Spinocerebellar ataxia type 1 (SCA1) is an adult onset, autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in ataxin-1, which encodes the ataxin-1 protein. SCA1 is one of nine polyQ-expansion gain-of-function diseases which includes Huntington's disease, spinal-bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and other ataxias. Clinical symptoms of SCA1 include ataxia, dysarthria, ophthalmoparesis, muscle wasting, and extrapyramidal and bulbar dysfunction. Cerebellar Purkinje cells (PCs), neurons in the inferior olive and nuclei of the brainstem are affected. No disease-modifying therapy exists for SCA1. The goals of my thesis were to assess the safety and efficacy of AAV-delivered artificial miRNAs targeting ataxin-1 to alleviate neuropathological and behavioral phenotypes in the knock-in and transgenic SCA1 mouse models. In the knock-in SCA1 mouse model AAVs expressing an artificial miRNA (miSCA1) targeting sequences conserved in mouse and human ataxin-1 were injected directly to the deep cerebellar nuclei. This achieved long term silencing of ataxin-1 mRNA and significantly improved rotarod performance, gait deficiencies, and neuropathology of the cerebellum. In the transgenic SCA1 mouse model the same method of delivery was executed with an artificial microRNA (miR) (miS1) designed to optimize potency, efficacy and safety to suppress Atxn1 expression. Additionally the therapeutic potential of continuous overexpression of ataxin-1-like was examined. Delivery of either ataxin-1-like or miS1 viral vectors to SCA1 mouse cerebellum resulted in widespread cerebellar Purkinje cell transduction. There was significant improvement to rotarod performance, gait deficiencies, coordination and balance, as well as the neuropathology of cerebellar Purkinje cells. In summary, these data indicate the utility of these approaches as possible therapies for SCA1 patients.

The 14-3-3 family of proteins are important signalling proteins involved in a number of cellular processes. These include cell cycle regulation, apoptosis, signal transduction and cell signalling. There is also considerable evidence in the literature that 14-3-3 proteins play a vital role in the pathology of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, Huntington’s and Prion disease. The neurodegenerative disease of focus in this research is Spinocerebellar Ataxia Type 1 (SCA1). SCA1 is a polyglutamine-repeat disease and the interaction of the disease protein ataxin-1 with 14-3-3 proteins leads to the toxic accumulation and subsequent protein aggregation which is characteristic of this disease. This study focused on attempting to elucidate the structure of various domains of the disease protein and also in identifying potential inhibitors of this deleterious interaction. Unfortunately, structural studies were not successful due to a number of caveats encountered in the expression and purification of the ataxin-1 protein domains. By utilising computational methods and small molecule inhibitors, a number of potential lead compounds which possess the ability to at least partly disrupt the interaction of 14- 3-3ζ have been identified. As 14-3-3 proteins play roles in other neurodegenerative diseases, successful identification of potential drug lead treatments can have far reaching benefits in a number of neurodegenerative diseases including SCA1. Lipid rafts are also involved in neurodegenerative disease pathology. Lipid rafts are cholesterol and sphingolipid rich domains which organise the plasma membrane into discrete microdomains and act as signalling platforms and processing centres which attach specific proteins and lipids. A number of disease proteins are processed at these membrane regions, including those involved in Alzheimer’s, Parkinson’s and Prion disease. This processing is a step which is critical in the pathology of disease and abnormal processing leads to the formation of toxic protein aggregates. Previous research in the lab identified the association of low levels of the five main brain isoforms of 14-3-3 proteins with rafts. This study expanded on this to positively identify the presence of the two phospho-forms of 14-3-3, α and δ. The mechanism by which 14-3-3 proteins associate with rafts was also investigated, indicating that 14-3-3 associates with rafts via an unidentified raftbound protein(s). In addition, the phosphorylation status and quaternary structure of 14-3-3 in the presence of sphingolipids has been explored.