A proteomic probing of the full-length huntingtin interactome based on a novel BAC transgenic mouse model of Huntington's disease

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting from an expanded CAG mutation in the gene that encodes the protein, huntingtin (Htt). Despite its ubiquitous expression, mutant Htt mainly affects only a small subpopulation of neurons in the cortex and striatum. Little was known until recently with regards to the biological function of Htt, but screening efforts using techniques such as yeast-two-hybrid assays and mass-spectrometry-based proteomics methods have successfully identified numerous Htt interacting proteins, revealing a glimpse into the complex and dynamic world of this protein.;The focus of my Ph.D. studies has been to further elucidate the function of Htt by deciphering how the Htt interactome changes between the early and late stages of disease, and within those brain regions that are affected compared to those that remain unaffected throughout the course of disease. Using immunoprecipitation techniques along with proteomics and mass spectrometry methods, we have identified over 750 high-confidence full-length Htt interactors (of which, more than 130 have previously been shown to interact with Htt) using a novel BAC transgenic mouse model of HD (BACHD), which expresses the entire mutant human huntingtin gene containing 97 glutamines, including the endogenous huntingtin promoter and the likely endogenous regulatory elements.;The dissertation contains an in-depth description of the BACHD mouse model, followed by a description of the techniques utilized to obtain the Htt interactomes. The results from my interactome studies are revealed along with examples of validation by Western blotting and reverse immunoprecipitation. The appendices contain additional information derived during the course of my interactome studies, including several bioinformatic algorithms I created in order decipher subtle differences between the interactomes. These results support the validity of using such a proteomics-based method to obtain an unbiased list of potential Htt interactors which may be involved in complexes possibly related to HD pathology. The utilization of such a method is not limited to only studying HD, but can be applied to a variety of diseases.