Distinctive alteration in the expression of autophagy genes in Drosophila models of amyloidopathy and tauopathy

ORIGINAL ARTICLE

Distinctive alteration in the expression of autophagy genes in Drosophila
models of amyloidopathy and tauopathy

Mehrnaz Haghia, Raheleh Masoudia and Seyed Morteza Najibib,c

aDepartment of Biology, College of Sciences, Shiraz University, Shiraz, Iran; bCenter for Molecular Protein Science, Lund University, Lund,
Sweden; cDepartment of Statistics, College of Sciences, Shiraz University, Shiraz, Iran

ABSTRACT
Background: Alzheimer’s disease (AD) is one the most common types of dementia. Plaques of amyl-
oid beta and neurofibrillary tangles of tau are two major hallmarks of AD. Metabolism of these two
proteins, in part, depends on autophagy pathways. Autophagy dysfunction and protein aggregation in
AD may be involved in a vicious circle. The aim of this study was to investigate whether tau or amyl-
oid beta 42 (Ab42) could affect expression of autophagy genes, and whether they exert their effects
in the same way or not.
Methods: Expression levels of some autophagy genes, Hook, Atg6, Atg8, and Cathepsin D, were meas-
ured using quantitative PCR in transgenic Drosophila melanogaster expressing either Ab42 or
Tau R406W.
Results: We found that Hook mRNA levels were downregulated in Ab42-expressing flies both 5 and
25 days old, while they were increased in 25-day-old flies expressing Tau R406W. Both Atg6 and Atg8
were upregulated at day 5 and then downregulated in 25-day-old flies expressing either Ab42 or Tau
R406W. Cathepsin D expression levels were significantly increased in 5-day-old flies expressing Tau
R406W, while there was no significant change in the expression levels of this gene in 5-day-old flies
expressing Ab42. Expression levels of Cathepsin D were significantly decreased in 25-day-old transgenic
flies expressing Tau R406W or Ab42.
Conclusion: We conclude that both Ab42 and Tau R406W may affect autophagy through dysregula-
tion of autophagy genes. Interestingly, it seems that these pathological proteins exert their toxic
effects on autophagy through different pathways and independently.

ARTICLE HISTORY
Received 6 January 2020
Revised 11 June 2020
Accepted 16 June 2020

KEYWORDS
Alzheimer’s disease;
amyloid beta; autophagy
genes; Drosophila
melanogaster; tau

Introduction

Alzheimer’s disease (AD) is characterized by progressive mem-
ory impairment and dementia. This neurodegenerative disease
is also one of the main causes of death in the elderly popula-
tion (1). Neurofibrillary tangles (intracellular inclusion of hyper-
phosphorylated tau) and Ab plaques (extracellular inclusion of
Ab42 peptide) are two major markers in the brain of AD
patients (2). The mechanism underlying AD pathology is not
fully understood. Some hypotheses point to these aggrega-
tions as the reason for developing AD symptoms. On the
other hand, recent investigations have shown that aggregate
formation is a defense mechanism against soluble and aggres-
sive forms of Ab and hyperphosphorylated tau (3,4).

Although these aggregates may exert protection against
their soluble oligomer forms, they block axonal/dendritic trans-
port (5) and cause damage to mitochondrial complexes (6,7),
leading to increased levels of reactive oxygen species (ROS) in
the brain of AD patients. Autophagy (self-eating) is one of the
pathways responsible for clearing these aggregations in order
to prevent their accumulation (8). To eliminate the aggregates,

they are transferred via a dynein/dynactin motor complex to a
perinuclear aggresome. Then, they are encapsulated by neuro-
filaments, and the aggresome is formed. Ultimately, aggregates
are cleared when the aggresome is fused to a lysosome (9).
Hook protein is an adaptor facilitating the association of motor
complex with its cargos (10).

Many stimuli can trigger the initiation of autophagy by
ULK1/Atg1 protein kinase complex (11). This serine/threonine
kinase phosphorylates Beclin1/Atg6 in the Beclin1-VPS34 PI3
kinase complex involved in nucleation of autophagosome
formation (phagophore). Following Atg6 phosphorylation,
VPS34 PI3 kinase can convert phosphatidylinositol (PtdIns or
PI) to PI 3-phosphate (PI3P), which is essential for expansion
of the autophagosome membrane (12,13). Next, elongation
phase and phagophore formation occur (14). Two ubiquitin-
like pathways, Atg5–Atg12 and the LC3 conjugation systems,
are involved in this phase. LC3 (I)/Atg8 is cleaved by Atg4 at
the carboxyl-terminal site. Later, LC3 is converted to its active
form, LC3II, through covalent bonding to phosphatidyl etha-
nolamine, which assists the fusion of the autophagosome
membrane with the lysosome (15,16). Ultimately, the internal

CONTACT Raheleh Masoudi rmasoudi@shirazu.ac.ir Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran
Supplemental data for this article can be accessed here.

� 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

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components are degraded by lysosome hydrolases such as
Cathepsin D (17–19).

Hyperphosphorylated tau and Ab aggregates can affect
mitochondrial complexes and its electron chain, leading to
increased levels of ROS and oxidative stress (6,7).
Interestingly, it has been shown that ROS is involved in the
induction of autophagy in AD. Lipinski et al. reported that
ROS can enhance autophagy through increase in type III PI3
kinase activity. They also confirmed that, unlike in normal
aging, autophagy genes are transcriptionally increased in AD
patients (20). On the other hand, many studies have shown
that ROS can lead to autophagy impairment by various path-
ways either directly or indirectly. ROS directly inhibits LC3 lip-
idation and also its translocation to the phagophore
membrane (21). ROS increases NF-jB and nitric oxide (NO)
synthase which, in turn, enhances NO levels (22). NO can
inhibit JNK1 via S-nitrosylation at C116, leading to reduction
of Bcl2 phosphorylation, which ultimately increases its inter-
action with Beclin1. This event disrupts the formation of the
hVps34/Beclin1 complex, and autophagy is eventually
impaired (23).

It seems that presence of protein aggregates and autoph-
agy dysfunction in AD create a vicious circle. The aim of this
study was to investigate the effect of pathological Tau
R406W and Ab42, two major causes of aggregate formation
in AD, on the expression of autophagy genes, Atg6, Atg8,
Hook, and Cathepsin D. These genes are involved in different
stages of autophagy. Considering that AD is an age-depend-
ent disorder, the expression levels of autophagy genes were
assessed at two different time points in Ab42 or Tau R406W
transgenic Drosophila melanogaster. Tau R406W is an auto-
somal dominant mutation that causes tau-positive fronto-
temporal dementia in human. Its expression in Drosophila
melanogaster, using a binary system, provides a proper tau-
opathy model (24).

Materials and methods

Chemicals

RNA extraction and cDNA synthesis kits were purchased from
CinnaGen and Parstous companies, Iran, respectively. Brilliant
II SYBR Green qPCR master mix was provided by Biofact,
Germany. Other materials used in this study were provided
from CinnaGen, Iran and Merck, Germany.

Fly strains

Drosophila melanogaster stocks were raised in standard rolled
oats-agar medium at 22 ± 1 �C, 60–70% humidity, and 12-h
light/12-h dark circadian cycle. MAPT R406W for tauopathy
(expression of this protein in transgenic fly is discussed in
Wittmann et al., in 2001) (24) and Ab42 for amyloidopathy
(Bloomington Stock No. 33769) (25) were expressed in neu-
rons using Dmel\P{GawB}elavC155 (Bloomington Stock No.
458) driver. While expressed tau remains in the cytoplasm,
presence of a signal peptide in the Ab42 construct (26)
causes Ab42 transportation to the endoplasmic reticulum

(27). To investigate the pathogenesis of Ab42 and Tau
R406W, GMR-Gal4 driver (Bloomington Stock No. 8605) (28)
was applied to express these proteins in the fly eyes.

Flies expressing UAS-Tau R406W were from Feany’s lab
(Harvard Medical School, Boston, MA, USA), and all other
lines were from Bloomington Drosophila Stock Centre. All
crosses and their counterpart controls (parental lines) were
applied in triplicate. All transgenic stocks were outcrossed to
w1118 for several generations to obtain the identical genetic
background of all lines, prior to the tests.

Climbing assay

For climbing assay analysis, nine groups of flies, with 10 flies
per group (mix of both genders), were prepared for every
single genotype. All 10 flies were transferred into a vial, and
the vial was tapped gently. After each tap, flies were
observed for the first 10 s to record the number of flies that
were able to climb above the 8-cm marked line on the vial.
This assay was repeated five times for each group with a 2-
min interval between each measurement (29,30).

Drosophila eye analysis

Transgenic (GMR-Gal4/Tau R406W and GMR-Gal4/Ab42) and
control (GMR-Gal4/+) flies from at least three independent
crosses (9 flies per group, per cross) were processed for light
microscopy and image analysis with three experimental repeats.

Imaging was performed using a Nikon 80i light micro-
scope to observe degeneration phenotype in the fly eyes. A
new plugin (FLEYE) in ImageJ software was used to analyze
the fly eyes. Regularity in the ommatidia was represented by
probability parameter (PP) including: PP0, green; PP1, blue;
PP2, yellow; PP3, orange; and finally PP4, red. Change in the
colour from green to red represents the degree of reduction
in eye regularity (28).

Quantitative real-time PCR

First filial generation (F1) and their parental lines (as control)
were collected at 5 and 25 days after eclosion. For RNA
extraction, flies were kept in acetone and stored in a �20 �C
freezer overnight (31,32). Then, flies were frozen at �80 �C.
After 10 min, flies were shaken harshly in the tube in order
to separate the heads. RNA was extracted from 100 heads on
liquid nitrogen by RNX plus kit following the manufacturer’s
protocol. RNA concentration was measured using Nano drop
(Thermo Fisher Scientific), and 3 mg of total RNA was reverse-
transcribed to cDNA. Amplification of cDNA was performed
using Biofact SYBER Green master mix in an ABI 7500 PCR
machine. All primers were designed as the exon–exon junc-
tion primer. RPL32 was applied as reference gene to deter-
mine the relative expression levels of Hook, Atg6, Atg8, and
Cathepsin D using a 2(-delta delta C(T)) method (33). Each
sample was run in triplicate. The sequences of primers are
given in Supplementary Table S1.

266 M. HAGHI ET AL.

https://doi.org/10.1080/03009734.2020.1785063

Statistical analysis

Here, we applied R (version 3.6.1) and SPSS (version 19) pro-
grammes to perform statistical analysis.

The number of samples in biological studies is usually small,
and the assumptions of parametric statistical models cannot
be achieved. One solution is to use alternative non-parametric
statistical models, which have lower power compared to the
parametric versions. Another solution is to use Bayesian infer-
ence, which is more powerful to infer reliable and reproducible
results (34–36). In this study, because Ab42 and Tau R406W
have two subgroups (cross and control), we used the Bayesian
hierarchical mixture model for testing. Our hypotheses were
that there are differential gene expression and climbing ability
in two biologic conditions (AD and control) at different time
points (5 and 25 days). The proposed model exploits available
position-specific read counts, minimizing required data pre-
processing and making maximum use of available information.
Our analysis has been done by Stan language that is using a
state-of-the-art algorithm known as Hamiltonian Monte Carlo
(HMC), which builds upon the Metropolis–Hastings algorithm
by incorporating many theoretical ideas from physics.
Specifically, we used the rstanarm package in R software,
which is a powerful package for Bayesian hierarchical models
by stan_lme4 for estimating the model parameters. In this
paper, we considered a full multilevel model for group/sub-
group�time�
(genes) that means we assume random intercept and slope for
each category. We had to have an estimation of different inter-
cepts and slopes for each experimental condition, which was
clearly needed based on the raw data (see Figures 1 and 2).
The number of samples is selected to be 4000 (2000 for warm-
up), and four independent chains ran for the sake of conver-
gency and posterior sampling evaluation. The median estima-
tion and the highest posterior density (HPD) intervals with
probability of 0.95 are reported based on 2000 samples from
posterior distribution of model parameters. The Bayesian con-
trast estimations are evaluated by the emmeans package in R.

Besides the Bayesian model, we have also presented our
data as fold changes when comparing crosses and their
counterpart controls. Independent samples t test was
employed to compare the mean values. p values less than
0.05 were considered as statistically significant.

Results

Decrease in the climbing ability of Ab42 or Tau R406W
transgenic flies

Locomotive defects can be examined in transgenic flies as
one of the AD symptoms. Here, we tested negative geotaxis
ability as a behavioural assay to show that the expression of
our target genes can affect the natural tendency of flies to
move against gravity (29,30). To assess the climbing ability,
5- and 25-day-old transgenic flies were examined. It was
found that the ability of climbing in both transgenic flies
was remarkably decreased compared to their controls. The
raw data and Bayesian estimation of difference between con-
trols and transgenic flies are depicted in Figure 1 (for more
details see Table 1; and also Supplementary Table S2).

There was 0.32- and 0.42-fold decrease (p values � 0.001)
in 5-day-old Ab42 or Tau R406W-expressing flies, respect-
ively; 25-day-old flies expressing Ab42 or Tau R406W showed
0.44- and 0.53-fold reduction (p values �0.001) in the climb-
ing ability, respectively. For more details see Supplementary
Figure S1 and Supplementary Table S3.

Eye degeneration was observed in flies expressing
either Tau R406W or Ab42

To screen the pathogenesis of our genes of interest
(Ab42 and Tau R406W), we investigated the degeneration of
Drosophila retina as a model system by expressing the trans-
genes using the GMR-Gal4 driver. As can be seen in Figure 3,
there was more irregularity (yellow and red colors) in the eye
ommatidia of transgenic flies compared to parental lines

Figure 1. (A) Representing the raw data of the percent of flies (control and cross) above the target line in three independent biological repeats for 5- and 25-day-
old flies and the fitted Bayesian hierarchical model lines for each group and subgroup and their shaded 95% highest posterior density (HPD) region based on 2000
posterior samplings of model parameters. (B) Bayesian estimation of contrast between medians (control–cross) and their 95% HPD region based on 2000 posterior
samplings of each contrast. Because zero is not included in any of the reported HPD’s intervals, there is significant difference between the control and cross nega-
tive geotaxis ability with 5% error type (a ¼ 0.05).

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with regular eye ommatidia (blue and green colour).
Interestingly, both Ab42 and Tau R406W transgenic flies
demonstrate irregularities in their eyes.

Quantitative real time PCR

Alteration in the expression of Hook, as a
pre-autophagy gene
As mentioned earlier, Hook is a mediator to facilitate inter-
action between motor proteins and their cargo as a pre-
autophagy protein (37). In the AD brain, reduction in Hook3
expression and increase in Hook2 mRNA levels have been
observed. Interestingly, when Hook3 is knocked down, there

is an increase in Ab production (38). While there are three
forms of Hook in humans, only one form of Hook has
been reported in Drosophila. Here, we assessed the effect of
Ab42 and Tau R406W on the expression levels of dHook
(Drosophila Hook) using transgenic flies. As can be seen in
Figure 2 and from Bayesian contrast estimations in Table 2,
although there was no significant change in the levels of
dHook expression in 5-day-old flies expressing Tau R406W, a
prominent increase was observed in the levels of this gene
in 25-day-old flies. It seems that Tau R406W exerts its effect
on the Hook expression at a later time point of the life cycle
of this fly. Regarding Ab42-expressing flies, there was a sig-
nificant decrease in the levels of Hook expression in both 5-

Figure 2. Representing the raw data of autophagy-related gene expression in three independent biological repeats of Ab42 or Tau R406W transgenic flies and the
fitted Bayesian hierarchical model lines for each group and subgroup shaded by their 95% HPD region based on 2000 posterior samplings of model parameters.

Table 1. Bayesian contrast estimation of medians (control–cross) for climbing assay in 5- and 25-day-old flies.

Time point Fly type Contrast estimate (control–cross) Lower HPD (2.5%) Upper HPD (97.5%)

5 days Ab42 29.92a 27.55 32.12
Tau R406W 39.49a 37.13 41.67

25 days Ab42 34.83a 32.44 37.04
Tau R406W 42.53a 40.15 44.74

aThe 95% highest posterior density (HPD) intervals for each contrast are reported. Therefore, there is a significant difference between
control and cross with 5% error type I (a ¼ 0.05) in all of the cases thus marked.

268 M. HAGHI ET AL.

and 25-day-old flies. For more details see Figure 4; and also
Supplementary Table S4.

Regarding differential expression, there was about a 2.26-
fold increase in the Hook expression in 25-day-old flies
expressing Tau R406W (p ¼ 0.018). The reduction in the
expression of Hook was around 0.5- and 0.59-fold for 5- and
25-day-old Ab42-expressing flies, respectively (p ¼ 0.001 and
0.002). More details are in the Supplementary data,
Supplementary Figure S2 and Supplementary Table S5.

Alteration in the expression of autophagy genes
Expression of autophagy genes in fly is influenced by various
factors like age (39) and oxidative stress (40). On the other
hand, it has been shown that suppression or enhancement
of some autophagy genes like Atg8 can increase the sensitiv-
ity to aging and oxidative stress (41). Here, the expression
levels of three autophagy markers including Atg6, Atg8, and
Cathepsin D were assessed to track the autophagy process in

Drosophila models of AD. Transgenic flies expressing either
Tau R406W or Ab42 were applied as tauopathy or amyloid-
opathy models at two different ages, 5- and 25-day-old flies.
This could provide some information on how time might
affect the expression of some autophagy genes in the pres-
ence of two different types of amyloid-like aggregates.

Atg6, a core protein in the nucleation stage
of autophagy

One of the main proteins for phagophore formation is Atg6.
According to the Bayesian model analysis, our data showed
(Figure 2 and Table 2) that Atg6 was upregulated in 5-day-
old flies expressing either Ab42 or Tau R406W while the
mRNA levels of this gene had a considerable decrease in 25-
day-old transgenic flies (for more details, see Figure 4; and
also Supplementary Table S4). It seems that both Tau R406W
and Ab42 aggregates exert the same effect on the expres-
sion levels of this gene at different time points.

Figure 3. Eye degeneration has been observed in flies expressing Tau R406W or Ab42. A and B, respectively, are analyses for eye ommatidium regularity in 5- and
25-day-old flies by ImageJ, and columns A, B, and C are GMR-GAL4/+, GMR-GAL4/Tau R406W, and GMR-GAL4/Ab42. (PP ¼ probability parameter)

Table 2. Bayesian contrast estimation for medians of genes expressions (control–cross) in 5- and 25-day-old flies and different genes and fly types.

Gene type Time point Fly type Contrast estimate (control–cross) Lower HPD (2.5%) Upper HPD (97.5%)

Hook 5 days Ab42 0.512a 0.3964 0.625
Tau R406W �0.0002 �0.114 0.128

25 days Ab42 0.647a 0.525 0.764
Tau R406W �1.354a �1.475 �1.235

Atg6 5 days Ab42 �3.414a �3.530 �3.296
Tau R406W �5.353a �5.470 �5.231

25 days Ab42 0.569a 0.444 0.681
Tau R406W 0.829a 0.716 0.951

Atg8 5 days Ab42 �2.164a �2.290 �2.046
Tau R406W �5.229a �5.343 �5.106

25 days Ab42 0.452a 0.335 0.567
Tau R406W 0.685a 0.567 0.803

Cathepsin D 5 days Ab42 0.058 �0.065 0.173
Tau R406W �1.196a �1.309 �1.078

25 days Ab42 0.683a 0.564 0.800
Tau R406W 0.858a 0.743 0.977

aThe 95% highest posterior density (HPD) intervals for each contrast are reported. Therefore, there is a significant difference between control and cross with 5%
error type I (a ¼ 0.05) in all of the cases thus marked.

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There was a 4.45- and 6.31-fold increase in the levels of
Atg6 in 5-day-old flies expressing Ab42 or Tau R406W,
respectively (p � 0.001). In contrast, 25 days after eclosion
these transgenic flies showed a significant decrease
(p � 0.001), around 0.58- and 0.85-fold, in the Atg6 expression
levels compared to their counterpart controls. For more
details see Supplementary Figure S2 and Supplementary
Table S6.

Atg8, as the main gene in the elongation phase

Atg8 is the marker protein in the elongation phase of the
autophagosome formation. This unique protein undergoes
two processing steps. In the first step, Atg8 is cleaved by
Atg4 and the next step is mediated by the
Atg5–Atg12–Atg16 complex to get conjugated with phos-
phatidylethanolamine. These two steps are necessary for
elongation and closure of the autophagic membrane (42).
The expression of this gene showed the same pattern as the
Atg6. There was a significant increase in the expression levels
of Atg8 in 5-day-old transgenic flies expressing either Tau
R406W or Ab42, followed by a prominent decline in the lev-
els of Atg8 mRNA expression in those flies 25 days after eclo-
sion (for more details on the Bayesian model see Figures 2
and 4, and Table 2; and also Supplementary Table S4).
Therefore, it seems that both forms of aggregates have simi-
lar impact on the Atg8 mRNA levels.

In the fold change analysis, 5-day-old Ab42 or Tau R406W
transgenic flies showed a 3.15- and 6.28-fold increase in the
levels of Atg8 (p � 0.001), while there was a significant
decline (p � 0.001) in the expression levels of this gene
(about 0.45- and 0.70-fold, respectively) in 25-day-old flies
expressing Ab42 or Tau R406W (See Supplementary Figure
S2 and Supplementary Table S7 for more details).

Expression of the lysosome enzyme Cathepsin D,
involved in the final step of autophagy

In the central nervous system, the activity of Cathepsin D is
essential to control neuronal homeostasis, cell migration, and
interneuron communication. Cathepsin D-mediated proteoly-
sis plays a significant role in neuronal survival by accomplish-
ing the degradation of aggregated proteins that reach the
lysosomes via autophagy (43). There is evidence to reveal
that this protein is involved in amyloidogenic processing of
the amyloid precursor protein (APP), as a critical component
of a, b, and c secretase (44). We found that the mRNA levels
of Cathepsin D have different patterns in 5-day-old flies
expressing Tau R406W or Ab42. According to the Bayesian
model, there was no significant change in the levels of
Cathepsin D mRNA in flies expressing Ab42, while
Tau R406W-expressing flies showed a prominent increase in
the mRNA levels of this gene at day 5 after eclosion.
Interestingly, at day 25, both transgenic flies showed a

Figure 4. Bayesian estimation of median contrasts (control–cross) for gene expression in 5- and 25-day-old flies. Hook gene has different expression pattern in
Ab42- or Tau R406W-expressing flies either 5 or 25 days old, while Atg6 and Atg8 mRNA levels changed with similar pattern in both transgenic lines either 5 or
25 days after eclosion.

270 M. HAGHI ET AL.

remarkable decline in the expression levels of Cathepsin D
mRNA (Figures 2 and 4, and Table 2; and also
Supplementary Table S4).

The fold change analysis showed that there was a 2.24-
fold increase (p ¼ 0.007) in the levels of Cathepsin D expres-
sion in 5-day-old flies expressing Tau R406W. In 25-day-old
Ab42 or Tau R406W-expressing flies, there was a 0.74- and
0.85-fold decrease, respectively (p � 0.001). More details are
included in Supplementary Figure S2 and Supplementary
Table S8.

Discussion

According to the amyloid cascade, proposed by Hardy and
Higgins in 1992, amyloid is the main culprit in AD pathology
(44). Other events in AD, including tau hyperphosphorylation
and subsequent neuronal death, were supposed to be down-
stream events of the amyloid pathway (45–47). Therefore,
most treatments for AD have been based on the removal of
Ab (reviewed in 46). Recently, there is increasing evidence to
support the idea that pathological tau can exert its own
toxic effect independently (48–50). On the other hand, there
are some studies showing that aggregations of Ab (senile
plaque, SP) and tau (neurofibrillary tangles, NFT) are, in fact,
a defensive mechanism against Ab monomers and the sol-
uble pathological tau (3,51). However, these aggregates, in
turn, block axonal/dendritic transport (5) and can lead to
mitochondrial damage and an increase in the levels of
ROS (6,7).

In order to shed light on the mechanism underlying the
relation between protein aggregates and autophagy dysfunc-
tion in AD, here, we investigated the differential expression
of autophagy genes in Ab42-expressing flies as an amyloid-
opathy model and flies expressing Tau R406W as a tauop-
athy model (24). We used Tau R406W transgenic flies as they
have been reported to exhibit AD-like phenotypes (52). On
the other hand, previous studies have shown that tau wild-
type transgenic flies have no aggregate formation (53).
Therefore, to clarify the effect of the aggregates of this pro-
tein on autophagy genes, this model was more suitable.

Our climbing assay and fly eye analysis showed that Ab42
and Tau R406W can cause defects in climbing ability and eye
regularity in transgenic flies. These data also showed that tau
can exert its toxicity independent of Ab42.

Neurons are highly dependent on the autophagy to
remove protein aggregates such as SP and NFT (8). Defects in
this pathway clearly lead to neurodegenerative disorders such
as AD. There is strong evidence to confirm the association of
autophagy dysregulation with AD (54). Herrmann and col-
leagues showed that Hook3 was co-localized with tau aggre-
gate and retained in those aggregates (38). Surprisingly, in our
current research, there was a remarkable increase in the levels
of dHook expression in 25-day-old flies expressing Tau R406W.
It is possible that Hook is the adaptor for tau aggregates in
the autophagy pathway and that is why the expression of
Hook is increased in Tau R406W-expressing flies. However, the
expression of this gene is downregulated in Ab42-expressing
flies. This may suggest that either the transportation of Ab

aggregates is mainly managed by another adaptor or Ab42
can affect autophagy through Hook downregulation. In sum-
mary, it seems that although both Tau R406W and Ab42 alter
the expression of Hook, their effects occur via differ-
ent mechanisms.

Several studies have shown that Beclin1 (ATG6), a main
protein to initiate autophagy, is downregulated in AD, while
some other evidence argues that autophagy genes are
increased during AD (20,55,56). Despite all the controversies,
it is clear that autophagy is deregulated in this disease.

Atg6 is downregulated in early and late stages of AD (55).
However, it has been shown that ATG8 expression is
increased in AD patients (56,57). According to our results,
while Atg6 and Atg8 showed upregulation in their mRNA lev-
els in 5-day-old flies expressing either Tau R406W or Ab42, a
remarkable downregulation was observed for both genes at
day 25 after eclosion. It appears that in earlier stages (5 day
after eclosion) autophagy genes are increased in order to
clear the aggregates. However, ultimately autophagy is
decreased at later time points, probably due to an increase
in the ROS production (6,7) or other mechanisms.

Association of autophagosome with lysosome is the last
step of autophagy. Finally, its internal components are
degraded by lysosome hydrolases like Cathepsin D (17).
Cathepsin D is the only proteolytic enzyme the expression of
which, in different tissues, is regulated in response to growth
factors, cytokines, and vitamins (58). Cathepsin D-mediated
proteolysis is essential to neurons because it degrades
unfolded/oxidized protein aggregates that continuously
reach the lysosomes via autophagy or endocytosis (43).
Many proteins produced in neurons are physiologic sub-
strates of Cathepsin D and will be abnormally accumulated if
they are not efficiently degraded (e.g. APP, a-synuclein, and
huntingtin). Therefore, dysfunction of Cathepsin D in the
lysosomal system is closely related to the mechanism under-
pinning neurodegeneration (43).

In 2006, Urbanelli et al. demonstrated that there was a
decrease in the Cathepsin D mRNA and protein levels in AD
(59). However, in 1995, Cataldo et al. demonstrated intense
Cathepsin D immunoreactivities and lowered Cathepsin D
mRNA levels in degenerating neurons (60). The upregulation
of Cathepsin D synthesis and accumulation of hydrolase-
laden lysosomes indicated an early activation of the endoso-
mal-lysosomal system in vulnerable neuronal populations,
possibly reflecting early regenerative or repair processes (60).
Urbanelli and colleagues provided evidence of altered bal-
ance of the Cathepsin D expression in skin fibroblasts from
patients with sporadic or familial forms of AD (59). In particu-
lar, they showed that the expression of this gene is downre-
gulated at both the transcriptional and translational levels
and its processing is altered in AD fibroblasts (59). High lev-
els of the constitutively active form of Ras in normal or AD
fibroblasts induce Cathepsin D downregulation. Furthermore,
the p38 MAPK signalling pathway also appears to downregu-
late the Cathepsin D levels. Urbanelli et al. proposed that the
impairment of lysosomes in AD can be one of the main fac-
tors for the progression of the disease (59).

UPSALA JOURNAL OF MEDICAL SCIENCES 271

https://doi.org/10.1080/03009734.2020.1785063
https://doi.org/10.1080/03009734.2020.1785063
https://doi.org/10.1080/03009734.2020.1785063
https://doi.org/10.1080/03009734.2020.1785063

Finally, in 2018 Chai et al. investigated the Cathepsin D
immunoreactivities in the temporal and parietal cortices of
well characterized AD brains. Their results showed an
increase in the Cathepsin D immunoreactivities in AD tissues
and its correlation with neuropathological NFT scores, and
phosphorylated pSer396 tau burden (17).

Our data showed that there was a significant increase in
the mRNA levels of Cathepsin D in Tau R406W-expressing
flies just 5 days after eclosion. This increase could be due to
regenerative or repair processes occurring at earlier stages of
the disease (60). However, in 25-day-old transgenic flies, this
gene showed significant reduction. Following the increase in
Tau R406W and Ab42 aggregations at later time points (25-
day-old flies), probably due to an increase in ROS production,
Cathepsin D is downregulated (43). Moreover, cystatin C, as
an inhibitor of cathepsins (cysteine protease), is upregulated
by ROS and has been shown to be co-localized with the
Ab42 peptide (61,62).

Conclusion

In summary, our results suggest that both Ab42 and Tau
R406W can affect the autophagy pathway through gene
expression dysregulation. Interestingly, they showed a similar
effect on the genes involved in the nucleation and elong-
ation steps of autophagy. However, Tau R406W and Ab42
exert different effects on the expression of the pre-autoph-
agy gene, Hook, and a gene involved in the last step of
autophagy, Cathepsin D. We conclude that although both
Tau R406W and Ab42 can alter the process of autophagy
during AD, it seems that they act independently, through dif-
ferent mechanisms. Therefore, AD treatment involving the
removal of merely Ab42, without considering pathological
tau, will not be sufficiently effective in ameliorating
AD symptoms.

Acknowledgements

We would like to show our gratitude and appreciation to Dr. M. Haddadi
for providing the desired Drosophila stock and sharing his pearls of wis-
dom with us during the course of working with the flies. We also
express our sincere thanks to Dr M. Ebrahimi for assistance with
FLEYE plugin.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Funding

This research was financially supported by Shiraz University Research
Council. Seyed Morteza Najibi was supported by a Swedish Research
Council grant (2016–06947) and a grant from eSSENCE@LU.

Notes on contributors

Mehrnaz Haghi, PhD student of Molecular and Cellular Biology,
Department of Biology, Shiraz University, Shiraz, Iran.

Raheleh Masoudi, PhD of Molecular Genetics, Assistant professor in
Department of Biology, Shiraz University, Shiraz, Iran.

Seyed Morteza Najibi, PhD of Statistics, Researcher at Center for
Molecular Protein Science, Lund University, Lund, 22362, Sweden.

References

1. Hayashi SI, Sato N, Yamamoto A, Ikegame Y, Nakashima S,
Ogihara T, et al. Alzheimer disease-associated peptide, amyloid
beta40, inhibits vascular regeneration with induction of endothe-
lial autophagy. Arterioscler Thromb Vasc Biol. 2009;29:1909–15.

2. Caccamo A, Maldonado MA, Majumder S, Medina DX, Holbein W,
Magr�ı A, et al. Naturally secreted amyloid-beta increases mamma-
lian target of rapamycin (mTOR) activity via a PRAS40-mediated
mechanism. J Biol Chem. 2011;286:8924–32.

3. Esparza TJ, Gangolli M, Cairns NJ, Brody DL. Soluble amyloid-beta
buffering by plaques in Alzheimer disease dementia versus high-
pathology controls. PLoS One. 2018;13:e0200251.

4. G€otz J, Ittner LM, F€andrich M, Schonrock N. Is tau aggregation
toxic or protective: a sensible question in the absence of sensitive
methods? J Alzheimers Dis. 2008;14:423–9.

5. Bendiske J, Bahr BA. Lysosomal activation is a compensatory
response against protein accumulation and associated synaptopa-
thogenesis-an approach for slowing Alzheimer disease? J
Neuropathol Exp Neurol. 2003;62:451–63.

6. Golpich M, Amini E, Mohamed Z, Azman Ali R, Mohamed Ibrahim
N, Ahmadiani A. Mitochondrial dysfunction and biogenesis in neu-
rodegenerative diseases: pathogenesis and treatment. CNS
Neurosci Ther. 2017;23:5–22.

7. Schulz KL, Eckert A, Rhein V, Mai S, Haase W, Reichert AS, et al. A
new link to mitochondrial impairment in tauopathies. Mol
Neurobiol. 2012;46:205–16.

8. Uddin M, Stachowiak A, Mamun AA, Tzvetkov NT, Takeda S,
Atanasov AG, et al. Autophagy and Alzheimer’s disease: from
molecular mechanisms to therapeutic implications. Front Aging
Neurosci 2018;10:4.

9. Chin LS, Olzmann JA, Li L. Aggresome formation and neurodege-
nerative diseases: therapeutic implications. Curr Med Chem. 2008;
15:47–60.

10. Olenick MA, Tokito M, Boczkowska M, Dominguez R, Holzbaur EL.
Hook adaptors induce unidirectional processive motility by
enhancing the dynein-dynactin interaction. J Biol Chem. 2016;291:
18239–51.

11. Hurley JH, Young LN. Mechanisms of autophagy initiation. Annu
Rev Biochem. 2017;86:225–44.

12. Fitzwalter BE, Thorburn A. Recent insights into cell death and
autophagy. Febs J. 2015;282:4279–88.

13. Shravage BV, Hill JH, Powers CM, Wu L, Baehrecke EH. Atg6 is
required for multiple vesicle trafficking pathways and hematopoi-
esis in Drosophila. Development. 2013;140:1321–9.

14. Reggiori F, Ungermann C. Autophagosome maturation and fusion.
J Mol Biol. 2017;429:486–96.

15. Badadani M. Autophagy mechanism, regulation, functions, and
disorders. ISRN Cell Biol. 2012;2012:1–11.

16. Barth S, Glick D, Macleod KF. Autophagy: assays and artifacts. J
Pathol. 2010;221:117–24.

17. Chai YL, Chong JR, Weng J, Howlett D, Halsey A, Lee JH, et al.
Lysosomal cathepsin D is upregulated in Alzheimer’s disease neo-
cortex and may be a marker for neurofibrillary degeneration. Brain
Pathol. 2019;29:63–74.

18. Kuchitsu Y, Fukuda M. Revisiting Rab7 functions in mammalian
autophagy: Rab7 knockout studies. Cells 2018;7:215.

19. Fujita N, Huang W, Lin TH, Groulx JF, Jean S, Nguyen J, et al.
Genetic screen in Drosophila muscle identifies autophagy-medi-
ated T-tubule remodeling and a Rab2 role in autophagy. Elife.
2017;6:e23367.

20. Lipinski MM, Zheng B, Lu T, Yan Z, Py BF, Ng A, et al. Genome-
wide analysis reveals mechanisms modulating autophagy in

272 M. HAGHI ET AL.

normal brain aging and in Alzheimer’s disease. Proc Natl Acad Sci
USA. 2010;107:14164–9.

21. Burgoyne JR. Oxidative stress impairs autophagy through oxida-
tion of ATG3 and ATG7. Autophagy 2018;14:1092–3.

22. Kaur U, Banerjee P, Bir A, Sinha M, Biswas A, Chakrabarti S.
Reactive oxygen species, redox signaling and neuroinflammation
in Alzheimer’s disease: the NF-jB connection. Curr Top Med
Chem. 2015;15:446–57.

23. Sarkar S, Korolchuk VI, Renna M, Imarisio S, Fleming A, Williams A,
et al. Complex inhibitory effects of nitric oxide on autophagy. Mol
Cell. 2011;43:19–32.

24. Wittmann CW, Wszolek MF, Shulman JM, Salvaterra PM, Lewis J,
Hutton M, et al. Tauopathy in Drosophila: neurodegeneration
without neurofibrillary tangles. Science. 2001;293:711–4.

25. Tare M, Modi RM, Nainaparampil JJ, Puli OR, Bedi S, Fernandez-
Funez P, et al. Activation of JNK signaling mediates amyloid-ss-
dependent cell death. PLOS One. 2011;6:e24361.

26. Chouhan AK, Guo C, Hsieh YC, Ye H, Senturk M, Zuo Z, et al.
Uncoupling neuronal death and dysfunction in Drosophila models
of neurodegenerative disease. Acta Neuropathol Commun. 2016;4:
62.

27. Finelli A, Kelkar A, Song HJ, Yang H, Konsolaki M. A model for
studying Alzheimer’s Abeta42-induced toxicity in Drosophila mela-
nogaster. Mol Cell Neurosci. 2004;26:365–75.

28. Diez-Hermano S, Valero J, Rueda C, Ganfornina MD, Sanchez D. An
automated image analysis method to measure regularity in bio-
logical patterns: a case study in a Drosophila neurodegenerative
model. Mol Neurodegener. 2015;10:9.

29. Madabattula ST, Strautman JC, Bysice AM, O’Sullivan JA,
Androschuk A, Rosenfelt C. Quantitative analysis of climbing
defects in a Drosophila model of neurodegenerative disorders. J
Vis Exp 2015;100:e52741.

30. Ali YO, Escala W, Ruan K, Zhai RG. Assaying locomotor, learning,
and memory deficits in Drosophila models of neurodegeneration.
J Vis Exp 2011;49:2504.

31. Koopmans M, Monroe SS, Coffield LM, Zaki SR. Optimization of
extraction and PCR amplification of RNA extracts from paraffin-
embedded tissue in different fixatives. J Virol Methods. 1993;43:
189–204.

32. Fukatsu T. Acetone preservation: a practical technique for molecu-
lar analysis. Mol Ecol. 1999;8:1935–45.

33. Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2(-delta delta C(T))
method. Methods. 2001;25:402–8.

34. Austin PC, Brunner LJ, Hux JE. Bayeswatch: an overview of
Bayesian statistics. J Eval Clin Pract. 2002;8:277–86.

35. Button KS, Ioannidis JP, Mokrysz C, Nosek BA, Flint J, Robinson ES,
et al. Power failure: why small sample size undermines the reliabil-
ity of neuroscience. Nat Rev Neurosci. 2013;14:365–76.

36. R Core Team. R: A language and environment for statistical com-
puting. Vienna, Austria: R Foundation for Statistical Computing;
2020. Available at: http://www.R-project.org/.

37. Szebenyi G, Hall B, Yu R, Hashim AI, Kr€amer H. Hook2 localizes to
the centrosome, binds directly to centriolin/CEP110 and contrib-
utes to centrosomal function. Traffic. 2007;8:32–46.

38. Herrmann L, Wiegmann C, Arsalan-Werner A, Hilbrich I, J€ager C,
Flach K, et al. Hook proteins: association with Alzheimer pathology
and regulatory role of hook3 in amyloid beta generation. PLOS
One. 2015;10:e0119423.

39. Rubinsztein DC, Shpilka T, Elazar Z. Mechanisms of autophago-
some biogenesis. Curr Biol. 2012;22:R29–R34.

40. Simonsen A, Cumming RC, Brech A, Isakson P, Schubert DR, Finley
KD. Promoting basal levels of autophagy in the nervous system
enhances longevity and oxidant resistance in adult Drosophila.
Autophagy 2008;4:176–84.

41. Wu H, Wang MC, Bohmann D. JNK protects Drosophila from oxi-
dative stress by trancriptionally activating autophagy. Mech Dev.
2009;126:624–37.

42. Shpilka T, Weidberg H, Pietrokovski S, Elazar Z. Atg8: an autoph-
agy-related ubiquitin-like protein family. Genome Biol. 2011;12:
226.

43. Di Domenico F, Tramutola A, Perluigi M. Cathepsin D as a thera-
peutic target in Alzheimer’s disease. Expert Opin Ther Targets.
2016;20:1393–5.

44. Straface E, Matarrese P, Gambardella L, Vona R, Sgadari A, Silveri
MC, et al. Oxidative imbalance and cathepsin D changes as per-
ipheral blood biomarkers of Alzheimer disease: a pilot study. FEBS
Lett. 2005;579:2759–66.

45. Hardy JA, Higgins GA. Alzheimer’s disease: the amyloid cascade
hypothesis. Science. 1992;256:184–5.

46. Lewis J, Dickson DW, Lin WL, Chisholm L, Corral A, Jones G, et al.
Enhanced neurofibrillary degeneration in transgenic mice express-
ing mutant tau and APP. Science. 2001;293:1487–91.

47. Rajmohan R, Reddy PH. Amyloid-beta and phosphorylated tau
accumulations cause abnormalities at synapses of Alzheimer’s dis-
ease neurons. J Alzheimers Dis. 2017;57:975–99.

48. Kametani F, Hasegawa M. Reconsideration of amyloid hypothesis
and tau hypothesis in Alzheimer’s Disease. Front Neurosci. 2018;
12:25.

49. Belrose JC, Masoudi R, Michalski B, Fahnestock M. Increased pro-
nerve growth factor and decreased brain-derived neurotrophic
factor in non-Alzheimer’s disease tauopathies. Neurobiol Aging.
2014;35:926–33.

50. Harrison JR, Owen MJ. Alzheimer’s disease: the amyloid hypothesis
on trial. Br J Psychiatry 2016;208:1–3.

51. Bretteville A, Planel E. Tau aggregates: toxic, inert, or protective
species? J Alzheimers Dis. 2008;14:431–6.

52. Nakamura M, Shiozawa S, Tsuboi D, Amano M, Watanabe H,
Maeda S, et al. Pathological progression induced by the fronto-
temporal dementia-associated R406W tau mutation in patient-
derived iPSCs. Stem Cell Reports. 2019;13:684–99.

53. Passarella D, Goedert M. Beta-sheet assembly of tau and neurode-
generation in Drosophila melanogaster. Neurobiol Aging. 2018;72:
98–105.

54. Funderburk SF, Marcellino BK, Yue Z. Cell “‘self-eating’ (autophagy)
mechanism in Alzheimer’s disease”. Mt Sinai J Med. 2010;77:59–68.

55. Lee JA, Gao FB. Regulation of Abeta pathology by beclin 1: a pro-
tective role for autophagy? J Clin Invest. 2008;118:2015–8.

56. Ihara Y, Morishima-Kawashima M, Nixon R. The ubiquitin–protea-
some system and the autophagic–lysosomal system in Alzheimer
disease. Cold Spring Harb Perspect Med. 2012;2:a006361.

57. Rajaguru P, Vaiphei K, Saikia B, Kochhar R. Increased accumulation
of dendritic cells in celiac disease associates with increased
expression of autophagy protein LC3. Indian J Pathol Microbiol.
2013;56:342.

58. Vidoni C, Follo C, Savino M, Melone MAB, Isidoro C. The role of
cathepsin D in the pathogenesis of human neurodegenerative dis-
orders. Med Res Rev. 2016;36:845–70.

59. Urbanelli L, Emiliani C, Massini C, Persichetti E, Orlacchio A, Pelicci
G, et al. Cathepsin D expression is decreased in Alzheimer’s dis-
ease fibroblasts. Neurobiol Aging. 2008;29:12–22.

60. Cataldo AM, Barnett JL, Berman SA, Li J, Quarless S, Bursztajn S,
et al. Gene expression and cellular content of cathepsin D in
Alzheimer’s disease brain: evidence for early up-regulation of the
endosomal-lysosomal system. Neuron 1995;14:671–80.

61. McGrath LT, McGleenon BM, Brennan S, McColl D, McIlroy S,
Passmore AP. Increased oxidative stress in Alzheimer’s disease as
assessed with 4-hydroxynonenal but not malondialdehyde. QJM.
2001;94:485–90.

62. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr.
2000;71:621S–9S.

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http://www.R-project.org/

Abstract
Introduction
Materials and methods
Chemicals
Fly strains
Climbing assay
Drosophila eye analysis
Quantitative real-time PCR
Statistical analysis

Results
Decrease in the climbing ability of Aβ42 or Tau R406W transgenic flies
Eye degeneration was observed in flies expressing either Tau R406W or Aβ42
Quantitative real time PCR
Alteration in the expression of Hook, as apre-autophagy gene
Alteration in the expression of autophagy genes

Atg6, a core protein in the nucleation stage of autophagy
Atg8, as the main gene in the elongation phase
Expression of the lysosome enzyme Cathepsin D, involved in the final step of autophagy

Discussion
Conclusion
Acknowledgements
Disclosure statement
Notes on contributors
References