Wild-type and A315T mutant TDP-43 exert differential neurotoxicity ...

Wild-type and A315T mutant TDP-43 exertdifferential neurotoxicity in a Drosophilamodel of ALS

Patricia S. Estes1, Ashley Boehringer1, Rebecca Zwick1, Jonathan E. Tang1, Brianna Grigsby1

and Daniela C. Zarnescu1,2,3,∗

1Department of Molecular and Cellular Biology, 2Department of Neuroscience and 3Department of Neurology,

University of Arizona, Tucson, AZ 85721, USA

Received October 22, 2010; Revised March 4, 2011; Accepted March 23, 2011

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causativelocus and as a marker of pathology. With several missense mutations being identified within TDP-43, effortshave been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms.Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapi-tulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here wereport a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression inDrosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts moresevere effects on neuromuscular junction architecture, viability and motor neuron loss compared with theA315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indi-cating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is thesole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and geneticinteraction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype andsuggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require thecytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 cha-perone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypicconsequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations arecritical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is an adult-onset, pro-gressive neurodegenerative disorder characterized by motorneuron dysfunction, which leads to paralysis and respiratoryfailure followed by death, generally within 5 years fromdiagnosis. About 20% of all ALS patients also exhibit fronto-temporal lobar degeneration, which is characterized byneurodegeneration of the frontal and temporal lobes (1).Approximately 10% of all ALS cases are inherited (familialALS, fALS) and have been linked to a number of loci, includ-ing superoxide dismutase (SOD1), alsin (a GPTase), senataxin

(a DNA/RNA helicase), VAMP/synaptobrevin-associatedprotein B, P150 dynactin, angiogenin, TAR DNA-bindingprotein (TDP-43) and FUsed in Sarcoma (Fus) (2–8). Theremaining 90% of ALS cases are sporadic (sALS) andremain poorly understood.

Extensive pathological studies have identified TDP-43 as acommon component of cytoplasmic inclusions found in almostall non-SOD1 cases of ALS studied to date (9–11) as well asin other neurodegenerative disorders (reviewed in 1). Histo-logical examinations of human tissue obtained at autopsyhave defined distinct subtypes of TDP-43-positive cytoplasmicinclusions ranging in shape from filamentous to round

∗To whom correspondence should be addressed at: Departments of Molecular and Cellular Biology, Neuroscience and Neurology, 1007 E LowellStreet, Life Sciences South 552, University of Arizona, Tucson, AZ 85721, USA. Tel: +1 5206261478; Fax: +1 5206213709; Email: [email protected]

# The Author 2011. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2011, Vol. 20, No. 12 2308–2321doi:10.1093/hmg/ddr124Advance Access published on March 26, 2011

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aggregates that are present in neurons and sometimes in thesurrounding glia (12). Recently, several articles reported theidentification of TDP-43 gene mutations in both fALS andsALS patients of diverse ethnicities (3,11,13–18). Thus,TDP-43 has emerged as a common denominator for themajority of ALS cases known to date, and studying its func-tion has the potential to provide valuable insights into thepathology of neurodegeneration.

TDP-43 protein consists of two RNA recognition motifs(RRM1 and 2) as well as a glycine-rich domain within the Cterminus (19). In vitro assays have demonstrated that TDP-43binds with high-affinity UG-rich sequences, consistent with arole in mRNA splicing (20). Except for a single mutationfound in the first RNA-binding domain of TDP-43, all othermutations found in ALS patients lie in the C-terminus, includ-ing the glycine-rich domain (3,17,18). These mutations areamino acid substitutions that are thought to increase TDP-43phosphorylation and target it for degradation by the proteasome(3). The TDP-43 protein is ubiquitously expressed andco-localizes with Survival of Motor Neuron (SMN) andgemin proteins in the nucleus. Its cellular functions are justbeginning to be understood and include transcriptional repres-sion, splicing, miRNA biogenesis, apoptosis and cell division(reviewed in 1). In cultured neurons, TDP-43 associates withRNA granules and co-purifies with beta-actin and CaMKIImRNAs. Furthermore, TDP-43 co-localizes with fragile Xmental retardation protein (FMRP) and Staufen in an activity-dependent manner, suggesting that TDP-43 may regulatesynaptic plasticity in vivo by controlling the transport andsplicing of synaptic mRNAs (21).

Recently, an avalanche of articles demonstrated the require-ment for TDP-43 function in various aspects of neuronaldevelopment and function in Drosophila neurons (22–26).Loss-of-function and overexpression studies showed thatboth a TDP-43 deficit and excess human TDP-43 (hTDP-43)lead to a decrease in the size of the larval neuromuscular junc-tion (NMJ) as well as reduced motility (23,25). In contrast,overexpression of hTDP-43 in the dendritic arborizationneurons leads to an overgrown dendritic arbor, a phenotypewhich is less pronounced when the M337V or Q331K ALSvariants of TDP-43 are overexpressed (26). Recently, a com-parison of cellular and functional phenotypes resulting fromexpression of various TDP-43 variants revealed a requirementfor TDP-43′s RNA-binding activity in neurons (22). Althoughthese and other recent reports demonstrate the presence ofseveral features of ALS pathology in Drosophila, worms,mice or zebrafish models (27–30), more clarity is neededregarding the phenotypic consequences of overexpressingwild-type versus missense mutations of TDP-43 in neurons.

Here we report the direct comparison of a range of pheno-types produced by expressing wild-type and A315T mutantTDP-43 in the Drosophila nervous system. We also showthat TDP-43 neurotoxicity is modulated by the proteasome,HSP70 chaperone and apoptotic pathways. We generatedtransgenic Drosophila expressing either fly or hTDP-43 var-iants in two neuronal models: the retina and motor neurons.We found that TDP-43 expression in photoreceptor neuronsleads to the formation of cytoplasmic and axonal aggregatesin developing retina. Adult eyes expressing TDP-43 variantsexhibit progressive neuronal loss and neurodegeneration in a

dose-dependent manner. When comparing transgenic linesthat express TDP-43 at similar levels, we found that the Dro-sophila variants are more potent than their human counter-parts. Similar differential effects between fly and hTDP-43as well as between wild-type and the A315T allele werefound in motor neurons. Notably, wild-type DrosophilaTDP-43 (TBPH) expression in motor neurons led to a reloca-lization from the nucleus, where it is normally found, to thecytoplasm, where it formed visible aggregates. In contrast,the TBPH A315T mutant as well as the hTDP-43 variantsremained restricted to the nucleus regardless of their level ofexpression (i.e. moderate versus high). For both Drosophilaand human variants, wild-type TDP-43 expression was moredetrimental than A315T to viability and motor neuron survi-val, as evidenced by earlier lethality and neuronal apoptosisphenotypes in the nervous system. When expressed at compar-able levels with wild-type TBPH, the A315T TBPH variantwas restricted to the nucleus and showed modest evidencefor neuronal death. Although these results might suggest adirect correlation between the amount of mislocalized (cyto-plasmic) TDP-43 and apoptosis in motor neurons, our analysesof the hTDP-43 transgenes indicate that cell death can occur inthe absence of cytoplasmic aggregation and is likely due toneurotoxic effects caused by excess wild-type TDP-43 or thepresence of the A315T allele. Interestingly, although boththe wild-type and A315T alleles of hTDP-43 remain restrictedto the nucleus, expression of wild-type hTDP-43 (hwt) resultsin a dramatic loss of motor neurons, but expression of thehA315T mutant leads to just a few cells expressing markersof apoptosis. Adult survival and climbing ability also appearto be more severely affected by overexpression of wild-typeTDP-43 than that of the A315T variant. In contrast, larvalturning behavior is significantly more affected by expressionof A315T than that of wild-type TDP-43. Taken together,our results demonstrate that wild-type and A315T mutantTDP-43 exert differential toxicity. The wild-type allele hasmore dramatic effects on neuronal death, adult survival andclimbing ability, whereas the A315T allele has a more pro-nounced effect on larval turning behavior, which requirescomplex motor neuron coordination across anterior–posteriorand dorsal–ventral axes. Our work also reveals a dose-dependent effect of TDP-43 expression as evidenced by thedirect correlation between TDP-43 protein levels and neuro-toxic phenotypes. RNA interference (RNAi) and geneticinteraction experiments show that wild-type and A315T over-expression mimic a loss-of-function phenotype and suggest adominant-negative effect. Finally, genetic manipulations ofproteasome function, the HSP70 chaperone and caspaseinhibitors suggest that TDP-43 toxicity is modulated byproteasome-mediated degradation, protein folding and theapoptosis pathways.

RESULTS

Overexpression of wild-type and A315T mutant TDP-43in the eye leads to neurodegeneration accompaniedby cell loss

To directly compare the in vivo phenotypes of wild-type TDP-43with the effects of individual missense mutations found in ALS

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patients (3), we generated transgenic flies expressing both wild-type and mutant TDP-43 under the control of the UAS promoter.This strategy allows for tissue-specific expression duringDrosophila development using the bipartite Gal4-UAS system(31). Using the GMR Gal4 driver, we expressed wild-type andA315T mutant human and TBPH variants in the developingretina. After surveying several transgenic lines for eachTDP-43 variant, we chose wild-type and A315Tmutant-expressing lines (one of each) that are comparable inlevels of expression (Fig. 1G and H for hTDP-43, and Sup-plementary Material, Fig. S1 for TBPH). In addition, wechose a higher expressing A315T line (hA315T HE, Fig. 1Gand H). As shown in Figure 1A–C, expression of hTDP-43(wild-type and A315T mutant) has no visible phenotype in theeye at 258C. Despite the absence of surface phenotypes,retinal sections through the adult eyes exhibit evidence of cellloss and neurodegeneration (Fig. 1A′ –C′), suggesting thathTDP-43 expression is toxic in the retina. These results weresubstantiated by our findings that when expressed at higherlevels (by raising the flies at 298C, which increases Gal4activity), both wild-type and A315T mutant hTDP-43expression led to visible signs of retinal neurodegeneration(Fig. 1D–F). Plastic sections through these retina showedclear evidence of cell loss (Fig. 1D′ –F′). Similar results wereobtained with additional hTDP-43 transgenic lines (includinghA315T HE), suggesting that the milder phenotypes at 258Care likely due to the presence of a threshold for hTDP-43 toxicityrather than a positional effect (due to the randomness of trans-gene insertion in the genome). A similar dose response wasobserved for wild-type TBPH, which leads to severe eye neuro-degeneration at 258C but is 100% lethal when overexpressed athigher levels (by raising the flies at 298C). We also found thatretinal cell loss has an aging component, as suggested by thefinding that hTDP-43-expressing adults (both wild-type andA315T), despite showing little or no visible phenotypes at258C when 1–3 days old (DO) (Fig. 1B and C), they all

exhibited a visible loss of pigmentation at 5 DO. This phenotypeworsens as animals age (compare Fig. 1B with 7A and Fig. 1Cwith 7E).

Taken together, these results indicate that when expressed atcomparable levels, Drosophila and hTDP-43 overexpressionhave similar consequences, although the fly transgenes appearto be more potent than their human equivalents. Thus, althoughthe TDP-43 gene, TBPH, is highly conserved in Drosophila, ourdata suggest that some differences may exist between the fly andhTDP-43 pathways in vivo. Indeed, flies harbor an additional,previously unreported TDP-43 homolog, namely CG7804,which is 41.6% identical to TBPH and 34.5% identical tohTDP-43 (Supplementary Material, Fig. S2). The presence ofCG7804 may account for the fact that the wild-type TBPH trans-gene is more toxic than hwt despite being expressed at compar-able levels (Supplementary Material, Fig. S1). Our results alsosupport the concept that TDP-43 and its toxicity in vivo aredose- and age-dependent.

Wild-type and mutant TDP-43 accumulate in axonalaggregates in the developing eye

One of the hallmarks of ALS pathology is the accumulation ofcytoplasmic inclusions containing TDP-43 (32). Previousreports include both the presence and absence of TDP-43aggregates in models ranging from yeast to flies, zebrafish,cultured neurons and mice (22–24,27–29,33,34). We alsoasked whether overexpression of wild-type TDP-43 and theA315T mutant transgenes alters the subcellular localizationof TDP-43 and leads to the formation of cytoplasmic aggre-gates. To this end, we compared the distribution of individual,YFP-tagged hTDP-43 transgenes (wild-type and A315T) withnuclear GFP. As seen in Figure 2, both TDP-43 variantsaccumulate in axonal aggregates when expressed in develop-ing eye discs (Fig. 2A–F′, arrowheads). When examiningtheir subcellular distribution in comparison with GFP-NLS

Figure 1. Overexpression of human wild-type and A315T mutant TDP-43 leads to neurodegeneration in the adult retina. (A–F) Expression of hwt and hA315Tresults in normal surface phenotypes at 258C (B and C compared with A). Both hTDP-43 transgenes exhibit strong surface phenotypes when expressed at higherlevels (298C, see E and F) compared with controls (D). Genotypes as indicated. Anterior right, dorsal up. (A′ –F′) Corresponding plastic sections indicate thatregardless of the surface phenotype, the retinas undergo cell loss. Note large areas of cell loss within the retina (arrows). Adult eyes shown are from 1–2-day-oldflies. (G) Western blot analyses showing hTDP-43 expression. Note similar levels of TDP-43 expression for the wild-type (hwt) and A315T (hA315T) transgenesas well as increased expression levels in an additional hA315T line (hA315T HE). Genotypes as indicated, on top. Blotting antibodies as indicated on the right.Tubulin was used as a loading control. (H) Quantification of relative protein levels from Western blot analysis. Scale bar (A′): 30 mm.

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at high magnification, we found that all TDP-43 variants usedin this study exhibit some nuclear as well as some cytoplasmicpresence (compare Fig. 2D′′ with E′′ and F′′). When comparingtheir subcellular localization, the A315T mutant appearsdiffuse (asterisk in Fig. 2F′′) in comparison with wild-typeTDP-43, which exhibits a more particulate distribution (arrow-head in Fig. 2E′′). Similar results were obtained expressingTBPH (Supplementary Material, Fig. S2). These data indicatethat all transgenes examined in this study exhibit some levelof redistribution from the nucleus into the axons of thedeveloping retina.

Wild-type TBPH accumulates in cytoplasmic aggregateswhen expressed in motor neurons

We next compared the subcellular distribution of TDP-43variants in motor neurons. Using the D42 Gal4 driver (35),we expressed YFP-tagged wild-type and A315T hTDP-43

(Fig. 3B–B′′ and C–C′′, respectively) as well as RFP-tagged,wild-type and A315T TBPH (Fig. 3D–D′′ and E–E′′, respect-ively) and then compared their localization with that ofnuclear GFP (GFP-NLS, Fig. 3A–A′′) or RFP (RFP-NLS,data not shown). These experiments showed that whenexpressed at similar levels, the hTDP-43 variants remainedrestricted to the nucleus (compare white arrows in Fig. 3A–C′).Although some cells expressing hwt exhibit a ring-like distri-bution at the edge of the nucleus (arrowheads in Fig. 3B andB′), hA315T is clearly restricted to the nucleus (arrows inFig. 3C and C′). Interestingly, a fraction of wild-type TBPHredistributed to the cytoplasm and formed visible aggregateswithin the neuropil (red arrows in Fig. 3D′), whereas theTBPH A315T allele was mostly restricted to the nucleus(arrows in Fig. 3E and E′). Thus, of all transgenes used inthis study, wild-type TBPH is the sole variant to exhibit a pro-nounced exit from the nucleus. These data indicate that motorneurons handle expression of Drosophila and human, both

Figure 2. Overexpression of TDP-43 in larval eye imaginal discs leads to altered cytoplasmic localization and axonal aggregates. (A–F) Single-confocal slices(1 mm each) showing hTDP-43 localization when expressed in the developing retina with GMR-Gal4. hTDP-43 visualized via individual fluorescent tags [asindicated (B, C and E–F′′), compare with GFP-NLS (A, D–D′′)]. Filamentous actin labeled with phalloidin (phall), and DNA stained with Hoechst (as indi-cated). Note TDP-43 aggregates in axons (arrowheads). (D′ –F′) High magnification views of optic stalk show TDP-43 aggregates in axons (white arrowheads,D′ –F′), whereas GFP-NLS remains restricted to nuclei (D′). (D′′ –F′′) High-magnification insets showing the localization of GFP-NLS (D′′) and TDP-43 wt andA315T in relation to the nucleus (E′′ –F′′). Stainings as indicated. Note that hwt forms more pronounced aggregates than hA315T, which appears more diffuselydistributed (asterisk). Individual nuclei are circled in red. Red arrowheads indicate some amount of depletion from the nucleus hwt. Scale bar (A): 50 mm.




wild-type and A315T mutant TDP-43 differently than photo-receptor neurons where all TDP-43 variants accumulated inaxonal aggregates.

Motor neurons expressing TDP-43 variants exhibitmorphological defects at the NMJ synapse

To determine whether TDP-43 overexpression has an impact onthe ability of motor neurons to form synaptic connections, weexamined the morphology of the larval NMJ (Fig. 4). Thelarval NMJ synapse at muscles 6/7 consists of structural varic-osities referred to as type 1s and type 1b synaptic boutons thatform when motor neuron terminals innervate the surface ofpost-synaptic muscles (36). We labeled the pre-synaptic motorneuron membrane at the NMJ with horseradish peroxidase(HRP) antibodies and the synaptic vesicles within boutonswith cysteine string protein (CSP) antibodies (37) (Fig. 4A–D′′ and E–H′′). Using these markers, we measured the effectof TDP-43 variants on the overall size of the NMJ synapse byquantifying the total number of boutons (as marked by HRP),the area occupied by synaptic vesicles (as indicated by CSP)as well as the number of satellite boutons and axonal branches(using both CSP and HRP labels) (Fig. 4I). First, quantificationof the number of type 1s and 1b as well as the total number ofboutons (1s plus 1b) per muscle area showed that overexpres-sion of hwt led to a significant decrease in the total numberof boutons (Fig. 4I). When equal levels of hA315T proteinwere expressed at the NMJ, we found no significant changesin bouton numbers (Fig. 4I). However, when higher levels ofA315T (hA315T HE; Fig. 1G and H) were expressed, therewas a significant decrease in the number of synaptic boutonsat the NMJ, similar to the effect of the lower expressinghuman wild-type TDP-43 line (Fig. 4I).

We also quantified the total area occupied by synapticvesicles and found that hwt leads to a significant decreasecompared with controls (Fig. 4I). In addition, our morphologi-cal analyses revealed the presence of supranumerary satelliteboutons (arrow in Fig. 4B′) due to expression of hwt. These

structures represent abnormal synaptic growths and suggestpotential defects in microtubule organization and intracellulartrafficking. As with the total number of boutons, comparablelevels of hA315T had no effect, but when the higherhA315T-expressing line (hA315T HE) was used, the areaoccupied by synaptic vesicles was decreased and the numberof satellite boutons was increased, similar to the effect ofhwt (Fig. 4I). These results suggest that TDP-43 may controlsynaptic function at the NMJ (Fig. 5A).

Additional quantifications included the number of axonalbranches per muscle area (Fig. 4I). hwt but not hA315T orhA315T HE led to a significant decrease in the number ofaxonal branches at the NMJ (Fig. 4I). These data are consistentwith a previous report indicating that hwt overexpression leadsto fewer boutons and branches at the NMJ compared withcontrols (23).

We also investigated whether TDP-43-overexpressinglarvae exhibit signs of motor neuron degeneration as reportedin ALS patients. Neurodegeneration at the larval NMJ hasbeen shown to manifest through the presence of synaptic ‘foot-prints’, i.e. structural remains of boutons that are positive forpost-synaptic Dlg but lack presynaptic markers such as CSPand HRP (38). Because no obvious ‘footprints’ were foundin the larval anterior segment A3 (data not shown), we alsoexamined the more posterior segment A6, which is thoughtto be more sensitive to motor neuron degeneration, presum-ably due to the increased length of the motor neuron axons.Although some thinning of the neuronal membrane (as indi-cated by HRP staining) was observed with all TDP-43 variants(arrowheads, Fig. 4E–H; Supplementary Material, Fig. S3D–F),no terminal boutons positive for Dlg but showing reduced CSPstaining were found (data not shown; Supplementary Material,Fig. S3). Experiments using fly transgenes also show thatwhen comparable levels of wild-type and A315T mutantTBPH are expressed in motor neurons, the wild-type allelehas a more pronounced effect on the size of the NMJ (as indi-cated by its effect on the total number of boutons per musclearea) and the number of satellite boutons (Supplementary

Figure 3. Subcellular localization of TDP-43 in motor neurons. (A–E) D42-Gal4-driven expression of GFP-NLS (A, control) and TDP-43 variants (B–E) inventral ganglia of third instar larvae. Genotypes indicated on the top, and stainings shown on the left. Images shown represent projections of 1 mm confocalslices. GFP (or YFP) and RFP indicate tags used to visualize TDP-43 variants, and DNA visualized using Hoechst. (A′ –E′′) High-magnification views ofventral ganglia shown in (A)–(E). Both human transgenes (wt and A315T) remained restricted to the nucleus (white arrows in B and B′, and C and C′).Note cell with peripheral nuclear localization in hTDP-43 wt (white arrowhead in B and B′). Wild-type TBPH translocates to the cytoplasm and formsaxonal aggregates (red arrows in D′). The fly A315T mutant protein is mostly restricted to the nucleus (white arrows in E and E′). Hoechst staining of thesamples shown in (A′)–(E′) labels DNA. Scale bars: 30 mm in (A), 15 mm in (A′).









Material, Fig. S4). Taken together, these data indicate thatTDP-43 regulates NMJ morphology and that despite somedifferences between the effects of Drosophila and hTDP-43,

the wild-type allele has more pronounced phenotypic effectsat the NMJ than the A315T variant when expressed atsimilar levels. Furthermore, our results indicate that by expres-sing it at higher levels, hA315T generates toxicity comparablewith that of hwt in regard to bouton numbers, synaptic vesiclearea and satellite boutons (Fig. 4I).

Locomotor activity, viability and survival are impairedregardless of the presence of detectable TDP-43cytoplasmic aggregates

The defects detected at the NMJ, in particular the decrease inthe area occupied by synaptic vesicles and the increasednumber of satellite boutons, led us to hypothesize that overex-pression of TDP-43 variants may lead to locomotor defects.To test this possibility, we assayed locomotor ability usinglarval turning assays (39). In brief, crawling third instarlarvae were gently rolled ventral side up, and the timeneeded to turn back to dorsal side up was recorded. As seenin Figure 5A, both hwt and the hA315T allele led to a signifi-cant increase in the time needed to turn over and resumecrawling on the ventral side. Notably, in this assay, theA315T mutant phenotype was significantly worse thanthat of hwt. In keeping with our previous observation thatTDP-43 toxicity is dose-dependent, we found that thehA315T HE transgene leads to a higher level of impairmentin larval motor coordination as measured by larval turningassays (Fig. 5A). Furthermore, upon comparison with theturning behavior of TBPH RNAi in motor neurons(D42::TBPHRNAi), we found that overexpression of bothhTDP-43 variants mimics a loss-of-function phenotype

Figure 4. NMJ morphology is altered by overexpression of TDP-43 variants. (A–H′′) Larval NMJs at muscles 6/7, abdominal segment A3 (A–D′′) and abdomi-nal segment A6 (E–H′′). Genotypes shown on the left, and stainings indicated on the top. Selected terminal type 1b boutons (marked with asterisks) are shown in(A′ –D′) and (A′ –D′, HRP only), and likewise for A6 in (E′ –H′) and (E′ –H′, HRP only). Arrowheads indicate thinning of the HRP-stained neuronal membrane.Arrows indicate satellite boutons. D42-Gal4-driven overexpression of TDP-43 (wild-type and A315T) affects various aspects of synaptic morphology [see (I) forquantitative analyses at A3, which include a high expressing hA315T transgene (A315T HE)]. Student’s t-test was used to determine statistical significance.∗∗∗P , 0.001; ∗∗P , 0.01; ∗P , 0.05. Scale bar (A): 45 mm.

Figure 5. Overexpression of TDP-43 variants acts as a dominant negative andaffects locomotor activity and survival. (A) Larvae expressing D42-drivenhuman wild-type, hA315T mutant TDP-43 (including hA315T HE) andTBPH RNAi transgenes (alone or in combination) take significantly longerto turn over following a ventral-up inversion. Genotypes as indicated.(B) hTDP-43 expression or TBPH RNAi in motor neurons cause a dramaticdecrease in adult survival. (C and D) Adult climbing assays performed onthe adult survivors show severe motor impairment at both 18 (C) and 258C(D). Student’s t-test was used to determine statistical significance.∗∗∗P , 0.001; ∗∗P , 0.01; ∗P , 0.05.




(Fig. 5A). Similar results were obtained with the TBPH trans-genes, except that no significant differences were notedbetween the effect of wild-type and A315T (SupplementaryMaterial, Fig. S5A).

An outstanding question in the field is whether the TDP-43variants act as loss- or gain-of function mutants. To furtheraddress this issue, we coexpressed our hTDP-43 variantstogether with TBPH RNAi (Fig. 5A), which results in asignificant downregulation of the endogenous DrosophilaTBPH expression (Supplementary Material, Fig. S5B; notethat TBPHRNAi targets both Drosophila homologs, TBPHand CG7804). These experiments show that the reduction ofTBPH by RNAi enhances the toxic effect of both wild-typeand A315T hTDP-43 expression in motor neurons andsupport the notion that hTDP-43 overexpression in motorneurons may act as a dominant negative, at least in regard tolarval turning behavior.

Expression of TDP-43 during development has dramaticeffects on the viability of the fly. Wild-type and A315TTBPH expression in motor neurons leads to lethality at boththe larval and pupal stages. Comparable levels of wild-typeand A315T hTDP-43 are less toxic than those of Drosophilacounterparts and exerted differential effects on viability andsurvival when expressed in motor neurons (Fig. 5B andTable 1). We found that expression of hwt was semilethal at258C but adults were viable at 188C. When expressed at com-parable levels, the hTDP-43 A315T mutant had no detectableeffect on viability (adults were obtained at both 18 and 258C).Higher levels of A315T, namely hA315T HE expression, inmotor neurons resulted in 100% pupal lethality at 258C.Although viable adults were obtained with both wild-typeand A315T mutant hTDP-43 expression, we did observe aneffect on survival. As seen in Figure 5B, control flies exhibiteda 30% decrease in survival 30 days after eclosion (at both 18and 258C). In contrast, expression of hwt at 188C led to a 78%

decrease in survival, whereas the A315T-expressing adultsexhibited a 47% reduction in survival at 188C and 98% at258C. Because only few adults expressing hwt were obtainedat 258C, both survival and adult locomotor assays with thewild-type transgene were performed at 188C only (Fig. 5Band C).

Next, we tested the effects of wild-type and A315T mutanthTDP-43 on adult locomotor function. Using climbing assays(40), we found that at 188C, control flies exhibited a progress-ive decline in their ability to climb (94.3% for 2 DO to 25.4%for 30 DO flies) (Fig. 5C), whereas expression of hTDP-43variants had a more significant impact. hwt-expressing adultsshowed a decline in climbing ability from 76.3% at 2 DO to3.1% at 30 DO. The A315T-expressing adults were compar-able with controls except for the last time point (83.7% at 2DO to 0% at 30 DO). At 258C, the A315T allele showed a pro-gressive and significant decline in climbing ability comparedwith controls (Fig. 5D). Just like the effect on survival, theimpairment in locomotor function due to TDP-43 overexpres-sion mimicked the effect of TDP-43 loss (using TBPH RNAi;Fig. 5D).

In keeping with our previous results, these data are consist-ent with a graded effect whereby the Drosophila variants havemore severe phenotypic consequences than the human alleles(compare Supplementary Material, Fig. S5A with Fig. 5A).Furthermore, these results indicate that when expressed atcomparable levels, hwt is more detrimental to viability, survi-val and adult climbing than the A315T allele. As with thelarval turning results, these effects mimic the phenotypesdue to TDP-43 loss of function in motor neurons, suggestingthat TDP-43 overexpression may also act as a dominant nega-tive in regard to survival and motor neuron function. Interest-ingly, hA315T is more detrimental than hwt for larval turningbut not adult climbing behavior. This finding supports thenotion that certain behaviors may require more complex

Table 1. Summary of TDP-43 phenotypes

Transgene/phenotypes TBPH wt TBPH A315T hTDP-43 wt hTDP-43 A315T

Adult eyesSurface phenotype Depigmentation (at 258C) Depigmentation (at 258C) Depigmentation (298C,

age-dependent at 258C)Depigmentation (298C,

age-dependent at 258C)Neuronal loss Yes Yes Yes Yes

Eye discsCellular localization Nuclear/cytoplasmic Nuclear/cytoplasmic Nuclear/cytoplasmic Nuclear/cytoplasmicAxonal aggregates Yes Yes Yes Yes

Motor neuronsCellular localization Nuclear/cytoplasmic Nuclear Nuclear NuclearNeurite aggregates Yes No No No

Locomotor functionLarval turning Impaired Impaired Impaired ImpairedAdult climbing ND (lethal) ND (lethal) Impaired Impaired

Motor neuron death Yes Yes, few cells Yes Yes, few cellsViability Larval/pupal lethal Pupal lethal Adult semilethal (258C),

viable (188C)Adult viable (188C, 258C)

HE line, pupal lethalNMJ synapse

Number of boutons Increased NC Decreased NC (decreased for HE)Synaptic area Decreased Decreased Decreased NCNumber of satellites Increased NC Increased NC (decreased for HE)Number of branches NC NC Decreased NC

Survival ND (due to larval/pupal lethality) ND (due to pupal lethality) Affected Affected

NC, no change; ND, not determined.







integration of neuromuscular function and thus may be moresensitive to the A315T mutation than other phenotypes.Taken together, our results suggest that A315T may impairneuromuscular function through a mechanism distinct fromthat of wild-type TDP-43.

Cytoplasmic aggregation of TDP-43 is not requiredfor apoptotic death in motor neurons

A hallmark of ALS pathology is motor neuron dysfunctionaccompanied by apoptotic death (41). Although in the retinathere is clear evidence for cell loss (Fig. 1), we wanted todetermine whether our transgenes also affect motor neuronalsurvival. To test this possibility, we performed terminaldUTP nick end labeling (TUNEL) assays in the larvalventral ganglia, where motor neurons reside. As seen inFigure 6, apoptosis was detected in several cells that coincidewith wild-type TBPH-expressing motor neurons [compareFig. 6C and C′ with A and A′ (positive control) and B andB′ (negative control)]. Apoptotic death due to A315T TBPHexpression in motor neurons was very modest (Fig. 6D andD′). The human transgenes expressing wild-type and A315T

mutant hTDP-43 at comparable levels did not show any evi-dence of apoptosis in the larval ventral ganglia. The larvaeexpressing hA315T HE contained a few TUNEL-positivecells and died as pupae (data not shown).

Given that the hTDP-43 variant expression produced viableadults as opposed to the TBPH variants that were lethal atearlier stages, it is possible that cell death in the animalsexpressing hTDP-43 at comparable levels may occur later indevelopment, in the adult nervous system. To this end, we per-formed TUNEL assays in adult thoracic ganglia expressingeither wild-type or A315T mutant hTDP-43. Indeed, asshown in Figure 6F–F′′, hwt expression results in a dramaticloss of motor neurons as evidenced by: (i) a visible reductionin the number of cells expressing the hTDP-43 transgene(Fig. 6F, compare with E) and the presence of TUNEL-positive cells that coincide with transgene expression(compare Fig. 6F′ and F′′ with controls in Fig. 6E1′ –E2′′).In contrast, no obvious signs of cell loss were present in thecontext of hA315T expression (Fig. 6G, compare with E).TUNEL assays, however, indicate the presence of a few apop-totic cells that coincide with hA315T-expressing cells(Fig. 6G′ and G′′). Taken together, our results with the fly

Figure 6. Motor neuron apoptosis due to TDP-43 overexpression. (A–D) TUNEL staining marks apoptotic cells. Genotypes/treatment indicated on top, andstainings shown on the left. (A and A′) RFP-NLS expressing ventral ganglia treated with HCl exhibits widespread TUNEL staining. (B and B′) RFP-NLS expres-sing ventral ganglia shows no indication of cell death (negative control). (C and C′) Wild-type TBPH expression results in some apoptotic cells within the ventralganglia (arrows, compare C′ with A′ and B′). (D and D′) Few apoptotic cells can be detected in A315T TBPH expressing ventral ganglia (arrows). (E) GFP NLSexpression in adult motor neurons using D42 Gal4. Thoracic segments T1, 2, 3 as shown. Arrows (E–G) indicate areas where motor neurons are located. Note:The region marked by red inset in (E) is shown at high magnification for different thoracic ganglia (assayed for apoptosis) in (E1′)–(G′′). (E1′ and E1′′) TUNELstaining in the T1/T2 region of a GFP-NLS adult thoracic ganglia treated with HCl (positive control). (E2′ and E2′′) GFP-NLS expression does not induce apop-tosis (negative control). (F′ –G′′) Adult thoracic ganglia expressing hwt (F′ and F′′) and hA315T mutant (G′ and G′′). Motor neurons visualized via fluorescentprotein tag when either hwt (F) or hA315T mutant TDP-43 (G) is expressed with the D42 driver. Note the dramatic reduction in motor neurons as a result of hwtexpression (F). TUNEL assays indicate the presence of apoptotic cells (arrows) when hwt (F′ and F′′) or, to a lesser extent, A315T mutant hTDP-43 (G′ and G′′)are expressed in adult motor neurons. Scale bar: 70 mm (A), 125 mm (E) and 70 mm (E1′).



and human transgenes support the notion that regardless ofits subcellular localization (nuclear or cytoplasmic), TDP-43overexpression leads to motor neuron death by apoptosis.These data also substantiate our previous observations thatwild-type TDP-43 exerts higher toxicity than the A315Tvariant (Table 1).

TDP-43 toxicity is modulated by the proteasomeand the HSP70 chaperone activities as wellas the apoptosis pathway

Previous studies have suggested that the mechanisms under-lying the toxicity of TDP-43 include hyperphosphorylation ofthe mutated variants, followed by excessive targeting to theproteasome, which may eventually become functionally over-whelmed (3,30). To test whether the proteasome contributesto TDP-43 neurotoxicity in vivo, we used a dominant-negative form of the b2 subunit of the 20S proteasomecomplex, namely prosb (42). Overexpression of prosbalone in the eye does not produce a visible phenotype(Fig. 7I); however, when coexpressed with wild-type andA315T mutant hTDP-43, it enhanced the depigmentationphenotype due to TDP-43 overexpression (Fig. 7A and E

with B and F, respectively). Although this enhancementwas observed throughout adult life, the data shown arefrom 15-day-old adults when the hTDP-43 phenotypes aremore clearly visible at 258C. Our results are consistentwith a scenario where TDP-43 overexpression may behandled by the proteasome, which when further impairedby our dominant-negative approach contributes to enhancedtoxicity.

To gain further insight into the mechanisms underlying theneurotoxic effects of TDP-43, we also tested whether overex-pression of the HSP70 chaperone can rescue the eye pheno-types due to wild-type and A315T overexpression. HSP70was chosen due to its ability to rescue the toxicity of polyglu-tamine, a-synuclein and RNA-mediated neurodegeneration inDrosophila models, in some instances by alleviating the pres-ence of protein aggregates (43–45). As seen in Figure 7, over-expression of human HSP70 alleviates the depigmentationphenotype due to hTDP-43 (wild-type and A315T) expressionin the eye (compare Fig. 7C and G with A and E, respectively).These data, together with the prosb interaction, suggest thatTDP-43 toxicity may be due to aggregates that pose an over-whelming stress on the proteasome but may be mitigated byexcess HSP70 chaperone activity.

Figure 7. TDP-43 toxicity is modulated by the proteasome, HSP70 activities and the apoptosis pathway. (A and E) Compound eyes expressing hwt (A) or A315Tmutant hTDP-43 (hA315T, E) at 258C, in 15-day-old adults, exhibit age-dependent depigmentation. Genotypes as indicated on the left, and interacting genes onthe top. All transgenes expressed with GMR-Gal4. (B and F) Coexpression of prosb enhances the depigmentation phenotype due to TDP-43 overexpression(arrows). (C, D, G and H) Coexpression of Hsp70 (C and G) or the p35 caspase inhibitor (D and H) alleviates the eye depigmentation phenotypes due toTDP-43 overexpression. All comparisons were performed with similarly aged flies. (I–K) Overexpression of prosb, Hsp70 or p35 alone does not lead tovisible eye phenotypes.



Although we have detected clear evidence of cell death inthe eye, we wanted to determine whether inhibition of apopto-sis can mitigate the eye phenotypes due to wild-type andA315T overexpression. As seen in Figure 7D and H(compare with Fig. 7A and E, respectively), overexpressionof the p35 baculovirus protein, a caspase inhibitor (46),rescues, although not perfectly, the TDP-43 phenotypes inthe adult eye. These results are consistent with the notionthat caspase-mediated apoptotic pathways mediates in partthe toxicity due to TDP-43 overexpression in the eye.

DISCUSSION

Despite the discovery of several missense mutations in theRNA-binding protein TDP-43, the mechanisms of neurode-generation involving the individual alleles remain poorlyunderstood. To uncover the consequences of TDP-43mutations linked to ALS, we performed a direct in vivo com-parison of the neuronal and functional phenotypes resultingfrom overexpression of wild-type and the A315T mutantTDP-43 in the Drosophila nervous system. Expression ofeither allele (wild-type or A315T) of TDP-43 in photoreceptorand motor neurons led to effects on viability, locomotor func-tion and survival. In the developing retina, overexpression ofthe TDP-43 variants used in this study resulted in the for-mation of cytoplasmic and axonal aggregates accompaniedby neuronal loss. The eye tissue showed the highest sensitivityto overexpression of TDP-43 and revealed the most dramaticalterations in the subcellular localization of TDP-43, resem-bling those found in postmortem ALS tissues (9,11). WhenTDP-43 variants were expressed in motor neurons, we alsofound several hallmark features of ALS, including cytoplasmicaggregates, cell death and defects in locomotor ability(Table 1). To our surprise, when expressed at comparablelevels, wild-type TDP-43 was more toxic than the A315Tmutant in regard to viability, survival, NMJ anatomy andadult climbing behavior but not for larval turning behavior.We also found that by simply increasing expression of themutant allele we were able to recapitulate several, althoughnot all, phenotypes resulting from overexpression of wild-typeTDP-43. These include effects on viability, and select NMJfeatures such as decreased synaptic area and malformedsynaptic boutons that may be more sensitive to mutations inTDP-43 than others (e.g. axon branching). This is particularlyinteresting because, on the one hand, it shows that TDP-43 isinherently toxic, consistent with previous in vivo and in vitrostudies (22,23,26,28,29,33,47,48), whereas, on the otherhand, our data show that wild-type and A315T mutantTDP-43 exert differential toxicity when evaluated by a reper-toire of cellular and behavioral assays. These results empha-size the need for detailed studies of individual missensemutations in TDP-43, in particular those that mimic themutations identified in ALS patients. Such studies areneeded to identify allele-specific neuronal and synapticdefects that could provide clues to their mechanism ofaction and lead to the development of personalized therapeuticinterventions.

We found that when expressed at comparable levels, theTBPH variants were more toxic than their human counterparts.

A possible explanation for this difference is provided by thefact that the Drosophila genome encodes an additional, pre-viously unreported homolog of TDP-43, namely CG7804.This locus encodes a second, highly homologous TDP-43protein in Drosophila that contains the RNA-bindingdomains RRM1/2 but lacks the C-terminal glycine-richregion. The function of this predicted, truncated, additionalTDP-43 protein, as well as the significance of its loss duringevolution, remains to be investigated. Although the fly andhTDP-43 pathways may not be identical, most differencesseem to be mitigated by the fact that by simply expressinghigher levels of hA315T (hA315T HE) we can recapitulateseveral of the neuronal and functional phenotypes resultingfrom wild-type TBPH overexpression, the most toxic of thetransgenes in this study. Thus, dosage appears to directlycorrelate with toxicity.

TDP-43 has been previously shown to be required for theproper morphology of the NMJ synapse (23,25) and ourfindings further support this. In addition, our morphologicalanalyses revealed novel phenotypes, including the reducedarea occupied by synaptic vesicles, which appears to bemost sensitive to alterations in TDP-43 (both wild-type andA315T) and suggests the presence of functional defects atthe synapse. Indeed, larval turning assays, which rely oncomplex motor coordination along the anterior–posteriorand dorsal–ventral axes of the larvae, showed that both thewild-type and A315T alleles affect locomotor ability. It isintriguing that larval turning is the only assay in which theA315T mutant shows higher toxicity than the wild-typeallele and it remains to be seen what are the molecular mech-anisms underlying this effect. Additional evidence forTDP-43′s effect on locomotor function is provided by theadult climbing assays. Although these results suggest thatTDP-43 overexpression affects synaptic function, more workis needed to determine the underlying defects at the NMJsynapse. For example, it would be interesting to determinewhat specific aspects of synaptic transmission may be affectedby altering TDP-43 function in presynaptic motor neurons.Knowing which parameters of synaptic function are impaireddue to expression of missense mutations in TDP-43 willprovide important clues to the mechanisms underlying thepathology of ALS.

Our results indicate that cytoplasmic aggregates are notrequired for motor neuron death, consistent with previous find-ings using hwt (24). Upon wild-type TBPH overexpression,motor neuron death was sparsely detected in larval ventralganglia before lethality ensued. When hwt was overexpressed,adult motor neuron numbers were significantly reduced inthoracic ganglia and we found strong evidence for apoptoticdeath. In contrast, overexpression of A315T TBPH led tobarely detectable apoptosis, and hA315T resulted in somecells positive for TUNEL staining. Surprisingly, apoptosisdetection was also marginal in the ventral ganglia of thehigher expressing hA315T HE larvae. Although our findingson the extent of apoptotic death may not be in full agreementwith previous reports (22,23), it is likely that differences inexpression levels and type of neuron or animal modelstudied may account for the differences observed. We wouldalso like to note that in the case of the Drosophila transgenes,their effect on viability may have overshadowed the motor



neuron phenotypes. For example, overexpression of TBPH,which overall has more pronounced effects than thehTDP-43 variants, may have led to lethality due to theinability of the transgenic larvae to feed properly. Indeed,the wild-type TBPH expressing larvae appeared ‘skinny’ anddevelopmentally delayed. The TBPH transgenics that diedbefore reaching adulthood may have succumbed to starvationand/or delayed growth, thus precluding us from detectingextensive motor neuron death, which may otherwise havebeen observed later in development. This scenario is furthersubstantiated by our findings with the hTDP-43 variants,which exhibited motor neuron apoptosis in the adult but notlarval nervous system.

Several questions remain regarding the molecular mechan-isms that mediate the dominant effect of TDP-43 overexpres-sion. How can overexpression of wild-type TDP-43 mimic aloss-of-function phenotype? One possibility is that excessprotein is sequestered in cytoplasmic or nuclear aggregates,which may affect the stoichiometry of physiological TDP-43complexes, thus mimicking a loss-of-function phenotype(49). Overall, our data suggest that the precise amount ofTDP-43 expression is critical to proper neuronal and synapticfunction and alterations in TDP-43 levels and/or function areresponsible for the observed phenotypes. The dominanteffects of TDP-43 overexpression are not unlike thoseobtained with other RNA-binding proteins including FMRPand Argonaute 1, which result in visible phenotypes whenoverexpressed in the eye (50,51). Thus, we suggest that thephenotypes resulting from wild-type and mutant TDP-43 over-expression stem from defects in gene expression, in particularthe dysregulation of RNA targets that are just beginning to bediscovered (52).

The genetic interactions with the proteasome support thenotion that the insoluble TDP-43 products that arise fromoverexpression or the presence of missense mutations(3,23,30,53) may normally be processed by the proteasomedegradation machinery. By impairing the function of the pro-teasome, insoluble TDP-43 products may not be cleared effi-ciently, which could account for the enhanced toxicity weobserved in the eye. Although this result is not entirely unex-pected and previous studies have shown that pharmacologicalinhibition of the proteasome enhances the accumulation ofC-terminal fragments of TDP-43 (3), our results provide anin vivo demonstration for the involvement of the proteasomein TDP-43 neurotoxicity. Future biochemical studies willfocus on whether the amount of insoluble TDP-43 productsis increased upon proteasome impairment or whetheradditional mechanisms account for the enhanced neurotoxicitywe observed in the eye. Although it remains to be seenwhether cytoplasmic and axonal TDP-43 aggregates arecleared by HSP70 coexpression, our discovery that excessHSP70 chaperone alleviates the phenotype of TDP-43 in theeye supports the notion that TDP-43 misfolding underlies itstoxicity in the eye. Not surprisingly, given the dramatic cellloss evident in the retina, we found that inhibition of caspasesthrough overexpression of the p35 baculovirus protein alsoalleviates, although does not completely rescue, the phenotypeof TDP-43 overexpression in the eye. Taken together, thesefindings indicate that commonalities may exist betweenTDP-43 and other known proteinopathies and suggest that

therapies based on targeting the proteasome function, HSP70chaperone activity or caspases may be effective for ALS aswell as other neurodegenerative disorders in which these path-ways have been implicated.

In summary, our results support previous findings andprovide further evidence that wild-type and mutant TDP-43overexpression in Drosophila recapitulates hallmark aspectsof ALS pathology. Perhaps the most surprising result is that,when expressed at comparable levels, wild-type TDP-43exerted higher toxicity than the A315T allele, in regard to via-bility, survival, neuronal loss and adult locomotor function butnot larval locomotor behavior. Although a mouse model ofA315T overexpression exhibits ubiquitin-positive cytoplasmicaggregates and other features of ALS pathology (28), a recentarticle reports the presence of rather weak pathological hall-marks in an ALS patient carrying this allele (54). These find-ings suggest that the genetic background may influenceTDP-43 phenotypes and are consistent with a recent reportthat ataxin 2 constitutes a risk factor for ALS (47). Takentogether, our results and these published data indicate thatdetailed phenotypic analyses of different mutants in animalmodels such as Drosophila are critical for elucidating thebiology of TDP-43 and the mechanisms of neurodegenerativedisease. In addition, our work suggests that perhaps animalmodels based on individual missense mutations that mimicthose found in human patients will serve as better modelsfor genetic and drug screens that can lead to novel effectiveand potentially personalized therapies.

MATERIALS AND METHODS

Drosophila genetics

All Drosophila stocks and crosses were kept on standard yeast/cornmeal/molasses food at 258C unless otherwise noted. TBPHcDNA GH09868 was obtained from the Drosophila GenomeProject in the pOT2 vector. Following PCR amplification andsite-directed mutagenesis (QuikChange II, Stratagene), theinserts (wild-type and A315T) were cloned into the pUASTdestination vectors pTGW and pTRW (pUAST with N-terminalGFP and RFP, respectively; Drosophila Genome ResourceCenter), using the Gateway Technology (Invitrogen).hTDP-43, wild-type and A315T with YFP C-terminal tag (inpRS416 yeast expression vector, from A. Gitler) were clonedinto the NotI and KpnI sites of the pUAST germline transform-ation vector. Transgenic lines were mapped and balanced usingstandard genetic techniques and include GFP TBPH wild-type,RFP TBPH wild-type and A315T, hTDP-43 wild-type YFP,hTDP-43 A315T YFP. Gal4 drivers used in this study includethe eye-specific GMR Gal4 R13 and the motor neuron driverD42 Gal4 (35). TBPH RNAi lines were obtained fromthe Vienna Drosophila RNAi Center (lines w1118;P{GD6943}v38377 and w1118;P{GD6943}v38377) and usedto generate a double-RNAi recombinant stock. GMR Gal4 wild-type hTDP-43 and GMR Gal4 hA315T stocks were generatedusing standard meiotic recombination techniques. For geneticinteractions, the following stocks were used: (i) w1118;P{w[+mC]¼UAS-Prosbeta2[1]}1B (dominant-negative form of the20S proteasome core subunit); (ii) w1118;P{w[+mC]¼UAS-Hsap\HSPA1L.W}53.1/CyO (human molecular chaperone



Hsp70) and (iii) w[∗];P{w[+mC]¼UAS-p35.H}BH2 (baculo-virus-derived apoptosis inhibitor).

Western blots

To determine the relative expression levels of the variousTDP-43 transgenes used in this study, Drosophila headswere collected from adults expressing wild-type and A315Tmutant, Drosophila or hTDP-43, using the GMR Gal4driver. Following homogenization in 2× Laemmli buffer,the lysates were resolved on SDS–PAGE and then transferredto a PVDF membrane (Millipore). Drosophila and hTDP-43transgenes were detected using rabbit polyclonal anti-GFP(Invitrogen) at 1/3000, rabbit polyclonal anti-TARDBP(Abcam) at 1/1250 or rabbit polyclonal anti-TBPH at1/3000. Tubulin was used as a loading control and wasdetected using a mouse monoclonal anti-tubulin antibody(Millipore) at 1/1000. The secondary antibody used was goatanti-mouse- or goat anti-rabbit-conjugated HRP, as appropri-ate, at 1/1000 (Thermo Scientific). Proteins of interest werevisualized using SuperSignal West Femto Substrate (ThermoScientific). Protein levels were quantified using NIH Imagesoftware.

Immunohistochemistry and imaging

Adult fly eyes were imaged with a Leica MZ6 microscopeequipped with an Olympus DP71 camera and controlled byOlympus DP Controller and Olympus DP Manager software.Individual images were processed using Adobe PhotoshopCS2 (Adobe).

Larval NMJ preparations have been described previously(55). Briefly, wandering third instar larvae were filleted,pinned out on Sylgard dishes, fixed in 3.5% formaldehyde inPBS, pH 7.2, for 20 min and then permeabilized with 0.1%Triton X-100. Following treatment in a blocking agent consist-ing of 2% BSA and 5% NGS, the fillets were stained withanti-DCSP2 at 1/300 (DSHB), anti-HRP-FITC at 1/50(Sigma) and anti-Dlg polyclonal at 1/900 (gift of PeterBryant). Secondary antibodies at 1/1000 were from MolecularProbes. Larval muscles 6 and 7 were imaged in abdominalsegments A3 and A6 on a Nikon PCM 2000 confocal micro-scope and were displayed as a projection of 1 mm serial sec-tions. Type 1b, 1s, and satellite boutons as well as brancheswere manually counted and total CSP area was determinedusing Metamorph Image Analysis software (UniversalImaging). All measurements were divided by total musclearea to take into account variations in the size of the individuallarvae.

Eye discs and larval ventral ganglia were dissected and pre-pared as above. Eye discs were stained with either rhodaminephalloidin (1/300), FITC phalloidin (1/150; Sigma) or Alexa647 phalloidin (1/300) (Molecular Probes) as well asHoechst 33342 (Invitrogen) at 1/10 000 and, if appropriate,anti-GFP-FITC (Rockland) at 1/200. Ventral ganglia werestained as above except that instead of phalloidin, they werestained with anti-HRP-FITC or TRITC (Sigma) at 1/50.They were imaged on a Zeiss Meta 510 confocal microscopeand displayed as projections of 1 mm serial Z sections.

Eye sections

Adult eyes were embedded and sectioned as described pre-viously (51). In brief, following the removal of the proboscis,adult heads were fixed in Trump’s fixative (4% paraformalde-hyde, 1% glutaraldehyde, 100 mM cacodylate buffer, pH 7.2,2 mM sucrose, 0.5 mM EGTA) overnight. The following day,the samples were washed three times in 100 mM cacodylatebuffer with 264 mM sucrose, then placed in a 0.5% OsO4 in100 mM cacodylate buffer for 1 h and rinsed for 10 min in100 mM cacodylate buffer with 264 mM sucrose. Following adehydration protocol using ethanol and propylene oxide,samples were embedded in Embed 812 and baked at 658Covernight. Using a Reichert Jung Ultracut E Ultramicrotome,1 mm thick plastic sections were cut and stained with 1% tolui-dine blue in 1% sodium borate. Images were acquired using aZeiss Axioplan microscope equipped with an Olympus DP71camera and controlled by Olympus DP Controller andOlympus DP Manager software.

TUNEL assay

TUNEL assays were performed on larval ventral and adultthoracic ganglia, using the In Situ Cell Death Detection Kit(Roche, Indianapolis, IN, USA). Briefly, ganglia were fixedin semi-intact preparations with 3.5% formaldehyde in PBS,pH 7.2, for 20 min. After permeabilization with 0.05%Triton X-100, the ganglia were treated with 10 mg/ml protein-ase K (Fermentas) for 10 min at 378 C. The ganglia were thendissected into microtiter dishes. The positive control for theTUNEL reaction consisted of ganglia treated with 2 N HClfor 30 min. The TUNEL reaction was carried out for 1.5 h at378 C as per manufacturer’s instructions using, as appropriate,a TMR-Red or an FITC label. Ganglia were then stained withHoechst 33342 (Invitrogen) at 1/10 000 and, if appropriate,anti-GFP-FITC (Rockland) at 1/200. Preps were mounted in4% N-propyl gallate in 95% glycerol and imaged on a ZeissMeta 510 confocal microscope. Ganglia were displayed asprojections of 1 mm serial Z sections.

Locomotor function assays

Larval turning assays. Third instar wandering larvae wereplaced on a grape juice plate at room temperature. Afterbecoming acclimated, crawling larvae were gently turnedonto their backs (ventral side up) and monitored until theywere able to turn back (dorsal side up) and continue theirforward movement. The amount of time that it took eachlarva to complete this task was recorded. Three to five trialsof 8 to 10 larvae were performed for each genotype. Student’st-test was performed to assess statistical significance.

Climbing assays. Expression of the Drosophila transgenesusing D42 Gal 4 was lethal before adulthood, primarily atthe pupal stage. Therefore, climbing assays were performedwith adults expressing wild-type and A315T mutanthTDP-43 in motor neurons. Owing to effects on viabilityfrom these transgenes as well, flies were raised at both 25and 188C. Ten (1-day-old) adult males of each genotypewere collected and tested for their climbing ability starting



when 2 DO and every 4 days thereafter, until they reached 30days after eclosion. Five to ten such cohorts (50–100 males)were tested for each genotype. To assess their climbingability, the flies were transferred to an empty vial marked at5 cm from the bottom (40). After being allowed to acclimateto the new environment (�30 s), flies were gently tappeddown to the bottom of the vial and then the time it tookeach fly to pass the 5 cm mark was recorded. All flies thatclimbed the 5 cm up the vial in 18 s or less passed, whereasthose that could not climb that high or took longer than 18 sfailed. Both the number of flies that passed and the numberof flies surviving were recorded each time the test was per-formed. The climbing index for each genotype was calculatedas the number of flies that passed the climbing test, normalizedto the number of survivors on the day of the test. Survivabilitywas calculated by dividing the number of flies alive on eachday by the number alive on day 2.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

We are indebted to the Himelic family for their generoussupport. We also thank Drs Bruce Coull, David Labiner andKatalin Scherer (Department of Neurology, University ofArizona) for their support and suggestions; Aaron Gitler (Uni-versity of Pennsylvania) for the human TDP-43 constructs;Fabian Feiguin and Francisco Baralle (ICGEB, Trieste) forthe TBPH antibody; Gabrielle Boulianne (University ofToronto) for helpful discussions and sharing unpublishedresults; Peter Bryant (University of California, Irvine) forthe polyclonal Dlg antibody; Drosophila Genome ResourceCenter, Bloomington Stock Center and the Vienna DrosophilaRNAi Center for reagents; Iowa Hybridoma Bank for CSPantibodies; Dan Marenda (Drexel University) for suggestionswith the climbing assays; Genetics Services for generatingtransgenic flies; Gio Bosco (Department of Molecular and Cel-lular Biology, University of Arizona) for access to theOlympus DP71 camera; John Hildebrand (Department ofNeuroscience, University of Arizona) for the use of theZeiss microscope/Olympus imaging system; Sam Ward(Department of Molecular and Cellular Biology, Universityof Arizona) for the MetaMorph software; Carl Boswell(Department of Molecular and Cellular Biology, Universityof Arizona) for help with confocal imaging; Patty Jansma(Department of Neuroscience, University of Arizona) forhelp with adult eye sections; Annette Estevez for helpful com-ments on the manuscript.

Conflict of Interest statement. None declared.

FUNDING

This work was supported by the Jim Himelic Foundation andby the Muscular Dystrophy Association (in part throughsupport from Mr and Mrs Dunham in memory of their daugh-ter Susan Hope Nearing Dunham; MDA173230 to D.C.Z.).

Partial support was also provided by the UndergraduateBiology Research Program funded by the Howard HughesMedical Institute (HHMI 5205889 to J.T. and B.G.).

REFERENCES

1. Banks, G.T., Kuta, A., Isaacs, A.M. and Fisher, E.M. (2008) TDP-43 is aculprit in human neurodegeneration, and not just an innocent bystander.Mamm. Genome, 19, 299–305.

2. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P.,Hentati, A., Donaldson, D., Goto, J., O’Regan, J.P., Deng, H.X. et al.(1993) Mutations in Cu/Zn superoxide dismutase gene are associated withfamilial amyotrophic lateral sclerosis. Nature, 362, 59–62.

3. Kabashi, E., Valdmanis, P.N., Dion, P., Spiegelman, D., McConkey, B.J.,Vande Velde, C., Bouchard, J.P., Lacomblez, L., Pochigaeva, K.,Salachas, F. et al. (2008) TARDBP mutations in individuals with sporadicand familial amyotrophic lateral sclerosis. Nat. Genet., 40, 572–574.

4. Beleza-Meireles, A. and Al-Chalabi, A. (2009) Genetic studies ofamyotrophic lateral sclerosis: controversies and perspectives. Amyotroph.Lateral Scler., 10, 1–14.

5. Vance, C., Rogelj, B., Hortobagyi, T., De Vos, K.J., Nishimura, A.L.,Sreedharan, J., Hu, X., Smith, B., Ruddy, D., Wright, P. et al. (2009)Mutations in FUS, an RNA processing protein, cause familialamyotrophic lateral sclerosis type 6. Science, 323, 1208–1211.

6. Valdmanis, P.N., Daoud, H., Dion, P.A. and Rouleau, G.A. (2009) Recentadvances in the genetics of amyotrophic lateral sclerosis. Curr. Neurol.Neurosci. Rep., 9, 198–205.

7. Lagier-Tourenne, C. and Cleveland, D.W. (2009) Rethinking ALS: theFUS about TDP-43. Cell, 136, 1001–1004.

8. Kwiatkowski, T.J. Jr, Bosco, D.A., Leclerc, A.L., Tamrazian, E.,Vanderburg, C.R., Russ, C., Davis, A., Gilchrist, J., Kasarskis, E.J.,Munsat, T. et al. (2009) Mutations in the FUS/TLS gene on chromosome16 cause familial amyotrophic lateral sclerosis. Science, 323, 1205–1208.

9. Maekawa, S., Leigh, P.N., King, A., Jones, E., Steele, J.C., Bodi, I., Shaw,C.E., Hortobagyi, T. and Al-Sarraj, S. (2009) TDP-43 is consistentlyco-localized with ubiquitinated inclusions in sporadic and Guamamyotrophic lateral sclerosis but not in familial amyotrophic lateralsclerosis with and without SOD1 mutations. Neuropathology, 29,672–683.

10. Tan, C.F., Eguchi, H., Tagawa, A., Onodera, O., Iwasaki, T., Tsujino, A.,Nishizawa, M., Kakita, A. and Takahashi, H. (2007) TDP-43immunoreactivity in neuronal inclusions in familial amyotrophic lateralsclerosis with or without SOD1 gene mutation. Acta Neuropathol., 113,535–542.

11. Van Deerlin, V.M., Leverenz, J.B., Bekris, L.M., Bird, T.D., Yuan, W.,Elman, L.B., Clay, D., Wood, E.M., Chen-Plotkin, A.S., Martinez-Lage,M. et al. (2008) TARDBP mutations in amyotrophic lateral sclerosis withTDP-43 neuropathology: a genetic and histopathological analysis. LancetNeurol., 7, 409–416.

12. Neumann, M., Sampathu, D.M., Kwong, L.K., Truax, A.C., Micsenyi,M.C., Chou, T.T., Bruce, J., Schuck, T., Grossman, M., Clark, C.M. et al.(2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration andamyotrophic lateral sclerosis. Science, 314, 130–133.

13. Daoud, H., Valdmanis, P.N., Kabashi, E., Dion, P., Dupre, N., Camu, W.,Meininger, V. and Rouleau, G.A. (2009) Contribution of TARDBPmutations to sporadic amyotrophic lateral sclerosis. J. Med. Genet., 46,112–114.

14. Gitcho, M.A., Baloh, R.H., Chakraverty, S., Mayo, K., Norton, J.B.,Levitch, D., Hatanpaa, K.J., White, C.L. III, Bigio, E.H., Caselli, R. et al.(2008) TDP-43 A315T mutation in familial motor neuron disease. Ann.Neurol., 63, 535–538.

15. Kuhnlein, P., Sperfeld, A.D., Vanmassenhove, B., Van Deerlin, V.,Lee, V.M., Trojanowski, J.Q., Kretzschmar, H.A., Ludolph, A.C. andNeumann, M. (2008) Two German kindreds with familial amyotrophiclateral sclerosis due to TARDBP mutations. Arch. Neurol., 65,1185–1189.

16. Pamphlett, R., Luquin, N., McLean, C., Jew, S.K. and Adams, L. (2009)TDP-43 neuropathology is similar in sporadic amyotrophic lateralsclerosis with or without TDP-43 mutations. Neuropathol. Appl.Neurobiol., 35, 222–225.




17. Rutherford, N.J., Zhang, Y.J., Baker, M., Gass, J.M., Finch, N.A., Xu,Y.F., Stewart, H., Kelley, B.J., Kuntz, K., Crook, R.J. et al. (2008) Novelmutations in TARDBP (TDP-43) in patients with familial amyotrophiclateral sclerosis. PLoS Genet., 4, e1000193.

18. Sreedharan, J., Blair, I.P., Tripathi, V.B., Hu, X., Vance, C., Rogelj, B.,Ackerley, S., Durnall, J.C., Williams, K.L., Buratti, E. et al. (2008)TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.Science, 319, 1668–1672.

19. Johnson, B.S., McCaffery, J.M., Lindquist, S. and Gitler, A.D. (2008) Ayeast TDP-43 proteinopathy model: exploring the molecular determinantsof TDP-43 aggregation and cellular toxicity. Proc. Natl Acad. Sci. USA,105, 6439–6444.

20. Buratti, E. and Baralle, F.E. (2001) Characterization and functionalimplications of the RNA binding properties of nuclear factor TDP-43,a novel splicing regulator of CFTR exon 9. J. Biol. Chem., 276,36337–36343.

21. Wang, I.F., Wu, L.S., Chang, H.Y. and Shen, C.K. (2008) TDP-43, thesignature protein of FTLD-U, is a neuronal activity-responsive factor.J. Neurochem., 105, 797–806.

22. Voigt, A., Herholz, D., Fiesel, F.C., Kaur, K., Muller, D., Karsten, P.,Weber, S.S., Kahle, P.J., Marquardt, T. and Schulz, J.B. (2010)TDP-43-mediated neuron loss in vivo requires RNA-binding activity.PLoS One, 5, e12247.

23. Li, Y., Ray, P., Rao, E.J., Shi, C., Guo, W., Chen, X., Woodruff, E.A. III,Fushimi, K. and Wu, J.Y. (2010) A Drosophila model for TDP-43proteinopathy. Proc. Natl Acad. Sci. USA, 107, 3169–3174.

24. Hanson, K.A., Kim, S.H., Wassarman, D.A. and Tibbetts, R.S. (2010)Ubiquilin modifies TDP-43 toxicity in a Drosophila model ofamyotrophic lateral sclerosis (ALS). J. Biol. Chem., 285, 11068–11072.

25. Feiguin, F., Godena, V.K., Romano, G., D’Ambrogio, A., Klima, R. andBaralle, F.E. (2009) Depletion of TDP-43 affects Drosophila motoneuronsterminal synapsis and locomotive behavior. FEBS Lett., 583, 1586–1592.

26. Lu, Y., Ferris, J. and Gao, F.B. (2009) Frontotemporal dementia andamyotrophic lateral sclerosis-associated disease protein TDP-43 promotesdendritic branching. Mol. Brain, 2, 30.

27. Ash, P.E., Zhang, Y.J., Roberts, C.M., Saldi, T., Hutter, H., Buratti, E.,Petrucelli, L. and Link, C.D. (2010) Neurotoxic effects of TDP-43overexpression in C. elegans. Hum. Mol. Genet., 19, 3206–3218.

28. Wegorzewska, I., Bell, S., Cairns, N.J., Miller, T.M. and Baloh, R.H.(2009) TDP-43 mutant transgenic mice develop features of ALS andfrontotemporal lobar degeneration. Proc. Natl Acad. Sci. USA, 106,18809–18814.

29. Kabashi, E., Lin, L., Tradewell, M.L., Dion, P.A., Bercier, V., Bourgouin,P., Rochefort, D., Bel Hadj, S., Durham, H.D., Velde, C.V. et al. (2009)Gain and loss of function of ALS-related mutations of TARDBP(TDP-43) cause motor deficits in vivo. Hum. Mol. Genet., 19, 671–683.

30. Liachko, N.F., Guthrie, C.R. and Kraemer, B.C. (2010) Phosphorylationpromotes neurotoxicity in a Caenorhabditis elegans model of TDP-43proteinopathy. J. Neurosci., 30, 16208–16219.

31. Brand, A.H. and Perrimon, N. (1993) Targeted gene expression as ameans of altering cell fates and generating dominant phenotypes.Development, 118, 401–415.

32. Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H.,Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y. et al. (2006)TDP-43 is a component of ubiquitin-positive tau-negative inclusions infrontotemporal lobar degeneration and amyotrophic lateral sclerosis.Biochem. Biophys. Res. Commun., 351, 602–611.

33. Johnson, B.S., Snead, D., Lee, J.J., McCaffery, J.M., Shorter, J. and Gitler,A.D. (2009) TDP-43 is intrinsically aggregation-prone and ALS-linkedmutations accelerate aggregation and increase toxicity. J. Biol. Chem.,284, 20329–20339.

34. Barmada, S.J., Skibinski, G., Korb, E., Rao, E.J., Wu, J.Y. and Finkbeiner,S. (2010) Cytoplasmic mislocalization of TDP-43 is toxic to neurons andenhanced by a mutation associated with familial amyotrophic lateralsclerosis. J. Neurosci., 30, 639–649.

35. Gustafson, K. and Boulianne, G.L. (1996) Distinct expression patternsdetected within individual tissues by the GAL4 enhancer trap technique.Genome, 39, 174–182.

36. Koh, Y.H., Gramates, L.S. and Budnik, V. (2000) Drosophila larvalneuromuscular junction: molecular components and mechanismsunderlying synaptic plasticity. Microsc. Res. Tech., 49, 14–25.

37. Ranjan, R., Bronk, P. and Zinsmaier, K.E. (1998) Cysteine string proteinis required for calcium secretion coupling of evoked neurotransmission inDrosophila but not for vesicle recycling. J. Neurosci., 18, 956–964.

38. Eaton, B.A. and Davis, G.W. (2005) LIM Kinase1 controls synapticstability downstream of the type II BMP receptor. Neuron, 47, 695–708.

39. Bodily, K.D., Morrison, C.M., Renden, R.B. and Broadie, K. (2001) Anovel member of the Ig superfamily, turtle, is a CNS-specific proteinrequired for coordinated motor control. J. Neurosci., 21, 3113–3125.

40. Le Bourg, E. and Lints, F.A. (1992) Hypergravity and aging in Drosophila

melanogaster. 4. Climbing activity. Gerontology, 38, 59–64.

41. Neumann, M. (2009) Molecular neuropathology of TDP-43proteinopathies. Int. J. Mol. Sci., 10, 232–246.

42. Belote, J.M. and Fortier, E. (2002) Targeted expression of dominantnegative proteasome mutants in Drosophila melanogaster. Genesis, 34,80–82.

43. Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L. andBonini, N.M. (1999) Suppression of polyglutamine-mediatedneurodegeneration in Drosophila by the molecular chaperone HSP70. Nat.

Genet., 23, 425–428.

44. Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M. and Bonini, N.M.(2002) Chaperone suppression of alpha-synuclein toxicity in a Drosophila

model for Parkinson’s disease. Science, 295, 865–868.

45. Jin, P., Zarnescu, D.C., Zhang, F., Pearson, C.E., Lucchesi, J.C., Moses,K. and Warren, S.T. (2003) RNA-mediated neurodegeneration caused bythe fragile X premutation rCGG repeats in Drosophila. Neuron, 39,739–747.

46. Zhou, L., Schnitzler, A., Agapite, J., Schwartz, L.M., Steller, H. andNambu, J.R. (1997) Cooperative functions of the reaper and headinvolution defective genes in the programmed cell death of Drosophila

central nervous system midline cells. Proc. Natl Acad. Sci. USA, 94,5131–5136.

47. Elden, A.C., Kim, H.J., Hart, M.P., Chen-Plotkin, A.S., Johnson, B.S.,Fang, X., Armakola, M., Geser, F., Greene, R., Lu, M.M. et al. (2010)Ataxin-2 intermediate-length polyglutamine expansions are associatedwith increased risk for ALS. Nature, 466, 1069–1075.

48. Ritson, G.P., Custer, S.K., Freibaum, B.D., Guinto, J.B., Geffel, D.,Moore, J., Tang, W., Winton, M.J., Neumann, M., Trojanowski, J.Q. et al.

(2010) TDP-43 mediates degeneration in a novel Drosophila modelof disease caused by mutations in VCP/p97. J. Neurosci., 30,7729–7739.

49. Yang, C., Tan, W., Whittle, C., Qiu, L., Cao, L., Akbarian, S. and Xu, Z.(2010) The C-terminal TDP-43 fragments have a high aggregationpropensity and harm neurons by a dominant-negative mechanism. PLoS

ONE, 5, e15878.

50. Jin, P., Zarnescu, D.C., Ceman, S., Nakamoto, M., Mowrey, J., Jongens,T.A., Nelson, D.L., Moses, K. and Warren, S.T. (2004) Biochemical andgenetic interaction between the fragile X mental retardation protein andthe microRNA pathway. Nat. Neurosci., 7, 113–117.

51. Zarnescu, D.C., Jin, P., Betschinger, J., Nakamoto, M., Wang, Y.,Dockendorff, T.C., Feng, Y., Jongens, T.A., Sisson, J.C., Knoblich, J.A.et al. (2005) Fragile X protein functions with lgl and the par complex inflies and mice. Dev. Cell, 8, 43–52.

52. Sephton, C.F., Cenik, C., Kucukural, A., Dammer, E.B., Cenik, B., Han,Y., Dewey, C.M., Roth, F.P., Herz, J., Peng, J. et al. (2011) Identificationof neuronal RNA targets of TDP-43-containing ribonucleoproteincomplexes. J. Biol. Chem., 286, 1204–1215.

53. Zhou, H., Huang, C., Chen, H., Wang, D., Landel, C.P., Xia, P.Y.,Bowser, R., Liu, Y.J. and Xia, X.G. (2010) Transgenic rat model ofneurodegeneration caused by mutation in the TDP gene. PLoS Genet., 6,e1000887.

54. Cairns, N.J., Perrin, R.J., Schmidt, R.E., Gru, A., Green, K.G., Carter, D.,Taylor-Reinwald, L., Morris, J.C., Gitcho, M.A. and Baloh, R.H. (2010)TDP-43 proteinopathy in familial motor neuron disease with TARDBPA315T mutation: a case report. Neuropathol. Appl. Neurobiol., 36,673–679.

55. Estes, P.S., Roos, J., van der Bliek, A., Kelly, R.B., Krishnan, K.S. andRamaswami, M. (1996) Traffic of dynamin within individual Drosophilasynaptic boutons relative to compartment-specific markers. J. Neurosci.,16, 5443–5456.



Wild-type and A315T mutant TDP-43 exert differential neurotoxicity ...

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