RESUMEN

Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of an uninterrupted polyglutamine (polyQ) repeat in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in fruit fly survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.

Asunto(s)

RESUMEN

Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of polyglutamine (polyQ) in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.

RESUMEN

Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of polyglutamine (polyQ) in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.

RESUMEN

Polyglutamine (polyQ) diseases are devastating, slowly progressing neurodegenerative conditions caused by expansion of polyQ-encoding CAG repeats within the coding regions of distinct, unrelated genes. In spinal and bulbar muscular atrophy (SBMA), polyQ expansion within the androgen receptor (AR) causes progressive neuromuscular toxicity, the molecular basis of which is unclear. Using quantitative proteomics, we identified changes in the AR interactome caused by polyQ expansion. We found that the deubiquitinase USP7 preferentially interacts with polyQ-expanded AR and that lowering USP7 levels reduced mutant AR aggregation and cytotoxicity in cell models of SBMA. Moreover, USP7 knockdown suppressed disease phenotypes in SBMA and spinocerebellar ataxia type 3 (SCA3) fly models, and monoallelic knockout of Usp7 ameliorated several motor deficiencies in transgenic SBMA mice. USP7 overexpression resulted in reduced AR ubiquitination, indicating the direct action of USP7 on AR. Using quantitative proteomics, we identified the ubiquitinated lysine residues on mutant AR that are regulated by USP7. Finally, we found that USP7 also differentially interacts with mutant Huntingtin (HTT) protein in striatum and frontal cortex of a knockin mouse model of Huntington's disease. Taken together, our findings reveal a critical role for USP7 in the pathophysiology of SBMA and suggest a similar role in SCA3 and Huntington's disease.

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RESUMEN

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.

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RESUMEN

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.

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RESUMEN

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.

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