Advances in the in vitro study of Huntington's disease mutagenesis and pathogenesis

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by the expansion of a CAG·CTG tract within exon 1 of the gene for the huntingtin protein. The mechanisms by which such expansions occur and how they lead to eventual neurodegeneration are poorly understood. This dissertation applies existing model systems, describes the creation of a new model system, and lays a foundation for the development of newer models to address the important mechanistic questions about HD mutagenesis and pathogenesis. I determined that two homologs of DNA repair proteins identified as modulators of trinucleotide repeat (TNR) mutagenesis in yeast screens do not contribute to TNR stability in cultured human cells. I did find, however, that several important cis-acting mechanistic features of HD genetics are recapitulated in a new selective system for detecting TNR expansions in cultured astrocytes, including length-dependence, the ability to form aberrant DNA secondary structures, TNR tract purity, and DNA replication. I also determined that the specific ROCK (Rho-associated coiled coil kinase) inhibitor Y-27632 enhances important single-cell culture techniques needed to make scientific and therapeutic use of human pluripotent stem cells as HD model systems or therapies. This process also led to several mechanistic insights into the function of ROCK inhibitors. Finally, I examined the transcriptional regulation of an important transcription regulator and oncogene implicated in HD pathogenesis, Bmi1. In so doing, I identified important cis-regulatory features of two Bmi1 promoters that may be exploitable for HD therapy or studying HD pathogenesis. Together these significant findings lay the foundation for further study of the mechanisms of mutation and pathology in HD using in vitro model systems.