New pathogenic mechanisms in SCA1 neurodegenerative disease revealed by the ataxin-1 interactome

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease in which a marked atrophy of neurons in the cerebellum and brainstem causes coordination and movement disabilities and ultimately death within 10-20 years of symptom onset. The molecular cause is an expanded polyglutamine (polyQ) sequence in the ataxin-1 protein. The resulting accumulation of the mutant ataxin-1 (polyQ-ataxin-1) protein as distinctive nuclear bodies (NBs) has been proposed as contributing to neuronal toxicity and/or dysfunction, but little is known about the biophysical properties of these NBs and their ultimate impact on neuronal cells. The studies presented in this thesis address these issues initially with proteomics approaches in Neuro-2a neuronal cells to identify the interacting protein partners of polyQ-ataxin-1, i.e. the polyQ-ataxin-1 interactome. The results from proximity labelling and affinity purification approaches were combined to improve confidence in the resulting lists of partner proteins. Further bioinformatics analysis identified enrichment of several protein functional groups; nuclear transport proteins and RNA helicases were prioritised for further study by biochemical and advanced imaging techniques. The expression of polyQ-ataxin-1 in Neuro-2a cells was shown to disrupt the localisation of multiple nuclear transport proteins. Nuclear transporters importin-α2, importin-β1, importin- 13, Hikeshi, exportin-1, and nucleoporin NUP98 have been mislocalized and partially co- localized with ataxin-1 NBs. The observations of altered nuclear/cytoplasmic distributions of model cargo proteins were also consistent with the disruption of the processes of nuclear transport in the presence of polyQ-ataxin-1. The physical properties of the polyQ-ataxin-1 NBs were also assessed, with specific consideration of the contributions by RNA/RNA helicases. Under standard conditions these NBs showed rapid exchange of the ataxin-1 protein consistent with dynamic liquid droplets; the down-regulation of RNA helicases DDX42, DDX46, and DHX15, the decreased ATP level, or altered environmental conditions were shown to slow exchange. These results thus reveal the phase transition to a less dynamic hydrogel or fibrillar phase and emphasize the tunable dynamics of these polyQ-ataxin-1 NBs as RNA/protein droplets. Taken together, these studies have revealed new insights into the impact and regulation of polyQ-ataxin-1 made possible by the identification of new proximal or interacting partners for polyQ-ataxin-1, and thus suggest new strategies for future interventions in the treatment of SCA1 and other neurodegenerative diseases.

Keywords

neurodegenerative disease, SCA1, ataxin-1, interactome, nuclear transport, phase separation, phase transition

Collections

Biochemistry and Pharmacology - Theses
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