Spinocerebellar ataxia type 3 (SCA3) is a progressive, fatal, and untreatable neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the ATXN3 gene. Misfolding and aggregation of mutant ataxin-3 (ATXN3) in the brain is believed to be central to the pathogenesis of this disorder. Several cell-culture and animal models of SCA3 recapitulate key disease features and have identified several factors that might contribute to mutant ATXN3 aggregation and SCA3 pathogenesis. However, existing models of SCA3 overexpress the mutant protein above physiological concentrations. Consequently, the factors that influence mutant ATXN3 aggregation and SCA3 pathogenesis in vivo, under physiological conditions, have not been explored. The objective of this proposal is to identify key factors that influence Atxn3 aggregation and SCA3 pathogenesis. The central hypothesis is that increased proteolytic cleavage of mutant Atxn3 critically enhances its accumulation and aggregation in vivo, with aberrant nuclear localization leading to detrimental transcriptional changes in neurons. To explore disease pathogenesis, our laboratory recently generated the first knock-in mouse model of SCA3 with 82 CAG repeats inserted in the endogenous murine Atxn3 locus. Preliminary results in the knock-in reveal the age-dependent accumulation and aggregation of Atxn3 with regional and subcellular specificity. Unexpectedly, the knock-in revealed extensive aggregation in the hippocampus, including large extranuclear deposits in the stratum radiatum and subiculum, highly active and synapse-rich regions of the hippocampus. My first specific aim is determine how neuronal excitation affects mutant Atxn3 cleavage, accumulation, and aggregation in vivo. I will use electron microscopy, biochemical analyses, and primary neuronal cultures from the knock-in to determine the degree of excitation-driven mutant Atxn3 cleavage and aggregation under physiological expression conditions. I will then pharmacologically induce hippocampal seizures in knock-in mice to directly determine the extent to which neuronal activity affects mutant Atxn3 cleavage and aggregation in vivo. I expect the results of this aim to show that increased neuronal activity enhances mutant Atxn3 cleavage and aggregation, with mutant Atxn3 fragments preferentially accumulating in neuronal nuclei. Since several studies have determined that the nuclear localization of mutant ATXN3 is an important determinant of toxicity, my second specific aim is to identify aberrant transcriptional changes in the SCA3 knock-in induced by mutant Atxn3 expression. I will use RNA-seq, a sensitive next- generation sequencing method, to identify transcriptional changes in disease-susceptible neurons from the SCA3 knock-in that aberrantly accumulate mutant Atxn3, the deep cerebellar nuclei. After analysis of the RNA- seq data, I will then confirm gene expression changes predicted to be important in SCA3 pathogenesis in knock-in mice of different ages and SCA3 human brain tissue. I expect to identify key candidate transcriptional changes that might contribute to neurotoxicity. Together these aims are expected to identify key pathogenic factors in SCA3. The results of this proposal could help in the future development of effective therapies for SCA3. Keywords: Neurodegeneration, triplet repeat expansion disorder, ataxin-3
This proposal aims to define critical pathogenic factors in the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) by utilizing a newly developed, physiologically precise, knock-in mouse model of the disease. The results are expected to have an important positive impact on our understanding of SCA3 and other related neurodegenerative disorders, which may share overlapping mechanisms. Importantly, findings from this proposal should provide insight into the future development of effective therapies for SCA3, which currently is an untreatable and fatal disease.