p80 coilin,a coiled body-specific protein,interacts with ataxin-1, the SCA1 gene product

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 is associated with an elongated polyglutamine tract in ataxin-1, the SCA1 gene product. Using the yeast two-hybrid system and co-immunoprecipitation experiments, we have found that p80 coilin, coiled body-specific protein, binds to ataxin-1. In further experiments with deletion mutants, we found that the C-terminal regions of ataxin-1 and p80 coilin were essential for this interaction. In HeLa cells that have been co-transfected with ataxin-1 and p80 coilin, the p80 coilin protein co-localizes with ataxin-1 aggregates in the nucleoplasm. However, immunohistochemical analysis and immunofluorescence assays showed that mutant ataxin-1 aggregates do not redistribute p80 coilin's dot-like structures in the Purkinje cells of SCA1 transgenic mice. This feature of the interaction between ataxin-1 and p80 coilin suggests that p80 coilin might be implicated in altering the function of ataxin-1.     

Conformational Behavior and Aggregation of Ataxin-3 in SDS

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases all characterized by the presence of intraneuronal inclusions that contain aggregated protein. Aggregation of ataxin-3, the causative protein of SCA3, has been well characterized in vitro, with both pathogenic and non-pathogenic length ataxin-3 undergoing fibrillogenesis. However, only ataxin-3 containing an expanded polyQ tract leads to SCA3. Therefore other cellular factors, not present in previous in vitro studies, may modulate aggregation during disease. The interactions between fibrillar species and cell membranes have been characterized in a number of amyloid diseases, including Huntington’s Disease, and these interactions affect aggregation and toxicity. We have characterized the effects of the membrane mimetic sodium dodecyl sulfate (SDS) on ataxin-3 structure and aggregation, to show that both micellar and non-micellar SDS have differing effects on the two stages of ataxin-3 aggregation. We also demonstrate that fibrillar ataxin-3 binds phospholipids, in particular phosphorylated phosphotidylinositols. These results highlight the effect of intracellular factors on the ataxin-3 misfolding landscape and their implications in SCA3 and polyQ diseases in general are discussed.     

Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is one of several neurological disorders caused by a CAG repeat expansion. In SCA1, this expansion produces an abnormally long polyglutamine tract in the protein ataxin-1. Mutant polyglutamine proteins accumulate in neurons, inducing neurodegeneration, but the mechanism underlying this accumulation has been unclear. We have discovered that the 14-3-3 protein, a multifunctional regulatory molecule, mediates the neurotoxicity of ataxin-1 by binding to and stabilizing ataxin-1, thereby slowing its normal degradation. The association of ataxin-1 with 14-3-3 is regulated by Akt phosphorylation, and in a Drosophila model of SCA1, both 14-3-3 and Akt modulate neurodegeneration. Our finding that phosphatidylinositol 3-kinase/Akt signaling and 14-3-3 cooperate to modulate the neurotoxicity of ataxin-1 provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention.     

Proteasome function is inhibited by polyglutamine-expanded ataxin-1, the SCA1 gene product

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. Using d2EGFP, a short-lived enhanced green fluorescent protein, we investigated whether polyglutamine-expanded ataxin-1 affects the function of the proteasome, a cellular multicatalytic protease that degrades most misfolded proteins and regulatory proteins. In Western blot analysis and immunofluorescence experiments, d2EGFP was less degraded in HEK 293T cells transfected with ataxin-1(82Q) than in cells transfected with lacZ or empty vector controls. To test whether the stability of the d2EGFP protein was due to aggregation of ataxin-1, we constructed a plasmid carrying ataxin-1-Delta114, lacking the self-association region (SAR), and examined degradation of the d2EGFP. Both the level of ataxin-1-Delta114 aggregates and the amount of d2EGFP were drastically reduced in cells containing ataxin-1-Delta114. Furthermore, d2EGFP localization experiments showed that polyglutamine-expanded ataxin-1 inhibited the general function of the proteasome activity. Taken together, these results demonstrate that polyglutamine-expanded ataxin-1 decreases the activity of the proteasome, implying that a disturbance in the ubiquitin-proteasome pathway is directly involved in the development of spinocerebellar ataxia type1.     

A Drosophila homolog of the polyglutamine disease gene SCA2 is a dosage-sensitive regulator of actin filament formation

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. In this study we have used genetic and molecular approaches to investigate the function of a Drosophila homolog of the SCA2 gene (Datx2). Like human ataxin-2, Datx2 is found throughout development in a variety of tissue types and localizes to the cytoplasm. Mutations that reduce Datx2 activity or transgenic overexpression of Datx2 result in female sterility, aberrant sensory bristle morphology, loss or degeneration of tissues, and lethality. These phenotypes appear to result from actin filament formation defects occurring downstream of actin synthesis. Further studies demonstrate that Datx2 does not assemble with actin filaments, suggesting that the role of Datx2 in actin filament formation is indirect. These results indicate that Datx2 is a dosage-sensitive regulator of actin filament formation. Given that loss of cytoskeleton-dependent dendritic structure defines an early event in SCA2 pathogenesis, our findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2.     

Molecular pathogenesis of spinocerebellar ataxia type 1 disease

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 is associated with an elongated polyglutamine tract in ataxin-1, the SCA1 gene product. As summarized in this review, recent studies have clarified the molecular mechanisms of SCA1 pathogenesis and provided direction for future therapeutic approaches. The nucleus is the subcellular site where misfolded mutant ataxin-1 acts to cause SCA1 disease in the cerebellum. The role of these nuclear aggregates is the subject of intensive study. Additional proteins have been identified, whose conformational alterations occurring through interactions with the polyglutamine tract itself or non-polyglutamine regions in ataxin-1 are the cause of SCA-1 cytotoxicity. Therapeutic hope comes from the observations concerning the reduction of nuclear aggregation and alleviation of the pathogenic phenotype by the application of potent inhibitors and RNA interference.     

Caspase-mediated proteolysis of the polyglutamine disease protein ataxin-3

Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease, is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated disease genes. Polyglutamine disorders are characterized by disease protein misfolding and aggregation; often within the nuclei of affected neurons. Although the precise mechanism of polyglutamine-mediated cell death remains elusive, evidence suggests that proteolysis of polyglutamine disease proteins by caspases contributes to pathogenesis. Using cellular models we now show that the endogenous spinocerebellar ataxia type-3 disease protein, ataxin-3, is proteolyzed in apoptotic paradigms, resulting in the loss of full-length ataxin-3 and the corresponding appearance of an approximately 28-kDa fragment containing the glutamine repeat. Broad-spectrum caspase inhibitors block ataxin-3 proteolysis and studies suggest that caspase-1 is a primary mediator of cleavage. Site-directed mutagenesis experiments eliminating three, six or nine potential caspase cleavage sites in the protein suggest redundancy in the site(s) at which cleavage can occur, as previously described for other disease proteins; but also map a major cleavage event to a cluster of aspartate residues within the ubiquitin-binding domain of ataxin-3 near the polyglutamine tract. Finally, caspase-mediated cleavage of expanded ataxin-3 resulted in increased ataxin-3 aggregation, suggesting a potential role for caspase-mediated proteolysis in spinocerebellar ataxia type-3 pathogenesis.     

Activity and cellular functions of the deubiquitinating enzyme and polyglutamine disease protein ataxin-3 are regulated by ubiquitination at lysine 117

Deubiquitinating enzymes (DUbs) play important roles in many ubiquitin-dependent pathways, yet how DUbs themselves are regulated is not well understood. Here, we provide insight into the mechanism by which ubiquitination directly enhances the activity of ataxin-3, a DUb implicated in protein quality control and the disease protein in the polyglutamine neurodegenerative disorder, Spinocerebellar Ataxia Type 3. We identify Lys-117, which resides near the catalytic triad, as the primary site of ubiquitination in wild type and pathogenic ataxin-3. Further studies indicate that ubiquitin-dependent activation of ataxin-3 at Lys-117 is important for its ability to reduce high molecular weight ubiquitinated species in cells. Ubiquitination at Lys-117 also facilitates the ability of ataxin-3 to induce aggresome formation in cells. Finally, structure-function studies support a model of activation whereby ubiquitination at Lys-117 enhances ataxin-3 activity independent of the known ubiquitin-binding sites in ataxin-3, most likely through a direct conformational change in or near the catalytic domain.     

Structural modeling of ataxin-3 reveals distant homology to adaptins

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3.     

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.     

Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat–containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.     

Expression of Expanded Polyglutamine Protein Induces Behavioral Changes in Drosophila (Polyglutamine-Induced Changes in Drosophila)

Spinocerebellar ataxia type-3 or Machado-Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disease caused by triplet nucleotide expansion. The expansion of the polyglutamine tract near the C terminus of the MJD1 gene product, ataxin-3, above a threshold of 40 glutamine repeats causes neuronal loss and degeneration. The expanded ataxin-3 forms aggregates, and nuclear inclusions, within neurons, possibly due to the misfolding of mutant proteins. Here we report upon the behavioral test changes related to truncated and expanded forms of MJD protein (MJDtr) in Drosophila, and show that expanded MJDtr, when expressed in the nervous system, causes characteristic locomotor dysfunction and anosmia. This phenomenon has not been previously reported in humans or in transgenic Drosophila models. In addition, the in vivo expression of the antiapoptotic gene bcl-2 showed no evidence of ameliorating the deleterious effect of MJDtr-Q78s, either in the eye or in the nervous system. The study shows that such Drosophila transgenic models express olfactory dysfunction and ataxic behavior as observed in human patients.     

Cerebellar Soluble Mutant Ataxin-3 Level Decreases during Disease Progression in Spinocerebellar Ataxia Type 3 Mice

Spinocerebellar Ataxia Type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominantly inherited neurodegenerative disease caused by an expanded polyglutamine stretch in the ataxin-3 protein. A pathological hallmark of the disease is cerebellar and brainstem atrophy, which correlates with the formation of intranuclear aggregates in a specific subset of neurons. Several studies have demonstrated that the formation of aggregates depends on the generation of aggregation-prone and toxic intracellular ataxin-3 fragments after proteolytic cleavage of the full-length protein. Despite this observed increase in aggregated mutant ataxin-3, information on soluble mutant ataxin-3 levels in brain tissue is lacking. A quantitative method to analyze soluble levels will be a useful tool to characterize disease progression or to screen and identify therapeutic compounds modulating the level of toxic soluble ataxin-3. In the present study we describe the development and application of a quantitative and easily applicable immunoassay for quantification of soluble mutant ataxin-3 in human cell lines and brain samples of transgenic SCA3 mice. Consistent with observations in Huntington disease, transgenic SCA3 mice reveal a tendency for decrease of soluble mutant ataxin-3 during disease progression in fractions of the cerebellum, which is inversely correlated with aggregate formation and phenotypic aggravation. Our analyses demonstrate that the time-resolved Förster resonance energy transfer immunoassay is a highly sensitive and easy method to measure the level of soluble mutant ataxin-3 in biological samples. Of interest, we observed a tendency for decrease of soluble mutant ataxin-3 only in the cerebellum of transgenic SCA3 mice, one of the most affected brain regions in Spinocerebellar Ataxia Type 3 but not in whole brain tissue, indicative of a brain region selective change in mutant ataxin-3 protein homeostasis.     

SUMO-1 interacts with mutant ataxin-1 and colocalizes to its aggregates in Purkinje cells of SCA1 transgenic mice

Spinocerebellar ataxia type 1 (SCA1) is one of several progressive neurodegenerative diseases caused by the expanded polyglutamine tract in ataxin-1, the SCA1 gene product. In SCA1 patients and transgenic mice, the affected neuronal cells contain a large ubiquitin-positive aggregate which is derived from the mutant ataxin-1. Small ubiquitin-like modifier-1 (SUMO-1) is one of the most intriguing ubiquitin-like modifiers being conjugated to target proteins and modulating a number of cellular pathways. Recent findings that the aggregates from several neurodegenerative diseases are SUMO-1-positive prompted us to examine the implication of SUMO-1 in SCA1 pathogenesis. In our yeast two-hybrid experiments using mutant ataxin-1 as bait, we identified a SUMO-1 protein that directly binds to ataxin-1 protein. Interestingly, we found that most of the mutant ataxin-1-derived aggregates were SUMO-1-positive both in Purkinje cells of SCA1 transgenic mice and in HeLa cells, but not wild-type ataxin-1 in HeLa cells. In addition, the aggregates in Purkinje cells of SCA1 transgenic mice were positive against both anti-SUMO-1 and anti-ubiquitin antibodies. These results show that the SUMO-1 protein interacts with mutant ataxin-1 and colocalizes with its aggregates which suggests the involvement of the SUMO-1 system in the pathogenesis of SCA1 disease.