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1
hSOD1 promotes Tau toxicity via elevated Tau
phosphorylation in the Drosophila model
Yunpeng Huang1, Zhihao Wu1, Bing Zhou1,2,*
1. State Key Laboratory of Biomembrane and Membrane Biotechnology,
School of Life Sciences, Tsinghua University, Beijing 100084, China;
2. Beijing Institute for Brain Disorders, Beijing, China
* Correspondence: Dr. Bing Zhou, School of Life Sciences, Tsinghua
University, Beijing, 100084, China. Email: [email protected].
2
Abstract
Tau hyperphosphorylation has been found in several neurodegenerative
diseases such as Alzheimer Disease (AD), Down Syndrome (DS), and
Amyotrophic Lateral Sclerosis (ALS). However, factors affecting Tau
hyperphosphorylation are not yet clearly understood. SOD1, a Cu/Zn
superoxide dismutase whose mutations can cause adult-onset ALS, is
believed to be involved in the pathology of Down Syndrome. In this work,
the model organism Drosophila was used to study the possible link
between hSOD1 and Tau. Our results show that hSOD1, and to a higher
degree hSOD1(A4V), can increase Tau toxicity in Drosophila and
exacerbate the corresponding neurodegeneration phenotype. The
increased Tau toxicity appears to be the result of elevated Tau
hyperphosphorylation. Tau(S2A), a Tau mutant with impaired
phosphorylation capabilities, does not respond to hSOD1 and
hSOD1(A4V)’s expression. We suggest that increased SOD1 expression
can lead to Tau hyperphsphorylation, which might serve as an important
contributing factor to the etiology of Down Syndrome and SOD1-related
ALS disease.
Key words: hSOD1; Tau; hyperphosphorylation; Drosophila
3
Introduction
Tau is a cytosolic protein which can bind to microtubules, which allows for
the stabilization and regulation of microtubule dynamics [1-2]. Under
physiological conditions, Tau phosphorylation plays an important role in
regulating the microtubule dynamics [3-5]. It is now understood that Tau
phosphorylation is regulated by several kinases including MARK
(MAP/microtubule affinity-regulating kinase), Par-1, JNK, and PKA [6-8].
Aberrant hyperphosphorylation, detectable by several phosphoepitope-specific
antiserums such as AT8, AT180, and PHF-1 [9-10], is found in several kinds of
neurodegenerative disease such as Alzheimer disease (AD), Pick’s disease,
progressive supranuclear palsy, Down syndrome, and amyotrophic lateral
sclerosis (ALS or Lou Gehrig's disease), and is believed to be a potential risk
factor in these diseases [10-14].
SOD1 is a Cu/Zn superoxide dismutase which converts superoxide anions
into hydrogen peroxide, protecting living cells from the harmful effects of
superoxide. SOD1 is also considered a risk factor in Down syndrome and ALS
[15-18]. Down syndrome is caused by the trisomy of chromosome 21, and
leads to mental retardation in patients. SOD1, located on chromosome 21, is
suggested as one of the risk elements in Down syndrome along with other
factors such as APP and CCT8. Mice that are trisomic for a region
encompassing the SOD1 loci (Ts65Dn) could re-produce some symptoms of
human Down syndrome, including learning deficits and neuronal degeneration
[19]; meanwhile, TgSOD1 mice also suffered from learning defects and
degeneration [16, 20-21]. With these considerations, it appears that SOD1
could be an important pathological factor in Down syndrome, but a concrete
4
mechanism of how SOD1 functions is still unclear.
There are more than 100 different reported SOD1 mutations such as A4V,
G73R, and G85R [22], that contribute to ~20% of all cases of ALS, a fatal
adult-onset neurodegenerative disease caused by motor neuron death [17].
Interestingly, Tau hyperphosphorylation has been found in both of these
SOD1-relevant diseases – Down Syndrome and ALS [10, 14]. From this
observation, a question naturally arises: is Tau hyperphosphorylation and
SOD1 connected? In this work, we used the Drosophila model organism to
explore whether Tau phosphorylation and its toxicity could be affected by
hSOD1 and the ALS related hSOD1 mutation A4V. We found that both the wild
type hSOD1 and hSOD1(A4V) could accelerate Tau pathological processes in
Drosophila, such as shortened lifespan, impaired movement ability, and
elevated neurodegeneration levels. The enhanced Tau toxicity appears to be a
result from increased Tau hyperphosophorylation, which was promoted by
hSOD1 and hSOD1(A4V) expression, while the hypophosphorylated Tau
mutant Tau(S2A) was insulated from the SOD1’s potentiating effect. Our
results suggest a possible link between hSOD1 and Tau toxicity, revealing a
likely pathologic pathway underlying Down Syndrome and ALS.
Materials and Methods
Fly stocks and genetics
Tau used in this study was Tau(R406W). UAS-Tau and UAS-Tau(S2A)
flies were kindly gifted to us by Dr. Bingwei Lu (Stanford University, USA).
UAS-hSOD1 and UAS-hSOD1(A4V), Gmr-Gal4, Elav-Gal4 flies were stocks
from the Bloomington Drosophila Stock center. All flies were maintained and
5
reared at 25 ℃ on standard Drosophila corn media. First, UAS-Tau,
UAS-Tau(S2A) flies were combined with Gmr-Gal4 or Elav-Gal4, and then
crossed with UAS-hSOD1 or UAS-hSOD1(A4V) flies to generate the
Gmr-Gal4/+;UAS-Tau/UAS-hSOD1, Gmr-Gal4/+;UAS-Tau/UAS-hSOD1(A4V),
Gmr-Gal4/UAS-Tau(S2A);+/UAS-hSOD1, or
Gmr-Gal4/UAS-Tau(S2A);+/UAS-hSOD1(A4V) flies that specifically expressed
Tau and hSOD1 in Drosophila eyes. Gmr-Gal4/+;UAS-Tau/+ and
Gmr-Gal4/UAS-GFP;UAS-Tau/+ flies were used as the control.
The Elav-Gal4/+;UAS-Tau/UAS-hSOD1,
Elav-Gal4/+;UAS-Tau/UAS-hSOD1(A4V),
Elav-Gal4/+;+/UAS-Tau(S2A);+/UAS-hSOD1, or
Elav-Gal4/+;+/UAS-Tau(S2A);+/UAS-hSOD1(A4V) flies that specifically
express Tau and hSOD1 in the Drosophila CNS were used in the lifespan and
climbing assays.
Lifespan and climbing ability assays
Flies’ lifespans were assayed at 25℃ on standard corn media. At least
100 newly eclosed adult flies were used for each individual lifespan recording.
Fresh food was changed every two days. For the movement assay, flies were
divided into 20 vials per group, and each genotype consisted of n=6 individual
repeats. The mobility index reflects the total number of flies that were able to
climb 7cm in 8 seconds divided by the total number of flies in the assayed
group.
H&E staining
6
For paraffin sections, fly heads were fixed in the Carnoy fixation solution
(ethanol: chloroform: acetic acid= 6: 3: 1) for 4 hours at room temperature,
dehydrated (in the following sequence) twice by 100% ethanol for 30 min, once
by dry ethanol (100% ethanol dried with desiccant) for 1 hour, and once by
methyl benzoate for 1 hour, and then embedded in melted paraffin. The fly
heads were sectioned into 8μm continuous sections. Hematoxylin & Eosin
(ZSGBBIO, China) staining was used to facilitate the observation of the
vacuoles in the brains.
Protein preparation and immunoblotting
To analyze the phosphorylation levels of Tau proteins, adult fly heads
were homogenized in the lysis buffer as described [23]. Protein extracts were
mixed with SDS loading buffer and heated to 60℃, and then centrifuged at
10,000 g for 5 min before they were loaded into a 12% SDS-PAGE. The
proteins were then transferred onto PVDF membranes (Millipore) and
incubated with antibodies at the following dilutions: Tau5 (mouse, 1:1500),
PHF-1 (mouse, 1:1500), CP13 (rabbit, 1:2000), AT180 (rabbit, 1 :1500), pS262
(rabbit, 1:1500), pS356 (rabbit, 1:1500), pJNK (rabbit, 1:1500), JNK (rabbit,
1:1000), pMAPK (mouse, 1:1000) and Actin (mouse, 1:1500). The PHF-1
antibody originated from the Hybridoma Bank (University of Iowa, USA); the
pJNK antibody was purchased from Cell Signaling; the JNK antibody was
purchased from Santa Cruz; the pMAPK was a kind gift from Dr. Hong Luo of
Tsinghua University; the other antibodies were purchased from Invitrogen.
Secondary antibodies were peroxidase-labeled anti-mouse IgG, or anti-rabbit
IgG. Immunoblot signals were developed by enhanced chemiluminescence
7
(Pierce).
Statistics
Data is presented as mean ± S.E.M.. Differences among groups were
analyzed by the IBM SPSS v13.0 with Student’s t (comparison of two groups)
or ANOVA test (3 groups or more). *: p<0.05; **: p<0.01.
Results
hSOD1 and hSOD1(A4V) could promote Tau toxicity in Drosophila eyes
In order to investigate whether the toxicity of Tau could be affected by
hSOD1 or not, we co-expressed Tau(R406W) and hSOD1 in Drosophila eyes
by crossing Gmr-Gal4; UAS-Tau(R406W) flies with UAS-hSOD1 flies [24-25].
Tau toxicity in flies’ eyes could be obviously observed and readily evaluated
[25]. Interestingly, instead of suppression, hSOD1 expression actually
worsened the Tau toxicity in Drosophila compound eyes: the rough eye
phenotype caused by Tau expression became more severe in Tau/hSOD1
flies, and the ommatidia were more severely fused and irregular (Fig. 1A,
hSOD1/Tau panel). Tangential sections of fly compound eyes also confirmed
such findings (Fig. 1B, hSOD1/Tau): Tau/hSOD1 combination caused
ommatidia to become more markedly disorganized when compared with Tau
alone (Fig. 1B, hSOD1/Tau). In parallel, the hSOD1(A4V) mutant was also
combined with Tau. The worsening effect of hSOD1(A4V) on Tau toxicity
seems more apparent (Fig. 1A and Fig. 1B, hSOD1(A4V)/Tau panel), as
demonstrated by a more severely damaged eye appearance, and more
irregular and degenerated ommatidial structures. As control, both hSOD1 and
8
hSOD1(A4V) alone did not develop the distinct rough eye phenotype in our
experiments, and the tangential sections of their ommatidia remained more or
less regular (Fig. 1A and Fig. 1B, Gmr-Gal4>hSOD1 and
Gmr-Gal4>hSOD1(A4V)). Thus, expression of hSOD1 and hSOD1(A4V) could
elevate Tau toxicity in Drosophila eyes.
Tau-related neurodegeneration was worsened by hSOD1 and
hSOD1(A4V)
In AD, Down Syndrome and other neurodegenerative diseases, the
degenerative process that causes the loss of neurons is one of the main
pathological features. It has been shown that Tau expression could lead to
neurodegenerative phenotypes in mice and Drosophila [24, 26], as well as
affect the animals’ lifespans. To further characterize the effect of hSOD1 and
hSOD1(A4V) on Tau toxicity, we next analyzed the flies’ lifespans and
mobilities in addition to the visual system reported above.
Expression of hSOD1/UAS-Tau(R406W) or hSOD1(A4V)/Tau(R406W) in
the CNS was driven by Elav-Gal4. hSOD1 and hSOD1(A4V) expression was
able to shorten the Tau flies’ lifespan by ~6-10 days (Fig. 2A), and
exacerbated movement impairment (Fig. 2C). Expression of these SOD1
versions alone did not reduce the lifespan. If anything, hSOD1 alone even
slightly elongated the lifespan. These results indicated that Tau-related
neurodegeneration could be aggravated by the expression of hSOD1 or
hSOD1(A4V).
Brain sectioning and a H&E staining study were further performed to
examine the neuronal pathology. Tau toxicity can damage brain neurons and
cause neurodegeneration associated with vacuole formation [24]. When Tau
9
and hSOD1 or hSOD1(A4V) were combined together, the number of vacuoles
significantly increased in flies’ brains (Fig. 2D and 2E) from 45 vacuoles per
brain (Tau flies) to 60 (Tau/hSOD1 flies) or 75 per brain (Tau/hSOD1(A4V)
flies). Therefore hSOD1 and its mutant form hSOD1(A4V) could both
significantly worsen the Tau flies’ phenotypes without similarly affecting the
phenotypes of the normal flies, indicating that Tau toxicity could be relatively
specifically promoted by hSOD1 and hSOD1(A4V).
Tau phosphorylation status were altered by hSOD1 and hSOD1(A4V)
Tau hyperphosphorylation is believed to be one major factor that relates to
its toxicity. In several cases of Down syndrome, hSOD1 was reported to be
overexpressed, leading us to consider the likelihood of hSOD1 affecting the
phosphorylation status of Tau.
To test this possibility, flies’ brains were dissected and total protein
extracts were made from these brains. Tau phosphorylation status was
analyzed with several phosphoepitope-specific antibodies, including AT180,
PHF-1, and pS262. hSOD1 was shown to elevate Tau phosphorylation levels,
especially on pS262 and PHF-1 (pS356 and pS404) sites (Fig. 3A).
hSOD1(A4V) had a similar effect on Tau hyperphosphorylation (Fig. 3A).
These results indicated that Tau phosphorylation could be affected by hSOD1
or hSOD1(A4V) expression, although the detailed mechanism remains unclear
at this stage.
Tau phsophorylation on pS262 and AT8 sites could be regulated by the
JNK kinase [7-8, 27]. We therefore tried to investigate whether
hSOD1-promoted Tau hyperphosphorylation is mediated by this pathway. We
10
examined whether the phophorylation level of JNK and related MAPK [27-30],
while using the pJNK and pMAPK antibodies, was affected by hSOD1
expression. The results showed that pJNK levels were slightly elevated in
Tau/hSOD1 and Tau/hSOD1(A4V) flies as compared with the control Tau flies
(Fig. 3B and 3C). In contrast, the phosphorylation level of MAPK and the total
JNK level was not obviously changed (Fig. 3B, 3C and 3D). This data suggests
that JNK kinase might be involved in mediating the effect of hSOD1 and
hSOD1(A4V) on Tau hyperphosphorylation. Furthermore, the JNK was also
slightly activated in hSOD1 and hSOD1(A4V) within flies (Fig. 3D), indicating
the JNK activation may be directly related with hSOD1 and hSOD1(A4V),
because H2O2 is the product of hSOD1 and hSOD1(A4V) catalatic activity, and
the H2O2 product can activate JNK in cell culture [31-32]. We then fed flies with
20 mM of Vitamine C daily in order to reduce the H2O2 in flies for one month.
The results showed that Vitamine C could sinificantly slow the eyes
degenerative process in the Tau/hSOD1 and Tau/hSOD1(A4V) flies (Fig. 3E),
but did not significantly alter the Tau alone phenotype (Fig. 3E), suggesting
that the catalytic activity of hSOD1 may at least be partly involved in the
process of hSOD1 enhanced Tau toxicity.
Hypophosphorylated Tau(S2A) resists hSOD1 and hSOD1(A4V)
potentiating effects
Tau(S2A), which carries the Ser262Ala and Ser356Ala changes,
eliminates some key phosphorylation sites and is associated with an overall
reduction in phosphorylation and a suppression of much of the toxicity [23].
Using the phosphorylation specific antibodies pS262 and pS356, we confirmed
11
that the phosphorylation on Ser262 and Ser356 was completely abolished for
Tau(S2A) (Fig. 4A).
We then wondered if hSOD1 could affect the toxicity of Tau(S2A). After
being introduced into Tau(S2A) flies, unlike their effects on Tau, hSOD1 and
hSOD1(A4V) could no longer influence Tau(S2A)’s toxicity on the eyes (Fig.
4A); the eyes’ appearance of hSOD1/Tau(S2A) or hSOD1(A4V)/Tau(S2A) was
indistinguishable from that of Tau(S2A,) and they were all largely normal (Fig.
4B). Subsequent lifespan assays revealed a similar scenario: Tau(S2A)’s
lifespan could not be affected by hSOD1 and its’ A4V mutant (Fig. 4C). Both
the lifespans of hSOD1/Tau(S2A) and hSOD1(A4V)/Tau(S2A) were normal
and similar to that of the Tau(S2A) control. In the climbing assay, hSOD1, as
well as hSOD1(A4V), could no longer significantly change the mobility of
Tau(S2A) flies (Fig. 4D). Likewise, neither hSOD1 nor hSOD1(A4V) were able
to dramatically affect the number of brain vacuoles in Tau(S2A) fly (Fig. 4E). In
conclusion, the phosphorylation-abnormal Tau(S2A) is able to insulate itself
from the effect of hSOD1 and hSOD1(A4V), suggesting the toxicity as a result
of coexpression of hSOD1 or hSOD1(A4V) together with Tau is related to Tau
hyperphosphorylation.
Discussion
Tau hyperphosphorylation is considered to be highly related to its’ toxicity
in several kinds of neurodegenerative diseases, and there is convincing
evidence showing that if Tau phosphorylation levels are reduced such as in
Tau(S2A) [33], its toxicity can be dramatically reduced. Hyperphosphorylation
is important to Tau toxicity not only in flies’ model, but also in mammalian
12
models such as in mice [23, 34-35]. In the present study, we found that Tau
phosphorylation could be promoted by hSOD1 and hSOD1(A4V) in the fruit fly
model, and the elevated phosphorylation levels correlated with Tau’s toxicity.
In Down Syndrome as well as ALS patients, Tau hyperphophorylation was
reported and studied [10, 14]. Despite the presence of many genes or loci on
chromosome 21, and the likelihood that some other components on
chromosome 21 are also able to cause Tau hyperphosphorylation [36-37], we
demonstrated SOD1 elevation alone is able to cause a certain degree of
increase in Tau hyperphosphorylation and promotes Tau toxicity. Therefore,
the increased hSOD1 expression in Down Syndrome as a result of trisomy 21
may at least partially contribute to Tau hyperphosphorylation and can lead to
Tau-related toxicity and neurodegeneration. Notably, Tau inclusion, associated
with the phosphorylation, was found at the adult stage instead of the infant
stage in the Down syndrome patients [38]. This suggests that Tau aggregation
might be a late event in the disease process and may play a critic role in the
late onset phenotypes that correlate with severe neurodegeneration and
neuron loss [39].
A significant portion of ALS is caused by mutations in hSOD1. The
hSOD1(A4V) mutant we used in the present study has been reported to cause
a rapidly progressing dominant form of familial ALS [40-41]. Besides findings
reported in this work, we also observed some deleterious effect of this mutant
on aged flies (data not shown). Although Tau hyperphosphorylation has not
been reported in SOD1(A4V)-related ALS patients, it has been found in other
ALS patients [42]. In our study, we found that hSOD1(A4V) could more
potently elevate Tau hyperphosphorylation which resulted in increased Tau
13
toxicity, suggesting the Tau hyperphosphorylation in ALS patient may be
caused by the presence of mutant hSOD1 [43-44]. As a result, the elevated
phosphorylation could lead to Tau toxicity and finally induce the neuron
damage in ALS patients. Considering that Tau may contribute to the
progression of Amyotrophic Lateral Sclerosis and Down Syndrome, the
blockage of Tau hyperphosphorylation and its inclusion formation might be a
helpful way to delay the disease progress.
Another interesting issue that is worth mentioning is how hSOD1 and
hSOD1(A4V) may affect Tau phosphorylation. Several candidate elements, as
described in previous studies, may hold the key. It was reported that
JNK/SAPK were activated in the Ts65Dn mice (the Down Syndrome mouse
that carries a smaller trisomic segment that includes SOD1) [45]. Consistently,
we found that JNK kinase was activated in hSOD1/Tau and hSOD1(A4V)/Tau
flies and suggested that this may have partially contributed the to Tau
hyperphosphorylation in those flies. Other components such as PP2A, one of
the major cellular serine-threonine phosphatases that can affect Tau
phosphorylation suggested to be related with ALS pathology [43], may also
play a role. Nevertheless, an exact functional delineation of the contribution of
these kinases or phosphotase to hSOD1 or hSOD1(A4V) effects on Tau
toxicity still requires further experimentation to elucidate. In our expriments, we
also observed that the hSOD1(A4V) could more significantly promote Tau
toxicity when compared with the wild type hSOD1, suggesting that besides the
catalytic activity of hSOD1(A4V), some other elements may also be involved
with its effect on Tau. Because the hSOD1(A4V) aggregates and therefore
also causes ER-stress responses [46-47], the related stress and stress
14
responses may also contribute to the activation of stress related kinase JNK.
However, whether it is the hSOD1(A4V) aggregation or its related ER-stress
responses that actually contributes to Tau hyperphosphorylation and its
toxicity still requires further study,
Acknowledgement
This study was supported by the National Basic Research Program of
China (2013CB910700) and by the National Science Foundation of China
(31123004). We are grateful to the Bloomington Drosophila Stock Center for
fly stocks, Biomedical Analysis Center of Tsinghua University for their help and
services, Dr. Bingwei Lu of Stanford University for his kind gifts of the Tau flies
and some reagents, and Hong Luo of Tsinghua University for his kind gifts of
some reagents.
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Figures and legends
Fig. 1. hSOD1 promotes Tau toxicity in Drosophila eyes.
Gmr-Gal4 was used to drive Tau and hSOD1 expression in Drosophila eyes.
A) Toxicity-promoting effects of hSOD1 and hSOD1(A4V) on Tau in fly eyes.
Scale bar: 100 μm. The green arrowhead indicates a severe ly damaged part in
the fly’s eye. B) Tangential sections of fly compound eyes. Scale bar: 20 μm.
Green arrowheads mark abnormal ommatidia.
Fig. 2. Tau-related neurodegeneration is enhanced by hSOD1.
Elav-Gal4 was used to drive Tau and hSOD1 expression in Drosophila CNS.
A) Effects of hSOD1 and hSOD1(A4V) on the lifespans of Tau flies. B) The
control indicating the effect of hSOD1 and hSOD1(A4V) on the lifespan of wild
type flies. C) Effects of hSOD1 and hSOD1(A4V) on Tau flies’ mobilities. Data
represent mean ± SEM, *: p<0.05, **: p<0.01. D) H&E stained brain sections of
Tau/hSOD1 or Tau/hSOD1(A4V) flies. The green arrows indicate the
degenerative vacuoles in flies’ brains. Scale bar: 50 μm. (E) The quantification
of (D). Data represent mean ± SEM, *: p<0.05, **: p<0.01.
Fig. 3. Tau phosphorylation level can be altered by hSOD1 and
hSOD1(A4V).
Tau phosphorylation levels in Gmr-Gal4>Tau/hSOD1 and
Gmr-Gal4>Tau/hSOD1(A4V) flies were tested by immunoblotting. AT180,
cp13, pS262, pS356 and PHF1 antibodies were used to detect different
specific phospho-epitopes on Tau. Tau5 was used to indicate the total Tau
protein level, and the Actin level was used as the loading control. B) The
21
phosphorylation levels of Tau-related kinase JNK and MAPK were analyzed by
immunoblotting using the pJNK and pMAPK antibodies. pJNK was slightly
elevated in hSOD1 and hSOD1(A4V) flies, but pMAPK was not significantly
affected. Gmr-Gal4 was used to express proteins in Drosophila eyes, Actin
level and total JNK were used as the loading control. C) The quantification of
(B). Data represent mean ± SEM, *: p<0.05, **: p<0.01, n.s: p>0.05. D) The
control of (B). The phosphorylation levels of JNK in hSOD1, hSOD1(A4V), and
Tau flies were analzed by immunoblotting, tubulin was used as the loading
control. E) The rescue effects of 20mM Vitamine C on Tau/hSOD1 and
Tau/hSOD1(A4V) flies. Shown here are the represented results of the eyes’
phenotypes of Tau, Tau/hSOD1, and Tau/hSOD1(A4V) flies that were fed
either 20mM of Vitamine C or the control food. Gmr-Gal4 was used to express
Tau and hSOD1 on fly eyes.
Fig. 4. Hypophosphorylated Tau(S2A) is insulated from the modifying
effect of hSOD1.
A) Phosphorylation levels of Tau and Tau(S2A) assayed by pS262 and
pS356 antibodies. Tau5 was used to indicate the total Tau levels and Actin
was used as an additional loading control. Gmr-Gal4 was used to express Tau,
Tau(S2A) in Drosophila eyes. B) Effects of hSOD1 and hSOD1(A4V) on the
eyes of Tau and Tau(S2A) flies. Gmr-Gal4 was used to drive the expression of
the target proteins in the eyes. C) Effects of hSOD1 and hSOD1(A4V) on
lifespans of Tau and Tau(S2A) flies. Elav-Gal4 was used to drive Tau in the
Drosophila CNS. D) Effects of hSOD1 and hSOD1(A4V) on Tau and S2A flies’
mobilities. Elav-Gal4 was used to drive Tau or Tau(S2A) specifically