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

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posted on 2017-03-03, 03:52 authored by Leck, Su-Ling
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.

History

Principal supervisor

Marie-Isabel Aguilar

Year of Award

2016

Department, School or Centre

Biomedical Sciences (Monash Biomedicine Discovery Institute)

Additional Institution or Organisation

Biochemistry and Molecular Biology

Campus location

Australia

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences

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