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In vivo characterization of caspase resistant huntingtin : insights into the pathogenic mechanism of Huntington disease Graham, Rona Kyrenia


Proteolytic cleavage of htt is regarded as a critical event in the pathogenesis of HD. Expression of htt fragments containing an expanded polyglutamine repeat are toxic in vitro and in vivo, and accumulation of N-terminal truncated products of htt are observed in human and mouse HD brain. Notably, the presence of htt fragments prior to clinical onset of HD suggests that htt cleavage may be a crucial, causal event in the pathogenesis of HD, rather than simply resulting from nonselective activation of proteolytic pathways in the late stage of disease. However the relationship between specific huntingtin fragments and the pathogenesis of HD is unknown. Mutagenesis of all caspase sites in mutant huntingtin prevents toxicity in cultured cells and caspase inhibitors improve survival of neurons transfected with mutant htt. Caspase resistant (CR) htt mouse models therefore would be ideal systems in which to assess whether creation of caspase generated fragments of htt underlye the pathogenesis of HD in vivo. To examine whether a specific caspase cleavage fragment of mutant huntingtin is responsible for the selective neurodegeneration observed in HD, we generated YAC transgenic mice expressing selective mutations of the caspase cleavage sites within mutant huntingtin. We show, using sequential mutagenesis, that caspase-6 and not caspase-3, mediated cleavage of mutant htt is responsible for the HD-related behavioural phenotype and selective striatal neurodegeneration observed in the YAC 128 model of HD. Activation of caspase-6 and nuclear translocation of htt fragments coincide with onset of motor dysfunction in the YAC 128 model, supporting a role for a specific nuclear htt fragment in initiating neuronal dysfunction. Furthermore, caspase-6 cleavage of mutant htt influences susceptibility to excitotoxic stress highlighting caspase-6 mediated proteolysis of htt and excitotoxicity as a primary mechanism underlying motor dysfunction and neuropathology in HD. The results presented in this thesis support and further refine the toxic fragment hypothesis by identifying a specific proteolytic cleavage site in htt that is required for initiating a sequence of events which culminate in the death of selective neurons affected in HD. This evidence demonstrates that generation of a specific fragment of mutant htt in vivo represents a primary, initiating event in the pathogenesis of HD and identifies novel approaches for inhibiting cell death in neurodegenerative disorders such as HD.

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