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Role of huntingtin phosphorylation at serine 421 in Huntington Disease Warby, Simon Christopher

Abstract

Huntington Disease (HD) is a lethal neurodegenerative disorder that results from polyglutamine-expansion in the huntingtin protein. Despite the widespread tissue expression pattern of huntingtin, there is selective degeneration of specific neurons populations in the brain. The regulation of huntingtin by posttranslational modifications, such as phosphorylation, is not well understood. The objective of this thesis was to conduct molecular and cell biological studies to determine whether huntingtin was regulated by phosphorylation and what role this might play in the disease. I found that huntingtin is phosphorylated on serine-421 (5421) by the pro-survival signalling protein kinase Akt (PKB) in the brain under normal physiological conditions. There are differences in the basal endogenous phosphorylation state of huntingtin in different regions of the brain and regions more susceptible in the disease have lower levels of S421 phosphorylation. Consistent with the hypothesis that S421 phosphorylation protects against the toxicity of polyglutamine-expanded huntingtin, 5421 phosphorylation is reduced in the presence of the HD mutation. Furthermore, I discovered significant relationships between the phosphorylation status of huntingtin and pathological processes in the disease, including the cleavage and nuclear localization of polyglutamine-expanded huntingtin. The S421 site is close to the proteolytic domain containing the caspase cleavage sites (aa500-600) and phosphorylation at S421 reduces the cleavage of huntingtin. The nuclear localization of huntingtin fragments is also known to be important in the disease. I directly assessed whether phosphorylation at S421 alters the nuclear localization of huntingtin using subcellular fractionation and immunofluorescent techniques. Phosphorylation at S421 reduces the nuclear localization of both full length huntingtin and huntingtin fragments. Further, the 1-586aa huntingtin fragment is specifically trafficked and concentrated in the nucleus, providing a functional link between cleavage and nuclear localization that may be mediated by S421 phosphorylation. Phosphorylation at S421 is a dynamic means of regulating huntingtin and is implicated in HD because it is reduced by the polyglutamine-expansion and modifies key pathogenic processes such as the cleavage and nuclear localization of huntingtin. The integration of signalling pathways on huntingtin and the phosphatase regulation of huntingtin phosphorylation are enticing therapeutic possibilities for modulating the pathogenesis of HD.

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