The polyglutamine diseases are a clinically heterogeneous group of inherited neurodegenerative disorders caused by expansion of polyglutamine-encoding (CAG)n trinucleotide repeats within the disease genes. It is increasingly clear that the amino acid sequences flanking the polyglutamine expansion in each disease protein, i.e. the specific protein context, contribute to selective neuronal toxicity by influencing the behavior of the disease protein within selectively vulnerable neuronal populations. In the studies described here, I explore the role that protein context plays in the polyglutamine disease, Spinocerebellar ataxia type 3 (SCA3). Toward this end, I utilize biochemical, cell-based, and animal models to gain a broader understanding of the SCA3 disease protein, ataxin-3, and generate tools for further exploration of the molecular properties of ataxin-3 that modulate its toxicity during disease. In Chapter 1, I provide an overview of the recognized polyglutamine diseases, emphasizing the elements of protein context that are distinct among the polyglutamine disease proteins and may contribute to the neuropathological and clinical heterogeneity within this family of diseases. Alternative splicing of the polyglutamine disease gene products adds an additional level of complexity to the tissue-specific protein context of expanded polyglutamine, yet this phenomenon has been underinvestigated. In Chapter 2, I examine the significance of ataxin-3 splice variation. Several minor 5' variants and both known 3' splice variants of ataxin-3, a deubiquitinating enzyme, are expressed at the mRNA level in brain. At the protein level, however, the C-terminal splice isoform with three ubiquitin interacting motifs (3UIM ataxin-3) is the predominant isoform in brain, independent of age or (CAG)n expansion. Although both C-terminal ataxin-3 splice isoforms display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and is more rapidly degraded by the proteasome. These observations demonstrate how alternative splicing of sequences distinct from the polyglutamine-encoding (CAG)n repeat can alter disease-related components of protein context. Knock-in models of polyglutamine diseases utilize pathogenic (CAG)n expansions within the endogenous genomic, transcript, and protein context to recreate key features of individual polyglutamine diseases. In chapter 3, I describe the creation of the first knock-in mouse model of SCA3. Hemizygous knock-in mice transmit the knock-in allele in Mendelian ratios and broadly express both the expanded Atxn3(Q3KQ82) protein and the wildtype murine Atxn3(Q6) protein. In this chapter, I also compare the gene targeting efficiencies and rates of chromosomal instability of a novel C57BL/6J ES cell line (UMB6JD7) and two well established ES cell lines (W4 and Bruce4.G9). Of these, Bruce4.G9 ES cells proved superior based on lower rates of aneuploidy and the production of germline transmitting chimeras. Finally, in Chapter 4 I discuss questions and concepts raised during the course of these studies, and suggest avenues of future research aimed at broadening our understanding of ataxin-3 physiology and of protein context-dependent elements in polyglutamine disease pathogenesis.

<p>Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and visual problems due to a progressive and selective loss of neurons within the cerebellum, brainstem and retina. The disease is caused by the expansion of a CAG repeat in the first coding exon of the SCA7 gene, resulting in an expanded polyglutamine domain in the N-terminal part of ataxin-7, a protein of unknown function.</p><p>To expand our knowledge of the ataxin-7 protein and the mechanism by which mutant ataxin-7 causes disease, we have studied the expression and function of both the normal and the mutated ataxin-7 protein. </p><p>Ataxin-7 expression was examination in brain and non-CNS tissues from SCA7 patients and age-matched controls. Expression was predominantly nuclear in neurons throughout the brain of both healthy and SCA7 individuals. We also observed aggregation of mutant ataxin-7 in the nuclei of neurons. No obvious difference in the expression level of ataxin-7 or the formation of aggregates could be observed between affected and non-affected brain regions in SCA7 patients. Based on these findings, we could conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression levels or to the formation of ataxin-7 aggregates.</p><p>To widen our studies on ataxin-7 expression, we isolated and characterized the mouse SCA7 gene homolog. Cloning of the mouse SCA7 gene revealed two SCA7 mRNA isoforms that were highly homologous to their human counterparts. Immunohistochemical analysis also revealed a conserved expression pattern of ataxin-7 in adult mouse brain. In addition, ataxin-7 expression was observed during embryonic development in brain as well as in several non-neuronal tissues such as heart, liver and lung. </p><p>Besides SCA7, eight neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1-3, 6 and 17, DRPLA, SBMA and Huntington’s disease. The polyglutamine disorders have many features in common and a common pathological disease mechanism involving transcriptional dysregulation has been proposed. To investigate the possible involvement of transcriptional dysregulation in SCA7 pathology, we analyzed the effects of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP, the Purkinje cell-expressed nuclear receptor RORα1 or a basic TATA promoter. As previously shown for other polyglutamine disease proteins, expansion of the polyglutamine domain in ataxin-7 leads to reduced transcription. Surprisingly, strong repression of CBP-mediated, RORα1-mediated and basal transcription was also observed with wild-type ataxin-7, suggesting that the normal ataxin-7 protein may have a role in transcriptional regulation. </p>

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and visual problems due to a progressive and selective loss of neurons within the cerebellum, brainstem and retina. The disease is caused by the expansion of a CAG repeat in the first coding exon of the SCA7 gene, resulting in an expanded polyglutamine domain in the N-terminal part of ataxin-7, a protein of unknown function. To expand our knowledge of the ataxin-7 protein and the mechanism by which mutant ataxin-7 causes disease, we have studied the expression and function of both the normal and the mutated ataxin-7 protein. Ataxin-7 expression was examination in brain and non-CNS tissues from SCA7 patients and age-matched controls. Expression was predominantly nuclear in neurons throughout the brain of both healthy and SCA7 individuals. We also observed aggregation of mutant ataxin-7 in the nuclei of neurons. No obvious difference in the expression level of ataxin-7 or the formation of aggregates could be observed between affected and non-affected brain regions in SCA7 patients. Based on these findings, we could conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression levels or to the formation of ataxin-7 aggregates. To widen our studies on ataxin-7 expression, we isolated and characterized the mouse SCA7 gene homolog. Cloning of the mouse SCA7 gene revealed two SCA7 mRNA isoforms that were highly homologous to their human counterparts. Immunohistochemical analysis also revealed a conserved expression pattern of ataxin-7 in adult mouse brain. In addition, ataxin-7 expression was observed during embryonic development in brain as well as in several non-neuronal tissues such as heart, liver and lung. Besides SCA7, eight neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1-3, 6 and 17, DRPLA, SBMA and Huntington’s disease. The polyglutamine disorders have many features in common and a common pathological disease mechanism involving transcriptional dysregulation has been proposed. To investigate the possible involvement of transcriptional dysregulation in SCA7 pathology, we analyzed the effects of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP, the Purkinje cell-expressed nuclear receptor RORα1 or a basic TATA promoter. As previously shown for other polyglutamine disease proteins, expansion of the polyglutamine domain in ataxin-7 leads to reduced transcription. Surprisingly, strong repression of CBP-mediated, RORα1-mediated and basal transcription was also observed with wild-type ataxin-7, suggesting that the normal ataxin-7 protein may have a role in transcriptional regulation.