Modulators of spinocerebellar ataxia type 3-associated ataxin-3 aggregation

Spinocerebellar ataxia type-3 (SCA3), also known as Machado-Joseph disease, is proposed to be the most common dominant autosomal ataxia. It is a progressive, fatal neurodegenerative disease with no known cure. Ataxin-3 protein, which is expressed ubiquitously in the brain, initiates the early onset of SCA3 with a polyglutamine (poly-Q) expansion within the protein. Non-expanded ataxin-3 (non-pathogenic) has a poly-Q repeat threshold between 12–46 residues, while disease expansion coincides with 51 residues and above . The pathological hallmark of SCA3 is the presence of nuclear inclusions of aggregated ataxin-3, co-localised with ataxin-3 fragments, ubiquitin and other interacting proteins. The aggregation of ataxin-3 is more complex than many other misfolded proteins that are associated with neurodegenerative diseases (ND). It has been shown to undergo a two-stage aggregation pathway, which relates to the Josephin domain and the length of the poly-Q tract. In addition, it has also been well documented that ataxin-3 aggregation can be modulated by a number of interacting partners or factors. This thesis therefore sought to investigate the interrelationship between the length of the poly-Q tract and the effects of a range of factors and modulators on ataxin-3 aggregation, including ataxin-3 fragments generated by proteolytic cleavage and also chemical chaperones. Proteolytic cleavage has been implicated in the development of toxic fragments that initiate the aggregation in ND-associated proteins. Since ataxin-3 has been shown to be a substrate for the protease, calpain-2, the possible role of toxic fragments of ataxin-3, (i.e. toxic fragment hypothesis), was investigated in this study, together with an investigation into whether expanded poly-Q ataxin-3 leads to a faster rate of proteolysis. Also, osmolytes or ‘chemical chaperones’ have been increasingly used in the study of protein folding and aggregation. The osmolyte, trimethyl-N-oxide (TMAO) has been shown to reduce misfolding of proteins, stabilising native conformations and in particular, reduce the aggregation of ataxin-3 fragments expressed in cultured cells. Thus, the modulation of ataxin-3 aggregation using a range of ataxin-3 variants in the presence of TMAO was also further explored. Overall, the results demonstrated that calpain-2 cleaved all ataxin-3 variants (poly-Q length of 15, 28, 50, 64 residues), as the cleavage sites of ataxin-3 were mostly located after the Josephin domain. Proteolysis of ataxin-3 variants by calpain-2 were analysed kinetically and it was found that the rate of proteolysis was independent of the length of the poly-Q tract. Subsequently, a C-terminal poly-Q containing fragment of ataxin-3 namely (242Q15) and (242Q64) were expressed as a recombinant fusion protein and purified. The aggregation of both fusion and fragment proteins were analysed in vitro. It was determined that the fragments (242Q15) and (242Q64) have a high propensity to aggregate. Importantly, the result that both fragments can accelerate the aggregation of full-length ataxin-3 (Q64) to form SDS-insoluble aggregates, confirms the toxic fragment hypothesis as an overarching mechanism for ataxin-3 induced disease. Lastly, non-pathological and pathological lengths of ataxin-3 rapidly aggregated in the presence of TMAO. Intriguingly, the de-ubiquitinating activity of ataxin-3 was also enhanced with TMAO. Taken together, the data in this thesis provides a deeper insight into the influence of modulators on ataxin-3 aggregation, which may benefit the development of potential therapeutics in targeting protein aggregation in SCA3 pathogenesis.