Interrogating alpha-synuclein aggregate-associated proteomes to identify modifiers of Lewy body disease.

1. ABSTRACT After Alzheimer’s disease, Lewy body dementia is the most common form of dementia afflicting aging individuals. The goal of this project is to advance our mechanistic understanding of the role that alpha-synuclein plays in disease pathogenesis and progression of Alzheimer disease related dementias (ADRDs) such as Lewy body dementia (LBD) by discovering molecular interactors of alpha-synuclein and defining the alpha-synuclein aggregate-associated interactome. We hypothesize that the cellular and molecular environment contributes to αSYN-associated Lewy pathology processes and that quantitative proteomics can be applied to capture the molecular microenvironment proximal to αSYN as it forms oligomers, protofibrils, and insoluble aggregates to reveal key cofactors in aggregation and neurodegeneration processes. In this application we will employ proximity-dependent biotin identification in live human neurons and mouse brain slices (BioID) (aim 1) and in situ probe-dependent proximity profiling in fixed human patient brain tissue (ProPPr) (aim 2) to enable biotinylation of αSYN-associated cofactors under physiological and pathogenic conditions. Proximal biotinylated proteins will be isolated via affinity purification and identified by quantitative mass spectrometry. We propose the following coordinated aims to accomplish this goal: In aim 1 we will capture, identify, and differentiate intracellular proteins in proximity to soluble alpha-synuclein in vitro in human iPSC-derived neurons and glia and ex vivo in murine organotypic brain slice cultures, or in proximity to insoluble aggregates of alpha- synuclein induced by human-derived proteopathic seeds. In aim 2 we will use in situ probe-dependent proximity profiling in human postmortem brain to probe the αSYN-associated proteome in different synucleinopathies (DLB, PD, MSA), over time (comparing brain regions affected early versus late in disease course), and with consideration of comorbid pathologies (tau, TDP-43, beta-amyloid) to reveal the underlying global and local proteomic landscape of synucleinopathies and shed light on the molecular heterogeneity and mechanisms underlying disease. Finally in aim 3 we will functionally investigate the pathophysiological role of select candidate proteins in human tissue, cell models, ex vivo brain slice cultures or in vivo in murine models. Successful completion of these aims will specifically identify molecular cofactors of αSYN monomers, oligomers, and aggregates and determine the processes at play during Lewy pathology formation, maturation, and progression.