Towards Improved Therapies, Model Systems and Understanding of Spinocerebellar Ataxia Type 3

Abstract

Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder and the most common dominantly inherited ataxia worldwide. The genetic cause of SCA3 was first identified over 25 years ago, yet there remains no effective therapies to slow or alter progression of this debilitating and ultimately fatal disorder. One of nine polyglutamine diseases, SCA3 is caused by a CAG trinucleotide repeat expansion in the ATXN3 gene leading to an extended polyglutamine sequence in the encoded disease protein. Polyglutamine-expanded mutant ATXN3 (mutATXN3) acts through a presumed dominant toxic mechanism that requires its sequestration into neuronal nuclei, but the underlying processes that contribute to neuronal toxicity in SCA3 are still poorly understood. This dissertation aims to address key gaps in our understanding of SCA3 disease mechanisms, and develop new tools and therapies to advance the field towards the ultimate goal of a disease-modifying therapy for SCA3 patients. Chapter 1 reviews SCA3 disease mechanisms, model systems, and progress towards oligonucleotide-based therapies for SCA3. In Chapters 2 and 3, I assess a gene silencing therapeutic approach for SCA3 in multiple cellular and mouse transgenic models of disease. I first perform a short-term proof-of-concept study assessing safety and target engagement of several candidate antisense oligonucleotides (ASO) targeting ATXN3 in two transgenic mouse models of SCA3. Next, I demonstrate in a longitudinal preclinical study that an anti-ATXN3 ASO can lead to sustained reduction in mutATXN3 protein and prevent molecular, neuropathological, electrophysiological and behavioral features of the disease in SCA3 transgenic mice. Together, these two preclinical studies establish efficacy of ATXN3-targeted ASOs as a disease-modifying therapeutic strategy for SCA3, and support further efforts to develop ASOs for human clinical trials in SCA3. In Chapter 4, I perform the first characterization of a novel SCA3 disease-specific human embryonic stem cell (SCA3-hESC) line, and identify disease-relevant phenotypes in SCA3-hESCs and differentiated cortical neurons. Robust ATXN3 aggregation and formation of aggresomes, two SCA3 phenotypes not observed in any other cell line expressing physiological levels of mutATXN3 protein, were completely rescued following ASO transfection into SCA3-hESCs. These studies validate the SCA3-hESC line as a unique and highly relevant human disease model that holds strong potential to advance understanding of SCA3 disease mechanisms and facilitate the evaluation of possible SCA3 therapies. Finally, Chapter 5 addresses key gaps in our understanding of SCA3, such as why is mutATXN3 transported into neuronal nuclei in SCA3 and how does this lead to neuronal toxicity. Using cellular and mouse models of SCA3, I reveal the first descriptive evidence of generalized impairments in nuclear trafficking as indicated by mislocalization of key proteins regulating nucleocytoplasmic transport (NCT) and formation of cytoplasmic puncta enriched in key NCT proteins. Furthermore, I demonstrate that ASO knockdown of mutATXN3 rescues abnormal subcellular localization of the master nuclear trafficking GTPase RAs-related Nuclear protein (Ran) and improves genomic integrity of neurons in disease-vulnerable brain regions of SCA3 mice. Together, this dissertation demonstrates the significant promise of ASO therapies for SCA3, establishes the utility of SCA3-hESCs for disease modeling and translational studies, and provides evidence for a novel disease mechanism that may contribute to aberrant mutATXN3 behavior and toxicity in SCA3.