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: zhoubing@mail.tsinghua.edu.cn.

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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

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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

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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

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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

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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

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(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

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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

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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

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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

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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

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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

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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

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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

22

expressed in the Drosophila CNS. E) Quantitative results of brain vacuoles in

the Tau(S2A)/hSOD1 or Tau(S2A)/hSOD1(A4V) flies. Brain sections were

stained with H&E and the numbers of vacuoles were counted. Data represent

mean ± SEM, *: p<0.05, **: p<0.01.