The Role of the Gut Microbiome in Neuromodulation Therapies as a Potential Treatment Adjunct for Multiple Sclerosis Abstract Berkeley Pharma Tech Journal of Medicine Correspondence: jwlarrick@gmail.com Keywords: Chloroquine, hydrochloroquine, autophagy, aging, geroprotection Submitted: March 10, 2023 Accepted: April 6, 2023 Published: June 30, 2023 Full Open Access Creative Commons Attribution License 4.0 Autophagy, the turnover of cellular components including organelles, declines with age. Thus, enhancement of this characteristic process is hypothesized to improve health and extend lifespan. Two recent papers present data indicating that contrary to expectation, chloroquine (CQ), a nominal inhibitor of autophagy, extended the lifespan of middle-aged mice and rats by ~10%. Details of these studies provide a cautionary tale regarding traditional reagents or “tool compounds” of “established” mechanisms often used in cellular biological research. However, these and earlier studies support a deeper investigation of CQ or its more commonly used clinical analog, hydroxychloroquine (HCQ), as potential drugs to increase health span and slow the aging process. Chloroquine defeats aging? By: James W. Larrick, M.D. Ph.D. and Jasmine W. Larrick Introduction 1.1 Autophagy Autophagy, literally “self-eating," was �rst described by Christian De Duve, who shared the 1974 Nobel prize for his discovery of lysosomes. Autophagy is an evolutionarily conserved catabolic process wherein lysosomes degrade various cellular components to maintain cytoplasmic quality control and thus cellular homeostasis. Several distinct forms of autophagy include macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Dysregulation of this critical process has been associated with many pathological processes including infectious, metabolic, and neurodegenerative disorders, as well as cancer and autoimmunity. Reduced autophagy was identi�ed by López-Otín et al. (2013) as a so-called "Hallmark of aging". 1.2 Chloroquine Native peoples of South America have long used an extract of the bark of the Cinchona tree ( Cinchona officinalis ) as a remedy for fevers. By the 1600s, this herbal medicine was being used in Europe for treatment of malaria. Quinine was isolated from the bark in 1820. Seeking an alternative to quinine, Bayer chemists discovered chloroquine, a synthetic analog with a similar mechanism of action. A related analogue, 3-methyl-chloroquine, was utilized by the Germans in World War II. Material captured by the Americans in North Africa was analyzed by the United States government, resulting in corroborative studies and subsequent clinical approval in 1947 of chloroquine as a prophylactic treatment for malaria. While chloroquine and its derivative hydroxychloroquine are widely used in rheumatology (SLE, RA, etc.) 1 , chloroquine-resistant malaria has mandated the development of newer antimalarials such as pyrimethamine, artemisinin and me�oquine 2 . Chloroquine inhibits hemozoin formation from the heme released by parasitic digestion of erythrocyte hemoglobin during Plasmodium infection. The free heme then lyses membranes and leads to parasite death 3, 4, 5 . Chloroquine passively di�uses through cell membranes and into Berkeley Pharma Tech Journal of Medicine | 2 endosomes, lysosomes, and Golgi vesicles which become protonated, trapping the chloroquine in the organelles and raising the surrounding pH. The raised pH in the endosomal compartments (a so-called lysosomotropic e�ect), inhibits viral infectivity and various cellular processes such as autophagy. Geroprotective activities of chloroquine Li et al. reported the geroprotective e�ects of low-dose CQ on aged rats 6 . These studies were inspired by a perspective article published at the height of the COVID-19 pandemic by Sargiacomo et al. in 2020 7 . This British group sought to explain the considerably higher mortality rate in COVID-19 patients with advanced chronological age prior to the development of safe vaccines. They pointed out that the proposed SARS-Cov-2 viral receptors, CD26 and ACE2, were associated with senescence 8, 9 . Furthermore, at the time, azithromycin, quercetin, and HCQ were proposed as "o�-the-shelf" treatments for SARS-CoV2 infection 10 . Quercetin has a senolytic activity 11 and HCQ is known to inhibit beta-galactosidase, a marker of senescence 12 . Although early and questionable trials demonstrated some bene�t of HCQ 13 for SARS-CoV2, subsequent well-controlled trials demonstrated limited, if any, bene�t 14, 15 . However, it is important to note that investigation of the role of the autophagy pathway in viral pathogenesis continues 16 . To investigate the geroprotective e�ects of HCQ, Li et al. treated 24-month-old Sprague Dawley male rats (n = 9) with CQ (0.1 mg/kg, in drinking water) twice weekly for 5 months 6 . Compared to controls (n = 13), the treated animals exhibited a 6% increase in medial survival and 13% increase in maximum lifespan (p = 0.02) 6 . Related to the work of Li et al., Doeppner et al. (2022) also reported a chloroquine-mediated increase in the lifespan of rodents, in this case mice 17 . Work by Fifan et al. 18 and others 19, 20 demonstrated that the polyamine spermidine increased the maximum life span of C. elegans and the median life span of mice (~10%). Because spermidine increases autophagy, they hypothesized that treatment with chloroquine, an inhibitor of autophagy, would shorten the lifespan of mice. Remarkably, addition of chloroquine (50 mg/kg) to the drinking water extended overall lifespan of middle-aged Berkeley Pharma Tech Journal of Medicine | 3 male NMRI mice (n = 28; treatment initiated at age 500 days) by 11.4% (786 days) compared to control mice (n = 28; 689 days, p = 0.0002). Median life span of the middle-aged mice increased by 11.4%. Studies of chloroquine by these two groups and other data dating back a couple decades suggest the e�ect is real, although the precise mechanism is not clear. Mechanistic studies carried out by both groups suggest a variety of mechanisms, though this "ball of yarn" is far from being unraveled. Mechanism studies As noted above, CQ treatment was initiated at age 500 days when the mice weighed on average ~35 gm. Over the next 100 days, the control animals gained weight, reaching a maximum of 45 gm. In contrast, the CQ-treated animals did not gain signi�cant weight over the duration of the experiment (almost 800 days). While the control animals consumed more liquid (p = 0.002), the food consumed by both groups was equal (~4 gm/day) ruling out the idea that CQ somehow reduced food intake through an arti�cial "calorie restriction" e�ect. Previously, CQ (at 60 mg/kg) was reported to impair autophagosome-lysosome fusion rather than a�ecting the acidity and/or degradative activities of lysosomes 21 . In addition, HCQ treatment was associated with "autophagy-independent" disorganization of the Golgi and endo-lysosomal systems, with predominant Golgi disorganization seen in kidney and intestinal tissues. The microtubule-associated protein I light chain (LC3) family of proteins (LC3A, B, C) 22 is the major structural protein family of autophagosome membranes. LC3-II is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine on the surface of nascent autophagosomes. Doeppner et al. showed that the CQ-treated mice exhibited a dose-dependent increase in LC3B-II as well as p62 in the liver and heart, as con�rmed by transmission electron microscopy 17 . Curiously, the treated animals exhibited increased liver glycogen and reduced serum insulin growth factor binding protein 3 (IGFBP3), though no di�erence in IGF-1, IRS, or growth hormone levels 23, 24 . CQ treatment elicited a decrease in glycogenolysis in the liver. Berkeley Pharma Tech Journal of Medicine | 4 IGFBP3 binds IGF1 and IGF2 to modulate their binding to the IGF-1 receptor. While IGFBP3 levels, like IGFs, are regulated by GH, expression in the liver is GH-independent 25 . IGFB3 mediates a plethora of other activities via its binding to a number of proteins and its interaction with various cell surface and nuclear signaling pathways. Several labs have demonstrated a bene�cial role for reduced IGFB3 in senescence 26, 27, 28 . Li et al. found that low-dose CQ (0.1 mg/kg given twice a week) extended the lifespan of aged rats when given in late middle age 6 . This dose is ~100-fold lower than doses typically given as a putative autophagy inhibitor in cancer 29 . Qian et al. attributed the geroprotective e�ect of CQ to ATM activation leading to enhanced DNA damage repair within their C. elegans and progeria mouse strain models 30 . Others have shown that activation of ATM by CQ can slow atherosclerosis, improve insulin sensitivity, and rescue glucose intolerance in type 2 diabetes (T2D) 31, 32, 33 . Li et al. studied the transcriptomes from CQ-treated and control old rats to de�ne CQ-induced, di�erentially expressed genes so-called “CQ DEGs” across multiple tissues. For example, 40% of kidney-speci�c aged genes, 30% of small intestine-speci�c aged genes, and 20% of liver-speci�c aged genes were "rescued" by CQ treatment. These changes were consistent with the reduced �brosis and improved histology of the kidneys compared to other tissues. Contrary to expectation, CQ treatment augmented a number of genes associated with various cardiac diseases (e.g. heart failure, cardiac arrhythmias, ischemic cardiomyopathy (Caps2), hypertrophic cardiomyopathy, and cardiac arrhythmias (Myh7). Thus, CQ treatment may actually mediate pro-aging activity in certain tissues 6 . Summary A number of labs have studied several animal species to generate data suggesting that CQ can mediate a pro-longevity e�ect. Mechanistic studies indicate that multiple mechanisms are at play, each addressing a hallmark of aging 34 . While CQ and HCQ are familiar to cell biologists as prototypic Berkeley Pharma Tech Journal of Medicine | 5 lysosomotropic e�ect tool compounds, numerous diverse studies highlight the plethora of other activities mediated by CQ. Isolated papers over the past 20 years have identi�ed non-autophagy-mediated e�ects of CQ. For example, CQ improves vasculogenesis 35 , insulin metabolism 31 , �brosis 36 , reduces oxidative stress 37 , modulates autophagic �ux 21 , boosts DNA repair via STM 30 , and attenuates atherogenesis 33 to name a few. When we began looking into this story, we sought a unifying mechanism, pathway, target, etc. to explain the increased lifespans. Unfortunately, at the present time, descriptive studies nominate many parallel mechanisms. The detailed transcriptome studies of Li et al. show quite variable tissue speci�c patterns, with some that are bene�cial for health span and others that are consistent with cardiac diseases 6 . Of course, investigating the geroprotective e�ects of CQ using Horvath or other methylation clocks might lead to interesting �ndings. Medical Implications As noted above, CQ and HCQ are widely used drugs 38 . The studies of Li et al. and Doeppner et al. support further, possibly human studies of these drugs. On October 8–13, 2013, a workshop entitled ‘Interventions to Slow Aging in Humans: Are We Ready?’ was held in Erice, Italy 39 . At the time, a number of interventions were deemed worthy of human clinical trials. Since then, some of these ideas have been put to the test or are being tested . With regard to development of CQ/HCQ, suitable biomarkers will need to be validated. At low doses, CQ or HCQ seem to qualify for evaluation in humans. However, the safety pro�le of this class of drugs will need to be considered. For instance, periodic eye exams and cardiac monitoring are routine 40 at doses commonly used in rheumatology 38 . Clearly these anti-malarials have come a long way from the rainforests of South America! Berkeley Pharma Tech Journal of Medicine | 6 References 1. Plantone D, Koudriavtseva T. Current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: A mini-review. Clinical Drug Investigation . 2018;38(8):653-671. doi:10.1007/s40261-018-0656-y 2. Rasmussen C, Alonso P, Ringwald P. Current and emerging strategies to combat antimalarial resistance. Expert Review of Anti-infective Therapy . 2021;20(3):353-372. doi:10.1080/14787210.2021.1962291 3. Chou AC, Fitch CD. Heme polymerase: Modulation by chloroquine treatment of a rodent malaria. Life Sciences . 1992;51(26):2073-2078. doi:10.1016/0024-3205(92)90158-l 4. Slater AF, Cerami A. Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. Nature . 1992;355(6356):167-169. doi:10.1038/355167a0 5. Coronado LM, Nadovich CT, Spadafora C. Malarial hemozoin: From target to tool. Biochimica et Biophysica Acta (BBA) - General Subjects . 2014;1840(6):2032-2041. doi:10.1016/j.bbagen.2014.02.009 6. Li W, Zou Z, Cai Y, et al. Low-dose chloroquine treatment extends the lifespan of aged rats. Protein & Cell . 2022;13(6):454-461. doi:10.1007/s13238-021-00903-1 7. Sargiacomo C, Sotgia F, Lisanti MP. Covid-19 and Chronological Aging: Senolytics and other anti-aging drugs for the treatment or prevention of Corona virus infection? Aging . 2020;12(8):6511-6517. doi:10.18632/aging.103001 8. Kim KM, Noh JH, Bodogai M, et al. Identi�cation of senescent cell surface targetable protein DPP4. Genes & Development . 2017;31(15):1529-1534. doi:10.1101/gad.302570.117 9. Guy JL, Lambert DW, Turner AJ, Porter KE. Functional angiotensin-converting enzyme 2 is expressed in human cardiac myo�broblasts. Experimental Physiology . 2008;93(5):579-588. doi:10.1113/expphysiol.2007.040139 10. Cavalcante MB, Saccon TD, Nunes ADC, et al. Dasatinib plus quercetin prevents uterine age-related dysfunction and �brosis in mice. Aging . 2020;12(3):2711-2722. doi:10.18632/aging.102772 11. Zoico E, Nori N, Darra E, et al. Senolytic e�ects of quercetin in an in vitro model of pre-adipocytes and adipocytes induced senescence. Scientific Reports . 2021;11(1). doi:10.1038/s41598-021-02544-0 12. Kurz DJ, Decary S, Hong Y, Erusalimsky JD. Senescence-associated (beta)-galactosidase re�ects an increase in lysosomal mass during replicative ageing of human endothelial cells. Journal of Cell Science . 2000;113(20):3613-3622. doi:10.1242/jcs.113.20.3613 13. Gautret P, Lagier J-C, Parola P, et al. Hydroxychloroquine and azithromycin as a treatment of COVID-19: Results of an open-label non-randomized clinical trial. International Journal of Antimicrobial Agents . 2020. doi:10.1101/2020.03.16.20037135 14. Boulware DR, Pullen MF, Bangdiwala AS, et al. A randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19. New England Journal of Medicine . 2020;383(6):517-525. doi:10.1056/nejmoa2016638 15. Tang W, Cao Z, Han M, et al. Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: Open label, Randomised Controlled Trial. BMJ . 2020:m1849. doi:10.1136/bmj.m1849 16.He W, Gao Y, Zhou J, Shi Y, Xia D, Shen H-M. Friend or foe? implication of the autophagy-lysosome pathway in SARS-COV-2 infection and COVID-19. International Journal of Biological Sciences . 2022;18(12):4690-4703. doi:10.7150/ijbs.72544 Berkeley Pharma Tech Journal of Medicine | 7 17. Doeppner TR, Coman C, Burdusel D, et al. Long-term treatment with chloroquine increases lifespan in middle-aged male mice possibly via autophagy modulation, proteasome inhibition and glycogen metabolism. Aging . 2022;14(10):4195-4210. doi:10.18632/aging.204069 18. Filfan M, Olaru A, Udristoiu I, et al. Long-term treatment with spermidine increases health span of middle-aged Sprague-Dawley male rats. GeroScience. 2020;42(3):937-949. doi:10.1007/s11357-020-00173-5 19. Eisenberg T, Knauer H, Schauer A, et al. Induction of autophagy by spermidine promotes longevity. Nature Cell Biology . 2009;11(11):1305-1314. doi:10.1038/ncb1975 20. Eisenberg T, Abdellatif M, Schroeder S, et al. Cardioprotection and lifespan extension by the natural polyamine spermidine. Nature Medicine . 2016;22(12):1428-1438. doi:10.1038/nm.4222 21. Mauthe M, Orhon I, Rocchi C, et al. Chloroquine inhibits autophagic �ux by decreasing autophagosome-lysosome fusion. Autophagy . 2018;14(8):1435-1455. doi:10.1080/15548627.2018.1474314 22. Yoshii SR, Mizushima N. Monitoring and measuring autophagy. International Journal of Molecular Sciences . 2017;18(9):1865. doi:10.3390/ijms18091865 23. Brown-Borg HM, Bartke A. GH and igf1: Roles in energy metabolism of long-living GH mutant mice. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences . 2012;67A(6):652-660. doi:10.1093/gerona/gls086 24. Junnila RK, List EO, Berryman DE, Murrey JW, Kopchick JJ. The GH/IGF-1 axis in ageing and longevity. Nature Reviews Endocrinology . 2013;9(6):366-376. doi:10.1038/nrendo.2013.67 25. Olivecrona H, Hilding A, Ekström Christina, et al. Acute and short-term e�ects of growth hormone on insulin-like growth factors and their binding proteins: Serum levels and hepatic messenger ribonucleic acid responses in humans1. The Journal of Clinical Endocrinology & Metabolism . 1999;84(2):553-560. doi:10.1210/jcem.84.2.5466 26. Hong S, Kim M-M. IGFBP-3 plays an important role in senescence as an aging marker. Environmental Toxicology and Pharmacology . 2018;59:138-145. doi:10.1016/j.etap.2018.03.014 27. Hong S, Kim M-M. IGFBP-3 plays an important role in senescence as an aging marker. Environmental Toxicology and Pharmacology . 2018;59:138-145. doi:10.1016/j.etap.2018.03.014 28. Yamada PM, Mehta HH, Hwang D, Roos KP, Hevener AL, Lee KW. Evidence of a role for insulin-like growth factor binding protein (IGFBP)-3 in metabolic regulation. Endocrinology . 2010;151(12):5741-5750. doi:10.1210/en.2010-0672 29. Solomon VR, Lee H. Chloroquine and its analogs: A new promise of an old drug for e�ective and safe cancer therapies. European Journal of Pharmacology . 2009;625(1-3):220-233. doi:10.1016/j.ejphar.2009.06.063 30. Qian M, Liu Z, Peng L, et al. Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria. eLife . 2018;7. doi:10.7554/elife.34836 31. Emami J, Gerstein HC, Pasutto FM, Jamali F. Insulin-sparing e�ect of hydroxychloroquine in diabetic rats is concentration dependent . Canadian Journal of Physiology and Pharmacology . 1999;77(2):118-123. doi:10.1139/y98-146 32. Schneider JG, Finck BN, Ren J, et al. ATM-dependent suppression of stress signaling reduces vascular disease in metabolic syndrome. Cell Berkeley Pharma Tech Journal of Medicine | 8 Metabolism . 2006;4(5):377-389. doi:10.1016/j.cmet.2006.10.002 33. Razani B, Feng C, Semenkovich CF. P53 is required for chloroquine-induced atheroprotection but not insulin sensitization. Journal of Lipid Research . 2010;51(7):1738-1746. doi:10.1194/jlr.m003681 34. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194-1217. doi:10.1016/j.cell.2013.05.039 35. Maes H, Kuchnio A, Peric A, et al. Tumor vessel normalization by chloroquine independent of autophagy. Cancer Cell . 2014;26(2):190-206. doi:10.1016/j.ccr.2014.06.025 36. He W, Wang B, Yang J, et al. Chloroquine improved carbon tetrachloride-induced liver �brosis through its inhibition of the activation of hepatic stellate cells: Role of autophagy. Biological and Pharmaceutical Bulletin . 2014;37(9):1505-1509. doi:10.1248/bpb.b14-00297 37. Shen H, Wu N, Wang Y, et al. Chloroquine attenuates paraquat-induced lung injury in mice by altering in�ammation, oxidative stress and �brosis. International Immunopharmacology . 2017;46:16-22. doi:10.1016/j.intimp.2017.02.020 38. Schrezenmeier E, Dörner T. Mechanisms of action of hydroxychloroquine and chloroquine: Implications for rheumatology. Nature Reviews Rheumatology. 2020;16(3):155-166. doi:10.1038/s41584-020-0372-x 39. Longo VD, Antebi A, Bartke A, et al. Interventions to Slow Aging in Humans: Are We Ready?. Aging Cell . 2015;14(4):497-510. doi:10.1111/acel.12338 40. Wozniacka A, Cygankiewicz I, Chudzik M, Sysa-Jędrzejowska A, Wranicz JK. The cardiac safety of chloroquine phosphate treatment in patients with systemic lupus erythematosus: The in�uence on arrhythmia, heart rate variability and repolarization parameters. Lupus . 2006;15(8):521-525. doi:10.1191/0961203306lu2345oa Berkeley Pharma Tech Journal of Medicine | 9