Genome-Wide Screen for Modifiers of Ataxin-3 Neurodegeneration in Drosophila 
[['Spinocerebellar ataxia type 3 ( SCA3 ) is the most common dominantly inherited ataxia worldwide and is caused by a CAG repeat expansion encoding glutamine within the ATXN3 gene [1 , 2] .', 'The expanded polyglutamine ( polyQ ) is thought to confer toxicity on the protein Ataxin-3 , leading to neural dysfunction and loss [3] .', "At least nine human diseases , including additional spinocerebellar ataxias and Huntington's disease , share this mechanism .", 'Studies on such pathogenic proteins reveal that the long polyQ domain alters protein conformation , causing an enriched beta sheet structure [4] .', 'Mutant polyQ protein also dynamically associates with chaperones and colocalizes with proteasome subunits , indicating that the protein is misfolded or abnormally folded [5 , 6] .', 'Such accumulation of misfolded protein is a common pathology of human degenerative disorders , including Alzheimer , Parkinson , and prion diseases [7–9] , indicating that these diseases may share molecular and cellular mechanisms .', 'Models for human neurodegenerative diseases in simple systems have provided valuable tools to dissect molecular mechanisms of disease pathology [10–13] .', 'Directed expression of pathogenic human Ataxin-3 in Drosophila recapitulates key features of disease , with late-onset neuronal dysfunction and degeneration accompanied by ubiquitinated inclusions [14 , 15] .', 'Neurotoxicity is more severe with increasing length of the polyQ repeat , similar to the human disease where longer repeats are associated with more severe and earlier onset disease [16 , 17] .', 'In the fly , increased levels of expression of the disease protein leads to more severe degeneration and earlier onset protein accumulation , suggesting that abnormal accumulation of the mutant protein is central to disease and degeneration .', 'A number of modifiers of select polyQ disease proteins have been identified using animal models , including chaperones , transcriptional coregulators , and microRNAs [18–22] .', 'Although these approaches have revealed genes that modulate polyQ toxicity , little is known regarding how the modifiers act biologically to modulate polyQ degeneration .', 'Among polyQ proteins , Ataxin-3 is unique because it has been implicated in ubiquitin pathways , and its normal activity may impinge on protein degradation pathways [23–26] .', 'Truncation of Ataxin-3 to remove the ubiquitin protease domain , or mutation of the ubiquitin protease activity , dramatically enhances toxicity [15] , indicating that the normal activity of Ataxin-3 may be critical in Ataxin-3-induced degeneration .', 'To reveal insight into pathways that modulate Ataxin-3 neurodegeneration , we performed a genetic modifier screen in Drosophila .', 'These studies revealed a range of modifiers that , despite some broadly diverse predicted molecular functions , converge on protein misfolding with a subset mitigating toxicity through proteasome and/or autophagy pathways .', 'These findings underscore the critical role of protein quality control in SCA3 pathogenesis and provide potential new targets toward therapeutics .'], ['To define modifiers that may reveal new insight into mechanisms of human SCA3 disease , we performed an overexpression screen for modifiers of Ataxin-3-induced neurodegeneration in Drosophila .', 'SCA3trQ78 causes late onset progressive degeneration characterized by loss of pigmentation and collapse of the eye ( Figure 1A and 1B ) [14] .', 'We initially screened a subset of 2 , 300 available EP-element insertion lines , each carrying a transposon engineered to direct expression of the downstream gene in the presence of the yeast GAL4 protein [27] .', 'Because reproducibility was variable with this collection , we then performed a screen de novo , selecting for new EP-insertions that modified SCA3trQ78 toxicity .', 'This approach identified 17 suppressors and one enhancer ( Figures 1C–1J and S1 ) , which affected both external and internal retinal degeneration .', 'Plasmid rescue was performed to identify the genes affected .', 'BLAST searches with genomic sequence from the integration sites revealed that the lines bore insertions in the 5′ regulatory region of select genes , and all were in an orientation to direct GAL4-dependent gene expression ( Text S1; Table S1 ) .', 'Northern and reverse transcription PCR analysis confirmed upregulation of the targeted genes; using a variety of tests we confirmed that the modifiers did not appear to affect transcription of the Ataxin-3 transgene or general GAL4-UAS transgene expression ( Figure S2 ) .', 'Both molecular and genetic analyses confirmed that the insertions were single insertions ( see Materials and Methods ) .', 'Reversion analysis proved that the EP-elements were causal in modification .', 'Taken together , these data indicated that the EP modifiers resulted in increased expression of the targeted downstream genes , which modulated SCA3trQ78 toxicity and neurodegeneration .', 'Analysis of the targeted genes revealed that the majority fell into two major classes of chaperones and ubiquitin-pathway components ( Table 1; Figures S1 and S3 ) .', 'The remaining modifiers were placed in a third category of miscellaneous functions .', 'Class 1 ( Figure 1C–1E ) included an Hsp70 family member , Hsp68E407; two Hsp40 genes , DnaJ-1B345 . 2 and mrjE1050; a small heat shock protein αB crystalline CG14207EP1348; and the cochaperone Tpr2EB7-1A .', 'The class 2 ubiquitin-pathway components included: polyubiquitin CR11700EP1384; a ubiquitin-specific protease Ubp64EE213-1A; two ubiquitin ligases , CG8209B3-Sa and Faf E659; and an F-box protein CG11033EP3093 , the only enhancer of the group .', 'Class 3 ( Figure 1F and 1G ) included genes with a variety of predicted molecular functions: the nuclear export protein Embargoed , embE2-1A and embE128-1A; three transcriptional regulators Sin3AB9-E , NFAT ( NFATEP1335 and NFATEP1508 ) , and debra ( dbrEP456 and dbrEP9 ) ; three translational regulators , including four alleles of the lin-41 homologue dappled ( dpldJM120 , dpldJM265 , dpldEP546 , and dpldEP2594 ) , a polyA binding protein orb2B8-S , and insulin growth factor II mRNA binding protein ImpEP1433; and finally a fatty acid oxidation enzyme palmitoyl co-A oxidase CG5009B227 . 2 .', 'This indicated that , although chaperone and ubiquitin-pathway components are major modifier categories , a variety of functional pathways are implicated in Ataxin-3 pathogenesis .', 'The screen selected for modifiers that , upon upregulation , affected toxicity .', 'To determine whether the activity of these genes may normally play a role in SCA3 toxicity , we examined whether reduction in the level of the modifier genes had an effect .', 'To do this , we reduced gene expression by 50% using loss-of-function alleles where available or deficiency lines .', 'Among these , reduction with a deficiency of DnaJ-1 and Ubp64E ( within the same deficiency; reduction of DnaJ-1 has previously been shown to enhance with a dominant-negative construct [16] ) , Trp2 , emb ( with an allele ) , and dbr dramatically enhanced degeneration ( Figure 1K–1O; Table S2 ) .', 'Although the deficiencies reduce the level of a number of genes , these data suggest that the endogenous activity of these genes may normally help to protect against degeneration .', 'To address whether morphological rescue correlated with functional rescue , we determined the ability of select suppressors to restore function in a phototaxis assay .', 'When flies bearing the SCA3trQ78 protein were given a choice between a light and dark chamber , they distributed randomly , indicating that they are functionally blind ( Figure 1P ) .', 'However , when mrjE1050 , which dramatically rescued degeneration , was co-expressed , normal vision was restored .', 'A milder suppressor that anatomically restored less retinal tissue , CG14207EP1348 , restored vision partially .', 'Thus , anatomical rescue correlated with functional rescue .', 'These and other studies indicated that the modifiers not only modulated toxicity of the external eye , but also that of the neuronal cells .', 'To confirm this in another situation , we tested select modifiers for the ability to mitigate polyQ toxicity when directed exclusively to the nervous system with elav-GAL4 ( Figure S4 ) .', 'These studies confirmed that the modifiers mitigated neuronal toxicity of the Ataxin-3 protein .', 'We performed select additional experiments with dpld , for which we obtained many independent EP overexpression alleles .', 'These detailed studies confirmed activity of the EP alleles of dpld with independent UAS-dpld transgenic lines .', 'Further , suppression by dpld was not limited to development but also extended to the adult timeframe ( Figures S2 , S5 , and S6 ) .', 'Because the genes were isolated as modifiers of a truncated Ataxin-3 protein , we tested whether they could mitigate toxicity of full-length Ataxin-3 , which is largely a neuronal toxicity [15] .', 'Modifiers that phenotypically strongly suppressed toxicity of truncated Ataxin-3 also strongly suppressed full-length Ataxin-3 toxicity; however , a number of modifiers that were weak or moderate suppressors of the truncated protein were , in contrast , strong suppressors of full-length Ataxin-3: CG14207EP1348 ( αB crystalline ) , CR11700EP1384 ( polyubiquitin ) , CG8209B3-Sa ( putative ubiquitin ligase ) , and Sin3AB9-E ( Figure S7 and unpublished data ) .', 'Consistent with strong anatomical rescue , CG14207EP1348 also robustly suppressed functional vision defects of full-length Ataxin-3 ( Figure 1Q ) .', 'The enhancer , however , CG11033EP3093 had a minimal effect on toxicity of full-length Ataxin-3 ( unpublished data ) .', 'This indicated that the modifiers varied in strength and selectivity depending upon whether the Ataxin-3 protein was intact or truncated .', 'As truncation may be a feature of SCA3 disease [28–30] , the effectiveness of modifiers against different forms of Ataxin-3 has implications for disease pathogenesis .', 'Previous studies have shown that the molecular chaperone Hsp70 is a potent suppressor of SCA3 toxicity [31] .', 'Therefore , we considered that the class 1 modifiers may function similar to Hsp70 to help cells handle the misfolded disease protein , whereas those of class 2 likely have a role in ubiquitin-dependent pathways that process misfolded proteins .', 'However , class 3 presented a range of potential activities .', 'To address how the modifiers were functioning biologically , we tested whether the modifiers could affect a general protein misfolding phenotype: compromised chaperone activity with a dominant-negative form of Hsp70 ( Hsp70 . K71E ) .', 'This situation results in an eye phenotype that resembles severe polyQ degeneration ( Figure 2A and [32] ) .', 'Thus , modifiers that affected both SCA3 and Hsp70 . K71E toxicity would likely include those whose mode of action was to modulate protein misfolding .', 'Strikingly , we found that most of the suppressors of polyQ toxicity also mitigated the Hsp70 . K71E phenotype , as well as or better than directed expression of Hsp70 itself ( Figure 2; Table 1 ) .', 'Interestingly , DnaJ-1B345 . 2 and Tpr2EB7-1A enhanced this phenotype; we interpreted this to indicate that these genes , which encode proteins thought to act as cochaperones of Hsp70 , may compromise residual Hsp70 in the dominant-negative situation .', 'The chaperone mrjE1050 , although an Hsp40 class chaperone , acted in a manner distinct from DnaJ-1B345 . 2 , as it suppressed rather than enhanced the Hsp70 . K17E phenotype .', 'Moreover , the enhancer of SCA3trQ78 toxicity , CG11033EP3093 , suppressed the misfolding phenotype .', 'Only one modifier did not affect this phenotype , the ubiquitin protease Ubp64EE213-1A; interestingly , normal Ataxin-3 also has ubiquitin protease activity that mitigates its own pathogenicity , and similarly , has no effect on Hsp70 . K71E [15] .', 'We considered that one mechanism by which the modifiers may mitigate the Hsp70 . K71E phenotype is by upregulating Hsp70/Hsc70 chaperones; however , none of the modifiers appeared to act in this way ( Figure 2K and unpublished data ) .', 'These results indicated that the majority of modifiers of SCA3trQ78 toxicity appeared to function biologically by aiding in situations of compromised chaperone activity and/or protein misfolding .', 'The degree of neurodegeneration induced by pathogenic polyQ protein is typically correlated with the level of accumulation of the protein in animals in vivo .', 'We reasoned , therefore , that the modifiers may affect protein levels .', 'We therefore examined protein accumulation by immunohistochemistry and western analysis .', 'Although nuclear inclusions may not be causal in disease [33] , later onset , smaller nuclear inclusions are typically reflective of reduced pathogenicity of the protein in vivo .', 'Hsp70 and Hsp40 have also been shown to increase the solubility of pathogenic polyQ protein by western blots , concomitant with reducing toxicity [16] .', 'We therefore examined protein accumulation using rh1-GAL4 or the full-length Ataxin-3 protein—both situations that allow sensitive analysis of protein accumulation [15 , 22] .', 'In these studies , we limited analysis to the strong and moderate modifiers .', 'Immunohistochemical analysis revealed that select modifiers had striking effects to reduce NIs .', 'These included the class I chaperones Hsp68E407 , DnaJ-1B345 . 2 , mrjE1050 , but did not include αB-crystalline CG14207EP1348 or Tpr2EB7-1A ( Figure 3; Table 1 ) .', 'Of the class 2 ubiquitin-pathway components , polyubiquitin CR11700EP1384 and the ubiquitin protease Ubp64EE213-1A reduced NIs , but the other strong modifiers of this class did not .', 'Of class 3 modifiers tested , all reduced NI except ImpEP1433 ( Figure 3B and 3F ) .', 'We then analyzed solubility of the pathogenic protein by immunoblot .', 'This approach revealed that , although the modifiers had no effect on protein levels at early time points prior to inclusion formation , all suppressors increased the level of monomeric protein over time , thus all increased the solubility properties of the pathogenic protein ( Figure 3E ) .', 'The effect was specific , as co-expression of a control protein ( green fluorescent protein [GFP] ) had no effect ( unpublished data ) .', 'Similarly , the enhancer reduced monomer levels ( unpublished data ) .', 'These findings indicate that the modifiers either affected pathogenic protein accumulation or altered the solubility properties of the toxic protein , concomitant with altering protein pathogenicity .', 'Given that many modifiers mitigated protein accumulation , we asked whether there were interactions with genes of protein degradation pathways .', 'A key pathway thought to modulate the pathogenic polyQ toxicity is the ubiquitin-proteasomal system ( UPS ) [34] .', 'We therefore tested whether suppression by modifiers that lowered protein levels was dependent upon a fully functional proteasome .', 'Proteasome activity can be reduced by a dominant temperature-sensitive mutation in a proteasome protein subunit ( DTS5 ) [35] .', 'In a situation where limiting proteasome activity using this mutation had no effect on SCA3 toxicity on its own , we found that a striking number of modifiers still suppressed polyQ toxicity , thus indicating that they were not sensitive to inhibition of the proteasome by this assay; these included dpld alleles ( Figure 4; Table 1 ) .', 'In contrast , a striking exception was the class 2 ubiquitin-pathway suppressors: all of these modifiers lost the ability to suppress upon proteasome inhibition with the DTS mutation .', 'Autophagy , or lysosome-mediated protein degradation , has also been implicated in polyQ toxicity and cell survival in situations of stress [36 , 37] .', 'We therefore asked whether the modifier genes had an effect on this process .', 'First , we determined whether normal or pathogenic Ataxin-3 itself induced lysosomal accumulation reflective of autophagy , by examining the fat body tissue from larvae , a standard assay for autophagy [38] .', 'Normally , well-fed animals show minimal lysosomal induction , whereas starved animals show a dramatic increase , reflected by the uptake of dye ( Figure 4F and 4G ) .', 'Whereas expression of normal Ataxin-3 ( SCA3Q27 ) had minimal effect , expression of pathogenic Ataxin-3-induced autophagy ( Figure 4J ) .', 'To further address the role of autophagy , we determined whether limiting the activity of autophagy genes affected SCA3 toxicity .', 'Key genes to which RNA interference transgenic lines are available include Atg5 .', 'Whereas reduction of Atg5 activity on its own had little effect , reducing Atg5 in the presence of pathogenic SCA3 protein appeared to enhance toxicity , with increased loss of retinal integrity ( Figure 4L–4N ) .', 'This suggests that , normally , autophagy may mitigate toxicity of the pathogenic protein .', 'Reduction of autophagy also enhanced aggregation of the protein by western immunoblot and enhanced cytoplasmic protein accumulation along photoreceptor axons ( Figure 4O–4Q ) .', 'We then determined whether strong modifier genes also modulated autophagy .', 'We examined two situations: ( 1 ) to determine whether strong modifier genes could induce autophagy in well-fed animals when autophagy is normally minimal; ( 2 ) to determine whether they could block autophagy under starvation , when autophagy is stimulated as a protective mechanism .', 'We tested these as we considered that a modifier may affect Ataxin-3 pathogenicity either by inducing autophagy-mediated lysosomal degradation of the pathogenic protein , or alternatively , by blocking autophagy if autophagy contributes to loss of the cells in response to mutant polyQ protein .', 'These studies revealed that select modifiers induced , whereas others mitigated , autophagy ( Figure 4H , 4I , and 4K ) .', 'Of the class 1 chaperone modifiers , the two Hsp40 genes ( DnaJ-1B345 . 2 and mrj E1050 ) increased autophagy , whereas Hsp70 and the class 3 modifier ImpEP1433 reduced autophagy .', 'Although Dpld showed no effect in these assays , limiting autophagy by reduction of Atg5 , Atg7 , or Atg12 mitigated Dpld suppression , suggesting that its activity was dependent on autophagy ( Figure 4R , 4S , and unpublished data ) .', 'These and other data ( Figures S8 and S9 ) suggested that Dpld may act upstream of autophagy genes to activate autophagy in select situations .', 'We also tested available GFP protein trap lines to examine localization of modifier proteins .', 'Although none of these lines showed GFP immunostaining , one line with a protein trap insertion in Hsc70Cb enhanced SCA3 neuronal toxicity and increased protein accumulation in the neurophil similar to autophagy genes ( Figure S8G–S8K ) .', 'Taken together , these studies indicated that the modifiers , despite broad molecular nature , mitigated situations of protein misfolding; in some cases their activity appeared dependent on the proteasome , whereas others may involve autophagy-based protein clearance or autophagy-related cell loss .', 'The modifiers were selected based on ability to modulate SCA3 degeneration; however , our studies suggested that the modifiers may have broader functions in protein misfolding .', 'We therefore determined whether they could modulate toxicity of tau .', 'Abnormal accumulation of tau in neurofibrillary tangles or mutations in tau are associated with Alzheimer disease and frontotemporal dementia [39] .', 'Tau-induced degeneration is mitigated by the caspase inhibitor P35 and DIAPs , implicating programmed cell death pathways in tau toxicity [40] .', 'Expression of normal ( tau . wt ) or mutant ( tau . R406 ) tau causes toxicity reflected in a reduced and degenerate eye [41] .', 'Co-expression of the class 1 chaperone Tpr2EB7-1A and the Hsc70Cb line class 2 modifier polyubiquitin CR11700EP1384 , and class 3 modifiers dpld JM265 , ImpEP1433 , and CG5009B227 . 2 strikingly suppressed toxicity of tau ( Figure 5; Figure S8 ) .', 'The class 3 modifier embE2-1A enhanced tau ( Figure 5 ) , whereas NFAT EP1335 enhanced tau . wt , but had no effect on mutant tau . R406W ( unpublished data ) .', 'We then examined the ability of the modifiers to affect programmed cell death .', 'Several class 3 modifiers had an effect on hid-induced eye loss: co-expression of embE2-1A and NFATEP1508 enhanced hid , whereas ImpEP1433 and dpldJM265 alleles suppressed hid-induced cell death ( Figure 5 and unpublished data ) .', 'Alleles of those genes that modulated tau and programmed cell death similarly ( emb , dpld , NFAT , and Imp ) may modulate tau toxicity by altering cell death .', 'In contrast , the others ( Tpr2 , polyubiquitin CR11700 , and Hsc70Cb ) likely modulate tau toxicity through other means .', 'Taken together , these data indicate that select modifiers that influence cell survival and protein misfolding may be common to both SCA3 and tau-induced degeneration .'], ['Our screen was designed to identify upregulation modifiers , targeting genes whose increased activity could modulate neuronal toxicity and degeneration of a pathogenic Ataxin-3 protein .', 'This screen identified activities that may become compromised or normally insufficient in the disease protein situation .', 'A number of modifiers appeared dosage sensitive , in that reduction of the genetic region encoding the gene enhanced degeneration .', 'This suggests that , at least for select modifiers , their normal activity is also critical for maintenance of neurons in disease .', 'Because of the severity of the degenerate eye , we may have selected for particularly strong modifiers .', 'Modifiers were also initially selected for the ability to mitigate an external eye degeneration , rather than direct neuronal toxicity .', 'Despite this , all of the modifiers mitigated internal neural degeneration .', 'This indicates that the degree of external eye degeneration is a reasonable predictor of internal neuronal integrity .', 'A number of modifiers were identified multiple times ( dpld , NFAT , emb , and dbr ) , although most were found only once , indicating that the screen is not saturated .', 'Additional screens using different mutagens or different types of screening approaches may reveal additional and different classes of modifiers .', 'Although the modifiers were selected for mitigation of toxicity of a truncated Ataxin-3 protein , nearly all effectively mitigated toxicity of the full-length pathogenic protein .', 'Our previous and other studies suggest that truncation may normally occur in disease to remove the N-terminal ubiquitin protease domain and would dramatically enhance degeneration [15 , 29 , 30] .', 'Therefore , these are efficacious modifiers that mitigate toxicity of various forms of the pathogenic protein .', 'The majority of modifiers were either chaperones or , by sequence and functional tests , modulated ubiquitin-mediated protein quality control pathways .', 'Whereas those that have sequence implications in this pathway might be anticipated , a surprise was that many genes with widely divergent molecular activities were functionally implicated in this process .', 'These data suggest that many genes can modulate situations of protein misfolding , and moreover , suggest that protein misfolding is central to polyQ pathogenesis .', 'It is also possible that , given the normal function of Ataxin-3 in ubiquitin-modulated pathways , the screen selected for modifiers that impinge on normal activities of Ataxin-3 .', 'Among the modifiers , some decreased protein accumulation , whereas others suppressed with no apparent change in Ataxin-3 inclusions .', 'Of those that decreased Ataxin-3 accumulation , some appeared dependent on functional proteasome activity , whereas others stimulated autophagy , or both , implicating various mechanisms that attenuate Ataxin-3 degeneration .', 'Among the modifiers was polyubiquitin , arguing that the level of ubiquitin itself may normally be insufficient to handle the misfolded protein over prolonged periods .', 'This is consistent with the lack of a robust stress response to pathogenic polyQ [42] .', 'In a Caenorhabditis elegans of model of polyQ , Gidalevitz et al . [43] reveal that polyQ expansions can cause temperature-sensitive alleles of various unrelated genes ( paramyosin , dynamin , and ras ) to display the mutant effect at what would normally be permissive conditions .', 'This finding suggests that long polyQ runs can cause a general disruption of the cellular protein-folding environment to affect the temperature-sensitive mutant proteins .', 'Our work used a pathogenic human disease protein Ataxin-3 and revealed that many modifiers that mitigate Ataxin-3 toxicity also modulate general protein misfolding .', 'Taken together , these results suggest that , in the absence of upregulation of the various components needed , the cell may become overwhelmed , triggering deleterious effects [32] .', 'Our detailed analysis of modifiers of Ataxin-3 have revealed a variety of biological processes that can help manage pathogenic polyQ protein , as well as specific genes of interest ( Figure 6A ) .', 'The precise molecular pathways by which those modifiers that are not clearly within protein folding pathways can modulate misfolding to affect neurodegeneration requires further study .', 'We envisage , however , that they function by their predicted molecular mechanisms , but on targets that impinge on cellular protein homeostasis .', 'For example , chromatin modifiers may tweak the expression of a variety of genes in such processes , whereas translational regulators may translationally modify such genes .', 'The finding that a variety of cellular functions are involved would be consistent with RNA interference screens in C . elegans that identified a variety of genes that impinge on cellular protein homeostasis as enhancers of the aggregation of polyQ protein [20] .', 'These findings , along with other studies including those we present here , underscore the critical importance of proper protein homeostasis to most—if not all—cellular functions , such that a variety of genes can influence this process .', 'Among our modifiers , some that might have been expected to act in a similar manner , appeared to have distinct biological effects .', 'The class 2 modifiers showed complete dependence on proper proteasome activity , while other modifiers , including alleles of DnaJ-1 and Imp , appeared to be insensitive to limiting proteasome activity , but rather affected autophagy in stress conditions .', 'Hsp70 suppresses multiple stress conditions including protein misfolding , starvation-induced autophagy , and paraquat-induced oxidative stress [11] , suggesting it may both facilitate UPS activity , but also block autophagy-dependent degeneration .', 'In vivo , these pathways interplay to maintain proper neuronal function in the face of disease , thus further study of the modifiers may allow greater molecular identification of the individual pathways , as well as their integration .', 'Although only select modifiers affected protein accumulation as assayed by immunohistochemistry , all affected the solubility of the disease protein by biochemical analysis .', 'The relationship between protein inclusions and proposed toxic oligomers is still under investigation , but it seems reasonable to suggest that the change in solubility may reflect activity of the modifiers to buffer or alter toxic conformations of the protein .', 'Our efforts to detect toxic oligomers of Ataxin-3 using available antibodies [33 , 44 , 45] are still in progress .', 'We note that , despite gross similarity , SCA3 degeneration is not identical to general misfolding: some suppressors of Ataxin-3 toxicity strikingly enhanced dominant-negative Hsp70 ( upregulation of DnaJ-1 and Tpr2 ) , whereas the enhancer CG11033E3093 suppressed it .', 'The UPS and autophagy pathways function in either degeneration or polyQ protein pathogenicity [46] .', 'Our findings indicate that these pathways at least in part function in the removal or decrease of the toxic protein , thereby reducing degeneration .', 'It is also possible that activities of these pathways may be inhibited during pathogenesis; such inhibition of normal activity would contribute to disease pathology [47–49] .', 'The normal function of Ataxin-3 is in ubiquitin-modulated pathways [23–26]; our data suggest the possibility that Ataxin-3 may also modulate autophagy .', 'Normal physiological levels of autophagy are critical to integrity of neurons , as loss of autophagy causes degeneration associated with ubiquitinated inclusions [50 , 51] .', 'Our studies also reveal that compromise of autophagy pathways strikingly increased cytoplasmic Ataxin-3 accumulations in the neurophil without an obvious change in NIs .', 'This may indicate that autophagy normally modulates cytoplasmic accumulation of the disease protein .', 'Previous studies show that cytoplasmic polyQ protein is very toxic and blocks axonal transport [52] .', 'These findings suggest that perturbations in autophagy may enhance cytoplasmic toxicity further .', 'In addition to regulation of the disease protein level and misfolding , results with select modifiers ( Hsp70 and Imp ) suggest that autophagy may also regulate loss of the cells .', 'Given that our previous findings failed to reveal a clear role of caspase-dependent cell death in SCA3 [22] , autophagy may be a mechanism of cell loss in this situation .', 'Identification of other components of UPS and autophagy pathway will give further insights into how the disease protein is degraded and mechanisms of neuronal loss .', 'Genetic screens have been performed for modifiers of Ataxin-1 [19] and of pure polyQ domains [18] , revealing similar components of protein folding and degradation: DnaJ-1 [18 , 19] and Tpr2 [18] , but also RNA binding proteins and transcription factors .', 'Although components of ubiquitin pathways that modulate Ataxin-1 appear distinct from Ataxin-3 , this may reflect different regulators required for modifying different proteins or lack of saturation of the screens .', 'Similarly several RNA binding proteins identified as modifiers of Ataxin-1 were suggested to reflect Ataxin-1 function as a putative RNA binding protein; our data suggest that it is also possible that these modifiers work more fundamentally to modulate protein solubility or levels .', 'Our other work , however , has revealed a role for microRNAs in modulating neuronal survival in neurodegenerative situations [22 , 53] , which has recently been extended to vertebrate neuronal integrity as well [54] .', 'A C . elegans screen revealed a large number of modifiers of a pure polyQ aggregation phenotype [20] .', 'Several modifiers identified in that screen affect RNA synthesis , processing , and protein synthesis as modifiers of misfolding .', 'It will be important to test these modifiers against various specific disease proteins , as the action of a pure polyQ repeat may be distinct from a pathogenic repeat within a host protein .', 'One reason for global commonality among modifiers of polyQ disease proteins may be that the proteins themselves fall within a common interacting protein network .', 'Recent studies using the yeast two-hybrid approach and proteomic databases reveal a protein interaction network of the ataxia proteins [55] .', 'A surprise was that many different ataxia proteins , including Ataxin-3 , fall within a few interaction steps from one another , suggesting that their common phenotypes may be a reflection of common interactions , which , when perturbed , contribute to disease .', 'In that study [55] , seven proteins were identified as direct interaction partners of Ataxin-3; however none of these appeared in our screen .', 'Another direct binding protein–VCP [56]—was also not identified .', 'However , the human orthologues of the majority of the genetic modifiers we defined fit into the protein interaction network at various points; some are direct interactors of other ataxia disease proteins , and others are one or more steps removed ( Table S3 ) .', 'Alzheimer disease and polyQ diseases are two unrelated human neurodegenerative diseases that cause neuronal degeneration in distinct brain regions [1 , 2 , 57] .', 'Therefore , no overlap was expected between modifiers of these disease proteins in flies; indeed previous studies suggest minimal overlapping genes [58] .', 'Surprisingly , we found that select modifiers of Ataxin-3 suppressed tau-degeneration , 
['Introduction', 'Results', 'Discussion', 'Materials and Methods', 'Supporting Information']
['Spinocerebellar ataxia type-3 ( SCA3 ) is among the most common dominantly inherited ataxias , and is one of nine devastating human neurodegenerative diseases caused by the expansion of a CAG repeat encoding glutamine within the gene .', 'The polyglutamine domain confers toxicity on the protein Ataxin-3 leading to neuronal dysfunction and loss .', 'Although modifiers of polyglutamine toxicity have been identified , little is known concerning how the modifiers function mechanistically to affect toxicity .', 'To reveal insight into spinocerebellar ataxia type-3 , we performed a genetic screen in Drosophila with pathogenic Ataxin-3-induced neurodegeneration and identified 25 modifiers defining 18 genes .', 'Despite a variety of predicted molecular activities , biological analysis indicated that the modifiers affected protein misfolding .', 'Detailed mechanistic studies revealed that some modifiers affected protein accumulation in a manner dependent on the proteasome , whereas others affected autophagy .', 'Select modifiers of Ataxin-3 also affected tau , revealing common pathways between degeneration due to distinct human neurotoxic proteins .', 'These findings provide new insight into molecular pathways of polyQ toxicity , defining novel targets for promoting neuronal survival in human neurodegenerative disease .']
['Spinocerebellar ataxia type-3 is the most common dominantly inherited movement disorder and is caused by a CAG repeat expansion within the gene ATXN3 , encoding the Ataxin-3 protein .', 'This leads to a protein with an expanded polyglutamine domain , which confers a dominant toxicity on the protein , leading to late onset , progressive neural degeneration in the brain .', 'Although some modifiers of Ataxin-3 toxicity have been defined , little was known about their molecular mechanisms of action .', 'The fruit fly Drosophila recapitulates fundamental aspects of the human disease .', 'Here , we performed a genome-wide screen for new modifiers of Ataxin-3 toxicity using the fly and defined 25 modifiers in 18 genes .', 'The majority of the genes belong to chaperone and ubiquitin proteasome pathways , which modulate protein folding and degradation , but the remaining modifiers have a broad range of predicted molecular functions .', 'Assays in vivo revealed that the biological activity of all modifiers converge on aiding in situations of protein misfolding , despite distinct predicted molecular functions .', 'Select modifiers of Ataxin-3 toxicity also modulated tau toxicity associated with Alzheimer disease .', 'These findings underscore the importance of protein homeostasis pathways to disease and provide the foundation for new therapeutic insight .']
['cell biology', 'neurological disorders', 'eukaryotes', 'drosophila', 'neuroscience', 'animals', 'genetics and genomics', 'insects']