PROJECT SUMMARY One major obstacle to halting neurodegeneration in synucleinopathies like Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB) is a lack of understanding of why vulnerable cell populations die. Although aggregated alpha-synuclein Lewy pathology occurs within specific neurons, and their dysfunction or degeneration leads to symptoms in these diseases, how Lewy pathology itself plays a role is unclear. Recent work by the Unni lab has discovered a previously unrecognized function for the protein alpha-synuclein in repairing nuclear DNA double-strand breaks (DSB), by using newly developed in vivo multiphoton imaging techniques to study alpha-synuclein aggregation in mice. These approaches have now been extended (for the first time) to study DNA repair in living mouse brain. The unexpected function for alpha-synuclein immediately suggests an exciting new hypothesis for the role of aggregated alpha-synuclein Lewy inclusions in neurodegeneration. This central hypothesis is that in disease, alpha-synuclein protein is sequestered in cytoplasmic Lewy bodies, decreasing its nuclear DSB repair function and leading to cell death of Lewy body- containing neurons. Furthermore, it is proposed that the source of fibrillar α-synuclein that initially seeds Lewy body formation comes from dysregulated DSB repair. The preliminary data suggest this complex interrelationship, with alpha-synuclein aggregation causing dysregulated DSB repair and vice versa. Although completely novel, this link between alpha-synuclein and DSB repair could explain previously poorly understood associations between human and mouse mutations in the DSB repair protein Ataxia-Telangiectasia Mutated and alpha-synuclein aggregation, and between specific synuclein family members and cancer (e.g. alpha- synuclein & melanoma, gamma-synuclein & breast cancer). This proposal will use a combination of advanced imaging approaches in purified, reconstituted in vitro systems, in mouse brain in vivo, and in mouse & human (PD, DLB) fixed tissue to test how alpha-synuclein mediates DSB repair (Aim 1), how loss of nuclear α- synuclein function after cytoplasmic Lewy inclusion formation dysregulates DSB repair and contributes to neuronal cell death (Aim 2), and how dysregulated DSB repair can lead to Lewy inclusion formation (Aim 3). Overall, this project is innovative because it uses powerful, new in vivo experimental approaches to test a fundamentally new hypothesis for how alpha-synuclein aggregation into Lewy pathology is related to neurodegeneration. This contribution will be significant because it will provide a completely new set of targets for treating these debilitating neurodegenerative disorders that focuses on the DSB repair pathway, and because understanding alpha-synuclein-mediated DSB repair in detail will increase our basic science knowledge of this important biological process.