A phenotypically robust model of spinal and bulbar muscular atrophy in Drosophila.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked disorder that affects males who inherit the androgen receptor (AR) gene with an abnormal CAG triplet repeat expansion. The resulting protein contains an elongated polyglutamine (polyQ) tract and causes motor neuron degeneration in an androgen-dependent manner. The precise molecular sequelae of SBMA are unclear. To assist with its investigation and the identification of therapeutic options, we report here a new model of SBMA in Drosophila melanogaster. We generated transgenic flies that express the full-length, human AR with a wild-type or pathogenic polyQ repeat. Each transgene is inserted into the same safe harbor site on the third chromosome of the fly as a single copy and in the same orientation. Expression of pathogenic AR, but not of its wild-type variant, in neurons or muscles leads to consistent, progressive defects in longevity and motility that are concomitant with polyQ-expanded AR protein aggregation and reduced complexity in neuromuscular junctions. Additional assays show adult fly eye abnormalities associated with the pathogenic AR species. The detrimental effects of pathogenic AR are accentuated by feeding flies the androgen, dihydrotestosterone. This new, robust SBMA model can be a valuable tool toward future investigations of this incurable disease.

Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when the enzyme is itself ubiquitinated at lysine (K) at position 117: in vitro, K117-ubiqutinated Atxn3 cleaves poly-ubiquitin markedly more rapidly compared to its unmodified counterpart. How polyQ expansion causes SCA3 remains unclear. To gather insights into the biology of disease of SCA3, here we posited the question: is K117 important for toxicity caused by pathogenic Atxn3? To answer this question, we generated transgenic Drosophila lines that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. We found that mutating K117 mildly enhances the toxicity and aggregation of pathogenic Atxn3. An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest that Atxn3 ubiquitination is a regulatory step of SCA3, in part by modulating its aggregation.

Phenotypic defects from the expression of wild-type and pathogenic TATA-binding proteins in new Drosophila models of Spinocerebellar Ataxia Type 17.

Spinocerebellar Ataxia Type 17 (SCA17) is the most recently identified member of the polyglutamine (polyQ) family of disorders, resulting from abnormal CAG/CAA expansion in the TATA box-binding protein (TBP), an initiation factor essential for of all eukaryotic transcription. A largely autosomal dominant inherited disease, SCA17, is unique in both its heterogeneous clinical presentation and low incidence of genetic anticipation, the phenomenon in which subsequent generations inherit longer polyQ expansions that yield earlier and more severe symptom onset. Like other polyQ disease family members, SCA17 patients experience progressive ataxia and dementia, and treatments are limited to preventing symptoms and increasing quality of life. Here, we report 2 new Drosophila models that express human TBP with polyQ repeats in either wild-type or SCA17 patient range. We find that TBP expression has age- and tissue-specific effects on neurodegeneration, with polyQ-expanded SCA17 protein expression generally having more severe effects. In addition, SCA17 model flies accumulate more aggregation-prone TBP, with a greater proportion localizing to the nucleus. These new lines provide a new resource for the biochemical characterization of SCA17 pathology and the future identification of therapeutic targets.

Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when it is ubiquitinated at lysine (K) at position 117. K117-ubiqutinated Atxn3 cleaves poly-ubiquitin more rapidly in vitro compared to its unmodified counterpart and this residue is also important for Atxn3 roles in cell culture and in Drosophila melanogaster . How polyQ expansion causes SCA3 remains unclear. To gather insight into the biology of disease of SCA3, here we posited the question: is K117 important for toxicity caused by Atxn3? We generated transgenic Drosophila lines that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. We found that K117 mutation mildly enhances the toxicity and aggregation of pathogenic Atxn3 in Drosophila . An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest Atxn3 ubiquitination as a regulatory step of SCA3, in part by modulating its aggregation.

Drosophila as a Model of Unconventional Translation in Spinocerebellar Ataxia Type 3.

RNA toxicity contributes to diseases caused by anomalous nucleotide repeat expansions. Recent work demonstrated RNA-based toxicity from repeat-associated, non-AUG-initiated translation (RAN translation). RAN translation occurs around long nucleotide repeats that form hairpin loops, allowing for translation initiation in the absence of a start codon that results in potentially toxic, poly-amino acid repeat-containing proteins. Discovered in Spinocerebellar Ataxia Type (SCA) 8, RAN translation has been documented in several repeat-expansion diseases, including in the CAG repeat-dependent polyglutamine (polyQ) disorders. The ATXN3 gene, which causes SCA3, also known as Machado-Joseph Disease (MJD), contains a CAG repeat that is expanded in disease. ATXN3 mRNA possesses features linked to RAN translation. In this paper, we examined the potential contribution of RAN translation to SCA3/MJD in Drosophila by using isogenic lines that contain homomeric or interrupted CAG repeats. We did not observe unconventional translation in fly neurons or glia. However, our investigations indicate differential toxicity from ATXN3 protein-encoding mRNA that contains pure versus interrupted CAG repeats. Additional work suggests that this difference may be due in part to toxicity from homomeric CAG mRNA. We conclude that Drosophila is not suitable to model RAN translation for SCA3/MJD, but offers clues into the potential pathogenesis stemming from CAG repeat-containing mRNA in this disorder.

Ubiquitin-binding site 1 of pathogenic ataxin-3 regulates its toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Spinocerebellar Ataxia Type 3 (SCA3) is a member of the family of polyglutamine (polyQ) diseases that are caused by anomalous CAG triplet repeat expansions in several genes. SCA3 results from abnormal polyQ expansion in the deubiquitinase (DUB), ataxin-3 (Atxn3). To understand the role of the different domains of mutant Atxn3 on its pathogenicity, with the hope that they can be explored for therapeutic interventions, we have systematically studied their individual and collective effects on its toxicity. One such domain is ubiquitin-binding site 1 (UbS1) on the catalytic domain of Atxn3; UbS1 is necessary for the enzymatic activity of Atxn3. Here, we investigated the importance of UbS1 on the toxicity of pathogenic Atxn3. We generated transgenic Drosophila melanogaster lines that express polyQ-expanded Atxn3 with and without a functional UbS1. We found that mutating UbS1 markedly exacerbates the toxicity of pathogenic Atxn3. Additional studies indicated that UbS1 regulates the toxicity of Atxn3 not by affecting its aggregation or sub-cellular localization, but by impacting its role in ubiquitin processing. Our findings provide additional insights into the role of Atxn3's domains in the pathogenicity of SCA3.

Targeting the VCP-binding motif of ataxin-3 improves phenotypes in Drosophila models of Spinocerebellar Ataxia Type 3.

Of the family of polyglutamine (polyQ) neurodegenerative diseases, Spinocerebellar Ataxia Type 3 (SCA3) is the most common. Like other polyQ diseases, SCA3 stems from abnormal expansions in the CAG triplet repeat of its disease gene resulting in elongated polyQ repeats within its protein, ataxin-3. Various ataxin-3 protein domains contribute to its toxicity, including the valosin-containing protein (VCP)-binding motif (VBM). We previously reported that VCP, a homo-hexameric protein, enhances pathogenic ataxin-3 aggregation and exacerbates its toxicity. These findings led us to explore the impact of targeting the SCA3 protein by utilizing a decoy protein comprising the N-terminus of VCP (N-VCP) that binds ataxin-3's VBM. The notion was that N-VCP would reduce binding of ataxin-3 to VCP, decreasing its aggregation and toxicity. We found that expression of N-VCP in Drosophila melanogaster models of SCA3 ameliorated various phenotypes, coincident with reduced ataxin-3 aggregation. This protective effect was specific to pathogenic ataxin-3 and depended on its VBM. Increasing the amount of N-VCP resulted in further phenotype improvement. Our work highlights the protective potential of targeting the VCP-ataxin-3 interaction in SCA3, a key finding in the search for therapeutic opportunities for this incurable disorder.

Ubiquitin-interacting motifs of ataxin-3 regulate its polyglutamine toxicity through Hsc70-4-dependent aggregation.

Spinocerebellar ataxia type 3 (SCA3) belongs to the family of polyglutamine neurodegenerations. Each disorder stems from the abnormal lengthening of a glutamine repeat in a different protein. Although caused by a similar mutation, polyglutamine disorders are distinct, implicating non-polyglutamine regions of disease proteins as regulators of pathogenesis. SCA3 is caused by polyglutamine expansion in ataxin-3. To determine the role of ataxin-3's non-polyglutamine domains in disease, we utilized a new, allelic series of Drosophila melanogaster. We found that ataxin-3 pathogenicity is saliently controlled by polyglutamine-adjacent ubiquitin-interacting motifs (UIMs) that enhance aggregation and toxicity. UIMs function by interacting with the heat shock protein, Hsc70-4, whose reduction diminishes ataxin-3 toxicity in a UIM-dependent manner. Hsc70-4 also enhances pathogenicity of other polyglutamine proteins. Our studies provide a unique insight into the impact of ataxin-3 domains in SCA3, identify Hsc70-4 as a SCA3 enhancer, and indicate pleiotropic effects from HSP70 chaperones, which are generally thought to suppress polyglutamine degeneration.

Degron capability of the hydrophobic C-terminus of the polyglutamine disease protein, ataxin-3.

Ataxin-3 is a deubiquitinase and polyglutamine disease protein whose cellular properties and functions are not entirely understood. Mutations in ataxin-3 cause spinocerebellar ataxia type 3 (SCA3), a neurodegenerative disorder that is a member of the polyglutamine family of diseases. Two major isoforms arise from alternative splicing of ATXN3 and are differently toxic in vivo as a result of faster proteasomal degradation of one isoform compared to the other. The isoforms vary only at their C-termini, suggesting that the hydrophobic C-terminus of the more quickly degraded form of ataxin-3 (here referred to as isoform 2) functions as a degron-that is, a peptide sequence that expedites the degradation of its host protein. We explored this notion in this study and present evidence that: (a) the C-terminus of ataxin-3 isoform 2 signals its degradation in a proteasome-dependent manner, (b) this effect from the C-terminus of isoform 2 does not require the ubiquitination of ataxin-3, and (c) the isolated C-terminus of isoform 2 can enhance the degradation of an unrelated protein. According to our data, the C-terminus of ataxin-3 isoform 2 is a degron, increasing overall understanding of the cellular properties of the SCA3 protein.

Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling.

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.

Druggable genome screen identifies new regulators of the abundance and toxicity of ATXN3, the Spinocerebellar Ataxia type 3 disease protein.

Spinocerebellar Ataxia type 3 (SCA3, also known as Machado-Joseph disease) is a neurodegenerative disorder caused by a CAG repeat expansion encoding an abnormally long polyglutamine (polyQ) tract in the disease protein, ataxin-3 (ATXN3). No preventive treatment is yet available for SCA3. Because SCA3 is likely caused by a toxic gain of ATXN3 function, a rational therapeutic strategy is to reduce mutant ATXN3 levels by targeting pathways that control its production or stability. Here, we sought to identify genes that modulate ATXN3 levels as potential therapeutic targets in this fatal disorder. We screened a collection of siRNAs targeting 2742 druggable human genes using a cell-based assay based on luminescence readout of polyQ-expanded ATXN3. From 317 candidate genes identified in the primary screen, 100 genes were selected for validation. Among the 33 genes confirmed in secondary assays, 15 were validated in an independent cell model as modulators of pathogenic ATXN3 protein levels. Ten of these genes were then assessed in a Drosophila model of SCA3, and one was confirmed as a key modulator of physiological ATXN3 abundance in SCA3 neuronal progenitor cells. Among the 15 genes shown to modulate ATXN3 in mammalian cells, orthologs of CHD4, FBXL3, HR and MC3R regulate mutant ATXN3-mediated toxicity in fly eyes. Further mechanistic studies of one of these genes, FBXL3, encoding a F-box protein that is a component of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex, showed that it reduces levels of normal and pathogenic ATXN3 in SCA3 neuronal progenitor cells, primarily via a SCF complex-dependent manner. Bioinformatic analysis of the 15 genes revealed a potential molecular network with connections to tumor necrosis factor-α/nuclear factor-kappa B (TNF/NF-kB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways. Overall, we identified 15 druggable genes with diverse functions to be suppressors or enhancers of pathogenic ATXN3 abundance. Among identified pathways highlighted by this screen, the FBXL3/SCF axis represents a novel molecular pathway that regulates physiological levels of ATXN3 protein.

Differential toxicity of ataxin-3 isoforms in Drosophila models of Spinocerebellar Ataxia Type 3.

The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein ataxin-3, a deubiquitinase. Two major full-length isoforms of ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. Most SCA3 studies have focused on isoform 1, the predominant version in mammalian brain, yet both isoforms are present in brain and a better understanding of their relative pathogenicity in vivo is needed. We took advantage of the fruit fly, Drosophila melanogaster to model SCA3 and to examine the toxicity of each ataxin-3 isoform. Our assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to our results, although both full-length, pathogenic ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. Our findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.

Expression and Regulation of Deubiquitinase-Resistant, Unanchored Ubiquitin Chains in Drosophila.

The modifier protein, ubiquitin (Ub) regulates various cellular pathways by controlling the fate of substrates to which it is conjugated. Ub moieties are also conjugated to each other, forming chains of various topologies. In cells, poly-Ub is attached to proteins and also exists in unanchored form. Accumulation of unanchored poly-Ub is thought to be harmful and quickly dispersed through dismantling by deubiquitinases (DUBs). We wondered whether disassembly by DUBs is necessary to control unanchored Ub chains in vivo. We generated Drosophila melanogaster lines that express Ub chains non-cleavable into mono-Ub by DUBs. These chains are rapidly modified with different linkages and represent various types of unanchored species. We found that unanchored poly-Ub is not devastating in Drosophila, under normal conditions or during stress. The DUB-resistant, free Ub chains are degraded by the proteasome, at least in part through the assistance of VCP and its cofactor, p47. Also, unanchored poly-Ub that cannot be cleaved by DUBs can be conjugated en bloc, in vivo. Our results indicate that unanchored poly-Ub species need not be intrinsically toxic; they can be controlled independently of DUB-based disassembly by being degraded, or through conjugation onto other proteins.

Toxicity and aggregation of the polyglutamine disease protein, ataxin-3 is regulated by its binding to VCP/p97 in Drosophila melanogaster.

Among the nine dominantly inherited, age-dependent neurodegenerative diseases caused by abnormal expansion in the polyglutamine (polyQ) repeat of otherwise unrelated proteins is Spinocerebellar Ataxia Type 3 (SCA3). SCA3 is caused by polyQ expansion in the deubiquitinase (DUB), ataxin-3. Molecular sequelae related to SCA3 remain unclear. Here, we sought to understand the role of protein context in SCA3 by focusing on the interaction between this DUB and Valosin-Containing Protein (VCP). VCP is bound directly by ataxin-3 through an arginine-rich area preceding the polyQ repeat. We examined the importance of this interaction in ataxin-3-dependent degeneration in Drosophila melanogaster. Our assays with new isogenic fly lines expressing pathogenic ataxin-3 with an intact or mutated VCP-binding site show that disrupting the ataxin-3-VCP interaction delays the aggregation of the toxic protein in vivo. Importantly, early on flies that express pathogenic ataxin-3 with a mutated VCP-binding site are indistinguishable from flies that do not express any SCA3 protein. Also, reducing levels of VCP through RNA-interference has a similar, protective effect to mutating the VCP-binding site of pathogenic ataxin-3. Based on in vivo pulse-chases, aggregated species of ataxin-3 are highly stable, in a manner independent of VCP-binding. Collectively, our results highlight an important role for the ataxin-3-VCP interaction in SCA3, based on a model that posits a seeding effect from VCP on pathogenic ataxin-3 aggregation and subsequent toxicity.

Interaction of the polyglutamine protein ataxin-3 with Rad23 regulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Polyglutamine (polyQ) repeat expansion in the deubiquitinase ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.