It is clear that the mutant huntingtin (mHtt) forms intracellular aggregates and induces apoptosis in Huntington's disease (HD). However, the underlying molecular mechanism remains elusive. The long-term goal of the project is to understand the molecular pathways that are altered by mHtt expression. Recent studies suggest a functional link between accumulation of mHtt and proteasomal dysfunction in context of mHtt-induced cell death in HD. However, it is unclear how mHtt-induced proteasomal dysfunction can be molecularly converted to neuronal death. In a series of pilot experiments, we began to discover that BimEL expression is significantly up-regulated in two different cell lines overexpressing mHtt. More interestingly, the up-regulation of BimEL is largely due to BimEL phosphorylation rather than the transcriptional regulation. Based on our preliminary findings, we specifically hypothesize that the pro-apoptotic BH3-only protein, BimEL, is the molecule that functionally links mHtt aggregates formation and apoptosis. In this application, we propose to test this hypothesis in the R6/2 mouse model of HD. In the first specific aim, we propose to confirm our preliminary findings in R6/2 mice. BimEL expression in the striatum of R6/2 and control mice will be examined at both transcriptional and post-translational levels using quantitative RT-PCR and western blot respectively. We next propose to determine the molecular mechanism that up- regulates BimEL expression in R6/2 mice with a special focus on the regulation of BimEL phosphorylation. BimEL phosphorylation and the related protein kinases activity will be examined by western blot analysis using specific phospho antibodies. Finally, we propose to determine the physiological relevance of BimEL phosphorylation and up-regulation in HD. mHtt-induced death in the presence or absence of silencing BimEL expression will be investigated by flow cytometry analysis. Moreover, Bax translocation to mitochondrial membrane will be analyzed by western blot analysis under different conditions that favor or prevent BimEL phosphorylation. Information derived from the proposed studies will improve the current understanding of the molecular pathways that are altered in response to mHtt in HD and may represent a universal mechanism in the pathogenesis of neurodegenerative diseases that are involved with protein misfolding and aggregation. PUBLIC HEALTH RELEVANCE: Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the huntingtin protein. Today, approximately 30,000 Americans are living with HD. In addition, a staggering 200,000 more are at risk. Identifying the pathways that are altered in response to the mutant protein is crucial for understanding the cellular processes impacted by the disease as well as for the rational development of effective pharmacological interventions.