The identification of the gene (IT-15) causing HD with its expanding CAG triplet repeat clarifies the genetics of HD, but raises many new questions regarding its pathophysiology. The peptide sequence of the putative HD gene protein product has no clear homology to any known protein. Furthermore, the widespread expression of the IT-15 transcript and the lack of notable change in its expression in HD patients raises the issue of how a widely expressed gene can cause selective neuronal vulnerability in restricted portions of the basal ganglia and the cerebral cortex. The studies in this project are designed to elucidate the basic cell biology of the HD protein product (Huntington). Our hypothesis is that the CAG repeats are translated into an expanded polyglutamine tract, causing a gain of function. We have made specific antipeptide affinity purified antibodies to several epitopes within the HD amino acid sequence, for immunochemical and immunohistochemical studies of the protein. We will determine its tissue and regional brain localization. We will study its cellular and subcellular sites of expression using immunohistochemistry and Western blots of subcellular fractions. We will use double label and EM immunohistochemistry in both brain and peripheral tissue. We will express truncated and full length cDNAs with normal and expanded glutamine repeats in expression vectors from which protein can be purified for biochemical studies. In collaborative experiments, we will study the structure of the HD protein. We will elucidate the functional role of the HD protein in stably transfected neuronal cell lines. We will cotransfect the HD protein vector with vectors coding for proteins which associate with it, as identified in the 3rd specific aim. We will use antisense oligonucleotide treated cells and ES cells from gene knockouts to study the function of the normal protein. Finally, we will identify proteins which interact with the HD protein, using coimmunoprecipitation an the yeast two-hybrid system. We will clone full length cDNAs, and study them using techniques described above. These studies taken together will clarity the cellular and tissue sites or expression of the expanding glutamine repeat.
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