Project Summary/ Abstract Huntington’s Disease (HD) is a devastating neurological disease with no available disease modifying therapies. Accordingly, there is a dire need to identify new therapeutic targets that can slow down disease progression and improve symptoms. HD is an inherited autosomal disease caused by expansion of the CAG repeat sequence in the Huntingtin gene, resulting in structural atrophy of the striatum and cortex. Approaches to reduce the levels of mutant Huntingtin protein in the brains of patients have so far been unsuccessful in clinical trials, so it is essential to identify new therapeutic targets. Studies have found genetic variants of multiple DNA damage repair proteins that affect the age of onset of HD. This, combined with the accumulation of DNA damage seen in HD brains and models, implicates defective DNA damage repair as a critical driver of the pathology of HD. Wild-type Huntingtin protein is part of a transcription coupled DNA damage repair complex in neurons, termed the transcription coupled non-homologous end joining (TC-NHEJ) complex. This complex is recruited to genes being actively transcribed, so that DNA double strand breaks can be quickly repaired, preserving genomic integrity and neuronal survival. Huntingtin interacts with numerous DNA damage repair proteins in this complex including PNKP, and DNA-PK. Interestingly, our preliminary data also show TAR DNA-binding protein 43 (TDP- 43) is associated with Huntingtin TC-NHEJ complex, and this interaction increases after DNA damage. TDP-43 is expelled from the nucleus and phosphorylated TDP-43 accumulates in the cytosol in several neurological diseases including HD. Another hallmark of HD is the increased level of protein SUMOylation. DNA damage repair proteins are also often regulated by SUMO, so it is important to learn how post translational modifications affect TC-NHEJ activity in unaffected and HD cells. The working hypothesis of this proposal is that incorporation of mutant Huntington into the TC-NHEJ DNA repair complex disrupts critical protein-protein interactions and promotes excess SUMOylation, resulting in decreased DNA damage repair and accumulation of DNA damage. Identifying critical protein interactions and post-translational modifications to boost DNA damage repair in HD will provide a new spectrum of potential therapeutic targets. To accomplish this, I will define the relationship between the SUMO E3 ligase PIAS1 and PNKP (Aim 1). I will identify the interface between these two proteins and design mutant iPSC lines to determine if disrupting binding between these two proteins improves DNA damage repair in HD lines. I will investigate whether TDP-43 is essential for activity of the TC-NHEJ complex (Aim 2), and how its phosphorylation and expulsion from the nucleus affects DNA damage repair. I will identify the E3 SUMO ligase for TDP-43 (Aim 3) to enable studies which modulate TDP-43 SUMOylation.