Huntington's disease causes motor and cognitive dysfunctions, the degeneration of striatal and cortical neurons in the brain, and death of its victim within 15-20 years. The genetic mutation is an expanded region of polyglutamines at the N-terminus of huntingtin. The function of wild-type huntingtin and the mechanism of HD pathogenesis caused by mutant huntingtin are unknown. We have observed an abnormal accumulation and transport of huntintin in affected neurons of the HD brain. Similar patterns of mutant huntintin accumulation appear in clonal striatal cells transfected with cDNAs encoding huntingtin with an expanded polyglutamine region. Published studies and our preliminary observations suggest that wild-type huntingtin may function in receptor- mediated endocytosis. Mutant huntintin, like wild-type huntingtin, associates with clathrin-enriched membranes. Our overall hypothesis is that mutant huntintin causes neuronal dysfunction through its direct effects on receptor-mediated endocytosis and by its abnormal accumulation and transport. We propose a series of studies in clonal striatal cells to explore wild-type huntingtin's association with endosomes (Aimsl), to analyze the consequences of polyglutamine expansion in huntintin on endocytic function (Aim 2), and to evaluate the subcellular compartments that accumulate mutant huntingtin and contribute to cell death (Aim 3). Our studies will include techniques in confocal immunofluorescence microscopy, immunogold/electron microscopy, subcellular membrane fractionation, immunoisolation and Western blot. The results will identify the subcellular processes involved in HD pathogenesis and will lead to a rational strategy for treatment of this devastating disorder.
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