440 reported different spectral properties of mitochondrial and non-mitochondrial muscular diseases; and gautam et al.441 showed the differences in the spectra of raman measurements from muscles of drosophila with certain mutations affecting the muscular system in comparison to healthy controls. sprs was used for in vivo identification of duchenne muscular dystrophy (dmd) affected muscles in a mouse model and human muscles affected with the same disease with ex vivo measurements showing similar raman peaks442. hentschel et al.443 evaluated the use of fibroblasts together with application of cars and other methods to study the etiology of neuromuscular diseases. blood sample testing for the diagnosis of dmd was proposed and successfully performed in a mouse model444; the comparison of spectral properties of the erythrocyte membrane in dmd patients and healthy controls demonstrated biochemical differences due to protein anomaly445. one of the potential domains of rs in the area of infectious diseases of the brain and meninges is the diagnostic detection of pathogens. it has already been capable of identifying viral strains446, changes in bacterial metabolism447, or differentiate/detect different types of bacteria related to meningitis448,449. although the diagnoses of tuberculous meningitis450 or neisseria meningitis451 as well as possible differentiation of blood cell types452 using rs on csf samples is reported, reliable detection of bacterial meningitis in csf was not yet sufficiently sensible; therefore, a combination of techniques was suggested453. another approach employs rs in neuroimmunology as a tool to monitor apoptotic changes in hippocampal progenitor cells454. rs has also been applied in psychiatric disorders; e.g. to visualize the drug mechanism of a serotonin reuptake inhibitor in mouse brain455 and to identify blood serum samples based on alterations in phospholipids and proteins of patients with affective disorders456–458. recently, chaichi et al.459 measured changes in brain lipidome spectroscopically in post-traumatic stress disorder (ptsd) rats, but also the vibrational spectroscopic properties within myalgic encephalomyelitis have been subjected to further analysis460,461. conclusions and outlook all studies and literature cited in this review focused on preclinical/clinical use of rs with the intention to provide the interested reader a general overview rather than a detailed account of each particular topic. before jumping into action and establishing rs as an additional research method in one’s own laboratory, taking a look on the methodological reviews by butler et al.28 (including concrete information about the general experimental setup and requirements for biological tissue), and guo et al.462 (analysis of raman data, machine learning algorithms) may prove useful. upcoming applications of rs potentially aim for in vivo prediction of progression risk463 or employment of vibrational spectroscopy for detection of epileptogenic brain regions464. advanced raman techniques such as spatially offset raman spectroscopy (sors)465 may potentially permit live insight into tissue biochemistry of deeper brain structures. alternatively, a future establishment of intraoperative raman imaging (in particular it may even be performed in vivo466) will potentially allow fast detection of both histomorphological features and tumor genetics; therefore producing an integrated diagnosis467 at an early stage of the diagnostic workflow468. extensive clinical studies aiming for approval of rs in neuroscience by regulatory authorities are still missing, even though a clinical need and a patient benefit has been demonstrated by a broad range of groups and laboratories. to translate promising results into clinical practice, several challenges should be considered. when vibrational spectroscopy is tested as a diagnostic method in a multicenter approach, experimental workflows of spectroscopic examination need to be standardized and facilitated; consensus within the spectroscopic community on a collaborative experimental setup and procedure prevents potential invariances due to different sample preparation protocols and hidden artifacts91. to maximize spectral output and enhance spectral intensity in a clinical setting, handheld probes / spectrometers with optimized design and in vivo parameters as well as a preferably low signal-to-noise and high signal-to-background ratio are currently under investigation by a growing number of companies stepping up their efforts in the interface of research and clinical implementation.117,469 since the use of rs on ffpe tissue allows direct comparison with the diagnostic gold standard of histology, rs is expected to expand its applications in neuropathological diagnostics in the future. upcoming studies will not only challenge the current use of rs on unstained ffpe tissue (is reliable diagnosis also achievable on h&e stained samples?) but also discuss a potential use of various raman substrates in a cost-oriented manner470. to reduce the cost factor (id est expensive substrates such as caf2 or low-e slides) future employment of rs on glass slides seems worthwhile; therefore, occurring autofluorescence during measurement needs to be addressed. within that approach, the use of a certain excitation wavelength or the detection of only a small spectral wavenumber range have been proposed60,471. in this sense, ibrahim et al.472 aimed to use glass as raman substrates by employing a digital processing method. in the field of neurodegenerative diseases, a major and highly anticipated impact of rs could be the early and non-surgical diagnosis of disorders in a reproducible manner. despite promising results, this application area is only beginning to develop. to maximize diagnostic reliability, a deeper understanding of raman features and their corresponding biochemical origin in biofluids is key. within the huge amount of obtained data, it remains necessary to address patient dependent spectral variation as well as variations related to a concrete experimental set up. close cooperation between different research groups and ensured data share470 potentially accelerate the development towards clinical implementation. an exemplary success story of clinical translation was reported in the field of dermatology, where rs had already been established as a diagnostic method for early detection of skin cancer; a handheld device was commercially produced in canada463,473,474. to speed up translation from research labs to commercialization and clinical use, several networks have been founded, e.g., international society for clinical spectroscopy (clirspec, clirspec.org) and raman4clinics (raman4clinics.eu), all aiming for exchange of expertise and creation of research collaboration117. to conclude, it highly likely that rs will continue to evolve as a method in the intersection of applied biophysics and medicine – and potentially make its way deeper into the field of life science, such as detection of plastic in zebrafish brain homogenates as a result of exposure to nanoplastic475 and even more clinical applications. where the journey will finally lead remains to be seen in the next years. acknowledgements figure 1 and figure 2 were created with biorender.com. mm thanks the fnr for funding support (fnr pearl p16/bm/11192868 grant). funding luxembourg national research fond, fnr (fnr pearl p16/bm/11192868 grant to m.m.) conflicts of interest we have no conflicts of interest to disclose. references 1. hollon tc, pandian b, adapa ar, et al. near real-time intraoperative brain tumor diagnosis using stimulated raman histology and deep neural networks. nat med. 2020;26(1):52-58. https://doi.org/10.1038/s41591-019-0715-9 2. devitt g, howard k, mudher a, mahajan s. raman spectroscopy: an emerging tool in neurodegenerative disease research and diagnosis. acs chem neurosci. 2018;9(3):404-420. https://doi.org/10.1021/acschemneuro.7b00413 3. jermyn m, mok k, mercier j, et al. intraoperative brain cancer detection with raman spectroscopy in humans. sci transl med. 2015;7(274):274ra19. https://doi.org/10.1126/scitranslmed.aaa2384 4. segura-uribe jj, farfán-garcía ed, guerra-araiza c, ciprés-flores fj, garcía-dela torre p, soriano-ursúa ma. differences in brain regions of three mice strains identified by label-free micro-raman. spectrosc lett. 2018;51(7):356-366. https://doi.org/10.1080/00387010.2018.1473883 5. payne td, moody as, wood al, pimiento pa, elliott jc, sharma b. raman spectroscopy and neuroscience: from fundamental understanding to disease diagnostics and imaging. analyst. 2020;145(10):346 1-3480. https://doi.org/10.1039/d0an00083c 6. tashibu k. [analysis of water content in rat brain using raman spectroscopy]. no to shinkei. 1990;42(10):999-1004. 7. kitajima t, tashibu k, tani s, mizuno a, nakamura n. [analysis of water content in young rats brain edema by raman spectroscopy]. no to shinkei. 1993;45(6):519-524. 8. mizuno a, hayashi t, tashibu k, maraishi s, kawauchi k, ozaki y. near-infrared ft-raman spectra of the rat brain tissues. neurosci lett. 1992;141(1):47-52. https://doi.org/10.1016/0304-3940(92)90331-z 9. mizuno a, kitajima h, kawauchi k, muraishi s, ozaki y. near-infrared fourier transform raman spectroscopic study of human brain tissues and tumours. j raman spectrosc. 1994;25(1):25-29. https://doi.org/10.1002/jrs.1250250105 10. pliss a, kuzmin an, prasad pn, mahajan sd. mitochondrial dysfunction: a prelude to neuropathogenesis of sars-cov-2. acs chem neurosci. 2022;13(3):308-312. https://doi.org/10.1021/acschemneuro.1c00675 11. raman c v, krishnan ks. a new type of secondary radiation. nature. 1928;121(3048):501-502. https://doi.org/10.1038/121501c0 12. raman c v. a change of wave-length in light scattering. nature. 1928;121(3051):619-619. https://doi.org/10.1038/121619b0 13. singh r. c. v. raman and the discovery of the raman effect. phys perspect. 2002;4(4):399-420. https://doi.org/10.1007/s000160200002 14. c. tm. raman spectra of crystalline lysozyme, pepsin, and alpha chymotrypsin. science (80). 1968;161(3836):68-69. https://doi.org/10.1126/science.161.3836.68 15. lord rc, yu nt. laser-excited raman spectroscopy of biomolecules. i. native lysozyme and its constituent amino acids. j mol biol. 1970;50(2):509-524. https://doi.org/10.1016/0022-2836(70)90208-1 16. antonio ka, schultz zd. advances in biomedical raman microscopy. anal chem. 2014;86(1):30-46. https://doi.org/10.1021/ac403640f 17. vlasov a v, maliar nl, bazhenov s v, et al. raman scattering: from structural biology to medical applications. crystals. 2020;10(1):38. https://doi.org/10.3390/cryst10010038 18. carey pr. biochemical applications of raman and resonance raman spectroscopy. london, s.1-70: academic press; 1982. 19. diem m, mazur a, lenau k, et al. molecular pathology via ir and raman spectral imaging. j biophotonics. 2013;6(11-12):855-886. https://doi.org/10.1002/jbio.201300131 20. auner gw, koya sk, huang c, et al. applications of raman spectroscopy in cancer diagnosis. cancer metastasis rev. 2018;37(4):691-717. https://doi.org/10.1007/s10555-018-9770-9 21. cialla-may d, schmitt m, popp j. 1. theoretical principles of raman spectroscopy. in: popp j, mayerhöfer t, eds. micro-raman spectroscopy. de gruyter; 2020:1-14. https://doi.org/10.1515/9783110515312-001 22. popp j, mayerhöfer t, eds. micro-raman spectroscopy: theory and application. de gruyter; 2020. https://doi.org/ https://doi.org/10.1515/9783110515312 23. hu f, shi l, min w. biological imaging of chemical bonds by stimulated raman scattering microscopy. nat methods. 2019;16(9):830-842. https://doi.org/10.1038/s41592-019-0538-0 24. shi l, fung aa, zhou a. advances in stimulated raman scattering imaging for tissues and animals. quant imaging med surg. 2021;11(3):1078-1101. https://doi.org/10.21037/qims-20-712 25. evans cl, xie xs. coherent anti-stokes raman scattering microscopy: chemical imaging for biology and medicine. annu rev anal chem. 2008;1(1):883-909. https://doi.org/10.1146/annurev.anchem.1.031207.112754 26. zheng x-s, jahn ij, weber k, cialla-may d, popp j. label-free sers in biological and biomedical applications: recent progress, current challenges and opportunities. spectrochim acta part a mol biomol spectrosc. 2018;197:56-77. https://doi.org/10.1016/j.saa.2018.01.063 27. synytsya a, judexova m, hoskovec d, miskovicova m, petruzelka l. raman spectroscopy at different excitation wavelengths (1064, 785 and 532 nm) as a tool for diagnosis of colon cancer. j raman spectrosc. 2014;45(10):903-911. https://doi.org/10.1002/jrs.4581 28. butler hj, ashton l, bird b, et al. using raman spectroscopy to characterize biological materials. nat protoc. 2016;11(4):664-687. https://doi.org/10.1038/nprot.2016.036 29. matousek p, morris m. emerging raman applications and techniques in biomedical and pharmaceutical fields. (matousek p, morris md, eds.). berlin heidelberg, s. 1-24: springer; 2010. https://doi.org/10.1007/978-3-642-02649-2 30. raman images explained. https://www.renishaw.de/de/raman-images-explained--25810. accessed november 14, 2021. 31. he r, xu y, zhang l, et al. dual-phase stimulated raman scattering microscopy for real-time two-color imaging. optica. 2017;4(1):44-47. https://doi.org/10.1364/optica.4.000044 32. zhang b, sun m, yang y, et al. rapid, large-scale stimulated raman histology with strip mosaicing and dual-phase detection. biomed opt express. 2018;9(6):2604-2613. https://doi.org/10.1364/boe.9.002604 33. wei l, shen y, xu f, et al. imaging complex protein metabolism in live organisms by stimulated raman scattering microscopy with isotope labeling. acs chem biol. 2015;10(3):901-908. https://doi.org/10.1021/cb500787b 34. hu f, lamprecht mr, wei l, morrison b, min w. bioorthogonal chemical imaging of metabolic activities in live mammalian hippocampal tissues with stimulated raman scattering. sci rep. 2016;6(1):39660. https://doi.org/10.1038/srep39660 35. shi l. raman imaging of metabolic activities in brain. in: proc.spie. vol 11497. ; 2020. https://doi.org/10.1117/12.2571112 36. lu f-k, calligaris d, olubiyi oi, et al. label-free neurosurgical pathology with stimulated raman imaging. cancer res. 2016;76(12):3451-3462. https://doi.org/10.1158/0008-5472.can-16-0270 37. pezzotti g. raman spectroscopy in cell biology and microbiology. j raman spectrosc. 2021;52(12):2348-2443. https://doi.org/10.1002/jrs.6204 38. czamara k, majzner k, pacia mz, kochan k, kaczor a, baranska m. raman spectroscopy of lipids: a review. j raman spectrosc. 2015;46(1):4-20. https://doi.org/10.1002/jrs.4607 39. rygula a, majzner k, marzec km, kaczor a, pilarczyk m, baranska m. raman spectroscopy of proteins: a review. j raman spectrosc. 2013;44(8):1061-1076. https://doi.org/10.1002/jrs.4335 40. wiercigroch e, szafraniec e, czamara k, et al. raman and infrared spectroscopy of carbohydrates: a review. spectrochim acta part a mol biomol spectrosc. 2017;185:317-335. https://doi.org/10.1016/j.saa.2017.05.045 41. wilkosz n, czaja m, seweryn s, et al. molecular spectroscopic markers of abnormal protein aggregation. molecules. 2020;25(11). https://doi.org/10.3390/molecules25112498 42. clemens g, hands jr, dorling km, baker mj. vibrational spectroscopic methods for cytology and cellular research. analyst. 2014;139(18):4411-4444. https://doi.org/10.1039/c4an00636d 43. benevides jm, overman sa, thomas gj. raman spectroscopy of proteins. curr protoc protein sci. 2003;33(1):1-35. https://doi.org/10.1002/0471140864.ps1708s33 44. kurouski d, van duyne rp, lednev ik. exploring the structure and formation mechanism of amyloid fibrils by raman spectroscopy: a review. analyst. 2015;140(15):4967-4980. https://doi.org/10.1039/c5an00342c 45. krafft c, neudert l, simat t, salzer r. near infrared raman spectra of human brain lipids. spectrochim acta part a mol biomol spectrosc. 2005;61(7):1529-1535. https://doi.org/10.1016/j.saa.2004.11.017 46. vedad j, mojica e-re, desamero rzb. raman spectroscopic discrimination of estrogens. vib spectrosc. 2018;96:93-100. https://doi.org/10.1016/j.vibspec.2018.02.011 47. pezzotti g, horiguchi s, boschetto f, et al. raman imaging of individual membrane lipids and deoxynucleoside triphosphates in living neuronal cells during neurite outgrowth. acs chem neurosci. 2018;9(12):3038-3048. https://doi.org/10.1021/acschemneuro.8b00235 48. gorelik vs, krylov as, sverbil vp. local raman spectroscopy of dna. bull lebedev phys inst. 2014;41(11):310-315. https://doi.org/10.3103/s1068335614110025 49. lee hj, zhang d, jiang y, et al. label-free vibrational spectroscopic imaging of neuronal membrane potential. j phys chem lett. 2017;8(9):1932-1936. https://doi.org/10.1021/acs.jpclett.7b00575 50. movasaghi z, rehman s, rehman iu. raman spectroscopy of biological tissues. appl spectrosc rev. 2007;42(5):493-541. https://doi.org/10.1080/05704920701551530 51. de gelder j, de gussem k, vandenabeele p, moens l. reference database of raman spectra of biological molecules. j raman spectrosc. 2007. https://doi.org/10.1002/jrs.1734 52. edwards hgm. spectra-structure correlations in raman spectroscopy. in: handbook of vibrational spectroscopy. ; 2006. https://doi.org/10.1002/0470027320.s4103 53. grasselli jg, snavely mk, bulkin bj. applications of raman spectroscopy. phys rep. 1980;65(4):231-344. https://doi.org/10.1016/0370-1573(80)90065-4 54. visser t, van der maas jh. systematic interpretation of raman spectra of organic compounds. ii-ethers. j raman spectrosc. 1977;6(3):114-116. https://doi.org/10.1002/jrs.1250060303 55. visser t, van der maas jh. systematic interpretation of raman spectra of organic compounds. iii—carbonyl compounds. j raman spectrosc. 1978;7(3):125-129. https://doi.org/10.1002/jrs.1250070304 56. visser t, van der maas jh. systematic interpretation of raman spectra of organic compounds. iv—nitrogen compounds. j raman spectrosc. 1978;7(5):278-281. https://doi.org/10.1002/jrs.1250070510 57. vankeirsbilck t, vercauteren a, baeyens w, et al. applications of raman spectroscopy in pharmaceutical analysis. trac trends anal chem. 2002;21(12):869-877. https://doi.org/10.1016/s0165-9936(02)01208-6 58. malgorzata baranska jan cz. dobrowolski, hartwig schulz, rafal baranski, mr. recent advances in raman analysis of plants: alkaloids, carotenoids, and polyacetylenes. curr anal chem. 2013;9(1):108-127. https://doi.org/http://dx.doi.org/10.2174/1573411011309010108 59. fullwood lm, griffiths d, ashton k, et al. effect of substrate choice and tissue type on tissue preparation for spectral histopathology by raman microspectroscopy. analyst. 2014;139(2):446-454. https://doi.org/10.1039/c3an01832f 60. kerr lt, byrne hj, hennelly bm. optimal choice of sample substrate and laser wavelength for raman spectroscopic analysis of biological specimen. anal methods. 2015;7(12):5041-5052. https://doi.org/10.1039/c5ay00327j 61. gee ar, o’shea dc, cummins hz. raman scattering and fluorescence in calcium fluoride. solid state commun. 1966;4(1):43-46. https://doi.org/10.1016/0038-1098(66)90102-5 62. cui l, butler hj, martin-hirsch pl, martin fl. aluminium foil as a potential substrate for atr-ftir, transflection ftir or raman spectrochemical analysis of biological specimens. anal methods. 2016;8(3):481-487. https://doi.org/10.1039/c5ay02638e 63. thomas p v, ramakrishnan v, vaidyan vk. oxidation studies of aluminum thin films by raman spectroscopy. thin solid films. 1989;170(1):35-40. https://doi.org/10.1016/0040-6090(89)90619-6 64. hou h-c, banadaki ym, basu s, sharifi s. a cost-efficient surface enhanced raman spectroscopy (sers) molecular detection technique for clinical applications. j electron mater. 2018;47(9):5378-5385. https://doi.org/10.1007/s11664-018-6429-9 65. shim mg, wilson bc. the effects of ex vivo handling procedures on the near-infrared raman spectra of normal mammalian tissues. photochem photobiol. 1996;63(5):662-671. https://doi.org/10.1111/j.1751-1097.1996.tb05671.x 66. ó faoláin e, hunter mb, byrne jm, et al. a study examining the effects of tissue processing on human tissue sections using vibrational spectroscopy. vib spectrosc. 2005;38(1):121-127. https://doi.org/10.1016/j.vibspec.2005.02.013 67. candefjord s, ramser k, lindahl oa. effects of snap-freezing and near-infrared laser illumination on porcine prostate tissue as measured by raman spectroscopy. analyst. 2009;134(9):1815-1821. https://doi.org/10.1039/b820931f 68. huang z, mcwilliams a, lam s, et al. effect of formalin fixation on the near-infrared raman spectroscopy of normal and cancerous human bronchial tissues. int j oncol. 2003;23(3):649-655. https://doi.org/10.3892/ijo.23.3.649 69. mariani mm, lampen p, popp j, wood br, deckert v. impact of fixation on in vitro cell culture lines monitored with raman spectroscopy. analyst. 2009;134(6):1154-1161. https://doi.org/10.1039/b822408k 70. faoláin eó, hunter mb, byrne jm, et al. raman spectroscopic evaluation of efficacy of current paraffin wax section dewaxing agents. j histochem cytochem. 2005;53(1):121-129. https://doi.org/10.1369/jhc.4a6536.2005 71. draux f, gobinet c, sulé-suso j, et al. raman spectral imaging of single cancer cells: probing the impact of sample fixation methods. anal bioanal chem. 2010;397(7):2727-2737. https://doi.org/10.1007/s00216-010-3759-8 72. galli r, uckermann o, koch e, schackert g, kirsch m, steiner g. effects of tissue fixation on coherent anti-stokes raman scattering images of brain. j biomed opt. 2013;19(7):1-8. https://doi.org/10.1117/1.jbo.19.7.071402 73. chan jw, taylor ds, thompson dl. the effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers raman spectroscopy. biopolymers. 2009;91(2):132-139. https://doi.org/10.1002/bip.21094 74. krishna cm, sockalingum gd, vadhiraja bm, et al. vibrational spectroscopy studies of formalin-fixed cervix tissues. biopolymers. 2007;85(3):214-221. https://doi.org/10.1002/bip.20631 75. mian sa, colley he, thornhill mh, rehman i. development of a dewaxing protocol for tissue-engineered models of the oral mucosa used for raman spectroscopic analysis. appl spectrosc rev. 2014;49(8):614-617. https://doi.org/10.1080/05704928.2014.882348 76. hackett mj, mcquillan ja, el-assaad f, et al. chemical alterations to murine brain tissue induced by formalin fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis. analyst. 2011;136(14):2941-2952. https://doi.org/10.1039/c0an00269k 77. mazur ai, marcsisin ej, bird b, miljković m, diem m. evaluating different fixation protocols for spectral cytopathology, part 1. anal chem. 2012;84(3):1259-1266. https://doi.org/10.1021/ac202046d 78. mazur ai, marcsisin ej, bird b, miljković m, diem m. evaluating different fixation protocols for spectral cytopathology, part 2: cultured cells. anal chem. 2012;84(19):8265-8271. https://doi.org/10.1021/ac3017407 79. stefanakis m, lorenz a, bartsch jw, et al. formalin fixation as tissue preprocessing for multimodal optical spectroscopy using the example of human brain tumour cross sections. severcan f, ed. j spectrosc. 2021;2021:1-14. https://doi.org/10.1155/2021/5598309 80. ali sm, bonnier f, tfayli a, et al. raman spectroscopic analysis of human skin tissue sections ex-vivo: evaluation of the effects of tissue processing and dewaxing. j biomed opt. 2013;18(6):61202. https://doi.org/10.1117/1.jbo.18.6.061202 81. gaifulina r, maher at, kendall c, et al. label-free raman spectroscopic imaging to extract morphological and chemical information from a formalin-fixed, paraffin-embedded rat colon tissue section. int j exp pathol. 2016;97(4):337-350. https://doi.org/10.1111/iep.12194 82. kirkby cj, gala de pablo j, tinkler-hundal e, wood hm, evans sd, west np. developing a raman spectroscopy-based tool to stratify patient response to pre-operative radiotherapy in rectal cancer. analyst. 2021;146(2):581-589. https://doi.org/10.1039/d0an01803a 83. lyng fm, faoláin eó, conroy j, et al. vibrational spectroscopy for cervical cancer pathology, from biochemical analysis to diagnostic tool. exp mol pathol. 2007;82(2):121-129. https://doi.org/10.1016/j.yexmp.2007.01.001 84. tan km, herrington cs, brown cta. discrimination of normal from pre-malignant cervical tissue by raman mapping of de-paraffinized histological tissue sections. j biophotonics. 2011;4(1-2):40-48. https://doi.org/10.1002/jbio.201000083 85. rehman s, movasaghi z, tucker at, et al. raman spectroscopic analysis of breast cancer tissues: identifying differences between normal, invasive ductal carcinoma and ductal carcinomain situ of the breast tissue. j raman spectrosc. 2007;38(10):1345-1351. https://doi.org/10.1002/jrs.1774 86. ning t, li h, chen y, zhang b, zhang f, wang s. raman spectroscopy based pathological analysis and discrimination of formalin fixed paraffin embedded breast cancer tissue. vib spectrosc. 2021;115:103260. https://doi.org/10.1016/j.vibspec.2021.103260 87. lazaro-pacheco d, shaaban am, titiloye na, rehman s, rehman iu. elucidating the chemical and structural composition of breast cancer using raman micro-spectroscopy. excli j. 2021;20:1118-1132. https://doi.org/10.17179/excli2021-3962 88. haka as, shafer-peltier ke, fitzmaurice m, crowe j, dasari rr, feld ms. identifying microcalcifications in benign and malignant breast lesions by probing differences in their chemical composition using raman spectroscopy. cancer res. 2002;62(18):5375-5380. 89. krishna cm, sockalingum gd, venteo l, et al. evaluation of the suitability of ex vivo handled ovarian tissues for optical diagnosis by raman microspectroscopy. biopolymers. 2005;79(5):269-276. https://doi.org/10.1002/bip.20346 90. devpura s, thakur js, sarkar fh, sakr wa, naik vm, naik r. detection of benign epithelia, prostatic intraepithelial neoplasia, and cancer regions in radical prostatectomy tissues using raman spectroscopy. vib spectrosc. 2010;53(2):227-232. https://doi.org/10.1016/j.vibspec.2010.03.009 91. jermyn m, desroches j, aubertin k, et al. a review of raman spectroscopy advances with an emphasis on clinical translation challenges in oncology. phys med biol. 2016;61(23):r370-r400. https://doi.org/10.1088/0031-9155/61/23/r370 92. jermyn m, desroches j, mercier j, et al. neural networks improve brain cancer detection with raman spectroscopy in the presence of operating room light artifacts. j biomed opt. 2016;21(9):094002. https://doi.org/10.1117/1.jbo.21.9.094002 93. desroches j, laurence a, jermyn m, et al. raman spectroscopy in microsurgery: impact of operating microscope illumination sources on data quality and tissue classification. analyst. 2017;142(8):1185-1191. https://doi.org/10.1039/c6an02061e 94. bury d, morais c, ashton k, dawson t, martin f. ex vivo raman spectrochemical analysis using a handheld probe demonstrates high predictive capability of brain tumour status. biosensors. 2019;9(2):49. https://doi.org/10.3390/bios9020049 95. dallaire f, picot f, tremblay j-p, et al. quantitative spectral quality assessment technique validated using intraoperative in vivo raman spectroscopy measurements. j biomed opt. 2020;25(4):1-8. https://doi.org/10.1117/1.jbo.25.4.040501 96. zhao j, short ma, braun ta, m.d. hl, m.d. dim, zeng h. clinical raman measurements under special ambient lighting illumination. j biomed opt. 2014;19(11):1-4. https://doi.org/10.1117/1.jbo.19.11.111609 97. beleites c, neugebauer u, bocklitz t, krafft c, popp j. sample size planning for classification models. anal chim acta. 2013;760:25-33. https://doi.org/10.1016/j.aca.2012.11.007 98. zhang l, henson mj. a practical algorithm to remove cosmic spikes in raman imaging data for pharmaceutical applications. appl spectrosc. 2007;61(9):1015-1020. http://opg.optica.org/as/abstract.cfm?uri=as-61-9-1015. 99. barton sj, hennelly bm. an algorithm for the removal of cosmic ray artifacts in spectral data sets. appl spectrosc. 2019;73(8):893-901. https://doi.org/10.1177/0003702819839098 100. bowie bt, chase db, lewis ir, griffiths pr. anomalies and artifacts in raman spectroscopy. in: handbook of vibrational spectroscopy. in: griffiths pr, ed. handbook of vibrational spectroscopy. chichester, s. 2355-2378: john wiley & sons; 2006:2355-2378. https://doi.org/10.1002/9780470027325.s3103 101. zhao j, lui h, mclean di, zeng h. automated autofluorescence background subtraction algorithm for biomedical raman spectroscopy. appl spectrosc. 2007;61(11):1225-1232. https://doi.org/10.1366/000370207782597003 102. cao a, pandya ak, serhatkulu gk, et al. a robust method for automated background subtraction of tissue fluorescence. j raman spectrosc. 2007;38(9):1199-1205. https://doi.org/10.1002/jrs.1753 103. smith zj, huser tr, wachsmann-hogiu s. raman scattering in pathology. anal cell pathol. 2012;35(3):145-163. https://doi.org/10.3233/acp-2011-0048 104. lieber ca, mahadevan-jansen a. automated method for subtraction of fluorescence from biological raman spectra. appl spectrosc. 2003;57(11):1363-1367. https://doi.org/10.1366/000370203322554518 105. trevisan j, angelov pp, carmichael pl, scott ad, martin fl. extracting biological information with computational analysis of fourier-transform infrared (ftir) biospectroscopy datasets: current practices to future perspectives. analyst. 2012;137(14):3202-3215. https://doi.org/10.1039/c2an16300d 106. savitzky a, golay mje. smoothing and differentiation of data by simplified least squares procedures. anal chem. 1964;36(8):1627-1639. https://doi.org/10.1021/ac60214a047 107. lasch p. spectral pre-processing for biomedical vibrational spectroscopy and microspectroscopic imaging. chemom intell lab syst. 2012;117:100-114. https://doi.org/10.1016/j.chemolab.2012.03.011 108. menges f. "spectragryph optical spectroscopy software”, version 1.2.14, 2020, http://www.effemm2.de/spectragryph/. 109. jollife it, cadima j. principal component analysis: a review and recent developments. philos trans r soc a math phys eng sci. 2016;374(2065). https://doi.org/10.1098/rsta.2015.0202 110. olson rs, cava w la, mustahsan z, varik a, moore jh. data-driven advice for applying machine learning to bioinformatics problems. pac symp biocomput. 2018;23:192-203. https://pubmed.ncbi.nlm.nih.gov/29218881. 111. meza ramirez ca, greenop m, ashton l, rehman i ur. applications of machine learning in spectroscopy. appl spectrosc rev. 2021;56(8-10):733-763. https://doi.org/10.1080/05704928.2020.1859525 112. ralbovsky nm, lednev ik. towards development of a novel universal medical diagnostic method: raman spectroscopy and machine learning. chem soc rev. 2020;49(20):7428-7453. https://doi.org/10.1039/d0cs01019g 113. hajian-tilaki k. receiver operating characteristic (roc) curve analysis for medical diagnostic test evaluation. casp j intern med. 2013;4(2):627-635. https://pubmed.ncbi.nlm.nih.gov/24009950. 114. zhang lei, shealey p, hayden l, xie c, li y-q. study of brain cells by near-infrared raman spectroscopy. j north carolina acad sci. 2005;121(1):41-44. http://www.jstor.org/stable/24336004. 115. banerjee h nath, zhang l. deciphering the finger prints of brain cancer astrocytoma in comparison to astrocytes by using near infrared raman spectroscopy. mol cell biochem. 2007;295(1):237-240. https://doi.org/10.1007/s11010-006-9278-4 116. hollon t, orringer da. label-free brain tumor imaging using raman-based methods. j neurooncol. 2021;151(3):393-402. https://doi.org/10.1007/s11060-019-03380-z 117. santos ip, barroso em, bakker schut tc, et al. raman spectroscopy for cancer detection and cancer surgery guidance: translation to the clinics. analyst. 2017;142(17):3025-3047. https://doi.org/10.1039/c7an00957g 118. iturrioz-rodríguez n, de pasquale d, fiaschi p, ciofani g. discrimination of glioma patient-derived cells from healthy astrocytes by exploiting raman spectroscopy. spectrochim acta a mol biomol spectrosc. 2022;269:120773. https://doi.org/10.1016/j.saa.2021.120773 119. aguiar rp, silveira lj, falcão et, pacheco mtt, zângaro ra, pasqualucci ca. discriminating neoplastic and normal brain tissues in vitro through raman spectroscopy: a principal components analysis classification model. photomed laser surg. 2013;31(12):595-604. https://doi.org/10.1089/pho.2012.3460 120. depaoli d, lemoine é, ember k, et al. rise of raman spectroscopy in neurosurgery: a review. j biomed opt. 2020;25(05):050901. https://doi.org/10.1117/1.jbo.25.5.050901 121. broadbent b, tseng j, kast r, et al. shining light on neurosurgery diagnostics using raman spectroscopy. j neurooncol. 2016;130(1):1-9. https://doi.org/10.1007/s11060-016-2223-9 122. brusatori m, auner g, noh t, scarpace l, broadbent b, kalkanis sn. intraoperative raman spectroscopy. neurosurg clin n am. 2017;28(4):633-652. https://doi.org/10.1016/j.nec.2017.05.014 123. shu c, zheng w, wang z, yu c, huang z. development and characterization of a disposable submillimeter fiber optic raman needle probe for enhancing real-time in vivo deep tissue and biofluids raman measurements. opt lett. 2021;46(20):5197-5200. https://doi.org/10.1364/ol.438713 124. brahimaj bc, kochanski rb, pearce jj, et al. structural and functional imaging in glioma management. neurosurgery. 2021;88(2):211-221. https://doi.org/10.1093/neuros/nyaa360 125. hollon t, stummer w, orringer d, suero molina e. surgical adjuncts to increase the extent of resection: intraoperative mri, fluorescence, and raman histology. neurosurg clin n am. 2019;30(1):65-74. https://doi.org/10.1016/j.nec.2018.08.012 126. luther e, matus a, eichberg dg, shah ah, ivan m. stimulated raman histology for intraoperative guidance in the resection of a recurrent atypical spheno-orbital meningioma: a case report and review of literature. cureus. 2019;11(10):e5905. https://doi.org/10.7759/cureus.5905 127. yuan y, shah n, almohaisin mi, saha s, lu f. assessing fatty acid-induced lipotoxicity and its therapeutic potential in glioblastoma using stimulated raman microscopy. sci rep. 2021;11(1):7422. https://doi.org/10.1038/s41598-021-86789-9 128. ricci m, ragonese f, gironi b, et al. glioblastoma single-cell microraman analysis under stress treatments. sci rep. 2018;8(1):7979. https://doi.org/10.1038/s41598-018-26356-x 129. kopec m, imiela a, abramczyk h. monitoring glycosylation metabolism in brain and breast cancer by raman imaging. sci rep. 2019;9(1):166. https://doi.org/10.1038/s41598-018-36622-7 130. abramczyk h, surmacki jm, brozek-pluska b, kopec m. revision of commonly accepted warburg mechanism of cancer development: redox-sensitive mitochondrial cytochromes in breast and brain cancers by raman imaging. cancers (basel). 2021;13(11). https://doi.org/10.3390/cancers13112599 131. hollon t, lewis s, freudiger cw, sunney xie x, orringer da. improving the accuracy of brain tumor surgery via raman-based technology. neurosurg focus foc. 2016;40(3):e9. https://doi.org/10.3171/2015.12.focus15557 132. soltani s, guang z, zhang z, olson j, robles f. label-free detection of brain tumors in a 9l gliosarcoma rat model using stimulated raman scattering-spectroscopic optical coherence tomography. j biomed opt. 2021;26(7). https://doi.org/10.1117/1.jbo.26.7.076004 133. ji m, orringer da, freudiger cw, et al. rapid, label-free detection of brain tumors with stimulated raman scattering microscopy. sci transl med. 2013;5(201):201ra119-201ra119. https://doi.org/10.1126/scitranslmed.3005954 134. pope i, masia f, ewan k, et al. identifying subpopulations in multicellular systems by quantitative chemical imaging using label-free hyperspectral cars microscopy. analyst. 2021;146(7):2277-2291. https://doi.org/10.1039/d0an02381g 135. gao x, yue q, liu z, et al. guiding brain-tumor surgery via blood-brain-barrier-permeable gold nanoprobes with acid-triggered mri/serrs signals. adv mater. 2017;29(21). https://doi.org/10.1002/adma.201603917 136. han l, duan w, li x, et al. surface-enhanced resonance raman scattering-guided brain tumor surgery showing prognostic benefit in rat models. acs appl mater interfaces. 2019;11(17):15241-15250. https://doi.org/10.1021/acsami.9b00227 137. uckermann o, galli r, tamosaityte s, et al. label-free delineation of brain tumors by coherent anti-stokes raman scattering microscopy in an orthotopic mouse model and human glioblastoma. plos one. 2014;9(9):e107115. https://doi.org/10.1371/journal.pone.0107115. 138. kast r, auner g, yurgelevic s, et al. identification of regions of normal grey matter and white matter from pathologic glioblastoma and necrosis in frozen sections using raman imaging. j neurooncol. 2015;125(2):287-295. https://doi.org/10.1007/s11060-015-1929-4 139. kalkanis sn, kast re, rosenblum ml, et al. raman spectroscopy to distinguish grey matter, necrosis, and glioblastoma multiforme in frozen tissue sections. j neurooncol. 2014;116(3):477-485. https://doi.org/10.1007/s11060-013-1326-9 140. koljenović s, schut tcb, wolthuis r, et al. raman spectroscopic characterization of porcine brain tissue using a single fiber-optic probe. anal chem. 2007;79(2):557-564. https://doi.org/10.1021/ac0616512 pm 17222020 m4 – citavi 141. depaoli dt, lapointe n, messaddeq y, parent m, côté dc. intact primate brain tissue identification using a completely fibered coherent raman spectroscopy system. neurophotonics. 2018;5(3):35005. https://doi.org/10.1117/1.nph.5.3.035005 142. buttolph ml, mejooli ma, sidorenko p, eom c-y, schaffer cb, wise fw. synchronously pumped raman laser for simultaneous degenerate and nondegenerate two-photon microscopy. biomed opt express. 2021;12(4):2496-2507. https://doi.org/10.1364/boe.421647 143. daković m, stojiljković as, bajuk-bogdanović d, et al. profiling differences in chemical composition of brain structures using raman spectroscopy. talanta. 2013;117:133-138. https://doi.org/10.1016/j.talanta.2013.08.058 144. santos lf, wolthuis r, koljenović s, almeida rm, puppels gj. fiber-optic probes for in vivo raman spectroscopy in the high-wavenumber region. anal chem. 2005;77(20):6747-6752. https://doi.org/10.1021/ac0505730 145. sajid j, elhaddaoui a, turrell s. fourier transform vibrational spectroscopic analysis of human cerebral tissue. j raman spectrosc. 1997;28(2-3):165-169. https://doi.org/10.1002/(sici)1097-4555(199702)28:2/33.0.co;2-k 146. zięba-palus j, wesełucha-birczyńska a, sacharz j, et al. 2d correlation raman microspectroscopy of chosen parts of rat’s brain tissue. j mol struct. 2017;1147:310-316. https://doi.org/10.1016/j.molstruc.2017.06.117 147. meyer t, bergner n, bielecki c, et al. nonlinear microscopy, infrared, and raman microspectroscopy for brain tumor analysis. j biomed opt. 2011;16(2):21113. https://doi.org/10.1117/1.3533268 148. guo t, ding f, li d, zhang w, cao l, liu z. full-scale label-free surface-enhanced raman scattering analysis of mouse brain using a black phosphorus-based two-dimensional nanoprobe. appl sci. 2019;9(3). https://doi.org/10.3390/app9030398 149. riva m, sciortino t, secoli r, et al. glioma biopsies classification using raman spectroscopy and machine learning models on fresh tissue samples. cancers (basel). 2021;13(5):1-14. https://doi.org/10.3390/cancers13051073 150. depciuch j, tołpa b, witek p, et al. raman and ftir spectroscopy in determining the chemical changes in healthy brain tissues and glioblastoma tumor tissues. spectrochim acta part a mol biomol spectrosc. 2020. https://doi.org/10.1016/j.saa.2019.117526 151. baria e, pracucci e, pillai v, pavone fs, ratto gm, cicchi r. in vivo detection of murine glioblastoma through raman and reflectance fiber-probe spectroscopies. neurophotonics. 2020;7(4):45010. https://doi.org/10.1117/1.nph.7.4.045010 152. kowalska aa, berus s, szleszkowski ł, et al. brain tumour homogenates analysed by surface-enhanced raman spectroscopy: discrimination among healthy and cancer cells. spectrochim acta part a mol biomol spectrosc. 2020;231:117769. https://doi.org/10.1016/j.saa.2019.117769 153. köhler m, machill s, salzer r, krafft c. characterization of lipid extracts from brain tissue and tumors using raman spectroscopy and mass spectrometry. anal bioanal chem. 2009;393(5):1513-1520. https://doi.org/10.1007/s00216-008-2592-9 154. jelke f, mirizzi g, borgmann fk, et al. intraoperative discrimination of native meningioma and dura mater by raman spectroscopy. sci rep. 2021;11(1):23583. https://doi.org/10.1038/s41598-021-02977-7 155. koljenović s, schut tb, vincent a, kros jm, puppels gj. detection of meningioma in dura mater by raman spectroscopy. anal chem. 2005;77(24):7958-7965. https://doi.org/10.1021/ac0512599 156. di l, eichberg dg, park yj, et al. rapid intraoperative diagnosis of meningiomas using stimulated raman histology. world neurosurg. 2021;150:108-116. https://doi.org/10.1016/j.wneu.2021.02.097 157. aydin o, altaş m, kahraman m, bayrak of, culha m. differentiation of healthy brain tissue and tumors using surface-enhanced raman scattering. appl spectrosc. 2009;63(10):1095-1100. https://doi.org/10.1366/000370209789553219 158. leslie dg, kast re, poulik jm, et al. identification of pediatric brain neoplasms using raman spectroscopy. pediatr neurosurg. 2012;48(2):109-117. https://doi.org/10.1159/000343285 159. gajjar k, heppenstall ld, pang w, et al. diagnostic segregation of human brain tumours using fourier-transform infrared and/or raman spectroscopy coupled with discriminant analysis. anal methods. 2013;5(1):89-102. https://doi.org/10.1039/c2ay25544h 160. galli r, meinhardt m, koch e, et al. rapid label-free analysis of brain tumor biopsies by near infrared raman and fluorescence spectroscopy—a study of 209 patients. front oncol. 2019;9:1165. https://doi.org/10.3389/fonc.2019.01165 161. krafft c, sobottka sb, schackert g, salzer r. near infrared raman spectroscopic mapping of native brain tissue and intracranial tumors. analyst. 2005;130(7):1070-1077. https://doi.org/10.1039/b419232j 162. aguiar rp, falcão et, pasqualucci ca, silveira l. use of raman spectroscopy to evaluate the biochemical composition of normal and tumoral human brain tissues for diagnosis. lasers med sci. 2020. https://doi.org/10.1007/s10103-020-03173-1 163. zhou y, liu c-h, sun y, et al. human brain cancer studied by resonance raman spectroscopy. j biomed opt. 2012;17(11):116021. https://doi.org/10.1117/1.jbo.17.11.116021 164. kopec m, błaszczyk m, radek m, abramczyk h. raman imaging and statistical methods for analysis various type of human brain tumors and breast cancers. spectrochim acta part a mol biomol spectrosc. 2021;262:120091. https://doi.org/10.1016/j.saa.2021.120091 165. anna i, bartosz p, lech p, halina a. novel strategies of raman imaging for brain tumor research. oncotarget. 2017;8(49):85290-85310. https://doi.org/10.18632/oncotarget.19668 166. bury d, morais clm, martin fl, et al. discrimination of fresh frozen non-tumour and tumour brain tissue using spectrochemical analyses and a classification model. br j neurosurg. 2020;34(1):40-45. https://doi.org/10.1080/02688697.2019.1679352 167. bergner n, bocklitz t, romeike bfm, et al. identification of primary tumors of brain metastases by raman imaging and support vector machines. chemom intell lab syst. 2012;117:224-232. https://doi.org/10.1016/j.chemolab.2012.02.008 168. bergner n, romeike bfm, reichart r, kalff r, krafft c, popp j. raman and ftir microspectroscopy for detection of brain metastasis. in: clinical and biomedical spectroscopy and imaging ii. optical society of america; 2011:80870x. https://doi.org/10.1364/ecbo.2011.80870x 169. krafft c, sobottka sb, schackert g, salzer r. raman and infrared spectroscopic mapping of human primary intracranial tumors: a comparative study. j raman spectrosc. 2006;37(1-3):367-375. https://doi.org/10.1002/jrs.1450 170. zhang q, yun kk, wang h, yoon sw, lu f, won d. automatic cell counting from stimulated raman imaging using deep learning. plos one. 2021;16(7):e0254586. https://doi.org/10.1371/journal.pone.0254586. 171. koljenović s, choo-smith l-p, bakker schut tc, kros jm, van den berge hj, puppels gj. discriminating vital tumor from necrotic tissue in human glioblastoma tissue samples by raman spectroscopy. lab invest. 2002;82(10):1265-1277. https://doi.org/10.1097/01.lab.0000032545.96931.b8 172. krafft c, belay b, bergner n, et al. advances in optical biopsy--correlation of malignancy and cell density of primary brain tumors using raman microspectroscopic imaging. analyst. 2012;137(23):5533-5537. https://doi.org/10.1039/c2an36083g pm 23050263 m4 citavi 173. kast re, auner gw, rosenblum ml, et al. raman molecular imaging of brain frozen tissue sections. j neurooncol. 2014;120(1):55-62. https://doi.org/10.1007/s11060-014-1536-9 174. amharref n, beljebbar a, dukic s, et al. discriminating healthy from tumor and necrosis tissue in rat brain tissue samples by raman spectral imaging. biochim biophys acta biomembr. 2007;1768(10):2605-2615. https://doi.org/10.1016/j.bbamem.2007.06.032 175. bae k, xin l, zheng w, tang c, ang b-t, huang z. mapping the intratumoral heterogeneity in glioblastomas with hyperspectral stimulated raman scattering microscopy. anal chem. 2021;93(4):2377-2384. https://doi.org/10.1021/acs.analchem.0c04262 176. eichberg dg, shah ah, di l, et al. stimulated raman histology for rapid and accurate intraoperative diagnosis of cns tumors: prospective blinded study. j neurosurg. 2021;134(1):137-143. https://doi.org/10.3171/2019.9.jns192075 177. neidert n, straehle j, erny d, et al. stimulated raman histology in the neurosurgical workflow of a major european neurosurgical center — part a. neurosurg rev. 2021. https://doi.org/10.1007/s10143-021-01712-0 178. yang y, chen l, ji m. stimulated raman scattering microscopy for rapid brain tumor histology. j innov opt health sci. 2017;10(05):1730010. https://doi.org/10.1142/s1793545817300105 179. lee m, herrington cs, ravindra m, et al. recent advances in the use of stimulated raman scattering in histopathology. analyst. 2021;146(3):789-802. https://doi.org/10.1039/d0an01972k 180. hollon tc, lewis s, pandian b, et al. rapid intraoperative diagnosis of pediatric brain tumors using stimulated raman histology. cancer res. 2018;78(1):278-289. https://doi.org/10.1158/0008-5472.can-17-1974 181. orringer da, pandian b, niknafs ys, et al. rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated raman scattering microscopy. nat biomed eng. 2017. https://doi.org/10.1038/s41551-016-0027 182. jiang c, bhattacharya a, linzey jr, et al. rapid automated analysis of skull base tumor specimens using intraoperative optical imaging and artificial intelligence. neurosurgery. 2022;90(6). https://journals.lww.com/neurosurgery/fulltext/2022/06000/rapid_automated_analysis_of_skull_base_tumor.14.aspx. 183. straehle j, erny d, neidert n, et al. neuropathological interpretation of stimulated raman histology images of brain and spine tumors: part b. neurosurg rev. december 2021. https://doi.org/10.1007/s10143-021-01711-1 184. di l, eichberg dg, huang k, et al. stimulated raman histology for rapid intraoperative diagnosis of gliomas. world neurosurg. 2021;150:e135-e143. https://doi.org/10.1016/j.wneu.2021.02.122 185. shin ks, francis at, hill ah, et al. intraoperative assessment of skull base tumors using stimulated raman scattering microscopy. sci rep. 2019;9(1):20392. https://doi.org/10.1038/s41598-019-56932-8 186. wolthuis r, van aken m, fountas k, robinson, bruining ha, puppels gj. determination of water concentration in brain tissue by raman spectroscopy. anal chem. 2001;73(16):3915-3920. https://doi.org/10.1021/ac0101306 187. hollon tc, pandian b, urias e, et al. rapid, label-free detection of diffuse glioma recurrence using intraoperative stimulated raman histology and deep neural networks. neuro oncol. 2021;23(1):144-155. https://doi.org/10.1093/neuonc/noaa162 188. kircher mf, de la zerda a, jokerst j v, et al. a brain tumor molecular imaging strategy using a new triple-modality mri-photoacoustic-raman nanoparticle. nat med. 2012;18(5):829-834. https://doi.org/10.1038/nm.2721 189. hubbard tje, shore a, stone n. raman spectroscopy for rapid intra-operative margin analysis of surgically excised tumour specimens. analyst. 2019;144(22):6479-6496. https://doi.org/10.1039/c9an01163c 190. daoust f, nguyen t, orsini p, et al. handheld macroscopic raman spectroscopy imaging instrument for machine-learning-based molecular tissue margins characterization. j biomed opt. 2021;26(2). https://doi.org/10.1117/1.jbo.26.2.022911 191. pekmezci m, morshed ra, chunduru p, et al. detection of glioma infiltration at the tumor margin using quantitative stimulated raman scattering histology. sci rep. 2021;11(1):12162. https://doi.org/10.1038/s41598-021-91648-8 192. neuschmelting v, harmsen s, beziere n, et al. dual-modality surface-enhanced resonance raman scattering and multispectral optoacoustic tomography nanoparticle approach for brain tumor delineation. small. 2018;14(23):e1800740. https://doi.org/10.1002/smll.201800740 193. duan w, yue q, liu y, et al. a ph ratiometrically responsive surface enhanced resonance raman scattering probe for tumor acidic margin delineation and image-guided surgery. chem sci. 2020;11(17):4397-4402. https://doi.org/10.1039/d0sc00844c 194. jin z, yue q, duan w, et al. intelligent sers navigation system guiding brain tumor surgery by intraoperatively delineating the metabolic acidosis. adv sci (weinheim, baden-wurttemberg, ger. january 2022:e2104935. https://doi.org/10.1002/advs.202104935 195. galli r, uckermann o, temme a, et al. assessing the efficacy of coherent anti-stokes raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples. j biophotonics. 2017;10(3):404-414. https://doi.org/10.1002/jbio.201500323 196. ji m, lewis s, camelo-piragua s, et al. detection of human brain tumor infiltration with quantitative stimulated raman scattering microscopy. sci transl med. 2015;7(309):309ra163-309ra163. https://doi.org/10.1126/scitranslmed.aab0195 197. tanahashi k, natsume a, ohka f, et al. assessment of tumor cells in a mouse model of diffuse infiltrative glioma by raman spectroscopy. biomed res int. 2014;2014:860241. https://doi.org/10.1155/2014/860241 198. jermyn m, desroches j, mercier j, et al. raman spectroscopy detects distant invasive brain cancer cells centimeters beyond mri capability in humans. biomed opt express. 2016;7(12):5129-5137. https://doi.org/10.1364/boe.7.005129 199. beljebbar a, dukic s, amharref n, manfait m. ex vivo and in vivo diagnosis of c6 glioblastoma development by raman spectroscopy coupled to a microprobe. anal bioanal chem. 2010;398(1):477-487. https://doi.org/10.1007/s00216-010-3910-6 200. bae k, zheng w, lin k, et al. epi-detected hyperspectral stimulated raman scattering microscopy for label-free molecular subtyping of glioblastomas. anal chem. 2018;90(17):10249-10255. https://doi.org/10.1021/acs.analchem.8b01677 201. lemoine é, dallaire f, yadav r, et al. feature engineering applied to intraoperative in vivo raman spectroscopy sheds light on molecular processes in brain cancer: a retrospective study of 65 patients. analyst. 2019;144(22):6517-6532. https://doi.org/10.1039/c9an01144g 202. livermore lj, isabelle m, bell im, et al. raman spectroscopy to differentiate between fresh tissue samples of glioma and normal brain: a comparison with 5-ala-induced fluorescence-guided surgery. j neurosurg. 2021;135(2):469-479. https://doi.org/10.3171/2020.5.jns20376 203. kairdolf ba, bouras a, kaluzova m, et al. intraoperative spectroscopy with ultrahigh sensitivity for image-guided surgery of malignant brain tumors. anal chem. 2016;88(1):858-867. https://doi.org/10.1021/acs.analchem.5b03453 204. livermore lj, isabelle m, bell i mac, et al. rapid intraoperative molecular genetic classification of gliomas using raman spectroscopy. neuro-oncology adv. 2019;1(1):vdz008. https://doi.org/10.1093/noajnl/vdz008 205. sciortino t, secoli r, d’amico e, et al. raman spectroscopy and machine learning for idh genotyping of unprocessed glioma biopsies. cancers (basel). 2021;13(16):1-13. https://doi.org/10.3390/cancers13164196 206. uckermann o, yao w, juratli ta, et al. idh1 mutation in human glioma induces chemical alterations that are amenable to optical raman spectroscopy. j neurooncol. 2018;139(2):261-268. https://doi.org/10.1007/s11060-018-2883-8 207. bukva m, dobra g, gomez-perez j, et al. raman spectral signatures of serum-derived extracellular vesicle-enriched isolates may support the diagnosis of cns tumors. cancers . 2021;13(6). https://doi.org/10.3390/cancers13061407 208. abramczyk h, brozek-pluska b, kopec m, surmacki j, błaszczyk m, radek m. redox imbalance and biochemical changes in cancer by probing redox-sensitive mitochondrial cytochromes in label-free visible resonance raman imaging. cancers (basel). 2021;13(5). https://doi.org/10.3390/cancers13050960 209. giardina g, micko a, bovenkamp d, et al. morpho-molecular metabolic analysis and classification of human pituitary gland and adenoma biopsies based on multimodal optical imaging. cancers (basel). 2021;13(13). https://doi.org/10.3390/cancers13133234 210. abramczyk h, imiela a, surmacki j. novel strategies of raman imaging for monitoring intracellular retinoid metabolism in cancer cells. j mol liq. 2021;334:116033. https://doi.org/10.1016/j.molliq.2021.116033 211. nair jb, mohapatra s, joseph mm, et al. tracking the footprints of paclitaxel delivery and mechanistic action via sers trajectory in glioblastoma cells. acs biomater sci eng. 2020;6(9):5254-5263. https://doi.org/10.1021/acsbiomaterials.0c00717 212. manciu fs, guerrero j, bennet ke, et al. assessing nordihydroguaiaretic acid therapeutic effect for glioblastoma multiforme. sensors (basel). 2022;22(7). https://doi.org/10.3390/s22072643 213. li j, wang c, yao y, et al. label-free discrimination of glioma brain tumors in different stages by surface enhanced raman scattering. talanta. 2020;216:120983. https://doi.org/10.1016/j.talanta.2020.120983 214. zhou y, liu c-h, wu b, et al. optical biopsy identification and grading of gliomas using label-free visible resonance raman spectroscopy. j biomed opt. 2019;24(09):095001. https://doi.org/10.1117/1.jbo.24.9.095001 215. morais clm, lilo t, ashton km, et al. determination of meningioma brain tumour grades using raman microspectroscopy imaging. analyst. 2019;144(23):7024-7031. https://doi.org/10.1039/c9an01551e 216. lilo t, morais clm, ashton km, et al. raman hyperspectral imaging coupled to three-dimensional discriminant analysis: classification of meningiomas brain tumour grades. spectrochim acta part a mol biomol spectrosc. 2022;273:121018. https://doi.org/10.1016/j.saa.2022.121018 217. zhang l, zhou y, wu b, et al. intraoperative detection of human meningioma using a handheld visible resonance raman analyzer. lasers med sci. 2021. https://doi.org/10.1007/s10103-021-03390-2 218. banerjee hn, banerji a, banerjee an, et al. deciphering the finger prints of brain cancer glioblastoma multiforme from four different patients by using near infrared raman spectroscopy. j cancer sci ther. 2015;7(2):44-47. https://doi.org/10.4172/1948-5956.1000323 219. bergner n, medyukhina a, geiger kd, et al. hyperspectral unmixing of raman micro-images for assessment of morphological and chemical parameters in non-dried brain tumor specimens. anal bioanal chem. 2013;405(27):8719-8728. https://doi.org/10.1007/s00216-013-7257-7 220. bergner n, krafft c, geiger kd, kirsch m, schackert g, popp j. unsupervised unmixing of raman microspectroscopic images for morphochemical analysis of non-dried brain tumor specimens. anal bioanal chem. 2012;403(3):719-725. https://doi.org/10.1007/s00216-012-5858-1 221. zhang j, fan y, he m, et al. accuracy of raman spectroscopy in differentiating brain tumor from normal brain tissue. oncotarget. 2017;8(22):36824-36831. https://doi.org/10.18632/oncotarget.15975 222. wills h, kast r, stewart c, et al. raman spectroscopy detects and distinguishes neuroblastoma and related tissues in fresh and (banked) frozen specimens. j pediatr surg. 2009;44(2):386-391. https://doi.org/10.1016/j.jpedsurg.2008.10.095 223. rabah r, weber r, serhatkulu gk, et al. diagnosis of neuroblastoma and ganglioneuroma using raman spectroscopy. j pediatr surg. 2008;43(1):171-176. https://doi.org/10.1016/j.jpedsurg.2007.09.040 224. ricciardi v, perna g, lasalvia m, et al. raman micro-spectroscopy investigation on the effects of x-rays and polyphenols in human neuroblastoma cells. in: clinical and preclinical optical diagnostics ii. optical society of america; 2019:11073_35. https://doi.org/10.1117/12.2526590 225. polis b, imiela a, polis l, abramczyk h. raman spectroscopy for medulloblastoma. childs nerv syst. 2018;34(12):2425-2430. https://doi.org/10.1007/s00381-018-3906-7 226. bovenkamp d, micko a, püls j, et al. line scan raman microspectroscopy for label-free diagnosis of human pituitary biopsies. molecules. 2019;24(19). https://doi.org/10.3390/molecules24193577 227. das d, bhattacharjee k, barman mj, et al. pathologic evidence of retinoblastoma seeds supported by field emission scanning electron microscopy and raman spectroscopy. indian j ophthalmol. 2021;69(12):3612-3617. https://doi.org/10.4103/ijo.ijo_436_21 228. kirsch m, schackert g, salzer r, krafft c. raman spectroscopic imaging for in vivo detection of cerebral brain metastases. anal bioanal chem. 2010;398(4):1707-1713. https://doi.org/10.1007/s00216-010-4116-7 229. klamminger gg, klein k, mombaerts l, et al. differentiation of primary cns lymphoma and glioblastoma using raman spectroscopy and machine learning algorithms. free neuropathol. 2021;2(se-original papers):26. https://doi.org/10.17879/freeneuropathology-2021-3458 230. doran ce, frank cb, mcgrath s, packer ra. use of handheld raman spectroscopy for intraoperative differentiation of normal brain tissue from intracranial neoplasms in dogs. front vet sci. 2022;8. https://doi.org/10.3389/fvets.2021.819200 231. jermyn m, mercier j, aubertin k, et al. highly accurate detection of cancer in situ with intraoperative, label-free, multimodal optical spectroscopy. cancer res. 2017;77(14):3942-3950. https://doi.org/10.1158/0008-5472.can-17-0668 232. desroches j, jermyn m, pinto m, et al. a new method using raman spectroscopy for in vivo targeted brain cancer tissue biopsy. sci rep. 2018;8(1):1792. https://doi.org/10.1038/s41598-018-20233-3 233. lakomkin n, hadjipanayis cg. the use of spectroscopy handheld tools in brain tumor surgery: current evidence and techniques. front surg. 2019;6:30. https://doi.org/10.3389/fsurg.2019.00030 234. desroches j, jermyn m, mok k, et al. characterization of a raman spectroscopy probe system for intraoperative brain tissue classification. biomed opt express. 2015;6(7):2380-2397. https://doi.org/10.1364/boe.6.002380 235. karabeber h, huang r, iacono p, et al. guiding brain tumor resection using surface-enhanced raman scattering nanoparticles and a hand-held raman scanner. acs nano. 2014;8(10):9755-9766. https://doi.org/10.1021/nn503948b 236. stevens oac, hutchings j, gray w, day jc. a low background raman probe for optical biopsy of brain tissue. in: proc.spie. vol 8939. ; 2014. https://doi.org/10.1117/12.2044139 237. desroches j, lemoine é, pinto m, et al. development and first in-human use of a raman spectroscopy guidance system integrated with a brain biopsy needle. j biophotonics. 2019;12(3):1-7. https://doi.org/10.1002/jbio.201800396 238. stables r, clemens g, butler hj, et al. feature driven classification of raman spectra for real-time spectral brain tumour diagnosis using sound. analyst. 2017;142(1):98-109. https://doi.org/10.1039/c6an01583b 239. nicolson f, andreiuk b, andreou c, hsu h-t, rudder s, kircher mf. non-invasive in vivo imaging of cancer using surface-enhanced spatially offset raman spectroscopy (sesors). theranostics. 2019;9(20):5899-5913. https://doi.org/10.7150/thno.36321 240. devpura s, thakur js, poulik jm, rabah r, naik vm, naik r. raman spectroscopic investigation of frozen and deparaffinized tissue sections of pediatric tumors: neuroblastoma and ganglioneuroma. j raman spectrosc. 2013;44(3):370-376. https://doi.org/10.1002/jrs.4223 241. fullwood lm, clemens g, griffiths d, et al. investigating the use of raman and immersion raman spectroscopy for spectral histopathology of metastatic brain cancer and primary sites of origin. anal methods. 2014;6(12):3948-3961. https://doi.org/10.1039/c3ay42190b 242. klamminger gg, gérardy j-j, jelke f, et al. application of raman spectroscopy for detection of histologically distinct areas in formalin-fixed paraffin-embedded glioblastoma. neuro-oncology adv. 2021;3(1):vdab077. https://doi.org/10.1093/noajnl/vdab077 243. mehta k, atak a, sahu a, srivastava s, c mk. an early investigative serum raman spectroscopy study of meningioma. analyst. 2018;143(8):1916-1923. https://doi.org/10.1039/c8an00224j 244. chen c, wu w, chen c, et al. rapid diagnosis of lung cancer and glioma based on serum raman spectroscopy combined with deep learning. j raman spectrosc. 2021;52(11):1798-1809. https://doi.org/10.1002/jrs.6224 245. le reste p, pilalis e, aubry m, et al. integration of raman spectra with transcriptome data in glioblastoma multiforme defines tumour subtypes and predicts patient outcome. j cell mol med. 2021;(august):10846-10856. https://doi.org/10.1111/jcmm.16902 246. selkoe dj. folding proteins in fatal ways. nature. 2003;426(6968):900-904. https://doi.org/10.1038/nature02264 247. chiti f, dobson cm. protein misfolding, functional amyloid, and human disease. annu rev biochem. 2006;75(1):333-366. https://doi.org/10.1146/annurev.biochem.75.101304.123901 248. paul tj, john h, h. fk. toxic proteins in neurodegenerative disease. science (80). 2002;296(5575):1991-1995. https://doi.org/10.1126/science.1067122 249. miller lm. chapter 5 infrared spectroscopy and imaging for understanding neurodegenerative protein-misfolding diseases. in: ozaki y, baranska m, lednev ik, wood brbt-vs in pr, eds. academic press; 2020:121-142. https://doi.org/10.1016/b978-0-12-818610-7.00005-0 250. paraskevaidi m, martin-hirsch pl, martin fl. vibrational spectroscopy: a promising approach to discriminate neurodegenerative disorders. mol neurodegener. 2018;13(1):20. https://doi.org/10.1186/s13024-018-0252-x 251. paraskevaidi m, martin-hirsch pl, martin fl. progress and challenges in the diagnosis of dementia: a critical review. acs chem neurosci. 2018;9(3):446-461. https://doi.org/10.1021/acschemneuro.8b00007 252. mitchell al, gajjar kb, theophilou g, martin fl, martin-hirsch pl. vibrational spectroscopy of biofluids for disease screening or diagnosis: translation from the laboratory to a clinical setting. j biophotonics. 2014;7(3-4):153-165. https://doi.org/10.1002/jbio.201400018 253. lopes j, correia m, martins i, et al. ftir and raman spectroscopy applied to dementia diagnosis through analysis of biological fluids. j alzheimers dis. 2016;52(3):801-812. https://doi.org/10.3233/jad-151163 254. paraskevaidi m, morais clm, lima kmg, et al. differential diagnosis of alzheimer’s disease using spectrochemical analysis of blood. proc natl acad sci u s a. 2017;114(38):e7929-e7938. https://doi.org/10.1073/pnas.1701517114 255. oladepo sa, xiong k, hong z, asher sa, handen j, lednev ik. uv resonance raman investigations of peptide and protein structure and dynamics. chem rev. 2012;112(5):2604-2628. https://doi.org/10.1021/cr200198a 256. lednev ik, ermolenkov v v, he w, xu m. deep-uv raman spectrometer tunable between 193 and 205 nm for structural characterization of proteins. anal bioanal chem. 2005;381(2):431-437. https://doi.org/10.1007/s00216-004-2991-5 257. barron ld, hecht l, blanch ew, bell af. solution structure and dynamics of biomolecules from raman optical activity. prog biophys mol biol. 2000;73(1):1-49. https://doi.org/10.1016/s0079-6107(99)00017-6 258. barron ld, buckingham ad. rayleigh and raman scattering from optically active molecules. mol phys. 1971;20(6):1111-1119. https://doi.org/10.1080/00268977100101091 259. martial b, lefèvre t, auger m. understanding amyloid fibril formation using protein fragments: structural investigations via vibrational spectroscopy and solid-state nmr. biophys rev. 2018;10(4):1133-1149. https://doi.org/10.1007/s12551-018-0427-2 260. summers kl, fimognari n, hollings a, et al. a multimodal spectroscopic imaging method to characterize the metal and macromolecular content of proteinaceous aggregates ("amyloid plaques”). biochemistry. 2017;56(32):4107-4116. https://doi.org/10.1021/acs.biochem.7b00262 261. luo z, xu h, liu l, ohulchanskyy ty, qu j. optical imaging of beta-amyloid plaques in alzheimer’s disease. biosensors. 2021;11(8):255. https://doi.org/10.3390/bios11080255 262. ghosh c, pramanik d, mukherjee s, dey a, dey sg. interaction of no with cu and heme-bound aβ peptides associated with alzheimer’s disease. inorg chem. 2013;52(1):362-368. https://doi.org/10.1021/ic302131n 263. moran sd, zanni mt. how to get insight into amyloid structure and formation from infrared spectroscopy. j phys chem lett. 2014;5(11):1984-1993. https://doi.org/10.1021/jz500794d 264. li h, lantz r, du d. vibrational approach to the dynamics and structure of protein amyloids. molecules. 2019;24(1). https://doi.org/10.3390/molecules24010186 265. ma j, pazos im, zhang w, culik rm, gai f. site-specific infrared probes of proteins. annu rev phys chem. 2015;66(1):357-377. https://doi.org/10.1146/annurev-physchem-040214-121802 266. bloem r, koziol k, waldauer sa, et al. ligand binding studied by 2d ir spectroscopy using the azidohomoalanine label. j phys chem b. 2012;116(46):13705-13712. https://doi.org/10.1021/jp3095209 267. wang z, ye j, zhang k, et al. rapid biomarker screening of alzheimer’s disease by interpretable machine learning and graphene-assisted raman spectroscopy. acs nano. 2022;16(4):6426-6436. https://doi.org/10.1021/acsnano.2c00538 268. hanlon eb, manoharan r, koo tw, et al. prospects for in vivo raman spectroscopy. phys med biol. 2000;45(2):r1-59. https://doi.org/10.1088/0031-9155/45/2/201 269. dong j, atwood cs, anderson ve, et al. metal binding and oxidation of amyloid-β within isolated senile plaque cores:  raman microscopic evidence. biochemistry. 2003;42(10):2768-2773. https://doi.org/10.1021/bi0272151 270. sudworth cd, m.d. nk. raman spectroscopy of alzheimer’s diseased tissue. in: proc.spie. vol 5321. ; 2004. https://doi.org/10.1117/12.552869 271. chen p, shen a, zhao w, baek s-j, yuan h, hu j. raman signature from brain hippocampus could aid alzheimer’s disease diagnosis. appl opt. 2009;48(24):4743-4748. https://doi.org/10.1364/ao.48.004743 272. paul tj, hoffmann z, wang c, et al. structural and mechanical properties of amyloid beta fibrils: a combined experimental and theoretical approach. j phys chem lett. 2016;7(14):2758-2764. https://doi.org/10.1021/acs.jpclett.6b01066 273. xiong j, jiji rd. insights into the aggregation mechanism of aβ(25-40). biophys chem. 2017;220:42-48. https://doi.org/10.1016/j.bpc.2016.11.003 274. popova la, kodali r, wetzel r, lednev ik. structural variations in the cross-β core of amyloid β fibrils revealed by deep uv resonance raman spectroscopy. j am chem soc. 2010;132(18):6324-6328. https://doi.org/10.1021/ja909074j 275. xiong j, roach ca, oshokoya oo, et al. role of bilayer characteristics on the structural fate of aβ(1–40) and aβ(25–40). biochemistry. 2014;53(18):3004-3011. https://doi.org/10.1021/bi4016296 276. cunha r, lafeta l, fonseca ea, et al. nonlinear and vibrational microscopy for label-free characterization of amyloid-β plaques in alzheimer’s disease model. analyst. 2021;146(9):2945-2954. https://doi.org/10.1039/d1an00074h 277. buividas r, dzingelevičius n, kubiliūtė r, et al. statistically quantified measurement of an alzheimer’s marker by surface-enhanced raman scattering. j biophotonics. 2015;8(7):567-574. https://doi.org/10.1002/jbio.201400017 278. park hj, cho s, kim m, jung ys. carboxylic acid-functionalized, graphitic layer-coated three-dimensional sers substrate for label-free analysis of alzheimer’s disease biomarkers. nano lett. 2020;20(4):2576-2584. https://doi.org/10.1021/acs.nanolett.0c00048 279. xia y, padmanabhan p, sarangapani s, gulyás b, vadakke matham m. bifunctional fluorescent/raman nanoprobe for the early detection of amyloid. sci rep. 2019;9(1):8497. https://doi.org/10.1038/s41598-019-43288-2 280. zhou y, liu j, zheng t, tian y. label-free sers strategy for in situ monitoring and real-time imaging of aβ aggregation process in live neurons and brain tissues. anal chem. 2020;92(8):5910-5920. https://doi.org/10.1021/acs.analchem.9b05837 281. yu x, hayden ey, xia m, et al. surface enhanced raman spectroscopy distinguishes amyloid β-protein isoforms and conformational states. protein sci. 2018;27(8):1427-1438. https://doi.org/10.1002/pro.3434 282. bonhommeau s, talaga d, hunel j, cullin c, lecomte s. tip-enhanced raman spectroscopy to distinguish toxic oligomers from aβ(1-42) fibrils at the nanometer scale. angew chem int ed engl. 2017;56(7):1771-1774. https://doi.org/10.1002/anie.201610399 283. d’andrea c, foti a, cottat m, et al. nanoscale discrimination between toxic and nontoxic protein misfolded oligomers with tip-enhanced raman spectroscopy. small. 2018;14(36):e1800890. https://doi.org/10.1002/smll.201800890 284. michael r, lenferink a, vrensen gfjm, gelpi e, barraquer ri, otto c. hyperspectral raman imaging of neuritic plaques and neurofibrillary tangles in brain tissue from alzheimer’s disease patients. sci rep. 2017;7(1):15603. https://doi.org/10.1038/s41598-017-16002-3 285. lochocki b, morrema thj, ariese f, hoozemans jjm, de boer jf. the search for a unique raman signature of amyloid-beta plaques in human brain tissue from alzheimer{’}s disease patients. analyst. 2020;145(5):1724-1736. https://doi.org/10.1039/c9an02087j 286. el khoury y, schirer a, patte-mensah c, et al. raman imaging reveals accumulation of hemoproteins in plaques from alzheimer’s diseased tissues. acs chem neurosci. 2021;12(15):2940-2945. https://doi.org/10.1021/acschemneuro.1c00289 287. röhr d, boon bdc, schuler m, et al. label-free vibrational imaging of different aβ plaque types in alzheimer’s disease reveals sequential events in plaque development. acta neuropathol commun. 2020;8(1):222. https://doi.org/10.1186/s40478-020-01091-5 288. palombo f, tamagnini f, jeynes jcg, et al. detection of aβ plaque-associated astrogliosis in alzheimer’s disease brain by spectroscopic imaging and immunohistochemistry. analyst. 2018;143(4):850-857. https://doi.org/10.1039/c7an01747b 289. kiskis j, fink h, nyberg l, thyr j, li j-y, enejder a. plaque-associated lipids in alzheimer’s diseased brain tissue visualized by nonlinear microscopy. sci rep. 2015;5(1):13489. https://doi.org/10.1038/srep13489 290. li s, luo z, zhang r, et al. distinguishing amyloid β-protein in a mouse model of alzheimer’s disease by label-free vibrational imaging. biosensors. 2021;11(10):365. https://doi.org/10.3390/bios11100365 291. fonseca ea, lafetá l, cunha r, et al. a fingerprint of amyloid plaques in a bitransgenic animal model of alzheimer{’}s disease obtained by statistical unmixing analysis of hyperspectral raman data. analyst. 2019;144(23):7049-7056. https://doi.org/10.1039/c9an01631g 292. lochocki b, boon bdc, verheul sr, et al. multimodal, label-free fluorescence and raman imaging of amyloid deposits in snap-frozen alzheimer’s disease human brain tissue. commun biol. 2021;4(1):474. https://doi.org/10.1038/s42003-021-01981-x 293. tabatabaei m, caetano fa, pashee f, ferguson ssg, lagugné-labarthet f. tip-enhanced raman spectroscopy of amyloid β at neuronal spines. analyst. 2017;142(23):4415-4421. https://doi.org/10.1039/c7an00744b 294. liu k, li j, raghunathan r, zhao h, li x, wong stc. the progress of label-free optical imaging in alzheimer’s disease screening and diagnosis. front aging neurosci. 2021;13:455. https://doi.org/10.3389/fnagi.2021.699024 295. ji m, arbel m, zhang l, et al. label-free imaging of amyloid plaques in alzheimer’s disease with stimulated raman scattering microscopy. sci adv. 2018;4(11):eaat7715. https://doi.org/10.1126/sciadv.aat7715 296. michael r, otto c, lenferink a, et al. absence of amyloid-beta in lenses of alzheimer patients: a confocal raman microspectroscopic study. exp eye res. 2014;119:44-53. https://doi.org/10.1016/j.exer.2013.11.016 297. fonseca ea, lafeta l, luiz campos j, et al. micro-raman spectroscopy of lipid halo and dense-core amyloid plaques: aging process characterization in the alzheimer{’}s disease appsweps1δe9 mouse model. analyst. 2021;146(19):6014-6025. https://doi.org/10.1039/d1an01078f 298. lee jh, kim dh, song wk, oh m-k, ko d-k. label-free imaging and quantitative chemical analysis of alzheimer’s disease brain samples with multimodal multiphoton nonlinear optical microspectroscopy. j biomed opt. 2015;20(5):1-7. https://doi.org/10.1117/1.jbo.20.5.056013 299. huang c-c, isidoro c. raman spectrometric detection methods for early and non-invasive diagnosis of alzheimer’s disease. j alzheimers dis. 2017;57(4):1145-1156. https://doi.org/10.3233/jad-161238 300. hrubešová k, fousková m, habartová l, et al. search for biomarkers of alzheimer’s disease: recent insights, current challenges and future prospects. clin biochem. 2019;72:39-51. https://doi.org/10.1016/j.clinbiochem.2019.04.002 301. eravuchira pj, banchelli m, d’andrea c, angelis m de, matteini p, gannot i. hollow core photonic crystal fiber-assisted raman spectroscopy as a tool for the detection of alzheimer’s disease biomarkers. j biomed opt. 2020;25(7):1-10. https://doi.org/10.1117/1.jbo.25.7.077001 302. oyarzún mp, tapia-arellano a, cabrera p, jara-guajardo p, kogan mj. plasmonic nanoparticles as optical sensing probes for the detection of alzheimer’s disease. sensors (basel). 2021;21(6). https://doi.org/10.3390/s21062067 303. cennamo g, montorio d, morra vb, et al. surface-enhanced raman spectroscopy of tears: toward a diagnostic tool for neurodegenerative disease identification. j biomed opt. 2020;25(8):1-12. https://doi.org/10.1117/1.jbo.25.8.087002 304. ralbovsky nm, halámková l, wall k, anderson-hanley c, lednev ik. screening for alzheimer’s disease using saliva: a new approach based on machine learning and raman hyperspectroscopy. j alzheimers dis. 2019;71(4):1351-1359. https://doi.org/10.3233/jad-190675 305. ryzhikova e, ralbovsky nm, sikirzhytski v, et al. raman spectroscopy and machine learning for biomedical applications: alzheimer’s disease diagnosis based on the analysis of cerebrospinal fluid. spectrochim acta part a mol biomol spectrosc. 2021;248:119188. https://doi.org/10.1016/j.saa.2020.119188 306. chou i-h, benford m, beier ht, et al. nanofluidic biosensing for β-amyloid detection using surface enhanced raman spectroscopy. nano lett. 2008;8(6):1729-1735. https://doi.org/10.1021/nl0808132 307. stiebing c, jahn ij, schmitt m, et al. biochemical characterization of mouse retina of an alzheimer’s disease model by raman spectroscopy. acs chem neurosci. 2020;11(20):3301-3308. https://doi.org/10.1021/acschemneuro.0c00420 308. carmona p, molina m, calero m, bermejo-pareja f, martínez-martín p, toledano a. discrimination analysis of blood plasma associated with alzheimer’s disease using vibrational spectroscopy. j alzheimers dis. 2013;34(4):911-920. https://doi.org/10.3233/jad-122041 309. carmona p, molina m, lópez-tobar e, toledano a. vibrational spectroscopic analysis of peripheral blood plasma of patients with alzheimer’s disease. anal bioanal chem. 2015;407(25):7747-7756. https://doi.org/10.1007/s00216-015-8940-7 310. paraskevaidi m, morais clm, halliwell de, et al. raman spectroscopy to diagnose alzheimer’s disease and dementia with lewy bodies in blood. acs chem neurosci. 2018;9(11):2786-2794. https://doi.org/10.1021/acschemneuro.8b00198 < 311. habartová l, hrubešová k, syslová k, et al. blood-based molecular signature of alzheimer’s disease via spectroscopy and metabolomics. clin biochem. 2019;72:58-63. https://doi.org/10.1016/j.clinbiochem.2019.04.004 312. ralbovsky nm, fitzgerald gs, mcnay ec, lednev ik. towards development of a novel screening method for identifying alzheimer’s disease risk: raman spectroscopy of blood serum and machine learning. spectrochim acta a mol biomol spectrosc. 2021;254:119603. https://doi.org/10.1016/j.saa.2021.119603 313. demeritte t, viraka nellore bp, kanchanapally r, et al. hybrid graphene oxide based plasmonic-magnetic multifunctional nanoplatform for selective separation and label-free identification of alzheimer’s disease biomarkers. acs appl mater interfaces. 2015;7(24):13693-13700. https://doi.org/10.1021/acsami.5b03619 314. yang j-k, hwang i-j, cha mg, et al. reaction kinetics-mediated control over silver nanogap shells as surface-enhanced raman scattering nanoprobes for detection of alzheimer’s disease biomarkers. small. 2019;15(19):e1900613. https://doi.org/10.1002/smll.201900613 315. yu d, yin q, wang j, et al. sers-based immunoassay enhanced with silver probe for selective separation and detection of alzheimer’s disease biomarkers. int j nanomedicine. 2021;16:1901-1911. https://doi.org/10.2147/ijn.s293042 316. ryzhikova e, kazakov o, halamkova l, et al. raman spectroscopy of blood serum for alzheimer’s disease diagnostics: specificity relative to other types of dementia. j biophotonics. 2015;8(7):584-596. https://doi.org/10.1002/jbio.201400060 317. hao n, wang z, liu p, et al. acoustofluidic multimodal diagnostic system for alzheimer’s disease. biosens bioelectron. 2022;196:113730. https://doi.org/10.1016/j.bios.2021.113730 318. carlomagno c, cabinio m, picciolini s, gualerzi a, baglio f, bedoni m. sers-based biosensor for alzheimer disease evaluation through the fast analysis of human serum. j biophotonics. 2020;13(3):e201960033. https://doi.org/10.1002/jbio.201960033 319. chen p, tian q, baek sj, et al. laser raman detection of platelet as a non-invasive approach for early and differential diagnosis of alzheimer’s disease. laser phys lett. 2011;8(7):547-552. https://doi.org/10.1002/lapl.201110016 320. monfared amt, tiwari vs, trudeau vl, anis h. surface-enhanced raman scattering spectroscopy for the detection of glutamate and γ-aminobutyric acid in serum by partial least squares analysis. ieee photonics j. 2015;7(3). https://doi.org/10.1109/jphot.2015.2423284 321. ardini m, huang j-a, sánchez cs, et al. live intracellular biorthogonal imaging by surface enhanced raman spectroscopy using alkyne-silver nanoparticles clusters. sci rep. 2018;8(1):12652. https://doi.org/10.1038/s41598-018-31165-3 322. lee w, kang b-h, yang h, et al. spread spectrum sers allows label-free detection of attomolar neurotransmitters. nat commun. 2021;12(1):159. https://doi.org/10.1038/s41467-020-20413-8 323. moody as, sharma b. multi-metal, multi-wavelength surface-enhanced raman spectroscopy detection of neurotransmitters. acs chem neurosci. 2018;9(6):1380-1387. https://doi.org/10.1021/acschemneuro.8b00020 324. moody as, baghernejad pc, webb kr, sharma b. surface enhanced spatially offset raman spectroscopy detection of neurochemicals through the skull. anal chem. 2017;89(11):5688-5692. https://doi.org/10.1021/acs.analchem.7b00985 325. moody as, payne td, barth ba, sharma b. surface-enhanced spatially-offset raman spectroscopy (sesors) for detection of neurochemicals through the skull at physiologically relevant concentrations. analyst. 2020;145(5):1885-1893. https://doi.org/10.1039/c9an01708a 326. cao x, qin m, li p, et al. probing catecholamine neurotransmitters based on iron-coordination surface-enhanced resonance raman spectroscopy label. sensors actuators b chem. 2018;268:350-358. https://doi.org/10.1016/j.snb.2018.04.117 327. zhou b, li x, tang x, li p, yang l, liu j. highly selective and repeatable surface-enhanced resonance raman scattering detection for epinephrine in serum based on interface self-assembled 2d nanoparticles arrays. acs appl mater interfaces. 2017;9(8):7772-7779. https://doi.org/10.1021/acsami.6b15205 328. ciubuc jd, bennet ke, qiu c, alonzo m, durrer wg, manciu fs. raman computational and experimental studies of dopamine detection. biosensors. 2017;7(4). https://doi.org/10.3390/bios7040043 329. silwal ap, yadav r, sprague je, lu hp. raman spectroscopic signature markers of dopamine–human dopamine transporter interaction in living cells. acs chem neurosci. 2017;8(7):1510-1518. https://doi.org/10.1021/acschemneuro.7b00048 330. manciu fs, manciu m, ciubuc jd, et al. simultaneous detection of dopamine and serotonin-a comparative experimental and theoretical study of neurotransmitter interactions. biosensors. 2018;9(1). https://doi.org/10.3390/bios9010003 331. shi l, liu m, zhang l, tian y. a liquid interfacial sers platform on a nanoparticle array stabilized by rigid probes for the quantification of norepinephrine in rat brain microdialysates. angew chem int ed engl. march 2022:e202117125. https://doi.org/10.1002/anie.202117125 332. miura t, suzuki k, kohata n, takeuchi h. metal binding modes of alzheimer’s amyloid β-peptide in insoluble aggregates and soluble complexes. biochemistry. 2000;39(23):7024-7031. https://doi.org/10.1021/bi0002479 333. miura t, suzuki k, takeuchi h. binding of iron(iii) to the single tyrosine residue of amyloid β-peptide probed by raman spectroscopy. j mol struct. 2001;598:79-84. https://doi.org/10.1016/s0022-2860(01)00807-9 334. yugay d, goronzy dp, kawakami lm, et al. copper ion binding site in β-amyloid peptide. nano lett. 2016;16(10):6282-6289. https://doi.org/10.1021/acs.nanolett.6b02590 335. ren h, zhang y, guo s, et al. identifying cu(ii)–amyloid peptide binding intermediates in the early stages of aggregation by resonance raman spectroscopy: a simulation study. phys chem chem phys. 2017;19(46):31103-31112. https://doi.org/10.1039/c7cp06206k 336. suzuki k, miura t, takeuchi h. inhibitory effect of copper(ii) on zinc(ii)-induced aggregation of amyloid beta-peptide. biochem biophys res commun. 2001;285(4):991-996. https://doi.org/10.1006/bbrc.2001.5263 337. miura t, mitani s, takanashi c, mochizuki n. copper selectively triggers β-sheet assembly of an n-terminally truncated amyloid β-peptide beginning with glu3. j inorg biochem. 2004;98(1):10-14. https://doi.org/10.1016/j.jinorgbio.2003.10.008 338. syme cd, blanch ew, holt c, et al. a raman optical activity study of rheomorphism in caseins, synucleins and tau. new insight into the structure and behaviour of natively unfolded proteins. eur j biochem. 2002;269(1):148-156. https://doi.org/10.1046/j.0014-2956.2001.02633.x 339. ramachandran g, milán-garcés ea, udgaonkar jb, puranik m. resonance raman spectroscopic measurements delineate the structural changes that occur during tau fibril formation. biochemistry. 2014;53(41):6550-6565. https://doi.org/10.1021/bi500528x 340. zengin a, tamer u, caykara t. a sers-based sandwich assay for ultrasensitive and selective detection of alzheimer’s tau protein. biomacromolecules. 2013;14(9):3001-3009. https://doi.org/10.1021/bm400968x 341. maurer v, frank c, porsiel jc, zellmer s, garnweitner g, stosch r. step‐by‐step monitoring of a magnetic and sers‐active immunosensor assembly for purification and detection of tau protein. j biophotonics. 2020;13(3). https://doi.org/10.1002/jbio.201960090 342. sereda v, lednev ik. polarized raman spectroscopy of aligned insulin fibrils. j raman spectrosc. 2014;45(8):665-671. https://doi.org/10.1002/jrs.4523 343. deckert-gaudig t, kurouski d, hedegaard mab, singh p, lednev ik, deckert v. spatially resolved spectroscopic differentiation of hydrophilic and hydrophobic domains on individual insulin amyloid fibrils. sci rep. 2016;6(1):33575. https://doi.org/10.1038/srep33575 344. kurouski d, deckert-gaudig t, deckert v, lednev ik. surface characterization of insulin protofilaments and fibril polymorphs using tip-enhanced raman spectroscopy (ters). biophys j. 2014;106(1):263-271. https://doi.org/10.1016/j.bpj.2013.10.040 345. kurouski d, sorci m, postiglione t, belfort g, lednev ik. detection and structural characterization of insulin prefibrilar oligomers using surface enhanced raman spectroscopy. biotechnol prog. 2014;30(2):488-495. https://doi.org/10.1002/btpr.1852 346. rivas-arancibia s, rodríguez-martínez e, badillo-ramírez i, lópez-gonzález u, saniger jm. structural changes of amyloid beta in hippocampus of rats exposed to ozone: a raman spectroscopy study. front mol neurosci. 2017;10:137. https://doi.org/10.3389/fnmol.2017.00137 347. maiti nc, apetri mm, zagorski mg, carey pr, anderson ve. raman spectroscopic characterization of secondary structure in natively unfolded proteins:  α-synuclein. j am chem soc. 2004;126(8):2399-2408. https://doi.org/10.1021/ja0356176 348. apetri mm, maiti nc, zagorski mg, carey pr, anderson ve. secondary structure of α-synuclein oligomers: characterization by raman and atomic force microscopy. j mol biol. 2006;355(1):63-71. https://doi.org/10.1016/j.jmb.2005.10.071 349. flynn jd, mcglinchey rp, walker rl 3rd, lee jc. structural features of α-synuclein amyloid fibrils revealed by raman spectroscopy. j biol chem. 2018;293(3):767-776. https://doi.org/10.1074/jbc.m117.812388 350. sevgi f, brauchle em, carvajal berrio da, et al. imaging of α-synuclein aggregates in a rat model of parkinson’s disease using raman microspectroscopy. front cell dev biol. 2021;9:664365. https://doi.org/10.3389/fcell.2021.664365 351. mensch c, konijnenberg a, van elzen r, lambeir am, sobott f, johannessen c. raman optical activity of human α-synuclein in intrinsically disordered, micelle-bound α-helical, molten globule and oligomeric β-sheet state. j raman spectrosc. 2017;48(7):910-918. https://doi.org/10.1002/jrs.5149 352. freitas a, aroso m, barros a, et al. characterization of the striatal extracellular matrix in a mouse model of parkinson’s disease. antioxidants (basel, switzerland). 2021;10(7). https://doi.org/10.3390/antiox10071095 353. palanisamy s, yan l, zhang x, he t. surface enhanced raman scattering-active worm-like ag clusters for sensitive and selective detection of dopamine. anal methods. 2015;7(8):3438-3447. https://doi.org/10.1039/c4ay03061c 354. lim jw, kang ij. fabrication of chitosan-gold nanocomposites combined with optical fiber as sers substrates to detect dopamine molecules. bull korean chem soc. 2014;35(1):25-29. https://doi.org/10.5012/bkcs.2014.35.1.25 355. an j-h, el-said wa, yea c-h, kim t-h, choi j-w. surface-enhanced raman scattering of dopamine on self-assembled gold nanoparticles. j nanosci nanotechnol. 2011;11(5):4424-4429. https://doi.org/10.1166/jnn.2011.3688 356. ranc v, markova z, hajduch m, et al. magnetically assisted surface-enhanced raman scattering selective determination of dopamine in an artificial cerebrospinal fluid and a mouse striatum using fe3o4/ag nanocomposite. anal chem. 2014;86(6):2939-2946. https://doi.org/10.1021/ac500394g 357. phung v-d, jung w-s, nguyen t-a, kim j-h, lee s-w. reliable and quantitative sers detection of dopamine levels in human blood plasma using a plasmonic au/ag nanocluster substrate. nanoscale. 2018;10(47):22493-22503. https://doi.org/10.1039/c8nr06444j 358. sharma a, müller j, schuetze k, et al. comprehensive profiling of blood coagulation and fibrinolysis marker reveals elevated plasmin-antiplasmin complexes in parkinson’s disease. biology (basel). 2021;10(8). https://doi.org/10.3390/biology10080716 359. carlomagno c, bertazioli d, gualerzi a, et al. identification of the raman salivary fingerprint of parkinson’s disease through the spectroscopiccomputational combinatory approach. front neurosci. 2021;15:704963. https://doi.org/10.3389/fnins.2021.704963 360. schipper hm, kwok cs, rosendahl sm, et al. spectroscopy of human plasma for diagnosis of idiopathic parkinson’s disease. biomark med. 2008;2(3):229-238. https://doi.org/10.2217/17520363.2.3.229 361. mammadova n, summers cm, kokemuller rd, et al. accelerated accumulation of retinal α-synuclein (pser129) and tau, neuroinflammation, and autophagic dysregulation in a seeded mouse model of parkinson’s disease. neurobiol dis. 2019;121:1-16. https://doi.org/10.1016/j.nbd.2018.09.013 362. tian f, yang w, mordes da, et al. monitoring peripheral nerve degeneration in als by label-free stimulated raman scattering imaging. nat commun. 2016;7(1):13283. https://doi.org/10.1038/ncomms13283 363. picardi g, spalloni a, generosi a, et al. tissue degeneration in als affected spinal cord evaluated by raman spectroscopy. sci rep. 2018;8(1):13110. https://doi.org/10.1038/s41598-018-31469-4 364. zhang q-j, chen y, zou x-h, et al. prognostic analysis of amyotrophic lateral sclerosis based on clinical features and plasma surface-enhanced raman spectroscopy. j biophotonics. 2019;12(8):e201900012. https://doi.org/10.1002/jbio.201900012 365. zhang qj, chen y, zou xh, et al. promoting identification of amyotrophic lateral sclerosis based on label-free plasma spectroscopy. ann clin transl neurol. 2020;7(10):2010-2018. https://doi.org/10.1002/acn3.51194 366. morasso cf, sproviero d, mimmi mc, et al. raman spectroscopy reveals biochemical differences in plasma derived extracellular vesicles from sporadic amyotrophic lateral sclerosis patients. nanomedicine nanotechnology, biol med. 2020;29:102249. https://doi.org/10.1016/j.nano.2020.102249 367. carlomagno c, banfi pi, gualerzi a, et al. human salivary raman fingerprint as biomarker for the diagnosis of amyotrophic lateral sclerosis. sci rep. 2020;10(1):10175. https://doi.org/10.1038/s41598-020-67138-8 368. miao k, wei l. live-cell imaging and quantification of polyq aggregates by stimulated raman scattering of selective deuterium labeling. acs cent sci. 2020;6(4):478-486. https://doi.org/10.1021/acscentsci.9b01196 369. xiong k, punihaole d, asher sa. uv resonance raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization. biochemistry. 2012;51(29):5822-5830. https://doi.org/10.1021/bi300551b 370. perney nm, braddick l, jurna m, et al. polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-stokes raman scattering microscopy. plos one. 2012;7(7):e40536. https://doi.org/10.1371/journal.pone.0040536. 371. huefner a, kuan w-l, mason sl, mahajan s, barker ra. serum raman spectroscopy as a diagnostic tool in patients with huntington{’}s disease. chem sci. 2020;11(2):525-533. https://doi.org/10.1039/c9sc03711j 372. muratore m. raman spectroscopy and partial least squares analysis in discrimination of peripheral cells affected by huntington’s disease. anal chim acta. 2013;793:1-10. https://doi.org/10.1016/j.aca.2013.06.012 373. shashilov v, xu m, makarava n, savtchenko r, baskakov i v, lednev ik. dissecting structure of prion amyloid fibrils by hydrogen–deuterium exchange ultraviolet raman spectroscopy. j phys chem b. 2012;116(27):7926-7930. https://doi.org/10.1021/jp2122455 374. mccoll ih, blanch ew, gill ac, et al. a new perspective on β-sheet structures using vibrational raman optical activity:  from poly(l-lysine) to the prion protein. j am chem soc. 2003;125(33):10019-10026. https://doi.org/10.1021/ja021464v 375. zhu f, davies p, thompsett ar, et al. raman optical activity and circular dichroism reveal dramatic differences in the influence of divalent copper and manganese ions on prion protein folding. biochemistry. 2008;47(8):2510-2517. https://doi.org/10.1021/bi7022893 376. miura t, hori-i a, mototani h, takeuchi h. raman spectroscopic study on the copper(ii) binding mode of prion octapeptide and its ph dependence. biochemistry. 1999;38(35):11560-11569. https://doi.org/10.1021/bi9909389 377. miura t, hori-i a, takeuchi h. metal-dependent α-helix formation promoted by the glycine-rich octapeptide region of prion protein. febs lett. 1996;396(2-3):248-252. https://doi.org/10.1016/0014-5793(96)01104-0 378. krasnoslobodtsev a v, portillo am, deckert-gaudig t, deckert v, lyubchenko yl. nanoimaging for prion related diseases. prion. 2010;4(4):265-274. https://doi.org/10.4161/pri.4.4.13125 379. carmona p, monleón e, monzón m, badiola jj, monreal j. raman analysis of prion protein in blood cell membranes from naturally affected scrapie sheep. chem biol. 2004;11(6):759-764. https://doi.org/10.1016/j.chembiol.2004.04.005 380. pezzotti g, adachi t, miyamoto n, et al. raman probes for in situ molecular analyses of peripheral nerve myelination. acs chem neurosci. 2020;11(15):2327-2339. https://doi.org/10.1021/acschemneuro.0c00284 381. carmona p, ramos jm, de cózar m, monreal j. conformational features of lipids and proteins in myelin membranes using raman and infrared spectroscopy. j raman spectrosc. 1987;18(7):473-476. https://doi.org/10.1002/jrs.1250180704 382. hu c-r, zhang d, slipchenko mn, cheng j, hu b. label-free real-time imaging of myelination in the xenopus laevis tadpole by in vivo stimulated raman scattering microscopy. j biomed opt. 2014;19(8):086005. https://doi.org/10.1117/1.jbo.19.8.086005 383. turcotte r, rutledge dj, bélanger e, dill d, macklin wb, côté dc. intravital assessment of myelin molecular order with polarimetric multiphoton microscopy. sci rep. 2016;6(1):31685. https://doi.org/10.1038/srep31685 384. huang j-r, cheng y-c, huang hj, chiang h-p. confocal mapping of myelin figures with micro-raman spectroscopy. appl phys a. 2017;124(1):58. https://doi.org/10.1007/s00339-017-1450-z 385. wang h, fu y, zickmund p, shi r, cheng j-x. coherent anti-stokes raman scattering imaging of axonal myelin in live spinal tissues. biophys j. 2005;89(1):581-591. https://doi.org/10.1529/biophysj.105.061911 386. fu y, huff tb, wang h-w, wang h, cheng j-x. ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-stokes raman scattering microscopy. opt express. 2008;16(24):19396-19409. https://doi.org/10.1364/oe.16.019396 387. lucas a, poleg s, klug a, mccullagh ea. myelination deficits in the auditory brainstem of a mouse model of fragile x syndrome. front neurosci. 2021;15:772943. https://doi.org/10.3389/fnins.2021.772943 388. poulen g, gerber yn, perez j-c, et al. coherent anti-stokes raman scattering microscopy: a label-free method to compare spinal cord myelin in different species. front phys. 2021;9:438. https://doi.org/10.3389/fphy.2021.665650 389. ramos ir, lyng fm, rehman iu, sharrack b, woodroofe mn. the use of vibrational spectroscopy to study the pathogenesis multiple sclerosis and other neurological conditions. appl spectrosc rev. 2017;52(10):868-882. https://doi.org/10.1080/05704928.2017.1336450 390. ozsvár a, szipőcs r, ozsvár z, et al. quantitative analysis of lipid debris accumulation caused by cuprizone induced myelin degradation in different cns areas. brain res bull. 2018;137:277-284. https://doi.org/10.1016/j.brainresbull.2018.01.003 391. poon kwc, brideau c, klaver r, schenk gj, geurts jj, stys pk. lipid biochemical changes detected in normal appearing white matter of chronic multiple sclerosis by spectral coherent raman imaging. chem sci. 2018;9(6):1586-1595. https://doi.org/10.1039/c7sc03992a 392. poon kw, brideau c, schenk gj, et al. quantitative biochemical investigation of various neuropathologies using high-resolution spectral cars microscopy. in: proc.spie. vol 9305. ; 2015. https://doi.org/10.1117/12.2076654 393. poon kw, brideau c, teo w, et al. investigation of human multiple sclerosis lesions using high resolution spectrally unmixed cars microscopy. in: proc.spie. vol 8565. ; 2013. https://doi.org/10.1117/12.2005504 394. imitola j, côté d, rasmussen s, et al. multimodal coherent anti-stokes raman scattering microscopy reveals microglia-associated myelin and axonal dysfunction in multiple sclerosis-like lesions in mice. j biomed opt. 2011;16(2):21109. https://doi.org/10.1117/1.3533312 395. dessai cvp, pliss a, kuzmin an, furlani ep, prasad pn. coherent raman spectroscopic imaging to characterize microglia activation pathway. j biophotonics. 2019;12(5):e201800133. https://doi.org/10.1002/jbio.201800133 396. fu y, frederick tj, huff tb, goings ge, miller sd, cheng j-x. paranodal myelin retraction in relapsing experimental autoimmune encephalomyelitis visualized by coherent anti-stokes raman scattering microscopy. j biomed opt. 2011;16(10):106006. https://doi.org/10.1117/1.3638180 397. gasecka p, jaouen a, bioud f-z, et al. lipid order degradation in autoimmune demyelination probed by polarized coherent raman microscopy. biophys j. 2017;113(7):1520-1530. https://doi.org/10.1016/j.bpj.2017.07.033 398. alba-arbalat s, andorra m, sanchez-dalmau b, et al. in vivo molecular changes in the retina of patients with multiple sclerosis. invest ophthalmol vis sci. 2021;62(6):11. https://doi.org/10.1167/iovs.62.6.11 399. stiebing c, schie iw, knorr f, et al. nonresonant raman spectroscopy of isolated human retina samples complying with laser safety regulations for in vivo measurements. neurophotonics. 2019;6(4):41106. https://doi.org/10.1117/1.nph.6.4.041106 400. rodionova nn, allakhverdiev es, maksimov g v. study of myelin structure changes during the nerve fibers demyelination. plos one. 2017;12(9):1-12. https://doi.org/10.1371/journal.pone.0185170 401. hajjar h, boukhaddaoui h, rizgui a, et al. label-free non-linear microscopy to measure myelin outcome in a rodent model of charcot-marie-tooth diseases. j biophotonics. 2018;11(12):e201800186. https://doi.org/10.1002/jbio.201800186 402. canta a, chiorazzi a, carozzi va, et al. age-related changes in the function and structure of the peripheral sensory pathway in mice. neurobiol aging. 2016;45:136-148. https://doi.org/10.1016/j.neurobiolaging.2016.05.014 403. shi y, zhang d, huff tb, et al. longitudinal in vivo coherent anti-stokes raman scattering imaging of demyelination and remyelination in injured spinal cord. j biomed opt. 2011;16(10):106012. https://doi.org/10.1117/1.3641988 404. bélanger e, henry fp, vallée r, et al. in vivo evaluation of demyelination and remyelination in a nerve crush injury model. biomed opt express. 2011;2(9):2698-2708. https://doi.org/10.1364/boe.2.002698 405. morisaki s, ota c, matsuda k, et al. application of raman spectroscopy for visualizing biochemical changes during peripheral nerve injury in vitro and in vivo. j biomed opt. 2013;18(11):1-9. https://doi.org/10.1117/1.jbo.18.11.116011 406. bae k, zheng w, huang z. quantitative assessment of spinal cord injury using circularly polarized coherent anti-stokes raman scattering microscopy. appl phys lett. 2017;111(6):63704. https://doi.org/10.1063/1.4991792 407. boissonnas a, louboutin f, laviron m, et al. imaging resident and recruited macrophage contribution to wallerian degeneration. j exp med. 2020;217(11). https://doi.org/10.1084/jem.20200471 408. blat a, dybas j, chrabaszcz k, et al. ftir, raman and afm characterization of the clinically valid biochemical parameters of the thrombi in acute ischemic stroke. sci rep. 2019;9(1):15475. https://doi.org/10.1038/s41598-019-51932-0 409. matthäus c, dochow s, bergner g, et al. in vivo characterization of atherosclerotic plaque depositions by raman-probe spectroscopy and in vitro coherent anti-stokes raman scattering microscopic imaging on a rabbit model. anal chem. 2012;84(18):7845-7851. https://doi.org/10.1021/ac301522d 410. lattermann a, matthäus c, bergner n, et al. characterization of atherosclerotic plaque depositions by raman and ftir imaging. j biophotonics. 2013;6(1):110-121. https://doi.org/10.1002/jbio.201200146 411. qin z, chon ch, lam akn, kwok jck, yuen mmf, lam dcc. feasibility examination of isolated zonal thrombolysis using raman spectroscopy. annu int conf ieee eng med biol soc ieee eng med biol soc annu int conf. 2015;2015:1353-1356. https://doi.org/10.1109/embc.2015.7318619 412. jiménez-altayó f, marzi j, galan m, et al. arachnoid membrane as a source of sphingosine-1-phosphate that regulates mouse middle cerebral artery tone. j cereb blood flow metab off j int soc cereb blood flow metab. september 2021:271678x211033362. https://doi.org/10.1177/0271678x211033362 413. jung gb, kang sw, lee g-j, kim d. biochemical characterization of the brain hippocampal areas after cerebral ischemia-reperfusion using raman spectroscopy. appl spectrosc. 2018;72(10):1479-1486. https://doi.org/10.1177/0003702818776627 414. liu j, liu z, wang w, tian y. real-time tracking and sensing of cu(+) and cu(2+) with a single sers probe in the live brain: toward understanding why copper ions were increased upon ischemia. angew chem int ed engl. 2021;60(39):21351-21359. https://doi.org/10.1002/anie.202106193 415. russo v, candeloro p, malara n, et al. key role of cytochrome c for apoptosis detection using raman microimaging in an animal model of brain ischemia with insulin treatment. appl spectrosc. 2019;73(10):1208-1217. https://doi.org/10.1177/0003702819858671 416. yamazoe s, naya m, shiota m, et al. large-area surface-enhanced raman spectroscopy imaging of brain ischemia by gold nanoparticles grown on random nanoarrays of transparent boehmite. acs nano. 2014;8(6):5622-5632. https://doi.org/10.1021/nn4065692 417. caine s, hackett mj, hou h, et al. a novel multi-modal platform to image molecular and elemental alterations in ischemic stroke. neurobiol dis. 2016;91:132-142. https://doi.org/10.1016/j.nbd.2016.03.006 418. fan y, chen c, xie x, et al. rapid noninvasive screening of cerebral ischemia and cerebral infarction based on tear raman spectroscopy combined with multiple machine learning algorithms. lasers med sci. 2022;37(1):417-424. https://doi.org/10.1007/s10103-021-03273-6 419. lee b-r, joo k-i, choi es, jahng j, kim h, kim e. evans blue dye-enhanced imaging of the brain microvessels using spectral focusing coherent anti-stokes raman scattering microscopy. plos one. 2017;12(10):e0185519. https://doi.org/10.1371/journal.pone.0185519 420. brazhe na, thomsen k, lønstrup m, et al. monitoring of blood oxygenation in brain by resonance raman spectroscopy. j biophotonics. 2018;11(6):e201700311. https://doi.org/10.1002/jbio.201700311 421. williamson mr, dietrich k, hackett mj, et al. rehabilitation augments hematoma clearance and attenuates oxidative injury and ion dyshomeostasis after brain hemorrhage. stroke. 2017;48(1):195-203. https://doi.org/10.1161/strokeaha.116.015404 422. zhao p, sun j, zhao s, et al. sers-based immunoassay based on gold nanostars modified with 5,5’-dithiobis-2-nitrobenzoic acid for determination of glial fibrillary acidic protein. mikrochim acta. 2021;188(12):428. https://doi.org/10.1007/s00604-021-05081-9 423. kim w, lee sh, ahn yj, et al. a label-free cellulose sers biosensor chip with improvement of nanoparticle-enhanced lspr effects for early diagnosis of subarachnoid hemorrhage-induced complications. biosens bioelectron. 2018;111:59-65. https://doi.org/10.1016/j.bios.2018.04.003 424. kawon k, setkowicz z, drozdz a, janeczko k, chwiej j. the methods of vibrational microspectroscopy reveals long-term biochemical anomalies within the region of mechanical injury within the rat brain. spectrochim acta part a mol biomol spectrosc. 2021;263:120214. https://doi.org/10.1016/j.saa.2021.120214 425. saxena t, deng b, hasenwinkel jm, stelzner d, chaiken j. raman spectroscopic investigation of spinal cord injury in a rat model. j biomed opt. 2011;16(2):1-14. https://doi.org/10.1117/1.3549700 426. saxena t, deng b, lewis-clark e, et al. near infrared raman spectroscopic study of reactive gliosis and the glial scar in injured rat spinal cords. in: biomedical vibrational spectroscopy iv: advances in research and industry. vol 7560. ; 2010:75600i. https://doi.org/10.1117/12.846897 427. banbury c, styles i, eisenstein n, et al. spectroscopic detection of traumatic brain injury severity and biochemistry from the retina. biomed opt express. 2020;11(11):6249-6261. https://doi.org/10.1364/boe.399473 428. surmacki jm, ansel-bollepalli l, pischiutta f, zanier er, ercole a, bohndiek se. label-free monitoring of tissue biochemistry following traumatic brain injury using raman spectroscopy. analyst. 2017;142(1):132-139. https://doi.org/10.1039/c6an02238c 429. khalenkow d, donche s, braeckman k, vanhove c, skirtach ag. added value of microscale raman chemical analysis in mild traumatic brain injury (tbi): a comparison with macroscale mri. acs omega. 2018;3(12):16806-16811. https://doi.org/10.1021/acsomega.8b02404 430. tay l-l, tremblay rg, hulse j, zurakowski b, thompson m, bani-yaghoub m. detection of acute brain injury by raman spectral signature. analyst. 2011;136(8):1620-1626. https://doi.org/10.1039/c0an00897d 431. li d, yang m, li h, mao l, wang y, sun b. sers based protocol using flow glass-hemostix for detection of neuron-specific enolase in blood plasma. new j chem. 2019;43(15):5925-5931. https://doi.org/10.1039/c8nj02561d 432. gao x, zheng p, kasani s, et al. paper-based surface-enhanced raman scattering lateral flow strip for detection of neuron-specific enolase in blood plasma. anal chem. 2017;89(18):10104-10110. https://doi.org/10.1021/acs.analchem.7b03015 433. rickard jjs, di-pietro v, smith dj, davies dj, belli a, oppenheimer pg. rapid optofluidic detection of biomarkers for traumatic brain injury via surface-enhanced raman spectroscopy. nat biomed eng. 2020;4(6):610-623. https://doi.org/10.1038/s41551-019-0510-4 434. gao x, boryczka j, zheng p, et al. a "hot spot”-enhanced paper lateral flow assay for ultrasensitive detection of traumatic brain injury biomarker s-100β in blood plasma. biosens bioelectron. 2021;177:112967. https://doi.org/10.1016/j.bios.2021.112967 435. wang y, zhao p, mao l, hou y, li d. determination of brain injury biomarkers by surface-enhanced raman scattering using hollow gold nanospheres. rsc adv. 2018;8(6):3143-3150. https://doi.org/10.1039/c7ra12410d 436. mowbray m, banbury c, rickard jjs, davies dj, goldberg oppenheimer p. development and characterization of a probe device toward intracranial spectroscopy of traumatic brain injury. acs biomater sci eng. 2021;7(3):1252-1262. https://doi.org/10.1021/acsbiomaterials.0c01156 437. kočović dm, bajuk-bogdanović d, pećinar i, nedeljković bb, daković m, andjus pr. assessment of cellular and molecular changes in the rat brain after gamma radiation and radioprotection by anisomycin. j radiat res. 2021;62(5):793-803. https://doi.org/10.1093/jrr/rrab045 438. stevens ar, stickland ca, harris g, et al. raman spectroscopy as a neuromonitoring tool in traumatic brain injury: a systematic review and clinical perspectives. cells. 2022;11(7). https://doi.org/10.3390/cells11071227 439. niedieker d, grosserüschkamp f, schreiner a, et al. label-free identification of myopathological features with coherent anti-stokes raman scattering. muscle nerve. 2018;58(3):456-459. https://doi.org/10.1002/mus.26140 440. alix jjp, plesia m, lloyd gr, et al. rapid identification of human muscle disease with fibre optic raman spectroscopy. analyst. 2022. https://doi.org/10.1039/d1an01932e 441. gautam r, vanga s, madan a, gayathri n, nongthomba u, umapathy s. raman spectroscopic studies on screening of myopathies. anal chem. 2015;87(4):2187-2194. https://doi.org/10.1021/ac503647x 442. plesia m, stevens oa, lloyd gr, et al. in vivo fiber optic raman spectroscopy of muscle in preclinical models of amyotrophic lateral sclerosis and duchenne muscular dystrophy. acs chem neurosci. 2021;12(10):1768-1776. https://doi.org/10.1021/acschemneuro.0c00794 443. hentschel a, czech a, münchberg u, et al. protein signature of human skin fibroblasts allows the study of the molecular etiology of rare neurological diseases. orphanet j rare dis. 2021;16(1):73. https://doi.org/10.1186/s13023-020-01669-1 444. ralbovsky nm, dey p, galfano a, dey bk, lednev ik. diagnosis of a model of duchenne muscular dystrophy in blood serum of mdx mice using raman hyperspectroscopy. sci rep. 2020;10(1):11734. https://doi.org/10.1038/s41598-020-68598-8 445. wallach df, verma sp, singer we. a protein anomaly in erythrocyte membranes of patients with duchenne muscular dystrophy. j exp med. 1983;157(6):2017-2028. https://doi.org/10.1084/jem.157.6.2017 446. driskell jd, zhu y, kirkwood cd, zhao y, dluhy ra, tripp ra. rapid and sensitive detection of rotavirus molecular signatures using surface enhanced raman spectroscopy. plos one. 2010;5(4):e10222. https://doi.org/10.1371/journal.pone.0010222. 447. palchaudhuri s, rehse sj, hamasha k, et al. raman spectroscopy of xylitol uptake and metabolism in gram-positive and gram-negative bacteria. appl environ microbiol. 2011;77(1):131-137. https://doi.org/10.1128/aem.01458-10 448. harz m, kiehntopf m, stöckel s, et al. direct analysis of clinical relevant single bacterial cells from cerebrospinal fluid during bacterial meningitis by means of micro-raman spectroscopy. j biophotonics. 2009;2(1-2):70-80. https://doi.org/10.1002/jbio.200810068 449. gracie k, correa e, mabbott s, et al. simultaneous detection and quantification of three bacterial meningitis pathogens by sers. chem sci. 2014;5(3):1030-1040. https://doi.org/10.1039/c3sc52875h 450. sathyavathi r, dingari nc, barman i, et al. raman spectroscopy provides a powerful, rapid diagnostic tool for the detection of tuberculous meningitis in ex vivo cerebrospinal fluid samples. j biophotonics. 2013;6(8):567-572. https://doi.org/10.1002/jbio.201200110 451. kamińska a, witkowska e, kowalska a, et al. highly efficient sers-based detection of cerebrospinal fluid neopterin as a diagnostic marker of bacterial infection. anal bioanal chem. 2016;408(16):4319-4327. https://doi.org/10.1007/s00216-016-9535-7 452. harz m, kiehntopf m, stöckel s, rösch p, deufel t, popp j. analysis of single blood cells for csf diagnostics via a combination of fluorescence staining and micro-raman spectroscopy. analyst. 2008;133(10):1416-1423. https://doi.org/10.1039/b716132h 453. steelman z, meng z, traverso aj, yakovlev v v. brillouin spectroscopy as a new method of screening for increased csf total protein during bacterial meningitis. j biophotonics. 2015;8(5):408-414. https://doi.org/10.1002/jbio.201400047 454. ladiwala u, bankapur a, barkur s, thakur b, santhosh c, mathur d. raman spectroscopic detection of rapid, reversible, early-stage inflammatory cytokine-induced apoptosis of adult hippocampal progenitors/stem cells: a preliminary study. proc indian natl sci acad. 2015;81(5):1223-1236. https://doi.org/10.16943/ptinsa/2015/v81i5/48343 455. tanuma m, kasai a, bando k, et al. direct visualization of an antidepressant analog using surface-enhanced raman scattering in the brain. jci insight. 2020;5(6):e133348. https://doi.org/10.1172/jci.insight.133348 456. pogocki d, kisała j, cebulski j. depression as is seen by molecular spectroscopy. phospholipidprotein balance in affective disorders and dementia. curr mol med. 2020;20(6):484-487. https://doi.org/10.2174/1566524020666191219102746 457. depciuch j, sowa-kućma m, nowak g, et al. phospholipid-protein balance in affective disorders: analysis of human blood serum using raman and ftir spectroscopy. a pilot study. j pharm biomed anal. 2016;131:287-296. https://doi.org/10.1016/j.jpba.2016.08.037 458. depciuch j, parlinska-wojtan m. comparing dried and liquid blood serum samples of depressed patients: an analysis by raman and infrared spectroscopy methods. j pharm biomed anal. 2018;150:80-86. https://doi.org/10.1016/j.jpba.2017.11.074 459. chaichi a, hasan sma, mehta n, et al. label-free lipidome study of paraventricular thalamic nucleus (pvt) of rat brain with post-traumatic stress injury by raman imaging. analyst. 2021;146(1):170-183. https://doi.org/10.1039/d0an01615b 460. xu j, potter m, tomas c, et al. a new approach to find biomarkers in chronic fatigue syndrome/myalgic encephalomyelitis (cfs/me) by single-cell raman micro-spectroscopy. analyst. 2019;144(3):913-920. https://doi.org/10.1039/c8an01437j 461. gonzález-cebrián a, almenar-pérez e, xu j, et al. diagnosis of myalgic encephalomyelitis/chronic fatigue syndrome with partial least squares discriminant analysis: relevance of blood extracellular vesicles. front med. 2022;9:842991. https://doi.org/10.3389/fmed.2022.842991 462. guo s, popp j, bocklitz t. chemometric analysis in raman spectroscopy from experimental design to machine learning–based modeling. nat protoc. 2021;16(12):5426-5459. https://doi.org/10.1038/s41596-021-00620-3 463. wang w, mcgregor h, short m, zeng h. clinical utility of raman spectroscopy: current applications and ongoing developments. adv heal care technol. june 2016:13. https://doi.org/10.2147/ahct.s96486 464. li z, sun w, duan w, et al. guiding epilepsy surgery with an lrp1-targeted spect/serrs dual-mode imaging probe. acs appl mater interfaces. may 2022. https://doi.org/10.1021/acsami.2c02540 465. mosca s, conti c, stone n, matousek p. spatially offset raman spectroscopy. nat rev methods prim. 2021;1(1):21. https://doi.org/10.1038/s43586-021-00019-0 466. liao c-s, wang p, huang cy, et al. in vivo and in situ spectroscopic imaging by a handheld stimulated raman scattering microscope. acs photonics. 2018;5(3):947-954. https://doi.org/10.1021/acsphotonics.7b01214 467. louis dn, perry a, wesseling p, et al. the 2021 who classification of tumors of the central nervous system: a summary. neuro oncol. 2021;23(8):1231-1251. https://doi.org/10.1093/neuonc/noab106 468. cicerone mt, camp ch. histological coherent raman imaging: a prognostic review. analyst. 2018;143(1):33-59. https://doi.org/10.1039/c7an01266g 469. stevens o, iping petterson ie, day jcc, stone n. developing fibre optic raman probes for applications in clinical spectroscopy. chem soc rev. 2016;45(7):1919-1934. https://doi.org/10.1039/c5cs00850f 470. baker mj, byrne hj, chalmers j, et al. clinical applications of infrared and raman spectroscopy: state of play and future challenges. analyst. 2018;143(8):1735-1757. https://doi.org/10.1039/c7an01871a 471. bassan p, mellor j, shapiro j, williams kj, lisanti mp, gardner p. transmission ft-ir chemical imaging on glass substrates: applications in infrared spectral histopathology. anal chem. 2014;86(3):1648-1653. https://doi.org/10.1021/ac403412n 472. ibrahim o, maguire a, meade ad, et al. improved protocols for pre-processing raman spectra of formalin fixed paraffin preserved tissue sections. anal methods. 2017;9(32):4709-4717. https://doi.org/10.1039/c6ay03308c 473. v f. optical scanners for melanoma detection [issues in emerging health technologies, issue 123]. ottawa: canadian agency for drugs and technologies in health; 2014. 474. cbc news. skin cancer detector approved | cbc news. https://www.cbc.ca/news/health/skin-cancer-detector-approved-1.1121389 . published 2011. accessed march 25, 2022. 475. sulukan e, baran a, şenol o, et al. the synergic toxicity of temperature increases and nanopolystrene on zebrafish brain implies that global warming may worsen the current risk based on plastic debris. sci total environ. 2021;808:152092. https://doi.org/10.1016/j.scitotenv.2021.152092 476. li s, li y, yi r, liu l, qu j. coherent anti-stokes raman scattering microscopy and its applications . front phys . 2020;8. https://www.frontiersin.org/article/10.3389/fphy.2020.598420. 477. robert b. resonance raman spectroscopy. photosynth res. 2009;101(2-3):147-155. https://doi.org/10.1007/s11120-009-9440-4 478. bruzas i, lum w, gorunmez z, sagle l. advances in surface-enhanced raman spectroscopy (sers) substrates for lipid and protein characterization: sensing and beyond. analyst. 2018;143(17):3990-4008. https://doi.org/10.1039/c8an00606g 479. campion a, kambhampati p. surface-enhanced raman scattering. chem soc rev. 1998;27(4):241-250. https://doi.org/10.1039/a827241z 480. muehlethaler c, leona m, lombardi jr. review of surface enhanced raman scattering applications in forensic science. anal chem. 2016;88(1):152-169. https://doi.org/10.1021/acs.analchem.5b04131 481. mirsadeghi s, dinarvand r, ghahremani mh, et al. protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process. nanoscale. 2015;7(11):5004-5013. https://doi.org/10.1039/c4nr06009a 482. nandakumar p, kovalev a, volkmer a. vibrational imaging based on stimulated raman scattering microscopy. new j phys. 2009;11. https://doi.org/10.1088/1367-2630/11/3/033026 483. lin h, lee hj, tague n, et al. microsecond fingerprint stimulated raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning. nat commun. 2021;12(1):3052. https://doi.org/10.1038/s41467-021-23202-z 484. w. fc, wei m, g. sb, et al. label-free biomedical imaging with high sensitivity by stimulated raman scattering microscopy. science (80). 2008;322(5909):1857-1861. https://doi.org/10.1126/science.1165758 485. topp mr. pulsed laser spectroscopy. appl spectrosc rev. 1978;14(1):1-100. https://doi.org/10.1080/05704927808060389 486. zumbusch a, holtom gr, xie xs. three-dimensional vibrational imaging by coherent anti-stokes raman scattering. phys rev lett. 1999;82(20):4142-4145. https://doi.org/10.1103/physrevlett.82.4142 487. potma eo, xie xs. cars microscopy for biology and medicine. opt photon news. 2004;15(11):40-45. https://doi.org/10.1364/opn.15.11.000040 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. postmortem changes in brain cell structure: a review feel free to add comments by clicking these icons on the sidebar free neuropathology 4:10 (2023) review postmortem changes in brain cell structure: a review margaret m. krassner1,2,3, justin kauffman1,2,3, allison sowa4, katarzyna cialowicz4, samantha walsh5, kurt farrell1,2,3, john f. crary1,2,3, andrew t. mckenzie1,2,3,6 department of neuroscience, icahn school of medicine at mount sinai, new york, new york, usa friedman brain institute, departments of pathology, neuroscience and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa microscopy and advanced bioimaging core, icahn school of medicine at mount sinai, new york, new york, usa hunter college libraries, cuny hunter college, new york, ny department of psychiatry, icahn school of medicine at mount sinai, new york, new york, usa corresponding author: andrew t. mckenzie · icahn school of medicine at mount sinai · icahn building 9th floor, room 20 · 1425 madison avenue · new york, ny 10029 · usa andrew.mckenzie@icahn.mssm.edu additional resources and electronic supplementary material: supplementary material submitted: 14 april 2023 accepted: 15 may 2023 copyedited by: georg haase published: 31 may 2023 https://doi.org/10.17879/freeneuropathology-2023-4790 keywords: postmortem changes, oncotic necrosis, autolysis, staining methods, species differences, brain mapping abstract brain cell structure is a key determinant of neural function that is frequently altered in neurobiological disorders. following the global loss of blood flow to the brain that initiates the postmortem interval (pmi), cells rapidly become depleted of energy and begin to decompose. to ensure that our methods for studying the brain using autopsy tissue are robust and reproducible, there is a critical need to delineate the expected changes in brain cell morphometry during the pmi. we searched multiple databases to identify studies measuring the effects of pmi on the morphometry (i.e. external dimensions) of brain cells. we screened 2119 abstracts, 361 full texts, and included 172 studies. mechanistically, fluid shifts causing cell volume alterations and vacuolization are an early event in the pmi, while the loss of the ability to visualize cell membranes altogether is a later event. decomposition rates are highly heterogenous and depend on the methods for visualization, the structural feature of interest, and modifying variables such as the storage temperature or the species. geometrically, deformations of cell membranes are common early events that initiate within minutes. on the other hand, topological relationships between cellular features appear to remain intact for more extended periods. taken together, there is an uncertain period of time, usually ranging from several hours to several days, over which cell membrane structure is progressively lost. this review may be helpful for investigators studying human postmortem brain tissue, wherein the pmi is an unavoidable aspect of the research. table of contents introduction review methods review framework eligibility criteria qualitative data analysis characteristics of included studies mechanisms and associated microscopic outcomes cell death by oncotic necrosis intracellular structures temporary versus permanent ischemia compacted or “dark” neurons apoptosis postmortem fluid shifts perivascular rarefaction pericellular rarefaction intracellular vacuolization neuropil and white matter vacuolization biomolecule degradation alterations of biomolecule distribution summary rates of postmortem decomposition time series correlational studies case reports summary differences in decomposition rates across assessment methods immunohistochemistry morphological staining electron microscopy summary selective vulnerability to decomposition models of selective vulnerability brain region and cell type heterogeneity alterations in cellular and subcellular volumes dendrites axons synapses myelin summary variables modifying decomposition rates temperature acidity and agonal damage hydration oxygen content putrefaction in situ versus ex situ brain storage variation across species in metabolic rate premortem metabolic state age premortem pathology summary interactions between postmortem changes and preservation methods morphological staging of decomposition implications for human brain mapping further studies of postmortem changes comparison to other reviews strengths and limitations of this review conclusions abbreviations author contributions acknowledgements funding data availability supplementary files references introduction in studying the structure, function, and etiopathology of neurobiological disorders in the human brain, direct examination of postmortem autopsy human brain tissue has an unparalleled role (buja et al., 2019). compared to postmortem autopsy tissue, biopsy tissue is limited in size and obtainable only in a narrow set of conditions justifying neurosurgical intervention, neuroimaging studies have limited spatial and biomolecular resolution, and animal models have lower fidelity to human neurobiology. however, studying autopsy tissue presents significant confounds, crucial among them alterations occurring during the postmortem interval (pmi). the pmi is defined as the time elapsed between the subject's death and the autopsy and preservation of the brain tissue. in practical brain banking settings, the pmi generally lasts from a period of hours to several days until the autopsy is completed and the tissue is processed (beach et al., 2015; henstridge et al., 2015; samarasekera et al., 2013; vonsattel et al., 2008). left unprocessed, postmortem brain tissue will eventually disintegrate and liquefy, which has the obvious potential to significantly confound neuropathologic investigation (gonzalez-riano et al., 2017; hayman and oxenham, 2017). because of the value of studying donated autopsy human brain tissue, there is a critical need to understand and account for the changes that occur during the pmi. a diverse set of assessments can be performed on autopsy brain tissue, which we can loosely bracket into three categories: functional, biomolecular, and morphological properties. functional properties such as electrophysiological activity or cellular viability tend to be lost relatively rapidly during the pmi but can also be maintained for a surprising amount of time, on a timescale of minutes to hours (bailey, 2019; charpak and audinat, 1998; madea, 1994). biomolecular properties vary widely regarding their maintenance during the pmi, depending on the biomolecule's class (e.g. rna, protein, or lipid), individual type (e.g. particular rna transcripts), and property (e.g. enzyme activity, conformation state, or subcellular location). several studies and reviews have discussed the rate of postmortem decomposition of biomolecules (beach et al., 2015; kretzschmar, 2009; nagy et al., 2015; samarasekera et al., 2013; stan et al., 2006). in contrast, morphological properties have received less attention. morphological properties of the brain can be macroscopic (i.e., gross) or microscopic. macroscopically, brains with the least decomposition have clear internal anatomy, no softening, and are able to be extracted from the calvaria without fragmentation (hayman and oxenham, 2017). as the brain begins to decompose, which generally occurs over a time course of days, it liquefies and eventually reaches a pasteor fluid-like consistency (hayman and oxenham, 2017). microscopically, a great deal of morphologic information can be measured in the postmortem brain. here, we focus on the decomposition of cell membrane morphology, also known as cell “morphometry,” which refers to the external shape of a cell. specifically, we focus on general morphometric properties such as the presence of visible dendritic spines, rather than on detailed properties such as cell membrane width. this focus is because numerous neurobiological disorders have been associated with morphometric alterations, including those in dendrites, synapses, or myelin (kulkarni and firestein, 2012; stadelmann et al., 2019). for example, in alzheimer’s disease, evidence suggests that disruptions in the actin cytoskeleton of dendritic spines is a key mediator of disease pathogenesis (pelucchi et al., 2020). as another example, dendritic spine density has been found to be lower in the cortical tissue of brain donors with a diagnosis of schizophrenia (berdenis van berlekom et al., 2020). thus, understanding how cell morphometry degrades in the pmi is a crucial consideration. cell membranes are made up of a multiple classes of biomolecules; by mass, about equal amounts of proteins and lipids with much more diversity in the types of proteins (lodish and rothman, 1979). because cell morphometry is dependent on a multitude of biomolecules, morphometric alterations may proceed at either a slower or faster rate than the decay of any one type of the constituent biomolecules. to keep the review tractable, we do not focus on intracellular morphologic features, such as nuclear shape, the presence of rough endoplasmic reticulum, or other aspects of organelles, except insofar as they affect cellular morphometry. to integrate diverse empirical findings of postmortem changes into a unified understanding, it is essential to have a model of how cell membranes decompose after death. brain cell membrane shape is largely maintained through the cytoskeleton, a gel-like network of proteins that exhibits both passive elastic and active viscous behavior (ananthakrishnan et al., 2006; eberhardt et al., 2022; mogilner and manhart, 2018). in particular, cell membrane biomolecules are tethered to the underlying actin cortex through interacting proteins (chugh and paluch, 2018; svitkina, 2020). during the postmortem interval, the cytoskeleton and other gel-like networks in the cell break down. this is initially due to fragmentation and diffusion associated with autolysis, and later putrefaction if microorganisms are present (hau et al., 2014). after an extended postmortem period, the cytoskeleton and other gel-like structures will ultimately liquefy. it is essential to distinguish the chronological pmi from the amount of biological decomposition that occurs during that time. several modifying factors may affect the rate of postmortem decomposition, such as the storage temperature, which need to be accounted for as well. there are three complementary approaches to address the confound of the pmi in the study of neurobiological disorders. the first is to match cohorts by the duration of the pmi; for example, to ensure that the cases and controls each have the same average pmi (swaab and bao, 2021). the second is to adjust any quantitative traits under investigation by the measured pmi prior to or alongside statistical inference. the third is to restrict the pmi to a relatively short amount of time. for example, one recent study on the size of the synaptic surface in alzheimer's disease limited the sample to autopsy brains with pmis of less than 4.5 hours, yielding very high quality ultrastructure of the samples (montero-crespo et al., 2021). another study also reported that restricting the pmi to four hours or less provided higher quality ultrastructural data (roberts et al., 1996). all these approaches may benefit from an improved understanding of changes in the pmi, which otherwise has the potential to confound inference about group differences due to disease (schwab et al., 1994). an accurate estimate of how long it is expected for a particular morphologic feature to degrade in the postmortem period can help in designing a study with the highest possible statistical power. a better understanding of postmortem changes may also help in addressing the possibility that postmortem changes interact with disease states or agonal factors, i.e. the terminal state before death or the manner of death. indeed, agonal factors are often thought to contribute more to donated brain tissue quality than relatively short pmis (vonsattel et al., 2008; williams et al., 1978). while there have been many empirical studies measuring the degree of histologic degradation after different pmis, to the best of our knowledge there has been no recent, large-scale review that attempts to summarize these studies and to construct a model of how cell membranes decompose during the postmortem period. here, we perform a comprehensive literature search to build a database of studies addressing this topic. we enumerate the mechanisms by which brain cells have been proposed to decompose and we build a database of the timescales over which cell morphometry has been found to degrade in different contexts. we discuss how variation in decomposition outcomes can be explained by different visualization methods, the aspect of cell membrane morphology under study, and modifying variables affecting the state of the brain tissue. our overarching goal is to review progress towards building a coherent model of how brain cell morphometry decomposes during the pmi, which investigators who are banking or studying autopsy brain tissue can use to guide their approaches. review methods review framework we adopted a “realist synthesis” approach which incorporates aspects of a systematic review but focuses on theoretical understanding and pragmatism (wong et al., 2013). we chose this review style because of the wide-ranging and variably defined nature of the topic. we report on our adherence to the associated rameses criteria (supplementary file 1) (wong et al., 2013). prior to the formal search method development as described below, scoping of the literature was performed primarily via searches on pubmed and google scholar, alongside discussions among the authors. additional methods, including the search query, can be found in supplementary file 2. eligibility criteria any scholarly publication such as a journal article that describes the effect of the pmi on cell membrane morphology in the brain was included. the pmi was defined as the amount of time that elapses between when (a) death is declared, which generally means that blood flow to the brain ceases, and (b) the brain tissue is preserved or otherwise processed. a wide range of durations of pmi, from minutes to weeks or months, were considered. cell morphometry could be evaluated with any form of histology. to be included, studies had to contain a measurement of shape rather than solely a quantification of biomolecules. additionally, the study needed to measure cell membranes, not solely intracellular features such as nuclear or other organellar morphology. studies on humans or non-human animals of any age were included. to exclude the archaeological literature, pmi lengths of a year or more were not considered (morton-hayward et al., 2020). review articles, studies on the retina, and non-english studies were also excluded. qualitative data analysis we performed an assessment of the degree to which cell membrane structural features tend to degrade. for qualitative synthesis, decomposition timelines were considered both as a whole and, where possible, grouped by structural features (e.g. dendrites, somata, and axons), visualization methods (e.g. morphological staining, immunohistochemistry, or electron microscopy), or modifying variables (e.g. storage temperature during the pmi). we also reviewed the decomposition mechanisms posited by the different included studies. building upon these, we attempted to describe a model of how cells in the brain degrade after death and how this affects the ability to visualize cell morphometry in autopsy brain tissue. characteristics of included studies screening identified 172 studies that met our inclusion criteria, including 22 outside of the formal search (figure 1; supplementary file 3; supplementary file 4). these studies were classified as correlational studies (n = 90), time series studies (n = 84), and case reports (n = 6). of the 172 included studies, 133 (77%) used only light microscopy, 33 (19%) used only electron microscopy, and 6 (3%) used a combination of the two. there was substantial heterogeneity in the methods. among the 84 time series studies, there was a diversity of species studied, with 31 (37%) studying rat brains, 11 (13%) human brains, 9 (11%) mouse brains, and the rest studying brains from other or multiple species. all but one of the correlational and case report studies were on human brains. figure 1. study selection flow diagram. studies were screened and selected using the web-based software covidence (available at https://www.covidence.org/). an export of the covidence database for this review containing the individual study screening decisions is available (supplementary file 3). mechanisms and associated microscopic outcomes cell death by oncotic necrosis a cell death pathway describes a stereotyped sequence of events by which the functions and structure of a cell are lost. delineating cell death is complex, as there are usually not exact boundaries for when a cell has undergone an irreversible cessation of its functions and is therefore considered dead (galluzzi et al., 2018). it is a widely replicated finding that brain cells do not necessarily “die” immediately after somatic death. neurons can retain electrophysiological functions for hours after somatic death (abbas et al., 2022; charpak and audinat, 1998). according to a consensus definition, cell death is mediated by the loss of cell membrane integrity (galluzzi et al., 2018). however, cell morphometry can potentially be visualized – or partially visualized – for a window of time even after the loss of cell membrane integrity. therefore, even if a cell is dead, it may still be possible to extract useful data from visualizing it. the major cell death pathway associated with global cerebral ischemia, a sine qua non of the postmortem brain, is oncotic cell death, also known as oncosis (fricker et al., 2018; loh et al., 2019; majno and joris, 1995; weerasinghe and buja, 2012). oncosis was coined by von recklinghausen in 1910 to describe cell death with swelling, from the greek root onkos, which refers to “mass” or “bulk” (majno and joris, 1995). subsequently oncosis fell out of favor as a concept, but in recent years has become more commonly used as cell death pathways are precisely dissected (fricker et al., 2018; majno and joris, 1995). the driver of oncotic cell death, which can also be triggered by causes other than ischemia, is the loss of cellular atp (fricker et al., 2018). global cerebral ischemia causes the depletion of atp because oxygen is no longer delivered to cells through the blood, thus halting oxidative phosphorylation, after which energy stores such as glycogen are rapidly consumed (pélissier-alicot et al., 2003). global cerebral ischemia also stops the process of metabolic waste product removal that is normally ensured by the blood flow (jenkins et al., 1979). oncosis is a non-regulated form of cell death, thus distinguishing it from the many different types of regulated cell death (galluzzi et al., 2018; lossi, 2022). in causes of death that directly affect the brain, such as death due to a toxin or traumatic brain injury, the postmortem cell death pathway may be much different. there are three stages of oncotic cell death (d’arcy, 2019; majno and joris, 1995; pélissier-alicot et al., 2003; weerasinghe and buja, 2012). in the first stage, loss of atp causes inactivation of the sodium–potassium atpase, resulting in increased intracellular sodium and chloride concentrations, a net gain in solute, and usually an associated osmotic influx of water leading to cell swelling (kramer and myers, 2013). there is a concomitant increase in intracellular calcium, leading to activation of catabolic enzymes (majno and joris, 1995; trump et al., 1997). in the second stage, there is a non-selective increase in membrane permeabilization, leading to vacuolization and cell membrane blebbing. in the third stage, there is physical disruption of the cell membrane, leading to a loss of membrane integrity. at this point, the cell is generally considered dead, initiating the necrosis phase. during the process of necrosis, cellular contents fragment, condense, convert from a gel-like phase to a liquid phase, and ultimately progress towards equilibrium with the environment (majno and joris, 1995; weerasinghe and buja, 2012). alternatively, some investigators use the term necrosis to describe the morphological alterations at this stage of cell death (fink and cookson, 2005; fricker et al., 2018). we describe this entire sequence of decomposition as cell death by oncotic necrosis. this cell death pathway has also been described as coagulative or ischemic necrosis (levin et al., 1999). among the articles included in our review, several mention cell death pathways. shepherd and colleagues, in their study of postmortem rat brains, provide the only article that specifically describes oncotic cell death (shepherd et al., 2009). they found a substantial increase in calpain-specific spectrin hydrolysis products at 1 and 4 h postmortem, which they attributed to catabolic molecular changes consistent with oncosis (shepherd et al., 2009). another frequently used term to describe postmortem cell death is autolysis, which is used to describe the self-destruction of a cell due to its own enzymes (nakabayashi et al., 2021; tafrali, 2019). for example, wenzlow and colleagues describe the postmortem cell death pathway observed in horse brains as cell autolysis (wenzlow et al., 2021). they note that the observed morphological changes are similar to in vivo necrosis, with the exception of inflammatory cell infiltrates in the latter. most of the early decomposition in the postmortem brain is due to autolysis as opposed to putrefaction, which is decomposition driven by microbial agents (ith et al., 2011). however, we prefer the term oncotic necrosis to describe the postmortem cell death pathway, in part because the term autolysis assumes an aseptic mechanism that is usually not directly tested. moreover, autolytic cell death can also occur in vivo and is not always associated with atp depletion, making it a less specific term than oncotic necrosis (fricker et al., 2018). intracellular structures several studies we identified note that cell membranes are generally more resistant to postmortem decomposition than intracellular organelles (karlsson and schultz, 1966; van nimwegen and sheldon, 1966). for example, schulz 1980 reported that after 22 h of pmi, there were significant intracellular changes including cytoplasm lysis, but there were no associated significant changes in the size and form of cells at this time point (schulz et al., 1980). the loss of cytoplasmic components can lead to cells becoming hypereosinophilic on h&e, which are sometimes then referred to as “red neurons” (garman, 2011; finnie et al., 2022). most authors refer to red neurons as distinct from postmortem changes, because the preferential breakdown of cell content leading to hypereosinophilia may be an active, atp-requiring process. because intracellular content can theoretically be lost without significant alterations of cell shape, we do not focus on red neurons or this distinction here. however, red neurons can also be associated with cell shrinkage, which would be a change in cell morphometry (finnie et al., 2022). temporary versus permanent ischemia a common area of confusion, which raises concern about the value of studying brain tissue after extended pmis, is the finding that just a few minutes of temporary cerebral ischemia can cause severe structural and functional damage to the brain. however, this brain damage is a delayed phenomenon that occurs hours to days following reperfusion, due to the triggering of atp-dependent cell death pathways (lipton, 1999; lee et al., 2019). this process diverges from the cell death mechanisms in the postmortem brain, in part because it is an active process requiring atp. on the other hand, after permanent ischemia of one or more (but not all) cerebral blood vessels, the resulting histologic changes are similar to those observed in the postmortem period (tao-cheng et al., 2007). for example, solenski and colleagues found that in the ischemic core of the cortex exposed to permanent ischemia due to occlusion of the middle cerebral artery, neuronal swelling was present by 3 h, became more severe by 5 h, and by 24 h neurons had shrunken with edema of the neuropil, broadly consistent with the expectations of the pathway of cell death by oncotic necrosis (solenski et al., 2002). this study also corroborated that cell death was more advanced in tissue that had been reperfused (solenski et al., 2002). as another example of permanent focal ischemia, garcia and colleagues permanently occluded the right middle cerebral artery in rats and found that acute shrinkage and swelling were prominent within 6 h, followed by delayed necrotic changes occurring onwards from 6 to 12 h (garcia et al., 1995). using a similar methodology, this same group reported that leukocyte invasion into the ischemic parenchyma was present by 12 h and peaked at 24 h after occlusion (garcia et al., 1994b). leukocyte invasion is one mechanism through which the rate of tissue decomposition after occlusion of an isolated cerebral blood vessel can be faster than during the global cerebral ischemia that occurs postmortem (lipton, 1999). compacted or “dark” neurons another form of postmortem histological damage that has been investigated is the formation of compacted neurons. also known as “dark neurons” or basophilic neurons, these are a common artifact in preserved brain tissue described by several studies (bywater et al., 1962; cammermeyer, 1978). compacted neurons have a shrunken cell body, shrunken dendrites, intact membranes, and a hyper-basophilic staining pattern on h&e (cammermeyer, 1978; kovács et al., 2007). they occur following of a diverse set of stressors including mechanical trauma of unfixed tissue. the compaction phenomenon is considered haphazard, as it does not affect all neurons but tends to occur in clusters. biophysically, there is strong evidence that compaction involves rapid loss of water and gel-gel phase transition (kovács et al., 2007). following this striking change in cell morphology, compacted neurons are not expected to follow the typical changes in oncotic necrosis, although they still will eventually undergo necrosis (cammermeyer, 1979). the preponderance of studies report that compacted neurons do not become more common during the pmi, and in fact, that they may become less frequent (garcia et al., 1995; kherani and auer, 2008). this may be because intracellular gel-like networks are weakened during the pmi, making a gel-gel phase transition less likely. however, one study noted that solitary compacted neurons could be stimulated due to a perfusion fixation delay of five to ten min (cammermeyer, 1978). additionally, another study postulated that one of two types of compacted neurons they studied was associated with 3 h of pmi, because these cells also had other morphological signs of postmortem decomposition, such as vacuoles and nuclear damage (badonic et al., 1992). thus, shorter but not longer periods of postmortem decomposition may be associated with the formation of compacted neurons following fixation (garman, 2011). apoptosis apoptosis, a form of programmed cell death, is a tightly regulated and controlled process by which cells undergo self-destruction, often in response to cellular damage (galluzzi et al., 2018). this cell death pathway is characterized by distinct morphological findings, such as chromatin condensation, cell shrinkage, and the formation of apoptotic bodies. apoptosis is not prominent in postmortem tissue, because it is an active, atp-dependent process, and cellular atp stores are rapidly depleted postmortem. caspase inhibitors, which prevent apoptosis, have been found to protect against cell death in focal but not global cerebral ischemia (fricker et al., 2018). the preponderance of studies report that apoptosis markers are not associated with the pmi (brück et al., 1996; del bigio et al., 2000; geiger et al., 2006; hausmann et al., 2007; lucassen et al., 1995; müller et al., 2001). for example, one study found that apoptotic morphology was not present in any of the postmortem human brains studied (lucassen et al., 1997). these authors also noted that necrotic cell death takes longer to complete than apoptotic cell death, the latter of which would be expected to take only a few hours to be completed. as another example, one study found that that positive neuronal ssdna immunostaining, a marker of apoptosis, was not correlated with the pmi in a large (n = 335) study of postmortem brains with a pmi range of 2.8 to 48 h (michiue et al., 2009). however, apoptotic markers were associated with certain causes of death such as drowning and drug intoxication. this finding is consistent with the notion that markers of apoptotic cell death beginning prior to death can be identified in postmortem brains but these markers are not expected to progress postmortem. in contrast to most studies, a few studies have reported increases in certain markers of apoptosis in the pmi. for example, in postmortem rat brains, one study noted an increase in immunostaining for the apoptosis marker caspase-3 that peaked at 9 h of pmi (sheleg et al., 2008). at that time, 2.5% of cortical neurons were found to stain strongly positive for caspase-3. as another example, schallock and colleagues reported that one marker of apoptosis, i.e. clusters of cells labeled with the in situ end-labeling technique, appeared after 48 h of pmi in mouse brains stored at room temperature (schallock et al., 1997). labeled cells were generally found in isolation, not in clusters, and moreover the cells did not consistently show morphological changes of apoptosis. as a result, the authors reasoned that this marker was more likely due to dna fragmentation associated with postmortem degeneration. because of the potential for certain antigens to be unmasked during the pmi, some markers of apoptosis could also be seen due to decomposition during oncotic necrosis. postmortem fluid shifts at the time of death, the brain is largely composed of gel-like networks of different strengths, organized at a range of scales from the extracellular matrix to sub-cellular structures as well as localized compartments of liquids. after death, oncotic necrosis begins wherein catabolic enzymes break down tissue macromolecules. as gel-like networks break down, their components solubilize, increasing the fluid content of the brain. at the same time there is a disruption of the blood brain barrier and other tissue barriers allowing fluid that is typically cordoned off to diffuse into the parenchyma, further increasing the free fluid content of the brain. this fluid is made up of water, lipids, and other soluble molecules. the end result is that the brain is transformed to the liquid state (miller and zachary, 2017). consistent with these theoretical expectations, the fluid content of the brain has been found to increase after death. one study of rat brains found that the fluid content increased from an average of 77.8% at the time of death to 79.4% at 3 h pmi, 79.6% at 6 h pmi, and 80% at 24 h pmi (leonard et al., 2016). notably, in this study, a head-down position was not associated with increased brain fluid content, suggesting that postmortem fluid increases are primarily due to processes within the brain rather than migration from other areas of the body. another study reported an increase in the water content of the brain by around 10% in the pmi (ansari et al., 1976b). macroscopically, in neuroimaging studies, a decline in grey-white matter differentiation occurs at pmis longer than 24 h, which also occurs in cerebral edema and has been attributed to autolysis and fluid shifts (wagensveld et al., 2017; martin et al., 2022). large-scale fluid shifts can also manifest across the postmortem brain. for example, as a result of gravity, blood has been reported to migrate to dependent parts of the calvarium during the pmi in a phenomenon known as hypostasis (takahashi et al., 2010). microscopically, postmortem fluid shifts also lead to many of the defining cellular changes that occur in the pmi, including circumscribed rarefaction and vacuolization, discussed below. perivascular rarefaction several studies using light microscopy have reported that perivascular rarefactions appear and increase in frequency and size during the pmi (bywater et al., 1962; garcia et al., 1978; liu and windle, 1950; schwarzmaier et al., 2022). morphologically, these non-staining areas generally appear as ellipsoid shapes around blood vessels (figure 2a). they have been described as “a rose-branch beset with thorns” because intertwining glial fibers frequently remain between the non-staining areas (bruce and dawson, 1911). figure 2. representative micrographs demonstrating rarefaction and vacuolization in the postmortem brain. tissue from the frontal cortex tissue of an 87-year-old man with a pmi of 28 hours. a: h&e/lfb (luxol fast blue)-stained image demonstrating a perivascular rarefaction in the grey matter. arrowheads denote examples of intertwining fibers. scale bar = 10 μm. b: h&e/lfb-stained image showing variable degrees of asymmetric pericellular rarefactions (asterisks). scale bar = 20 μm. c: h&e/lfb-stained image of white matter vacuoles (asterisks) and pericellular rarefactions. white matter vacuoles are found haphazard in distribution, but appear more common near blood vessels, likely because of postmortem fluid extravasation. scale bar = 20 μm. d: electron photomicrograph in grey matter demonstrates prominent electron lucent areas that appear to be swollen cellular processes (asterisks). scale bar = 500 nm. the underlying nature of perivascular rarefactions seen on light microscopy has caused controversy for generations of neuroanatomists (bruce and dawson, 1911; maynard et al., 1957; weller et al., 2018). frequently described as a “space” or “cleft”, we do not favor these terms because they imply an underlying mechanism. similarly, the term “retraction artifact” implies a physical separation of the blood vessel from the parenchyma occurring during the tissue processing procedure. instead, we prefer the more agnostic term “rarefaction” to describe the non-staining areas. another potential point of confusion is that the perivascular rarefactions that develop in the pmi can be distinct from the dilated perivascular spaces seen on neuroimaging in vivo (kwee and kwee, 2007; weller et al., 2018). for example, in one correlative postmortem study, these were found to stain positive for collagen (haider et al., 2022). sometimes, these perivascular rarefactions are described as resulting from a fixation artifact, although this is considered less likely, as they also occur in postmortem brain tissue preserved via freezing in liquid nitrogen (de groot et al., 1995). instead, electron microscopy data from ischemic or postmortem states and detailed analysis of light microscopy data suggest that perivascular rarefactions are actually due to swollen astrocyte processes (maynard et al., 1957; arsénio-nunes et al., 1973; garcia et al., 1994a; garman, 2011; weller et al., 2018; dehghani et al., 2018). other than postulating that perivascular rarefactions on light microscopy are swollen astrocyte processes, or other swollen tissue elements, perhaps the only other explanation for the divergence between light and electron microscopy data would be differences in tissue processing between the two techniques, which is less consistent with the available data. these swollen areas of astrocytes have been described as vacuoles and contain predominantly fluid, so it makes sense that they are electron lucent and do not stain positive on light microscopy for astrocyte markers such as gfap (schultz et al., 1957; gibson and tomlinson, 1979; lafrenaye and simard, 2019). consistent with this, gfap staining decreases in brain tissue following a hypoxic/ischemic period (sullivan et al., 2010). protoplasmic astrocyte processes can have irregular geometries, allowing them to fit into narrow spaces (schultz et al., 1957). pericellular rarefaction as with perivascular rarefactions, pericellular rarefactions have also been found to develop in the pmi (figure 2b) (bywater et al., 1962; de groot et al., 1995; dehghani et al., 2018; liu and windle, 1950; shepherd et al., 2009). similarly to perivascular areas, electron microscopy data in grey matter shows that astrocyte processes, including end feet, swell in the perineuronal area in postmortem and ischemic conditions, thereby accounting for the change observed on light microscopy (figure 2d) (kuroiwa et al., 1998; suzuki, 1987). these non-staining areas are heterogenous, as they do not occur in all cells to the same degree and are more pronounced in some brain regions than others (snyder et al., 2021). in white matter, oligodendrocyte cell bodies have also been found to swell (kuroiwa et al., 1998). this accounts for the frequently described pericellular “halos” of oligodendrocytes, leading to an overall “fried egg” appearance on light microscopy (snyder et al., 2021). it is generally supported that the major mechanism of perivascular and pericellular swelling of tissue elements in the postmortem period is fluid shifts. for example, pericellular and perivascular swelling is a microscopic finding seen in brain edema (dreier et al., 2013). it is well-described that astrocyte processes become dramatically enlarged in ischemia, mediated via passive aquaporin-4 channels, which is thought to be a protective response to minimize brain damage (nahirney and tremblay, 2021). the observed astrocyte process swelling at 30 min of complete global cerebral ischemia has been found to be reversible with recirculation, consistent with the idea that this is a homeostatic process (arsénio-nunes et al., 1973). it is still undetermined why astrocyte processes are more liable to swell around cells and around blood vessels than in other areas of brain tissue. in breast cancer, where the presence of pericellular areas on histology correlates with an unfavorable prognosis, similar areas have been attributed to functional pre-lymphatic spaces (acs et al., 2012). the predilection for swelling in these areas in the postmortem brain may reflect aspects of the in vivo functioning of the glymphatic system, which operates by shuttling water via aquaporin-4 channels on astrocytes (silva et al., 2021). regardless, the extent to which fluid shifts into astrocytes or other cells will depend on the local anatomic and biochemical context. intracellular vacuolization the formation of vacuoles is a common finding in oncotic cell death and is also associated with fluid shifts in the postmortem brain (shubin et al., 2016; weerasinghe and buja, 2012). vacuoles are homogenous, spherical areas visualized under the microscope. vacuolization is a common endpoint of a heterogeneous set of physiologic or pathologic processes, such as water accumulation, lipid droplet formation, dilatation of organelles, fusion of small vesicles, invagination of the plasma membrane, degradation of cytoplasmic components, or artifact formation during slide preparation (henics and wheatley, 1999; ilse et al., 1979; wohlsein et al., 2013). as with perivascular and pericellular rarefactions, the precise underlying nature of vacuolization is often unknown. many studies describe vacuoles as a characteristic morphologic finding in postmortem brain cells (haines and jenkins, 1968; hilbig et al., 2004; koenig and koenig, 1952). for example, lindenberg 1956 found that in cat brains stored at 37° c, small vacuole-like transparencies were first seen at 30 min pmi, while at 6 h pmi larger vacuoles could be seen in some cells (lindenberg, 1956). in this study, the rate of development of the vacuoles was slower at lower temperatures: when the brains were stored at 18 °c, it took until 12 h pmi for vacuoles to appear. albrechtsen 1977 noted that small vacuoles were commonly seen in cerebellar granule cells, preceding necrotic changes as one of the first signs of decomposition, and that only minimal vacuole formation was seen in cerebella without necrosis of the granule layer (albrechtsen, 1977a). in an early paper about postmortem degeneration, koenig 1952 reported that cytoplasmic vacuoles appeared at 3 h of pmi, increase in size and frequency throughout the pmi, and stated that their nature was not known (koenig and koenig, 1952). subsequent electron microscopy studies have attributed the vacuoles in brain cells that accumulate postmortem or in ischemia to swollen mitochondria and dilatations of the endoplasmic reticulum or golgi apparatus (badonic et al., 1992; shibayama and kitoh, 1976; suzuki, 1987). it is not clear if all the vacuoles are due to the swelling of these organelles, or if some vacuoles could be due to other factors. also using electron microscopy, sele and colleagues found that the number of vacuoles increased during the pmi and speculated that they resulted from degraded cellular compartments (sele et al., 2019). intracellular vacuoles have the clear potential to affect cell morphometry by causing asymmetric cell membrane distortions. neuronal somata have also been found to have lateral expansions, or blisters, during the pmi (williams et al., 1978). the presence of organelle-free membrane blisters or blebs is also a commonly described finding in oncotic cell death that may be associated with vacuolization (weerasinghe and buja, 2012). gibson and colleagues report that there is an increase in electron translucent vacuoles in human cortical tissue during the pmi (gibson and tomlinson, 1979). they associate vacuolization with the swelling of cell processes, especially astrocytic processes, and found a highly significant correlation between the pmi and the degree of vacuolization up to 33 h, followed by no significant change up to 69 h pmi. while they did not detect any obvious structural degeneration of membranes during the pmi range studied, this vacuolization led to a severe compression of the tissue. for example, there was a significant decrease in the number of recognizable synapses during the pmi, even though there was no structural disintegration of synapses observed, indicating that the presence of large vacuoles could prevent the visualization of other cellular structures. neuropil and white matter vacuolization vacuolization can also occur during the pmi in parts of brain tissue that are not as clearly associated with a single cell, including the white matter (figure 2c) (hilbig et al., 2004). white matter vacuoles often look like randomly distributed holes and have been reported to occur in densely myelinated areas such as the corpus callosum or the internal capsule (snyder et al., 2021). in ischemia, electron microscopy shows that white matter vacuoles can result from swollen astrocyte processes, swollen axons, or the separation of the myelin sheath from the axon, with fluid filling the resulting potential space (pantoni et al., 1996). vacuoles can also be seen in the neuropil during the pmi. in their study of horse brains stored at 22 °c, wenzlow and colleagues found a non-linear trend for neuropil vacuolization: it increased up to 24 h pmi and then decreased until 72 h pmi (wenzlow et al., 2021). however, they did not find a significant change in cytoplasmic vacuolization over the pmi range studied. garcia 1978 reported that the sponginess seen in the neuropil on light microscopy in regional ischemia was due to extensive swelling of astrocytic processes and presynaptic terminals seen on electron microscopy (garcia et al., 1978). consistent with the idea that neuropil vacuoles result from fluid shifts, incubating brains in saline prior to fixation has also been found to lead to vacuolization of the neuropil (garman, 1990). biomolecule degradation the primary macromolecules making up brain cells are proteins and lipids while nucleic acids and carbohydrates account for only a small percentage (susaki et al., 2020). therefore, understanding how proteins and lipids disintegrate in the pmi is critical for charting how cell structure changes postmortem. in the postmortem brain, proteins are thought to be primarily broken down by enzyme-mediated proteolysis. this is consistent with findings that the uncatalyzed hydrolysis of peptide bonds at neutral ph is extremely slow, with reaction half-lives on the order of hundreds of years (mahesh et al., 2018; radzicka and wolfenden, 1996). calpains and cathepsins are two of the major enzyme families that contribute to autolytic proteolysis. calpain activation is not dependent upon atp, but thought to be triggered by the postmortem increase in intracellular calcium concentration (sorimachi et al., 1996; zissler et al., 2020). empirical results have indicated that calpain enzymes are indeed active in the postmortem brain (geddes et al., 1995; harada et al., 1997; sorimachi et al., 1996). cathepsin family enzymes, which are activated by acidic ph, also have activity in the postmortem brain (compaine et al., 1995). cathepsin family enzymes are usually cordoned in lysosomes, but lysosomal membrane integrity is lost during the pmi, which is thought to allow cathepsin-catalyzed proteolysis to catabolize cytoplasmic proteins (compaine et al., 1995). if lysosomes rupture or are permeabilized during the pmi, the release of catabolic enzymes would accelerate the breakdown of cell structure. indeed, one study defines lysosomal cell death, i.e. cell death resulting from lysosomal membrane permeabilization and the resulting activity of cathepsins and other proteases, as a synonym for autolysis (fricker et al., 2018). multiple studies have reported that lysosomal enzymes contribute substantially to decomposition in the postmortem brain (albrechtsen, 1977a; shibayama and kitoh, 1976). while some studies report that the number or size of lysosomes increase during the pmi, the evidence for this is mixed, the effect size is not strong, and lysosomal expansion is not required for lysosomal enzymes to contribute to decomposition (sheleg et al., 2008; tafrali, 2019; van nimwegen and sheldon, 1966). different protein substrates can have differential sensitivity to enzyme-mediated proteolysis. for example, geddes and colleagues report that map2, nf-m, and nf-l are relatively more sensitive to calpain-mediated proteolysis, while tau and nf-h are more resistant (geddes et al., 1995). as another example, sarnat and colleagues report that neun immunoreactivity tends to degrade within 6 or 12 h of pmi, while synaptophysin is much more resistant to postmortem autolysis and can be detected even in brains with pmis of more than 96 h (sarnat et al., 2010). different regions of the same protein can also have different rates of postmortem proteolysis. for example, li and colleagues studied the postmortem degradation of different epitopes of glutamate transporters in rat brains after 0-72 h pmi (li et al., 2012). they found that the termini of glt-1 degrade much faster than the central parts of the protein. they also found that the proportions of immunolabeling signal observed for different epitopes varies by the pmi. if the region of a protein recognized by an antibody is proteolyzed first, then the protein could appear to be totally absent when immunolabeled with that antibody, even though other parts of the protein could still be present. even if a biomolecule does not disintegrate into component parts during the early pmi, it could still undergo a change in conformation, which could also lead to a false negative result when attempting to measure the protein’s distribution with a particular label. postmortem proteolysis does not always decrease antigenicity, but instead is often found to increase the immunolabeling signal for certain proteins. for example, multiple studies reported that postmortem proteolysis leads to more accessible epitopes of the astrocyte marker gfap (de groot et al., 1995; hilbig et al., 2004). similarly, immunoreactivity for the vascular basement membrane marker laminin increased during the pmi, which has been attributed to unmasking by proteolytic processes (mori et al., 1992; szöllősi et al., 2018). hayes and colleagues found that during the pmi, staining for somatostatin 28 decreased, while staining for its breakdown products somatostatin 14 and somatostatin 281-12 increased, suggesting post-mortem proteolytic processing of somatostatin 28 (hayes et al., 1991). monroy-gomez and colleagues found that immunostaining for rabies antigens increased substantially in the postmortem period, which they attributed to autolytic disintegration of the intracytoplasmic viral inclusions and subsequent dispersion of the viral antigen (monroy-gómez et al., 2020). as with protein processing, lipid breakdown is also thought to be enzyme-mediated, via the activity of lipases such as phospholipase a2 (jernerén et al., 2015). lipids can be divided into metabolic and structural classes. metabolic lipids such as endocannabinoids are often rapidly degraded in the pmi (palkovits et al., 2008). however, the levels of structural lipids are liable to be stable for longer periods. for example, one study in rat brains found that the composition of glycerophospholipid, a major structural lipid component of cell membranes, did not change significantly up to a pmi of 18 h (pearce and komoroski, 2000). many studies have been performed on the biomolecular composition of the brain in the early pmi. while a full analysis is outside of the scope of this review, a general trend is that the levels of proteins in the brain, including synaptic proteins, tend to be surprisingly well maintained for many hours and often up to one or two days of pmi (fountoulakis et al., 2001; halim et al., 2003; knudsen and pallesen, 1986; siew et al., 2004; stan et al., 2006). it is worth noting, however, that the effect of the pmi on protein breakdown is highly variable depending upon the protein considered (ferrer et al., 2007). another source of variability in postmortem proteolysis is across cell types. li and colleagues reported that there was a significant heterogeneity of postmortem proteolysis rates across cells, resulting in patchy immunolabeling patterns (li et al., 2012). in the cerebellum, postmortem proteolysis has been suggested to occur earlier in the granule cells than in the purkinje cells (albrechtsen, 1977a). alterations of biomolecule distribution for this review, we are interested in the extent to which the postmortem spatial distribution of populations of biomolecules matches their in vivo distribution. we are not focused on the precise location of individual biomolecules, which are thought to be exchangeable in their functions and are often diffusing within a given cellular region in vivo regardless. we define two spatial scales when discussing altered location of biomolecules: the structural feature level, which considers alterations in the location of objects made of many biomolecules seen under the microscope, and the biomolecule level, which considers alteration of locations of individual types of biomolecules. cells themselves can be thought of as structural features and can also move in the pmi, although many non-blood cells are tethered to one another and to the extracellular matrix by connections that are relatively resistant to postmortem decomposition. although the structure of individual biomolecules is often stable in the early pmi, if the biomolecules that comprise cell membranes move sufficiently far from their original locations, then cell morphometry can still be lost. indeed, cell membrane morphology is often reported to break down prior to the breakdown of its biomolecular constituents. for example, hukkanen 1987 reported that myelin ultrastructure in surgical specimens had degenerated after 24 h of pmi at 25 °c, even though the levels of the major myelin glycoprotein were unaltered (hukkanen and röyttä, 1987). subcellular structural features such as organelles are frequently reported to move in the pmi. this can occur due to mechanical forces that arise by swelling, shrinkage, or other fluid shifts, leading to an increase in local pressure and thereby causing the intermolecular bonds stabilizing structural features to break. it can also occur due to catabolism of the biomolecules that typically maintain the structure in place. for example, several studies have found that myelin lamellae tend to split during the pmi, which is likely due to a combination of mechanical forces and the breakdown of the biomolecules typically connecting the lamellae (ansari et al., 1976a; hukkanen and röyttä, 1987; rees, 1976; shibayama and kitoh, 1976). as another example, one study found that cilia on ependymal cells had fused together and sunk down onto the ependymal surface after 1 h of pmi (hetzel, 1980). this may be due to a postmortem fluid shift within ventricular spaces. at the level of individual biomolecules, the situation can be more complex, requiring us first to understand how biomolecules are organized during life before we can understand how this organization is lost postmortem. there are three types of biomolecular organization patterns that we will consider, which are not mutually exclusive: confinement in compartments, inclusion in gel-like networks, and maintenance by active transport mechanisms. the most obvious form of biomolecular organization results from containment within cell compartments, such as within a cell or organelle membrane. in living cells, diffusion is often constrained to a local area, such as by an organelle membrane or cytoskeletal structure. the nature of the confinement depends on the size and properties of the molecule as well as the properties of the confining material. after death, membranes will eventually become permeable and therefore there will be a loss of confinement. the next form of biomolecular organization is the gel-like network. structural features seen under the microscope are often composed of densely aggregated, gel-like networks (douglas, 2018). for example, the nucleolus is made up in part by a concentrated gel of enmeshed rrna (lafontaine et al., 2021; riback et al., 2022). gel-like networks can be formed by covalent and non-covalent interactions that lead to the aggregation of biomolecules. during the pmi, intraand inter-molecular bonds will degrade due to hydrolysis and loss of active maintenance, causing these gel-like networks to break down. however, many of the constituent biomolecules may still be present in the local area, just no longer densely aggregated enough to be seen under the microscope. as a result, changes in the pmi can cause gel-like structures such as the nucleolus to no longer be visualized under the microscope, even though the local levels of their constituent biomolecules, such as rrna, may be relatively stable. notably, the ability to visualize gel-like networks under the microscope can also depend on the strength of crosslinking fixation, which stabilizes them (wang and minassian, 1987). another major form of in vivo biomolecular organization is active transport. in active transport, atp is expended to move biomolecules and ions against a concentration gradient. after death, atp will be depleted, so active transport will cease, causing a potential loss of the spatial distribution of biomolecules maintained in vivo. an example of this is the loss of sodium–potassium atpase activity very early in the pmi. we can next consider what happens to individual biomolecules once these organizing structures and functions are lost in the pmi. molecular diffusion is the primary driving force of individual biomolecule movement in the pmi, due to thermal fluctuations of molecules in the liquid, which causes constituent biomolecules to be displaced in random directions (schavemaker et al., 2018). each biomolecule has its own diffusion coefficient, resulting from its size, lipophilicity, the temperature, how it interacts with the solvent, and the extent to which it is bound to other molecules (schavemaker et al., 2018). if a population of biomolecules is localized to a particular location or “point” in vivo and the organizing factors maintaining the localization are lost postmortem, then its distribution is expected to increasingly spread out during the pmi as a result of diffusion (schavemaker et al., 2018; ślęzak and burov, 2021). in the absence of barriers, postmortem diffusion would be expected to cause the spread of biomolecules to follow a gaussian displacement distribution, with the degree of displacement depending on the pmi (manzo and garcia-parajo, 2015). however, in biological systems, barriers to diffusion, such as the actin cytoskeleton and extracellular matrix, are ubiquitous, meaning that non-gaussian spread of biomolecules is commonplace (manzo and garcia-parajo, 2015; ślęzak and burov, 2021). diffusion speeds are substantially decreased in the crowded cytoplasm compared to pure solution, often to an order of magnitude less (kekenes-huskey et al., 2016). as the cytoplasm becomes less crowded and structures break down during the pmi, effective diffusion coefficients will accelerate. because of the complex factors that affect the rate and spread of postmortem diffusion, the outcome of biomolecular diffusion after a given pmi is largely an empirical question about which it is difficult to make precise predictions a priori. many studies describe the phenomenon in which biomolecules redistribute from a localized to diffuse distribution in the pmi. sex steroid receptors in brain cells have been found to diffuse from a nuclear to a perinuclear distribution after 24 h of pmi (fodor et al., 2002). oehmichen reported that enzymes redistribute from localized to diffuse distributions at different postmortem time courses, beginning for several enzymes at 32 h pmi (oehmichen, 1980). hilbig and colleagues found that during the pmi, localized synaptophysin immunoreactivity was lost after 4 h when brain tissue was stored at 22 °c and after 12 h when it was stored at 4 °c (hilbig et al., 2004). it is also a well-known phenomenon that biomolecules eventually leak into the extracellular space as autolysis progresses (pélissier-alicot et al., 2003). as a result, cell membrane visualization will become less distinct when the tissue is labeled by a biomolecule that has undergone postmortem diffusion. another complicating factor for predicting the outcome of biomolecular diffusion is that population level movement patterns can be non-random due to sinks that capture biomolecules. as a possible example of this, mori 1991 studied changes in the distribution of immunostained immunoglobulin during the pmi (mori et al., 1991). they noted that immunoglobulin leaked out of blood vessels, evolving from a focal to diffuse pattern as the pmi increased, as expected based on random diffusion. additionally, they found that immunoglobulin underwent a selective neuronal uptake phenomenon where it was incorporated into shrunken, hyperchromatic neurons that had been damaged by trauma. because such compacted neurons result from a gel-gel phase transition, the uptake may occur as a result of the gel matrix in these neurons capturing diffusing immunoglobulins (kovács et al., 2007). while this is a plausible mechanism for the postmortem localization patterns of immunoglobulin, it requires further study. it is also critical to distinguish between the population level biomolecule distribution that is observed under the microscope and the changes in the tissue that led to that observation. as discussed in the previous section, postmortem metabolism can also cause dramatic changes to the composition and/or conformation of individual biomolecules. if postmortem metabolism is not uniform during the pmi, this can affect the observed localization of populations of biomolecules in the pmi. we can call this phenomenon differential metabolism. for example, there could be different concentrations of proteases, phospholipases, or endonucleases in different parts of the cell or across cells. alternatively, there could be a differential change in epitope accessibility between compartments during the pmi, for example due to a decrease in cytoplasmic crowding. as a term agnostic to mechanisms, we will use the term redistribution to describe an observed change in the spatial distribution of a type of biomolecule measured at a particular postmortem timepoint. several articles described redistribution of certain labeled biomolecules from neurites to cell bodies (i.e. the perikarya) (d’andrea et al., 2017; gärtner et al., 1998; geddes et al., 1995; irving et al., 1997; kitamura et al., 2005; schwab et al., 1994). among these, geddes and colleagues reported the redistribution of several different neurofilament proteins, including nf-h, nh-m, map2, and tau, from neurites to cell bodies in the pmi, which they call perikaryal accumulation (geddes et al., 1995). in their discussion, they posed the question of whether this redistribution was due to (a) a shift in the cellular localization of the proteins themselves or (b) a change in the antigenicity of neurofilament epitopes in different compartments, for example due to calpain activation, which we would consider a type of differential metabolism. gärtner and colleagues studied the spatial distribution of tau at baseline and after 30 min of pmi in rat brains using seven different tau-specific antibodies, which label for different epitopes of the protein (gärtner et al., 1998). they found that labeling with two of the antibodies, tau-1 and 12e8, led to a perikaryal accumulation of staining intensity following the pmi. on the other hand, labeling with three of the antibodies for tau led to no change in the distribution of tau immunostaining in the pmi. they reasoned that their results could be explained by differential dephosphorylation of tau in different compartments of neurons, as opposed to movement of the tau protein itself. irving and colleagues also reported that the postmortem redistribution of tau was dependent upon the antibody used (irving et al., 1997). taking these studies together, the observed perikaryal redistribution of many neurofilament proteins is more likely due to differential metabolism in different neuronal compartments, rather than a shift in the location of the individual protein molecules. a special type of postmortem differential metabolism is postmortem synthesis. during the pmi, new biomolecules can be synthesized, leading to population-level shifts in the distribution of the synthesized biomolecule. for example, certain rna transcripts have been suggested to be transcribed postmortem in the mouse brain (pozhitkov et al., 2017). while some of these findings could also be attributed to complex differential metabolism, the possibility of postmortem synthesis remains valid and has the potential to alter the spatial distribution of biomolecules (schwab et al., 1994). as an insight from an adjacent field, the postmortem redistribution of small molecule drugs across body compartments is a commonly studied phenomenon in forensic pathology (pélissier-alicot et al., 2003). mechanistically, drug redistribution can result from diffusion away from drug reservoirs, cell death, putrefaction, and postmortem metabolism (yarema and becker, 2005). more basic and more lipophilic drugs are more likely to move in the postmortem interval. one source notes that the brain is not clearly affected by the postmortem redistribution of drugs (pélissier-alicot et al., 2003). while many biomolecules are observed to have postmortem redistribution, these are not omnipresent findings. many of the included studies that measured the spatial distribution of biomolecules postmortem did not find a significant degree of postmortem redistribution (blair et al., 2016; quartu et al., 2005; serra et al., 2005). for example, blair and colleagues found that the expected neuronal localization of neun immunostaining localization in isolated human brain tissue did not change during the pmi time points analyzed, up to 53 h of pmi (blair et al., 2016). as another example, serra and colleagues found that immunostaining patterns for gfralpha-1, gfralpha-2, gfralpha-3 and ret receptor molecules were not substantially altered up to 72 h of pmi in rat brains (serra et al., 2005). summary there are numerous distinct mechanisms that lead to changes in observed cell morphometry during the pmi (table 1). these include the initiation of cell death by oncotic necrosis, fluid shifts causing swelling of tissue elements and vacuolization, biomolecular breakdown by catabolic enzymes, and biomolecular redistribution. by characterizing the mechanisms of postmortem decomposition, our goal is to allow predictions to be made about how cell membrane morphometry will degrade during the pmi. these mechanisms have the potential to differ substantially from biological processes occurring in vivo. they also have implications for which methodologies are likely to be most robust for use in postmortem brains. for example, individual biomolecules may have artifactual disintegration or redistribution during the pmi. as a result, using individual biomolecular labeling as a proxy for cell membrane morphology is not expected to be as robust of a method as staining nonspecifically for classes of biomolecules, unless the labeled biomolecule is highly stable in the pmi. mechanism or associated outcome role in postmortem brain decomposition cell death by oncotic necrosis the main cell death pathway in the postmortem brain, wherein the loss of atp leads to a sequence of morphological changes and eventual necrosis autolysis primary role in the early postmortem decomposition of brain cells apoptosis not a substantial contributor to postmortem decomposition, because this cell death pathway requires atp compacted neurons (a.k.a. dark neurons) may become more common after short periods of pmi, but less common with longer periods of pmi; is not thought to contribute to postmortem decomposition temporary ischemia temporary cerebral ischemia followed by reperfusion has much different mechanisms of decomposition (e.g. requiring atp) than the permanent global cerebral ischemia that occurs postmortem postmortem fluid shifts biomolecular breakdown and blood extravasation leads to dramatic fluid shifts in the early postmortem brain; eventually, the brain is transformed to a pasteor fluid-like consistency perivascular and pericellular rarefactions these areas expand and become more frequent in the pmi, which appears to be due to the swelling of astrocyte processes and other cells, causing those areas to not stain on light microscopy vacuolization common finding in postmortem brain cells and tissue spaces such as the neuropil that evolves throughout the pmi and can cause alteration of cell morphometry or compression of other structures biomolecule degradation largely driven by enzymes, biomolecule breakdown is a key driving force of postmortem decomposition, varying in rate based on many factors, such as the substrate and the temperature biomolecule redistribution the breakdown of the factors stabilizing biomolecules in place in vivo leads them to eventually diffuse away from their original locations, with degrees of both gaussian and non-gaussian spread table 1. summary of the role of postmortem brain cell decomposition mechanisms. rates of postmortem decomposition time series describing the mechanisms of postmortem decomposition does not tell us about the kinetics over which the decomposition occurs. there are three types of empirical studies that we will review next to address this question: time series studies, correlational studies, and case reports. in a time series study, the pmi is experimentally controlled, thus allowing an estimate of the effect of a given pmi on cell membrane morphology. because the time series study design is less susceptible to confounding biases, it allows for the most reliable estimates of the three study types. as an example of a time series study, we highlight the study by haines and jenkins (haines and jenkins, 1968). in this study, the authors measured the effect of pmi on cell structures in the habenulopeduncular tract and nucleus of adult dogs. after death, the brains were kept inside of the head (i.e., in situ) and stored at around 25°c. at multiple pmi time points – 0, 6, 12, 18, 24, 38, and 48 h – they extracted the brain, isolated the habenula, and fixed it via immersion in 10% buffered formalin. the tissue was morphologically stained with a method designed to distinguish myelin, axons, and general cell architecture. they reported that general cell architecture began to become indistinct by 18 h of pmi, while myelin and axons were relatively well preserved over the course of the study up until 48 h of pmi. this is an example of how one study can make multiple descriptions about the degree of decomposition of cell morphometry at different time points or for different structural features, which we call “observations”. to build a database and draw comparisons across studies, we extracted the observations from the text of each included time series study (supplementary file 5). for each observation, multiple raters independently graded the severity of decomposition on a subjective 0-3 scale. note that we are using the term “grade” in the sense of severity rating, as opposed to the use of the term in neoplastic grading. we next calculated inter-rater reliability scores based on these decomposition grades, using the intraclass correlation (icc) statistic. the icc value was calculated as 0.721, with a 95% confidence interval of 0.657 to 0.775 (f-test p-value = 7.9 * 10-43). this score is considered to be of moderate reliability, which likely reflects limitations in both the clarity of the categories we defined as well as the precision by which the observations were described in the included studies (koo and li, 2016). to elucidate the variability in outcomes between studies, we plotted these decomposition grades at different pmis (figure 3). we found a wide range of pmis after which histology reaches different decomposition severities across studies, experimental designs, and structural features of focus. because the studies were very heterogeneous, it was not possible to perform a detailed quantitative meta-analysis of decomposition kinetics across studies. however, as expected, we did identify a significant positive rank correlation between the pmi and the decomposition severity grade when pooling all observations (ρ = 0.29, p-value = 4.0 x 10-7). figure 3. temporal progression of structural decomposition in time series studies. correlational studies rather than making observations at defined time points, correlational studies make observations about how the appearance of a structural feature varies over a range of pmis. because these studies are correlative, before discussing them further, it is important to consider the limitations when using correlational studies to estimate the effect of pmi on cell membrane degradation (lewis, 2002; palmer et al., 1988). these are not limitations of the original studies, which often do not have this task as the primary goal, but rather limitations of using this data for our purposes in this review. the first limitation is the potential for confounding variables, such as agonal state and tissue ph (glausier et al., 2019). a second limitation is that many studies correlate multiple structural features with the pmi but do not perform an adjustment for multiple hypothesis testing, raising the probability of a spurious correlation. a third limitation are statistical floor or ceiling effects, based on the range of pmis. if the shortest pmi is several hours after death, or the longest pmi is relatively short, this restricts our ability to infer what occurs in brain tissue outside of the pmi range sampled. a fourth limitation in the many included studies that treat the pmi as a nuisance variable that must be considered is the possibility of publication bias. finally, a fifth limitation is selection bias if tissue requires a quality standard for inclusion in a study. this would clearly affect any naïve correlations measured with the pmi. as an example of quality selection bias, one study included 16 brains with pmi < 24 h (jacobs and scheibel, 1993). this study also included 4 brains with pmi of longer than 24 h because those brains had been refrigerated and the tissue appeared to be in good condition. while the study found no correlation between pmi and total dendritic length as measured by golgi-cox staining, this result is more difficult to interpret in terms of pmi effects given that brains with longer pmis were included because of relatively better markers of tissue quality. despite the potential limitations of correlational studies, they are still helpful, especially for exploring large effect sizes over the pmi range studied. correlational studies can aid in bounding the range of plausible postmortem effects over the time ranges studied. evaluating this literature is helpful for investigators studying tissue stored in brain banks, because it is most representative of the brain tissue that is available for study. we found as an overall trend from correlational studies that the pmi often does not have a substantial effect on cell morphometry (figure 4; supplementary file 6). some studies even find this with pmis of several days. for example, garey and colleagues used rapid golgi impregnation to study cell morphometry in 24 brains with a pmi range of 4 to 120 h, finding that there was no significant effect of this time range on the observed dendritic spine density of pyramidal neurons (garey et al., 1998). however, other studies have found that even relatively shorter pmis can have a substantial effect. for example, booze and colleagues found that there was a loss of staining for fine varicose axons over a pmi range from 1 to 6 h (booze et al., 1993). the effect of pmi likely depends on characteristics of the cohort studied, such as whether the bodies were refrigerated, as well as the visualization methods and structural feature investigated. figure 4. cell morphometry changes associated with the postmortem interval in the included correlational studies. in this plot, each correlational study is a data point. if the study describes an association between the postmortem interval (pmi) and a change in any type of cell morphometry, based either on statistical significance or the qualitative impression of the authors, then that data point is considered positive or “yes”, and vice versa for “no”. if it is unclear whether there was an association reported in the study, then the data for that study is not included in the plot. if there are multiple structural changes tested and at least one of them has an association with the pmi, then that is considered positive for the purposes of this visualization. both the sample size of the study (i.e., the number of brains considered, y-axis) and the highest recorded pmi (x-axis) are plotted on log scales. case reports case report studies, of which six were identified, describe the degree of preservation of cell morphometry in relation to the pmi of a single brain (supplementary file 7). for example, mackenzie reported a case of a body that was immersed in water in late autumn and winter for 10 weeks prior to autopsy (mackenzie, 2014). the histology quality of this brain was reportedly “good”, with morphological changes of anoxic neuronal injury and axonal spheroids still able to be identified. as another example, gelpi and colleagues described a case of where a body was stored in a mortuary cooling chamber for 2 months after death prior to autopsy (gelpi et al., 2007). histomorphology was described as “well preserved”, with all cell types, neuropil, and axons appropriately visualized, although they did note moderate vacuolization of brain parenchyma. on the other hand, suárez-pinilla and fernández-vega described a case with severe autolysis of granule and purkinje cells in the cerebellum after only 6 h of pmi, which was associated with a metabolic insult (suárez-pinilla and fernández-vega, 2015). notably, the rest of the brain was found to have essentially normal microscopic findings. case reports demonstrate exceptions from the typical way that practitioners in the field are perceived to think about the problem, thereby making these findings especially interesting for publication. in this sense, the case reports collectively show that it is considered surprising when cell membrane structure is maintained after a pmi of several weeks, or when it is lost in one brain region after a pmi of only 6 h. summary the studies we identified had a wide range of pmis, over which cell morphometry was altered to different degrees. time series studies offer experimental control but create artificial contexts divergent from naturalistic studies relevant in brain banking. correlational studies have several confounds limiting their interpretation, but they represent the practical contexts that investigators encounter. finally, the few case reports identified may illustrate the limits of our understanding and they may challenge paradigms. as an attempt to explain the high heterogeneity between studies, we next turn our attention to potentially modifying variables. differences in decomposition rates across assessment methods changes identified due to the pmi depend in complex ways on the methods used to visualize them. for example, there might be selective degradation or inaccessibility of the label measured in the visualization procedure, rather than the structure itself. intracellular regions could also become more difficult or easier to stain if the cell membranes are partially degraded, for example due to changes in membrane permeability. immunohistochemistry immunohistochemistry (ihc) is a method commonly used as a proxy of cell membrane morphology. ihc involves the use of specific antibodies that can bind to target antigens of biomolecules, which are then labeled with a detectable marker, allowing for the visualization of the antigen under the microscope. in ihc, an abnormal staining pattern could result if the labeled biomolecule (a) is totally lost from the tissue, (b) has been fragmented, (c) has undergone a conformation change, (d) has bound to another biomolecule that blocks the epitope, or (e) the antibody is not able to diffuse closely enough to the biomolecule due to accessibility issues. for example, gärtner and colleagues used different anti-tau antibodies to study how tau immunostaining is affected by 30 min of ex situ postmortem storage at 37° c (gärtner et al., 1998). they found that three antibodies were not affected by the pmi, two others revealed alterations in the labeling patterns identified, and two others showed complete loss of immunoreactivity. this is an example of how different epitopes of the same protein can be variably affected by the pmi. while caution is warranted in the interpretation of ihc signals for antigens that are unstable in the pmi used as a proxy for the structure of cell membranes more broadly, ihc staining for certain biomolecules that do not undergo changes postmortem can be useful. for example, one study found that immunostaining for calbindin-d28k was more sensitive in postmortem brain tissue than nissl staining for detecting purkinje cells, although neither of these methods were found to vary with the pmi range studied of 3 to 48 h (whitney et al., 2008). another study found that neuronal morphology was lost with toluidine blue staining at 30 h pmi, but that immunostaining for rabies antigen was still able to delimit the profile of the soma of infected cells at this time point (monroy-gómez et al., 2020). morphological staining the most common morphological stain is h&e. hematoxylin stains basophilic regions of tissue such as the nucleus and rough endoplasmic reticulum, while eosin stains acidophilic regions such as the cell membrane, extracellular matrix, proteins, and most organelles (chan, 2014). nissl staining is also common, wherein basic dyes such as cresyl violet or thionine bind to nucleic acids in the cell including rna in the rough endoplasmic reticulum or in free ribosomes (kádár et al., 2009). because certain neurons have high concentrations of rough endoplasmic reticulum, nissl staining can highlight neuronal cytoplasm, but morphology is poorly visualized, although basic structural aspects of somata may be discerned. while h&e and nissl stains are not effective at visualizing neuronal processes, other specialized chromogenic dyes can be helpful. for example, das and colleagues used staining with the lipophilic dye dii and found that dendritic spines can be visualized for up to 28 h of pmi in cases of sudden death (das et al., 2019). they conclude that this is a longer period of dendritic spine stability during the pmi than previously thought, and that they were able to detect dendritic spines at extended time points because of their novel method. the golgi impregnation is a different technique that completely stains a subset of cells including their entire dendritic tree (kang et al., 2017). there are differences in the sensitivity of different golgi stains to the pmi. specifically, rapid golgi stains are more sensitive to changes in the pmi than golgi-cox. one study found that a pmi of more than 4 h leads to a reduction in visualized dendrite length in rapid golgi stained tissue (de ruiter and uylings, 1987). another study suggested that golgi-cox impregnation had robust visualization of dendritic spines with longer pmis (up to 28 h tested), while rapid golgi staining only had similarly good visualization for one case with a shorter pmi of 6 h (buell, 1982). in contrast to golgi methods, the bielschowsky method uses silver to stain essentially all types of cells and neural processes in the brain (switzer, 2000). it is most often used to visualize axons and has been reported to be robust to changes in the pmi. for example, one study found that there was no change with bielschowsky staining after 24 h of pmi (as cited in (haines and jenkins, 1968)). in a case report of a brain with a pmi of 2 months, another study reported that bielschowsky silver impregnation was more robust for the visualization of axons than staining with anti-neurofilament antibodies (gelpi et al., 2007). however, as the likelihood of putrefaction increases with extended pmis, bielschowsky stained tissue needs to be examined with more caution, because colonies of microorganisms can also stain positive and mimic neuritic plaques (mackenzie, 2014). taken together, as opposed to ihc for potentially unreliable antigens, morphological stains are reported to be more robust to changes in the pmi, because they label classes of biomolecules rather than specific ones. electron microscopy the electron microscopy (em) studies included in this review generally stain tissue with osmium tetroxide, which primarily labels unsaturated lipids in membranes, and uranyl acetate, which has binding affinity towards proteins and provides contrast (hua et al., 2015; moscardini et al., 2020). however, staining protocols for em are fastidious and require precise optimization (tapia et al., 2012). because the properties of brain tissue might be substantially altered during the pmi, additional optimization of the staining protocol for postmortem conditions might be required, which is not necessarily always performed. as a result, as with all stains, it is possible that with increasing pmi, what is lost when visualizing tissue with em is not the structural components of cell membranes themselves, but rather the ability to identify them with the particular methods employed. for example, as previously discussed, gibson and tomlinson claim that the decrease in visualized synapses on em over the first day or two of the pmi is largely due to cell process swelling which compresses synapses and makes them more difficult to visualize in 2d sections (gibson and tomlinson, 1979). if this is the case, then the compressed synaptic membranes could still potentially be visualized by an alternative approach such as a high-resolution volumetric em method or an immuno-em method that labeled for a particular synaptic protein that is relatively resistant to postmortem decomposition. consistent with the sensitivity of em to changes in the pmi, sarnat and colleagues claim that em is more sensitive to pmi artifacts than their light microscopy method of visualizing synapses by immunostaining with synaptophysin (sarnat et al., 2010). other authors have also claimed that light microscopy is more robust than em to changes in the pmi in the visualization of synapses (peroski et al., 2016). summary taken together, various methods of visualizing brain cell membranes differ in their robustness to changes in the pmi (table 2). as a result, investigators need to be aware of the potential for bias when using methods that are not as robust to postmortem changes. method reported robustness to postmortem decomposition immunohistochemistry often sensitive to postmortem changes, but highly dependent on the antigen labeled h&e and nissl relatively robust to postmortem changes, but unable to visualize cell processes specialized chromogenic dye some methods are relatively more robust to postmortem decomposition(e.g., dii staining) golgi golgi-cox is more reliable than rapid golgi bielschowsky reported to be generally robust to changes in the pmi, but can show false positive staining for microorganisms electron microscopy may be more sensitive than light microscopy to postmortem changes. structures may be not visualized on two-dimensional images due to compression table 2. summary of the relative robustness of different visualization methods to postmortem decomposition in the brain. selective vulnerability to decomposition during the pmi, some aspects of cell membranes decompose faster than others. these structural features include various aspects of cell membranes, such as dendrites or synapses. each instance of a feature is generally composed of millions or billions of individual biomolecules, which make up the underlying biochemical material. our attempt to summarize these collections of biomolecules by describing them with a single name is meant to be a useful abstraction, although there is a significant heterogeneity of biomolecular content within features of the same type. models of selective vulnerability as an initial way to think about how quickly different features might degrade, we can consider a naïve model in which: (a) the density or composition of biomolecules does not vary significantly between features and (b) the decomposition rate of each biomolecule is independent of its local context. under this model, features will degrade at a rate proportional to their size. of course, both assumptions of this model are false. different features are clearly composed of different types of biomolecules; for example, the cytoskeleton of axons tends to be made of more longitudinal neurofilaments, whereas the cytoskeleton of dendrites tends to be made of microtubules, which may degrade at different rates postmortem (schwab et al., 1994). similarly, the decomposition rate of each biomolecule will clearly depend on its local context, such as the density of catabolic enzymes. a size-proportional decomposition model, under which structural features decompose at a rate proportional to their size, may still be a helpful first approximation. smaller features will have fewer biomolecules, a lower probability of a label binding to that biomolecule, and therefore a lower probability of being visualized as its constituent biomolecules begin to degrade and it eventually disintegrates during the pmi. brain region and cell type heterogeneity before discussing sub-cellular features, it is important to consider how rates of decomposition vary across brain regions and cell types. certain brain regions, such as the hippocampus, are well-known to be more susceptible to degeneration due to temporary cerebral ischemia followed by reperfusion injury. however, damage following temporary ischemia is an active process and therefore caused by a different decomposition mechanism than what occurs postmortem. multiple studies reported that postmortem decomposition generally progresses at equal rates in the different brain regions studied, instead of progressing faster in brain regions more susceptible to temporary ischemia (garcia et al., 1978; spector, 1963). the main brain region that has been associated with a faster rate of postmortem decomposition is the cerebellum (averback, 1980; furukawa et al., 2015; finnie et al., 2016). bywater and colleagues reported that the cerebellum is the most sensitive region to postmortem autolysis, with significant changes occurring within 20 min, which they attribute to the high enzyme content of the granular layer (bywater et al., 1962). ikuta and colleagues also reported that there is a selective disintegration of the granular layer of the cerebellum in the pmi, without a change in glia or adjacent purkinje cells, due to similar enzyme-mediated autolysis in granular cells (ikuta et al., 1963). albrechtsen noted that there is a significant correlation between the ph of the brain tissue and the degree of decomposition of the granule cell layer of the cerebellum, consistent with a role of enzyme-mediated autolysis (albrechtsen, 1977a). aside from the cerebellum, some sources described the relative vulnerability of other brain regions, although not as consistently. multiple sources reported that white matter tends to be better preserved than other regions of the brain (furukawa et al., 2015; mackenzie, 2014). one source noted that the cortex tends to be better preserved than the basal ganglia and thalamus (mackenzie, 2014). oehmichen noted that the pons and certain thalamic nuclei are the first to show damage in the pmi (oehmichen, 1980). yeung and colleagues, studying an extended pmi of 30 d, found that outer layers of the cortex (i-iii) were less well preserved than the inner layers of the cortex (iv-vi) (yeung et al., 2010). notably, there can also be a patchy heterogeneity of decomposition within a local area of the brain (lesnikova et al., 2018). some regional differences may also be confounded by immersion-based preservation methods, which is discussed below. in terms of cell types, one source noted that ventral thalamic nucleus neurons and small cells of the striatum are the most vulnerable to postmortem decomposition, while large pyramidal cells in the parietal cortex are the least vulnerable (as cited in (choe et al., 1995)). several other sources also report that larger neurons are better preserved and/or that smaller neurons have more rapid postmortem changes (irving et al., 1997; lindenberg, 1956; williams et al., 1978). it has been reported that there is a large heterogeneity in the decomposition rate within a cell class (as cited in (choe et al., 1995)). in the anterior pituitary, significant variability of decomposition rate has also been reported within cell type classes, attributed to the functional state of the cell at the time of death (ilse et al., 1979). overall, decompensation kinetics are highly variable even within a cell type class, but certain types of smaller cells may be relatively more vulnerable. alterations in cellular and subcellular volumes in cell death by oncotic necrosis, cells are expected to initially swell in the first stage, and eventually shrink as necrosis progresses. however, the presence or absence of cell swelling depends on the local environment, including any fluid take-up by other nearby cells. indeed, cells may even shrink initially if nearby cells take up relatively more fluid. moreover, swelling may also be transient and not captured by the histological techniques at any given timepoint (majno and joris, 1995). therefore, the potential effects of pmi on cell volume are challenging to predict. cell swelling is one of the most common morphometric findings early in the pmi. schulz and colleagues reported cell swelling after 30 seconds of pmi that disappeared with continued postmortem time (schulz et al., 1980). they explain the mechanism as due to an increase in intracellular cations, leading to a rise in intracellular oncotic pressure. shibayama and kitoh noted swelling of pyramidal cells at 1 h pmi, which they reported was more pronounced near the white matter (shibayama and kitoh, 1976). in their study of isolated biopsy brain tissue, dachet and colleagues found that neuronal swelling first appeared by 2 h of pmi and that by 4-8 h, a majority of neurons were swollen (dachet et al., 2021). cell volume changes can have complex evolution patterns over the pmi. hayes and colleagues found that average neuron size in the white matter of monkey brains increased only slightly following a 2 h pmi, then increased substantially at a 12 h pmi, and then there was no additional increase at 24 or 48 h (hayes et al., 1991). they note that the inclusion of brains with relatively longer pmis in human cohorts may be helpful to factor out the effect of pmi on neuron size. in their study of horse brains, wenzlow and colleagues noted a linear decrease in the size of the cytoplasm up to 3 d of pmi when brains were stored at stored at 8 °c, although no volume changes were detected when brains were stored at 22 °c (wenzlow et al., 2021). tafrali found that neurons shrank at 12 h pmi, while astrocytes and to a lesser extent oligodendrocytes increased in size (tafrali, 2019). as opposed to swelling of many cell types, capillaries have been found to have decreased volume at 22 h pmi (hunziker and schweizer, 1977). consistent with the previously described mechanism of fluid shifts, many studies describe swelling of astrocytes and astrocyte processes (dachet et al., 2021; gibson and tomlinson, 1979; shibayama and kitoh, 1976; tafrali, 2019; williams et al., 1978). jenkins 1979 noted that astrocytic cell bodies and processes were more dramatically swollen than neurons or oligodendrocytes, starting at 5 min of pmi and continuing until 25 min of pmi (jenkins et al., 1979). arsénio-nunes and colleagues described pronounced astrocyte process swelling in the molecular layer and at the level of vascular end feet at 30 min pmi (arsénio-nunes et al., 1973). finnie and colleagues reported that bergmann glia had cytoplasmic swelling much earlier than purkinje cells in the pmi (finnie et al., 2016). del bigio and colleagues described pronounced swelling of astrocytes and oligodendrocytes during the pmi that could be spatially associated with a hemorrhagic lesion prior to death (del bigio et al., 2000). they attributed the postmortem etiology of this swelling as due to the uptake of leaked plasma proteins from the extracellular space by these cell types during the pmi. rees found that astrocyte processes were “markedly swollen” at 2 h of pmi and that this was especially evident in astrocytes around blood vessels (rees, 1976). in summary, postmortem cell volume changes very frequently occur in the pmi. swelling can occur very early in the pmi and may either persist, revert, or convert to shrinkage. generally, the volume of a given cell or cell sub-region assessed after a given period of pmi cannot be assumed to be the same as it was in vivo. cell volume changes are also heterogenous across cell type, between brain regions, and dependent on local events such as hemorrhage. dendrites dendrites play a critical role in information processing in the brain and have a wide variety of shapes and configurations. they can be very narrow, with diameters of less than 500 nm in certain areas (harris and spacek, 2016). dendritic spines have especially narrow necks connecting them to dendritic shafts, with diameters of 50-400 nm (adrian et al., 2014). based on the size-proportional decomposition model, dendrites are expected to be among the most vulnerable features to postmortem decomposition. several studies using immunohistochemical approaches have noted that immunoreactivity is lost from dendrites relatively early in the pmi. boekhoorn and colleagues found that doublecortin immunoreactivity was greatly diminished in dendrites after a pmi of only 1 h in rodent brains and was effectively lost by 8 h pmi, while soma immunoreactivity was retained for a longer time (boekhoorn et al., 2006). using calretinin staining in the monkey hippocampus, lavenex and colleagues found that dendrite staining became coarser over a pmi of 2 to 48 h (lavenex et al., 2009). specifically, while dendrites could still be visualized in the later pmi brains, they appeared as a string of discontinuous agglomerates as opposed to a series of ovals connected by a continuous fiber. multiple studies used the antibody smi-32, which binds to neurofilament h and can be used to visualize neuronal processes including dendrites. gonzalez-riano and colleagues did not find any changes in the distribution of staining for smi-32 in mouse brains at a pmi of 5 h (gonzalez-riano et al., 2017). hilbig 2004 noted that details of the dendritic tree in large pyramidal cells were visible up to a pmi of 12 h with immunostaining for smi-32. with increasing pmi after 12 h, a granular-like immunostaining of disintegrated dendritic neurofilaments was found instead. there is a substantial literature on postmortem map2 immunostaining, which has also been generally found to degrade relatively rapidly during the pmi, thus reducing visualization of dendrites in map2-stained sections (irving et al., 1997; kitamura et al., 2005; lingwood et al., 2008; schwab et al., 1994). for example, nakabayashi 2021 performed immunostaining for map2 in rat brains, reporting a fragmentation of secondary dendrites at 6 h pmi and a disappearance of primary dendrites at 1 d pmi (nakabayashi et al., 2021). map2 postmortem redistribution has been speculated to be due to the sensitivity of the map2 protein to postmortem proteolysis, although the precise mechanism is unclear (d’andrea et al., 2017). some studies using morphological staining have also found that dendrite structure is altered relatively early in the pmi. for example, in a study using the golgi rapid method to visualize pyramidal neurons in mouse brains, williams and colleagues noted that dendrites degenerated in a “moniliform” fashion, i.e. with alternating varicosities and constrictions, which was first noticed at 6 h pmi (williams et al., 1978). at 6 h of pmi, there were also patchy areas of dendrites that had a decreased density of spines. they also reported a centripetal loss of dendrite visualization with increasing pmi, wherein distal segments were lost first followed by more proximal areas. using an alternative visualization method of differential interference contrast optics, they found that this loss was at least partially attributable to a failure of silver impregnation of distal dendritic segments with increasing pmi. as another example of early dendritic changes in the pmi, bywater and colleagues used a silver impregnation method and reported that dendrites began to “curl up in a corkscrew manner” by 4.5 h of pmi in monkey brains (bywater et al., 1962). in their em study, rees 1976 found that dendrites were swollen after 30 min of pmi (rees, 1976). roberts and colleagues, also using em, noted that dendrites, along with axon terminals, spines, and mitochondria, became “bloated and/or irregular in contour” at pmis of greater than several hours, and therefore they restricted their pmi range to less than 4 h (roberts et al., 1996). other studies using morphological staining methodologies found that dendrite morphology is stable for longer pmis. for example, as previously discussed in the visualization section, das and colleagues found that dendritic spines can be visualized via staining with the dye dil for up to a pmi of 28 h (das et al., 2019). within their sample, they found that the pmi was not associated with spine density, spine head diameter, or spine length. jacobs and scheibel reported that dendrite morphology was well-preserved in their cohort of human brains, with pmis up to 32 h (jacobs and scheibel, 1993). they used the golgi-cox staining method because it had been reported to be less sensitive to postmortem autolysis. they did not find the markers of autolysis (e.g., irregular varicose enlargements and loss of spines) that had been previously reported with the rapid golgi method by williams and colleagues (williams et al., 1978). yeung 2010 found that dendritic branching patterns could be visualized with bielschowsky silver staining in a subset of pyramidal cells from human brains after even 30 d of pmi with storage at 4 °c (yeung et al., 2010). much of the correlational literature in human brain cohorts suggests a relative stability of dendritic structures in the pmi, including studies with a pmi range up to 9 h using em (kolomeets et al., 2005), up to 28 h or 78 h using golgi-cox staining (boros et al., 2017; buell, 1982), up to 74 h using immunostaining for glutamate receptor subunits (benes et al., 2001), or up to 120 h using the rapid golgi method (garey et al., 1998). an exception is the study of tóth and colleagues that used ihc and found that dendrites on calretinin-immunoreactive cells appeared to be shorter, varicose, and degenerating with increasing pmi over a range of 2 to 10 h (tóth et al., 2010). it is challenging to reconcile differences in the decomposition rate of dendrites observed across these studies. however, problems with dendrite visualization in studies finding that dendrites degrade relatively faster could help to explain this divergence, whereas it is more difficult to imagine how the literature suggesting dendrites are stable for longer periods of time could be systematically flawed. in summary, dendritic swelling can occur early in the pmi (rees, 1976; roberts et al., 1996). additionally, there is evidence for clumping or beading of dendritic components (lavenex et al., 2009; williams et al., 1978). however, total loss of dendritic cell membranes is not a commonly reported early phenomenon in the pmi. as a result, dendrite tracing in volumetric microscopy data may be possible even in brains with relatively longer pmis if robust visualization methods are used. certain morphological staining methods, such as the golgi-cox impregnation method, are more resistant to pmi artifacts than others. experiments labeling for particular biomolecules, such as map2 or smi32, should not be considered dispositive of the state of dendrite morphology more generally. axons like dendrites, axons are narrow, and have been found to have similar sized or lower average diameters compared to dendrites (faitg et al., 2021). this makes axons similarly vulnerable to postmortem changes under a size-dependent model. in studies using morphological stains, axons tend to be relatively well preserved, albeit with volume changes at early pmis. using rapid golgi staining, williams and colleagues found that axons tend to exhibit bead-like changes and become more lightly impregnated after a pmi of more than 6 h, which was a slower change than they reported for dendrites (williams et al., 1978). in the em studies by rees and roberts and colleagues, it was reported that axons were swollen at the same early time periods as dendrites (rees, 1976; roberts et al., 1996). in their study using light microscopy to visualize the habenular nuclei of dog brains, haines and jenkins found that a few large axons started to show areas of slight dilation and constriction at 12 h pmi (haines and jenkins, 1968). at 36 h pmi, these areas of constriction and dilation were found in most axons, and almost every axon started to have areas with short loops. generally, they found that axons retained their integrity up to 48 h pmi and were relatively stable postmortem, which corroborated the previous reports they cited. segmental axon swelling has also been reported as a consequence of focal ischemia in ultrastructural studies (pantoni et al., 1996). notably, this postmortem artifact is distinct from axonal spheroids, which are marked by accumulation of amyloid precursor protein (app) and can occur in the context of blockage of fast axonal transport (coleman, 2005). in immunostaining studies, axon definition can be lost after an extended pmi (sarnat et al., 2010). for example, blair and colleagues found that immunostaining for α-tubulin was lost at 48 h pmi in 3/6 of the human brains profiled (blair et al., 2016). on the other hand, in this study, neurofilament proteins were still detectable at these extended pmis and axons were still able to be thereby visualized. eggan and lewis studied the immunoreactivity of cannabinoid receptor 1 in axons of monkey brain tissue sections with light microscopy (eggan and lewis, 2007). they found that after a 24 h pmi, axons became less distinct, and axon terminals became swollen. booze and colleagues found that immunostaining for fine varicose axons with tyrosine hydrolase was lost between 1 and 6 h of pmi (booze et al., 1993). taken together, axons have similar postmortem decomposition patterns as dendrites, with early swelling and patches of alternating constrictions and varicosities that become more common in the pmi. some evidence suggests that axons may degrade slightly slower than dendrites postmortem, although this is highly uncertain and may relate to visualization methodology. synapses time series studies using em and short pmis have found that aspects of synaptic morphometry can be altered very rapidly in the pmi, within minutes. routtenberg and tarrant found that after 1 min of pmi, there was a substantially diminished concavity of the synapse and a thickening of the postsynaptic membrane (routtenberg and tarrant, 1974). tao-cheng and colleagues also found that after 6.5 min of pmi, there was a change in curvature and an increased thickness of the postsynaptic density (tao-cheng et al., 2007). karlsson and schultz found that after 60 min of pmi there were fewer and flattened synaptic vesicles, as well as occasional plasma membrane separation and an increase in extracellular space (karlsson and schultz, 1966). roberts and colleagues noted that as the pmi extends beyond several hours in human brains, the postsynaptic density becomes thicker, thus obscuring whether the synapse is asymmetric or symmetric (roberts et al., 1996). huttenlocher noted that in human postmortem brains, presynaptic and postsynaptic markers are less sharply demarcated, and intracleft areas cannot always be seen (huttenlocher, 1979). while morphometric properties of synapses are rapidly altered, they are usually found to be demonstrable with em following a more extended pmi. rees found that synapses had not disintegrated by 4 h of pmi in cats or 5.5 h of pmi in monkeys (rees, 1976). vrselja and colleagues found that post-synaptic densities were preserved at 10 h of pmi, although the presynaptic vesicle pool was not maintained (vrselja et al., 2019). tang and colleagues found that there was no significant decrease in visualized synaptic density at 2 d of pmi in mammalian brains (tang et al., 2001). shibayama and kitoh found that synaptic contacts were preserved for pmi of up to 10 h although vesicles decreased in number (shibayama and kitoh, 1976). in isolated rat brains stored at 37 °c, de wolf and colleagues found that synapses were able to be visualized at a pmi of 13.5 h, but the ability to visualize them was lost by a pmi of 24 h (de wolf et al., 2020). when stored at 0 °c, this study found that synapses could be visualized at 48 h of pmi but were lost at the next time point studied of 168 h of pmi. one clear exception to the trend of postmortem synapse maintenance is the study of petit and leboutillier which analyzed rat brain tissue with two em staining methods, measuring both synaptic density and morphometry (petit and leboutillier, 1990). the study required the presence of synaptic vesicles to identify a synapse and only counted synapses had a clear preand postsynaptic density. with an osmium-based staining method, it was found that synapse density dropped markedly from 0 to 1 h of pmi, and then markedly once again from 10 to 15 h of pmi. with an ethanol phosphotungstic acid method of staining, synapse density dropped more slowly from 1 h to 15 h of pmi, with a total of 60-70% of synapses lost by the latest pmi time point tested. notably, synaptic structure within identified synapses was found to be “remarkably stable” up to the longest pmi they studied of 15 h, suggestive that synapses in an intermediate disintegrating state were not frequently found. in the osmium-stained tissue, the decline in the number of synapses from 1 h to 15 h of pmi mirrored an increase in the number of contacts without synaptic vesicles. the authors suggest that the observed decline in synaptic density in the pmi may be due to a loss of synaptic vesicles and therefore the ability to identify a connection as a synapse, although they noted that other explanations were also possible. correlational studies using em in human brain cohorts have had mixed results regarding synapse preservation. glausier and colleagues found that there was a significant decrease in the number of postsynaptic densities identified as the pmi increased up to 24 h (glausier et al., 2019). however, glausier and colleagues did not find a significant correlation between the pmi and the postsynaptic density length or with the total number of neuronal profiles identified. roberts and colleagues found that preand postsynaptic membrane structures were lost or distorted with pmi of greater than 7 h (roberts et al., 1996). on the other hand, roberts and colleagues reported that synapse classification and morphology was equivalent in cases with pmi of up to 7 h (roberts et al., 2005). scheff and colleagues found that there was no association between pmi up to 13 h and the synaptic features they studied (scheff et al., 1990; scheff and price, 1993). kolomeets and colleagues found that there was no correlation between the pmis of their cohort, which had an average of 6-7 h, and the number of synaptic contacts identified per mossy fiber terminal (kolomeets et al., 2007). gibson and tomlinson found that the numbers of recognizable synapses decreased by 77% at 33 h of pmi, at which point it stabilized until up to 69 h of pmi (gibson and tomlinson, 1979). as previously discussed, these authors suggested that the loss of recognizable synapses could be accounted for by the compression of synapses due to astrocyte vacuolization. it is not clear how to reconcile the differences between studies regarding the postmortem decomposition of synapses. several studies suggest that synapse visualization can be lost within the first hours of the pmi, while other studies suggest that the delay is longer and that the apparent loss of synapses is due to technical problems rather than synaptic disintegration. the latter prospect is consistent with the finding that partially disintegrated synapses are rarely reported, whereas one would expect to see these if synapses were disintegrating in the early pmi as opposed to becoming not recognizable with the techniques used. in summary, certain synaptic morphometric changes, such as thickening of the postsynaptic membrane and changes in curvature, can begin in the earliest minutes of the pmi. however, the preponderance of evidence suggests that synapses can remain generally intact for an extended period, up to many hours or even multiple days of pmi. certain methods of visualizing synapses are more robust in the postmortem period, suggesting that technical approaches are crucial to consider carefully when designing these experiments. myelin one of the major myelin artifacts that is consistently noted in the pmi is myelin lamellae separation. shibayama and kitoh noted that myelin lamellae are partially distorted at 30 mins of pmi (shibayama and kitoh, 1976). choe and colleagues described concentric unraveling of myelin at 1 h of pmi (choe et al., 1995). karlsson and schultz noted that there are infrequent myelin membrane separation defects after 1 h of pmi (karlsson and schultz, 1966). rees noted marked separation of lamellae in some myelin sheaths in monkey brains at 4 h of pmi (rees, 1976). hukkanen noted the formation of network-like structures in myelin lamellae at 24 h (hukkanen and röyttä, 1987). de wolf and colleagues noted unravelling of myelin sheaths at 9 h pmi with storage at 37 °c (de wolf et al., 2020). ansari and colleagues noted that myelin lamellae splitting can also occur in cerebral edema and that its occurrence may be associated with the postmortem increase in the water content of the brain (ansari et al., 1976b). another phenomenon, which may be related to lamellae splitting, is volume changes in myelin sheaths. haines and jenkins noted a slight swelling of myelin in some areas, first noticed at 18 h of pmi (haines and jenkins, 1968). on the other hand, de wolf and colleagues described myelin thinning, first noticed at 3.86 h of pmi with storage at 37 °c (de wolf et al., 2020). aside from these artifacts, myelin is generally reported to be stable in the pmi. myelin lamellae splitting is not associated with a break in the continuity of the separated lamellae (ansari et al., 1976a). several studies note that myelin is a relatively robust structure to postmortem decomposition (haines and jenkins, 1968; karlsson and schultz, 1966; sele et al., 2019; vrselja et al., 2019). summary among brain cell-specific membrane structures, volume and geometry changes are a frequent occurrence early in the pmi, and there are several types of common postmortem artifacts in these structures (table 3; figure 5). however, studies using robust visualization methods tend to find that approximate cell membrane location information is still able to be delineated in the early postmortem period. this suggests that the basic connectivity scheme of brain cells can likely be mapped even after postmortem decomposition has begun. feature key points of decomposition patterns brain region differences the cerebellum can have faster decomposition than other regions cell type differences smaller cells may decompose faster than larger cells cell volume cell volumes changes are rapid and occur within minutes. ongoing swelling, shrinkage, and compression processes evolve throughout the pmi dendrite segmental swelling within hours. highly sensitive to visualization method, and appear to be more stable with more robust methods of visualization axon segmental swelling and volume changes, similar to dendrites synapse morphometric changes, including postsynaptic membrane thickening and changes in curvature, occur within minutes. variability based on visualization method, but their overall presence is relatively stable in the early pmi myelin lamellar separation without a break in continuity of the separated lamellae. relatively slow decomposition overall table 3. summary of relative postmortem decomposition rates of different structural features of cell membranes in the brain. figure 5. possible morphometric changes of structural features in the postmortem brain. a-b: ultrastructural diagram of a human synapse at an earlier pmi (1 hour; a) with insignificant postmortem changes. compared to a later pmi (12 hours; b), at which point there are several changes, including vesicle clumping and loss, increased synaptic density with possible difficulty identifying the cleft, swelling of surrounding processes, and mitochondrial damage. these diagrams were adapted from (anders, 1977) (figure 2). c: segmental swelling of axons, adapted from an image of bielschowsky silver-stained ischemic rat brain tissue from (pantoni et al., 1996) (figure 6). d: myelin lamellae splitting, adapted from an electron microscopy image of postmortem human brain tissue from (glausier et al., 2019) (figure 1). variables modifying decomposition rates temperature the temperature at which the brain is stored during the pmi has a very well-replicated role in mediating the rate of postmortem decomposition. mechanistically, cold storage temperature slows down diffusion, decreases enzymatic activity, and inhibits microbial growth, all of which delay decomposition (schavemaker et al., 2018). low temperature storage refers to temperatures down to 0 °c, as below causes ice crystal formation and accompanying tissue damage. storage temperature has been reported to have the strongest effect on the rate of brain protein breakdown of any modifying variable (ferrer et al., 2007). one study in sheep brains shows that decreasing the storage temperature dramatically delays the average onset time of a sudden concentration change in several metabolites, attributed to the initiation of putrefaction, from 30 h pmi at 26 °c to 700 h pmi at 4 °c (ith et al., 2011). many studies find that the storage temperature plays a substantial role in the retention of cell membrane structure during the pmi. in their study of synapses in human brains, kay and colleagues report that they can perform effective ultrastructural analyses on tissue with pmis of up to 100 h (kay et al., 2013). they note that a key factor for ultrastructure tissue preservation is whether the cadaver is stored at cold temperature of 4-6 °c to reduce structural degradation. de wolf and colleagues found that advanced necrosis developed in isolated rat brain tissue at 36 h pmi when stored at 37 °c, compared to 2 months pmi when stored at 0 °c (de wolf et al., 2020). hukkanen and röyttä found that the white matter of isolated human surgical specimens showed degradation patterns at 6 h of pmi when stored at 25 °c, resembling those seen at 24 h of pmi when stored at 4 °c (hukkanen and röyttä, 1987). the environmental temperature at the time of death can also be relevant which has practical implications. for example, more rapid postmortem changes in the brain have been associated with death in summer months, while hypothermia at death is associated with slower postmortem decomposition (albrechtsen, 1977b; kitamura et al., 2005). some brain bankers record the refrigeration delay in addition to the pmi, which is the interval of time between death and when the body was stored at 4 °c (torres-platas et al., 2014; vonsattel et al., 2008). a formula that predicts the amount of brain decomposition based on storage temperature could allow for more accurate estimation of the impact of pmi. for example, the q10 rule suggests that the speed of chemical reactions in biological systems increases two-fold or slightly more with each 10 °c rise in temperature (vass, 2011). in the forensic literature, there is a related metric of “accumulated degree days” (vass, 2011). if the q10 rule holds, then reducing the storage temperature from a typical room temperature of 20-24 °c to 0-4 °c would be expected to decrease the decomposition rate by approximately 4-fold. a challenge is that brain temperature does not immediately equilibrate with the environment. even when the brain is stored at refrigerator temperatures of 8 °c, it can take 20-30 h for the tissue itself to actually reach that temperature, because of the slow rate of heat conduction (perry et al., 1977). as a result, the effects of refrigerator storage are nonlinear. consistent with this, studies investigating postmortem effects at earlier pmi time points, such as 4-12 h of pmi, tend to report smaller effects of refrigeration (hilbig et al., 2004). for the same reason of the low thermal conductivity of brain tissue, refrigerator storage is also expected to have a larger effect on the surface of the brain tissue, in smaller brains, and in isolated brain tissue. acidity and agonal damage across tissues, high acidity is strongly associated with faster decomposition rates in the pmi. for example, in a study on fish, postmortem decomposition was the fastest in the gastrointestinal tract and slowest in muscle tissue, with the brain somewhere in the middle, as expected based on their relative phs (george et al., 2016). across donated brains, those with relatively lower ph values have been found to have more postmortem structural breakdown following a given pmi (albrechtsen, 1977a; glausier et al., 2019). a major reason that lower ph is associated with faster postmortem decomposition is that acidic conditions activate certain autolytic enzymes, such as those in capthesin family (kies and schwimmer, 1942; albrechtsen, 1977a; compaine et al., 1995). postmortem acidic conditions in the brain are usually attributed to an increase in the production of lactic acid as a result of anaerobic glycolysis in the absence of oxygen delivery (powers, 2005). anaerobic glycolysis, in turn, has been reported to occur predominantly in two time periods: (a) the agonal phase and (b) the first few hours of the pmi. the agonal phase is the period during which a person is gravely ill and under profound physiologic stress, usually associated with cerebral hypoxia (lewis, 2002). for some brain donors, the agonal phase can last for only minutes or less prior to death, while for others, it can last for hours or days (li et al., 2004). numerous studies have found that a lower brain tissue ph is strongly correlated with a longer agonal phase (hardy et al., 1985; johnston et al., 1997; li et al., 2004). the agonal phase can contribute to damage measured postmortem in two ways. first, brain tissue can decompose during the agonal phase itself, leading to a higher total amount of decomposition after any given amount of pmi. damage associated with prolonged agonal states can be immense, which is why it is considered extremely important in mediating the degree of tissue decomposition in brain banking (ohm and diekmann, 1994; waldvogel et al., 2006; mccullumsmith et al., 2014; glausier et al., 2019). second, because brain cells are hypoxic during the agonal phase, they will produce excess lactic acid during this period, leading to lower ph levels and a faster rate of autolysis once the pmi commences. in addition to the agonal phase, ph has also been found to decrease over the first several hours of the pmi. for example, one study found that there was a significant inverse relationship of pmis between 1.5 and 4.5 h and brain tissue ph (beach et al., 2008). this is likely because some brain cells still have intact metabolic function in this early time range and therefore are still able to perform anaerobic glycolysis. on the other hand, it has been reported that brain ph does not continue to decrease in the pmi after the initial several hours (albrechtsen, 1977a; lewis, 2002). taken together, because of the complex associations of low ph with postmortem decomposition, tissue acidity is a critical variable to be cognizant of when evaluating the literature on postmortem decomposition. for example, ph has been proposed to be the most decisive determinant of postmortem necrosis in the granule cell layer of the cerebellum (albrechtsen, 1977a). hydration the water content of the brain has implications for postmortem decomposition rates. mechanistically, an increased water content can increase biochemical reaction rates (vass, 2011). across tissues, atmospheric humidity rates of greater than 85% are associated with an increase in decomposition rates (vass, 2011). because the brain is encased in the calvaria, atmospheric humidity likely does not substantially affect decomposition speed in the relatively early pmi ranges most relevant to brain banking. however, when the brain is taken out of the calvaria, either as a part of the preservation procedure or for ex situ storage in a time series study, investigators need to choose how to store the tissue. the brain is typically moist in its in situ location, bathed in cerebrospinal fluid (csf). one option for storage is to submerse tissue in a solution that matches the composition of the csf, although this risks worsening fluid shifts and associated artifacts such as vacuolization (fix and garman, 2000). alternatively, the brain can be stored in the dry air, although this is likely to lead to severe tissue damage due to dehydration (budday et al., 2015). additionally, the brain’s relatively high water content, which has been found to increase in the postmortem period, may play a role in its relatively faster rate of postmortem autolysis compared to some other tissues (ansari et al., 1976b; zhou and byard, 2011). as a result, premortem conditions in brain donors that promote water accumulation, such as cerebral edema, may predispose to a faster decomposition rate during the pmi. oxygen content the atmospheric oxygen content also affects the rate of postmortem decomposition across tissues (cockle and bell, 2015; vass, 2011). mechanistically, high oxygen content leads to oxidative damage to biomolecules as well as increased bacterial growth, thus promoting putrefaction (cockle and bell, 2015). the brain is relatively insensitive to changes in atmospheric oxygen content in the early stages of the pmi, due to its protection in the calvaria and the relative isolation of the brain from bacteria in the gastrointestinal tract. however, the brain is likely much more susceptible to changes in atmospheric oxygen content in the later postmortem period when bacteria have had time to proliferate in the brain tissue. this may help to explain the relatively good long-term preservation of brains stored in environments with low oxygen content, such as the body described in a case study that was immersed underwater for 10 weeks prior to autopsy (mackenzie, 2014). additionally, when the brain is taken out of the calvaria without protection from oxygen, it is likely to be more susceptible to oxidative damage and putrefaction due to the increased exposure to atmospheric oxygen. putrefaction to what extent there are microbes present in the brain under normal conditions remains an open question (link, 2021). regardless, there is evidence that proliferation of putrefactive bacteria is not a typical occurrence in the early pmi. instead, autolysis is thought to be the main contributor to early decomposition within at least the first day of the pmi (ith et al., 2011). various factors could modify the rate at which putrefaction initiates, such as isolation from the gastrointestinal tract, the presence of premortem fever, sepsis, or bacterial encephalitis, and environmental storage conditions (zhou and byard, 2011). at later pmis, the potential for putrefaction is a critical variable to consider. for example, finnie and colleagues noted that there was no confounding bacterial invasion for the full 4 weeks of postmortem decomposition that they studied (finnie et al., 2016). in situ versus ex situ brain storage one of the key distinctions in the methodologies of the studies we identified is whether the brain is stored in situ or excised to be stored ex situ during the pmi. although the decomposition rates between these two conditions were not directly compared among the included studies, this has been found to affect the rate of liver decomposition. specifically, removal of liver fragments from its original location has been found to substantially accelerate the postmortem decomposition kinetics of the liver compared to leaving it in situ (nunley et al., 1972). in studies of neuronal ischemia, morphological damage and disruption of the cytoskeleton have also been found to occur more quickly and intensely in slices than cells in situ (lipton, 1999). in addition to differences in the other modifying variables between the in situ and ex situ conditions, other factors will also affect the rate of decomposition in these two much different environmental contexts. one possible mechanism is that removal of the brain could significantly affect the tissue’s mechanical state, such as its hydraulic permeability, which may affect decomposition rates (jamal et al., 2021). variation across species in metabolic rate several studies noted differences in the decomposition rates between species (lucassen et al., 1995; martin et al., 2003). one notes that postmortem decomposition is likely to be faster in the smaller brain of a rodent and slower in the larger brain of a human (roberts et al., 2014). others suggest that human or bovine brain tissue appear to degrade relatively more slowly postmortem than rodent brain tissue (ansari et al., 1975; schwab et al., 1994). the most likely reason for species differences is that the metabolic rate varies, for example being much faster in rodents, which means that there may be a higher density of catabolic enzymes present in the postmortem period (demetrius, 2005). consistent with this, lesion evolution following cerebral infarction also appears to be markedly faster in rats than humans (mena et al., 2004). the infarct process in general has been reported to occur faster in animals that are smaller with higher metabolic rates (kloner et al., 2018). across tissues, autolysis has been suggested to be faster in malignant tissues with higher metabolic rates (lesnikova et al., 2018). as a result, studies using rodent models of human decomposition may overestimate the rate. this may account for the generally faster rates of decomposition reported in time series studies, which are primarily on rodents, compared with correlational studies, which are predominantly on human brains. premortem metabolic state lindenberg suggest that premortem metabolic state plays an important role in mediating postmortem decomposition rates (lindenberg, 1956). they report that if antemortem hypoxia lasted for more than 60 min, then brain cells retained their microscopic structure for at least 18 h pmi, even if kept at the relatively high temperature of 37 °c. on the other hand, if there was no antemortem hypoxia, then brain cells lost their structure much more quickly. their explanation is that cells exposed to antemortem hypoxia have already depleted and eliminated metabolic products that might otherwise contribute to structural damage. this hypothesis is inconsistent with many other studies finding that more severe agonal damage is associated with worse brain cell morphology (williams et al., 1978; glausier et al., 2019). however, because the underlying biology is complex and poorly understood, it remains true that certain antemortem metabolic states could be protective against postmortem decomposition, requiring further investigation. for example, premortem administration of metformin, a chemical that alters metabolism, has also been shown to decrease the rate of postmortem nuclear swelling (dehghani et al., 2018). age the evidence for the interaction of age with pmi effects is mixed and may depend on the structural feature considered. williams and colleagues found that in general, the effects of the pmi were the same on neuronal morphology in juvenile (14 d old) and adult (60 d old) mice (williams et al., 1978). buell also reported that neuronal morphology in human brains with substantial pmis was not dependent on age (buell, 1982). however, itoyama and colleagues, comparing young (7 d old) and adult rats, found that young rats had more perikaryal swelling of oligodendrocytes, more fragmentation of oligodendrocyte processes, and more myelin vacuolization at the same pmi (itoyama et al., 1980). additionally, mori and colleagues found that blood vessels from older (32-month-old) rats were found to leak proteins at shorter pmis than younger (3-month-old) rats (mori et al., 1991). myelin is known to have delayed development, while blood vessels are known to accumulate damage with age, which may make these features more vulnerable to differential decomposition in the pmi based on age. premortem pathology the presence of premortem brain pathology may affect the rate of postmortem decomposition. this could occur due to factors affecting the cohesiveness or catabolic rate in the brain. in one case study, severe autolysis of granule cells and purkinje cells in the cerebellum occurred after only 6 h of pmi, enormously faster than typical, which was attributed to severe diabetic ketoacidosis (suárez-pinilla and fernández-vega, 2015). death due to uncontrolled diabetes has been associated with higher rates of necrosis of the granule cell layer of the cerebellum in other studies as well (albrechtsen, 1977b). more generally, obesity, hyperglycemia, and ingestion of certain substances have been reported to promote the rate of postmortem decomposition of cadavers and may also therefore influence the rate of brain decomposition (zhou and byard, 2011). on the other hand, some conditions have been found to not increase the rate of postmortem decomposition, such as rabies infection (monroy-gómez et al., 2020). some types of premortem pathology could even predispose for stability in the pmi. certain pathologic proteins in the brain, such as abnormal prion protein and amyloid beta, can be detected via ihc for months after death (scudamore et al., 2011). in the paleoanthropology literature, tissue from a 2600-year-old brain was found to be partially intact, likely due to an aggregate of intermediate filaments, which may have been related to premortem brain pathology (petzold et al., 2020). the possibility of brain pathology affecting postmortem decomposition rates is especially important if investigators adjust for the effects of pmi on cell morphometry with a linear model. in this setting, if there is an interaction effect that is not considered, the investigator might conclude that brain pathology is associated with a particular aspect of cell morphometry, when the difference between groups could instead be due to an artifact of the pmi. summary there are many ways that modifying variables can interact with pmi to affect the visualization of cell morphometry (table 4). well-controlled studies will account for this possibility. these factors also can help to explain heterogeneity between studies. modifying variable reported effect on the rate of postmortem changes storage temperature lower temperatures (above 0°c) are clearly associated with slower decomposition tissue acidity lower ph values are associated with faster decomposition and may be partially a marker for more damage in the agonal state tissue hydration atypically high or low hydration levels may be associated with faster decomposition rates oxygen content higher oxygen content is associated with oxidative damage to biomolecules and more rapid putrefaction putrefaction generally thought to make minimal contributions in the early days of the pmi, but can be more rapid in certain circumstances storage location there is evidence that brains stored outside of the calvaria (ex situ) have more rapid decomposition compared to being stored inside the calvaria (in situ) species there is evidence that smaller animals with faster metabolic rates, such as rodents, have faster decomposition rates premortem metabolic state complex effects of premortem metabolic state on decomposition rate that require further study before conclusions can be drawn age mixed evidence depending on the structural feature studied, with some showing no age dependency, and others, such as myelin and blood vessels, having some indication for an interaction premortem pathology certain pathologies, such as uncontrolled diabetes, are associated with faster postmortem decomposition rates table 4. summary of variables potentially modifying the postmortem decomposition rate. interactions between postmortem changes and preservation methods many studies have noted that the quality of perfusion as compared to immersion fixation could vary based on the pmi. indeed, there is good reason to think that perfusion quality can be limited in the pmi, for example due to a loss of vascular patency, the perivascular accumulation of water, and/or postmortem clot formation (cammermeyer, 1960; de la torre et al., 1992; garcia et al., 1978; hansma et al., 2015; kloner et al., 2018). vascular abnormalities are thought to be the key limitation preventing the brain from tolerating ischemic episodes, thus being a primary cause of death, which makes the difficulty of postmortem perfusion-based preservation unsurprising (jenkins et al., 1979). one study that performed postmortem perfusion, routtenberg and tarrant acknowledged that theoretically perfusion fixation may be lower quality in postmortem cases, and therefore delays in time prior to perfusion fixation may lead to changes in fixation quality rather than pure decomposition effects (routtenberg and tarrant, 1974). in their case, perfusing brains at up to 10 min pmi, they reported that perfusion quality was unlikely to have affected their results, because markers of high-quality perfusion, namely tissue hardness and clearing of blood vessels, were still observed. koenig and koenig used perfusion fixation on guinea pig brains at up to 23.5 h pmi and did not note decreased perfusion quality as an issue that affected their results (koenig and koenig, 1952). consistent with the known benefits of perfusion fixation on tissue quality, several studies note that this method yields substantially fewer postmortem artifacts than immersion fixation (mcfadden et al., 2019). bywater and colleagues, in a study of monkey brains, performed both perfusion and immersion fixation at the different time points studied (bywater et al., 1962). they noted that brains with a pmi of 1 h preserved via perfusion fixation had similar postmortem artifacts as brains immersion fixed immediately after death. liu 1950 noted that immersion fixed brains demonstrate widespread artifacts similar to postmortem artifacts found in brains with extended pmis, which they attribute to inadequate fixation by immersion (liu and windle, 1950). lavenex and colleagues reported that the difference between immunostaining patterns for smi-32 was striking between perfusion fixed brains at 0 h pmi and immersion fixed brains at 2 h of pmi, but that there was no substantial difference between immersion fixed brains preserved at 2 h and 48 h of pmi, with postmortem storage at 4 °c (lavenex et al., 2009). as a result, studies of pmi effects that do not account for the possibility of worse preservation with immersion as compared to perfusion fixation, for example using perfusion fixation as the baseline timepoint of zero pmi and immersion fixation at subsequent pmi timepoints, are susceptible to bias (airaksinen et al., 1991; garcia et al., 1978; geddes et al., 1995; terstege et al., 2022). another important distinction is the differences in fixation as compared to freezing, with several studies noting relative advantages of each. for example, schulz and colleagues noted that dark neurons, a well-known fixation artifact, are seen in small tissue blocks that were fixed but not ones that were frozen, while frozen tissue blocks had freezing artifacts such as cytoplasmic vacuolization (schulz et al., 1980). itoyama and colleagues found a differential effect of a postmortem delay of 20 h when immunostaining for myelin basic protein in frozen as compared to immersion fixed tissue (itoyama et al., 1980). they reported that myelin sheaths were slightly distorted following the pmi in immersion fixed tissue but had substantially worse quality in frozen tissue following the same pmi compared to the baseline state. freezing artifacts could theoretically be worse in cases of longer pmi because more free water and weaker cellular structures may predispose to more mechanical ice damage, which warrants further investigation. finally, the fixative used can also influence observed pmi artifacts. one study found differential effects of a 24 h pmi on immunostaining properties for several antigens in blocks of human brain tissue based on the fixative used for preservation (sillevis smitt et al., 1993). when the tissue was preserved using bouin and b5 fixatives, they found no effect of a 24 h pmi on the quality of immunostaining with smi-32 and bf-10, which are two antibodies that recognize neurofilament. on the other hand, the immunostaining with these antibodies was diminished by a 24 h pmi when the tissue was preserved with a different fixative, sensofix. it may be that fixation needs to be stronger in the presence of a longer pmi to prevent damage during dehydration and embedding. morphological staging of decomposition we present a model of how cell morphometry changes during different stages of the oncotic necrosis pathway in the postmortem brain (figure 6). of note, the timeline of cellular events depicted here likely varies between cells, as these are rough guidelines, not absolute. additionally, the intracellular changes shown, which are not a focus on this review, are primarily based on other sources (choe et al., 1995; majno and joris, 1995; suzuki, 1987). figure 6. model of the oncotic necrosis pathway of cell membrane deterioration in the brain. stage zero is the antemortem cell, which shows intact cell membrane and normal sized structures. stage one is oncosis, wherein ischemia leads to loss of atp, loss of ion pump activity, breakdown of biomolecules, bleb formatting, vacuolization, and other changes. the asterisk (*) indicates that while cell volume increases are possible, no change or shrinkage is also possible. stage two is early necrosis, wherein the physical disruption of the cell membrane causes focal loss in cell membrane shape and leakage of intracellular contents. there is also severe swelling of surrounding processes, potentially compressing cellular structures such as neurites and synapses. from this point on, the cell has necrotic morphology. stage three is late necrosis, wherein gel-like networks break down, eventually leading to a complete cellular dissolution, and only cell fragments or debris may remain. the main figures used for adaptation in building this model were: (majno and joris, 1995) (figure 9), (suzuki, 1987) (figure 2.7), (fricker et al., 2018) (figure 3), and (trump et al., 1984) (figure 8). in this model, oncosis is initiated by a lack of blood flow, leading to atp depletion, a rise in the intracellular concentrations of na+ and ca2+, fluid shifts, and the initiation of biomolecular breakdown and redistribution. while cell swelling is a classical finding in oncosis, it may not occur, or shrinkage can occur instead, depending upon the cell and surrounding milieu. cell volume can also evolve over the course of the pmi. processes surrounding cells also tend to swell, which is an early event in the pmi. finally, cell membranes can form blebs as well. early necrosis is initiated by focal disruptions of the cell membrane, leading to leakage of intracellular contents. the remaining cell membranes tend to become indistinct, although still visible. processes around cells can become extremely swollen, in some cases larger than many cells themselves. note that while these swollen cell processes are usually astrocyte processes, other studies also describe swelling of dendrites, axons, and oligodendrocyte processes (rees, 1977; gibson and tomlinson, 1979). as a result, we describe these agnostically as cell processes. early necrosis is followed by late necrosis, which is characterized by further disruption of gel-like networks leading to the loss of the original cell shape. the ultimate speed of dissolution of cell structures likely depends upon the initial strength of the gel-like networks. for example, the strong compaction of myelin may help to explain why this structure tends to be highly stable in the postmortem period. the structures that last the longest are also the ones that are the most slowly catabolized in the postmortem period. the ultimate outcome of late necrosis is cellular liquefaction and coming to an equilibrium with the environment. an implication of this model is that the transition which leads to loss of cell morphometry information – making the postmortem tissue no longer useful for studying cell morphometric alterations in neurobiological disorders – is most likely the transition from early necrotic to late necrotic, when the gel-like networks maintaining cell shapes are degraded. the transitions between these stages are not discrete but continuous, which may be a useful model for investigators studying postmortem brain tissue. we also present the histologic findings expected regarding cell morphometry at each of the stages (table 5). stage of decomposition cell shape cell size cell membrane findings common artifacts antemortem intact no change not applicable not applicable oncotic largely intact mild to moderate swelling or shrinkage is likely may have blebbing or compression pericellular and perivascular rarefactions; vacuolization early necrotic largely intact some cells, especially astrocytes, are excessively enlarged, while others begin to shrink cell membranes may be indistinct, blurry, or partially damaged above artifacts, but more widespread and severe; intracellular contents may be seen in extracellular space late necrotic altered cells shrink as they dissolve cells are dissolved washed out tissue areas devoid of cells table 5. typical cell morphometric findings at different stages of postmortem decomposition. implications for human brain mapping to map the brain across large areas of brain tissue, surgically extracted tissue will not be sufficient. this draws attention to the inevitable pmi in autopsy brain samples which are of particular interest for designing brain mapping studies. one relevant lesson is that postmortem brain mapping will have higher fidelity to in vivo states for some structural features compared to others. cell volumes and feature morphometry such as synapse size are likely to be altered rapidly in the pmi. cell membrane topography can also be altered relatively early due to the heterogeneous formation of vacuoles, blisters, and compression. on the other hand, cell membrane topological relationships are likely to be maintained across longer pmis. as a result, it may be possible to map connectivity and circuitry, as well as circuit properties such as the rough degree of axon myelination, even after a relatively longer degree of postmortem decomposition. however, there is considerable uncertainty about how broadly this applies across brain regions and cell types. it is unknown what degree of postmortem decomposition can be tolerated before topological cell membrane relationships are also lost, requiring further research. another lesson for postmortem brain mapping is that, compared with animal brains preserved at the time of death, some visualization procedures will be substantially more robust than others. as previously discussed, ihc studies can be sensitive to the pmi, because certain antigens can be lost relatively early in the pmi. if ihc is to be used in postmortem tissue, then labeling antigens that are robust to postmortem decomposition is essential. based on the studies we reviewed, synaptophysin (liu and brun, 1995; sarnat et al., 2010), gfap (blair et al., 2016), and neurofilaments (lavenex et al., 2009; blair et al., 2016) are relatively stable in the postmortem period in visualizing cell morphometry, although this certainly warrants further investigation. some morphological stains have also been shown to be resistant to postmortem decomposition, although there remain questions of how widely they can be used and the degree to which their neuronal mapping is unbiased. for example, golgi-cox staining only visualizes a small subset of neurons, although its resistance to postmortem decomposition makes it a valuable staining method. while some antigens are sensitive to postmortem decomposition, others may be particularly stable in ways that could prove valuable. for example, ischemic fluid shifts such as severe dendrite swelling have been found to be partially reversible if there is a physiological correction within 20-60 minutes, which has been attributed to the resilience of the cytoskeleton (zhang et al., 2005). this suggests that staining and mapping cytoskeletal biomolecules may be a way to infer cellular morphometry even in the setting of volume changes that might affect morphological stains. studies using electron microscopy to map the postmortem brain require particular attention to sample preparation. for example, some studies have found that synapses decrease in number during extended pmis, although this is certainly not a universal finding (huttenlocher, 1979; petit and leboutillier, 1990; roberts et al., 1996). as discussed, several lines of evidence suggest the loss of synapse visualization may be due to compression or loss of synaptic markers, rather than frank synaptic fragmentation or dissolution (gibson and tomlinson, 1979; petit and leboutillier, 1990). as a result, the identification of synapses would likely be less affected by postmortem decomposition if they were visualized with a robust staining procedure and/or volumetric imaging. multiple authors reported optimizing the standard electron microscopy procedures for using on postmortem human brains (sele et al., 2019; tang et al., 2001). in addition to optimizing tissue processing, data analysis methods can also aid in reconstructing the original state of structures affected by postmortem artifacts. in reconstructive brain mapping, common artifacts could be computationally reversed – with some degree of fidelity to the original state – via deterministic or stochastic algorithms. for example, if myelin lamellae have split, it may be possible to infer the shape in which they were originally compacted. as an early example in this field, one study performed a 3d reconstruction of brain tissue based on em images while using a set of models to correct the extracellular space volume fraction for shrinkage attributed to hypoxia, fixation, and dehydration (kinney et al., 2013). it may also be possible to map the decomposed biomolecules that originally comprised a structural feature if the structure itself has fragmented. this could never be done perfectly but could potentially infer the original structure to a sufficient degree of accuracy in many cases. however, because of the ubiquity of non-gaussian diffusion, computational reversal of postmortem biomolecule redistribution is likely a very challenging problem. it would require mapping multiple biomolecules and triangulating their predicted diffusion patterns, while accounting for fluid shifts, biomolecular degradation, and other factors. decomposed biomolecules that have diffused about and partially degraded may be among the last remnants of an in vivo structure before the information about it is totally lost, thereby defining classical physical limits to the potential for reconstructive brain mapping. further studies of postmortem changes a time series study in which the pmi is experimentally manipulated yields the most precise measurements of postmortem changes. however, when time series studies are used as a proxy for what happens in postmortem human brains, they often lack ecological validity. for example, studies on isolated surgical biopsy tissue introduce a dramatically different metabolic and biophysical state than that of the postmortem brain, which may limit the validity of these studies in predicting how decomposition occurs in an intact human brain postmortem. to address this, time series studies could measure and/or manipulate modifying variables, such as the location (in situ or ex situ), temperature, metabolic state, and tissue humidity. it would also be beneficial to perform time series studies that can help to account for variability across the existing studies, as reviewed here. one of the least widely studied areas is variability across brain regions. as we approach the ability to achieve brain-wide cellular mapping in the coming decades, there is a clear need to know whether some brain regions are more susceptible to postmortem decomposition than others, but this knowledge is currently limited. regarding correlational studies, scaling the sample size is likely to be helpful. larger studies will be helpful to account for measured and unmeasured confounders, in addition to including these variables as covariates where available. with larger sample sizes also comes the possibility of unsupervised machine learning studies. most of our existing knowledge of postmortem changes is based on subjective semi-quantitative scoring, but this is biased towards pre-existing knowledge. unsupervised machine learning studies, as have recently begun to be performed in studying the neurobiology of disease, may also help to uncover biomarkers of postmortem decomposition (mckenzie et al., 2022). developing an objective measure of histologic decomposition, analogous to the rna integrity number (rin) for rna preservation quality, would be valuable as a quality metric in brain banking. the status quo involves investigators judgments subject to reporting bias. for example, the same cell could look “distorted” to one person but “largely preserved” to another, partially reflecting their prior expectations. this may be one reason for the heterogeneity in the decomposition outcomes we observed between studies. more quantitative histologic metrics, for example using morphology comparison algorithms, would help to mitigate reporting biases and make comparisons across studies more precise (costa et al., 2016). among the most reliable histologic findings available today to measure the extent of postmortem decomposition are perivascular rarefaction, pericellular rarefaction, and vacuolization on morphologically stained tissue, such as h&e. several studies describe these as common postmortem changes that become more prominent with increasing pmi (de groot et al., 1995; hilbig et al., 2004; shepherd et al., 2009; monroy-gómez et al., 2020). however, the appearance and enlargement of these postmortem artifacts appears to have a non-linear relationship with pmi. moreover, the kinetics of their appearance and the variables modifying them are poorly mapped out. therefore, there is a need for additional research and validation prior to the routine use of these findings as measures of postmortem decomposition. comparison to other reviews to our knowledge, there has not been a previous comprehensive review focused solely on the topic of brain cell morphology changes in the pmi, although several publications have touched on it as a part of a broader discussion. oehmichen reviewed the extant literature on postmortem alterations in the histochemistry of cns tissue (oehmichen, 1980). they concluded that cell structures seen under the light microscope were effectively unchanged up to 6-8 h pmi and that after this, autolytic processes commence. lewis, in a broader review of human brain research, included a discussion of pmi (lewis, 2002). they noted that any pmi threshold for inclusion in a study, such as less than 24 h, would be arbitrary, and that tissue quality is highly dependent on antemortem factors. they also pointed out that postmortem effects can vary based on the feature measured and the brain region. finally, this review noted that it can be difficult to distinguish differences between brains due to disease as opposed to postmortem artifact. ravid and colleagues noted that the distribution of receptors tends to be stable in the postmortem period (ravid et al., 1992). they also discussed the importance of developing profiling techniques for longer pmis. wohlsein and colleagues described common postmortem artifacts in animal brains, such as vacuolization, shrinkage of glial cells, hemolysis, and putrefactive decomposition in advanced stages (wohlsein et al., 2013). hostiuc and colleagues performed a systematic review on ultrastructural changes as a marker of the pmi in various organs; however, they used different search and selection criteria than our review and identified just two studies from the brain (hostiuc et al., 2018). a forensic neuropathology textbook by oehmichen and colleagues discusses histologic changes in the pmi (oehmichen et al., 2006, p. 74). in their assessment, neuronal swelling is the most fundamental postmortem change, causing cells to lose their contours and become spherical. they note that oligodendrocytes and astrocytes can also have postmortem swelling, but that these changes are slight compared to neuronal changes and less specific. ramirez and colleagues reported that there is no correlation between the pmi and tissue quality in brain banking samples (ramirez et al., 2018). they note that many investigators are rigid about the pmi, despite this number not accurately reflecting the degree of metabolic changes that have occurred in the preand post-mortem tissue. there have also been reviews on cell morphology changes in brain death, which results from the same underlying process as in the pmi, namely a global lack of blood flow to the brain (oehmichen, 1994; folkerth et al., 2022). notably, the kinetics of decomposition are faster in brain death because a relatively higher body temperature is maintained. strengths and limitations of this review a strength of this review is that, to the best of our knowledge, this is the largest collection of studies yet assembled on this topic. another strength is that a semi-quantitative grading system was developed and implemented on the time series studies. finally, we have attempted to integrate the literature from studies using both light and electron microscopy to share insights between these two interrelated fields. in addition, this review has several limitations. first, the decomposition grades in the time series studies may give a false sense of precision. instead, the purpose of these grades is to aid in conceptualization and visualization. second, we largely did not consider disease state among the correlational studies which may have contributed to variations in the effects of pmi. finally, there is no doubt that we missed a substantial set of the literature. this includes the non-english literature, especially the relatively large german and russian language literatures. also, we did not systematically evaluate references or citing articles of all included articles, which further limited the literature identified. nevertheless, we expect that the studies included are a representative sample. conclusions a reductionist focus on the numerical pmi, while convenient, does not account for variability based on the visualization method used, feature analyzed, region or cell type, and variables that modify the decomposition speed. indeed, there is no obvious single pmi threshold at which cell morphometry is clearly lost. instead, research is likely to be more fruitful in understanding how the actual amount of decomposition in each brain can be measured, which structural features are likely to be altered sooner than others, and which visualization methods are most robust to postmortem changes. in biorepository contexts, it may make the most sense to cast a wider net on the inclusion of brain tissue, as opposed to having strict pmi inclusion requirements. on the other hand, it is essential to recognize that decomposition starts in the first minutes and is clearly occurring throughout the pmi. therefore, there is a need to minimize postmortem decomposition by expediting processing procedures to the extent possible. more research into knowledge gaps regarding the postmortem decomposition of brain cells will help to further elucidate this critical barrier to studying human neurobiology using donated brains. abbreviations atpase adenosine triphosphatase, csf cerebrospinal fluid, d day, em electron microscopy, er endoplasmic reticulum, gfap glial fibrillary acidic protein, gfralpha glial cell line-derived neurotrophic factor family receptor alpha, h hour, h&e hematoxylin and eosin, icc intraclass correlation, ihc immunohistochemistry, lfb luxol fast blue, map2 microtubule associated protein 2, min minute, nf-h neurofilament heavy chain, nf-l neurofilament light chain, nf-m neurofilament medium chain, neun neuronal nuclear protein, pmi postmortem interval, rameses realist and meta-narrative evidence syntheses evolving standards, rin rna integrity number, rrna ribosomal rna, ssdna single-stranded dna. author contributions m.k., k.f., j.c., and a.m. conceptualized the study. s.w. advised on the search strategy and performed the database searches. a.m. performed abstract and article screening. m.k. and a.m. performed data extraction and review from the individual studies. m.k., j.k., and a.m. performed grading of the studies. a.s. and k.c. performed electron microscopy studies. a.m. wrote the initial draft of the manuscript. all authors reviewed the manuscript and approved the final manuscript. acknowledgements we would like to thank rebecca folkerth, etty cortes, and thomas beach for helpful personal communications regarding this topic. the icahn school of medicine at mount sinai provided access to library resources. electron microscopy tissue preparation and imaging were performed at the microscopy and advanced bioimaging core at the icahn school of medicine at mount sinai. funding the study was supported by nih grants p30ag066514, rf1mh128969, r01ag054008, rf1ns095252, rf1ag060961, r01ns086736, r01ag062348, and the rainwater charitable foundation. the funders had no role in the design of the study or in the collection or interpretation of the data. data availability the generated database of studies studying postmortem changes in brain cell morphometry is freely available at https://github.com/andymckenzie/postmortem_interval. this repository also contains the code required to reproduce the figures created in r. supplementary files supplementary file 1. rameses checklist for the review (pdf) supplementary file 2. supplementary methods including database search methods (pdf) supplementary file 3. export of covidence files containing study screening results (zip file) supplementary file 4. table with information about the included studies (pdf) supplementary file 5. table with extracted data from the time series studies (pdf) supplementary file 6. table with extracted data from the correlational studies (pdf) supplementary file 7. table with extracted data from the case report studies (pdf) references abbas, f., becker, s., jones, b.w., mure, l.s., panda, s., hanneken, a., vinberg, f., 2022. revival of light signalling in the postmortem mouse and human retina. nature 606, 351–357. https://doi.org/10.1038/s41586-022-04709-x acs, g., paragh, g., rakosy, z., laronga, c., zhang, p.j., 2012. the extent of retraction clefts correlates with lymphatic vessel density and vegf-c expression and predicts nodal metastasis and poor prognosis in early-stage breast carcinoma. mod. pathol. off. j. u. s. can. acad. pathol. inc 25, 163–177. https://doi.org/10.1038/modpathol.2011.138 adrian, m., kusters, r., wierenga, c.j., storm, c., hoogenraad, c.c., kapitein, l.c., 2014. barriers in the brain: resolving dendritic spine morphology and compartmentalization. front. neuroanat. 8, 142. https://doi.org/10.3389/fnana.2014.00142 airaksinen, m.s., paetau, a., paljärvi, l., reinikainen, k., riekkinen, p., suomalainen, r., panula, p., 1991. histamine neurons in human hypothalamus: anatomy in normal and alzheimer diseased brains. neuroscience 44, 465–481. https://doi.org/10.1016/0306-4522(91)90070-5 albrechtsen, r., 1977a. naphthylamidase used as a lysosome marker in the study of acute selective necrosis of the internal granular layer of cerebellum. acta pathol. microbiol. scand. [a] 85, 875–888. https://doi.org/10.1111/j.1699-0463.1977.tb03904.x albrechtsen, r., 1977b. the incidence of the so-called acute selective necrosis of the granular layer of cerebellum in 1000 autopsied patients. acta pathol. microbiol. scand. [a] 85a, 193–202. https://doi.org/10.1111/j.1699-0463.1977.tb00417.x ananthakrishnan, r., guck, j., wottawah, f., schinkinger, s., lincoln, b., romeyke, m., moon, t., käs, j., 2006. quantifying the contribution of actin networks to the elastic strength of fibroblasts. j. theor. biol. 242, 502–516. https://doi.org/10.1016/j.jtbi.2006.03.021 anders, v.n., 1977. [human brain synapses during postmortem autolysis (electron-microscopic and electron-cytochemical studies)]. zhurnal nevropatol. psikhiatrii im. ss korsakova mosc. russ. 1952 77, 1080–1084. ansari, k.a., hendrickson, h., rand, a., 1976a. electrophoretic and morphologic studies on normal human white matter obtained at surgery with special reference to its basic protein component. j. neuropathol. exp. neurol. 35, 606–612. https://doi.org/10.1097/00005072-197611000-00002 ansari, k.a., hendrickson, h., sinha, a.a., rand, a., 1975. myelin basic protein in frozen and unfrozen bovine brain: a study of autolytic changes in situ. j. neurochem. 25, 193–195. https://doi.org/10.1111/j.1471-4159.1975.tb06952.x ansari, k.a., rand, a., hendrickson, h., bentley, m.d., 1976b. qualitative and quantitative studies on human myelin basic protein in situ with respect to time interval between death and autopsy. j. neuropathol. exp. neurol. 35, 180–90. https://doi.org/10.1097/00005072-197603000-00005 arsénio-nunes, m.l., hossmann, k.a., farkas-bargeton, e., 1973. ultrastructural and histochemical investigation of the cerebral cortex of cat during and after complete ischaemia. acta neuropathol. (berl.) 26, 329–344. https://doi.org/10.1007/bf00688080 averback, p., 1980. a study of the rate of cell depletion in solid tissue. j. pathol. 130, 173–178. https://doi.org/10.1002/path.1711300306 badonic, t., frumkina, l.j., jakovleva, n.i., hornáková, a., 1992. ultrastructural changes of neurons in dependence on the death cause in human brain. funct. dev. morphol. 2, 231–234. bailey, d.m., 2019. oxygen and brain death; back from the brink. exp. physiol. 104, 1769–1779. https://doi.org/10.1113/ep088005 beach, t.g., adler, c.h., sue, l.i., serrano, g., shill, h.a., walker, d.g., lue, l., roher, a.e., dugger, b.n., maarouf, c., birdsill, a.c., intorcia, a., saxon-labelle, m., pullen, j., scroggins, a., filon, j., scott, s., hoffman, b., garcia, a., caviness, j.n., hentz, j.g., driver-dunckley, e., jacobson, s.a., davis, k.j., belden, c.m., long, k.e., malek-ahmadi, m., powell, j.j., gale, l.d., nicholson, l.r., caselli, r.j., woodruff, b.k., rapscak, s.z., ahern, g.l., shi, j., burke, a.d., reiman, e.m., sabbagh, m.n., 2015. arizona study of aging and neurodegenerative disorders and brain and body donation program. neuropathology 35, 354–389. https://doi.org/10.1111/neup.12189 beach, t.g., sue, l.i., walker, d.g., roher, a.e., lue, l., vedders, l., connor, d.j., sabbagh, m.n., rogers, j., 2008. the sun health research institute brain donation program: description and eexperience, 1987–2007. cell tissue bank. 9, 229–245. https://doi.org/10.1007/s10561-008-9067-2 benes, f.m., todtenkopf, m.s., kostoulakos, p., 2001. glur5,6,7 subunit immunoreactivity on apical pyramidal cell dendrites in hippocampus of schizophrenics and manic depressives. hippocampus 11, 482–491. https://doi.org/10.1002/hipo.1065 berdenis van berlekom, a., muflihah, c.h., snijders, g.j.l.j., macgillavry, h.d., middeldorp, j., hol, e.m., kahn, r.s., de witte, l.d., 2020. synapse pathology in schizophrenia: a meta-analysis of postsynaptic elements in postmortem brain studies. schizophr. bull. 46, 374–386. https://doi.org/10.1093/schbul/sbz060 blair, j.a., wang, c., hernandez, d., siedlak, s.l., rodgers, m.s., achar, r.k., fahmy, l.m., torres, s.l., petersen, r.b., zhu, x., casadesus, g., lee, h.g., 2016. individual case analysis of postmortem interval time on brain tissue preservation. plos one 11, e0151615. https://doi.org/10.1371/journal.pone.0151615 boekhoorn, k., joels, m., lucassen, p.j., 2006. increased proliferation reflects glial and vascular-associated changes, but not neurogenesis in the presenile alzheimer hippocampus. neurobiol. dis. 24, 1–14. https://doi.org/10.1016/j.nbd.2006.04.017 booze, r.m., mactutus, c.f., gutman, c.r., davis, j.n., 1993. frequency analysis of catecholamine axonal morphology in human brain. i. effects of postmortem delay interval. j. neurol. sci. 119, 99–109. https://doi.org/10.1016/0022-510x(93)90197-7 boros, b.d., greathouse, k.m., gentry, e.g., curtis, k.a., birchall, e.l., gearing, m., herskowitz, j.h., 2017. dendritic spines provide cognitive resilience against alzheimer’s disease. ann. neurol. 82, 602–614. https://doi.org/10.1002/ana.25049 bruce, a., dawson, j.w., 1911. on the relations of the lymphatics of the spinal cord. j. pathol. bacteriol. 15, 169–178. brück, y., brück, w., kretzschmar, h.a., lassmann, h., 1996. evidence for neuronal apoptosis in pontosubicular neuron necrosis. neuropathol. appl. neurobiol. 22. https://doi.org/10.1111/j.1365-2990.1996.tb00842.x budday, s., nay, r., de rooij, r., steinmann, p., wyrobek, t., ovaert, t.c., kuhl, e., 2015. mechanical properties of gray and white matter brain tissue by indentation. j. mech. behav. biomed. mater. 46, 318–330. https://doi.org/10.1016/j.jmbbm.2015.02.024 buell, s.j., 1982. golgi-cox and rapid golgi methods as applied to autopsied human brain tissue: widely disparate results. j. neuropathol. exp. neurol. 41, 500–507. https://doi.org/10.1097/00005072-198209000-00003 buja, l.m., barth, r.f., krueger, g.r., brodsky, s.v., hunter, r.l., 2019. the importance of the autopsy in medicine: perspectives of pathology colleagues. acad. pathol. 6, 2374289519834041. https://doi.org/10.1177/2374289519834041 bywater, j.e., glees, p., hauffe, h., 1962. the variability of neuron structure caused by the type of fixation or by the autolytic changes. ii. silver method. j. hirnforsch. 5, 147–161. https://doi.org/10.1515/9783112519806-003 cammermeyer, j., 1979. argentophil neuronal perikarya and neurofibrils induced by postmortem trauma and hypertonic perfusates. acta anat. (basel) 105, 9–24. https://doi.org/10.1159/000145102 cammermeyer, j., 1978. is the solitary dark neuron a manifestation of postmortem trauma to the brain inadequately fixed by perfusion? histochemistry 56, 97–115. https://doi.org/10.1007/bf00508437 cammermeyer, j., 1960. the post-mortem origin and mechanism of neuronal hyperchromatosis and nuclear pyknosis. exp. neurol. 2, 379–405. https://doi.org/10.1016/0014-4886(60)90022-4 chan, j.k.c., 2014. the wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology. int. j. surg. pathol. 22, 12–32. https://doi.org/10.1177/1066896913517939 charpak, s., audinat, e., 1998. cardiac arrest in rodents: maximal duration compatible with a recovery of neuronal activity. proc. natl. acad. sci. u. s. a. 95, 4748–4753. https://doi.org/10.1073/pnas.95.8.4748 choe, b.y., gil, h.j., suh, t.s., shinn, k.s., 1995. postmortem metabolic and morphologic alterations of the dog brain thalamus with use of in vivo 1h magnetic resonance spectroscopy and electron microscopy. invest. radiol. 30, 269–274. https://doi.org/10.1097/00004424-199505000-00001 chugh, p., paluch, e.k., 2018. the actin cortex at a glance. j. cell sci. 131, jcs186254. https://doi.org/10.1242/jcs.186254 cockle, d.l., bell, l.s., 2015. human decomposition and the reliability of a “universal” model for post mortem interval estimations. forensic sci. int. 253, 136.e1–9. https://doi.org/10.1016/j.forsciint.2015.05.018 coleman, m., 2005. axon degeneration mechanisms: commonality amid diversity. nat. rev. neurosci. 6, 889–898. https://doi.org/10.1038/nrn1788 compaine, a., schein, j.d., tabb, j.s., mohan, p.s., nixon, r.a., 1995. limited proteolytic processing of the mature form of cathepsin d in human and mouse brain: postmortem stability of enzyme structure and activity. neurochem. int. 27, 385–396. https://doi.org/10.1016/0197-0186(95)00020-9 costa, m., manton, j.d., ostrovsky, a.d., prohaska, s., jefferis, g.s.x.e., 2016. nblast: rapid, sensitive comparison of neuronal structure and construction of neuron family databases. neuron 91, 293–311. https://doi.org/10.1016/j.neuron.2016.06.012 dachet, f., brown, j.b., valyi-nagy, t., narayan, k.d., serafini, a., boley, n., gingeras, t.r., celniker, s.e., mohapatra, g., loeb, j.a., 2021. selective time-dependent changes in activity and cell-specific gene expression in human postmortem brain. sci. rep. 11, 6078. https://doi.org/10.1038/s41598-021-85801-6 d’andrea, m.r., howanski, r.j., saller, c.f., 2017. map2 ihc detection: a marker of antigenicity in cns tissues. biotech. histochem. off. publ. biol. stain comm. 92, 363–373. https://doi.org/10.1080/10520295.2017.1295169 d’arcy, m.s., 2019. cell death: a review of the major forms of apoptosis, necrosis and autophagy. cell biol. int. 43, 582–592. https://doi.org/10.1002/cbin.11137 das, s.c., chen, d., callor, w.b., christensen, e., coon, h., williams, m.e., 2019. dii-mediated analysis of presynaptic and postsynaptic structures in human postmortem brain tissue. j. comp. neurol. 527, 3087–3098. https://doi.org/10.1002/cne.24722 de groot, c.j., theeuwes, j.w., dijkstra, c.d., van der valk, p., 1995. postmortem delay effects on neuroglial cells and brain macrophages from lewis rats with acute experimental allergic encephalomyelitis: an immunohistochemical and cytochemical study. j. neuroimmunol. 59, 123–134. https://doi.org/10.1016/0165-5728(95)00034-y de la torre, j.c., fortin, t., saunders, j.k., butler, k., richard, m.t., 1992. the no-reflow phenomenon is a post-mortem artifact. acta neurochir. (wien) 115, 37–42. https://doi.org/10.1007/bf01400588 de ruiter, j.p., uylings, h.b., 1987. morphometric and dendritic analysis of fascia dentata granule cells in human aging and senile dementia. brain res. 402, 217–229. https://doi.org/10.1016/0006-8993(87)90028-x de wolf, a., phaedra, c., perry, r.m., maire, m., 2020. ultrastructural characterization of prolonged normothermic and cold cerebral ischemia in the adult rat. rejuvenation res. 23, 193–206. https://doi.org/10.1089/rej.2019.2225 dehghani, a., karatas, h., can, a., erdemli, e., yemisci, m., eren-kocak, e., dalkara, t., 2018. nuclear expansion and pore opening are instant signs of neuronal hypoxia and can identify poorly fixed brains. sci. rep. 8, 14770. https://doi.org/10.1038/s41598-018-32878-1 del bigio, m.r., deck, j.h., davidson, g.s., 2000. glial swelling with eosinophilia in human post-mortem brains: a change indicative of plasma extravasation. acta neuropathol. (berl.) 100, 688–694. https://doi.org/10.1007/s004010000236 demetrius, l., 2005. of mice and men. when it comes to studying ageing and the means to slow it down, mice are not just small humans. embo rep. 6 spec no, s39-44. https://doi.org/10.1038/sj.embor.7400422 douglas, j.f., 2018. weak and strong gels and the emergence of the amorphous solid state. gels basel switz. 4, e19. https://doi.org/10.3390/gels4010019 dreier, j.p., victorov, i.v., petzold, g.c., major, s., windmüller, o., fernández-klett, f., kandasamy, m., dirnagl, u., priller, j., 2013. electrochemical failure of the brain cortex is more deleterious when it is accompanied by low perfusion. stroke 44, 490–496. https://doi.org/10.1161/strokeaha.112.660589 eberhardt, f., bushong, e.a., phan, s., peltier, s., monteagudo, p., weinkauf, t., herz, a.v.m., stemmler, m., ellisman, m., 2022. a uniform and isotropic cytoskeletal tiling fills dendritic spines. eneuro eneuro.0342-22.2022. https://doi.org/10.1523/eneuro.0342-22.2022 eggan, s.m., lewis, d.a., 2007. immunocytochemical distribution of the cannabinoid cb1 receptor in the primate neocortex: a regional and laminar analysis. cereb. cortex 17. https://doi.org/immunocytochemical distribution of the cannabinoid cb1 receptor in the primate neocortex: a regional and laminar analysis faitg, j., lacefield, c., davey, t., white, k., laws, r., kosmidis, s., reeve, a.k., kandel, e.r., vincent, a.e., picard, m., 2021. 3d neuronal mitochondrial morphology in axons, dendrites, and somata of the aging mouse hippocampus. cell rep. 36, 109509. https://doi.org/10.1016/j.celrep.2021.109509 ferrer, i., santpere, g., arzberger, t., bell, j., blanco, r., boluda, s., budka, h., carmona, m., giaccone, g., krebs, b., limido, l., parchi, p., puig, b., strammiello, r., ströbel, t., kretzschmar, h., 2007. brain protein preservation largely depends on the postmortem storage temperature: implications for study of proteins in human neurologic diseases and management of brain banks: a brainnet europe study. j. neuropathol. exp. neurol. 66, 35–46. https://doi.org/10.1097/nen.0b013e31802c3e7d fink, s.l., cookson, b.t., 2005. apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. infect. immun. 73, 1907–1916. https://doi.org/10.1128/iai.73.4.1907-1916.2005 finnie, j.w., blumbergs, p.c., manavis, j., 2016. temporal sequence of autolysis in the cerebellar cortex of the mouse. j. comp. pathol. 154, 323–328. https://doi.org/10.1016/j.jcpa.2016.03.005 finnie, j.w., jerrett, i.v., manavis, j., 2022. red neurons in ovine polioencephalomalacia (cerebrocortical necrosis) are strongly amyloid precursor protein immunopositive. vet. res. commun. 46, 289–293. https://doi.org/10.1007/s11259-022-09888-6 fix, a.s., garman, r.h., 2000. practical aspects of neuropathology: a technical guide for working with the nervous system. toxicol. pathol. 28, 122–131. https://doi.org/10.1177/019262330002800115 fodor, m., van leeuwen, f.w., swaab, d.f., 2002. differences in postmortem stability of sex steroid receptor immunoreactivity in rat brain. j. histochem. cytochem. off. j. histochem. soc. 50, 641–650. https://doi.org/10.1177/002215540205000505 folkerth, r.d., crary, j.f., shewmon, d.a., 2022. neuropathologic findings in a young woman 4 years following declaration of brain death: case analysis and literature review. j. neuropathol. exp. neurol. nlac090. https://doi.org/10.1093/jnen/nlac090 fountoulakis, m., hardmeier, r., höger, h., lubec, g., 2001. postmortem changes in the level of brain proteins. exp. neurol. 167, 86–94. https://doi.org/10.1006/exnr.2000.7529 fricker, m., tolkovsky, a.m., borutaite, v., coleman, m., brown, g.c., 2018. neuronal cell death. physiol. rev. 98, 813–880. https://doi.org/10.1152/physrev.00011.2017 furukawa, s., nishi, k., morita, s., hitosugi, m., wingenfeld, l., 2015. the damaging in the granular cells and expression patterns of cirbp, rbm3, hsp70, hif-1, aif1, sirt1, ngb, cfos, p53 and ccc9 in the postmortem human cerebellums obtained from individuals who died due to hanging, strangulation, drowning, or asphyxia by anaphylaxis or food aspiration may be closely linking with agonal duration. anil aggrawals internet j. forensic med. toxicol. 16. galluzzi, l., vitale, i., aaronson, s.a., abrams, j.m., adam, d., agostinis, p., alnemri, e.s., altucci, l., amelio, i., andrews, d.w., annicchiarico-petruzzelli, m., antonov, a.v., arama, e., baehrecke, e.h., barlev, n.a., bazan, n.g., bernassola, f., bertrand, m.j.m., bianchi, k., blagosklonny, m.v., blomgren, k., borner, c., boya, p., brenner, c., campanella, m., candi, e., carmona-gutierrez, d., cecconi, f., chan, f.k.-m., chandel, n.s., cheng, e.h., chipuk, j.e., cidlowski, j.a., ciechanover, a., cohen, g.m., conrad, m., cubillos-ruiz, j.r., czabotar, p.e., d’angiolella, v., dawson, t.m., dawson, v.l., de laurenzi, v., de maria, r., debatin, k.-m., deberardinis, r.j., deshmukh, m., di daniele, n., di virgilio, f., dixit, v.m., dixon, s.j., duckett, c.s., dynlacht, b.d., el-deiry, w.s., elrod, j.w., fimia, g.m., fulda, s., garcía-sáez, a.j., garg, a.d., garrido, c., gavathiotis, e., golstein, p., gottlieb, e., green, d.r., greene, l.a., gronemeyer, h., gross, a., hajnoczky, g., hardwick, j.m., harris, i.s., hengartner, m.o., hetz, c., ichijo, h., jäättelä, m., joseph, b., jost, p.j., juin, p.p., kaiser, w.j., karin, m., kaufmann, t., kepp, o., kimchi, a., kitsis, r.n., klionsky, d.j., knight, r.a., kumar, s., lee, s.w., lemasters, j.j., levine, b., linkermann, a., lipton, s.a., lockshin, r.a., lópez-otín, c., lowe, s.w., luedde, t., lugli, e., macfarlane, m., madeo, f., malewicz, m., malorni, w., manic, g., marine, j.-c., martin, s.j., martinou, j.-c., medema, j.p., mehlen, p., meier, p., melino, s., miao, e.a., molkentin, j.d., moll, u.m., muñoz-pinedo, c., nagata, s., nuñez, g., oberst, a., oren, m., overholtzer, m., pagano, m., panaretakis, t., pasparakis, m., penninger, j.m., pereira, d.m., pervaiz, s., peter, m.e., piacentini, m., pinton, p., prehn, j.h.m., puthalakath, h., rabinovich, g.a., rehm, m., rizzuto, r., rodrigues, c.m.p., rubinsztein, d.c., rudel, t., ryan, k.m., sayan, e., scorrano, l., shao, f., shi, y., silke, j., simon, h.-u., sistigu, a., stockwell, b.r., strasser, a., szabadkai, g., tait, s.w.g., tang, d., tavernarakis, n., thorburn, a., tsujimoto, y., turk, b., vanden berghe, t., vandenabeele, p., vander heiden, m.g., villunger, a., virgin, h.w., vousden, k.h., vucic, d., wagner, e.f., walczak, h., wallach, d., wang, y., wells, j.a., wood, w., yuan, j., zakeri, z., zhivotovsky, b., zitvogel, l., melino, g., kroemer, g., 2018. molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. cell death differ. 25, 486–541. https://doi.org/10.1038/s41418-017-0012-4 garcia, j.h., liu, k.f., ho, k.l., 1995. neuronal necrosis after middle cerebral artery occlusion in wistar rats progresses at different time intervals in the caudoputamen and the cortex. stroke 26, 636–642; discussion 643. https://doi.org/10.1161/01.str.26.4.636 garcia, j.h., liu, k.f., yoshida, y., chen, s., lian, j., 1994a. brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (wistar rat). am. j. pathol. 145, 728–740. garcia, j.h., liu, k.f., yoshida, y., lian, j., chen, s., del zoppo, g.j., 1994b. influx of leukocytes and platelets in an evolving brain infarct (wistar rat). am. j. pathol. 144, 188–199. garcia, j.h., lossinsky, a.s., kauffman, f.c., conger, k.a., 1978. neuronal ischemic injury: light microscopy, ultrastructure and biochemistry. acta neuropathol. (berl.) 43, 85–95. https://doi.org/10.1007/bf00685002 garey, l.j., ong, w.y., patel, t.s., kanani, m., davis, a., mortimer, a.m., barnes, t.r., hirsch, s.r., 1998. reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia. j. neurol. neurosurg. psychiatry 65, 446–453. https://doi.org/10.1136/jnnp.65.4.446 garman, r.h., 2011. common histological artifacts in nervous system tissues, in: fundamental neuropathology for pathologists and toxicologists. john wiley & sons, ltd, pp. 191–201. https://doi.org/10.1002/9780470939956.ch13 garman, r.h., 1990. artifacts in routinely immersion fixed nervous tissue. toxicol. pathol. 18, 149–153. https://doi.org/10.1177/019262339001800120 gärtner, u., janke, c., holzer, m., vanmechelen, e., arendt, t., 1998. postmortem changes in the phosphorylation state of tau-protein in the rat brain. neurobiol. aging 19, 535–543. https://doi.org/10.1016/s0197-4580(98)00094-3 geddes, j.w., bondada, v., tekirian, t.l., pang, z., siman, r.g., 1995. perikaryal accumulation and proteolysis of neurofilament proteins in the post-mortem rat brain. neurobiol. aging 16, 651–660. https://doi.org/10.1016/0197-4580(95)00062-j geiger, k.d., stoldt, p., schlote, w., derouiche, a., 2006. ezrin immunoreactivity reveals specific astrocyte activation in cerebral hiv. j. neuropathol. exp. neurol. 65, 87–96. https://doi.org/10.1097/01.jnen.0000195943.32786.39 gelpi, e., preusser, m., bauer, g., budka, h., 2007. autopsy at 2 months after death: brain is satisfactorily preserved for neuropathology. forensic sci. int. 168, 177–182. https://doi.org/10.1016/j.forsciint.2006.07.017 george, j., van wettere, a.j., michaels, b.b., crain, d., lewbart, g.a., 2016. histopathologic evaluation of postmortem autolytic changes in bluegill (lepomis macrohirus) and crappie (pomoxis anularis) at varied time intervals and storage temperatures. peerj 4, e1943. https://doi.org/10.7717/peerj.1943 gibson, p.h., tomlinson, b.e., 1979. vacuolation in the human cerebral cortex and its relationship to the interval between death and autopsy and to synapse numbers: an electron microscopic study. neuropathol. appl. neurobiol. 5, 1–7. https://doi.org/10.1111/j.1365-2990.1979.tb00608.x glausier, j.r., konanur, a., lewis, d.a., 2019. factors affecting ultrastructural quality in the prefrontal cortex of the postmortem human brain. j. histochem. cytochem. off. j. histochem. soc. 67, 185–202. https://doi.org/10.1369/0022155418819481 gonzalez-riano, c., tapia-gonzález, s., garcía, a., muñoz, a., defelipe, j., barbas, c., 2017. metabolomics and neuroanatomical evaluation of post-mortem changes in the hippocampus. brain struct. funct. 222, 2831–2853. https://doi.org/10.1007/s00429-017-1375-5 haider, l., hametner, s., endmayr, v., mangesius, s., eppensteiner, a., frischer, j.m., iglesias, j.e., barkhof, f., kasprian, g., 2022. post-mortem correlates of virchow-robin spaces detected on in vivo mri. j. cereb. blood flow metab. off. j. int. soc. cereb. blood flow metab. 42, 1224–1235. https://doi.org/10.1177/0271678x211067455 haines, d.e., jenkins, t.w., 1968. studies on the epithalamus. i. morphology of post-mortem degeneration: the habenular nucleus in dog. j. comp. neurol. 132, 405–417. https://doi.org/10.1002/cne.901320304 halim, n.d., weickert, c.s., mcclintock, b.w., hyde, t.m., weinberger, d.r., kleinman, j.e., lipska, b.k., 2003. presynaptic proteins in the prefrontal cortex of patients with schizophrenia and rats with abnormal prefrontal development. mol. psychiatry 8, 797–810. https://doi.org/10.1038/sj.mp.4001319 hansma, p., powers, s., diaz, f., li, w., 2015. agonal thrombi at autopsy. am. j. forensic med. pathol. 36, 141–144. https://doi.org/10.1097/paf.0000000000000162 harada, k., sorimachi, y., yoshida, k., 1997. proteolysis of ankyrin and na+/k(+)-atpase in postmortem rat brain: is calpain involved? forensic sci. int. 86, 77–85. https://doi.org/10.1016/s0379-0738(97)02120-8 hardy, j.a., wester, p., winblad, b., gezelius, c., bring, g., eriksson, a., 1985. the patients dying after long terminal phase have acidotic brains; implications for biochemical measurements on autopsy tissue. j. neural transm. 61, 253–264. https://doi.org/10.1007/bf01251916 harris, k.m., spacek, j., 2016. dendrite structure, in: stuart, g., spruston, n., häusser, m. (eds.), dendrites. oxford university press, p. 0. https://doi.org/10.1093/acprof:oso/9780198745273.003.0001 hau, t.c., hamzah, n.h., lian, h.h., hamzah, s., 2014. decomposition process and post mortem changes. sains malays. 43, 1873–1882. hausmann, r., seidl, s., betz, p., 2007. hypoxic changes in purkinje cells of the human cerebellum. int. j. legal med. 121, 175–183. https://doi.org/10.1007/s00414-006-0122-x hayes, t.l., cameron, j.l., fernstrom, j.d., lewis, d.a., 1991. a comparative analysis of the distribution of prosomatostatin-derived peptides in human and monkey neocortex. j. comp. neurol. 303, 584–599. https://doi.org/10.1002/cne.903030406 hayman, j., oxenham, m., 2017. estimation of the time since death in decomposed bodies found in australian conditions. aust. j. forensic sci. 49, 31–44. https://doi.org/10.1080/00450618.2015.1128972 henics, t., wheatley, d.n., 1999. cytoplasmic vacuolation, adaptation and cell death: a view on new perspectives and features. biol. cell 91, 485–498. https://doi.org/10.1016/s0248-4900(00)88205-2 henstridge, c.m., jackson, r.j., kim, j.m., herrmann, a.g., wright, a.k., harris, s.e., bastin, m.e., starr, j.m., wardlaw, j., gillingwater, t.h., smith, c., mckenzie, c.-a., cox, s.r., deary, i.j., spires-jones, t.l., 2015. post-mortem brain analyses of the lothian birth cohort 1936: extending lifetime cognitive and brain phenotyping to the level of the synapse. acta neuropathol. commun. 3, 53. https://doi.org/10.1186/s40478-015-0232-0 hetzel, w., 1980. post-mortem modifications of the ependyma of the lateral ventricular wall. acta neuropathol. (berl.) 51, 15–22. https://doi.org/10.1007/bf00688845 hilbig, h., bidmon, h.-j., oppermann, o.t., remmerbach, t., 2004. influence of post-mortem delay and storage temperature on the immunohistochemical detection of antigens in the cns of mice. exp. toxicol. pathol. off. j. ges. toxikol. pathol. 56, 159–171. https://doi.org/10.1016/j.etp.2004.08.002 hostiuc, s., rusu, m.c., mănoiu, v.s., vrapciu, a.d., negoi, i., popescu, m.v., 2018. usefulness of ultrastructure studies for the estimation of the postmortem interval. a systematic review. romanian j. morphol. embryol. rev. roum. morphol. embryol. hua, y., laserstein, p., helmstaedter, m., 2015. large-volume en-bloc staining for electron microscopy-based connectomics. nat. commun. 6, 7923. https://doi.org/10.1038/ncomms8923 hukkanen, v., röyttä, m., 1987. autolytic changes of human white matter: an electron microscopic and electrophoretic study. exp. mol. pathol. 46, 31–9. https://doi.org/10.1016/0014-4800(87)90028-1 hunziker o., schweizer a., 1977. postmortem changes in stereological parameters of cerebral capillaries. beitr pathol. 161, 244-255. https://doi.org/10.1016/s0005-8165(77)80080-2 huttenlocher, p.r., 1979. synaptic density in human frontal cortex developmental changes and effects of aging. brain res. 163, 195–205. https://doi.org/10.1016/0006-8993(79)90349-4 ikuta, f., hirano, a., zimmerman, h.m., 1963. an experimental study of post-mortem alterations in the granular layer of the cerebellar cortex. j. neuropathol. exp. neurol. 22, 581–593. https://doi.org/10.1097/00005072-196310000-00002 ilse, g., kovacs, k., ryan, n., horvath, e., ilse, d., 1979. autolytic changes in the rat adenohypophysis. a histologic, immunocytologic and electron microscopic study. exp. pathol. (jena) 17, 185–95. https://doi.org/10.1016/s0014-4908(79)80011-3 irving, e.a., mcculloch, j., dewar, d., 1997. the effect of postmortem delay on the distribution of microtubule-associated proteins tau, map2, and map5 in the rat. mol. chem. neuropathol. 30, 253–271. https://doi.org/10.1007/bf02815102 ith, m., scheurer, e., kreis, r., thali, m., dirnhofer, r., boesch, c., 2011. estimation of the postmortem interval by means of 1h mrs of decomposing brain tissue: influence of ambient temperature. nmr biomed. 24, 791–798. https://doi.org/10.1002/nbm.1623 itoyama, y., sternberger, n.h., kies, m.w., cohen, s.r., richardson, e.p., webster, h., 1980. immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system. ann. neurol. 7, 157–166. https://doi.org/10.1002/ana.410070211 jacobs, b., scheibel, a.b., 1993. a quantitative dendritic analysis of wernicke’s area in humans. i. lifespan changes. j. comp. neurol. 327, 83–96. https://doi.org/10.1002/cne.903270107 jamal, a., mongelli, m.t., vidotto, m., madekurozwa, m., bernardini, a., overby, d.r., de momi, e., rodriguez y baena, f., sherwood, j.m., dini, d., 2021. infusion mechanisms in brain white matter and their dependence on microstructure: an experimental study of hydraulic permeability. ieee trans. biomed. eng. 68, 1229–1237. https://doi.org/10.1109/tbme.2020.3024117 jenkins, l.w., povlishock, j.t., becker, d.p., miller, j.d., sullivan, h.g., 1979. complete cerebral ischemia. an ultrastructural study. acta neuropathol. (berl.) 48, 113–125. https://doi.org/10.1007/bf00691152 jernerén, f., söderquist, m., karlsson, o., 2015. post-sampling release of free fatty acids effects of heat stabilization and methods of euthanasia. j. pharmacol. toxicol. methods 71, 13–20. https://doi.org/10.1016/j.vascn.2014.11.001 johnston, n.l., cervenak, j., shore, a.d., torrey, e.f., yolken, r.h., cerevnak, j., 1997. multivariate analysis of rna levels from postmortem human brains as measured by three different methods of rt-pcr. stanley neuropathology consortium. j. neurosci. methods 77, 83–92. https://doi.org/10.1016/s0165-0270(97)00115-5 kádár, a., wittmann, g., liposits, z., fekete, c., 2009. improved method for combination of immunocytochemistry and nissl staining. j. neurosci. methods 184, 115–118. https://doi.org/10.1016/j.jneumeth.2009.07.010 kang, h.w., kim, h.k., moon, b.h., lee, seo jun, lee, se jung, rhyu, i.j., 2017. comprehensive review of golgi staining methods for nervous tissue. appl. microsc. 47, 63–69. https://doi.org/10.9729/am.2017.47.2.63 karlsson, u., schultz, r.l., 1966. fixation of the central nervous system for electron microscopy by aldehyde perfusion: iii. structural changes after exsanguination and delayed perfusion. j. ultrastruct. res. 14, 47–63. https://doi.org/10.1016/s0022-5320(66)80034-5 kay, k.r., smith, c., wright, a.k., serrano-pozo, a., pooler, a.m., koffie, r., bastin, m.e., bak, t.h., abrahams, s., kopeikina, k.j., mcguone, d., frosch, m.p., gillingwater, t.h., hyman, b.t., spires-jones, t.l., 2013. studying synapses in human brain with array tomography and electron microscopy. nat. protoc. 8, 1366–1380. https://doi.org/10.1038/nprot.2013.078 kekenes-huskey, p., scott, c., atalay, s., 2016. quantifying the influence of the crowded cytoplasm on small molecule diffusion. j. phys. chem. b 120, 8696–8706. https://doi.org/10.1021/acs.jpcb.6b03887 kherani, z.s., auer, r.n., 2008. pharmacologic analysis of the mechanism of dark neuron production in cerebral cortex. acta neuropathol. (berl.) 116, 447–452. https://doi.org/10.1007/s00401-008-0386-y kies, m.w., schwimmer, s., 1942. observations on proteinase in brain. j. biol. chem. 145, 685–691. https://doi.org/10.1016/s0021-9258(18)51311-9 kinney, j.p., spacek, j., bartol, t.m., bajaj, c.l., harris, k.m., sejnowski, t.j., 2013. extracellular sheets and tunnels modulate glutamate diffusion in hippocampal neuropil. j. comp. neurol. 521, 448–464. https://doi.org/10.1002/cne.23181 kitamura, o., gotohda, t., ishigami, a., tokunaga, i., kubo, s., nakasono, i., 2005. effect of hypothermia on postmortem alterations in map2 immunostaining in the human hippocampus. leg. med. 7, 340–344. https://doi.org/10.1016/j.legalmed.2005.08.006 kloner, r.a., king, k.s., harrington, m.g., 2018. no-reflow phenomenon in the heart and brain. am. j. physiol. heart circ. physiol. 315, h550–h562. https://doi.org/10.1152/ajpheart.00183.2018 knudsen, l.m., pallesen, g., 1986. the preservation and loss of various non-haematopoietic antigens in human post-mortem tissues as demonstrated by monoclonal antibody immunohistological staining. histopathology 10, 1007–1014. https://doi.org/10.1111/j.1365-2559.1986.tb02537.x koenig, r.s., koenig, h., 1952. an experimental study of post mortem alterations in neurons of the central nervous system. j. neuropathol. exp. neurol. 11, 69–78. https://doi.org/10.1097/00005072-195201000-00008 kolomeets, n.s., orlovskaya, d.d., rachmanova, v.i., uranova, n.a., 2005. ultrastructural alterations in hippocampal mossy fiber synapses in schizophrenia: a postmortem morphometric study. synap. n. y. n 57. https://doi.org/10.1002/syn.20153 kolomeets, n.s., orlovskaya, d.d., uranova, n.a., 2007. decreased numerical density of ca3 hippocampal mossy fiber synapses in schizophrenia. synap. n. y. n 61, 615–621. https://doi.org/10.1002/syn.20405 koo, t.k., li, m.y., 2016. a guideline of selecting and reporting intraclass correlation coefficients for reliability research. j. chiropr. med. 15, 155–163. https://doi.org/10.1016/j.jcm.2016.02.012 kovács, b., bukovics, p., gallyas, f., 2007. morphological effects of transcardially perfused sds on the rat brain. biol. cell 99, 425–432. https://doi.org/10.1042/bc20060128 kramer, e.m., myers, d.r., 2013. osmosis is not driven by water dilution. trends plant sci. 18, 195–197. https://doi.org/10.1016/j.tplants.2012.12.001 kretzschmar, h., 2009. brain banking: opportunities, challenges and meaning for the future. nat. rev. neurosci. 10, 70–78. https://doi.org/10.1038/nrn2535 kulkarni, v.a., firestein, b.l., 2012. the dendritic tree and brain disorders. mol. cell. neurosci. 50, 10–20. https://doi.org/10.1016/j.mcn.2012.03.005 kuroiwa, t., nagaoka, t., ueki, m., yamada, i., miyasaka, n., akimoto, h., 1998. different apparent diffusion coefficient: water content correlations of gray and white matter during early ischemia. stroke 29, 859–865. https://doi.org/10.1161/01.str.29.4.859 kwee, r.m., kwee, t.c., 2007. virchow-robin spaces at mr imaging. radiogr. rev. publ. radiol. soc. n. am. inc 27, 1071–1086. https://doi.org/10.1148/rg.274065722 lafontaine, d.l.j., riback, j.a., bascetin, r., brangwynne, c.p., 2021. the nucleolus as a multiphase liquid condensate. nat. rev. mol. cell biol. 22, 165–182. https://doi.org/10.1038/s41580-020-0272-6 lafrenaye, a.d., simard, j.m., 2019. bursting at the seams: molecular mechanisms mediating astrocyte swelling. int. j. mol. sci. 20, 330. https://doi.org/10.3390/ijms20020330 lavenex, p., lavenex, p.b., bennett, j.l., amaral, d.g., 2009. postmortem changes in the neuroanatomical characteristics of the primate brain: hippocampal formation. j. comp. neurol. 512, 27–51. https://doi.org/10.1002/cne.21906 lee, t.-k., kim, hyunjung, song, m., lee, j.-c., park, j.h., ahn, j.h., yang, g.e., kim, hyeyoung, ohk, t.g., shin, m.c., cho, j.h., won, m.-h., 2019. time-course pattern of neuronal loss and gliosis in gerbil hippocampi following mild, severe, or lethal transient global cerebral ischemia. neural regen. res. 14, 1394–1403. https://doi.org/10.4103/1673-5374.253524 leonard, a., vink, r., byard, r.w., 2016. brain fluid content related to body position and postmortem interval an animal model. j. forensic sci. 61, 671–673. https://doi.org/10.1111/1556-4029.13038 lesnikova, i., schreckenbach, m.n., kristensen, m.p., papanikolaou, l.l., hamilton-dutoit, s., 2018. usability of immunohistochemistry in forensic samples with varying decomposition. am. j. forensic med. pathol. 39, 185–191. https://doi.org/10.1097/paf.0000000000000408 levin, s., bucci, t.j., cohen, s.m., fix, a.s., hardisty, j.f., legrand, e.k., maronpot, r.r., trump, b.f., 1999. the nomenclature of cell death: recommendations of an ad hoc committee of the society of toxicologic pathologists. toxicol. pathol. 27, 484–490. https://doi.org/10.1177/019262339902700419 lewis, d.a., 2002. the human brain revisited: opportunities and challenges in postmortem studies of psychiatric disorders. neuropsychopharmacol. off. publ. am. coll. neuropsychopharmacol. 26, 143–154. https://doi.org/10.1016/s0893-133x(01)00393-1 li, j.z., vawter, m.p., walsh, d.m., tomita, h., evans, s.j., choudary, p.v., lopez, j.f., avelar, a., shokoohi, v., chung, t., mesarwi, o., jones, e.g., watson, s.j., akil, h., bunney, w.e., jr, myers, r.m., 2004. systematic changes in gene expression in postmortem human brains associated with tissue ph and terminal medical conditions. hum. mol. genet. 13, 609–616. https://doi.org/10.1093/hmg/ddh065 li, y., zhou, y., danbolt, n.c., 2012. the rates of postmortem proteolysis of glutamate transporters differ dramatically between cells and between transporter subtypes. j. histochem. cytochem. off. j. histochem. soc. 60, 811–821. https://doi.org/10.1369/0022155412458589 lindenberg, r., 1956. morphotropic and morphostatic necrobiosis; investigations on nerve cells of the brain. am. j. pathol. 32, 1147–1177. lingwood, b.e., healy, g.n., sullivan, s.m., pow, d.v., colditz, p.b., 2008. map2 provides reliable early assessment of neural injury in the newborn piglet model of birth asphyxia. j. neurosci. methods 171, 140–146. https://doi.org/10.1016/j.jneumeth.2008.02.011 link, c.d., 2021. is there a brain microbiome? neurosci. insights 16, 26331055211018708. https://doi.org/10.1177/26331055211018709 lipton, p., 1999. ischemic cell death in brain neurons. physiol. rev. 79, 1431–1568. https://doi.org/10.1152/physrev.1999.79.4.1431 liu, c.n., windle, w.f., 1950. effects of inanition on the central nervous system; an experimental study on the guinea pig. arch. neurol. psychiatry 63, 918–927. https://doi.org/10.1001/archneurpsyc.1950.02310240077004 liu, x., brun, a., 1995. synaptophysin immunoreactivity is stable 36 h postmortem. dement. basel switz. 6, 211–217. https://doi.org/10.1159/000106949 lodish, h.f., rothman, j.e., 1979. the assembly of cell membranes. sci. am. 240, 48–63. https://doi.org/10.1038/scientificamerican0179-48 loh, k.y., wang, z., liao, p., 2019. oncotic cell death in stroke. rev. physiol. biochem. pharmacol. 176, 37–64. https://doi.org/10.1007/112_2018_13 lossi, l., 2022. the concept of intrinsic versus extrinsic apoptosis. biochem. j. 479, 357–384. https://doi.org/10.1042/bcj20210854 lucassen, p.j., chung, w.c., kamphorst, w., swaab, d.f., 1997. dna damage distribution in the human brain as shown by in situ end labeling; area-specific differences in aging and alzheimer disease in the absence of apoptotic morphology. j. neuropathol. exp. neurol. https://doi.org/10.1097/00005072-199708000-00007 lucassen, p.j., chung, w.c., vermeulen, j.p., van lookeren campagne, m., van dierendonck, j.h., swaab, d.f., 1995. microwave-enhanced in situ end-labeling of fragmented dna: parametric studies in relation to postmortem delay and fixation of rat and human brain. j. histochem. cytochem. off. j. histochem. soc. 43, 1163–1171. https://doi.org/10.1177/43.11.7560899 mackenzie, j.m., 2014. examining the decomposed brain. am. j. forensic med. pathol. 35, 265–270. https://doi.org/10.1097/paf.0000000000000111 madea, b., 1994. importance of supravitality in forensic medicine. forensic sci. int. 69, 221–241. https://doi.org/10.1016/0379-0738(94)90386-7 mahesh, s., tang, k.-c., raj, m., 2018. amide bond activation of biological molecules. mol. basel switz. 23, e2615. https://doi.org/10.3390/molecules23102615 majno, g., joris, i., 1995. apoptosis, oncosis, and necrosis. an overview of cell death. am. j. pathol. 146, 3–15. manzo, c., garcia-parajo, m.f., 2015. a review of progress in single particle tracking: from methods to biophysical insights. rep. prog. phys. phys. soc. g. b. 78, 124601. https://doi.org/10.1088/0034-4885/78/12/124601 martin, m. da g.m., paes, v.r., cardoso, e.f., neto, c.e.b.p., kanamura, c.t., leite, c. da c., otaduy, m.c.g., monteiro, r.a. de a., mauad, t., da silva, l.f.f., castro, l.h.m., saldiva, p.h.n., dolhnikoff, m., duarte-neto, a.n., 2022. postmortem brain 7t mri with minimally invasive pathological correlation in deceased covid-19 subjects. insights imaging 13, 7. https://doi.org/10.1186/s13244-021-01144-w martin, s.b., waniewski, r.a., battaglioli, g., martin, d.l., 2003. post-mortem degradation of brain glutamate decarboxylase. neurochem. int. 42, 549–554. https://doi.org/10.1016/s0197-0186(02)00189-4 maynard, e.a., schultz, r.l., pease, d.c., 1957. electron microscopy of the vascular bed of rat cerebral cortex. am. j. anat. 100, 409–433. https://doi.org/10.1002/aja.1001000306 mccullumsmith, r.e., hammond, j.h., shan, d., meador-woodruff, j.h., 2014. postmortem brain: an underutilized substrate for studying severe mental illness. neuropsychopharmacology 39, 65–87. https://doi.org/10.1038/npp.2013.239 mcfadden, w.c., walsh, h., richter, f., soudant, c., bryce, c.h., hof, p.r., fowkes, m., crary, j.f., mckenzie, a.t., 2019. perfusion fixation in brain banking: a systematic review. acta neuropathol. commun. 7, 146. https://doi.org/10.1186/s40478-019-0799-y mckenzie, a.t., marx, g.a., koenigsberg, d., sawyer, m., iida, m.a., walker, j.m., richardson, t.e., campanella, g., attems, j., mckee, a.c., stein, t.d., fuchs, t.j., white, c.l., part working group, farrell, k., crary, j.f., 2022. interpretable deep learning of myelin histopathology in age-related cognitive impairment. acta neuropathol. commun. 10, 131. https://doi.org/10.1186/s40478-022-01425-5 mena, h., cadavid, d., rushing, e.j., 2004. human cerebral infarct: a proposed histopathologic classification based on 137 cases. acta neuropathol. (berl.) 108. https://doi.org/10.1007/s00401-004-0918-z michiue, t., quan, l., ishikawa, t., zhu, b.-l., maeda, h., 2009. quantitative analysis of single-stranded dna immunoreactivity as a marker of neuronal apoptosis in hippocampus with regard to the causes of death in medicolegal autopsy. leg. med. tokyo jpn. 11 suppl 1, s168-170. https://doi.org/10.1016/j.legalmed.2009.01.105 miller, m.a., zachary, j.f., 2017. mechanisms and morphology of cellular injury, adaptation, and death. pathol. basis vet. dis. 2-43.e19. https://doi.org/10.1016/b978-0-323-35775-3.00001-1 mogilner, a., manhart, a., 2018. intracellular fluid mechanics: coupling cytoplasmic flow with active cytoskeletal gel. annu. rev. fluid mech. 50, 347–370. https://doi.org/10.1146/annurev-fluid-010816-060238 monroy-gómez, j., santamaría, g., sarmiento, l., torres-fernández, o., 2020. effect of postmortem degradation on the preservation of viral particles and rabies antigens in mice brains. light and electron microscopic study. viruses 12, e938. https://doi.org/10.3390/v12090938 montero-crespo, m., domínguez-álvaro, m., alonso-nanclares, l., defelipe, j., blazquez-llorca, l., 2021. three-dimensional analysis of synaptic organization in the hippocampal ca1 field in alzheimer’s disease. brain 144, 553–573. https://doi.org/10.1093/brain/awaa406 mori, s., sternberger, n.h., herman, m.m., sternberger, l.a., 1992. variability of laminin immunoreactivity in human autopsy brain. histochemistry 97, 237–241. https://doi.org/10.1007/bf00267633 mori, s., sternberger, n.h., herman, m.m., sternberger, l.a., 1991. leakage and neuronal uptake of serum protein in aged and alzheimer brains. a postmortem phenomenon with antemortem etiology. lab. investig. j. tech. methods pathol. 64, 345–351. morton-hayward, a.l., thompson, t., thomas-oates, j.e., buckley, s., petzold, a., ramsøe, a., o’connor, s., collins, m.j., 2020. a conscious rethink: why is brain tissue commonly preserved in the archaeological record? commentary on: petrone p, pucci p, niola m, et al. heat-induced brain vitrification from the vesuvius eruption in c.e. 79. n engl j med 2020;382:383-4. doi: 10.1056/nejmc1909867. star sci. technol. archaeol. res. 6, 87–95. https://doi.org/10.1080/20548923.2020.1815398 moscardini, a., di pietro, s., signore, g., parlanti, p., santi, m., gemmi, m., cappello, v., 2020. uranium-free x solution: a new generation contrast agent for biological samples ultrastructure. sci. rep. 10, 11540. https://doi.org/10.1038/s41598-020-68405-4 müller, m.b., lucassen, p.j., yassouridis, a., hoogendijk, w.j., holsboer, f., swaab, d.f., 2001. neither major depression nor glucocorticoid treatment affects the cellular integrity of the human hippocampus. eur. j. neurosci. 14, 1603–1612. https://doi.org/10.1046/j.0953-816x.2001.01784.x nagy, c., maheu, m., lopez, j.p., vaillancourt, k., cruceanu, c., gross, j.a., arnovitz, m., mechawar, n., turecki, g., 2015. effects of postmortem interval on biomolecule integrity in the brain. j. neuropathol. exp. neurol. 74, 459–469. https://doi.org/10.1097/nen.0000000000000190 nahirney, p.c., tremblay, m.-e., 2021. brain ultrastructure: putting the pieces together. front. cell dev. biol. 9, 629503. https://doi.org/10.3389/fcell.2021.629503 nakabayashi, y., nabeka, h., kuwahara, n., matsuda, s., asano, m., 2021. postmortem interval estimation by evaluating saposin d levels and morphological alterations in hippocampal neurons. albanian j. med. health sci. 55. nunley, w.c., schuit, k.e., dickie, m.w., kinlaw, j.b., 1972. delayed, in vivo hepatic post-mortem autolysis. virchows arch. b cell pathol. 11, 289–302. https://doi.org/10.1007/bf02889410 oehmichen, m., 1994. brain death: neuropathological findings and forensic implications. forensic sci. int. 69, 205–219. https://doi.org/10.1016/0379-0738(94)90385-9 oehmichen, m., 1980. enzyme alterations in brain tissue during the early postmortal interval with reference to the histomorphology: review of the literature. z. rechtsmed. 85, 81–95. https://doi.org/10.1007/bf02092198 oehmichen, m., auer, r.n., könig, h.g., 2006. forensic neuropathology and associated neurology. springer, berlin, heidelberg. https://doi.org/10.1007/3-540-28995-x ohm, t.g., diekmann, s., 1994. the use of lucifer yellow and mini-ruby for intracellular staining in fixed brain tissue: methodological considerations evaluated in rat and human autopsy brains. j. neurosci. methods 55, 105–110. https://doi.org/10.1016/0165-0270(94)90046-9 palkovits, m., harvey-white, j., liu, j., kovacs, z.s., bobest, m., lovas, g., bagó, a.g., kunos, g., 2008. regional distribution and effects of postmortal delay on endocannabinoid content of the human brain. neuroscience 152, 1032–1039. https://doi.org/10.1016/j.neuroscience.2008.01.034 palmer, a.m., lowe, s.l., francis, p.t., bowen, d.m., 1988. are post-mortem biochemical studies of human brain worthwhile? biochem. soc. trans. 16, 472–475. https://doi.org/10.1042/bst0160472 pantoni, l., garcia, j.h., gutierrez, j.a., 1996. cerebral white matter is highly vulnerable to ischemia. stroke 27, 1641–1646; discussion 1647. https://doi.org/10.1161/01.str.27.9.1641 pearce, j.m., komoroski, r.a., 2000. analysis of phospholipid molecular species in brain by (31)p nmr spectroscopy. magn. reson. med. 44, 215–223. https://doi.org/10.1002/1522-2594(200008)44:23.0.co;2-n pélissier-alicot, a.-l., gaulier, j.-m., champsaur, p., marquet, p., 2003. mechanisms underlying postmortem redistribution of drugs: a review. j. anal. toxicol. 27, 533–544. https://doi.org/10.1093/jat/27.8.533 pelucchi, s., stringhi, r., marcello, e., 2020. dendritic spines in alzheimer’s disease: how the actin cytoskeleton contributes to synaptic failure. int. j. mol. sci. 21, 908. https://doi.org/10.3390/ijms21030908 peroski, m., proudan, n., grignol, g., merchenthaler, i., dudas, b., 2016. corticotropin-releasing hormone (crh)-immunoreactive (ir) axon varicosities target a subset of growth hormone-releasing hormone (ghrh)-ir neurons in the human hypothalamus. j. chem. neuroanat. 78, 119–124. https://doi.org/10.1016/j.jchemneu.2016.09.005 perry, r.h., tomlinson, b.e., taylor, m.j., perry, e.k., 1977. human brain temperature at necropsy: a guide in post-mortem biochemistry. lancet lond. engl. 1, 38. https://doi.org/10.1016/s0140-6736(77)91669-5 petit, t.l., leboutillier, j.c., 1990. quantifying synaptic number and structure: effects of stain and post-mortem delay. brain res. 517, 269–275. https://doi.org/10.1016/0006-8993(90)91037-h petzold, a., lu, c.-h., groves, m., gobom, j., zetterberg, h., shaw, g., o’connor, s., 2020. protein aggregate formation permits millennium-old brain preservation. j. r. soc. interface 17, 20190775. https://doi.org/10.1098/rsif.2019.0775 powers, r.h., 2005. the decomposition of human remains, in: rich, j., dean, d.e., powers, r.h. (eds.), forensic medicine of the lower extremity: human identification and trauma analysis of the thigh, leg, and foot, forensic science and medicine. humana press, totowa, nj, pp. 3–15. https://doi.org/10.1385/1-59259-897-8:003 pozhitkov, a.e., neme, r., domazet-lošo, t., leroux, b.g., soni, s., tautz, d., noble, p.a., 2017. tracing the dynamics of gene transcripts after organismal death. open biol. 7, 160267. https://doi.org/10.1098/rsob.160267 quartu, m., serra, m.p., manca, a., mascia, f., follesa, p., del fiacco, m., 2005. neurturin, persephin, and artemin in the human preand full-term newborn and adult hippocampus and fascia dentata. brain res. 1041, 157–166. https://doi.org/10.1016/j.brainres.2005.02.007 radzicka, a., wolfenden, r., 1996. rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. j. am. chem. soc. 118, 6105–6109. https://doi.org/10.1021/ja954077c ramirez, e.p.c., keller, c.e., vonsattel, j.p., 2018. the new york brain bank of columbia university: practical highlights of 35 years of experience. handb. clin. neurol. 150, 105–118. https://doi.org/10.1016/b978-0-444-63639-3.00008-6 ravid, r., van zwieten, e.j., swaab, d.f., 1992. brain banking and the human hypothalamus--factors to match for, pitfalls and potentials. prog. brain res. 93, 83–95. https://doi.org/10.1016/s0079-6123(08)64565-3 rees, s., 1977. the incidence of ultrastructural abnormalities in the cortex of two retarded human brains (down’s syndrome). acta neuropathol. (berl.) 37, 65–68. https://doi.org/10.1007/bf00684542 rees, s., 1976. a quantitative electron microscopic study of the ageing human cerebral cortex. acta neuropathol. (berl.) 36, 347–362. https://doi.org/10.1007/bf00699640 riback, j.a., eeftens, j.m., lee, d.s.w., quinodoz, s.a., beckers, l., becker, l.a., brangwynne, c.p., 2022. viscoelastic rna entanglement and advective flow underlie nucleolar form and function. https://doi.org/10.1101/2021.12.31.474660 roberts, r.c., gaither, l.a., peretti, f.j., lapidus, b., chute, d.j., 1996. synaptic organization of the human striatum: a postmortem ultrastructural study. j. comp. neurol. 374, 523–534. https://doi.org/10.1002/(sici)1096-9861(19961028)374:43.0.co;2-3 roberts, r.c., roche, j.k., conley, r.r., 2005. synaptic differences in the postmortem striatum of subjects with schizophrenia: a stereological ultrastructural analysis. synap. n. y. n 56, 185–197. https://doi.org/10.1002/syn.20144 roberts, r.c., roche, j.k., mccullumsmith, r.e., 2014. localization of excitatory amino acid transporters eaat1 and eaat2 in human postmortem cortex: a light and electron microscopic study. neuroscience 277, 522–540. https://doi.org/10.1016/j.neuroscience.2014.07.019 routtenberg, a., tarrant, s., 1974. synaptic morphology and cytoplasmic densities: rapid post-mortem effects. tissue cell 6, 777–788. https://doi.org/10.1016/0040-8166(74)90015-9 samarasekera, n., al-shahi salman, r., huitinga, i., klioueva, n., mclean, c.a., kretzschmar, h., smith, c., ironside, j.w., 2013. brain banking for neurological disorders. lancet neurol. 12, 1096–1105. https://doi.org/10.1016/s1474-4422(13)70202-3 sarnat, h.b., flores-sarnat, l., trevenen, c.l., 2010. synaptophysin immunoreactivity in the human hippocampus and neocortex from 6 to 41 weeks of gestation. j. neuropathol. exp. neurol. 69, 234–245. https://doi.org/10.1097/nen.0b013e3181d0151f schallock, k., schulz-schaeffer, w.j., giese, a., kretzschmar, h.a., 1997. postmortem delay and temperature conditions affect the in situ end-labeling (isel) assay in brain tissue of mice. clin. neuropathol. 16, 133–136. schavemaker, p.e., boersma, a.j., poolman, b., 2018. how important is protein diffusion in prokaryotes? front. mol. biosci. 5, 93. https://doi.org/10.3389/fmolb.2018.00093 scheff, s.w., dekosky, s.t., price, d.a., 1990. quantitative assessment of cortical synaptic density in alzheimer’s disease. neurobiol. aging 11, 29–37. https://doi.org/10.1016/0197-4580(90)90059-9 scheff, s.w., price, d.a., 1993. synapse loss in the temporal lobe in alzheimer’s disease. ann. neurol. 33, 190–199. https://doi.org/10.1002/ana.410330209 schultz, r.l., maynard, e.a., pease, d.c., 1957. electron microscopy of neurons and neuroglia of cerebral cortex and corpus callosum. am. j. anat. 100, 369–407. https://doi.org/10.1002/aja.1001000305 schulz, u., hunziker, o., frey, h., schweizer, a., 1980. postmortem changes in stereological parameters of cerebral neurons. pathol. res. pract. 166, 260–270. https://doi.org/10.1016/s0344-0338(80)80134-8 schwab, c., bondada, v., sparks, d.l., cahan, l.d., geddes, j.w., 1994. postmortem changes in the levels and localization of microtubule-associated proteins (tau, map2 and map1b) in the rat and human hippocampus. hippocampus 4, 210–225. https://doi.org/10.1002/hipo.450040212 schwarzmaier, s.m., knarr, m.r.o., hu, s., ertürk, a., hellal, f., plesnila, n., 2022. perfusion pressure determines vascular integrity and histomorphological quality following perfusion fixation of the brain. j. neurosci. methods 372, 109493. https://doi.org/10.1016/j.jneumeth.2022.109493 scudamore, c.l., hodgson, h.k., patterson, l., macdonald, a., brown, f., smith, k.c., 2011. the effect of post-mortem delay on immunohistochemical labelling—a short review. comp. clin. pathol. 20, 95–101. https://doi.org/10.1007/s00580-010-1149-4 sele, m., wernitznig, s., lipovšek, s., radulović, s., haybaeck, j., birkl-toeglhofer, a.m., wodlej, c., kleinegger, f., sygulla, s., leoni, m., ropele, s., leitinger, g., 2019. optimization of ultrastructural preservation of human brain for transmission electron microscopy after long post-mortem intervals. acta neuropathol. commun. 7, 144. https://doi.org/10.1186/s40478-019-0794-3 serra, m.p., quartu, m., mascia, f., manca, a., boi, m., pisu, m.g., lai, m.l., del fiacco, m., 2005. ret, gfralpha-1, gfralpha-2 and gfralpha-3 receptors in the human hippocampus and fascia dentata. int. j. dev. neurosci. off. j. int. soc. dev. neurosci. 23, 425–438. https://doi.org/10.1016/j.ijdevneu.2005.05.003 sheleg, s.v., lobello, j.r., hixon, h., coons, s.w., lowry, d., nedzved, m.k., 2008. stability and autolysis of cortical neurons in post-mortem adult rat brains. int. j. clin. exp. pathol. 1, 291–299. shepherd, t.m., flint, j.j., thelwall, p.e., stanisz, g.j., mareci, t.h., yachnis, a.t., blackband, s.j., 2009. postmortem interval alters the water relaxation and diffusion properties of rat nervous tissue--implications for mri studies of human autopsy samples. neuroimage 44, 820–826. https://doi.org/10.1016/j.neuroimage.2008.09.054 shibayama, h., kitoh, j., 1976. the postmortem changes of pyramidal neurons in the hippocampus of rats. folia psychiatr. neurol. jpn. 30, 73–91. https://doi.org/10.1111/j.1440-1819.1976.tb00113.x shubin, a.v., demidyuk, i.v., komissarov, a.a., rafieva, l.m., kostrov, s.v., 2016. cytoplasmic vacuolization in cell death and survival. oncotarget 7, 55863–55889. https://doi.org/10.18632/oncotarget.10150 siew, l.k., love, s., dawbarn, d., wilcock, g.k., allen, s.j., 2004. measurement of preand post-synaptic proteins in cerebral cortex: effects of post-mortem delay. j. neurosci. methods 139, 153–159. https://doi.org/10.1016/j.jneumeth.2004.04.020 sillevis smitt, p.a., van der loos, c., vianney de jong, j.m., troost, d., 1993. tissue fixation methods alter the immunohistochemical demonstrability of neurofilament proteins, synaptophysin, and glial fibrillary acidic protein in human cerebellum. acta histochem. 95, 13–21. https://doi.org/10.1016/s0065-1281(11)80381-8 silva, i., silva, j., ferreira, r., trigo, d., 2021. glymphatic system, aqp4, and their implications in alzheimer’s disease. neurol. res. pract. 3, 5. https://doi.org/10.1186/s42466-021-00102-7 ślęzak, j., burov, s., 2021. from diffusion in compartmentalized media to non-gaussian random walks. sci. rep. 11, 5101. https://doi.org/10.1038/s41598-021-83364-0 snyder, j.m., gibson-corley, k.n., radaelli, e., 2021. nervous system, in: pathology of genetically engineered and other mutant mice. john wiley & sons, ltd, pp. 462–492. https://doi.org/10.1002/9781119624608.ch21 solenski, n.j., dipierro, c.g., trimmer, p.a., kwan, a.-l., helm, g.a., helms, g.a., 2002. ultrastructural changes of neuronal mitochondria after transient and permanent cerebral ischemia. stroke 33, 816–824. https://doi.org/10.1161/hs0302.104541 sorimachi, y., harada, k., yoshida, k., 1996. involvement of calpain in postmortem proteolysis in the rat brain. forensic sci. int. 81, 165–174. https://doi.org/10.1016/s0379-0738(96)01981-0 spector, r.g., 1963. selective changes in dehydrogenase enzymes and pyridine nucleotides in rat brain in anoxic-ischaemic encephalopathy. br. j. exp. pathol. 44, 312–316. stadelmann, c., timmler, s., barrantes-freer, a., simons, m., 2019. myelin in the central nervous system: structure, function, and pathology. physiol. rev. 99, 1381–1431. https://doi.org/10.1152/physrev.00031.2018 stan, a.d., ghose, s., gao, x.-m., roberts, r.c., lewis-amezcua, k., hatanpaa, k.j., tamminga, c.a., 2006. human postmortem tissue: what quality markers matter? brain res. 1123, 1–11. https://doi.org/10.1016/j.brainres.2006.09.025 suárez-pinilla, m., fernández-vega, i., 2015. an acute metabolic insult highly increased postmortem cerebellar autolysis: an autopsy case. clin. neuropathol. 34, 166–168. https://doi.org/10.5414/np300809 sullivan, s.m., björkman, s.t., miller, s.m., colditz, p.b., pow, d.v., 2010. morphological changes in white matter astrocytes in response to hypoxia/ischemia in the neonatal pig. brain res. 1319, 164–174. https://doi.org/10.1016/j.brainres.2010.01.010 susaki, e.a., shimizu, c., kuno, a., tainaka, k., li, x., nishi, k., morishima, k., ono, h., ode, k.l., saeki, y., miyamichi, k., isa, k., yokoyama, c., kitaura, h., ikemura, m., ushiku, t., shimizu, y., saito, t., saido, t.c., fukayama, m., onoe, h., touhara, k., isa, t., kakita, a., shibayama, m., ueda, h.r., 2020. versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues. nat. commun. 11, 1982. https://doi.org/10.1038/s41467-020-15906-5 suzuki, j., 1987. histological study, in: suzuki, j. (ed.), treatment of cerebral infarction: experimental and clinical study. springer, vienna, pp. 23–53. https://doi.org/10.1007/978-3-7091-8861-3_3 svitkina, t.m., 2020. actin cell cortex: structure and molecular organization. trends cell biol. 30, 556–565. https://doi.org/10.1016/j.tcb.2020.03.005 swaab, d.f., bao, a.-m., 2021. matching of the postmortem hypothalamus from patients and controls. handb. clin. neurol. 179, 141–156. https://doi.org/10.1016/b978-0-12-819975-6.00007-8 switzer, r.c., 2000. application of silver degeneration stains for neurotoxicity testing. toxicol. pathol. 28, 70–83. https://doi.org/10.1177/019262330002800109 szöllősi, d., tóth, l., kálmán, m., 2018. postmortem immunohistochemical alterations following cerebral lesions: a possible pathohistological importance of the β-dystroglycan immunoreactivity. neuropathol. off. j. jpn. soc. neuropathol. 38. https://doi.org/10.1111/neup.12447 tafrali, d., 2019. post-mortem changes and autolysis in frontal lobe cells of sus scrofa. medical university of graz, graz, austria. https://doi.org/10.13140/rg.2.2.31901.87520 takahashi, n., satou, c., higuchi, t., shiotani, m., maeda, h., hirose, y., 2010. quantitative analysis of intracranial hypostasis: comparison of early postmortem and antemortem ct findings. ajr am. j. roentgenol. 195, w388-393. https://doi.org/10.2214/ajr.10.4442 tang, y., nyengaard, j.r., de groot, d.m., gundersen, h.j., 2001. total regional and global number of synapses in the human brain neocortex. synap. n. y. n 41, 258–273. https://doi.org/10.1002/syn.1083 tao-cheng, j.-h., gallant, p.e., brightman, m.w., dosemeci, a., reese, t.s., 2007. structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. j. comp. neurol. 501, 731–740. https://doi.org/10.1002/cne.21276 tapia, j.c., kasthuri, n., hayworth, k.j., schalek, r., lichtman, j.w., smith, s.j., buchanan, j., 2012. high-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. nat. protoc. 7, 193–206. https://doi.org/10.1038/nprot.2011.439 terstege, d.j., addo-osafo, k., campbell teskey, g., epp, j.r., 2022. new neurons in old brains: implications of age in the analysis of neurogenesis in post-mortem tissue. mol. brain 15, 38. https://doi.org/10.1186/s13041-022-00926-7 torres-platas, s.g., comeau, s., rachalski, a., bo, g.d., cruceanu, c., turecki, g., giros, b., mechawar, n., 2014. morphometric characterization of microglial phenotypes in human cerebral cortex. j. neuroinflammation 11. https://doi.org/10.1186/1742-2094-11-12 tóth, k., eross, l., vajda, j., halász, p., freund, t.f., maglóczky, z., 2010. loss and reorganization of calretinin-containing interneurons in the epileptic human hippocampus. brain j. neurol. 133, 2763–2777. https://doi.org/10.1093/brain/awq149 trump, b.f., berezesky, i.k., chang, s.h., phelps, p.c., 1997. the pathways of cell death: oncosis, apoptosis, and necrosis. toxicol. pathol. 25, 82–88. https://doi.org/10.1177/019262339702500116 trump, b.f., berezesky, i.k., sato, t., laiho, k.u., phelps, p.c., declaris, n., 1984. cell calcium, cell injury and cell death. environ. health perspect. 57, 281–287. https://doi.org/10.1289/ehp.8457281 van nimwegen, d., sheldon, h., 1966. early postmortem changes in cerebellar neurons of the rat. j. ultrasructure res. 14, 36–46. https://doi.org/10.1016/s0022-5320(66)80033-3 vass, a.a., 2011. the elusive universal post-mortem interval formula. forensic sci. int. 204, 34–40. https://doi.org/10.1016/j.forsciint.2010.04.052 vonsattel, j.p.g., amaya, m. del p., cortes, e.p., mancevska, k., keller, c.e., 2008. 21st century brain banking practical prerequisites and lessons from the past: the experience of new york brain bank – taub institute columbia university. cell tissue bank. 9, 247–258. https://doi.org/10.1007/s10561-008-9079-y vrselja, z., daniele, s.g., silbereis, j., talpo, f., morozov, y.m., sousa, a.m.m., tanaka, b.s., skarica, m., pletikos, m., kaur, n., zhuang, z.w., liu, z., alkawadri, r., sinusas, a.j., latham, s.r., waxman, s.g., sestan, n., 2019. restoration of brain circulation and cellular functions hours post-mortem. nature 568, 336–343. https://doi.org/10.1038/s41586-019-1099-1 wagensveld, i.m., blokker, b.m., wielopolski, p.a., renken, n.s., krestin, g.p., hunink, m.g., oosterhuis, j.w., weustink, a.c., 2017. total-body ct and mr features of postmortem change in in-hospital deaths. plos one 12, e0185115. https://doi.org/10.1371/journal.pone.0185115 waldvogel, h.j., curtis, m.a., baer, k., rees, m.i., faull, r.l.m., 2006. immunohistochemical staining of post-mortem adult human brain sections. nat. protoc. 1, 2719–2732. https://doi.org/10.1038/nprot.2006.354 wang, n.s., minassian, h., 1987. the formaldehyde-fixed and paraffin-embedded tissues for diagnostic transmission electron microscopy: a retrospective and prospective study. hum. pathol. 18, 715–727. https://doi.org/10.1016/s0046-8177(87)80243-5 weerasinghe, p., buja, l.m., 2012. oncosis: an important non-apoptotic mode of cell death. exp. mol. pathol. 93, 302–308. https://doi.org/10.1016/j.yexmp.2012.09.018 weller, r.o., sharp, m.m., christodoulides, m., carare, r.o., møllgård, k., 2018. the meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human cns. acta neuropathol. (berl.) 135, 363–385. https://doi.org/10.1007/s00401-018-1809-z wenzlow, n., neal, d., stern, a.w., prakoso, d., liu, j.j., delcambre, g.h., beachboard, s., long, m.t., 2021. feasibility of using tissue autolysis to estimate the postmortem interval in horses. j. vet. diagn. investig. off. publ. am. assoc. vet. lab. diagn. inc 33, 825–833. https://doi.org/10.1177/10406387211021865 whitney, e.r., kemper, t.l., rosene, d.l., bauman, m.l., blatt, g.j., 2008. calbindin-d28k is a more reliable marker of human purkinje cells than standard nissl stains: a stereological experiment. j. neurosci. methods 168, 42–47. https://doi.org/10.1016/j.jneumeth.2007.09.009 williams, r.s., ferrante, r.j., caviness, v.s., 1978. the golgi rapid method in clinical neuropathology: the morphologic consequences of suboptimal fixation. j. neuropathol. exp. neurol. 37, 13–33. https://doi.org/10.1097/00005072-197801000-00002 wohlsein, p., deschl, u., baumgärtner, w., 2013. nonlesions, unusual cell types, and postmortem artifacts in the central nervous system of domestic animals. vet. pathol. 50, 122–143. https://doi.org/10.1177/0300985812450719 wong, g., greenhalgh, t., westhorp, g., buckingham, j., pawson, r., 2013. rameses publication standards: realist syntheses. bmc med. 11, 21. https://doi.org/10.1186/1741-7015-11-21 yarema, m.c., becker, c.e., 2005. key concepts in postmortem drug redistribution. clin. toxicol. phila. pa 43, 235–241. https://doi.org/10.1081/clt-58950 yeung, l.y., wai, m.s.m., yew, d.t., 2010. silver impregnation of the prefrontal cortices in the brains with long postmortem delay. int. j. neurosci. 120, 314–317. https://doi.org/10.3109/00207450903243507 zhang, s., boyd, j., delaney, k., murphy, t.h., 2005. rapid reversible changes in dendritic spine structure in vivo gated by the degree of ischemia. j. neurosci. off. j. soc. neurosci. 25, 5333–5338. https://doi.org/10.1523/jneurosci.1085-05.2005 zhou, c., byard, r.w., 2011. factors and processes causing accelerated decomposition in human cadavers an overview. j. forensic leg. med. 18, 6–9. https://doi.org/10.1016/j.jflm.2010.10.003 zissler, a., stoiber, w., steinbacher, p., geissenberger, j., monticelli, f.c., pittner, s., 2020. postmortem protein degradation as a tool to estimate the pmi: a systematic review. diagn. basel switz. 10, e1014. https://doi.org/10.3390/diagnostics10121014 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. strategies to gain novel alzheimer’s disease diagnostics and therapeutics using modulators of abca transporters feel free to add comments by clicking these icons on the sidebar free neuropathology 2:33 (2021) review strategies to gain novel alzheimer’s disease diagnostics and therapeutics using modulators of abca transporters jens pahnke1,2,3, pablo bascuñana1, mirjam brackhan1,2, katja stefan1, vigneshwaran namasivayam4, radosveta koldamova5, jingyun wu1, luisa möhle1, sven marcel stefan1 1 department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, oslo, norway 2 lied, university of lübeck, lübeck, germany 3 department of pharmacology, faculty of medicine, university of latvia, rīga, latvia 4 department of pharmaceutical and cellbiological chemistry, pharmaceutical institute, university of bonn, bonn, germany 4 department of environmental and occupational health, school of public health, university of pittsburgh, pittsburgh, pa, united states of america corresponding author: sven marcel stefan · department of pathology · section of neuropathology · translational neurodegeneration research and neuropathology lab · www.pahnkelab.eu · university of oslo and oslo university hospital · sognsvannsveien 20 · 0372 oslo · norway s.m.stefan@medisin.uio.no submitted: 07 september 2021 accepted: 12 november 2021 copyedited by: biswarathan ramani published: 13 december 2021 https://doi.org/10.17879/freeneuropathology-2021-3528 keywords: abc transporter, abcb1 (p-gp), abcc1 (mrp1), abcg2 (bcrp), abca1 (abc1), abca2, abca5, abca7, multitarget inhibitor (panabc), broad-spectrum modulator, alzheimer’s disease, amyloid-beta (aβ / abeta), inhibition, activation, induction, downregulation, pet tracer (petabc), pattern analysis, polypharmacology, rational drug design and development abstract adenosine-triphosphate-(atp)-binding cassette (abc) transport proteins are ubiquitously present membrane-bound efflux pumps that distribute endoand xenobiotics across intraand intercellular barriers. discovered over 40 years ago, abc transporters have been identified as key players in various human diseases, such as multidrug-resistant cancer and atherosclerosis, but also neurodegenerative diseases, such as alzheimer’s disease (ad). most prominent and well-studied are abcb1, abcc1, and abcg2, not only due to their contribution to the multidrug resistance (mdr) phenotype in cancer, but also due to their contribution to ad. however, our understanding of other abc transporters is limited, and most of the 49 human abc transporters have been largely neglected as potential targets for novel small-molecule drugs. this is especially true for the abca subfamily, which contains several members known to play a role in ad initiation and progression. this review provides up-to-date information on the proposed functional background and pathological role of abca transporters in ad. we also provide an overview of small-molecules shown to interact with abca transporters as well as potential in silico, in vitro, and in vivo methodologies to gain novel templates for the development of innovative abc transporter-targeting diagnostics and therapeutics. introduction from mdr to neurodegeneration: abc transporters in human disease abc transporters, aβ proteins, and ad part i: status quo abca transporters: physiological function and implications for ad abca1 abca2 abca3 abca4 abca5 abca6 abca7 abca8–abca10 abca12 abca13 modulators of abca transporter function, trafficking, and regulation small-molecule interactors of abca transporters small-molecule regulators of abca transporters part ii: pipeline development to gain novel diagnostics and therapeutics in silico methodologies to predict novel lead structures structure-based drug design ligand-based drug design in vitro methodologies to assess novel lead structures host system of abca transporters functional assessment of abca transporters in vivo assessment of clinical candidates knock-out mouse models rnai models overexpression models humanized abc transporter mouse models disease models imaging techniques concluding remarks: where do we go from here? appendix abbreviations 5-fu 5-fluorouracil, aβ amyloid-β, abca atp-binding cassette transporter subfamily a, acat acyl coenzyme a cholesteryl acyl transferase, ad alzheimer’s disease, adma asymmetric dimethylarginine, adp adenosine-diphosphate, als amyotrophic lateral sclerosis, ampk camp-activated protein kinase, apoa1/e3/e4 apolipoprotein a1/e3/e4, app amyloid precursor protein, atp adenosine-triphosphate, bbb blood-brain barrier, bcsfb blood-cerebrospinal fluid barrier, bhk baby hamster kidney, big1 brefeldin 1-inhibited guanine nucleotide exchange protein, bodipy 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, camp cyclic adenosine monophosphate, cftr cystic fibrosis transmembrane conductance regulator, cho chinese hamster ovary, cns central nervous system, cpt-camp 8-(4-chlorophenylthio)-camp, cryo-em cryogenic electron microscopy, csf cer-ebral spinal fluid, dids 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid, ec50 half-maximal effect concentration, ecd extracellular domain, ecgc epigallocatechin gallate, ed50 half-maximal effective dose, eoad early-onset ad, fpd5 fluorescigenic pyrazoline derivative 5, fxr farnesoid-x-receptor, gfp green fluorescent protein, ggpp geranylgeraniol pyrophosphate, gsh reduced glutathione, gwas genome-wide association study, hd huntington’s disease, hdac2 histone deacetylase 2, hdl high-density lipoprotein, hmg-coa-reductase 3-hydroxyl-3-methyl glutaryl-coenzyme a reductase, hts high-throughput screening, ic50 half-maximal inhibition concentration, lamp1 lysosomal-associated membrane protein 1, ldlr ldr receptor, lncrna long non-coding rna, load late-onset ad, ltc4 leukotriene c4, lxr liver-x-receptor, mdr multidrug resistance, mrna messenger rna, ms multiple sclerosis, msd membrane-spanning domain, nbd 7-nitro-2,1,3-benzooxadiazole or nucleotide binding domain, ndea n-nitrosodiethylamine, nem n-ethylmaleimide, (ox)ldl (oxidized) low density lipoprotein, pcb29-pq 2,3,5-trichloro-6-phenyl-[1,4]-benzoquinone, pd parkinson’s disease, pdb protein data bank, pg-j2 prostaglandin j2, pma phorbol 12-myristate 13-acetate, ppar peroxisome proliferator-activated receptor, prdx1 peroxiredoxin 1, rar retinoic acid receptor, rna ribonucleic acid, rxr retinoid-x-receptor, sar structure-activity relationships, shrna short-hairpin rna, sirna small interfering rna, snp single nucleotide polymorphism, sr-bi (srb1) scavenger receptor b1 (also hdl receptor), srepb sterol regulation element-binding protein, tki tyrosine kinase inhibitor, tki tyrosine kinase inhibitor, tm transmembrane helix introduction from mdr to neurodegeneration: abc transporters in human disease abc transporters are membrane-bound transport proteins that are ubiquitously present in the human body.1-4 they play a major role in determining the distribution of intrinsic and xenobiotic drugs between intraand intercellular compartments.5,6 the clinical relevance of abc transporters became pronounced when their expression was correlated to cross-resistance of cancer cells to antineoplastic agents.3,7-13 this phenomenon is called ‘multidrug resistance’ (mdr). however, despite enormous efforts and countless clinical trials to target these efflux pumps,14-17 mdr is still a major unresolved obstacle in cancer chemotherapy. to date, most abc transporters have been associated with mdr,3,7-9,11,12 but only a small minority has been studied properly and can be addressed by small-molecule modulators.18-22 amongst these are abcb1,1,18-27 abcc1,1,18,19,23,24,26,27 and abcg2.18,19,25 apart from their role in multidrug-resistant cancer, many abc transporters have been identified as key players in neurological disorders. evidence for this includes their high abundance at the blood-brain barrier (bbb) and blood-cerebrospinal fluid barrier (bcsfb) in the central nervous system (cns).28-32 additionally, their expression is altered in many pathological conditions in the brain.28-30,33-40 important players are, again, abcb1,28-30,34-36,39-44 abcc1,28-30,39,41,43,45 and abcg228,30,34,36,39-41,43in diseases like ad,28-30,41 amyotrophic lateral sclerosis (als),34,36,44 encephalopathy,45,46 epilepsy,39,40 multiple sclerosis (ms),35 and parkinson’s disease (pd).42,47 furthermore, abc transporters were also found to be associated with certain genetic neurological and psychiatric diseases such as huntington’s disease (hd),38 bipolar disorder,48,49 depression,48 or schizophrenia.48,49 table 1 summarizes the involvement of abc transporters in neurological diseases. table 1. abc transporters and related neurological and psychiatric diseases. abc transporter associated diseases abca1 ad50 hd51 abca2 ad52 abnormal sphingolipid metabolism53,54 abca4 cone-rod dystrophy55 fundus flavimaculatus56 retinitis pigmentosa57,58 stargardt disease59-62 abca5 ad28 abca7 ad63 abca13 lewy body disease64 psychiatric disorders48,65,66 stroke in mice67 abcb1 ad28 brain tumors68 hiv-associated depression and schizophrenia69,70 hiv-associated encephalopathy46 epilepsy71 ischemic stroke72 ms35 multiple systems atrophy73 pd74 progressive supranuclear palsy75 creutzfeldt-jakob disease76 abcb7 pd77 abcb9 pd78 abcc1 ad28 brain tumors79 epilepsy39 hiv-associated encephalopathy45 ischemic stroke80 abcc2 brain tumors79 epilepsy39 abcc3 brain tumors79 epilepsy39 abcc8 als81 abcc9 als81 limbic-predominant age-related tdp-43 encephalopathy (late)82 hippocampal sclerosis of aging and depression83 abcd1 cerebral adrenoleukodystrophy84 abcg1 ad85 brain metabolic disorder86 abcg2 ad87 als88 brain tumors89 epilepsy90 ms91 pd47 traumatic brain injury92 abcg4 ad93 hd51 abc transporters, aβ proteins, and ad since 2001, abc transporters have been implicated in ad pathogenesis.28-30,41,43,94,95 specifically abcb1,94 abcc1,96 and abcg297 have been suggested to directly transport amyloid-β (aβ) proteins, being involved in aβ clearance from the brain to the blood stream.94,96,97 in light of the failure of the first immunological treatment studies,98 it was already proposed that abc transporter dysfunction could explain the clearance problem of aβ.99,100 cerebral accumulation of aβ proteins interferes with neuronal metabolite homeostasis and leads to interruption of cortico-cortical circuits and hampered synaptic communication. this results in an irreversible atrophy and degeneration of specific brain regions, which further causes behavioral, cognitive, and visuospatial impairments in the progression of ad.101 the most prominent abc transporter subfamily involved in ad is the abca subfamily of cholesterol and phospholipid transporters, in which particularly abca1, abca2, abca5, and abca7 have been associated with ad.28-30,41,43,95,102 for abca1,28,41,95,103 and specifically for abca7,28,41,95,104-107 genetic variant28,41,108-111 and genome-wide association studies (gwas)28,41,106,107,112 have suggested that these transporters are risk factors in ad. these discoveries give the members of the abca subfamily a special standing within the group of ad-related abc transporters. cholesterol metabolism in the context of ad has been discussed extensively before.95,102,104,105,113-116 the contribution of cholesterol and phosphilipid transport to membrane constitution, composition, fluidity, and lipid raft formation mediated by abca transporters has already been proposed,6 presenting a putative pharmacological target.117 targeting cholesterol and lipid distribution impacts aβ production by differential activities between α-, β-, and γ-secretases, but also amyloid precursor protein (app) processing106,118-122 and aβ degradation.106,119,123-126 a contribution of abca transporters to aβ clearance from the brain was also proposed,103,106,119,124,127 but not through direct aβ transport.128,129 although abca transporters have been reviewed for the last two decades,3,130,131 little is known about their specific contribution to ad pathogenesis and their mode of action. this is mainly due to a lack of small-molecules that can be used to track, study, and impact the function of these under-studied abc transporters. the present review consists of two parts: part i provides the status quo of abca transporters in ad and small-molecule modulators – in particular intrinsic substrates, natural compounds, pharmacological drugs, and synthetic molecules – that have been reported to influence abca transporter function and expression; part ii outlines the necessary drug development pipeline for the discovery of novel lead structures as potential innovative diagnostics and therapeutics against ad. this pipeline includes cutting-edge in silico methodologies, established in vitro cell assays, and necessary in vivo models. collectively, this review contributes to a deeper understanding of small-molecule ligands that influence abca transporter function, potentially leading to the development of novel ad diagnostics and therapeutics. part i: status quo abca transporters: physiological function and implications for ad abca transporters are ubiquitously present in the human body,3,10,13 although differentially expressed.10 all of the 12 subfamily members have been associated with cholesterol and/or phospholipid transport and homeostasis,3,13,132 except for abca4, which is primarily a transporter of retinoids.133-138 in addition to the diseases listed in table 1, abca transporters have been described as key proteins in several other human disorders, including neonatal respiratory distress syndrome (abca3),139 chronic interstitial lung disease (abca3),140 cataract-microcornea syndrome (abca3),141 hypertrichosis terminalis (abca5),142 or harlequin ichtyosis (abca12).143 however, one major clinical implication for abca transporters, particularly abca1, abca2, abca5, and abca7, relates to ad.28,50,52,63 their suggested roles in this major burdensome neurodegenerative disease as well as general physiological aspects are summarized in the following sections. abca1 abca1 is the prototype of the abca subfamily,144 was first identified in 1994, and is located on human chromosome 9.145 the complete genomic sequence of human abca1 was reported in 2000. the abca1 gene spans 149 kb comprising 50 exons, and the resulting protein is 2261 amino acids long.146 abca1 is located in the plasma membrane and is also present intracellularly in the endoplasmic reticulum and golgi apparatus, where it mediates the efflux of cholesterol and phospholipids from intracellular compartments to extracellular lipid-free apolipoproteins, mainly apolipoprotein a1 (apoa1) and to a lesser extend apoa2 and apoe, to form high-density lipoprotein (hdl) particles.3,147,148 the lipidation of apoa1 is preceded by abca1 dimerization.149 abca1 thus represents the first and rate-limiting step in the reverse cholesterol transport pathway, which removes excess cholesterol from peripheral tissues via hdl and delivers it to the liver for conversion into bile acids and subsequent excretion. in contrast to peripheral tissues, the physiological role of abca1 in the brain, where it is expressed in all cell types, is not well defined.103 it has been suggested that abca1 is required for cholesterol transport from glial cells to neurons via apoe, which is secreted by glial cells and serves as the main lipid acceptor in the brain.103,125 in vitro and in vivo studies in abca1 knock-out models demonstrated that abca1 is essential for normal apoe secretion and lipidation in the cns.150,151 glial cells deficient for abca1 showed reduced lipid efflux with concurrent lipid accumulation as well as decreased apoe secretion, with apoe particles being small and poorly lipidated. in mice, abca1 knock-out resulted in dramatically decreased brain levels of apoe. moreover, examination of the hippocampi of abca1-deficient mice revealed a decrease in neurite length and number of neurite segments and branches, pointing to an importance of abca1 for neurite integrity.152 the major genetic risk factor for sporadic ad is the allelic state of the apoe genotype, with inheritance of the apoe4 allele markedly increasing disease risk.153,154 recently, rawat et al. investigated how apoe4 affected abca1 expression and function in vitro in astrocytes.155 the authors found that apoe4 decreased abca1 plasma membrane levels and increased abca1 co-localization with late endosomes via activation of adp-ribosylation factor 6, thereby reducing cholesterol efflux and lipidation of apoe particles. they corroborated their findings in blood-cerebrospinal fluid (csf) showing that csf from homozygous carriers of the apoe4 allele was less efficient in stimulating abca1-mediated cholesterol efflux compared to csf from homozygous carriers of the apoe3 allele. a recent study assessed cholesterol efflux capacity of csf by analyzing ad patients, non-ad patients, and control subjects.156 the results demonstrated that abca1-mediated csf-cholesterol efflux capacity was markedly reduced in ad but not in non-ad demented patients. however, this difference did not depend on apoe4 status. interestingly, abca1-mediated csf-cholesterol efflux capacity inversely correlated with total and phosphorylated protein tau, suggesting a link between the dysfunction of hdl-like particle in csf and neurodegeneration. apart from the indirect link via apoe, a direct link between abca1 and ad has also been subject to investigation. expression of hippocampal abca1 was elevated on both the mrna and protein levels and was positively correlated with neuro-pathological changes and dementia severity in ad patients.157 the authors of this study suggested that the observed upregulation of abca1 could be interpreted as a compensatory attempt to clear aβ from the brain. moreover, a variety of studies investigated associations between single nucleotide polymorphisms (snp) in the abca1 gene and the risk for ad,28,108-111 reporting inconclusive results.95,103 a meta-analysis of several studies identified the abca1 rs2422493 (c477t) polymorphism as a risk factor for ad while no association was found for the rs2066718 (v771m) or rs1800977 (c14t) polymorphisms.111 this risk effect for rs2422493 was confirmed in a recent genetic variant association study that, in contrast to the meta-analysis, also reported an increased ad risk for rs2066718 and a decreased ad risk for rs1800977.109 further genetic association studies and meta-analyses are necessary to search for potential associations between abca1 polymorphisms and ad risk. in a recent ad gwas, the rs1800978 polymorphism in the abca1 gene was identified as the lead snp in a new genome-wide significant locus.158 the association of genetic variants of the abca1 gene with ad risk was confirmed by exome sequencing data analysis from 32,558 individuals.158 the study identified around 120 variants that have an increased frequency in early-onset ad (eoad; 1.5%) and late-onset ad (load; 1.1%) cases, compared to 0.5% of all controls. the data demonstrated that ad-association was mainly explained by extremely rare variants, but also by a smaller number of more common variants, e.g., n1800h.159 intriguingly, loss of function and missense variants in the abca1 gene were respectively associated with a 4.7-fold (95%ci 2.2-10.3) and 2.7-fold (95%ci 1.9-3.8) increased eoad risk, and this was lower for load cases suggesting that the burden of damaging abca1 variants was concentrated in younger ad patients. additionally, some long non-coding (lnc) rnas such as lncrna loc286367 have been shown to affect abca1 expression.160 lncrna loc286367 and abca1 are located on the same chromosome but are transcribed in opposite directions. a recent study demonstrated that loc286367 reduces abca1 expression in thp-1 macrophages and increases the levels of proinflammatory cytokines.160 the role of abca1 in aβ deposition and clearance as well as in aβ deposits-related memory deficits has been extensively investigated in app-transgenic mouse models of ad. the lack of abca1 decreased brain apoe levels and either did not affect or increased aβ load.161-163 a recent study utilizing shotgun lipidomics experiments demonstrated a common apoe isoform-specific phospholipid signature between human apoe3/3 and apoe4/4 ad brains and lipoproteins isolated from astrocyte-conditioned media of apoe3 and apoe4 mice.164 interestingly, the lipoproteins derived from wild-type and abca1het mice had phospholipid content apoe3 > apoe4 > apoe3het > apoe4het suggesting that the combination of abca1 insufficiency and apoe4 genotype decreases apoe lipidation even further, thus aggravating apoe4 effect. these findings suggest that poorly lipidated apoe may promote aβ aggregation.129,161-163 in contrast, overexpression of abca1 in an app-transgenic mouse model resulted in increased lipidation, albeit reduced brain levels of apoe and decreased aβ load, implying that highly lipidated apoe may reduce aβ aggregation propensity.127 this is supported by findings of deane et al., who showed that different apoe isoforms may differentially disrupt aβ clearance from mice brains.165 a stable isotope-labelling kinetic study in an app-transgenic mouse model either lacking abca1 or overexpressing abca1 demonstrated increased apoe clearance in both abca1 knock-out and abca1-overexpressing mice, but did not reveal any effect on aβ clearance or production, suggesting that abca1 may regulate aβ deposition by a mechanism other than altering aβ metabolism.166 in contrast, a study assessing the clearance of intracerebrally injected 125i-aβ from the brain reported that abca1-deficiency decreased aβ clearance in non-app-transgenic mice.167 furthermore, knock-out of abca1 was found to augment the dissemination of intracerebrally injected, brain-derived aβ seeds in app-transgenic mice.167 haplodeficiency of abca1 led to decreased brain apoe levels and increased aβ oligomer levels but did not affect aβ deposition in app-transgenic mice.168 however, both haplodeficiency and homozygous knock-out of abca1 aggravated cognitive deficits in app-transgenic mice.152,167,168 lastly, the lack of one copy of abca1 exacerbated memory deficits, decreased aβ clearance, and increased aβ load in app-transgenic mice expressing human apoe4 but not in app-transgenic mice expressing human apoe3.169 abca2 abca2 is predominantly, but not exclusively, expressed in the brain, where it can be found in glial cells and neurons.170-173 on the subcellular level, abca2 is located in endoand lysosomal membranes, facilitating the sequestration of waste substances into intracellular vesicles.172 in addition, it is involved in myelin lipid transport, neural development, and macrophage activation.30,174,175 genetic variations of abca2 were identified as a risk factor for eoad and sporadic ad.52,176 these two studies showed a strong correlation between rs908832 and ad.52,176 however, a later study could not find a link between this snp and any form of ad.177 in addition, abca2 mrna expression was upregulated in ad patients compared to controls suggesting abca2 as a biomarker for differential diagnosis of ad.178 preclinical studies of abca2 suggested that this transporter modulates aβ production via the ldl receptor (ldlr).179,180 abca2 overexpression increased ldlr density, and ldlr deficiency has been described to enhance aβ deposition.181 chen et al. reported a co-localization of abca2 and aβ as well as aβ upregulation in cells overexpressing abca2. in addition, impairment of abca2 expression using small interfering rna (sirna) was accompanied by a decrease in aβ production.182 abca2 depletion has been shown to induce a shift from βto α-secretases and thus, a reduction of app processing by γ-secretase.182 furthermore, abca2 has been proposed to play a role in aβ production as it has been reported to upregulate sphingosine in murine cells and, therefore, to induce app transcription.183 however, another study in human cells could not confirm the modulation of aβ production or cholesterol efflux by abca2.184 thus, further research on the role of abca2 in ad pathogenesis and its potential as a therapeutic target is necessary. abca3 despite its initial report of exclusive lung expression,185 abca3 is also found in other tissues including the brain.186,187 within the brain, the highest levels of abca3 were found in oligodendrocytes.188 abca3 plays a role in producing surfactants in the lung, suggesting that the transporter may also be involved in lipid metabolism in the brain, specifically phosphatidylcholine and phosphatidylglycerol transport. interestingly, phosphatidylcholine has also been discussed in the context of ad.189 a genetic study revealed that mutations in abca3 can also cause cataract-microcornea syndrome, a rare congenital malformation of the eye.141 the actual implications of the potential connection between altered abca3 functionality and ad need to be addressed in future studies. abca4 abca4 is mainly expressed in the retina with very little presence in other tissues of the cns.190 abca4 mutation causes stargardt disease, characterized by macular dystrophy, retinal alterations, and lipofuscin accumulation.60,61,190,191 other retinal diseases, such as fundus flavimaculatus, retinitis pigmentosa, or cone-rod dystrophy, have also been associated with mutations of abca4.55,57,58,192 abca4 is expressed in brain capillary endothelial cells, as well.193 however, no link between abca4 and ad has been suggested to date. abca5 abca5 is a little-known member of the abca subfamily expressed mainly in skeletal muscle with unknown function in the brain.194 studies in peripheral tissues suggest that the function of abca5 is associated with cellular lipid metabolism.195 abca5 knock-out in mice induced signs of lysosomal storage disease in the heart and the thyroid gland.131 in the brain, abca5 is expressed in neurons and, to a lesser extent, in microglia, astrocytes, and oligodendrocytes.195 fu et al. showed that abca5 stimulated cholesterol efflux in neurons and induced a decrease in aβ production probably affecting app processing but not its expression.195 abca6 abca6 is ubiquitously expressed with high levels in liver, lung, heart, brain, and ovaries. this transporter is probably involved in macrophage lipid homeostasis as it is upregulated during macrophage differentiation and is responsive to cholesterol treatment.196 although certain missense variants of abca6 have been correlated with blood cholesterol levels,197 no link between abca6 and ad has yet been found. abca7 abca7 was first identified in the year 2000, and is located on human chromosome 19.198-200 analysis of abca7 mrna expression levels has shown that this transporter is mainly confined to the brain and the immune system.3 due to its high homology to abca1 (54%),200 abca7 was first hypothesized to play an important role in lipid trafficking, mediating cholesterol and phospholipid efflux. abca7 actively transports phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasm to the exocytoplasmic leaflet of membranes.198,199,201 however, in contrast to abca1, abca7 generates only small hdl particles.202 recent research has shown that lipid trafficking by abca7 plays a secondary role. studies in abca7 knock-out models have demonstrated that abca7 is involved in the phagocytotic activity of macrophages and fibroblasts198,203-205 but not in cell cholesterol release.206-208 in 2011, hollingworth et al. identified the abca7 gene as an ad risk locus.198,209 in multiple studies, variants of abca7 have been associated with an increased risk of developing ad.198,210-212 in 2015, steinberg et al. reported that rare loss-of-function variants of abca7 confer a risk of ad in icelanders (odds ratio: 2.12; p = 2.2 ∙ 10-13), and found a similar association in study groups from europe and the united states (combined odds ratio: 2.03; p = 6.8 ∙ 10-15).213 in particular, the rare ad-related polymorphism rs200538373 was associated with an ad risk odds ratio of 1.9.210 these studies suggest that reduced levels of abca7 may increase the risk of ad. nonetheless, it is not clear how these polymorphisms affect abca7 function and contribute to ad progression. increased levels of abca7 expression were described in ad patients and were also positively correlated with cognitive decline.198,211 this finding is consistent with abca7 mrna transcription levels in j20 mice.123 the increase of abca7 may be a compensatory defense mechanism that is insufficient to stop disease progression. furthermore, the rs3764650g allele has been associated with increased neuritic plaques in human patients198,214 and a limitation of the neuroprotective effects of exercise intervention.215 these studies support a potential protective role of abca7 in ad. to date, three potential roles have been identified for abca7 contribution to ad: app processing, immune response, and lipid metabolism. chan et al. proposed an inhibitory effect of abca7 on aβ deposition after showing in vitro inhibition of aβ production independent of β-secretase activity.120 other authors proposed that abca7 is not directly linked to aβ production, but rather through lipid metabolism as abca7 mediates the transport of lipids across the bbb and abca7 loss of function may alter cholesterol transport by decreasing apoe secretion and abca1 expression. this alteration in cholesterol metabolism can also contribute to ad development.216 however, abca7 knock-out induced an increase of aβ load with no difference in clearance rate and an increase of β-secretase expression. on the other hand, abca7 overexpression led to diminished aβ production and improved cognitive function.217,218 nevertheless, abca7 is highly expressed in phagocytic cells, including macrophages and microglia, suggesting a role of the transporter in phagocytosis.188,198 phagocytosis is crucial to maintain brain homeostasis. indeed, ineffective phagocytosis may induce neuroinflammation, which is a risk factor in ad. in addition, microglial cells are involved in phagocytosis and degradation of aβ. thus, an involvement of abca7 in microglial phagocytosis of aβ may explain the contribution of this transporter to ad pathogenesis. in ad patients, increased abca7 transcription has been found in areas with plaques but not in unaltered regions such as the cerebellum.123 this increase in transcription was paralleled by microglia recruitment supporting the contribution of abca7 to microglia-mediated phagocytosis of aβ. in addition, abca7 knock-out mice showed a reduced microglia response after intracerebral aβ injection.123 kim et al. demonstrated an increased aβ load in j20/a7 knock-out mice compared to j20 mice, potentially due to an altered phagocytic function.124,198 furthermore, it has recently been shown that abca7 haplodeficiency disturbs the microglial immune response and causes enhanced aβ accumulation in microglia, probably due to alterations in endolysosomal trafficking.219 last, a new hypothesis has emerged recently, assigning abca7 a prominent role in the altered lipidostasis hypothesis in ad.104 the authors of this study proposed the existence of a neurodegenerative lipid that is naturally removed by abca7. a loss of abca7 function due to the described polymorphisms might accelerate accumulation of this lipid, inducing aβ aggregation. in fact, a link between cholesterol metabolism and abca7-mediated phagocytosis has been reported, which may also explain the protective properties of statin treatment in the development of ad.105,198,203,220 despite recent findings, the role of abca7 in ad pathogenesis remains unclear. according to in vitro and preclinical research, it may be associated with phagocytic activity by microglia, which could be linked to cell cholesterol metabolism.105,198,203 thus, further investigation is required to reveal the role of abca7 in ad pathogenesis and its potential use as a therapeutic target for this neurodegenerative disease. abca8–abca10 so far, no obvious role of abca8–10 has been elucidated for ad, neurodegenerative diseases, nor any human disease. however, several potential intrinsic substrates of abca8 have been identified.10,221,222 furthermore, a significant number of abca transporter modulators have been identified on this target.222 hence, abca8 represents a good model system for the development of potential therapeutics targeting other abca transporters taking the scarce knowledge on this transporter subclass into account. abca12 abca12 is expressed predominantly in the epidermis, and its main function is the transport of lipids.223 it is hypothesized that abca12 plays a role in skin lipid homeostasis. mutations in this gene are associated with lamellar ichthyosis type 2 and harlequin ichthyosis.143,224,225 however, a japanese study investigated common polymorphisms of abca12 and did not find an association with sporadic ad.226 abca13 abca13 is the largest abc transporter with 576 kda.227 it has been reported to be highly expressed in the brain as well as in peripheral tissues.227 a very small study found reduced neuroinflammation and altered abca13 expression in post mortem analyses of brains from patients with lewy body dementia.64 in addition, increased abca13 expression has been reported after stroke in mice.67 furthermore, two studies showed enhanced abca13 mrna expression in schizophrenic patients after different antipsychotic treatments, suggesting a role of this transporter in psychiatric disorders.48,65,66 however, no association between abca13 and ad has been found. figure 1. molecular formulas of prominent interactors of abca transporters. modulators of abca transporter function, trafficking, and regulation ‘modulation’ is a widely used term to summarize actions of small-molecules that have been reported to alter abca transporter function, trafficking, and/or regulation. modulators can be divided into ‘interactors’ and ‘regulators’. interactors summarize compounds that directly bind to abca transports, which can have either inhibiting or activating effects on the transporters. substrates are also included in this category. in terms of abca transporters, however, a direct interaction of these agents with their target(s) has in most cases not yet been comprehensively proven. therefore, compounds that are believed to directly interact with abca transporters extend the category of interactors. figure 1 represents the most prominent interactors of abca transporters and provides additional information about their mode of modulation. regulators are compounds that change abca transporter expression (transcription and/or translation) in terms of induction and/or downregulation. in addition, compounds that regulate abca transporter trafficking can be included into the category of regulators, as this effect was often observed as ‘pseudo-protein increase’ at the cell membrane. figure 2 depicts the most prominent regulators of abca transporters including proposed mode of modulations. figure 2. molecular formulas of prominent regulators of abca transporters. it must be stated that the term ‘inhibitor’ and ‘activator’ are often misused in the literature, as in most cases studies describe a downregulation or induction. in the present review, this mislabeling has been taken into account and the present review and the respective compounds have been allocated into the correct groups. as established earlier,23,24 the compounds are sorted according to their origin: (i) intrinsic substrates and substrate-like molecules, (ii) (other) natural compounds, (iii) pharmacological drugs, (iv) high-throughput screening-(hts)-derived candidates, as well as (v) compounds from synthetic/medicinal chemistry approaches. figure 3 gives a general overview of specific interactors and their postulated mode of modulation. table 2 summarizes all modulators of abca1, the most studied abca transporter, while table 3 summarizes all known modulators in terms of the other abca transporters. the stated concentration values are indicators of bioactivities of the respective compound and are strongly dependent on the testing system utilized. hence, the respective data must be interpreted with caution. figure 3. general overview of proteins participating in abca1 regulation and interaction. small-molecule interactors of abca transporters endoand xenobiotic substrates the most genuine interactors of abca transporters are intrinsic substrates of these transporters. these include cholesterol (figure 1) and other sterol derivatives,10,221,222,228 but also phospholipids (figure 1), sphingolipids228,229 and retinoids (e.g., all-trans-retinal; figure 1).133-138 in addition, certain intrinsic molecules were demonstrated to interact with abca transporters, in particular with abca1230 and abca8.10,221,222 α-tocopherol (vitamin e) was demonstrated to be transported by abca1,230 and to interfere with abca1 regulation.231 the sterol derivatives estradiol-β-glucuronide, estrone sulfate, and taurocholic acid (figure 1), but also the physiological substrate leukotriene c4 (ltc4), the natural compound ochratoxin a, as well as the chemical p-amino hippuric acid were discovered as (potential) abca8 substrates.10,221,222 specifically the abca8-mediated taurocholate export from various human pancreatic cancer cell lines was suggested as the major mechanism behind gemcitabine resistance in these cells,221 which was corroborated in hek293 cells stably expressing abca8.10 in addition, a small body of evidence suggests that abca2 and abca3 contribute to the subcellular sequestration of certain antineoplastic agents into endoand lysosomes.232-235 these agents include cytarabine (abca3),235 daunorubicin (abca3),232,233,235 etoposide (abca3),235 imatinib (abca2 and abca3; figure 1),234,236 mitoxantrone (abca3),235 and vincristine (abca3; figure 1).235 furthermore, several antineoplastic agents were described to have less effect when abca2 was overexpressed in vitro171,237,238 and in vivo.239 for example, the anticancer drug estramustine (figure 1) was effluxed from abca2-overexpressing human ovary carcinoma cells, which were less susceptible to estramustine treatment than the sensitive cell line.171,238 antisense nucleotide treatment against abca2 re-sensitized the carcinoma cells, further demonstrating a role for abca2 in mediating drug efflux.238 furthermore, abca2 knock-out mice had elevated estradiol and estrone levels when treated with estramustine.239 a similar effect in terms of susceptibility and re-sensitization was observed for abca3-mediated transport of miltefosine in leishmania,240 doxorubicin resistance in acute myeloid leukemia cells,237 and cisplatin as well as paclitaxel resistance in several lung cancer cell lines.241 table 2. currently known modulators of abca1. mode of modulation name of modulator effect concentration; concentration range; ec50; dose; ed50 (potential) substrates cholesterol phospholipids β-sitosterol sphingomyelin α-tocopherol activators ati-5261 cs-6253 1.07 µm; 30 mg/kg body weight in mice 0.73 µm; 20 mg/kg body weight in mice inhibitors blt-4 bromosulfophthaleine bumetanide cyclosporine a dids diphenylamine 2-carboxylic acid flufenamic acid furosemide glibenclamide pimecrolimus probucol sirolimus tacrolimus valspodar 150 µm 500 µm 200 µm 1–20 µm; ic50 = 5.1–7.6 µm 40–500 µm 500 µm 500 µm 200 µm 50–1000 µm 20 µm; ic50 = 7.0 µm 1.9–20 µm 20 µm; ic50 = 18.8 µm 20 µm; ic50 = 13.6 µm 5 µm; ic50 = 1.9 µm inducers a-769662 aclarubicin allicin camp butyryl-camp 8-br-camp cpt-camp atorvastatin atra az1–az9 az-1 az-2 az10606120 az876 bcd1 n-benzothiazolyl-2-benzenesulfonamides berberine bergapten bexarotene bezafibrate bms-852927 sodium-butyrate cholesterol cholic acid analog 14b celastrol chalcone derivatives chromene derivatives 2, 3, and 5 chromone analog 6 cl2-57 curcumin daidzein danthron 1,6-o,o-diacetylbritannilactone digoxin doxazosin doxorubicin efatutazone e3317 egcg homo-eriodictyol ethyl 2,4,6-trihydroxybenzoate f1 f4 fargesin fenofibrate fluvastatin fpd5 fucosterol geniposide ginsenoside (derivatives) ginsenoside compound k glycyrrhizine gq-11 gw3965 gw7845 gypenosides hesperetin-7-o-β-d-glucopyranoside hesperetin-7-o-rutinosid 20-(s)-hydroxycholesterol 4-hydroxycholesterol 22-(r)-hydroxycholesterol 22-(s)-hydroxycholesterol 24-hydroxycholesterol 24-(s)-hydroxycholesterol 25-hydroxycholesterol 27-hydroxycholesterol 3-hydroxytyrosol idarubicin kaempferol l836,978 kuwanon g l-839,867 lxr623 lycopene m2 maslinic acid metformin mevalonate mevastatin mitotane naringenin obeticholic acid ondansetron orlistat ouabain paeonol pcb29-pq pemafibrate pestalotioquinoside c phenethyl isothiocyanate tadehagi triquetrum-derived glycosides pioglitazone pitavastatin platycodin d pma ponasterone a pratensein propofol prostaglandin j2 pyrrole-imidazole-polyamide pyrromycin quercetin 9-cis-retinoic acid ro0721957/5 ro0264456 rosiglitazone rpr-5 rutaecarpine and derivatives saikosaponin a 24-(s)-saringosterol sb203580 scutellarein selenium serdemetan simvastatin spf1 spf2 soraphene a 24-(s)-stigmast-5-ene-3β,24-diol cannabis sativa-derived stilbenoids sulfoxaflor tanshindiol c taraxasterol testosterone tetradecylthioacetic acid to901317 tr1 trichostatin a troglitazone ttnpb urolithin a urolithin b urolithin b sulfate vitamin d3 vitexin way-254011 wy14643 bexarotene derivatives z10 and z36 zafirlukast 250 µm ec50 = 0.49 µm 2.5–10 µm 0.1–10 µm 300 µm 0.3–1000 µm 300–500 µm 5–10 µm; 4 mg/kg body weight in mice 0.25–10 µm ed50 = 1.49–341 µmol/kg body weight in mice 10 µm 10 µm 10 µm ed50 = 0.956 µmol/kg body weight in mice ec50 = 0.035 µm ec50 = 0.37–33.42 µm 5–20 µm 12.5–50.0 mg/kg body weight in rats 0.1–1 µm 10–200 µm ed50 = 2.10 µmol/kg body weight in mice 1000–10.000 µm; 200–400 mg/kg body weight in mice 12.9–100 µm 5–40 µm 0.1–1.0 µm; 0.5–1 mg/kg body weight in mice 5–10 µm; 20 mg/kg body weight in mice 25 µm 25 µm 10 µm; 10 mg/kg body weight in mice 5–40 µm ec50 = 3.17 µm 10–40 µm; 60 mg/kg body weight in mice 8–10 µm; 10 ml/kg body weight in mice 0.010 µm 10 µm 0.0316–1 µm; 20 mg/kg body weight in mice 40 µm 0.01–1 µm; ec50 = 0.2 µm 40 mg/kg body weight in mice 41.4–165 µm 50–100 µm ed50 = 10 µm 20 µm; 50 mg/kg body weight in mice 2.77–40 µm 1–20 µm 1 µm; 0.005–0.02 mg/kg body weight in mice 100-200 µm 515 µm; 50–100 mg/kg body weight in mice 10–30 µm 1.25 µm 60.8–243 µm 20 mg/kg body weight in mice 0.5–50 µm; ed50 = 0.969 µmol/kg body weight in mice 5 µm 5 µg/ml 107–431 µm 100 µm; 3 mg/kg body weight in mice 5–20 µm 1–20 µm 1–25 µm; ec50 = 1.0 µm 5–20 µm 20 µm 0.5–1.5 µm 2–12.4 µm 6.21 µm–10 µm 2–5 µm 0.1 µm 2.5–10 µm u.c.a 20 µm 0.1–1 µm 0.1–1 µm; ed50 = 31.5 µmol/kg body weight in mice 2.2–6.6 mg/kg body weight in ferrets 10 µm 20 µm 10 µm 5–500 µm 50 µm 20–50 µm 25–100 µm 40 mg/kg body weight in mice 1 µm 50 µm 0.010 µm 100 µm 5–10 µm 0.1–10 µm; 0.3 mg/kg body weight in mice 50 µm 30–75 mg/kg body weight in mice 10 µm 5–10 µm; ec50 = 1.28–7.474 µm; 20 mg/kg body weight in mice 0.1–10 µm 5–20 µm 0.32 µm 2–5 µm ec50 = 2.91 µm 50 µm 1–20 µm 1 µm; 1 mg/kg body weight in mice ec50 = 0.85 µm 20 µm; 12.5 mg/kg body weight in mice 0.04–10 µm; ec50 = 0.29 µm 0.050 µm 0.005 µm 0.05–10 µm; ec50 = 1.49 µm 5 µm 0.035–34.98 µm; ec50 = 0.27 µm 2–8 µm 10 µm 20 µm 50 mg/kg body weight in mice 2.5–5 µm 2–5 µm 10 µm 1 µm 1 µm 0.03–20 µm; ec50 = 0.01391 µm 10 µm 2.5–3 µm u.d.b in aphis gossypii 10 µm 3–12 µm 0.001–0.01 µm 0.75% of high-fat diet in mice 0.1–25 µm; ed50 = 4.11 µmol/kg body weight in mice 10 µm 99.2 µm; 0.5 mg/kg body weight in mice 1 µm 0.25–10 µm 20 µm 0.1–10 µm 10 µm 1 µm 50 µm ed50 = in mice 0.05–100 µm 1 µm; 40 mg/kg body weight in mice 2.5–5 µm downregulators 5cppss-50 acrolein 8-br-camp angiotensin ii asymmetric dimethylarginine atorvastatin atr-101 bisphenol a chalcone derivatives 4-{[4-(4-chlorophenyl)-2-thiazolyl]amino}phenol cholesterol dexamethasone dibutyl phthalate egcg fluvastatin ggpp gsk2033 gw6471 gw9662 desulfated holothurin a homocysteine lipopolysaccharides lovastatin ly294002 methionine mevalonate mevastatin mitotane ndea 1,2,3,4,6-penta-o-galloyl-β-d-glucose phenylalanine-proline pitavastatin pravastatin raloxifene rosuvastatin simvastatin sr9243 tamoxifene α-tocopherol γ-tocopherol toremifene troglitazone valproic acid varenicline 20 µm 5–20 µm 0.3 µm 0.0001–0.100 µm 0.5–1 µm 0.1–100 µm 10–30 µm 100 µm 10 µm 5 µm 150 µm 0.1–2.5 µm; 8 mg/kg body weight in rats 0.1 µm 100 mg/kg body weight in mice 0.1–100 µm 10 µm–200 µm 0.05–5 µm 10 µm 10 µm 2.68–4.47 µm 50–200 µm 1 mg/ml 0.1–100 µm 20 µm 17 g/kg food in mice 100 µm 0.05–50 µm 50 µm 100 mg/kg body weight in rats 25–300 mg/kg body weight in mice 1000 µm; 600 mg/kg body weight in rats 10 µm 50 µm 10 µm 5–50 µm 0.1–100 µm 1 µm 2.5–10 µm 50–100 µm 50–100 µm 10 µm 10 µm 1000 µm 10 µm; 0.5 mg/kg body weight in mice stabilizers cyclosporine a diphenoquinone erythrodiol alln leupeptin probucol spiroquinone testosterone wogonin 10 µm 0.0001–0.0005 µm 10–15 µm 50 µm 1170 µm u.c.a 0.025–0.050 µm 0.01 µm 10–40 µm destabilizers brefeldin a 2-bromopalmitate cycloheximide gö6976 monensin a serdemetan tunicamycin 17.8–36 µm 7.5–60 µm; ic50 = 15 µm 355 µm 10 µm 10 µm 2–5 µm 2.41 µm a u.c. = unspecified concentration b u.d. = unspecified dose strikingly, abca2 co-localized with the lysosomal-associated membrane protein 1 (lamp1) – an endolysosomal marker – as well as the fluorescence probe dansyl-estramustine. this co-localization indicates a direct sequestration of this antineoplastic drug into endoand/or lysosomes.171 on the other hand, the susceptibility of abca3-overexpressing ccrf-cem leukemia cells to the antineoplastic agents cytarabine, methotrexate (figure 1), vincristine, but also the anti-inflammatory drug dexamethasone, was reduced compared to their parental counterparts.242 taken together, abca2 and abca3 are contributors to mdr, and the number of potential abca2 and abca3 substrates may be even higher than currently suggested. interestingly, missense mutations of abca4 were associated with chloroquineand hydroxychloroquine-associated retinopathy,243 although contradictory studies exist.244 a direct interaction was postulated, however, not proven. nevertheless, these results suggest chloroquine and hydroxychloroquine as potential abca4 substrates. table 3. currently known modulators of abca transporters other than abca1. mode of modulation name of modulator effect concentration; concentration range; ec50; dose; ed50 abca2 (potential) substrates cytarabine dexamethasone estramustine estradiol estrone imatinib methotrexate inducers imatinib methotrexate progesterone sulfoxaflor u18666a u.c.b 1.28 µm 31.8 µm u.d.c in aphis gossypii 5 µm downregulators celecoxib 10 µm abca3 (potential) substrates cisplatin cytarabine dasatinib daunorubicin dexamethasone doxorubicin etoposide imatinib methotrexate miltefosine mitoxantrone nilotinib paclitaxel vincristine inducers dasatinib 5-fu imatinib methotrexate nilotinib vitamin c u.c.b 50 µm 0.1–12.5 µm 1.28 µm u.c.b 56.78 µm downregulators genistein indomethacin lipopolysaccharides pk11195 sirolimus 3–9 µm 2 µm 10 µg/ml; 100 µg/ml in chicken lungs u.c.b 2 µm stabilizers c13 c14 c17 genistein ivacaftor 10 µm 10 µm 10 µm 10 µm 1 µm abca4 (potential) substrates chloroquine hydroxychloroquine β-ionone 11-cis-retinal 13-cis-retinal all-trans-retinal all-trans-retinoic acid all-trans-retinol n-retinylidene-phosphatidyl-ethanolamine phosphatidyl-ethanolamine stabilizers c3 c4 c18 lumacaftor 10–20 µm 1–20 µm 10–20 µm 10–20 µm abca5 inducers atorvastatin bezafibrate cholesterol gw3965 rosiglitazone tacrolimus troglitazone 20 µm 10 µm 100–150 µm 0.5 µm 10 µm 0.04 µm 10 µm downregulators digoxin 2.5 g/kg body weight in mice abca6 inducers acitretin lovastatin mevastatin 1–10 mg/kg body weight in pigs 10 µm 10 µm downregulators lovastatin mevastatin 10 µm 10 µm abca7 inducers ponasterone a pravastatin rosuvastatin 1–5 µm 50 µm 5 µm downregulators cholesterol digoxin 25-hydroxycholesterol 2 mm 2.5 g/kg body weight in mice 2.48 µm abca8 (potential) substrates p-aminohippuric acid estradiol-β-glucuronide estrone sulfate glibenclamide leukotriene c4 ochratoxin a taurocholic acid (potential) inhibitors digoxin dofequidar glibenclamide ochratoxin a probenecid verapamil verlukast 250 µm 10 µm 250 µm 50 µm 1000 µm 1000 µm 100 µm inducers gemcitabine polyethyleneglycol-block-polyactide nanoparticles 0.05–0.8 µm 42.04 g/kg body weight in rats downregulators digoxin 2.5 g/kg body weight in mice abca9 downregulators digoxin 2.5 g/kg body weight in mice abca12 inducers ceramide n-hexanoyl-d-erythro-sphingosine ciglitazone d609 xanthate d-ddmp gi 251929x gw610742 d-mapp d-nmappd d-ppmp d-pppp 22-(r)-hydroxycholesterol to901317 troglitazone 5 µm 7.5 µm 25 µm u.c.b 10 µm 8 µm 10 µm 5 µm 5 µm 10 µm 10 µm 10 µm 7.5 µm stabilizers acitretin 1–10 mg/kg body weight in pigs a apart from cholesterol and/or phospholipids b u.c. = unspecified concentration c u.d. = unspecified dose inhibitors to date, the number of small-molecules that (are believed to) directly interact with abca transporters is very low. for example, only 14 inhibitors can be found in the literature regarding the most studied prototype of abca transporters, abca1.245-248 only four of these inhibitors are associated with half-maximal inhibition concentrations (ic50),245,249 which is the ‘golden surrogate’ to evaluate and judge inhibitory activities of small-molecules. the following section will highlight these small-molecules as well as inhibitors of other abca transporters. abca1 glibenclamide and 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid (dids) as outlined above, abca1 is the most studied and understood abca transporter, although its particular role in neurodegenerative diseases in general51,103 – and in ad in particular – is not well understood.28-30,43,95,102 however, over time, several agents were found to impact abca1 transport function. the most prominent examples are glibenclamide and dids (both figure 1), which were first shown to inhibit abca1 in 1997.247,248 these drugs blocked the abca1-mediated 125i efflux from murine peritoneal macrophages247 as well as human abca1-transfected xenopus laevis oocytes.248 glibenclamide and dids inhibited the abca1-mediated transport of cholesterol and other sterols as well as phosphoand sphingolipids. thus, these agents became the ‘standard abca1 inhibitors’ and have frequently been used in abca1 studies ever since.229,250-269 glibenclamide and dids were preferred over other discovered abca inhibitors, such as bumetanide, diphenylamine 2-carboxylic acid, flufenamic acid, furosemide, and bromosulfophthaleine.248 specifically glibenclamide was rigorously evaluated regarding its mechanism of action. it was demonstrated that glibenclamide prevented cross-linking of 125i-marked apoa1 to abca1,267,270 not interfering with abca1 location at the cell surface.267 in essence, glibenclamide and dids may play a significant role in the development of future modulators of abca transporters in general. probucol and cyclosporine a less prominent but also well characterized are the antilipidemic drug probucol246,271-278 and the immunosuppressant cyclosporine a245,249,258,279-281 (both figure 1). probucol was demonstrated to reduce the cholesterol efflux from different abca1-overexpressing murine and human macrophages,275-278 and total lipid release (cholesterol + phospholipids) from human wi-38 fibroblasts.246 vice versa, probucol increased accumulation of free cholesterol, cholesterol esters, phosphatidylcholine, and sphingomyelin in human fibroblasts.246 additionally, probucol was reported to prevent cell surface-specific binding of 125i marked apoa1 to abca1.246,278 similarly, this effect has already been demonstrated for glibenclamide before.267,270 interestingly, it was shown that total abca1 protein levels were increased after exposure to probucol due to decreased degradation.246,275 this qualifies probucol also as a stabilizer. however, as its inhibiting effect is far more pronounced, we have included it as an inhibitor here. the immunosuppressant cyclosporine a has been characterized as an abca1 inhibitor in multiple studies.245,249,258,279-281 this inhibition was shown to be direct through a radiolabeled variant of cyclosporine a and purified abca1.245 cyclosporine a not only functionally inhibited abca1-mediated cholesterol and phospholipid efflux,245,249 and caused intracellular accumulation of cholesterol,258 but also inhibited the abca1-dependent binding of alexa 546or 125i-labeled apoa1,245,249 as demonstrated for glibenclamide267,270 and probucol246,278 before. interestingly, toxicity assays demonstrated that cyclosporine a negated the positive effect of an abca1 inducer on cell viability when cells were exposed to aβ proteins.280 this was confirmed in vivo in c57bl/6 mice that had reduced hdl levels.249 interestingly, cyclosporine a was shown to decrease abca1 turnover, increasing its presence at the cell surface by a factor of two as demonstrated with a gfp-labeled abca1 variant,249 suggesting a similar mode of inhibition as for probucol.275 thus, as for probucol,246,275 cyclosporine a also appears to have a stabilizer function,275 but is included in the current section due to its pronounced inhibitory role. morevover, the cyclosporine a analog valspodar (psc833) inhibited direct binding of radiolabeled cyclosporine a to abca1, revealing that valspodar also acts as an abca1 inhibitor.245,282 furthermore, several other calmodulin antagonists inhibited abca1-mediated cholesterol efflux and binding of apoa1.245 these include pimecrolimus,245 sirolimus,245 and tacrolimus,245 suggesting these molecules as potential scaffolds for the development of future abca1 modulators. other abca1 inhibitors in terms of other small-molecules that were suggested to inhibit abca1 function, blt-4 has been demonstrated to inhibit cholesterol and phospholipid export from adipocytes and macrophages,255 and to decrease cholesterol efflux from abca1-transfected hek293 cells. blt-4 was also shown to inhibit 125i-marked apoa1-binding to abca1,270 as demonstrated for glibenclamide,267,270 probucol,246,278 and cyclosporine.245,249 other abca transporters while abca1 can be considered a less-studied abc transporter with certain knowledge about its function and interfering small-molecules,18 all other abca transporters belong to the group of under-studied abc transporters that cannot be addressed by small-molecules with very rare exceptions.18 one rare example is abca8. using the xenopus laevis oocytes model in vitro testing system,248 tsuruoka et al. reported inhibitors of this transport protein.222 while digoxin, probenecid, and verapamil (all figure 1) could be identified as very weak inhibitors of abca8-mediated estradiol-β-glucuronide transport, dofequidar (ms-209), ochratoxin a, and verlukast (mk-571; figure 1) were discovered as moderately potent inhibitors.222 in addition, glibenclamide was also suggested to (partially) inhibit abca8 function.266 activators although activators of abc transporters have been reported, as for example, for abcb123 and abcc transporters,23,283-288 these reports are somewhat scarce compared with other classified modulators of abc transporters. in terms of a subclass abc transporters, no small-molecule activators are known. however, it is well established and has been extensively demonstrated that abca1 activity depends on (co)-administration of hdl and/or apoa1.117 hdl and apoa1 are not small-molecules but peptides, and therefore fall outside of the scope of the present review. similarly, it has been shown in several reports that hdl-mimics consisting of 26 amino acids are able to increase abca1-mediated transport.289 although these molecules are also not small-molecules, the scarceness of activators of abca transporters warrants the inclusion of these middle-sized molecules here. in 2004, structural elements of apoa1 were discovered to promote abca1-mediated cholesterol efflux.290 in 2007, vedhachalam et al. discovered that the c-terminus of apoe promoted abca1-mediated efflux from murine j774.a1 macrophages.291 the latter discovery led to the development of two short-length peptides, ati-5261 and cs-6253, consisting of 26 amino acids each.289 their amino acid sequences expressed in single-letter code are evrskleewfaafrefaeeflarlks289 and evcitskleewlaalcitelaeellacit-lks (cit = citrulline),292 respectively, which is of particular interest for the development of novel lead structures. both peptides increased abca1-mediated cholesterol and phospholipid transport in murine and human macrophages.289,292 interestingly, cs-6253 decreased 125i-labed apoa1 binding to abca1,292 as demonstrated for glibenclamide,267,270 probucol,246,278 cyclosporine a,245,249 and blt-4270 before. however, cs-6253 was shown to compete with apoa1 to promote abca1-mediated transport.292 both ati-5261 and cs-6253 have a high practical relevance regarding ad and other neurodegenerative diseases, as these agents demonstrated in vivo efficacy.289,293 ati-5261 treatment of high fat diet-fed apoe knock-out mice decreased cholesterol levels in both plasma and feces and reduced atherosclerotic lesions.289 for cs-6253, a reduction of aβ42 levels and tau protein phosphorylation in transgenic humanized apoe4 mice was demonstrated, which was accompanied by improved cognitive functions.293 interestingly, an elevation of abca1 protein was also observed in treated mice.293 indeed, a stabilization and/or induction may also have contributed to the observed effects. however, the proven direct binding of these agents suggested that activation takes place as the major mode of action. nonetheless, cs-6253 has not been tested in ad mouse models so far, and being a peptide, it would not be suitable for oral application in patients. small-molecule regulators of abca transporters the herein discussed regulators interfere with abca transporter expression and/or trafficking. important representatives are depicted in figure 2 and additional information is given in terms of their mode of modulation. since many different pathways are involved in abca transporter regulation, figure 3 provides a general overview of participating proteins and protein families in terms of the most studied abca transporter, abca1. inducers abca1 lxr and rxr pathways given the findings in ad mouse models with knock-out of abca1/abca1 or overexpression of abca1, upregulating abca1 activity may be a therapeutic strategy for decreasing aβ pathology in ad. abca1 is under the transcriptional control of the nuclear receptors liver-x-receptor (lxr) and retinoid-x-receptor (rxr),294-296 which can be targeted by small-molecule agonists of lxr and rxr to induce abca1 expression (figure 3). numerous studies reported that treatment of app-transgenic mice with lxr or rxr agonists decreased aβ load126,297-301 and/or improved cognitive impairment.126,297,298,300 other studies reported cognitive improvement without significant changes in aβ load in app-transgenic mice treated with lxr agonists.302,303 lxr and rxr agonists have already been described extensively as potential therapeutics in the literature, also with respect to ad.304 the present review will focus on those agonists that were reported in clear association with abca1. oxysterols and retinoic acids 22-(r)-hydroxycholesterol (figure 2) has been established as the natural gold standard for abca1/abca1 induction through lxr activation,122,205,249,252,259,262-264,268,277,278,305-315 while 9-cis retinoic acid (figure 2) became the natural gold standard for rxr activation.122,245,249,259,262,264,277,278,309,311,313,316 the inducing effects were described both on abca1/abca1 mrna122,205,252,263,264,305,307-311,313,315-317 and abca1 protein levels.122,252,263,264,306,309-311,316,318 other oxysterols like 4-hydroxycholesterol, 20-(s)-hydroxycholesterol, 22-(s)-hydroxycholesterol, 24-hydroxycholesterol, 24-(s)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and cholesterol itself also induced abca1/abca1 mrna205,305,313,315,319-327 and abca1 protein levels.321,328 the increase in abca1 protein was functionally confirmed by an enhanced cholesterol305,306,313,315,318 and phospholipid efflux,311,318 as well as reduced total cholesterol influx.305 specifically 22-(r)-hydroxycholesterol and cholesterol induced both lxra/lxra and lxrb/lxrb.310,321 additionally, cholesterol also induced murine peroxisome proliferator-activated receptor γ (ppar-γ) mrna (pparg),321 which represents an important alternative pathway for abca1/abca1 induction. furthermore, 24-(s)-hydroxycholesterol reduced in parallel the sterol regulation element-binding protein 2 (srebp2) gene expression (srebp2).323 the sreb protein family also represents another important pathway in abca1/abca1 regulation. the 9-cis-retinoic acid derivative all-trans¬-retinoic acid (atra) significantly increased abca1/abca1 mrna and abca1 protein content in murine and human macrophages, which was paralleled by increased lxra mrna levels in human macrophages.329 this increase resulted in a subsequently enhanced cholesterol efflux from murine macrophages. atra is an agonist of the retinoic acid receptor (rar),329 which is in close relation to the rxr receptor and a potential target of retinoic acid derivatives. to901317 and gw3965 the synthetic gold standard and most studied abca1/abca1 inducer in the literature is to901317 (often referred to as ‘t0901317’; figure 2).205,245,250,252,259,260,262,264,271,272,279,280,282,308,310,317,319,322,324,326,328-345 to901317 targeted both the lxr α250,310,328,330,332,335,337-340,342 and lxr β pathways,250,310,338,342 which correlated to abca1/abca1 induction on mrna and abca1 protein levels.205,250,279,282,310,319,322,324,326,328,330-335,337-340,342,343 in addition, an induction of srebp1c/srebp1c has also been observed.336,342 functionally, to901317 increased cholesterol efflux,250,259,260,262,264,282,319,324,329,331,342 decreased intracellular aβ content, and increased aβ secretion from different murine brain cells.126,345 further, it reduced aβ25-35-mediated toxicity toward cells by induction of abca1.280 in addition, to901317 mitigated memory deficits in high-fat diet-fed app23 mice, reducing both plaque and soluble aβ protein levels.344 besides, to901317 reduced methionine-(homocysteine)-induced atherosclerotic lesions in apoe knock-out c57bl/6 mice.335 these findings were paralleled by an increase of abca1 mrna and abca1 protein content,335 suggesting a potential relevance of to901217 in ad therapy, although it must be taken into account that lxr activators, in particular to901317, were demonstrated to have severe side effects in mice, such as neutropenia, hypertriacylglycerolemia, hepatic triacylglycerol accumulation, and hepatic steatosis.271,346,347 the second most common synthetic lxr-α and lxr-β agonist is gw3965 (figure 2).255,272,317,319,321,334,348-352 gw3965 increased mrna317,319,321,348,349,351,352 and protein levels255,272,351 in different abca1-expressing cells. functionally, increased abca1 mrna and abca1 protein levels correlated with enhanced cholesterol efflux.255,351 strikingly, exposure of murine bv2 microglia to gw3965 reduced aβ42 levels due to an enhanced degradation of aβ42,126 suggesting that abca1 contributes to general aβ degradation. finally, gw3965 significantly increased abca1 transcription in c57bl/6 mice,334,351 and improved contextual memory as well as aβ pathology in tg2576 mice,126 emphasizing its high relevance in ad therapy. abca1 other lxr agonists and inducers sterane and sterane-like natural compounds several sterane derivatives were demonstrated to target lxr-α and lxr-β activation253,307,310,353 and/or lxra/lxra and lxrb/lxrb upregulation,330,332,354,355,356,357 resulting in induction of abca1/abca1. celastrol,330,332 digoxin,253 fuco-sterol,308 certain gypenosides,354 ouabain,253 platy-codin d,355 saikosaponin a,356 24-(s)-saringosterol,307 24-(s)-stigmast-5-ene-3β,24-diol,307 taxarasterol,353 testosterone,357 and tr1310 increased abca1/abca1 mrna307,308,310,330,332,353,354,356,357 and/or abca1 protein content310,253,353,354,355,357 leading to an enhanced efflux of cholesterol in vitro253,308,330,332 and decreased intracellular cholesterol and/or phospholipid levels in vitro330,332,354,356,357 and in vivo in mice.253 the effect of fucosterol was comparable to that of the standard abca1/abca1 inducer to901317.308 a correlation to srebp1(c) upregulation308,307,357 and srebp1 protein expression357 could be determined in case of fucosterol,308 24-(s)-saringosterol,307 24-(s)-stigmast-5-ene-3β,24-diol,307 and testosterone.357 in case of celastrol, the regulation of intracellular cholesterol was pinned to an activation of autophagy330,332 and lipophagy,330 which are processes that may be associated with aβ degradation. flavonoids the flavonoids naringenin,339 quercetin,358 and vitexin359 increased abca1/abca1 mrna339,359 and abca1 protein levels339,360,358 by induction of lxra/lxra mrna358,359 and lxr-α protein.339,360 the effect of naringenin and the standard abca1/abca1 inducer to901317 were additive. naringenin was shown to be dependent on the camp-activated protein kinase (ampk) regulation (ampk), as well as srebp1c regulation.339 the ampk pathway is another very important regulator of abca1 expression. functionally, cholesterol efflux from human339,360 and murine360 macrophages was increased in the presence of naringenin.339,360 in vivo, naringenin and quercetin induced abca1360 and abca1,361,362 as well as abca1-mediated cholesterol transport,360 which was reflected in reduced atherosclerotic lesions in the aorta of high-fat diet-fed c57bl/6 mice.360 in terms of quercetin, a protein increase of lxr-α and ppar-γ was observed.361 chalcones, the precursors of flavonoid biosynthesis, were also demonstrated to intervene with abca1 expression. the chalcone derivatives 1h,363 1m,363,364 and 1m-6364 were demonstrated to increase abca1 mrna and abca1 protein levels in thp-1 macrophages,363,364 which was accompanied by an increase in lxra mrna and lxr-α protein levels.363 the intracellular lipid content was decreased, while the cholesterol efflux was increased after exposure of thp1-cells to 1m-6.364 in addition, srebp1 mrna was increased by 1m-6,364 and aortic atherosclerotic plaques were reduced in ldlr knock-out c57bl/6 mice.364 polyphenols and diterpenoid natural compounds the polyphenols kuwanon g,365 paeonol,252 the celtis biondii-derived compound ethyl 2,4,6-trihydroxybenzoate,342 and the diterpenoid farnesin366 increased abca1/abca1 mrna252,342,365,366 and abca1 protein252,342,365,366 content in an lxr-α-252,366 and lxr-β-dependent342 manner, which in parallel reduced cholesterol content252 and increased abca1-mediated cholesterol efflux in various cell lines.252,342,366 in vivo, farnesin increased abca1 protein content and cholesterol efflux in apoe knock-out c57bl/6 mice in primary peritoneal macrophages and the aorta, which was reflected in reduced atherosclerotic plaques.366 other natural compounds several other natural compounds induced abca1/abca1 targeting lxr-α and lxr-β activation256,272,256,349,367 and/or lxra/lxra and lxrb/lxrb induction.331,348,350,368,369,370,371,372,373,374 the garlic ingredient allicin,350 the alkaloid berberine,256 the coumarin bergapten a,368 certain pestalotiopsis neglecta-derived chromene derivatives,348 the rheum palmatum-derived anthra-quinone danthron,369 the lacton 1,6-o,o-diacetylbritannilactone,371 epigallocatechin gallate (egcg),370 the glycoside geniposide,375 the vegetable ingredient phenethyl isothiocyanate,373 the carotenoid lycopene,372 the pestalotiopsis neglecta-derived hydroquinone pestalotioquinoside c,349 the alkaloid rutaecarpine,367 selenium,374 the macro-lactone soraphene a,272 and vitamin d3331 led to increased abca1/abca1 mrna256,272,331,348,369,256,367,370,372,373 and abca1 protein256,272,331,349,350,368,369,256,367,371,373,374 content in vitro331,349,350,369,375,374 and in vivo,368,369,370,371,372,373 enhancing cellular cholesterol efflux256,272,256,367,369 and reducing intracellular cholesterol con-tent.331,350,369,256,367,375,372,374 danthron also increased ampk protein levels,369 while egcg downregulated srebp1 mrna and srebp1 protein content.370 lycopene induced ppara mrna in tobacco carcinogenand cigarette smoke-exposed ferrets,372 while isothiocyanate induced pparg mrna as well as ppar-γ protein content in high fat diet-fed c57bl/6 mice.373 the inducing effects on abca1 expression of vitamin d3 and to901317 were additive.331 danthron, egcg, geniposide, and rutaecarpine demonstrated also reduced atherosclerotic lesions in apoe knock-out c57bl/6 mice,369,370,375,367 and isothiocyanate ameliorated the aortic injury of the high-fat diet in the same mice.373 pharmacological drugs several pharmacological drugs also demonstrated an induction of abca1/abca1 through lxr-α and/or lxr-β, including the α1-blocker doxazosin,376 the 5-ht3 receptor antagonist ondansetron,279 and the anesthetic propofol.377 consequently, increased abca1 mrna279,376 and abca1 protein279,376 levels were observed in human279,377 and murine279,376 macrophages376,377 as well as astrocytes.279 functionally, ondansetron induced apoe efflux,279 while propofol led to increased cholesterol efflux.377 in addition, propofol increased pparg mrna and ppar-γ protein content in human macrophages.377 furthermore, certain antineoplastic agents interfered with abca1 expression via lxr-α and/or lxr-β. doxorubicin demonstrated an lxr activation with subsequent induction of abca1 mrna and abca1 protein in vitro and in vivo.250 functionally, doxorubicin elevated cholesterol export in vitro. it was shown that intraand extracellular levels of cholesterol, cholesterol precursors, and several oxysterols were elevated after exposure to doxorubicin. these precursors included lathosterol, lanosterol, and desmosterol, while the oxysterols included 7-α-hydroxycholesterol, 7-β-hydroxycholesterol, 7-ketocholesterol, 24-hydroxycholesterol, and 27-hydroxycholesterol. the authors suggested that doxorubicin exposure induced cholesterol metabolism subsequently leading to an induction of abca1. besides, idarubicin augmented also abca1 mrna levels in vitro. synthetic compounds and hts hits other synthetic compounds have been shown to induce abca1/abca1 expression by lxr-α and/or lxr-β induction. the polymer pyrrole-imidazole-polyamide activated a promoter region for abca1 expression and thereby increased cholesterol and lipid efflux from raw264.7 cells.376 the authors confirmed their findings in vivo, revealing increased abca1 mrna and abca1 protein content in peripheral blood mononuclear cells and the liver in c57bl/6 mice after exposure to pyrrole-imidazole-polyamide. in addition, the lxr agonist lxr623 induced abca1 mrna and abca1 protein levels in two human renal adenocarcinoma cell lines334 as well as abca1 mrna levels in vivo in c57bl/6 mice.378 this induction was reflected in reduced intracellular cholesterol and triglyceride levels. it must be noted that several other synthetic lxr-α and lxr-β agonists induced abca1 expression in vivo: az1–az9, az876, bms-852927, f1, way254011.378 finally, an hts approach discovered two lxr-α and lxr-β agonists as novel small-molecule abca1/abca1 inducers: f4 and m2.319 synthetic approaches a few synthetic approaches have aimed toward the development of abca1/abca1 inducers.271,336,352,379-382 the cholic acid analog 14b,336 the thiophene derivative cl2-57,271 as well as derivatives of n-benzothiazolyl-2-benzene-sulfonamide,379 ginsenoside,352 and rutaecarpine,367 all induced abca1/abca1 mrna336,352,381 and abca1 protein271,336,379,381 content in vitro271,336,379 and in vivo,271 targeting the lxr-α/lxr-β pathway352 by activation271 or induction336 of lxr-α/lxra/lxra and/or lxr-β/lxrb/lxrb. in vitro, cholesterol efflux increased379,381 and intracellular cholesterol as well as lipid content were reduced,336,352 while plasma and liver triglycerides levels were reduced in vivo in high fat diet-fed c57bl/6 mice.271 interestingly, 14b induced farnesoid-x-receptor (fxr) transcription (fxr),336 and cl2-57 inhibited rxr-β, ppar-γ, and ppar-δ,271 finally, singh et al. described highly potent lxr-α and lxr-β agonists with effect at concentrations in the nanomolar range.382 the described podocarpic acid derivatives have not yet been demonstrated to induce abca1. however, these compounds were designated as potential abca1 inducers by the authors,382 and their high potency makes them interesting candidates for further evaluation. such synthetic approaches should be highlighted,271,336,352,379-382 as chemical derivatization of abca1 inducers and elucidation of their structure-activity relationships (sar) have not yet been comprehensively assessed. more reports are needed to gain innovative molecules that can be considered clinically for the treatment of various abca1-related diseases. abca1 other rxr agonists and inducers in terms of synthetic rxr agonists, the 4-chromanon derivatives spf1 and spf2 increased abcb1 mrna and abca1 protein levels and lowered aβ25–35-mediated cell toxicity in vitro.280 the same effect was observed for the rxr agonist bexarotene,280 an fda approved drug against t-cell lymphoma-related cutaneous malformations. bexarotene was used as a standard inducer of abca1/abca1 via the rxr pathway in several studies.271,272,280,319,380 induction of abca1 mrna and abca1 protein levels was maximal for bexarotene in combination with to901317.280 bexarotene is of particular practical relevance as a potential treatment against ad due to its in vivo effects. in different ad mouse models, bexarotene increased abca1 mrna and abca1 protein levels, but also reduced cerebral load of aβ and hyperphosphorylated protein tau, which is also a histological marker in ad and other dementias.297,383 this prospect led to synthetic bexarotene derivatives, specifically z10 and z36.380 both candidates induced abca1 protein expression by rxr-α activation and reduced aβ burden in the hippocampus of female app/ps1 mice. this coincided with an enhanced abca1 protein expression in bv2 cells. moreover, the pan-rar agonist ttnpb also increased abca1 protein content in murine macrophages in an rxr-α-dependent manner. however, the effect was generally smaller compared to the effect of atra.329 finally, a combination of the lxr and rxr agonists ro0721957 and ro0264456 increased abca1 mrna in thp-1 macrophages accompanied by increased cholesterol efflux.384 ro0264456 was demonstrated to increase abca1 protein content in combination with to901317.260 abca1 – protein kinase c (pkc), ampk, and p38 mitogen-activated protein kinase (mapk) an alternative approach to induce abca1 is targeting the pkc pathway (figure 3). pkc agonists were extensively used to induce abca1/abca1 mrna and abca1 protein levels.230,248,249,255,265,266,273,278,289-292,384-387 prominent pkc agonists include camp313 as well as synthetic derivatives, such as 8-bromo-camp (8-br-camp; figure 2),230,249,255,266,290,292 8-(4-chlorophenylthio)-camp (cpt-camp),273,291,384 and dibutyryl-camp.385-387 the observed effects ranged in the same order of magnitude as the combination of 22-(r)-hydroxycholesterol and 9-cis-retoic acid.313 the increase in abca1/abca1 mrna and abca1 protein levels was reflected in an enhancement of abca1-mediated cholesterol and phospholipid efflux,249,255,386 and increased apoa1 binding to murine raw264.7 macrophages.385-387 similar observations have been made for the pkc stimulant phorbol 12-myristate 13-acetate (pma), which induced abca1 protein expression and abca1-mediated cholesterol and phospholipid release.386 pma is also the standard substance used to differentiate human monocytic leukemia cells into thp-1 macrophages – a standard host system for abca transporter evaluation.231,245,249,256,268,272,275,292,308,310,312-316,321,328,335,338, ​339,341,342,360,363,364,366,377,384,388-397 regarding the ampk pathway (figure 3), the natural compound curcumin induced abca1/abca1 mrna338,388 and abca1 protein levels388,394 as well as cholesterol efflux338,388,394 in thp-1338,388,394 and raw264.7394 macrophages, which was also mediated through lxr-α activation.338 however, these lxr-α activating effects were much more pronounced in combination with the gold standard to901317.338 other ampk-targeting agents are a 769662 and metformin,398 which induced abca1/abca1,398 lxra/lxra,396,398 and lxrb/ lxrb396,398 in human398 and murine (primary) macrophages,398 leading to increased cholesterol efflux.396 concerning the mapk pathway (figure 3), the sterane glycoside ginsenoside compound k increased abca1 mrna and abca1 protein levels in murine macrophages, reducing intracellular lipid content and promoting autophagy.399 these effects were pinned to a negative impact on the mapk pathway. finally, a synthetic inhibitor of mapk, sb203580, was shown to induce abca1 protein in combination with the above mentioned geniposide in vitro in murine macrophages.375 abca1 the ppar pathway another well-known approach to induce abca1 involves the ppar pathway (figure 3).268,272,295,309,315,321,326, 327,337,343,395,400-409 certain ppar/ppar inducers and/or ppar activators have been described above, as these modulators also have effects on the lxr pathway.321,361,372,373,377 several natural compounds target the ppar pathway, such as the flavonoids homo-eriodictyol,402 hesperetin-7-o-β-d-glucopyrano-side,402 scutellarein,403 and the antimycotic trichostatin a.410 these compounds increased abca1402 and pparg402 mrna as well as abca1,402,410 ppar-α,403 and ppar-γ402,410 protein levels in vitro402,410 and in vivo.403 decreased intracellular cholesterol levels were also observed.402 trichostatin a reduced aortic atherosclerotic plaques in high-fat diet-fed apoe knock-out mice,410 and an upregulation of abca1, ppar-γ, and lxr-α/β protein levels was observed in aortic cells as well as peritoneal macrophages.410 several drugs and drug-like ppar agonists were revealed to induce abca1/abca1 mrna and/or abca1 protein content, including the ppar-α agonists fenofibrate,326,400,404 pemafibrate (k 877),405 wy14643,268,343 and rpr-5,268 as well as the ppar-γ agonists efatutazone,337 pioglitazone,272,309,326,395,407 pitavastatin,343 prostaglandin j2 (pg-j2),268,327 rosiglitazone (figure 2),268,309,315,408,409 troglitazone,268 and gw7845,315 but also the broad-spectrum ppar-α, ppar-β, and ppar-γ agonist bezafibrate268,327 and the multitarget ppar-α, ppar-γ, and ppar-δ agonist tetradecylthioacetic acid.401 this induction was observed for abca1/abca1 mrna268,315,343,401,405 as well as abca1 protein levels,268,337,343,395,405,409 and was functionally confirmed by increased cholesterol efflux.268,315 a connection between the ppar and lxr pathways has also been drawn,268,326,327,337,400 highlighting the importance of both pathways for abca1/abca1 induction. furthermore, fenofibrate had a positive impact on both the lxr-α and ampk pathways400 certain ppar agonists have been used as standard inducers of abca1, e.g., pioglitazone407 and rosiglitazone.408 synthetic ppar agonists were also reported to induce abca1.406 the benzothiazole derivative e3317 dose-dependently increased abca1/abca1 mrna and abca1 protein levels though ppar-γ activation in several cell lines.406 this was reflected in decreased cholesterol efflux and reduced intracellular cholesterol content. finally, a molecular docking approach to discover novel ppar agonists has yielded gq-11, which induced abca1 mrna in livers of c57bl/6 ldlr knock-out mice.407 abca1 the 3-hydroxyl-3-methyl glutaryl-(hmg)-coa-reductase pathway other targets for abca1/abca1 induction are the 3-hydroxyl-3-methylglutaryl-(hmg)-coa-reduc-tase and cellular cholesterol synthesis (figure 3).318,343 several hmg-coa-reductase inhibitors such as atorvastatin (figure 2),330,343,362 fluvastatin,312,411 mevastatin (compactin),318 pitavastatin,318,343 and simvastatin312,343 increased abca1/abca1 mrna312,343 and abca1 protein levels,362,411 as well as abca1-mediated cholesterol efflux.318 these data are surprising, as one might expect the loss-of-function of an enzyme in the cholesterol synthesis pathway to induce a decrease of abca1, preventing cholesterol depletion from cells.314384,412 conversely, the overproduction of cholesterol leads to the opposite effect, as demonstrated for mevalonate, which is a building block of cholesterol synthesis413 and has been demonstrated to increase abca1/abca1 mrna312,314 and to abrogate abca1 downregulation.312 pitavastatin addressed srebp-driven promotor regions upregulating abca1 mrna levels,343 and atorvastatin reduced atherosclerotic plaques in apoe knocked-out c57bl/6 mice by induction of abca1 protein content in the murine aorta.362 other abca1 inducers sterane and sterane-like natural compounds several other agents were reported to induce abca1/abca1 mrna and/or abca1 protein level(s), with some studies reporting a unique mechanism of action for these agents. such compounds include the sterane derivative ponasterone a (ecdysone; abca1 protein; abca1-mediated cholesterol and phospholipid transport),202 and the enoxolone derivative glycyrrhizine (abca1 protein).414 in addition, the sterane derivative and farnesoid-x-receptor (fxr) activator obeticholic acid induced abca1 mrna levels in vivo in the ileum of srb1-deficient c57bl/6 mice.415 in thp-1 macrophages, the sterane-like maslinic acid induced abca1 mrna levels, paralleled with an increased cholesterol efflux from these cells.390 finally, the salvia miltiorrhiza-derived tanshindiol c was demonstrated to induce peroxiredoxin 1 mrna (prdx1) and protein (prdx1) content in murine raw264.7 cells.416 prdx1 was demonstrated to regulate abca1 mrna and abca1 protein expression. a reduction of intracellular cholesterol levels in murine peritoneal macrophages could also be observed. flavonoids the flavonoids daidzein (figure 2),309 kaempferol,397 and pratensein309 induced abca1 mrna309,397 and abca1 protein levels309 as well as abca1-mediated cholesterol efflux.397in addition, hesperetin-7-o-rutinosid (hesperidin) abrogated the negative effect of varenicline on abca1 protein expression in raw264.7 macrophages.417 the authors could underpin their findings with a reduction of aortic atherosclerotic plaques in apoe knock-out c57bl/6 mice along with reduced lipid levels in peritoneal macrophages derived from these mice. polyphenols and polyphenol-like natural compounds several polyphenols and polyphenol-like compounds induced abca1 mrna408,418 and abca1 protein393,404 levels in murine393,404,408,418 and human393 macrophages, leading to an increased cholesterol efflux.404,408,418 these include certain cannabis sativa-derived stilbenoids404 as well as the tadehagi triquetrum-derived phenylpropanoid glycosides urolithin a418 and urolithin b (sulfate).393 in vivo, atherosclerotic plaques were reduced after urolothin b treatment. one phenylpropanoid glycoside was demonstrated to increase lxra, but none of the other compounds could confirm these results. given that the effect of all compounds on abca1 expression was similar, it is likely that another, yet unknown pathway was the major contributor to the observed effects. other natural compounds sodium butyrate induced abca1 mrna and abca1 protein levels in murine raw264.7 cells, accompanied by an increased efflux of cholesterol from these cells.419 this induction was reflected by increased abca1 protein content in vivo, reduced plasma cholesterol and triglyceride levels, and reduced aortic atherosclerotic lesions and hepatic steatosis in high fat diet-fed apoe knock-out c57bl/6 mice. pharmacological drugs several pharmacological drugs induced abcb1/abca1 mrna309,420,421 and abca1 protein,309,391,421 including the anti-obesity drug orlistat,391 the antibiotic sulfoxaflor,420 the leukotriene receptor antagonist zafirlukast,421 as well as the anthracyclines aclarubicin309 and pyrromycin.309 zafirlukast in particular reduced intracellular cholesterol and lipid content in oxidized ldl-(oxldl)-induced lipid-overloaded raw264.7 macrophages, and increased cholesterol efflux from these cells.421 finally, it should be highlighted that mifepristone has frequently been used in a mifepristone-inducible transfection system to stabilize and increase abca1 expression in abca1-transfected baby hamster kidney (bhk)-21 cells. this abca1 induction could be functionally confirmed by increased abca1-mediated cholesterol and phospholipid efflux.245,273,422 synthetic compounds, hts hits, and synthetic approaches the purinergic p2y7 receptor antagonists az-1, az-2, and az10606120 increased abca1 mrna and abca1 protein levels and resulted in enhanced cholesterol efflux from human ccfsttg1 astrocytoma cells.423 the polychlorinated biphenyl quinone 2,3,5-trichloro-6-phenyl-[1,4]-benzo-quinone (pcb29-pq)424 and the fluorescigenic pyrazoline derivative 5 (fpd5)425 increased abca1 mrna424 and abca1 protein425 content in raw264.7 macrophages and reduced cholesterol content in these cells.424,425 in vivo, fpd5 reduced aortic lipid and cholesterol content and atherosclerotic lesions in apoe knock-out c57bl/6 mice. inducers of other abca transporters abca2 and abca3 as detailed above, abca2 and abca3 are believed to contribute to multidrug resistance in cancer.171,232-239,241,242 in human k562 leukemia cells, it was demonstrated that the tyrosine kinase inhibitor (tki) imatinib induced increased levels of abca2 mrna and abca2 protein.236 furthermore, the tkis dasatinib, imatinib, and nilotinib increased abca3 mrna levels in various cancer cell lines as well as in tki-treated leukemia patients.426 the antimetabolite 5-fluorouracil (5-fu) induced expression of abca3 mrna in a cholangiocarcinoma cell line,427 and methotrexate increased abca2 and abca3 mrna in a leukemia cell line.242 finally, the steroid hormone progesterone,179 the antibiotic sulfoxaflor,420 and the endosomal cholesterol transport inhibitor u18666a179 induced abca2/ abca2 transcripts420 in aphis gossypii420 as well as in abca2-transfected chinese hamster ovary (cho) cells and hepg2 cells179 abca5 and abca6 as discussed earlier, cholesterol and its derivatives have been shown to induce abca1/abca1 mrna and/or abca1 protein levels.122,205,249,252,259,262-264,268,277,278,305-315,319-328 induction by cholesterol has also been demonstrated for abca5 mrna and abca5 protein levels in raw264.7 macrophages.321 this effect relied on the induction of lxra, lxrb, and pparg. consequently, several lxr and ppar agonists increased abca5 expression, including bezafibrate (ppar-α, ppar-β, and ppar-γ; abca5 mrna and abca5 protein), gw3965 (lxr; abca5 mrna), rosiglitazone (ppar-γ; abca5 mrna), and troglitazone (ppar-γ; abca5 mrna) in murine raw264.7 macrophages.321 in addition, the hmg-coa-reductase inhibitor atorvastatin increased abca5 mrna and abca5 protein levels.321 interestingly, the abca1 inhibitor tacrolimus245 showed induction of abca5 mrna in human brain microvascular endothelial cells.428 the hmg-coa-reductase inhibitors lovastatin and mevastatin resulted in an induction of abca6 mrna in the human endothelial cell line ea.hy926.429 finally, in an abca12 pig model of the rare and lethal skin disease harlequin ichthyosis, it was demonstrated that treatment with the synthetic retinoid acitretin leads to a compensatory induction of abca6 mrna.430 abca7 similarly to abca1,202 the sterane derivative ponasterone a increased both abca7 protein expression and abca7-mediated transport, mainly of phospholipids, but also of cholesterol to a small extent.202 hmg-coa-reductase inhibitors were described above to interfere with abca1/ abca1312,318,330,343,362,411 and abca5321 expression. in addition, certain compounds were also demonstrated to interfere with abca7 expression.205,431 these include pravastatin205,431 and rosuvastatin (figure 2).431 these agents increased abca7 mrna and abca7 protein levels in vitro,205,431 whilst pravastatin had the same effects in vivo in murine peritoneal macrophages.431 surprisingly, this increase of abca7 mrna and abca7 protein levels was accompanied by a downregulation of lxra and upregulation of srebp2 in vitro.431 functionally, pravastatin and rosuvastatin reduced intracellular cholesterol content431 and induced phagocytosis in vitro and in vivo.431 these effects occurred in response to an abca1 downregulation by hmg-coa-reductase inhibitors as described earlier.312,321,384,432,439,429 due to their functional similarity, the upregulation of abca7 could be a compensatory mechanism to counteract the loss of abca1.198 similarly, the observed lxra downand srebp up-regulation may be a compensatory mechanism to counteract the loss of intracellular cholesterol. finally, as described for abca1,422 exposure of abca7-transfected bhk-21 cells to mifepristone increased abca7 protein content and abca7-mediated transport of phospholipids and, to a much lesser extent, of cholesterol.422 abca8 abca8 mrna and abca8 protein content were induced by gemcitabine in panc-1 and cfpac-1 human pancreatic cancer cells.221 in rat liver, an induction of abca8 was demonstrated via microarray analysis of cdna when the rats were exposed to polyethyleneglycol-block-polylactide nanoparticles.433 abca12 several lxr and ppar agonists induced abca12/abca12 expression, such as 22-(r)-hydroxycholesterol (lxr),434 to901317 (lxr),430,434 ciglitazone (ppar-γ),434 gi 251929x (ppar-γ),434 troglitazone (ppar-γ),434 ceramide n-hexanoyl-d-erythro-sphingosine (ppar-δ),435 and gw610742 (ppar-δ).434 interestingly, inhibition of certain enzymes to prevent ceramide processing elevated intracellular ceramide content and subsequently abca12 mrna levels.435 these enzymes include, for example, the glycosyl-ceramide-transferase synthase [d threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-pro-panol (d-ppmp), d-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (d-pppp / p4) and dl-threo-1-phenyl-2-de-canoylamino-3-morpholino-1-propanol (d-ddmp)], the sphingomyelin synthase [tricyclo[5.2.1.02,6]decanyl)ethanedithioic acid (d609 xanthate)], as well as the ceramidase [d-erythro-2-tetradecanoylamino-1-phenyl-1-propanol (d-mapp) and (d nmappd / b13)].435 downregulators abca1 lxr and rxr pathways – intrinsic substrates the intrinsic metabolite asymmetric dimethylarginine (adma) reduced abca1 mrna and abca1 protein levels in human and murine j744 macrophages in combination with oxldl, resulting in increased intracellular cholesterol and triglyceride levels.392 this was accompanied by decreased efflux of cholesterol from these cells. the authors suggested a negative effect on the lxr-α pathway. in this regard, the lxr-α downregulator homocysteine significantly reduced abca1/abca1 mrna and abca1 protein expression in vitro in thp-1 macrophages as well as in vivo in macrophages from apoe knock-out c57bl/6 mice.335 the cattle metabolite dipeptide phenylalanine-proline decreased abca1 mrna and abca1 protein levels in human colorectal adenocarcinoma-derived caco-2 cells.436 the observed downregulation of lxrb mrna could explain the negative impact on abca1 expression. in vivo, the jejunal abca1 mrna levels were decreased in wistar rats.436 the abca1 substrate α-tocopherol230 reduced abca1/abca1 mrna levels in vitro and in vivo.231 the same effects were observed for γ-tocopherol in vitro, most likely through the same mechanism. the authors suggested a negative impact on the lxr pathway due to deprived oxycholesterol derivatives after α-tocopherol treatment both in vitro in hep3b cells and in vivo in rat liver.231 lxr and rxr pathways sterane and sterane-like natural compounds cholesterol and its derivatives have extensively been used to induce abca1/abca1 expression122,205,249,252,259,262-264,268,277,278,305-315,319-328 however, mid-term exposure to excess cholesterol decreased abca1 expression though a negative impact on lxra, lxrb, and pparg expression.321 similar observations have been made for the sterol derivative dexamethasone, which also reduced abca1/abca1 mrna and abca1 protein expression in vitro and in vivo by downregulation of lxra/lxra mrna and lxr-α protein levels as well as upregulation of srebp2 and hmg-coa-reductase gene expression (hmgcr).437 finally, an abca1 mrna reduction was observed in murine raw264.7 macrophages for the thelenota ananas-derived saponin desulfated holothurin a.438 interestingly, hmgcr was downregulated after exposure to desulfated holothurin a, which contradicts other findings.437 lxr and rxr pathways – other natural compounds certain chalcone derivatives also caused reduced expression of abca1 protein.363 in addition, lipopolysaccharides reduced abca1 protein content in endometrial endothelial cells from c57bl/6 mice, which was accompanied by increased cholesterol levels in these cells.374 a parallel reduction in lxr-α protein was also observed. finally, the carcinogenic agent n-nitrosodiethylamine (ndea) demonstrated in vivo in wistar albino rats a downregulation of lxra and lxrb mrna as well as lxr-α and lxr-β protein levels and, subsequently, abca1 protein.368 lxr and rxr pathways – synthetic compounds and hts hits in terms of other lxr antagonists and downregulators, gsk2033 (figure 2),272,330,333 5cppss-50,357 and sr9243333 reduced abca1 mrna and abca1 protein expression.272,330,333,357 hmg-coa-reductase pathways – intrinsic substrates and pharmacological drugs the peptide hormone angiotensin ii reduced cholesterol efflux from murine peritoneal macrophages.439 this reduction could be reversed by the angiotensin ii receptor antagonist losartan. the authors concluded that abca1 was not involved in this process, as no concurrent change in abca1 expression was observed.439 however, in another report, angiotensin ii indeed demonstrated a reduction of abca1 mrna and abca1 protein levels in human podocytes.440 the authors concluded a contribution of the hmg-coa-reductase, srebp1, and srebp2.440 geranylgeraniol pyrophosphate (ggpp; figure 2), a product of the mevalonate pathway, reduced abca1 mrna expression in human macrophages, which was blocked by the prenylation inhibitors l836,978 and l-839,867.314 in addition, a reduction of abca1-mediated cholesterol export was observed, which is also true for mevalonate itself.318 ggpp was used as a standard abca1 downregulator in certain studies.279,354,366 as discussed above, atorvastatin,343 fluvastatin,312 pitavastatin,318,343 and simvasta-tin312,343 have been shown to increase abca1/abca1 mrna levels,312,343 and to enhance abca1-mediated cholesterol efflux.318 however, atorvastatin,312,321,384 fluvastatin,312 pitavastatin432 and simvastatin312,384 have also been reported to reduce abca1/abca1 transcription312,321,384,432 and abca1-mediated cholesterol efflux.384,431 these observations are in agreement with other reports on hmg-coa-reductase inhibitors that downregulated abca1.312,431 in particular, lovastatin,312 mevastatin (compactin),412 pravastatin,431 and rosuvastatin431,441 reduced abca1/abca1 mrna312,431 and abca1 protein431 levels. these findings are expected given that the loss of cholesterol by interruption of cholesterol synthesis leads to a compensatory reduction of cholesterol efflux.314384,412 the contradictory results relating to abca1 may be caused by the use of variable experimental conditions between studies, such as different cell lines, assay methodologies, or small-molecule-related aspects, such as concentration, distribution, and protein binding. finally, a similar interconnection between hmg-coa and abca1 was drawn for the antineoplastic agent mitotane, which downregulated abca1 mrna441 and increased intracellular cholesterol levels.333,441 however, mitotane in combination with lxr antagonists and lxr downregulators had an inverse effect on mrna regulation, increasing abca1 expression.327 pkc pathway intrinsic substrates interestingly, it was also demonstrated that long-term exposure to low concentrations of 8-br-camp, a standard abca1/abca1 inducer,230,249,255,266,290,292 led to decreased apoe secretion from human monocyte-derived macrophages.266 apoe secretion can be considered as a surrogate marker for abca1-mediated cholesterol transport. ppar pathway – pharmacological drugs and synthetic compounds regarding the important ppar pathway, it must be noted that troglitazone, indicated above as an abca1 inducer,268 was also reported to downregulate abca1 transcription.321 these inconsistent effects may be explained partially by the different concentrations used (1 μm vs 10 μm),268,321 but may also be related to cross-talk between the ppar, lxr, and mevalonate pathways. the ppar-γ antagonist gw9662 (figure 2) reduced abca1 protein levels.406 other abca1 downregulators – natural compounds other small-molecules have been reported to act as abca1/abca1 downregulators, acting independently of the previously mentioned lxr, rxr, ppar, and hmg-coa-reductase pathways. natural compounds such as α,β-unsaturated carbonyl derivative acrolein,442 the polyphenol bisphenol a,443 and the polyphenol 1,2,3,4,6 penta-o-galloyl-β-d-glucose444 demonstrated an abca1 mrna443,444 and abca1 protein442 downregulation in vitro443,442 and in vivo.444 the effect of acrolein could be abrogated by 3-hydroxytyrosol,442 an inducer of abca1 protein content.445 srebp2 has been demonstrated to be targeted by egcg in high fat diet-fed transgenic srebp+/+ wistar rats, resulting in abca1 mrna downregulation, while an abca1 mrna upregulation could be observed under the same conditions in srebp knock-out wistar rats.446 other abca1 downregulators – pharmacological drugs exposure of the human non-small cell lung cancer lines a549 and h358 to the antiepileptic drug valproate led to downregulation of abca1 mrna and abca1 protein levels through a histone deacetylase 2-(hdac2)-mediated mechanism. in parallel, the authors observed an increased sensitivity of these cells to cisplatin.447 the selective estrogen receptor modulators raloxifene, tamoxifen, and toremifene were reported to reduce abca1 protein content in thp-1 macrophages along with decreased cholesterol efflux and increased intracellular cholesterol levels.341 tamoxifen and raloxifene treatment decreased serum hdl-cholesterol levels in mice. in addition, tamoxifen reduced cholesterol levels in serum, liver, and feces of mice after injection with cholesterol-loaded macrophages.341 interestingly, the downregulation of abca1 protein content by these estrogen receptor modulators could not be demonstrated for murine liver, indicating a macrophage-specific effect.341 varenicline, a drug used in smoking cessation, was shown in vivo to promote aortic atherosclerotic lesions in apoe knock-out c57bl/6 mice.417,448 the authors demonstrated that intracellular lipid content in peritoneal macrophages was increased, and a decreased abca1 protein expression was confirmed in vitro in raw264.7 macrophages. finally, the antineoplastic agent gefitinib reduced abca1 protein content in various non-small cell lung cancer cell lines.400 other abca1 downregulators – synthetic compounds the plasticizer dibutyl phthalate389 and the pi3k/akt inhibitor ly294002421 reduced abca1 mrna389 and abca1 protein389,421 expression and increased cellular cholesterol and lipid levels389 in human389 and murine421 macrophages. the sphingosine kinase 1 and 2 inhibitor 4-{[4-(4-chlorophenyl)-2-thiazolyl]amino}phenol was demonstrated to downregulate abca1 protein expression in murine primary macrophages, which was dependent on the sphingosine kinase 2 as well as the sphingosine-1-phosphate receptor.306 this abca1 protein downregulation was accompanied by a reduced cholesterol efflux. the acyl coenzyme a cholesteryl acyl transferase (acat) inhibitor atr-101 reduced abca1 mrna levels and induced an increase in intracellular cholesterol content in h295r cells.251 the authors suggested that this was caused by inhibition of abca1 but provided no clear proof of direct inhibition of abca1. therefore, this compound was classified as a downregulator. other abca transporters abca2 and abca3 compared to abca1, knowledge relating to downregulators of the other abca transporters is very limited. as discussed above, human leukemia cells exposed to imatinib displayed increased abca2 mrna and abca2 protein expression.236 celecoxib abrogated this effect.236 a similar observation was reported for abca3, where the anti-inflammatory drug indomethacin and the abca1 inhibitor sirolimus245 (figure 2) downregulated abca3 mrna in various cancer cell lines.426,449,450 this treatment also resulted in a sensitization of these cell lines toward the tkis dasatinib, imatinib, and nilotinib when treated with indomethacin.426 other compounds were also reported to downregulate abca3/abca3 including the flavonoid genistein,451 lipopolysaccharides452 – already demonstrated above as abca1 protein downregulators374 – and the translocator protein ligand pk11195.453 the effect of lipopolysaccharides could be abrogated by ascorbic acid (vitamin c). abca5–abca9 interestingly, the abca8 inhibitor222 and abca1 protein inducer253 digoxin downregulated abca5 and abca7–9 in murine liver.454 the hmg-coa-reductase inhibitors lovastatin and mevastatin downregulated abca6 mrna in human umbilical vein endothelial cells.429 the cholesterol derivative 25-hydroxycholesterol, which was introduced above as an abca1 mrna inducer,327 showed the opposite effect on abca7 mrna.324 this finding is in agreement with a report stating that excess cholesterol reduced abca7 protein content in both human and murine fibroblasts.205 stabilizers of abca transporters stabilizers are compounds that promote functional activity of abc transporters through increasing their presence at the site of action (e.g., the cell membrane) either without interfering with mrna or protein levels, or in addition to these effects. the categorization is difficult, as the necessary information regarding many modulators of abca transporters is lacking and the underlying mode of modulation cannot be precisely identified. in this section, we consider only those modulators which predominantly interfere with abca1 trafficking, with relatively minor or no additional modes of action/modulation. stabilizers are of particular interest, as they may represent a novel generation of functional abc transporter activators, expanding treatment options for several diseases, particularly ad. abca1 probucol and cyclosporine a were demonstrated above to decrease abca1 turnover and increasing abca1 protein content at the cell membrane.246,275 arakawa et al. demonstrated that the probucol metabolites spiroquinone and diphenoquinone did not inhibit abca1-mediated transport like their parent compound but rather increased the fraction of functional abca1 in the cell membrane.275 this stabilization led to increased cholesterol and phospholipid efflux. both effects were observed at very low nanomolar concentrations,275 while abca1 mrna remained stable.275 strikingly, spiroquinone and diphenoquinone decreased vascular lipid deposits in vivo in cholesterol-fed rabbits,275 which may be of relevance for ad and potentially other neurodegenerative diseases. a similar mode of stabilization, albeit with less potency and no in vivo confirmation, has been observed for the flavonoid wogonin,254 the olive oil-derived compound erythrodiol,395 and certain thiol proteinase inhibitors, in particular n-acetyl-leu-leu-norleucinal and leupeptin.316,386 finally, the abca1 mrna and abca1 protein inducer testosterone was demonstrated to promote abca1 trafficking to the cell membrane.357 other abca transporters the cystic fibrosis transmembrane conductance regulator (cftr; abcc7) correctors c13,455 c14,455 c17,455 genistein,456 and ivacaftor (figure 2)456 were demonstrated to rescue abca3 mutants by increasing total abca3 mutant protein levels,455 promoting subcellular targeting of abca3 into vesicular bodies,455 and improving lipid transport function of abca3.456 furthermore, the correctors lumacaftor (vx-809; figure 2), c3, and c4, and c18 increased the presence of abca4 at the cell membrane in abca4-overexpressing hek293 cells, indicating promotion of abca4 trafficking to the plasma membrane.457,458 promotion of trafficking has already been demonstrated for other abc transporters, such as abcc123,24 and abcc7.459 hence, this mechanism represents a new potential therapeutic option for abca transporter-related ad. as proposed for abcc7,460 the authors suggested a direct binding of the correctors to the abca4 protein,457 which has not yet been proven. in an abca12 pig model of harlequin ichthyosis, acitretin (figure 2) treatment resulted in a redistribution of abca12 in the skin compared to wild-type pigs, and thus, a higher survival rate.430 destabilizers of abca transporters natural compounds in contrast to compounds that promote trafficking of functional abca1 to the plasma membrane, other compounds that have the opposite effect have been named ‘destablizers’. so far, only agents targeting abca1 are known. the lactone antibiotic brefeldin a (figure 2) interfered with abca1 cell-surface localization, recycling, and intracellular trafficking.387,461-463 these effects were at least in part dependent on the interaction with brefeldin 1-inhibited guanine nucleotide exchange protein (big1).461 this interference reduced the functional fraction of abca1 and, consequently, abca1-mediated cholesterol and phospholipid transport.255 similar observations have been made for the polyether-antibiotics monensin, which reduced abca1 turnover and trapped it inside endoand lysosomes. subsequently, monensin reduced the functional presence of abca1 at the cell surface,464 lowered cholesterol efflux,463 and increased intracellular cholesterol content.463,464 the same was demonstrated for nigericin, another polyether-antibiotic, which increased intracellular cholesterol concentration,463 and inhibited abca1-mediated cholesterol efflux from raw264.7 macrophages.385 inhibition of intracellular organelle transport as suggested for brefeldin a387,461-463 and monensin463,464 likely applies to nigericin as well.463,465 in addition, the endoplasmic reticulum stress promotor, tunicamycin, also reduced abca1 protein levels.360,466 this ‘downregulation’ is most likely mediated though stress-induced impaired abca1 trafficking and/or increased abca1 degradation.466 however, in terms of selective targeting of abca1 in particular, or abca transporters in general, these agents are less suitable as in vivo agents and serve better as in vitro controls. the palmitic acid derivative 2-bromopalmitate (figure 2) inhibited trafficking of abca1 to the plasma membrane and reduced abca1-mediated cholesterol efflux.273,467 however, the observed effect that abca1 did not translocate to the cell membrane in hek293/abca1 cells467 has not been demonstrated in bhk-21/abca1 cells.273 pharmacological drugs interestingly, the experimental anticancer drug serdemetan (jnj-26854165) was demonstrated to induce abca1 mrna levels but reduce abca1-mediated cholesterol efflux.468 the abca1 mrna induction was due to induction of lxra and lxrb. the abca1 mrna increase was also reflected at the protein level, which increased within 48 hours of exposure to serdemetan before a sudden decrease occurred. the authors also showed that abca1 turnover and degradation were increased. thus, serdemetan can be considered a destabilizer. synthetic compounds cycloheximide was frequently used to interrupt intracellular trafficking of vesicles, including abca1 containing endoand lysosomes.387,464,468 as mentioned earlier, abca1 is stabilized by n-acetyl-leu-leu-norleucinal.316,386 this stabilization could be abrogated by the protein kinase c inhibitor gö6976, which affected not only abca1 protein content, but also cholesterol and phospholipid transport.386 part ii: pipeline development to gain novel diagnostics and therapeutics in silico methodologies to predict novel lead structures rational drug design is the innovative process of identifying pharmaceutically relevant drug candidates. it is based on the information obtained in association with the drug target, e.g., abc transporters. in the following section, we will discuss computational approaches for in silico operations that help to identify novel lead molecules for potential diagnostic and therapeutic application. structure-based drug design the development of computational methodologies for structure-based drug design to understand the relationship between transporter sequence/structure and function depends on the availability of structural as well as biological information. recent advances in experimental approaches for structure determination have facilitated high-quality depictions of the structures of a growing number of abc transporters in different conformational states.469 these experimental approaches include in particular x-ray crystallography and cryo-electron microscopy (cryo-em). recently, the cryo-em structures of human abca1470 and human abca4471-473 with resolutions of 4.1 å and 3.3–3.6 å, respectively, were reported. in addition, a cryo-em structure of human abca7 has been announced474 on biorxiv (biorxiv.org), which was, however, not published to this date (pdb id: 7kqc). nevertheless, a homology model of abca7 has been recently developed.475 figure 4 shows the structures of abca1, abca4, and abca7 as determined by cryo-em as well as homology modelling. figure 4. available structures of abca transporters: the cryo-em structures of human abca1470 (very left; pdb id 5xjy) and abca4 [left (pdb id 7lkp, middle (pdb id 7e7i), and right (pdb id 7m1q)]471-473 as well as the homology model developed for human abca7 (very right).475 all three transporters are typical abca transporters with three crucial structural parts: two nucleotide-binding domains (nbds; intracellular), two membrane-spanning domains [msds (2 x 6 transmembrane helices tms); inter-membrane space], and two large extracellular domains (ecds; extracellular). considering the available structural knowledge, a ‘common’ abca transporter possesses a very long amino acid sequence (>2000 amino acids) and consists of two membrane-spanning domains (msd1 and msd2) each composed of six transmembrane helices (tm1–6 and tm7–12). these msds are followed by a cytoplasmic region comprising a nucleotide-binding domain (nbd1 and nbd2) and a small regulatory (r1 and r2) domain, which have been proposed to stabilize the interaction between nbd1 and nbd2470,473 and were found to strongly interact with each another in the absence of atp.471,472 abca transporters are ‘type ii transporters’ in which the msds indeed form a tunnel for substrate translocation from the cytosol to the lumen, however, represent separate entities without swapping/twisting of the msds, as this is the case with classical ‘type i transporters’ like abcb1.476 most tms are completely exposed to the hydrophobic environment of the membrane, which could promote the attraction and binding of fat-soluble cholesterol as well as phospholipids before guidance to and through the substrate translocation tunnel, and which hosts several cholesterol and phospholipid binding sites.470-474 a unique feature amongst abca transporters in comparison to other abc transporters is the existence of two large extracellular domains (ecd1 and ecd2). these domains together form a channel embedded in hydrophobic amino acids470-472 and are believed to facilitate intermediate storage of cholesterol470 and phospholipids. they have also been suggested as the primary binding site of apoa1,471,477 as indicated by the latest data on abca4.471 a large gap exists between the ecds and msds, pointing to strong conformational changes that are required for abca transporter function.470 another common feature amongst abca transporters are four intracellular and extracellular helices (ih1–4 and eh1–4), which are believed to provide the necessary flexibility for interaction between the msds and nbds in the substrate translocation process,478 and were suggested to enable proper folding and function of these transporters.471 of important note is that abca1 and abca4 share sequential and structural similarities with the abcg family, in particular with abcg5/abcg8,470 which is the model type ii transporter.478 this similarity suggests an evolutionary relevance amongst various abc transporter subfamilies. more importantly, conserved sequential and structural similarities also support the translation of knowledge gained on other abc transporter subfamilies to abca transporters.470,472 this is of particular interest when novel lead structures for new pharmacological targets, in this case under-studied abc transporters,18 are focused,6,18 and specific binding sites located within the msds or nbds are targeted. based on the sequence information of abc transporters within the same family, homology-modeling techniques are the preferred choice for structure determination and binding site elucidation if these subtypes do not yield x-ray or cryo-em structures. this methodology is of particular relevance for closely related homologs with high medical relevance,198 such as abca7 (similarity a1/a7: 54%; similarity a4/a7: 49%).200 the generated homology models can be refined further by molecular dynamics simulation, in which the transporter movement (‘trajectory’) is simulated to potentially unravel relevant transporter conformations. very recently, potential abca1 drug binding sites have been proposed by this methodology,479 and an abca7 homology model has been developed for molecular docking experiments.475 molecular docking is a very popular method for predicting binding orientations or poses of small-molecules within the transporter. most often, the docking programs account for full conformational flexibility of ligands within the binding site, treating the protein as a rigid body. binding site identification is an important prerequisite in the structure-based drug design implementation. in terms of abc transporters, the search for binding hot spots and cavities on the entire volume of the protein (e.g., through blind docking) is necessary due to the general lack of information on binding sites of abc transporters. recently, in search of highly effective modulators addressing abcg2-mediated mdr, derivatives of quinazolines were synthesized and biologically assessed using a hoechst 33342 accumulation assay.480 by utilizing the cryo-em structure of abcg2,481 molecular docking studies were performed using a fragment-based approach.482 this approach was used to gain insights into the molecular determinants involved in the formation of the transporter-substrate complex.480 based on the docking studies, the putative binding site of the abcg2 substrate, hoechst 33342, and its interaction with the amino acids in the binding pocket was proposed.480 the predicted binding pose was rationalized based on the mutagenesis data reported in the literature483-487 and further confirmed with kinetic studies to determine the mode of inhibition.480 this subsequent structure-based approach led to the discovery of highly potent pyrimidine-based abcg2 inhibitors,488,489 specifically by identifying a novel binding pocket of this transporter.488 in terms of abca transporters, molecular docking experiments with the newly derived abca7 homology model applying a set of diverse pan-abc transporter inhibitors revealed a putative common ‘multitarget binding site’ identified within the transmembrane domains of abca7. it must be noted that the nucleotide binding domains are the most highly conserved regions amongst all abc transporters, and hence, may also represent a(nother) multitarget binding site for certain drugs. however, the vast majority of data reported in the past hint to the transmembrane domains as the actual venue of bioactivity in terms of abc transporter modulation.472 these results as described above475,480,488,489 give this methodology a high relevance in the drug development process in terms of novel lead molecules in general, and provide the basis for rationally designed structure-guided approaches for the identification of modulators of abca transporters in particular, as recently demonstrated for abca7.475 ligand-based drug design similarity search the analysis of structure-activity relationships using ligand-based approaches is an essential component of medicinal chemistry and pharmacology of abc transporters. this becomes evident as x-ray or cryo-em structures of most abc transporter subtypes are lacking to serve as suitable templates with sufficient similarity for generating homology models. ligand-based approaches establish a correlation between the molecular structure of a small-molecule and the triggered biological response of the target. the chemical representation of the molecules is often expressed using descriptors, which are attributes that conserve the physicochemical information of the molecule. these descriptors refer to generic properties such as logp, molecular weight, polar surface area, rotatable bonds, or molar refractivity. alternatively, structural representations of the molecules can form fingerprints that portray existent molecular features of the molecule in a binary code. these fingerprints are, for example, path-like,490 or circular-based,491,492 such as maccs or ecfp4, respectively. utilizing these representations of molecules, similarity-driven virtual screenings can be applied. here, molecules are extracted from a virtual library of millions or billions of compounds compared to the bioactive template molecule(s) according to the similarity principle. the abstract representation of molecules enables clustering of compounds, which is a methodology to categorize a diverse set of molecules. moreover, these abstract representations can be used in different machine learning (artificial intelligence) approaches. pharmacophore modelling another common approach is pharmacophore modelling, which analyzes a number of ligands with a common mechanism of action. the model is the ensemble of common chemical features that are required to ensure the molecular interaction of the ligands with the target, such as hydrogen bond donors and acceptors as well as aromatic and hydrophobic centers. the pharmacophore models are generated by extracting common molecular features through flexible alignment of the active biomolecules.493,494 this can be achieved by generating all possible conformations of the ligand and aligning them to determine the essential chemical features and molecular orientation to construct the pharmacophore model. the conformational flexibility of the ligands representing the chemical features is the key factor in the pharmacophore model generation. pattern analysis in addition to these classical computational approaches, similarity search and pharmacophore modelling, a pattern analysis approach (‘c@pa’ = computer-aided pattern analysis’) has been reported recently.18,19,495 pattern analysis extracts both basic scaffolds and the statistical distribution of substructural elements amongst the template ligands. it works similarly to non-physicochemical properties-related fingerprints and conserves substructural features as they are present in the molecules. pattern analysis has specifically been derived for the development of novel potent multitarget abc transporter inhibitors. the basic operations were the categorization of bioactive molecules according to their inhibitory power against specific abc transporters and their classification according to their selectivity profile. the respective classes can statistically be analyzed for both their basic scaffolds and/or their substructural composition to extract the desired pharmacological profile and target preferences. the generated model focused multitargeting of abc transporters, and resulted in a biological hit rate of 21.7%.19 adaption of the model (‘c@pa_1.2’) through additional non-statistical and exploratory measures increased the biological hit rate to 40%,18 and an additional extension of the model enabled the discovery of the ‘outer multitarget modulator landscape’, which represented weak multitarget bioactivities (>10 μm) supporting the discovery of a larger number of multitarget agents.495 the hit rates are impressive considering that this approach takes several targets with individual ‘ligand preferences’ into account. furthermore, as several abc transporters of distinct subfamilies were considered (abcb1, abcc1, abcg2), the resultant multitarget agents open up the possibility to explore under-studied abc transporters,18 in particular abca transporters in terms of ad.6,14 combined approaches apart from the individual use of these methodologies, combined approaches may lead to improved hit rates and better prediction capabilities with respect to bioactivity of small-molecules. this has in particular been demonstrated for a combined virtual screening approach using similarity search and pharmacophore modelling for the discovery of novel abcc1 inhibitors.493 also, certain pattern analysis approaches have used a data set derived from a similarity search and pharmacophore modelling approach, and hence, can also be considered a combined computational approach.18,495 in vitro methodologies to assess novel lead structures the previous sections have already outlined the diverse testing systems that have been used to assess the modulatory effects of effectors toward abca transporters. the following section will highlight the abca transporter-expressing host systems and the related assays that can be implemented into the pipeline for the assessment of novel lead molecules as potential abca transporter diagnostics or therapeutics. host system of abca transporters the transporter host system (abca transporter carrying unit) can be categorized into (i) living-cell-based or (ii) membrane preparation-/vesicle-based (including isolated and reconstituted proteins). the vast majority of biological investigations used living cells. here, two different living cell-based transporter host systems can be differentiated: (i) native/induced/selected cells and (ii) transfected cells. native abca transporters-expressing living cells native/induced/selected cells naturally express the respective abca transporter or have been exposed to a ‘standard’ inducer, for example, the abca1 inducers 22-(r)-hydroxycholesterol,122,205,249,252,259,262-264,268,277,278,305-315 to901317,205,245,250,252,259,260,262,264,271,272,279, 280,282,308,310,317,319,322,324,326,328-345 or 8-br camp,230,249,255,266,290,292 and overexpress the respective transporter in response (e.g., abca1). most commonly, human or murine cells have been used. table 4 summarizes the cell lines used to assess the abca transporter modulators discussed in the previous sections. it must be noted that the addressed pathways regulate also the overexpression of other abc transporters. in terms of the studies of abca1, the co-expression (i.e., co-upregulation and co-downregulation) of other members, such as abcg1, has frequently been observed.160,320,335,364,366,402,410,418,421,448 table 4. non-exhaustive list of native abca transporters-expressing cell lines that have been established in the assessment of small-molecule modulators of abca transporters. cell type cell line name origin references abca1 colorectal adenocarcinoma cells caco-2 human 262,264,308,314,342,436 lung adenocarcinoma cells hcc827-gr pc9-g2 human 337 337 renal adenocarcinoma cells 786-o a498 achn hk-2 sn12c os-rc-2 human human human human human human 334 330 334,349 330 330 330 adipocytes 3t3 l-1 mouse 255 adrenocortical carcinoma cells h295r muc-1 human human 333,441 333 astrocytes human mouse rat 279 229,279 281 astrocytoma ccfsttg1 human 423 peripheral blood mononuclear cells pbmc human 411 breast cancer cells mcf-7 human 331 pancreatic β-cells ins-1 mouse 409 cardiomyocytes h9c2 hl-1 rat mouse 253 250 aortic endothelial cells haec human 263,269 endometrial endothelial cells mouse mouse umbilical vein endothelial cells huvec human 269,364,442,496 epithelial cells beas-b2 human 322 lung epithelial cells mouse 311 pigment epithelial cells human 257 mouse mammalian epithelial cells mmec mouse 350 aortic smooth muscle cells smc human 269 vascular smooth muscle cells vsmc unspecified origin 332 fibroblasts primary hip skin wi-38 (embryonic) wi38va13 (embryonic) balb/3t3 swiss 3t3 human human human mouse mouse 230,260 205,246 277 275 312 granulosa cells rat 443 hair follicles human 282 hepatoma fu5ah hep3b hepg2 mcarh7777 rat rat human rat rat 318 231 309,342,348,379 280,312,317,367,381 343 insulinoma cells ins-1 rat 405 keratinocytes human 282 embryonic kidney cells human 312 non-small cell lung cancer cells a549 h1650 h1975 h358 pc-9/gr human human human human human 322,447 400 400 447 400 liver cells l02 human 406 mantle cell lymphoma mcl human 468 macrophages primary human 268,305,339,396,398 mouse 306,312,313,320,329,341,360,366,439,448 hd11 chicken 356 j774.a1 mouse 252,254,255,259,265,271,278,289-292,384,392,393 raw264.7 mouse 249,312,313,321,336,339,342,352,360,365,367,369,375, 376,381,385,399,402,404,406,408,410,416-419,421,424, 425,438,442,448,497 thp-1 human 231,245,249,256,268,272,275,292,308,310,312-316,321, 328,335,338,339,341,342,360,363,364,366,377,384, 388-397 u937 human 307 microglia primary bv2 retinal (müller cells) rat mouse mouse 355 126,353,380 323 multiple myeloma mm human 468 neuroblastoma neuro-2a murine 359 neutrophils primary human 339 nephron cells a6 frog 258 periodontal ligament stem cells human 325 pheochromocytoma pc12 rat 280 podocytes human 440 retina cells arpe-19 human 354 oral squamous cell carcinoma cells cal27 human 371 trophoblasts bewo human 437 abca2 hepatoma hepg2 rat 179 ovary carcinoma skem human 238 abca3 cholangiocarcinoma m214-5fur human 427 lung epithelial cells mle-12 mouse 452 hepatoma hepg2 rat 451 leukemia primary (acute myeloid) bv173 k562 lama83 human human human human 234 234,236 234 235 lung cancer a549 nci-h1650 nci-h1975 human human human 241 241 241 abca5 brain microvascular endothelial cells hbmec human 428 macrophages raw264.7 thp mouse human 321 321 abca7 fibroblasts balb/3t3 wi-38 mouse human 205 205 macrophages j774.a1 mouse 431 in terms of abca1, most studies have been conducted with human thp1,231,245,249,256,268,272,275,292,308,310,312-316,321,328,335,338,339,341,342,360,363,364,366,377,384,388-397 murine j774.a1,252,254,255,259,265,271,278,289-292,384,392,393 or murine raw264.7 macrophages. 230,249,312,313,321,336,339, 342,352,360,365,367,369,375,376,381,385,399,402,404,406,408,410,416-419,421,424,425,438,442,448 in the set-up of a drug development pipeline, these cell lines are the backbone of the in vitro assessment of potential candidates. regarding other abca transporters, the situation is much more complicated due to the lack of cell lines that naturally (and almost exclusively) express the respective abca transporter. consequently, these abca transporters are much less studied and well-established. however, transfected cell lines are of great help to study one particular transporter instead of using native cell lines that may co-express several members. abca transporters-transfected living cells in terms of abca1, cell lines transfected with human abca1 have often been used, e.g., human embryonic kidney (hek) cells (hek293/abca1)171,201,202,249,260,267,270,275,329,352,386,464,467,498,499 and baby hamster kidney (bhk) cells (bhk-21/abca1).230,245,273,292,422 these transporter host systems have also been used to study other transporters, abca2,498,500 abca3,235,241,498 abca4,133-136,201,457,458,501,502 abca5,503 abca7,201,202,386,422,498 abca8,10 abca12,498 and abca13.48 transfected cells often express lower levels of the introduced transporter than native cell lines, which is a problem if the host cell lines (e.g., hek or bhk-21) naturally express other abc transporters as well. however, these transporter host systems are suitable to confirm results, and might be the only possibility to address abca transporters other than abca1. isolated abca transport proteins finally, apart from intact cells, vesicles of enriched or purified/reconstituted abca transporters have also been used to assess transporter function. compared with living-cell based assays, this kind of host system is rarely represented in the literature regarding abca transporters.133-139,201,499-502,504-506 specifically atpase assays are popular to assess functional abc transporter modulation.23,24,507-510 while transport protein purification and reconstitution in vesicles or nano discs requires advanced engineering, and is expensive and resource-consuming, membrane preparations of transporters, in particular for atpase assays, are much more feasible. however, this method has been used somewhat scarcely for abca transporter function assessment.133-135,137-139,201,499-502,504-506 functional assessment of abca transporters two groups of tracers have been established in terms of abca transporter function: (i) radiolabeled substrates,250,272,305,306,338,339,354,364,366,393,395,404,419,511,512,136,222,230,245,249,253,255,259,260,262,264,265,267-270, 273,276,278,289-292,311,313,315,318,329,341,367,377,381,384,385,464,467,499,513 and (ii) fluorescent substrates.171,201,238, 251,252,254,256,258,261,271,282,308,319,321,330,332,335,342,360,379,389,390,392,397,402,406,455,456,468,514-518  radiolabeled tracers of abca transport function in terms of radiolabeled substrates, cholesterol is by far the most frequently used genuineabca1 substrate,230,245,249,255,260,264,265,267-270,272,273,276,278,289-292,305,306,313,315,318,329,338,339,354,366,367,381,384,385, 393-395,404,408,419,464,467,499,512 followed by phospholipid(-components).249,255,267,269,273,311,464,467,514 however, other substrates have also been used. these substrates include mostly molecules with sterane scaffold, such as β-sitosterol (abca1)262 and estradiol-β-glucuronide (abca8).222 moreover, lipid-like substrates have attracted attention, like sphingosine-1-phosphate (abca1),229,496 α-tocopherol (abca1),230 and atra (abca4).136 notably, radiolabeled substrates are very effective in terms of accurate tracing of protein function, as these molecules are not changed in their molecular integrity in contrast to fluorescence probes.171,201,238,251,252,254,256,258,261,271,282,308,319,321,330,332,335,342,360,379,389,390,392,397,402,406,455,456,468, 514-518 on the downside, conducting these experiments is constrained to regulatory requirements and requires extensive staff training as well as expensive safety measures and laboratory equipment. fluorescent tracers of abca transport function regarding fluorescent derivatives of cholesterol and phospholipids, two major types can be differentiated: (i) 7-nitro-2,1,3-benzooxadiazole (nbd) derivatives201,251,252,254,256,258,261,308,335,342,360,379,389,390,392,394,397,402, 406,408,468 and (ii) 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (bodipy) derivatives.271,282,319,321,330,332,455,456, 515-517 other fluorophore-labeled dyes have been reported, too, including the sterane analog dansyl-estramustine,171,238,518 and propargyl choline, which is processed in vitro into propargylated phospholipids.514 in addition to the stated fluorescent tracers of abca transport function, several other derivatives of other substrates can be proposed. for example, n-3-oxododecanoyl-l-homoserine lactone (3oc12-hsl) was suggested as abca1 substrate, but final proof was missing.519 thus, it may be a suitable candidate for validation in a new set-up in vitro assay for abca1 (and potentially other abca transporters). other examples of potential probes are fluorescenct dyes that stand in association with cellular cholesterol and phospholipid distribution and abca1-mediated cholesterol and phospholipid transport.516 these include, for example, β bodipy fl c5-hpc, β-bodipy fl c12-hpc, bodipy tr ceramide, and red/green bodipy pc-a2, amongst many others.520-522 fluorescenct dyes are well-established tracers of abc transporter function,18,19,23,24,284,480,488,489,493,507, 523-525 and the knowledge that has accumulated regarding the well-studied abc transporters abcb1, abcc1, and abcg2 can be transferred to abca transporters as well. however, the added fluorophore changes the molecular composition of the tracing molecules. this alteration inheres the potential risk of changing affinities and even the binding site(s) of these molecules, undermining functional-kinetic analyses regarding binding site determination and elucidation of the mode of action. nevertheless, fluorescence probes are – if used and established correctly – extremely reliable, and can be used without regulatory restrictions and necessity of special equipment, except for microplate readers and/or flow cytometers. colorimetric determination of abca transport function – atpase assays as mentioned above, atpase assays have also been used to functionally analyze abca transporter function, in particular for abca1,201,499,505,506 abca2,500 abca3139,504 abca4,133-135,137,138,201,501,502 and abca7,201 although this methodology has been used somewhat rarely compared to other functional approaches. atpase assays are based on the principle that the active transport of any substrate of abc transporters consumes energy. this energy is derived from the cleavage of atp to adp and pi, and can be detected by different methodologies.23,24,507-510,526 table 5 highlights known atpase modulators of abca transporters and the associated literature reports. table 5. summary of known atpase modulators of abca transporters. transporter modulator mode of modulation references abca1 ceramide (30 mol–%) cholesterol (30 mol–%) phosphatidylcholine (30 mol–%) phosphatidylethanolamine (30 mol–%) phosphatidylinositol (30 mol–%) phosphatidylserine (30 mol–%) sphingomyelin (30 mol–%) inhibition inhibition activation inhibition inhibition activation activation 201 201,499 201 201 201 201 201 abca2 methyl-β-cyclodextrin (u.c.a) activation 500 abca4 amiodarone (20–75 µm) 2-tert-butylanthraquinone (20–50 µm) ceramide (30 mol–%) cholesterol (30 mol–%) dehydroabietylacetate (10–50 µm) digitonin (10–180 µm) n-ethylmaleimide (nem; 1000 µm) reduced glutathione (gsh; 1000 µm) β-ionone (50–100 µm) phosphatidylethanolamine (30 mol–%) phosphatidylglycerol (30 mol–%) phosphtidylinositol (30 mol–%) 11-cis-retinal (5–100 µm) 13-cis-retinal (5–100 µm) atra (5–100 µm; ec50 = 10 µm) all-trans-retinoic acid (20–100 µm) all-trans-retinol (20–100 µm) n-retinylidenephosphatidylethanolamine (40 µm) activation activation inhibition inhibition activation activation inhibition activation activation activation activation inhibition activation activation activation activation activation activation 138 138 201 201 138 138 137 137 138 201 201 201 137,138 138 133-135,137,138 138 133,138 133 abca7 ceramide (30 mol–%) cholesterol (30 mol–%) phosphatidylcholine (30 mol–%) phosphatidylethanolamine (30 mol–%) phosphatidylserine (30 mol–%) inhibition inhibition activation activation activation 201 201 201 201 201 a u.c. = unspecified concentration atpase assays have been and still are popular in terms of functional abc transporter modulation in general.23,24,507-510 strikingly, the nbds of abc transporters are – in contrast to the various binding sites identified within the transmembrane domains of abc transporters475 – highly conserved. this conservation enables targeting of abca nbds by known atpase modulators of other abc transporters. therefore, abca transporter function can be detected by methodologies that have already been established for other abc transporters.23,24,507-510,526 this transfer of knowledge will be of great use to confirm obtained results from other functional abca transporter analyses. colorimetric determination of abca transport function – other detection methodologies as a final note, it must be mentioned that other colorimetric analyses were also used to quantify the abca transporter-mediated function, specifically for transport of cholesterol or choline-containing lipids, using commercially available assay kits.202,205,246,251,268,272,275,329-332,334,336,354,365,366,369,372,374-376,386-389,392,405, 406,416,418,421,422,424,425,441,511 however, these methodologies require time-consuming extraction processes of the lipids, and hence, are less suitable to track the function of abca transporters in real-time and to determine kinetic aspects of their cholesterol and lipid transport. in rare instances, the extraction of lipid components was accomplished after incubation with a radioactive marker.246 while this is a valid methodology to accurately determine lipid components within cells, it increases workload and attracts regulatory constraints. gas-liquid chromatography has also been used in some reports.353,355 an extraction-free staining of cholesterol inside of cells was also demonstrated (filipin iii251,331,333,341,358 or oil red o staining 330,332,364,366,369,375,388,389,392,393,399,402,410,417,421,425,448). however, these systems are not suitable to track single-cell abca-mediated cholesterol or phospholipid transport. quantification of abca transporter regulation besides qpcr and western blotting, abca transporter expression was reported in several studies using fluorimetric assays. this was accomplished with either (i) green fluorescent protein-(gfp)tagged/labelled abca transporters235,241,261,275,386,422,464,504 or (ii) luciferase promotor-(luc)-transfected271,309,319,352,367,379,381,405, 406,419,436,447 abca transporter cells in luciferase reporter gene assays. in vivo assessment of clinical candidates in vivo models play a key role in drug discovery. although in vitro and cellular models are less expensive and less time consuming, in vivo models are needed to test abca modulators under physiological conditions. safety, toxicity, and efficacy of a drug candidate must be tested in an in vivo model as a last step before transferring it to clinical evaluation. however, these models also have disadvantages. animal studies are time consuming and require advanced personnel training and resources for maintaining the animals. in addition, although they are closer to humans than in vitro models, there are considerable physiological differences between species with respect to drug absorption, metabolism, and excretion, which may impede translatability. furthermore, the use of animals in research has its ethical concerns. thus, in recent years, research has been directed to reduce animal use and increase animal welfare. in vivo models have previously been used to study the role of abca transporters in physiology and disease as described above. thus, there are already available animal models for testing of abca modulators for the most prominent subtypes (table 6). as stated above, these models represent the last step before clinical evaluation of potential small-molecule therapeutics in humans. thus, after in silico identification and in vitro assessment, these in vivo models are the third column in the development of novel abca transporter diagnostics and therapeutics. in the following section, different in vivo models will be described in more detail. table 6. animal models to study the functional and pathological role of abca transporters. transporter type species phenotype references abca1 knock-out mouse reduced cholesterol and plasma phospholipid levels decreased brain apoe levels poorly lipidated apoe 161-163 https://www.jax.org/strain/003897 overexpression mouse increased lipidation of apoe 127 abca2 knock-out mouse reduced body weight, limb tremor, reduced sphingomyelin https://www.jax.org/strain/033139 54,527 abca3 knock-out mouse knocked-out pups die within 1h after birth 186,528,529 missense mutation mouse early macrophage predominant alveolitis which peaked at 8 weeks of age 530 abca4 knock-out mouse abnormal phospholipid composition, delayed dark adaptation 531,532 abca5 knock-out mouse exophthalmos and collapsed thyroid gland, early death due to cardiac insufficiency 123,131 abca7 knock-out humanized mouse mouse reduced microglia response altered phagocytosis increased β-secretase under characterization, increase aβ load 124 abca7tm1.1(abca7)pahnk mgi:6258226 abca8 knock-out mouse reduced plasma hdl 533 adenoviral overexpression mouse increased plasma hdl and cholesterol 533 abca12 not described https://www.jax.org/strain/033630 abca13 knock-out mouse monkey deficits of prepulse inhibition impaired neuronal formation, neurotransmitter alterations 48 534 knock-out mouse models a genetic knock-out mouse model is an animal model in which one or more genes of interest have been deactivated or removed by means of gene targeting. knock-out animals allow for direct investigation of the effect of a specific gene in an organism, as the loss of gene activity often causes phenotypic changes uncovering the function and biological mechanism of the targeted gene.535 knock-out mice have become one of the most useful scientific tools to analyze the human genome and its potential roles in many diseases.535 thus, knock-out animals are currently essential experimental tools for the investigation of genetic disorders and the evaluation of novel drugs.536 furthermore, the current knowledge on genome editing using the crispr/cas9 system makes generation of knock-out lines considerably faster than with the use of embryonic stem cells. to no surprise, this method has quickly become the most powerful tool for generating genetic models.537 knock-out animal models are designed with two variables in mind: (i) where and (ii) when is the gene of interest deactivated. the simplest and most common approach is a constitutive, ubiquitous knock-out, i.e., the product protein is absent permanently in all cells of an organism. to overcome limitations of this broad approach, more refined models have been developed. these conditional models use cre-lox recombination to target a gene either in specific cell populations, at specific time points, or a combination of both. here, the target gene is modified by inserting two loxp sites. the flanked gene segment can then be excised by the cre recombinase. cre activity, i.e., gene knock-out, can be limited to certain cell populations by appropriate promotor choice and/or linked to a tamoxifen-responsive element to control the exact time point at which the knock-out is induced. until now, several abca animal knock-out models have been described, which are summarized in table 6. these models are mainly mouse lines, except for abca13 (monkey).534 these animal models have contributed fundamentally to identifying the role of abca transporters in physiological conditions as well as in disease pathogenesis. in addition, these models can be used for novel drug testing, as they provide information about target specificity. if a drug is 100% specific for a transporter, knock-out of this transporter should completely abolish the drug’s effects observed in naïve animals. however, gene knock-outs often have phenotypical effects per se that need to be taken into account when evaluating drug effects. rnai models the use of rna interference (rnai) is an alternative to knock-out models. this technique is based on post-transcriptional silencing of the targeted gene using sirna molecules that are designed to bind to the target mrna.538 this process will deactivate the mrna using the cell’s own defense mechanism against pathogens. in contrast to standard knock-out models, this silencing is temporary as the sirna molecule will be degraded but the gene transcription continues.527 to avoid this temporal limitation, short-hairpin rna (shrna) has been developed. this method is based on the use of vectors that incorporate into the cell dna and encode for shrna. after transcription, these vectors are processed into sirna. these shrnas are continuously transcribed, increasing reproducibility of results.539 overexpression models similar to knock-out models, overexpression models can be used to investigate the function of a gene by evaluating the resultant phenotype. in addition, overexpression models have long been used for modeling diseases such as ad540 or pd.541 in the investigation of abca transporters, these models can resemble the effect of chronic activation of the transporters and may help to identify its physiological functions by evaluating the pathways upregulated in comparison to control animals.127 humanized abc transporter mouse models before it can be translated into clinical practice, each novel drug candidate must be tested in an in vivo model. however, the translational value of the animal model largely depends on whether the disease pathway under investigation is conserved between the two species. therefore, replacing the original (e.g., murine) gene by the respective human gene likely improves the animal model, and thus, is beneficial for evaluating a novel drug’s efficacy and specificity in clinical practice.542 with this approach, mice can be used as tools for pre-clinical screening and efficacy evaluation of new drugs, given their improved ability to predict human responses to treatments. our group has previously established a humanized abcc1 mouse model,543 and an abca7 model is under characterization. here, we generated knock-in mouse models producing a chimeric protein that is completely human except for one amino acid.543 in addition, as this gene was flanked by loxp sites, this humanized model can be knocked out in specific cell populations and at a specific age.543 models such as these represent the future of pre-clinical drug candidate evaluation. in addition, dallas et al. successfully generated a humanized abcg2 mouse model.544 however, other models, such as humanized abcb1 mice, were not successful despite multiple attempts.545 disease models in addition, all the models described above can also be used to study the role of a gene for the pathophysiology of specific diseases. for example, abca knock-out models have been crossed with transgenic mice in order to study their potential role in ad.54,123,131,161-163,527 these studies have elucidated potential disease mechanisms involving abca transporters that cannot be studied in patients. moreover, once a drug is developed and its specificity is proven, disease models enable evaluation of the role of that specific transporter in the pathophysiology of the disease. at the same time, these results may be the first step to evaluate the potential of novel transporter modulators as therapy for the respective disease. imaging techniques lastly, in vivo imaging can be used for the development of new drugs. on the one hand, labeling drug candidates with radioactive isotopes can give information about the drug distribution, drug target, and drug metabolism in vivo. in addition, it can also show whether a drug is able to cross specific natural barriers, such as the bbb. in vivo imaging can help to select candidates that appear successful or to discard drugs that seem likely to fail.546 on the other hand, drug candidates can also be used to develop new radiotracers (e.g., pet tracers) targeting abca transporters that could then be used in clinical diagnostics. radiotracers would facilitate the study of the specific gene and/or its product protein in human patients in vivo and in a longitudinal fashion, enabling a much better understanding of the role of abca transporters in human (patho)physiology.547 in this regard, knock-out animals can be used as negative controls for the development of new abca radiotracers to evaluate the specificity of the radiotracer.548 furthermore, these very same radiotracers can be used in animal disease models, enabling longitudinal studies and reducing the number of animals required.549-551 concluding remarks: where do we go from here? several in vivo studies demonstrated that modulators of abca transporters, in particular abca1, have systemic effects.231,249,250,253,271,275,289,293,297,330,335,344,350,361,362,366,368-370,376,378,383,410,415,417-419,425,431,436,448 however, the vast majority of these modulators were regulators,231,250,253,271,297,330,335,344,350,361,362,366,368-370, 376,378,383,410,415,417-419,425,431,436,448 specifically inducers,250,253,271,297,330,344,361,362,366,368-370,376,378,383,410, 415,418,419,425,431 and only very few interactors demonstrated in vivo effects.249,289,293 mostly emphasizing atherosclerosis,249,275,289,366,369,370,378,410,417-419,425,431,448 these regulators were able to demonstrate that cellular and plasma lipid content249,271,275,289,330,366,369,378,419,425,431 as well as atherosclerotic plaque formation275,289,366,369,370,410, 417-419,425,448 could be changed compared to controls (enhanced or reduced) after treatment with the respective drug. only very few in vivo approaches targeted for ad.293,297,344,383 taking the challenge of cns penetration of these drugs into account, drugs active in atherosclerosis models could generally be suggested to also have certain therapeutic relevance regarding ad. nevertheless, so far, none of these drugs has made it into clinical evaluation in humans. the underlying cause can be pinned to the fact that the principal mechanism by which abca transporters contribute to ad is still unknown. while a rationale can be found in atherosclerosis (efflux of cellular lipid to apoe and hdl resulting in lower lipid burden in the vascular system), the translation of this rationale to ad can only be achieved to a very limited extent. several questions need addressing in future evaluations: (i) what is the general function of abca transporters in the brain to ameliorate (or exacerbate) ad in patients; (ii) when does this development start; and (iii) at which stage of development can a pharmacological intervention with abca transporter modulators lead to a positive therapeutic effect? in this regard, more in vitro tests are needed with new lead structures that are rigorously assessed for their particular mechanism of action – to study vice versa the mechanism of action of abca transporters in general. one possibility to gain novel lead structures is the screening of huge analog compound libraries. however, the number of existing compounds is limited, and blind in vitro testing is resource-consuming, especially regarding time and funds. computational methodologies may help to generate novel lead structures based on the knowledge of existing modulators of abca transporters. this has led to new lead molecules in the past.18,19,493,495 particularly the knowledge on abca1 and abca8 inhibitors and substrates is of interest, because these compounds inherit the molecular-structural information that is critical for direct binding to these transporters. considering the newly developed pattern analysis methodology, c@pa,18,19,495 the scaffolds and substructural composition of this set of molecules may reveal the critical necessities for direct interaction with abca transporters. c@pa is therefore of high relevance because it was specifically developed to gain multitargeting pan-abc transporter modulators18,19,495 – molecules that particularly interact with different abc transporters of different subfamilies. assuming that a conserved multitarget binding site exists as proposed earlier,6,14,475 multitargeting may be the key to explore under-studied abc transporters in general and abca transporters in particular.6,14,18,19 several thousands of these molecules have already been predicted,18,19,493,495 and the predictions were in part biologically confirmed.18,19,493,495 additionally, selected pan-abc transporter inhibitors were analyzed in molecular docking studies, which revealed the potential existence of the multitarget binding site.475 hence, combining the existent knowledge of abca transporter modulators with (sub)structural elements of these pan-abc transporter modulators and powerful computational approaches (e.g., molecular docking or molecular dynamics simulations) could ultimately lead to the successful exploration of abca transporters in general, as well as abca1 and abca7 in particular.28,95,103-112 several drugs and drug-like compounds have already been demonstrated to be pan-abc transporter modulators interacting also with abca transporters. these drugs and drug-like compounds are, for example, cyclosporine a (9 targets of 4 subfamilies: abca1,245 abcb1,20 abcb4,552 abcb11,553 abcc1–2,24,554 abcc10,26 and abcg1–2555,556), glibenclamide (8 targets of 4 subfamilies: abca1,270 abcb11,553 abcc1,24 abcc5,557 abcc7–9,558-560 and abcg2554), imatinib (6 targets of 4 subfamilies: abca3,426 abcb1,561 abcb11,553 abcc1,561 abcc10,561 and abcg2561), probenecid (8 targets of 2 subfamilies: abca8,222 abcc1–6,24,26,562-564 abcc10565), verapamil (9 targets of 4 subfamilies: abca8,222 abcb1,20 abcb4–5,552,566 abcb11,567 abcc1,24 abcc4,568 abcc10,565 and abcg2554), and verlukast (11 targets of 4 subfamilies: abca8,222 abcb4,552 abcb11,553 abcc1–5,24,554,557,564,569 abcc10–11,26,570 abcg2554). in silico analyses with verapamil and verlukast supported the notion of addressing the multitarget binding site in abca7.475 taking their structural peculiarities in a pattern-based rational drug design approach into account may yield novel lead structures for functional in vitro studies of abca transporters. this may ultimately result in the development of innovative ad diagnostics and therapeutics. appendix author information corresponding author sven marcel stefan, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0002-2048-8598 email: s.m.stefan@medisin.uio.no authors jens pahnke, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; lied, university of lübeck, ratzeburger allee 160, 23538 lübeck, germany; department of pharmacology, faculty of medicine, university of latvia, jelgavas iela 1, 1004 rīga, latvia; orcid: 0000-0001-7355-4213 pablo bascuñana, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0003-2186-8899 mirjam brackhan, department of pathology, section of neuropathology, translational neurodeg-eneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; lied, university of lübeck, ratzenburger allee 160, 23538 lübeck, germany; orcid: 0000-0002-0753-6292 katja stefan, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0003-3544-2477 vigneshwaran namasivayam, department of pharmaceutical and cellbiological chemistry, pharmaceutical institute, university of bonn, an der immenburg 4, 53121 bonn, germany; orcid: 0000-0003-3031-3377 radosveta koldamova, department of environmental and occupational health, school of public health, university of pittsburgh, 130 de soto street, pittsburgh, pa 15261, united states of america; orcid: 0000-0002-6761-0984 jingyun wu, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0002-5137-4614 luisa möhle, department of pathology, section of neuropathology, translational neurodege-neration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0002-4535-9952 conflict of interest the authors declare that they have no conflict of interest. funding jp received funding from deutsche forschungsgemeinschaft (dfg, german research foundation; germany; 263024513); latvian council of science (latvia; lzp-2018/1-0275); helsesø (norway; 2019054, 2019055); barnekreftforeningen (norway; 19008); eea grant/norway grants kappa programme (iceland, liechtenstein, norway; tačr tarimad to100078); norges forskningsråd [norway; 260786 (prop-ad), 295910 (napi), and 327571 (petabc)]; european commission (european union; 643417). prop-ad and petabc are eu joint programme neurodegenerative disease research (jpnd) projects. prop-ad is supported through the following funding organizations under the aegis of jpnd – www.jpnd.eu: aka #301228 – finland, bmbf #01ed1605 – germany; cso-moh #30000-12631 – israel; nfr #260786 – norway; src #2015-06795 – sweden). petabc is supported through the following funding organizations under the aegis of jpnd – www.jpnd.eu: nfr #327571 – norway; ffg #882717 – austria; bmbf #01ed2106 – germany; msmt #8f21002 – czech republic; viaa #es rtd/2020/26 – latvia; anr #20-jpw2-0002-04 – france, src #2020-02905 – sweden. the projects receive funding from the european union’s horizon 2020 research and innovation programme under grant agreement #643417 (jpco-fund). ks receives a walter benjamin fellowship of the dfg (germany; 466106904). rk is funded by the national institute of health (nih; united states; ag056371, ag057565; ag066198). lm is supported by the norwegian health association (nasjonalforeningen for folkehelsen; norway; #16154). sms receives a walter benjamin fellowship of the dfg (germany; 446812474). acknowledgement the authors would like to cordially thank joseph mark robertson (napi / department of immunology, university of oslo and oslo university hospital) for proofreading the manuscript. references wang, j. q.; yang, y.; cai, c. y.; teng, q. x.; cui, q.; lin, j.; assaraf, y. g.; chen, z. s. multidrug resistance proteins (mrps): structure, function and the overcoming of cancer multidrug resistance. drug resist updat 2021, 54, 100743. gil-martins, e.; barbosa, d. j.; silva, v.; remiao, f.; silva, r. dysfunction of abc transporters at the blood-brain barrier: role in neurological disorders. pharmacol ther 2020, 213, 107554. pasello, m.; giudice, a. m.; scotlandi, k. the abc subfamily a transporters: multifaceted players with incipient potentialities in cancer. semin cancer biol 2020, 60, 57-71. sodani, k.; patel, a.; kathawala, r. j.; chen, z. s. multidrug resistance associated proteins in multidrug resistance. chin j cancer 2012, 31, 58-72. szakacs, g.; abele, r. an inventory of lysosomal abc transporters. febs lett 2020, 594, 3965-85. stefan, k.; leck, l. y. w.; namasivayam, v.; bascuñana, p.; huang, m. l.-h.; riss, p. j.; pahnke, j.; jansson, p. j.; stefan, s. m. vesicular atp-binding cassette transporters in human disease: relevant aspects of their organization for future drug development. future drug discov 2020, 2, fdd51. domenichini, a.; adamska, a.; falasca, m. abc transporters as cancer drivers: potential functions in cancer development. biochim biophys acta gen subj 2019, 1863, 52-60. adamska, a.; falasca, m. atp-binding cassette transporters in progression and clinical outcome of pancreatic cancer: what is the way forward? world j gastroenterol 2018, 24, 3222-38. robey, r. w.; pluchino, k. m.; hall, m. d.; fojo, a. t.; bates, s. e.; gottesman, m. m. revisiting the role of abc transporters in multidrug-resistant cancer. nat rev cancer 2018, 18, 452-64. sasaki, k.; tachikawa, m.; uchida, y.; hirano, s.; kadowaki, f.; watanabe, m.; ohtsuki, s.; terasaki, t. atp-binding cassette transporter a subfamily 8 is a sinusoidal efflux transporter for cholesterol and taurocholate in mouse and human liver. mol pharm 2018, 15, 343-55. pan, s. t.; li, z. l.; he, z. x.; qiu, j. x.; zhou, s. f. molecular mechanisms for tumour resistance to chemotherapy. clin exp pharmacol physiol 2016, 43, 723-37. ween, m. p.; armstrong, m. a.; oehler, m. k.; ricciardelli, c. the role of abc transporters in ovarian cancer progression and chemoresistance. crit rev oncol hematol 2015, 96, 220-56. wenzel, j. j.; piehler, a.; kaminski, w. e. abc a-subclass proteins: gatekeepers of cellular phosphoand sphingolipid transport. front biosci 2007, 12, 3177-93. stefan, s. m. multi-target abc transporter modulators: what next and where to go? future med chem 2019, 11, 2353-58. yu, m.; ocana, a.; tannock, i. f. reversal of atp-binding cassette drug transporter activity to modulate chemoresistance: why has it failed to provide clinical benefit? cancer metastasis rev 2013, 32, 211-27. amiri-kordestani, l.; basseville, a.; kurdziel, k.; fojo, a. t.; bates, s. e. targeting mdr in breast and lung cancer: discriminating its potential importance from the failure of drug resistance reversal studies. drug resist updat 2012, 15, 50-61. tamaki, a.; ierano, c.; szakacs, g.; robey, r. w.; bates, s. e. the controversial role of abc transporters in clinical oncology. essays biochem 2011, 50, 209-32. namasivayam, v.; silbermann, k.; pahnke, j.; wiese, m.; stefan, s. m. scaffold fragmentation and substructure hopping reveal potential, robustness, and limits of computer-aided pattern analysis (c@pa). comput struct biotechnol j 2021, 19, 3269-83. namasivayam, v.; silbermann, k.; wiese, m.; pahnke, j.; stefan, s. m. c@pa: computer-aided pattern analysis to predict multitarget abc transporter inhibitors. j med chem 2021, 64, 3350-66. zhang, h.; xu, h.; ashby, c. r., jr.; assaraf, y. g.; chen, z. s.; liu, h. m. chemical molecular-based approach to overcome multidrug resistance in cancer by targeting p-glycoprotein (p-gp). med res rev 2021, 41, 525-55. dong, j.; qin, z.; zhang, w. d.; cheng, g.; yehuda, a. g.; ashby, c. r., jr.; chen, z. s.; cheng, x. d.; qin, j. j. medicinal chemistry strategies to discover p-glycoprotein inhibitors: an update. drug resist updat 2020, 49, 100681. palmeira, a.; sousa, e.; vasconcelos, m. h.; pinto, m. m. three decades of p-gp inhibitors: skimming through several generations and scaffolds. curr med chem 2012, 19, 1946-2025. wiese, m.; stefan, s. m. the a-b-c of small-molecule abc transport protein modulators: from inhibition to activation-a case study of multidrug resistance-associated protein 1 (abcc1). med res rev 2019, 39, 2031-81. stefan, s. m.; wiese, m. small-molecule inhibitors of multidrug resistance-associated protein 1 and related processes: a historic approach and recent advances. med res rev 2019, 39, 176-264. pena-solorzano, d.; stark, s. a.; konig, b.; sierra, c. a.; ochoa-puentes, c. abcg2/bcrp: specific and nonspecific modulators. med res rev 2017, 37, 987-1050. zhou, s. f.; wang, l. l.; di, y. m.; xue, c. c.; duan, w.; li, c. g.; li, y. substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development. curr med chem 2008, 15, 1981-2039. norman, b. h. inhibitors of mrp1-mediated multidrug resistance. drugs fut 1998, 23(9), 1001. pereira, c. d.; martins, f.; wiltfang, j.; da cruz, e. s. o. a. b.; rebelo, s. abc transporters are key players in alzheimer's disease. j alzheimers dis 2018, 61, 463-85. abuznait, a. h.; kaddoumi, a. role of abc transporters in the pathogenesis of alzheimer's disease. acs chem neurosci 2012, 3, 820-31. wolf, a.; bauer, b.; hartz, a. m. abc transporters and the alzheimer's disease enigma. front psychiatry 2012, 3, 54. redzic, z. molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences. fluids barriers cns 2011, 8, 3. kortekaas, r.; leenders, k. l.; van oostrom, j. c.; vaalburg, w.; bart, j.; willemsen, a. t.; hendrikse, n. h. blood-brain barrier dysfunction in parkinsonian midbrain in vivo. ann neurol 2005, 57, 176-9. jha, n. k.; kar, r.; niranjan, r. abc transporters in neurological disorders: an important gateway for botanical compounds mediated neuro-therapeutics. curr top med chem 2019, 19, 795-811. jablonski, m. r.; markandaiah, s. s.; jacob, d.; meng, n. j.; li, k.; gennaro, v.; lepore, a. c.; trotti, d.; pasinelli, p. inhibiting drug efflux transporters improves efficacy of als therapeutics. ann clin transl neurol 2014, 1, 996-1005. kooij, g.; kroon, j.; paul, d.; reijerkerk, a.; geerts, d.; van der pol, s. m.; van het hof, b.; drexhage, j. a.; van vliet, s. j.; hekking, l. h.; van buul, j. d.; pachter, j. s.; de vries, h. e. p-glycoprotein regulates trafficking of cd8(+) t cells to the brain parenchyma. acta neuropathol 2014, 127, 699-711. jablonski, m. r.; jacob, d. a.; campos, c.; miller, d. s.; maragakis, n. j.; pasinelli, p.; trotti, d. selective increase of two abc drug efflux transporters at the blood-spinal cord barrier suggests induced pharmacoresistance in als. neurobiol dis 2012, 47, 194-200. westerlund, m.; belin, a. c.; olson, l.; galter, d. expression of multi-drug resistance 1 mrna in human and rodent tissues: reduced levels in parkinson patients. cell tissue res 2008, 334, 179-85. valenza, m.; carroll, j. b.; leoni, v.; bertram, l. n.; bjorkhem, i.; singaraja, r. r.; di donato, s.; lutjohann, d.; hayden, m. r.; cattaneo, e. cholesterol biosynthesis pathway is disturbed in yac128 mice and is modulated by huntingtin mutation. hum mol genet 2007, 16, 2187-98. dombrowski, s. m.; desai, s. y.; marroni, m.; cucullo, l.; goodrich, k.; bingaman, w.; mayberg, m. r.; bengez, l.; janigro, d. overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy. epilepsia 2001, 42, 1501-6. sisodiya, s. m.; lin, w. r.; harding, b. n.; squier, m. v.; thom, m. drug resistance in epilepsy: expression of drug resistance proteins in common causes of refractory epilepsy. brain 2002, 125, 22-31. katzeff, j. s.; kim, w. s. atp-binding cassette transporters and neurodegenerative diseases. essays biochem 2021 ebc20210012. dinda, b.; dinda, m.; kulsi, g.; chakraborty, a.; dinda, s. therapeutic potentials of plant iridoids in alzheimer's and parkinson's diseases: a review. eur j med chem 2019, 169, 185-99. pahnke, j.; langer, o.; krohn, m. alzheimer's and abc transporters--new opportunities for diagnostics and treatment. neurobiol dis 2014, 72 pt a, 54-60. susa, m.; iyer, a. k.; ryu, k.; choy, e.; hornicek, f. j.; mankin, h.; milane, l.; amiji, m. m.; duan, z. inhibition of abcb1 (mdr1) expression by an sirna nanoparticulate delivery system to overcome drug resistance in osteosarcoma. plos one 2010, 5, e10764. robillard, k. r.; hoque, m. t.; bendayan, r. expression of atp-binding cassette membrane transporters in a hiv-1 transgenic rat model. biochem biophys res commun 2014, 444, 531-6. hayashi, k.; pu, h.; tian, j.; andras, i. e.; lee, y. w.; hennig, b.; toborek, m. hiv-tat protein induces p-glycoprotein expression in brain microvascular endothelial cells. j neurochem 2005, 93, 1231-41. matsuo, h.; tomiyama, h.; satake, w.; chiba, t.; onoue, h.; kawamura, y.; nakayama, a.; shimizu, s.; sakiyama, m.; funayama, m.; nishioka, k.; shimizu, t.; kaida, k.; kamakura, k.; toda, t.; hattori, n.; shinomiya, n. abcg2 variant has opposing effects on onset ages of parkinson's disease and gout. ann clin transl neurol 2015, 2, 302-6. nakato, m.; shiranaga, n.; tomioka, m.; watanabe, h.; kurisu, j.; kengaku, m.; komura, n.; ando, h.; kimura, y.; kioka, n.; ueda, k. abca13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking. j biol chem 2020, 296, 100166. dwyer, s.; williams, h.; jones, i.; jones, l.; walters, j.; craddock, n.; owen, m. j.; o'donovan, m. c. investigation of rare non-synonymous variants at abca13 in schizophrenia and bipolar disorder. mol psychiatry 2011, 16, 790-1. nordestgaard, l. t.; tybjaerg-hansen, a.; nordestgaard, b. g.; frikke-schmidt, r. loss-of-function mutation in abca1 and risk of alzheimer's disease and cerebrovascular disease. alzheimers dement 2015, 11, 1430-8. gonzalez-guevara, e.; cardenas, g.; perez-severiano, f.; martinez-lazcano, j. c. dysregulated brain cholesterol metabolism is linked to neuroinflammation in huntington's disease. mov disord 2020, 35, 1113-27. macé, s.; cousin, e.; ricard, s.; génin, e.; spanakis, e.; lafargue-soubigou, c.; génin, b.; fournel, r.; roche, s.; haussy, g.; massey, f.; soubigou, s.; bréfort, g.; benoit, p.; brice, a.; campion, d.; hollis, m.; pradier, l.; benavides, j.; deleuze, j. f. abca2 is a strong genetic risk factor for early-onset alzheimer's disease. neurobiol dis 2005, 18, 119-25. davis, w., jr. the atp-binding cassette transporter-2 (abca2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism. biochim biophys acta 2014, 1841, 168-79. sakai, h.; tanaka, y.; tanaka, m.; ban, n.; yamada, k.; matsumura, y.; watanabe, d.; sasaki, m.; kita, t.; inagaki, n. abca2 deficiency results in abnormal sphingolipid metabolism in mouse brain. j biol chem 2007, 282, 19692-9. maugeri, a.; klevering, b. j.; rohrschneider, k.; blankenagel, a.; brunner, h. g.; deutman, a. f.; hoyng, c. b.; cremers, f. p. mutations in the abca4 (abcr) gene are the major cause of autosomal recessive cone-rod dystrophy. am j hum genet 2000, 67, 960-6. cremers, f. p. m.; lee, w.; collin, r. w. j.; allikmets, r. clinical spectrum, genetic complexity and therapeutic approaches for retinal disease caused by abca4 mutations. prog retin eye res 2020, 79, 100861. cremers, f. p.; van de pol, d. j.; van driel, m.; den hollander, a. i.; van haren, f. j.; knoers, n. v.; tijmes, n.; bergen, a. a.; rohrschneider, k.; blankenagel, a.; pinckers, a. j.; deutman, a. f.; hoyng, c. b. autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the stargardt's disease gene abcr. hum mol genet 1998, 7, 355-62. martínez-mir, a.; paloma, e.; allikmets, r.; ayuso, c.; del rio, t.; dean, m.; vilageliu, l.; gonzàlez-duarte, r.; balcells, s. retinitis pigmentosa caused by a homozygous mutation in the stargardt disease gene abcr. nat genet 1998, 18, 11-2. srisuwanwattana, p.; vachiramon, v. necrolytic acral erythema in seronegative hepatitis c. case rep dermatol 2017, 9, 69-73. conley, s. m.; cai, x.; makkia, r.; wu, y.; sparrow, j. r.; naash, m. i. increased cone sensitivity to abca4 deficiency provides insight into macular vision loss in stargardt's dystrophy. biochim biophys acta 2012, 1822, 1169-79. cideciyan, a. v.; aleman, t. s.; swider, m.; schwartz, s. b.; steinberg, j. d.; brucker, a. j.; maguire, a. m.; bennett, j.; stone, e. m.; jacobson, s. g. mutations in abca4 result in accumulation of lipofuscin before slowing of the retinoid cycle: a reappraisal of the human disease sequence. hum mol genet 2004, 13, 525-34. allikmets, r. a photoreceptor cell-specific atp-binding transporter gene (abcr) is mutated in recessive stargardt macular dystrophy. nat genet 1997, 17, 122. kjeldsen, e. w.; tybjaerg-hansen, a.; nordestgaard, b. g.; frikke-schmidt, r. abca7 and risk of dementia and vascular disease in the danish population. ann clin transl neurol 2018, 5, 41-51. rajkumar, a. p.; bidkhori, g.; shoaie, s.; clarke, e.; morrin, h.; hye, a.; williams, g.; ballard, c.; francis, p.; aarsland, d. postmortem cortical transcriptomics of lewy body dementia reveal mitochondrial dysfunction and lack of neuroinflammation. am j geriatr psychiatry 2020, 28, 75-86. qian, l.; qin, y.; chen, x.; zhang, f.; yang, b.; dong, k.; wang, z.; zhang, k. atp-binding cassette transporter 13 mrna expression level in schizophrenia patients. sci rep 2020, 10, 21498. crespo-facorro, b.; prieto, c.; sainz, j. schizophrenia gene expression profile reverted to normal levels by antipsychotics. int j neuropsychopharmacol 2014, 18, pyu066. dongsheng, h.; zhuo, z.; jiamin, l.; hailan, m.; lijuan, h.; fan, c.; dan, y.; he, z.; yun, x. proteomic analysis of the peri-infarct area after human umbilical cord mesenchymal stem cell transplantation in experimental stroke. aging dis 2016, 7, 623-34. ginguene, c.; champier, j.; maallem, s.; strazielle, n.; jouvet, a.; fevre-montange, m.; ghersi-egea, j. f. p-glycoprotein (abcb1) and breast cancer resistance protein (abcg2) localize in the microvessels forming the blood-tumor barrier in ependymomas. brain pathol 2010, 20, 926-35. bernstein, h. g.; hildebrandt, j.; dobrowolny, h.; steiner, j.; bogerts, b.; pahnke, j. morphometric analysis of the cerebral expression of atp-binding cassette transporter protein abcb1 in chronic schizophrenia: circumscribed deficits in the habenula. schizophr res 2016, 177, 52-58. de klerk, o. l.; willemsen, a. t.; roosink, m.; bartels, a. l.; hendrikse, n. h.; bosker, f. j.; den boer, j. a. locally increased p-glycoprotein function in major depression: a pet study with [11c]verapamil as a probe for p-glycoprotein function in the blood-brain barrier. int j neuropsychopharmacol 2009, 12, 895-904. seegers, u.; potschka, h.; loscher, w. transient increase of p-glycoprotein expression in endothelium and parenchyma of limbic brain regions in the kainate model of temporal lobe epilepsy. epilepsy res 2002, 51, 257-68. patak, p.; hermann, d. m. atp-binding cassette transporters at the blood-brain barrier in ischaemic stroke. curr pharm des 2011, 17, 2787-92. bartels, a. l.; willemsen, a. t.; kortekaas, r.; de jong, b. m.; de vries, r.; de klerk, o.; van oostrom, j. c.; portman, a.; leenders, k. l. decreased blood-brain barrier p-glycoprotein function in the progression of parkinson's disease, psp and msa. j neural transm (vienna) 2008, 115, 1001-9. vautier, s.; fernandez, c. abcb1: the role in parkinson's disease and pharmacokinetics of antiparkinsonian drugs. expert opin drug metab toxicol 2009, 5, 1349-58. bernstein, h. g.; holzl, g.; dobrowolny, h.; hildebrandt, j.; trubner, k.; krohn, m.; bogerts, b.; pahnke, j. vascular and extravascular distribution of the atp-binding cassette transporters abcb1 and abcc1 in aged human brain and pituitary. mech ageing dev 2014, 141-142, 12-21. vogelgesang, s.; glatzel, m.; walker, l. c.; kroemer, h. k.; aguzzi, a.; warzok, r. w. cerebrovascular p-glycoprotein expression is decreased in creutzfeldt-jakob disease. acta neuropathol 2006, 111, 436-43. chi, h.; tang, w.; bai, y. molecular evidence of impaired iron metabolism and its association with parkinson's disease progression. 3 biotech 2020, 10, 173. chuang, y. h.; paul, k. c.; bronstein, j. m.; bordelon, y.; horvath, s.; ritz, b. parkinson's disease is associated with dna methylation levels in human blood and saliva. genome med 2017, 9, 76. decleves, x.; fajac, a.; lehmann-che, j.; tardy, m.; mercier, c.; hurbain, i.; laplanche, j. l.; bernaudin, j. f.; scherrmann, j. m. molecular and functional mdr1-pgp and mrps expression in human glioblastoma multiforme cell lines. int j cancer. 2002, 98, 173-80. kilic, e.; spudich, a.; kilic, u.; rentsch, k. m.; vig, r.; matter, c. m.; wunderli-allenspach, h.; fritschy, j. m.; bassetti, c. l.; hermann, d. m. abcc1: a gateway for pharmacological compounds to the ischaemic brain. brain 2008, 131, 2679-89. vidal-taboada, j. m.; pugliese, m.; salvado, m.; gamez, j.; mahy, n.; rodriguez, m. j. katp channel expression and genetic polymorphisms associated with progression and survival in amyotrophic lateral sclerosis. mol neurobiol 2018, 55, 7962-72. crary, j. f. top ten discoveries of the year: neurodegeneration. free neuropathol 2020, 1, 12. nelson, p. t.; jicha, g. a.; wang, w. x.; ighodaro, e.; artiushin, s.; nichols, c. g.; fardo, d. w. abcc9/sur2 in the brain: implications for hippocampal sclerosis of aging and a potential therapeutic target. ageing res rev 2015, 24, 111-25. berger, j.; forss-petter, s.; eichler, f. s. pathophysiology of x-linked adrenoleukodystrophy. biochim 2014, 98, 135-42. tansley, g. h.; burgess, b. l.; bryan, m. t.; su, y.; hirsch-reinshagen, v.; pearce, j.; chan, j. y.; wilkinson, a.; evans, j.; naus, k. e.; mcisaac, s.; bromley, k.; song, w.; yang, h. c.; wang, n.; demattos, r. b.; wellington, c. l. the cholesterol transporter abcg1 modulates the subcellular distribution and proteolytic processing of beta-amyloid precursor protein. j lipid res 2007, 48, 1022-34. burgess, b. l.; parkinson, p. f.; racke, m. m.; hirsch-reinshagen, v.; fan, j.; wong, c.; stukas, s.; theroux, l.; chan, j. y.; donkin, j.; wilkinson, a.; balik, d.; christie, b.; poirier, j.; lutjohann, d.; demattos, r. b.; wellington, c. l. abcg1 influences the brain cholesterol biosynthetic pathway but does not affect amyloid precursor protein or apolipoprotein e metabolism in vivo. j lipid res 2008, 49, 1254-67. shen, s.; callaghan, d.; juzwik, c.; xiong, h.; huang, p.; zhang, w. abcg2 reduces ros-mediated toxicity and inflammation: a potential role in alzheimer's disease. j neurochem 2010, 114, 1590-604. van vliet, e. a.; iyer, a. m.; mesarosova, l.; colakoglu, h.; anink, j. j.; van tellingen, o.; maragakis, n. j.; shefner, j.; bunt, t.; aronica, e. expression and cellular distribution of p-glycoprotein and breast cancer resistance protein in amyotrophic lateral sclerosis patients. j neuropathol exp neurol 2020, 79, 266-276. bleau, a. m.; hambardzumyan, d.; ozawa, t.; fomchenko, e. i.; huse, j. t.; brennan, c. w.; holland, e. c. pten/pi3k/akt pathway regulates the side population phenotype and abcg2 activity in glioma tumor stem-like cells. cell stem cell 2009, 4, 226-35. van vliet, e. a.; redeker, s.; aronica, e.; edelbroek, p. m.; gorter, j. a. expression of multidrug transporters mrp1, mrp2, and bcrp shortly after status epilepticus, during the latent period, and in chronic epileptic rats. epilepsia 2005, 46, 1569-80. kooij, g.; mizee, m. r.; van horssen, j.; reijerkerk, a.; witte, m. e.; drexhage, j. a.; van der pol, s. m.; van het hof, b.; scheffer, g.; scheper, r.; dijkstra, c. d.; van der valk, p.; de vries, h. e. adenosine triphosphate-binding cassette transporters mediate chemokine (c-c motif) ligand 2 secretion from reactive astrocytes: relevance to multiple sclerosis pathogenesis. brain 2011, 134, 555-70. adams, s. m.; conley, y. p.; ren, d.; okonkwo, d. o.; puccio, a. m.; dixon, c. e.; clark, r. s. b.; kochanek, p. m.; empey, p. e. abcg2 c.421c>a is associated with outcomes after severe traumatic brain injury. j neurotrauma 2018, 35, 48-53. do, t. m.; noel-hudson, m. s.; ribes, s.; besengez, c.; smirnova, m.; cisternino, s.; buyse, m.; calon, f.; chimini, g.; chacun, h.; scherrmann, j. m.; farinotti, r.; bourasset, f. abcg2and abcg4-mediated efflux of amyloid-beta peptide 1-40 at the mouse blood-brain barrier. j alzheimers dis 2012, 30, 155-66. lam, f. c.; liu, r.; lu, p.; shapiro, a. b.; renoir, j. m.; sharom, f. j.; reiner, p. b. beta-amyloid efflux mediated by p-glycoprotein. j neurochem 2001, 76, 1121-8. piehler, a. p.; ozcurumez, m.; kaminski, w. e. a-subclass atp-binding cassette proteins in brain lipid homeostasis and neurodegeneration. front psychiatry 2012, 3, 17. krohn, m.; lange, c.; hofrichter, j.; scheffler, k.; stenzel, j.; steffen, j.; schumacher, t.; bruning, t.; plath, a. s.; alfen, f.; schmidt, a.; winter, f.; rateitschak, k.; wree, a.; gsponer, j.; walker, l. c.; pahnke, j. cerebral amyloid-beta proteostasis is regulated by the membrane transport protein abcc1 in mice. j clin invest 2011, 121, 3924-31. xiong, h.; callaghan, d.; jones, a.; bai, j.; rasquinha, i.; smith, c.; pei, k.; walker, d.; lue, l. f.; stanimirovic, d.; zhang, w. abcg2 is upregulated in alzheimer's brain with cerebral amyloid angiopathy and may act as a gatekeeper at the blood-brain barrier for abeta(1-40) peptides. j neurosci 2009, 29, 5463-75. holmes, c.; boche, d.; wilkinson, d.; yadegarfar, g.; hopkins, v.; bayer, a.; jones, r. w.; bullock, r.; love, s.; neal, j. w.; zotova, e.; nicoll, j. a. long-term effects of abeta42 immunisation in alzheimer's disease: follow-up of a randomised, placebo-controlled phase i trial. lancet 2008, 372, 216-23. pahnke, j.; walker, l. c.; scheffler, k.; krohn, m. alzheimer's disease and blood-brain barrier function-why have anti-beta-amyloid therapies failed to prevent dementia progression? neurosci biobehav rev 2009, 33, 1099-108. pahnke, j.; wolkenhauer, o.; krohn, m.; walker, l. c. clinico-pathologic function of cerebral abc transporters implications for the pathogenesis of alzheimer's disease. curr alzheimer res 2008, 5, 396-405. walker, l. c. abeta plaques. free neuropathol 2020, 1, 31. behl, t.; kaur, i.; sehgal, a.; kumar, a.; uddin, m. s.; bungau, s. the interplay of abc transporters in abeta translocation and cholesterol metabolism: implicating their roles in alzheimer's disease. mol neurobiol 2021, 58, 1564-82. koldamova, r.; fitz, n. f.; lefterov, i. atp-binding cassette transporter a1: from metabolism to neurodegeneration. neurobiol dis 2014, 72 pt a, 13-21. lyssenko, n. n.; praticò, d. abca7 and the altered lipidostasis hypothesis of alzheimer's disease. alzheimers dement 2020, 17, 164-174. abe-dohmae, s.; yokoyama, s. abca7 links sterol metabolism to the host defense system: molecular background for potential management measure of alzheimer's disease. gene 2021, 768, 145316. aikawa, t.; holm, m. l.; kanekiyo, t. abca7 and pathogenic pathways of alzheimer's disease. brain sci 2018, 8, 27. li, h.; karl, t.; garner, b. understanding the function of abca7 in alzheimer's disease. biochem soc trans 2015, 43, 920-3. andrews, s. j.; fulton-howard, b.; goate, a. protective variants in alzheimer's disease. curr genet med rep 2019, 7, 1-12. teresa, j. c.; fernado, c.; nancy, m. r.; gilberto, v. a.; alberto, c. r.; roberto, r. r. association of genetic variants of abca1 with susceptibility to dementia: (sadem study). metab brain dis 2020, 35, 915-22. jiang, s.; zhang, c. y.; tang, l.; zhao, l. x.; chen, h. z.; qiu, y. integrated genomic analysis revealed associated genes for alzheimer's disease in apoe4 non-carriers. curr alzheimer res 2019, 16, 753-63. chen, q.; liang, b.; wang, z.; cheng, x.; huang, y.; liu, y.; huang, z. influence of four polymorphisms in abca1 and ptgs2 genes on risk of alzheimer's disease: a meta-analysis. neurol sci 2016, 37, 1209-20. piaceri, i.; nacmias, b.; sorbi, s. genetics of familial and sporadic alzheimer's disease. front biosci (elite ed) 2013, 5, 167-77. agarwal, m.; khan, s. plasma lipids as biomarkers for alzheimer's disease: a systematic review. cureus 2020, 12, e12008. picard, c.; julien, c.; frappier, j.; miron, j.; théroux, l.; dea, d.; breitner, j. c. s.; poirier, j. alterations in cholesterol metabolism-related genes in sporadic alzheimer's disease. neurobiol aging 2018, 66, 180.e1-180.e9. beel, a. j.; sakakura, m.; barrett, p. j.; sanders, c. r. direct binding of cholesterol to the amyloid precursor protein: an important interaction in lipid-alzheimer's disease relationships? biochim biophys acta 2010, 1801, 975-82. shobab, l. a.; hsiung, g. y.; feldman, h. h. cholesterol in alzheimer's disease. lancet neurol 2005, 4, 841-52. sviridov, d.; mukhamedova, n.; miller, y. i. lipid rafts as a therapeutic target. j lipid res 2020, 61, 687-695. satoh, k.; abe-dohmae, s.; yokoyama, s.; st george-hyslop, p.; fraser, p. e. atp-binding cassette transporter a7 (abca7) loss of function alters alzheimer amyloid processing. j biol chem 2015, 290, 24152-65. zhao, q. f.; yu, j. t.; tan, m. s.; tan, l. abca7 in alzheimer's disease. mol neurobiol 2015, 51, 1008-16. chan, s. l.; kim, w. s.; kwok, j. b.; hill, a. f.; cappai, r.; rye, k. a.; garner, b. atp-binding cassette transporter a7 regulates processing of amyloid precursor protein in vitro. j neurochem 2008, 106, 793-804. sun, y.; yao, j.; kim, t. w.; tall, a. r. expression of liver x receptor target genes decreases cellular amyloid beta peptide secretion. j biol chem 2003, 278, 27688-94. koldamova, r. p.; lefterov, i. m.; ikonomovic, m. d.; skoko, j.; lefterov, p. i.; isanski, b. a.; dekosky, s. t.; lazo, j. s. 22r-hydroxycholesterol and 9-cis-retinoic acid induce atp-binding cassette transporter a1 expression and cholesterol efflux in brain cells and decrease amyloid beta secretion. j biol chem 2003, 278, 13244-56. fu, y.; hsiao, j. h.; paxinos, g.; halliday, g. m.; kim, w. s. abca7 mediates phagocytic clearance of amyloid-beta in the brain. j alzheimers dis 2016, 54, 569-84. kim, w. s.; li, h.; ruberu, k.; chan, s.; elliott, d. a.; low, j. k.; cheng, d.; karl, t.; garner, b. deletion of abca7 increases cerebral amyloid-beta accumulation in the j20 mouse model of alzheimer's disease. j neurosci 2013, 33, 4387-94. koldamova, r.; fitz, n. f.; lefterov, i. the role of atp-binding cassette transporter a1 in alzheimer's disease and neurodegeneration. biochim biophys acta 2010, 1801, 824-30. jiang, q.; lee, c. y.; mandrekar, s.; wilkinson, b.; cramer, p.; zelcer, n.; mann, k.; lamb, b.; willson, t. m.; collins, j. l.; richardson, j. c.; smith, j. d.; comery, t. a.; riddell, d.; holtzman, d. m.; tontonoz, p.; landreth, g. e. apoe promotes the proteolytic degradation of abeta. neuron 2008, 58, 681-93. wahrle, s. e.; jiang, h.; parsadanian, m.; kim, j.; li, a.; knoten, a.; jain, s.; hirsch-reinshagen, v.; wellington, c. l.; bales, k. r.; paul, s. m.; holtzman, d. m. overexpression of abca1 reduces amyloid deposition in the pdapp mouse model of alzheimer disease. j clin invest 2008, 118, 671-82. saint-pol, j.; vandenhaute, e.; boucau, m. c.; candela, p.; dehouck, l.; cecchelli, r.; dehouck, m. p.; fenart, l.; gosselet, f. brain pericytes abca1 expression mediates cholesterol efflux but not cellular amyloid-beta peptide accumulation. j alzheimers dis 2012, 30, 489-503. akanuma, s.; ohtsuki, s.; doi, y.; tachikawa, m.; ito, s.; hori, s.; asashima, t.; hashimoto, t.; yamada, k.; ueda, k.; iwatsubo, t.; terasaki, t. atp-binding cassette transporter a1 (abca1) deficiency does not attenuate the brain-to-blood efflux transport of human amyloid-beta peptide (1-40) at the blood-brain barrier. neurochem int 2008, 52, 956-61. albrecht, c.; viturro, e. the abca subfamily--gene and protein structures, functions and associated hereditary diseases. pflugers arch 2007, 453, 581-9. kubo, y.; sekiya, s.; ohigashi, m.; takenaka, c.; tamura, k.; nada, s.; nishi, t.; yamamoto, a.; yamaguchi, a. abca5 resides in lysosomes, and abca5 knockout mice develop lysosomal disease-like symptoms. mol cell biol 2005, 25, 4138-49. takahashi, k.; kimura, y.; nagata, k.; yamamoto, a.; matsuo, m.; ueda, k. abc proteins: key molecules for lipid homeostasis. med mol morphol 2005, 38, 2-12. garces, f. a.; scortecci, j. f.; molday, r. s. functional characterization of abca4 missense variants linked to stargardt macular degeneration. int j mol sci 2020, 22, 185. curtis, s. b.; molday, l. l.; garces, f. a.; molday, r. s. functional analysis and classification of homozygous and hypomorphic abca4 variants associated with stargardt macular degeneration. hum mutat 2020, 41, 1944-1956. garces, f.; jiang, k.; molday, l. l.; stohr, h.; weber, b. h.; lyons, c. j.; maberley, d.; molday, r. s. correlating the expression and functional activity of abca4 disease variants with the phenotype of patients with stargardt disease. invest ophthalmol vis sci 2018, 59, 2305-2315. quazi, f.; lenevich, s.; molday, r. s. abca4 is an n-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer. nat commun 2012, 3, 925. ahn, j.; wong, j. t.; molday, r. s. the effect of lipid environment and retinoids on the atpase activity of abcr, the photoreceptor abc transporter responsible for stargardt macular dystrophy. j biol chem 2000, 275, 20399-405. sun, h.; molday, r. s.; nathans, j. retinal stimulates atp hydrolysis by purified and reconstituted abcr, the photoreceptor-specific atp-binding cassette transporter responsible for stargardt disease. j biol chem 1999, 274, 8269-81. wambach, j. a.; yang, p.; wegner, d. j.; heins, h. b.; kaliberova, l. n.; kaliberov, s. a.; curiel, d. t.; white, f. v.; hamvas, a.; hackett, b. p.; cole, f. s. functional characterization of atp-binding cassette transporter a3 mutations from infants with respiratory distress syndrome. am j respir cell mol biol 2016, 55, 716-21. nathan, n.; berdah, l.; delestrain, c.; sileo, c.; clement, a. interstitial lung diseases in children. presse med 2020, 49, 103909. chen, p.; dai, y.; wu, x.; wang, y.; sun, s.; xiao, j.; zhang, q.; guan, l.; zhao, x.; hao, x.; wu, r.; xie, l. mutations in the abca3 gene are associated with cataract-microcornea syndrome. invest ophthalmol vis sci 2014, 55, 8031-43. destefano, g. m.; kurban, m.; anyane-yeboa, k.; dall'armi, c.; di paolo, g.; feenstra, h.; silverberg, n.; rohena, l.; lopez-cepeda, l. d.; jobanputra, v.; fantauzzo, k. a.; kiuru, m.; tadin-strapps, m.; sobrino, a.; vitebsky, a.; warburton, d.; levy, b.; salas-alanis, j. c.; christiano, a. m. mutations in the cholesterol transporter gene abca5 are associated with excessive hair overgrowth. plos genetics 2014, 10, e1004333. elkhatib, a. m.; omar, m. ichthyosis fetalis. in statpearls, treasure island (fl), 2021. zarubica, a.; trompier, d.; chimini, g. abca1, from pathology to membrane function. pflugers arch 2007, 453, 569-79. luciani, m. f.; denizot, f.; savary, s.; mattei, m. g.; chimini, g. cloning of two novel abc transporters mapping on human chromosome 9. genomics 1994, 21, 150-9. santamarina-fojo, s.; peterson, k.; knapper, c.; qiu, y.; freeman, l.; cheng, j. f.; osorio, j.; remaley, a.; yang, x. p.; haudenschild, c.; prades, c.; chimini, g.; blackmon, e.; francois, t.; duverger, n.; rubin, e. m.; rosier, m.; denefle, p.; fredrickson, d. s.; brewer, h. b., jr. complete genomic sequence of the human abca1 gene: analysis of the human and mouse atp-binding cassette a promoter. proc natl acad sci u s a 2000, 97, 7987-92. tang, c.; oram, j. f. the cell cholesterol exporter abca1 as a protector from cardiovascular disease and diabetes. biochim biophys acta 2009, 1791, 563-72. oram, j. f.; lawn, r. m. abca1. the gatekeeper for eliminating excess tissue cholesterol. j lipid res 2001, 42, 1173-9. hafiane, a.; gianopoulos, i.; sorci-thomas, m. g.; daskalopoulou, s. s. current models of apolipoprotein a-i lipidation by adenosine triphosphate binding cassette transporter a1. curr opin lipidol 2021. hirsch-reinshagen, v.; zhou, s.; burgess, b. l.; bernier, l.; mcisaac, s. a.; chan, j. y.; tansley, g. h.; cohn, j. s.; hayden, m. r.; wellington, c. l. deficiency of abca1 impairs apolipoprotein e metabolism in brain. j biol chem 2004, 279, 41197-207. wahrle, s. e.; jiang, h.; parsadanian, m.; legleiter, j.; han, x.; fryer, j. d.; kowalewski, t.; holtzman, d. m. abca1 is required for normal central nervous system apoe levels and for lipidation of astrocyte-secreted apoe. j biol chem 2004, 279, 40987-93. fitz, n. f.; carter, a. y.; tapias, v.; castranio, e. l.; kodali, r.; lefterov, i.; koldamova, r. abca1 deficiency affects basal cognitive deficits and dendritic density in mice. j alzheimers dis 2017, 56, 1075-85. corder, e. h.; saunders, a. m.; strittmatter, w. j.; schmechel, d. e.; gaskell, p. c.; small, g. w.; roses, a. d.; haines, j. l.; pericak-vance, m. a. gene dose of apolipoprotein e type 4 allele and the risk of alzheimer's disease in late onset families. science 1993, 261, 921-3. poirier, j.; davignon, j.; bouthillier, d.; kogan, s.; bertrand, p.; gauthier, s. apolipoprotein e polymorphism and alzheimer's disease. lancet 1993, 342, 697-9. rawat, v.; wang, s.; sima, j.; bar, r.; liraz, o.; gundimeda, u.; parekh, t.; chan, j.; johansson, j. o.; tang, c.; chui, h. c.; harrington, m. g.; michaelson, d. m.; yassine, h. n. apoe4 alters abca1 membrane trafficking in astrocytes. j neurosci 2019, 39, 9611-22. marchi, c.; adorni, m. p.; caffarra, p.; ronda, n.; spallazzi, m.; barocco, f.; galimberti, d.; bernini, f.; zimetti, f. abca1and abcg1-mediated cholesterol efflux capacity of cerebrospinal fluid is impaired in alzheimer's disease. j lipid res 2019, 60, 1449-56. akram, a.; schmeidler, j.; katsel, p.; hof, p. r.; haroutunian, v. increased expression of cholesterol transporter abca1 is highly correlated with severity of dementia in ad hippocampus. brain res 2010, 1318, 167-77. holstege, h.; hulsman, m.; charbonnier, c.; grenier-boley, b.; quenez, o.; grozeva, d.; van rooij, j. g. j.; sims, r.; ahmad, s.; amin, n.; norsworthy, p. j.; dols-icardo, o.; hummerich, h.; kawalia, a.; database, a. s. d. n. i.; amouyel, p.; beecham, g. w.; berr, c.; bis, j. c.; boland, a.; bossù, p.; bouwman, f.; bras, j.; campion, d.; cochran, j. n.; daniele, a.; dartigues, j.-f.; debette, s.; deleuze, j.-f.; denning, n.; destefano, a. l.; farrer, l. a.; fernandez, m. v.; fox, n. c.; galimberti, d.; genin, e.; gille, h.; guen, y. l.; guerreiro, r.; haines, j. l.; holmes, c.; ikram, m. a.; ikram, m. k.; jansen, i. e.; kraaij, r.; lathrop, m.; lemstra, a. w.; lleó, a.; luckcuck, l.; mannens, m. m. a. m.; marshall, r.; martin, e. r.; masullo, c.; mayeux, r.; mecocci, p.; meggy, a.; mol, m. o.; morgan, k.; myers, r. m.; nacmias, b.; naj, a. c.; napolioni, v.; pasquier, f.; pastor, p.; pericak-vance, m. a.; raybould, r.; redon, r.; reinders, m. j. t.; richard, a.-c.; riedel-heller, s. g.; rivadeneira, f.; rousseau, s.; ryan, n. s.; saad, s.; sanchez-juan, p.; schellenberg, g. d.; scheltens, p.; schott, j. m.; seripa, d.; seshadri, s.; sie, d.; sistermans, e.; sorbi, s.; van spaendonk, r.; spalletta, g.; tesi, n.; tijms, b.; uitterlinden, a. g.; van der lee, s. j.; de visser, p. j.; wagner, m.; wallon, d.; wang, l.-s.; zarea, a.; clarimon, j.; van swieten, j. c.; greicius, m. d.; yokoyama, j. s.; cruchaga, c.; hardy, j.; ramirez, a.; mead, s.; van der flier, w. m.; van duijn, c. m.; williams, j.; nicolas, g.; bellenguez, c.; lambert, j.-c. exome sequencing identifies rare damaging variants in the atp8b4 and abca1 genes as novel risk factors for alzheimer’s disease. medrxiv 2021, 2020.07.22.20159251. alrasadi, k.; ruel, i. l.; marcil, m.; genest, j. functional mutations of the abca1 gene in subjects of french-canadian descent with hdl deficiency. atheroscler 2006, 188, 281-91. ma, x.; wang, t.; zhao, z. l.; jiang, y.; ye, s. propofol suppresses proinflammatory cytokine production by increasing abca1 expression via mediation by the long noncoding rna loc286367. mediators inflamm 2018, 2018, 8907143. koldamova, r.; staufenbiel, m.; lefterov, i. lack of abca1 considerably decreases brain apoe level and increases amyloid deposition in app23 mice. j biol chem 2005, 280, 43224-35. wahrle, s. e.; jiang, h.; parsadanian, m.; hartman, r. e.; bales, k. r.; paul, s. m.; holtzman, d. m. deletion of abca1 increases abeta deposition in the pdapp transgenic mouse model of alzheimer disease. j biol chem 2005, 280, 43236-42. hirsch-reinshagen, v.; maia, l. f.; burgess, b. l.; blain, j. f.; naus, k. e.; mcisaac, s. a.; parkinson, p. f.; chan, j. y.; tansley, g. h.; hayden, m. r.; poirier, j.; van nostrand, w.; wellington, c. l. the absence of abca1 decreases soluble apoe levels but does not diminish amyloid deposition in two murine models of alzheimer disease. j biol chem 2005, 280, 43243-56. fitz, n. f.; nam, k. n.; wolfe, c. m.; letronne, f.; playso, b. e.; iordanova, b. e.; kozai, t. d. y.; biedrzycki, r. j.; kagan, v. e.; tyurina, y. y.; han, x.; lefterov, i.; koldamova, r. phospholipids of apoe lipoproteins activate microglia in an isoform-specific manner in preclinical models of alzheimer's disease. nat commun 2021, 12, 3416. deane, r.; sagare, a.; hamm, k.; parisi, m.; lane, s.; finn, m. b.; holtzman, d. m.; zlokovic, b. v. apoe isoform-specific disruption of amyloid beta peptide clearance from mouse brain. j clin invest 2008, 118, 4002-13. basak, j. m.; kim, j.; pyatkivskyy, y.; wildsmith, k. r.; jiang, h.; parsadanian, m.; patterson, b. w.; bateman, r. j.; holtzman, d. m. measurement of apolipoprotein e and amyloid beta clearance rates in the mouse brain using bolus stable isotope labeling. mol neurodegener 2012, 7, 14. fitz, n. f.; tapias, v.; cronican, a. a.; castranio, e. l.; saleem, m.; carter, a. y.; lefterova, m.; lefterov, i.; koldamova, r. opposing effects of apoe/apoa1 double deletion on amyloid-beta pathology and cognitive performance in app mice. brain 2015, 138, 3699-715. lefterov, i.; fitz, n. f.; cronican, a.; lefterov, p.; staufenbiel, m.; koldamova, r. memory deficits in app23/abca1+/mice correlate with the level of abeta oligomers. asn neuro 2009, 1, e00006. fitz, n. f.; cronican, a. a.; saleem, m.; fauq, a. h.; chapman, r.; lefterov, i.; koldamova, r. abca1 deficiency affects alzheimer's disease-like phenotype in human apoe4 but not in apoe3-targeted replacement mice. j neurosci 2012, 32, 13125-36. broccardo, c.; nieoullon, v.; amin, r.; masmejean, f.; carta, s.; tassi, s.; pophillat, m.; rubartelli, a.; pierres, m.; rougon, g.; nieoullon, a.; chazal, g.; chimini, g. abca2 is a marker of neural progenitors and neuronal subsets in the adult rodent brain. j neurochem 2006, 97, 345-55. vulevic, b.; chen, z.; boyd, j. t.; davis, w., jr.; walsh, e. s.; belinsky, m. g.; tew, k. d. cloning and characterization of human adenosine 5'-triphosphate-binding cassette, sub-family a, transporter 2 (abca2). cancer res 2001, 61, 3339-47. zhou, c.; zhao, l.; inagaki, n.; guan, j.; nakajo, s.; hirabayashi, t.; kikuyama, s.; shioda, s. atp-binding cassette transporter abc2/abca2 in the rat brain: a novel mammalian lysosome-associated membrane protein and a specific marker for oligodendrocytes but not for myelin sheaths. j neurosci 2001, 21, 849-57. zhao, l. x.; zhou, c. j.; tanaka, a.; nakata, m.; hirabayashi, t.; amachi, t.; shioda, s.; ueda, k.; inagaki, n. cloning, characterization and tissue distribution of the rat atp-binding cassette (abc) transporter abc2/abca2. biochem j 2000, 350 pt 3, 865-72. tanaka, y.; yamada, k.; zhou, c. j.; ban, n.; shioda, s.; inagaki, n. temporal and spatial profiles of abca2-expressing oligodendrocytes in the developing rat brain. j comp neurol 2003, 455, 353-67. kaminski, w. e.; piehler, a.; püllmann, k.; porsch-ozcürümez, m.; duong, c.; bared, g. m.; büchler, c.; schmitz, g. complete coding sequence, promoter region, and genomic structure of the human abca2 gene and evidence for sterol-dependent regulation in macrophages. biochem biophys res commun 2001, 281, 249-58. wollmer, m. a.; kapaki, e.; hersberger, m.; muntwyler, j.; brunner, f.; tsolaki, m.; akatsu, h.; kosaka, k.; michikawa, m.; molyva, d.; paraskevas, g. p.; lütjohann, d.; von eckardstein, a.; hock, c.; nitsch, r. m.; papassotiropoulos, a. ethnicity-dependent genetic association of abca2 with sporadic alzheimer's disease. am j med genet b neuropsychiatr genet 2006, 141b, 534-6. minster, r. l.; dekosky, s. t.; kamboh, m. i. no association of dapk1 and abca2 snps on chromosome 9 with alzheimer's disease. neurobiol aging 2009, 30, 1890-1. hu, w.; lin, x.; zhang, h.; zhao, n. atp binding cassette subfamily a member 2 (abca2) expression and methylation are associated with alzheimer's disease. med sci monit 2017, 23, 5851-61. davis, w., jr.; boyd, j. t.; ile, k. e.; tew, k. d. human atp-binding cassette transporter-2 (abca2) positively regulates low-density lipoprotein receptor expression and negatively regulates cholesterol esterification in chinese hamster ovary cells. biochim biophys acta 2004, 1683, 89-100. chen, z. j.; vulevic, b.; ile, k. e.; soulika, a.; davis, w., jr.; reiner, p. b.; connop, b. p.; nathwani, p.; trojanowski, j. q.; tew, k. d. association of abca2 expression with determinants of alzheimer's disease. faseb j 2004, 18, 1129-31. katsouri, l.; georgopoulos, s. lack of ldl receptor enhances amyloid deposition and decreases glial response in an alzheimer's disease mouse model. plos one 2011, 6, e21880. michaki, v.; guix, f. x.; vennekens, k.; munck, s.; dingwall, c.; davis, j. b.; townsend, d. m.; tew, k. d.; feiguin, f.; de strooper, b.; dotti, c. g.; wahle, t. down-regulation of the atp-binding cassette transporter 2 (abca2) reduces amyloid-beta production by altering nicastrin maturation and intracellular localization. j biol chem 2012, 287, 1100-11. davis, w., jr. the atp-binding cassette transporter-2 (abca2) overexpression modulates sphingosine levels and transcription of the amyloid precursor protein (app) gene. curr alzheimer res 2015, 12, 847-59. kim, w. s.; rahmanto, a. s.; kamili, a.; rye, k. a.; guillemin, g. j.; gelissen, i. c.; jessup, w.; hill, a. f.; garner, b. role of abcg1 and abca1 in regulation of neuronal cholesterol efflux to apolipoprotein e discs and suppression of amyloid-beta peptide generation. j biol chem 2007, 282, 2851-61. yamano, g.; funahashi, h.; kawanami, o.; zhao, l. x.; ban, n.; uchida, y.; morohoshi, t.; ogawa, j.; shioda, s.; inagaki, n. abca3 is a lamellar body membrane protein in human lung alveolar type ii cells. febs lett 2001, 508, 221-5. fitzgerald, m. l.; xavier, r.; haley, k. j.; welti, r.; goss, j. l.; brown, c. e.; zhuang, d. z.; bell, s. a.; lu, n.; mckee, m.; seed, b.; freeman, m. w. abca3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol. j lipid res 2007, 48, 621-32. stahlman, m. t.; besnard, v.; wert, s. e.; weaver, t. e.; dingle, s.; xu, y.; von zychlin, k.; olson, s. j.; whitsett, j. a. expression of abca3 in developing lung and other tissues. j histochem cytochem 2007, 55, 71-83. kim, w. s.; guillemin, g. j.; glaros, e. n.; lim, c. k.; garner, b. quantitation of atp-binding cassette subfamily-a transporter gene expression in primary human brain cells. neuroreport 2006, 17, 891-6. whiley, l.; sen, a.; heaton, j.; proitsi, p.; garcía-gómez, d.; leung, r.; smith, n.; thambisetty, m.; kloszewska, i.; mecocci, p.; soininen, h.; tsolaki, m.; vellas, b.; lovestone, s.; legido-quigley, c. evidence of altered phosphatidylcholine metabolism in alzheimer's disease. neurobiol aging 2014, 35, 271-8. allikmets, r.; singh, n.; sun, h.; shroyer, n. f.; hutchinson, a.; chidambaram, a.; gerrard, b.; baird, l.; stauffer, d.; peiffer, a.; rattner, a.; smallwood, p.; li, y.; anderson, k. l.; lewis, r. a.; nathans, j.; leppert, m.; dean, m.; lupski, j. r. a photoreceptor cell-specific atp-binding transporter gene (abcr) is mutated in recessive stargardt macular dystrophy. nat genet 1997, 15, 236-46. kaway, c. s.; adams, m. k. m.; jenkins, k. s.; layton, c. j. a novel abca4 mutation associated with a late-onset stargardt disease phenotype: a hypomorphic allele? case rep ophthalmol 2017, 8, 180-4. muller, p. l.; treis, t.; odainic, a.; pfau, m.; herrmann, p.; tufail, a.; holz, f. g. prediction of function in abca4-related retinopathy using ensemble machine learning. j clin med 2020, 9. ohtsuki, s.; watanabe, y.; hori, s.; suzuki, h.; bhongsatiern, j.; fujiyoshi, m.; kamoi, m.; kamiya, n.; takanaga, h.; terasaki, t. mrna expression of the atp-binding cassette transporter subfamily a (abca) in rat and human brain capillary endothelial cells. biol pharm bull 2004, 27, 1437-40. petry, f.; kotthaus, a.; hirsch-ernst, k. i. cloning of human and rat abca5/abca5 and detection of a human splice variant. biochem biophys res commun 2003, 300, 343-50. fu, y.; hsiao, j. h.; paxinos, g.; halliday, g. m.; kim, w. s. abca5 regulates amyloid-beta peptide production and is associated with alzheimer's disease neuropathology. j alzheimers dis 2015, 43, 857-69. kaminski, w. e.; wenzel, j. j.; piehler, a.; langmann, t.; schmitz, g. abca6, a novel a subclass abc transporter. biochem biophys res commun 2001, 285, 1295-301. van leeuwen, e. m.; karssen, l. c.; deelen, j.; isaacs, a.; medina-gomez, c.; mbarek, h.; kanterakis, a.; trompet, s.; postmus, i.; verweij, n.; van enckevort, d. j.; huffman, j. e.; white, c. c.; feitosa, m. f.; bartz, t. m.; manichaikul, a.; joshi, p. k.; peloso, g. m.; deelen, p.; van dijk, f.; willemsen, g.; de geus, e. j.; milaneschi, y.; penninx, b. w.; francioli, l. c.; menelaou, a.; pulit, s. l.; rivadeneira, f.; hofman, a.; oostra, b. a.; franco, o. h.; mateo leach, i.; beekman, m.; de craen, a. j.; uh, h. w.; trochet, h.; hocking, l. j.; porteous, d. j.; sattar, n.; packard, c. j.; buckley, b. m.; brody, j. a.; bis, j. c.; rotter, j. i.; mychaleckyj, j. c.; campbell, h.; duan, q.; lange, l. a.; wilson, j. f.; hayward, c.; polasek, o.; vitart, v.; rudan, i.; wright, a. f.; rich, s. s.; psaty, b. m.; borecki, i. b.; kearney, p. m.; stott, d. j.; adrienne cupples, l.; jukema, j. w.; van der harst, p.; sijbrands, e. j.; hottenga, j. j.; uitterlinden, a. g.; swertz, m. a.; van ommen, g. j.; de bakker, p. i.; eline slagboom, p.; boomsma, d. i.; wijmenga, c.; van duijn, c. m. genome of the netherlands population-specific imputations identify an abca6 variant associated with cholesterol levels. nat commun 2015, 6, 6065. dib, s.; pahnke, j.; gosselet, f. role of abca7 in human health and in alzheimer's disease. int j mol sci 2021, 22, 4603. kaminski, w. e.; piehler, a.; schmitz, g. genomic organization of the human cholesterol-responsive abc transporter abca7: tandem linkage with the minor histocompatibility antigen ha-1 gene. biochem biophys res commun 2000, 278, 782-9. kaminski, w. e.; orso, e.; diederich, w.; klucken, j.; drobnik, w.; schmitz, g. identification of a novel human sterol-sensitive atp-binding cassette transporter (abca7). biochem biophys res commun 2000, 273, 532-8. quazi, f.; molday, r. s. differential phospholipid substrates and directional transport by atp-binding cassette proteins abca1, abca7, and abca4 and disease-causing mutants. j biol chem 2013, 288, 34414-26. hayashi, m.; abe-dohmae, s.; okazaki, m.; ueda, k.; yokoyama, s. heterogeneity of high density lipoprotein generated by abca1 and abca7. j lipid res 2005, 46, 1703-11. tanaka, n.; abe-dohmae, s.; iwamoto, n.; yokoyama, s. roles of atp-binding cassette transporter a7 in cholesterol homeostasis and host defense system. j atheroscler thromb 2011, 18, 274-81. jehle, a. w.; gardai, s. j.; li, s.; linsel-nitschke, p.; morimoto, k.; janssen, w. j.; vandivier, r. w.; wang, n.; greenberg, s.; dale, b. m.; qin, c.; henson, p. m.; tall, a. r. atp-binding cassette transporter a7 enhances phagocytosis of apoptotic cells and associated erk signaling in macrophages. j cell biol 2006, 174, 547-56. iwamoto, n.; abe-dohmae, s.; sato, r.; yokoyama, s. abca7 expression is regulated by cellular cholesterol through the srebp2 pathway and associated with phagocytosis. j lipid res 2006, 47, 1915-27. meurs, i.; calpe-berdiel, l.; habets, k. l.; zhao, y.; korporaal, s. j.; mommaas, a. m.; josselin, e.; hildebrand, r. b.; ye, d.; out, r.; kuiper, j.; van berkel, t. j.; chimini, g.; van eck, m. effects of deletion of macrophage abca7 on lipid metabolism and the development of atherosclerosis in the presence and absence of abca1. plos one 2012, 7, e30984. kim, w. s.; fitzgerald, m. l.; kang, k.; okuhira, k.; bell, s. a.; manning, j. j.; koehn, s. l.; lu, n.; moore, k. j.; freeman, m. w. abca7 null mice retain normal macrophage phosphatidylcholine and cholesterol efflux activity despite alterations in adipose mass and serum cholesterol levels. j biol chem 2005, 280, 3989-95. linsel-nitschke, p.; jehle, a. w.; shan, j.; cao, g.; bacic, d.; lan, d.; wang, n.; tall, a. r. potential role of abca7 in cellular lipid efflux to apoa-i. j lipid res 2005, 46, 86-92. hollingworth, p.; harold, d.; sims, r.; gerrish, a.; lambert, j. c.; carrasquillo, m. m.; abraham, r.; hamshere, m. l.; pahwa, j. s.; moskvina, v.; dowzell, k.; jones, n.; stretton, a.; thomas, c.; richards, a.; ivanov, d.; widdowson, c.; chapman, j.; lovestone, s.; powell, j.; proitsi, p.; lupton, m. k.; brayne, c.; rubinsztein, d. c.; gill, m.; lawlor, b.; lynch, a.; brown, k. s.; passmore, p. a.; craig, d.; mcguinness, b.; todd, s.; holmes, c.; mann, d.; smith, a. d.; beaumont, h.; warden, d.; wilcock, g.; love, s.; kehoe, p. g.; hooper, n. m.; vardy, e. r.; hardy, j.; mead, s.; fox, n. c.; rossor, m.; collinge, j.; maier, w.; jessen, f.; ruther, e.; schurmann, b.; heun, r.; kolsch, h.; van den bussche, h.; heuser, i.; kornhuber, j.; wiltfang, j.; dichgans, m.; frolich, l.; hampel, h.; gallacher, j.; hull, m.; rujescu, d.; giegling, i.; goate, a. m.; kauwe, j. s.; cruchaga, c.; nowotny, p.; morris, j. c.; mayo, k.; sleegers, k.; bettens, k.; engelborghs, s.; de deyn, p. p.; van broeckhoven, c.; livingston, g.; bass, n. j.; gurling, h.; mcquillin, a.; gwilliam, r.; deloukas, p.; al-chalabi, a.; shaw, c. e.; tsolaki, m.; singleton, a. b.; guerreiro, r.; muhleisen, t. w.; nothen, m. m.; moebus, s.; jockel, k. h.; klopp, n.; wichmann, h. e.; pankratz, v. s.; sando, s. b.; aasly, j. o.; barcikowska, m.; wszolek, z. k.; dickson, d. w.; graff-radford, n. r.; petersen, r. c.; alzheimer's disease neuroimaging, i.; van duijn, c. m.; breteler, m. m.; ikram, m. a.; destefano, a. l.; fitzpatrick, a. l.; lopez, o.; launer, l. j.; seshadri, s.; consortium, c.; berr, c.; campion, d.; epelbaum, j.; dartigues, j. f.; tzourio, c.; alperovitch, a.; lathrop, m.; consortium, e.; feulner, t. m.; friedrich, p.; riehle, c.; krawczak, m.; schreiber, s.; mayhaus, m.; nicolhaus, s.; wagenpfeil, s.; steinberg, s.; stefansson, h.; stefansson, k.; snaedal, j.; bjornsson, s.; jonsson, p. v.; chouraki, v.; genier-boley, b.; hiltunen, m.; soininen, h.; combarros, o.; zelenika, d.; delepine, m.; bullido, m. j.; pasquier, f.; mateo, i.; frank-garcia, a.; porcellini, e.; hanon, o.; coto, e.; alvarez, v.; bosco, p.; siciliano, g.; mancuso, m.; panza, f.; solfrizzi, v.; nacmias, b.; sorbi, s.; bossu, p.; piccardi, p.; arosio, b.; annoni, g.; seripa, d.; pilotto, a.; scarpini, e.; galimberti, d.; brice, a.; hannequin, d.; licastro, f.; jones, l.; holmans, p. a.; jonsson, t.; riemenschneider, m.; morgan, k.; younkin, s. g.; owen, m. j.; o'donovan, m.; amouyel, p.; williams, j. common variants at abca7, ms4a6a/ms4a4e, epha1, cd33 and cd2ap are associated with alzheimer's disease. nat genet 2011, 43, 429-35. vasquez, j. b.; simpson, j. f.; harpole, r.; estus, s. alzheimer's disease genetics and abca7 splicing. j alzheimers dis 2017, 59, 633-41. karch, c. m.; jeng, a. t.; nowotny, p.; cady, j.; cruchaga, c.; goate, a. m. expression of novel alzheimer's disease risk genes in control and alzheimer's disease brains. plos one 2012, 7, e50976. allen, m.; zou, f.; chai, h. s.; younkin, c. s.; crook, j.; pankratz, v. s.; carrasquillo, m. m.; rowley, c. n.; nair, a. a.; middha, s.; maharjan, s.; nguyen, t.; ma, l.; malphrus, k. g.; palusak, r.; lincoln, s.; bisceglio, g.; georgescu, c.; schultz, d.; rakhshan, f.; kolbert, c. p.; jen, j.; haines, j. l.; mayeux, r.; pericak-vance, m. a.; farrer, l. a.; schellenberg, g. d.; petersen, r. c.; graff-radford, n. r.; dickson, d. w.; younkin, s. g.; ertekin-taner, n.; alzheimer's disease genetics, c.; apostolova, l. g.; arnold, s. e.; baldwin, c. t.; barber, r.; barmada, m. m.; beach, t.; beecham, g. w.; beekly, d.; bennett, d. a.; bigio, e. h.; bird, t. d.; blacker, d.; boeve, b. f.; bowen, j. d.; boxer, a.; burke, j. r.; buros, j.; buxbaum, j. d.; cairns, n. j.; cantwell, l. b.; cao, c.; carlson, c. s.; carney, r. m.; carroll, s. l.; chui, h. c.; clark, d. g.; corneveaux, j.; cotman, c. w.; crane, p. k.; cruchaga, c.; cummings, j. l.; de jager, p. l.; decarli, c.; dekosky, s. t.; demirci, f. y.; diaz-arrastia, r.; dick, m.; dombroski, b. a.; duara, r.; ellis, w. d.; evans, d.; faber, k. m.; fallon, k. b.; farlow, m. r.; ferris, s.; foroud, t. m.; frosch, m.; galasko, d. r.; gallins, p. j.; ganguli, m.; gearing, m.; geschwind, d. h.; ghetti, b.; gilbert, j. r.; gilman, s.; giordani, b.; glass, j. d.; goate, a. m.; green, r. c.; growdon, j. h.; hakonarson, h.; hamilton, r. l.; hardy, j.; harrell, l. e.; head, e.; honig, l. s.; huentelman, m. j.; hulette, c. m.; hyman, b. t.; jarvik, g. p.; jicha, g. a.; jin, l. w.; jun, g.; kamboh, m. i.; karlawish, j.; karydas, a.; kauwe, j. s.; kaye, j. a.; kennedy, n.; kim, r.; koo, e. h.; kowall, n. w.; kramer, p.; kukull, w. a.; lah, j. j.; larson, e. b.; levey, a. i.; lieberman, a. p.; lopez, o. l.; lunetta, k. l.; mack, w. j.; marson, d. c.; martin, e. r.; martiniuk, f.; mash, d. c.; masliah, e.; mccormick, w. c.; mccurry, s. m.; mcdavid, a. n.; mckee, a. c.; mesulam, m.; miller, b. l.; miller, c. a.; miller, j. w.; montine, t. j.; morris, j. c.; myers, a. j.; naj, a. c.; nowotny, p.; parisi, j. e.; perl, d. p.; peskind, e.; poon, w. w.; potter, h.; quinn, j. f.; raj, a.; rajbhandary, r. a.; raskind, m.; reiman, e. m.; reisberg, b.; reitz, c.; ringman, j. m.; roberson, e. d.; rogaeva, e.; rosenberg, r. n.; sano, m.; saykin, a. j.; schneider, j. a.; schneider, l. s.; seeley, w.; shelanski, m. l.; slifer, m. a.; smith, c. d.; sonnen, j. a.; spina, s.; st george-hyslop, p.; stern, r. a.; tanzi, r. e.; trojanowski, j. q.; troncoso, j. c.; tsuang, d. w.; van deerlin, v. m.; vardarajan, b. n.; vinters, h. v.; vonsattel, j. p.; wang, l. s.; weintraub, s.; welsh-bohmer, k. a.; williamson, j.; woltjer, r. l. novel late-onset alzheimer disease loci variants associate with brain gene expression. neurol 2012, 79, 221-8. steinberg, s.; stefansson, h.; jonsson, t.; johannsdottir, h.; ingason, a.; helgason, h.; sulem, p.; magnusson, o. t.; gudjonsson, s. a.; unnsteinsdottir, u.; kong, a.; helisalmi, s.; soininen, h.; lah, j. j.; demgene; aarsland, d.; fladby, t.; ulstein, i. d.; djurovic, s.; sando, s. b.; white, l. r.; knudsen, g. p.; westlye, l. t.; selbaek, g.; giegling, i.; hampel, h.; hiltunen, m.; levey, a. i.; andreassen, o. a.; rujescu, d.; jonsson, p. v.; bjornsson, s.; snaedal, j.; stefansson, k. loss-of-function variants in abca7 confer risk of alzheimer's disease. nat genet 2015, 47, 445-7. shulman, j. m.; chen, k.; keenan, b. t.; chibnik, l. b.; fleisher, a.; thiyyagura, p.; roontiva, a.; mccabe, c.; patsopoulos, n. a.; corneveaux, j. j.; yu, l.; huentelman, m. j.; evans, d. a.; schneider, j. a.; reiman, e. m.; de jager, p. l.; bennett, d. a. genetic susceptibility for alzheimer disease neuritic plaque pathology. jama neurol 2013, 70, 1150-7. sinha, n.; berg, c. n.; shaw, a.; gluck, m. a. abca7 genotype moderates the effect of aerobic exercise intervention on generalization of prior learning in healthy older african americans. j alzheimers dis 2020, 74, 309-18. lamartiniere, y.; boucau, m. c.; dehouck, l.; krohn, m.; pahnke, j.; candela, p.; gosselet, f.; fenart, l. abca7 downregulation modifies cellular cholesterol homeostasis and decreases amyloid-beta peptide efflux in an in vitro model of the blood-brain barrier. j alzheimers dis 2018, 64, 1195-1211. li, m.; yuan, y.; hu, b.; wu, l. study on lentivirus-mediated abca7 improves neurocognitive function and related mechanisms in the c57bl/6 mouse model of alzheimer's disease. j mol neurosci 2017, 61, 489-97. sakae, n.; liu, c. c.; shinohara, m.; frisch-daiello, j.; ma, l.; yamazaki, y.; tachibana, m.; younkin, l.; kurti, a.; carrasquillo, m. m.; zou, f.; sevlever, d.; bisceglio, g.; gan, m.; fol, r.; knight, p.; wang, m.; han, x.; fryer, j. d.; fitzgerald, m. l.; ohyagi, y.; younkin, s. g.; bu, g.; kanekiyo, t. abca7 deficiency accelerates amyloid-beta generation and alzheimer's neuronal pathology. j neurosci 2016, 36, 3848-59. aikawa, t.; ren, y.; yamazaki, y.; tachibana, m.; johnson, m. r.; anderson, c. t.; martens, y. a.; holm, m. l.; asmann, y. w.; saito, t.; saido, t. c.; fitzgerald, m. l.; bu, g.; kanekiyo, t. abca7 haplodeficiency disturbs microglial immune responses in the mouse brain. proc natl acad sci u s a 2019, 116, 23790-23796. zissimopoulos, j. m.; barthold, d.; brinton, r. d.; joyce, g. sex and race differences in the association between statin use and the incidence of alzheimer disease. jama neurolog 2017, 74, 225-32. yang, c.; yuan, h.; gu, j.; xu, d.; wang, m.; qiao, j.; yang, x.; zhang, j.; yao, m.; gu, j.; tu, h.; gan, y. abca8-mediated efflux of taurocholic acid contributes to gemcitabine insensitivity in human pancreatic cancer via the s1pr2-erk pathway. cell death discov 2021, 7, 6. tsuruoka, s.; ishibashi, k.; yamamoto, h.; wakaumi, m.; suzuki, m.; schwartz, g. j.; imai, m.; fujimura, a. functional analysis of abca8, a new drug transporter. biochem biophys res commun 2002, 298, 41-5. akiyama, m.; sugiyama-nakagiri, y.; sakai, k.; mcmillan, j. r.; goto, m.; arita, k.; tsuji-abe, y.; tabata, n.; matsuoka, k.; sasaki, r.; sawamura, d.; shimizu, h. mutations in lipid transporter abca12 in harlequin ichthyosis and functional recovery by corrective gene transfer. j clin invest 2005, 115, 1777-84. kelsell, d. p.; norgett, e. e.; unsworth, h.; teh, m. t.; cullup, t.; mein, c. a.; dopping-hepenstal, p. j.; dale, b. a.; tadini, g.; fleckman, p.; stephens, k. g.; sybert, v. p.; mallory, s. b.; north, b. v.; witt, d. r.; sprecher, e.; taylor, a. e.; ilchyshyn, a.; kennedy, c. t.; goodyear, h.; moss, c.; paige, d.; harper, j. i.; young, b. d.; leigh, i. m.; eady, r. a.; o'toole, e. a. mutations in abca12 underlie the severe congenital skin disease harlequin ichthyosis. am j hum genet 2005, 76, 794-803. lefévre, c.; audebert, s.; jobard, f.; bouadjar, b.; lakhdar, h.; boughdene-stambouli, o.; blanchet-bardon, c.; heilig, r.; foglio, m.; weissenbach, j.; lathrop, m.; prud'homme, j. f.; fischer, j. mutations in the transporter abca12 are associated with lamellar ichthyosis type 2. hum mol genet 2003, 12, 2369-78. ohkubo, t.; shibata, n.; ohnuma, t.; higashi, s.; usui, c.; ueki, a.; nagao, m.; arai, h. no genetic association between atp binding cassette proteins and japanese sporadic alzheimer's disease. dement geriatr cogn disord 2005, 20, 95-8. prades, c.; arnould, i.; annilo, t.; shulenin, s.; chen, z. q.; orosco, l.; triunfol, m.; devaud, c.; maintoux-larois, c.; lafargue, c.; lemoine, c.; denèfle, p.; rosier, m.; dean, m. the human atp binding cassette gene abca13, located on chromosome 7p12.3, encodes a 5058 amino acid protein with an extracellular domain encoded in part by a 4.8-kb conserved exon. cytogenet genome res 2002, 98, 160-8. kim, w. s.; weickert, c. s.; garner, b. role of atp-binding cassette transporters in brain lipid transport and neurological disease. j neurochem 2008, 104, 1145-66. sato, k.; malchinkhuu, e.; horiuchi, y.; mogi, c.; tomura, h.; tosaka, m.; yoshimoto, y.; kuwabara, a.; okajima, f. critical role of abca1 transporter in sphingosine 1-phosphate release from astrocytes. j neurochem 2007, 103, 2610-9. zhao, x.; murata, t.; ohno, s.; day, n.; song, j.; nomura, n.; nakahara, t.; yokoyama, k. k. protein kinase calpha plays a critical role in mannosylerythritol lipid-induced differentiation of melanoma b16 cells. j biol chem 2001, 276, 39903-10. olivier, m.; bott, g. r.; frisdal, e.; nowick, m.; plengpanich, w.; desmarchelier, c.; roi, s.; quinn, c. m.; gelissen, i.; jessup, w.; van eck, m.; guerin, m.; le goff, w.; reboul, e. abcg1 is involved in vitamin e efflux. biochim biophys acta 2014, 1841, 1741-51. stefan, s. m.; jansson, p. j.; kalinowski, d. s.; anjum, r.; dharmasivam, m.; richardson, d. r. the growing evidence for targeting p-glycoprotein in lysosomes to overcome resistance. future med chem 2020, 12, 473-477. zhitomirsky, b.; assaraf, y. g. lysosomes as mediators of drug resistance in cancer. drug resist updat 2016, 24, 23-33. chapuy, b.; panse, m.; radunski, u.; koch, r.; wenzel, d.; inagaki, n.; haase, d.; truemper, l.; wulf, g. g. abc transporter a3 facilitates lysosomal sequestration of imatinib and modulates susceptibility of chronic myeloid leukemia cell lines to this drug. haematol 2009, 94, 1528-36. chapuy, b.; koch, r.; radunski, u.; corsham, s.; cheong, n.; inagaki, n.; ban, n.; wenzel, d.; reinhardt, d.; zapf, a.; schweyer, s.; kosari, f.; klapper, w.; truemper, l.; wulf, g. g. intracellular abc transporter a3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration. leuk 2008, 22, 1576-86. dharmapuri, g.; doneti, r.; philip, g. h.; kalle, a. m. celecoxib sensitizes imatinib-resistant k562 cells to imatinib by inhibiting mrp1-5, abca2 and abcg2 transporters via wnt and ras signaling pathways. leuk res 2015, 39, 696-701. steinbach, d.; gillet, j. p.; sauerbrey, a.; gruhn, b.; dawczynski, k.; bertholet, v.; de longueville, f.; zintl, f.; remacle, j.; efferth, t. abca3 as a possible cause of drug resistance in childhood acute myeloid leukemia. clin cancer res 2006, 12, 4357-63. laing, n. m.; belinsky, m. g.; kruh, g. d.; bell, d. w.; boyd, j. t.; barone, l.; testa, j. r.; tew, k. d. amplification of the atp-binding cassette 2 transporter gene is functionally linked with enhanced efflux of estramustine in ovarian carcinoma cells. cancer res 1998, 58, 1332-7. mack, j. t.; brown, c. b.; garrett, t. e.; uys, j. d.; townsend, d. m.; tew, k. d. ablation of the atp-binding cassette transporter, abca2 modifies response to estrogen-based therapies. biomed pharmacother 2012, 66, 403-8. dohmen, l. c.; navas, a.; vargas, d. a.; gregory, d. j.; kip, a.; dorlo, t. p.; gomez, m. a. functional validation of abca3 as a miltefosine transporter in human macrophages: impact on intracellular survival of leishmania (viannia) panamensis. j biol chem 2016, 291, 9638-47. overbeck, t. r.; hupfeld, t.; krause, d.; waldmann-beushausen, r.; chapuy, b.; guldenzoph, b.; aung, t.; inagaki, n.; schondube, f. a.; danner, b. c.; truemper, l.; wulf, g. g. intracellular atp-binding cassette transporter a3 is expressed in lung cancer cells and modulates susceptibility to cisplatin and paclitaxel. oncol 2013, 84, 362-70. aberuyi, n.; rahgozar, s.; pourabutaleb, e.; ghaedi, k. selective dysregulation of abc transporters in methotrexate-resistant leukemia t-cells can confer cross-resistance to cytarabine, vincristine and dexamethasone, but not doxorubicin. curr res transl med 2021, 69, 103269. shroyer, n. f.; lewis, r. a.; lupski, j. r. analysis of the abcr (abca4) gene in 4-aminoquinoline retinopathy: is retinal toxicity by chloroquine and hydroxychloroquine related to stargardt disease? am j ophthalmol 2001, 131, 761-6. mack, h. g.; kowalski, t.; lucattini, a.; symons, r. a.; wicks, i.; hall, a. j. genetic susceptibility to hydroxychloroquine retinal toxicity. ophthalmic genet 2020, 41, 159-70. nagao, k.; maeda, m.; manucat, n. b.; ueda, k. cyclosporine a and psc833 inhibit abca1 function via direct binding. biochim biophys acta 2013, 1831, 398-406. wu, c. a.; tsujita, m.; hayashi, m.; yokoyama, s. probucol inactivates abca1 in the plasma membrane with respect to its mediation of apolipoprotein binding and high density lipoprotein assembly and to its proteolytic degradation. j biol chem 2004, 279, 30168-74. hamon, y.; luciani, m. f.; becq, f.; verrier, b.; rubartelli, a.; chimini, g. interleukin-1beta secretion is impaired by inhibitors of the atp binding cassette transporter, abc1. blood 1997, 90, 2911-5. becq, f.; hamon, y.; bajetto, a.; gola, m.; verrier, b.; chimini, g. abc1, an atp binding cassette transporter required for phagocytosis of apoptotic cells, generates a regulated anion flux after expression in xenopus laevis oocytes. j biol chem 1997, 272, 2695-9. le goff, w.; peng, d. q.; settle, m.; brubaker, g.; morton, r. e.; smith, j. d. cyclosporin a traps abca1 at the plasma membrane and inhibits abca1-mediated lipid efflux to apolipoprotein a-i. arterioscler thromb vasc biol 2004, 24, 2155-61. monzel, j. v.; budde, t.; meyer zu schwabedissen, h. e.; schwebe, m.; bien-moller, s.; lutjohann, d.; kroemer, h. k.; jedlitschky, g.; grube, m. doxorubicin enhances oxysterol levels resulting in a lxr-mediated upregulation of cardiac cholesterol transporters. biochem pharmacol 2017, 144, 108-119. burns, v. e.; kerppola, t. k. atr-101 inhibits cholesterol efflux and cortisol secretion by atp-binding cassette transporters, causing cytotoxic cholesterol accumulation in adrenocortical carcinoma cells. br j pharmacol 2017, 174, 3315-32. zhao, j. f.; jim leu, s. j.; shyue, s. k.; su, k. h.; wei, j.; lee, t. s. novel effect of paeonol on the formation of foam cells: promotion of lxralpha-abca1-dependent cholesterol efflux in macrophages. am j chin med 2013, 41, 1079-96. campia, i.; sala, v.; kopecka, j.; leo, c.; mitro, n.; costamagna, c.; caruso, d.; pescarmona, g.; crepaldi, t.; ghigo, d.; bosia, a.; riganti, c. digoxin and ouabain induce the efflux of cholesterol via liver x receptor signalling and the synthesis of atp in cardiomyocytes. biochem j 2012, 447, 301-11. chen, c. y.; shyue, s. k.; ching, l. c.; su, k. h.; wu, y. l.; kou, y. r.; chiang, a. n.; pan, c. c.; lee, t. s. wogonin promotes cholesterol efflux by increasing protein phosphatase 2b-dependent dephosphorylation at atp-binding cassette transporter-a1 in macrophages. j nutr biochem 2011, 22, 1015-21. howard, a. d.; verghese, p. b.; arrese, e. l.; soulages, j. l. characterization of apoa-i-dependent lipid efflux from adipocytes and role of abca1. mol cell biochem 2010, 343, 115-24. lee, t. s.; pan, c. c.; peng, c. c.; kou, y. r.; chen, c. y.; ching, l. c.; tsai, t. h.; chen, s. f.; lyu, p. c.; shyue, s. k. anti-atherogenic effect of berberine on lxralpha-abca1-dependent cholesterol efflux in macrophages. j cell biochem 2010, 111, 104-10. duncan, k. g.; hosseini, k.; bailey, k. r.; yang, h.; lowe, r. j.; matthes, m. t.; kane, j. p.; lavail, m. m.; schwartz, d. m.; duncan, j. l. expression of reverse cholesterol transport proteins atp-binding cassette a1 (abca1) and scavenger receptor bi (sr-bi) in the retina and retinal pigment epithelium. br j ophthalmol 2009, 93, 1116-20. wang, j.; zhang, z. r.; chou, c. f.; liang, y. y.; gu, y.; ma, h. p. cyclosporine stimulates the renal epithelial sodium channel by elevating cholesterol. am j physiol renal physiol 2009, 296, f284-90. pirillo, a.; uboldi, p.; pappalardo, g.; kuhn, h.; catapano, a. l. modification of hdl3 by mild oxidative stress increases atp-binding cassette transporter 1-mediated cholesterol efflux. cardiovasc res 2007, 75, 566-74. nofer, j. r.; remaley, a. t.; feuerborn, r.; wolinnska, i.; engel, t.; von eckardstein, a.; assmann, g. apolipoprotein a-i activates cdc42 signaling through the abca1 transporter. j lipid res 2006, 47, 794-803. alder-baerens, n.; muller, p.; pohl, a.; korte, t.; hamon, y.; chimini, g.; pomorski, t.; herrmann, a. headgroup-specific exposure of phospholipids in abca1-expressing cells. j biol chem 2005, 280, 26321-9. field, f. j.; born, e.; mathur, s. n. lxr/rxr ligand activation enhances basolateral efflux of beta-sitosterol in caco-2 cells. j lipid res 2004, 45, 905-13. reddy, s. t.; hama, s.; ng, c.; grijalva, v.; navab, m.; fogelman, a. m. atp-binding cassette transporter 1 participates in ldl oxidation by artery wall cells. arterioscler thromb vasc biol 2002, 22, 1877-83. murthy, s.; born, e.; mathur, s. n.; field, f. j. lxr/rxr activation enhances basolateral efflux of cholesterol in caco-2 cells. j lipid res 2002, 43, 1054-64. huang, z. h.; lin, c. y.; oram, j. f.; mazzone, t. sterol efflux mediated by endogenous macrophage apoe expression is independent of abca1. arterioscler thromb vasc biol 2001, 21, 2019-25. von eckardstein, a.; langer, c.; engel, t.; schaukal, i.; cignarella, a.; reinhardt, j.; lorkowski, s.; li, z.; zhou, x.; cullen, p.; assmann, g. atp binding cassette transporter abca1 modulates the secretion of apolipoprotein e from human monocyte-derived macrophages. faseb j 2001, 15, 1555-61. wang, n.; silver, d. l.; thiele, c.; tall, a. r. atp-binding cassette transporter a1 (abca1) functions as a cholesterol efflux regulatory protein. j biol chem 2001, 276, 23742-7. chinetti, g.; lestavel, s.; bocher, v.; remaley, a. t.; neve, b.; torra, i. p.; teissier, e.; minnich, a.; jaye, m.; duverger, n.; brewer, h. b.; fruchart, j. c.; clavey, v.; staels, b. ppar-alpha and ppar-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the abca1 pathway. nat med 2001, 7, 53-8. fielding, p. e.; nagao, k.; hakamata, h.; chimini, g.; fielding, c. j. a two-step mechanism for free cholesterol and phospholipid efflux from human vascular cells to apolipoprotein a-1. biochemistry 2000, 39, 14113-20. nieland, t. j.; chroni, a.; fitzgerald, m. l.; maliga, z.; zannis, v. i.; kirchhausen, t.; krieger, m. cross-inhibition of sr-biand abca1-mediated cholesterol transport by the small molecules blt-4 and glyburide. j lipid res 2004, 45, 1256-65. lewandowski, c. t.; khan, m. w.; benaissa, m.; dubrovskyi, o.; ackerman-berrier, m.; ladu, m. j.; layden, b. t.; thatcher, g. r. j. metabolomic analysis of a selective abca1 inducer in obesogenic challenge provides a rationale for therapeutic development. ebiomedicine 2021, 66, 103287. wang, d.; hiebl, v.; schachner, d.; ladurner, a.; heiss, e. h.; atanasov, a. g.; dirsch, v. m. soraphen a enhances macrophage cholesterol efflux via indirect lxr activation and abca1 upregulation. biochem pharmacol 2020, 177, 114022. quach, d.; vitali, c.; la, f. m.; xiao, a. x.; millar, j. s.; tang, c.; rader, d. j.; phillips, m. c.; lyssenko, n. n. cell lipid metabolism modulators 2-bromopalmitate, d609, monensin, u18666a and probucol shift discoidal hdl formation to the smaller-sized particles: implications for the mechanism of hdl assembly. biochim biophys acta 2016, 1861, 1968-79. de la llera-moya, m.; drazul-schrader, d.; asztalos, b. f.; cuchel, m.; rader, d. j.; rothblat, g. h. the ability to promote efflux via abca1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages. arterioscler thromb vasc biol 2010, 30, 796-801. arakawa, r.; tsujita, m.; iwamoto, n.; ito-ohsumi, c.; lu, r.; wu, c. a.; shimizu, k.; aotsuka, t.; kanazawa, h.; abe-dohmae, s.; yokoyama, s. pharmacological inhibition of abca1 degradation increases hdl biogenesis and exhibits antiatherogenesis. j lipid res 2009, 50, 2299-305. adorni, m. p.; zimetti, f.; billheimer, j. t.; wang, n.; rader, d. j.; phillips, m. c.; rothblat, g. h. the roles of different pathways in the release of cholesterol from macrophages. j lipid res 2007, 48, 2453-62. duong, m.; collins, h. l.; jin, w.; zanotti, i.; favari, e.; rothblat, g. h. relative contributions of abca1 and sr-bi to cholesterol efflux to serum from fibroblasts and macrophages. arterioscler thromb vasc biol 2006, 26, 541-7. favari, e.; zanotti, i.; zimetti, f.; ronda, n.; bernini, f.; rothblat, g. h. probucol inhibits abca1-mediated cellular lipid efflux. arterioscler thromb vasc biol 2004, 24, 2345-50. shinohara, m.; shinohara, m.; zhao, j.; fu, y.; liu, c. c.; kanekiyo, t.; bu, g. 5-ht3 antagonist ondansetron increases apoe secretion by modulating the lxr-abca1 pathway. int j mol sci 2019, 20. wang, w.; nakashima, k. i.; hirai, t.; inoue, m. neuroprotective effect of naturally occurring rxr agonists isolated from sophora tonkinensis gagnep. on amyloid-beta-induced cytotoxicity in pc12 cells. j nat med 2019, 73, 154-62. kheirollah, a.; ito, j.; nagayasu, y.; lu, r.; yokoyama, s. cyclosporin a inhibits apolipoprotein a-i-induced early events in cellular cholesterol homeostasis in rat astrocytes. neuropharmacol 2006, 51, 693-700. palmer, m. a.; smart, e.; haslam, i. s. localisation and regulation of cholesterol transporters in the human hair follicle: mapping changes across the hair cycle. histochem cell biol 2021, 155, 529-45. bardin, e.; pastor, a.; semeraro, m.; golec, a.; hayes, k.; chevalier, b.; berhal, f.; prestat, g.; hinzpeter, a.; gravier-pelletier, c.; pranke, i.; sermet-gaudelus, i. modulators of cftr. updates on clinical development and future directions. eur j med chem 2021, 213, 113195. schmitt, s. m.; stefan, k.; wiese, m. pyrrolopyrimidine derivatives and purine analogs as novel activators of multidrug resistance-associated protein 1 (mrp1, abcc1). biochim biophys acta 2017, 1859, 69-79. trechot, p.; conart, j. b.; trechot, f. atp sensitive potassium channel openers: a new class of ocular hypotensive agents. exp eye res 2019, 178, 223-4. csandl, m. a.; conseil, g.; cole, s. p. cysteinyl leukotriene receptor 1/2 antagonists nonselectively modulate organic anion transport by multidrug resistance proteins (mrp1-4). drug metab dispos 2016, 44, 857-66. wang, h.; tang, y.; wang, l.; long, c. l.; zhang, y. l. atp-sensitive potassium channel openers and 2,3-dimethyl-2-butylamine derivatives. curr med chem 2007, 14, 133-55. moreau, c.; prost, a. l.; derand, r.; vivaudou, m. sur, abc proteins targeted by katp channel openers. j mol cell cardiol 2005, 38, 951-63. bielicki, j. k.; zhang, h.; cortez, y.; zheng, y.; narayanaswami, v.; patel, a.; johansson, j.; azhar, s. a new hdl mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice. j lipid res 2010, 51, 1496-503. natarajan, p.; forte, t. m.; chu, b.; phillips, m. c.; oram, j. f.; bielicki, j. k. identification of an apolipoprotein a-i structural element that mediates cellular cholesterol efflux and stabilizes atp binding cassette transporter a1. j biol chem 2004, 279, 24044-52. vedhachalam, c.; narayanaswami, v.; neto, n.; forte, t. m.; phillips, m. c.; lund-katz, s.; bielicki, j. k. the c-terminal lipid-binding domain of apolipoprotein e is a highly efficient mediator of abca1-dependent cholesterol efflux that promotes the assembly of high-density lipoproteins. biochemistry 2007, 46, 2583-93. hafiane, a.; bielicki, j. k.; johansson, j. o.; genest, j. novel apoe-derived abca1 agonist peptide (cs-6253) promotes reverse cholesterol transport and induces formation of prebeta-1 hdl in vitro. plos one 2015, 10, e0131997. boehm-cagan, a.; bar, r.; liraz, o.; bielicki, j. k.; johansson, j. o.; michaelson, d. m. abca1 agonist reverses the apoe4-driven cognitive and brain pathologies. j alzheimers dis 2016, 54, 1219-33. wang, l.; schuster, g. u.; hultenby, k.; zhang, q.; andersson, s.; gustafsson, j. a. liver x receptors in the central nervous system: from lipid homeostasis to neuronal degeneration. proc natl acad sci u s a 2002, 99, 13878-83. santamarina-fojo, s.; remaley, a. t.; neufeld, e. b.; brewer, h. b., jr. regulation and intracellular trafficking of the abca1 transporter. j lipid res 2001, 42, 1339-45. repa, j. j.; turley, s. d.; lobaccaro, j. a.; medina, j.; li, l.; lustig, k.; shan, b.; heyman, r. a.; dietschy, j. m.; mangelsdorf, d. j. regulation of absorption and abc1-mediated efflux of cholesterol by rxr heterodimers. science 2000, 289, 1524-9. cramer, p. e.; cirrito, j. r.; wesson, d. w.; lee, c. y.; karlo, j. c.; zinn, a. e.; casali, b. t.; restivo, j. l.; goebel, w. d.; james, m. j.; brunden, k. r.; wilson, d. a.; landreth, g. e. apoe-directed therapeutics rapidly clear beta-amyloid and reverse deficits in ad mouse models. science 2012, 335, 1503-6. terwel, d.; steffensen, k. r.; verghese, p. b.; kummer, m. p.; gustafsson, j. a.; holtzman, d. m.; heneka, m. t. critical role of astroglial apolipoprotein e and liver x receptor-alpha expression for microglial abeta phagocytosis. j neurosci 2011, 31, 7049-59. lefterov, i.; bookout, a.; wang, z.; staufenbiel, m.; mangelsdorf, d.; koldamova, r. expression profiling in app23 mouse brain: inhibition of abeta amyloidosis and inflammation in response to lxr agonist treatment. mol neurodegener 2007, 2, 20. riddell, d. r.; zhou, h.; comery, t. a.; kouranova, e.; lo, c. f.; warwick, h. k.; ring, r. h.; kirksey, y.; aschmies, s.; xu, j.; kubek, k.; hirst, w. d.; gonzales, c.; chen, y.; murphy, e.; leonard, s.; vasylyev, d.; oganesian, a.; martone, r. l.; pangalos, m. n.; reinhart, p. h.; jacobsen, j. s. the lxr agonist to901317 selectively lowers hippocampal abeta42 and improves memory in the tg2576 mouse model of alzheimer's disease. mol cell neurosci 2007, 34, 621-8. koldamova, r. p.; lefterov, i. m.; staufenbiel, m.; wolfe, d.; huang, s.; glorioso, j. c.; walter, m.; roth, m. g.; lazo, j. s. the liver x receptor ligand t0901317 decreases amyloid beta production in vitro and in a mouse model of alzheimer's disease. j biol chem 2005, 280, 4079-88. donkin, j. j.; stukas, s.; hirsch-reinshagen, v.; namjoshi, d.; wilkinson, a.; may, s.; chan, j.; fan, j.; collins, j.; wellington, c. l. atp-binding cassette transporter a1 mediates the beneficial effects of the liver x receptor agonist gw3965 on object recognition memory and amyloid burden in amyloid precursor protein/presenilin 1 mice. j biol chem 2010, 285, 34144-54. vanmierlo, t.; rutten, k.; dederen, j.; bloks, v. w.; van vark-van der zee, l. c.; kuipers, f.; kiliaan, a.; blokland, a.; sijbrands, e. j.; steinbusch, h.; prickaerts, j.; lutjohann, d.; mulder, m. liver x receptor activation restores memory in aged ad mice without reducing amyloid. neurobiol aging 2011, 32, 1262-72. bunay, j.; fouache, a.; trousson, a.; de joussineau, c.; bouchareb, e.; zhu, z.; kocer, a.; morel, l.; baron, s.; lobaccaro, j. a. screening for liver x receptor modulators: where are we and for what use? br j pharmacol 2020, 178, 3277-93. salonurmi, t.; nabil, h.; ronkainen, j.; hyotylainen, t.; hautajarvi, h.; savolainen, m. j.; tolonen, a.; oresic, m.; kansakoski, p.; rysa, j.; hakkola, j.; hukkanen, j. 4beta-hydroxycholesterol signals from the liver to regulate peripheral cholesterol transporters. front pharmacol 2020, 11, 361. vaidya, m.; jentsch, j. a.; peters, s.; keul, p.; weske, s.; graler, m. h.; mladenov, e.; iliakis, g.; heusch, g.; levkau, b. regulation of abca1-mediated cholesterol efflux by sphingosine-1-phosphate signaling in macrophages. j lipid res 2019, 60, 506-15. castro navas, f. f.; giorgi, g.; maggioni, d.; pacciarini, m.; russo, v.; marinozzi, m. c24-hydroxylated stigmastane derivatives as liver x receptor agonists. chem phys lipids 2018, 212, 44-50. hoang, m. h.; jia, y.; jun, h. j.; lee, j. h.; lee, b. y.; lee, s. j. fucosterol is a selective liver x receptor modulator that regulates the expression of key genes in cholesterol homeostasis in macrophages, hepatocytes, and intestinal cells. j agric food chem 2012, 60, 11567-75. gao, j.; xu, y.; yang, y.; yang, y.; zheng, z.; jiang, w.; hong, b.; yan, x.; si, s. identification of upregulators of human atp-binding cassette transporter a1 via high-throughput screening of a synthetic and natural compound library. j biomol screen 2008, 13, 648-56. huang, t. h.; razmovski-naumovski, v.; salam, n. k.; duke, r. k.; tran, v. h.; duke, c. c.; roufogalis, b. d. a novel lxr-alpha activator identified from the natural product gynostemma pentaphyllum. biochem pharmacol 2005, 70, 1298-308. agassandian, m.; mathur, s. n.; zhou, j.; field, f. j.; mallampalli, r. k. oxysterols trigger abca1-mediated basolateral surfactant efflux. am j respir cell mol biol 2004, 31, 227-33. sone, h.; shimano, h.; shu, m.; nakakuki, m.; takahashi, a.; sakai, m.; sakamoto, y.; yokoo, t.; matsuzaka, k.; okazaki, h.; nakagawa, y.; iida, k. t.; suzuki, h.; toyoshima, h.; horiuchi, s.; yamada, n. statins downregulate atp-binding-cassette transporter a1 gene expression in macrophages. biochem biophys res commun 2004, 316, 790-4. zhang, y.; beyer, t. p.; bramlett, k. s.; yao, s.; burris, t. p.; schmidt, r. j.; eacho, p. i.; cao, g. liver x receptor and retinoic x receptor mediated abca1 regulation and cholesterol efflux in macrophage cells-messenger rna measured by branched dna technology. mol genet metab 2002, 77, 150-8. gan, x.; kaplan, r.; menke, j. g.; macnaul, k.; chen, y.; sparrow, c. p.; zhou, g.; wright, s. d.; cai, t. q. dual mechanisms of abca1 regulation by geranylgeranyl pyrophosphate. j biol chem 2001, 276, 48702-8. chawla, a.; boisvert, w. a.; lee, c. h.; laffitte, b. a.; barak, y.; joseph, s. b.; liao, d.; nagy, l.; edwards, p. a.; curtiss, l. k.; evans, r. m.; tontonoz, p. a ppar gamma-lxr-abca1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. mol cell 2001, 7, 161-71. arakawa, r.; yokoyama, s. helical apolipoproteins stabilize atp-binding cassette transporter a1 by protecting it from thiol protease-mediated degradation. j biol chem 2002, 277, 22426-9. di, d.; wang, z.; liu, y.; luo, g.; shi, y.; berggren-soderlund, m.; nilsson-ehle, p.; zhang, x.; xu, n. abca1 upregulating apolipoproein m expression mediates via the rxr/lxr pathway in hepg2 cells. biochem biophys res commun 2012, 421, 152-6. zanotti, i.; favari, e.; sposito, a. c.; rothblat, g. h.; bernini, f. pitavastatin increases abca1-mediated lipid efflux from fu5ah rat hepatoma cells. biochem biophys res commun 2004, 321, 670-4. ben aissa, m.; lewandowski, c. t.; ratia, k. m.; lee, s. h.; layden, b. t.; ladu, m. j.; thatcher, g. r. j. discovery of nonlipogenic abca1 inducing compounds with potential in alzheimer's disease and type 2 diabetes. acs pharmacol transl sci 2021, 4, 143-54. ma, l.; wang, l.; nelson, a. t.; han, c.; he, s.; henn, m. a.; menon, k.; chen, j. j.; baek, a. e.; vardanyan, a.; shahoei, s. h.; park, s.; shapiro, d. j.; nanjappa, s. g.; nelson, e. r. 27-hydroxycholesterol acts on myeloid immune cells to induce t cell dysfunction, promoting breast cancer progression. cancer lett 2020, 493, 266-83. ray, a. g.; choudhury, k. r.; chakraborty, s.; chakravarty, d.; chander, v.; jana, b.; siddiqui, k. n.; bandyopadhyay, a. novel mechanism of cholesterol transport by abca5 in macrophages and its role in dyslipidemia. j mol biol 2020, 432, 4922-41. he, p.; smith, a.; gelissen, i. c.; ammit, a. j. the effect of statins and the synthetic lxr agonist t0901317 on expression of abca1 transporter protein in human lung epithelial cell lines in vitro. pharmacol rep: pr 2019, 71, 1219-26. leger-charnay, e.; masson, e. a. y.; morala, t.; martine, l.; buteau, b.; leclere, l.; bretillon, l.; gambert, s. is 24(s)-hydroxycholesterol a potent modulator of cholesterol metabolism in muller cells? an in vitro study about neuron to glia communication in the retina. exp eye res 2019, 189, 107857. wu, c. a.; wang, n.; zhao, d. h. an evaluation of the mechanism of abca7 on cellular lipid release in abca7-hec293 cell. chin med j 2013, 126, 306-10. lee, j. s.; kim, e.; han, s.; kang, k. l.; heo, j. s. evaluating the oxysterol combination of 22(s)-hydroxycholesterol and 20(s)-hydroxycholesterol in periodontal regeneration using periodontal ligament stem cells and alveolar bone healing models. stem cell res ther 2017, 8, 276. panzenboeck, u.; kratzer, i.; sovic, a.; wintersperger, a.; bernhart, e.; hammer, a.; malle, e.; sattler, w. regulatory effects of synthetic liver x receptorand peroxisome-proliferator activated receptor agonists on sterol transport pathways in polarized cerebrovascular endothelial cells. int j biochem cell biol 2006, 38, 1314-29. ruan, x. z.; moorhead, j. f.; fernando, r.; wheeler, d. c.; powis, s. h.; varghese, z. ppar agonists protect mesangial cells from interleukin 1beta-induced intracellular lipid accumulation by activating the abca1 cholesterol efflux pathway. j am soc nephrol 2003, 14, 593-600. kim, b. y.; son, y.; cho, h. r.; lee, d.; eo, s. k.; kim, k. 27-hydroxycholesterol induces macrophage gene expression via lxr-dependent and -independent mechanisms. korean j physiol pharmacol 2021, 25, 111-8. costet, p.; lalanne, f.; gerbod-giannone, m. c.; molina, j. r.; fu, x.; lund, e. g.; gudas, l. j.; tall, a. r. retinoic acid receptor-mediated induction of abca1 in macrophages. mol cell biol 2003, 23, 7756-66. zhang, c. j.; zhu, n.; long, j.; wu, h. t.; wang, y. x.; liu, b. y.; liao, d. f.; qin, l. celastrol induces lipophagy via the lxralpha/abca1 pathway in clear cell renal cell carcinoma. acta pharmacol sin 2020, 42, 1472-85. munir, m. t.; ponce, c.; santos, j. m.; sufian, h. b.; al-harrasi, a.; gollahon, l. s.; hussain, f.; rahman, s. m. vd3 and lxr agonist (t0901317) combination demonstrated greater potency in inhibiting cholesterol accumulation and inducing apoptosis via abca1-chop-bcl-2 cascade in mcf-7 breast cancer cells. mol biol rep 2020, 47, 7771-82. shi, y.; jiang, s.; zhao, t.; gong, y.; liao, d.; qin, l. celastrol suppresses lipid accumulation through lxralpha/abca1 signaling pathway and autophagy in vascular smooth muscle cells. biochem biophys res commun 2020, 532, 466-74. warde, k. m.; schoenmakers, e.; ribes martinez, e.; lim, y. j.; leonard, m.; lawless, s. j.; o'shea, p.; chatterjee, k. v.; gurnell, m.; hantel, c.; dennedy, m. c. liver x receptor inhibition potentiates mitotane-induced adrenotoxicity in acc. endocr relat cancer 2020, 27, 361-73. wu, g.; wang, q.; xu, y.; li, j.; zhang, h.; qi, g.; xia, q. targeting the transcription factor receptor lxr to treat clear cell renal cell carcinoma: agonist or inverse agonist? cell death dis 2019, 10, 416. jin, p.; bian, y.; wang, k.; cong, g.; yan, r.; sha, y.; ma, x.; zhou, j.; yuan, z.; jia, s. homocysteine accelerates atherosclerosis via inhibiting lxralpha-mediated abca1/abcg1-dependent cholesterol efflux from macrophages. life sci 2018, 214, 41-50. luo, g.; qian, z.; qiu, r.; you, q.; xiang, h. lipid reducing activity of novel cholic acid (ca) analogs: design, synthesis and preliminary mechanism study. bioorg chem 2018, 80, 396-407. ni, j.; zhou, l. l.; ding, l.; zhang, x. q.; zhao, x.; li, h.; cao, h.; liu, s.; wang, z.; ma, r.; wu, j.; feng, j. efatutazone and t0901317 exert synergistically therapeutic effects in acquired gefitinib-resistant lung adenocarcinoma cells. cancer med 2018, 7, 1955-66. saenz, j.; alba, g.; reyes-quiroz, m. e.; geniz, i.; jimenez, j.; sobrino, f.; santa-maria, c. curcumin enhances lxralpha in an amp-activated protein kinase-dependent manner in human macrophages. j nutr biochem 2018, 54, 48-56. saenz, j.; santa-maria, c.; reyes-quiroz, m. e.; geniz, i.; jimenez, j.; sobrino, f.; alba, g. grapefruit flavonoid naringenin regulates the expression of lxralpha in thp-1 macrophages by modulating amp-activated protein kinase. mol pharm 2018, 15, 1735-45. lei, c.; lin, r.; wang, j.; tao, l.; fu, x.; qiu, y.; lei, b. amelioration of amyloid beta-induced retinal inflammatory responses by a lxr agonist to901317 is associated with inhibition of the nf-kappab signaling and nlrp3 inflammasome. neurosci 2017, 360, 48-60. fernandez-suarez, m. e.; escola-gil, j. c.; pastor, o.; davalos, a.; blanco-vaca, f.; lasuncion, m. a.; martinez-botas, j.; gomez-coronado, d. clinically used selective estrogen receptor modulators affect different steps of macrophage-specific reverse cholesterol transport. sci rep 2016, 6, 32105. hoang, m. h.; jia, y.; jun, h. j.; lee, j. h.; lee, d. h.; hwang, b. y.; kim, w. j.; lee, h. j.; lee, s. j. ethyl 2,4,6-trihydroxybenzoate is an agonistic ligand for liver x receptor that induces cholesterol efflux from macrophages without affecting lipid accumulation in hepg2 cells. bioorg med chem lett 2012, 22, 4094-9. maejima, t.; sugano, t.; yamazaki, h.; yoshinaka, y.; doi, t.; tanabe, s.; nishimaki-mogami, t. pitavastatin increases abca1 expression by dual mechanisms: srebp2-driven transcriptional activation and pparalpha-dependent protein stabilization but without activating lxr in rat hepatoma mcarh7777 cells. j pharmacol sci 2011, 116, 107-15. fitz, n. f.; cronican, a.; pham, t.; fogg, a.; fauq, a. h.; chapman, r.; lefterov, i.; koldamova, r. liver x receptor agonist treatment ameliorates amyloid pathology and memory deficits caused by high-fat diet in app23 mice. j neurosci 2010, 30, 6862-72. fukumoto, h.; deng, a.; irizarry, m. c.; fitzgerald, m. l.; rebeck, g. w. induction of the cholesterol transporter abca1 in central nervous system cells by liver x receptor agonists increases secreted abeta levels. j biol chem 2002, 277, 48508-13. van riel, n. a. w.; tiemann, c. a.; hilbers, p. a. j.; groen, a. k. metabolic modeling combined with machine learning integrates longitudinal data and identifies the origin of lxr-induced hepatic steatosis. front bioeng biotechnol 2020, 8, 536957. chisholm, j. w.; hong, j.; mills, s. a.; lawn, r. m. the lxr ligand t0901317 induces severe lipogenesis in the db/db diabetic mouse. j lipid res 2003, 44, 2039-48. liang, z.; gu, t.; wang, j.; she, j.; ye, y.; cao, w.; luo, x.; xiao, j.; liu, y.; tang, l.; zhou, x. chromene and chromone derivatives as liver x receptors modulators from a marine-derived pestalotiopsis neglecta fungus. bioorg chem 2021, 112, 104927. wang, j.; liang, z.; li, k.; yang, b.; liu, y.; fang, w.; tang, l.; zhou, x. ene-yne hydroquinones from a marine-derived strain of the fungus pestalotiopsis neglecta with effects on liver x receptor alpha. j nat prod 2020, 83, 1258-64. chen, y.; wang, y.; yang, m.; guo, m. y. allicin inhibited staphylococcus aureus -induced mastitis by reducing lipid raft stability via lxralpha in mice. j agric food chem 2019, 67, 10863-70. grinman, d. y.; careaga, v. p.; wellberg, e. a.; dansey, m. v.; kordon, e. c.; anderson, s. m.; maier, m. s.; burton, g.; maclean, p. s.; rudolph, m. c.; pecci, a. liver x receptor-alpha activation enhances cholesterol secretion in lactating mammary epithelium. american journal of physiology. endocrinol metab 2019, 316, e1136-e1145. huang, y.; liu, h.; zhang, y.; li, j.; wang, c.; zhou, l.; jia, y.; li, x. synthesis and biological evaluation of ginsenoside compound k derivatives as a novel class of lxralpha activator. molecules 2017, 22, 1232. liu, b.; he, z.; wang, j.; xin, z.; wang, j.; li, f.; fu, y. taraxasterol inhibits lps-induced inflammatory response in bv2 microglia cells by activating lxralpha. front pharmacol 2018, 9, 278. biswas, l.; zeng, z.; graham, a.; shu, x. gypenosides mediate cholesterol efflux and suppress oxidized ldl induced inflammation in retinal pigment epithelium cells. exp eye res 2020, 191, 107931. fu, y.; xin, z.; liu, b.; wang, j.; wang, j.; zhang, x.; wang, y.; li, f. platycodin d inhibits inflammatory response in lps-stimulated primary rat microglia cells through activating lxralpha-abca1 signaling pathway. front immunol 2017, 8, 1929. shuai-cheng, w.; xiu-ling, c.; jian-qing, s.; zong-mei, w.; zhen-jiang, y.; lian-tao, l. saikosaponin a protects chickens against pullorum disease via modulation of cholesterol. poult sci 2019, 98, 3539-47. kilby, e. l.; kelly, d. m.; jones, t. h. testosterone stimulates cholesterol clearance from human macrophages by activating lxralpha. life sci 2021, 269, 119040. li, s.; cao, h.; shen, d.; jia, q.; chen, c.; xing, s. l. quercetin protects against oxldlinduced injury via regulation of abcal, lxralpha and pcsk9 in raw264.7 macrophages. mol med rep 2018, 18, 799-806. malar, d. s.; suryanarayanan, v.; prasanth, m. i.; singh, s. k.; balamurugan, k.; devi, k. p. vitexin inhibits abeta25-35 induced toxicity in neuro-2a cells by augmenting nrf-2/ho-1 dependent antioxidant pathway and regulating lipid homeostasis by the activation of lxr-alpha. toxicol in vitro 2018, 50, 160-171. xu, x.; lei, t.; li, w.; ou, h. enhanced cellular cholesterol efflux by naringenin is mediated through inhibiting endoplasmic reticulum stress atf6 activity in macrophages. biochim biophys acta mol cell biol lipids 2019, 1864, 1472-82. jia, q.; cao, h.; shen, d.; li, s.; yan, l.; chen, c.; xing, s.; dou, f. quercetin protects against atherosclerosis by regulating the expression of pcsk9, cd36, ppargamma, lxralpha and abca1. int j mol med 2019, 44, 893-902. li, s. s.; cao, h.; shen, d. z.; chen, c.; xing, s. l.; dou, f. f.; jia, q. l. effect of quercetin on atherosclerosis based on expressions of abca1, lxr-alpha and pcsk in apoe(-/-) mice. chin j integr med 2020, 26, 114-21. teng, i. j.; tsai, m. c.; shih, s. f.; tsuei, b. f.; chang, h.; chuang, y. p.; lin, c. s.; chern, c. y.; chen, s. j. chalcone derivatives enhance atp-binding cassette transporters a1 in human thp-1 macrophages. molecules 2018, 23, 1620. chen, l. w.; tsai, m. c.; chern, c. y.; tsao, t. p.; lin, f. y.; chen, s. j.; tsui, p. f.; liu, y. w.; lu, h. j.; wu, w. l.; lin, w. s.; tsai, c. s.; lin, c. s. a chalcone derivative, 1m-6, exhibits atheroprotective effects by increasing cholesterol efflux and reducing inflammation-induced endothelial dysfunction. br j pharmacol 2020, 177, 5375-92. liu, x. x.; zhang, x. w.; wang, k.; wang, x. y.; ma, w. l.; cao, w.; mo, d.; sun, y.; li, x. q. kuwanon g attenuates atherosclerosis by upregulation of lxralpha-abca1/abcg1 and inhibition of nfkappab activity in macrophages. toxicol appl pharmacol 2018, 341, 56-63. wang, g.; gao, j. h.; he, l. h.; yu, x. h.; zhao, z. w.; zou, j.; wen, f. j.; zhou, l.; wan, x. j.; tang, c. k. fargesin alleviates atherosclerosis by promoting reverse cholesterol transport and reducing inflammatory response. biochim biophys acta mol cell biol lipids 2020, 1865, 158633. xu, y.; liu, q.; xu, y.; liu, c.; wang, x.; he, x.; zhu, n.; liu, j.; wu, y.; li, y.; li, n.; feng, t.; lai, f.; zhang, m.; hong, b.; jiang, j. d.; si, s. rutaecarpine suppresses atherosclerosis in apoe-/mice through upregulating abca1 and sr-bi within rct. j lipid res 2014, 55, 1634-47. pattanayak, s. p.; bose, p.; sunita, p.; siddique, m. u. m.; lapenna, a. bergapten inhibits liver carcinogenesis by modulating lxr/pi3k/akt and idol/ldlr pathways. biomed pharmacother 2018, 108, 297-308. shi, x.; zhang, y.; lin, b.; zhou, y.; suo, w.; wei, j.; zhang, l.; lin, j.; xiao, f.; zhao, l.; lin, y. danthron attenuates experimental atherosclerosis by targeting foam cell formation. exp physiol 2021, 106, 653-62. wang, w.; zhang, z. z.; wu, y.; wang, r. q.; chen, j. w.; chen, j.; zhang, y.; chen, y. j.; geng, m.; xu, z. d.; dai, m.; li, j. h.; pan, l. l. (-)-epigallocatechin-3-gallate ameliorates atherosclerosis and modulates hepatic lipid metabolic gene expression in apolipoprotein e knockout mice: involvement of ttc39b. front pharmacol 2018, 9, 195. zheng, s.; li, l.; li, n.; du, y.; zhang, n. 1, 6-o, o-diacetylbritannilactone from inula britannica induces anti-tumor effect on oral squamous cell carcinoma via mir-1247-3p/lxralpha/abca1 signaling. onco targets ther 2020, 13, 11097-109. mustra rakic, j.; liu, c.; veeramachaneni, s.; wu, d.; paul, l.; chen, c. o.; ausman, l. m.; wang, x. d. lycopene inhibits smoke-induced chronic obstructive pulmonary disease and lung carcinogenesis by modulating reverse cholesterol transport in ferrets. cancer prev res (phila) 2019, 12, 421-32. gwon, m. h.; im, y. s.; seo, a. r.; kim, k. y.; moon, h. r.; yun, j. m. phenethyl isothiocyanate protects against high fat/cholesterol diet-induced obesity and atherosclerosis in c57bl/6 mice. nutrients 2020, 12, 3657. chen, y.; zhao, y. f.; yang, j.; jing, h. y.; liang, w.; chen, m. y.; yang, m.; wang, y.; guo, m. y. selenium alleviates lipopolysaccharide-induced endometritis via regulating the recruitment of tlr4 into lipid rafts in mice. food funct 2020, 11, 200-10. shen, d.; zhao, d.; yang, x.; zhang, j.; he, h.; yu, c. geniposide against atherosclerosis by inhibiting the formation of foam cell and lowering reverse lipid transport via p38/mapk signaling pathways. eur j pharmacol 2019, 864, 172728. tsunemi, a.; ueno, t.; fukuda, n.; watanabe, t.; tahira, k.; haketa, a.; hatanaka, y.; tanaka, s.; matsumoto, t.; matsumoto, y.; nagase, h.; soma, m. a novel gene regulator, pyrrole-imidazole polyamide targeting abca1 gene increases cholesterol efflux from macrophages and plasma hdl concentration. j mol med (berl) 2014, 92, 509-21. ma, x.; li, s. f.; qin, z. s.; ye, j.; zhao, z. l.; fang, h. h.; yao, z. w.; gu, m. n.; hu, y. w. propofol up-regulates expression of abca1, abcg1, and sr-b1 through the ppargamma/lxralpha signaling pathway in thp-1 macrophage-derived foam cells. cardiovasc pathol 2015, 24, 230-5. belorusova, a. y.; evertsson, e.; hovdal, d.; sandmark, j.; bratt, e.; maxvall, i.; schulman, i. g.; akerblad, p.; lindstedt, e. l. structural analysis identifies an escape route from the adverse lipogenic effects of liver x receptor ligands. commun biol 2019, 2, 431. liu, h.; jiang, x.; gao, x.; tian, w.; xu, c.; wang, r.; xu, y.; wei, l.; cao, f.; li, w. identification of n-benzothiazolyl-2-benzenesulfonamides as novel abca1 expression upregulators. rsc med chem 2020, 11, 411-8. ren, g.; bao, w.; zeng, z.; zhang, w.; shang, c.; wang, m.; su, y.; zhang, x. k.; zhou, h. retinoid x receptor alpha nitro-ligand z-10 and its optimized derivative z-36 reduce beta-amyloid plaques in alzheimer's disease mouse model. mol pharm 2019, 16, 480-8. li, y.; feng, t.; liu, p.; liu, c.; wang, x.; li, d.; li, n.; chen, m.; xu, y.; si, s. optimization of rutaecarpine as abca1 up-regulator for treating atherosclerosis. acs med chem lett 2014, 5, 884-8. singh, s. b.; ondeyka, j. g.; liu, w.; chen, s.; chen, t. s.; li, x.; bouffard, a.; dropinski, j.; jones, a. b.; mccormick, s.; hayes, n.; wang, j.; sharma, n.; macnaul, k.; hernandez, m.; chao, y. s.; baffic, j.; lam, m. h.; burton, c.; sparrow, c. p.; menke, j. g. discovery and development of dimeric podocarpic acid leads as potent agonists of liver x receptor with hdl cholesterol raising activity in mice and hamsters. bioorg med chem lett 2005, 15, 2824-8. boehm-cagan, a.; michaelson, d. m. reversal of apoe4-driven brain pathology and behavioral deficits by bexarotene. j neurosci 2014, 34, 7293-301. niesor, e. j.; schwartz, g. g.; perez, a.; stauffer, a.; durrwell, a.; bucklar-suchankova, g.; benghozi, r.; abt, m.; kallend, d. statin-induced decrease in atp-binding cassette transporter a1 expression via microrna33 induction may counteract cholesterol efflux to high-density lipoprotein. cardiovasc drugs ther 2015, 29, 7-14. allen, a. m.; graham, a. mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apo)a-i from murine raw 264.7 macrophages. lipids health dis 2012, 11, 169. abe-dohmae, s.; ikeda, y.; matsuo, m.; hayashi, m.; okuhira, k.; ueda, k.; yokoyama, s. human abca7 supports apolipoprotein-mediated release of cellular cholesterol and phospholipid to generate high density lipoprotein. j biol chem 2004, 279, 604-11. abe-dohmae, s.; suzuki, s.; wada, y.; aburatani, h.; vance, d. e.; yokoyama, s. characterization of apolipoprotein-mediated hdl generation induced by camp in a murine macrophage cell line. biochemistry 2000, 39, 11092-9. zhong, y.; liu, c.; feng, j.; li, j. f.; fan, z. c. curcumin affects ox-ldl-induced il-6, tnf-alpha, mcp-1 secretion and cholesterol efflux in thp-1 cells by suppressing the tlr4/nf-kappab/mir33a signaling pathway. exp ther med 2020, 20, 1856-70. wang, y.; wen, y.; xiao, p.; sun, j.; chen, m.; gu, c.; kong, y.; gu, a.; zhang, j.; wang, y. di-n-butyl phthalate promotes lipid accumulation via the mir200c-5p-abca1 pathway in thp-1 macrophages. environ pollut 2020, 264, 114723. phang, s. w.; ooi, b. k.; ahemad, n.; yap, w. h. maslinic acid suppresses macrophage foam cells formation: regulation of monocyte recruitment and macrophage lipids homeostasis. vascul pharmacol 2020, 128-129, 106675. lou, k.; huang, p.; ma, h.; wang, x.; xu, h.; wang, w. orlistat increases arsenite tolerance in thp-1 derived macrophages through the up-regulation of abca1. drug chem toxicol 2019, 1-9. chen, c. h.; zhao, j. f.; hsu, c. p.; kou, y. r.; lu, t. m.; lee, t. s. the detrimental effect of asymmetric dimethylarginine on cholesterol efflux of macrophage foam cells: role of the nox/ros signaling. free radic biol med 2019, 143, 354-65. zhao, w.; wang, l.; haller, v.; ritsch, a. a novel candidate for prevention and treatment of atherosclerosis: urolithin b decreases lipid plaque deposition in apoe(-/-) mice and increases early stages of reverse cholesterol transport in ox-ldl treated macrophages cells. mol nutr food res 2019, 63, e1800887. zhong, y.; feng, j.; fan, z.; li, j. curcumin increases cholesterol efflux via heme oxygenase1mediated abca1 and srbi expression in macrophages. mol med rep 2018, 17, 6138-43. wang, l.; wesemann, s.; krenn, l.; ladurner, a.; heiss, e. h.; dirsch, v. m.; atanasov, a. g. erythrodiol, an olive oil constituent, increases the half-life of abca1 and enhances cholesterol efflux from thp-1-derived macrophages. front pharmacol 2017, 8, 375. kemmerer, m.; wittig, i.; richter, f.; brune, b.; namgaladze, d. ampk activates lxralpha and abca1 expression in human macrophages. int j biochem cell biol 2016, 78, 1-9. li, x. y.; kong, l. x.; li, j.; he, h. x.; zhou, y. d. kaempferol suppresses lipid accumulation in macrophages through the downregulation of cluster of differentiation 36 and the upregulation of scavenger receptor class b type i and atp-binding cassette transporters a1 and g1. int j mol med 2013, 31, 331-8. seneviratne, a.; cave, l.; hyde, g.; moestrup, s. k.; carling, d.; mason, j. c.; haskard, d. o.; boyle, j. j. metformin directly suppresses atherosclerosis in normoglycaemic mice via haematopoietic adenosine monophosphate-activated protein kinase. cardiovasc res 2021, 117, 1295-308. lu, s.; luo, y.; sun, g.; sun, x. ginsenoside compound k attenuates ox-ldl-mediated macrophage inflammation and foam cell formation via autophagy induction and modulating nf-kappab, p38, and jnk mapk signaling. front pharmacol 2020, 11, 567238. wang, m. s.; han, q. s.; jia, z. r.; chen, c. s.; qiao, c.; liu, q. q.; zhang, y. m.; wang, k. w.; wang, j.; xiao, k.; ding, x. s. pparalpha agonist fenofibrate relieves acquired resistance to gefitinib in non-small cell lung cancer by promoting apoptosis via pparalpha/ampk/akt/foxo1 pathway. acta pharmacol sin 2021. lundasen, t.; pedrelli, m.; bjorndal, b.; rozell, b.; kuiper, r. v.; burri, l.; pavanello, c.; turri, m.; skorve, j.; berge, r. k.; alexson, s. e. h.; tillander, v. the ppar pan-agonist tetradecylthioacetic acid promotes redistribution of plasma cholesterol towards large hdl. plos one 2020, 15, e0229322. shen, c. y.; lin, j. j.; jiang, j. g.; wang, t. x.; zhu, w. potential roles of dietary flavonoids from citrus aurantium l. var. amara engl. in atherosclerosis development. food funct 2020, 11, 561-71. lin, y.; ren, n.; li, s.; chen, m.; pu, p. novel anti-obesity effect of scutellarein and potential underlying mechanism of actions. biomed pharmacother 2019, 117, 109042. guo, t.; liu, q.; hou, p.; li, f.; guo, s.; song, w.; zhang, h.; liu, x.; zhang, s.; zhang, j.; ho, c. t.; bai, n. stilbenoids and cannabinoids from the leaves of cannabis sativa f. sativa with potential reverse cholesterol transport activity. food funct 2018, 9, 6608-17. dong, t.; lyu, j.; imachi, h.; kobayashi, t.; fukunaga, k.; sato, s.; ibata, t.; yoshimoto, t.; yonezaki, k.; iwama, h.; zhang, g.; murao, k. selective peroxisome proliferator-activated receptor-alpha modulator k-877 regulates the expression of atp-binding cassette transporter a1 in pancreatic beta cells. eur j pharmacol 2018, 838, 78-84. wang, x.; luo, j.; li, n.; liu, l.; han, x.; liu, c.; zuo, x.; jiang, x.; li, y.; xu, y.; si, s. e3317 promotes cholesterol efflux in macrophage cells via enhancing abca1 expression. biochem biophys res commun 2018, 504, 68-74. silva, j. c.; de oliveira, e. m.; turato, w. m.; trossini, g. h. g.; maltarollo, v. g.; pitta, m. g. r.; pitta, i. r.; de las heras, b.; bosca, l.; rudnicki, m.; abdalla, d. s. p. gq-11: a new ppar agonist improves obesity-induced metabolic alterations in ldlr(-/-) mice. int j obes (lond) 2018, 42, 1062-72. wang, s.; zhang, x.; li, x.; liu, q.; zhou, y.; guo, p.; dong, z.; wu, c. phenylpropanoid glucosides from tadehagi triquetrum inhibit oxldl-evoked foam cell formation through modulating cholesterol homeostasis in raw264.7 macrophages. nat prod res 2019, 33, 893-6. brunham, l. r.; kruit, j. k.; pape, t. d.; timmins, j. m.; reuwer, a. q.; vasanji, z.; marsh, b. j.; rodrigues, b.; johnson, j. d.; parks, j. s.; verchere, c. b.; hayden, m. r. beta-cell abca1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment. nat med 2007, 13, 340-7. gao, q.; wei, a.; chen, f.; chen, x.; ding, w.; ding, z.; wu, z.; du, r.; cao, w. enhancing ppargamma by hdac inhibition reduces foam cell formation and atherosclerosis in apoe deficient mice. pharmacol res 2020, 160, 105059. farzanegan gharabolagh, a.; bamdad, t.; hedayati, m.; dehghan manshadi, s. a. the synergistic effect of fluvastatin and ifn-lambda on peripheral blood mononuclear cells of chronic hepatitis c virus (hcv) patients with il-28b rs12979860 cc genotype. iran j allergy asthma immunol 2019, 18, 533-42. wong, j.; quinn, c. m.; brown, a. j. statins inhibit synthesis of an oxysterol ligand for the liver x receptor in human macrophages with consequences for cholesterol flux. arterioscler thromb vasc biol 2004, 24, 2365-71. pirmoradi, l.; seyfizadeh, n.; ghavami, s.; zeki, a. a.; shojaei, s. targeting cholesterol metabolism in glioblastoma: a new therapeutic approach in cancer therapy. j investig med 2019, 67, 715-9. fu, y.; zhou, e.; wei, z.; song, x.; liu, z.; wang, t.; wang, w.; zhang, n.; liu, g.; yang, z. glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing tlr4 recruitment into lipid rafts in raw264.7 cells. biochim biophys acta 2014, 1840, 1755-64. singh, a. b.; dong, b.; kraemer, f. b.; liu, j. fxr activation promotes intestinal cholesterol excretion and attenuates hyperlipidemia in sr-b1-deficient mice fed a high-fat and high-cholesterol diet. physiol rep 2020, 8, e14387. yang, y.; li, x.; peng, l.; an, l.; sun, n.; hu, x.; zhou, p.; xu, y.; li, p.; chen, j. tanshindiol c inhibits oxidized low-density lipoprotein induced macrophage foam cell formation via a peroxiredoxin 1 dependent pathway. biochim biophys acta mol basis dis 2018, 1864, 882-90. koga, m.; kanaoka, y.; inada, k.; omine, s.; kataoka, y.; yamauchi, a. hesperidin blocks varenicline-aggravated atherosclerotic plaque formation in apolipoprotein e knockout mice by downregulating net uptake of oxidized low-density lipoprotein in macrophages. j pharmacol sci 2020, 143, 106-11. han, q. a.; su, d.; shi, c.; liu, p.; wang, y.; zhu, b.; xia, x. urolithin a attenuated ox-ldl-induced cholesterol accumulation in macrophages partly through regulating mir-33a and erk/ampk/srebp1 signaling pathways. food funct 2020, 11, 3432-40. du, y.; li, x.; su, c.; xi, m.; zhang, x.; jiang, z.; wang, l.; hong, b. butyrate protects against high-fat diet-induced atherosclerosis via up-regulating abca1 expression in apolipoprotein e-deficiency mice. br j pharmacol 2020, 177, 1754-72. wang, l.; zhu, j.; cui, l.; wang, q.; huang, w.; ji, x.; yang, q.; rui, c. overexpression of atp-binding cassette transporters associated with sulfoxaflor resistance in aphis gossypii glover. pest manag sci 2021, 77, 4064-72. song, q.; hu, z.; xie, x.; cai, h. zafirlukast prevented ox-ldl-induced formation of foam cells. toxicol appl pharmacol 2020, 409, 115295. tomioka, m.; toda, y.; manucat, n. b.; akatsu, h.; fukumoto, m.; kono, n.; arai, h.; kioka, n.; ueda, k. lysophosphatidylcholine export by human abca7. biochim biophys acta mol cell biol lipids 2017, 1862, 658-65. fan, j.; zhao, r. q.; parro, c.; zhao, w.; chou, h. y.; robert, j.; deeb, t. z.; raynoschek, c.; barichievy, s.; engkvist, o.; maresca, m.; hicks, r.; meuller, j.; moss, s. j.; brandon, n. j.; wood, m. w.; kulic, i.; wellington, c. l. small molecule inducers of abca1 and apoe that act through indirect activation of the lxr pathway. j lipid res 2018, 59, 830-42. liu, j.; yang, b.; wang, y.; wu, y.; fan, b.; zhu, s.; song, e.; song, y. polychlorinated biphenyl quinone promotes macrophage polarization to cd163(+) cells through nrf2 signaling pathway. environ pollut 2020, 257, 113587. chen, x.; su, l.; yang, y.; qv, j.; wei, t.; cui, x.; shao, j.; liu, s.; xu, z.; zhao, b.; miao, j. a new activator of esterase d decreases blood cholesterol level through esd/jab1/abca1 pathway. j cell physiol 2021, 236, 4750-63. hupfeld, t.; chapuy, b.; schrader, v.; beutler, m.; veltkamp, c.; koch, r.; cameron, s.; aung, t.; haase, d.; larosee, p.; truemper, l.; wulf, g. g. tyrosinekinase inhibition facilitates cooperation of transcription factor sall4 and abc transporter a3 towards intrinsic cml cell drug resistance. br j haematol 2013, 161, 204-13. sribenja, s.; natthasirikul, n.; vaeteewoottacharn, k.; sawanyawisuth, k.; wongkham, c.; jearanaikoon, p.; wongkham, s. thymosin beta10 as a predictive biomarker of response to 5-fluorouracil chemotherapy in cholangiocarcinoma. ann hepatol 2016, 15, 577-85. quezada, c. a.; garrido, w. x.; gonzalez-oyarzun, m. a.; rauch, m. c.; salas, m. r.; san martin, r. e.; claude, a. a.; yanez, a. j.; slebe, j. c.; carcamo, j. g. effect of tacrolimus on activity and expression of p-glycoprotein and atp-binding cassette transporter a5 (abca5) proteins in hematoencephalic barrier cells. biol pharm bull 2008, 31, 1911-6. gai, j.; ji, m.; shi, c.; li, w.; chen, s.; wang, y.; li, h. foxo regulates expression of abca6, an intracellular atp-binding-cassette transporter responsive to cholesterol. int j biochem cell biol 2013, 45, 2651-9. wang, x.; cao, c.; li, y.; hai, t.; jia, q.; zhang, y.; zheng, q.; yao, j.; qin, g.; zhang, h.; song, r.; wang, y.; shui, g.; lam, s. m.; liu, z.; wei, h.; meng, a.; zhou, q.; zhao, j. a harlequin ichthyosis pig model with a novel abca12 mutation can be rescued by acitretin treatment. j mol cell biol 2019, 11, 1029-41. tanaka, n.; abe-dohmae, s.; iwamoto, n.; fitzgerald, m. l.; yokoyama, s. hmg-coa reductase inhibitors enhance phagocytosis by upregulating atp-binding cassette transporter a7. atheroscler 2011, 217, 407-14. akisato, y.; ishii, i.; kitahara, m.; tamaki, t.; saito, y.; kitada, m. [effect of pitavastatin on macrophage cholesterol metabolism]. yakugaku zasshi 2008, 128, 357-63. zhang, y.; hu, z.; ye, m.; pan, y.; chen, j.; luo, y.; zhang, y.; he, l.; wang, j. effect of poly(ethylene glycol)-block-polylactide nanoparticles on hepatic cells of mouse: low cytotoxicity, but efflux of the nanoparticles by atp-binding cassette transporters. eur j pharm biopharm 2007, 66, 268-80. jiang, y. j.; lu, b.; kim, p.; paragh, g.; schmitz, g.; elias, p. m.; feingold, k. r. ppar and lxr activators regulate abca12 expression in human keratinocytes. j invest dermatol 2008, 128, 104-9. jiang, y. j.; uchida, y.; lu, b.; kim, p.; mao, c.; akiyama, m.; elias, p. m.; holleran, w. m.; grunfeld, c.; feingold, k. r. ceramide stimulates abca12 expression via peroxisome proliferator-activated receptor {delta} in human keratinocytes. j biol chem 2009, 284, 18942-52. banno, a.; wang, j.; okada, k.; mori, r.; mijiti, m.; nagaoka, s. identification of a novel cholesterol-lowering dipeptide, phenylalanine-proline (fp), and its down-regulation of intestinal abca1 in hypercholesterolemic rats and caco-2 cells. sci rep 2019, 9, 19416. huang, w.; zhou, j.; zhang, g.; zhang, y.; wang, h. decreased h3k9 acetylation level of lxralpha mediated dexamethasone-induced placental cholesterol transport dysfunction. biochim biophys acta mol cell biol lipids 2019, 1864, 158524. han, q. a.; li, k.; dong, x.; luo, y.; zhu, b. function of thelenota ananas saponin desulfated holothurin a in modulating cholesterol metabolism. sci rep 2018, 8, 9506. kaplan, m.; aviram, m.; knopf, c.; keidar, s. angiotensin ii reduces macrophage cholesterol efflux: a role for the at-1 receptor but not for the abc1 transporter. biochem biophys res commun 2002, 290, 1529-34. yang, y.; yang, q.; yang, j.; ma, y.; ding, g. angiotensin ii induces cholesterol accumulation and injury in podocytes. sci rep 2017, 7, 10672. boulate, g.; amazit, l.; naman, a.; seck, a.; paci, a.; lombes, a.; pussard, e.; baudin, e.; lombes, m.; hescot, s. potentiation of mitotane action by rosuvastatin: new insights for adrenocortical carcinoma management. int j oncol 2019, 54, 2149-56. wu, x.; li, c.; mariyam, z.; jiang, p.; zhou, m.; zeb, f.; haq, i. u.; chen, a.; feng, q. acrolein-induced atherogenesis by stimulation of hepatic flavin containing monooxygenase 3 and a protection from hydroxytyrosol. j cell physiol 2018, 234, 475-85. samardzija, d.; pogrmic-majkic, k.; fa, s.; stanic, b.; jasnic, j.; andric, n. bisphenol a decreases progesterone synthesis by disrupting cholesterol homeostasis in rat granulosa cells. mol cell endocrinol 2018, 461, 55-63. kant, r.; lu, c. k.; nguyen, h. m.; hsiao, h. h.; chen, c. j.; hsiao, h. p.; lin, k. j.; fang, c. c.; yen, c. h. 1,2,3,4,6 penta-o-galloyl-beta-d-glucose ameliorates high-fat diet-induced nonalcoholic fatty liver disease and maintains the expression of genes involved in lipid homeostasis in mice. biomed pharmacother 2020, 129, 110348. zhang, x.; qin, y.; wan, x.; liu, h.; iv, c.; ruan, w.; lu, l.; he, l.; guo, x. hydroxytyrosol plays antiatherosclerotic effects through regulating lipid metabolism via inhibiting the p38 signal pathway. biomed res int 2020, 2020, 5036572. li, y.; wu, s. epigallocatechin gallate suppresses hepatic cholesterol synthesis by targeting srebp-2 through sirt1/foxo1 signaling pathway. mol cell biochem 2018, 448, 175-85. chen, j. h.; zheng, y. l.; xu, c. q.; gu, l. z.; ding, z. l.; qin, l.; wang, y.; fu, r.; wan, y. f.; hu, c. p. valproic acid (vpa) enhances cisplatin sensitivity of non-small cell lung cancer cells via hdac2 mediated down regulation of abca1. biol chem 2017, 398, 785-92. koga, m.; kanaoka, y.; okamoto, m.; nakao, y.; inada, k.; takayama, s.; kataoka, y.; yamauchi, a. varenicline aggravates atherosclerotic plaque formation in nicotine-pretreated apoe knockout mice due to enhanced oxldl uptake by macrophages through downregulation of abca1 and abcg1 expression. j pharmacol sci 2020, 142, 9-15. aung, t.; chapuy, b.; vogel, d.; wenzel, d.; oppermann, m.; lahmann, m.; weinhage, t.; menck, k.; hupfeld, t.; koch, r.; trumper, l.; wulf, g. g. exosomal evasion of humoral immunotherapy in aggressive b-cell lymphoma modulated by atp-binding cassette transporter a3. proc natl acad sci u s a 2011, 108, 15336-41. song, j. h.; kim, s. h.; kim, h. j.; hwang, s. y.; kim, t. s. alleviation of the drug-resistant phenotype in idarubicin and cytosine arabinoside double-resistant acute myeloid leukemia cells by indomethacin. int j oncol 2008, 32, 931-6. dai, w.; wang, f.; he, l.; lin, c.; wu, s.; chen, p.; zhang, y.; shen, m.; wu, d.; wang, c.; lu, j.; zhou, y.; xu, x.; xu, l.; guo, c. genistein inhibits hepatocellular carcinoma cell migration by reversing the epithelial-mesenchymal transition: partial mediation by the transcription factor nfat1. mol carcinog 2015, 54, 301-11. long, y.; wang, g.; li, k.; zhang, z.; zhang, p.; zhang, j.; zhang, x.; bao, y.; yang, x.; wang, p. oxidative stress and nf-kappab signaling are involved in lps induced pulmonary dysplasia in chick embryos. cell cycle 2018, 17, 1757-71. mendonca-torres, m. c.; roberts, s. s. the translocator protein (tspo) ligand pk11195 induces apoptosis and cell cycle arrest and sensitizes to chemotherapy treatment in preand post-relapse neuroblastoma cell lines. cancer biol ther 2013, 14, 319-26. wakaumi, m.; ishibashi, k.; ando, h.; kasanuki, h.; tsuruoka, s. acute digoxin loading reduces abca8a mrna expression in the mouse liver. clin exp pharmacol physio 2005, 32, 1034-41. kinting, s.; hoppner, s.; schindlbeck, u.; forstner, m. e.; harfst, j.; wittmann, t.; griese, m. functional rescue of misfolding abca3 mutations by small molecular correctors. hum mol genet 2018, 27, 943-53. kinting, s.; li, y.; forstner, m.; delhommel, f.; sattler, m.; griese, m. potentiation of abca3 lipid transport function by ivacaftor and genistein. j cell mol med 2019, 23, 5225-34. liu, q.; sabirzhanova, i.; bergbower, e. a. s.; yanda, m.; guggino, w. g.; cebotaru, l. the cftr corrector, vx-809 (lumacaftor), rescues abca4 trafficking mutants: a potential treatment for stargardt disease. cell physiol biochem 2019, 53, 400-12. sabirzhanova, i.; lopes pacheco, m.; rapino, d.; grover, r.; handa, j. t.; guggino, w. b.; cebotaru, l. rescuing trafficking mutants of the atp-binding cassette protein, abca4, with small molecule correctors as a treatment for stargardt eye disease. j biol chem 2015, 290, 19743-55. lopes-pacheco, m.; sabirzhanova, i.; rapino, d.; morales, m. m.; guggino, w. b.; cebotaru, l. correctors rescue cftr mutations in nucleotide-binding domain 1 (nbd1) by modulating proteostasis. chembiochem 2016, 17, 493-505. wang, y.; loo, t. w.; bartlett, m. c.; clarke, d. m. correctors promote maturation of cystic fibrosis transmembrane conductance regulator (cftr)-processing mutants by binding to the protein. j biol chem 2007, 282, 33247-51. lin, s.; zhou, c.; neufeld, e.; wang, y. h.; xu, s. w.; lu, l.; wang, y.; liu, z. p.; li, d.; li, c.; chen, s.; le, k.; huang, h.; liu, p.; moss, j.; vaughan, m.; shen, x. big1, a brefeldin a-inhibited guanine nucleotide-exchange protein modulates atp-binding cassette transporter a-1 trafficking and function. arterioscler thromb vasc biol 2013, 33, e31-8. remaley, a. t.; schumacher, u. k.; stonik, j. a.; farsi, b. d.; nazih, h.; brewer, h. b., jr. decreased reverse cholesterol transport from tangier disease fibroblasts. acceptor specificity and effect of brefeldin on lipid efflux. arterioscler thromb vasc biol 1997, 17, 1813-21. mendez, a. j. monensin and brefeldin a inhibit high density lipoprotein-mediated cholesterol efflux from cholesterol-enriched cells. implications for intracellular cholesterol transport. j biol chem 1995, 270, 5891-900. neufeld, e. b.; remaley, a. t.; demosky, s. j.; stonik, j. a.; cooney, a. m.; comly, m.; dwyer, n. k.; zhang, m.; blanchette-mackie, j.; santamarina-fojo, s.; brewer, h. b., jr. cellular localization and trafficking of the human abca1 transporter. j biol chem 2001, 276, 27584-90. field, f. j.; born, e.; chen, h.; murthy, s.; mathur, s. n. esterification of plasma membrane cholesterol and triacylglycerol-rich lipoprotein secretion in caco-2 cells: possible role of p-glycoprotein. j lipid res 1995, 36, 1533-43. castilho, g.; okuda, l. s.; pinto, r. s.; iborra, r. t.; nakandakare, e. r.; santos, c. x.; laurindo, f. r.; passarelli, m. er stress is associated with reduced abca-1 protein levels in macrophages treated with advanced glycated albumin reversal by a chemical chaperone. int j biochem cell biol 2012, 44, 1078-86. singaraja, r. r.; kang, m. h.; vaid, k.; sanders, s. s.; vilas, g. l.; arstikaitis, p.; coutinho, j.; drisdel, r. c.; el-husseini ael, d.; green, w. n.; berthiaume, l.; hayden, m. r. palmitoylation of atp-binding cassette transporter a1 is essential for its trafficking and function. circ res 2009, 105, 138-47. jones, r. j.; gu, d.; bjorklund, c. c.; kuiatse, i.; remaley, a. t.; bashir, t.; vreys, v.; orlowski, r. z. the novel anticancer agent jnj-26854165 induces cell death through inhibition of cholesterol transport and degradation of abca1. j pharmacol exp ther 2013, 346, 381-92. roberts, a. g. the structure and mechanism of drug transporters. methods in molecular biology (clifton, n.j) 2021, 2342, 193-234. qian, h.; zhao, x.; cao, p.; lei, j.; yan, n.; gong, x. structure of the human lipid exporter abca1. cell 2017, 169, 1228-1239 e10. scortecci, j. f.; molday, l. l.; curtis, s. b.; garces, f. a.; panwar, p.; van petegem, f.; molday, r. s. cryo-em structures of the abca4 importer reveal mechanisms underlying substrate binding and stargardt disease. nat commun 2021, 12, 5902. liu, f.; lee, j.; chen, j. molecular structures of the eukaryotic retinal importer abca4. elife 2021, 10, e63524. xie, t.; zhang, z.; fang, q.; du, b.; gong, x. structural basis of substrate recognition and translocation by human abca4. nat commun 2021, 12, 3853. my le, l. t.; thompson, j. r.; aikawa, t.; kanikeyo, t.; alam, a. cryo-em structure of lipid embedded human abca7 at 3.6å resolution. biorxiv 2021, 2021.03.01.433448. namasivayam, v. s., k.; pahnke, j.; stefan, s. m. . binding mode analysis of pan-abc transporter inhibitors within human abca7 to target alzheimer’s disease. comput struct biotechnol j 2021, 19, 6490-6504. alam, a.; kowal, j.; broude, e.; roninson, i.; locher, k. p. structural insight into substrate and inhibitor discrimination by human p-glycoprotein. science 2019, 363, 753-6. fitzgerald, m. l.; morris, a. l.; rhee, j. s.; andersson, l. p.; mendez, a. j.; freeman, m. w. naturally occurring mutations in the largest extracellular loops of abca1 can disrupt its direct interaction with apolipoprotein a-i. j biol chem 2002, 277, 33178-87. lee, j. y.; kinch, l. n.; borek, d. m.; wang, j.; wang, j.; urbatsch, i. l.; xie, x. s.; grishin, n. v.; cohen, j. c.; otwinowski, z.; hobbs, h. h.; rosenbaum, d. m. crystal structure of the human sterol transporter abcg5/abcg8. nature 2016, 533, 561-4. adzhubei, a. a.; kulkarni, a.; tolstova, a. p.; anashkina, a. a.; sviridov, d.; makarov, a. a.; bukrinsky, m. i. direct interaction between abca1 and hiv-1 nef: molecular modeling and virtual screening for inhibitors. comput struct biotechnol j 2021, 19, 3876-84. krapf, m. k.; gallus, j.; namasivayam, v.; wiese, m. 2,4,6-substituted quinazolines with extraordinary inhibitory potency toward abcg2. j med chem 2018, 61, 7952-76. jackson, s. m.; manolaridis, i.; kowal, j.; zechner, m.; taylor, n. m. i.; bause, m.; bauer, s.; bartholomaeus, r.; bernhardt, g.; koenig, b.; buschauer, a.; stahlberg, h.; altmann, k. h.; locher, k. p. structural basis of small-molecule inhibition of human multidrug transporter abcg2. nat struct mol biol 2018, 25, 333-40. rarey, m.; kramer, b.; lengauer, t. time-efficient docking of flexible ligands into active sites of proteins. proc int conf intell syst mol biol 1995, 3, 300-8. ni, z.; bikadi, z.; cai, x.; rosenberg, m. f.; mao, q. transmembrane helices 1 and 6 of the human breast cancer resistance protein (bcrp/abcg2): identification of polar residues important for drug transport. am j physiol cell physiol 2010, 299, c1100-9. tamura, a.; watanabe, m.; saito, h.; nakagawa, h.; kamachi, t.; okura, i.; ishikawa, t. functional validation of the genetic polymorphisms of human atp-binding cassette (abc) transporter abcg2: identification of alleles that are defective in porphyrin transport. mol pharmacol 2006, 70, 287-96. ozvegy, c.; varadi, a.; sarkadi, b. characterization of drug transport, atp hydrolysis, and nucleotide trapping by the human abcg2 multidrug transporter. modulation of substrate specificity by a point mutation. j biol chem 2002, 277, 47980-90. volk, e. l.; farley, k. m.; wu, y.; li, f.; robey, r. w.; schneider, e. overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance. cancer res 2002, 62, 5035-40. honjo, y.; hrycyna, c. a.; yan, q. w.; medina-perez, w. y.; robey, r. w.; van de laar, a.; litman, t.; dean, m.; bates, s. e. acquired mutations in the mxr/bcrp/abcp gene alter substrate specificity in mxr/bcrp/abcp-overexpressing cells. cancer res 2001, 61, 6635-9. silbermann, k.; li, j.; namasivayam, v.; stefan, s. m.; wiese, m. rational drug design of 6-substituted 4-anilino-2-phenylpyrimidines for exploration of novel abcg2 binding site. eur j med chem 2021, 212, 113045. silbermann, k.; li, j.; namasivayam, v.; baltes, f.; bendas, g.; stefan, s. m.; wiese, m. superior pyrimidine derivatives as selective abcg2 inhibitors and broad-spectrum abcb1, abcc1, and abcg2 antagonists. j med chem 2020, 63, 10412-32. kim, s.; thiessen, p. a.; bolton, e. e.; chen, j.; fu, g.; gindulyte, a.; han, l.; he, j.; he, s.; shoemaker, b. a.; wang, j.; yu, b.; zhang, j.; bryant, s. h. pubchem substance and compound databases. nucleic acids res 2016, 44, d1202-13. rogers, d.; hahn, m. extended-connectivity fingerprints. j chem inf model 2010, 50, 742-54. bender, a.; mussa, h. y.; glen, r. c.; reiling, s. molecular similarity searching using atom environments, information-based feature selection, and a naive bayesian classifier. j chem inf comput sci 2004, 44, 170-8. silbermann, k.; stefan, s. m.; elshawadfy, r.; namasivayam, v.; wiese, m. identification of thienopyrimidine scaffold as an inhibitor of the abc transport protein abcc1 (mrp1) and related transporters using a combined virtual screening approach. j med chem 2019, 62, 4383-400. danish, a.; namasivayam, v.; schiedel, a. c.; muller, c. e. interaction of approved drugs with synaptic vesicle protein 2a. arch pharm (weinheim) 2017, 350. namasivayam, v. s., k.; pahnke, j.; wiese, m.; stefan, s. m. feature-driven pattern analysis for multitarget modulator landscapes. bioinformatics 2021, https://www.doi.org/10.1093/bioinformatics/btab832 lee, y. m.; venkataraman, k.; hwang, s. i.; han, d. k.; hla, t. a novel method to quantify sphingosine 1-phosphate by immobilized metal affinity chromatography (imac). prostaglandins other lipid mediat 2007, 84, 154-62. oram, j. f.; vaughan, a. m.; stocker, r. atp-binding cassette transporter a1 mediates cellular secretion of alpha-tocopherol. j biol chem 2001, 276, 39898-902. haller, j. f.; cavallaro, p.; hernandez, n. j.; dolat, l.; soscia, s. j.; welti, r.; grabowski, g. a.; fitzgerald, m. l.; freeman, m. w. endogenous beta-glucocerebrosidase activity in abca12(-)/(-)epidermis elevates ceramide levels after topical lipid application but does not restore barrier function. j lipid res 2014, 55, 493-503. reboul, e.; dyka, f. m.; quazi, f.; molday, r. s. cholesterol transport via abca1: new insights from solid-phase binding assay. biochim 2013, 95, 957-61. beljanski, v.; soulika, a.; tucker, j. m.; townsend, d. m.; davis, w., jr.; tew, k. d. characterization of the atpase activity of human atp-binding cassette transporter-2 (abca2). in vivo (athens, greece) 2005, 19, 657-60. tsybovsky, y.; wang, b.; quazi, f.; molday, r. s.; palczewski, k. posttranslational modifications of the photoreceptor-specific abc transporter abca4. biochem 2011, 50, 6855-66. zhong, m.; molday, l. l.; molday, r. s. role of the c terminus of the photoreceptor abca4 transporter in protein folding, function, and retinal degenerative diseases. j biol chem 2009, 284, 3640-9. petry, f.; ritz, v.; meineke, c.; middel, p.; kietzmann, t.; schmitz-salue, c.; hirsch-ernst, k. i. subcellular localization of rat abca5, a rat atp-binding-cassette transporter expressed in leydig cells, and characterization of its splice variant apparently encoding a half-transporter. biochem j 2006, 393, 79-87. hu, j. y.; yang, p.; wegner, d. j.; heins, h. b.; luke, c. j.; li, f.; white, f. v.; silverman, g. a.; sessions cole, f.; wambach, j. a. functional characterization of four atp-binding cassette transporter a3 gene (abca3) variants. hum mutat 2020, 41, 1298-1307. trompier, d.; alibert, m.; davanture, s.; hamon, y.; pierres, m.; chimini, g. transition from dimers to higher oligomeric forms occurs during the atpase cycle of the abca1 transporter. j biol chem 2006, 281, 20283-90. takahashi, k.; kimura, y.; kioka, n.; matsuo, m.; ueda, k. purification and atpase activity of human abca1. j biol chem 2006, 281, 10760-8. gameiro, m.; silva, r.; rocha-pereira, c.; carmo, h.; carvalho, f.; bastos, m. l.; remiao, f. cellular models and in vitro assays for the screening of modulators of p-gp, mrp and bcrp. molecules 2017, 22. kraege, s.; stefan, k.; kohler, s. c.; wiese, m. optimization of acryloylphenylcarboxamides as inhibitors of abcg2 and comparison with acryloylphenylcarboxylates. chemmedchem 2016, 11, 2547-58. spindler, a.; stefan, k.; wiese, m. synthesis and investigation of tetrahydro-beta-carboline derivatives as inhibitors of the breast cancer resistance protein (abcg2). j med chem 2016, 59, 6121-35. kraege, s.; stefan, k.; juvale, k.; ross, t.; willmes, t.; wiese, m. the combination of quinazoline and chalcone moieties leads to novel potent heterodimeric modulators of breast cancer resistance protein (bcrp/abcg2). eur j med chem 2016, 117, 212-29. gelissen, i. c.; brown, a. j. methods mol biol 2017, 1583, 1–6. yang, a.; gelissen, i. c. abc-transporter mediated sterol export from cells using radiolabeled sterols. methods in molecular biology (clifton, n.j) 2017, 1583, 275-85. cattelotte, j.; andre, p.; ouellet, m.; bourasset, f.; scherrmann, j. m.; cisternino, s. in situ mouse carotid perfusion model: glucose and cholesterol transport in the eye and brain. j cereb blood flow metab 2008, 28, 1449-59. li, y.; kinting, s.; hoppner, s.; forstner, m. e.; uhl, o.; koletzko, b.; griese, m. metabolic labelling of choline phospholipids probes abca3 transport in lamellar bodies. biochim biophys acta mol cell biol lipids 2019, 1864, 158516. hoppner, s.; kinting, s.; torrano, a. a.; schindlbeck, u.; brauchle, c.; zarbock, r.; wittmann, t.; griese, m. quantification of volume and lipid filling of intracellular vesicles carrying the abca3 transporter. biochim biophys acta mol cell res 2017, 1864, 2330-5. cox, j. v.; abdelrahman, y. m.; peters, j.; naher, n.; belland, r. j. chlamydia trachomatis utilizes the mammalian cla1 lipid transporter to acquire host phosphatidylcholine essential for growth. cell microbiol 2016, 18, 305-18. sankaranarayanan, s.; kellner-weibel, g.; de la llera-moya, m.; phillips, m. c.; asztalos, b. f.; bittman, r.; rothblat, g. h. a sensitive assay for abca1-mediated cholesterol efflux using bodipy-cholesterol. j lipid res 2011, 52, 2332-40. stearns, m. e.; jenkins, d. p.; tew, k. d. dansylated estramustine, a fluorescent probe for studies of estramustine uptake and identification of intracellular targets. proc natl acad sci u s a 1985, 82, 8483-7. bryan, a.; watters, c.; koenig, l.; youn, e.; olmos, a.; li, g.; williams, s. c.; rumbaugh, k. p. human transcriptome analysis reveals a potential role for active transport in the metabolism of pseudomonas aeruginosa autoinducers. microbes infect 2010, 12, 1042-50. chaudhuri, a.; anand, d. cholesterol: revisiting its fluorescent journey on 200th anniversary of chevruel’s “cholesterine”. biomed spectrosc imaging 2017, 6, 1-24. solanko, k. a.; modzel, m.; solanko, l. m.; wustner, d. fluorescent sterols and cholesteryl esters as probes for intracellular cholesterol transport. lipid insights 2015, 8, 95-114. invitrogen. chapter 13 probes for lipids and membranes. in molecular probes handbook: a guide to fluorescent probes and labeling technologies, thermofischer, ed. 2010. silbermann, k.; shah, c. p.; sahu, n. u.; juvale, k.; stefan, s. m.; kharkar, p. s.; wiese, m. novel chalcone and flavone derivatives as selective and dual inhibitors of the transport proteins abcb1 and abcg2. eur j med chem 2019, 164, 193-213. stefan, k.; schmitt, s. m.; wiese, m. 9-deazapurines as broad-spectrum inhibitors of the abc transport proteins p-glycoprotein, multidrug resistance-associated protein 1, and breast cancer resistance protein. j med chem 2017, 60, 8758-80. schmitt, s. m.; stefan, k.; wiese, m. pyrrolopyrimidine derivatives as novel inhibitors of multidrug resistance-associated protein 1 (mrp1, abcc1). j med chem 2016, 59, 3018-33. stefan, k. etablierung und anwendung unterschiedlicher kolorimetrischer detektionsmethoden zur aktivitätsbestimmung von modulatoren der abc-transporter abcb1, abcc1 und abcg2. rheinische friedrich-wilhelms-universität bonn, 2020. mack, j. t.; beljanski, v.; soulika, a. m.; townsend, d. m.; brown, c. b.; davis, w.; tew, k. d. "skittish" abca2 knockout mice display tremor, hyperactivity, and abnormal myelin ultrastructure in the central nervous system. mol cell biol 2007, 27, 44-53. ban, n.; matsumura, y.; sakai, h.; takanezawa, y.; sasaki, m.; arai, h.; inagaki, n. abca3 as a lipid transporter in pulmonary surfactant biogenesis. j biol chem 2007, 282, 9628-34. hammel, m.; michel, g.; hoefer, c.; klaften, m.; müller-höcker, j.; de angelis, m. h.; holzinger, a. targeted inactivation of the murine abca3 gene leads to respiratory failure in newborns with defective lamellar bodies. biochem biophys res commun 2007, 359, 947-51. beers, m. f.; knudsen, l.; tomer, y.; maronn, j.; zhao, m.; ochs, m.; mulugeta, s. aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the abca3 gene. ann anat 2017, 210, 135-46. weng, j.; mata, n. l.; azarian, s. m.; tzekov, r. t.; birch, d. g.; travis, g. h. insights into the function of rim protein in photoreceptors and etiology of stargardt's disease from the phenotype in abcr knockout mice. cell 1999, 98, 13-23. molday, r. s. atp-binding cassette transporter abca4: molecular properties and role in vision and macular degeneration. j bioenerg biomembr 2007, 39, 507-17. trigueros-motos, l.; van capelleveen, j. c.; torta, f.; castaño, d.; zhang, l. h.; chai, e. c.; kang, m.; dimova, l. g.; schimmel, a. w. m.; tietjen, i.; radomski, c.; tan, l. j.; thiam, c. h.; narayanaswamy, p.; wu, d. h.; dorninger, f.; yakala, g. k.; barhdadi, a.; angeli, v.; dubé, m. p.; berger, j.; dallinga-thie, g. m.; tietge, u. j. f.; wenk, m. r.; hayden, m. r.; hovingh, g. k.; singaraja, r. r. abca8 regulates cholesterol efflux and high-density lipoprotein cholesterol levels. arterioscler thromb vasc biol 2017, 37, 2147-55. iritani, s.; torii, y.; habuchi, c.; sekiguchi, h.; fujishiro, h.; yoshida, m.; go, y.; iriki, a.; isoda, m.; ozaki, n. the neuropathological investigation of the brain in a monkey model of autism spectrum disorder with abca13 deletion. int j dev neurosci 2018, 71, 130-9. lepage, d. f.; conlon, r. a. animal models for disease: knockout, knock-in, and conditional mutant mice. methods mol med 2006, 129, 41-67. navabpour, s.; kwapis, j. l.; jarome, t. j. a neuroscientist's guide to transgenic mice and other genetic tools. neurosci biobehav rev 2020, 108, 732-48. campenhout, c. v.; cabochette, p.; veillard, a. c.; laczik, m.; zelisko-schmidt, a.; sabatel, c.; dhainaut, m.; vanhollebeke, b.; gueydan, c.; kruys, v. guidelines for optimized gene knockout using crispr/cas9. biotechniques 2019, 66, 295-302. lovett-racke, a. e.; cravens, p. d.; gocke, a. r.; racke, m. k.; stüve, o. therapeutic potential of small interfering rna for central nervous system diseases. arch neurol 2005, 62, 1810-3. moore, c. b.; guthrie, e. h.; huang, m. t.; taxman, d. j. short hairpin rna (shrna): design, delivery, and assessment of gene knockdown. methods mol biol (clifton, n.j) 2010, 629, 141-58. drummond, e.; wisniewski, t. alzheimer's disease: experimental models and reality. acta neuropathol 2017, 133, 155-75. mochizuki, h.; yamada, m.; mizuno, y. alpha-synuclein overexpression model. j neural transm 2006, 281-4. peltz, g. can 'humanized' mice improve drug development in the 21st century? trends pharmacol sci 2013, 34, 255-60. krohn, m.; zoufal, v.; mairinger, s.; wanek, t.; paarmann, k.; brüning, t.; eiriz, i.; brackhan, m.; langer, o.; pahnke, j. generation and characterization of an abcc1 humanized mouse model (habcc1(flx/flx)) with knockout capability. mol pharmacol 2019, 96, 138-147. dallas, s.; salphati, l.; gomez-zepeda, d.; wanek, t.; chen, l.; chu, x.; kunta, j.; mezler, m.; menet, m. c.; chasseigneaux, s.; decleves, x.; langer, o.; pierre, e.; diloreto, k.; hoft, c.; laplanche, l.; pang, j.; pereira, t.; andonian, c.; simic, d.; rode, a.; yabut, j.; zhang, x.; scheer, n. generation and characterization of a breast cancer resistance protein humanized mouse model. mol pharmacol 2016, 89, 492-504. krohn, m.; wanek, t.; menet, m. c.; noack, a.; decleves, x.; langer, o.; loscher, w.; pahnke, j. humanization of the blood-brain barrier transporter abcb1 in mice disrupts genomic locus lessons from three unsuccessful approaches. eur j microbiol immunol (bp) 2018, 8, 78-86. willmann, j. k.; van bruggen, n.; dinkelborg, l. m.; gambhir, s. s. molecular imaging in drug development. nat rev drug discov 2008, 7, 591-607. mairinger, s.; erker, t.; muller, m.; langer, o. pet and spect radiotracers to assess function and expression of abc transporters in vivo. curr drug metab 2011, 12, 774-92. zoufal, v.; mairinger, s.; krohn, m.; wanek, t.; filip, t.; sauberer, m.; stanek, j.; traxl, a.; schuetz, j. d.; kuntner, c.; pahnke, j.; langer, o. influence of multidrug resistance-associated proteins on the excretion of the abcc1 imaging probe 6-bromo-7-[(11)c]methylpurine in mice. mol imaging biol 2019, 21, 306-16. wanek, t.; zoufal, v.; brackhan, m.; krohn, m.; mairinger, s.; filip, t.; sauberer, m.; stanek, j.; pekar, t.; pahnke, j.; langer, o. brain distribution of dual abcb1/abgc2 substrates is unaltered in a beta-amyloidosis mouse model. int j mol sci 2020, 21, 8245. zoufal, v.; wanek, t.; krohn, m.; mairinger, s.; filip, t.; sauberer, m.; stanek, j.; pekar, t.; bauer, m.; pahnke, j.; langer, o. age dependency of cerebral p-glycoprotein function in wild-type and appps1 mice measured with pet. j cereb blood flow metab 2018, 40, 150-62. campbell, b. r.; gonzalez trotter, d.; hines, c. d.; li, w.; patel, m.; zhang, w.; evelhoch, j. l. in vivo imaging in pharmaceutical development and its impact on the 3rs. ilar j 2016, 57, 212-20. bieczynski, f.; burkhardt-medicke, k.; luquet, c. m.; scholz, s.; luckenbach, t. chemical effects on dye efflux activity in live zebrafish embryos and on zebrafish abcb4 atpase activity. febs lett 2021, 595, 828-43. pedersen, j. m.; matsson, p.; bergstrom, c. a.; hoogstraate, j.; noren, a.; lecluyse, e. l.; artursson, p. early identification of clinically relevant drug interactions with the human bile salt export pump (bsep/abcb11). toxicol sci 2013, 136, 328-43. matsson, p.; pedersen, j. m.; norinder, u.; bergstrom, c. a.; artursson, p. identification of novel specific and general inhibitors of the three major human atp-binding cassette transporters p-gp, bcrp and mrp2 among registered drugs. pharm res 2009, 26, 1816-31. cserepes, j.; szentpetery, z.; seres, l.; ozvegy-laczka, c.; langmann, t.; schmitz, g.; glavinas, h.; klein, i.; homolya, l.; varadi, a.; sarkadi, b.; elkind, n. b. functional expression and characterization of the human abcg1 and abcg4 proteins: indications for heterodimerization. biochem biophys res commun 2004, 320, 860-7. ivnitski-steele, i.; larson, r. s.; lovato, d. m.; khawaja, h. m.; winter, s. s.; oprea, t. i.; sklar, l. a.; edwards, b. s. high-throughput flow cytometry to detect selective inhibitors of abcb1, abcc1, and abcg2 transporters. assay drug dev technol 2008, 6, 263-76. borst, p.; de wolf, c.; van de wetering, k. multidrug resistance-associated proteins 3, 4, and 5. pflugers arch 2007, 453, 661-73. berghaus, a.; jovanovic, s. technique and indications of extended sublabial rhinotomy ("midfacial degloving"). rhinol 1991, 29, 105-10. lim, j. g.; lee, h. y.; yun, j. e.; kim, s. p.; park, j. w.; suh, s. i.; jang, b. c.; cho, c. h.; bae, j. h.; kim, s. s.; han, j.; park, m. j.; song, d. k. taurine block of cloned atp-sensitive k+ channels with different sulfonylurea receptor subunits expressed in xenopus laevis oocytes. biochem pharmacol 2004, 68, 901-10. york, n. w.; parker, h.; xie, z.; tyus, d.; waheed, m. a.; yan, z.; grange, d. k.; remedi, m. s.; england, s. k.; hu, h.; nichols, c. g. kir6.1and sur2-dependent katp over-activity disrupts intestinal motility in murine models of cantu syndrome. jci insight 2020, 5, e141443. beretta, g. l.; cassinelli, g.; pennati, m.; zuco, v.; gatti, l. overcoming abc transporter-mediated multidrug resistance: the dual role of tyrosine kinase inhibitors as multitargeting agents. eur j med chem 2017, 142, 271-89. horikawa, m.; kato, y.; sugiyama, y. reduced gastrointestinal toxicity following inhibition of the biliary excretion of irinotecan and its metabolites by probenecid in rats. pharm res 2002, 19, 1345-53. smeets, p. h.; van aubel, r. a.; wouterse, a. c.; van den heuvel, j. j.; russel, f. g. contribution of multidrug resistance protein 2 (mrp2/abcc2) to the renal excretion of p-aminohippurate (pah) and identification of mrp4 (abcc4) as a novel pah transporter. j am soc nephrol 2004, 15, 2828-35. dalpiaz, a.; pavan, b. nose-to-brain delivery of antiviral drugs: a way to overcome their active efflux? pharm 2018, 10, 39. zhou, y.; hopper-borge, e.; shen, t.; huang, x. c.; shi, z.; kuang, y. h.; furukawa, t.; akiyama, s.; peng, x. x.; ashby, c. r., jr.; chen, x.; kruh, g. d.; chen, z. s. cepharanthine is a potent reversal agent for mrp7(abcc10)-mediated multidrug resistance. biochem pharmacol 2009, 77, 993-1001. saeed, m. e. m.; boulos, j. c.; elhaboub, g.; rigano, d.; saab, a.; loizzo, m. r.; hassan, l. e. a.; sugimoto, y.; piacente, s.; tundis, r.; yagi, s.; khalid, h.; efferth, t. cytotoxicity of cucurbitacin e from citrullus colocynthis against multidrug-resistant cancer cells. phytomedicine 2019, 62, 152945. horikawa, m.; kato, y.; tyson, c. a.; sugiyama, y. potential cholestatic activity of various therapeutic agents assessed by bile canalicular membrane vesicles isolated from rats and humans. drug metab pharmacokinet 2003, 18, 16-22. bai, j.; lai, l.; yeo, h. c.; goh, b. c.; tan, t. m. multidrug resistance protein 4 (mrp4/abcc4) mediates efflux of bimane-glutathione. int j biochem cell biol 2004, 36, 247-57. videmann, b.; mazallon, m.; prouillac, c.; delaforge, m.; lecoeur, s. abcc1, abcc2 and abcc3 are implicated in the transepithelial transport of the myco-estrogen zearalenone and its major metabolites. toxicol lett 2009, 190, 215-23. tun-yhong, w.; chinpaisal, c.; pamonsinlapatham, p.; kaewkitichai, s. tenofovir disoproxil fumarate is a new substrate of atp-binding cassette subfamily c member 11. antimicrob agents chemother 2017, 61, e01725-16. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. alzheimer’s disease is an inherent, natural part of human brain aging: an integrated perspective feel free to add comments by clicking these icons on the sidebar free neuropathology 3:17 (2022) review alzheimer’s disease is an inherent, natural part of human brain aging: an integrated perspective isidro ferrer department of pathology and experimental therapeutics, university of barcelona; emeritus researcher of the bellvitge institute of biomedical research (idibell); biomedical research network of neurodegenerative diseases (ciberned); institute of neurosciences, university of barcelona; hospitalet de llobregat, barcelona, spain corresponding author: i. ferrer · department of pathology and experimental therapeutics · university of barcelona · campus bellvitge · carrer feixa llarga sn · 08907 hospitalet de llobregat · spain 8082ifa@gmail.com submitted: 25 february 2022 accepted: 21 june 2022 copyedited by: félicia jeannelle published: 08 july 2022 https://doi.org/10.17879/freeneuropathology-2022-3806 keywords: alzheimer’s disease, human brain aging, genetics, epigenetics, new therapies abstract alzheimer disease is one of the most challenging demons in our society due to its very high prevalence and its clinical manifestations which cause deterioration of cognition, intelligence, and emotions – the very capacities that distinguish homo sapiens from other animal species. besides the personal, social, and economical costs, late stages of ad are vivid experiences for the family, relatives, friends, and general observers of the progressive ruin of an individual who turns into a being with lower mental and physical capacities than less evolved species. a human brain with healthy cognition, conscience, and emotions can succeed in dealing with most difficulties that life may pose. without these capacities, the same person probably cannot. due, in part, to this emotional impact, the absorbing study of ad has generated, over the years, a fascinating and complex story of theories, hypotheses, controversies, fashion swings, and passionate clashes, together with tremendous efforts and achievements geared to improve understanding of the pathogenesis and treatment of the disorder. familal ad is rare and linked to altered genetic information associated with three genes. sporadic ad (sad) is much more common and multifactorial. a major point of clinical discussion has been, and still is, establishing the differences between brain aging and sad. this is not a trivial question, as the neuropathological and molecular characteristics of normal brain aging and the first appearance of early stages of sad-related pathology are not easily distinguishable in most individuals. another important point is confidence in assigning responsibility for the beginning of sad to a few triggering molecules, without considering the wide number of alterations that converge in the pathogenesis of aging and sad. genetic risk factors covering multiple molecular signals are increasing in number. in the same line, molecular pathways are altered at early stages of sad pathology, currently grouped under the aegis of normal brain aging, only to increase massively at advanced stages of the process. sporadic ad is here considered an inherent, natural part of human brain aging, which is prevalent in all humans, and variably present or not in a few individuals in other species. the progression of the process has devastating effects in a relatively low percentage of human beings eventually evolving to dementia. the continuum of brain aging and sad implies the search for a different approach in the study of human brain aging at the first stages of the biological process, and advances in the use of new technologies aimed at slowing down the molecular defects underlying human brain aging and sad at the outset, and transfering information and tasks to ai and coordinated devices. index summary 1. introduction 2. β-amyloid and tau 2a. β-amyloid (aβ) 2b. tau 3. familial ad (fad; early-onset familial alzheimer’s disease: eofad), and the β-amyloid cascade hypothesis 4. sporadic ad (sad; late-onset alzheimer disease: load) 5. nfts and sps in non-human brain aging 6. synapses 7. neurotransmitters, neuromodulators, and related receptors 7a. acetylcholine (ach) and acetylcholine receptors (achr) 7b. glutamate and glutamate receptors (glurs) 7c. γ-aminobutyric acid (gaba) and gaba receptors 7d. serotonin and 5-hydroxytryptamine (5-ht) receptors 7e. noradrenergic system 7f. adenosine receptors 7g. endocannabinoids and cannabinoid receptors (cbrs) 8. trophic factors and receptors 9. endoplasmic reticulum stress 10. failure to remove debris: the ubiquitin-proteasome system (ups) and autophagy in sad 11. granulovacuolar degeneration (gvd) 12. glial alterations in aging and sad 12a. astrocytes 12b. microglia 12c. oligodendrocytes 13. the neurovascular system in ad 14. purine and pyrimidine metabolism in sad 15. epigenetics in brain aging and sad 15a. histone modifications, dna methylation, and hydroxymethylation 15b. non-coding rnas 16. microorganisms and sad 16a. microorganisms in the brain and oral cavity 16b. gut microbiota 17. seeding and spreading of β-amyloid and tau 17a. seeding β-amyloid 17b. tau seeding 17c. multiple seeding foci of β-amyloid and tau pathology; vulnerable and resistant populations to tau seeding in brain aging and sad 18. neuronal death 19. neuronal connectivity networks in brain aging and sad 20. human brain aging and preclinical ad 21. primary age-related tauopathy (part), rapidly progressive sad, and sad resilience 21a. part 21b. rapidly progressive ad 21c. sad resilience 22. biochemical changes beyond tau and β-amyloid at the the first stages of nft pathology 22a. aberrant cell-cycle re-entry, and altered adult neurogenesis 22b. brain lipids 22c. lipid rafts and cell membranes 22d. mitochondria 22e. oxidative stress damage 22f. inflammation 22g. protein synthesis impairment 22h. dysregulated protein phosphorylation 23. concluding comments abbreviations funding acknowledgements references summary this is a comprehensive historical and up-dated review on the pathogenesis of alzheimer’s disease (ad) in relation to intrinsic process of natural brain aging. the study covers, in addition to β-amyloid and tau pathology, alterations in multiple merging molecular pathways and sub-cellular structures underpinning brain aging and ad. familial ad (fad) is rare and linked to altered genetic information associated with three genes. sporadic ad (sad) is much more common and multifactorial. a major point of clinical discussion has been, and remains, establishing the differences between brain aging and sad. this is not a trivial question, as the neuropathological and molecular characteristics of normal brain aging and the first appearance of early stages of sad-related pathology are not easily distinguishable in most individuals. another important point is confidence in assigning responsibility for the beginning of sad to a few triggering molecules, without considering the wide number of alterations that converge in the pathogenesis of aging and sad. recognized genetic risk factors covering multiple molecular signals are increasing in number. molecular alterations of lipid rafts, protein synthesis from the nucleolus to the ribosome, protein phosphorylation, kinase activation, purine metabolism, epigenetic regulation of dna and rna, mitochondria and energy metabolism, inflammation, oxidative stress, cell-cycle re-entry, and cell death precede, in some regions (i.e., frontal cortex), abnormal tau deposition and amyloid plaques. human brain aging and sad do not follow a linear logic based on the assumption that a cause results in one or several effects; several separate alterations converge and potentiate each other to incorporate anomalies in additional pathways. tau seeding and spreading are active intercellular and intracellular processes that explain, only in part, disease progression. cell and region vulnerability are essential elements. brain aging with neurofibrillary tangles (nfts) restricted to the temporal lobe and selected nuclei of the brain stem, primary age-related tauopathy, preclinical ad, mild cognitive impairment (mci) of alzheimer type, typical ad, rapid progressive ad, and ad subtypes, are forms of sad modulated by individual genetic and molecular factors. as in atherosclerosis, the progression of the process has devastating effects in a relatively low percentage of human beings. future modulation of human brain aging and sad will require the combined application of artificial intelligence, brain dna editing, external electrical or wave-based signals to reduce energy consumption, and optimization of mitochondrial function, together with implantation of microdevices, to facilitate cooperative human-machine operation, pharmacological protection of lipid-protein interactions, high-throughput molecular technology, and resetting during sleep stages. 1. introduction the clinical and neuropathological characteristics and clinical correlates of alzheimer disease (ad) have been described in several recent reviews (1-9). however, the study of ad has generated a fascinating and complex compendium of theories, hypotheses, controversies, fashion swings, and passionate clashes, together with tremendous efforts and achievements geared to improve understanding of the pathogenesis and treatment of the disorder. the present paper is a critical review of brain aging and ad that includes molecular abnormalities and early metabolic alterations beyond β-amyloid and tau pathology. these changes, together with genetic factors, converge in the pathogenesis of ad. learning about early molecular modifications preceding by many years the appearance of clinical symptoms, when present, will serve to improve understanding of brain aging and the ad continuum. figure 1: dystrophic neurites of sps and nfts in the frontal cortex of a 76-year-old woman with dementia. paraffin section, gros-bielschowsky silver method without counterstaining, black and white figure, bar = 25μm. until the beginning of the last century, cognitive impairment and dementia were considered natural features of old age. multiple brain infarcts were common in old people, and vascular dementia due to arteriopathy was thought to be the main cause of senile dementia. however, microscopic study of post-mortem brains stained with the dyes available at that time revealed the presence of certain structural anomalies in aged individuals. paul block and georges marinesco (10) described “amas ronds”, and emil redlich (11) “miliare sklerose” in the neuropil, interpreted at that time as nodules of glial sclerosis, which we now know as senile plaques (sps). the introduction of the max bielschowsky silver method allowed visualisation of argyrophilic structures in neurons. using this method, alois alzheimer described for the first time large numbers of argyrophilic neurofibrillary tangles (nfts) and aggregates of dystrophic neurites in the brain of a 51-year-old woman who had suffered from progressive dementia and hallucinations in the previous four and half years (12). other cases were published shortly afterwards (13). the term alzheimer’s pre-senile dementia was introduced by emil kraepelin (14) to define the combination of pre-senile (before the age of 65) dementia in individuals with the morphological lesions described by alzheimer. oskar fischer (15), using the same method, described the presence of ‘drusen’ or ‘drusige nekrosen’ in 16 cases of senile dementia characterized by loss of memory and sense of location, disorientation, and confabulation. subsequent fischer reports (16, 17) detailed the morphology of abnormal fibrils and abnormal neurites, and their stages of formation, in a large series of older individuals. the term ‘‘senile plaque’’ (sp) for these structures was proposed by simchowitz (18). fischer also described “drusige entartung der gefässe” which corresponds to amyloid angiopathy. interestingly, fischer also reported and illustrated the presence of nfts in the same cases with dementia (19). hundreds of articles appeared in the succeeding years. alzheimer focused on nfts as the main cause of dementia, whereas fischer thought that sps were the main substrate of dementia in older cases. moreover, alzheimer contemplated nfts as aggregates of abnormal neurofibrils, while fischer considered dystrophic neurites of sps composed of abnormal neurofibrils, and nfts a particular abnormality of nerve cells (19). bielschowsky proposed a link between tangles and neuritic changes (20) (figures 1 and 2). nfts and sps are now considered ad-related pathology or ad-neuropathologic change (adnc) (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). the term pick’s disease (pid) was coined in 1926 to distinguish ad from pid primary frontotemporal degenerative atrophy (21). as late as the 1960s, ad and pid were considered early dementias, whereas pure senile dementia, vascular dementias, and mixed (vascular and degenerative) were classified as dementias in old age (22). the frontiers between ad and pure senile dementia were not clear, as the onset of clinical symptoms in many cases classified as ad was after the age of sixty (23). it was not until the 1970s that alzheimer’s pre-senile dementia and senile dementia with changes of alzheimer type were considered to be within the same spectrum (24-27). the inclusive term “alzheimer-fischer dementia” was never contemplated. figure 2: neurofibrillary tangles in the ca1 region of the hippocampus of a man aged 69 years with no apparent cognitive impairment. paraffin section, gallyas staining, lightly counterstained with haemtoxylin, bar = 25μm. the first approach toward a clinical consensus on ad was made in 1984; clinical diagnosis of ad was set up in three categories – possible, probable, and definite (requiring neuropathological verification) (28). definite ad was fixed as a neurodegenerative disease manifested by progressive dementia with a neuropathological substrate characterized by brain atrophy, neuronal death, and a particular distribution of abundant sps and nfts in the brain. box 1: clinical classification of alzheimer’s disease (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). preclinical alzheimer’s disease in this phase, individuals may have measurable brain changes that indicate the earliest signs of ad (biomarkers), but they have not yet developed symptoms such as memory loss. mild cognitive impairment due to alzheimer’s disease people with mci due to ad have biomarker evidence of alzheimer’s brain changes plus new but subtle symptoms such as problems with memory, language and thinking. these cognitive problems may be noticeable to the individual, family members and friends, but not to others, and they may not interfere with individuals’ ability to carry out everyday activities. mild alzheimer’s dementia in the mild stage of alzheimer’s dementia, most people are able to function independently in many areas but are likely to require assistance with some activities to maximize independence and remain safe. handling money and paying bills may be especially challenging, and they may need more time to complete common daily tasks. they may still be able to drive, work and participate in their favorite activities. moderate alzheimer’s dementia in the moderate stage of alzheimer’s dementia, which is often the longest stage, individuals experience more problems with memory and language, are more likely to become confused, and find it harder to complete multistep tasks such as bathing and dressing. they may become incontinent at times, and they may start having personality and behavioral changes, including suspiciousness and agitation. they may also begin to have problems recognizing loved ones. severe alzheimer’s dementia in the severe stage of alzheimer’s dementia, individuals’ ability to communicate verbally is greatly diminished, and they are likely to require around-the-clock care. because of damage to areas of the brain involved in movement, individuals become bed-bound. being bed-bound makes them vulnerable to physical complications including blood clots, skin infections and sepsis, which triggers body-wide inflammation that can result in organ failure. damage to areas of the brain that control swallowing makes it difficult to eat and drink. because of this, food particles may be deposited in the lungs and cause lung infection. in contrast to ad dementia, well-tolerated progressive slower processing, memory loss particularly related to recent events, more trouble multitasking, slight cognitive decline, sleep disorder, emotional changes, slight or moderate depression, and bilateral brain activation for memory functions developing around the sixties are all consistent with “normal brain aging”. neuropathological alterations in normal old-aged individuals are nfts in the hippocampus, entorhinal cortex, and inferior temporal cortex, and very rarely in the frontal neocortex; the distribution of sps, if present, is more heterogeneous (29-35). clinical and neuropathological criteria to identify borderline cases between ad and cognitive impairment due to normal brain aging yielded only a limited consensus (36, 37). a few years later, cerad proposed a neuritic plaque score based on the number of plaques per mm2 and the age of the individual to categorize ad in comparison to normal brain aging (38, 39). evidence of a clinical progression and post-mortem neuropathological observations showing a concatenation of ad-related changes in old age and sad (29-32, 40-43) prompted a clinical redefinition of ad at the beginning of the second decade of this century. a crucial approach was the combination of clinical criteria, biochemical biomarkers in body fluids, and neuroimaging techniques to define the diagnosis of preclinical ad, mild cognitive impairment (mci) due to ad, and ad (44-50). more precise clinical definitions have been proposed to categorize different stages of ad (51). the classification shown in box 1 is a summarized transcription of the alzheimer’s association report: 2022 alzheimer’s disease facts and figures (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). the american academy of neurology estimates that mci is present in about 8% of people age 65 to 69, in 15% of 75to 79-year-olds, in 25% of those age 80 to 84, and in about 37% of people 85 years of age and older. about 7.5% will develop dementia in the first year after diagnosis of mci; about 15% will develop dementia in the second year; about one third will develop dementia due to ad within five years (52, 53). the prevalence of dementia in 65-69-year-olds is approximately 0.01% of individuals; the prevalence of dementia doubles with increments of five years; thereby, between 25% and 50% of individuals over the age of 85 suffer from dementia (2). it is estimated that between 50% and 80% of cases with dementia have ad (2). age, gender, race, living conditions, and genetic factors mark differences in the duration of preclinical and dementia stages in sad (54, 55). 2. β-amyloid and tau electron microscopic studies revealed that nfts were composed of paired helical filaments (phfs) that disrupted the architecture of the cyto-skeleton. sps were forged from a core of compact fibrils consistent with amyloids surrounded by dystrophic neurites filled with altered mitochondria, vesicles, numerous pleomorphic residual bodies, and phfs (56-61) (figure 3). figure 3: electron microscopy of an sp showing the central core of amyloid fibrils (asterisk) and peripheral dystrophic neurites (black arrows) filled with vesicles, dense bodies, abnormal mitochondria, and paired helical filaments; bar = 5μm. 2a. β-amyloid (aβ) subsequently, molecular studies identified β-amyloid as the main component of cerebral amyloid in β-amyloid angiopathy and sps (62-66). the amyloid precursor protein (app) is a transmembrane protein which modulates brain cell adhesion, synaptic plasticity, and multiple intracellular signaling through the small endodomain of the molecule. app processing is regulated by cytoplasmic phosphorylation (67). cleavage of app occurs through the combined action of α-, β-, and δ-secretases. β-secretase (bace) is a gpi-anchored aspartyl protease (68). γ-secretase is a coprotein complex mainly composed of presenilin 1 (psen1) and presenilin 2 (psen2); components of the γ-secretase complex aph-1 homolog a; γ-secretase subunit (aph1a); aph1b; nicastrin (nct/ncstn); and presenilin enhancer γ-secretase subunit (pen2/psenen), together with the modulators neprilysin (nep/mme) and insulin-degrading enzyme (ide). the γ-secretase complex is considered the “proteasome of the membrane” because of its capacity to act as a protelytic enzyme on more than 90 substrates (69-71). cleavage of app through αand δ-secretase leads to the non-amyloidogenic pathway of app degradation, whereas the combined action of βand δ-secretases generates small truncated c-teminal peptides at positions 42 (aβ1-42 or aβ42) or 40 (aβ1-40 or aβ40), depending on the thickness of the membrane, and many other small forms are amyloidogenic as well (72-75). local cholesterol content affects the various secretase activities (76), including cholesterol derived from astrocytes (77). low physiological concentrations of aβ seem necessary for long-term potentiation induction and for memory formation, probably acting on camp and cgmp (78). however, in aging and ad there is not only abnormal production of β-amyloid. aβ is aggregated and accumulates in the extracellular space due to its hydrofobicity, facility for oligomerization, and transformation from an α-helix to a β-sheet conformation (66, 73, 74). several enzymes can degrade β-amyloid such as neprilysin, plasmin, endothelin-converting enzymes, angiotensin-converting enzymes, insulin-degrading enzyme, several matrix proteinases, and cathepsins a and b (79, 80). soluble aβ is drained across the lymphatic wall, binding to low-density lipoprotein receptor-related protein (lrp-1). the expression levels of some of these enzymes and transporters are reduced in ad (81-85). impaired lymphatic drainage and altered blood vessel walls impair the elimination of soluble β-amyloid via the circulatory system (86, 87) (see section 13). astrocytes followed by neurons are the main source of clusterin in brain; clusterin is then released to the extracellular space (88). clusterin expression is increased in ad (87, 88), and co-localizes with β-amyloid deposits (91), more specifically with aβ1-40 (92). clusterin may act as an extracellular chaperone (93) and it contributes to early stages of β-amyloid plaque pathology (94). in addition to being involved in aβ aggregation and clearance and in the modulation of aβ transport across the blood brain barrier (bbb) (95-97), clusterin is also known to reduce aβ toxicity (98, 99). in addition, clusterin seems to interact with bridging integrator protein 1 (bin1) and tau (100). the main β-amyloid that circulates in brain interstitial fluid and cerebrospinal fluid (csf) is soluble aβ40. β-amyloid deposits in ad are categorized as primitive or immature plaques, mature or neuritic plaques (classical sps), compact or burned-out, cotton-wool plaques, diffuse plaques, subpial β-amyloid deposits, β-amyloid angiopathy, and perivascular plaques (dyshoric angiopathy) (figure 4). β-amyloid can also be found in the cytoplasm of neurons, and in astrocytes at the periphery of sps. β-amyloid is composed of a mixture of peptides of different molecular weight: aβ40 and aβ42 are predominant in sps, while aβ40 is mainly located at the core of sps and aβ42 at their periphery. diffuse plaques contain aβ42 and truncated forms aβ17-42. subpial β-amyloid deposits are mainly composed of amino-terminal truncated species. β-amyloid species have different aggregation properties. n-terminal truncated aβ with pyroglutamate modification at position 3 and aβ phosphorylated at serine 8 show enhanced aggregation into oligomers and fibrils. these forms appear at late stages (biochemical stages 2 and 3) of β-amyloid formation, whereas soluble and insoluble aggregates composed of non-modified aβ are found at early stages (stage 1) (101, 102). figure 4: β-amyloid deposits in the temporal cortex. paraffin section, β-amyloid immunohistochemistry, slight hematoxylin counterstaing, bar = 50μm. soluble β-amyloid oligomers (aβos) and amyloid-β derived diffusible ligands (addls), acting through specific cell surface receptors rather than fibrils, are toxic and cause neurodegeneration (103-115). high-molecular-weight β-amyloid oligomer levels are elevated in the csf in ad (116). yet in the cerebral tissue, the ratio of aβ oligomer levels to plaque density distinguishes demented from non-demented patients (117). several membrane receptors can bind to aβ oligomers. these receptors include the cellular prion protein (prpc); the α7 nicotinic acetylcholine receptor (α7nachr); fcγ receptor ii-b (fcγriib); the p75 neurotrophin receptor (p75ntr); the paired immunoglobulin-like receptor b (pirb); the pirb human orthologue receptor (lilrb2); the β-adrenergic receptors (β-ars); and the eph receptors (118), among others. prpc is one of the binding partners for aβ oligomers (119-125), and prpc mediates impairment of synaptic plasticity by aβ oligomers (124). besides β-amyloid species, several molecules are also components of sps including metal ions, lipids, mucopolysaccharides, immunoglobulins, members of the complement system, molecules linked to lipid metabolism and lipid transport, blood coagulation/haemostasis factors, proteins linked to metabolism and molecular transport, neural, cell adhesion and extracellular matrix proteins, proteoglycans, and other cellular proteins (126, 127). the large amount of proteins in sps is likely the consequence of co-aggregation and alteration of associated biochemical processes by which β-amyloid formation leads to neurodegeneration (127). moreover, prpc co-localizes with aβ in sps (128). β-amyloid plaques are associated with variable alteration of neuronal processes, reactive astrocytes, and microglia. altered synaptic protein deposition with a granular pattern is found in diffuse plaques (129). neurotransmitter-containing and peptidergic dystrophic neurites precede those containing paired helical filaments within sps (130-132). altered neuronal structure, accumulation of abnormal molecules, and abnormal organelles and debris are characteristic of dystrophic neurites of mature sps (133, 135). in addition to synaptic proteins, components of dense-core vesicles accumulate in dystrophic neurites of sps (129, 135-138). immunohistochemical studies have shown that dystrophic neurites of sps contain 3rtau and 4rtau; several phospho-tau species; map2-p; phosphorylated neurofilaments light; medium and heavy chains; and active kinases p38, sapk/jnk, gsk3β, and ck1-δ, in addition to markers of the ubiquitin-proteasome system (ups) and autophagy (139). mitochondria are altered in dystrophic neurites of sps with variable vulnerability of the mitochondrial complexes of the respiratory chain (140, 141). dystrophic neurites are likely derived from axons arising from diverse neuronal populations, as revealed by specific neuronal markers (130-132, 142-146) therefore indicating that neuronal vulnerability is not restricted to a single cellular population. pyramidal cells in the vicinity of sps show distorted dendrites and loss of dendritic spines (147-149). 2b. tau the microtubule-associated protein tau, encoded by mapt, participates in microtubule stability, cellular polarity, and anterograde and retrograde axonal transport of organelles and vesicles. in addition to microtubules and actin, tau interacts with a large number of proteins and lipids in the cytoplasm, cell membranes, and synapses, and with dna and proteins involved in dna protection, among many other substrates (150, 151). the various functions of tau require interaction with multiple partners (151-157). figure 5: neurofibrillary tangles in the ca1 region of the hippocampus. paraffin section, at8 immunohistochemistry, slight haematoxylin counterstaining, bar = 25μm. the main constituent of nfts dystrophic neurites of sps and neuropil threads is abnormal tau (158-173) (figures 5, 6, and 7). a combination of all six hyper-phosphorylated brain tau isoforms (3rtau and 4rtau expressed in brain), generated from alternative tau splicing, is characteristic of ad tau (163, 174, 175). the amount of 3rtau is similar to 4rtau in the human adult brain and in ad. however, possible variations in the ratio of 3rtau/4rtau among cell types in the human brain have not been adequately assessed. abnormal tau in ad includes several species resulting from hyper-phosphorylation at different sites, acetylation, glycosylation, altered confor mation, truncation at glutamic acid 391 and at aspartic acid 421 (mediated by caspase 3), oligomerization, and β-sheet-rich fibril aggregation, among others (171-173, 176-196). the site of tau phosphorylation and other post-translational modifications in tau have commonalities and differences among tauopathies (197, 198). tau inclusions in glial cells are not found in ad, unless accompanied by other tau co-morbidities including aging-related tau astrogliopathy (artag) and argyrophilic grain disease (agd) which are 4rtau-only tauopathies. figure 6: dystrophic neurites of sps in the entorhinal cortex containing hyper-phosphorylated tau. paraffin section, at8 immunohistochemistry, slight haematoxylin counterstaing, bar = 25μm figure 7: immunoelectron microscopy showing phospho-tau deposits (black dots) in paired helical filaments. at8 antibody, bar = 0.2μm. tau hyper-phosphorylation, the first step in nft formation, is geared by the activation of specific kinases, and probably also by accompanying inhibition of phosphatases (171, 199). several kinases are implicated in both the physiological and the pathological phosphorylation of tau, including glycogen synthase kinase 3β (gsk3β); cyclin-dependent kinase 5 (cdk5); protein kinase a (pka); jun n-terminal kinase (jnk); p38; and others (200). co-localization of selected active kinases and tau deposits can be visualized in brain tissue (201-203). g-protein-coupled receptor (gpcr) kinases are also associated with nfts and β-amyloid plaques in ad (204). these alterations are cumulative but not homogeneous. more than one tau species may be present in a particular neuron. furthermore, distinct defects may result depending on the type of accumulated tau, ranging from reversible dysfunction to irreversible disruption of the cytoskeleton, altered axonal transport, undermined cell signaling, synaptic dysfunction, and cell death. tau-linked alterations can be the direct result of toxic species or the interactions of multiple partners (156). soluble and insoluble tau oligomers, both phosphorylated and non-phosphorylated, may be involved in neurodegeneration (191, 205). the association of tau with the plasma membrane is determined by its phosphorylation pattern. tau associated with the plasma membrane can move to the cytosol upon tau hyper-phosphorlation (206, 207). the phosphorylation of tau also depends on phosphatidyl choline and phosphatidyl serine (208). therefore, the composition of lipids at the membrane may modify the phosphorylation of tau and its capacity to shift its binding with actin and cytosolic proteins (209-211). tau interactions with the membrane have several implications (155, 212). in addition to stabilizing membrane-cytosol interactions, tau is secreted associated to vesicles, or vesicle-free, key features in tau transmission (213-215). morphologically, abnormal neuronal tau deposits in ad are manifested as perinuclear tau deposits, granular cytoplasmic deposits, diffuse cytoplasmic deposits (all considered pre-tangle stages), neurofibrillary tangles (classical nfts), ghost tangles (remains of nfts in the neuropil), dystrophic neurites of sps, and neuropil threads. the redistribution of abnormal tau from axons to the somatodendritic compartment of neurons and dendritic spines is a characteristic consequence of tau pathology in ad and other tauopathies. phfs induce tau accumulation into aggresomes that gather misfolded proteins when the protein degradation system is overloaded (216). the structure of tau filaments in the different tauopathies largely depends on tau composition (3rtau and 4rtau) and on post-translational modifications including conformation and truncation, as revealed by transmission electron microscopy, and more recently by optimized cryo-electronmicroscopy and mass spectrometry (193, 217-222). the different structure of tau aggregates in tauopathies indicates the formation of different tau strains which are specific to each tauopathy (223). the accumulation rate of tau aggregates is greater in females and younger β-amyloid-positive subjects (224). increased expression of 19 genes in chromosome x is associated with tau burden and slower cognitive decline in women but not in men, suggesting that specific x chromosome factors could confer risk or resilience in aging and ad (225). in addition to abnormal tau, nfts contain numerous proteins. total tau interacts with a good number of proteins in ad (226, 227). laser-capture micro-dissection of nfts and liquid chromatography-/tandem mass spectrometry (lc-ms/ms) analysis in sad followed by affinity purification mass spectrometry revealed that seventy-five proteins present in nfts interacted with phf1-immunoreactive phosphorylated tau (228). nfts also contain markers of the sequestosome/p62, ubiquitin, and mutant ubiquitin (229, 230). increased prpc expression downregulates tau protein (231-234). conversely, reduction or ablation of prpc levels induces an increase in tau 3rtau/4rtau balance through downregulation of gsk3β activity, thus indicating that prpc plays a role in tau exon 10 inclusion through the inhibitory capacity of gsk3β (235). increased prpc levels at early and middle stages of nft pathology yields lower tau and hosphor-tau. in contrast, prpc levels decrease at advanced stages of nft pathology, which correlates with increased amounts of tau and hosphor-tau. taken together, these observations suggest a protective role for prpc in early stages of ad (236). these observations linking an interaction of prion protein and tau may have implications in certain familial prion diseases grouped under the term gerstmann-sträussler-scheinker disease, in which abundant prpres-amyloid deposits are accompanied by extensive tau pathology (see section 3). 3. familial ad (fad; early-onset familial alzheimer’s disease: eofad), and the β-amyloid cascade hypothesis from the early nineties, mutations in the genes app (β-amyloid precursor protein), psen1 (presenilin1), and psen2 (presenilin2), all of them involved in the production of β-amyloid, have been identified in several families with pre-senile dementia of alzheimer’s type (early-onset familial alzheimer disease: eofad, or fad); increased app dosage was also causative of ad and β-amyloid angiopathy (237-244). recent genetic studies of the first alzheimer’s case identified that the patient carried a mutation in psen1. these groundbreaking discoveries led to the “β-amyloid cascade hypothesis”, which supports the idea that the production of β-amyloid species is the primary factor triggering nft formation and ad progression (245). the amyloid cascade hypothesis was further supported by the production of β-amyloid in transgenic mice bearing human mutations causative of ad. yet mutations in these genes did not result in nft formation in transgenic mice, although a few small hyper-phosphorylated tau deposits did appear in dystrophic neurites around β-amyloid plaques. at most, the joint production of sps and tau deposits similar to nfts in mice requires the cumulative expression of different mutated genes involved in human ad and tauopathies (246-248). however, in vitro and in vivo studies have shown the capacity of β-amyloid to phosphorylate tau and enhance tau aggregation, thus giving a boost to the β-amyloid cascade hypothesis (249). the β-amyloid cascade hypothesis fits with fad linked to mutations of app, psen1, and psen2. another genetic condition linked to increased risk of ad is down syndrome. middle-aged individuals (ma) with down syndrome have neuropathological lesions of ad. amyloid deposits may start as early as 12 years of age and they are universal by the age of 31. nfts appear later in the entorhinal cortex, hippocampus, and neocortex (250-253). the discovery of β-oligomers and cumulative evidence of their toxicity has led to modification of the “β-amyloid cascade” hypothesis, leading to the “amyloid-β oligomer hypothesis” (103-108). according to the new proposal, it is not the presence of fibrillar β-amyloid and deposition into definite aggregates, but rather soluble β-oligomers that are causative of cell damage and that trigger the process of neurodegeneration in ad (103-106, 254-257). however, several points are still obscure. the level of insoluble aβ rises with age and is further increased in ad whereas the total level of aβ40 in both soluble and insoluble fractions and the level of aβ42 in the soluble fraction decline with age before about 50 years. differential production or retention of aβ40 and aβ42 likely contributes to the influence of age on the risk of sporadic ad, but the levels of soluble aβ concentrations, higher in young adults than in older individuals and in subjects with ad, do not match the proposed toxic role of oligomers in ad (258, 259). tau deposits, other than those located in dystrophic neurites of sps, are largely independent of β-amyloid. other factors, including apolipoprotein e (apoe), the endocytic system, cholesterol metabolism, and microglial activation, are regulators of tau pathology (260). neurons derived from induced pluripotent stem cell (ipsc) lines from sad and fad linked to psen1 mutations show increased phosphorylation of tau at different sites, increased levels of active gsk3β, and a significant upregulation of app synthesis and app carboxy-terminal fragment cleavage. however, significantly increased aβ1-42/aβ1-40 ratios are observed in fad but not in sad (261). other amyloids are the main constituents, in combination with nfts, of different genetic neurodegenerative diseases causing dementia. familial british dementia (fbd) and familial danish dementia (fdd) are linked to specific mutations in the bri2 gene; the cleavage of integral membrane protein 2b (bri2) produces abri and adan amyloidogenic peptides, respectively. amyloid plaques and amyloid angiopathy, and nfts with a tau composition identical to ad tau, are found in both diseases (262, 263). gerstmann-sträussler-scheinker disease (gss) is linked to mutations in the prion protein gene (prnp) that cause a prionopathy. depending on the muta-tion, gss is manifested pathologically by a combination of abundant prion-immunoreactive plaques surrounded by dystrophic neurites, together with numerous nfts indistinguishable from ad-nfts (264-266). interestingly, app, bri2, and prion are proteins located at the cell membrane, and they interact with each other in normal conditions. the non-fibrillar, soluble bri2-derived amyloids are also toxic, and probably play a central role in the pathogenesis of bri2-linked dementias (267). the common structure of soluble amyloid oligomers suggests a common mechanism of pathogenesis (109, 113). despite the differing genetic nature of these disorders, plaques and nfts do not appear until middle age. understanding of the mechanisms that control the metabolic pathways, that delay the beginning of the molecular and clinical manifestations of the disease for years, is a major challenge in neurodegenerative diseases linked to mutations in specific genes. in contrast to mutations linked to β-amyloid production, mutations in mapt are causative of familial tauopathy and are never associated with β-amyloid or other amyloid deposits (170, 171). 4. sporadic ad (sad; late-onset alzheimer disease: load) most cases of ad (more than 95%) are sporadic (sad) and occur in older individuals (late-onset alzheimer’s disease: load). sad has an insidious onset and a progressive course leading to death about 10-15 years after the first clinical symptoms of dementia. aging is the main contributory factor. sad is favoured by individual or combined low penetrating genetic factors, mainly allele ε4 of apoe (268-271). genome-wide association studies (gwas) have identified other risk genes of sad: ldl receptor related protein 1 (lrp1); low density lipoprotein protein receptor 1 (ldlr); interleukin 1a; clusterin (clu); phosphatidylinositol binding clathrin assembly protein (picalm); complement component (3b/4b) receptor 1 (cr1); bridging integrator 1 (bin1), involved in synaptic vesicles and endocytosis; triggering receptor expressed on myeloid cells 2 (trem2); sortilin-related receptor 1 (sorl1), involved in endocytosis and sorting; adam metallopeptidase domain 10 (adam10), involved in the cleavage of several proteins; atp binding cassette subfamily a member 7 (abca7); spi-1 proto-oncogene (spi1); paired immunoglobin like type 2 receptor alpha (pilra); membrane-spanning 4-domains subfamily a (msa4), linked to inflammation; cd2-associated protein (cd2ap) that regulates actin cytoskeleton; and ephrin receptor a1 (epha1), among others (272-288). the regional and areal distribution of nfts and sps in the cerebral cortex is not homogeneous. in the hippocampal complex, nfts predominate in the ca1 region and subiculum, the ca2, ca3 and hilus are less affected, and the dentate gyrus is spared in pure sad cases. in the entorhinal cortex, nfts are more abundant in layers ii and v, whereas in the neocortex, nfts predominate in layers iii and v, with marked regional variations (the primary motor and sensory cortices have fewer nfts than the association areas). nfts are found more abundant in the temporal cortex, followed by the frontal and parietal cortex, and the occipital cortex. in subcortical regions, nfts are localized in the basal nucleus of meynert and nuclei of the basal forebrain, amygdala, hypothalamic nuclei, relay neurons within intralaminar and limbic thalamic nuclei, ventral tegmental area, raphe nuclei, locus ceruleus, and olfactory bulb. the cerebellar cortex is spared of nft pathology. cortical neurons with nfts are mainly subpopulations of large pyramidal glutamatergic neurons (289-291). this is consistent with the observation that neurons with high content of neurofilaments are more susceptible to nft formation (290-295). gabaergic neurons are more resistant to nft pathology, although the density of gabaergic neurons decreases and gaba-uptake is impaired in sad (296-300) (see section 6 for details). somatostain, which is expressed in a subpopulation of inhibitory neurons, and somatostatin receptors are also reduced in sad (296, 301, 302). calcium-binding proteins parvalbumin (pv), calbindin d28k (cb) and caretinin (cr) are expressed in subpopulations of gabaergic neurons (303-305). pv-positive neuron numbers in the temporal, visual, and prefrontal cortex are preserved in ad (306-309), but pv-immunoreactive neurons are decreased in the entorhinal cortex and hippocampus in sad (310-316). cb-immunoreactive neurons in the hippocampus, entorhinal cortex, and cortical layers v and vi are vulnerable, whereas cb-positive neurons in the occipital cortex and upper layers of the frontal cortex are resistant (309, 314, 314, 317). cr-positive neurons are not affected in the prefrontal, temporal, and visual cortices (309, 319, 320), but their number is reduced in the hippocampus and entorhinal cortex (314, 321). pv and somatostin, together with neuropeptide y, cholecystokinin and substance p, are found in dystrophic neurites of sps, thus evidencing the involvement of inhibitory and peptidergic neurons in sps (130, 144). neuron loss is negligible in cognitively normal subjects, but the number of neurons decreases in the hippocampus and entorhinal cortex with nft progression (322-324). the rate of this process is extremely variable among individuals. a major achievement in improving our understanding of the progression of sad pathology was the staging of nfts and sps in the post-mortem brain of large cohorts of non-demented and demented individuals covering a natural human population. in the telencephalon, the first nfts appear in the entorhinal and transentorhinal cortex (stages i-ii), followed by the hippocampus, temporal cortex, and other nuclei of limbic system (stages iii-iv), and then continue on to most areas of the neocortex (stages v-vi). the spreading of nfts is accompanied by a dramatic increase in the number of neurons with nft pathology across stage progression (41, 325-327). about 85% of individuals aged 65 have nft pathology, at least restricted to stages i-iii (41, 325, 328, 329). all of them, excluding those having concomitant pathologies, are considered “cognitively normal for age” (330). some individuals at stage iv-v suffer from moderate cognitive impairment; only about 5% have dementia. however, dementia of ad type accounts for about 25%-30% of the population at the age of 85 years, all of them categorized as nft stages v-vi (331). regionaland stage-dependent neuropathological alterations in sad are accompanied by specified patterns of altered gene expression, that extend beyond the genes implicated in tau and β-amyloid pathology (332). the braak staging scheme does not rule out the occurrence of exceptions that do not fulfil the strict neuropathological criteria. these untypical cases are considered ad subtypes: hippocampal sparing, limbic-predominant, and minimal atrophy sad subtypes might account for about 25% of cases (333). in addition, several clinical sad variants including non-amnestic, corticobasal syndromal, primary progressive aphasia, posterior cortical atrophy, behavioral/dysexecutive, and mild dementia variants have been categorized (334). the olfactory bulb and tract, and several nuclei of the brain stem including the raphe nuclei and the locus coeruleus, are affected by nft formation at the first stages of nft pathology. the involvement of the olfactory bulb and tracts may contribute to the altered olfaction arising in sad. damage to selected nuclei of the brainstem, which are the origin of major serotoninergic and noradrenergic innervation of the entire brain, underlies a large series of clinical symptoms including impaired arousal, loss of attention and memory, impaired decision making, apathy, depression, anxiety, and altered reward processing, among others (335-340). considered together, nft generation and neuron loss largely depend on the specific cell and regional vulnerability of specific neuronal populations. moreover, the simultaneous presence of nft lesions in separate brain regions indicates that there is no single origin of nft pathology that spreads through the brain, but rather various and cumulative original sources of tau pathology in the aging brain. additionally, the rates of nft progression, although variable from one individual to another, appear slowly at early nft stages and progress rapidly at advanced stages of the disease (41). the distribution of sps differs from nfts in the cerebral cortex in patients with sad (341). assessing the same series of cases for the study of nft progression evidenced that the localization and distribution of sps largely differ from nft staging. the majority of individuals at nft stages i-ii and almost half of those at stage iii do not have sps or β-amyloid deposits (41, 342). stages 0, a, b, c of braak define the progression of sps through the neocortex. the phases of thal represent, from phase 1 to 6, the progressive and cumulative appearance of sps from the neocortex, allocortex, diencephalic nuclei, striatum, and cholinergic nuclei of the basal forebrain, the brainstem, and the cerebellum (343). the early appearance of tau pathology compared with the later appearance of β-amyloid plaques in a series of 2366 cases from children to centenarians has been recently revisited (344). based on the results of these observations and many previous studies, the paper hypothesizes that tau pathology is an initiating factor in sad (344). indeed, the lack of temporal and regional concordance between nfts and sps in sad is intuitively barely consistent with the β-amyloid cascade hypothesis, unless non-identified soluble or other species of β-amyloid interact with neurons, thus triggering nft pathology (345). these comments do not mean that there is no interaction between the two proteins. tauopathy fuelled by β-amyloid in a synergetic mechanism is well documented in ad (346-348). the arguments between supporters of β-amyloid and of tau as the primal origin of sad have consumed a great deal of effort, time, and financial investment (349). there is no doubt about the new acquisition of knowledge generated regarding sad pathogenesis, but exclusive hypotheses have not produced the anticipated unequivocal results. cognitive impairment and dementia correlate with tau deposition and nft pathology rather than with β-amyloid deposits and sps (331, 350-358). neuron loss occurs largely in parallel with tau pathology rather than with sps in most regions (359). however, neuron degeneration and neuron loss are not restricted to neurons with nfts (see section 10). recently, a classification of ad has been proposed: ad autosomal dominant (fad), apoeε4 sad, and non-apoeε4 sad (360). this categorization is not new, but rather recovers and further emphasizes the well-known relative importance of β-amyloid deposition in the different ad categories depending on genetic factors involved in the production of β-amyloid. 5. nfts and sps in non-human brain aging β-amyloid plaques and β-amyloid angiopathy may be found in old-aged animals in some species including non-human primates, monkeys, dolphins and other cetaceans, dogs, cats, bears, and pinniped species, among others; deposits are usually diffuse whereas core plaques surrounded by tau-containing dystrophic neurites are exceptional (361-372). phosphorylated-tau deposits in neurons are rarely encountered in most mammals, and they usually have the characteristics of pre-tangles rather than nfts, as in a few vulnerable aged mouse lemurs (362). intracytoplasmic tau inclusions in neurons, astrocytes, and oligodendrocytes may occur in aged baboons (373, 374), aged gorillas (369), and chimpanzees (371). tau accumulation in the brain of old sea lions, seals, and walruses forms argyrophilic fibrillar 3rtau and 4rtau aggregates in the neuronal somata and neurites, and olny few tau aggregates are found in oligodendrocytes and microglia (372). importantly, these changes are linked to aging, but they are not the only expression of brain aging (375, 376). hyperphosphorylated tau accumulation in neurons, intraneuronal β-amyloid deposits, and diffuse amyloid plaques may occur in the brain of aged domestic cats (377, 378). the characteristics and distribution of tau lesions in a few cats are reminiscent of sad including the deposition of 4rtau and 3rtau (379). a unique 4rtauopathy without β-amyloid deposits mainly involving neurons of the neocortex but not the hippocampus, accompanied by widespread coiled bodies in the cerebral white matter, has been reported in aged domestic cats (380). these observations show that β-amyloid deposition and tau pathology may occur with high species variability, in aged mammals, and, particularly, in non-human primates and pinnipeds. however, we do not have evidence at present on whether these species show changes in the same way as human beings. in aged cynomolgus monkeys, β-amyloid plaques combine with 4rtau deposits in pre-tangle neurons and coiled bodies in glial cells with a regional pattern reminiscent of progressive supranuclear palsy (370). therefore, the old hypothesis suggesting that sad is a phylogenetic disease (381) has a relative relevance unless applied to the search for mechanisms modulating similarities and differences between non-humans and humans regarding the tremendous prevalence, widespread localization, particular regional distribution, and composition and structure of tau deposits in humans in comparison with other species. 6. synapses synaptic alterations were described in the 1960s in the seminal electron microscopic studies of ad (382). these findings were followed by the observation of decreased numbers of dendritic spines on cortical neurons assessed with the golgi method in post-mortem and biopsy samples at a time when cerebral biopsies were still considered appropriate tools for diagnosis of dementia (383-389). synaptic loss is the major morphological correlate of cognitive impairment (390). for this reason, ad is considered as the consequence of a synaptic failure (391). subsequent studies have refined synaptic alterations using different methods (392; 393), including the use of intraneuronal dyes in post-mortem tissues (394, 395). the golgi method also provided evidence of dendritic degeneration and dendritic sprouting and re-growth in several brain regions in ad (396-401). dendritic sprouting is reinforced by the presence of growth-associated protein 43 (gap-43), a marker of neuritic growth and sprouting around sps (143, 402). aberrant sprouting seems to be triggered by pre-amyloid species and neurotrophic factors (401). aberrant sprouting involves neurites, dendrites, and synapses, and it affects distinct connections in ad (401). cycles of aberrant synaptic sprouting and neurodegeneration are common in ad (403). spine loss occurs mainly in clusters linked to tau pathology (404, 405). immunohistochemistry also reveals altered expression of synaptic markers not only around sps but also in diffuse plaques, suggesting a close relationship between synapses and β-amyloid deposition (129, 406, 407). abnormal preand post-synaptic tau and tau oligomers damage the synapses and produce altered synaptic function (150, 408, 409). double-labeling of neurons also shows a direct relationship between tau deposition and loss of dendritic spines on cortical pyramidal neurons in ad (404). abnormal tau and β-amyloid oligomers act synergistically to disrupt synaptic function (409). however, synaptic loss also appears not to be dependent on fibrillar β-amyloid in a murine model of β-amyloid deposition (149). abnormal neuronal expression of app and cytoskeletal proteins in early stages of the disease might be involved in the mechanisms of synaptic pathology in ad (410). synaptic proteins are important components associated with β-amyloid in sps (127). it has recently been postulated that altered synapses are the origin of amyloid plaques in ad (411; see also section 2a). both β-amyloid and abnormal tau are accumulated at the synapses (412-421). recent neuroimaging studies further support the association of tau pathology, synaptic loss, and altered synaptic function (422). it has been proposed that synaptic tau pathology is an early event, and synaptic tau seeding precedes tau pathology in sad (423). other factors are also important such as cytoskeletal actin dysregulation (424), and oxidative stress lipid and protein damage (425). no less significant is the association between cell-cycle dysfunction and failure of synaptic plasticity in ad (426). synaptic alterations include abnormalities in the synaptic and postsynaptic delivery of neurotransmitters and neuromodulators, and the selective vulnerability and responses of their receptors (see section 7). finally, lipid and protein alterations at the cell membrane, and altered cytoskeletal proteins, may affect synaptic integrity and function (see sections 22c and 22h). in addition, synapses are organelles with high energy consumption, and therefore they are vulnerable to deficits in energy production linked to mitochondrial failure (427) (see section 22d). 7. neurotransmitters, neuromodulators, and related receptors an early relevant biochemical observation was the discovery of the involvement of the meynert nucleus in ad, the correlation of this involvement with the number of plaques and cognitive impairment, and the accompanying impairment of cholinergic innervation in the cerebral cortex (428-430). the “cholinergic hypothesis” stated that ad was a disorder of cholinergic innervations (430, 431). the enthusiasm for the cholinergic theory was supported by the prior discovery of dopamine deficiency in the substantia nigra pars compacta in parkinson’s disease, and the success of l-dopa treatment for this disorder which is still in use 50 years later (432). cholinergic drugs were used although their benefits were clearly lower than initially expected. later, the glutamatergic theory stated that excitotoxicity resulting from excessive synaptic or extrasynaptic activation of n-methyl-d-aspartate (nmda) subtype of ionotropic l-glutamate receptors might enhance vulnerability of neurons in ad (433, 434). the role of glutamate in the pathogenesis of ad was driven, in part, by the discovery of altered glutamate transport and increased excitotoxicity in amyotrophic lateral sclerosis (435), and the interest at that time in excitatory amino acid neurotoxicity in the pathogenesis of neurodegenerative diseases (436). glutamate overload increases mitochondrial ca2+ influx and oxidative stress and leads to mitochondrial dysfunction (437). however, nmda receptor blockers may also have undesirable effects due to their double effects on cell death as well as cell survival and plasticity (438). now, neuroprotective therapies aim to both enhance the effect of synaptic activity and disrupt extrasynaptic nmdar-dependent death signaling (438). cholinergic and glutamatergic neurotransmitter alterations play a significant role in the pathogenesis of brain aging and sad (439-442). other neurotransmitters and receptors are involved as well. the important point is that alterations are not homogeneous; they depend on the type of neurotransmitter, the cells of origin, and the kind of receptor; not all receptors of a given neurotransmitter are equally vulnerable to aging and sad. in addition to the neurotransmitters, neuromodulators, and receptors discussed below, general aspects of gpcr, amylin receptors, netrin receptors, and dopamine receptors in sad are detailed in other reviews (443, 444). endorphins, enkephalins, dynorphins, and endomorphins are endogenous opioid peptides that bind to opioid receptors. β-endorphin has opioid activity through μ-receptors, but α-endorphin and γ-endorphin lack affinity for opiate receptors. endorphins interact with the γaminobutyric acid (gaba), which in turn modulates the release of dopamine. the expression of endogenous opioids and receptors is altered in human brain aging and sad (445-448). many olfactory and taste receptors and molecules involved downstream are expressed in the human brain (449). their ligands and functions remain unknown, although both olfactory and taste receptors might contribute to intercellular and intracellular cell signaling. the expression of some olfactory receptors is altered in sad and other neurodegenerative diseases (450). 7a. acetylcholine (ach) and acetylcholine receptors (achr) ach is synthesized in neurons by choline acetyl transferase (chat). ach acts upon nicotinic acetylcholine receptors (nachrs) and muscarinic acetylcholine receptors (machrs). ach is degraded by acetylcholinesterase (ache). nicotinic receptors are ionotropic ligand-gated receptors, and muscarinic receptors are gpcr (451). the nachrs are arranged into homomeric or heteromeric subunits consisting of a diverse set of complex subtypes including α1-7, α9-10, β14, γ, δ, and ε. allosteric modulation of nachrs increases pre-synaptic ach levels and enhances the cholinergic nicotinic neurotransmission. α7 and α4β2 nachr mediate the presynaptic release of ach. nachrs are also expressed in astrocytes and microglia (452, 453). through its receptors trka and p75ntr, the nerve growth factor (ngf) plays an essential role in the survival and maintenance of cholinergic neurons in the basal forebrain (454-457). loss of basal forebrain cholinergic neurons occurs at early stages of nft pathology (428, 458, 459). pretangle pathology within cholinergic nucleus basalis neurons coincides with local neurotrophic and neurotransmitter receptor gene dysregulation (460). loss of cholinergic neurons in the basal forebrain leads to reduced ach levels and chat upregulation (461). in addition, nachrs, particularly α7nachrs, are altered in sad (462-465). importantly, nicotine and nachrs also participate in the regulation of aβ. on the one hand, nicotine inhibits the formation of aβ1-42 fibrils and disrupts preformed aβ fibrils (466). on the other hand, aβ1-42 binds to α7nachr and inhibits the release of ach (467). finally, α7 nachrs mediate aβ-induced neurotoxicity (468, 469). and aβ-induced tau phosphorylation (470). α7nachrs also participate in microglial activation (471). astrocytic and microglial nachrs modulate aβ phagocytosis and degradation, aβ-related oxidative stress, and neurotoxicity (472). the metabotropic machrs are classified into five m1-m5 subtypes (473). m1, m3, and m5 receptors interact with the gq/11 protein, stimulate phospholipase c (plc), phosphatidylinositol trisphosphate (pi3p), and activate protein kinase c (pkc). m2 and m4 receptors interact with go/i proteins, inhibit adenylyl cyclase (ac) and protein kinase a (pka), and decrease camp levels (474-476). there are no changes in the number of machrs in sad; however, the interaction of gq/11 protein is altered in sad compared with controls (477). m1 muscarinic agonists reduce β-amyloid and tau pathology, whereas m1 muscarinic antagonists or deletion of m1 subtype augment β-amyloid and tau pathology in in vitro and in vivo murine models of ad (478-480). sps have low levels of ache, but the activity of ache increases around β-amyloid plaques (481, 482). ache inhibitors such as donepezil, galantamine, tacrine, and rivastigmine are administered at the initial stages of sad. 7b. glutamate and glutamate receptors (glurs) glutamate is released from synaptic terminals and acts on post-synaptic ionotropic glutamate receptors (iglurs). this mechanism mediates fast excitatory synaptic transmission. glutamate can also act on metabotropic glutamate receptors (mglurs). the mechanism modulates various effects by coupling to g proteins with subsequent recruitment of second messenger systems. there are three families of iglurs: nmda (n-methyl-d-aspartate receptor), ampa (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), and ka (kainate) receptors: nmdar, ampar, and kar, respectively. nmdars are composed of different subunits encoded by glun1 (nr1), glun2a (nr2a), glun2b (nr2b), glun2c (nr2c), glun2d (nr2d), glun3a (nr3a), and glun3b (nr3b). ampars are composed of combinations of glua1 (glur1), glua2 (glur2), glua3 (glur3) and glua4 (glur4); and kars by gluk1 (glur5), gluk2 (glur6), gluk3 (glur7), gluk4 (ka-1) and gluk5 (ka-2). in nmdars, the binding of glutamate and glycine is necessary to activate glutamate-gated ion channels. the removal of magnesium ions (mg2+) block permits the entry of calcium ions (ca2+) and synaptic signaling (483, 484). both iglurs and mglurs are also localized pre-synaptically acting as auto-receptors and hetero-receptors. this localization facilitates neurotransmission in the short term and depresses neurotransmission in the long term (485). glurs are localized non-synaptically and are also expressed by astrocytes and oligodendrocytes (486). glutamate binding to nmdars, ampars, and kars is reduced in the aging brain and sad mainly in the cerebral cortex and hippocampus. these changes are receptor-, region-, and layer-dependent, thus indicating variable vulnerability. altered expression is likely dependent on several factors and not necessarily correlated with local nfts and sps, although loss of neurons and reduced neuronal connectivity may account, in part, for the decreased receptor expression (440). the localization of the receptors also plays a cardinal role. nmdars containing glun2a subunits are located at synaptic sites and are implicated in the protective pathways. in contrast, glun2b subunits are located mainly at extra-synaptic sites, and they increase neuronal vulnerability. β-amyloid activates glun2b-containing nmdars (487-489). in addition, nmdars are necessary for synaptic targeting of aβ oligomers (490) and neuronal aβ production (491). nmdar alterations are implicated in synaptic dysfunction in sad (492, 493). nmdars also participate in redox-mediated synaptic function impairment in brain aging and sad (494). memantine, an nmdar antagonist, is currently used at the middle clinical stages of ad to reduce the hyperactivity of glutamate, resulting in transient and limited success. ampars are consistently endocytosed. the increase in the rate of ampa endocytosis induces long-term depression and synaptic degeneration (495). soluble aβ oligomers are involved in synaptic damage via the subunit glua3 ampar (496). aβ also induces ampar ubiquitination and degradation (497). glutamate abnormalities in aging and sad are not restricted to alterations in glutamate production and its effects on synaptic receptors in neurons and glial cells. furthermore, glutamate effects in normal and pathological conditions also depend on glutamate transport by specific neuronal and glial transporters (498). excitatory amino acid transporters (eaats) re-uptake glutamate from the synaptic cleft and extra-synaptic sites, and transfer glutamate to glial cells and neurons. vesicular glutamate transporters (vgluts) move glutamate from the cell cytoplasm into synaptic vesicles. eaats can also transport l-aspartate and d-aspartate. eaat1 and eaat2 (slc1a3 and slc1a2, respectively) are localized in astrocytes whereas eeat3 (slc1a1), eaat4 (slc1a6) and vgluts 1/2/3 (slc17a7, slc17a6, slc17a8, respectively) are found in neurons. loss of eaat2 occurs in many neurodegenerative diseases, including sad (see section 12a). abnormal expression of vgluts and eaats may contribute to neuronal excitotoxicity and neuron demise in sad (499-501). mglurs are g-coupled proteins that act upon different effector systems, including plc and ac. mglurs are classified into three groups based on their pharmacological profiles, molecular properties, and transduction mechanisms. group i receptors (mglur1, mglur5) are coupled to plc activation through gq/11 proteins, whereas groups ii and iii are coupled to ac inhibition through gi/o proteins. mglur1/5 are primarily excitatory, and mglur2/3 and mglur4/5/6,7/8 are inhibitory (502-505). mglurs, determined by radioligand binding assays, and expression levels of mglur1, detected by western blotting, are significantly decreased in the frontal cortex in sad. this decrease is already observed at nft stages i-ii and iii-iv not involving the frontal cortex, and further decrease with the appearance of nfts in the frontal cortex with disease progression. the expression levels of phospholipase cβ1 (plcβ1) isoform, which is the effector of group i mglurs, is decreased in parallel. plcβ1 decrease, in turn, is associated with reduced gtpand l-glutamate-stimulated plc activity in sad. these results show that group i mglurs/plc signaling is downregulated and desensitized in the frontal cortex at the first stages of nft pathology, and that these modifications worsen with the progression of sad (506). 7c. γ-aminobutyric acid (gaba) and gaba receptors the inhibitory neurotransmitter gaba is generated by α-decarboxylation from l-glutamate in neurons by the action of glutamic acid decarboxylase (gad). gaba is then incorporated into the synaptic vesicles by vesicular gaba transporter (vgat). after release from synaptic vesicles, gaba binds to ionotropic gabaa and metabotropic gabab receptors. gabaa receptor activation opens chloride ion channels; gabab acts through g proteins, reduces calcium ion channels, and inhibits ac and intracellular production of camp (507-510). gabaa receptors are composed of combinations of five different subunits, α1-α6, β1-β3, γ1-γ3, δ, ε, θ, π, and ρ1-ρ2. the most frequent pentamers are 2α:2β:1γ (507). gabab receptors are heterodimers composed of r1 and r2 subunits (508). gaba at the synapses is picked up by astrocytes that catalyze it to glutamine, which is then transported into neurons and converted to glutamate (511, 512). the involvement of astrocytes in gaba metabolism has suggested a potential role of astrocytes in gaba gliotransmission (513). several studies have shown inconsistent results regarding total gaba levels in sad but there is a trend toward stressing abnormal gabaergic function (514-516). reduced gaba, glutamate, and glutamine levels are observed in individuals with mci and ad as revealed by magnetic resonance spectroscopy (517-520). regarding gaba receptors, electrophysiological studies reveal a reduction in gaba currents in the temporal cortex in sad. this is associated with mrna downregulation of α1 and γ2 subunits and upregulation of α2, β1, and γ1 transcripts (521). α1 and α5 subunit protein-immunoreactive levels are decreased in the ca1 region of the hippocampus (521-524), whereas α3, β1, β2, β3, and γ2 subunits are unaffected. in contrast, α1 subunits are increased in the ca3 region, granule cell layer, and hilus of the dentate gyrus in sad (523-527). β3 subunit expression is decreased in the stratum oriens, radiatum of ca2 and ca3, and stratum moleculare (527), whereas γ1/3 subunits are upregulated in the hippocampus in sad (525, 527). aβ induces the downregulation of gabaa receptors, inhibitory dysfunction, and sprouting of gabaergic axons (528-532). 7d. serotonin and 5-hydroxytryptamine (5-ht) receptors 5-hydroxytryptamine (5-ht) derives from the amino acid tryptophan via the intermediate 5-hydroxytryptophan and decarboxylation to form serotonin. in the brain, 5-ht is mainly produced in the raphe nuclei of the brain stem that constitutes part of the reticular formation. ascending serotoninergic fibres innervate the whole telencephalon. descending projections innervate the cerebellum and the spinal cord (443). serotonin is stored at the synaptic vesicles and released into the synapse, where it binds to post-synaptic and auto-pre-synaptic receptors. serotonin is then re-uptaken via serotonin transporters and reused or degraded by monoamine oxidase. 5-ht receptors are categorized into metabotropic and ionotropic receptors. metabotropic gpcr are 5-ht1, 5-ht2, 5-ht4, 5-ht5, 5-ht6, and 5-ht7. the only ionotropic receptor is 5-ht3, permeable to sodium, potassium, and calcium ions. 5-ht1 and 5-ht5 receptors bind to gαi/o proteins, inhibit ac, and decrease camp levels. 5-ht2 receptors bind with gαq/11, activate plc, generate pi3p, and activate pkc (533-541). 5-ht and receptors interact with the cholinergic, glutamatergic, noradrenergic, gabaergic, endocannabinoid, and glial cell systems (542-545). orexins regulate serotonin neurons in the raphe nucleus (546). the serotoninergic system is altered in aging and sad (443, 547-549). the main alteration of the serotoninergic system in brain aging and sad results from neuronal damage and nft formation in the raphe nuclei in the independent origin of nft pathology at early stages of ad-related pathology. damage to the serotoninergic system contributes to mood changes and depression which are characteristic non-cognitive clinical manifestations of brain aging and sad (443, 550-553). serotonin is linked to decreased β-amyloid production and modulation of soluble β-amyloid precursor protein (sappβ) (554-557). in addition, 5-ht4 receptors inhibit the secretion of β-amyloid peptides (558-560). due to the multiple facets of serotonin, serotonin receptors, and their interaction with other neurotransmitters, agonists, antagonists of the different 5-ht receptors, and principally selective serotonin re-uptake inhibitors are useful pharmacological agents to improve cognition and reduce depression in aging and sad (444, 552, 561-568). 7e. noradrenergic system norepinephrine or noradrenaline is synthesized from dopamine by the enzyme dopamine β-hydroxylase (dbh). norepinephrine is metabolized by mono-amino oxidase (mao) and catechol-o-methyltransferase (comt). norepinephrine is transported from the cytosol to the synaptic vesicles by the vesicular monoamino transporter (vmat) (569, 570). norepinephrine can bind both to metabotropic preand post-synaptic α1, α2, β1, β2, and β3 receptors. α1 are gq-coupled and activate plc; α2 are coupled to gi/g0 proteins and inhibit ac; β1, β2, and β3 are coupled to gs proteins and activate ac (571-574). the locus coeruleus, which contains about 15,000 neurons in primates, is the principal source of brain noradrenaline. noradrenergic terminals innervate the hippocampus, amygdala, cerebral neocortex, and hypothalamus (575-577). post-synaptic α1 receptors are excitatory, whereas perisomatic and pre-synaptic α2 receptors are inhibitory (578). adrenergic receptors are widely distributed in the brain (579, 580). neurons of the locus coeruleus excite the cerebral cortex principally through α1 receptor signaling (581, 582). the locus coeruleus-noradrenergic system has a major role in arousal, attention, and stress responses. in the brain, norepinephrine may also contribute to long-term synaptic plasticity, pain modulation, motor control, and energy homeostasis (583). noradrenergic terminals are also in contact with glial cells and blood vessels (584-586). due to these connections, noradrenergic innervations also modulate inflammation and cerebral blood flow (583, 587-589). in addition, the noradrenergic system interacts with the cholinergic and gabaergic systems (576, 590-592). moreover, the locus coeruleus and the raphe nuclei are interconnected (593, 594). finally, orexin/hypocretin, histamine and noradrenaline converge in the dorsal raphe nucleus (593). the locus coeruleus is damaged at early stages of ad-related pathology (443, 595-598) (see also section 4). at these first stages, nfts accompany neuron loss, but sps only appear in some cases at advanced thal phases of β-amyloid deposition. in contrast to the massive loss of noradrenergic neurons, calbindin-immunoreactive neurons are preserved in the locus coeruleus even at advanced stages of nft pathology (599). early neuronal alterations in the locus coeruleus are accompanied by abnormalities in the ascending noradrenergic system (600-602). increased α2a adrenergic receptor protein occurs in the amygdala and hippocampus in parallel with early nft pathology in the locus coeruleus (338). in contrast, reduced dbh activity is found in the post-mortem hippocampus and neocortex, probably as a compensatory mechanism to noradrenaline lessening (603, 604). dbh levels are also reduced in plasma at early stages of ad (605). moreover, connectivity between norepinephrine and dopamine brainstem centers is disrupted in sad (606). the orexin system is compromised in sad, thus contributing, in combination with the noradrenergic and serotonin decay, to altered sleep in sad (607-611). 7f. adenosine receptors adenosine is transported across the plasma membrane based on: a) its concentration gradients, and b) active na+-dependent transporters that carry adenosine against its concentration gradient. adenosine receptors are purinergic gpcr classified into a1, a2a, a2b, and a3 receptors. a1 and a3 receptors inhibit ac through gi/o proteins, while a2a and a2b receptors stimulate ac through gs proteins (612, 613). adenosine receptors are present in subpopulations of neurons, astrocytes, oligodendrocytes, and microglia (614-616). adenosine receptors modulate the release of glutamate, gaba, acetylcholine, noradrenaline, and serotonin (616-623). early autoradiographic studies showed decreased a1 expression in the hippocampus at advanced stages of sad (624-627). however, a1 receptors accumulate in neurons with nfts in sad (628). more recent studies at first stages of nft pathology have shown upregulation of adenosine receptors and sensitization of their specific signaling pathways preceding nfts and sps in the frontal cortex (629). 7g. endocannabinoids and cannabinoid receptors (cbrs) cbrs are classified as type 1 (cb1r) and type 2 (cb2r) (630). anandamide (n-arachidonoyl ethanolamine, aea) and 2-arachidonoyl glycerol (2-ag) are the main endogenous ligands of cbrs (631-634). both endocannabinoids derive from arachidonic acid (aa). they are synthesized and metabolized by different pathways and induce specific biological functions. in the human brain, cb1rs are mainly expressed in the limbic system. cb1rs localize in the pre-synapses modulating glutamate and gaba neurotransmission (635-641). cb2rs are expressed in microglia, and participate in inflammation and phagocytosis (642, 643). low levels of cb2r expression have also been identified in some neurons (644-646). cannabinoid compounds may also bind to other receptors, such as gpr55, peroxisome proliferator-activated receptors pparα and pparγ, and transient receptor potential vannilloid-1 channels (647, 648). the study of expression of cb1rs and mediators in sad has yielded variable results (649). in contrast, increased cb2r expression in microglia surrounding sps is consistently documented (650, 651). a few studies have shown altered expression levels of endocannabinoids and enzymes linked to their metabolism in sad (652-654). therefore, the endocannabinoid system plays a role in sad although its precise contribution remains largely unknown. treatment with exogenous cannabinoids and modulation of cbrs in murine models of ad has shown beneficial effects including reduction of β-amyloid plaques and β-amyloid burden, reduced tau phosphorylation, reduced inflammation, excitoxicity, mitochondrial dysfunction, and oxidative stress, and relief of cognitive impairment (649). 8. trophic factors and receptors the expression of trophic factors, particularly brain-derived neutrophic factor (bdnf) and nerve growth factor (ngf) and their receptors, is altered in sad (655-659). bdnf mrna and protein are decreased in the frontal cortex and hippocampus in ad (660-662). bdnf immunoreactivity is reduced in tangle-bearing and non-tangle-bearing neurons, whereas immunoreactivity to full-length trkb (the high affinity receptor for bdnf, neurotrophin-3 and neurotrophin-4) is reduced in tangle-bearing neurons. strong bdnf immunoreactivity is observed in dystrophic neurites surrounding sps, and strong trkb in reactive glial cells, including those surrounding sps. truncated trkb immunoreactivity occurs in individual neurons and reactive glial cells in the cerebral cortex and white matter in sad (661). the cause of abnormal trkb immunoreactivity in sad is not known but β-amyloid modulates trkb alternative transcript expression (663). probdnf is increased in sad, and it is modified by reactive oxygen species (ros)-derived advanced glycation end products, which prevent the processing of probdnf to mature bdnf (664). abnormal probdnf/bdnf signaling impairs axonal transport, decreases trophic effects, and increases pathogenicity and cell death (661, 664, 665). in contrast, the expression of ngf is increased in sad (655, 666), but ngf-containing neurons in the basal forebrain are lost in sad (454, 667). prongf is also increased in sad (668, 669). this has relevant implications as prongf induces processing and nuclear translocation of the intracellular domain of p75ntr (the low affinity neurotrophin receptor for ngf, bdnf, neurotrophin 3, and neurotrophin 4) and induces cell death in association with cofactor sortilin, a member of the vps10p sorting receptor family (670-672). 9. endoplasmic reticulum stress the accumulation of abnormally misfolded proteins in the endoplasmic reticulum (er) causes er stress which is manifested by activation of one or more of the three signaling pathways of the misfolded protein response (upr) (673). glucose-related protein 78 (grp78/bip) is the master protein that regulates the upr (674). the cytosolic domains of the transmembrane er proteins pkr-like endoplasmic reticulum kinase (perk), inositol-requiring protein 1 (ire1), and activating transcription factor (atf)-6, trigger specific pathways once activated. perk activates atf-4, and the activation of atf6 involves its displacement from the er to the golgi apparatus to be cleaved into atf6c. ire1 dimers phosphorylate and lead to the production of x-box binding protein 1 (xbp-1) which increases the er capacity of protein folding and the degradation of abnormally folded proteins in the er, thus reducing er stress. ire1 may also bind to the traf2 (tnf receptor-associated factor 2) adaptor molecule and activate the apoptosis signal-regulating kinase 1 (ask1), which in turn causes the phosphorylation of c-jun n-terminal kinase (jnk), thereby triggering cell death. atf-4, truncated atf6, and xbp-1, through downstream target genes, modulate er homeostasis or apoptosis, depending on the saturation of the system (673, 675). the expression of several upr components is altered in aging and sad (676-679). er stress also generates ros which, together with mitochondrial ros, is a major cause of oxidative stress damage. mitochondrial dysfunction and er stress are relevant promoters of apoptosis in sad (680). er stress is also linked to brain inflammation (681, 682). moreover, β-amyloid may activate the upr, either mediated by glutamate receptors and calcium uptake, or linked to mitochondrial dysfunction and ros production; er stress may also be induced by abnormal tau (683-685). moreover, perk, ire1 and atf6 signaling pathways activate autophagy (686-688). 10. failure to remove debris: the ubiquitin-proteasome system (ups) and autophagy in sad autophagy and ups are the two main mechanisms of intracellular protein degradation. there is a certain relationship between these two mechanisms, and there are some molecules in common that initiate compensatory effects to prevent disease progression (689, 690). autophagy includes macroautophagy, microautophagy, and chaperone-mediated autophagy (691). microautophagy and chaperone-mediated autophagy involve lysosomal membrane invagination and chaperon recognition (692). macroautophagy is mediated by autophagosome protein assembly of beclin 1-vps34 lipid kinase, atg9-wipi-1, atg12 conjugation system, microtubule-associated protein light chain 3 (lc3), and unc-51-like autophagy activating kinase 1 protein kinase. lysosomal-associated membrane protein 1 (lamp-1) is a transmembrane glycoprotein enriched in the membrane of lysosomes (693-695). material digested by autophagy is incorporated into lysosomes. autophagy is impaired in brain aging and sad (696-706). altered macroautophagy in sad is manifested in dystrophic neurites of sps, in which mitochondria, dense bodies, and vesicles are common targets, and in synapses and granulovacuolar degeneration (see section 11). autophagy is also involved in β-amyloid metabolism and clearance (707). ups activity is initiated by the conjugation of ubiquitin to the substrate following a three-step cascade to tag the protein into the proteasome. the 20s proteasome is a hetero-oligomer formed by heptameric rings organized into a structure resembling a hollow cylinder which has three main peptidase activities: chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. the 20s proteasome can associate, in the presence of atp, with two caps or 19s complexes, thereby forming the 26s proteasome complex. the 19s complex serves in the recognition of ubiquitylated proteins, protein unfolding, and translocation of the unfolded polypeptide to the inner chambers of the 20s proteasome for hydrolysis. the 20s proteasome can interact with other complexes. the pa28α/β activator (11s regulator) can also bind to 20s proteasome to form the pa28-proteasome complex. additionally, the three catalytic β subunits of the 20s proteasome in response to γ-interferon are replaced by inducible homologous proteins lmp2, lmp7, and mecl1, forming the immunoproteasome (689). the immunoproteasome has a role in peptide production for antigen presentation by the major histocompatibility complex in most settings, but it is also involved in the clearance of oxidatively damaged proteins (708). the ups is altered in brain aging and ad (709-712). impaired removal of altered proteins is manifested by the deposition of hyper-phospohorylated, abnormally conformed, and truncated ubiquitinated tau species resistant to ups degradation in nfts, dystrophic neurites, and threads (197). in addition to ubiquitin, mutant ubiquitin is expressed in the aging brain and at early stages of sad, whereas misframed ubiquitin contributes to the blockade of the proteasome by nfts and other tau inclusions (229, 230, 713-716). yet, mutant ubiquitin reduces aβ plaque formation (717, 718). the immunoproteasome is activated in ad and app/ps1 double-transgenic mice (719-721). the reasons for immunoproteasome activation are not fully understood, but the presence of advanced glycation end products (ages) appears to induce activation of the immunoproteasome; the inhibition of age receptor (rage), and the downstream signalling jak2 (janus kinase 2)/stat1 (signal transducer and activator of transcription 1) abolishes age-induced activation of the immunoproteasome (722). 11. granulovacuolar degeneration (gvd) gvd is characterized by the presence of vacuolar cytoplasmic lesions with a dense central core, mainly in ca1 hippocampal neurons (18). gvd is common in the hippocampus in the aging human brain and present in the majority, if not all cases, of sad (figure 8). gvd first appears in neurons of the hippocampal subfields ca1 and ca2, and the subiculum; this is followed by the entorhinal cortex, and ca4 neurons in stage 2, temporal neocortex in stage 3, amygdala and/or the hypothalamus in stage 4, and cingulate, frontal, and parietal cortices in stage 5 (723). gvd appears in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, particularly ad (724). the most widely accepted model of agd generation follows the scheme: abnormal tau in pretangles induces abnormal reticulum stress responses, which in turn trigger endocytic and autophagic pathways in the face of impaired proteolysis and altered function of the ups. this scenario is consistent with the idea that gvd is the final stage of an active process linked to failed degradation of abnormal protein aggregates in the cytoplasm of a subpopulation of neurons (677, 725-727). immunohistochemistry to casein kinase 1δ (ck1-δ) and at8 and laser microdissection have identified the proteomes of neurons containing gvd and nfts, respectively, using label-free lc-ms/ms (728). a significant change in the abundance of 115 proteins in gvd-containing neurons and 197 in nft-containing neurons was observed compared to control neurons (728). differences in protein composition between nfts and neurons with gvd are further supported by the demonstration of different activation of kinases and different profiles of phosphorylated proteins (139). nonetheless, the same study of phosphoprotein expression showed that abnormal phosphorylation of various substrates is common at the first stages of nft and gvd generation (139). these observations suggest that gvd is not restricted to tau pathology but rather involves a varied number of proteins. since gvd is common in the older human population, it is not surprising to find a correlation between gvd pathology and cognitive impairment. however, when analysis is controlled for other associated neuropathologies, the associations between gvd and dementia lose significance (729). figure 8: granulovacuolar degeneration (black arrows) in neurons of the hippocampus. paraffin section, p38-p immunohistochemistry, slight haematoxylin counterstaining, bar = 25 μm. 12. glial alterations in aging and sad glial cells are altered in aging and sad. changes in glial cells have multiple facets, including cell senescence, astrocytic gliosis, microgliosis, activated inflammatory responses, and calcium homeostasis, among others. astrocytes are key players in ad modulating β-amyloid turnover, calcium homeosthasis, tripartite synaptic function, neuroinflammation, oxidative stress responses, and bbb dysfunction (730-734). microglia and astrocytes, together with neurons and blood vessels, participate in the process of activation of inflammatory responses in aging and sad (735-737). moreover, microglia have the capacity to transform a subset of reactive astrocytes through the combination of il-1α, tnf, and c1q (738). both microglia and astrocytes are key participants in neuroinflammation in ad (739). 12a. astrocytes astrocytes are key elements in the maintenance of the central nervous system (cns) due to their role in brain homeostasis at all levels of organization from molecular to the whole organ (740). astrocytes express neurotransmitter receptors, pumps, and transporters at their plasmalemma, along with transporters in the endoplasmic reticulum and mitochondria that regulate the cytosolic levels of ions which underlie most, if not all, astroglial homeostatic functions (741). with aging, astrocytes show accumulation of lipofuscin, hypertrophy of cytoplasmic filaments, increased expression of glial fibrillary acidic protein (gfap), s100β and vimentin, and modifications in morphology and number (742-744). senescent astrocytes also exhibit senescence-associated secretory phenotype manifested by increased production of pro-inflammatory cytokines together with oxidative damage and increased superoxide production (745). perivascular astrocyte senescence leads to altered bbb (746,747). this is accompanied by reduced expression of efflux transporter and increased expression of influx transporter receptors for ages. abnormal transport of proteins through blood vessels affects the transfer of β-amyloid, leading to its accumulation in blood vessels (748, 749). water aquaporin 1 (aqp1) expression in astrocytes is also altered in the frontal cortex at nft stages i-ii, suggesting early impairment of water transport linked to ad-related pathology (750). aqp4 expression is altered with aging and sad; loss of aqp4 is associated with increased levels of β-amyloid and tau, suggesting that loss of aqp4 impairs the bbb and the gliolymphatic barrier (751). astrogliosis and astrocyte atrophy are relatively early events in ad (752-756). reduced branching and reduced connexin 43 expression occur in sad (757) which may compromise the extent of coverage domain and synaptic function in neighboring neurons (758). β-amyloid peptides also induce mitochondrial dysfunction and oxidative stress in astrocytes (759). transcriptomics of laser-captured microdissection using gfap as a marker revealed marked dysregulation of insulin, phosphatidylinositol 3-kinase (pi3k)/akt, and mitogen-activated protein kinase (mapk) signaling pathways at advanced braak stages of the disease; minor and different abnormalities were observed at earlier stages, indicating different responses of astrocytes along disease progression (760). recent transcriptomic studies in sad have shown upregulation of genes related to perisynaptic astrocytic processes and downregulation of genes encoding endolysosomal and mitochondrial proteins; downregulation of astrocytic mitochondrial genes inversely correlates with the disease stages defined by braak and cerad scoring (761). reactive astrocytes are found mainly around aβ deposits in sps and blood vessels (752, 762, 763) and in areas without plaques then distributed in a layered pattern (762, 764). in response to vascular β-amyloid deposition, astrocytes produce cytokines, metabolizing enzymes, and ros which in turn contribute to altered bbb and perivascular astrocytic function (765-769). reactive astrocytes may contain aβ (770-772) and n-terminal truncated β-amyloid (773), and they have the capacity to internalize and degrade β-amyloid fibrils (774). activation of metalloproteinases (765, 775) and lysosomal degradation (776, 777) are the main complementary mechanisms by which astrocytes degrade β-amyloid. in addition, bace may be expressed in astrocytes under appropriate conditions, facilitating the generation of β-amyloid in these cells (752, 778). acquisition of a pro-inflammatory profile along with activation of the β-amyloidogenic pathway further potentiates toxicity of a subpopulation of astrocytes in ad (779). curiously, β-amyloid seems to impair the phagocytosis of dystrophic synapses by astrocytes (780). astrocytes bearing β-amyloid show abnormal calcium homeostasis (758, 759, 781-783). in addition, β-amyloid-induced glutamate release by astrocytes may contribute to neuronal excitatory damage (784). de-regulation of specific metabotropic glutamate receptors in astroglia is also a putative harmful effect of β-amyloid (785). nmdars are expressed in a sub-population of astrocytes in the cortex and spinal cord. these receptors are composed of two glun1, one glun2c or d, and one glun3 subunits. this composition makes astroglial nmdars operational, modulating resting membrane potential (786). eaat2 clears excess extracellular glutamate from the synaptic cleft and extrasynaptic sites via glutamate re-uptake by glial cells and neurons to prevent neuronal hyperexcitability and excitotoxicity. oxidative damage, splice variants, and altered solubility of eaat2 may lead to functional alterations of glutamate transporters in ad (787-789). moreover, eaat2 expression is reduced with disease progression in parallel with increased gfap expression in sad (762, 790, 791). furthermore, astrocytes surrounding sps show altered immunoproteasome markers, and augmented expression of cytokines and mediators of the immune response (721, 735, 792, 793). reactive astrocytes in sad also have higher levels of complement component c3 which is required for both classical and alternative complement activation pathways (794). changes in the morphology of astrocytes are accompanied by alterations in the regulation and expression of gfap and other astrocyte markers in cases with mci and in pre-clinical ad (791, 795-797). these changes are preceded by expression of various cytokines and components of the inflammatory response (793, 798). astrocytic responses are not homogeneous but rather variable, even in the same region (799-801). as for m1 and m2 suggested phenotypes for microglia (see section 12b), a1 and a2 astrocyte phenotypes have been proposed depending on their transcriptional profiles: a1 are neurotoxic and a2 neuroprotective (738, 802). however, this categorization is difficult to apply in the context of sad (801-803). glucose hypometabolism is a characteristic metabolic feature associated with cognitive impairment in ad (804-807). furthermore, neurons utilize lactate as a source of energy, and astrocytes are the main local source of neuronal lactate (808-811). lactate levels are reduced in the brain of ad transgenic murine models (812). in the brain, the local source of ketone bodies results from fatty acid oxidation in astrocytes (813). in addition, astrocytes can produce ketones from amino acids (814). considering that about 20% of cerebral atp is generated from fatty acids (815), it may be inferred that impaired energy metabolism in astrocytes negatively impacts on neuronal energy metabolism, including maintenance of the energy requirements of the synapses (803, 816-818). 12b. microglia microglial cells are multifunctional cells that respond to different stimuli leading to either beneficial or harmful effects depending on the production of specific molecules. several studies have dealt with a proposed polarization of microglia into two types. m1 phenotype is stimulated by interferon-γ (ifn-γ) for the expression of pro-inflammatory cytokines, and m2 phenotype by il-4/il-13 for resolution of inflammation and tissue repair (819). however, the division between m1 and m2 microglial phenotypes with opposing effects in pathological conditions is probably a simplification, as complex microglial responses occur in the same setting, particularly in ad (820, 821). the term neuroinflammation, although widely used, sheds little light on the molecular consequences of microglial (and astrocytic) responses in any particular setting (822-824). microglia are modified in aging and ad (825-828). dystrophic (senescent) microglia precede activated microglia in aging and sad (826). increased numbers of activated microglial cells occur in parallel with the production of β-amyloid and tau pathology (829). microglial cells are found associated with sps in contact with dystrophic neurites and internal to reactive astrocytes (7). diffuse microgliosis involving the cerebral cortex and subcortical white matter occurs as well. microglial activation in ad is associated with upregulation of a large number of cytokines, chemokines, members of the complement system, and other mediators of the immune system (736, 830-837). importantly, the inflammatory response is not homogeneous but is largely region and stage dependent (838). inflammatory responses also occur in transgenic mouse models of ad with specific region and stage profiles. yet, the regulation of different components of brain inflammation and the immune system differ in transgenic mice when compared with sad (838, 839). this is an important point, as inflammatory responses vary in different regions with disease progression; protective and deleterious microglia-associated effects may occur simultaneously in any individual at any stage of the disease. another relevant point is the role of lipids in activated microglia; increased expression of apoe, triggering receptor expressed on myeloid cells 2 (trem2), and lipoprotein lipase (lp2), is found in activated microglia (840). a link between β-amyloid and microglia is well documented (829, 841, 842). a cell surface receptor complex for fibrillary-amyloid mediates microglial activation (843). microglia, in turn, mediates the clearance of soluble aβ through fluid phase macropinocytosis (844). microglia-mediated synapsis loss in ad is enhanced by fibrillar aβ and oligomeric aβ aggregation onto neuronal post-synaptic terminals, complement deposition, c3 receptor (cd11b/cd18) activation, microglial activation, and synapsis phagocytosis (821, 845-849). microglia activation also correlates with tau pathology and nft staging (829, 850-852). positron emission tomography (pet)-based studies have also shown a correlation between tau pathology progression and microglial activation across braak stages (853, 854). abnormal tau at the synapsis also favors synapsis pruning and elimination by micoglial phagocytosis (855). the notion that two types of microglial cells, unrelated to the proposed m1 and m2 subtypes, may play different roles in sad is supported by recent observations. using single nuclei rna sequencing (snrnaseq) of isolated microglial nuclei in sad brains, the abundance of phagocytic/activated named ad1-microglia correlates with tissue β-amyloid load and localizes with β-amyloid plaques; ad2-microglia are more abundant in association with tau pathology (856). microglial and altered expression of inflammatory markers is observed in children with down syndrome, and it is modified in parallel with the appearance of tau and β-amyloid pathology and through disease progression. microglial responses in down syndrome with ad pathology differ from those of sad (857-859). the profile of brain inflammatory responses also differs in human cases with ad-resilient pathology; levels of trophic factors are increased, whereas expression levels of chemokines are decreased (860). the inflammasome is a multi-protein complex containing a member of the nod-like family, such as pyrin-domain containing 3 (nlrp3r) that interacts with the inflammasome-adaptor protein asc. the immune response elicited by β-amyloid functions through complex crosstalk between the toll-like receptor 4 (tlr4), complement, and inflammasome signaling pathways (861). the activation of the inflammasome in microglia triggers a cascade that involves caspase 1, and maturation of several cytokines including il-1β and il-18. the inflammasome in microglia participates in the nucleation of β-amyloid plaques and enhances tau pathology (862, 863). aβ aggregates, and soluble aβ oligomers and protofibrils, activate the nlrp3 inflammasome (864-866). conversely, activation of nlrp3-asc inflammasome aggravates amyloid pathology (867). aggregated tau also activates nlrp3/asc and exacerbates tau pathology (868). loss of nlrp3 inflammasome function reduces tau hyperphosphorylation and aggregation by regulating tau kinases and phosphatases. conversely, tau activates the nlrp3 inflammasome. moreover, the intracerebral injection of fibrillar β-containing brain homogenates has been shown to induce tau pathology in an nlrp3-dependent manner (869). microglial and inflammatory responses are dependent on genetic factors in sad (280, 870-872). apoe status is a modifier of the inflammatory response (873, 874). cd33, trem2, and other genes linked with inflammation are expressed in microglia (871, 875, 876). carriers of ad-associated risk variants in trem2 show a reduction in plaque-associated microglia, and an increase in dystrophic neurites and overall pathological tau compared with ageand disease stage-matched sad patients without trem2 risk variants (877). another study shows that trem+ control cases have no pathological hallmarks of sad, whereas trem2+ sad cases show amoeboid microglia and upregulation of inflammatory markers when compared with trem2+ controls and trem2sad cases. these findings suggest that trem2 influences, but does not trigger, the microglial responses in sad (878). 12c. oligodendrocytes myelin generation and maintenance, and axonal nurture in the cns, are carried by oligodendrocytes. oligodendrocytes may contain abnormal protein deposits in various neurodegenerative diseases with abnormal protein aggregates. yet alterations in oligodendrocytes may occur without accompanying abnormal deposits. oligodendrogliopathy is used to stress the role of altered oligodendrocytes in the pathogenesis of certain neurological diseases with or without abnormal oligodendroglial deposits (879). oligodendrocytes suffer a functional decline in aging and ad as revealed by combined morphological, biochemical, and neuroradiological methods. neuroimaging and neuropathological studies have shown reduced white matter (wm) volume, wm lesions, and altered wm integrity and cortical disconnection in aging human brain (880-884). wm changes are associated with disruption of myelin and axons (885, 886). alterations in the number of oligodendrocytes and oligodendroglial precursor cells (opcs/ng2-positive cells) have been reported in aged primates and rodents (887). neuroimaging studies show reduced wm size, wm hyper-lucencies, and myelin and axon damages in patients with mci and dementia of alzheimer’s type (883, 888-896). wm atrophy, decreased myelin density, and demyelination are also observed in post-mortem neuropathological studies (881, 896, 897). breakdown of wm integrity is considered a contributor to the loss of neuronal tract connectivity in aging and sad (898, 899). recent neuropathological studies have shown preservation of mbp, plp1, cnp, mag, mal, mog, and mobp mrna expression levels in the wm of the frontal cortex at nft stages i-ii/0-a when compared with ma individuals without nft pathology, but a significant decrease at stages iii-iv/0-c. this is accompanied by reduced expression of ng2 and pdgfra (platelet-derived growth factor receptor a) mrna, reduced numbers of ng2-, olig2-, and hdac2 (histone deacetylase 2)-immunoreactive cells, and reduced glucose transporter immunoreactivity at stages iii-iv/0-c. curiously, partial recovery of some of these markers occurs at stages v-vi/b-c. these changes show that myelin loss is accompanied by reduced transcription of myelin-related proteins in the wm of the frontal cortex at middle-stages of ad (342). early myelin loss, decreased numbers of oligodendrocytes, and region-specific alterations, followed by partial reparative responses, also occur in transgenic mouse models of ad (900-903). 13. the neurovascular system in ad the first highlighting of the role of vascular pathology in ad was in 1989 (904). the “vascular hypothesis” of sad has been supported more recently (905-908). interestingly, denervation probably due to the loss of inputs from the locus coeruleus and basal forebrain was initially considered a key factor (909); altered innervation of the cerebral blood vessels linked to cholinergic deficiency is still postulated as contributing to cerebral blood flow (cbf) reduction (910, 911). there are hundreds of papers dealing with β-amyloid angiopathy and its effects on neurovascular function, as well as the role of lipid transporters and vascular receptors in the clearing of β-amyloid in the cerebral blood vessels (912-923). yet, there is also overwhelming evidence of altered neurovascular functioning in sad beyond that expected in association with β-amyloid angiopathy. neurovascular dysfunction in individuals with mci and advanced ad is manifested by reduced cbf (cerebral hypoperfusion), reduced cerebral glucose transport, impaired bbb function, altered lymphatic function, and altered structure of the capillaries (80, 910, 924-935). in addition to atherosclerosis and small blood vessel disease that are common in the elderly, primary involvement of arterioles and capillaries is common in brain aging and sad. primary blood vessel damage in sad is characterized by atrophy and irregularities of capillaries and arterioles, edema and increased numbers of pynocytotic vesicles in endothelial cells and pericytes, atrophy of smooth muscle fibers, thickening and focal disruption of the basal membrane, increase in collagen iv, heparan sulfate, proteoglycans, and laminin in the basal membrane, increased aquaporin expression in perivascular astrocytes, and gliovascular dysfunction, among other defects (910, 936-941). degeneration of endothelial cells is further supported by reduced staining of endothelial cell markers (942) accompanied by aberrant angiogenesis (943). pericytes are decreased in number and show abnormal mitochondria, pinocytotic vesicles, and disorganization and accumulation of osmiophilic material in aging with cumulative damage with sad progression (944-947). aβ oligomers constrict human capillaries in ad by signaling to pericytes (948). pericyte degeneration and impaired bbb function reduce aβ clearance and increase β-amyloid accumulation in the brain. importantly, pericyte alteration occurs at early stages of sad and probably it has a determining role in the altered capillary permeability (949-950). it is difficult to ascertain the weight of each one of the vascular pathologies in the eventual neurovascular failure in a particular individual. atherosclerosis, small blood vessel disease, β-amyloid angiopathy, brain hypoperfusion, altered bbb, and primary ad-linked non-amyloidotic angiopathy have cumulative effects (951, 952). the timing and development of neurovascular failure in sad is not a simple process. the “two-hit vascular hypothesis” suggests that early vascular damage leads to increased accumulation of aβ deposits in the brain, which in turn provokes additional vascular damage. apoe is one of the factors that modulate cerebrovascular integrity (953). impaired glucose uptake is an early event in pre-symptomatic fad (954), and bbb breakdown is an early biomarker of human cognitive dysfunction (955), suggesting that the neurovascular system is dysfunctional at early stages of ad. reduced expression of glucose transporter 1 (glut1) is manifested at early stages of sad, and it impairs blood glucose uptake and vasculo-neuronal function (956, 957). altered glucose transport accounts, in part, for the more complex brain glucose metabolism dysregulation in ad (958). more precise information has been obtained from the study of mouse models of cerebral β-amyloidosis mimicking β-amyloid deposits in ad (959). in the majority of these mice, cerebral blood vessels are altered and there is often impaired bbb at early stages of aβ plaque deposition, usually preceding or in the absence of β-amyloid angiopathy (959). these observations are in line with the role of aβ in the pathogenesis of neurovascular damage in ad (928). unfortunately, little is known about the occurrence of different soluble, insoluble, and oligomeric amyloid species in these models. the possible deleterious effects of abnormal tau on the integrity of the cerebral blood vessels have also been assessed (960). tau expression is accompanied by bbb breakdown in tetracycline-regulable tau-transgenic mice; bbb function is recovered once tau levels are normalized (961). non-structural but functional bbb dysfunction mediated by altered modulation of vasoactive factors including vasoconstrictor endothelin-1 has been suggested (962). altered cbf, impaired bbb, and structural anomalies in the blood vessels also occur in other neurodegenerative diseases such as parkinson’s and huntington’s diseases, amyotrophic lateral sclerosis, and multiple sclerosis (933, 934, 963), in which aβ and ad-tau have no chance to play a causative role. neurons and astrocytes also play a role via glutamate in the regulation of cbf. arteriole constriction depends on the metabolism of arachidonic acid (aa). upon mglur activation, increased aa in the plasma membrane is converted into 20-hydroxyeicosatetraenoic acid and induces vasoconstriction, or it is converted into prostaglandin 2 and produces vasodilatation (964). astrocytes have cardinal functions in the maintenance of bbb (see section 12a). 14. purine and pyrimidine metabolism in sad purines are heterocyclic double-ring aromatic organic molecules. primary purines adenine and guanosine, together with one-ring primary pyrimidine nucleobases cytosine, thymidine, and uracil, are the core of dna, rna, nucleosides, and nucleotides. adenosine and guanosine are purine ribonucleosides resulting from the binding of adenine or guanine to ribose, respectively. when adenine and guanine are attached to a deoxyribose ring, the resulting compounds are deoxyadenosine and deoxyguanosine, respectively. nucleotides result from the incorporation of phosphate groups in nucleosides: adenosine monophosphate (amp), adenosine diphosphate, adenosine triphosphate (atp), guanosine monophosphate (gmp), guanosine diphosphate, guanosine triphosphate, and cyclic forms camp and cgmp are primary purine-derived nucleotides. modified purine nucleobases hypoxantine and xanthine result from the replacement of the amino-group by a carbonyl-group from adenine and guanine, respectively, whereas methyl-guanine results from the incorporation of a methyl group to guanine. corresponding modified purine nucleosides are inosine, xanthosine, and methyl-guanosine, respectively. nucleotides participate in a wide variety of crucial metabolic pathways including energy metabolism and cell signaling. in addition, purine bases are incorporated to other molecules to form cofactors of several enzymatic reactions such as coenzyme a, flavin adenine dinucleotide (fad), nicotinamide adenine dinucleotide (nadþ), nicotinamide adenine dinucleotide phosphate (nadpþ), and the corresponding reduced forms fadh2, nadh, and nadph. s-adenosyl methionine is made from atp and methionine by methionine adenosyltransferase and is involved in the transfer of methyl groups to distinct substrates, including nucleic acids, proteins, lipids, and metabolites (965, 966). in addition, adenosine may act as neuromodulator on specific adenosine receptors (967) (see section 7f). adenosine receptors also modulate the bbb (968). significantly decreased levels of adenosine, guanosine, hypoxanthine, and xanthine are found in the frontal cortex at stages i-ii of nft pathology, but the parietal cortex and temporal cortex show an opposing pattern at advanced stages of sad. the activity of 5’-nucleotidase, which hydrolizes adenosine amp to generate adenosine, is reduced in the frontal cortex mainly at nft stages i-ii, and only at nft stages v-vi in the temporal cortex. adenosine deaminase activity, which synthetizes inosine from adenosine, is decreased in the frontal cortex in sad but is increased at nft stages i-ii in the temporal cortex. finally, purine nucleoside phosphorylase activity, which metabolizes guanosine to guanine, is increased only in the temporal cortex at nft stages i-ii. purine metabolism alterations are regionand stage-dependent and occur independently of nfts and β-amyloid plaques (969). in another study, 23 purine metabolism genes were analyzed with rt-pcr in the entorhinal cortex, frontal cortex area 8, and precuneus in ma individuals without nft pathology and in cases at nft stages i-ii, iii-iv, and v-vi (966). the mrna expression levels of several enzymes were dysregulated at stages iii-iv and v-vi in a region-dependent manner when compared with ma individuals. in addition, liquid chromatography mass spectrometry-based metabolomics in the entorhinal cortex identified decreased levels of xanthosine, guanine, and deoxyguanosine at stages i-ii, followed by dgmp, inosine diphosphate, and glycine at nft stages iii-iv in sad (966). some years ago, studies pointed to s-adenosylmethionine as a complementary candidate for therapeutic intervention in sad (208, 970). more recently, several studies have scrutinized the modulation of purinergic signaling to ameliorate sad (971-976). considering the large number of different enzymes altered, this represents a tremendous endeavor. 15. epigenetics in brain aging and sad epigenetic regulation plays a crucial role in the final transcription of genes (977, 978). three central mechanisms are briefly discussed: modifications of histones, dna methylation and hydroxymethylation, and non-coding rnas. 15a. histone modifications, dna methylation, and hydroxymethylation nucleosomes consist of dna wound around a protein octamer composed of two molecules of the histones h2a, h2b, h3, and h4. the n-terminal of histones (histone tail) extends out from the surface of the nucleosome. acetylations by histone acetyltransferases (hats) and histone deacetylases (hdacs), and methylation mediated by histone methyltransferases (hmts) and histone demethylases (hdms), respectively, occur at the histone tails (979, 980). histone acetylation decreases the interactions of histones with dna, relaxes heterochromatin to euchromatin, and enables gene transcription (981-984). methylation of histones can either increase or decrease gene transcription, depending on which amino acids in the histones are methylated, and on the number of methyl groups involved. methylations, which permit dna uncoiling facilitate the access of transcription factors and rna polymerase. the trimethylation of histone 3 at lysine 4 (h3k4m3) activates transcription, whereas dimethylation of histone h3 at lysine 9 (h3k9me2) is inhibitory. likewise, methylation of lysine 4 of histone 3 (h3k4me1) facilitates gene transcription (985-988). dna methylation and dna hydroxymethylation are biological processes by which methyl or hydroxymethyl groups are added to the dna. methylation and hydroxymethylation can change the activity of a dna segment without changing the sequence. the methylation of a 5′-cytosine in cytosine-guanine-rich regions (cpg islands) of dna promoters results mainly in the inability of transcription factors to bind dna and compact heterochromatin; as a result, gene transcription is silenced. four types of dna methyltransferases, dnmt1, dnmt2, dnmt3a, and dnmt3b, carry out the process of dna methylation. s-adenosyl-l-methionine (sam) is the donor of methyl groups. dna hydroxymethylation can occur as a result of oxidative stress or the action of ten-eleven-translocation-1 (tet1) proteins (989-994). sam which is generated by adding adenosine to methionine, a reaction catalyzed by s-adenosyl-methionine transferase, transfers the methyl group to dna methylation by dna methyltransferase. brain aging and sad is accompanied by epigenetic dna modifications (995-1002). dna modifications involve genes linked to β-amyloid production (1003-1006), apoe-ε4 (996), tau phosphorylation (1007-1009), ribosomes (1010), bdnf (1011), and inflammation (1012, 1013). a large number of unrelated genes has also been assessed, showing either changes or no modifications in the methylation of dna promoters (1014-1023). the expression of adenosine receptor a2a is modulated by dna methylation in the gene promoter region (1024-1026). therefore, epigenetic changes in dna in aging and sad are not widespread but selective for particular genes. in monozygotic twins discordant for ad, significantly reduced levels of dna methylation were observed in the neuronal nuclei of temporal neocortex in the ad twin (991). unfortunately, most studies are carried out on samples at advanced stages of sad which precludes learning whether epigenetic changes in dna are primary or secondary events (1027). in addition to modifications in genomic dna, increased mitochondrial 5-methylcytosine in most cpg and non-cpg sites in the d-loop region of mitochondrial dna (mtdna) has been identified in the entorhinal cortex at nft stages i-ii and iii-iv compared with control samples (1028). these are relevant data as they indicate early mtdna methylation linked to the pathogenesis of ad-related neuropathologic change (1028). 15b. non-coding rnas non-coding rnas do not encode proteins, but most modulate protein translation targeting mrnas. transfer rnas (trnas) and ribosomal rnas (rrnas); small non-coding (snrnas) such as micrornas (mirnas), small interfering rnas (sirnas), piwi-interacting rnas (pirnas), small nuclear rnas (snrnas), small nucleolar rnas (snornas), extracellular rnas (exrnas), small cajal-body specific rnas (scarnas); and long non-coding rnas (lncrnas) are the principal types. mirnas bind to complementary un-translated regions (3’-utrs) of mrnas to regulate target genes, resulting in translational repression or degradation (1029-1031). in animals, mirnas are synthesized from primary mirnas by the action of two rnase iii-type proteins: drosha, in the nucleus, and dicer in the cytoplasm. argonaute (ago) subfamily proteins bind mature mirnas to target mrnas (1032, 1033). thousands of human genes are mirna targets (1034, 1035). ago2:rna interactions using hits-clip have been used to generate a transcriptome-wide map of mirna binding sites in the human brain. about 7,000 stringent ago2 binding sites were highly enriched for conserved sequences corresponding to abundant brain mirnas. this interactome points to functional mirna: target pairs across about 3,000 genes (1036). dysregulation of mirnas is involved in the pathogenesis of sad (1037-1040). several mirnas target genes, many of them abnormally regulated in ad, are associated with β-amyloid (1041-1060), tau phosphorylation, and cytoskeleton (1061-1074). it is difficult to ascertain which mirna alterations are primarily altered or secondary to β-amyloid and tau pathology in sad (1075). several mirnas can bind to specific mrnas, modulating gene transcription differentially depending on the triggering factor (1038). mirna 163, mirna30a 5p, and mirna206 bind to bdnf mrna and modulate altered bdnf expression in sad (1040, 1055, 1076). furthermore, one mirna can bind to different mrnas resulting in various functions. for example, reduced mirna 132 is associated with downregulation of chat immunoreactivity in the nucleus basalis of meynert (1061) and to ache upregulation (1077, 1078). in addition, mirna 132 targets creb and promotes neuritogenesis and synaptic activity (1079). finally, mirna dysregulation is stageand region-dependent. for example, in the locus coeruleus, mirna-27a-3p, mirna-124-3p, and mirna-143-3p show a trend to increase at nft stages i-ii and are significantly upregulated at nft stages iii-iv when compared with ma individuals without nft pathology. in the entorhinal cortex of the same cases, only mirna-143-3p is upregulated at stages iii-iv. the expression levels of mirna-27a-3p, mirna-124-3p, and mirna-143-3p are not modified in ca1 at any stage, whereas mirna-124-3p is significantly downregulated in the dentate gyrus at nft stages i-ii (1080). the interpretation of these results is puzzling: downregulation of mirna124 results in β-amyloid accumulation (1007); mirna143-3p inhibition promotes neuronal survival in sad (1081); and mirna 27a-3p regulates the expression of intercellular junction proteins at the brain endothelium, while its downregulation increases bbb permeability (1082, 1083). 16. microorganisms and sad the possibility that microorganisms participate in the pathogenesis of ad was suggested in a seminal publication in 1907 (1084). the role of bacteria, viruses, and fungi has been proposed to the present (1085-1088). 16a. microorganisms in the brain and oral cavity more than thirty years ago, borrelia burgdorferi was reported in the brains of sad cases (1089), although other studies did not validate this finding (1090). however, spirochetes, including borrelia and treponema, were postulated as contributing factors in the pathogenesis of the disease (1091-1094). rrna sequencing has shown the presence of bacteria in alzheimer’s post-mortem brain (1095). β-amyloid exhibits potent antimicrobial activity against candida albicans and some bacteria, pointing to the possibility that secretion of aβ is triggered by microbial infection (1096). numerous periodontal bacteria have also been implicated in sad in the context of chronic periodontitis (1097-1099). the association between periodontal inflammatory disease and sad has raised much concern mainly because of the possibility of rapid therapeutic intervention (1100-1102). increased levels of anti-helicobacter pylori-specific antibodies were also reported in the csf and serum of sad patients (1099). histological and immunohistochemical techniques have detected fungi and bacteria in the brain of sad (1103-1105). pcr analysis revealed various fungal species in the brain and csf in sad (1106-1108). a combination of bacteria and fungi was observed in several cases, and several fungi were identified in different brain regions in the same individual, suggesting multi-fungal infection (1107). the corpora amylacea are particularly enriched in fungal and bacterial peptides, acting as scavengers of microbial debris (1109). the presence of fungi is not restricted to sad as they have also been identified in the cns and csf in amyotrophic lateral sclerosis (1110, 1111). herpes viruses were proposed as participants in the pathogenesis of sad about four decades ago (1112, 1113). since then, several studies have implicated herpes viruses in sad (11141116). herpes simplex virus 1 (hsv-1) has received much attention due to the presence of high expression levels in the brain and hsv-1 igg antibodies in the csf (1117-1121). interestingly, hsv-1 dna is localized in senile plaques (1122). the link between herpes zoster and sad is still controversial (1123). human cytomegalovirus (cmv) seropositivity was associated with a risk of sad (1124). human herpes virus 6 (hhv-6) has focused on several studies (1125-1128). viral associations have also been reported, including hsv-1 and cmv, hsv-1 and hhv-6, hhv-6 and ebv (epstein-barr virus), and hhv-6 and hhv-7 (1115, 1126, 1129, 1130). a relationship is established between β-amyloid and herpes virus, suggesting a mechanism against brain infection (1131). since differences in sad and controls are not always significant, it is still difficult to conclude that a direct causal relationship exists between herpes virus and sad. the hypothesis of multi-pathogen infections in the pathogenesis of sad has recently been discussed (1085). moreover, exposure to systemic infections in 5xfad transgenic mice, carrying mutated genes associated with fad, causes neurodegeneration in brain regions displaying β-amyloid pathology and high local microglia density (1132). together, the available information shows that the human brain, particularly the aged brain, accommodates viruses, bacteria, and fungi, together with the debris of these microorganisms over time. whether brain and oral microorganisms might contribute to “neuroinflammation” and facilitate the course of sad remains obscure. nonetheless, a recent review postulates a link between the β-amyloid cascade hypothesis and chronic infection in sad: β-amyloid deposition is produced in response to brain microbial inflammation (1133). 16b. gut microbiota in recent years, several studies have implicated the “gut-brain-microbiota axis” in the pathogenesis of sad; altered gut microbiota may promote neuroinflammation, aβ aggregation, and oxidative stress (1134-1140). the putative mechanisms producing effects include changes in the systemic metabolism that may influence brain functions, transfer of microbiota metabolites to the brain through the bbb, and direct impact of microbiota reaching the brain parenchyma. serotonin may play a role in the gut-microbiota axis (1141). the opioid system has been postulated to be contributory as well (1142). conversely, the infusion of tracers into the lateral ventricle of rats has permitted their visualization in the nasal cavity, nasal pharynx, soft palate, and esophagus, thus suggesting that at least rats can swallow waste from the brain (1143). 17. seeding and spreading of β-amyloid and tau 17a. seeding β-amyloid the intracerebral injection of diluted extracts from ad brains or from old ad-like transgenic mice accelerates β-amyloid deposition in transgenic mice bearing app and/or psen1 mutations (1144-1148). soluble forms of aβ are particularly effective at inducing plaque formation (1149). plaques not only develop locally but extend as well to the cerebral cortex distant from the injection site. these findings support the possibility that once the first β-amyloid deposits appear they may accelerate the accumulation of additional seeding in other parts of the brain, thereby contributing to the exponential deposition of β-amyloid (1150). neuronal activity regulates the regional vulnerability to β-amyloid deposition (1151). β-amyloid seeding and spreading may also occur following peripheral inoculation of β-amyloid seeds (1152). however, the intracellular mechanisms linked to the formation and fibrillisation of host β-amyloid and recruited β-amyloid spreading need clarification. intraventricular injection of aβ oligomers in cynomolgus macaques leads to diffusion into the brain, causing tau hyperphosphorylation, nft formation, synaptic loss, and astrocyte and microglial activation in regions of the macaque brain where aβ oligomers are abundant (1153). β-amyloid deposits were also observed in the brains of patients with iatrogenic creutzfeldt-jabob disease (icjd) secondary to cadaveric dura mater grafts, treatment with cadaveric human growth hormone obtained from hypophysis of cjd-affected donors, or contaminated neurosurgery early in life (1154). 17b. tau seeding physiological release of neuronal tau is stimulated by neuronal activity and extracellular tau in vivo (1155-1158). intercellular tau transmission may have physiological functions that permit the transfer of tau status information from one neuron to another, enabling them to modify the status of the host tau according to this information. this mechanism may lie behind the activation of post-translational modifications of tau in the host neuron following the transfer of pathological tau in models of tau seeding and spreading. tau is secreted by exosomes, and the extracellular appearance of tau does not depend on neuronal death, but rather physiological tau transfer from one neuron to another (1159). tau phosphorylation facilitates tau transmission and propagation (1160). several studies have suggested that the progression of sad and other neurodegenerative diseases with abnormal protein aggregates occurs in a similar way to prions in prion diseases (1161-1164). in favor of this hypothesis, many experimental designs in mouse and rat models have provided evidence that cells have the capacity to transfer tau from one cell to another. the over-expression of human tau p301l restricted to the entorhinal cortex shows the progression of tau without detectable transgene expression anterogradely from the entorhinal cortex to the dentate gyrus and ca1 region of the hippocampus and subiculum, and retrogradely to scattered neurons in the perirhinal and secondary somatosensory cortex (1165, 1166). using a lentiviral-mediated rat model, tau protein is axonally transferred from hippocampus neurons to neurons of distant brain regions such as the olfactory and limbic systems (1167) several mechanisms of tau release and uptake have been proposed, mainly in the context of synaptic transmission. however other processes may occur as well. tau release may occur via (snap: soluble nsf attachment protein receptor) snare-mediated exocytosis, release by secretory vesicles from lysosomes, microvesicle shedding, and direct plasma membrane crossing. tau uptake may occur via endocytosis, adsorptive endocytosis, macropinocytosis, transmembrane diffusion, and nanotunneling. binding of phosphorylated tau to ampa and nmda receptors is another putative mechanism (1168-1179). tau seeding and spreading is produced following the intracerebral inoculation of synthetic tau fibrils (1180, 1181), and the inoculation of fibrillar-enriched fractions from human and mouse brain homogenates of tauopathies, including sad, containing hyper-phosphorylated tau (1182-1186). tau seeding and spreading in neurons also occur following intracerebral inoculation of similar tau aggregates in wild type mice (wt) and in transgenic wt mice (1183, 1187-1194). propagation in these models occurs through connectivity rather than proximity (1182). in addition to neurons, deposits may also occur in astrocytes, and the morphology of glial inclusions appears to mimic the glial aggregates of the corresponding human tauopathies (1183, 1188, 1189, 1195). differences in the type of inoculum that produce different protein aggregate deposits in the host and variable involvement of astrocytes have led to the proposal that tau strains are behind the different phenotypes and progression of human tauopathies (1161, 1196-1198). more refined situations probably occur in human neurodegenerative diseases. for example, sad brain contains different tau strains with particular properties (1199); different tau strains may contribute to the clinical heterogeneity of ad (1200). moreover, tau seeding and spreading also occur in oligodendrocytes in wt mice following inoculation of sarkosyl-insoluble fractions in the hippocampus and corpus callosum of human brain homogenates from sad and cases with primary age-related tauopathy (part) (1191-1193). yet, tau deposits in oligodendrocytes never occur in humans affected by sad and part without co-morbidities (see section 21 for part details). these discrepancies between human diseases and mouse models may be explained by the differences between human and murine tau (1201, 1202). whether different types of tau occur in neurons and glial cells, or even in different neuron populations granting selective vulnerability, deserves further research. tau seeding and spreading have also been generated in rhesus monkeys after the inoculation of adeno-associated virus expressing a double-4rtau mutation in the left hemisphere (1203). tau spreading was accompanied by robust trem2+ microglial proliferation (1203), an unexpected observation not seen in other models. another curious observation that deserves validation is the triggering of tau deposits in tau transgenic mice following peripheral administration of tau aggregates (1204). neuropathological studies in humans reveal the beginning of tau seeding in the telencephalon in sad in the transentorhinal and entorhinal cortex, and from these brain regions tau spreads to others (1205, 1206). neuroimaging analysis further support tau spreading along functional connectivity networks (1207). however, combined post-mortem regional seed amplification and pet studies in living individuals suggest that from nft braak stage iii onward, local replication rather than spreading between regions is the main mechanism to explain progressive tau burden in sad (1208). together, tau seeding and spreading differ from seeding and spreading of prions. grossly, tau seeding and spreading circumvent certain regions that should be involved according to the hypotheses of either neuronal connectivity or neuronal proximity. most importantly, once up-taken by the receptive neuron, abnormal tau triggers a series of molecular processes that include activation of several tau kinases, post-translational modifications of tau—for instance, hyperphosphorylation, nitration, and altered conformation; recruitment of additional substrates in tau deposits; and modifications in the ratio of 3rtau/4rtau in the host neuron (1209), which implies the capacity of foreign abnormal tau to modulate exon 10 splicing in mapt in the host (1190, 1191, 1202, 1209). 17c. multiple seeding foci of β-amyloid and tau pathology; vulnerable and resistant populations to tau seeding in brain aging and sad another important point is the characteristics of tau spreading in the human brain that seem to skip over obligate regions on the basis of cell connectivity. the dentate gyrus does not show tau pathology in ad and other 3r+4r tauopathies without co-morbidities, but the dentate gyrus is affected in 3r-tauopathies and 4r-tauopathies. alternative pathways from the entorhinal cortex to the hippocampus proper are possible but there is no clear explanation for this particular pattern in ad. it may be suggested that certain populations are able to transfer abnormal tau from one neuron to the next in the connecting pathway without recruiting host tau and forming local aggregates within themselves. regional vulnerability plays a cardinal role in tau spreading in ad-related animal models (1210). in summary, tau seeding and spreading is not the only cause of tau progression in sad and other tauopathies. cellular vulnerability, including neuronal, astroglial, and oligodendroglial, together with regional vulnerability, already highlighted several years ago, are also key points to understanding tau progression in sad and other tauopathies. finally, several studies implicate microglia in the process of tau seeding with apparent enhancing and mitigating effects (868, 1211-1216). another study concludes that microglia have a complex role; they are capable of taking up and breaking down seed-competent tau, but do so inefficiently and could hardly play a role in the spread of tau pathology (1217). the capacity for seeding of β-amyloid and tau does not imply a unique origin of β-amyloid and tau pathology in sad. neuropathological studies at early stages of nft and sp pathology have shown that independent tau deposits are localized in separate regions such as the raphe nuclei and locus ceruleus, transentorhinal and entorhinal cortex, and olfactory bulb, in addition to isolated tau-positive neurons in other parts of the brain. similarly, β-amyloid deposits in the brain parenchyma are not connected; likewise there is not an intimate connection of blood vessels affected by β-amyloid angiopathy. aβ and tau pathology, like other molecular alterations in brain aging and sad, have multiple and separate foci; lesions in aging and sad do not start in a single region only to progress from that site to the whole brain. 18. neuronal death neuronal depletion in ad is the result of cell death and lack of neuronal renewal. apoptosis was postulated as a major mechanism of neuronal death in ad and most neurodegenerative diseases based on the positivity of cells stained with the method of in situ end-labeling of nuclear fragmentation, and immunoreactivity to cleaved caspase 3 (1218-1220). caspase 3-mediated and caspase 3-non-mediated apoptosis is also supported by studies in cell lines (1221-1224). however, dna fragmentation occurs during the agonic state and post-mortem delay, and active caspase-3 participates in tau truncation during the genesis of nfts. therefore, the weight of apoptosis as a cause of neuron demise in ad is probably unevenly represented. necroptosis is another type of programmed cell death, frequently linked to inflammation that depends on the kinase activity of receptor interacting protein 3 (rip3) and rip1, and the subsequent activation of the mixed lineage kinase domain-like protein (mlkl). this pathway is activated in ad and other neurodegenerative diseases (1225-1227). neuronal degeneration and demise in ad is not correlated with tau pathology (1228, 1229), although ghost tangles are not rare in advanced stages of ad. intraneuronal accumulation of β-amyloid is involved in synaptic dysfunction, cognitive impairment, and the formation of amyloid plaques in ad (416, 1230, 1231). in addition, excessive production of aβ peptides and app activates death signaling pathways including apoptosis, necrosis, necroptosis, oxytosis (gsh-mediated oxidative cell death), pyroptosis (inflammasome-mediated cell death), and autophagy in vitro (1232). in summary, although β-amyloid and tau pathology may induce cell death in sad, they are not the only factors triggering active cell death pathways. other alterations, such as aberrant cell-cycle re-entry, energy metabolism failure, oxidative stress damage, and altered cell membrane signaling, are relevant contributory factors (see section 21). 19. neuronal connectivity networks in brain aging and sad the notion that clinical manifestations in brain aging may correspond solely to early stages of ad-related pathology restricted to the hippocampus, inner regions of the temporal cortex and selected nuclei of the brain stem is simplistic, and probably untrue. functional neuroimaging techniques have shown selective modifications of the intrinsic connectivity network in aging that exceed the domains of these regions. the default mode, salience, dorsal attention, fronto-parietal control, and auditory, visual and motor networks decline by middle adulthood, but the motor network shows increased connectivity in middle adulthood, followed by a lessening (1233-1237). disruption of neuronal connectivity networks appears in aging and is intensified in ad (1237-1243). however, functional connectivity alterations do not always correlate locally to tau and β-amyloid deposition; hyperand hypo-connectivity cycles can occur repeatedly at different stages of the disease (1244). moreover, greater segregation of functional connections into distinct large-scale networks is associated with cognitive resilience at early stages of sad (1245, 1246). additional studies suggest a compensation phase followed by a degenerative phase in aging, and in early, preclinical ad (1247). this is important information, as plasticity changes may circumvent, at least temporarily, deficient functioning during brain aging and early stages of sad. plasticity may explain the particular capacities of the adult brain to manage information in creating new networks, manifested as “experience”. considering the areas affected in functional neuroimaging studies, it may be inferred that molecular changes take place in the aging brain in regions other than those affected by tau and β-amyloid pathology. 20. human brain aging and preclinical ad the combination of clinical manifestations and complementary biomarkers has established consensus criteria to define different clinical phases of ad (see box 1). at preclinical stages, individuals may have measurable brain changes that indicate the earliest signs of ad (biomarkers), but they have not yet developed symptoms such as memory loss (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). the preclinical phase is further categorized into three stages. preclinical ad stage 1 includes cognitively normal individuals with abnormal β-amyloid markers and no neurodegeneration; stage 2, individuals with abnormal β-amyloid and neurodegeneration as revealed by magnetic resonance imaging (mri); and stage 3, individuals with abnormal β-amyloid deposition and neurodegeneration, and “subtle” cognitive changes (2, 48, 1248-1252). the refinement of the diagnosis of preclinical stages has improved with the acquisition of new neuroradiological methods, and optimization of csf and blood biomarkers (1253-1256). however, improvement in detection using csf, blood, and plasma biomarkers largely depends on the availability of optimized instruments and markers. current csf and plasma biomarkers used in the diagnosis of ad are levels of β-amyloid, phospho-tau, tau, and phospho-tau ratio, neurofilaments, synaptic proteins, markers of activated astrocytes, and inflammatory markers. at present, the available methods cannot detect differential levels of tau, phospho-tau, β-amyloid, and structural and synaptic proteins unless the degenerative process is at least at undetermined middle stages of ad neuropathological change. hippocampal atrophy, as revealed by computed tomography (ct) and mri, and currently used to correlate cognitive impairment and altered neuropathological substrate, is a late marker of ad that is only positive when there is advanced nft pathology and neuron loss in the hippocampus. a recent meta-analysis to estimate the prevalence of aβ pathology as measured with biomarkers in participants with normal cognition, subjective cognitive impairment, or mild cognitive impairment revealed that about 25-35% of cognitively normal older adults harbored a significant amount of β-amyloid (1257). further studies have shown that β-amyloid csf-based estimates using adjusted data-driven cutoffs are up to 10% higher than pet-based estimates in people without dementia thus suggesting that preclinical ad may be more prevalent than previously suspected (1258). in contrast, tau pet imaging using optimized tracers provides more precise information in vivo at the different stages of nft progression (855, 1259-1267). improved neuroimaging methods corroborate earlier neuropathological observations in post-mortem brains. individuals without cognitive impairment may have tau or β-amyloid positive signal alone, or β-amyloid and tau pathology, or tau and β-amyloid pet negativity (1268-1270). neuroimaging studies have also identified tau pathology in the entorhinal region and mesial temporal cortex in normal aged individuals with or without minimal cognitive impairment (1271-1276). tau pet observations are also useful to show that tau deposits appear before β-amyloid deposits in most individuals. finally, neuroimaging allows the visualization of a continuum of lesions from “normal” brain aging to alzheimer’s type dementia in sad and fad (1277, 1278). therefore, neuroimaging observations further suggest that tau pathology is predominant in normal brain aging that overlaps with tau pathology in individuals with preclinical ad. clinical criteria of ad are biased by the consideration that aβ pathology precedes tau pathology in sad. in other words, the conception of the β-amyloid cascade hypothesis permeates the diagnosis of sad. in addition to clinical and biomarker criteria, new neuropathological standards, based on the study of a wide number of selected brain regions stained with selected immunohistochemical markers, take into consideration ad pathology progression and the presence of comorbid pathologies were proposed by the national institute on aging–alzheimer’s association (nia-aa) in 2012 (49, 50). the main considerations are a) recognition that ad neuropathologic changes may occur in the apparent absence of cognitive impairment, (b) consideration of an “abc” score for ad neuropathologic change that incorporates histopathologic assessments of β-amyloid deposits (named a, based on thal phases), staging of neurofibrillary tangles (named b, based on braak stages), and scoring of neuritic plaques (named c, based on cerad), and (c) assessment of co-morbid conditions such as lewy body disease, vascular brain injury, hippocampal sclerosis, and tdp-43 proteinopathy; also including artag and agd. at present, the post-mortem neuropathological examination is still the most precise approach to identify the morphological changes linked to a brain aging and sad (49, 50, 325) (table 1). “a” thal phase for aβ plaques “b” braak and braak nft stage “c” cerad neuritic plaque score 0 0 0 none 0 none 1 1 or 2 1 i or ii 1 sparse 2 3 2 iii or iv 2 moderate 3 4 or 5 3 v or vi 3 frequent table 1: “abc” score for ad neuropathologic change modified from ref. 49 and 50. national institute on aging–alzheimer’s association (nia-aa) guidelines for the neuropathologic assessment of alzheimer’s disease. following this classification, the score levels of ad neuropathologic change are classified as “not”, “low”, “intermediate”, and “high” (table 2). the presence of aβ is mandatory. it is also stated that “medial temporal lobe nfts in the absence of significant aβ or neuritic plaques occurs in older people and may be seen in individuals without cognitive impairment, with mild impairment, or with cognitive impairment from causes other than ad” (49, 50). to support this assertion, a previous work is cited: “brains that have many nfts in medial temporal lobe structures (braak stage iii or iv) but no cortical sps may be a diagnostic dilemma; they also raise questions about the amyloid cascade hypothesis of ad in which nft development is thought to occur downstream of the development of amyloid plaques” (1279). more recently, the introduction of the term part has been used to name cases with nft pathology without sps (see section 21a). ad neuropathologic change b a c 0 or 1 2 3 0 0 not not not 1 0 or 1 low low low 2 or 3 low intermediate intermediate 2 any c low intermediate intermediate 3 0 or 1 low intermediate intermediate 2 or 3 low intermediate high table 2: “abc” score level of ad neuropathologic change. aβ/amyloid plaques (a), nft stage (b), and neuritic plaque score (c). the combination of a, b, and c scores is designated as “not”, “low”, “intermediate” or “high” ad neuropathologic change. “intermediate” or “high” ad neuropathologic change is considered sufficient for dementia. national institute on aging–alzheimer’s association (nia-aa) guidelines for the neuropathologic assessment of alzheimer’s disease. a key point of this neuropathologic classification is the notion that β-amyloid deposition is the first “sine-qua-non” condition to consider the possibility of ad. the occurrence of nfts without β-amyloid plaques is not consistent with ad at any stage. the neuropathological proposal of the national institute on aging-alzheimer’s association assumes the “amyloid cascade hypothesis” as the cause of sad. yet the cognitive status correlates with nft burden rather than with β-amyloid plaques (331). 21. primary age-related tauopathy (part), rapidly progressive sad, and sad resilience 21a. part in 2014, the term part, a common pathology associated with human aging, was proposed to categorize a subpopulation of individuals with normal cognition or with mci showing nft pathology at stages i-iv of braak and no sps, and slower progression to the clinical stage of dementia in some individuals (1280-1283). the concept of part derives from the interpretation of the “β-amyloid cascade hypothesis” as the cause of ad. in other words, it is assumed that the presence of nfts without aβ deposition is not ad and therefore, cases with nfts and without sps do not match with the neuropathologic assessment of ad proposed by the nia-aa. part includes the majority of individuals aged 65 or older with first stages of nft pathology and without sps. the individuals represent about 85% of human beings at the age of 65 (328, 329, 1284), and are considered clinically affected by “normal brain aging”. therefore, part appears to be a predominant disorder until the emergence of sps (41, 329, 336, 1284). once a substantial number of sps appears in the brain, ad is overriding, whereas dementia with only tangles (or tangle-predominant dementia) which would be the logical progression of part decays and becomes rare (1285). it has been stated that the frequency of part increases at the age of 85 years (1286). however, most individuals with nft pathology at stages i-iii without sps are younger in largest series of cases (41, 329, 1284). the distribution of tau pathology in ca1 and ca3 regions of the hippocampus is reported to be different in part and sad. however, in one series, the mean age of the individuals was about 88 but the nft stages varied in the samples with part and sad (1287). in another series (mean age 84.3 ± 9.4 years), tau pathology was high in both ca1 and subiculum, followed by ca2/3, entorhinal cortex, ca4, and dentate gyrus in sad. in part, the severity of tau pathology in ca1 and subiculum was high, followed by enthorinal cortex, ca2/3, ca4, and dentate gyrus (1288). differences appear to be minimal and were probably modified by the presence of tau pathology in sps in sad. the proposal of part as a new tauopathy is not widely accepted. part has also been interpreted as being within the spectrum of sad (a part of sad) in cases with a particular genetic background characterized by lower prevalence of apoeε4, ptk2b, bin1, and cr1 genes, and higher prevalence of apoeε2 (1282, 1289, 1290). in contrast, tangle-predominant dementia has been associated with mapt h1 haplotype (1291). 21b. rapidly progressive ad patients with rapidly progressive ad (rpad) are younger and have a median survival time after diagnosis of about 7-10 months (1292); the neuropathological hallmarks and peripheral biomarkers are similar to those seen in current sad excepting for a low frequency of apoeε4 allele and increased serum levels of specific pro-inflammatory cytokines (1293, 1294). yet aβ42 oligomers in rpad have distinct properties which promote the faster spread of aβ42 pathology (1295). the composition of sps also differs in rpad, with significantly higher levels of neuronal proteins, decreased levels of astrocytic proteins, and particular abundance of synaptic-derived proteins (1296). moreover, high-density oligomers of the prion protein and a significant 1.2-fold decrease in di-glycosylated prp isoforms occur in rpad. fifteen proteins appear to interact with prpc while only two proteins, 3/4histone h2b-type1-b and zinc alpha-2 protein3/4, are specifically bound with the prp isoform isolated from rpad cases (1297). abnormal prp isoforms in rpad are accompanied by altered localization of distinct interactors including the growth arrest-specific 2-like protein and associated end-binding protein 1, α-tubulin, and β-actin (1298). molecular profiles of β-amyloid proteoforms differ in rpad when compared with typical sad (1299). moreover, the presence of highly hydrophobic aβ seeds in rpad brains that seeded reactions at a slower pace in comparison to typical sad has been validated (1299). another anomaly is the downregulation and dislocalization from the nucleus to the cytoplasm of the splicing factor proline and glutamine rich (sfpq), its colocalization with tia-1 in stress granules, and its association with tau oligomers in the brain of rpad (1300). 21c. sad resilience centenarians have resistance to sad or very low progression to advanced braak stages of tau and β-amyloid pathology (1301-1303). curiously, ad-related changes in the oldest-old population also show a particular neuropathological distribution including high densities of nfts, mainly in the hippocampus, without apparent major clinical deficiencies (1302-1307). multiple factors are associated with increased or reduced structural and behavioral patterns linked to cognition in the elderly and sad. the cognitive reserve, linked to educational and occupational acquisition, social networks, and leisure activities in later life, have a protective effect (1308-1312). glycolitic dysfunction reduces resilience (1313) whereas coffee and cacao favour protection from sad (1314). comorbidities, including tdp-43 proteinopathy and mesial sclerosis, reduce resilience (1315-1317). genetic factors also participate in cognitive resilience (1318, 1319). a greater number of genes apply in women (225, 1320). a top variant on chromosome 18 upstream of atp8b1 is significantly associated with methylation in prefrontal cortex tissue at multiple cpg sites, including one just upstream of atpb81, and present in individuals with unimpaired cognition (1321). a rare variant in the 3'-utr of rab10 is protective for sad (1322). hdac4, rest, and gi intracellular signaling are sad-specific pathways involved in regulating the onset of memory deficits (1323). molecular modulators of neuronal vulnerability, such as rar related orphan receptor b (rorb), and glial dysfunctions are also involved in neuronal vulnerability (1324). the myocyte-specific enhancer factor 2c (mef2c) is upregulated in a subpopulation of glutamatergic neurons in resilient individuals. over-expression of mef2a/c in the ps19 transgenic mouse model of tauopathy improves cognitive flexibility and reduces hyperexcitability (1325). the profiles of brain inflammatory responses also differ in resilient sad cases, with expression levels of chemokines decreasing and trophic factors increasing (860, 1326). yet a consensus is needed on how to define the concepts of cognitive reserve, resilience, and resistance linked to cognition in aged individuals (1327). the genetic and molecular factors linked to the ad spectrum are listed in table 3. fad mutations in the genes app, psen1 (presenilin1), and psen2; increased app dosage sad ε4 of apoe, lrp1, ldlr, interleukin 1a, clu, picalm, cr1, bin1, trem2, sorl1, adam10, abca7, spi1, pilra, msa4, cd2ap, and epha1 part lower prevalence of apoeε4, ptk2b, bin1, and cr1 genes, and higher prevalence of apoeε2 rpad low frequency of apoeε4 allele; increased inflammation; different aβ oligomers; different amyloid‑β proteoforms; different seeding capacities of β-amyloid; high-density prp oligomers; decreased prp di-glycosylated isoforms; specific prp isoform; altered localization of the growth arrest-specific 2-like 2 protein (g2l2), α-tubulin and β-actin; downregulation and dislocalization from the nucleus to the cytoplasm of sfpq, their colocalization with tia-1 in stress granules, and their association with tau oligomers resilient ad variant of chromosome 18 upstream of atp8b1; rare variant in the 3'-utr of rab10; mef2c upregulation in a subpopulation of glutamatergic neurons; decreased expression levels of chemokines and increased levels of trophic factors tangle-predominant dementia association with mapt h1 haplotype table 3: genetic and molecular factors linked to the ad spectrum (see sections 3, 4 and 21 for references). considering the previous data, and the preponderance of tau pathology over β-amyloid deposits at the first and middle stages of ad-related pathology (344), a modification to the nia-aa “abc” score level of ad neuropathologic is hereby proposed. the added value is the reflection of tau pathology at the same diagnostic value as β-amyloid (table 4). ad neuropathologic change a c b 0 0 0 or 1 2 3 0 0 low low intermediate 1 0 or 1 low low intermediate 2 or 3 low intermediate intermediate 2 any c low intermediate intermediate 3 0 or 1 low intermediate intermediate 2 or 3 low intermediate high table 4: modified “abc” score level of ad neuropathologic change. aβ/amyloid plaques (a), nft stage (b), and neuritic plaque score (c). the combination of a, b, and c scores is designated as “not”, “low”, “intermediate” or “high” ad neuropathologic change. “intermediate” or “high” ad neuropathologic change is considered sufficient for dementia. here, nfts are considered with equal relevance to sps in the progression from normal brain aging with nft changes, part, preclinical ad, and sad (including ad variants, rpad and adnc in centenarians). 22. biochemical changes beyond tau and β-amyloid at the the first stages of nft pathology in addition to the classical neuropathological hallmarks restricted to the inner region of the temporal lobe and selected nuclei of the brain stem which characterize the first stages of nft pathology, there is cumulative evidence of molecular and biochemical alterations in other regions in the same brains. several brain regions such as the frontal cortex, which does not show nft pathology at stages i-iii (and very rarely, if present, β-amyloid deposition at these stages), show altered lipid and protein membrane composition, abnormal mitochondrial function, oxidative stress damage, altered protein synthesis, activation of kinases, dysregulated protein phosphorylation, and abnormal inflammatory responses, among other disruptions. all of these alterations advance in parallel with the progression of nft pathology; most of them have implications in tau and β-amyloid pathology, and they all persist or increase at middle and advanced nft stages and sad. the terms nft and sad in these sections are those used in the original publications. 22a. aberrant cell-cycle re-entry, and altered adult neurogenesis the nervous system has plastic capacities and this is manifested in many ways in sad. one of these is the activation of pathways geared to activate cell cycle re-entry. however, neurons are post-mitotic cells that activate cell death programs at g1/a and g2/m points in response to cell cycle reactivation (1328). aberrant neuronal cell-cycle re-entry, as revealed by the expression of various proteins involved in the activation and progression of the cell cycle, is produced in a subpopulation of neurons in sad (1329-1340). the expression of cell-cycle-related proteins occurs before the appearance of nfts and sps and may trigger programmed cell death or the activation of kinases leading to oxidative stress damage, tau hyperphosphorylation, and activation of β-amyloid pathways (1341-1346). the expression of p75ntr in doublecortin (dcx)-immunoreactive dentate gyrus progenitors is reduced in ad and related transgenic models. the inoculation of pro-ngf neutralizing antibodies into the dentate gyrus restores memory performance of app/ps1 animals and significantly increases the percentage of dcx+ progenitors in the dentate gyrus of these animals, thus suggesting that impaired prongf-p75ntr signaling blocks adult neurogenesis in ad (1347). moreover, the prongf/p75ntr signaling pathway blocks adult neurogenesis and neuron cell death in ad (670-672). altered adult neurogenesis is not restricted to sad, as it occurs as well in the dentate gyrus in other neurodegenerative diseases such as huntington’s disease, amyotrophic lateral sclerosis, lewy body disease, and frontotemporal dementia (1348). 22b. brain lipids the brain is the second richest organ in lipids after the adipose tissue. cholesterol accounts for 20-25% of the total lipids in the plasma membrane of neurons; most local cholesterol is synthesized by astrocytes in the adult brain (1349). glycerophospholipids are the main phospholipid components ubiquitously found in cell membranes, and they are found in abundance in membranes from neural cells. lipids carry out, in addition to structural functions, the roles of mediators or second messengers. these are lipophilic molecules involved in signal transduction processes. lipid mediators are derived from the enzymatic degradation of glycerosphingolipids, sphingolipids, and cholesterol by phospholipases, sphingomyelinases, and cytochrome p50 hydroxylases, respectively. eicosanoids such as prostaglandins, leukotrienes, and lipoxins are derived from oxidation of the aa. docosanoids, including neuroprotectins, resolvins, and maresin, are mediators of docosahexanoic acid (dha). these mediators are important modulators of oxidative damage, inflammation, and apoptosis. other glycerophospholipid-derived lipid mediators are diacylglycerols (dags) phosphatidylinositol 1,4,5-triphosphates, platelet-activating factor, lysophosphatidic acid, and endocannabinoids (1350). degradation of sphingolipids also results in the generation of mediators, such as ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. these mediators are involved in differentiation, growth, cell migration, and apoptosis. cholesterol-derived lipid mediators, including 24and 25-hydroxycholesterol, produce apoptosis (1351-1354). the physical-chemical properties of the membrane bilayer and the chemical reactivity of fatty acids determine their susceptibility to oxidative damage (1355-1357). ros and reactive nitrogen species (rns) are more soluble in the fluid lipid bilayer than in the aqueous solution (1358, 1359). more importantly, polyunsaturated fatty acid (pufa) residues of phospholipids are very sensitive to oxidation (1360). as a result, neural cell membrane lipids become primary targets of oxidative damage and lipid peroxidation. in the aging brain, there is a progressive decrease in the levels of cholesterol, phosphatidylethanolamine, phosphatidyl inositol, phospholipid, ethanolamine plasmalogen, and sphingomyelin (1361-1366). increasing age is associated with progressive modifications in the composition of pufa, including dha and aa levels (1367). the age-related reduction in pufas is inversely correlated with stearoyl-coa desaturase expression and activity, resulting in higher levels of monounsaturated fatty acids (mufas). lipofuscin located in secondary lysosomes increases with age in neurons and glial cells (1368, 1369). the “aging pigment” is composed of two-thirds protein and one third lipids (1370, 1371). proteins in lipofuscin belong to the cytoskeleton, mitochondrial bioenergetics, synapse, and membrane receptors (1372). this proteome is practically identical to the proteome derived from lipoxidation reactions identified in the frontal cortex of aged humans (1373). levels of glycerophospholipids, sphingolipids, dags, and plasmalogens are altered in the brain in sad (1374-1379). the disturbance of human brain lipid content in sad pathology may be categorized into four main groups (i) decreased expression of phospholipids, specifically plasmalogen pe and plasmalogen pc, due in part to abnormal peroxisome activity; (ii) reduced sulfatide content; iii) increased levels of ceramides; and (iv) increased lipoxidative stress (1354). plasmalogen pe plays a particular role as anti-oxidant (1380). moreover, in gray matter, the major ppe molecular species are enriched in dha and aa. modifications in dha, aa, and pufas in sad produce an imbalance between their protective role (the adaptive responses derived from their lipid mediators) and a deleterious role (derived from their susceptibility to oxidation) (1381, 1382). lipid peroxidation is an early event and a major cause of oxidative stress damage in sad progression (1373, 1381). β-amyloid plays an important part since sps are always surrounded by oxidized lipids, as revealed using fourier transform infrared microscopy (1383). one mechanism to modulate oxidative damage is mediation by the upregulation of dha synthesis, which occurs in selected regions at early stages of sad; this is followed by an adaptation, and then decreased dha contents. this change is not uniform, but rather regionand stage-dependent. dha content in sad progression is like a shock wave manifested in three steps: upregulation, adaptation, and depletion. crucial information connecting lipid alterations and sad comes from genetic data. in addition to allele ε4 of apoe (268-271, 1384, 1385), many genes involved in sad are linked to cholesterol and lipid metabolism (284, 1386-1391). most of them increase the risk of β-amyloid deposition but apoeε2 allele appears to decrease it (1392, 1393). 22c. lipid rafts and cell membranes lipid rafts are microdomains of cell membranes enriched in glycosphingolipids, cholesterol, and protein receptors which favor multiple cell signaling interactions at the cell membrane, necessary for signal transduction (1394, 1395). the exoplasmic leaflet is enriched with glycosphingolipids and sphingomyelin, the cytoplasmic leaflet is enriched with glycerolipids; cholesterol is present in both. the lipid composition makes this system highly dynamic in that several proteins act as structural proteins, transmembrane signaling proteins, and protein anchors linked to protein-protein interactions (1396). cholesterol in lipid rafts facilitates the clustering of αand β-secretases. although cholesterol does not have a direct effect on γ-secretases, the increment of local cholesterol facilitates the production of β-amyloid (1397, 1398). the lipid composition of membranes is altered in sad. β-amyloid oligomers and peptides are recruited in lipid rafts (1399-1402). altered lipid raft composition also occurs in app/ps1 double-transgenic mice, a model of familial cerebral β-amyloidopathy. altered composition of lipid rafts is observed at the age of three months in parallel with the appearance of the first plaques in these mice (1403). prion protein expression in sps further supports alteration of lipid raft-enriched cell membrane in association with β-amyloid deposition at advanced stages of sad (128, 1404, 1405). lipid raft alteration in ad does not occur abruptly. the composition of lipid rafts changes with age in a gender-dependent manner. the main changes affect levels of plasmalogens, polyunsaturated fatty acids (especially dha and aa), total polar lipids (mainly phosphatidylinositol, sphingomyelin, sulfatides, and cerebrosides), and total neutral lipids (particularly cholesterol and sterol esters) (1406). importantly, the lipid composition of lipid rafts in the entorhinal cortex and frontal cortex, but not the cerebellum, is already altered in human brain at nft stages i-ii without β-amyloid deposits; lipid rafts at these stages display higher anisotropy, indicating that lipid changes in brain at nft stages i-ii increase membrane order and viscosity in these domains (1407). among other alterations, the structure of lipid rafts at stages i-ii is associated with increased bace1/aβpp interaction (1408). there is also a close functional relationship between cytoskeletal proteins and cell membranes through protein-protein interactions, electrostatic interactions with lipid membranes, and lipid tails. these complementary interactions are ruined once one of the components is altered. altered composition of lipid rafts and membrane proteins increases aβ pathways and tau fibrilisation. at the same time, tau monomers and β-sheet-rich tau structures disrupt cell membranes (156, 212, 1389, 1409-1413). tau connections with lipid tails depend on electrostatic interactions and phospholipid composition with high affinity for anionic lipids and anionic vesicles (1414, 1416). tau monomers may concentrate at the membrane and form oligomers and fibrils under pathological conditions. these β-sheet-rich tau structures are capable of disrupting membrane organization and function (212). deposition of abnormal tau at the preand post-synaptic membranes may appear prior to the appearance of nfts, and it contributes to early synaptic dysfunction (409). conversely, there is also the possibility that alterations at the cell membrane involving the lipid composition and post-translational modifications of membrane proteins trigger abnormal phosphorylation of tau and induce formation of tau fibrils (1417) (see section 21h). plasma membrane specializations containing caveolin are invaginated and form caveolae which are closely related to lipid rafts (1418). caveolae in sad participate in a wide number of processes including internalization of tau oligomers and β-amyloid metabolism (1419-1421). other factors influence membrane damage, in particular, microglial pro-inflammatory mediators generating membrane damages (1422). 22d. mitochondria mitochondrial alterations were identified in the pioneering ultrastructural research of ad and were later sustained by functional studies (1423-1427). mitochondrial alterations in sad led to the formulation of the “mitochondrial cascade hypothesis”, proposing that mitochondrial alterations and failed energy metabolism trigger sad (1428, 1429). recent reviews have stressed the importance of mitochondrial failure in the pathogenesis of ad (1430-1432). functional defects in atp-synthase are considered a main contributory factor to explain failure of energy production in mitochondria (1433). other studies have stressed the impairment of complex i in mild cases of ad (1434). besides energy metabolism deterioration, abnormal mitochondrial function also causes increased production of ros (1435-1437). mitochondrial atp-synthase is a target of oxidative damage in the entorhinal cortex at stages i-ii of nft pathology; total levels of atp-synthase are preserved but atpase function is impaired (1438). altered mitochondrial dna methylation is manifested in the entorhinal cortex at stages i-ii of nft pathology and advances with disease progression (1028). increased mitochondrial 5-methylcytosine levels are found in the d-loop region of mtdna in the entorhinal cortex. interestingly, this region shows a dynamic pattern in the content of mitochondrial 5-methylcytosine in app/ps1 transgenic mice in parallel with the progression of β-amyloid pathology in these mice (1028). aβ deposits have been considered a main cause of mitochondrial dysfunction in ad (1439). additionally, mitochondrial abnormalities are observed in tau transgenic mice (1440). aβ and tau pathology have synergistic effects on mitochondria in triple transgenic mice (1441). it has been proposed that there are mitochondrial links between brain aging and ad (1442). the mitochondria-associated lipid raft-domain of the er in close contact with the mitochondria, called mam (mitochondria-associated er membrane), facilitates functional and biochemical interaction between these structures, mainly linked to metabolism of the cholesterol, phospholipids, glucose, fatty acids, and calcium signaling (1443, 1444). mam functions are altered at early stages of sad (1445, 1446), thus prompting the “mam-hypothesis” as a determinant in the pathogenesis of sad (1447). there is growing evidence of impaired physical and proteome crosstalk between er and mitochondria in sad (1444). 22e. oxidative stress damage the mitochondria respiratory chain generates ros which participate in cell signalling under physiological conditions. peroxisomes, er, microsomes, nucleus and plasma membrane are potential sources of ros. excess production of ros and deficient anti-oxidant responses lead to oxidative stress damage to dna, rna, carbohydrates, lipids, and proteins. ion-catalyzed oxidation of some amino acid residues may result in the production of carbonyl derivatives such as glutamic semialdehyde and aminoadipic semialdehyde. in addition to direct effects, oxidative modifications may induce the production of reactive carbonyl species such as glyoxal, glycoaldehyde, methylglyoxal, malondialdehyde (mda), and 4-hydroxynonenal (hne), derived from the oxidation of carbohydrates and lipids. carbonyl species react with lysine, arginine, and cysteine residues, leading to the formation of advanced glycation and lipoxidation end-products (age/ales) in proteins. typical ages/ales adducts are carboxymethyl-lysine (cml), carboxyethyl-lysine (cel), and mda-lysine (mdal), among others (1357, 1373, 1381, 1448, 1449). regarding rns, nitric oxide damage to thiols, amines, and hydroxyls leads to nitrosative damage. reactions with rns lead to the formation of 3-nitrotyrosine (nitration) and to oxidation of distinct substrates. reactive peroxinitrite is able to nitrate tyrosine residues and to oxidize methionine residues of proteins (1450). oxidative stress damage is a major component of brain aging. protein oxidative and glycoxidative damage significantly increases with age; 60 years of age is the breakpoint of human frontal cortex aging (1451). oxidative stress damage in the aging brain is regionand age-dependent (1452). regional vulnerability to neurodegeneration based on energy demands, oxidative stress, and other metabolic factors can predict neurodegeneration (1453). for example, lower energy demand, lower mitochondrial stress, and one-carbon metabolism (particularly restricted to the methionine cycle), together with lower target of rapamycin (tor) signaling and better antioxidant capacity, occur in the frontal cortex compared with the entorhinal cortex and the hippocampus. these differences suggest that the frontal cortex is relatively resistant to stress compared to the entorhinal cortex and hippocampus (1454). however, this hypothesis does not apply universally when assessing different brain regions of individual brains in parallel (1455). the results are more complex and their interpretation is more difficult. vulnerable cortical and diencephalic regions are in fact more resistant to degeneration in aging (1455). however, differences in vulnerability to protein oxidation are dependent on the subcellular localization, secondary structure, and external exposition of certain amino acids. lipoxidized proteins are mainly those involved in energy metabolism, cytoskeleton, proteostasis, neurotransmission, and o2/co2/heme metabolism (1452). ages are produced in aging and sad, but their levels and the levels of their receptor (rage) do not correlate with aβ levels, tau levels, or dementia (1456, 1457). most studies of oxidative stress damage in sad are centered on stages v-vi (cases with dementia) and iii-iv (cases with mci). proteins involved in glycolysis and energy metabolism, in electron transport chain, oxidative phosphorylation, and other mitochondrial components; structural proteins; chaperones; stress proteins, and stress responses; and proteins of the ups are all targets of oxidative damage (1373, 1458-1467). protein oxidative damage is usually accompanied by decreased functional activity (1468). altered enzymatic activity has been demonstrated for the oxidized protein creatine kinase bb, enolase 1, glutamine synthetase, pin-1, carbonic anhydrase 2, uchl-1, α-enolase, gapdh, gdh, h+ transporting atpase, ldh, atp synthase, and pyruvate kinase in sad. a few studies have included the identification of oxidatively damaged protein, the quantification of total protein levels, and the reduction of enzymatic activity (1381). in an attempt to correlate oxidative stress damage and regional vulnerability in sad, a meta-analysis of mda, hne, protein carbonylation, 8-hydroxyguanine levels and superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase activities showed that changes linked to oxidative stress were variable from one region to another and dependent on the type of adduct. no correlation was seen between oxidative damage and regional vulnerability (1469). oxidative damage was advocated to be the earliest event in sad (1470). further research revealed that oxidative damage was more marked in younger cases, in cases with rapid disease progression, and in neurons without nfts when compared with neurons with tangles in the same individual (1471). moreover, oxidative stress precedes β-amyloid deposition in pre-symptomatic fad and down syndrome (1472, 1473). early observations suggested that oxidative post-translational modifications might play a role in the formation of sps and nfts (1474). following this argument, aggregation of aβ and tau was considered a compensatory response to underlying oxidative stress (1475). however, β-amyloid is also a cause of oxidative stress, thereby potentiating the loop (1476). oxidative stress generates mitochondrial dysfunction by damaging structural proteins and components of the mitochondrial respiratory chain in sad (1438, 1477-1479). oxidative damage also causes synaptic dysfunction (425). as indicated in previous paragraphs, oxidative damage of atp-synthase resulting in the loss of its function occurs at stages i-ii of nft pathology (1438). mitochondrial dysfunction, abnormalities in lipid rafts, and oxidative stress damage potentiate each other and are major players in neuronal energy failure at the first stages of nft pathology (1480). another relevant consequence of oxidative stress is the effect of advanced glycation end products on cell-cycle re-entry and arrest, also occurring at the first stages of sad (1481). 22f. inflammation aging is accompanied by low levels of activated innate inflammatory responses (1482, 1483). the role of microglia and inflammation in the aging brain and sad has been discussed in previous paragraphs. here, the focus is on the relevance of inflammatory changes in brain aging and at early stages of ad pathology. several reports in the 1990s described a protective effect of non-steroidal anti-inflammatory drugs used for the treatment of autoimmnune diseases on the manifestation and progression of sad (1484-1490). further studies delineated their positive effects when the treatment was initiated before the appearance of cognitive impairment, but the effects of the same treatments were minimal when administered in advanced ad. these observations show that inflammatory responses modulate the history of ad at the early stages of the process. more importantly, there is also a chance for anti-inflammatory drugs in the treatment of sad when administered at the appropriate time (1491-1493). molecular studies disclosed that multiple cytokines are involved at the early/middle stages of ad (1494). more detailed studies in ma and in cases at first (i-ii), middle (iii-iv), and advanced (v-vi) nft stages examined the expression of cytokines and mediators of the immune system in different regions progressively affected in sad: the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 (838). changes in mrna expression correlated with the corresponding protein levels as revealed by immunohistochemistry and western blotting (838). moreover, gene regulation at first stages of ad pathology (nft i-ii, sps: 0) was not related to nfts, β-amyloid plaques, concentration of aβ40 and aβ42, or membrane-bound fibrillar β-amyloid in the frontal cortex (838). these observations do not contradict previous studies showing a relationship between membrane-associated β-amyloid and inflammatory changes in cases with more advanced preclinical ad and sps in the cerebral cortex (101), but they highlight the observations that: (a) inflammatory markers appear at the first stages of nft pathology (stages i-iii) in regions with no nfts and sps; (b) inflammatory changes are modified with disease progression; and (c) different inflammatory responses occur simultaneously in different regions in the same individual. 22g. protein synthesis impairment protein synthesis is altered in sad, and this is due to multiple alterations at different subcellular levels from the nucleolus to the ribosome. the relation nuclear organizer region (nor) surface/total nucleus surface is reduced; the rdna promoter is hyper-methylated, and dimethylated histone h3k9 and acetylated histone h3k12 are decreased in the ca1 region of the hippocampus; nuclear tau declines; specific transcription factors are abnormally regulated; rrna levels are decreased and rna is oxidatively damaged; the expression of nucleolar proteins as well as the expression of rnas involved in the generation of ribosomal proteins decreases; the expression levels of translation initiation and elongation factor of the protein synthesis in ribosomes is dysregulated; and the capacity of isolated ribosomes to incorporate s35 methionine into protein is impaired (1010, 1473, 1495-1506). tau protein disrupts nucleocytoplasmic transport in ad (1507), but the role of tau, if any, in the other steps of the protein synthesis pathway is not known. alterations of protein synthesis pathways are already identified in the hippocampus at the first stages of nft pathology. nucleophosmin 1 (npm1) mrna is significantly increased, and upstream binding transcription factor rna polymerase i gene (ubtf) mrna and 28s rrna significantly decreased in ca1, but not in the dentate gyrus at nft stages i-ii. dimethylated histone h3k9 (h3k9m2) immunoreactivity is reduced in neurons of the dentate gyrus and ca1 at nft stages i-ii. mrna expression of ribosomal proteins rpl23a, rpl26, rpl31rps5, rps6, rps10, and rps13 is significantly reduced in the dentate gyrus at stages i-ii when compared with ma individuals without nft pathology. in contrast, rpl5 and rpl26 mrna expression is increased, and rps5 and rps6 mrna decreased in ca1 at the same stages i-ii (1508). these results show early alterations not only in the ca1 region, which will be involved in nft pathology, but also in the dentate gyrus, which does not contain abnormal tau deposits at any time in the process. seven upregulated mirnas (mir-125b, mir-146a, mir-200c, mir-26b, mir-30e, mir-34a, and mir-34c) and three downregulated mirnas (mir-107, mir-210, and mir-485), all of which associated with oxidative stress, are found in vulnerable brain regions of sad at the clinical prodromal stage (1501). together, these observations show that alterations in protein synthesis pathways appear at early stages of ad-related pathology and they are not linked to tau and β-amyloid deposits. disruption at many steps of the protein synthesis pathway increases in all regions with sad progression (1508), making it difficult to establish a causative relationship between tau and β-amyloid pathology and the progressive decay of protein synthesis at advanced stages of sad. 22h. dysregulated protein phosphorylation early phosphoproteomics studies identified a few abnormally phosphorylated proteins in the hippocampus and cerebral cortex in small numbers of cases with sad compared with controls (1509-1511). subsequent work with more accurate methods in advanced sad identified a large number of abnormally phosphorylated proteins, some of them with increased phosphorylation, and others with decreased phosphorylation. abnormally phosphorylated proteins corresponded to cytoskeletal proteins, integral membrane proteins, synaptic proteins, adhesion molecules, serine/threonine kinases, transport/cargo proteins, heat-shock proteins, and others mostly involved in cell growth and/or maintenance, cell communication, and metabolism (1417, 1513, 1514). multi-omics integration highlighted relevant altered networks including amyloid cascade, inflammation, complement, wnt protein signaling, transforming growth factor-β and bone morphogenic protein signaling, lipid metabolism, iron homeostasis, and membrane transport (1515). recent studies performed in the entorhinal cortex and frontal cortex in human brain aging and sad at different nft stages identified sixty-five dysregulated phosphoproteins in the entorhinal cortex, and eighty-one phosphoproteins in the frontal cortex at nft stages i-ii when compared with ma individuals without nft pathology. dysregulated protein phosphorylation of selected proteins occurs in parallel with the appearance of nfts in the entorhinal cortex but precedes the appearance of nfts and sps in the frontal cortex. the number of dysregulated phosphoproteins increases in both regions with nft stage, most of them added to those already dysregulated at stages i-ii. considering the total number of identified dysregulated phosphoproteins, the most active period corresponds to nft stages iii-iv, at a time when a subpopulation of people might be clinically categorized as suffering from mci (1417). the main group of dysregulated phosphoproteins at nft stages i-ii are membrane proteins; proteins of the cytoskeleton; proteins of the synapses and dense core vesicles; proteins linked to membrane transport and ion channels; kinases; proteins linked to dna and protein deacetylation; proteins linked to gene transcription and protein synthesis, and proteins involved in energy metabolism (1417). altered phosphorylation of selected proteins, accomplished by activation of several kinases, may alter membrane and cytoskeletal function, among these synaptic transmission and membrane/cytoskeleton signaling, in addition to energy metabolism, protein synthesis, and dna homeostasis (1417). dags are constituents of cell membranes that participate in intermediate lipid metabolism, and they are key components in lipid-mediated signaling. in neurons, dags modulate several signal transduction proteins linked to the activation of protein kinases, traffic and fusion of synaptic vesicles, ion channels, axonal guidance, and cytoskeletal homeostasis, among others (1516-1518). tau and β-amyloid phosphorylation may also be mediated by dags and pkc (1519). dag levels are increased in the sad frontal cortex (1520, 1521). further studies are needed to learn about possible links between dags and abnormal phosphorylation of cell membrane proteins. 23. concluding comments linear logic based on the assumption that a cause results in one or several effects does not explain complex biological phenomena such as brain aging and sad; there is no single cause of aging and sad. rather, the biological processes involved in generation, development, living, decline, and death are complex concatenations of complementary, disruptive, and adaptive responses. not surprisingly, the mutually exclusive hypotheses formulated to explain sad are not satisfactory. genetic studies in sad have also provided a wealth of information identifying genetic risk factors which principally cover cholesterol and lipid metabolism, inflammation, and cell membranes and synapses (1390). a scheme of the several factors involved in brain aging and sad is presented in figure 9. in addition to genetic factors, there is an interaction between molecular alterations in cellular structures that may favor the production of β-amyloid and abnormal tau. conversely, the presence of β-amyloid and abnormal tau has a negative effect on the majority of cellular structures, contributing to a detrimental loop in sad pathogenesis. figure 9: factors involved in brain aging and sad. in addition to genetic factors, molecular alterations in cellular structures may induce the production of β-amyloid and abnormal tau. conversely, the presence of β-amyloid and abnormal tau has a negative effect on the majority of cellular structures, contributing to a harmful loop in sad pathogenesis. molecular alterations are similar and cumulative in brain aging and sad. therefore, a continuum of brain aging and sad primarily based on an archetypal distribution of nfts usually followed by the deposition of β-amyloid plaques is a factual possibility. the pathogenesis and evolution of arteriosclerosis is another example of cumulative age-related degenerative changes in blood vessels. almost all people are affected to some degree by the age of 65. however, the first lesions in atherosclerosis, characterized by macrophage infiltration and intracellular lipid accumulation in the blood vessel wall, may occur in the first or second decade; inflammation, endothelial and perycite damage, altered metabolism of muscle fibers, extracellular lipid cores and atheromatous plaques develop later. clinical manifestations may appear at advanced stages of fibroatheroma, as well as complicated lesions in ma individuals. thus, the atheromatous plaque, the characteristic lesion of atherosclerosis, is not the first requirement to identify atherosclerosis as a biological process which is expressed by cumulative stages of blood vessel damage leading or not to clinical manifestations of variable severity. searching possible therapies for sad has been constant over the years. cholinesterase inhibitors, nmda receptor antagonists, membrane protectors, anti-oxidants, and anti-inflammatory agents, all assayed at middle and advanced clinical stages of sad, have proven poorly effective. other treatments are directed to reducing β-amyloid or tau accumulation. several aβ-protein-targeted drugs, including β-secretase inhibitors, γ-secretase inhibitors and modulators, α-secretase activators, direct inhibitors of aβ aggregation, and immunotherapy have been assayed or are under different phases of clinical trial (1522). β-amyloid immunization in humans has been successful in the sense that sps are largely reduced in the brain of treated patients; yet the abundance of tau deposits and the progression of the disease are not substantially modified by β-amyloid immunization (719, 1523, 1524). the clinical improvement seen with β-amyloid immunotherapy has been null or negligible, or at best arguable (1525-1530). treatment with a bace1 inhibitor has yielded very limited neuropathological improvement (1531). trials with new generation aβ immunotherapy are in progress. following the same line of thinking, tau immunization, although putatively preventing tau accumulation, is unlikely to be a unique alternative treatment (1532-1534). various tau-based therapies have been developed or are under development. these therapies include the use of inhibitors of tau phosphorylation, glycosylation, and acetylation; microtubule stabilizers; inhibitors of tau aggregation; and anti-tau immunotherapy (1535-1538). various tau therapies based on active and passive immunization are effective in murine and primate models. however, some of these attempts have failed in sad (1539). although encouraging, it is not clear how such treatments will reduce the different forms of abnormal tau and decrease aβ burden. in short, there is no evidence that merely lessening the abnormal levels and deposits of tau or β-amyloid at middle and advanced stages of the illness will cure sad. therefore, it is crucial to re-consider the optimal age to start combined anti-tauand anti-β-amyloid-based treatments to combat two main representative components of ad. the identification of new putative targets for therapeutic intervention before the appearance of tau and amyloid deposits is a promising endeavor. gene therapy has also been assessed in transgenic mouse models with variable success using both vector-based therapies and genetically modified cell replacement (1540-1542). studies in humans are limited, at this time, to upregulated ngf in sad patients, with little evidence of benefit (1543-1545). exosomes are a class of membrane vesicles derived from endolysosomal compartment implicated in cell-cell communication by shuttling different lipids, protein, and rnas between cells. the use of shuttles including exosomes to deliver selected molecules into the cells is still at the early stages. the meager success of available therapies to curb the effects of human brain aging and sad is a matter of concern. besides disease modification and symptomatic therapies for sad (1546-1549), other relevant and long-term ventures may also be considered. one of these is a revision of animal models used to test sad therapies, because they have failed when applied to humans (1550). to make this clear: for 25 years, many ad-like mouse models have been “cured” in the lab, only for the treatments to fail in clinical trials in humans (1551, 1552). it seems that mice have the ability to remodel complex metabolic defects once one of the factors involved in the process is normalized. this does not occur in humans. these considerations are made yet more compulsory because a number of molecular function derangements take place before or concurrent with the appearance of β-amyloid and tau pathology morphologically manifested as nfts and sps. altered lipid and protein composition of cellular membranes accompanied by impaired subcellular cell signaling from the cell membrane to the er, mitochondrial membranes, altered synapses, altered mitochondria and energy metabolism, and reactivation and abortion of developmental programs leading to neuronal death, together with dysfunctional bbb and singular brain inflammatory expression, all point to sad as a human brain age-related disorder of convergent mechanisms that shatters brain self-organization (329, 1343, 1553, 1554). a great data analysis covering the tremendous body of information on ad must be updated in real time. we need to think of the coming decades as an opportunity to take advantage of the rapid growth of artificial intelligence (ai) and cell reprogramming. we will be able to redesign some aspects of the human brain in the near future using advanced technologies. to this end, we also need to identify the main targets and appropriate intervention times. timing is crucial since it will be difficult to reprogram molecular pathways in old-adult individuals that have already suffered brain deterioration. brain reprogramming would likely be undertaken before the beginning of the slow-pace functional decline in ma adults. brain reprogramming may cover different areas including brain dna editing, utilization of external electrical or wave-based signals to reduce energy consumption of basic neuronal networks, optimization of mitochondrial function, implanting of microdevices to facilitate cooperative human-machine function, pharmacological combined protection of lipid-protein interactions, and program resetting during stages of sleep. improvement of brain function in aging and sad has a chance in the application of high-throughput molecular technology, ai, and robotics (1394, 1555-1561). this new era is certain to present formidable ethical challenges. abbbreviations 2-ag arachidonoyl glycerol, 5-ht 5-hydroxytriptamine, 3’-utr complementary un-translated regions, aa arachidonic acid, aβ beta amyloid, abca7 atp binding cassette subfamily a member 7, aβos soluble β-amyloid oligomers, ac adenylyl cyclise, ach acetylcholine, ache acetylcholinesterase, ad alzheimer disease, adam10 adam metallopeptidase domain 10, addls amyloid-β derived diffusible ligands, adnc ad-neuropathologic change, aea n-arachidonoyl ethanolamine, agd argyrophylic grain disease, ages advanced glycation end-products, ago argonaute subfamily proteins, ai artificial intelligence, ales advanced lipoxidation end-products, amp adenosine monophosphate, ampa α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, ampar α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, aphh1a α-secretase subunit a, apoe apolipoprotein e, app amyloid precursor protein, aqp aquaporin, artag aging-related tau astrogliopathy, asc inflammasome-adaptor protein, atf-6 activating transcription factor 6, atp8b1 atpase phospholipid transporting 8b1, atp adenosine triphosphate, bace β-secretase, β-ar β-adrenergic receptor, bbb blood brain barrier, bdnf brain-derived neurotrophic factor, bin1 bridging integrator 1, c1qtnf7 complement c1q tumor necrosis factor-related protein 7, c3ar1 complement c3a receptor 1, ca2+ calcium ion, camp cyclic adenosine monophosphate, cb calbindin d28k, cbf cerebral blood flow, cbr cannabinoid receptor, cd2ap cd2-associated protein, cdk5 cyclin-dependent kinase 5, cerad consortium to establish a registry for alzheimer’s disease, cel carboxyethyl-lysine, cgmp cyclic guanosine monophosphate, chat choline acetyl transferase, ck1-δ casein kinase i isoform delta, clu clusterin, cml carboxymethyl-lysine, cmv human cytomegalovirus, cnp 2',3'-cyclic-nucleotide 3'-phosphodiesterase, cns central nervous system, comt catechol-o-methyltransferase, cpg cytosine-guanine-rich regions , cr calretinin, cr1 complement component (3b/4b) receptor 1, creb camp response element-binding protein, csf cerebrospinal fluid, csf1r colony stimulating factor 1 receptor, csf3r colony stimulating factor 3 receptor, ct computed tomography, dag diacylglycerol, dbh dopamine β-hydroxylase, dcx doublecortin, dha docosahexanoic acid, dnmt dna methyl transferases, gsh glutathione, eaat excitatory amino acid transporter, ebv epstein-barr virus, eofad early onset alzheimer disease, eph ephrin, epha1 ephrin receptor a1, er endoplasmic reticulum, exrnas extracellular rnas, fad familial alzheimer disease, fad flavin adenine dinucleotide, fbd familial british dementia, fcγriib fcγ, receptor ii-b, fdd familial danish dementia, gaba γaminobutyric acid, gabaa receptors ionotropic gabaa receptors, gabab receptors metabotropic gabab receptors, gad glutamic acid decarboxylase, gap-43 growth-associated protein 43, gdh glutamate dehydrogenase, gfap glial fibrillary acidic protein, glurs glutamate receptors, glut 1 glucose transporter 1, gmp guanosine monophosphate, gpcr g-protein-coupled receptor, grp78/bip glucose-related protein 78, gsk3β glycogen synthase kinase 3β, gss gerstmann-sträussler-scheinker, gvd granulovacuolar degeneration, gwas genome-wide association studies, h3k4me1 methylation of lysine 4 of histone 3, h3k4m3 trimethylation of histone 3 at lysine 4, h3k9me2 dimethylation of histone h3 at lysine 9, hat histone acetyltransferase, hdac histone deacetylase, hdm histone demethylase, hhv-6 human herpes virus 6, hmt histone methyltransferase, hne 4-hydroxynonenal, hsv-1 herpes simplex virus 1, icjd iatrogenic creutzfeldt-jabob disease, ide insulin degrading enzyme, ifn-γ interferon γ, iglur ioinotropic glutamate receptor, il interleukin, ipsc indiuced pluripotent stem cells, ire 1 inositol-requiring protein 1, jak2 janus kinase 2, ka kainate, kar kainate receptor, lamp-1 lysosomal-associated membrane protein 1, lc3 protein light chain 3, lc-ms/ms liquid chromatography (lc) tandem mass spectrometry (ms), lcnrnas long non-coding rnas, ldh lactate dehydrogenase, ldlr low density lipoprotein protein receptor 1, lilrb2 pirb human orthologue receptor , load late onset alzheimer disease, lp2 lipoprotein lipase 2, lrp1 hldl receptor related protein 1, ma middle-aged, machr muscarinic acetylcholine receptor, mag myelin associated glycoprotein, mal myelkin and lymphocyte protein, mams mitochondria-associated er membranes, map2 microtubule associated protein 2, mapk mitogen-activated protein kinase, mapt microtubule associated protein tau, mapt h1 microtubule-associated protein tau haplotype 1, mao mono-amino oxidase, mbp myelin basic protein, mci mild cognitive impairment, mda malondialdehyde, mdal mda-lysine, mef2c myocyte-specific enhancer factor 2c, mg2+ magnesium ion, mglur metabotropic glutamate receptor, mirna microrna, mlkl mixed lineage kinase domain-like protein, mobp myelin-associated oligodendrocyte basic protein, mog myelin-oligodendrocyte glycoprotein, mri magnetic resonance imaging, msa4 membrane-spanning 4-domains subfamily a, mtrnas mitochondrial rnas, mufa monounsaturated fatty acids, nachr nicotinic acetylcholine receptor, nadþ nicotinamide adenine dinucleotide, nadpþ nicotinamide adenine dinucleotide phosphate, nct/ncstn nicastrin, nep/nme neprelysin, nft neurofibrillary tangle, ng2 neural/glial antigen 2, ngf nerve growth factor, nia-aa national institute on aging–alzheimer’s association, nlrp3r pyrin-domain containing 3, nmda n-methyl d-aspartate, nmdar n-methyl-d-aspartate receptor, nor nuclear iorganizer region, npm1 nucleophosmin 1, nrnas small non-coding rnas, opc oligodendroglial precurosor cells, part primary age-related tauopathy, p75ntr p75 neurotrophin receptor, low affinity nerve growth factor receptor, pdgfra platelet-derived growth factor receptor a, pen2/psenen presenilin enhancer γ-secretase subunit, perk pkr-like endoplasmic reticulum kinase, pet positron emission tomography, phf paired helical filament, pi3k phosphatidylinositol 3-kinase, pi3p phosphatidylinositol trisphosphate, picalm phosphatidylinositol binding clathrin assembly protein, pid pick disease, pilra paired immunoglobin like type 2 receptor alpha, pirb paired immunoglobulin-like receptor b, pirnas piwi-interacting rnas, pka protein kinase a, pkc protein kinase c, plc phospholipase c, plp1 proteolipid protein 1, ppar peroxisome proliferator-activated receptors, prnp prion protein gene, prpc cellular prion protein, psen1 presenilin 1, psen2 presenilin 2, ptk2b protein tyrosine kinase 2 beta, pufa polyunsaturated fatty acids, pv parvalbumin, rab10 ras-related protein rab-10, rage age receptor, rest re1-silencing transcription factor, rip3 receptor interacting protein 3, rns reactive nitrogen species, rorb rar related orphan receptor b, ros reactive oxygen species, rp ribosomal protein, rpad rapidly progressive ad, rrna ribosomal rna, sad sporadic alzheimer disease, sam s-adenosyl-l-methionine, sapk/jnk stress-activated protein kinase/jun n-terminal kinase, scarnas small cajal-body specific rnas, sfpq splicing factor proline and glutamine rich, sirnas small interfering rnas, snap soluble nsf attachment protein receptor, snare snap receptor protein, snrnaseq single nuclei rna sequencing, snrna small nuclear rnas, snornas small nucleolar rnas, sorl1 sortilin-related receptor 1, spi1 spi-1 proto-oncogene, sp senile plaque, stat1 signal transducer and activator of transcription 1, tet1 ten-eleven-translocation-1 protein, tgf transforming growth factor, tlr toll like receptor, tnf tumor necrosis factor, tor target of rapamycin, traf2 tnf receptor-associated factor 2, trem2 triggering receptor expressed on myeloid cells 2, trka specific ngf receptor, trna transfer rnas, ubtf upstream binding transcription factor rna polymerase i gene, uchl-1 ubiquitin c-terminal hydrolase l1, upr misfolded protein response, ups ubiquitin proteasome system, vgat vesicular gaba transporter, vglut vesicular glutamate transporter, vmat vesicular monoamino transporter, wm white matter, wt wild type, xbp-1 x-box binding protein 1. funding the project leading to these results received funding from the “la caixa” foundation (id 100010434) under the agreement lcf/pr/hr19/52160007, hr18-00452. i thank the cerca programme of the generalitat de catalunya for institutional support. acknowledgements this work is, in part, the result of the personal experience of 40 years in the study of ad and other tauopathies. to name all the people i would like to thank for their help, support, and satisfactory collaboration is practically impossible. i appreciate the aid of ciberned, and i am extremely grateful to tom yohannan for his continual editorial assistance. references duyckaerts c, dickson dw. neuropathology of alzheimer’s disease and its variants. in: neurodegeneration: the molecular pathology of dementia and and movement disorders; second edition. dickson dw, wetter ro (eds) international society of neuropathology, blackwell publish ltd, 2011, pp: 62-91. https://doi.org/10.1002/9781444341256.ch10 knopman d. clinical aspects of alzheimer’s disease. in: neurodegeneration, the molecular pathology of dementia and movement disorders. dickson dw, weller ro (eds) wileyblackwell, 2011, pp: 39-50. https://doi.org/10.1038/s41572-021-00269-y serrano-pozo a, frosch mp, masliah e, hyman bt. neuropathological alterations in alzheimer disease. cold spring harb perspect med 2011;1:a006189. https://doi.org/10.1101/cshperspect.a006189 love j, kalaria r. dementia. in: greenfield’s neuropathology, vol 1, nnth edition. love s, budka h, ironside jw, perry a (eds). crc press, 2015, pp: 858-973. lowe j, kalaria r. dementia. in: greenfield’s neuropathology. ninth edition. loe s, budka h, ironside jw, perry a (eds). crc press 2015, pp: 858-973. masters cl, bateman r, blennow k, et al. alzheimer's disease. nat rev dis primers 2015;1:15056. https://doi.org/10.1038/nrdp.2015.56 deture ma, dickson dw. the neuropathological diagnosis of alzheimer’s disease. mol neurodeg 2019;14:32. https://doi.org/10.1186/s13024-019-0333-5 jellinger ka. neuropathology of the alzheimer’s continuum: an update. free neuropathol 2020;1:32. https://doi.org/10.17879/freeneuropathology-2020-3050 avila j, perry g. a multilevel view of the development of alzheimer’s disease. neuroscience 2021; 457: 283-293. https://doi.org/10.1016/j.neuroscience.2020.11.015 blocq p, marinesco g. sur les lésions et la pathogénie de l’épilepsie dite essentielle. sem méd 1892; 12:445-446. redlich e. uber miliare sklerose der hirnrinde bei seniler atrophie. jahrb psychiat neurol 1898;17:208-216. alzheimer a. über eine eigenartige erkrankung der hirnrinde. allg z psychiat 1907; 64: 146-148. perusini g. über klinisch und histologisch eigenartige psychische erkrankungen des spaateren lebensalters. in: nissl f, alzheimer a (eds) histologische und histopathologische arbeiten. fischer, jena, 1910 kraepelin e. psychiatrie: ein lehrbuch für studierende und ärzte. ii. band, klinische psychiatrie. verlag johann ambrosius barth, leipzig, 1910, vol.ii. fischer o. miliare nekrosen mit drusigen wucherungen der neurofibrillen, eine regelmässige veränderung der hirnrinde bei seniler demenz. monatsschr psychiat neurol 1907; 22: 361-372. https://doi.org/10.1159/000211873 fischer o. die presbyophrene demenz, deren anatomische grundlage und klinische abgrenzung. z ges neurol psychiat 1910;3: 371-471. https://doi.org/10.1007/bf02893605 fischer o. ein weiterer beitrag zur klinik und pathologie der presbyophrenen demenz. z ges neurol psychiat 1912; 12: 99-135. https://doi.org/10.1007/bf02866372 simchowitz t. histologische studien uber die senile demenz. in: nissl f, alzheimer a (eds) histologische und histopathologische arbeiten über die grosshirnrindem, vol. 4. fischer, jena, 1911; pp 267-444. goedert m. oskar fischer and the study of dementia. brain 2009; 132;1102-1111. https://doi.org/10.1093/brain/awn256 bielschowsky m. zur kenntnis der alzheimerschen krankheit (präsenilen demenz mit herdsymptomen). j psychol neurol 1911;18:1-20. karenberg a. early history of pick's disease. fortschr neurol psychiatr 2001;69: 545-50. https://doi.org/10.1055/s-2001-18378 delay j, brion s. les démences tardives. masson, paris, 1962 jervis ga, soltz se. alzheimer’s disease: the so-called juvenile form. am j psychiatr 1936; 93: 39-56. https://doi.org/10.1176/ajp.93.1.39 blessed g, tomlinson b, roth m. the association between quantitative measures of dementia and of senile changes in the cerebral grey matter of elderly subjects. br j psych 1968;114: 797-811. https://doi.org/10.1192/bjp.114.512.797 katzman r. editorial: the prevalence and malignancy of alzheimer disease: a major killer. arch neurol 1976; 33:217-218. https://doi.org/10.1001/archneur.1976.00500040001001 tomlinson be, blessed g, roth m. observations on the brains of demented old people. j neurol sci 1970; 11:205-242. https://doi.org/10.1016/0022-510x(70)90063-8 terry rd, katzman r. senile dementia of the alzheimer type. ann neurol 1983;14: 497-506. https://doi.org/10.1002/ana.410140502 mckhann gm, knopman ds, chertkow h, et al. the diagnosis of dementia due to alzheimer's disease: recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease. alzheimers dement 2011;7:263-269. https://doi.org/10.1016/j.jalz.2011.03.005 crystal h, dickson d, fuld p, et al. clinico-pathologic studies in dementia: nondemented subjects with pathologically confirmed alzheimer's disease neurology 1988;38:1682-1687. https://doi.org/10.1212/wnl.38.11.1682 price jl, davis pb, morris jc, et al. the distribution of tangles, plaques and related immunohistochemical markers in healthy aging and alzheimer's disease. neurobiol aging 1991;12:295-312. https://doi.org/10.1016/0197-4580(91)90006-6 rriagada pv, marzloff k, hyman bt. distribution of alzheimer-type pathologic changes in nondemented elderly individuals matches the pattern in alzheimer's disease. neurology 1992;42:1681-1688. https://doi.org/10.1212/wnl.42.9.1681 bouras c, hof pr, morrison jh. neurofibrillary tangle densities in the hippocampal formation in a non-demented population define subgroups of patients with differential early pathologic changes. neurosci lett 1993;153:131-135. https://doi.org/10.1016/0304-3940(93)90305-5 bouras c, hof pr, giannakopoulos p, et al. regional distribution of neurofibrillary tangles and senile plaques in the cerebral cortex of elderly patients: a quantitative evaluation of a one-year autopsy population from a geriatric hospital. cereb cortex 1994;4:138-150. https://doi.org/10.1093/cercor/4.2.138 knopman ds, parisi je, salviati a, floriach-robert m, boeve bf, ivnik rj, et al. neuropathology of cognitively normal elderly. j neuropathol exp neurol 2003;62:1087-1095. https://doi.org/10.1093/jnen/62.11.1087 bennett da, schneider ja, arvanitakis z, et al. neuropathology of older persons without cognitive impairment from two community-based studies. neurology 2006;66:1837-1844. https://doi.org/10.1212/01.wnl.0000219668.47116.e6 khachaturian zs. diagnosis of alzheimer’s disease. arch neurol 1985;42: 1097–1105. https://doi.org/10.1001/archneur.1985.04060100083029 wisniewski hm, robe a, zigmanw, et al. neuropathological diagnosis of alzheimer disease. j neuropathol exp neurol 1989;48: 606-609. https://doi.org/10.1097/00005072-198911000-00001 mirra ss, heyman a, mckeel d, et al. the consortium to establish a registry for alzheimer’s disease (cerad). part ii. standardization of the neuropathologic assessment of alzheimer’s disease. neurology 1991;41:479-486. https://doi.org/10.1212/wnl.41.4.479 mirra ss, hart mn, terry rd. making the diagnosis of alzheimer's disease. a primer for practicing pathologists. arch pathol lab med 1993;117:132-144. pmid: 8427562 hubbard bm, fenton gw, anderson jm. a quantitative histological study of early clinical and preclinical alzheimer’s disease. neuropathol appl neurobiol 1990;16:111–121. https://doi.org/10.1111/j.1365-2990.1990.tb00940.x braak h, braak e. frequency of stages of alzheimer-related lesions in different age categories. neurobiol aging 1997; 18:351-357. https://doi.org/10.1016/s0197-4580(97)00056-0 hulette c, welsh-bohmer k, murray m, et al. neuropathological and neuropsychological changes in "normal" aging: evidence for preclinical alzheimer disease in cognitively normal individuals. j neuropathol exp neurol 1998;57:1168-1274. https://doi.org/10.1097/00005072-199812000-00009 pmid: 9862640. price j, morris jc. tangles and plaques in non demented aging and "preclinical" alzheimer's disease. ann neurol 1999;45:358-368. https://doi.org/10.1002/1531-8249(199903)45:3<358::aid-ana12>3.0.co;2-x khachaturian zs. diagnosis of alzheimer's disease: two-decades of progress. j alzheimers dis 2006;9:409-415. https://doi.org/10.3233/jad-2006-9s346 dubois b, feldman hh, jacova c, et al. revising the definition of alzheimer's disease: a new lexicon. lancet neurol 2010;9:1118-1127. https://doi.org/10.1016/s1474-4422(10)70223-4 albert ms, dekosky st, dickson d, et al. the diagnosis of mild cognitive impairment due to alzheimer’s disease: recommendations from the national institute on aging-alzheimer’s association workgroups on diagnostic guidelines for alzheimer’s disease. alzheimers dement 2011; 7:270–279. https://doi.org/10.1016/j.jalz.2011.03.008 mckhann gm, knopman ds, chertkow h, et al. the diagnosis of dementia due to alzheimer's disease: recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease. alzheimers dement 2011;7:263-269. https://doi.org/10.1016/j.jalz.2011.03.005 sperling ra, aisen ps, beckett la, et al. toward defining the preclinical stages of alzheimer's disease: recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease. alzheimers dement 2011;7:280-292. https://doi.org/10.1016/j.jalz.2011.03.003 hyman bt, phelps ch, beach tg, et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease. alzheimers dement 2012;8:1-13. https://doi.org/10.1016/j.jalz.2011.10.007 montine tj, phelps ch, beach tg, et al. national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol 2012;123:1-11. https://doi.org/10.1007/s00401-011-0910-3 jack cr, bennett da, blennow k, et al. nia-aa research framework: toward a biological definition of alzheimer’s disease. alzheimers dement 2018;14:535-562. https://doi.org/10.1016/j.jalz.2018.02.018 ward a, tardiff s, dye c, et al. rate of conversion from prodromal alzheimer’s disease to alzheimer’s dementia: a systematic review of the literature. dement geriatr cogn disord extra 2013;3:320-332. https://doi.org/10.1159/000354370 petersen rc, lopez o, armstrong mj, et al. practice guideline update summary: mild cognitive impairment. neurology 2018;90:126-135. https://doi.org/10.1212/wnl.0000000000004826 gao s, hendrie hc, hall ks, et al. the relationships between age, sex, and the incidence of dementia and alzheimer disease: a meta-analysis. arch gen psych 1998; 55:809-815. https://doi.org/10.1001/archpsyc.55.9.809 vermunt l, sikkes sam, van den hout a, et al. duration of preclinical, prodromal, and dementia stages of alzheimer’s disease in relation to age, sex, and apoe genotype. alzheimers dement 2019;15:888-898. https://doi.org/10.1016/j.jalz.2019.04.001 kidd m. paired helical filaments in electron microscopy of alzheimer's disease. nature 1963; 197: 192-193. pmid: 14032480. kidd m. alzheimer’s diseasean alectron microscopical study. brain 1964;87:307-320. https://doi.org/10.1093/brain/87.2.307 terry rd. the fine structure of neurofibrillary tangles in alzheimer’s disease. j neuropathol exp neurol 1963;22:629-642. https://doi.org/10.1097/00005072-196310000-00005 terry rd, gonatas nk, weiss m. ultrastructural studies in alzheimer’s presenile dementia. am j pathol 1964;44: 269-297. pmid: 14119171. luse sa, smith kr. the ultrastructure of senile plaques. am j pathol 1964;44:553-563. pmid: 5877505. wisniewski h, terry rd, hirano a. neurofibrillary pathology. j neuropathol exp neurol 1970; 29: 163-176. pmid: 5435819. glenner gg, wong cw. alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. biochem biophys res commun 1984; 120: 885-890. https://doi.org/10.1016/s0006-291x(84)80190-4 glenner gg, wong cw, quaranta v, et al. the amyloid deposits in alzheimer's disease: their nature and pathogenesis. appl pathol 1984; 12:357-369. pmid: 6242724. masters cl, simms g, weinman na, et al. amyloid plaque core protein in alzheimer disease and down syndrome. proc nat acad sci usa 1985; 82:4245-4249. https://doi.org/10.1073/pnas.82.12.4245 iwatsubo t, odaka a, suzuki n, et al. visualization of a beta 42(43) and a beta 40 in senile plaques with end-specific a beta monoclonals: evidence that an initially deposited species is a beta 42(43). neuron 1994; 13:45-53. https://doi.org/10.1016/0896-6273(94)90458-8 masters cl, beyreuther k. amyloid-β production. in: dickson dw, weller ro, eds. neurodegeneration: the molecular pathology of dementia and movement disorders, 2nd ed. blackwell publishing co; 2011:92–6. lee ms, kao sc, lemere ca, et al. app processing is regulated by cytoplasmic phosphorylation. j cell biol 2013;163:83-95. https://doi.org/10.1083/jcb.200301115 vassar r, kovacs dm, yan r, et al. the beta-secretase enzyme bace in health and alzheimer's disease: regulation, cell biology, function, and therapeutic potential j neurosci 2009;29:12787-12794. https://doi.org/10.1523/jneurosci.3657-09.2009 zhang x, li y, xu h, zhang yw. the gamma-secretase complex: from structure to function. front cell neurosci 2014;8:427. https://doi.org/10.3389/fncel.2014.00427 barthet g, georgakopoulos a, robakis nk. cellular mechanisms of γ-secretase substrate selection, processing and toxicity. prog neurobiol 2012;98:166-175. https://doi.org/10.1016/j.pneurobio.2012.05.006 wolfe ms. structure and function of the γ-secretase complex. biochemistry 2019;58:2953-2966. https://doi.org/10.1021/acs.biochem 9b00401. beyreuther k, masters cl. amyloid precursor protein (app) and beta a4 amyloid in the etiology of alzheimer's disease: precursor-product relationships in the derangement of neuronal function. brain pathol 1991;1:241-251. https://doi.org/10.1111/j.1750-3639.1991.tb00667.x masters cl, beyreuther k. alzheimer's centennial legacy: prospects for rational therapeutic intervention targeting the abeta amyloid pathway. brain 2006;129:2823-2839. https://doi.org/10.1093/brain/awl251 masters cl, selkoe dj. biochemistry of amyloid β-protein and amyloid deposits in alzheimer disease. cold spring harb perspect med 2012;2:a006262. https://doi.org/10.1101/cshperspect.a006262 chen gf, xu th, yan y, et al. amyloid beta: structure, biology and structure-based therapeutic treatment. acta pharmacol sin 2017;38:1205-1235. https://doi.org/10.1038/aps.2017.28 fassbender k, simons m, bergmann c, et al. simvastatin strongly reduces levels of alzheimer's disease beta-amyloid peptides abeta 42 and abeta 40 in vitro and in vivo. proc natl acad sci usa 2001;98:5856-5861. https://doi.org/10.1073/pnas.081620098 wang h, kulas ja, wang c, et al. regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol. proc natl acad sci usa 2021;118:e2102191118. https://doi.org/10.1073/pnas.2102191118 ricciarelli r, fedele e. camp, cgmp and amyloid β: three ideal partners for memory formation. trends neurosci 2018;41:255-266. https://doi.org/10.1016/j.tins.2018.02.001 eckman ea, eckman cb. abeta-degrading enzymes: modulators of alzheimer's disease pathogenesis and targets for therapeutic intervention. biochem soc trans 2005;33:1101-1105. https://doi.org/10.1042/bst20051101 miners js, baig s, palmer j, et al. abeta-degrading enzymes in alzheimer's disease. brain pathol 2008;18:240-252. https://doi.org/10.1111/j.1750-3639.2008.00132.x love s, miners s, palmer j, et al. insights into the pathogenesis and pathogenicity of cerebral amyloid angiopathy. front biosci 2009;14:4778-4792. https://doi.org/10.2741/3567 bell rd, zlokovic bv. neurovascular mechanisms and blood-brain barrier disorder in alzheimer’s disease. acta neuropathol 2009;118:103-113. https://doi.org/10.1007/s00401-009-0522-3 miners js, baig s, tayler h, et al. neprilysin and insulin-degrading enzyme levels are increased in alzheimer disease in relation to disease severity. j neuropathol exp neurol 2009;68:902-914. https://doi.org/10.1097/nen.0b013e3181afe475 zlokovic bv, deane r, sagare ap, et al. low-density lipoprotein receptor-related protein-1: a serial clearance homeostatic mechanism controlling alzheimer's amyloid beta-peptide elimination from the brain. j neurochem 2010;115:1077-1089. https://doi.org/10.1111/j.1471-4159.2010.07002.x pacheco-quinto j, clausen d, pérez-gonzález r, et al. intracellular metalloprotease activity controls intraneuronal aβ aggregation and limits secretion of aβ via exosomes. faseb j 2019;33:3758-3771. https://doi.org/10.1096/fj.201801319r weller ro, djuanda e, yow hy, et al. lymphatic drainage of the brain and the pathophysiology of neurological disease. acta neuropathol 2009;117:1-14. https://doi.org/10.1007/s00401-008-0457-0 gouveia-freitas k, bastos-leite aj. perivascular spaces and brain waste clearance systems: relevance for neurodegenerative and cerebrovascular pathology. neuroradiology 2021;63:1581-1597. https://doi.org/10.1007/s00234-021-02718-7 foster em, dangla-valls a, lovestone s, et al. clusterin in alzheimer's disease: mechanisms, genetics, and lessons from other pathologies. front neurosci 2019;13:164. https://doi.org/10.3389/fnins.2019.00164 baig s, palmer le, owen mj, et al. clusterin mrna and protein in alzheimer's disease. j alzheimers dis 2012;28:337-344. https://doi.org/10.3233/jad-2011-110473 miners js, clarke p, love s. clusterin levels are increased in alzheimer's disease and influence the regional distribution of aβ. brain pathol 2017;27:305-313. https://doi.org/10.1111/bpa.12392 kida e, choi-miura nh, wisniewski ke. deposition of apolipoproteins e and j in senile plaques is topographically determined in both alzheimer's disease and down's syndrome brain. brain res 1995;685:211-216. https://doi.org/10.1016/0006-8993(95)00482-6 howlett dr, hortobágyi t, francis pt. clusterin associates specifically with aβ1-40 in alzheimer’s disease brain tissue. brain pathol 2013;23:623-632. https://doi.org/10.1111/bpa.12057 humphreys dt, carver ja, easterbrook-smith sb, et al. clusterin has chaperone-like activity similar to that of small heat shock proteins. j biol chem 1999; 274: 6875-6881. https://doi.org/10.1074/jbc.274.11.6875 oh sb, kim ms, park s, et al. clusterin contributes to early stage of alzheimer's disease pathogenesis. brain pathol 2019;29:217-231. https://doi.org/10.1111/bpa.12660 bell rd, sagare ap, friedman ae, et al. transport pathways for clearance of human alzheimer’s amyloid β-peptide and apolipoproteins e and j in the mouse central nervous system. j cereb blood flow metab 2007;27: 909-918. https://doi.org/10.1038/sj.jcbfm.9600419 tarasoff-conway jm, carare ro, osorio rs, et al. clearance systems in the brain—implications for alzheimer disease. nat rev neurol 2015;11:457-470. https://doi.org/10.1038/nrneurol.2015.119 calero m, rostagno a, matsubara e, et al. apolipoprotein j (clusterin) and alzheimer's disease. microsc res tech 2000;50:305-315. https://doi.org/10.1002/1097-0029(20000815)50:4<305::aid-jemt10>3.0.co;2-l matsubara e, soto c, governale s, frangione b, ghiso j. apolipoprotein j and alzheimer’s amyloid beta solubility. biochem j 1996;316:671-679. https://doi.org/10.1042/bj3160671 robbins jp, perfect l, ribe em, et al. clusterin is required for β-amyloid toxicity in human ipsc-derived neurons. front neurosci 2018;12:504. https://doi.org/10.3389/fnins.2018.00504 zhou y, hayashi i, wong j, et al. intracellular clusterin interacts with brain isoforms of the bridging integrator 1 and with the microtubule-associated protein tau in alzheimer's disease. plos one 2014;9:e103187. https://doi.org/10.1371/journal.pone.0103187 rijal upadhaya a, kosterin i, kumar s, et al. biochemical stages of amyloid-β peptide aggregation and accumulation in the human brain and their association with symptomatic and pathologically preclinical alzheimer's disease. brain 2014;137:887-903. https://doi.org/10.1093/brain/awt362 thal dr, walter j, saido tc, fändrich m. neuropathology and biochemistry of abeta and its aggregates in alzheimer's disease. acta neuropathol 2015;129:167-182. https://doi.org/10.1007/s00401-014-1375-y lambert mp, barlow ak, chromy ba, et al. diffusible, nonfibrillar ligands derived from abeta1-42 are potent central nervous system neurotoxins. proc natl acad sci usa 1998;95:6448-53. https://doi.org/10.1073/pnas.95.11.6448 gong y, chang l, viola kl, et al. alzheimer’s disease-affected brain: presence of oligomeric a beta ligands (addls) suggests a molecular basis for reversible memory loss. proc natl acad sci usa 2003;100:10417-10422. https://doi.org/10.1073/pnas.1834302100 hayden ey, teplow db. amyloid beta-protein oligomers and alzheimer’s disease, alzheimers res ther 2013; 5: 60. https://doi.org/10.1186/alzrt226 viola kl, klein wl. amyloid β oligomers in alzheimer's disease pathogenesis, treatment, and diagnosis. acta neuropathol 2015;129:183-206. https://doi.org/10.1007/s00401-015-1386-3 mroczko b, groblewska m, litman-zawadzka a, et al. cellular receptors of amyloid β oligomers (aβos) in alzheimer's disease. int j mol sci 2018;19:1884. https://doi.org/10.3390/ijms19071884 mroczko b, groblewska m, litman-zawadzka a, et al. amyloid β oligomers (aβos) in alzheimer's disease. j neural transm 2018;125:177-191. https://doi.org/10.1007/s00702-017-1820-x kayed r, head e, thompson jl, et al. common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. science 2003;300:486-489. https://doi.org/10.1126/science.1079469 deshpande a, mina e, glabe c, et al. different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. j neurosci 2006;26:6011-6018. https://doi.org/10.1523/jneurosci.1189-06.2006 glabe cg. common mechanisms of amyloid oligomer pathogenesis in degenerative disease. neurobiol aging 2006;27:570-575. https://doi.org/10.1016/j.neurobiolaging.2005.04.017 walsh dm, selkoe dj. a beta oligomers a decade of discovery. j neurochem 2007;101:1172-1184. https://doi.org/10.1111/j.1471-4159.2006.04426.x glabe cg, kayed r. common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis. neurology 2006;66:s74-8. https://doi.org/10.1212/01.wnl.0000192103.24796.42 shankar gm, li s, mehta th, et al. amyloid-beta protein dimers isolated directly from alzheimer's brains impair synaptic plasticity and memory. nat med 2008;14:837-842. https://doi.org/10.1038/nm1782 sengupta u, nilson an, kayed r. the role of amyloid-β oligomers in toxicity, propagation, and immunotherapy. ebiomedicine 2016;6:42-49. https://doi.org/10.1016/j.ebiom.2016.03.035 fukumoto h, tokuda t, kasai t, et al. high-molecular-weight beta-amyloid oligomers are elevated in cerebrospinal fluid of alzheimer patients. faseb j 2010;24:2716-2726. https://doi.org/10.1096/fj.09-150359 esparza tj, zhao h, cirrito jr, et al. amyloid-β oligomerization in alzheimer dementia versus high-pathology controls. ann neurol 2013;73:104-119. https://doi.org/10.1002/ana.23748 del río ja, ferrer i, gavín r. role of celular prion protein in interneuronal amyloid transmission. prog neurobiol 2018;165-167:87-102. https://doi.org/10.1016/j.pneurobio.2018.03.001 gunther ec, strittmatter sm. beta-amyloid oligomers and cellular prion protein in alzheimer’s disease. j mol. med 2010; 88: 331-338. https://doi.org/10.1007/s00109-009-0568-7 dohler f, sepulveda-falla d, krasemann s, et al. high molecular mass assemblies of amyloid-beta oligomers bind prion protein in patients with alzheimer’s disease. brain 2014; 137: 873-886. https://doi.org/10.1093/brain/awt375 fluharty br, biasini e, stravalaci m, et al. an n-terminal fragment of the prion protein binds to amyloid-beta oligomers and inhibits their neurotoxicity in vivo. j biol chem 2013;288:7857-7866. https://doi.org/10.1074/jbc.m112.423954 freir db, nicoll aj, klyubin i, et al. interaction between prion protein and toxic amyloid beta assemblies can be therapeutically targeted at multiple sites. nat commun 2011;2:336. https://doi.org/10.1038/ncomms1341 ganzinger ka, narayan p, qamar ss, et al. single-molecule imaging reveals that small amyloid-beta1-42 oligomers interact with the cellular prion protein (prp(c)). chembiochem 2014;15:2515-2521. https://doi.org/10.1002/cbic.201402377 lauren j, gimbel da, nygaard hb, et al. cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. nature 2009, 457, 1128-1132. https://doi.org/10.1038/nature07761 hartmann a, muth c, dabrowski o, et al. exosomes and the prion protein: more than one truth. front neurosci 2017;11:194. https://doi.org/10.3389/fnins.2017.00194 chaudhary h, meister sw, zetterberg h, et al. dissecting the structural organization of multiprotein amyloid aggregates using a bottom-up approach. acs chem neurosci 2020;11:1447-1457. https://doi.org/10.1021/acschemneuro.0c00110 rahman mm, lendel c. extracellular protein components of amyloid plaques and their roles in alzheimer's disease pathology. mol neurodegener 2021;16:59. https://doi.org/10.1186/s13024-021-00465-0 ferrer i, blanco r, carmona m, et al. prion protein expression in senile plaques in alzheimer's disease. acta neuropathol 2001;101:49-56. https://doi.org/10.1007/s004010000271 ferrer i, martí e, tortosa a, et al. dystrophic neurites of senile plaques are defective in proteins involved in exocytosis and neurotransmission. j neuropathol exp neurol 1998;57:218-225. https://doi.org/10.1097/00005072-199803000-00002 lenders mb, peers mc, tramu g, et al. dystrophic peptidergic neurites in senile plaques of alzheimer's disease hippocampus precede formation of paired helical filaments. brain res 1989;481:344-349. https://doi.org/10.1016/0006-8993(89)90812-3 benzing wc, brady dr, mufson ej, et al. evidence that transmitter-containing dystrophic neurites precede those containing paired helical filaments within senile plaques in the entorhinal cortex of nondemented elderly and alzheimer's disease patients. brain res 1993;619:55-68. https://doi.org/10.1016/0006-8993(93)91595-j benzing wc, ikonomovic md, brady dr, et al. evidence that transmitter-containing dystrophic neurites precede paired helical filament and alz-50 formation within senile plaques in the amygdala of nondemented elderly and patients with alzheimer's disease. j comp neurol 1993;334:176-191. https://doi.org/10.1002/cne.903340203 dickson dw, farlo j, davies p, et al. alzheimer's disease. a double-labeling immunohistochemical study of senile plaques. am j pathol 1988;132:86-101. pmid: 2456021. spires tl, hyman bt. neuronal structure is altered by amyloid plaques. rev neurosci. 2004;15:267-278. https://doi.org/10.1515/revneuro 2004.15.4.267. brion jp, couck am, bruce m, et al. synaptophysin and chromogranin a immunoreactivities in senile plaques of alzheimer's disease. brain res 1991;539:143-150. https://doi.org/10.1016/0006-8993(91)90697-t munoz dg. chromogranin a-like immunoreactive neurites are major constituents of senile plaques. lab invest 1991;64:826-832. pmid: 1710735. marksteiner j, kaufmann wa, gurka p, et al. synaptic proteins in alzheimer's disease. j mol neurosci 2002;18:53-63. https://doi.org/10.1385/jmn:18:1-2:53 barranco n, plá v, alcolea d, et https://doi.org/10.1385/jmn:18:1-2:53.al dense core vesicle markers in csf and cortical tissues of patients with alzheimer's disease. transl neurodegener 2021;10:37. https://doi.org/10.1186/s40035-021-00263-0 andrés-benito p, carmona m, jordán pirla m, et al. dysregulated protein phosphorylation as main contributor of granulovacuolar degeneration at the first stages of nft pathology. neuroscience 2021 oct 30;s0306-4522(21)00534-0. https://doi.org/10.1016/j.neuroscience.2021.10.023 woodhouse a, vickers jc, dickson tc. cytoplasmic cytochrome c immunolabelling in dystrophic neurites in alzheimer's disease. acta neuropathol 2006;112:429-437. https://doi.org/10.1007/s00401-006-0107-3 pérez-gracia e, torrejón-escribano b, ferrer i. dystrophic neurites of senile plaques in alzheimer's disease are deficient in cytochrome c oxidase. https://doi.org/10.1007/s00401-008-0370-6 armstrong dm, bruce g, hersh lb, et al. choline acetyltransferase immunoreactivity in neuritic plaques of alzheimer brain. neurosci lett 1986;71:229-234. https://doi.org/10.1016/0304-3940(86)90564-1 masliah e, mallory m, deteresa r, et al. differing patterns of aberrant neuronal sprouting in alzheimer's disease with and without lewy bodies. brain res 1993;617:258-266. https://doi.org/10.1016/0006-8993(93)91093-8 ferrer i, zújar mj, rivera r, et al. parvalmunin-immunoreactive dystrophic neurites and aberrant sprouts in the cerebral cortex of patients with alzheimer’s disease. neurosci lett 1993;158:163-166. https://doi.org/10.1016/0304-3940(93)90254-i adams la, munoz dg. differential incorporation of processes derived from different classes of neurons into senile plaques in alzheimer's disease. acta neuropathol 1993;86:365-370. https://doi.org/10.1007/bf00369449 dickson tc, king ce, mccormack gh, et al. neurochemical diversity of dystrophic neurites in the early and late stages of alzheimer’s disease. exp neurol 1999;156:100-110. https://doi.org/10.1006/exnr.1998.7010 adlard pa, vickers jc. morphologically distinct plaque-types differentially affect dendritic structure and organisation in the early and late stages of alzheimer’s disease. acta neuropathol 2002;103:377-383. https://doi.org/10.1007/s00401-001-0476-6 spires tl, meyer-luehmann m, stern ea, et al. dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. j neurosci 2005;25:7278-7287. https://doi.org/10.1523/jneurosci.1879-05.2005 knafo s, alonso-nanclares l, gonzalez-soriano j, et al. widespread changes in dendritic spines in a model of alzheimer's disease. cereb cortex. 2009;19:586-592. https://doi.org/10.1093/cercor/bhn111 robbins m, clayton e, kaminski schierle gs. synaptic tau: a pathological or physiological phenomenon? acta neuropathol commun 2021;9:149. https://doi.org/10.1186/s40478-021-01246-y alquezar c, arya s, kao aw. tau post-translational modifications: dynamic transformers of tau function, degradation, and aggregation. front neurol 2021;11:595532. https://doi.org/10.3389/fneur.2020.595532 hernández f, avila j. intraand extracellular protein interactions with tau. curr alzheimer res 2010;7:670-676. https://doi.org/10.2174/156720510793611583 rauch jn, olson sh, gestwicki je. interactions between microtubule-associated protein tau (mapt) and small molecules. cold spring harb perspect med 2017;7:a024034. https://doi.org/10.1101/cshperspect.a024034 borna h, assadoulahei k, riazi g, et al. structure, function and interactions of tau: particular focus on potential drug targets for the treatment of tauopathies. cns neurol disord drug targets 2018;17:325-337. https://doi.org/10.2174/1871527317666180525112008 trushina ni, bakota l, mulkidjanian ay, et al. the evolution of tau phosphorylation and interactions. front aging neurosci 2019;11:256. https://doi.org/10.3389/fnagi.2019.00256 brandt r, trushina ni, bakota l. much more than a cytoskeletal protein: physiological and pathological functions of the non-microtubule binding region of tau. front neurol 2020;11:590059. https://doi.org/10.3389/fneur.2020.590059 sinsky j, pichlerova k, hanes j. tau protein interaction partners and their roles in alzheimer's disease and other tauopathies. int j mol sci 2021;22:9207. https://doi.org/10.3390/ijms22179207 delacourte a, defossez a. alzheimer's disease: tau proteins, the promoting factors of microtubule assembly, are major components of paired helical filaments. j neurol sci 1986;76:173-186. https://doi.org/10.1016/0022-510x(86)90167-x kosik ks, joachim cl, selkoe dj. microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in alzheimer disease. proc natl acad sci usa 1986;83:4044-4048. https://doi.org/10.1073/pnas.83.11.4044 wood jg, mirra ss, pollock nj, et al. neurofibrillary tangles of alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (tau). proc natl acad sci usa 1986;83:4040-4043. https://doi.org/10.1073/pnas.83.11.4040 grundke-iqbal i, iqbal k, quinlan m, et al. microtubule-associated protein tau. a component of alzheimer paired helical filaments. j biol chem 1986;261: 6084-6089. pmid: 3084478. iqbal k, grundke-iqbal i, wisniewski hm. neuronal cytoskeleton in aging and dementia. prog brain res 1986;70:279-288. https://doi.org/10.1016/s0079-6123(08)64310-1 goedert m, wischik cm, crowther ra, et al. cloning and sequencing of the cdna encoding a core protein of the paired helical filament of alzheimer disease: identification as microtubule-associated protein tau. proc natl acad sci usa 1988;85: 4051-4055. https://doi.org/10.1073/pnas.85.11.4051 wischik cm, novak m, edwards pc, et al. structural characterization of the core of the paired helical filament of alzheimer disease. proc natl acad sci usa 1988;85:4884-4888. https://doi.org/10.1073/pnas.85.13.4884 kosik ks, orecchio ld, binder l, et al. epitopes that span the tau molecule are shared with paired helical filaments. neuron 1988;1:817-825. https://doi.org/10.1016/0896-6273(88)90129-8 delacourte a, buée l. normal and pathological tau proteins as factors of microtubule assembly. int rev cytol 1997;171:167-224. https://doi.org/10.1016/s0074-7696(08)62588-7 buée l, bussière t, buée-scherrer v, et al. tau protein isoforms, phosphorylation and role in neurodegenerative disorders. brain res brain res rev 2000; 33:95-130. https://doi.org/10.1016/s0165-0173(00)00019-9 iqbal k, grundke-iqbal i. discoveries of tau, abnormally hyperphosphorylated tau and others of neurofibrillary degeneration: a personal historical perspective. j alzheimers dis 2006;9:219-242. https://doi.org/10.3233/jad-2006-9s325 mandelkow em, mandelkow e. biochemistry and cell biology of tau protein in neurofbrillary degeneration. cold spring harb perspect med 2012;2: a006247. https://doi.org/10.1101/cshperspect.a006247 spillantini mg, goedert m. tau pathology and neurodegeneration. lancet neurol 2013;12: 609-622. https://doi.org/10.1016/s1474-4422(13)70090-5 arendt t, stieler jt, holzer m. tau and tauopathies. brain res bull 2016, 26: 238-292. https://doi.org/10.1016/j.brainresbull.2016.08.018 iqbal k, liu f, gong cx. tau and neurodegenerative disease: the story so far. nat rev neurol 2016;12:15-27. https://doi.org/10.1038/nrneurol.2015.225 goedert m, spillantini mg. ordered assembly of tau protein and neurodegeneration. adv exp med biol 2019;1184:3-21. https://doi.org/10.1007/978-981-32-9358-8_1 goedert m, spillantini mg, cairns nj, et al. tau proteins in alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms. neuron 1992; 8: 159-168. https://doi.org/10.1016/0896-6273(92)90117-v delacourte a, david jp, sergeant n, et al. the biochemical pathway of neurofibrillary degeneration in aging and alzheimer’s disease. neurology 1999;52: 1158-1165. https://doi.org/10.1212/wnl.52.6.1158 novak m, kabat j, wischik cm. molecular characterization of the minimal protease resistant tau unit of the alzheimer's disease paired helical filament. embo j 1993;12:365-370. https://doi.org/10.1002/j.1460-2075.1993.tb05665.x jicha ga, berenfeld b, davies p. sequence requirements for formation of conformational variants of tau similar to those found in alzheimer's disease. j neurosci res 1999; 55:713-723. https://doi.org/10.1002/(sici)1097-4547(19990315)55:6<713::aid-jnr6>3.0.co;2-g weaver cl, espinoza m, kress y, et al. conformational change as one of the earliest alterations of tau in alzheimer's disease. neurobiol aging 2000;21:719-727. https://doi.org/10.1016/s0197-4580(00)00157-3 gamblin tc, chen f, zambrano a, et al. caspase cleavage of tau: linking amyloid and neurofibrillary tangles in alzheimer's disease. proc natl acad sci usa 2003;100:10032-10037. https://doi.org/10.1073/pnas.1630428100 garcía-sierra f, ghoshal n, quinn b, et al. conformational changes and truncation of tau protein during tangle evolution in alzheimer's disease. j alzheimers dis 2003;5:65-77. https://doi.org/10.3233/jad-2003-5201 rissman ra, poon ww, blurton-jones m, et al. caspase-cleavage of tau is an early event in alzheimer disease tangle pathology. j clin invest 2004;114:121-130. https://doi.org/10.1172/jci20640 hyman bt, augustinack jc, ingelsson m. transcriptional and conformational changes of the tau molecule in alzheimer's disease. biochim biophys acta 2005;1739:150-157. https://doi.org/10.1016/j.bbadis.2004.06.015 andreadis a. tau gene alternative splicing: expression patterns, regulation and modulation of function in normal brain and neurodegenerative diseases. biochim biophys acta 2005;1739:91-103. https://doi.org/10.1016/j.bbadis.2004.08.010 avila j. tau phosphorylation and aggregation in alzheimer's disease pathology. febs lett 2006;580:2922-2927. https://doi.org/10.1016/j.febslet.2006.02.067 zilka n, filipcik p, koson p, et al. truncated tau from sporadic alzheimer's disease suffices to drive neurofibrillary degeneration in vivo. febs lett 2006;580:3582-3588. https://doi.org/10.1016/j.febslet.2006.05.029 luna-muñoz j, chávez-macías l, garcía-sierra f, et al. earliest stages of tau conformational changes are related to the appearance of a sequence of specific phospho-dependent tau epitopes in alzheimer's disease. j alzheimers dis 2007;12:365-375. https://doi.org/10.3233/jad-2007-12410 garcía-sierra f, mondragón-rodríguez s, basurto-islas g. truncation of tau protein and its pathological significance in alzheimer's disease. https://doi.org/10.3233/jad-2008-14407 mondragón-rodríguez s, basurto-islas g, santa-maria i, et al. cleavage and conformational changes of tau protein follow phosphorylation during alzheimer's disease. int j exp pathol 2008;89:81-90. https://doi.org/10.1111/j.1365-2613.2007.00568.x martin l, latypova x, terro f. post-translational modifications of tau protein: implications for alzheimer's disease. neurochem int 2011;58:458-471. https://doi.org/10.1016/j.neuint.2010.12.023 cohen tj, guo jl, hurtado de, et al. the acetylation of tau inhibits its function and promotes pathological tau aggregation. nat commun 2011;2:252. https://doi.org/10.1038/ncomms1255 takashima a. tauopathies and tau oligomers. j alzheimers dis 2013;37:565-568. https://doi.org/10.3233/jad-130653 hernández f, pérez m, gómez de barreda e et al. tau as a molecular marker of development, aging and neurodegenerative disorders. curr aging sci 2008;1:56-61. https://doi.org/10.2174/1874609810801010056 fitzpatrick awp, falcon b, he s, et al. cryo-em structures of tau filaments from alzheimer's disease. nature 2017; 547:185-190. https://doi.org/10.1038/nature23002 ramesh m, gopinath p, govindaraju t. role of post-translational modifications in alzheimer's disease. chembiochem 2020;21:1052-1079. https://doi.org/10.1002/cbic.201900573 wang d, huang x, yan l, et al. the structure biology of tau and clue for aggregation inhibitor design. protein j 2021;40:656-668. https://doi.org/10.1007/s10930-021-10017-6 moloney cm, lowe vj, murray me. visualization of neurofibrillary tangle maturity in alzheimer's disease: a clinicopathologic perspective for biomarker research. alzheimers dement 2021;17:1554-1574. https://doi.org/10.1002/alz.12321 ferrer i, lópez-gonzález i, carmona m, et al. glial and neuronal tau pathology in tauopathies: characterization of disease-specific phenotypes and tau pathology progression. j neuropathol exp neurol 2014;73:81-97. https://doi.org/10.1097/nen.0000000000000030 kametani f, yoshida m, matsubara t, et al. comparison of common and disease-specific post-translational modifications of pathological tau associated with a wide range of tauopathies. front neurosci 2020; 14: 581936. https://doi.org/10.3389/fnins.2020.581936 wang jz, xia yy, grundke-iqbal i, et al. abnormal hyperphosphorylation of tau: sites, regulation, and molecular mechanism of neurofibrillary degeneration. j alzheimers dis 2013;33:s123-139. https://doi.org/10.3233/jad-2012-129031 hanger dp, anderton bh, noble w. tau phosphorylation: the therapeutic challenge for neurodegenerative disease. trends mol med 2009;15:112-119. https://doi.org/10.1016/j.molmed.2009.01.003 ferrer i, blanco r, carmona m, et al. phosphorylated map kinase (erk1, erk2) expression is associated with early tau deposition in neurones and glial cells, but not with increased nuclear dna vulnerability and cell death, in alzheimer disease, pick's disease, progressive supranuclear palsy and corticobasal degeneration. brain pathol 2001;11:144-158. https://doi.org/10.1111/j.1750-3639.2001.tb00387.x ferrer i, blanco r, carmona m, et al. phosphorylated mitogen-activated protein kinase (mapk/erk-p), protein kinase of 38 kda (p38-p), stress-activated protein kinase (sapk/jnk-p), and calcium/calmodulin-dependent kinase ii (cam kinase ii) are differentially expressed in tau deposits in neurons and glial cells in tauopathies. j neural transm 2001;108:1397-415. https://doi.org/10.1007/s007020100016 ferrer i, gomez-isla t, puig b, et al. current advances on different kinases involved in tau phosphorylation, and implications in alzheimer's disease and tauopathies. curr alzheimer res 2005;2:3-18. https://doi.org/10.2174/1567205052772713 guimaraes tr, swanson e, kofler j, et al. g-protein-coupled receptor kinases are associated with alzheimer’s disease pathology. neuropathol appl neurobiol 2021;47:942-957. https://doi.org/10.1111/nan.12742 cowan cm, quraishe s, mudher a what is the pathological significance of tau oligomers? biochem soc trans 2012;40:693-697. https://doi.org/10.1042/bst20120135 moraga dm, nunez p, garrido j, et al. a tau fragment containing a repetitive sequence induces bundling of actin filaments. j neurochem 1993;61:979-986. https://doi.org/10.1111/j.1471-4159.1993 tb03611.x. maas t, eidenmüller j, brandt r. interaction of tau with the neural membrane cortex is regulated by phosphorylation at sites that are modified in paired helical filaments. j biol chem 2000;275:15733-15740. https://doi.org/10.1074/jbc.m00038920 shea tb, chan a. s-adenosyl methionine: a natural therapeutic agent effective against multiple hallmarks and risk factors associated with alzheimer's disease. j alzheimers dis 2008;13:67-70. https://doi.org/10.3233/jad-2008-13107 schneider a, biernat j, von bergen m, et al. phosphorylation that detaches tau protein from microtubules (ser262, ser214) also protects it against aggregation into alzheimer paired helical filaments. biochemistry 1999;38:3549-3558. https://doi.org/10.1021/bi981874p farias ga, muñoz jp, garrido j, et al. tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies. j cell biochem 2002;85:315-324. https://doi.org/10.1002/jcb.10133 mohan r, john a. microtubule-associated proteins as direct crosslinkers of actin filaments and microtubules. iubmb life 2015;67:395-403. https://doi.org/10.1002/iub.1384 sallaberry ca, voss bj, majewski j, et al. tau and membranes: interactions that promote folding and condensation. front cell develop biol 2021;9:725241. https://doi.org/10.3389/fcell.2021.725241 brunello ca, merezhko m, uronen rl, et al. mechanisms of secretion and spreading of pathological tau protein. cell mol life sci 2020;77:1721-1744. https://doi.org/10.1007/s00018-019-03349-1 bok e, leem e, lee br, et al. role of the lipid membrane and membrane proteins in tau pathology. front cell dev biol 2021;9:653815. https://doi.org/10.3389/fcell.2021.653815 annadurai n, de sanctis jb, hajdúch m, et al. tau secretion and propagation: perspectives for potential preventive interventions in alzheimer's disease and other tauopathies. exp neurol 2021;343:113756. https://doi.org/10.1016/j.expneurol.2021.113756 santa-maria i, varghese m, ksiéżak-reding h, et al. paired helical filaments from alzheimer disease brain induces intracellular accumulation of tau protein in aggresomes. j biol chem 2012: 287: 2052220533. https://doi.org/10.1074/jbc.m111.323279 falcon b, zhang w, schweighauser m, et al. tau filaments from multiple cases of sporadic and inherited alzheimer's disease adopt a common fold. acta neuropathol 2018;136:699-708. https://doi.org/10.1007/s00401-018-1914-z falcon b, zhang w, murzin ag, et al. structures of filaments from pick's disease reveal a novel tau protein fold. nature 2018;561:137-140. https://doi.org/10.1038/s41586-018-0454-y falcon b, zivanov j, zhang w, et al. novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules. nature 2019;568:420-423. https://doi.org/10.1038/s41586-019-1026-5 zhang w, tarutani a, newell kl, et al. novel tau filament fold in corticobasal degeneration. nature 2020;580:283-287. https://doi.org/10.1038/s41586-020-2043-0 arakhamia t, lee ce, carlomagno y, et al. posttranslational modifications mediate the structural diversity of tauopathy strains. cell 2020;180:633-644.e12. https://doi.org/10.1016/j.cell.2020.01.027 shi y, murzin ag, falcon b, et al. cryo-em structures of tau filaments from alzheimer's disease with pet ligand apn-1607. acta neuropathol 2021;141: 697-708. https://doi.org/10.1007/s00401-021-02294-3 shi y, zhang w, yang y, et al. structure-based classification of tauopathies. nature 2021;598:359-363. https://doi.org/10.1038/s41586-021-03911-7 smith r, strandberg o, mattsson-carlgren n, et al. the accumulation rate of tau aggregates is higher in females and younger amyloid-positive subjects. brain 2020;143:3805-3815. https://doi.org/10.1093/brain/awaa327 davis ej, carolinew. solsberg cw, et al. sex-specific association of the x chromosome with cognitive change and tau pathology in aging and alzheimer disease. jama neurol 2021; 78:1249-1254. https://doi.org/10.1001/jamaneurol.2021.2806 meier s, bell m, lyons dn, et al. identification of novel tau interactions with endoplasmic reticulum proteins in alzheimer’s disease brain. j alzheimers dis 2015; 48: 687-702. https://doi.org/10.3233/jad-150298 ayyadevara s, balasubramaniam m, parcon pa, et al. proteins that mediate protein aggregation and cytotoxicity distinguish alzheimer’s hippocampus from normal controls. aging cell 2016; 15: 924-939. https://doi.org/10.1111/acel.12501 drummond e, pires g, macmurray c, et al. phosphorylated tau interactome in the human alzheimer’s disease brain. brain 2020;143:2803-2817. https://doi.org/10.1093/brain/awaa223 van leeuwen fw, fischer df, kamel d, et al. molecular misreading: a new type of transcript mutation expressed during aging. neurobiol aging 2000;21:879-891. https://doi.org/10.1016/s0197-4580(00)00151-2 van leeuwen fw, hol em, fischer df. frameshift proteins in alzheimer's disease and in other conformational disorders: time for the ubiquitin-proteasome system. j alzheimers dis 2006;9:319-325. https://doi.org/10.3233/jad-2006-9s336 vergara c, ordonez-gutierrez l, wandosell f, et al. role of prp(c) expression in tau protein levels and phosphorylation in alzheimer’s disease evolution. mol neurobiol 2015;51:1206-1220. https://doi.org/10.1007/s12035-014-8793-7 chen rj, chang ww, lin yc, et al. alzheimer’s amyloid-beta oligomers rescue cellular prion protein induced tau reduction via fyn pathways. acs chem neurosci 2013; 4:1287-1296. https://doi.org/10.1021/cn400085q schmitz m, wulf k, signore sc, et al. impact of the cellular prion protein on amyloid-beta and 3po-tau processing. j alzheimers dis 2014;38: 551-565. https://doi.org/10.3233/jad-130566 lidón l, vergara c, ferrer i, et al. tau protein as a new regulator of cellular prion protein transcription. mol neurobiol 2020;57:4170-4186. https://doi.org/10.1007/s12035-020-02025-x lidón l, llaó-hierro l, nuvolone m, et al. tau exon 10 inclusion by prpc through down-regulating gsk3β activity. int j mol sci 2021;22: 5370. https://doi.org/10.3390/ijms22105370 gavín r, lidón l, ferrer i, del río ja. the quest for cellular prion protein functions in the aged and neurodegenerating brain. cells 2020;9:591. https://doi.org/10.3390/cells9030591 goate a, chartier-harlin mc, mullan m, et al. segregation of a missense mutation in the amyloid precursor protein gene with familial alzheimer's disease. nature 1991;349:704-706. https://doi.org/10.1038/349704a0 chartier-harlin mc, crawford f, houlden h, et al. early-onset alzheimer's disease caused by mutations at codon 717 of the β-amyloid precursor protein gene. nature 1991; 353: 844-846. https://doi.org/10.1038/353844a0 murrell j, farlow m, ghetti b, et al. a mutation in the amyloid precursor protein associated with hereditary alzheimer's disease. science 1991;254:97-99. https://doi.org/10.1126/science.1925564 levy-lahad e, wasco w, poorkaj p, et al. candidate gene for the chromosome 1 familial alzheimer's disease locus. science 1995; 18;269:973-977. https://doi.org/10.1126/science.7638622 sherrington r, rogaev ei, liang y, et al. cloning of a gene bearing missense mutations in early-onset familial alzheimer's disease. nature 1995;375:754-760. https://doi.org/10.1038/375754a0 rogaev ei, sherrington r, rogaeva ea, et al. familial alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the alzheimer's disease type 3 gene. nature 1995;376:775-778. https://doi.org/10.1038/376775a0 goedert m, spillantini mg. a century of alzheimer’s disease. science 2006; 314: 777-781. https://doi.org/10.1126/science.1132814 bertram l, tanzi re. genetics of alzheimer’s disease. in: dickson dw, weller ro (eds) the molecular pathology of dementias and movement disorders, 2nd edn. wiley-blackwell, oxford, 2011; pp 51-61 hardy ja, higgins ga. alzheimer’s disease: the amyloid cascade hypothesis. science 1992; 256:184-185. https://doi.org/10.1126/science.1566067 lewis j, dickson dw, lin wl, et al. enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and app. science 2001;293:1487-1491. https://doi.org/10.1126/science.1058189 ribé em, pérez m, puig b, et al. accelerated amyloid deposition, neurofibrillary degeneration, and neuronal loss in double mutant app/tau transgenic mice. neurobiol dis 2005;20:814-822. https://doi.org/10.1016/j.nbd.2005.05.027 samura e, shoji m, kawarabayashi t, et al. enhanced accumulation of tau in doubly transgenic mice expressing mutant betaapp and presenilin-1. brain res 2006;1094:192-199. https://doi.org/10.1016/j.brainres.2005.12.134 bennett re, devos sl, dujardin s, et al. enhanced tau aggregation in the presence of amyloid β. am j pathol 2017;187:1601-1612. https://doi.org/10.1016/j.ajpath.2017.03.011 hyman bt, west hl, rebeck gw, et al. neuropathological changes in down's syndrome hippocampal formation. effect of age and apolipoprotein e genotype. arch neurol 1995; 52:373-378. https://doi.org/10.1001/archneur.1995.00540280059019 hof pr, bouras c, perl dp, et al. age-related distribution of neuropathological changes in the cerebral cortex of patients with down's syndrome. arch neurol 1995; 52:379-391. https://doi.org/10.1001/archneur.1995.00540280065020 lemere ca, blusztajn jk, yamaguchi h, et al. sequence of deposition of heterogeneous amyloid beta-peptides and apo e in down syndrome: implications for initial events in amyloid plaque formation. neurobiol dis 1996;3:16-32. https://doi.org/10.1006/nbdi.1996.0003 hartley d, blumenthal t, carrillo m, et al. down syndrome and alzheimer’s disease: common pathways, common goals. alzheimers dement 2015;11:700-709. https://doi.org/10.1016/j.jalz.2014.10.007 tu s, okamoto s, lipton sa, et al. oligomeric aβ-induced synaptic dysfunction in alzheimer's disease. mol neurodegener 2014;9:48. https://doi.org/10.1186/1750-1326-9-48 hillen h. the beta-amyloid dysfunction (bad) hypothesis for alzheimer’s disease. front neurosci 2019; 13:1154. https://doi.org/10.3389/fnins.2019.01154 selkoe dj, hardy j. the amyloid hypothesis of alzheimer's disease at 25 years. embo mol med 2016; 8:595-608. https://doi.org/10.15252/emmm.201606210 cline en, bicca ma, viola kl, et al. the amyloid-β oligomer hypothesis: beginning of the third decade. j alzheimers dis 2018;64:s567-s610. https://doi.org/10.3233/jad-179941 van helmond z, scott miners j, kehoe pg, et al. higher soluble amyloid beta concentration in frontal cortex of young adults than in normal elderly or alzheimer's disease. brain pathol 2010;20:787-793. https://doi.org/10.1111/j.1750-3639.2010.00374.x miners js, jones r, love s. differential changes in aβ42 and aβ40 with age. j alzheimers dis 2014;40:727-735. https://doi.org/10.3233/jad-132339 van der kant r, goldstein lsb, ossenkoppele r. amyloid-beta-independent regulators of tau pathology in alzheimer disease. nat rev neurosci 2020;21:21-35. https://doi.org/10.1038/s41583-019-0240-3 ochalek a, mihalik b, avci hx, et al. neurons derived from sporadic alzheimer's disease ipscs reveal elevated tau hyperphosphorylation, increased amyloid levels, and gsk3b activation. alzheimers res ther 2017;9:90. https://doi.org/10.1186/s13195-017-0317-z holton jl, ghiso j, lashley t, et al. regional distribution of amyloid-bri deposition and its association with neurofibrillary degeneration in familial british dementia. am j pathol 2001;158:515-526. https://doi.org/10.1016/s0002-9440(10)63993-4 garringer hj, murrell j, sammeta n, et al. increased tau phosphorylation and tau truncation, and decreased synaptophysin levels in mutant bri2/tau transgenic mice. plos one 2013;8:e56426. https://doi.org/10.1371/journal.pone.0056426 alzualde a, indakoetxea b, ferrer i, et al. a novel prnp y218n mutation in gerstmann–sträussler–scheinker disease with neurofibrillary degeneration j neuropathol exp neurol 2010; 69: 789-800. https://doi.org/10.1097/nen.0b013e3181e85737 ghetti b, piccardo p, zanusso g. dominantly inherited prion protein cerebral amyloidoses a modern view of gerstmann-sträussler-scheinker. handb clin neurol 2018;153:243-269. https://doi.org/10.1016/b978-0-444-63945-5.00014-3 hallinan gi, hoq mr, ghosh m, et al. structure of tau filaments in prion protein amyloidoses. acta neuropathol 2021;142:227-241. https://doi.org/10.1007/s00401-021-02336-w gibson g, gunasekera n, lee m, et al. oligomerization and neurotoxicity of the amyloid adan peptide implicated in familial danish dementia. j neurochem 2004;88:281-290. https://doi.org/10.1046/j.1471-4159.2003.02134.x corder eh, saunders am, strittmatter wj, et al. gene dose of apolipoprotein e type 4 allele and the risk of alzheimer’s disease in late onset families. science 1993; 261, 921–923. https://doi.org/10.1126/science.8346443 saunders am, strittmatter wj, schmechel d, et al. association of apolipoprotein e allele epsilon 4 with late-onset familial and sporadic alzheimer's disease. neurology 1993; 43: 1467-1472. https://doi.org/10.1212/wnl.43.8.1467 strittmatter wj, saunders am, schmechel d, et al. apolipoprotein e: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial alzheimer disease. proc natl acad sci usa 1993; 90: 1977-1981. https://doi.org/10.1073/pnas.90.5.1977 farrer la, cupples la, haines jl, et al. effects of age, sex, and ethnicity on the association between apolipoprotein e genotype and alzheimer disease. a meta-analysis. apoe and alzheimer disease meta analysis consortium. jama 1997; 27, 1349-1356. pmid: 9343467 harold d, abraham r, hollingworth p, et al. genome-wide association studies identifies variants clu and picalm associated with alzheimer’s disease. nat genet 2009;41:1088-1093. https://doi.org/10.1038/ng.440 lambert jc, heath s, even g, et al. genome-wide association study identifies variants at clu and cr1 associated with alzheimer's disease. nat genet 2009;41:1094-1099. https://doi.org/10.1038/ng.439 seshadri s, fitzpatrick al, ikram ma, et al. genome-wide analysis of genetic loci associated with alzheiomer’s disease. jama 2010;303:1832-1840. https://doi.org/10.1001/jama.2010.574 jun g, naj ac, beecham gw, et al. meta-analysis confirms cr1, clu, and picalm as alzheimer disease risk loci and reveals interactions with apoe genotypes. arch neurol 2010;67:1473-1484. https://doi.org/10.1001/archneurol.2010.201 jones l, holmans pa, hamshere ml, et al. genetic evidence implicates the immune system and cholesterol metabolism in the aetiology of alzheimer’s disease. plos one. 2010;5:e13950. https://doi.org/10.1371/journal.pone.0013950 hollingworth p, harold d, sims r, et al. common variants at abca7, ms4a6a/ms4a4e, epha1, cd33 and cd2ap are associated with alzheimer’s disease. nat genet 2011;43:429-435. https://doi.org/10.1038/ng.803 lambert jc, ibrahim-verbaas ca, harold d, et al. meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for alzheimer's disease. nat genet 2013; 45: 1452-1458. https://doi.org/10.1038/ng.2802 naj ac, jun g, reitz c, et al. effects of multiple genetic loci on age at onset in late-onset alzheimer disease: a genome-wide association study. jama neurol 2014;71:1394-1404. https://doi.org/10.1001/jamaneurol.2014.1491 sims r, van der lee sj, naj ac et al. rare coding variants in plcg2, abi3, and trem2 implicate microglial-mediated innate immunity in alzheimer's disease. nat genet 2017;49:1373-1384. https://doi.org/10.1038/ng.3916 marioni re, harris se, zhang q, et al. gwas on family history of alzheimer's disease. transl psychiat 2018; 8: 99. https://doi.org/10.1038/s41398-018-0150-6 pimenova aa, raj t, goate am. untangling genetic risk for alzheimer's disease. biol psychiat 2018; 83: 300-310. https://doi.org/10.1016/j.biopsych.2017.05.014 jansen ie, savage je, watanabe k, et al. genome-wide meta-analysis identifies new loci and functional pathways influencing alzheimer's disease risk. nat genet 2019; 51: 404-413. https://doi.org/10.1038/s41588-018-0311-9 kunkle bw, grenier-boley b, sims r, et al. genetic meta-analysis of diagnosed alzheimer’s disease identifies new risk loci and implicates aβ, tau, immunity and lipid processing. nat genet 2019; 51: 414-430. https://doi.org/10.1038/s41588-019-0358-2 andrews sj, fulton-howard b, goate a. interpretation of risk loci from genome-wide association studies of alzheimer's disease. lancet neurol 2020;19:326-335. https://doi.org/10.1016/s1474-4422(19)30435-1 tansey ke, cameron d, hill mj. genetic risk for alzheimer's disease is concentrated in specific macrophage and microglial transcriptional networks. genome med 2018;10:14. https://doi.org/10.1186/s13073-018-0523-8 bellenguez c, grenier-boley b, lambert jc. genetics of alzheimer's disease: where we are, and where we are going. curr opin neurobiol 2020;61:40-48. https://doi.org/10.1016/j.conb.2019.11.024 bellenguez c, küçükali f, jansen ie, et al. new insights into the genetic etiology of alzheimer’s disease and related dementias. nat genet 2022;54:412-436. https://doi.org/10.1038/s41588-022-01024-z vogt ba, vogt lj, hof pr. patterns of cortical neurodegeneration in alzheimer’s disease : subgroups, subtypes, and implications for staging strategies. in functional neurobiology of aging. hof pr, mobbs cv (eds). academic press 2001, pp: 111-129 hof pr, cox k, morrison jh. quantitative analysis of a vulnerable subset of pyramidal neurons in alzheimer's disease: i. superior frontal and inferior temporal cortex. j comp neurol 1990;301:44-54. https://doi.org/10.1002/cne.903010105 hof pr, morrison jh. quantitative analysis of a vulnerable subset of pyramidal neurons in alzheimer's disease: ii. primary and secondary visual cortex. j comp neurol 1990;30:55-64. https://doi.org/10.1002/cne.903010106 morrison jh, lewis da, campbell mj, et al. a monoclonal antibody to non-phosphorylated neurofilament protein marks the vulnerable cortical neurons in alzheimer's disease. brain res 1987;416:331-336. https://doi.org/10.1016/0006-8993(87)90914-0 vickers jc, riederer bm, marugg ra, et al. alterations in neurofilament protein immunoreactivity in human hippocampal neurons related to normal aging and alzheimer's disease. neuroscience 1994;62:1-13. https://doi.org/10.1016/0306-4522(94)90310-7 morrison jh, hof pr. life and death of neurons in the aging brain. science 1997;278:412-419. https://doi.org/10.1126/science.278.5337 412. vickers jc, dickson tc, adlard pa, et al. the cause of neuronal degeneration in alzheimer's disease. prog neurobiol 2000;60:139-165. https://doi.org/10.1016/s0301-0082(99)00023-4 chan-palay v. somatostatin immunoreactive neurons in the human hippocampus and cortex shown by immunogold/silver intensification on vibratome sections: coexistence with neuropeptide y neurons, and effects in alzheimer-type dementia. j comp neurol 1987; 260: 201-223. https://doi.org/10.1002/cne.902600205 rossor mn, emson pc, mountjoy cq, et al. reduced amounts of immunoreactive somatostatin in the temporal cortex in senile dementia of alzheimer type. neurosci lett 1980; 20: 373-377. https://doi.org/10.1016/0304-3940(80)90177-9 rossor mn, garrett nj, johnson al, et al. a post-mortem study of the cholinergic and gaba systems in senile dementia. brain 1982;105:313-330. https://doi.org/10.1093/brain/105.2.313 hardy j, cowburn r, barton a, et al. a disorder of cortical gabaergic innervation in alzheimer's disease. neurosci lett 1987;73:192-196. https://doi.org/10.1016/0304-3940(87)90016-4 simpson md, cross aj, slater p, deakin jf. loss of cortical gaba uptake sites in alzheimer's disease. j neural transm 1988;71:219-226. https://doi.org/10.1007/bf01245715 gabriel sm, bierer lm, harotunian v, et al. widespread deficits in somatostatin but not neuropeptide y concentrations in alzheimer's disease cerebral cortex. neurosci lett 1993;155:116-120. https://doi.org/10.1016/0304-3940(93)90686-f beal mf, mazurek mf, tran vt, et al. reduced numbers of somatostatin receptors in the cerebral cortex in alzheimer's disease. science 1985;229:289-291. https://doi.org/10.1126/science.2861661 baimbridge kg, celio mr, rogers jh. calcium-binding proteins in the nervous system. trends neurosci 1992;15:303-308. https://doi.org/10.1016/0166-2236(92)90081-i klausberger t, somogyi p. neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. science 2008;321:53-57. https://doi.org/10.1126/science.1149381 tremblay r, lee s, rudy b. gabaergic interneurons in the neocortex: from cellular properties to circuits. neuron 2016;91:260-292. https://doi.org/10.1016/j.neuron.2016.06.033 ferrer i, soriano e, tuñón t, et al. parvalbumin immunoreactive neurons in normal human temporal neocortex and in patients with alzheimer's disease. j neurol sci 1991;106:135-141. https://doi.org/10.1016/0022-510x(91)90250-b hof pr, cox k, young wg, et al. parvalbumin-immunoreactive neurons in the neocortex are resistant to degeneration in alzheimer's disease. j neuropathol exp neurol 1991;50:451-462. https://doi.org/10.1097/00005072-199107000-00006 sampson vl, morrison jh, vickers jc. the cellular basis for the relative resistance of parvalbumin and calretinin immunoreactive neocortical neurons to the pathology of alzheimer's disease. exp neurol 1997;145:295-302. https://doi.org/10.1006/exnr.1997.6433 leuba g, kraftsik r, saini k. quantitative distribution of parvalbumin, calretinin, and calbindin d-28k immunoreactive neurons in the visual cortex of normal and alzheimer cases. exp neurol 1998;152:278-291. https://doi.org/10.1006/exnr.1998.6838 arai h, emson pc, mountjoy cq, et al. loss of parvalbumin-immunoreactive neurones from cortex in alzheimer-type dementia. brain res 1987;418:164-169. https://doi.org/10.1016/0006-8993(87)90974-7 brady dr, mufson ej. parvalbumin-immunoreactive neurons in the hippocampal formation of alzheimer's diseased brain. neuroscience 1997;80:1113-1125. https://doi.org/10.1016/s0306-4522(97)00068-7 fonseca m, soriano e, ferrer i, et al. chandelier cell axons identified by parvalbumin-immunoreactivity in the normal human temporal cortex and in alzheimer's disease. neuroscience 1993; 55: 1107-1116. https://doi.org/10.1016/0306-4522(93)90324-9 inaguma y, shinohara h, inagaki t, et al. immunoreactive parvalbumin concentrations in parahippocampal gyrus decrease in patients with alzheimer's disease. j neurol sci 1992;110:57-61. https://doi.org/10.1016/0022-510x(92)90009-a mikkonen m, alafuzoff i, tapiola t, et al. subfieldand layer-specific changes in parvalbumin, calretinin and calbindin-d28k immunoreactivity in the entorhinal cortex in alzheimer's disease. neuroscience 1999;92:515-532. https://doi.org/10.1016/s0306-4522(99)00047-0 satoh j, tabira t, sano m, et al. parvalbumin-immunoreactive neurons in the human central nervous system are decreased in alzheimer's disease. acta neuropathol 1991; 81:388-395. https://doi.org/10.1007/bf00293459 solodkin a, veldhuizen sd, van hoesen gw. contingent vulnerability of entorhinal parvalbumin-containing neurons in alzheimer's disease. j neurosci 1996;16:3311-3321. hof pr, morrison jh. neocortical neuronal subpopulations labeled by a monoclonal antibody to calbindin exhibit differential vulnerability in alzheimer's disease. exp neurol 1991;111:293-301. https://doi.org/10.1523/jneurosci.16-10-03311.1996 ferrer i, tuñon t, soriano e, et al. calbindin d-28k immunoreactivity in the temporal neocortex in patients with alzheimer's disease. clin neuropathol 1993;12:53-58. pmid: 8440080 fonseca m, soriano e. calretinin-immunoreactive neurons in the normal human temporal cortex and in alzheimer's disease. brain res 1995;691:83-91. https://doi.org/10.1016/0006-8993(95)00622-w hof pr, nimchinsky ea, celio mr, et al. calretinin-immunoreactive neocortical interneurons are unaffected in alzheimer's disease. neurosci lett 1993;152:145-148. https://doi.org/10.1016/0304-3940(93)90504-e brion jp, resibois a. a subset of calretinin-positive neurons are abnormal in alzheimer's disease. acta neuropathol 1994;88:33-43. https://doi.org/10.1007/bf00294357 west mj, coleman pd, flood dg, et al. differences in the pattern of hippocampal neuronal loss in normal ageing and alzheimer's disease. lancet 1994;344:769-772. https://doi.org/10.1016/s0140-6736(94)92338-8 gómez-isla t, price jl, mckeel dw jr, et al. profound loss of layer ii entorhinal cortex neurons occurs in very mild alzheimer's disease. j neurosci 1996;16:4491-500. https://doi.org/10.1523/jneurosci.16-14-04491.1996 simić g, kostović i, winblad b, et al. volume and number of neurons of the human hippocampal formation in normal aging and alzheimer's disease. j comp neurol 1997;379:482-494. https://doi.org/10.1002 (sici)1096-9861(19970324)379:4<482::aid-cne2>3.0.co;2-z. braak h, braak e. neuropathological stageing of alzheimer-related changes. acta neuropathol 1991; 82: 239-259. https://doi.org/10.1007/bf00308809 braak h, braak e. staging of alzheimer's disease-related neurofibrillary changes. neurobiol aging 1995;16:271-278. https://doi.org/10.1016/0197-4580(95)00021-6 braak h, alafuzoff i, arzberger t, et al. staging of alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. acta neuropathol 2006;112:389-404. https://doi.org/10.1007/s00401-006-0127-z braak h, del tredici k. the pathological process underlying alzheimer's disease in individuals under thirty. acta neuropathol 2011;121:171-181. https://doi.org/10.1007/s00401-010-0789-4 ferrer i. defining alzheimer as a common age-related neurodegenerative process not inevitably leading to dementia. prog neurobiol 2012; 97: 38-51. https://doi.org/10.1016/j.pneurobio 2012.03.005. tsartsalis s, aikaterini xekardaki a, hof pr, et al. early alzheimer-type lesions in cognitively normal subjects. neurobiol aging 2018; 62: 34-44. https://doi.org/10.1016/j.neurobiolaging.2017.10.002 nelson pt, alafuzoff i, bigio eh, et al. correlation of alzheimer disease neuropathologic changes with cognitive status: a review of the literature. j neuropathol exp neurol 2012; 71:362-381. https://doi.org/10.1097/nen.0b013e31825018f7 liang ws, dunckley t, beach tg, et al. altered neuronal gene expression in brain regions differentially affected by alzheimer’s disease: a reference data set. physiol genomics 2008; 33: 240-256. https://doi.org/10.1152/physiolgenomics.00242.2007 murray me, graff-radford nr, ross oa, et al. neuropathologically defined subtypes of alzheimer's disease with distinct clinical characteristics: a retrospective study. lancet neurol 2011;10:785-796. https://doi.org/10.1016/s1474-4422(11)70156-9 jellinger ka. pathobiological subtypes of alzheimer disease. dement geriatr cogn disord 2020a;49:321-333. https://doi.org/10.1159/000508625 grinberg lt, rüb u, ferretti re, et al. the dorsal raphe nucleus shows phospho-tau neurofibrillary changes before the transentorhinal region in alzheimer's disease. a precocious onset? neuropathol appl neurobiol 2009;35:406-416. https://doi.org/10.1111/j.1365-2990.2009.00997.x braak h, del tredici k. the preclinical phase of the pathological process underlying sporadic alzheimer's disease. brain 2015; 138:2814-2833. https://doi.org/10.1093/brain/awv236 arendt t, bruckner mk, morawski m, et al. early neurone loss in alzheimer’s disease: cortical or subcortical? acta neuropathol commun 2015; 3:10. https://doi.org/10.1186/s40478-015-0187-1 andrés-benito p, fernández-dueñas v, carmona m, et al. locus coeruleus at asymptomatic early and middle braak stages of neurofibrillary tangle pathology. neuropathol appl neurobiol 2017;43:373-392. https://doi.org/10.1111/nan.12386 eser ra, ehrenberg aj, petersen c, et al. selective vulnerability of brainstem nuclei in distinct tauopathies: a postmortem study. j neuropathol exp neurol 2018;77:149-161. https://doi.org/10.1093/jnen/nlx113 matchett bj, grinberg lt, theofilas p, et al. the mechanistic link between selective vulnerability of the locus ceruleus and neurodegeneration in alzheimer’s disease. acta neuropathol 2021;141:631-650. https://doi.org/10.1007/s00401-020-02248-1 arnold se, hyman bt, flory j, et al. the topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with alzheimer's disease. cereb cortex 1991;1:103-116. https://doi.org/10.1093/cercor/1.1.103 ferrer i, andrés-benito p. white matter alterations in alzheimer’s disease without concomitant pathologies. neuropathol appl neurobiol 2020;46:654-672. https://doi.org/10.1111/nan.12618 thal dr, rüb u, orantes m, et al. phases of aβ-deposition in the human brain and its relevance for the development of ad. neurology 2002;58:1791-1800. https://doi.org/10.1212/wnl.58.12.1791 arnsten aft, datta d, del tredici k, et al. hypothesis: tau pathology is an initiating factor in sporadic alzheimer's disease. alzheimers dement 2021;17:115-124. https://doi.org/10.1002/alz.12192 armstrong ra, myers d, smith cu. the spatial patterns of plaques and tangles in alzheimer's disease do not support the 'cascade hypothesis'. dementia 1993;4:16-20. https://doi.org/10.1159/000107291 delacourte a, sergeant n, champain d, et al. nonoverlapping but synergetic tau and app pathologies in sporadic alzheimer’s disease. neurology 2002;59:398-407. https://doi.org/10.1212/wnl.59.3.398 delacourte a. alzheimer’s disease: a true tauopathy fueled by amyloid precursor protein dysfunction. in: hanin i, cacabelos r, fisher a (eds) recent progress in alzheimer’s and parkinson’s diseases. taylor & francis new york, 2005, pp 301–307 busche ma, hyman bt. synergy between amyloid-β and tau in alzheimer’s disease. nat neurosci 2020;23:1183-1193. https://doi.org/10.1038/s41593-020-0687-6 mudher a, lovestone s. alzheimer's disease-do tauists and baptists finally shake hands? trends neurosci 2002;25:22-26. https://doi.org/10.1016/s0166-2236(00)02031-2 bierer lm, hof pr, purohit dp, et al. neocortical neurofibrillary tangles correlate with dementia severity in alzheimer's disease. arch neurol 1995;52:81-88. https://doi.org/10.1001/archneur.1995 00540250089017. bancher c, braak h, fischer p, et al. neuropathological staging of alzheimers lesions and intellectual status in alzheimer’s disease and parkinson’s disease patients. neurosci lett 1993;162:179-182. https://doi.org/10.1016/0304-3940(93)90590-h hof pr, morrison jh. hippocampal and neocortical involvement in normal brain aging and dementia: morphological and neurochemical profile of the vulnerable circuits. j am geriatr soc 1996;44:857-864. https://doi.org/10.1111/j.1532-5415.1996.tb03748.x bancher c, jellinger k, lassmann h, et al. correlations between mental state and quantitative neuropathology in the vienna longitudinal study on dementia. eur arch psychiatry clin neurosci 1996;246:137-146. https://doi.org/10.1007/bf02189115 duyckaerts c, bennecib m, grignon y, et al. modeling the relation between neurofibrillary tangles and mental status. neurobiol aging 1997;18:267-273. https://doi.org/10.1016/s0197-4580(97)80306-5 haroutunian v, purohit dp, perl dp, et al. neurofibrillary tangles in nondemented elderly subjects and mild alzheimer disease. arch neurol 1999;56:713-718. https://doi.org/10.1001/archneur.56.6.713 guillozet al, weintraub s, mash dc, et al. neurofibrillary tangles, amyloid, and memory in aging and mild cognitive impairment. arch neurol 2003;60:729-736. https://doi.org/10.1001/archneur.60.5.729 giannakopoulos p, herrmann fr, bussière t, et al. tangle and neuron numbers, but not amyloid load, predict cognitive status in alzheimer's disease. neurology 2003;60:1495-500. https://doi.org/10.1212/01.wnl.0000063311.58879.01 imhof a, kövari e, von gunten a, et al. morphological substrates of cognitive decline in nonagenarians and centenarians: a new paradigm? j neurol sci 2007;257:72-79. https://doi.org/10.1016/j.jns.2007.01.025 hof pr. regional and laminar patterns of selective neuronal vulnerability in alzheimer’s disease. in: functional neurobiology of aging., hof pr, mobbs cv (eds). academic press, new york, 2001:95-109 frisoni gb, altomare d, thal dr, et al. the probabilistic model of alzheimer disease: the amyloid hypothesis revised. nature rev neurosci 2021;23:53-66. https://doi.org/10.1038/s41583-021-00533-w price dl, martin lj, sisodia ss, et al. aged non-human primates: an animal model of age-associated neurodegenerative disease. brain pathol 1991;1: 287-296. https://doi.org/10.1111/j.1750-3639.1991.tb00672.x giannakopoulos p, silhol s, jallageas v, et al. quantitative analysis of tau protein-immunoreactive accumulations and beta-amyloid protein deposits in the cerebral cortex of the mouse lemur, microcebus murinus. acta neuropathol 1997;94:131-139. https://doi.org/10.1007/s004010050684 head e. neurobiology of the aging dog. age 2012;33:485-496. https://doi.org/10.1007/s11357-010-9183-3 morelli l, wei l, amorim a, et al. cerebrovascular amyloidosis in squirrel monkeys and rhesus monkeys: apolipoprotein e genotype. febs lett 1996; 379:132-134. https://doi.org/10.1016/0014-5793(95)01491-8 yu ch, song gs, yhee jy, et al. histopathological and immunohistochemical comparison of the brain of human patients with alzheimer’s disease and the brain of aged dogs with cognitive dysfunction. j comp pathol 2011;145:45-58. https://doi.org/10.1016/j.jcpa.2010.11.004 mutsuga m, chambers jk, uchida k, et al. binding of curcumin to senile plaques and cerebral amyloid angiopathy in the aged brain of various animals and to neurofibrillary tangles in alzheimer’s brain. j vet med sci 2012;74:51-57. https://doi.org/10.1292/jvms.11-0307 finch ce, austad sn. commentary: is alzheimer's disease uniquely human? neurobiol aging 2015 feb;36:553-5. https://doi.org/10.1016/j.neurobiolaging.2014.10.025 languille s, blanc s, blin o, et al. the grey mouse lemur: a non-human primate model for ageing studies. ageing res rev 2012;11:150-162. https://doi.org/10.1016/j.arr.2011.07.001 pérez se, raghanti ma, hof pr, et al. alzheimer's disease pathology in the neocortex and hippocampus of the weste.rn lowland gorilla (gorilla gorilla gorilla). j comp neurol 2013;521:4318-4338. https://doi.org/10.1002/cne.23428 uchihara t, endo k, kondo h, et al. tau pathology in aged cynomolgus monkeys is progressive supranuclear palsy/corticobasal degeneration, but not alzheimer disease-like. ultrastructural mapping of tau by edx. acta neuropathol commun 2016;4:118. https://doi.org/10.1186/s40478-016-0385-5 edler mk, sherwood cc, meindl rs, et al. aged chimpanzees exhibit pathologic hallmarks of alzheimer's disease. neurobiol aging 2017;59:107-120. https://doi.org/10.1016/j.neurobiolaging.2017.07.006 takaichi y, chambers jk, takahashi k, et al. amyloid β and tau pathology in brains of aged pinniped species (sea lion, seal, and walrus). amyloid β and tau pathology in brains of aged pinniped species (sea lion, seal, and walrus). acta neuropathol commun 2021;9:10. https://doi.org/10.1186/s40478-020-01104-3 schultz c, dehghani f, hubbard gb, et al. filamentous tau pathology in nerve cells, astrocytes, and oligodendrocytes of aged baboons. j neuropathol exp neurol 2000:59:39-52. https://doi.org/10.1093/jnen/59.1.39 schultz c, hubbard gb, rüb u, et al. age-related progression of tau pathology in brains of baboons. neurobiol aging 2000;21: 905-912. https://doi.org/10.1016/s0197-4580(00)00176-7 abdel rassoul r, alves s, pantesco v, et al. distinct transcriptome expression of the temporal cortex of the primate microcebus murinus during brain aging versus alzheimer’s disease like pathology. plos one 2010;5: e12770. https://doi.org/10.1371/journal.pone.0012770 edler mk, munger el, meindl rs, et al. neuron loss associated with age but not alzheimer's disease pathology in the chimpanzee brain. philos trans r soc lond b biol sci 2020;375:20190619. https://doi.org/10.1098/rstb.2019.0619 head e, moffat k, das p, et al. beta-amyloid deposition and tau phosphorylation in clinically characterized aged cats. neurobiol aging 2005;26:749–763. https://doi.org/10.1016/j.neurobiolaging.2004.06.015 gunn-moore da, mcvee j, bradshaw jm, et al. ageing changes in cat brains demonstrated by β-amyloid and at8-immunoreactive phosphorylated tau deposits j feline med surg 2006;8:234-242. https://doi.org/10.1016/j.jfms.2006.01.003 chambers jk, tokuda t, uchida k, et al. the domestic cat as a natural animal model of alzheimer's disease. acta neuropathol commun 2015;3:78. https://doi.org/10.118 /s40478-015-0258-3. poncelet l, ando k, vergara c, et al. a 4r tauopathy develops without amyloid deposits in aged cat brains. neurobiol aging 2019;81:200e212. https://doi.org/10.1016/j.neurobiolaging.2019.05 024. rapoport si. hypothesis: alzheimer's disease is a phylogenetic disease. med hypotheses 1989; 29: 147-150. https://doi.org/10.1016/0306-9877(89)90185-0 gonatas nk, anderson w, evangelista i. the contribution of altered synapses in the senile plaques: an electron microscopic study in alzheimer’s disease. j neuropathol exp neurol 1967;26:25-39. https://doi.org/10.1097/00005072-196701000-00003 scheibel me, lindsay rd, tomiyasu u, et al. progressive dendritic changes in aging human cortex. exp neurol 1975;47:392-403. https://doi.org/10.1016/0014-4886(75)90072-2 buell sj, coleman pd. dendritic growth in the aged human brain and failure of growth in senile dementia. science 1979; 206:854-856. https://doi.org/10.1126/science.493989 buell sj, coleman pd. quantitative evidence for selective dendritic growth in normal human aging but not in senile dementia. brain res 1981;214:23-41. https://doi.org/10.1016/0006-8993(81)90436-4 flood dg, buell sj, defiore ch, et al. age-related dendritic growth in dentate gyrus of human brain is followed by regression in the 'oldest old'. brain res 1985;345:366-368. https://doi.org/10.1016/0006-8993(85)91018-2 flood dg, buell sj, horwitz gj, et al. dendritic extent in human dentate gyrus granule cells in normal aging and senile dementia. brain res 1987;402:205-216. https://doi.org/10.1016/0006-8993(87)90027-8 catalá i, ferrer i, galofré e, et al. decreased numbers of dendritic spines on cortical pyramidal neurons in dementia. a quantitative golgi study on biopsy samples. hum neurobiol 1988;6:255-259. pmid: 3350705. ferrer i, guionnet n, cruz-sánchez f, et al. neuronal alterations in patients with dementia: a golgi study on biopsy samples. neurosci lett 1990;114:11-16. https://doi.org/10.1016/0304-3940(90)90420-e terry rd, masliah e, salmon dp, et al. physical basis of cognitive alterations in alzheimer's disease: synapse loss is the major correlate of cognitive impairment. ann neurol 1991;30: 572-580. https://doi.org/10.1002/ana.410300410 selkoe dj. alzheimer's disease is a synaptic failure. science 2002; 298:789-791. https://doi.org/10.1126/science.1074069 scheff sw, price da. synaptic pathology in alzheimer’s disease: a review of ultrastructural studies. neurobiol aging 2003;24:1029-1046. https://doi.org/10.1016/j.neurobiolaging.2003.08.002 arendt t. synaptic degeneration in alzheimer's disease. acta neuropathol 2009;118: 167-179. https://doi.org/10.1007/s00401-009-0536-x domínguez-álvaro m, montero-crespo m, blazquez-llorca l, et al. three-dimensional analysis of synapses in the transentorhinal cortex of alzheimer's disease patients. acta neuropathol commun 2018;6:20. https://doi.org/10.1186/s40478-018-0520-6 montero-crespo m, domínguez-álvaro m, alonso-nanclares l, et al. three-dimensional analysis of synaptic organization in the hippocampal ca1 field in alzheimer's disease. brain 2021;144:553-573. https://doi.org/10.1093/brain/awaa406 cheibel ab, tomiyasu u. dendritic sprouting in alzheimer's presenile dementia. exp neurol 1978;60:1-8. https://doi.org/10.1016/0014-4886(78)90164-4 paula-barbosa mm, cardoso rm, guimaraes ml, et al. dendritic degeneration and regrowth in the cerebral cortex of patients with alzheimer's disease. j neurol sci 1980;45:129-134. https://doi.org/10.1016/s0022-510x(80)80014-1 probst a, basler v, bron b, et al. neuritic plaques in senile dementia of alzheimer type: a golgi analysis in the hippocampal region. brain res 1983;268:249-254. https://doi.org/10.1016/0006-8993(83)90490-0 ferrer i, aymami a, rovira a, et al. growth of abnormal neurites in atypical alzheimer’s disease. a study with the golgi method. acta neuropathol 1983;59:167-170. https://doi.org/10.1007/bf00703200 arendt t, zvegintseva hg, leontovich ta. dendritic changes in the basal nucleus of meynert and in the diagonal band nucleus in alzheimer's disease--a quantitative golgi investigation. neuroscience 1986;19:1265-1278. https://doi.org/10.1016/0306-4522(86)90141-7 masliah e, mallory m, hansen l, et al. patterns of aberrant sprouting in alzheimer's disease. neuron 1991;6:729-739. https://doi.org/10.1016/0896-6273(91)90170-5 masliah e, mallory m, hansen l, et al. localization of amyloid precursor protein in gap43-immunoreactive aberrant sprouting neurites in alzheimer's disease. brain res 1992;574:312-316. https://doi.org/10.1016/0006-8993(92)90831-s hashimoto m, masliah e. cycles of aberrant synaptic sprouting and neurodegeneration in alzheimer's and dementia with lewy bodies. neurochem res 2003;28:1743-1756. https://doi.org/10.1023/a:1026073324672 merino-serrais p, benavides-piccione r, blazquez-llorca l, et al. the influence of phospho-tau on dendritic spines of cortical pyramidal neurons in patients with alzheimer's disease. brain 2013;136:1913-1928. https://doi.org/10.1093/brain/awt088 mijalkov m, volpe g, fernaud-espinosa i, et al. dendritic spines are lost in clusters in alzheimer's disease. sci rep 2021;11:12350. https://doi.org/10.1038/s41598-021-91726-x tsai j, grutzendler j, duff k, et al. fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. nat neurosci 2004;7:1181-1183. https://doi.org/10.1038/nn1335 garcia-marin v, blazquez-llorca l, rodriguez jr, et al. diminished perisomatic gabaergic terminals on cortical neurons adjacent to amyloid plaques. front neuroanat 2009;3:28. https://doi.org/10.3389/neuro.05.028.2009 hoover br, reed mn, su s, et al. tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. neuron 2010;68:1067-1081. https://doi.org/10.1016/j.neuron.2010.11.030 yin x, zhao c, qiu y, et al. dendritic/post-synaptic tau and early pathology of alzheimer's disease. front mol neurosci 2021;14:671779. https://doi.org/10.3389/fnmol.2021.671779 masliah e, mallory m, hansen l, et al. synaptic and neuritic alterations during the progression of alzheimer’s disease. neurosci lett 1994;174:67-72. https://doi.org/10.1016/0304-3940(94)90121-x gouras gk, willén k, faideau m. the inside-out amyloid hypothesis and synapse pathology in alzheimer's disease. neurodegener dis 2014;13:142-146. https://doi.org/10.1159/000354776 muntané g, dalfó e, martinez a, et al. phosphorylation of tau and alpha-synuclein in synaptic-enriched fractions of the frontal cortex in alzheimer's disease, and in parkinson's disease and related alpha-synucleinopathies. neuroscience 2008;152:913-923. https://doi.org/10.1016/j.neuroscience.2008.01.030 gouras gk, tampellini d, takahashi rh, et al. intraneuronal beta-amyloid accumulation and synapse pathology in alzheimer's disease. acta neuropathol 2010;119:523-541. https://doi.org/10.1007/s00401-010-0679-9 palop jj, mucke l. amyloid-beta-induced neuronal dysfunction in alzheimer's disease: from synapses toward neural networks. nat neurosci 2010;13:812-818. https://doi.org/10.1038/nn.2583 sheng m, sabatini bl, südhof tc. synapses and alzheimer's disease. cold spring harb perspect biol 2012;4:a005777. capetillo-zarate e, gracia l, tampellini d, et al. intraneuronal aβ accumulation, amyloid plaques, and synapse pathology in alzheimer's disease. neurodegener dis 2012;10:56-59. https://doi.org/10.1101/cshperspect.a005777 spires-jones tl, hyman bt. the intersection of amyloid beta and tau at synapses in alzheimer's disease. neuron 2014;82:756-771. https://doi.org/10.1016/j.neuron.2014.05.004 rajmohan r, reddy ph. amyloid-beta and phosphorylated tau accumulations cause abnormalities at synapses of alzheimer's disease neurons. j alzheimers dis 2017;57: 975-999. https://doi.org/10.3233/jad-160612 li k, wei q, liu ff, et al. synaptic dysfunction in alzheimer's disease: abeta, tau, and epigenetic alterations. mol neurobiol 2018;55:3021-3032. https://doi.org/10.1007/s12035-017-0533-3 chen y, fu aky, ip ny. synaptic dysfunction in alzheimer’s disease: mechanisms and therapeutic strategies. pharmacol ther 2019;195:186-198. https://doi.org/10.1016/j.pharmthera.2018.11.006 john a, reddy ph. synaptic basis of alzheimer's disease: focus on synaptic amyloid beta, p-tau and mitochondria. ageing res rev 2021;65:101208. https://doi.org/10.1016/j.arr.2020.101208 coomans em, schoonhoven dn, tuncel h, et al. in vivo tau pathology is associated with synaptic loss and altered synaptic function. alzheimers res ther 2021;13:35. https://doi.org/10.1186/s13195-021-00772-0 de vos sl, corjuc bt, oakley dh, et al. synaptic tau seeding precedes tau pathology in human alzheimer’s disease brain. front neurosci 2018;12: 267. https://doi.org/10.3389/fnins.2018.00267 pelucchi s, stringhi r, marcello e. dendritic spines in alzheimer's disease: how the actin cytoskeleton contributes to synaptic failure. int j mol sci 2020;21:908. https://doi.org/10.3390/ijms21030908 kamat pk, kalani a, rai s, et al. mechanism of oxidative stress and synapse dysfunction in the pathogenesis of alzheimer's disease: understanding the therapeutics strategies. mol neurobiol 2016;53:648-661. https://doi.org/10.1007/s12035-014-9053-6 arendt t, brückner mk. linking cell-cycle dysfunction in alzheimer's disease to a failure of synaptic plasticity. biochim biophys acta 2007;1772:413-421. https://doi.org/10.1016/j.bbadis.2006.12.005 li s, sheng zh. energy matters: presynaptic metabolism and the maintenance of synaptic transmission. nat rev neurosci 2022;23:4-22. https://doi.org/10.1038/s41583-021-00535-8 davies p, maloney aj. selective loss of central cholinergic neurons in alzheimer's disease. lancet 1976;2:1403. https://doi.org/10.1016/s0140-6736(76)91936-x perry ek, tomlinson be, blessed g, et al. correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. br med j 1978;2:1457-1459. https://doi.org/10.1136/bmj.2.6150.1457 coyle jt, price dl, delong mr. alzheimer’s disease: a disorder of cortical cholinergic innervation. science 1983;219:1184-1190. https://doi.org/10.1126/science.6338589 francis pt, palmer am, snape m, et al. the cholinergic hypothesis of alzheimer’s disease: a review of progress. j neurol neurosurg psychiatry 1999;66:137-147. https://doi.org/10.1136/jnnp.66.2.137 lees aj, tolosa e, olanow cw. four pioneers of l-dopa treatment: arvid carlsson, oleh hornykiewicz, george cotzias, and melvin yahr. mov disord 2015;30:19-36. https://doi.org/10.1002/mds.26120 hynd mr, scott hl, dodd pr. glutamate-mediated excitotoxicity and neurodegeneration in alzheimer’s disease. neurochem int 2004;45:583-595. https://doi.org/10.1016/j.neuint.2004.03.007 parsons mp, raimond la. extrasynaptic nmda receptor involvement in central nervous system disorders. neuron 2014;82: 279-293. https://doi.org/10.1016/j.neuron.2014.03.030 rothstein jd, martin lj, kuncl rw. decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. n engl j med 1992;326:1464-1468. https://doi.org/10.1056/nejm199205283262204 meldrum b, garthwaite j. excitatory amino acid neurotoxicity and neurodegenerative disease. trends pharmacol sci 1990;11:379-387. https://doi.org/10.1016/0165-6147(90)90184-a song j, yanga x, zhanga m, et al. glutamate metabolism in mitochondria is closely related to alzheimer’s disease. j alzheimers dis 2021;84:557-578. https://doi.org/10.3233/jad-210595 hardingham ge, bading h. synaptic versus extrasynaptic nmda receptor signalling: implications for neurodegenerative disorders. nat rev neurosci 2010; 11: 682-696. https://doi.org/10.1038/nrn2911 mufson ej, kordower jh. cholinergic basal forebtrain system in the primate central nervous system: anatomy, connectivity, neurochemistry, aging, dementia, and experimental therapeutics. in: functional neurobiology of aging. hof pr, mobbs cv (eds.) academic press 2001, pp: 243-281 mishizen a, ikonomovic m, armstrong dm. glutamate receptors in aging and alzheimer’s disease. in: functional neurobiology of aging. hof pr, mobbs cv (eds.). academic press 2001, pp: 283-314 wang r, reddy ph. role of glutamate and nmda receptors in alzheimer’s disease. j alzheimers dis 2017;57:1041-1048. https://doi.org/10.3233/jad-160763 armada-moreira a, gomes ji, pina cc, et al. going the extra (synaptic) mile: excitotoxicity as the road toward neurodegenerative diseases. front cell neurosci 2020;14:90. https://doi.org/10.3389/fncel.2020.00090 šimić g, babić leko m, wray s, et al. monoaminergic neuropathology in alzheimer’s disease. prog neurobiol 2017;151:101-138. https://doi.org/10.1016/j.pneurobio.2016.04.001 sharma k, pradhan s, duffy l, et al. role of receptors in relation to plaques and tanglers in alzheimer’s disease pathology. int j mol sci 2021;22:12987. https://doi.org/10.3390/ijms222312987 cai z, ratka a. opioid system and alzheimer's disease. neuromolecular med 2012;14:91-111. https://doi.org/10.1007/s12017-012-8180-3 morley je. peptides and aging: their role in anorexia and memory. peptides 2015;72:112-118. https://doi.org/10.1016/j.peptides 2015.04.007. henry ms, gendron l, tremblay me, et al. enkephalins: endogenous analgesics with an emerging role in stress resilience. neural plast 2017;2017:1546125. https://doi.org/10.1155/2017/154612 valentino rj, volkow nd. untangling the complexity of opioid receptor function. neuropsychopharmacology 2018;43:2514-2520. https://doi.org/10.1038/s41386-018-0225-3 garcia-esparcia p, schlüter a, carmona m, et al. functional genomics reveals dysregulation of cortical olfactory receptors in parkinson disease: novel putative chemoreceptors in the human brain. j neuropathol exp neurol 2013;72:524-539. https://doi.org/10.1097/nen.0b013e318294fd76 ferrer i, garcia-esparcia p, carmona m, et al. olfactory receptors in non-chemosensory organs: the nervous system in health and disease front aging neurosci 2016; 8:163. https://doi.org/10.3389/fnagi.2016.00163 carlson ab, kraus gp. physiology, cholinergic receptors. in statpearls; statpearls publishing: treasure island, fl, usa, 2021. available online: https://www.ncbi.nlm.nih.gov/books/nbk526134 maelicke a. allosteric modulation of nicotinic receptors as a treatment strategy for alzheimer’s disease. dement geriatr cogn disord 2000;11:11-18. https://doi.org/10.1159/000051227 cheng yj, lin ch, lane hy. involvement of cholinergic, adrenergic, and glutamatergic network modulation with cognitive dysfunction in alzheimer's disease. int j mol sci 2021; 22:2283. https://doi.org/10.3390/ijms22052283 mufson ej, bothwell m, hersh lb, et al. nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: colocalization with cholinergic neurons. j comp neurol 1989;285:196-217. https://doi.org/10.1002/cne.902850204 mufson ej, bothwell m, kordower jh. loss of nerve growth factor receptor-containing neurons in alzheimer's disease: a quantitative analysis across subregions of the basal forebrain. exp neurol 1989;105:221-232. https://doi.org/10.1016/0014-4886(89)90124-6 kordower jh, gash dm, bothwell m, et al. nerve growth factor receptor and choline acetyltransferase remain colocalized in the nucleus basalis (ch4) of alzheimer's patients. neurobiol aging 1989;10:67-74. https://doi.org/10.1016/s0197-4580(89)80013-2 mufson ej, kordower ej. nerve growth factor in alzheimer’s disease. in cerebral cortex, peters aa, morrison jh, eds. kluver academic/plenum, new york, 1999;14:681-731 whitehouse pj, price dl, struble rg, et al. alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. science 1982; 215:1237-1239. https://doi.org/10.1126/science.7058341 dekosky st, ikonomovic md, styren sd, et al. upregulation of choline acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment. ann neurol 2002;51:145-155. https://doi.org/10.1002/ana.10069 tiernan ct, ginsberg sd, he b, et al. pretangle pathology within cholinergic nucleus basalis neurons coincides with neurotrophic and neurotransmitter receptor gene dysregulation during the progression of alzheimer's disease. neurobiol dis 2018;117:125-136. https://doi.org/10.1016/j.nbd.2018.05.021 ferreira-vieira th, guimaraes im, silva fr, et al. alzheimer’s disease: targeting the cholinergic system. curr neuropharmacol 2016;14:101-115. https://doi.org/10.2174/1570159x13666150716165726 perry ek, morris cm, court ja, et al. alteration in nicotine binding sites in parkinson’s disease, lewy body dementia and alzheimer’s disease: possible index of early neuropathology. neuroscience 1995;64:385-395. https://doi.org/10.1016/0306-4522(94)00410-7 court j, martin-ruiz c, piggott m, et al. nicotinic receptor abnormalities in alzheimer’s disease. biol psychiatry 2001;49:175-184. https://doi.org/10.1016/s0006-3223(00)01116-1 pimlott sl, piggott m, owens j, et al. nicotinic acetylcholine receptor distribution in alzheimer’s disease, dementia with lewy bodies, parkinson’s disease, and vascular dementia: in vitro binding study using 5-[125i]-a-85380. neuropsychopharmacology 2004;29:108-116. https://doi.org/10.1038/sj.npp.1300302 shen j, wu j. nicotinic cholinergic mechanisms in alzheimer’s disease. int rev neurobiol 2015;124:275-292. https://doi.org/10.1016/bs.irn.2015.08.002 ono k, hasegawa k, yamada m, et al. nicotine breaks down preformed alzheimer’s β-amyloid fibrils in vitro. biol psychiatry 2002; 52:880-886. https://doi.org/10.1016/s0006-3223(02)01417-8 wang hy, lee dh, d’andrea mr, et al. β-amyloid1-42 binds to α7 nicotinic acetylcholine receptor with high affinity: implications for alzheimer’s disease pathology. j biol chem 2000; 275: 5626-5632. https://doi.org/10.1074/jbc.275.8.5626 liu q, xie x, lukas rj, et al. a novel nicotinic mechanism underlies beta-amyloidinduced neuronal hyperexcitation. j neurosci 2013;33:7253-7263. https://doi.org/10.1523/jneurosci.3235-12.2013 liu q, xie x, emadi s, et al. a novel nicotinic mechanism underlies beta-amyloid induced neurotoxicity. neuropharmacology 2015;97:457-463. https://doi.org/10.1016/j.neuropharm.2015.04.025 wang hy, li w, benedetti nj, et al. alpha 7 nicotinic acetylcholine receptors mediate beta-amyloid peptide-induced tau protein phosphorylation. j biol chem 2003; 278:31547-31553. https://doi.org/10.1074/jbc.m212532200 shytle rd, mori t, townsend k, et al. cholinergic modulation of microglial activation by alpha 7 nicotinic receptors. j neurochem 2004; 89:337-343. https://doi.org/10.1046/j.1471-4159.2004.02347.x sadigh-eteghad s, majdi a, mahmoudi j, et al. astrocytic and microglial nicotinic acetylcholine receptors: an overlooked issue in alzheimer's disease. j neural transm 2016;1231359-1367. https://doi.org/10.1007/s00702-016-1580-z lebois ep, thorn c, edgerton jr, et al. muscarinic receptor subtype distribution in the central nervous system and relevance to aging and alzheimer’s disease. neuropharmacology 2018; 136: 362-373. https://doi.org/10.1016/j.neuropharm.2017.11.018 volpicelli la, allan il. muscarinic acetylcholine receptor subtypes in cerebral cortex and hippocampus. prog brain res 2004;145:59-66. https://doi.org/10.1016/s0079-6123(03)45003-6 ishii m, kurachi y. muscarinic acetylcholine receptors. curr pharm des 2006;12: 3573-3581. https://doi.org/10.2174/138161206778522056 kudlak m, tadi p. physiology, muscarinic receptor. in statpearls; statpearls publishing: treasure island, fl, usa, 2021. pavía j, de ceballos ml, sanchez de la cuesta f. alzheimer's disease: relationship between muscarinic cholinergic receptors, beta-amyloid and tau proteins. fundam clin pharmacol 1998;12:473-481. https://doi.org/10.1111/j.1472-8206.1998.tb00975.x clader jw, wang y. muscarinic receptor agonists and antagonists in the treatment of alzheimer’s disease. curr pharm des 2005;11:3353-3361. https://doi.org/10.2174/138161205774370762 fisher a. cholinergic treatments with emphasis on m1 muscarinic agonists as potential disease-modifying agents for alzheimer’s disease. neurotherapeutics 2008; 5:433-442. https://doi.org/10.1016/j.nurt.2008.05.002 davis aa, fritz jj, wess j, et al. deletion of m1 muscarinic acetylcholine receptors increases amyloid pathology in vitro and in vivo. j neurosci 2010;30:4190-4196. https://doi.org/10.1523/jneurosci.6393-09.2010 geula c, mesulam m. special properties of cholinesterases in the cerebral cortex of alzheimer’s disease. brain res 1989;498: 185-189. https://doi.org/10.1016/0006-8993(89)90419-8 sberna g, sáez-valero j, beyreuther k, et al. the amyloid beta-protein of alzheimer’s disease increases acetylcholinesterase expression by increasing intracellular calcium in embryonal carcinoma p19 cells. j neurochem 1997;69:1177-1184. https://doi.org/10.1046/j.1471-4159.1997.69031177.x furukawa h, singh sk, mancusso r, et al. subunit arrangement and function in nmda receptors. nature 2005;438:185-192. https://doi.org/10.1038/nature04089 yamamoto h, hagino y, kasai s, et al. specific roles of nmda receptor subunits in mental disorders. curr mol med 2015; 15: 193-205. https://doi.org/10.2174/1566524015666150330142807 pinheiro ps, mulle c. presynaptic glutamate receptors: physiological functions and mechanisms of action. nature rev neurosci 2008; 9:423. https://doi.org/10.1038/nrn2379 steinhäuser c, gallo v. news on glutamate receptors in glial cells. trends neurosci 1996;19: 339-345. https://doi.org/10.1016/0166-2236(96)10043-6 shemer i, holmgren c, min r, et al. non-fibrillar beta-amyloid abates spike-timing-dependent synaptic potentiation at excitatory synapses in layer 2/3 of the neocortex by targeting postsynaptic ampa receptors. eur j neurosci 2006;23:2035-47. https://doi.org/10.1111/j.1460-9568.2006.04733.x parameshwaran k, dhanasekaran m, suppiramaniam v. amyloid beta peptides and glutamatergic synaptic dysregulation. exp neurol 2008;210:7-13. https://doi.org/10.1016/j.expneurol.2007.10.008 ferreira il, bajouco lm, mota si, et al. amyloid beta peptide 1-42 disturbs intracellular calcium homeostasis through activation of glun2b-containing n-methyl-d-aspartate receptors in cortical cultures. cell calcium 2012;51:95-106. https://doi.org/10.1016/j.ceca.2011.11.008 decker h, jürgensen s, adrover mf, et al. n-methyl-d-aspartate receptors are required for synaptic targeting of alzheimer's toxic amyloid-β peptide oligomers. j neurochem 2010;115:1520-1529. https://doi.org/10.1111/j.1471-4159.2010.07058.x epub 2010 nov 11. bordji k, becerril-ortega j, buisson a. synapses, nmda receptor activity and neuronal abeta production in alzheimer's disease. rev neurosci 2011;22:285-294. https://doi.org/10.1515/rns.2011.029 mota si, ferreira il, rego ac. dysfunctional synapse in alzheimer's disease a focus on nmda receptors. neuropharmacology 2014;76:16-26. https://doi.org/10.1016/j.neuropharm.2013.08.013 zhang y, li p, feng j, wu m. dysfunction of nmda receptors in alzheimer's disease. neurol sci 2016;37:1039-1047. https://doi.org/10.1007/s10072-016-2546-5 foster tc, kyritsopoulos c, kumar a. central role for nmda receptors in redox mediated impairment of synaptic function during aging and alzheimer's disease. behav brain res 2017;322:223-232. https://doi.org/10.1016/j.bbr.2016.05.012 babaei p. nmda and ampa receptors dysregulation in alzheimer's disease. eur j pharmacol 2021;908:174310. https://doi.org/10.1016/j.ejphar.2021.174310 reinders nr, pao y, renner mc, et al. effects of amyloid-β require ampar subunit glua3. proc natl acad sci usa 2016;113: e6526-e6534. https://doi.org/10.1073/pnas.1614249113 zhang y, guo o, huo y, et al. amyloid-β induces ampa receptor ubiquitination and degradation in primary neurons and human brains of alzheimer’s disease. j alzheimer’s dis 2018;62:1789-1801. https://doi.org/10.3233/jad-170879 danbolt nc. glutamate uptake. prog neurobiol 2001;65:1-105. https://doi.org/10.1016/s0301-0082(00)00067-8 kirvell sl, esiri m, francis pt. down-regulation of vesicular glutamate transporters precedes cell loss and pathology in alzheimer's disease. j neurochem 2006;98:939-950. https://doi.org/10.1111/j.1471-4159.2006.03935.x jacob cp, koutsilieri e, bartl j, et al. alterations in expression of glutamatergic transporters and receptors in sporadic alzheimer's disease. j alzheimers dis 2007; 11:97-116. https://doi.org/10.3233/jad-2007-11113 pow dv, cook dg.neuronal expression of splice variants of "glial" glutamate transporters in brains afflicted by alzheimer's disease: unmasking an intrinsic neuronal property. neurochem res 2009;34:1748-1757. https://doi.org/10.1007/s11064-009-9957-0 conn pj, pin jp. pharmacology and functions of metabotropic glutamate receptors. annu rev pharmacol toxicol 1997; 37: 205-237. https://doi.org/10.1146/annurev.pharmtox.37.1.205 pin jp, duvoisin r. the metabotropic glutamate receptors: structure and functions. neuropharmacology 1995;3:1-26. https://doi.org/10.1016/0028-3908(94)00129-g crupi r, impellizeri d, cuzzocrea s. role of metabotropic glutamate receptors in neurological disorders. front mol neurosci 2019;12:20. https://doi.org/10.3389/fnmol.2019.00020 mao lm, bodepudi a, chu xp, et al. group i metabotropic glutamate receptors and interacting partners: an update. int j mol sci 2022;23:840. https://doi.org/10.3390/ijms23020840 albasanz jl, dalfó e, ferrer i, et al. impaired metabotropic glutamate receptor/phospholipase c signaling pathway in the cerebral cortex in alzheimer's disease and dementia with lewy bodies correlates with stage of alzheimer's-disease-related changes. neurobiol dis 2005;20:685-693. https://doi.org/10.1016/j.nbd.2005.05.001 goetz t, arslan a, wisden w, et al. gaba(a) receptors: structure and function in the basal ganglia. prog brain res 2007;160:21-41. https://doi.org/10.1016/s0079-6123(06)60003-4 padgett cl, slesinger pa. gabab receptor coupling to g-proteins and ion channels. adv pharmacol sci 2010;58:123-147. https://doi.org/10.1016/s1054-3589(10)58006-2 jembrek mj, vlainic j. gaba receptors: pharmacological potential and pitfalls. curr pharm des 2015;21:4943-4959. https://doi.org/10.2174/1381612821666150914121624 terunuma m. diversity of structure and function of gaba(b) receptors: a complexity of gaba(b)-mediated signaling. proc jpn acad ser b phys biol sci 2018;94:390-411. https://doi.org/10.2183/pjab.94.026 bak lk, schousboe a, waagepetersen hs. the glutamate/gaba-glutamine cycle: aspects of transport, neurotransmitter homeostasis and ammonia transfer. j neurochem 2006;98:641-653. https://doi.org/10.1111/j.1471-4159.2006.03913.x leke r, schousboe a. the glutamine transporters and their role in the glutamate/gaba-glutamine cycle. adv neurobiol 2016;13:223-257. https://doi.org/10.1007/978-3-319-45096-4_8 yoon be, lee cj. gaba as a rising gliotransmitter. front neural circuits 2014;8:141. https://doi.org/10.3389/fncir.2014.00141 ambrad giovannetti e, fuhrmann m. unsupervised excitation: gabaergic dysfunctions in alzheimer's disease. brain res 2019;1707:216-226. https://doi.org/10.1016/j.brainres.2018.11.042 calvo-flores guzmán b, vinnakota c, govindpani k, et al. the gabaergic system as a therapeutic target for alzheimer's disease. j neurochem 2018;146:649-669. https://doi.org/10.1111/jnc.14345 govindpani k, calvo-flores guzmán b, et al. towards a better understanding of gabaergic remodeling in alzheimer's disease. int j mol sci 2017;18: 1813. https://doi.org/10.3390/ijms18081813 riese f, gietl a, zölch n, et al. posterior cingulate γ-aminobutyric acid and glutamate/glutamine are reduced in amnestic mild cognitive impairment and are unrelated to amyloid deposition and apolipoprotein e genotype. neurobiol aging 2015;36:53-59. https://doi.org/10.1016/j.neurobiolaging.2014.07.030 huang d, liu d, yin j, et al. glutamate-glutamine and gaba in brain of normal aged and patients with cognitive impairment. eur radiol 2017;27:2698-2705. https://doi.org/10.1007/s00330-016-4669-8 murari g, liang dr, ali a, et al. prefrontal gaba levels correlate with memory in older adults at high risk for alzheimer's disease. cereb cortex commun 2020;1:tgaa022. https://doi.org/10.1093/texcom/tgaa02 liu h, zhang d, lin h, et al. meta-analysis of neurochemical changes estimated via magnetic resonance spectroscopy in mild cognitive impairment and alzheimer's disease. front aging neurosci 2021;13:738971. https://doi.org/10.3389/fnagi.2021.738971 limon a, reyes-ruiz jm, miledi r. loss of functional gaba(a) receptors in the alzheimer diseased brain. proc natl acad sci usa 2012;109:10071-10076. https://doi.org/10.1073/pnas.1204606109 mizukami k, ikonomovic md, grayson dr, et al. immunohistochemical study of gabaa receptor alpha1 subunit in the hippocampal formation of aged brains with alzheimer-related neuropathologic changes. brain res 1998;799:148-155. https://doi.org/10.1016/s0006-8993(98)00437-5 rissman ra, mishizen-eberz aj, carter tl, et al. biochemical analysis of gaba(a) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of patients with alzheimer's disease neuropathology. neuroscience 2003;120:695-704. https://doi.org/10.1016/s0306-4522(03)00030-7 rissman ra, bennett da, armstrong dm. subregional analysis of gaba(a) receptor subunit mrnas in the hippocampus of older persons with and without cognitive impairment. j chem neuroanat 2004;28:17-25. https://doi.org/10.1016/j.jchemneu.2004.05.003 iwakiri m, mizukami k, ikonomovic dm, et al. an immunohistochemical study of gaba a receptor gammasubunits in alzheimer's disease hippocampus: relationship to neurofibrillary tangle progression. neuropathology 2009;29:263-269. https://doi.org/10.1111/j.1440-1789.2008.00978.x mizukami k, ikonomovic md, grayson dr, et al. immunohistochemical study of gaba(a) receptor beta2/3 subunits in the hippocampal formation of aged brains with alzheimer-related neuropathologic changes. exp neurol 1997;147:333-345. https://doi.org/10.1006/exnr.1997.6591 kwakowsky a, calvo-flores guzmán b, pandya m, et al. gabaa receptor subunit expression changes in the human alzheimer's disease hippocampus, subiculum, entorhinal cortex and superior temporal gyrus. j neurochem 2018;145:374-392. https://doi.org/10.1111/jnc.14325 villette v, poindessous-jaza f, simon a, et al. decreased rhythmic gabaergic septal activity and memory-associated theta oscillations after hippocampal amyloid-beta pathology in the rat. j neurosci 2010;30:10991-11003. https://doi.org/10.1523/jneurosci.6284-09.2010 verret l, mann eo, hang gb, et al. inhibitory interneuron deficit links altered network activity and cognitive dysfunction in alzheimer model. cell 2012;149:708-721. https://doi.org/10.1016/j.cell.2012.02.046 ulrich d. amyloid-beta impairs synaptic inhibition via gaba(a) receptor endocytosis. j neurosci 2015;35: 9205-9210. https://doi.org/10.1523/jneurosci.0950-15.2015 hamm v, héraud c, bott jb, et al. differential contribution of app metabolites to early cognitive deficits in a tgcrnd8 mouse model of alzheimer's disease. sci adv 2017;3:e1601068. https://doi.org/10.1126/sciadv.1601068 palop jj, chin j, roberson ed, et al. aberrant excitatory neuronal activity and compensatory remodelling of inhibitory hippocampal circuits in mouse models of alzheimer's disease. neuron 2007;55:697-711. https://doi.org/10.1016/j.neuron.2007.07.025 hoyer d, martin g. 5-ht receptor classification and nomenclature: towards a harmonization with the human genome. neuropharmacology 1997;36:419-428. https://doi.org/10.1016/s0028-3908(97)00036-1 barnes nm, sharp t. a review of central 5-ht receptors and their function. neuropharmacology 1999;38:1083-1152. https:/ doi.org/10.1016/s0028-3908(99)00010-6. leysen je. 5-ht2 receptors. curr drug targets-cns neurol disord 2004;3:11-26. https://doi.org/10.2174/1568007043482598 fink kb, göthert m. 5-ht receptor regulation of neurotransmitter release. pharmacol rev 2007;59:360-417. https://doi.org/10.1124/pr.107.07103 nichols de, nichols cd. serotonin receptors. chem rev 2008;108:1614-1641. https://doi.org/10.1021/cr078224o barnes nm, hales tg, lummis sc, peters ja. the 5-ht3 receptor: the relationship between structure and function. neuropharmacology 2009;56:273-284. https://doi.org/10.1016/j.neuropharm.2008.08.003 berumen lc, rodríguez a, miledi r, et al. serotonin receptors in hippocampus. sci world j 2012; 2012:823493. https://doi.org/10.1100/2012/823493 palacios jm. serotonin receptors in brain revisited. brain res 2016;1645:46-49. https://doi.org/10.1016/j.brainres.2015.12.042 wirth a, holst k, ponimaskin e. how serotonin receptors regulate morphogenic signalling in neurons. prog neurobiol 2017;151:35-56. https://doi.org/10.1016/j.pneurobio.2016.03.007 consolo s, arnaboldi s, giorgi s, et al. 5-ht4 receptor stimulation facilitates acetylcholine release in rat frontal cortex. neuroreport 1994;5:1230-1232. https://doi.org/10.1097/00001756-199406020-00018 woods s, clarke nn, layfield r, et al. 5-ht (6) receptor agonists and antagonists enhance learning and memory in a conditioned emotion response paradigm by modulation of cholinergic and glutamatergic mechanisms. br j pharmacol 2012;167: 436-449. https://doi.org/10.1111/j.1476-5381.2012.02022.x seyedabadi m, fakhfouri g, ramezani v, et al. the role of serotonin in memory: interactions with neurotransmitters and downstream signaling. exp brain res 2014;232:723-738. https://doi.org/10.1007/s00221-013-3818-4 de deurwaerdere p, di giovanni g. 5-ht interaction with other neurotransmitters: an overview. prog brain res 2021;259:1-5. https://doi.org/10.1016/bs.pbr.2021.01.001 liu rj, van den pol an, aghajanian gk. hypocretins (orexins) regulate serotonin neurons in the dorsal raphe nucleus by excitatory direct and inhibitory indirect actions. j neurosci 2002;22:9453-9464. https://doi.org/10.1523/jneurosci.22-21-09453.2002 noristani hn, olabarria m, verkhratsky a, et al. serotonin fibre sprouting and increase in serotonin transporter immunoreactivity in the ca1 area of hippocampus in a triple transgenic mouse model of alzheimer's disease. eur j neurosci 2010;32:71-79. https://doi.org/10.1111/j.1460-9568.2010.07274.x rodríguez jj, noristani hn, verkhratsky a. the serotonergic system in ageing and alzheimer's disease. prog neurobiol 2012;99:15-41. https://doi.org/10.1016/j.pneurobio.2012.06.010 švob štrac d, pivac n, mück-šeler d. the serotoninergic system and cognitive function. transl neurosci 2016;7:35-49. https://doi.org/10.1515/tnsci-2016-0007 meltzer cc, smith g, dekosky st, et al. serotonin in aging, late-life depression, and alzheimer’s disease: the emerging role of functional imaging. neuropsychopharmacology 1998;18:407-430. https://doi.org/10.1016/s0893-133x(97)00194-2 michelsen ka, prickaerts j, steinbusch hw. the dorsal raphe nucleus and serotonin: implications for neuroplasticity linked to major depression in alzheimer’s disease. prog brain res 2008;172:233-264. https://doi.org/10.1016/s0079-6123(08)00912-6 yun hm, park kr, kim ec, et al. serotonin 6 receptor controls alzheimer's disease and depression. oncotarget 2015;6:26716-26728. https://doi.org/10.18632/oncotarget.5777 burke ad, goldfarb d, bollam p, et al. diagnosing and treating depression in patients with alzheimer's disease. neurol ther 2019;8:325-350. https://doi.org/10.1007/s40120-019-00148-5 cachard-chastel m, lezoualc’h f, dewachter i, et al. 5-ht4 receptor agonists increase sappalpha levels in the cortex and hippocampus of male c57bl/6j mice. br j pharmacol 2007;150:883-892. https://doi.org/10.1038/sj.bjp.0707178 cirrito jr, disabato bm, restivo jl, et al. serotonin signaling is associated with lower amyloid-β levels and plaques in transgenic mice and humans. proc natl acad sci usa 2011;108:14968-14973. https://doi.org/10.1073/pnas.1107411108 cochet m, donneger r, cassier e, et al. 5-ht4 receptors constitutively promote the non-amyloidogenic pathway of app cleavage and interact with adam10. acs chem neurosci 2013;4: 130-140. https://doi.org/10.1021/cn300095t giannoni p, gaven f, de bundel d, et al. early administration of rs 67333, a specific 5-ht4 receptor agonist, prevents amyloidogenesis and behavioral deficits in the 5xfad mouse model of alzheimer’s disease. front aging neurosci 2013;5:96. https://doi.org/10.3389/fnagi.2013.00096 moser pc, bergis oe, jegham s, et al. sl65.0155, a novel 5-hydroxytryptamine(4) receptor partial agonist with potent cognition-enhancing properties. j pharmacol exp ther 2002;302:731-741. https://doi.org/10.1124/jpet.102.034249 lezoualc'h f. 5-ht4 receptor and alzheimer's disease: the amyloid connection. exp neurol 2007;205:325-329. https://doi.org/10.1016/j.expneurol.2007.02.001 cho s, hu y. activation of 5-ht4 receptors inhibits secretion of beta-amyloid peptides and increases neuronal survival. exp neurol 2007;203:274-278. https://doi.org/10.1016/j.expneurol.2006.07.021 geldenhuys wj, van der schyf cj. role of serotonin in alzheimer’s disease: a new therapeutic target? cns drugs 2011;25:765-781. https://doi.org/10.2165/11590190-000000000-00000 werner fm, covenas r. serotonergic drugs: agonists/antagonists at specific serotonergic subreceptors for the treatment of cognitive, depressant and psychotic symptoms in alzheimer's disease. curr pharm des 2016;22:2064-2071. https://doi.org/10.2174/1381612822666160127113524 magierski r, sobow t. serotonergic drugs for the treatment of neuropsychiatric symptoms in dementia. expert rev neurother 2016;16:375-387. https://doi.org/10.1586/14737175.2016.1155453 ferrero h, solas m, francis pt, et al. serotonin 5-ht(6) receptor antagonists in alzheimer's disease: therapeutic rationale and current development status. cns drugs 2017;31:19-32. https://doi.org/10.1007/s40263-016-0399-3 brendel m, sauerbeck j, greven s, et al. serotonin selective reuptake inhibitor treatment improves cognition and grey matter atrophy but not amyloid burden during two-year follow-up in mild cognitive impairment and alzheimer's disease patients with depressive symptoms. j alzheimers dis 2018;65:793-806. https://doi.org/10.3233/jad-170387 manzoor s, hoda n. a comprehensive review of monoamine oxidase inhibitors as anti-alzheimer's disease agents: a review. eur j med chem 2020;206:112787. https://doi.org/10.1016/j.ejmech.2020 112787. mdawar b, ghossoub e, khoury r. selective serotonin reuptake inhibitors and alzheimer’s disease. regen res 2020;15:41-46. https://doi.org/10.4103/1673-5374.264445 kucwaj-brysz k, baltrukevich h, czarnota k, et al. chemical update on the potential for serotonin 5-ht(6) and 5-ht(7) receptor agents in the treatment of alzheimer's disease. bioorg med chem lett 2021;49:128275. https://doi.org/10.1016/j.bmcl.2021.128275 pacholczyk t, blakely rd, amara sg. expression cloning of a cocaineand antidepressant-sensitive human noradrenaline transporter. nature 1991;350:350-354. https://doi.org/10.1038/350350a0 eiden le, schäfer mk, weihe e, et al. the vesicular amine transporter family (slc18): amine/proton antiporters required for vesicular accumulation and regulated exocytotic secretion of monoamines and acetylcholine. pflügers arch. 2004;447: 636-640. https://doi.org/10.1007/s00424-003-1100-5 ruffolo rr, hieble jp. alpha-adrenoreceptors. pharmacol ther 1994;61:1-64. https://doi.org/10.1016/0163-7258(94)90058-2 musacchio jm. enzymes involved in the biosynthesis and degradation of catecholamines. in: biochemistry of biogenic amines. iverson l (ed). springer 2013, pp:1-35. pickel vm. extrasynaptic distribution of monoamine transporters and receptors. prog brain res 2000;125:267-276. https://doi.org/10.1016/s0079-6123(00)25016-4 perez dm. α1-adrenergic receptors in neurotransmission, synaptic plasticity, and cognition. front pharmacol 2020;11:581098. https://doi.org/10.3389/fphar.2020.581098 moore ry, bloom fe. central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. annu rev neurosci 1979; 2: 113-168. https://doi.org/10.1146/annurev.ne.02.030179.000553 szabadi e. functional neuroanatomy of the central noradrenergic system. j psychopharmacol 2013;27:659-693. https://doi.org/10.1177/0269881113490326 poe gr, foote s, eschenko o, et al. locus coeruleus: a new look at the blue spot. nat rev neurosci 2020;21:644-659. https://doi.org/10.1038/s41583-020-0360-9 starke k. presynaptic autoreceptors in the third decade: focus on alpha2-adrenoceptors. j neurochem 2001;78: 685-693. https://doi.org/10.1046/j.1471-4159.2001.00484.x nicholas ap, pieribone v, hökfelt t. distributions of mrnas for alpha-2 adrenergic receptor subtypes in rat brain: an in situ hybridization study. j comp neurol 1993;328: 575-594. https://doi.org/10.1002/cne.903280409 day he, campeau s, watson sj jr, et al. distribution of alpha 1a-, alpha 1band alpha 1d-adrenergic receptor mrna in the rat brain and spinal cord. j chem neuroanat 1997;13:115-139. https://doi.org/10.1016/s0891-0618(97)00042-2 pieribone va, nicholas ap, dagerlind a, et al. distribution of alpha 1 adrenoceptors in rat brain revealed by in situ hybridization experiments utilizing subtype-specific probes. j neurosci 1994;14:4252-4268. https://doi.org/10.1523/jneurosci.14-07-04252.1994 domyancic av, morilak da. distribution of alpha1a adrenergic receptor mrna in the rat brain visualized by in situ hybridization. j comp neurol 1997;386:358-378. https://doi.org/10.1002/(sici)1096-9861(19970929)386:3<358::aid-cne3>3.0.co;2-0 benarroch ee. locus coeruleus. cell tissue res 2018;373:221-232. https://doi.org/10.1007/s00441-017-2649-1 paspalas cd papadopoulos gc. ultrastructural relationships between noradrenergic nerve fibers and non-neuronal elements in the rat cerebral cortex. glia 1996;17:133-146. https://doi.org/10.1002 (sici)1098-1136(199606)17:2<133::aid-glia5>3.0.co;2-3. yoshioka y, negoro r, kadoi h, et al. noradrenaline protects neurons against h2o2-induced death by increasing the supply of glutathione from astrocytes via beta3-adrenoceptor stimulation. j neurosci res. 2021 feb;99(2):621-637. https://doi.org/10.1002/jnr.24733 giorgi fs, galgani a, puglisi-allegra s, et al., locus coeruleus and neurovascular unit: from its role in physiology to its potential role in alzheimer’s disease pathogenesis. j neurosci res 2020;98:2406-2434. https://doi.org/10.1002/jnr.24718 feinstein dl, heneka mt, gavrilyuk v, et al. noradrenergic regulation of inflammatory gene expression in brain. neurochem int 2002;41:357-365. https://doi.org/10.1016/s0197-0186(02)00049-9 bekar lk, wei hs, nedergaard m. the locus coeruleus-norepinephrine network optimizes coupling of cerebral blood volume with oxygen demand. j cereb blood flow metab 2012;32:2135-2145. https://doi.org/10.1038/jcbfm.2012.115 wirth kj. role of noradrenergic brain nuclei in the regulation of carotid artery blood flow: pharmacological evidence from anesthetized pigs with alpha-2 adrenergic receptor modulator drugs. j alzheimers dis 2018;66:407-419. https://doi.org/10.3233/jad-180340 descarries l, mechawar n. ultrastructural evidence for diffuse transmission by monoamine and acetylcholine neurons of the central nervous system. prog brain res 2000;125:27-47. https://doi.org/10.1016/s0079-6123(00)25005-x watabe am, zaki pa, o'dell tj. coactivation of β-adrenergic and cholinergic receptors enhances the induction of long-term potentiation and synergistically activates mitogen-activated protein kinase in the hippocampal ca1 region. j neurosci 2000;20: 5924-5931. https://doi.org/10.1523/jneurosci.20-16-05924.2000 giorgi fs, biagioni f, galgani a, et al. locus coeruleus modulates neuroinflammation in parkinsonism and dementia. int j mol sci 2020; 21: 8630. https://doi.org/10.3390/ijms21228630 brown re, sergeeva oa, eriksson ks, et al. convergent excitation of dorsal raphe serotonin neurons by multiple arousal systems (orexin/hypocretin, histamine and noradrenaline). j neurosci 2002;22: 8850-8859. https://doi.org/10.1523/jneurosci.22-20-08850.2002 morilak da, barrera g, echevarria dj, et al. role of brain norepinephrine in the behavioral response to stress. prog neuropsychopharmacol biol psychiatry 2005;29:1214-1224. https://doi.org/10.1016/j.pnpbp.2005.08.007 zarow c, lyness sa, mortimer ja, et al. neuronal loss is greater in the locus coeruleus than nucleus basalis and substantia nigra in alzheimer and parkinson diseases. arch neurol 2003; 60:337-341. https://doi.org/10.1001/archneur.60.3.337 mravec b, lejavova k, cubinkova v. locus (coeruleus) minoris resistentiae in pathogenesis of alzheimer’s disease. curr alzheimer res 2014; 11: 992-1001. https://doi.org/10.2174/1567205011666141107130505 ross ja, mcgonigle p, van bockstaele ej. locus coeruleus, norepinephrine and aβ peptides in alzheimer's disease. neurobiol stress 2015;2:73-84. https://doi.org/10.1016/j.ynstr.2015.09.002 bekdash ra. the cholinergic system, the adrenergic system and the neuropathology of alzheimer's disease. int j mol sci 2021;22:1273. https://doi.org/10.3390/ijms22031273 lamerand s, shahidehpour r, ayala i, et al. calbindin-d28k, parvalbumin, and calretinin in young and aged human locus coeruleus. neurobiol aging 2020;94:243-249. https://doi.org/10.1016/j.neurobiolaging.2020.06.006 samuels er, szabadi e. functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part ii: physiological and pharmacological manipulations and pathological alterations of locus coeruleus activity in humans. curr neuropharmacol 2008;6:254-285. https://doi.org/10.2174/157015908785777193 trillo l, das d, hsieh w, et al. ascending monoaminergic systems alterations in alzheimer's disease: translating basic science into clinical care. neurosci biobehav rev 2013;37:1363-1679. https://doi.org/10.1016/j.neubiorev.2013.05.008 patthy á, murai j, hanics j, et al. neuropathology of the brainstem to mechanistically understand and to treat alzheimer's disease. j clin med 2021;10:1555. cross aj, crow tj, perry ek, et al. reduced dopamine beta-hydroxylase activity in alzheimer's disease. br med j 1981;282:93-94. https://doi.org/10.1136/bmj.282.6258.93 perry ek, tomlinson be, blessed g, et al. neuropathological and biochemical observations on the noradrenergic system in alzheimer's disease. j neurol sci 1981;51:279-287. https://doi.org/10.1016/0022-510x(81)90106-4 mustapić m, presečki p, pivac n, et al. genotype-independent decrease in plasma dopamine β-hydroxylase activity in alzheimer's disease. prog neuropsychopharmacol biol psychiatry 2013;44:94-99. https://doi.org/10.1016/j.pnpbp.2013.02.002 serra l, d’amelio m, di domenico c, et al. in vivo mapping of brainstem nuclei functional connectivity disruption in alzheimer’s disease. neurobiol aging 2018;72:72-82. https://doi.org/10.1016/j.neurobiolaging.2018.08.012 schöne c, burdakov d. orexin/hypocretin and organizing principles for a diversity of wake-promoting neurons in the brain. curr top behav neurosci 2017;33:51-74. https://doi.org/10.1007/7854_2016_45 sakurai t, saito yc, yanagisawa m. interaction between orexin neurons and monoaminergic systems. front neurol neurosci 2021;45:11-21. https://doi.org/10.1159/000514955 liguori c. orexin and alzheimer's disease. curr top behav neurosci 2017;33:305-322. https://doi.org/10.1007/7854_2016_50 um yh, lim hk. orexin and alzheimer's disease: a new perspective. psychiatry investig 2020;17:621-626. https://doi.org/10.30773/pi.2020.0136 dauvilliers y. hypocretin/orexin, sleep and alzheimer's disease. front neurol neurosci 2021;45:139-149. https://doi.org/10.1159/000514967 fredholm bb, ijzerman ap, jacobson ka, et al. international union of pharmacology. xxv. nomenclature and classification of adenosine receptors. pharmacol rev 2001;53:527-552. pmid: 11734617 fredholm bb, ijzerman ap, jacobson ka, et al. international union of basic and clinical pharmacology. lxxxi. nomenclature and classification of adenosine receptors: an update. pharmacol rev 2011;63:1-34. https://doi.org/10.1124/pr.110.003285 cunha ra. adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors. neurochem int 2001;38:107-125. https://doi.org/10.1016/s0197-0186(00)00034-6 fredholm bb, chen jf, cunha ra, et al. adenosine and brain function. int rev neurobiol 2005;63:191-270. https://doi.org/10.1016/s0074-7742(05)63007-3 sheth s, brito r, mukherjea d, et al. adenosine receptors: expression, function and regulation. int j mol sci 2014; 15: 2024-2052. https://doi.org/10.3390/ijms15022024 sebastiao am, ribeiro ja. adenosine a2 receptor-mediated excitatory actions on the nervous system. prog neurobiol 1996;48:167-189. https://doi.org/10.1016/0301-0082(95)00035-6 cunha ra, almeida t, ribeiro ja. modification by arachidonic acid of extracellular adenosine metabolism and neuromodulatory action in the rat hippocampus. j biol chem 2000;275:37572-37581. https://doi.org/10.1074/jbc.m003011200 brand a, vissiennon z, eschke d, et al. adenosine a(1) and a(3) receptors mediate inhibition of synaptic transmission in rat cortical neurons. neuropharmacology 2001;40:85–95. https://doi.org/10.1016/s0028-3908(00)00117-9 hettinger bd, lee a, linden j, et al. ultrastructural localization of adenosine a2a receptors suggests multiple cellular sites for modulation of gabaergic neurons in rat striatum. j comp neurol 2001;431:331-346. https://doi.org/10.1002/1096-9861(20010312)431:3<331::aid-cne1074>3.0.co;2-w lopes lv, cunha ra, kull b, et al. adenosine a(2a) receptor facilitation of hippocampal synaptic transmission is dependent on tonic a(1) receptor inhibition. neuroscience 2002;112:319-329. https://doi.org/10.1016/s0306-4522(02)00080-5 rebola n, oliveira cr, cunha ra. transducing system operated by adenosine a(2a) receptors to facilitate acetylcholine release in the rat hippocampus. eur j pharmacol 2002;454:31-38. https://doi.org/10.1016/s0014-2999(02)02475-5 rebola n, rodrigues rj, oliveira cr, et al. different roles of adenosine a1, a2a and a3 receptors in controlling kainate-induced toxicity in cortical cultured neurons. neurochem int 2005;47:317-325. https://doi.org/10.1016/j.neuint.2005.05.009 kalaria rn, sromek s,wilcox bj, et al. hippocampal adenosine a1 receptors are decreased in alzheimer’s disease. neurosci lett 1990;118:257-260. https://doi.org/10.1016/0304-3940(90)90641-l ulas j, brunner lc, nguyen l, et al. reduced density of adenosine a1 receptors and preserved coupling of adenosine a1 receptors to g proteins in alzheimer hippocampus: a quantitative autoradiographic study. neuroscience 1993;52:843-854. https://doi.org/10.1016/0306-4522(93)90533-l streit wj, braak h, xue qs, et al. dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in alzheimer's disease. acta neuropathol. 2009;118:475-485. https://doi.org/10.1007/s00401-009-0556-6 deckert j, abel f, kunig g, et al. loss of human hippocampal adenosine a1 receptors in dementia: evidence for lack of specificity. neurosci lett 1998;244:1-4. https://doi.org/10.1016/s0304-3940(98)00108-6 angulo e, casado v, mallol j, et al. a1 adenosine receptors accumulate in neurodegenerative structures in alzheimer disease and mediate both amyloid precursor protein processing and tau phosphorylation and translocation. brain pathol 2003;13:440-451. https://doi.org/10.1111/j.1750-3639.2003.tb00475.x albasanz jl, perez s, barrachina m, et al. up-regulation of adenosine receptors in the frontal cortex in alzheimer’s disease brain pathology brain pathol 2008;18:211-219. https://doi.org/10.1111/j.1750-3639.2007.00112 howlett ac. the cannabinoid receptors. prostaglandins other lipid mediat 2002;68-69: 619-631. https://doi.org/10.1016/s0090-6980(02)00060-6 devane wa, axelrod j. enzymatic synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, by brain membranes. proc natl acad sci usa 1994;91:6698-6701. https://doi.org/10.1073/pnas.91.14.6698 sugiura t, waku k. 2-arachidonoylglycerol and the cannabinoid receptors. chem phys lipids 2000;108:89-106. https://doi.org/10.1016/s0009-3084(00)00189-4 sugiura t, waku k. cannabinoid receptors and their endogenous ligands. j biochem 2002; 132:7-12. https://doi.org/10.1093/oxfordjournals.jbchem.a003200 sugiura t. physiological roles of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand. biofactors 2009;35:88-97. https://doi.org/10.1002/biof.18 glass m, dragunow m, faull rl. cannabinoid receptors in the human brain: a detailed anatomical and quantitative autoradiographic study in the fetal, neonatal and adult human brain. neuroscience 1997;77:299-318. https://doi.org/10.1016/s0306-4522(96)00428-9 biegon a, kerman ia. autoradiographic study of preand postnatal distribution of cannabinoid receptors in human brain. neuroimage 2001;14:1463-1468. https://doi.org/10.1006/nimg.2001.0939 katona i, rancz ea, acsady l, et al. distribution of cb1 cannabinoid receptors in the amygdala and their role in the control of gabaergic transmission. j neurosci 2001; 21: 9506-9518. https://doi.org/10.1523/jneurosci.21-23-09506.2001 katona i, urban gm, wallace m, et al. molecular composition of the endocannabinoid system at glutamatergic synapses. j neurosci 2006; 26: 5628-5637. https://doi.org/10.1523/jneurosci.0309-06.2006 ohno-shosaku t, tsubokawa h, mizushima i, et al. presynaptic cannabinoid sensitivity is a major determinant of depolarization-induced retrograde suppression at hippocampal synapses. j neurosci 2002; 22:3864-3872. https://doi.org/10.1523/jneurosci.22-10-03864.2002 wilson ri, kunos g, nicoll ra. presynaptic specificity of endocannabinoid signaling in the hippocampus. neuron 2001;31:453-462. https://doi.org/10.1016/s0896-6273(01)00372-5 wilson ri, nicoll ra. endocannabinoid signaling in the brain. science 2002; 296:678-682. https://doi.org/10.1126/science.1063545 cabral ga, griffin-thomas l. emerging role of the cannabinoid receptor cb2 in immune regulation: therapeutic prospects for neuroinflammation. expert rev mol med 2009;11:e3. https://doi.org/10.1017/s1462399409000957 mecha m, feliu a, carrillo-salinas fj, et al. endocannabinoids drive the acquisition of an alternative phenotype in microglia. brain behav immun 2015;49:233-245. https://doi.org/10.1016/j.bbi.2015.06.002 van sickle md, duncan m, kingsley pj, et al. identification and functional characterization of brainstem cannabinoid cb2 receptors. science 2005;310: 329-332. https://doi.org/10.1126/science.1115740 brusco a, tagliaferro pa, saez t, et al. ultrastructural localization of neuronal brain cb2 cannabinoid receptors. ann ny acad sci 2008;1139:450-457. https://doi.org/10.1196/annals.1432.037 onaivi es, ishiguro h, gong jp, et al. functional expression of brain neuronal cb2 cannabinoid receptors are involved in the effects of drugs of abuse and in depression. ann ny acad sci 2008;1139: 434-449. https://doi.org/10.1196/annals.1432.036 maccarrone m, dainese e, oddi s. intracellular trafficking of anandamide: new concepts for signaling. trends biochem sci 2010; 35: 601-608. https://doi.org/10.1016/j.tibs.2010.05.008 pertwee rg, howlett ac, abood me, et al. cannabinoid receptors and their ligands: beyond cb1 and cb2. pharmacol rev 2010;62:588-631. https://doi.org/10.1124/pr.110.003004 aso e, ferrer i. cannabinoids for treatment of alzheimer's disease: moving toward the clinic. front pharmacol 2014;5:37. https://doi.org/10.3389/fphar.2014.00037 ramírez bg, blázquez c, gómez del pulgar t, et al. prevention of alzheimer’s disease pathology by cannabinoids: neuroprotection mediated by blockade of microglial activation. j neurosci 2005;25: 1904-1913. https://doi.org/10.1523/jneurosci.4540-04.2005 solas m, francis pt, franco r, et al. cb2 receptor and amyloid pathology in frontal cortex of alzheimer’s disease patients. neurobiol aging 2013;34: 805-808. https://doi.org/10.1016/j.neurobiolaging 2012.06.005. benito c, núñez e, tolón rm, et al. cannabinoid cb2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in alzheimer’s disease brains. j neurosci 2003;23:11136-11141. https://doi.org/10.1523/jneurosci.23-35-11136.2003 mulder j, zilberter m, pasquare sj, et al. molecular reorganization of endocannabinoid signalling in alzheimer’s disease. brain 2011;134: 1041-1060. https://doi.org/10.1093/brain/awr046 jung km, astarita g, yasar s, et al. an amyloid β42-dependent deficit in anandamide mobilization is associated with cognitive dysfunction in alzheimer’s disease. neurobiol aging 2012;33: 1522-1532. https://doi.org/10.1016/j.neurobiolaging.2011.03.012 hock c, heese k, hulette c, et al. region-specific neurotrophin imbalances in alzheimer disease: decreased levels of brain-derived neurotrophic factor and increased levels of nerve growth factor in hippocampus and cortical areas. arch neurol 2000;57:846-851. https://doi.org/10.1001/archneur.57.6.846 garzon d, yu g, fahnestock m. a new brain-derived neurotrophic factor transcript and decrease in brain-derived neurotrophic factor transcripts 1, 2 and 3 in alzheimer’s disease parietal cortex. j neurochem 2002; 82: 1058-1064. https://doi.org/10.1046/j.1471-4159.2002.01030.x budni j, bellettini-santos t, mina f, et al. the involvement of bdnf, ngf and gdnf in aging and alzheimer's disease. aging dis 2015;6:331-341. https://doi.org/10.14336/ad.2015.0825 mohammadi a, ghasem amooeian v, rashidi e. dysfunction in brain-derived neurotrophic factor signaling pathway and susceptibility to schizophrenia, parkinson's and alzheimer's diseases. curr gene ther 2018;18:45-63. https://doi.org/10.2174/1566523218666180302163029 numakawa t, odaka h. brain-derived neurotrophic factor signaling in the pathophysiology of alzheimer's disease: beneficial effects of flavonoids for neuroprotection. int j mol sci 2021;22:5719. https://doi.org/10.3390/ijms22115719 phillips hs, hains jm, armanini m, et al. bdnf mrna is decreased in the hippocampus of individuals with alzheimer’s disease. neuron 1991;7:695-702. https://doi.org/10.1016/0896-6273(91)90273-3 ferrer i, marín c, rey mj, et al. bdnf and full-length and truncated trkb expression in alzheimer disease: implications in therapeutic strategies. j neuropathol exp neurol 1999;58:729-739. https://doi.org/10.1097/00005072-199907000-00007 holsinger rm, schnarr j, henry p, et al. quantitation of bdnf mrna in human parietal cortex by competitive reverse transcription-polymerase chain reaction: decreased levels in alzheimer's disease. brain res mol brain res 2000;76:347-354. https://doi.org/10.1016/s0169-328x(00)00023-1 wong j, higgins m, halliday g, et al. amyloid beta selectively modulates neuronal trkb alternative transcript expression with implications for alzheimer's disease. neuroscience 2012;210:363-374. https://doi.org/10.1016/j.neuroscience.2012.02.037 fleitas c, piñol-ripoll g, marfull p, et al. probdnf is modified by advanced glycation end-products in alzheimer’s disease, and causes neuronal apoptosis by inducing p75 neutrophin receptor processing. mol brain 2018;11:68. https://doi.org/10.1186/s13041-018-0411-6 ye x, tai w, zhang d. the early events of alzheimer's disease pathology: from mitochondrial dysfunction to bdnf axonal transport deficits. neurobiol aging 2012;33:1122.e1-10. https://doi.org/10.1016/j.neurobiolaging.2011.11.004 hellweg r, gericke ca, jendroska k, et al. ngf content in the cerebral cortex of non-demented patients with amyloid-plaques and in symptomatic alzheimer’s disease. int j dev neurosci 1998; 16:787-794. https://doi.org/10.1016/s0736-5748(98)00088-4 mufson ej, kordower jh. nerve growth factor receptor expressing human basal forebrain neurons: pathologic alterations in alzheimer's and parkinson's disease. prog clin biol res 1989;317:401-414. pmid: 2557638. fahnestock m, michalski b, xu b, et al. the precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in alzheimer’s disease. mol cell neurosci 2001; 18:210-220. https://doi.org/10.1006/mcne.2001.1016 peng s, wuu j, mufson ej, et al. increased prongf levels in subjects with mild cognitive impairment and mild alzheimer disease. j neuropathol exp neurol 2004; 63:641-649. https://doi.org/10.1093/jnen/63.6.641 pedraza ce, podlesniy p, vidal n, et al. pro-ngf isolated from the human brain affected by alzheimer’s disease induces neuronal apoptosis mediated by p75ntr. am j pathol 2005; 166:533-543. https://doi.org/10.1016/s0002-9440(10)62275-4 podlesniy p, kichev a, pedraza c, et al. pro-ngf from alzheimer’s disease and normal human brain displays distinctive abilities to induce processing and nuclear translocation of intracellular domain of p75ntr and apoptosis. am j pathol 2006; 169:119-131. https://doi.org/10.2353/ajpath.2006.050787 al-shawi r, hafner a, olsen j, et al. neurotoxic and neurotrophic roles of prongf and the receptor sortilin in the adult and ageing nervous system. eur j neurosci 2008; 27:2103-2114. https://doi.org/10.1111/j.1460-9568.2008.06152.x lin jh, walter p, yen tsb. endoplasmic reticulum stress in disease pathogenesis. annu rev pathol 2008;3:399-425. https://doi.org/10.1146/annurev.pathmechdis.3.121806.151434 kozutsumi y, segal m, normington k, et al. the presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. nature 1998;332:462-464. https://doi.org/10.1038/332462a0 oakes sa, papa fr. the role of endoplasmic reticulum stress in human pathology. annu rev pathol. 2015;10:173-194. https://doi.org/10.1146/annurev-pathol-012513-104649 hoozemans jjm, veerhuis r, van haastert es, et al. the unfolded protein response is activated in alzheimer’s disease. acta neuropathol 2005; 110:165-172. https://doi.org/10.1007/s00401-005-1038-0 hoozemans jj, van haastert es, nijholt da, et al. the unfolded protein response is activated in pretangle neurons in alzheimer’s disease hippocampus. am j pathol 2009;174:1241-1251. https://doi.org/10.2353/ajpath.2009.080814 huang hc, tang d, lu sy, et al. endoplasmic reticulum stress as a novel neuronal mediator in alzheimer's disease. neurol res 2015;37:366-374. https://doi.org/10.1179/1743132814y.0000000448 li jq, yu jt, jiang t, tan l. endoplasmic reticulum dysfunction in alzheimer’s disease. mol neurobiol 2015;51:383-395. https://doi.org/10.1007/s12035-014-8695-8 takuma k, yan ss, stern dm, et al. mitocondrial dysfunction, endoplasmic reticulum stress, and apoptosis in alzheimer’s disease. j pharmacol sci 2005;97:312-316. https://doi.org/10.1254/jphs cpj04006x. santos le, ferreira st. crosstalk between endoplasmic reticulum stress and brain inflammation in alzheimer's disease. neuropharmacology 2018;136:350-360. https://doi.org/10.1016/j.neuropharm.2017.11.016 salminen a, kaarniranta k, kauppinen a. er stress activates immunosuppressive network: implications for aging and alzheimer's disease. j mol med 2020;98:633-650. https://doi.org/10.1007/s00109-020-01904-z liu xj, wei j, shang yh, et al. modulation of aβpp and gsk3β by endoplasmic reticulum stress and involvement in alzheimer's disease. j alzheimers dis 2017;57:1157-1170. https://doi.org/10.3233/jad-161111 uddin ms, mamun a, rahman ma, et al. emerging proof of protein misfolding and interactions in multifactorial alzheimer's disease. curr top med chem 2020;20:2380-2390. https://doi.org/10.2174/1568026620666200601161703 uddin ms, tewari d, sharma g, et al. molecular mechanisms of er stress and upr in the pathogenesis of alzheimer's disease. mol neurobiol 2020;57:2902-2919. https://doi.org/10.1007/s12035-020-01929-y nijholt da, de graaf tr, van haastert es, et al. endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells: implications for alzheimer's disease. cell death differ 2011;18:1071-1081. https://doi.org/10.1038/cdd.2010.176 scheper w, nijholt da, hoozemans jj. the unfolded protein response and proteostasis in alzheimer disease: preferential activation of autophagy by endoplasmic reticulum stress. autophagy 2011;7:910-911. https://doi.org/10.4161/auto.7.8.15761 cai y, arikkath j, yang l, et al. interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders. autophagy 2016;12:225-244. https://doi.org/10.1080/15548627.2015.1121360 nandi d, tahiliani p, kumar a, et al. the ubiquitin-proteasome system. j biosci 2006;31:137-155. https://doi.org/10.1007/bf02705243 wang y, le wd. autophagy and ubiquitin proteasome system. adv exp med biol 2019;1206:527-550. https://doi.org/10.1007/978-981-15-0602-4_25 yu l, chen y, tooze sa. autophagy pathway: cellular and molecular mechanisms. autophagy 2018;14:207-215. https://doi.org/10.1080/15548627.2017.1378838 tekirdag k, cuervo am. chaperone-mediated autophagy and endosomal microautophagy: joint by a chaperone. j biol chem 2018; 293:5414-5424. https://doi.org/10.1074/jbc.r117.818237 he c, klionsky dj. regulation mechanisms and signaling pathways of autophagy. annu rev genet 2009; 43:67-93. https://doi.org/10.1146/annurev-genet-102808-114910 mehrpour m, esclatine a, beau i, et al. overview of macroautophagy regulation in mammalian cells. cell res 2010; 20:748-762. https://doi.org/10.1038/cr.2010.82 tanida i. autophagosome formation and molecular mechanism of autophagy. antioxid redox signal 2011; 14:2201-2214. https://doi.org/10.1089/ars.2010.3482 cataldo am, paskevich pa, kominami e, et al. lysosomal hydrolases of different classes are abnormally distributed in brains of patients with alzheimer disease. proc natl acad sci usa 1991;88:10998-101002. https://doi.org/10.1073/pnas.88.24.10998 yu wh, cuervo am, kumar a, et al. macroautophagy—a novel b-amyloid peptide-generating pathway activated in alzheimer’s disease. j cell biol 2005;171:87-98. https://doi.org/10.1083/jcb.200505082 nixon ra, wegiel j, kumar a, et al. extensive involvement of autophagy in alzheimer disease: an immunoelectron microscopy study. j neuropathol exp neurol 2005;64:113-122. https://doi.org/10.1093/jnen/64.2.113 barrachina m, maes t, buesa c, et al. up-regulation of lysosome associated-membrane protein 1 (lamp-1) in alzheimer’s disease. neuropathol appl neurobiol 2006;32:505-516. https://doi.org/10.1111/j.1365-2990.2006.00756.x cecarini v, ding q, keller jn. oxidative inactivation of the proteasome in alzheimer’s disease. free radic res 2007; 41: 673-680. https://doi.org/10.1080/10715760701286159 pickford f, masliah e, britschgi m, et al. the autophagy-related protein beclin 1 shows reduced expression in early alzheimer disease and regulates amyloid β accumulation in mice. j clin invest 2008; 118:2190–2199. https://doi.org/10.1172/jci33585 lipinski mm, zheng b, lu t, et al. genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in alzheimer's disease. proc natl acad sci usa 2010;107:14164-14169. https://doi.org/10.1073/pnas.1009485107 ghavami s, shojaei s, yeganeh b, et al. autophagy and apoptosis dysfunction in neurodegenerative disorders. prog neurobiol 2014;112:24-49. https://doi.org/10.1016/j.pneurobio.2013.10.004 uddin ms, stachowiak a, mamun aa, et al. autophagy and alzheimer's disease: from molecular mechanisms to therapeutic implications. front aging neurosci 2018;10:04. https://doi.org/10.3389/fnagi.2018.00004 guo f, liu x, cai h, le w. autophagy in neurodegenerative diseases: pathogenesis and therapy. brain pathol 2018;28:3-13. https://doi.org/10.1111/bpa.12545 pradeepkiran ja, reddy ph. defective mitophagy in alzheimer's disease. ageing res rev 2020;64:101191. https://doi.org/10.1016/j.arr.2020.101191 salminen a, kaarniranta k, kauppinen a, et al. impaired autophagy and app processing in alzheimer's disease: the potential role of beclin 1 interactome. prog neurobiol 2013;106-107:33-54. https://doi.org/10.1016/j.pneurobio.2013.06.002 johnston-carey hk, pomatto lc, davies kj.the immunoproteasome in oxidative stress, aging, and disease. crit rev biochem mol biol 2015;51:268-281. https://doi.org/10.3109/10409238 2016.1172554. lee mj, lee jh, rubinsztein dc. tau degradation: the ubiquitin-proteasome system versus the autophagy-lysosome system. prog neurobiol 2013; 105: 49–59. https://doi.org/10.1016/j.pneurobio 2013.03.001. riederer bm, leuba g, vernay a, et al. the role of the ubiquitin proteasome system in alzheimer’s disease. exp biol med 2011;236:268-276. https://doi.org/10.1258/ebm.2010.010327 tramutola a, di domenico f, barone e, et al. polyubiquitinylation profile in down syndrome brain before and after the development of alzheimer neuropathology. antioxid redox signal 2017;26:280-298. https://doi.org/10.1089/ars.2016.6686 weng fl, he l. disrupted ubiquitin proteasome system underlying tau accumulation in alzheimer's disease. neurobiol aging 2021;99:79-85. https://doi.org/10.1016/j.neurobiolaging.2020.11.015 ferrer i, santpere g, van leeuwen fw. argyrophilic grain disease. brain 2008;131:1416-1432. https://doi.org/10.1093/brain/awm305 tank em, true hl. disease-associated mutant ubiquitin causes proteasomal impairment and enhances the toxicity of protein aggregates. plos genet 2009;5:e1000382. chadwick l, gentle l, strachan j, et al. review: unchained maladie a reassessment of the role of ubb(+1) -capped polyubiquitin chains in alzheimer's disease. neuropathol appl neurobiol 2012;38:118-131. https://doi.org/10.1111/j.1365-2990.2011.01236.x gentier rj, van leeuwen fw. misframed ubiquitin and impaired protein quality control: an early event in alzheimer's disease. front mol neurosci 2015;8:47. https://doi.org/10.3389/fnmol.2015.00047 van tijn p, dennissen fj, gentier rj, et al. mutant ubiquitin decreases β-amyloid plaque formation in a transgenic mouse model of alzheimer’s disease. neurochem int 2012;61:739-748. https://doi.org/10.1016/j.neuint.2012.07.007 verheijen bm, stevens jaa, gentier rjg, et al. paradoxical effects of mutant ubiquitin on aβ plaque formation in an alzheimer mouse model. neurobiol aging 2018;72:62-71. https://doi.org/10.1016/j.neurobiolaging.2018.08.011 ferrer i, boada rovira m, sanchez guerra ml, et al. neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in alzheimer’s disease. brain pathol 2004;14:11-20. https://doi.org/10.1111/j.1750-3639.2004.tb00493.x aso e, lomoio s, lópez-gonzález i, et al. amyloid generation and dysfunctional immunoprotasome activation with disease progression in animal model of familial alzheimer’s disease. brain pathol 2012;22:636-653. https://doi.org/10.1111/j.1750-3639.2011.00560.x orre m, kamphuis w, dooves s, et al. reactive glia show increased immunoproteasome activity in alzheimer's disease. brain 2013;136: 1415-1431. https://doi.org/10.1093/brain/awt083 grimm s, ott c, hörlacher m, et al. advanced-glycation-end-product-induced formation of immunoproteasomes: involvement of rage and jak2/stat1. biochem j 2012;448:127-139. https://doi.org/10.1042/bj20120298 thal dr, del tredici k, ludolph ac, et al. stages of granulovacuolar degeneration: their relation to alzheimer's disease and chronic stress response. acta neuropathol 2011;122:577-589. https://doi.org/10.1007/s00401-011-0871-6 yamazaki y, matsubara t, takahashi t, et al. granulovacuolar degenerations appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders. plos one. 2011;6:e26996. https://doi.org/10.1371/journal.pone.0026996 funk ke, mrak re, kuret j. granulovacuolar degeneration (gvd) bodies of alzheimer’s disease (ad) resemble late-stage autophagic organelles. neuropathol appl neurobiol 2011;37:295-306. https://doi.org/10.1111/j.1365-2990.2010.01135.x köhler c. granulovacuolar degeneration: a neurodegenerative change that accompanies tau pathology. acta neuropathologica 2016; 132: 339-359. https://doi.org/10.1007/s00401-016-1562-0 wiersma vi, van ziel am, vazquez‑sanchez s, et al. granulovacuolar degeneration bodies are neuron‑selective lysosomal structures induced by intracellular tau pathology. acta neuropathologica 2019;138:943-970. https://doi.org/10.1007/s00401-019-02046-4 hondius dc, koopmans f, leistner c, et al. the proteome of granulovacuolar degeneration and neurofibrillary tangles in alzheimer’s disease. acta neuropathol 2021; 141: 341-358. https://doi.org/10.1007/s00401-020-02261-4 hunter s, minett t, polvikoski t, et al. re-examining tau-immunoreactive pathology in the population: granulovacuolar degeneration and neurofibrillary tangles. alzheimers res ther 2015;7:57. https://doi.org/10.1186/s13195-015-0141-2 rudy cc, hunsberger hc, weitzner ds, et al. the role of the tripartite glutamatergic synapse in the pathophysiology of alzheimer's disease. aging dis 2015;6:131-148. https://doi.org/10.14336/ad.2014.0423 garwood cj, radcliffe le, simpson je, et al. review: astrocytes in alzheimer’s disease and other age-associated dementias: the supporting player with a central role. neuropathol appl neurobiol 2017; 43:281-298. https://doi.org/10.1111/nan.12338 ferrer i. diversity of astroglial responses across human neurodegenerative disorders and brain aging. brain pathol 2017;27:645-674. https://doi.org/10.1111/bpa.12538 verkhratsky a, zorec r, rodriguez-arellano jj, et al. neuroglia in ageing. adv exp med biol 2019;1175:181-197. https://doi.org/10.1007/978-981-13-9913-8_8 price br, johnson la, norris cm. reactive astrocytes: the nexus of pathological and clinical hallmarks of alzheimer's disease. ageing res rev 2021;68:101335. https://doi.org/10.1016/j.arr.2021.101335 phillips ec, croft cl, kurbatskaya k, et al. astrocytes and neuroinflammation in alzheimer's disease. biochem soc trans 2014; 42: 1321-1325. https://doi.org/10.1042/bst20140155 heneka mt, carson mj, el khoury j, et al. o, neuroinflammation in alzheimer's disease. lancet neurol 2015;14: 388-405. https://doi.org/10.1016/s1474-4422(15)70016-5 de strooper bd, karran e. the cellular phase of alzheimer’s disease. cell 2016;164: 603-615. https://doi.org/10.1016/j.cell.2015.12.056 liddelow sa, guttenplan ka, clarke le, et al. neurotoxic reactive astrocytes are induced by activated microglia. nature 2017;541: 481-487. https://doi.org/10.1038/nature21029 kaur d, sharma v, deshmukh r. activation of microglia and astrocytes: a roadway to neuroinflammation and alzheimer's disease. inflammopharmacology 2019;27:663-677. https://doi.org/10.1007/s10787-019-00580-x verkhratsky a, nedergaard m. physiology of astroglia. physiol rev 2018;98:239-389. https://doi.org/10.1152/physrev.00042.2016 verkhratsky a, parpura v, li b, et al. astrocytes: the housekeepers and guardians of the cns. adv neurobiol 2021;26:21-53. https://doi.org/10.1007/978-3-030-77375-5_2 cotrina ml, nedergaard m. astrocytes in the aging brain. j neurosci res 2002;67:1-10. https://doi.org/10.1002/jnr.10121 porchet r, probst a, bouras c, et al. analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in alzheimer's disease. proteomics 2003;3: 1476-1485. https://doi.org/10.1002/pmic.200300456 lynch am, murphy kj, deighan bf, et al. the impact of glial activation in the aging brain. aging dis 2010;1: 262-278. pmid: 22396865. salminen a, ojala j, kaarniranta k, et al. astrocytes in the aging brain express characteristics of senescence-associated secretory phenotype. eur j neurosci 2011;34: 3-11. https://doi.org/10.1111/j.1460-9568.2011.07738.x farrall aj, wardlaw jm. blood-brain barrier: ageing and microvascular disease--systematic review and meta-analysis. neurobiol aging 2009;30:337-352. https://doi.org/10.1016/j.neurobiolaging 2007.07.015. popescu bo, toescu ec, popescu lm, et al. blood-brain barrier alterations in ageing and dementia. j neurol sci 2009;283: 99-106. https://doi.org/10.1016/j.jns.2009.02.321 silverberg gd, messier aa, miller mc, et al. amyloid efflux transporter expression at the blood-brain barrier declines in normal aging. j neuropathol exp neurol 2010;69: 1034-1043. https://doi.org/10.1097/nen.0b013e3181f46e25 silverberg gd, miller mc, messier aa, et al. amyloid deposition and influx transporter expression at the blood-brain barrier increase in normal aging. j neuropathol exp neurol 2010b;69: 98-108. https://doi.org/10.1097/nen.0b013e3181c8ad2f pérez e, barrachina m, rodriguez a, et al. aquaporin expression in the cerebral cortex is increased at early stages of alzheimer's disease. brain res 2007;1128:164-174. https://doi.org/10.1016/j.brainres 2006.09.109. zeppenfeld dm, simon m, haswell jd, et al. association of perivascular localization of aquaporin-4 with cognition and alzheimer disease in aging brains. jama neurol 2017;74:91-99. https://doi.org/10.1001/jamaneurol.2016.4370 heneka mt, sastre m, dumitrescu-ozimek l, et al. focal glial activation coincides with increased bace1 activation and precedes amyloid plaque deposition in app[v717i] transgenic mice. j neuroinflammation 2005;2: 22. https://doi.org/10.1186/1742-2094-2-22 rodríguez jj, olabarria m, chvatal a, et al. astroglia in dementia and alzheimer's disease. cell death differ 2009;16: 378-385. https://doi.org/10.1038/cdd.2008.172 rodríguez-arellano jj, parpura v, zorec r, et al. astrocytes in physiological aging and alzheimer's disease. neuroscience 2016;323:170-182. https://doi.org/10.1016/j.neuroscience.2015.01.007 verkhratsky a, rodríguez jj, parpura v. neuroglia in ageing and disease. cell tissue res 2014;357: 493-503. https://doi.org/10.1007/s00441-014-1814-z verkhratsky a, zorec r, rodríguez jj, et al. astroglia dynamics in ageing and alzheimer's disease. curr opin pharmacol 2016;26: 74-79. https://doi.org/10.1016/j.coph.2015.09.011 nagy ji, li w, hertzberg el, marotta ca. elevated connexin43 immunoreactivity at sites of amyloid plaques in alzheimer's disease. brain res 1996;717: 173-178. https://doi.org/10.1016/0006-8993(95)01526-4 kuchibhotla kv, lattarulo cr, hyman bt, et al. synchronous hyperactivity and intercellular calcium waves in astrocytes in alzheimer mice. science 2009; 323: 1211-1215. https://doi.org/10.1126/science.1169096 abramov ay, canevari l, duchen mr. calcium signals induced by amyloid beta peptide and their consequences in neurons and astrocytes in culture. biochim biophys acta 2004;1742: 81-87. https://doi.org/10.1016/j.bbamcr.2004.09.006 simpson je, ince pg, shaw pj, et al. microarray analysis of the astrocyte transcriptome in the aging brain: relationship to alzheimer's pathology and apoe genotype. neurobiol aging 2011;32: 1795-1807. https://doi.org/10.1016/j.neurobiolaging.2011.04.013 galea e, weinstock ld, larramona-arcas r, et al. multi-transcriptomic analysis points to early organelle dysfunction in human astrocytes in alzheimer's disease. neurobiol dis 2022;166:105655. https://doi.org/10.1016/j.nbd.2022.105655 simpson je, ince pg, lace g, et al. astrocyte phenotype in relation to alzheimer-type pathology in the ageing brain. neurobiol aging 2010;31: 578-590. https://doi.org/10.1016/j.neurobiolaging 2008.05.015. kamphuis w, middeldorp j, kooijman l, et al. glial fibrillary acidic protein isoform expression in plaque related astrogliosis in alzheimer's disease. neurobiol aging 2014;35: 492-510. https://doi.org/10.1016/j.neurobiolaging.2013.09.035 beach tg, mcgeer eg. lamina-specific arrangement of astrocytic gliosis and senile plaques in alzheimer's disease visual cortex. brain res 1988;463: 357-361. https://doi.org/10.1016/0006-8993(88)90410-6 yin kj, cirrito jr, yan p, et al. matrix metalloproteinases expressed by astrocytes mediate extracellular amyloid-beta peptide catabolism. j neurosci 2006;26:10939-10948. https://doi.org/10.1523/jneurosci 2085-06.2006. yang y, estrada ey, thompson jf, et al. matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. j cereb blood flow metab 2007;27:697-709. https://doi.org/10.1038/sj.jcbfm.9600375 miners js, van helmond z, kehoe pg, et al. changes with age in the activities of beta-secretase and the abeta-degrading enzymes neprilysin, insulin-degrading enzyme and angiotensin-converting enzyme. brain pathol 2010;20:794-802. https://doi.org/10.1111/j.1750-3639.2010.00375.x carrano a, hoozemans jj, van der vies sm, et al. neuroinflammation and blood-brain barrier changes in capillary amyloid angiopathy. neurodegener dis 2012;10:329-331. https://doi.org/10.1159/000334916 han bh, zhou ml, johnson aw, et al. contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged tg2576 mice. proc natl acad sci usa 2015;112:e881–890. https://doi.org/10.1073/pnas.1414930112 funato h, yoshimura m, yamazaki t, et al. astrocytes containing amyloid beta-protein (abeta)-positive granules are associated with abeta40-positive diffuse plaques in the aged human brain. am j pathol 1988;152: 983-992. pmid: 9546359. guénette sy. astrocytes: a cellular player in abeta clearance and degradation. trends mol med 2003;9: 279-280. https://doi.org/10.1016/s1471-4914(03)00112-6 nagele rg, wegiel j, venkataraman v, et al. contribution of glial cells to the development of amyloid plaques in alzheimer's disease. neurobiol aging 2004;25: 663-674. https://doi.org/10.1016/j.neurobiolaging 2004.01.007. thal dr, schultz c, dehghani f, et al. amyloid beta-protein (abeta)-containing astrocytes are located preferentially near n-terminal-truncated abeta deposits in the human entorhinal cortex. acta neuropathol 2000;100: 608-617. https://doi.org/10.1007/s004010000242 koistinaho m, lin s, wu x, esterman m, et al. apolipoprotein e promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides. nat med 2004;10: 719-726. https://doi.org/10.1038/nm1058 yan p, hu x, song h, yin k, et al. matrix metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact plaques in situ. j biol chem 2006;281: 24566-24574. https://doi.org/10.1074/jbc.m602440200 xiao q, yan p, ma x, et al. enhancing astrocytic lysosome biogenesis facilitates aβ clearance and attenuates amyloid plaque pathogenesis. j neurosci 2014;34: 9607-9620. https://doi.org/10.1523/jneurosci.3788-13.2014 basak jm, verghese pb, yoon h, et al. low-density lipoprotein receptor represents an apolipoprotein e-independent pathway of aβ uptake and degradation by astrocytes. j biol chem 2012; 287:13959-13971. https://doi.org/10.1074/jbc.m111.288746 rossner s, lange-dohna c, zeitschel u, et al. alzheimer's disease beta-secretase bace1 is not a neuron-specific enzyme. j neurochem 2005;92: 226-234. https://doi.org/10.1111/j.1471-4159.2004.02857.x zhao j, o'connor t, vassar r. the contribution of activated astrocytes to aβ production: implications for alzheimer's disease pathogenesis. j neuroinflammation 2011;8: 150. https://doi.org/10.1186/1742-2094-8-150 sanchez-mico mv, jimenez s, gomez-arboledas a, et al. amyloid beta impairs phagocytosis of dystrophic synapses by astrocytes in alzheier’s disease. glia 2021;69:997-1011. https://doi.org/10.1002/glia.23943 grolla aa, sim ja, lim d, et al. amyloid-β and alzheimer's disease type pathology differentially affects the calcium signalling toolkit in astrocytes from different brain regions. cell death dis 2013; 4: e623. https://doi.org/10.1038/cddis.2013.145 lim d, ronco v, grolla aa, et al. glial calcium signalling in alzheimer's disease. rev physiol biochem pharmacol 2014;167: 45-65. https://doi.org/10.1007/112_2014_19 verkhratsky a, rodríguez-arellano jj, parpura v, et al. astroglial calcium signalling in alzheimer's disease. biochem biophys res commun 2017;483:1005-1012. https://doi.org/10.1016/j.bbrc.2016.08.088 talantova m, sanz-blasco s, zhang x, et al. aβ induces astrocytic glutamate release, extrasynaptic nmda receptor activation, and synaptic loss. proc natl acad sci usa 2013;110: e2518-2527. https://doi.org/10.1073/pnas.1306832110 lim d, iyer a, ronco v, et al. amyloid beta deregulates astroglial mglur5-mediated calcium signaling via calcineurin and nf-kb. glia 2013;61: 1134-1145. https://doi.org/10.1002/glia.22502 verkhratsky a, chvátal a. nmda receptors in astrocytes. neurochem res 2020;45:122-133. https://doi.org/10.1007/s11064-019-02750-3 lauderback cm, hackett jm, huang ff, et al. the glial glutamate transporter, glt-1, is oxidatively modified by 4-hydroxy-2-nonenal in the alzheimer's disease brain: the role of abeta1-42. j neurochem 2001;78: 413-416. https://doi.org/10.1046/j.1471-4159.2001.00451.x scott ha, gebhardt fm, mitrovic ad, et al. glutamate transporter variants reduce glutamate uptake in alzheimer's disease. neurobiol aging 2011;32: 553.e1-11. https://doi.org/10.1016/j.neurobiolaging 2010.03.008. woltjer rl, duerson k, fullmer jm, et al. aberrant detergent-insoluble excitatory amino acid transporter 2 accumulates in alzheimer disease. j neuropathol exp neurol 2010;69: 667-676. https://doi.org/10.1097/nen.0b013e3181e24adb masliah e, alford m, deteresa r, et al. deficient glutamate transport is associated with neurodegeneration in alzheimer’s disease. ann neurol 1996;40: 759-766. https://doi.org/10.1002/ana.410400512 abdul hm, sama ma, furman jl, et al. cognitive decline in alzheimer’s disease is associated with selective changes in calcineurin/nfat signaling. j neurosci 2009;29:12957-12969. https://doi.org/10.1523/jneurosci.1064-09.2009 orre m, kamphuis w, osborn lm, jansen ah, et al. isolation of glia from alzheimer's mice reveals inflammation and dysfunction. neurobiol aging 2014;35: 2746-2760. https://doi.org/10.1016/j.neurobiolaging 2014.06.004. dansokho c, heneka mt. neuroinflammatory responses in alzheimer’s disease. j neural transm 2018;125:771-779. https://doi.org/10.1007/s00702-017-1831-7 tomimoto h, akiguchi i, wakita h, et al. regressive changes of astroglia in white matter lesions in cerebrovascular disease and alzheimer’s disease patients. acta neuropathol 1997;94:146-152. https://doi.org/10.1007/s004010050686 schipper hm, bennett da, liberman a, et al. glial heme oxygenase-1 expression in alzheimer disease and mild cognitive impairment. neurobiol aging 2006;27:252-261. https://doi.org/10.1016/j.neurobiolaging.2005.01.016 assaraf mi, diaz z, liberman a, et al. brain erythropoietin receptor expression in alzheimer disease and mild cognitive impairment. j neuropathol exp neurol 2007;66:389-398. https://doi.org/10.1097/nen.0b013e3180517b28 owen jb, di domenico f, sultana r, et al. proteomics-determined differences in the concanavalin-a-fractionated proteome of hippocampus and inferior parietal lobule in subjects with alzheimer’s disease and mild cognitive impairment: implications for progression of ad. j proteome res 2009;8: 471-482. https://doi.org/10.1021/pr800667a serrano-pozo a, gomez-isla t, growdon jh, et al. a phenotypic change but not proliferation underlies glial responses in alzheimer disease. am j pathol 2013;182:2332-2344. https://doi.org/10.1016/j.ajpath.2013.02.031 perez-nievas bg, serrano-pozo a. deciphering the astrocyte reaction in alzheimer’s disease. front aging neurosci 2018;10:114. https://doi.org/10.3389/fnagi.2018.00114 batiuk my, martirosyan a, wahis j, et al. identification of region-specific astrocyte subtypes at single cell resolution. nat commun 2020;11: 1220. https://doi.org/10.1038/s41467-019-14198-8 sofroniew mv. astrocyte reactivity: subtypes, states, and functions in cns innate immunity. trends immunol 2020;41:758-770. https://doi.org/10.1016/j.it.2020.07.004 zamanian jl, xu l, foo lc, et al. genomic analysis of reactive astrogliosis. j neurosci 2012;32:6391-6410. https://doi.org/10.1523/jneurosci.6221-11.2012 escartin c, galea e, lakatos a, et al. reactive astrocyte nomenclature, definitions and future directions. nat neurosci 2021;24:312-325. https://doi.org/10.1038/s41593-020-00783-4 haxby jv, grady cl, koss e, et al. longitudinal study of cerebral metabolic asymmetries and associated neuropsychological patterns in early dementia of the alzheimer type. arch neurol 1990;47: 753-760. https://doi.org/10.1001/archneur.1990.00530070043010 small gw, ercoli lm, silverman dh, et al. cerebral metabolic and cognitive decline in persons at genetic risk for alzheimer’s disease. proc natl acad sci usa 2000;97:6037-6042. https://doi.org/10.1073/pnas.090106797 de leon mj, convit a, wolf ot, et al. prediction of cognitive decline in normal elderly subjects with 2-[(18)f]fluoro-2-deoxy-d-glucose/poitron-emission tomography (fdg/pet). proc natl acad sci usa 2001;98:10966-10971. https://doi.org/10.1073/pnas.191044198 mosconi l, de santi s, li j, et al. hippocampal hypometabolism predicts cognitive decline from normal aging. neurobiol aging 2008;29:676-692. https://doi.org/10.1016/j.neurobiolaging.2006.12.008 van hall g, stromstad m, rasmussen p, et al. blood lactate is an important energy source for the human brain. j cereb blood flow metab 2009;29:1121-1129. https://doi.org/10.1038/jcbfm.2009.35 wyss mt, jolivet r, buck a, et al. in vivo evidence for lactate as a neuronal energy source. j neurosci 2011;31:7477-7485. https://doi.org/10.1523/jneurosci.0415-11.2011 proia p, di liegro cm, schiera g, et al. lactate as a metabolite and a regulator in the central nervous system. int j mol sci 2016;17:1450. https://doi.org/10.3390/ijms17091450 alberini cm, cruz e, descalzi g, et al. astrocyte glycogen and lactate: new insights into learning and memory mechanisms. glia 2018;66:1244-1262. https://doi.org/10.1002/glia.23250 zhang m, cheng x, dang r, et al. lactate deficit in an alzheimer disease mouse model: the relationship with neuronal damage. j neuropathol exp neurol 2018;77:1163-1167. https://doi.org/10.1093/jnen/nly102 le foll c, levin be. fatty acid-induced astrocyte ketone production and the control of food intake. am j physiol regul integr comp physiol 2016;310:r1186-1192. https://doi.org/10.1152/ajpregu.00113.201 . guzman m, blazquez c. is there an astrocyte-neuron ketone body shuttle? trends endocrinol metab 2001;12:169-173. https://doi.org/10.1016/s1043-2760(00)00370-2 panov a, orynbayeva z, vavilin v, et al. fatty acids in energy metabolism of the central nervous system. biomed res int 2014;2014:472459. https://doi.org/10.1155/2014/472459 bélanger m, allaman i, magistretti pj. brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. cell metab 2011;14:724-38. https://doi.org/10.1016/j.cmet.2011.08.016 steele ml, robinson sr. reactive astrocytes give neurons less support: implications for alzheimer's disease. neurobiol aging 2012;33:423.e1-13. https://doi.org/10.1016/j.neurobiolaging.2010.09.018 zulfiqar s, garg p, nieweg k. contribution of astrocytes to metabolic dysfunction in the alzheimer's disease brain. biol chem 2019;400:1113-1127. https://doi.org/10.1515/hsz-2019-0140 orihuela r, mcpherson ca, harry gj. microglial m1/m2 polarization and metabolic states. br j pharmacol 2016;173:649-665. https://doi.org/10.1111/bph.13139 tang y, le w. differential roles of m1 and m2 microglia in neurodegenerative diseases. mol neurobiol 2016;53:1181-1194. https://doi.org/10.1007/s12035-014-9070-5 ransohoff rm. a polarizing question: do m1 and m2 microglia exist? nat neurosci 2016;19:987-991. https://doi.org/10.1038/nn.4338 graeber mb, streit wj. microglia: biology and pathology. acta neuropathol 2010; 119:89-105. https://doi.org/10.1007/s00401-009-0622-0 svahn aj, becker ts, graeber mb. emergent properties of microglia. brain pathol 2014;24:665-670. https://doi.org/10.1111/bpa.12195 hashemiaghdam a, mroczek m. microglia heterogeneity and neurodegeneration: the emerging paradigm of the role of immunity in alzheimer's disease. j neuroimmunol 2020;341:577185. https://doi.org/10.1016/j.jneuroim.2020.577185 streit wj, samkons nw, kuhns aj, et al. dystrophic microglia in the aging human brain. glia 2004;45:208-212. https://doi.org/10.1002/glia.10319 yuan c, aierken a, xie z, et al. the age-related microglial transformation in alzheimer's disease pathogenesis. neurobiol aging 2020;92:82-91. https://doi.org/10.1016/j.neurobiolaging.2020.03.024 saez-atienzar s, masliah e. cellular senescence and alzheimer disease: the egg and the chicken scenario. nat rev neurosci 2020;21:433-444. https://doi.org/10.1038/s41583-020-0325-z albrecht ds, sagare a, pachicano m, et al. early neuroinflammation is associated with lower amyloid and tau levels in cognitively normal older adults. brain behav immun 2021;94:299-307. https://doi.org/10.1016/j.bbi.2021.01.010 dani m, wood m, mizoguchi r, et al. microglial activation correlates in vivo with both tau and amyloid in alzheimer's disease. brain 2018;141:2740-2754. https://doi.org/10.1093/brain/awy188 akiyama h, barger s, barnum s, et al. inflammation and alzheimer’s disease. neurobiol aging 2000;21:383-421. https://doi.org/10.1016/s0197-4580(00)00124-x streit wj, conde jr, harrison jk. chemokines and alzheimer’s disease. neurobiol aging 2001;22:909-913. https://doi.org/10.1016/s0197-4580(01)00290-1 mcgeer eg, mcgeer pl. neuroinflammation in alzheimer’s disease and mild cognitive impairment: a field in its infancy. j alzheimers dis 2010;19:355-361. https://doi.org/10.3233/jad-2010-1219 morimoto k, horio j, satoh h, et al. expression profiles of cytokines in the brains of alzheimer’s disease (ad) patients, compared to the brains of non-demented patients with and without increasing ad pathology. j alzheimers dis 2011;25:59-76. https://doi.org/10.3233/jad-2011-101815 nagae t, araki k, shimoda y, et al. cytokines and cytokine receptors involved in the pathogenesis of alzheimer's disease. j clin cell immunol 2016;7:441. https://doi.org/10.4172/2155-9899.1000441 prokop s, miller kr, heppner fl. microglia actions in alzheimer’s disease. acta neuropathol 2013;126:461-477. https://doi.org/10.1007/s00401-013-1182-x heneka mt, kummer mp, latz e. innate immune activation in neurodegenerative disease. nat rev immunol 2014;14:463-477. https://doi.org/10.1038/nri3705 jorda a, campos-campos j, iradi a, et al. the role of chemokines in alzheimer's disease. endocr metab immune disord drug targets 2020;20:1383-1390. https://doi.org/10.2174/1871530320666200131110744 lópez-gonzález i, schlüter a, aso e, et al. neuroinflammatory signals in alzheimer disease and app/ps1 transgenic mice: correlations with plaques, tangles, and oligomeric species. j neuropathol exp neurol 2015;74: 319-344. https://doi.org/10.1097/nen.0000000000000176 lópez-gonzález i, aso e, carmona m, et al. neuroinflammatory gene regulation, mitochondrial function, oxidative stress, and brain lipid modifications with disease progression in tau p301s transgenic mice as a model of frontotemporal lobar degeneration-tau. j neuropathol exp neurol 2015;74: 975-999. https://doi.org/10.1097/nen.0000000000000241 loving ba, bruce kd. lipid and lipoprotein metabolism in microglia. front physiol. 2020;11:393. https://doi.org/10.3389/fphys.2020.00393 mcgeer pl, mcgeer eg. the amyloid cascade/inflammatory hypothesis of alzheimer disease: implications for therapy. acta neuropathol 2013;126:479-497. https://doi.org/10.1007/s00401-013-1177-7 cai z, hussain md, yan lj. microglia, neuroinflammation, and beta-amyloid protein in alzheimer's disease. int j neurosci 2014;124:307-321. https://doi.org/10.3109/00207454.2013.833510 bamberger me, harris me, mcdonald dr, et al. a cell surface receptor complex for fibrillary-amyloid mediates microglial activation. j neurosci 2003;23:2665-2674. https://doi.org/10.1523/jneurosci.23-07-02665.2003 mandrekar s, jiang q, lee cy, et al. microglia mediate the clearance of soluble aβ through fluid phase macropinocytosis. j neurosci 2009;29:4252-4262. https://doi.org/10.1523/jneurosci.5572-08.2009 carroll mc. the complement system in b cell regulation. mol immunol 2004;41:141-146. https://doi.org/10.1016/j.molimm.2004.03.017 gasque p. complement: a unique innate immune sensor for danger signals. mol immunol 2004;41:1089-1098. https://doi.org/10.1016/j.molimm.2004.06.011 reichwald j, danner s, wiederhold kh, et al. expression of complement system components during aging and amyloid deposition in app transgenic mice. j neuroinflammation 2009; 6:35. https://doi.org/10.1186/1742-2094-6-35 pan xd, zhu yg, lin n, et al. microglial phagocytosis induced by fibrillar β-amyloid is attenuated by oligomeric β-amyloid: implications for alzheimer’s disease. mol neurodegener 2011;6:45. https://doi.org/10.1186/1750-1326-6-45 vogels t, murgoci an, hromádka t. intersection of pathological tau and microglia at the synapse. acta neuropathol commun 2019;7:109. https://doi.org/10.1186/s40478-019-0754-y sheffield lg, marquis jg, berman ne. regional distribution of cortical microglia parallels that of neurofibrillary tangles in alzheimer’s disease. neurosci lett 2000;285: 165-168. https://doi.org/10.1016/s0304-3940(00)01037-5 serrano-pozo a, mielke ml, gómez-isla t, et al. reactive glia not only associates with plaques but also parallels tangles in alzheimer’s disease. am j pathol 2011;179:1373-1384. https://doi.org/10.1016/j.ajpath.2011.05.04 perea jr, bolós m, avila j. microglia in alzheimer’s disease in the context of tau pathology. biomolecules 2020;10:1439. https://doi.org/10.3390/biom10101439 zhou r, ji b, kong y, et al. pet imaging of neuroinflammation in alzheimer's disease. front immunol 2021;12:739130. https://doi.org/10.3389/fimmu.2021.739130 pascoal ta, benedet al, ashton nj, et al. microglial activation and tau propagate jointlyacross braak stages. nat med 2021;27:1592-1599. https://doi.org/10.1038/s41591-021-01456-w vogels t, leuzy a, cicognola c, et al. propagation of tau pathology: integrating insights from postmortem and in vivo studies. biol psychiatry 2020; 87: 808-818. https://doi.org/10.1016/j.biopsych.2019.09.019 gerrits e, brouwer n, kooistra sm, et al. distinct amyloid-β and tau-associated microglia profiles in alzheimer's disease. acta neuropathol 2021;141:681-696. https://doi.org/10.1007/s00401-021-02263-w wilcock dm, hurban j, helman am, et al. down syndrome individuals with alzheimer's disease have a distinct neuroinflammatory phenotype compared to sporadic alzheimer's disease. neurobiol aging 2015;36:2468-2474. https://doi.org/10.1016/j.neurobiolaging.2015.05.016 flores-aguilar l, iulita mf, kovecses o, et al. evolution of neuroinflammation across the lifespan of individuals with down syndrome. brain 2020;143:3653-3671. https://doi.org/10.1093/brain/awaa326 ahmed mm, johnson nr, boyd td, et al. innate immune system activation and neuroinflammation in down syndrome and neurodegeneration: therapeutic targets or partners? front aging neurosci 2021;13:718426. https://doi.org/10.3389/fnagi.2021.718426 barroeta-espar i, weinstock ld, perez-nievas bg, et al. distinct cytokine profiles in human brains resilient to alzheimer’s disease. neurobiol dis 2019;121:327-337. https://doi.org/10.1016/j.nbd.2018.10.009 yang j, wise l, fukuchi ki. tlr4 cross-talk with nlrp3 inflammasome and complement signaling pathways in alzheimer's disease. front immunol 2020;11:724. https://doi.org/10.3389/fimmu.2020.00724 hanslik kl, ulland tk. the role of microglia and the nlrp3 inflammasome in alzheimer's disease. front neurol 2020;11:570711. https://doi.org/10.3389/fneur.2020.570711 zhang y, zhao y, zhang j, et al. mechanisms of nlrp3 inflammasome activation: its role in the treatment of alzheimer's disease. neurochem res 2020;45:2560-2572. https://doi.org/10.1007/s11064-020-03121-z halle a, hornung v, petzold gc, et al. the nalp3 inflammasome is involved in the innate immune response to amyloid-beta. nat immunol 2008;9:857-865. https://doi.org/10.1038/ni.1636 lučiūnaitė a, mcmanus rm, jankunec m, et al. soluble aβ oligomers and protofibrils induce nlrp3 inflammasome activation in microglia. j neurochem 2020;155:650-661. https://doi.org/10.1111/jnc.14945 van zeller m, dias d, sebastião am, et al. nlrp3 inflammasome: a starring role in amyloid-βand tau-driven pathological events in alzheimer's disease. j alzheimers dis 2021;83:939-961. https://doi.org/10.3233/jad-210268 heneka mt, kummer mp, stutz a, et al. nlrp3 is activated in alzheimer's disease and contributes to pathology in app/ps1 mice. nature 2013;493:674-678. https://doi.org/10.1038/nature11729 stancu ic, cremers n, vanrusselt h, et al. aggregated tau activates nlrp3-asc inflammasome exacerbating exogenously seeded and non-exogenously seeded tau pathology in vivo. acta neuropathol 2019;137:599-617. https://doi.org/10.1007/s00401-018-01957-y ising c, venegas c, zhang s, et al. nlrp3 inflammasome activation drives tau pathology. nature 2019;575:669-673. https://doi.org/10.1038/s41586-019-1769-z guerreiro r, wojtas a, bras j, et al. trem2 variants in alzheimer’s disease. n engl j med 2013;368:117-127. https://doi.org/10.1056/nejmoa1211851 takatori s, wang w, iguchi a, tomita t. genetic risk factors for alzheimer disease: emerging roles of microglia in disease pathomechanisms. adv exp med biol 2019;1118:83-116. https://doi.org/10.1007/978-3-030-05542-4_5 leng f, edison p. neuroinflammation and microglial activation in alzheimer disease: where do we go from here? nat rev neurol 2021;17:157-172. https://doi.org/10.1038/s41582-020-00435-y egensperger r, kösel s, von eitzen u, et al. microglial activation in alzheimer disease: association with apoe genotype. brain pathol 1998;8:439-447. https://doi.org/10.1111/j.1750-3639.1998.tb00166.x kloske cm, dugan aj, weekman em, et al. inflammatory pathways are impaired in alzheimer disease and differentially associated with apolipoprotein e status. j neuropathol exp neuro 2021; 80: 922-932. https://doi.org/10.1093/jnen/nlab085 singh ak, mishra g, maurya a, et al. role of trem2 in alzheimer's disease and its consequences on β-amyloid, tau and neurofibrillary tangles. curr alzheimer res 2019;16:1216-1229. https://doi.org/10.2174/1567205016666190903102822 griciuc a, tanzi re. the role of innate immune genes in alzheimer’s disease. curr opin neurol 2021;34: 228-236. https://doi.org/10.1097/wco.0000000000000911 prokop s, miller kr, labra sr, et al. impact of trem2 risk variants on brain region-specific immune activation and plaque microenvironment in alzheimer's disease patient brain samples. acta neuropathol 2019;138:613-630. https://doi.org/10.1007/s00401-019-02048-2 toomey ce, heywood w, benson bc, et al. investigation of pathology, expression and proteomic profiles in human trem2 variant postmortem brains with and without alzheimer's disease. brain pathol 2020;30:794-810. https://doi.org/10.1111/bpa.12842 ferrer i. oligodendrogliopathy in neurodegenerative diseases with abnormal protein aggregates: the forgotten pattern. prog neurobiol 2018;169:24-54. https://doi.org/10.1016/j.pneurobio.2018.07.004 hachinski vc, potter p, merskey h. leuko-araiosis. arch neurol 1987;44:21-23. https://doi.org/10.1001/archneur.1987.00520130013009 scheltens p, barkhof f, leys d, et al. histopathologic correlates of white matter changes on mri in alzheimer's disease and normal aging. neurology 1995;45:883-888. https://doi.org/10.1212/wnl.45.5.883 salat dh, tuch ds, greve dn, et al. age-related alterations in white matter microstructure measured by diffusion tensor imaging. neurobiol aging 2005; 26:1215-1227. https://doi.org/10.1016/j.neurobiolaging.12004.09.017 holland cm, smith ee, csapo i, et al. spatial distribution of white-matter hyperintensities in alzheimer disease, cerebral amyloid angiopathy, and healthy aging. stroke 2008; 39: 1127-1133. https://doi.org/10.1161/strokeaha.107.497438 liu h, yang y, xi y, et al. aging of cerebral white matter. ageing res rev 2017;34:64-76. https://doi.org/10.1016/j.arr.2016.11.006 marner l, nyengaard jr, tang y, et al. marked loss of myelinated nerve fibers in the human brain with age. https://doi.org/10.1016/j.arr.2016.11.006 erten-lyons d, woltjer r, kaye j, et al. neuropathologic basis of white matter hyperintensity accumulation with advanced age. neurology 2013; 81: 977-983. https://doi.org/10.1016/j.arr.2016.11.006 kohama sg, rosene dl, sherman ls. age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. age 2012; 34: 1093-1110. https://doi.org/10.1007/s11357-011-9357-7 bartzokis g, cummings jl, sultzer d, et al. white matter structural integrity in healthy aging adults and patients with alzheimer disease: a magnetic resonance imaging study. arch neurol 2003; 60: 393-398. https://doi.org/10.1001/archneur.60.3.393 wang l, goldstein fc, levey ai, et al. white matter hyperintensities and changes in white matter integrity in patients with alzheimer’s disease. neuroradiology 2011;53:373-381. https://doi.org/10.1007/s00234-010-0806-2 radanovic m, pereira fr, stella f, et al. white matter abnormalities associated with alzheimer's disease and mild cognitive impairment: a critical review of mri studies. expert rev neurother 2013; 13:483-493. https://doi.org/10.1586/ern.13.45 selnes p, fjell am, gjerstad l, et al. white matter imaging changes in subjective and mild cognitive impairment. alzheimers dement 2012;8:s112-s121. https://doi.org/10.1016/j.jalz.2011.07.001 molinuevo jl, ripolles p, simo m, et al. white matter changes in preclinical alzheimer's disease: a magnetic resonance imaging diffusion tensor imaging study on cognitively normal older people with positive amyloid beta protein 42 levels. neurobiol aging 2014; 35: 2671-2680. https://doi.org/10.1016/j.neurobiolaging.2014.05.027 hoy ar, ly m, carlsson cm, okonkwo oc, et al. microstructural white matter alterations in preclinical alzheimer's disease detected using free water elimination diffusion tensor imaging. plos one 2017; 12: e0173982. https://doi.org/10.1371/journal.pone.0173982 bouhrara m, reiter da, bergeron cm, et al. evidence of demyelination in mild cognitive impairment and dementia using a direct and specific magnetic resonance imaging measure of myelin content. alzheimers dement 2018; 14: 998-1004. https://doi.org/10.1016/j.jalz.2018.03.007 zhang x, sun y, li w, et al. characterization of white matter changes along fibers by automated fiber quantification in the early stages of alzheimer's disease neuroimage: clinical 2019; 22: 101723. https://doi.org/10.1016/j.nicl.2019.101723 gouw aa, seewann a, vrenken h, et al. heterogeneity of white matter hyperintensities in alzheimer's disease: post-mortem quantitative mri and neuropathology. brain 2008; 131: 3286-3298. https://doi.org/10.1093/brain/awn265 ihara m, polvikoski tm, hall r, et al. quantification of myelin loss in frontal lobe white matter in vascular dementia, alzheimer’s disease, and dementia with lewy bodies. acta neuropathol 2010; 119: 579-589. https://doi.org/10.1007/s00401-009-0 35-8. rose se, chen f, chalk jb, et al. loss of connectivity in alzheimer's disease: an evaluation of white matter tract integrity with colour coded mr diffusion tensor imaging. j neurol neurosurg psychiatry 2000;69:528-530. https://doi.org/10.1136/jnnp.69.4.528 bennett ij, madden dj. disconnected aging: cerebral white matter integrity and age-related differences in cognition. neuroscience 2014; 276: 187-205. https://doi.org/10.1016/j.neuroscience.2013.11.026 desai mk, sudol kl, janelsins mc, et al. triple-transgenic alzheimer’s disease mice exhibit region-specific abnormalities in brain myelination patterns prior to appearance of amyloid and tau pathology. glia 2009; 57: 54-65. https://doi.org/10.1002/glia.20734 kamphuis w, orre m, kooijman l, et al. differential cell proliferation in the cortex of the appsweps1de9 alzheimer’s disease mouse model. glia 2012; 60: 615-629. https://doi.org/10.1002/glia.22295 dong xx, zhang hy, li hy, et al. association between alzheimer’s disease pathogenesis and early demyelination and oligodendrocyte dysfunction. neural regen res 2018;13:908-914. https://doi.org/10.4103/1673-5374.232486 gu l, wu d, tang x, et al. myelin changes at early stage of 5xfad mice. brain res bull 2018; 137:285-293. https://doi.org/10.1016/j.brainresbull.2017.12.013 scheibel ab, duong th, jacobs r. alzheimer's disease as a capillary dementia. ann med 1989;21:103-107. https://doi.org/10.3109/07853898909149194 de la torre jc. is alzheimer’s disease a neurodegenerative or a vascular disorder? data, dogma, and dialectics. lancet neurol 2004;3:184-190. https://doi.org/10.1016/s1474-4422(04)00683-0 de la torre jc. alzheimer’s disease is a vasocognopathy: a new term to describe its nature. neurol res 2004;26:517-524. https://doi.org/10.1179/016164104225016254 henry-feugeas mc. alzheimer's disease in late-life dementia: a minor toxic consequence of devastating cerebrovascular dysfunction. med hypotheses 2008;70:866-875. https://doi.org/10.1016/j.mehy.2007.07.027 østergaard l, aamand r, gutiérrez-jiménez e, et al. the capillary dysfunction hypothesis of alzheimer's disease. neurobiol aging 2013; 34:1018-1031. https://doi.org/10.1016/j.neurobiolaging.2012.09.011 scheibel ab, duong th, tomiyasu u. denervation microangiopathy in senile dementia, alzheimer type. alzheimer dis assoc disord 1987;1:19-37. https://doi.org/10.1097/00002093-198701000-00004 farkas e, luiten pg. cerebral microvascular pathology in aging and alzheimer’s disease. prog neurobiol 2001;64:575-611. https://doi.org/10.1016/s0301-0082(00)00068-x jellinger k. alzheimer disease and cerebrovascular pathology: an update. j neural transm 2002;109; 813-836. https://doi.org/10.1007/s007020200068 vinters hv, gilbert jj. cerebral amyloid angiopathy: incidence and complications in the aging brain. ii. the distribution of amyloid vascular changes. stroke 1983;14:924-928. https://doi.org/10.1161/01.str.14.6.924 kalaria rn, premkumar dr, pax ab, et al. production and increased detection of amyloid beta protein and amyloidogenic fragments in brain microvessels, meningeal vessels and choroids plexus in alzheimer's disease. mol brain res 1996;35:58–68. https://doi.org/10.1016/0169-328x(95)00180-z buée l, hof pr, bouras c, et al. pathological alterations of the cerebral microvasculature in alzheimer's disease and related dementing disorders. acta neuropathol 1994;87:469-480. https://doi.org/10.1007/bf00294173 castellani rj, smith ma, perry g, et al. cerebral amyloid angiopathy: major contributor or decorative response to alzheimer's disease pathogenesis. neurobiol aging 2004;25:599-602. https://doi.org/10.1016/j.neurobiolaging.2003.12.019 thal dr, griffin wst, de vos rai, et al. cerebral amyloid angioapthy and its relationship to alzheimer’s disease. acta neuropathol 2008;115:599-609. https://doi.org/10.1007/s00401-008-0366-2 weller ro, subash m, preston sd, et al. perivascular drainage of amyloid-beta peptides from the brain and its failure in cerebral amyloid angiopathy and alzheimer’s disease. brain pathol 2008;18:253-266. https://doi.org/10.1111/j.1750-3639.2008.00133.x weller ro, boche d, nicoll ja. microvasculature changes and cerebral amyloid angiopathy in alzheimer’s diseaseand their potential impact on therapy. acta neuropathol 2009;118:87-102. https://doi.org/10.1007/s00401-009-0498-z soontornniyomkij v, choi c, pomakian j, et al. high-definition characterization of cerebral beta-amyloid angiopathy in alzheimer's disease. hum pathol 2010;41:1601-1608. https://doi.org/10.1016/j.humpath.2010.04.011 hunter jm, kwan j, malek-ahmadi m, et al. morphological and pathological evolution of the brain microcirculation in aging and alzheimer's disease. plos one 2012;7:e36893. https://doi.org/10.1371/journal.pone.0036893 magaki s, tang z, tung s, et al. the effects of cerebral amyloid angiopathy on integrity of the blood-brain barrier. neurobiol aging 2018;70:70-77. https://doi.org/10.1016/j.neurobiolaging.2018.06.004 jäkel l, de kort am, klijn cjm, et al. prevalence of cerebral amyloid angiopathy: a systematic review and meta-analysis. alzheimers dement 2022;18:10-28. https://doi.org/10.1002/alz.12366 apátiga-pérez r, soto-rojas lo, campa-córdoba bb, et al. neurovascular dysfunction and vascular amyloid accumulation as early events in alzheimer's disease. metab brain dis 2022;37:39-50. https://doi.org/10.1007/s11011-021-00814-4 iadecola c. neurovascular regulation in the normal brain and in alzheimer's disease. nat rev neurosc 2004;5:347-360. https://doi.org/10.1038/nrn1387 johnson na, jahng gh, weiner mw, pattern of cerebral hypoperfusion in alzheimer disease and mild cognitive impairment measured with arterial spin-labeling mr imaging: initial experience. radiology 2005;234:851-859. https://doi.org/10.1148/radiol.23 3040197. sagare ap, bell rd, zhao z, et al. pericyte loss influences alzheimer-like neurodegeneration in mice. nat commun 2013;4:2932. https://doi.org/10.1038/ncomms3932 franzblau m, gonzales-portillo c, gonzales-portillo gs, et al. vascular damage: a persisting pathology common to alzheimer’s disease and traumatic brain injury. med hypotheses 2013;81:842-845. https://doi.org/10.1016/j.mehy.2013.09.012 nelson ar, sweeney md, sagare ap, et al. neurovascular dysfunction and neurodegeneration in dementia and alzheimer’s disease. biochim biophys acta 2016;1862:887-900. https://doi.org/10.1016/j.bbadis.2015.12.016 kisler k, nelson ar, montagne a, et al. cerebral blood flow regulation and neurovascular dysfunction in alzheimer disease. nat rev neurosci 2017;18:419-434. https://doi.org/10.1038/nrn.2017.48 leijenaar jf, van maurik is, kuijer jpa, et al. lower cerebral blood flow in subjects with alzheimer’s dementia, mild cognitive impairment, and subjective cognitive decline using two-dimensional phase-contrast magnetic resonance imaging. alzheimers dement 2017;9:76-83. https://doi.org/10.1016/j.dadm.2017.10.001 yamazaki y, kanekiyo t. blood-brain barrier dysfunction and the pathogenesis of alzheimer's disease. int j mol sci 2017;18:1965. https://doi.org/10.3390/ijms18091965 rasmussen mk, mestre h, nedergaard m. the glymphatic pathway in neurological disorders. lancet neurol 2018;17:1016-1024. https://doi.org/10.1016/s1474-4422(18)30318-1 ahmad a, patel v, xiao j, et al. the role of neurovascular system in neurodegenerative diseases. mol neurobiol 2020;57: 4373-4393. https://doi.org/10.1007/s12035-020-02023-z sweeney md, montagne a, sagare ap, et al. vascular dysfunction: the disregarded partner of alzheimer's disease. alzheimers dement 2019;15:158-167. https://doi.org/10.1016/j.jalz.2018.07.222 kurz c, walker l,rauchmann bs, et al. dysfunction of the blood-brain barrier in alzheimer’s disease: evidence from human studies. neuropathol appl neurobiol neuropathol appl neurobiol 2022;e12782. https://doi.org/10.1111/nan.12782 kalaria rn, pax ab. increased collagen content of cerebral microvessels in alzheimer’s disease. brain res 1995;705:349-352. https://doi.org/10.1016/0006-8993(95)01250-8 verbeek mm, otte-holler i, van den born j, et al. agrin is a major heparan sulfate proteoglycan accumulating in alzheimer's disease brain. am j pathol 1999;155:2115-2125. https://doi.org/10.1016/s0002-9440(10)65529-0 berzin tm, zipser bd, rafii ms, et al. agrin and microvascular damage in alzheimer’s disease. neurobiol aging 2000;21:349-355. https://doi.org/10.1016/s0197-4580(00)00121-4 grammas p, yamada m, zlokovic b. the cerebromicrovasculature: a key player in the pathogenesis of alzheimer’s disease. j alzheimers dis 2002;4:217-223. https://doi.org/10.3233/jad-2002-4311 ervin jf, pannell c, szymansky m, et al. vascular smooth muscle actin is reduced in alzheimer disease brain: a quantitative analysis. j neuropathol exp neurol 2004;63:735-741. https://doi.org/10.1093/jnen/63.7.735 bailey tl, rivara cb, rocher ab, et al. the nature and effects of cortical microvascular pathology in aging and alzheimer’s disease. neurol res 2004;26:573-578. https://doi.org/10.1179/016164104225016272 kalaria rn, hedera p. differential degeneration of the cerebral microvasculature in alzheimer’s disease. neuroreport 1995;6:477-480. https://doi.org/10.1097/00001756-199502000-00018 wu z, guo h, chow n, sallstrom j, et al. role of the meox2 homeobox gene in neurovascular dysfunction in alzheimer disease. nat med 2005;11:959-965. https://doi.org/10.1038/nm1287 hashimura t, kimura t, miyakawa t. morphological changes of blood vessels in the brain with alzheimer’s disease. jpn j psychiatry neurol 1991;45:661-665. https://doi.org/10.1111/j.1440-1819.1991.tb01187.x sagare ap, bell rd, zlokovic bv. neurovascular defects and faulty amyloid-beta vascular clearance in alzheimer's disease. j alzheimers dis 2013;33:s87-100. https://doi.org/10.3233/jad-2012-129037 ma q, zhao z, sagare ap, et al. blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-beta42 by lrp1-dependent apolipoprotein e isoform-specific mechanism. mol neurodegener 2018;13:57. https://doi.org/10.1186/s13024-018-0286-0 lendahl u, nilsson p, betsholtz c. emerging links between cerebrovascular and neurodegenerative diseases-a special role for pericytes. embo rep 2019;20:e48070. https://doi.org/10.15252/embr.201948070 nortley r, korte n, izquierdo p, et al. β oligomers constrict human capillaries in alzheimer's disease via signaling to pericytes. science 2019;365:eaav9518. https://doi.org/10.1126/science.aav9518 winkler ea, sengillo jd, bell rd, et al. blood-spinal cord barrier pericyte reductions contribute to increased capillary permeability. j cereb blood flow metab 2012;32:1841-1852. https://doi.org/10.1038/jcbfm.2012.113 winkler ea, sagare ap, zlokovic bv. the pericyte: a forgotten cell type with important implications for alzheimer’s disease? brain pathol 2014;24:371-386. https://doi.org/10.1111/bpa.12152 kalback w, esh c, castano em, et al. atherosclerosis, vascular amyloidosis and brain hypoperfusion in the pathogenesis of sporadic alzheimer's disease. neurol res 2004;26:525-539. https://doi.org/10.1179/016164104225017668 shabir o, berwick j, francis se. neurovascular dysfunction in vascular dementia, alzheimer's and atherosclerosis. bmc neurosci 2018;19:62. https://doi.org/10.1186/s12868-018-0465-5 bell rd, winkler ea, singh i, et al. apolipoprotein e controls cerebrovascular integrity via cyclophilin a. nature 2012;485:512-516. https://doi.org/10.1038/nature11087 mosconi l, sorbi s, de leon mj, et al. hypometabolism exceeds atrophy in presymptomatic early-onset familial alzheimer’s disease. j nucl med 2006;47:1778–1786. pmid: 17079810. nation da, sweeney md, montagne a, et al. blood-brain barrier breakdown is an early biomarker of human cognitive dysfunction. nat med 2019; 25: 270-276. https://doi.org/10.1038/s41591-018-0297-y winkler ea, nishida y, sagare ap, et al. glut1 reductions exacerbate alzheimer's disease vasculo-neuronal dysfunction and degeneration. nat neurosci 2015;18:521-530. https://doi.org/10.1038/nn.3966 koepsell h. glucose transporters in brain in health and disease. pflugers arch 2020;472:1299-1343. https://doi.org/10.1007/s00424-020-02441-x an y, varma vr, varma s, et al. evidence for brain glucose dysregulation in alzheimer's disease. alzheimers dement 2018;14:318-329. https://doi.org/10.1016/j.jalz.2017.09.011 szu ji, obenaus a.. cerebrovascular phenotypes in mouse models of alzheimer's disease. cereb blood flow metab 2021;41:1821-1841. https://doi.org/10.1177/0271678x21992462 canepa e, fossati s. impact of tau on neurovascular pathology in alzheimer's disease. front neurol 2021;11:573324. https://doi.org/10.3389/fneur.2020.573324 blair lj, frauen hd, zhang b, et al. tau depletion prevents progressive blood-brain barrier damage in a mouse model of tauopathy. acta neuropathol commun 2015;3:8. https://doi.org/10.1186/s40478-015-0186-2 love s, miners j. cerebrovascular disease in ageing and alzheimer's disease acta neuropathol 2016;131:645-658. https://doi.org/10.1007/s00401-015-1522-0 yu x, ji c, shao a. neurovascular unit dysfunction and neurodegenerative disorders. front neurosci 2020;14:334. https://doi.org/10.3389/fnins.2020.00334 gordon gr, choi hb, rungta rl, et al. brain metabolism dictates the polarity of astrocyte control over arterioles. nature 2008; 456: 745-749. https://doi.org/10.1038/nature07525 ipata pl, camici m, micheli v, et al. metabolic network of nucleosides in the brain. curr top med chem 2011;11:909-922. https://doi.org/10.2174/156802611795347555 ansoleaga b, jove m, schluter a, et al. deregulation of purine metabolism in alzheimer’s disease. neurobiol aging 2015;36:68-80. https://doi.org/10.1016/j.neurobiolaging.2014.08.004 rahman a. the role of adenosine in alzheimer’s disease. curr neuropharmacol 2009; 7: 207-216. https://doi.org/10.2174/157015909789152119 carman aj, mills jh, krenz a, et al. adenosine receptor signaling modulates permeability of the blood-brain barrier. j neurosci 2011; 31: 13272-13280. https://doi.org/10.1523/jneurosci.3337-11.201 alonso-andrés p, albasanz jl, ferrer i, et al. purine-related metabolites and their converting enzymes are altered in frontal, parietal and temporal cortex at early stages of alzheimer’s disease pathology. brain pathol 2018;28: 933-946. https://doi.org/10.1111/bpa.12592 panza f, frisardi v, capurso c, et al. polyunsaturated fatty acid and s-adenosylmethionine supplementation in predementia syndromes and alzheimer’s disease: a review. scientificworldjournal 2009;9:373-389. https://doi.org/10.1100/tsw.2009.48 ruan q, ruan j, zhang w, qian f, et al. targeting nad(+) degradation: the therapeutic potential of flavonoids for alzheimer's disease and cognitive frailty. pharmacol res 2018;128:345-358. https://doi.org/10.1016/j.phrs.2017.08.010 erb l, woods lt, khalafalla mg, et al. purinergic signalling in alzheimer’s disease. brain res bull 2019;151:25-37. https://doi.org/10.1016/j.brainresbull.2018.10.014 sharma vk, singh tg, singh s. cyclic nucleotides signaling and phosphodiesterase inhibition: defying alzheimer's disease. curr drug targets 2020;21:1371-1384. https://doi.org/10.2174/1389450121666200727104728 merighi s, poloni te, terrazzan a, et al. alzheimer and purinergic signaling: just a matter of inflammation? cells 2021;10:1267. https://doi.org/10.3390/cells10051267 pan l, ma y, li y, wu h, et al. the therapeutic potential of purinergic receptors in alzheimer's disease and promising therapeutic modulators. mini rev med chem 2021;21:1288-1302. https://doi.org/10.2174/1389557520999201209211610 kundu d, dubey vk. purines and pyrimidines: metabolism, function and potential as therapeutic options in neurodegenerative diseases. curr protein pept sci 2021;22:170-189. https://doi.org/10.2174/1389203721999201208200605 allis cd, caparros ml, jenuwein t, et al. epigenetics second edit. cold springer arbor laboratory press 2015. paro r, grossniklaus u, santoro r, wutz a. introduction to epigenetics, springer, 2021 wu h, tao j, sun ye. regulation and function of mammalian dna methylation patterns: a genomic perspective. brief funct genomics. 2012;11:240-250. https://doi.org/10.1093/bfgp/els01 qazi tj, quan z, mir a, qing h. epigenetics in alzheimer's disease: perspective of dna methylation. mol neurobiol 2018;55:1026-1044. https://doi.org/10.1007/s12035-016-0357-6 grunstein m. histone acetylation in chromatin structure and transcription. nature 1997;389: 349-352. https://doi.org/10.1038/38664 kuo mh, allis cd. roles of histone acetyltransferases and deacetylases in gene regulation. bioessays 1998; 20: 615-626. https://doi.org/10.1002/(sici)1521-1878(199808)20:8<615::aid-bies4>3.0.co;2-h de ruijter aj, van gennip ah, caron hn, et al. histone deacetylases (hdacs): characterization of the classical hdac family. biochem j 2003;370:737-749. https://doi.org/10.1042/bj20021321 torok ms, grant pa. histone acetyltransferase proteins contribute to transcriptional processes at multiple levels. adv protein chem 2004;67:181-199. https://doi.org/10.1016/s0065-3233(04)67007-0 cheung p, lau p. epigenetic regulation by histone methylation and histone variants. mol endocrin 2005;19:563-573. https://doi.org/10.1210/me.2004-0496 imhof a. epigenetic regulators and histone modification. brief funct genomic proteomic 2006;5:222-227. https://doi.org/10.1093/bfgp/ell030 gupta s, kim sy, artis s, et al. histone methylation regulates memory formation. j neurosci 2010; 30:3589–3599. https://doi.org/10.1523/jneurosci.3732-09.2010 greer el, shi y. histone methylation: a dynamic mark in health, disease and inheritance. nature rev genet 2012; 13:343-357. https://doi.org/10.1038/nrg3173 morrison ld, smith dd, kish sj. brain s-adenosylmethionine levels are severely decreased in alzheimer’s disease. j neurochem 1996;67:1328-1331. https://doi.org/10.1046/j.1471-4159.1996.67031328.x tahiliani m, koh kp, shen y, et al. conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian dna by mll partner tet1. science 2009;324:930–935. https://doi.org/10.1126/science.1170116 mastroeni d, mckee a, grover a, et al. epigenetic differences in cortical neurons from a pair of monozygotic twins discordant for alzheimer’s disease. plos one 2009; 4 e6617. https://doi.org/10.1371/journal.pone.0006617 tan l, shi yg. tet family proteins and 5hydroxymethylcytosine in development and disease. development 2012;139:1895–1902. https://doi.org/10.1242/dev.070771 irier ha, jin p. dynamics of dna methylation in aging and alzheimer’s disease. dna cell biol 2012;31:s42–s48. https://doi.org/10.1089/dna.2011.1565 wu x, zhang y. tet-mediated active dna demethylation: mechanism, function and beyond. nat rev genet 2017;18:517-534. https://doi.org/10.1038/nrg.2017.33 siegmund kd, connor cm, campan m, et al. dna methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons. plos one 2007; 2:e895. https://doi.org/10.1371/journal.pone.0000895 wang sc, oeize b, schumacher a. age-specific epigenetic drift in late-onset alzheimer’s disease. plos one 2008; 2008;3:e2698. https://doi.org/10.1371/journal.pone.0002698 mastroeni d, grover a, delvaux e, et al. epigenetic mechanisms in alzheimer’s disease. neurobiol aging 2011;32:1161-1180. https://doi.org/10.1016/j.neurobiolaging.2010.08.017 bakulski km, dolinoy dc, sartor ma et al. genome-wide dna methylation differences between late-onset alzheimer’s disease and cognitively normal controls in human frontal cortex. j alzheimers dis 2012;29:571-588. https://doi.org/10.3233/jad-2012-111223 watson ct, roussos p, garg p, et al. genome-wide dna methylation profiling in the superior temporal gyrus reveals epigenetic signatures associated with alzheimer’s disease. genome med 2016;8:5. https://doi.org/10.1186/s13073-015-0258-8 smith ar, smith rg, pishva e, et al. parallel profiling of dna methylation and hydroxymethylation highlights neuropathology-associated epigenetic variation in alzheimer's disease. clin epigenetics 2019;11:52. https://doi.org/10.1186/s13148-019-0636-y sanchez-mut jv, aso e, panayotis n, et al. dna methylation map of mouse and human brain identifies target genes in alzheimer’s disease. brain 2013;136:3018–3027. https://doi.org/10.1093/brain/awt237 sanchez-mut jv, heyn h, vidal e, et al. human dna methylomes of neurodegenerative diseases show common epigenomic patterns. transl psychiatry 2016;6:e718. https://doi.org/10.1038/tp.2015.214 rao js, keleshian vl, klein s, et al. epigenetic modifications in frontal cortex from alzheimer’s disease and bipolar disorder patients. transl psychiatry 2012;2: e132. https://doi.org/10.1038/tp.2012.55 scarpa s, fuso a, d’anselmi f, et al. presenilin 1 gene silencing by s-adenosylmethionine: a treatment for alzheimer disease? febs lett 2003;541:145-148. https://doi.org/10.1016/s0014-5793(03)00277-1 fuso a, seminara l, cavallaro ra, et al. s-adenosylmethionine/homocysteine cycle alterations modify dna methylation status with consequent deregulation of ps1 and bace and beta-amyloid production. mol cell neurosci 2005;28:195–204. https://doi.org/10.1016/j.mcn.2004.09.007 monti n, cavallaro ra, stoccoro a, et al. cpg and non-cpg presenilin1 methylation pattern in course of neurodevelopment and neurodegeneration is associated with gene expression in human and murine brain. epigenetics 2020;15: 781-799. https://doi.org/10.1080/15592294.2020.1722917 sontag e, nunbhakdi-craig v, sontag j-m, et al. protein phosphatase 2a methyltransferase links homocysteine metabolism with tau and amyloid precursor protein regulation. j neurosci 2007; 27:2751-2759. https://doi.org/10.1523/jneurosci.3316-06.2007 zhou xw, gustafsson ja, tanila h, et al. tau hyperphosphorylation correlates with reduced methylation of protein phosphatase 2a. neurobiol dis 2008;31:386-394. https://doi.org/10.1016/j.nbd.2008.05.013 bernstein ai, lin y, street rc, et al. 5-hydroxymethylation associated epigenetic modifiers of alzheimer’s disease modulates tau induced neurotoxicity. hum mol genet 2016;25:2437-2450. https://doi.org/10.1093/hmg/ddw109 pietrzak m, rempala g, nelson pt, et al. epigenetic silencing of nucleolar rrna genes in alzheimer's disease. plos one 2011;6:e22585. https://doi.org/10.1371/journal.pone.0022585 nagata t, kobayashi n, ishii j, et al. association between dna methylation of the bdnf promoter region and clinical presentation in alzheimer's disease. dement geriatr cogn dis extra 2015;5:64-73. https://doi.org/10.1159/000375367 wilson ag. epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases. j periodontol 2008;79:1514-1519. https://doi.org/10.1902/jop.2008.080172 smith ar, smith rg, condliffe d, et al. increased dna methylation near trem2 is consistently seen in the superior temporal gyrus in alzheimer’s disease brain. neurobiol aging 2016;47:35-40. https://doi.org/10.1016/j.neurobiolaging.2016.07.008 nuutinen t, suuronen t, kyrylenko s, et al. induction of clusterin/apoj expression by histone deacetylase inhibitors in neural cells. neurochem int 2005;647:528-538. https://doi.org/10.1016/j.neuint.2005.07.007 silva pno, gigek co, leal mf, et al. promoter methylation analysis of sirt3, smarca5, htert and cdh1 genes in aging and alzheimer’s disease. j alzheimers dis 2008;13:173-176. https://doi.org/10.3233/jad-2008-13207 smith ar, smith rg, condliffe d, et al. increased dna methylation near trem2 is consistently seen in the superior temporal gyrus in alzheimer’s disease brain. neurobiol aging 2016;47:35-40. https://doi.org/10.1016/j.neurobiolaging.2016.07.008 smith rg, pishva e, shireby g, et al. a meta-analysis of epigenome-wide association studies in alzheimer's disease highlights novel differentially methylated loci across cortex. nat commun 2021; 12:3517. https://doi.org/10.1038/s41467-021-23243-4 furuya tk, silva pno, payão slm, et al. analysis of snap25 mrna expression and promoter dna methylation in brain areas of alzheimer’s disease patients. neuroscience 2012;220:41-46. https://doi.org/10.1016/j.neuroscience.2012.06.035 furuya tk, da silva pno, payão slm, et al. sorl1 and sirt1 mrna expression and promoter methylation levels in aging and alzheimer’s disease. neurochem int 2012;61:973-975. https://doi.org/10.1016/j.neuint.2012.07.014 sanchez-mut jv, aso e, heyn h, et al. promoter hypermethylation of the phosphatase dusp22 mediates pka-dependent tau phosphorylation and creb activation in alzheimer's disease. hippocampus 2014;24:363-368. https://doi.org/10.1002/hipo.22245 de jager pl, srivastava g, burgess j, et al. alzheimer’s disease: early alterations in brain dna methylation at ank1, bin1, rhbdf2 and other loci. nat neurosci 2014 ;17:1156-1163. https://doi.org/10.1038/nn.3786 lunnon k, smith r, hannon e, et al. methylomic profiling implicates cortical deregulation of ank1 in alzheimer’s disease. nat neurosci 2014;17:1164–1170. https://doi.org/10.1038/nn.3782 shinagawa s, kobayashi n, nagata t, et al. dna methylation in the ncaph2/lmf2 promoter region is associated with hippocampal atrophy in alzheimer’s disease and amnesic mild cognitive impairment patients. neurosci lett 2016;629:33–37. https://doi.org/10.1016/j.neulet.2016.06.055 buira sp, albasanz jl, dentesano g, et al. dna methylation regulates adenosine a(2a) receptor cell surface expression levels. j neurochem 2010;112:1273-1285. https://doi.org/10.1111/j.1471-4159.2009.06538.x buira sp, dentesano g, albasanz jl, et al. dna methylation and yin yang-1 repress adenosine a2a receptor levels in human brain. j neurochem 2010;115:283-295. https://doi.org/10.1111/j.1471-4159.2010.06928.x villar-menéndez i, nuñez f, díaz-sánchez s, et al. striatal adenosine a2a receptor expression is controlled by s-adenosyl-l-methionine-mediated methylation. purinergic signal 2014;10:523-528. https://doi.org/10.1007/s11302-014-9417-4 piaceri i, raspanti b, tedde a, et al. epigenetic modifications in alzheimer’s disease: cause or effect? j alzheimers dis 2015;43:1169-1173. https://doi.org/10.3233/jad-141452 blanch m, mosquera jl, ansoleaga b, et al. altered mitochondrial dna methylation pattern in alzheimer disease-related pathology and in parkinson disease. am j pathol 2016;186:385-397. https://doi.org/10.1016/j.ajpath.2015.10.004 eddy sr. non-coding rna genes and the modern rna world. nat rev genet 2001;2:919-929. https://doi.org/10.1038/35103511 he l, hannon gj. micrornas: small rnas with a big role in gene regulation. nat rev genet 2004;5:522-531. https://doi.org/10.1038/nrg1379 bartel dp. micrornas: target recognition and regulatory functions. cell 2009;136: 215-233. https://doi.org/10.1016/j.cell.2009.01.002 kim vn, han j, siomi mc. biogenesis of small rnas in animals. nat rev mol biol 2009;10:126-139. https://doi.org/10.1038/nrm2632 wahid f, shezhad a, khan t, et al. micrornas: synthesis, mechanism, function, and recent clinical trials. bioch biophys acta mol cell res 2010;1803:1231-1243. https://doi.org/10.1016/j.bbamcr.2010.06.013 lewis bp, burge cb, bartel dp. conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets. cell 2005;120:15-20. https://doi.org/10.1016/j.cell.2004.12.035 lewis bp, shih ih, jones-rhoades mw, et al. prediction of mammalian microrna targets. cell 2003;115: 787-798. https://doi.org/10.1016/s0092-8674(03)01018-3 boudreau rl, jiang p, gilmore bl, et al. transcriptome-wide discovery of microrna binding sites in human brain. neuron 2014;81: 294-305. https://doi.org/10.1016/j.neuron.2013.10.062 tan l, yu jt, hu n, et al. non-coding rnas in alzheimer’s disease. mol neurobiol 2013;47:382-393. https://doi.org/10.1007/s12035-012-8359-5 dehgani r, rahmani f, rezaei n. microrna in alzheimer’s disease revisited: implications for major neuropathological mechanisms. rev neurosci 2018;29:161-182. https://doi.org/10.1515/revneuro-2017-0042 wang m, qin l, tang b. micrornas in alzheimer’s disease. front genet 2019;10: 153. https://doi.org/10.3389/fgene.2019.00153 lee st, chu k, jung kh, et al. mir-206 regulates brain-derived neurotrophic factor in alzheimer disease model. ann neurol 2012;72:269-277. https://doi.org/10.1002/ana.23588 hébert ss, horre k, nicolai l, et al. microrna regulation of alzheimer’s amyloid precursor protein expression. neurobiol dis 2009;33:422-428. https://doi.org/10.1016/j.nbd.2008.11.009 hébert ss, horré k, nicolai l, et al. loss of microrna cluster mir-29a/b-1 in sporadic alzheimer’s disease correlates with increased bace1/β-secretase expression. proc natl acad sci usa 2008;105, 6415-6420. https://doi.org/10.1073/pnas.0710263105 patel n, hoang d, miller n, et al. micrornas can regulate human app levels. mol neurodegener 2008; 3:10. https://doi.org/10.1186/1750-1326-3-10 jarvis ci, goncalves mb, clarke e, et al. retinoic acid receptor-alpha signalling antagonizes both intracellular and extracellular amyloid-beta production and prevents neuronal cell death caused by amyloid-beta. eur j neurosci 2010;32:1246-1255. https://doi.org/10.1111/j.1460-9568.2010.07426.x vilardo e, barbato c, ciotti m, et al. microrna-101 regulates amyloid precursor protein expression in hippocampal neurons. j biol chem 2010; 285: 18344-18351. https://doi.org/10.1074/jbc.m110.112664 liu cg, wang jl, li l, et al. microrna-384 regulates both amyloid precursor protein and beta-secretase expression and is a potential biomarker for alzheimer’s disease. int j mol med 2014;34:160-166. https://doi.org/10.3892/ijmm.2014.1780 liu cg, wang jl, li l, et al. microrna-135a and -200b, potential biomarkers for alzheimers disease, regulate beta secretase and amyloid precursor protein. brain res 2014;1583: 55-64. https://doi.org/10.1016/j.brainres.2014.04.026 liang c, zhu h, xu y, et al. microrna-153 negatively regulates the expression of amyloid precursor protein and amyloid precursor-like protein 2. brain res 2012;1455: 103-113. https://doi.org/10.1016/j.brainres.2011.10.051 long jm, ray b, lahiri dk. microrna-153 physiologically inhibits expression of amyloid-beta precursor protein in cultured human fetal brain cells and is dysregulated in a subset of alzheimer disease patients. j biol chem 2012;287: 31298-31310. https://doi.org/10.1074/jbc.m112.366336 long jm, ray b, lahiri dk. microrna-339-5p down-regulates protein expression of beta-site amyloid precursor protein-cleaving enzyme 1 (bace1) in human primary brain cultures and is reduced in brain tissue specimens of alzheimer disease subjects. j biol chem 2014; 289: 5184-5198. https://doi.org/10.1074/jbc.m113.518241 cheng c, li w, zhang z, et al. microrna-144 is regulated by activator protein-1 (ap-1) and decreases expression of alzheimer disease-related a disintegrin and metalloprotease 10 (adam10). j biol chem 2013;288:13748-13761. https://doi.org/10.1074/jbc.m112.381392 deng y, ding y, hou d. research status of the regulation of mirna on bace1. int j neurosci 2014;124:474-477. https://doi.org/10.3109/00207454.2013.858249 lei x, lei l, zhang z, et al. downregulated mir-29c correlates with increased bace1 expression in sporadic alzheimer’s disease. int j clin exp pathol 2015;8:1565-1574. pmid: 25973041. li q, li x, wang l, et al. mir-98-5p acts as a target for alzheimer’s disease by regulating aβ production through modulating snx6 expression. j mol neurosci 2016; 60:413-420. https://doi.org/10.1007/s12031-016-0815-7 li w, li x, xin x, et al. microrna-613 regulates the expression of brain-derived neurotrophic factor in alzheimer’s disease. biosci trends 2016;10: 372-377. https://doi.org/10.5582/bst.2016.01127 kim j, yoon h, chung de, et al. mir-186 is decreased in aged brain and suppresses bace1 expression. j neurochem 2016;137: 436-445. https://doi.org/10.1111/jnc.13507 an f, gong g, wang y, et al. mir-124 acts as a target for alzheimer’s disease by regulating bace1. oncotarget 2017;8:114065-114071. https://doi.org/10.18632/oncotarget.23119 gong g, an f, wang y et al. mir-15b represses bace1 expression in sporadic alzheimer's disease. oncotarget 2017;8:91551-91557. https://doi.org/10.18632/oncotarget.21177 chopra n, wang r, maloney b, et al. microrna-298 reduces levels of human amyloid-beta precursor protein (app), beta-site app-converting enzyme 1 (bace1) and specific tau protein moieties. mol psychiatry 2020;26: 5636-5657. https://doi.org/10.1038/s41380-019-0610-2 zhu hc, wang lm, wang m, et al. microrna-195 downregulates alzheimer’s disease amyloid-beta production by targeting bace1. brain res bull 2012;88:596-601. https://doi.org/10.1016/j.brainresbull.2012.05.018 zhu qb, unmehopa u, bossers k, et al. microrna-132 and early growth response-1 in nucleus basalis of meynert during the course of alzheimer’s disease. brain 2016;139: 908-921. https://doi.org/10.1093/brain/awv383 hu yk, wang x, li l, et al. microrna-98 induces an alzheimer’s disease-like disturbance by targeting insulin-like growth factor 1. neurosci bull 2013;29: 745-751. https://doi.org/10.1007/s12264-013-1348-5 absalon s, kochanek dm, raghavan v, et al. mir-26b, upregulated in alzheimer’s disease, activates cell cycle entry, tau-phosphorylation, and apoptosis in postmitotic neurons. j neurosci 2013;33:14645-14659. https://doi.org/10.1523/jneurosci.1327-13.2013 dickson jr, kruse c, montagna dr, et al. alternative polyadenylation and mir-34 family members regulate tau expression. j neurochem 2013;127:739-749. https://doi.org/10.1111/jnc.12437 gong j, zhang j, li b, et al. microrna-125b promotes apoptosis by regulating the expression of mcl-1, bcl-w and il-6r. oncogene 2013;32:3071-3079. https://doi.org/10.1038/onc.2012.318 zhao y, bhattacharjee s, jones bm, et al. regulation of neurotropic signaling by the inducible, nf-κb-sensitive mirna-125b in alzheimer’s disease (ad) and in primary human neuronal-glial (hng) cells. mol neurobiol 2014; 50: 97-106. https://doi.org/10.1007/s12035-013-8595-3 zhang x, tan l, lu y, et al. microrna-138 promotes tau phosphorylation by targeting retinoic acid receptor alpha. febs lett 2015;589:726-729. https://doi.org/10.1016/j.febslet.2015.02.001 salta e, sierksma a, vanden eynden e, et al. mir-132 loss de-represses itpkb and aggravates amyloid and tau pathology in alzheimer’s brain. embo mol med 2016;8:1005-1018. https://doi.org/10.15252/emmm.201606520 edbauer d, neilson jr, foster ka, et al. regulation of synaptic structure and function by fmrp-associated micrornas mir-125b and mir-132. neuron 2010;65: 373-384. https://doi.org/10.1016/j.neuron.2010.01.005 cohen je, lee pr, chen s, et al. microrna regulation of homeostatic synaptic plasticity. proc natl acad sci usa 2011;108:11650-11655. https://doi.org/10.1073/pnas.1017576108 giusti sa, vogl am, brockmann mm, et al. microrna-9 controls dendritic development by targeting rest. elife 2014:3:e02755. https://doi.org/10.7554/elife.02755 hu z, zhao j, hu t, et al. mir-501-3p mediates the activity-dependent regulation of the expression of ampa receptor subunit glua1. j cell biol 2015;208:949-959. https://doi.org/10.1083/jcb.201404092 olde loohuis nf, ba w, stoerchel ph, et al. microrna-137 controls ampa-receptor-mediated transmission and mglur-dependent ltd. cell rep 2015;11:1876-1884. https://doi.org/10.1016/j.celrep.2015.05.040 sim se, lim cs, kim ji, et al. the brain-enriched microrna mir-9-3p regulates synaptic plasticity and memory. j neurosci 2016;36:8641-8652. https://doi.org/10.1523/jneurosci.0630-16.2016 sierksma a, lu a, salta e, et al. deregulation of neuronal mirnas induced by amyloid-beta or tau pathology. mol neurodegener 2018;13:54. https://doi.org/10.1186/s13024-018-0285-1 croce n, gelfo f, ciotti mt, et al. npy modulates mir-30a-5p and bdnf in opposite direction in an in vitro model of alzheimer disease: a possible role in neuroprotection? mol cell biochem 2013;376: 189-195. https://doi.org/10.1007/s11010-013-1567-0 shaked i, meerson a, wolf y, et al. microrna-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase. immunity 2009;31: 965-973. https://doi.org/10.1016/j.immuni.2009.09.019 shaltiel g, hanan m, wolf y, et al. hippocampal microrna-132 mediates stress-inducible cognitive deficits through its acetylcholinesterase target. brain struct funct 2013;218: 59-72. https://doi.org/10.1007/s00429-011-0376-z vo n, klein me, varlamova o, et al. a camp-response element binding protein-induced microrna regulates neuronal morphogenesis. proc natl acad sci usa 2005;102:16426-16431. https://doi.org/10.1073/pnas.0508448102 llorens f, thüne k, andrés-benito p, et al. microrna expression in the locus ceruleus, entorhinal cortex, and hippocampus at early and middle stages of braak neurofibrillary tangle pathology. j mol neurosci 2017;63:206-215. https://doi.org/10.1007/s12031-017-0971-4 sun c, jia n, li r, zhang z, et al. mir-143-3p inhibition promotes neuronal survival in an alzheimer's disease cell model by targeting neuregulin-1. folia neuropathol 2020;58:10-21. https://doi.org/10.5114/fn.2020.94002 hammad s, mabondzo a, hamoudi r, et al. regulation of p-glycoprotein by mir-27a-3p at the brain endothelial barrier. j pharm sci 2022;111:1470-1479. https://doi.org/10.1016/j.xphs.2021.10.021 harati r, hammad s, tlili a, et al. mir-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability. plos one 2022;17:e0262152. https://doi.org/10.1371/journal.pone.0262152 sochocka m, zwolińska k, leszek j. the infectious etiology of alzheimer's disease. curr neuropharmacol. 2017;15:996-1009. https://doi.org/10.2174/1570159x15666170313122937 vigasova d, nemergut m, liskova b, et al. multi-pathogen infections and alzheimer's disease. microb cell fact 2021;20:25. https://doi.org/10.1186/s12934-021-01520-7 licastro f, carbone i, raschi e, et al. the 21st century epidemic: infections as inductors of neurodegeneration associated with alzheimer’s disease. immun ageing 2014;11:22. https://doi.org/10.1186/1742-4933-11-8 maheshwari p, eslick gd. bacterial infection and alzheimer’s disease: a meta-analysis. j alzheimers dis 2015;43:957-966. https://doi.org/10.3233/jad-140621 ashraf gm, tarasov vv, makhmutovа a, et al. the possibility of an infectious etiology of alzheimer disease. mol neurobiol 2019;56:4479-4491. https://doi.org/10.1007/s12035-018-1388-y macdonald ab, miranda jm. concurrent neocortical borreliosis and alzheimer’s disease. hum pathol 1987;18:759-761. https://doi.org/10.1016/s0046-8177(87)80252-6 marques ar, weir sc, fahle ga, et al. lack of evidence of borrelia involvement in alzheimer's disease. j infect dis 2000;182:1006-1007. https://doi.org/10.1086/315792 riviere gr, riviere kh, smith ks. molecular and immunological evidence of oral treponema in the human brain and their association with alzheimer’s disease. oral microbiol immunol. 2002;17:113-118. https://doi.org/10.1046/j.0902-0055.2001.00100.x miklossy j. alzheimer’s disease: a neurospirochetosis. analysis of the evidence following koch’s and hill’s criteria. j neuroinfammation 2011;8:90. https://doi.org/10.1186/1742-2094-8-90 miklossy j. historic evidence to support a causal relationship between spirochetal infections and alzheimer’s disease. front aging neurosci 2015;7:46. https://doi.org/10.3389/fnagi.2015.00046 senejania ag, maghsoudloua j, el-zohirya d, et al. borrelia burgdorferi co-localizing with amyloid markers in alzheimer’s disease brain tissues. j alzheimers dis 2022;85: 889-903. https://doi.org/10.3233/jad-215398 emery dc, shoemark dk, batstone te, et al. 16s rrna next generation sequencing analysis shows bacteria in alzheimer’s post-mortem brain. front aging neurosci. 2017;9:195. https://doi.org/10.3389/fnagi.2017.00195 soscia sj, kirby je, washicosky kj, et al. the alzheimer’s disease-associated amyloid beta-protein is an antimicrobial peptide. plos one 2010;5:e9505. https://doi.org/10.1371/journal.pone.0009505 kamer ar, dasanayake ap, craig rg, et al. alzheimer’s disease and peripheral infections: the possible contribution from periodontal infections, model and hypothesis. j alzheimers dis 2008;13:437-449. https://doi.org/10.3233/jad-2008-13408 sparks stein p, stefen mj, smith c, et al. serum antibodies to periodontal pathogens are a risk factor for alzheimer’s disease. alzheimer’s dement 2012;8:196-203. https://doi.org/10.1016/j.jalz.2011.04.006 beydoun ma, beydoun ha, weiss j, et al. helicobacter pylori, periodontal pathogens, and their interactive association with incident all-cause and alzheimer’s disease dementia in a large national survey. mol psychiatry 2021;26:6038-6053. https://doi.org/10.1038/s41380-020-0736-2 kamer ar, craig rg, pirraglia e, et al. tnf-α and antibodies to periodontal bacteria discriminate between alzheimer’s disease patients and normal subjects. j neuroimmunol. 2009;216:92-97. https://doi.org/10.1016/j.jneuroim.2009.08.013 abbayya k, puthanakar ny, naduwinmani s, et al. association between periodontitis and alzheimer’s disease. j neuroimmunol 2009;216:92-7.doi: 10.1016/j.jneuroim.2009.08.013. ide m, harris m, stevens a, et al. periodontitis and cognitive decline in alzheimer’s disease. plos one 2016;11:e0151081. https://doi.org/10.1016/j.jneuroim.2009.08.013 alonso r, pisa d, rabano a, et al. alzheimer’s disease and disseminated mycoses. eur j clin microbiol infect dis 2014b;33:1125-1132. https://doi.org/10.1007/s10096-013-2045-z pisa d, alonso r, rábano a, et al. different brain regions are infected with fungi in alzheimer's disease. sci rep 2015;5:15015. https://doi.org/10.1038/srep15015 pisa d, alonso r, fernández-fernández am, et al. polymicrobial infections in brain tissuie from alzheimer’s disease patients. sci rep 2017;7:5559. https://doi.org/10.1038/s41598-017-05903-y alonso r, pisa d, marina a, et al. fungal infection in patients with alzheimer’s disease. j alzheimers dis 2014;41:301-311. https://doi.org/10.3233/jad-132681 alonso r, pisa d, marina a, et al. evidence for fungal infection in cerebrospinal fluid and brain tissue from patients with amyotrophic lateral sclerosis. int j biol sci 2015;11:546-558. https://doi.org/10.7150/ijbs.11084 alonso r, pisa d, aguado b, et al. identification of fungal species in brain tissue from alzheimer’s disease by next-generation sequencing. j alzheimers dis 2017;58: 55-67. https://doi.org/10.3233/jad-170058 pisa d, alonso r, marina ai, et al. human and microbial proteins from corpora amylacea of alzheimer's disease. sci rep 2018;8:9880. https://doi.org/10.1038/s41598-018-28231-1 alonso r, pisa d, fernández-fernández am, et al. fungal infection in neural tissue of patients with amyotrophic lateral sclerosis. neurobiol dis 2017;108:249-260. https://doi.org/10.1016/j.nbd.2017.09.001 alonso r, pisa d, rábano a, et al. cerebrospinal fuid from alzheimer’s disease patients contains fungal proteins and dna. j alzheimers dis 2015;47:873-876. https://doi.org/10.3233/jad-150382 ball mj. limbic predilection in alzheimer dementia: is reactivated herpesvirus involved? can j neurol sci j 1982;9:303-306. https://doi.org/10.1017/s0317167100044115 gannicliffe a, sutton rn, itzhaki rf. viruses, brain and immunosuppression. psychol med 1986;16:247-249. https://doi.org/10.1017/s0033291700009053 lin w-r, wozniak ma, cooper rj, et al. herpesviruses in brain and alzheimer’s disease. j pathol 2002;197:395-402. https://doi.org/10.1002/path.1127 hemling n, röyttä m, rinne j, et al. herpesviruses in brains in alzheimer’s and parkinson’s diseases. ann neurol 2003; 54: 267-271. https://doi.org/10.1002/ana.10662 warren-gash c, forbes hj, williamson e, et al. human herpesvirus infections and dementia or mild cognitive impairment: a systematic review and meta-analysis. sci rep 2019;9:4743. https://doi.org/10.1038/s41598-019-41218-w jamieson ga, maitland nj, wilcock gk, et al. latent herpes simplex virus type 1 in normal and alzheimer’s disease brains. j med virol 1991;33:224-227. https://doi.org/10.1002/jmv.1890330403 itzhaki rf, lin wr, shang d, et al. herpes simplex virus type 1 in brain and risk of alzheimer’s disease. lancet 1997;349:241-244. https://doi.org/10.1016/s0140-6736(96)10149-5 harris sa, harris ea. herpes simplex virus type 1 and other pathogens are key causative factors in sporadic alzheimer's disease. j alzheimers dis 2015;48:319-353. https://doi.org/10.3233/jad-142853 lövheim h, gilthorpe j, johansson a, et al. herpes simplex infection and the risk of alzheimer’s disease: a nested case-control study. alzheimers dement 2015;11:587-592. https://doi.org/10.1016/j.jalz.2014.07.157 itzhaki rf. corroboration of a major role for herpes simplex virus type 1 in alzheimer’s disease. front aging neurosci 2018;10:324. https://doi.org/10.3389/fnagi.2018.00324 wozniak m, mee a, itzhaki r. herpes simplex virus type 1 dna is located within alzheimer’s disease amyloid plaques. j pathol 2009;217:131-138. https://doi.org/10.1002/path.2449 lai sw, kuo yh, liao kf. herpes zoster and alzheimer's disease. eur arch psychiatry clin neurosci 2021 jul 23. https://doi.org/10.1007/s00406-021-01311-6 barnes ll, capuano aw, aiello ae, et al. cytomegalovirus infection and risk of alzheimer disease in older black and white individuals. j infect dis 2015;211:230-237. https://doi.org/10.1093/infdis/jiu437 fotheringham j, akhyani n, vortmeyer a, et al. detection of active human herpesvirus–6 infection in the brain: correlation with polymerase chain reaction detection in cerebrospinal fluid. j infect dis 2007;195:450-454. https://doi.org/10.1086/510757 readhead b, haure-mirande jv, funk cc, et al. multiscale analysis of independent alzheimer’s cohorts finds disruption of molecular, genetic, and clinical networks by human herpesvirus. neuron 2018;99:64-82.e7. https://doi.org/10.1016/j.neuron.2018.05.023 allnutt ma, johnson k, bennett da, et al. human herpesvirus 6 detection in alzheimer’s disease cases and controls across multiple cohorts. neuron 2000; 105:1027-1035. https://doi.org/10.1016/j.neuron.2019.12.031 santpere g, telford m, andrés-benito p, et al. the presence of human herpesvirus 6 in the brain in health and disease. biomolecules 2020;10:1520. https://doi.org/10.3390/biom10111520 rodriguez jd, royall d, daum lt, et al. amplification of herpes simplex type 1 and human herpes type 5 viral dna from formalin-fxed alzheimer brain tissue. neurosci lett 2005;390:37-41. https://doi.org/10.1016/j.neulet.2005.07.052 carbone i, lazzarotto t, ianni m, et al. herpes virus in alzheimer’s disease: relation to progression of the disease. neurobiol aging 2014;35:122-129. https://doi.org/10.1016/j.neurobiolaging.2013.06.024 eimer wa, vijaya kumar dk, navalpur shanmugam nk, et al. alzheimer’s disease-associated β-amyloid is rapidly seeded by herpesviridae to protect against brain infection. neuron 2018;99:56-63.e3. https://doi.org/10.1016/j.neuron.2018.06.030 ganz t, fainstein n, elad a, et al. microbial pathogens induce neurodegeneration in alzheimer's disease mice: protection by microglial regulation. j neuroinflammation 2022;19:5. https://doi.org/10.1186/s12974-021-02369-8 phuna zx, madhavan p. a reappraisal on amyloid cascade hypothesis: the role of chronic infection in alzheimer's disease. int j neurosci 2022 mar 14;1-19. https://doi.org/10.1080/00207454.2022.2045290 jiang c, li g, huang p, et al. the gut microbiota and alzheimer's disease. j alzheimers dis 2017;58:1-15. https://doi.org/10.3233/jad-161141 szablewski l. human gut microbiota in health and alzheimer's disease. j alzheimers dis 2018;62:549-560. https://doi.org/10.3233/jad-170908 megur a, baltriukienė d, bukelskienė v, et al. the microbiota-gut-brain axis and alzheimer's disease: neuroinflammation is to blame? nutrients 2020;13:37. https://doi.org/10.3390/nu13010037 pluta r, ułamek-kozioł m, januszewski s, et al. gut microbiota and pro/prebiotics in alzheimer's disease. aging 2020;12:5539-5550. https://doi.org/10.18632/aging.102930 kesika p, suganthy n, sivamaruthi bs, et al. role of gut-brain axis, gut microbial composition, and probiotic intervention in alzheimer's disease. life sci 2021;264:118627. https://doi.org/10.1016/j.lfs.2020.118627 de la fuente m. the role of the microbiota-gut-brain axis in the health and illness condition: a focus on alzheimer's disease. j alzheimers dis 2021;81:1345-1360. https://doi.org/10.3233/jad-201587 łuc m, misiak b, pawłowski m, et al. gut microbiota in dementia. critical review of novel findings and their potential application. prog neuropsychopharmacol. biol psychiatry 2021;104: 110039. https://doi.org/10.1016/j.pnpbp.2020.110039 aaldijk e, vermeiren y. the role of serotonin within the microbiota-gut-brain axis in the development of alzheimer's disease: a narrative review. ageing res rev 2022;3: 101556. https://doi.org/10.1016/j.arr.2021.101556 rueda-ruzafa l, cruz f, cardona d, et al. opioid system influences gut-brain axis: dysbiosis and related alterations. pharmacol res 2020;159:104928. https://doi.org/10.1016/j.phrs.2020.104928 leaston j, kulkarni p, gharagouzloo c, et al. do we swallow the waste from our brain? front neurosci 2021; 15:763780. https://doi.org/10.3389/fnins.2021.763780 walker lc, callahan mj, bian f, et al. exogenous induction of cerebral beta-amyloidosis in betaapp-transgenic mice. peptides 2002;23: 1241-1247. meyer-luehmann m, coomaraswamy j, bolmont t, et al. exogenous induction of cerebral b-amyloidogenesis is governed by agent and host. science 2006;313:1781-1784. https://doi.org/10.1126/science.1131864 walker lc, levine h, mattson mp, et al. inducible proteopathies. trends neurosci 2006;29:438-443. https://doi.org/10.1016/j.tins.2006.06.010 rosen rf, fritz jj, dooyema j, et al. exogenous seeding of cerebral b-amyloid deposition in bapp-transgenic rats. j neurochem 2012;120:660-666. https://doi.org/10.1111/j.1471-4159.2011.07551.x morales r, duran-aniotz c, castilla j, et al. de novo induction of amyloid-b deposition in vivo. mol psychiatry 2012;17:1347-1353. https://doi.org/10.1038/mp.2011.120 langer f, eisele ys, fritschi sk, et al. soluble aβ seeds are potent inducers of cerebral b-amyloid deposition. j neurosci 2011;31: 14488-14495. https://doi.org/10.1523/jneurosci.3088-11.2011 hamaguchi t, eisele y, varvel n, et al. the presence of ab seeds, and not age per se, is critical to the initiation of ab deposition in the brain. acta neuropathol 2012;123:31-37. https://doi.org/10.1007/s00401-011-0912-1 bero aw, yan p, roh jh, et al. neuronal activity regulates the regional vulnerability to amyloid-β deposition. nat neurosci 2011;14:750-756. https://doi.org/10.1038/nn.2801 eisele ys, obermüller u, heilbronner g et al. peripherally applied abeta-containing inoculates induce cerebral beta-amyloidosis. science 2010;330:980-982. https://doi.org/10.1126/science.1194516 forny-germano l, lyra de silva nm, batista af, et al. alzheimer's disease-like pathology induced by amyloid-beta oligomers in nonhuman primates. j neurosci 2014;34:13629-13643. https://doi.org/10.1523/jneurosci.1353-14.2014 lauwers e, lalli g, brandner s, et al. potential human transmission of amyloid-beta pathology: surveillance and risks. lancet neurol 2020;19:872-878. https://doi.org/10.1016/s1474-4422(20)30238-6 pooler am, phillips ec, lau dhw, et al. physiological release of endogenous tau is stimulated by neuronal activity. embo rep 2013;14:389-394. https://doi.org/10.1038/embor.2013.15 yamada k, holth jk, liao f, et al. neuronal activity regulates extracellular tau in vivo. j exp med 2014; 211:387-393. https://doi.org/10.1084/jem.2013168 wu jw, hussaini sa, bastille im, et al. neuronal activity enhances tau propagation and tau pathology in vivo. nat neurosci 2016;19:1085-1092. https://doi.org/10.1038/nn.4328 schultz mk, gentzel r, usenovic m, et al. pharmacogenetic neuronal stimulation increases human tau pathology and trans-synaptic spread of tau to distal brain regions in mice. neurobiol dis 2018;118:161-176. https://doi.org/10.1016/j.nbd.2018.07.003 saman s, kim wh, raya m, et al. exosome-associated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid in early alzheimer disease. j biol chem 2012;287:3842-3849. https://doi.org/10.1074/jbc.m111.277061 takeda s, wegmann s, cho h, et al. neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from alzheimer’s disease brain. nat commun 2015;6: 8490. https://doi.org/10.1038/ncomms9490 mudner a, colin m, dujardin s, et al. what is the evidence that tau pathology spreads through prion-like propagation? acta neuropathol commun 2017; 5:99. https://doi.org/10.1186/s40478-017-0488-7 dujardin s, hyman bt. tau prion-like propagation: state of the art and current challenges. adv exp med biol 2019;1184:305-325. https://doi.org/10.1007/978-981-32-9358-8_23 goedert m. tau proteinopathies and the prion concept. prog mol biol transl sci 2020;175: 239-259. https://doi.org/10.1016/bs.pmbts.2020.08.003 peng c, trojanowski jq, lee vmy. protein transmission in neurodegenerative disease. nat rev neurol 2020;16:199-212. https://doi.org/10.1038/s41582-020-0333-7 de calignon a, polydoro m, suárez-calvet m, et al. propagation of tau pathology in a model of early alzheimer's disease. neuron 2012;73:685-697. https://doi.org/10.1016/j.neuron.2011.11.033 liu l, drouet v, wu jw, et al. trans-synaptic spread of tau pathology in vivo. plos one 2012; 7:e31302. https://doi.org/10.1371/journal.pone.0031302 dujardin s, lécolle k, caillierez r, et al. neuron-to-neuron wild-type tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies. acta neuropathol commun 2014; 2:14. https://doi.org/10.1186/2051-5960-2-14 fauré j, lachenal g, court m, et al. exosomes are released by cultured cortical neurones. mol cell neurosci 2006;31, 642-648. https://doi.org/10.1016/j.mcn.2005.12.003 smalheiser nr. exosomal transfer of proteins and rnas at synapses in the nervous system. biol direct 2007;2:35. https://doi.org/10.1186/1745-6150-2-35 frost b, jacks rl, diamond mi. small misfolded tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons. j biol chem 2009; 284:12845-12852. https://doi.org/10.1074/jbc.m808759200 simons m, raposo g. exosomes--vesicular carriers for intercellular communication. curr opin cell biol 2009;21:575-581. https://doi.org/10.1016/j.ceb.2009.03.007 wu jw, herman m, liu l, et al. small misfolded tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons. j biol chem 2013; 288:1856-1870. https://doi.org/10.1074/jbc.m112.394528 holmes bb, devos sl, kfoury n, et al. heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. proc natl acad sci usa 2013;110:e3138-e3147. https://doi.org/10.1073/pnas.1301440110 saman s, lee nc, inoyo i, et al. proteins recruited to exosomes by tau overexpression implicate novel cellular mechanisms linking tau secretion with alzheimer's disease. j alzheimers dis 2014;40:s47-70. https://doi.org/10.3233/jad-132135 polanco jc, scicluna bc, hill af, et al. extracellular vesicles isolated from the brains of rtg4510 mice seed tau protein aggregation in a threshold-dependent manner. j biol chem 2016;291:12445-12466. https://doi.org/10.1074/jbc.m115.709485 wang y, balaji v, kaniyappan s, et al. the release and trans-synaptic transmission of tau via exosomes. mol neurodegener 2017;12: 5. https://doi.org/10.1186/s13024-016-0143-y demaegd k, schymkowitz j, rousseau f. transcellular spreading of tau in tauopathies. chembiochem 2018;19:2424-2432. https://doi.org/10.1002/cbic.201800288 jiang l, dong h, cao h, et al. exosomes in pathogenesis, diagnosis, and treatment of alzheimer's disease. med sci monit 2019;25:3329-3335. https://doi.org/10.12659/msm.914027 pérez m, avila j, hernández f. propagation of tau via extracellular vesicles. front neurosci 2019;13:698. https://doi.org/10.3389/fnins.2019.00698 iba m, guo jl, mcbride jd, et al. synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of alzheimer's-like tauopathy. j neurosci 2013; 33: 1024-1037. https://doi.org/10.1523/jneurosci.2642-12.2013 peeraer e, bottelbergs a, van kolen k, et al. intracerebral injection of preformed synthetic fibrils initiates widesprad tauopathy and neuronal loss in the brains of tau transgenic mice. neurobiol dis 2015;73:83-95. https://doi.org/10.1016/j.nbd.2014.08.032 ahmed z, cooper j, murray tk, et al. a novel in vivo model of tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity. acta neuropathol 2014;127:667-683. https://doi.org/10.1007/s00401-014-1254-6 clavaguera f, bolmont t, crowther ra, et al. transmission and spreading of tauopathy in transgenic mouse brain. nat cell biol 2009; 111:909-913. https://doi.org/10.1038/ncb1901 clavaguera f, akatsu h, fraser g, et al. brain homogenates from human tauopathies induce tau inclusions in mouse brain. proc natl acad sci usa 2013; 110:9535-9540. https://doi.org/10.1073/pnas.1301175110 boluda s, iba m, zhang b, et al. differential induction and spread of tau pathology in young ps19 tau transgenic mice following intracerebral injections of pathological tau from alzheimer’s disease or corticobasal degeneration brains. acta neuropathol 2015;129:221–237. https://doi.org/10.1007/s00401-014-1373-0 hu w, zhang x, tung yc, et al. hyperphosphorylation determines both the spread and the morphology of tau pathology. alzheimers dement 2016;12:1066-1077. https://doi.org/10.1016/j.jalz.2016.01.014 audouard e, houben s, masaracchia c, et al. high-molecular weight paired helical filaments from alzheimer brain induces seeding of wild-type mouse tau into argyrophilic 4r tau pathology in vivo. am j pathol 2016; 186:2709-2722. https://doi.org/10.1016/j.ajpath.2016.06.008 guo jl, narasimhan s, changolkar l, et al. unique pathological tau conformers from alzheimer’s brains transmit tau pathology in nontransgenic mice. j exp med 2016; 213:2635–2654. https://doi.org/10.1084/jem.20160833 narashima s, guo jl, changolkar l, et al. pathological tau strains from human brains recapitulate the diversity of tauopathies in non-transgenic mouse brain. j neurosci 2017;37: 11406-11423. https://doi.org/10.1523/jneurosci.1230-17.2017 ferrer i, aguiló garcía m, lópez gonzález i, et al. aging-related tau astrogliopathy (artag): not only tau phosphorylation in astrocytes. brain pathol 2018; 28: 965-985. https://doi.org/10.1111/bpa.12593 ferrer i, andrés-benito p, sala-jarque j, et al. capacity for seeding and spreading of argyrophilic grain disease in a wild-type murine model; comparisons with primary age-related tauopathy. front mol neurosci 2020; 13:101. https://doi.org/10.3389/fnmol.2020.00101 ferrer i, andrés-benito p, zelaya mv, et al. familial globular glial tauopathy linked to mapt mutations: molecular neuropathology and seeding capacity of a prototypical mixed neuronal and glial tauopathy. acta neuropathol 2020;139:735-771. https://doi.org/10.1007/s00401-019-02122-9 ferrer i, aguiló garcía m, carmona m, et al. involvement of oligodendrocytes in tau seeding and spreading in tauopathies. front aging neurosci 2019; 11:112. https://doi.org/10.3389/fnagi.2019.00112 weitzman sa, narasimhan s, he z, et al. insoluble tau from human ftdp-17 cases exhibit unique transmission properties in vivo. j neuropathol exp neurol 2020;79: 941-949. https://doi.org/10.1093/jnen/nlaa086 clavaguera f, hench j, goedert m, et al. invited review: prion-like transmission and spreading of tau pathology. neuropathol appl neurobiol 2015;41:47-58. https://doi.org/10.1111/nan.12197 goedert m, spillantini mg. propagation of tau aggregates. mol brain 2017; 10:18. https://doi.org/10.1186/s13041-017-0298-7 goedert m, eisenberg ds, crowther ra. propagation of tau aggregates and neurodegeneration. annu rev neurosci 2017; 40:189-210. https://doi.org/10.1146/annurev-neuro-072116-031153 vaquer-alicea j, diamond mi, joachimiak la. tau strains shape disease. acta neuropathologica 2021;142:57-71. https://doi.org/10.1007/s00401-021-02301-7 li l, shi r, gu j, et al. alzheimer's disease brain contains tau fractions with differential prion-like activities. acta neuropathol commun 2021;9:28. https://doi.org/10.1186/s40478-021-01127-4 dujardin s, commins c, lathuiliere a, et al. tau molecular diversity contributes to clinical heterogeneity in alzheimer's disease. nat med 2020;26:1256-1263. https://doi.org/10.1038/s41591-020-0938-9 janke c, beck m, sthal t, et al. phylogenetic diversity of the expression of the microtubule-associated protein tau: implications for neurodegenerative disorders. brain res mol brain res 1999;68:119-128. https://doi.org/10.1016/s0169-328x(99)00079-0 ferrer i, zelaya v, aguio m, et al. relevance of host tau in tau seeding and spreading in tauopathies. brain pathol 2020;30:298-318. https://doi.org/10.1111/bpa.12778 beckman d, chakrabarty p, ott s, et al. a novel tau-based rhesus monkey model of alzheimer's pathogenesis. alzheimers dement 2021;17:933-945. https://doi.org/10.1002/alz.12318 clavaguera f, hench j, lavenir i, et al. peripheral administration of tau aggregates triggers intracerebral tauopathy in transgenic mice. acta neuropathol 2014; 127: 299-301. https://doi.org/10.1007/s00401-013-1231-5 furman jl, vaquer-alicea j, white cl, et al. widespread tau seeding activity at early braak stages. acta neuropathol 2017;133: 91-100. https://doi.org/10.1007/s00401-016-1644-z kaufman sk, del tredici k, thomas tl, et al. tau seeding activity begins in the transentorhinal/entorhinal regions and anticipates phospho-tau pathology in alzheimer’s disease and part. acta neuropathol 2018; 136:57-67. https://doi.org/10.1007/s00401-018-1855-6 seemiller j, bischof gn, hoenig mc, et al. indication of retrograde tau spreading along braak stages and functional connectivity pathways. eur j nucl med mol imaging 2021; 48: 2272-2282. https://doi.org/10.1007/s00259-020-05183-1 meisl g, hidari e, allinson k, et al. in vivo rate-determining steps of tau seed accumulation in alzheimer's disease. sci adv 2021;7:eabh1448. https://doi.org/10.1126/sciadv.abh1448 andrés-benito p, carmona m, jordán m, et al. host tau genotype specifically designs and regulates tau seeding and spreading and host tau transformation following intrahippocampal injection of identical tau ad inoculum. int j mol sci 2022;23:718. https://doi.org/10.3390/ijms23020718 detrez jr, maurin h, van kolen k, et al. regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain. neurobiol dis 2019;127:398-409. https://doi.org/10.1016/j.nbd.2019.03.010 maphis n, xu g, kokiko-cochran on, et al. reactive microglia drive tau pathology and contribute to the spreading of pathological tau in the brain. brain 2015;138:1738-1755. https://doi.org/10.1093/brain/awv081 bolós m, llorens-martín m, jurado-arjona j, et al. direct evidence of internalization of tau by microglia in vitro and in vivo. j alzheimers dis 2016;50:77-87. https://doi.org/10.3233/jad-150704 perea jr, llorens-martín m, ávila j, et al. the role of microglia in the spread of tau: relevance for tauopathies. front cell neurosci 2018;12:172. https://doi.org/10.3389/fncel.2018.00172 španić e, langer horvat l, hof pr, et al. role of microglial cells in alzheimer's disease tau propagation. front aging neurosci 2019;11:271. https://doi.org/10.3389/fnagi.2019.00271 brelstaff jh, mason m, katsinelos t, et al. microglia become hypofunctional and release metalloproteases and tau seeds when phagocytosing live neurons with p301s tau aggregates. sci adv 2021;7:eabg4980. https://doi.org/10.1126/sciadv.abg4980 gratuze m, chen y, parhizkar s, et al. activated microglia mitigate aβ-associated tau seeding and spreading. j exp med 2021;218:e20210542. https://doi.org/10.1084/jem.20210542 hopp sc, lin y, oakley d, et al. the role of microglia in processing and spreading of bioactive tau seeds in alzheimer's disease. j neuroinflammation 2018;15:269. https://doi.org/10.1186/s12974-018-1309-z jellinger ka, stadelmann c. problems of cell death in neurodegeneration and alzheimer's disease. j alzheimers dis 2001;3:31-40. https://doi.org/10.3233/jad-2001-3106 roth ka. caspases, apoptosis, and alzheimer disease: causation, correlation, and confusion. j neuropathol exp neurol 2001;60:829-838. https://doi.org/10.1093/jnen/60.9.829 obulesu m, lakshmi mj. apoptosis in alzheimer's disease: an understanding of the physiology, pathology and therapeutic avenues. neurochem res 2014;39:2301-2312. https://doi.org/10.1007/s11064-014-1454-4 fasulo l, ugolini g, visintin m, et al. the neuronal microtubule-associated protein tau is a substrate for caspase-3 and an effector of apoptosis. j neurochem 2000;75:624-633. https://doi.org/10.1046/j.1471-4159.2000.0750624.x chung cw, song yh, kim kk, et al. proapoptotic effects of tau cleavage product generated by caspase-3. neurobiol dis 2001;8:162-172. https://doi.org/10.1006/nbdi.2000.0335 zilkova m, zilka n, kovac a, et al. hyperphosphorylated truncated protein tau induces caspase-3 independent apoptosis-like pathway in the alzheimer's disease cellular model. alzheimers dis 2011;23:161-169. https://doi.org/10.3233/jad-2010-101434 gómez-ramos a, díaz-hernández m, cuadros r, et al. extracellular tau is toxic to neuronal cells. febs lett 2006;580:4842-4850. https://doi.org/10.1016/j.febslet.2006.07.078 caccamo a, branca c, piras is, et al. necroptosis activation in alzheimer's disease. nat neurosci 2017;20:1236-1246. https://doi.org/10.1038/nn.4608 royce gh, brown-borg hm, deepa ss. the potential role of necroptosis in inflammaging and aging. geroscience 2019;41:795-811. https://doi.org/10.1007/s11357-019-00131-w dionísio pa, amaral jd, rodrigues cmp. molecular mechanisms of necroptosis and relevance for neurodegenerative diseases. int rev cell mol biol 2020;353:31-82. https://doi.org/10.1016/bs.ircmb.2019.12.006 gómez-isla t, hollister r, west h, et al. neuronal loss correlates with but exceeds neurofibrillary tangles in alzheimer's disease. ann neurol 1997;41:17-24. https://doi.org/10.1002/ana.410410106 van de nes jap, nafe r, schlote w. non-tau based neuronal degeneration in alzheimer's disease -an immunocytochemical and quantitative study in the supragranular layers of the middle temporal neocortex. brain res 2008;1213:152-165. https://doi.org/10.1016/j.brainres.2008.03.043 takahashi rh, nagao t, gouras gk. plaque formation and the intraneuronal accumulation of β-amyloid in alzheimer's disease. pathol int 2017;67:185-193. https://doi.org/10.1111/pin.12520 jeong s. molecular and cellular basis of neurodegeneration in alzheimer's disease. mol cells 2017;40:613-620. https://doi.org/10.14348/molcells.2017.0096 leong yq, ng ky, chye sm, et al. mechanisms of action of amyloid-beta and its precursor protein in neuronal cell death. metab brain dis 2020;35:11-30. https://doi.org/10.1007/s11011-019-00516-y siman-tov t, bosak n, sprecher e, et al. early age-related functional connectivity decline in high-order cognitive networks. front aging neurosci 2017;8:330. https://doi.org/10.3389/fnagi.2016.00330 tsvetanov ka, henson rn, tyler lk, et al. extrinsic and intrinsic brain network connectivity maintains cognition across the lifespan despite accelerated decay of regional brain activation. j neurosci 2016;36:3115-3126. https://doi.org/10.1523/jneurosci.2733-15.2016 buckley rf, schultz ap, hedden t, et al. functional network integrity presages cognitive decline in preclinical alzheimer disease. neurology 2017;89:29-37. https://doi.org/10.1212/wnl.0000000000004059 jockwitz c, caspers s. resting-state networks in the course of aging-differential insights from studies across the lifespan vs. amongst the old. pflugers arch 2021;473:793-803. https://doi.org/10.1007/s00424-021-02520-7 zhang h, gertel vh, cosgrove al, et al. age-related differences in resting-state and task-based network characteristics and cognition: a lifespan sample. neurobiol aging 2021;101:262-272. https://doi.org/10.1016/j.neurobiolaging.2020.10.025 hoenig mc, bischof gn, seemiller j, et al. networks of tau distribution in alzheimer's disease. brain 2018;141:568-581. https://doi.org/10.1093/brain/awx353 zheng w, yao z, xie y, et al. identification of alzheimer's disease and mild cognitive impairment using networks constructed based on multiple morphological brain features. biol psychiatry cogn neurosci neuroimaging. 2018;3:887-897. https://doi.org/10.1016/j.bpsc.2018.06.004 pelkmans w, ossenkoppele r, dicks e, et al. tau-related grey matter network breakdown across the alzheimer's disease continuum. alzheimers res ther 2021;13:138. https://doi.org/10.1186/s13195-021-00876-7 shigemoto y, sone d, okita k, et al. gray matter structural networks related to (18)f-thk5351 retention in cognitively normal older adults and alzheimer's disease patients. neurological sci 2021;22:100309. https://doi.org/10.1016/j.ensci.2021.100309 smith rx, strain jf, tanenbaum a, et al. resting-state functional connectivity disruption as a pathological biomarker in autosomal dominant alzheimer disease. brain connect 2021;11:239-249. https://doi.org/10.1089/brain.2020.0808 wu z, gao y, potter t, et al. interactions between aging and alzheimer’s disease on structural brain networks. aging neurosci 2021;13:639795. https://doi.org/10.3389/fnagi.2021.639795 hasani sa, mayeli m, salehi ma, et al. a systematic review of the association between amyloid-beta and tau pathology with functional connectivity alterations in the alzheimer dementia spectrum utilizing pet scan and rsfmri. dement geriatr cogn dis extra 2021;11:78-90. https://doi.org/10.1159/000516164 ewers m, luan y, frontzkowski l, et al. segregation of functional networks is associated with cognitive resilience in alzheimer's disease. brain 2021;144:2176-2185. https://doi.org/10.1093/brain/awab112 king-robson j, wilson h, politis m, et al. associations between amyloid and tau pathology, and connectome alterations, in alzheimer's disease and mild cognitive impairment. j alzheimers dis 2021;82:541-560. https://doi.org/10.3233/jad-201457 cassady ke, adams jn, chen x, et al. alzheimer's pathology is associated with dedifferentiation of intrinsic functional memory networks in aging. cereb cortex 2021;31:4781-4793. https://doi.org/10.1093/cercor/bhab122 vos sj, xiong c, visser pj, et al. preclinical alzheimer's disease and its outcome: a longitudinal cohort study. lancet neurol 2013;12:957-965. https://doi.org/10.1016/s1474-4422(13)70194-7 dubois b, feldman hh, jacova c, et al. advancing research diagnostic criteria for alzheimer’s disease: the iwg-2 criteria. lancet neurol 2014;13:614-629. https://doi.org/10.1016/s1474-4422(14)70090-0 olsson b, lautner r, andreasson u, et al. csf and blood biomarkers for the diagnosis of alzheimer's disease: a systematic review and meta-analysis. lancet neurol 2016;15:673-684. https://doi.org/10.1016/s1474-4422(16)00070-3 sperling ra, donohue mc, raman r, et al. association of factors with elevated amyloid burden in clinically normal older individuals. jama neurol 2020;77:735-745. https://doi.org/10.1001/jamaneurol.2020.0387 zhang k, mizuma h, zhang x, et al. pet imaging of neural activity, beta-amyloid, and tau in normal brain aging. eur j nucl med mol imaging 2021;48:3859-3871. https://doi.org/10.1007/s00259-021-05230-5 dubois b, hampel h, feldman hh, et al. preclinical alzheimer's disease: definition, natural history, and diagnostic criteria. alzheimers dement 2016;12: 292-323. https://doi.org/10.1016/j.jalz.2016.02.002 rossini pm, di iorio r, vecchio f, et al. early diagnosis of alzheimer's disease: the role of biomarkers including advanced eeg signal analysis. report from the ifcn-sponsored panel of experts. clin neurophysiol 2020;131:1287-1310. https://doi.org/10.1016/j.jalz.2016.02.002 zetterberg h, bendlin bb. biomarkers for alzheimer’s disease-preparing for a new era of disease-modifying therapies. mol psychiatry 2021;26:296-308. https://doi.org/10.1038/s41380-020-0721-9 teunissen ce, verberk imw, thijssen eh, et al. blood-bassed biomarkers for alzheimer’s disease: towards clinical implementation. lancet neurol 2022;21:66-77. https://doi.org/10.1016/s1474-4422(21)00361-6 jansen wj, ossenkoppele r, knol dl, et al. prevalence of cerebral amyloid pathology in persons without dementia: a meta-analysis. jama 2015;313:1924-1938. https://doi.org/10.1001/jama.2015.4668 jansen wj, janssen o, tijms bm, et al. prevalence estimates of amyloid abnormality across the alzheimer disease clinical spectrum. jama neurol 2022;79:228-243. https://doi.org/10.1001/jamaneurol.2021.5216 okamura n, harada r, furumoto s, et al. tau pet imaging in alzheimer's disease. curr neurol neurosci rep 2014;14:500. https://doi.org/10.1007/s11910-014-0500-6 schöll m, lockhart sn, schonhaut dr, et al. pet imaging of tau deposition in the aging human brain. neuron 2016;89:971-982. https://doi.org/10.1007/s11910-014-0500-6 lockhart sn, baker sl, okamura n, et al. dynamic pet measures of tau accumulation in cognitively normal older adults and alzheimer’s disease patients measured using [18f] thk-5351. plos one 2016;11:e0158460. https://doi.org/10.1371/journal.pone.0158460 maass a, landau s, baker sl, et al. comparison of multiple tau-pet measures as biomarkers in aging and alzheimer’s disease. neuroimage 2017; 157:448-463. https://doi.org/10.1016/j.neuroimage.2017.05.058 chotipanich c, jantarato a, kunawudhi a, et al. 11c-pittsburgh compound b and 18f-thk 5351 positron emission tomography brain imaging in cognitively normal individuals. world j nucl med 2020;20:133-138. https://doi.org/10.4103/wjnm.wjnm_57_20 mueller a, bullich s, barret o, et al. tau pet imaging with 18f-pi-2620 in patients with alzheimer disease and healthy controls: a first-in-humans study. j nucl med 2020;61:911-919. https://doi.org/10.2967/jnumed.119.236224 sintini i, whitwell jl. update on neuroimaging in alzheimer's disease. curr opin neurol 2021;34:525-531. https://doi.org/10.1097/wco.0000000000000947 ossenkoppele r, smith r, mattsson-carlgren n, et al. accuracy of tau positron emission tomography as a prognostic marker in preclinical and prodromal alzheimer disease: a head-to-head comparison against amyloid positron emission tomography and magnetic resonance imaging. jama neurol 2021;78:961-971. https://doi.org/10.1001/jamaneurol.2021.1858 jack cr, wiste hj, weigand sd, et al. age-specific and sex-specific prevalence of cerebral β-amyloidosis, tauopathy, and neurodegeneration in cognitively unimpaired individuals aged 50-95 years: a cross-sectional study. lancet neurol 2017;16:435-444. https://doi.org/10.1016/s1474-4422(17)30077-7 lowe vj, wiste hj, senjem ml, et al. widespread brain tau and its association with ageing, braak stage and alzheimer's dementia. brain 2018;141:271-287. https://doi.org/10.1093/brain/awx320 yoon b, guo t, provost k, et al. abnormal tau in amyloid pet negative individuals. neurobiol aging 2022;109:125-134. https://doi.org/10.1016/j.neurobiolaging.2021.09.019 groot c, doré v, robertson j, et al. mesial temporal tau is related to worse cognitive performance and greater neocortical tau load in amyloid-beta-negative cognitively normal individuals. neurobiol aging 2021;97:41-48. https://doi.org/10.1016/j.neurobiolaging.2020.09.017 wisse lem, xie l, das sr, et al. tau pathology mediates age effects on medial temporal lobe structure. neurobiol aging 2022;109:135-144. https://doi.org/10.1016/j.neurobiolaging.2021.09.017 chen sd, lu jy, li hq, et al. staging tau pathology with tau pet in alzheimer's disease: a longitudinal study. transl psychiatry 2021;11:483. https://doi.org/10.1038/s41398-021-01602-5 rubinski a, tosun d, franzmeier n, et al. lower cerebral perfusion is associated with tau-pet in the entorhinal cortex across the alzheimer's continuum. neurobiol aging 2021;102:111-118. https://doi.org/10.1016/j.neurobiolaging.2021.02.003 berron d, vogel jw, insel ps, et al. early stages of tau pathology and its associations with functional connectivity, atrophy and memory. brain 2021;144:2771-2783. https://doi.org/10.1093/brain/awab114 shen xn, kuo k, yang yx, et al. subtle cognitive impairment as a marker of alzheimer's pathologies and clinical progression in cognitively normal individuals. alzheimers dement 2021;13:e12198. https://doi.org/10.1002/dad2.12198 sanchez js, becker ja, jacobs hil, et al. the cortical origin and initial spread of medial temporal tauopathy in alzheimer's disease assessed with positron emission tomography. sci transl med 2021;13:eabc0655. https://doi.org/10.1126/scitranslmed.abc0655 sanchez js, hanseeuw bj, lopera f, et al. longitudinal amyloid and tau accumulation in autosomal dominant alzheimer's disease: findings from the colombia-boston (colbos) biomarker study. alzheimers res ther 2021;13:27. https://doi.org/10.1186/s13195-020-00765-5 nelson pt, abner el, schmitt fa, et al. brains with medial temporal lobe neurofibrillary tangles but no neuritic amyloid plaques are a diagnostic dilemma but may have pathogenetic aspects distinct from alzheimer disease. j neuropathol exp neurol 2009;68:774–784. https://doi.org/10.1097/nen.0b013e3181aacbe9 crary jf, trojanowski jq, schneider ja, et al. primary age-related tauopathy (part): a common pathology associated with human aging. acta neuropathol 2014; 128: 755-766. https://doi.org/10.1007/s00401-014-1349-0 jellinger ka, alafuzoff i, attems j, et al. part, a distinct tauopathy, different from classical sporadic alzheimer disease. acta neuropathol 2015; 129: 757-762. https://doi.org/10.1007/s00401-015-1407-2 bell wr, an y, kageyama y, et al. neuropathologic, genetic, and longitudinal cognitive profiles in primary age-related tauopathy (part) and alzheimer’s disease. alzheimers dement 2019; 15:8-16. https://doi.org/10.1016/j.jalz.2018.07.215 teylan m, mock c, gauthreaux k, et al. cognitive trajectory in mild cognitive impairment due to primary age-related tauopathy. brain 2020; 143; 611-621. https://doi.org/10.1093/brain/awz403 braak h, thal dr, ghebremedhin e, et al. stages of the pathologic process in alzheimer disease: age categories from 1 to 100 years. j neuropathol exp neurol 2011; 70: 960-969. https://doi.org/10.1097/nen.0b013e318232a379 jellinger ka, bancher c. senile dementia with tangles (tangle predominant form of senile dementia). brain pathol 1998; 8: 367-376. https://doi.org/10.1111/j.1750-3639.1998.tb00160.x jellinger ka, attems j. prevalence of dementia disorders in the oldest-old: an autopsy study. acta neuropathol 2010: 119:421-433. https://doi.org/10.1007/s00401-010-0654-5 jellinger ka. different patterns of hippocampal tau pathology in alzheimer's disease and part. acta neuropathol 2018; 136:811-813. https://doi.org/10.1007/s00401-018-1894-z zhang l, jiang y, zhu j, et al. quantitative assessment of hippocampal tau pathology in ad and part. j mol neurosci 2020;70:1808-1811. https://doi.org/10.1007/s12031-020-01573-0 duyckaerts c, braak h, brion jp, et al. part is part of alzheimer disease. acta neuropathol 2015; 129:749-756. https://doi.org/10.1007/s00401-015-1390-7 mcmillan ct, lee eb, jefferson-george k, et al. alzheimer’s genetic risk is reduced in primary age-related tauopathy: a potential model of resistance? annals of clin transl neurol 2018; 5: 927-934. https://doi.org/10.1002/acn3.581 santa-maria i, haggiagi a, liu x, et al. the mapt h1 haplotype is associated with tangle-predominant dementia. acta neuropathol 2012;124:693–704. https://doi.org/10.1007/s00401-012-1017-1 schmidt c, wolff m, weitz m, et al. rapidly progressive alzheimer disease. arch neurol 2011; 68:1124-1130. https://doi.org/10.1001/archneurol.2011.189 stoeck k, schmitz m, ebert e, et al. immune responses in rapidly progressive dementia: a comparative study of neuroinflammatory markers in creutzfeldt-jakob disease, alzheimer’s disease and multiple sclerosis. j neuroinflammation 2014;11:170. https://doi.org/10.1186/s12974-014-0170-y schmidt c, haik s, satoh k, et al. rapidly progressive alzheimer’s disease: a multicenter update. j alzheimer’s dis 2012;30:751-756. https://doi.org/10.3233/jad-2012-120007 cohen m, appleby b, safar jg. distinct prion-like strains of amyloid beta implicated in phenotypic diversity of alzheimer’s disease. prion 2016;10:9-17. https://doi.org/10.1080/19336896.2015.1123371 drummond e, nayak s, faustin a, et al. proteomic differences in amyloid plaques in rapidly progressive and sporadic alzheimer's disease. acta neuropathol 2017;133:933-954. https://doi.org/10.1007/s00401-017-1691-0 zafar s, shafiq m, younas n, et al. prion protein interactome: identifying novel targets in slowly and rapidly progressive forms of alzheimer's disease. j alzheimers dis 2017;59:265-275. https://doi.org/10.3233/jad-170237 shafiq m, zafar s, younas n, et al. prion protein oligomers cause neuronal cytoskeletal damage in rapidly progressive alzheimer's disease. mol neurodegener 2021;16:11. https://doi.org/10.1186/s13024-021-00422-x noor a, zafar s, shafiq m, et al. molecular profiles of amyloid‑β proteoforms in typical and rapidly progressive alzheimer’s disease. mol neurobiol 2022 59:17-34. https://doi.org/10.1007/s12035-021-02566-9 younas n, zafar s, shafiq m, et al. sfpq and tau: critical factors contributing to rapid progression of alzheimer’s disease. acta neuropathol 2020;140:317-339. https://doi.org/10.1007/s00401-020-02178-y arenaza-urquijo em, vemuri p. resistance vs resilience to alzheimer disease: clarifying terminology for preclinical studies. neurology 2018;90:695-703. https://doi.org/10.1212/wnl.0000000000005303 andersen sl. centenarians as models of resistance and resilience to alzheimer's disease and related dementias. adv geriatr med res 2020;2:e200018. https://doi.org/10.20900/agmr20200018 beker n, ganz a, hulsman m, et al. association of cognitive function trajectories in centenarians with postmortem neuropathology, physical health, and other risk factors for cognitive decline. jama 2021;4:e2031654. https://doi.org/10.1001/jamanetworkopen.2020.31654 giannakopoulos p, hof pr, surini m, et al. quantittaive immunohistochemical analysis of the distribution of neurofibrillary tangles and senile plaques in the cerebral cortex of nonagenarians and centenerians. acta neuropathol 1993;85:602-610. https://doi.org/10.1007/bf00334669 giannakopoulos p, hof pr, vallet pg, et al. quantitative analysis of neuropathologic changes in the cerebral cortex of centenarians. prog neuropsychopharmacol biol psychiatry 1995;19:577-592. https://doi.org/10.1016/0278-5846(95)00103-3 rogalski e, gefen t, mao q, et al. cognitive trajectories and spectrum of neuropathology in superagers: the first 10 cases. hippocampus 2019; 29:458-467. https://doi.org/10.1002/hipo.22828 xuereb jh, brayne c, dufouil c, et al. neuropathological findings in the very old. results from the first 101 brains of a population-based longitudinal study of dementing disorders. ann n y acad sci 2000;903:490-496. https://doi.org/10.1111/j.1749-6632.2000.tb06404.x stern y. cognitive reserve in ageing and alzheimer's disease. lancet neurol 2012;11:1006-1012. https://doi.org/10.1016/s1474-4422(12)70191-6 lesuis sl, hoeijmakers l, korosi a, et al. vulnerability and resilience to alzheimer's disease: early life conditions modulate neuropathology and determine cognitive reserve. alzheimers res ther 2018;10:95. https://doi.org/10.1186/s13195-018-0422-7 montine tj, cholerton ba, corrada mm, et al. concepts for brain aging: resistance, resilience, reserve, and compensation. alzheimers res ther 2019;11:22. https://doi.org/10.1186/s13195-019-0479-y weisenbach sl, kim j, hammers d, et al. linking late life depression and alzheimer's disease: mechanisms and resilience. curr behav neurosci rep 2019;6:103-112. https://doi.org/10.1007/s40473-019-00180-7 peng s, roth ar, apostolova lg, et al. cognitively stimulating environments and cognitive reserve: the case of personal social networks. neurobiol aging 2022;112: 197-203. https://doi.org/10.1016/j.neurobiolaging.2022.01.004 zhang x, alshakhshir n, zhao l. glycolytic metabolism, brain resilience, and alzheimer's disease. front neurosci 2021;15:662242. https://doi.org/10.3389/fnins.2021.662242 camandola s, plick n, mattson mp. impact of coffee and cacao metabolites on neuroplasticity and neurodegenerative disease. neurochem res 2019;44:214-227. https://doi.org/10.1007/s11064-018-2492-0 aiello bowles ej, crane pk, walker rl, et al. cognitive resilience to alzheimer’s disease pathology in the human brain. j alzheimers dis 2019;68:1071-1083. https://doi.org/10.3233/jad-180942 buciuc m, wennberg am, weigand sd, et al. effect modifiers of tdp-43-associated hippocampal atrophy rates in patients with alzheimer's disease neuropathological changes. j alzheimers dis 2020a;73:1511-1523. https://doi.org/10.3233/jad-191040 buciuc m, whitwell jl, tosakulwong n, et al. association between transactive response dna-binding protein of 43 kda type and cognitive resilience to alzheimer's disease: a case-control study. neurobiol aging 2020b;92:92-97. https://doi.org/10.1016/j.neurobiolaging.2020.04.001 nygaard hb, erson-omay ez, wu x, et al. whole-exome sequencing of an exceptional longevity cohort. j gerontol a biol sci med sci 2019;74:1386-1390. https://doi.org/10.1093/gerona/gly098 seto m, weiner rl, dumitrescu l, et al. protective genes and pathways in alzheimer's disease: moving towards precision interventions. mol neurodegener 2021; 16:29. https://doi.org/10.1186/s13024-021-00452-5 mcdermott kl, mcfall gp, andrews sj, et al. memory resilience to alzheimer’s genetic risk: sex effects in predictor profiles. j gerontol b psychol sci soc sci 2017;72:937-946. https://doi.org/10.1093/geronb/gbw161 dumitrescu l, mahoney er, mukherjee s, et al. genetic variants and functional pathways associated with resilience to alzheimer’s disease. brain 2020;143:2561-2575. https://doi.org/10.1093/brain/awaa209 tavana jp, rosene m, jensen no, et al. rab10 an alzheimer’s disease resilience locus and potential drug target. clin interv aging 2018; 14: 73-79. https://doi.org/10.2147/cia.s159148 neuner sm, wilmott la, hoffmann br, et al. hippocampal proteomics defines pathways associated with memory decline and resilience in normal aging and alzheimer's disease mouse models. behav brain res 2017;322:288-298. https://doi.org/10.1016/j.bbr.2016.06.002 leng k, li e, eser r, et al. molecular characterization of selectively vulnerable neurons in alzheimer's disease. nat neurosci 2021;24:276-287. https://doi.org/10.1038/s41593-020-00764-7 barker sj, raju rm, milman nep, et al. mef2 is a key regulator of cognitive potential and confers resilience to neurodegeneration. sci transl med 2020;13:eabd7695. https://doi.org/10.1126/scitranslmed.abd7695 tesi n, van der lee sj, hulsman m, et al. immune response and endocytosis pathways are associated with the resilience against alzheimer's disease. transl psychiatry 2020;10:332. https://doi.org/10.1038/s41398-020-01018-7 perneczky r, kempermann g, korczyn ad, et al. translational research on reserve against neurodegenerative disease: consensus report of the international conference on cognitive reserve in the dementias and the alzheimer's association reserve, resilience and protective factors professional interest area working groups. bmc med 2019;17:47. https://doi.org/10.1186/s12916-019-1283-z frade jm, ovejero-benito mc. neuronal cell cycle: the neuron itself and its circumstances. cell cycle 2015;14:712-720. https://doi.org/10.1080/15384101.2015.1004937 arendt t, rödel l, gärtner u, et al. expression of the cyclin-dependent kinase inhibitor p16 in alzheimer's disease. neuroreport 1996;7:3047-3049. https://doi.org/10.1097/00001756-199611250-00050 mcshea a, harris pl, webster kr, et al. abnormal expression of the cell cycle regulators p16 and cdk4 in alzheimer's disease. am j pathol 1997;150:1933-1939. pmid: 9176387. nagy z, esiri mm, cato am, et al. cell cycle markers in the hippocampus in alzheimer's disease. acta neuropathol 1997;94:6-15. https://doi.org/10.1007/s004010050665 smith mz, nagy z, esiri mm. cell cycle-related protein expression in vascular dementia and alzheimer's disease. neurosci lett 1999;271:45-48. https://doi.org/10.1016/s0304-3940(99)00509-1 arendt t, holzer m, gärtner u. neuronal expression of cycline dependent kinase inhibitors of the ink4 family in alzheimer's disease. j neural transm 1998;105:949-960. https://doi.org/10.1007/s007020050104 raina ak, zhu x, rottkamp ca, et al. cyclin toward dementia: cell cycle abnormalities and abortive oncogenesis in alzheimer disease. j neurosci res 2000;61:128-133. https://doi.org/10.1002/1097-4547(20000715)61:2<128::aid-jnr2>3.0.co;2-h arendt t. alzheimer's disease as a loss of differentiation control in a subset of neurons that retain immature features in the adult brain. neurobiol aging 2000;21:783-796. https://doi.org/10.1016/s0197-4580(00)00216-5 ueberham u, arendt t. the expression of cell cycle proteins in neurons and its relevance for alzheimer's disease. curr drug targets cns neurol disord 2005;4:293-306. https://doi.org/10.2174/1568007054038175 lee hg, casadesus g, zhu x, et al. cell cycle re-entry mediated neurodegeneration and its treatment role in the pathogenesis of alzheimer's disease. neurochem int 2009;54:84-88. https://doi.org/10.1016/j.neuint.2008.10.013 moh c, kubiak jz, bajic vp, et al. cell cycle deregulation in the neurons of alzheimer's disease. results probl cell differ 2011;53:565-576. https://doi.org/10.1007/978-3-642-19065-0_23 koseoglu mm, norambuena a, sharlow er, et al. aberrant neuronal cell cycle re-entry: the pathological confluence of alzheimer's disease and brain insulin resistance, and its relation to cancer. j alzheimers dis 2019;67:1-11. https://doi.org/10.3233/jad-180874 mcshea a, wahl af, smith ma. re-entry into the cell cycle: a mechanism for neurodegeneration in alzheimer disease. med hypotheses 1999;52:525-527. https://doi.org/10.1054/mehy.1997.0680 nagy z. cell cycle regulatory failure in neurones: causes and consequences. neurobiol aging 2000;21:761-769. https://doi.org/10.1016/s0197-4580(00)00223-2 arendt t. alzheimer's disease as a disorder of mechanisms underlying structural brain self-organization. neuroscience 2001;102:723-765. https://doi.org/10.1016/s0306-4522(00)00516-9 lopes jp, oliveira cr, agostinho p. cell cycle re-entry in alzheimer's disease: a major neuropathological characteristic? curr alzheimer res 2009;6:205-212. https://doi.org/10.2174/156720509788486590 bonda dj, lee hp, kudo w, et al. pathological implications of cell cycle re-entry in alzheimer disease. expert rev mol med 2010;12:e19. https://doi.org/10.1017/s146239941000150x barrio-alonso e, hernández-vivanco a, walton cc, et al. cell cycle reentry triggers hyperploidization and synaptic dysfunction followed by delayed cell death in differentiated cortical neurons. sci rep 2018;8:14316. https://doi.org/10.1038/s41598-018-32708-4 olabiyi bf, fleitas c, zammou b et al. prongf involvement in the adult neurogenesis dysfunction in alzheimer's disease. int j mol sci 2021;22:10744. https://doi.org/10.3390/ijms221910744 terreros-roncal j, moreno-jiménez ep, flor-garcía m, et al. impact of neurodegenerative diseases on human adult hippocampal neurogenesis. science 2021;374:1106-1113. https://doi.org/10.1126/science.abl516 dietschy jm, turley sd. cholesterol metabolism in the brain. curr opin lipidol 2001; 12: 105-112. https://doi.org/10.1097/00041433-200104000-00003 naudí a, cabré r, jové m, et al. lipidomics of human brain aging and alzheimer’s disease pathology. int rev neurobiol 2015;122:133-189. https://doi.org/10.1016/bs.irn.2015.05.008 farooqui aa, horrocks la, farooqui t. glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders. chem phys lipids 2000;106:1-29. https://doi.org/10.1016/s0009-3084(00)00128-6 farooqui aa. lipid mediators in the neural cell nucleus: their metabolism, signaling, and association with neurological disorders. neuroscientist 2009;15:392-407. https://doi.org/10.1177/1073858409337035 piomelli d. the challenge of brain lipidomics. prostaglandins lipid mediat 2005;77: 23-34. https://doi.org/10.1016/j.prostaglandins.2004.09.006 piomelli d, astarita g, rapaka r. a neuroscientist's guide to lipidomics. nature rev neurosci 2007; 8:743-754. https://doi.org/10.1038/nrn2233 pamplona r, barja g, portero-otín m. membrane fatty acid unsaturation, protection against oxidative stress, and maximum life span: a homeoviscous-longevity adaptation? ann n y acad sci 2002;959:475-490. https://doi.org/10.1111/j.1749-6632.2002.tb02118.x hulbert aj, pamplona r, buffenstein r, et al. life and death: metabolic rate, membrane composition, and life span of animals. physiol rev 2007; 87:1175-1213. https://doi.org/10.1152/physrev.00047.2006 pamplona r, ilieva e, ayala v, bellmunt mj, et al. maillard reaction versus other nonenzymatic modifications in neurodegenerative processes. ann ny acad sci 2008;1126:315-319. https://doi.org/10.1196/annals.1433.014 moller m, botti h, batthyany c, et al. direct measurement of nitric oxide and oxygen partitioning into liposomes and low density lipoprotein. j biol chem 2005;280:8850-8854. https://doi.org/10.1074/jbc.m413699200 gamliel a, afri m, frimer aa. determining radical penetration of lipid bilayers with new lipophilic spin traps. free rad biol med 2008;44:1394-1405. https://doi.org/10.1016/j.freeradbiomed.2007.12.028 bielski bh, arudi rl, sutherland mw. a study of the reactivity of ho2/o2with unsaturated fatty acids. j biol chem 1983;258:4759-4761. pmid: 6833274. söderberg m, edlund c, kristensson k, et al. lipid compositions of different regions of the human brain during aging. j neurochem 1990;54:415-423. https://doi.org/10.1111/j.1471-4159.1990.tb01889.x svennerholm l, boström k, helander cg, et al. membrane lipids in the aging human brain. j neurochem 1991;56:2051-2059. https://doi.org/10.1111/j.1471-4159.1991.tb03466.x svennerholm l. distribution and fatty acid composition of phosphoglycerides in normal human brain. j lipid res 1968;9:570-579. pmid: 4302302. horrocks la, vanrollins m, yates aj. lipid changes in the ageing brain. in: the molecular basis of neuropathology. davinson an, thompson rhs (eds), edward arnold, 1981 de diego i, peleg s, fuchs b. the role of lipids in aging-related metabolic changes. chem phys lipids 2019;222:59-69. https://doi.org/10.1016/j.chemphyslip.2019.05.005 johnson aa, stolzing a. the role of lipid metabolism in aging, lifespan regulation, and age-related disease. aging cell 2019;516:e13048. https://doi.org/10.1111/acel.13048 mcnamarark, liu y, jandacek r, rider t, et al. the aging human orbitofrontal cortex: decreasing polyunsaturated fatty acid composition and associated increases in lipogenic gene expression and stearoyl-coa desaturase activity. prostaglandins, leukotrienes, essent fatty acids 2008;78:293-304. https://doi.org/10.1016/j.plefa.2008.04.00 keller jn, dimayuga e, chen q, et al. autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain. int j biochem cell biol 2004;36: 2376-2391. https://doi.org/10.1016/j.biocel.2004.05.003 double kl, dedov vn, fedorow h, et al. the comparative biology of neuromelanin and lipofuscin in the human brain. cell mol life sci 2008;65:1669-1682. porta ea. advances in age pigment research. arch gerontol geriat 1991;12:303-320. https://doi.org/10.1016/0167-4943(91)90036-p jolly rd, douglas bv, davey pm, et al. lipofuscin in bovine muscle and brain: a model for studying age pigment. gerontology 1965;41:s283-s295. https://doi.org/10.1159/000213750 ottis p, koppe k, onisko b, et al. human and rat brain lipofuscin proteome. proteomics 2012;12:2445-2454. https://doi.org/10.1002/pmic.201100668 pamplona r, dalfó e, ayala v, et al. proteins in human brain cortex are modified by oxidation, glycoxidation, and lipoxidation: effects of alzheimer disease and identification of lipoxidation targets. j biol chem 2005; 280:21522-21530. https://doi.org/10.1074/jbc.m502255200 haughey nj, bandaru vv, bae m, et al. roles for dysfunctional sphingolipid metabolism in alzheimer's disease neuropathogenesis. biochim biophys acta 2010; 1801: 878-886. https://doi.org/10.1016/j.bbalip.2010.05.003 frisardi v, panza f, seripa d, et al. glycerophospholipids and glycerophospholipid-derived lipid mediators: a complex meshwork in alzheimer's disease pathology. progr lipid res 2011;50:313-330. https://doi.org/10.1016/j.plipres.2011.06.001 wood pl. lipidomics of alzheimer's disease: current status. alzheimer res ther 2012; 4: 5. https://doi.org/10.1186/alzrt103 kosicek m, hecimovic s. phospholipids and alzheimer's disease: alterations, mechanisms and potential biomarkers. int j mol sci 2013;14:1310-1322. https://doi.org/10.3390/ijms14011310 touboul d, gaudin m. lipidomics of alzheimer's disease. bioanalysis 2014; 6: 541-561. https://doi.org/10.4155/bio.13.346 senanayake v, goodenowe db. plasmalogen deficiency and neuropathology in alzheimer’s disease: causation or coincidence? alzheimers dement 2019;5:524-532. https://doi.org/10.1016/j.trci.2019.08.003 brosche t, platt d. the biological significance of plasmalogens in defense against oxidative damage. exp gerontol 1998;33:363-369. https://doi.org/10.1016/s0531-5565(98)00014-x martínez a, portero-otin m, pamplona r, et al. protein targets of oxidative damage in human neurodegenerative diseases with abnormal protein aggregates. brain pathol 2010; 20:281-297. https://doi.org/10.1111/j.1750-3639.2009.00326.x sultana r, perluigi m, butterfield ad. lipid peroxidation triggers neurodegeneration: a redox proteomics view into the alzheimer disease brain. free rad biol med 2013;62:157-169. https://doi.org/10.1016/j.freeradbiomed.2012.09.027 benseny-cases n, klementieva o, cotte m, et al. microspectroscopy (μftir) reveals co-localization of lipid oxidation and amyloid plaques in human alzheimer disease brains. anal chem 2014;86:12047-12054. https://doi.org/10.1021/ac502667b evans rm, hui s, perkins a, et al. cholesterol and apoe genotype interact to influence alzheimer disease progression. neurology 2004;62: 1869-1871. https://doi.org/10.1212/01.wnl.0000125323.15458.3f bu g. apolipoprotein e and its receptors in alzheimer’s disease: pathways, pathogenesis and therapy. nat rev neurosci 2009;10:333-344. https://doi.org/10.1038/nrn2620 hope c, mettenburg j, gonias sl, et al. functional analysis of plasma α2-macroglobulin from alzheimer’s disease patients with the a2m intronic deletion. neurobiol dis 2003;14:504-512. https://doi.org/10.1016/j.nbd.2003.08.005 zappia m, manna i, serra p, et al. increased risk for alzheimer disease with the interaction of mpo and a2m polymorphisms. arch neurol 2004;61:341-344. https://doi.org/10.1001/archneur.61.3.341 marzolo mp, bu g. lipoprotein receptors and cholesterol in app trafficking and proteolytic processing, implications for alzheimer’s disease. semin cell dev biol 2009;20:191-200. https://doi.org/10.1016/j.semcdb.2008.10.005 jones em, dubey m, camp pj, et al. interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption. biochemistry 2012;51:2539-2550. https://doi.org/10.1021/bi201857v morgan k. the three new pathways leading to alzheimer’s disease. neuropathol appl neurobiol 2011;37:353-357. https://doi.org/10.1111/j.1365-2990.2011.01181.x d bruce k, tang m, reigan p, et al. genetic variants of lipoprotein lipase and regulatory factors associated with alzheimer's disease risk. int j mol sci 2020;21:8338 https://doi.org/10.3390/ijms21218338 robinson jl, lee eb, xie sx, et al. neurodegenerative disease concomitant proteinopathies are prevalent, age-related and apoe4-associated. brain 2018;141:2181-2193. https://doi.org/10.1093/brain/awy146 robinson ac, davidson ys, roncaroli f, et al. influence of apoe genotype in primary age-related tauopathy. acta neuropathol commun 2020; 8:215. https://doi.org/10.1186/s40478-020-01095-1 lingwood d, simons k. lipid rafts as a membrane-organizing principle. science 2010;327:46-50. https://doi.org/10.1126/science.1174621 sezgin e, levental i, mayor s, et al. the mystery of membrane organization: composition, regulation and roles of lipid rafts. nat rev mol cell biol 2017;18:361-374. https://doi.org/10.1038/nrm.2017.16 tatulian sa, qin s, pande ah, et al. positioning membrane proteins by novel protein engineering and biophysical approaches. j mol biol 2005;351:939-947. https://doi.org/10.1016/j.jmb.2005.06.080 tun h, marlow l, pinnix i, et al. lipid rafts play an important role in aβ biogenesis by regulating the β-secretase pathway. j mol neurosci 2002;19:31-35. https://doi.org/10.1007/s12031-002-0007-5 ehehalt r, keller p, haass c, et al. amyloidogenic processing of the alzheimer β-amyloid precursor protein depends on lipid rafts. j cell biol 2003;160:113-123. https://doi.org/10.1083/jcb.200207113 kawarabayashi t, shoji m, younkin lh, et al. dimeric amyloid beta protein rapidly accumulates in lipid rafts followed by apoilipoprotein e and phosphorylated tau accumulation in the tg2576 mouse model of alzheimer’s disease. j neurosci 2004;24:3801-3809. https://doi.org/10.1523/jneurosci.5543-03.2004 williamson r, usardi a, hanger dp, et al. membrane-bound β-amyloid oligomers are recruited into lipid rafts by a fyn-dependent mechanism. faseb j 2008;22:1552-1559. https://doi.org/10.1096/fj.07-9766com williamson r, sutherland c. neuronal membranes are key to the pathogenesis of alzheimer's disease: the role of both raft and non-raft membrane domains. curr alzheimer res 20118:213-221. https://doi.org/10.2174/156720511795256008 fabiani c, antollini ss. alzheimer's disease as a membrane disorder: spatial cross-talk among β-amyloid peptides, nicotinic acetylcholine receptors and lipid rafts. front cell neurosci 2019;13:309. https://doi.org/10.3389/fncel.2019.00309 fabelo n, martín v, marín r, et al. evidence for premature lipid raft aging in app/ps1 double-transgenic mice, a model of familial alzheimer disease. j neuropathol exp neurol. 2012;71:868-881. https://doi.org/10.1097/nen.0b013e31826be03c takahashi rh, tobiume m, sato y, et al. accumulation of cellular prion protein within dystrophic neurites of amyloid plaques in the alzheimer's disease brain. neuropathology 2011;31:208-214. https://doi.org/10.1111/j.1440-1789.2010.01158.x takahashi rh, yokotsuka m, tobiume m, et al. accumulation of cellular prion protein within β-amyloid oligomer plaques in aged human brains. brain pathol 2021;31:e12941. https://doi.org/10.1111/bpa.12941 díaz m, fabelo n, ferrer i, et al. “lipid raft aging” in the human frontal cortex during nonpathological aging: gender influences and potential implications in alzheimer's disease. neurobiol aging 2018;67:42-52. https://doi.org/10.1016/j.neurobiolaging.2018.02.022 fabelo n, martín v, marín r, et al. altered lipid composition in cortical lipid rafts occurs at early stages of sporadic alzheimer's disease and facilitates app/bace1 interactions. neurobiol aging 2014;35:1801-1812. https://doi.org/10.1016/j.neurobiolaging.2014.02.005 díaz m, fabelo n, martín v, et al. biophysical alterations in lipid rafts from human cerebral cortex associate with increased bace1/aβpp interaction in early stages of alzheimer's disease. j alzheimers dis 2015;43:1185-1198. https://doi.org/10.3233/jad-141146 chi ey, ege c, winans a, et al. lipid membrane templates the ordering and induces the fibrillogenesis of alzheimer's disease amyloid-beta peptide. proteins 2008;72:1-24. https://doi.org/10.1002/prot.21887 vetrivel ks, thinarkaran g. membrane rafts in alzheimer's disease beta-amyloid production. biochem biophys acta molecular and cell biology of lipids 2010;1801:860-867. https://doi.org/10.1016/j.bbalip.2010.03.007 relini a, marano n, gliozzi a. probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers. adv colloid interface sci 2014;207:81-92. https://doi.org/10.1016/j.cis.2013.10.015 georgieva er, xiao s, borbat pp, et al. tau binds to lipid membrane surfaces via short amphipathic helices located in its microtubule-binding repeats. biophys j 2014;107:1441-1452. https://doi.org/10.1016/j.bpj.2014.07.046 majewski j, jones em, vander zanden cm, et al. lipid membrane templated misfolding and self-assembly of intrinsically disordered tau protein. sci rep 2020;10:13324. https://doi.org/10.1038/s41598-020-70208-6 künze g, barré p, scheidt ha, et al. binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein. biochim biophys acta biomembr 2012; 1818: 2302–2313. https://doi.org/10.1016/j.bbamem.2012.03.019 elbaum-garfinkle s, ramlall t, rhoades e. the role of the lipid bilayer in tau aggregation. biophys j 2010; 98: 2722-2730. https://doi.org/10.1016/j.bpj.2010.03.013 chad a sallaberry ca, voss bj, et al. tau and membranes: interactions that promote folding and condensation. front cell dev biol 2021;9:725241. https://doi.org/10.3389/fcell.2021.725241 ferrer i, andrés-benito p, ausín k, et al. dysregulated protein phosphorylation: a determining condition in the continuum of brain aging and alzheimer’s disease. brain pathol 2021; 31:e12996. https://doi.org/10.1111/bpa.12996 quest af, leyton l, párraga m. caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease. biochem cell biol 2004;82:129-144. https://doi.org/10.1139/o03-071 van helmond zk, scott miners j, bednall e, et al. caveolin-1 and -2 and their relationship to cerebral amyloid angiopathy in alzheimer's disease. neuropathol appl neurobiol 2007;33:317-327. https://doi.org/10.1111/j.1365-2990.2006.00815.x ramírez cm, gonzález m, díaz m, et al. vdac and eralpha interaction in caveolae from human cortex is altered in alzheimer's disease. mol cell neurosci 2009;42:172-183. https://doi.org/10.1016/j.mcn.2009.07.001 puangmalai n, bhatt n, montalbano m, et al. internalization mechanisms of brain-derived tau oligomers from patients with alzheimer's disease, progressive supranuclear palsy and dementia with lewy bodies. cell death dis 2020;11:314. https://doi.org/10.1038/s41419-020-2503-3 de groot ns, burgas mt. is membrane homeostasis the missing link between inflammation and neurodegenerative diseases? cell mol life sci 2015;72:4795-805. https://doi.org/10.1007/s00018-015-2038-4 krigman mr, feldman rg, bensch k. alzheimer’s presenile dementia. a histochemical and electron microscopic study. lab invest 1965;14:381-396. pmid: 14281440. hirai k, aliev g, nunomura a, et al. mitochondrial abnormalities in alzheimer's disease. j neurosci 2001;21:3017-3023. https://doi.org/10.1523/jneurosci.21-09-03017.2001 baloyannis sj. mitochondrial alterations in alzheimer’s disease. j alzheimer’s dis 2006; 9:119-126. https://doi.org/10.3233/jad-2006-9204 chen jx, yan ss. role of mitochondrial amyloid-beta in alzheimer’s disease. j alzheimers dis 2010; 20: 20:s569-578. https://doi.org/10.3233/jad-2010-100357 cheng y, bai f. the association of tau with mitochondrial dysfunction in alzheimer’s disease. front neurosci 2018;12:163. https://doi.org/10.3389/fnins.2018.00163 swerdlow rh, khan sm. a “mitochondrial cascade hypothesis” for sporadic alzheimer’s disease. med hypotheses 2004; 63:8-20. https://doi.org/10.1016/j.mehy.2003.12.045 swerdlow rh. mitochondria and mitochondrial cascades in alzheimer’s disease. j alzheimers dis 2018; 62:1403-1416. https://doi.org/10.3233/jad-170585 hauptmann s, keil u, scherping i, et al. mitochondrial dysfunction in sporadic and genetic alzheimer's disease. exp gerontol 2006;41:668-673. https://doi.org/10.1016/j.exger.2006.03.012 grimm a, friedland k, eckert a. mitochondrial dysfunction: the missing link between aging and sporadic alzheimer's disease. biogerontology 2016;17:281-296. https://doi.org/10.1007/s10522-015-9618-4 wang w, zhao f, ma x, perry g, zhu x. mitochondria dysfunction in the pathogenesis of alzheimer’s disease: recent advances. mol neurodegener 2020;15:30. https://doi.org/10.1186/s13024-020-00376-6 banks b, ingram tl, chakrabarti l. atp synthase and alzheimer's disease: putting a spin on the mitochondrial hypothesis aging (albany ny) 2020;12:16647-16662. https://doi.org/10.18632/aging.103867 terada t, therriault j, kang msp, et al. mitochondrial complex i abnormalities is associated with tau and clinical symptoms in mild alzheimer's disease. mol neurodegener 2021;16:28. https://doi.org/10.1186/s13024-021-00448-1 kawamata h, manfredi g. proteinopathies and oxphos dysfunction in neurodegenerative diseases. j cell biol 2017;216:3917-3929. https://doi.org/10.1083/jcb.201709172 swerdlow rh, burns jm, khan sm. the alzheimer’s disease mitochondrial cascade hypothesis: progress and perspectives. biochim biophys acta mol basis dis 2014; 1842:1219-1231. https://doi.org/10.1016/j.bbadis.2013.09.010 hoekstra jg, hipp mj, montine tj, et al. mitochondrial dna mutations increase in early stage alzheimer disease and are inconsistent with oxidative damage. ann neurol 2016;80:301-306. https://doi.org/10.1002/ana.24709 terni b, boada j, portero-otin m, pamplona r, et al. mitochondrial atp-synthase in the entorhinal cortex is a target of oxidative stress at stages i/ii of alzheimer's disease pathology. brain pathol 2010;20:222-233. https://doi.org/10.1111/j.1750-3639.2009.00266.x busciglio j, pelsman a, wong c, et al. altered metabolism of the amyloid precursor protein is associated with mitochondrial dysfunction in down’s syndrome. neuron 2002;33: 677-688. https://doi.org/10.1016/s0896-6273(02)00604-9 kandimalla r, manczak m, yin x, et al. hippocampal phosphorylated tau induced cognitive decline, dendritic spine loss and mitochondrial abnormalities in a mouse model of alzheimer's disease. hum mol genet 2018;27:30-40. https://doi.org/10.1093/hmg/ddx381 eckert a, schulz kl, rhein v, et al. convergence of amyloid-beta and tau on mitochondria in vivo. mol neurobiol 2010;41:107-114. https://doi.org/10.1007/s12035-010-8109-5 wilkins hm, swerdlow rh. mitochondrial links between brain aging and alzheimer's disease. transl neurodegener 2021;10:33. https://doi.org/10.1186/s40035-021-00261-2 hayashi t, rizzuto r, hajnoczky g, su tp. mam: more than just a housekeeper. trends cell biol 2000;19:81-88. https://doi.org/10.1186/s40035-021-00261-2 eysert f, kinoshita pf, mary a, et al. molecular dysfunctions of mitochondria-associated membranes (mams) in alzheimer's disease. int j mol sci 2020;21:9521. https://doi.org/10.3390/ijms21249521 schon ea, area-gomez e. mitochondria-associated er membranes in alzheimer disease. mol cell neurosci 2013;55:26-36. https://doi.org/10.1016/j.mcn.2012.07.011 area-gomez e, schon e a. mitochondria-associated er membranes and alzheimer disease. curr opin genet dev 2016;38:90-96. https://doi.org/10.1016/j.gde.2016.04.006 area-gomez e, schon ea. on the pathogenesis of alzheimer’s disease: the mam hypothesis. faseb j 2017;31:864-867. https://doi.org/10.1096/fj.201601309 pamplona r. membrane phospholipids, lipoxidative damage and molecular integrity: a causal role in aging and longevity. biochim biophys acta 2008; 1777:1249-1262. https://doi.org/10.1016/j.bbabio.2008.07.003 zhao y, zhao b. oxidative stress and the pathogenesis of alzheimer’s disease. oxid med cell longev 2013; 2013:316523. https://doi.org/10.1155/2013/316523 thomas dd, ridnour l, donzelli s, et al. the chemistry of protein modifications elicited by nitric oxide and related nitrogen oxides. in: redox proteomics: from protein modifications to cellular dysfunction and diseases. dalle-done i, scaloni a, butterfield da (eds). johnwiley & sons, 2006: 25-58. cabré r, naudí a, dominguez-gonzalez m, et al. sixty years old is the breakpoint of human frontal cortex aging. free radic biol med 2017;103:14-22. https://doi.org/10.1016/j.freeradbiomed.2016.12.010 domínguez m, de oliveira e, odena ma, et al. redox proteomic profiling of neuroketal-adducted proteins in human brain: regional vulnerability at middle age increases in the elderly. free radic biol med 2016;95:1-15. https://doi.org/10.1016/j.freeradbiomed.2016.02.034 choi wt, tosun m, jeong hh, et al. metabolomics of mammalian brain reveals regional differences. bmc syst biol 2018;12:127. https://doi.org/10.1186/s12918-018-0644-0 cabré r, jové m, naudí a, et al. specific metabolomics adaptations define a differential regional vulnerability in the adult human cerebral cortex. front mol neurosci 2016;9:138. https://doi.org/10.3389/fnmol.2016.00138 domínguez-gonzález m, puigpinós m, jové m, et al. regional vulnerability to lipoxidative damage and inflammation in normal human brain aging. exp gerontol 2018;111:218-228. https://doi.org/10.1016/j.exger.2018.07.023 srikanth v, maczurek a, phan t, et al. advanced glycation endproducts and their receptor rage in alzheimer's disease. neurobiol aging 2011;32:763-777. https://doi.org/10.1016/j.neurobiolaging.2009.04.016 chambers a, bury jj, minett t, et al. advanced glycation end product formation in human cerebral cortex increases with alzheimer-type neuropathologic changes but is not independently associated with dementia in a population-derived aging brain cohort. j neuropathol exp neurol 2020;79:950-958. https://doi.org/10.1093/jnen/nlaa064 korolainen ma, auriola s, nyman ta, et al. proteomic analysis of glial fibrillary acidic protein in alzheimer’s disease and aging brain. neurobiol dis 2005;20:858-870. https://doi.org/10.1016/j.nbd.2005.05.021 korolainen ma, goldsteins g, nyman ta, et al. oxidative modification of proteins in the frontal cortex of alzheimer’s disease brain. neurobiol aging 2006;27:42-53. https://doi.org/10.1016/j.neurobiolaging.2004.11.010 butterfield da, sultana r. redox proteomics: understanding oxidative stress in the progression of age-related neurodegenerative disorders. expert rev proteomics 2008; 5:157-160. https://doi.org/10.1586/14789450.5.2.157 butterfield da, abdul hm, newman s, et al. redox proteomics in some age-related neurodegenerative disorders or models thereof. neurorx 2006; 3:344-357. https://doi.org/10.1016/j.nurx.2006.05.003 butterfield da, gnjec a, poon hf, et al. redox proteomics identification of oxidatively modified brain proteins in inherited alzheimer’s disease: an initial assessment. j alzheimers dis 2006;10:391-397. https://doi.org/10.3233/jad-2006-10407 butterfield da, poon hf, st clair d, et al. redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of alzheimer’s disease. neurobiol dis 2006;22:223-232. https://doi.org/10.1016/j.nbd.2005.11.002 sultana r, boyd-kimball d, poon hf, et al. redox proteomics identification of oxidized proteins in alzheimer’s disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in ad. neurobiol aging 2006;27:1564-1576. https://doi.org/10.1016/j.neurobiolaging.2005.09.021 sultana r, poon hf, cai j, et al. identification of nitrated proteins in alzheimer’s disease brain using a redox proteomics approach. neurobiol dis 2006;22:76-87. https://doi.org/10.1016/j.nbd.2005.10.004 reed t, perluigi m, sultana r, pierce wm, et al. proteomic identification of 4-hydroxy-2-nonenal modified brain proteins in amnestic mild cognitive impairment: insight into the role of lipid peroxidation in the progression and pathogenesis of alzheimer’s disease. neurobiol dis 2008;30:107-120. https://doi.org/10.1016/j.nbd.2007.12.007. reed tt, pierce wm jr, turner dm, et al. proteomic identification of nitrated brain proteins in early alzheimer’s disease inferior parietal lobule. j cell mol med 2009;13:2019-2029. https://doi.org/10.1111/j.1582-4934.2008.00478.x cabiscol e, ros j. oxidative damage to proteins: structural modifications and consequences in cell function. in: redox proteomics: from protein modifications to cellular dysfunction and diseases. i dalle-donne, a scaloni, da butterfield (eds), john wiley & sons: hoboken, nj. 2006; pp. 399-471. zabel m, nackenoff a, kirsch wm, et al. markers of oxidative damage to lipids, nucleic acids and proteins and antioxidant enzymes activities in alzheimer's disease brain: a meta-analysis in human pathological specimens. free radic biol med 2018;115:351-360. https://doi.org/10.1016/j.freeradbiomed.2017.12.016 nunomura a, perry g, aliev g, et al. oxidative damage is the earliest event in alzheimer disease. j neuropathol exp neurol 2001;60:759-767. https://doi.org/10.1093/jnen/60.8.759 nunomura a, castellani rj, zhu x, et al. involvement of oxidative stress in alzheimer disease. j neuropathol exp neurol 2006;65:631-641. https://doi.org/10.1097/01.jnen.0000228136.58062.bf nunomura a, perry g, pappolla ma, et al. neuronal oxidative stress precedes amyloid-beta deposition in down syndrome. j neuropathol exp neurol 2000;59:1011-1017. https://doi.org/10.1093/jnen/59.11.1011 nunomura a, chiba s, lippa cf, et al. neuronal rna oxidation is a prominent feature of familial alzheimer's disease. neurobiol dis 2004;17:108-113. https://doi.org/10.1016/j.nbd.2004.06.003 smith ma, sayre lm, monnier vm, et al. oxidative posttranslational modifications in alzheimer disease. a possible pathogenic role in the formation of senile plaques and neurofibrillary tangles. mol chem neuropathol 1996;28:41-48. https://doi.org/10.1007/bf02815203 nunomura a, honda k, takeda a, et al. oxidative damage to rna in neurodegenerative diseases. j biomed biotechnol 2006;2006: 82323. https://doi.org/10.1155/jbb/2006/82323 tamagno e, guglielmotto m, vasciaveo v, et al. oxidative stress and betaamyloid in alzheimer's disease. which comes first: the chicken or the egg? antioxidants 2021;10:1479. https://doi.org/10.3390/antiox10091479 wang x, wang w, li l, et al. oxidative stress and mitochondrial dysfunction in alzheimer’s disease. biochim biophys acta 2014; 1842:1240-1247. https://doi.org/10.1016/j.bbadis.2013.10.015 jové m, pradas i, dominguez-gonzalez m, et al. lipids and lipoxidation in human brain aging. mitochondrial atp-synthase as a key lipoxidation target. redox biol 2019;23:101082. https://doi.org/10.1016/j.redox.2018.101082 jové m, mota-martorell n, torres p, et al. the causal role of lipoxidative damage in mitochondrial bioenergetic dysfunction linked to alzheimer's disease pathology. life 2021;11:388. https://doi.org/10.3390/life11050388 ferrer i. altered mitochondria, energy metabolism, voltage-dependent anion channel, and lipid rafts converge to exhaust neurons in alzheimer's disease. j bioenerg biomembr 2009;41:425-431. https://doi.org/10.1007/s10863-009-9243-5 münch g, gasic-milenkovic j, arendt t. effect of advanced glycation endproducts on cell cycle and their relevance for alzheimer's disease. j neural transm suppl 2003;65:63-71. https://doi.org/10.1007/978-3-7091-0643-3_4 yankner ba, lu t, loerch p. the aging brain. annu rev pathol 2008;3:41-66. https://www.doi.org/10.1146/annurev.pathmechdis.2.010506.092044 lucin km, wyss-coray t. immune activation in brain aging and neurodegeneration: too much or too little? neuron 2009;64:110-122. https://doi.org/10.1016/j.neuron.2009.08.039 jenkinson ml, bliss mr, brain at, scott dl. rheumatoid arthritis and senile dementia of the alzheimer’s type. br j rheumatol 1989;28:86-88. https://doi.org/10.1093/rheumatology/28.1.86-b mcgeer pl, mcgeer e, rogers j, sibley j. anti-inflammatory drugs and alzheimer disease. lancet 1990;335:1037. https://doi.org/10.1016/0140-6736(90)91101-f myllykangas-luosujarvi r, isomaki h. alzheimer’s disease and rheumatoid arthritis. br j rheumatol 1994;33:501-502. https://doi.org/10.1093/rheumatology/33.5.501 breitner jc, welsh ka, helms mj, et al. delayed onset of alzheimer’s disease with non-steroidal anti-inflammatory and histamine h2 blocking drugs. neurobiol aging 1995;16:523-530. https://doi.org/10.1016/0197-4580(95)00049-k stewart wf, kawas c, corrada m, et al. risk of alzheimer’s disease and duration of nsaid use. neurology 1997;48:626-632. https://doi.org/10.1212/wnl.48.3.626 mackenzie ira, muñoz dg. nonsteroidal anti-inflammatory drug use and alzheimer-type pathology in aging. neurology 1998;50:986-990. https://doi.org/10.1212/wnl.50.4.986 zandi pp, anthony jc, hayden km, et al. reduced incidence of ad with nsaid but not h2 receptor antagonists: the cache county study. neurology 2002;59:880-886. https://doi.org/10.1212/wnl.59.6.880 vlad sc, miller dr, kowall nw, et al. protective effects of nsaids on the development of alzheimer disease. neurology 2008;70:1672-1677. 10.1212/01.wnl.0000311269.57716.63. terzi m, altun g, şen s, et al. the use of non-steroidal anti-inflammatory drugs in neurological diseases. j chem neuroanat 2018;87:12-24. https://doi.org/10.1016/j.jchemneu.2017.03.003 kaduševičius e. novel applications of nsaids: insight and future perspectives in cardiovascular, neurodegenerative, diabetes and cancer disease therapy. int j mol sci 2021;22:6637. https://doi.org/10.3390/ijms22126637 wilberding a, morimoto k, satoh h, et al. multiple cytokines are involved in the early events leading to the alzheimer's disease pathology.tottori rinsho kagaku kenkyukai shi 2008;1:359-373. pmid: 22586434. sajdel-sulkowska em, marotta ca. alzheimer's disease brain: alterations in rna levels and in a ribonuclease-inhibitor complex. science 1984;225: 947-949. https://doi.org/10.1126/science.6206567 langstrom ns, anderson jp, lindroos hg, et al. alzheimer's disease-associated reduction of polysomal mrna translation. brain res mol brain res 1989;5:259-269. https://doi.org/10.1016/0169-328x(89)90060-0 da silva am, payão sl, borsatto b, et al. quantitative evaluation of rrna in alzheimer’s disease. mech ageing dev 2000;120: 57-64. https://doi.org/10.1016/s0047-6374(00)00180-9 ferrer i. differential expression of phosphorylated translation initiation factor 2 alpha in alzheimer's disease and creutzfeldt-jakob's disease. neuropathol appl neurobiol 2002;28: 441-451. https://doi.org/10.1046/j.1365-2990.2002.t01-1-00410.x shan x, tashiro h, lin cl. the identification and characterization of oxidized rnas in alzheimer's disease. j neurosci 2003;23: 4913-4921. https://doi.org/10.1523/jneurosci.23-12-04913.2003 li x, an wl, alafuzoff i, et al. phosphorylated eukaryotic translation factor 4e is elevated in alzheimer brain. neuroreport 2004;15: 2237-2240. https://doi.org/10.1097/00001756-200410050-00019 nunomura a, perry g. rna and oxidative stress in alzheimer's disease: focus on micrornas. oxid med cell longev 2020;2020:2638130. https://doi.org/10.1155/2020/2638130 dönmez-altuntas h, akalain h, karaman y, et al. evaluation of the nucleolar organizer regions in alzheimer’s disease. gerontology 2005;51: 297-301. https://doi.org/10.1159/000086365 honda k, smith ma, zhu x, et al. ribosomal rna in alzheimer disease is oxidized by bound redox-active iron.j biol chem 2005;280:20978-20986. https://doi.org/10.1074/jbc.m500526200 ding q, markesbery wr, chen q, et al. ribosome dysfunction is an early event in alzheimer’s disease. j neurosci 2005;25: 9171-9175. https://doi.org/10.1523/jneurosci.3040-05.2005 ding q, markesbery wr, cecarini v, et al. decreased rna, and increased rna oxidation, in ribosomes from early alzheimer’s disease. neurochem res 2006;31:705-710. https://doi.org/10.1007/s11064-006-9071-5 shan x, lin cl. quantification of oxidized rnas in alzheimer's disease. neurobiol aging 2006 27: 657-662. https://doi.org/10.1016/j.neurobiolaging.2005.03.022 eftekharzadeh b, daigle jg, kapinos le, et al. tau protein disrupts nucleocytoplasmic transport in alzheimer disease. neuron 2018;99:925-940.e7. https://doi.org/10.1016/j.neuron.2018.07.039 hernández-ortega k, garcia-esparcia p, gil l, et al. altered machinery of protein synthesis in alzheimer’s: from the nucleolus to the ribosome. brain pathol 2016;26: 593-605. https://doi.org/10.1111/bpa.12335 di domenico f, sultana r, barone e, et al. quantitative proteomics analysis of phosphorylated proteins in the hippocampus of alzheimer's disease subjects. j proteomics 2011;74:1091-1103. https://doi.org/10.1016/j.jprot.2011.03.033 xia q, cheng d, duong dm, et al. phosphoproteomic analysis of human brain by calcium phosphate precipitation and mass spectrometry. j proteome res 2008;7:2845-2851. https://doi.org/10.1021/pr8000496 tan h, wu z, wang h, et al. refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome. proteomics 2015;15:500-507. https://doi.org/10.1002/pmic.201400171 triplett jc, swomley am, cai j, et al. quantitative phosphoproteomic analyses of the inferior parietal lobule from three different pathological stages of alzheimer's disease. j alzheimers dis 2016;49:45-62. https://doi.org/10.3233/jad-150417 dammer eb, lee ak, duong dm, et al. quantitative phosphoproteomics of alzheimer's disease reveals cross-talk between kinases and small heat shock proteins. proteomics 2015;15:508-519. https://doi.org/10.1002/pmic.201400189 sathe g, mangalaparthi kk, jain a, et al. multiplexed phosphoproteomic study of the brain in patients with alzheimer's disease and age-matched cognitively healthy controls. omics 2020;24:2016-2227. https://doi.org/10.1089/omi.2019.0191 bai b, wang x, li y, et al. deep multilayer brain proteomics identifies molecular networks in alzheimer's disease progression. neuron 2020;105: 975-991.e7. https://doi.org/10.1016/j.neuron.2019.12.015 mérida i, avila-flores a, merino e. diacylglycerol kinases: at the hub of cell signalling. biochem j 2008;409:1-18. https://doi.org/10.1042/bj20071040 almena m, mérida i. shaping up the membrane: diacylglycerol coordinates spatial orientation of signalling. trends biochem sci 2011;36:593-603. https://doi.org/10.1016/j.tibs.2011.06.005 sakane f, hoshino f, murakami c. new era of diacylglycerol kinase, phosphatidic acid and phosphatidic acid-binding protein. int j mol sci 2020;21:6794. https://doi.org/10.3390/ijms21186794 ekinci fj, shea tb. free pkc catalytic subunits (pkm) phosphorylate tau via a pathway distinct from that utilized by intact pkc. brain res 1999;850:207-216. https://doi.org/10.1016/s0006-8993(99)02146-0 chan rb, oliveira tg, cortes ep, et al. comparative lipidomic analysis of mouse and human brain with alzheimer disease. j biol chem 2012;287:2678-2688. https://doi.org/10.1074/jbc.m111.274142 wood pl, medicherla s, sheikh n, et al. targeted lipidomics of frontal cortex and plasma diacylglycerols (dag) in mild cognitive impairment and alzheimer's disease: validation of dag accumulation early in the pathophysiology of alzheimer's disease. j alzheimers dis 2015;48:537-546. https://doi.org/10.3233/jad-150336 pinheiro l, faustino c. therapeutic strategies targeting amyloid-β in alzheimer's disease. curr alzheimer res 2019;16:418-452. https://doi.org/10.2174/1567205016666190321163438 icoll ja, wilkinson d, holmes c, et al. neuropathology of human alzheimer disease after immunization with amyloid-beta peptide: a case report. nat med 2003;9: 448-452. https://doi.org/10.1038/nm840 nicoll jar, buckland gr, harrison ch, et al. persistent neuropathological effects 14 years following amyloid-β immunization in alzheimer's disease. brain 2019; 142: 2113-2126. https://doi.org/10.1093/brain/awz142 loureiro jc, pais mv, stella f, et al. passive antiamyloid immunotherapy for alzheimer’s disease. curr opin psychiatry 2020; 33: 284-291. https://doi.org/10.1097/yco.0000000000000587 honig ls, vellas b, woodward m, et al. trial of solanezumab for mild dementia due to alzheimer’s disease. n engl j med 2018; 378, 321-330. https://doi.org/10.1056/nejmoa1705971 van bokhoven p, de wilde a, vermunt l, et al. the alzheimer’s disease drug development landscape. alzheimers res ther 2021;13:186. https://doi.org/10.1186/s13195-021-00927-z abushouk ai, elmaraezy a, aglan a, et al. bapineuzumab for mild to moderate alzheimer’s disease: a meta-analysis of randomized controlled trials. bmc neurol 2017;17:66. https://doi.org/10.1186/s12883-017-0850-1 ratti e, kong j, o’gorman j, rajagovindan r, et al. baseline characteristics from tango: phase 2 study to evaluate gosuranemab (biib092) in patients with early alzheimer’s disease. alzheimers dement 2020;16, e044910. walsh s, merrick r, milne r, et al. aducanumab for alzheimer’s disease? bmj 2021;374: n1682. https://doi.org/10.1136/bmj.n1682 querol-vilaseca m, sirisi ms, molina-porcel l, et al. neuropathology of a patient with alzheimer disease treated with low doses of verubecestat. neuropathol appl neurobiol 2022;48:e12781. https://doi.org/10.1111/nan.12781 han p, shi j.a. theoretical analysis of the synergy of amyloid and tau in alzheimer's disease. j alzheimers dis 2016;52:1461-70. https://doi.org/10.3233/jad-151206 sherman ma, lacroix m, amar f, et al. soluble conformers of aβ and tau alter selective proteins governing axonal transport. j neurosci 2016;36:9647-9658. https://doi.org/10.1523/jneurosci.1899-16.2016 kim cm, montal v, diez i, et al. network interdigitations of tau and amyloid-beta deposits define cognitive levels in aging. hum brain mapp 2021;42:2990-3004. https://doi.org/10.1002/hbm.25350 braczynski ak, schulz jb, bach jp. vaccination strategies in tauopathies and synucleinopathies. j neurochem 2017;143:467-488. https://doi.org/10.1111/jnc.14207 congdon ee, sigurdson em. tau-targeting therapies for alzheimer’s disease. nature rev 2018;14: 399-415. https://doi.org/10.1038/s41582-018-0013-z medina m. an overview on the clinical development of tau-based therapies. int j mol sci 2018;19:1160. https://doi.org/10.3390/ijms19041160 jadhav s, avila j, schöll m, et al. a walk through tau therapeutic strategies. acta neuropathol commun 2019;7:22. https://doi.org/10.1186/s40478-019-0664-z mullard a. failure of first anti-tau antibody in alzheimer disease highlights risks of history repeating. nat rev drug discov 2020;20:3-5. https://doi.org/10.1038/d41573-020-00217-7 loera-valencia r, piras a, ismail mam, et al.targeting alzheimer’s disease with gene and cell therapies. j intern med 2018; 284:2-36. https://doi.org/10.1111/joim.12759 dunbar ce, high ka, joung jk, et al. gene therapy comes of age. science 2018; 359:eaan4672. https://doi.org/10.1126/science.aan4672 lennon mj, rigney g, raymont v, et al. genetic therapies for alzheimer’s disease: a scoping review. j alz dis 2021; 84: 491-504. https://doi.org/10.3233/jad-215145 tuszynski mh, thal l, pay m, et al. a phase 1 clinical trial of nerve growth factor gene therapy for alzheimer disease. nat med 2005; 11: 551-555. https://doi.org/10.1038/nm1239 eriksdotter-jönhagen m, linderoth b, lind g, et al. encapsulated cell biodelivery of nerve growth factor to the basal forebrain in patients with alzheimer’s disease. dement geriatr cogn disord 2012;33:18-28. https://doi.org/10.1159/000336051 rafii ms, tuszynski mh, thomas rg, et al. adeno-associated viral vector (serotype 2)-nerve growth factor for patients with alzheimer disease: a randomized clinical trial. jama neurol 2018; 75: 834-841. https://doi.org/10.1001/jamaneurol.2018.0233 huang lk, chao sp, hu cj. clinical trials of new drugs for alzheimer disease. j biomed sci 2020; 27:18. https://doi.org/10.1186/s12929-019-0609-7 cummings j, lee g, zhong k, fonseca j, et al. alzheimer’s disease drug development pipeline: 2021. alzheimers dement 2021;7:e12179. https://doi.org/10.1002/trc2.12179 mcdade e, llibre-guerra jj, holtzman dm, et al. the informed road map to prevention of alzheimer disease: a call to arms. mol neurodegener 2021;16:49. https://doi.org/10.1186/s13024-021-00467-y cummings j. disease modification is not all — we need symptomatic therapies for alzheimer disease. nature rev neurol 2022;18:3-4. https://doi.org/10.1038/s41582-021-00591-9 gotz j, bodea lg, goedert m. rodent models for alzheimer disease. nat rev neurosci 2018;9:583-598. https://doi.org/10.1038/s41583-018-0054-8 vitek mp, araujo ja, fossel m, et al. translational animal models for alzheimer's disease: an alzheimer's association business consortium think tank. alzheimers dement 2021;6:e12114. https://doi.org/10.1002/trc2.12114 mckean ne, handley rr, snell rg. a review of the current mammalian models of alzheimer’s disease and challenges that need to be overcome. int j mol sci 2021;22:13168. https://doi.org/10.3390/ijms222313168 arendt t. alzheimer's disease as a disorder of dynamic brain self-organization. prog brain res 2005;147:355-378. https://doi.org/10.1016/s0079-6123(04)47025-3 swerdlow rh. alzheimer's disease pathologic cascades: who comes first, what drives what. neurotox res 2012;22:182-194. https://doi.org/10.1007/s12640-011-9272-9 sharpee to, destexhe a, kawato m, et al. 25th annual computational neuroscience meeting: cns-2016. bmc neurosci 2016;17 suppl 1:54. https://doi.org/10.1186/s12868-016-0283-6 bashtrykov p, jeltsch a. epigenome editing in the brain. adv exp med biol 2017;978:409-424. https://doi.org/10.1007/978-3-319-53889-1_21 mancioppi g, fiorini l, timpano sportiello m, et al. novel technological solutions for assessment, treatment, and assistance in mild cognitive impairment. front neuroinform 2019;13:58. https://doi.org/10.3389/fninf.2019.00058 romanella sm, roe d, paciorek r, et al. sleep, noninvasive brain stimulation, and the aging brain: challenges and opportunities. ageing res rev 2020;61:101067. https://doi.org/10.1016/j.arr.2020.101067 el-atab n, shaikh sf, hussain mm. nano-scale transistors for interfacing with brain: design criteria, progress and prospect. nanotechnology 2019;30:442001. https://doi.org/10.1088/1361-6528/ab3534 koutentakis d, pilozzi a, huang x. designing socially assistive robots for alzheimer’s disease and related dementia patients and their caregivers: where we are and where we are headed. healthcare 2020;8:73. https://doi.org/10.3390/healthcare8020073 ling ts, chandrasegaran s, xuan lz, et al. the potential benefits of nanotechnology in treating alzheimer's disease. biomed res int 2021;2021:5550938. https://doi.org/10.1155/2021/5550938 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.