Project Summary / Abstract: Huntington’s disease (HD) is a devastating neurodegenerative disease that affects movement, psychiatric well- being and cognition. HD is caused by a CAG mutation expansion (≥40 repeats) in exon1 of the Huntingtin gene (HTT), and expansion of a polyglutamine repeat in the mutated protein (mHTT). Currently, there are no approved disease modifying treatments. An effective and rational clinical treatment of HD will rely on the comprehensive understanding of HTT functions and disruption of those functions by the HD mutation. HTT interactor studies have revealed interactions with RNA-binding proteins (RBPs) that are involved in chemically modifying the RNA base Adenosine to N6-Methyladenosine (m6A). Specific to the brain, m6A regulates neurodevelopment, neurogenesis, synaptic plasticity, and cognition. m6A is a new area of study for the field of neurodegeneration, with implications that may provide novel mechanistic insights into mHTT induced toxicity. Preliminary data in a HD R6/2 mouse model shows a general trend of less m6A installation protein expression, suggesting a decrease in m6A levels. m6A/RNA-sequencing performed on striatal tissue from HD and non-transgenic (NT) mice revealed a ~20% increase in m6A sites detected in HD over NT. Analysis of m6A modified transcripts unique to HD reveals enrichment of an RBP binding motif involved in mRNA decay. This proposal is based on the hypothesis that m6A modifications are dysregulated in HD and contribute to HD pathogenesis through a) decreasing gene expression and b) causing dysregulation of mRNA decay. In Aim 1 the applicant sets out to establish the role of m6A-dependent mRNA decay contributing to transcriptional dysregulation in HD using a well-established phenotypic readout and will attempt to understand how mHTT may directly cause changes in m6A modifications. To achieve this, the applicant has developed and optimized a protein- protein labeling strategy in induced pluripotent stem cells (iPSCs), that can be differentiated into the affected neuronal population in HD, to enrich for HTT and mHTT RBP interactors and their RNAs. Aim 2 will determine if increases in m6A modifications directly contribute to the HD transcriptional profile in an in vivo system and provide a proof of concept for therapeutic intervention. This proposal will provide the scientific community with a first look at the m6A machinery in Huntington’s disease, provide a comprehensive annotation of HTT RBP interactors with associated RNAs, new insights into HTT/mHTT function, and address questions of whether HTT’s interaction with RBPs leads to changes in RNA processing, directly causing transcriptional dysregulation.