PROJECT SUMMARY. Accumulation of α-synuclein (α-syn) protein aggregates in brain neurons is thought to play a causal role in Parkinson's disease (PD). There is mounting evidence that the gut is a source of α-syn aggregates in the brain; however, the factors involved remain incompletely understood, limiting development of treatments to prevent PD. We propose a gut microbiota-mediated mechanism of α-syn aggregation. In the brain, α-syn aggregation results when oxidative stress within the host transforms iron and dopamine into a toxic pair. This toxic pair kills dopamine-producing neurons, decreasing dopamine levels and causing motor dysfunction. We hypothesize that in the gut, the microbiota—not the host—creates the oxidizing redox potential that modulates α-syn aggregation. Our preliminary experiments in vitro and in a C. elegans model of PD indicate that when the gut microbe E. coli performs nitrate respiration, yielding the oxidizing agent nitrite, a formerly innocuous trio of gut molecules—Fe2+, dopamine, and α-syn monomers—transforms into one that generates toxic α-syn aggregates. What remains unknown is the impact of bacterial nitrate respiration on α-syn aggregation in the mammalian gut. As accumulation of α-syn aggregates in intestinal tissue foreshadows neurodegeneration in the brain, there is a critical need to discover the factors that initiate aggregation of α-syn in the GI tract so that early interventions can be developed to stop PD before neurons die. This application’s objective is to determine the molecular mechanism underlying nitrite-induced α-syn aggregation in intestinal epithelial cells and to examine this mechanism as a gateway for α-syn aggregates to spread to neurons and cause motor impairment. Such work would represent a major step toward demonstrating the relevance of this phenomenon in human PD. The proposed approach to address these knowledge gaps entails (1) revealing the role of dopamine in α-syn aggregation in host intestinal cells by using chemical and genetic tools, (2) determining the impacts of nearly 100 gut microbes (in isolation and in combination) on α-syn aggregation in intestinal cells and mapping biochemical responses via proteomics, and (3) tracking the formation and fate of α-syn aggregates in intestinal cells by using bioluminescent reporters. Upon completing this research, our contribution will be a detailed molecular mechanism for how the gut microbiome initiates α-syn aggregation in PD. Our 3 complementary but independent specific aims interrogate this process at the level of molecules (Aim 1), the microbial ecosystem (Aim 2), and host physiology (Aim 3), providing a holistic roadmap for clinical translation. This work is innovative because it employs an unprecedented systems-level approach to define and control the gut microbiome’s role in PD. It is significant because it will likely reveal new therapeutic targets for PD, potentially including intestinal dopamine and common species of gut bacteria. ...