Project Summary Decades of genetic and experimental evidence has placed α-synuclein (αS) as a central player in the pathogenesis of several neurodegenerative diseases including Parkinson’s Disease (PD), Alzheimer’s Disease (AD) and related dementias (ADRD). αS protein levels closely correlate with PD and ADRD risk, and reduction has been shown to be protective in multiple disease models, making lowering αS levels a target for therapeutic intervention. Despite that, there is very poor understanding of how the abundance and turnover of endogenous αS protein is regulated. We recently found, using unbiased CRISPR screens in human cell lines and iPSC- derived neurons, that disruption of the NatB complex resulted in a stark reduction in endogenous protein levels and is well tolerated in human iPSC derived neurons. NatB installs N-terminal acetylation (AcN) of αS. While AcN is a prevalent post-translational modification, its functional consequences are not well understood. AcN has been implicated in several processes specific to αS, such as membrane binding, cell uptake, and aggregation. Indeed, our own preliminary data support the importance of these effects and more. These results led us to hypothesize that lack of αS AcN compromises interactions that are fundamental to its molecular function and that pharmacological targeting of AcN represents a novel therapeutic strategy for αS’s involvement in PD and ADRDs. We propose three aims that will rigorously investigate the implications of these findings on αS biology and therapeutics. In Aim 1, we will identify the molecular interactions that are lost when αS lacks AcN. We believe that this will provide critical insight into its biological function. We will also investigate the mechanisms responsible for the rapid degradation of non-AcN αS compared to AcN αS. This may provide additional tools for therapeutic reduction of pathological αS in disease and insight into αS turnover mechanisms. In Aim 2, we will focus on understanding the outcome of NatB inhibition on the proteome and N-terminal acetylome of human cells and neurons using quantitative proteomics approaches. Such understanding will be essential for the development of NatB as a therapeutic target. Lastly, in Aim 3 we will develop the first NatB pharmacological inhibitors for evaluation in vitro, and in iPSC derived neurons. We use state-of-the-art computational approaches in combination with experimental high-throughput screening to identify molecules that can inhibit NatB acetylation of αS with minimal effects on AcN of other proteins. These molecules will serve as essential research tools and as lead therapeutic candidates for PD and ADRDs.