Search

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.

Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (i) MJDtr-76Q, containing both α-helical coiled-coil and β-sheet hairpin structures in the Q repeat region; (ii) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of β-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (iii) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.

O-GlcNAcylation, a protein posttranslational modification defined by the O-linked attachment of the monosaccharide N-acetylglucosamine (O-GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O-GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O-GlcNAc transferase (OGT), which attaches O-GlcNAc to target proteins, in Machado-Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O-GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasomedependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O-GlcNAc cycle as a promising target for the development of therapeutics in the yet incurableMJD. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, we investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila, and mouse models, we focused on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-α3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila, KPNA3/Kap-α3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3. [ABSTRACT FROM AUTHOR]

Aberrant O -GlcNAcylation, a protein posttranslational modification defined by the O -linked attachment of the monosaccharide N -acetylglucosamine ( O -GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O -GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O -GlcNAc transferase (OGT), which attaches O -GlcNAc to target proteins, in Machado-Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O -GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasome-dependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O -GlcNAc cycle as a promising target for the development of therapeutics in the yet incurable MJD., Competing Interests: The authors declare no competing interest.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, we investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila , and mouse models, we focused on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-α3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila , KPNA3/Kap-α3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3., Competing Interests: The authors declare no conflict of interest.

To investigate the functions of circadian neurons, we added two strategies to the standard Drosophila behavioral genetics repertoire. The first was to express a polyglutamine-expanded neurotoxic protein (MJDtr78Q; MJD, Machado-Joseph disease) in the major timeless (tim)-expressing cells of the adult brain. These Tim-MJD flies were viable, in contrast to the use of cell-death gene expression for tim neuron inactivation. Moreover, they were more arrhythmic than flies expressing other neurotoxins and had low but detectable tim mRNA levels. The second extended standard microarray technology from fly heads to dissected fly brains. By combining the two approaches, we identified a population of Tim-MJD-affected mRNAs. Some had been previously identified as sex-specific and relevant to courtship, including mRNAs localized to brain-proximal fat-body tissue and brain courtship centers. Finally, we found a decrease in the number of neurons that expressed male-specific forms of the fruitless protein in the laterodorsal region of the brain. The decrease was not a consequence of toxic protein expression within these specialized cells but a likely effect of communication with neighboring TIM-expressing neurons. The data suggest a functional interaction between adjacent circadian and mating circuits within the fly brain, as well as an interaction between circadian circuits and brain-proximal fat body.

Spinocerebellar ataxia type 3 is a human neurodegenerative disease resulting from polyglutamine tract expansion. The affected protein, ataxin-3, which contains an N-terminal Josephin domain followed by tandem ubiquitin (Ub)-interacting motifs (UIMs) and a polyglutamine stretch, has been implicated in the function of the Ub proteasome system. NMR-based structural analysis has now revealed that the Josephin domain binds Ub and has a papain-like fold that is reminiscent of that of other deubiquitinases, despite primary sequence divergence but consistent with its deubiqutinating activity. Mutation of the catalytic Cys enhances the stability of a complex between ataxin-3 and polyubiquitinated proteins. This effect depends on the integrity of the UIM region, suggesting that the UIMs are bound to the substrate polyubiquitin during catalysis. We propose that ataxin-3 functions as a polyubiquitin chain-editing enzyme.

The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity. By using the Josephin structure and the availability of NMR chemical shift assignments, we have mapped the enzyme active site by using the typical cysteine protease inhibitors, transepoxysuccinyl-L-eucylamido-4-guanidino-butane (E-64) and [L-3-trans-(propylcarbamyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline (CA-074). We also demonstrate that the specific interaction of Josephin with the ubiquitin-like domain of the ubiquitin- and proteasome-binding factor HHR23B involves complementary exposed hydrophobic surfaces. The structural similarity with other deubiquitinating enzymes suggests a model for the proteolytic enzymatic activity of ataxin-3.

The polyglutamine-containing neurodegenerative protein ataxin 3 (AT3) has deubiquitylating activity and binds ubiquitin chains with a preference for chains of four or more ubiquitins. Here we characterize the deubiquitylating activity of AT3 in vitro and show it trims/edits K48-linked ubiquitin chains. AT3 also edits polyubiquitylated (125)I-lysozyme and decreases its degradation by proteasomes. Cellular studies show that endogenous AT3 colocalizes with aggresomes and preaggresome particles of the misfolded cystic fibrosis transmembrane regulator (CFTR) mutant CFTRDeltaF508 and associates with histone deacetylase 6 and dynein, proteins required for aggresome formation and transport of misfolded protein. Small interfering RNA knockdown of AT3 greatly reduces aggresomes formed by CFTRDeltaF508, demonstrating a critical role of AT3 in this process. Wild-type AT3 restores aggresome formation; however, AT3 with mutations in the active site or ubiquitin interacting motifs cannot restore aggresome formation in AT3 knockdown cells. These same mutations decrease the association of AT3 and dynein. These data indicate that the deubiquitylating activity of AT3 and its ubiquitin interacting motifs as well play essential roles in CFTRDeltaF508 aggresome formation.

A hallmark of most neurodegenerative diseases, including those caused by polyglutamine expansion, is the formation of ubiquitin (Ub)-positive protein aggregates in affected neurons. This finding suggests that the Ub system may be involved in common mechanisms underlying these otherwise unrelated diseases. Here we report the finding of ataxin-3 (Atx-3), whose mutation is implicated in the neurodegenerative disease spinocerebellar ataxia type 3, in a bioinformatics search of the human genome for components of the Ub system. We show that wild-type Atx-3 is a Ub-binding protein and that the interaction of Atx-3 with Ub is mediated by motifs homologous to those found in a proteasome subunit. Both wild-type Atx-3 and the otherwise unrelated Ub-binding protein p62/Sequestosome-1 have been shown to be sequestered into aggregates in affected neurons in several neurodegenerative diseases, but the mechanism for this recruitment has remained unclear. In this article, we show that functional Ub-binding motifs in Atx-3 and p62 proteins are required for the localization of both proteins into aggregates in a cell-based assay that recapitulates several features of polyglutamine disease. We propose that the Ub-mediated sequestration of essential Ub-binding protein(s) into aggregates may be a common mechanism contributing to the pathogenesis of neurodegenerative diseases.

Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent protein-tagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders.

The protein ataxin-3 contains a polyglutamine region; increasing the number of glutamines beyond 55 in this region gives rise to the neurodegenerative disease spinocerebellar ataxia type 3. This disease and other polyglutamine expansion diseases are characterized by large intranuclear protein aggregates (nuclear inclusions). By using full-length human ataxin-3, we have investigated the changes in secondary structure, aggregation behavior, and fibril formation associated with an increase from the normal length of 27 glutamines (Q27 ataxin-3) to a pathogenic length of 78 glutamines (Q78 ataxin-3). Q78 ataxin-3 aggregates strongly and could be purified only when expressed with a solubility-enhancing fusion-protein partner. A marked decrease in alpha-helical secondary structure accompanies expansion of the polyglutamine tract, suggesting destabilization of the native protein. Proteolytic removal of the fusion partner in the Q78 protein, but not in the Q27 protein, leads to the formation of SDS-resistant aggregates and Congo-red reactive fibrils. Infrared spectroscopy of fibrils reveals a high beta-sheet content and suggests a parallel, rather than an antiparallel, sheet conformation. We present a model for a polar zipper composed of parallel polyglutamine beta-sheets. Our data show that intact ataxin-3 is fully competent to form aggregates, and posttranslational cleavage or other processing is not necessary to generate a misfolding event. The data also suggest that the protein aggregation phenotype associated with glutamine expansion may derive from two effects: destabilization of the native protein structure and an inherent propensity for beta-fibril formation on the part of glutamine homopolymers.