Huntington's disease (HD) is an autosomal-dominant progressive neurodegenerative disorder, which results from a CAG expansion in exon 1 of the huntingtin gene (htt). Currently it is unknown how the mutant huntingtin protein (Htt) promotes the selective neurodegeneration of HD. Multiple lines of evidence support the hypothesis that production of small N-terminal Htt fragments plays a key role in the pathogenesis of the disease. Preliminary evidence in our laboratory shows that calpain cleaves Htt at multiple sites to produce small N-terminal fragments and that the active form of calpain is substantially increased in the caudate of HD patients. In order to address the hypothesis that production of calpain-derived Htt fragments contributes to the specific neurodegeneration observed in HD, the predicted calpain cleavage sites in normal and expanded htt will be mutated to see if changes in production of calpain-delved Htt fragments influence cellular viability. In addition normal and expanded calpain-derived Htt fragments will be produced and expressed in striatal neurons to correlate the cytotoxicity of the fragments with nuclear migration and aggregation. Immunoprecipitation with a Htt antibody will also be utilized to determine whether the normal and/or expanded form of Htt physically associates with calpain to produce calpain-derived Htt fragments. Lastly, the cell types in the caudate of HD displaying changes in sub-cellular distribution of calpain will be identified and it will be determined if calpain colocalizes with Htt through double labeling of striatal neurons overexpressing calpain-derived Htt fragments.
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