Toward understanding the role of protein context in the polyglutamine disease, SCA3

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.