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Human Molecular Genetics Advance Access originally published online on February 19, 2007
Human Molecular Genetics 2007 16(5):555-566; doi:10.1093/hmg/ddm011
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Cytoskeleton proteins are modulators of mutant tau-induced neurodegeneration in Drosophila

Olivier Blard{dagger}, Sébastien Feuillette{dagger}, Jacqueline Bou, Boris Chaumette, Thierry Frébourg, Dominique Campion and Magalie Lecourtois*

Inserm U614 (IFRMP), University of Rouen & Department of Genetics, Rouen University Hospital, Institute for Biomedical Research, Rouen, France

* To whom correspondence should be addressed. Tel: +33 235148304; fax: +33 235148237; Email: magalie.lecourtois{at}univ-rouen.fr

Received December 22, 2006; Revised January 19, 2007; Accepted February 2, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tauopathies, including Alzheimer's disease and fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of aberrantly phosphorylated-tau. Tau is a neuronal microtubule-associated protein involved in microtubule assembly and stabilization. Currently, the molecular mechanisms underlying tau-mediated cellular toxicity remain elusive. To address the determinants of tau neurotoxicity, we first characterized the cellular alterations resulting from the over-expression of a mutant form of human tau associated with FTDP-17 (tau V337M) in Drosophila. We found that the over-expression of tau V337M, in Drosophila larval motor neurons, induced disruption of the microtubular network at presynaptic nerve terminals and changes in neuromuscular junctions morphological features. Secondly, we performed a misexpression screen to identify genetic modifiers of the tau V337M-mediated rough eye phenotype. The screening of 1250 mutant Drosophila lines allowed us to identify several components of the cytoskeleton, and particularly from the actin network, as specific modifiers of tau V337M-induced neurodegeneration. Furthermore, we found that numerous tau modulators identified in our screen were involved in the maintenance of synaptic function. Taken together, these findings suggest that disruption of the microtubule network in presynaptic nerve terminals could constitute early events in the pathological process leading to synaptic dysfunction in tau V337M pathology.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tauopathies are neurodegenerative disorders resulting from dysfunction of the tau/MAPT protein. Tau is a microtubule-associated protein (MAP), which is prominent in neurons and particularly localized in axons. Physiologically, tau plays a key role in regulating microtubule dynamics, axonal transport and neurite outgrowth (1). In tauopathies, tau is abnormally hyperphosphorylated and accumulates as intracellular neuronal filamentous inclusions. Tauopathies can be split into primary tauopathies (e.g. fronto-temporal dementia and parkinsonism linked to chromosome 17 or FTDP-17), which result from mutations in the tau gene, and secondary tauopathies such as Alzheimer's disease, in which only aberrant post-translational modifications of tau protein are detectable. Despite extensive research, the molecular mechanisms underlying tau-mediated cellular toxicity in these conditions remain elusive. Therefore, to address the mechanisms of tau neurotoxicity, we first characterized the cellular alterations resulting from the over-expression of mutant human tau in Drosophila. Then, we conducted a misexpression screen to identify genetic modifiers of the tau-induced neurodegeneration.

To facilitate the recovery of both suppressors or enhancers of tau toxicity, this study was performed using the V337M mutant form of tau, since this mutation has been previously shown to induce a mild neurodegeneration phenotype in Drosophila (2). In human, the V337M mutation within the tau gene had originally been described in a family (the Seattle family A) presenting an FTDP-17 dementia (3). This mutation is associated with predominantly neuronal pathology including neuronal loss and neurofibrillary tangles composed of aggregated forms of phosphorylated-tau. In vitro, the tau V337M mutant protein exhibits a moderate reduction of its affinity for microtubule (4,5), a decreased ability to promote microtubules polymerization (5–7) and a higher rate of tau filament formation than wild-type (8–10). When over-expressed in Drosophila, the human tau V337M mutant gene leads to an accumulation of abnormally phosphorylated and folded tau, age-dependent neurodegeneration and early death (11).

In this study, we found that when over-expressed in larval motor neurons, the mutant tau V337M protein results in abnormal morphological features, impairment of microtubule stability at nerve terminals, but no disruption of axonal transport. Furthermore, the screening of 1250 mutant Drosophila lines allowed us to identify several components of the cytoskeleton, and particularly from the actin network, as specific modifiers of tau V337M-induced rough eye phenotype. Moreover, we found that numerous tau modulators identified in our screen were involved in the maintenance of synaptic function. Taken together, these findings suggest that disruption of the microtubule network in presynaptic nerve terminals could constitute early events in the pathological process leading to synaptic dysfunction in tau V337M pathology.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of tau V337M toxicity in Drosophila
In this study, we used a Drosophila tauopathy model based on the GAL4-UAS expression system (12). The GMR-Gal4 driver was used to drive expression of the mutant form of human tau V337M (h-tauV337M) in all retinal cell types. Expression of h-tauV337M in the retina causes a moderately rough eye phenotype at 25°C, characterized by fused and disordered ommatidia with missing mechanosensory bristles (Fig. 1A–C).


Figure 1
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  		Figure 1. Eye degeneration phenotypes caused by over-expression of human tau V337M and analysis of the phosphorylation status of human tau V337M in Drosophila (A–C) SEM images of transgenic flies expressing h-tauV337M under the control of the eye-specific promoter element GMR-Gal4 (B, female; C, male) and control flies bearing only the GMR-Gal4 transgene (A). Flies over-expressing h-tauV337M developed rough eye phenotype with disorganized ommatidial structure, while flies bearing GMR-Gal4 alone showed normal external eye morphology. (D) The phosphorylation status of h-tauV337M was determined by western blot analysis using phosphorylation-dependent antibodies. The h-tauV337M levels were determined using a mix including T14 and T46, or Tau5 and 5A6, phosphorylation-independent anti-tau antibodies reacting with total h-tau. The AT8, AD2 and PHF1 antibodies recognize phosphorylated h-tau proteins, whereas Tau-1 recognizes non-phosphorylated h-tau proteins. Actin was used as loading control. (E) Western blot analysis of h-tauWT and h-tauV337M expression in transgenic flies. Actin was used as loading control.

 
Since tau toxicity in human is closely related to its phosphorylation status, we first analysed h-tauV337M phosphorylation state in our experimental model. Western blot analyses were performed on soluble and insoluble fractions extracted from adult heads of transgenic flies over-expressing h-tauV337M. Using phosphorylation-independent antibodies specific to human tau (T14/T46 or Tau5/5A6), we detected three main bands of 50–60 kDa in the soluble fraction (Fig. 1D), corresponding to monomeric h-tauV337M proteins. Phosphorylation-specific anti-tau antibodies which are commonly used to detect abnormal tau in patients' brains recognized h-tauV337M expressed in transgenic flies. Indeed, as shown in Figure 1D, the upper-bands of the triplet were recognized by the AT8 (phosphoserine 202 and phosphothreonine 205), AD2 (phosphoserines 396 and 404) and PHF1 (phosphoserines 396 and 404) antibodies. In contrast, the lower migrating species were detected with the Tau-1 antibody that recognizes tau dephosphorylated at serine-195, -198, -199 and -202. No immunoreactivity was detected in the insoluble fraction.

Recently, it has been shown that over-expression of wild-type human tau (h-tauWT) in Drosophila larval motor neurons causes changes in neuromuscular junctions (NMJs) morphological features, disruption of axonal transport and synaptic transmission (13,14). Therefore, we examined the effects of h-tauV337M over-expression on axonal transport and NMJs morphological features. We used the OK6-Gal4 driver line to selectively target the expression of human tau in larval motor neurons. Axons and nerve terminals innervating larval body-wall muscles were visualized by immunodetection of HRP, an insect neuronal marker. To evaluate the effect of human tau over-expression on axonal transport, we analysed the distribution of vesicles in motor axons, using the synaptic vesicle-associated cysteine-string protein (CSP) as a marker. As previously reported (13), we found that h-tauWT expressing nerves exhibited organelle accumulations indicative of axonal transport defects (Fig. 2A, A3, A4) compared with controls (Fig. 2A, A1, A2). Interestingly, although h-tauWT and h-tauV337M transgenes were expressed at similar levels (Fig. 1E), h-tauV337M expressing nerves did not contain prominently stained accumulations of CSP (Fig. 2A, A5, A6). These data showed that the V337M tau mutant, in contrast to the wild-type form of tau, does not cause disruption of axonal transport when over-expressed in larval motor neurons.


Figure 2
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  		Figure 2. Characterization of human tau V337M toxicity on Drosophila larval NMJ. Confocal images projections showing segmental nerves (A) or a portion of NMJ (B) in third instar larvae. The segmental nerves and NMJs were visualized using anti-HRP antibody (red). (A) Expression of h-tauWT resulted in organelle accumulations (anti-CSP, green) within larval segmental nerves (A3–A4). In contrast, expression of h-tauV337M (A5–A6) resulted in a phenotype indistinguishable from control (A1–A2). (B) Microtubules were visualized using an anti-acetylated {alpha}-tubulin antibody, which recognizes only stably polymerized microtubules (green). Compared with control animal (B1–B3), h-tauWT (B4–B6) and h-tauV337M (B7–B12) over-expression impaired microtubule stability as reflected by loss of acetylated {alpha}-tubulin immunoreactivity at motor axon terminals. B3, B6, B9, B11 and B12 are higher magnification of the dashed square in B2, B5, B8 and B10, respectively.

 
Regarding morphological features of NMJ, we found that both h-tauWT (Fig. 2B, B4–B6) and h-tauV337M (Fig. 2B, B7–B12) over-expression resulted in abnormal boutons undergoing more frequent branching, compared with controls (Fig. 2B, B1–B3). However, h-tauV337M over-expression led to a phenotype significantly stronger than that caused by h-tauWT. Moreover, we observed in h-tauV337M expressing larvae that the boutons often formed dense clusters, particularly at the ends of NMJ branches (Fig. 2B, B7–B12). This morphology was rarely observed in wild-type or h-tauWT expressing NMJs, where boutons were arranged more linearly. Interestingly, the boutons clumps that are characteristic of h-tauV337M NMJs exhibited an impairment of microtubule stability, reflected by a loss of acetylated {alpha}-tubulin immunoreactivity (Fig. 2B, B7–B12). In contrast, in wild-type or h-tauWT expressing NMJs, the microtubule network extended into the terminal boutons (Fig. 2B, B1–B6). This finding suggested that the h-tauV337M over-expressing NMJs phenotype could arise from alterations in microtubule networks.

Identification of modifiers of tau V337M toxicity
We crossed the GMR-Gal4 < UAS-h-tauV337M flies with 1250 insertion lines containing P{Mae-UAS.6.11}-transposable elements (15) and assessed the F1 progeny for suppression or enhancement of the eye phenotype. In order to discard any lines that caused rough eye phenotype on their own, all the candidate modifiers were crossed to the GMR-Gal4 line. Finally, 30 lines were identified as containing modifiers of the tau V337M-induced eye degeneration in heterozygous flies (Table 1, Fig. 3). PCR rescue experiments were performed to identify the insertion point of the transposon and its orientation. When possible, UAS transgenic stocks and/or mutant alleles of the candidate loci were tested for modifier activity. In several cases, RT–PCR experiments were performed to demonstrate enhanced expression of the locus under the control of GAL4.


Figure 3
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  		Figure 3. Modifiers of h-tauV337M toxicity. SEM images of genetic modifiers of h-tauV337M toxicity. (A) Eye phenotype of h-tauV337M transgenic fly (UAS-h-tauV337M/+GMR-Gal4/+). Reduction of h-tauV337M toxicity by UY4043Ptp4E (B). Enhancement of h-tauV337M toxicity by UY1135CG13942 (C), UY1501DnaJ-1 (D), UY1152Pax (E) and UY3173jar (F).

 


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  		Table 1. Genetic modifiers of tau V337M-induced toxicity

 
Structural constituents of cytoskeleton
As shown in Table 1, we identified four genes (cheerio, Chd64, jaguar and Paxillin) encoding structural constituents of cytoskeleton. In agreement with previously published data (16), we identified cheerio, a Drosophila ortholog of the actin-binding protein Filamin (FLN), as a tau enhancer. In mammals, the three FLN isoforms play important role in cross-linking cortical actin filaments into a dynamic three-dimensional structure. They also function as signaling scaffold by connecting a large variety of cellular processes to the dynamic regulation of actin cytoskeleton (17). Two other actin-binding proteins were identified in our screen, Chd64 and jaguar. The transposon UY1264 that acts as a tau enhancer is predicted to inactivate Chd64, the fly ortholog of transgelin3 (TAGLN3), which is a neuron-specific protein of unknown function. In rat and human, TAGLN3 has been shown to co-localize with elements of the microtubule and microfilament cytoskeleton, including filamentous actin, {alpha}-tubulin, tau and microtubule-associated protein 2 (MAP2), in processes of neurons (18,19). Human TAGLN3 was also shown to bind directly to microtubules in human brain (19). In addition, we found that UY3173 that is predicted to activate jaguar (jar) expression enhances tau toxicity. We validated this candidate locus using a jaguar loss-of-function allele. Indeed, the mutant allele jar1 suppressed the tau-induced rough eye. Jar is the founding member of class VI myosins (20). Mammalian Myosin VI (MYO6) proteins are motor proteins that move toward the slow growing (minus) ends of actin filaments, transporting vesicles and organelles. Interestingly, it has been recently reported in mice that MYO6 is highly expressed throughout the brain, localized to synapses and enriched at the post-synaptic density (21). Finally, we identified Paxillin (Pax) as an enhancer of tau toxicity. The UY1152 P element is inserted within the Pax transcription unit. We found that over-expression of Pax, using UAS-Pax transgene (22), also enhances tau toxicity. Pax is a focal adhesion-associated protein that functions as a multi-domain adaptor at the interface between the plasma membrane and the actin cytoskeleton. Furthermore, Pax has been shown to directly interact with {alpha}-tubulin (23). This interaction, combined with the ability of Pax to interact with several actin-binding proteins, raises the possibility that paxillin can facilitate cross-talk between these two filament systems.

Molecular chaperones
Three of the modifiers that enhance tau toxicity alter the expression of molecular co-chaperones. Unexpectedly, in all cases, the transposon was inserted proximal of a transcription unit and in the orientation expected to allow ectopic expression. RT–PCR experiments confirmed over-expression of the loci under the control of GAL4. The transposon UY1501 is associated with over-expression of DnaJ-like-1 (DnaJ-1). Using two independent transgenic lines UAS-DnaJ-1 (24) or the DnaJ-1EP411 allele, we confirmed that the over-expression of DnaJ-1 enhances tau toxicity (Fig. 4A–F). This gene encodes a protein homologous to the human co-chaperone HSP40/HDJ-1. The HSP40 co-chaperones represent a large family that functions to specify the cellular action of the stress-induced HSP70 chaperones proteins. The UY3148 insertion identified Cysteine string protein (Csp), another member of HSP40 family. Using the allele CspEP3141, we confirmed that the enhancement of tau-dependent rough eye phenotype was indeed due to the action of Csp (Fig. 4G–I). The synaptic vesicle protein Csp is an essential component of the neurotransmitter release machinery and is crucial for the integrity of synaptic nerve terminals. In Drosophila, analysis of Csp null-mutant revealed a variety of presynaptic defects (25). Biochemical and genetic studies provided compelling evidence that CSP might act as molecular chaperone in cooperation with HSC70 to direct the assembly or dissociation of multi-protein complexes (26,27). Interestingly, the third insertion, UY5268, is associated with the over-expression of the heat-shock protein cognate 70Cb (Hsc70Cb). Hsc70 are constitutively expressed members of the Hsp70 proteins family. Hsc70 has been shown to co-localize with Hsp40 at post-synaptic sites (28).


Figure 4
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  		Figure 4. Enhancement of tauV337M-induced degeneration upon co-expression of chaperones. (A) Eye phenotype of control fly bearing GMR-Gal4/+ alone. (B and C) Flies over-expressing h-tauV337M (UAS-h-tauV337M/+; GMR-Gal4/+) developed rough eye phenotype—female (B) and male (C). (D) Enhancement of tau toxicity by UY1501DnaJ-1 in h-tauV337M transgenic female. (E and F) DnaJ-1 over-expression in h-tauV337M transgenic female bearing EP411 (E) or UAS-DnaJ-1 (F) enhanced tau toxicity. (G) Enhancement of tau toxicity by UY3148Csp in h-tauV337M transgenic male. (H and I) Csp over-expression in h-tauV337M transgenic male bearing EP3141 (H) or EP659 (I) enhanced tau toxicity. (J) Western blot analysis of h-tauV337M expression. The h-tauV337M levels were determined using a mix of T14 and T46 phosphorylation-independent anti-tau antibodies reacting with total h-tau. Actin was used as loading control.

 
Identification of molecular chaperones as enhancers of tau toxicity in our screen was unexpected. Actually, it is generally assumed that molecular chaperones facilitate tau degradation (29–32). Therefore, we performed western blot analysis and found that over-expression of these molecular chaperones did not reduce tau accumulation (Fig. 4J).

Other genetic modifiers of tau V337M toxicity
Only one tyrosine phosphatase was recovered in our screen. The transposon UY4043, which acts as a tau suppressor, is predicted to activate expression of Protein tyrosine phosphatase 4E (Ptp4E). We also found one gene involved in the ubiquitin–proteolytic pathway: Ubiquitin activating enzyme 1 (Uba1). The transposon UY3010 is expected to allow over-expression of Uba1. Uba1 encodes a predicted ubiquitin-activating enzyme (E1) that shares strong sequence similarities with the mammalian UBE1. In Drosophila, Uba1 activity has been shown to be required for axon pruning during metamorphosis (33).

Three of the identified enhancers are genes encoding predicted ion transporting ATPase, including Vha44. This gene encodes a component of vacuolar ATPase (V-ATPase), a multi-subunit enzyme that mediates acidification of intracellular compartments of eukaryotic cells. V-ATPase-dependent acidification is necessary for such intracellular processes as protein sorting, receptor-mediated endocytosis and synaptic vesicle proton gradient generation. We also identified ATP{alpha}, a gene encoding the alpha subunit of the Na+/K+ ATPase. Two insertions affecting the ATP{alpha} locus were recovered in the screen. Both were predicted to activate ATP{alpha} expression. Using two independent transgenic lines UAS-ATP{alpha} (34), we confirmed that ATP{alpha} over-expression modulates tau toxicity. In Drosophila, mutations in ATP{alpha}, cause reduced lifespan, severe neuronal hyperexcitability and pronounced age-dependent neurodegeneration in the central nervous system (35). It is interesting to notice that mutations in ATP1A3, the human homolog of ATP{alpha}, have been recently associated with rapid-onset dystonia parkinsonism. Another gene involved in neuron integrity was also identified as a tau enhancer. The transposon UY1301 is inserted proximal to purity of essence (poe) in the same orientation. Mutations in the Drosophila poe gene confer increased neuronal excitability and spontaneous synaptic vesicle fusion.

Another group of enhancers are proteins that function as chromatin remodeling factors or transcriptional cofactors. This includes CG33097, the fly ortholog of TCERG1 that has been shown to interact with Huntingtin. Notably, it has been reported that specific alleles in TCERG1 locus were only observed in Huntington's disease patients (36). We also identified Enhancer of bithorax (E(bx)), the fly ortholog of Fetal Alzheimer Antigen (FALZ). Using the two alleles E(bx)EP3643 and E(bx)EP637, we confirmed that E(bx) over-expression enhances tau toxicity. We also identified one gene encoding an RNA-binding protein, mushroom-body expressed (mub). The P element acts as a suppressor of tau toxicity and is predicted to allow ectopic expression of mub. Using the allele mubEP3623, we confirmed that over-expression of mub rescues tau toxicity. Mub has been already identified as a modulator of Sca1-induced toxicity (37). Last, our screen has identified eight novel genes with unknown function, including suppressors and enhancers.

Effect of tau V337M modifiers on tau WT toxicity
In order to test the specificity of the h-tauV337M modifiers identified in our screen for this particular mutated form of tau, we tested the ability of these genetic interactors to module the h-tauwt-induced neurodegeneration eye phenotype. As indicated in Table 2, we found that 19/24 modifiers tested act similarly in both Drosophila models. Five of them (5/24) had no effect on the eye phenotype produced by the expression of h-tauwt. Interestingly, most of the modifiers specific to h-tauV337M toxicity encode structural constituents of cytoskeleton or molecular chaperones.


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  		Table 2. Modifiers of tau V337M-induced toxicity tested for tau WT and polyglutamine (SCA3) modification

 
Effect of tau V337M modifiers on polyglutamine toxicity
In this study, we identified three molecular chaperones as h-tauV337M enhancer. Intriguingly, previous studies showed that molecular chaperones act as common suppressors of polyglutamine-induced toxicity. In particular, over-expression of HSP40 suppressed the poly(Q)-induced neurodegeneration phenotype in various Drosophila models (24,37,38). To address this apparent discrepancy, we tested the effect of DnaJ-1 over-expression in a Drosophila polyglutamine disease model, the spinocerebellar ataxia type 3 also known as Machado–Joseph disease (MJD). Expression of truncated and expanded forms of the human MJD protein (MJDtr-Q78) in Drosophila eye causes progressive degeneration reflected externally by a loss of pigmentation (39). In accordance with previous works, we found that DnaJ-1 over-expression, using the UY1501 element or UAS-DnaJ-1 transgenes, totally suppressed the MJDtr-Q78-induced neurodegeneration eye phenotype (Fig. 5E–H). Hence, regarding tau and MJDtr-Q78 toxicity, DnaJ-1 acts in opposite ways.


Figure 5
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  		Figure 5. Molecular chaperones were implicated both in the response in vivo to tau and polyglutamine toxicity, but in a distinct mechanism. (A–D) SEM images illustrating modifications of h-tauV337M-induced eye phenotype upon co-expression of DnaJ-1. (A) Eye phenotype of control fly bearing GMR-Gal4/+ alone. (B) Fly over-expressing h-tauV337M (UAS-h-tauV337M/+; GMR-Gal4/+) developed rough eye phenotype. (C and D) Enhancement of tau toxicity by UY1501DnaJ-1 (C) and UAS-DnaJ-1 (D). (E–H) Dissecting microscope pictures presenting modifications of MJDtr-Q78-induced eye phenotype upon co-expression of DnaJ-1. (E) Eye phenotype of control fly bearing GMR-Gal4/+ alone. (F) MJDtr-Q78 over-expression (GMR-Gal4/+; UAS-MJDtr-Q78/+) resulted in adult eye degeneration, with loss of pigmentation. (G and H) Rescue of tau toxicity by UY1501DnaJ-1 (G) and UAS-DnaJ-1 (H).

 
Therefore, we tested the activity of the 30 h-tauV337M modifiers identified in this study in the Drosophila MJD model (Table 2). Ten modifiers had no effect on the eye phenotype produced by expression of mutant human MJDtr-Q78, suggesting that these genes are not determinants of polyglutamine toxicity. Seventeen act similarly in both Drosophila models: 15 modifiers identified as tau enhancer also enhanced MJDtr-Q78 toxicity, whereas two interactors identified as tau suppressor rescued MJDtr-Q78 toxicity. And finally, we found that the tau modifiers mub and CG4449 had opposite effect in the MJDtr-Q78 model.

Effect of specific tau V337M-induced neurodegeneration modifiers on tau V337M-induced NMJs phenotype
Finally, we tested the effect of the specific h-tauV337M-induced neurodegeneration modifiers, Chd64, Pax, DnaJ-1 and Csp, on the NMJs phenotype. First, we examined the phenotype associated with over-expression of these cytoskeleton or molecular chaperones interactors alone. We found that the over-expression of these interactors in NMJs, under the control of Gal4, resulted in abnormal NMJs morphological features. However, this phenotype was different from that observed in tau V337M over-expressing larvae. Indeed, we neither observed boutons forming dense clusters at the ends of NMJ branches nor impairment of microtubule stability at terminal nerves. When combined with h-tauV337M, we observed a complex phenotype sharing some aspects of the tau V337M-induced phenotype and some others resulting from the over-expression of the interactor (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we first explored the mechanisms underlying the human mutant tau V337M-mediated cellular toxicity in Drosophila. We found that the over-expression of tau V337M, but not of the wild-type form of tau, in Drosophila larval motor neurons induced disruption of the microtubular network at nerve terminals and changes in neuromuscular junctions morphological features. This phenotype, resulting from the over-expression of tau V337M only during four to five days, is partially concordant to that found in 11-month-old transgenic mice expressing V337M human tau, in which a complete loss of microtubules has been described (40). Therefore, the disruption of microtubule network in axon terminals could constitute early events in tau V337M pathology. Our data also showed that the V337M tau mutant, in contrast to the wild-type form of tau (13), does not cause disruption of axonal transport when over-expressed in larval motor neurons. This could be due to its reduced affinity for microtubules (4,5).

Secondly, in our genetic screen, performed on 1250 mutant Drosophila lines, we identified several components of the cytoskeleton, and particularly from the actin network, as modifiers of tau V337M-mediated rough eye phenotype. Cooperation between microtubules and actin cytoskeleton in transport of organelles is an emerging field (41). In neurons, microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Single vesicles can simultaneously possess both microtubules and actin-based motors. For example, it has been shown that the actin-based vesicle-transport motor myosin Va binds to organelles that are transported in axons along microtubules. In addition, myosin Va activity is necessary for local movements or processing of organelles in presynaptic terminals (42,43). Consistent with the expected impact of disruption of cytoskeleton network in presynaptic terminals on synaptic integrity, we found that numerous tau modulators identified in our screen were involved in the maintenance of synaptic function. This includes the genes Csp, poe and ATP{alpha}, whose activities have been shown to be strictly required to prevent synaptic degeneration in Drosophila, and jar/MYO6 that has been already involved in synaptic transmission.

Therefore, we examined if specific modifiers of tau V33M-mediated rough eye phenotype also modified the NMJ phenotype. Unfortunately, these experiments remained inconclusive because the over-expression of these interactors alone also resulted in abnormal NMJ morphological features. Therefore, it was impossible to determine if the NMJ phenotype observed in larvae co-expressing tau V337M and these interactors reflected additive genetic effects or true genetic interaction.

Interestingly, we found that most of the interactors specific for tau V337M toxicity (i.e. with no effect on the eye phenotype produced by the over-expression of the wild-type form of tau) encode structural constituents of cytoskeleton or molecular chaperones. The specificity of the interactors encoding structural constituents of cytoskeleton for tau V337M toxicity was also supported by our data obtained in a Drosophila MJD model. Indeed, most of them had no effect on the eye phenotype produced by expression of mutant human MJDtr-Q78. On the other hand, 17/30 of the tau V337M modifiers identified in this study showed an effect of MJDtr-Q78 toxicity. These data, which differ from those of Shulman and Feany (16), are in accordance with the proposal that different classes neurodegenerative disorders like tauopathies, Parkinson's disease and Huntington's disease, share some pathogenic features (44) but also have specific determinants.

In contrast to a previous Drosophila genetic screen for modifiers of tau toxicity (16), our screen did not allow us to identify modifiers encoding Drosophila homologs of kinases or phosphatases known to phosphorylate or dephosphorylate tau (i.e. GSK-3ß, JNK, Cdk5, Par-1). However, in our experimental condition, we found that the over-expression of GSK-3ß or Par-1 alone, under the control of the GMR-Gal4 driver line, is sufficient to induce a pronounced degeneration eye phenotype. Therefore, we cannot exclude that we eliminated such candidates due to our experimental strategy.

Taken together, these findings suggest that disruption of the microtubule network in nerve terminals could be a key event in the pathological process culminating in neurodegeneration, as exemplified by the rough eye phenotype, in V337M mutants. The contribution of microtubule disruption in tau pathology would require further investigation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Fly stocks and genetics
The enhancer–promoter (EP) strains and mutant stocks were obtained from the Bloomington Drosophila stock center and from the Szeged Drosophila stock center. The following transgenic Drosophila strains were used in this study: UAS-Pax (22), UAS-DnaJ-1 (24), UAS-ATP{alpha} (34), UAS-MJDtr-Q78 (39) and UAS-tauV337M (11). The UAS-tauV337M transgene expresses the V337M mutant form of the human 0N4R tau protein. OK6-Gal4 and GMR-Gal4 lines were kindly provided by Dr C. O'Kane (University of Cambridge, Cambridge, UK) and Dr M.O. Fauvarque (CEA, Grenoble, France), respectively. Drosophila strains were raised on standard cornmeal–yeast agar medium. Fly cultures and crosses were carried out at 25°C.

Scanning electron microscopy
Flies were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 2 h. After three 30 min washes in cacodylate buffer, they were dehydrated in successive baths of 50, 70, 95 and 100% ethanol, for 30 min each. Samples were dried by the critical point method with liquid CO2, sputter-coated with gold and observed with a JEOL JSM 840-A scanning electron microscope (SEM).

Immunoblot analysis
Protein extraction and western blotting were performed according to (45). Briefly, 30 adult fly heads from two-day-old flies were homogenized in 100 µl of RIPA buffer with 1% Nonidet-P40 (v/v). After 1 h at 4°C under agitation, samples were centrifuged. The supernatant was collected as the ‘soluble fraction’ that contains soluble human tau and detergent-extractable insoluble h-tau. The pellet was homogenized in 50 µl of 70% formic acid (FA) and centrifuged. The supernatant was collected, and after evaporation of the FA by Speed Vac, the pellet was resuspended in 50 µl SDS–PAGE sample buffer. This ‘insoluble fraction’ contains detergent-insoluble h-tau that can be extracted with 70% FA. The following anti-human tau antibodies were used in this study: T14 (Zymed Laboratories; South San Francisco, CA, USA; amino acids 83–120, diluted 1:3000) T46 (Zymed Laboratories; amino acids 395–432, diluted 1:3000), Tau5 (Chemicon, Temecula, CA, USA; amino acids 210–230, diluted 1:1000) and 5A6 (Developmental Studies Hybridoma Bank, Ames, IA, USA; amino acids 19–46, diluted 1:6000) which are phosphorylation-independent antibodies that recognize total tau, AT8 (Innogenetics, Gand, Belgium; diluted 1:500) which specifically detect tau phosphorylated at epitopes serine 202 and threonine 205, AD2 (Dr André Delacourte; diluted 1:1000) and PHF1 (Dr Peter Davies; diluted 1:200) which specifically detect tau-phosphorylated at serine 396 and serine 404 and Tau-1 (Dr Lester Binder, diluted 1:2000) which specifically detect tau-phosphorylated dephosphorylated at serines 195, 198, 199 and 202. The JLA20 antibody (Developmental Studies Hybridoma Bank; diluted 1:200) was used to detect actin. The membranes were incubated with peroxidase-labeled anti-mouse IgG (1:10 000) from Jackson Immunoresearch Laboratories (WestGrove, PA, USA), and signals were detected with chemiluminescence reagents (Amersham Biosciences, Buckinghamshire, UK).

Immunochemistry and confocal microscopy
Wandering third instar larvae were dissected in phosphate buffered-saline solution, and then fixed in 4% paraformaldehyde for 20 min. The following antibodies were used: mouse monoclonal anti-CSP (1:10, K. Zinsmaier, University of Arizona, Tucson, AZ, USA), mouse monoclonal anti-acetylated tubulin (clone 6-11B-1, 1:1000, Sigma–Aldrich, St Louis, MO, USA) and rabbit polyclonal anti-horse radish peroxidase (HRP) (1:1000; Sigma–Aldrich). Fluorescent-conjugated antibodies were obtained from Molecular Probes (Eugene, OR, USA). Confocal images were acquired using a CLSM Leica laser-scanning confocal microscope.

Real-time quantitative RT–PCR
For each fly sample, 20 heads (10 male and 10 female heads) were first homogenized with an Ultra Turrax homogenizer (Ika Labortechnik, Staufen, Germany) and then mRNA were extracted using the QuickPrep MicromRNA Purification Kit (Amersham Biosciences). mRNA (50 ng) were first treated with Deoxyribonuclease I Amplification Grade (Sigma–Aldrich) and then reverse-transcribed into cDNA, using the First Strand cDNA Synthesis Kit (Amersham Biosciences). PCR reactions were performed in a final volume of 25 µl, using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with 300 nM primers (primer sequences available upon request) designed using the Primer Express 2.0 Software (Applied Biosystems). PCR amplifications were performed using an Applied Biosystems 7300 Real Time PCR System. The comparative Ct method (Applied Biosystems) was then used to determine quantitative values for gene expression levels in each sample using Rpl32 gene to normalize for different starting template amounts.


   ACKNOWLEDGEMENTS
 
We thank Drs Nancy Bonini, Marie-Odile Fauvarque, Mel Feany, Parsa Kazemi, Cahir O'Kane, Paul Salvaterra and Jeffrey Settleman, the Bloomington Drosophila stock center and the Szeged Drosophila stock center for reagents and fly stocks. O.B. was supported by the ‘Conseil Régional de Haute Normandie’ and ‘France Alzheimer’ association. S.F. was supported by Sanofi-Aventis and the ‘France Alzheimer Finistère’ association.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Johnson G.V. and Stoothoff W.H. (2004) Tau phosphorylation in neuronal cell function and dysfunction. J. Cell. Sci. 117:5721–5729.[Abstract/Free Full Text]

  2. Chau K.W., Chan W.Y., Shaw P.C., Chan H.Y. (2006) Biochemical investigation of Tau protein phosphorylation status and its solubility properties in Drosophila. Biochem. Biophys. Res. Commun. 346:150–159.[CrossRef][ISI][Medline]

  3. Poorkaj P., Bird T.D., Wijsman E., Nemens E., Garruto R.M., Anderson L., Andreadis A., Wiederholt W.C., Raskind M., Schellenberg G.D. (1998) Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol. 43:815–825.[CrossRef][ISI][Medline]

  4. Nagiec E.W., Sampson K.E., Abraham I. (2001) Mutated tau binds less avidly to microtubules than wild-type tau in living cells. J. Neurosci. Res. 63:268–275.[CrossRef][ISI][Medline]

  5. Hong M., Zhukareva V., Vogelsberg-Ragaglia V., Wszolek Z., Reed L., Miller B.I., Geschwind D.H., Bird T.D., McKeel D., Goate A., et al. (1998) Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282:1914–1917.[Abstract/Free Full Text]

  6. Hasegawa M., Smith M.J., Goedert M. (1998) Tau proteins with FTDP-17 mutations have a reduced ability to promote microtubule assembly. FEBS Lett. 437:207–210.[CrossRef][ISI][Medline]

  7. Dayanandan R., Van Slegtenhorst M., Mack T.G., Ko L., Yen S.H., Leroy K., Brion J.P., Anderton B.H., Hutton M., Lovestone S. (1999) Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation. FEBS Lett. 446:228–232.[CrossRef][ISI][Medline]

  8. Nacharaju P., Lewis J., Easson C., Yen S., Hackett J., Hutton M., Yen S.H. (1999) Accelerated filament formation from tau protein with specific FTDP-17 missense mutations. FEBS Lett. 447:195–199.[CrossRef][ISI][Medline]

  9. Goedert M., Jakes R., Crowther R.A. (1999) Effects of frontotemporal dementia FTDP-17 mutations on heparin-induced assembly of tau filaments. FEBS Lett. 450:306–311.[CrossRef][ISI][Medline]

  10. Gamblin T.C., King M.E., Dawson H., Vitek M.P., Kuret J., Berry R.W., Binder L.I. (2000) In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants. Biochemistry 39:6136–6144.[CrossRef][Medline]

  11. Wittmann C.W., Wszolek M.F., Shulman J.M., Salvaterra P.M., Lewis J., Hutton M., Feany M.B. (2001) Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science 293:711–714.[Abstract/Free Full Text]

  12. Brand A.H. and Perrimon N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415.[Abstract]

  13. Mudher A., Shepherd D., Newman T.A., Mildren P., Jukes J.P., Squire A., Mears A., Drummond J.A., Berg S., MacKay D., et al. (2004) GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila. Mol. Psychiatry 9:522–530.[CrossRef][ISI][Medline]

  14. Chee F.C., Mudher A., Cuttle M.F., Newman T.A., MacKay D., Lovestone S., Shepherd D. (2005) Over-expression of tau results in defective synaptic transmission in Drosophila neuromuscular junctions. Neurobiol. Dis. 20:918–928.[CrossRef][ISI][Medline]

  15. Monnier V., Girardot F., Cheret C., Andres O., Tricoire H. (2002) Modulation of oxidative stress resistance in Drosophila melanogaster by gene overexpression. Genesis 34:76–79.[CrossRef][ISI][Medline]

  16. Shulman J.M. and Feany M.B. (2003) Genetic modifiers of tauopathy in Drosophila. Genetics 165:1233–1242.[Abstract/Free Full Text]

  17. Feng Y. and Walsh C.A. (2004) The many faces of filamin: a versatile molecular scaffold for cell motility and signalling. Nat. Cell. Biol. 6:1034–1038.[CrossRef][ISI][Medline]

  18. Depaz I.M., de Las Heras R., Kroon P.A., Wilce P.A. (2005) Changes in neuronal protein 22 expression and cytoskeletal association in the alcohol-dependent and withdrawn rat brain. J. Neurosci. Res. 81:253–260.[CrossRef][ISI][Medline]

  19. de Las Heras R., Depaz I., Jaquet V., Kroon P., Wilce P.A. (2007) Neuronal protein 22 colocalises with both the microtubule and microfilament cytoskeleton in neurite-like processes. Brain Res. 1128:12–20.[CrossRef][ISI][Medline]

  20. Kellerman K.A. and Miller K.G. (1992) An unconventional myosin heavy chain gene from Drosophila melanogaster. J. Cell. Biol. 119:823–834.[Abstract/Free Full Text]

  21. Osterweil E., Wells D.G., Mooseker M.S. (2005) A role for myosin VI in postsynaptic structure and glutamate receptor endocytosis. J. Cell. Biol. 168:329–338.[Abstract/Free Full Text]

  22. Chen G.C., Turano B., Ruest P.J., Hagel M., Settleman J., Thomas S.M. (2005) Regulation of Rho and Rac signaling to the actin cytoskeleton by paxillin during Drosophila development. Mol. Cell. Biol. 25:979–987.[Abstract/Free Full Text]

  23. Herreros L., Rodriguez-Fernandez J.L., Brown M.C., Alonso-Lebrero J.L., Cabanas C., Sanchez-Madrid F., Longo N., Turner C.E., Sanchez-Mateos P. (2000) Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton. J. Biol. Chem. 275:26436–26440.[Abstract/Free Full Text]

  24. Kazemi-Esfarjani P. and Benzer S. (2000) Genetic suppression of polyglutamine toxicity in Drosophila. Science 287:1837–1840.[Abstract/Free Full Text]

  25. Bronk P., Nie Z., Klose M.K., Dawson-Scully K., Zhang J., Robertson R.M., Atwood H.L., Zinsmaier K.E. (2005) The multiple functions of cysteine-string protein analyzed at Drosophila nerve terminals. J. Neurosci. 25:2204–2214.[Abstract/Free Full Text]

  26. Chamberlain L.H. and Burgoyne R.D. (2000) Cysteine-string protein: the chaperone at the synapse. J. Neurochem. 74:1781–1789.[CrossRef][ISI][Medline]

  27. Zinsmaier K.E. and Bronk P. (2001) Molecular chaperones and the regulation of neurotransmitter exocytosis. Biochem. Pharmacol. 62:1–11.[CrossRef][ISI][Medline]

  28. Suzuki T., Usuda N., Murata S., Nakazawa A., Ohtsuka K., Takagi H. (1999) Presence of molecular chaperones, heat shock cognate (Hsc) 70 and heat shock proteins (Hsp) 40, in the postsynaptic structures of rat brain. Brain Res. 816:99–110.[CrossRef][ISI][Medline]

  29. Shimura H., Miura-Shimura Y., Kosik K.S. (2004) Binding of tau to heat shock protein 27 leads to decreased concentration of hyperphosphorylated tau and enhanced cell survival. J. Biol. Chem. 279:17957–17962.[Abstract/Free Full Text]

  30. Dou F., Netzer W.J., Tanemura K., Li F., Hartl F.U., Takashima A., Gouras G.K., Greengard P., Xu H. (2003) Chaperones increase association of tau protein with microtubules. Proc. Natl Acad. Sci. USA 100:721–726.[Abstract/Free Full Text]

  31. Shimura H., Schwartz D., Gygi S.P., Kosik K.S. (2004) CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J. Biol. Chem. 279:4869–4876.[Abstract/Free Full Text]

  32. Petrucelli L., Dickson D., Kehoe K., Taylor J., Snyder H., Grover A., De Lucia M., McGowan E., Lewis J., Prihar G., et al. (2004) CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum. Mol. Genet. 13:703–714.[Abstract/Free Full Text]

  33. Watts R.J., Hoopfer E.D., Luo L. (2003) Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system. Neuron 38:871–885.[CrossRef][ISI][Medline]

  34. Sun B., Xu P., Wang W., Salvaterra P.M. (2001) In vivo modification of Na(+),K(+)-ATPase activity in Drosophila. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 130:521–536.[CrossRef][Medline]

  35. Palladino M.J., Bower J.E., Kreber R., Ganetzky B. (2003) Neural dysfunction and neurodegeneration in Drosophila Na+/K+ ATPase alpha subunit mutants. J. Neurosci. 23:1276–1286.[Abstract/Free Full Text]

  36. Chattopadhyay B., Ghosh S., Gangopadhyay P.K., Das S.K., Roy T., Sinha K.K., Jha D.K., Mukherjee S.C., Chakraborty A., Singhal B.S., et al. (2003) Modulation of age at onset in Huntington's disease and spinocerebellar ataxia type 2 patients originated from eastern India. Neurosci. Lett. 345:93–96.[CrossRef][ISI][Medline]

  37. Fernandez-Funez P., Nino-Rosales M.L., de Gouyon B., She W.C., Luchak J.M., Martinez P., Turiegano E., Benito J., Capovilla M., Skinner P.J., et al. (2000) Identification of genes that modify ataxin-1-induced neurodegeneration. Nature 408:101–106.[CrossRef][Medline]

  38. Takeyama K., Ito S., Yamamoto A., Tanimoto H., Furutani T., Kanuka H., Miura M., Tabata T., Kato S. (2002) Androgen-dependent neurodegeneration by polyglutamine-expanded human androgen receptor in Drosophila. Neuron 35:855–864.[CrossRef][ISI][Medline]

  39. Warrick J.M., Paulson H.L., Gray-Board G.L., Bui Q.T., Fischbeck K.H., Pittman R.N., Bonini N.M. (1998) Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila. Cell 93:939–949.[CrossRef][ISI][Medline]

  40. Tanemura K., Murayama M., Akagi T., Hashikawa T., Tominaga T., Ichikawa M., Yamaguchi H., Takashima A. (2002) Neurodegeneration with tau accumulation in a transgenic mouse expressing V337M human tau. J. Neurosci. 22:133–141.[Abstract/Free Full Text]

  41. Goode B.L., Drubin D.G., Barnes G. (2000) Functional cooperation between the microtubule and actin cytoskeletons. Curr. Opin. Cell. Biol. 12:63–71.[CrossRef][ISI][Medline]

  42. Huang J.D., Brady S.T., Richards B.W., Stenolen D., Resau J.H., Copeland N.G., Jenkins N.A. (1999) Direct interaction of microtubule- and actin-based transport motors. Nature 397:267–270.[CrossRef][Medline]

  43. Bridgman P.C. (1999) Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex. J. Cell. Biol. 146:1045–1060.[Abstract/Free Full Text]

  44. Trojanowski J.Q. and Lee V.M. (2000) ‘Fatal attractions’ of proteins:)aa comprehensive hypothetical mechanism underlying Alzheimer's disease and other neurodegenerative disorders. Ann. NY Acad. Sci. 924:62–67.[Abstract/Free Full Text]

  45. Blard O., Frebourg T., Campion D., Lecourtois M. (2006) Inhibition of proteasome and Shaggy/Glycogen synthase kinase-3beta kinase prevents clearance of phosphorylated tau in Drosophila. J. Neurosci. Res. 84:1107–1115.[CrossRef][ISI][Medline]


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