Parkinson's disease (PD) is a heterogeneous neurodegenerative disorder affecting an estimated 7 million to 10 million people worldwide. While its exact etiology remains largely unknown, PD is likely to be multifactorial with contributions from both genetic and environmental factors. Recent evidence suggests that the gut-brain axis plays a large role in the development of PD, and gastrointestinal dysfunction and microbiome dysbiosis have been linked to the onset and progression of PD pathology. Despite these advances, how a dysfunctional microbiome initiates or contributes to the disease progression of PD has yet to be determined. Using advanced microbial sequencing studies in the gut microbiomes of PD patients, we recently uncovered that the microbial pathways for synthesis of a neurotoxic chemical, trimethylamine (TMA) was highly elevated in PD patients. Microbiota-derived TMA is metabolized in the liver to trimethylamine N-oxide (TMAO), which enters systemic circulation and crosses the blood-brain barrier and has been shown to accelerate the rate of a-synuclein (aSyn) fibril formation. Additionally, microbial TMA can also be converted into toxic metabolites formaldehyde (HCHO) and ammonia (NH3). Our preliminary studies also found significantly elevated plasma TMAO and HCHO production in PD patients. Our subsequent mechanistic studies further revealed that TMAO treatment promotes aSyn aggregation and stabilizes its conformational changes and triggers inflammasome activation. Recently, our team successfully established a new biomarker assay called real-time quaking-induced conversion (RT-QuIC) for ultrasensitive detection and quantification of misfolded proteins in CSF, plasma exosome, submandibular gland (SMG), and skin samples from PD subjects. These exciting findings and existing literature lead to our hypothesis that chronic gut microbial dysbiosis in PD generates neurotoxic metabolic precursor TMA, which triggers and augments PD neuropathology through mechanisms involving aSyn aggregation and NLRP3 inflammasome activation by its metabolites TMAO and HCHO. Thus, therapeutic inhibition of microbial TMA generation provides a radical new treatment approach by which to achieve disease modification in PD. Our proposed studies will test the following three objectives: In Objective 1, we will identify the TMA-generating bacterial species and strains that are elevated in human PD and establish the link between neurotoxic TMAO and HCHO metabolites, aggregated aSyn load and gut and peripheral inflammatory markers. In Objective 2, we will evaluate the therapeutic potential of pharmacological inhibitors of bacterial TMA production for disease modification in PD using well-established transgenic and humanized germ-free mouse models of PD. In Objective 3, we will evaluate the translational potential of the clinically tested, healthy probiotic strain E. coli Nissle in restoring gut microbial homeostasis and mitigating bacterial TMA production, aSyn aggregation, and proinflammatory cascades in PD mouse models. Overall, we anticipate that our study will provide the basis for developing new treatment strategies targeting TMAO-mediated pathological processes for disease modification in PD.