Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are complex diseases involving gene- environment interplay that lead to alpha-synuclein (a-synuclein aggregation). Recent investigations suggest that the gut microbiome can modulate neuroinflammatory responses to hallmark a-synuclein aggregation, but little is known about specific gut microbial mediators. Studying rare kindreds with inherited forms of neurodegeneration that closely mimic sporadic disease has proved invaluable to the field, offering a window to understand idiopathic disease in a more controlled genetic and environmental background. We are investigating environmental determinants of disease penetrance utilizing microbiome samples from such a family harboring an alpha- synuclein (a-synuclein) E46>K mutation and exhibiting a spectrum of clinical PD dementia (PDD)/DLB outcomes, that pathologically mimic sporadic forms of these diseases. We hypothesize that gut microbiota contributes to the development of PDD/DLB through the secretion of metabolites that affect neuroinflammatory signaling and neuronal function. In our preliminary data, first we identified specific bacterial species associated with PDD/DLB disease penetrance, some of which have been previously reported to be altered in PD. Second, we found that transfer of the PDD/DLB gut microbiota into a PD mouse model (with amplified E46>K mutation) enhanced motor and cognitive deficits. Third, PDD/DLB microbiome transfer altered microglial transcriptional profiles suggesting that changes in microbiota could contribute to disease pathogenesis through neuroimmune modulation. In this proposal, we aim to identify microbes and metabolites which are linked with PDD/DLB pathogenesis using in vivo models and evaluate whether these metabolites directly affect microglial function and neuronal survival in vitro in PDD/DLB induced pluripotent stem cell (iPSC)-derived cultures. First, to investigate potential gut-brain axis mechanisms associated with altered motor and cognitive function, we will identify changes in the gut metagenome and metabolites, analyze altered microglia transcriptional profiles and evaluate a-synuclein pathology in PDD/DLB-microbiota colonized mice, when compared to mice colonized with either healthy control or CNS asymptomatic E46K carrier microbiome. Second, to determine the impact of microbial metabolites on pathways involved in PDD/DLB, we will treat E46K PDD/DLB patient and gene corrected iPSC-derived microglia and neuronal cultures with either bacterial supernatants from in-house isolated microbial species abundant in E46K PDD/DLB individuals, or media from bacteria-sensitized microglia. We will evaluate the neuroinflammatory cytokine and chemokine profile, microglia transcriptional profiles, neurite outgrowth and survival, and a-synuclein pathology to determine the mechanistic impact of PDD/DLB microbial metabolites on variable disease penetrance in vitro. The knowledge gained and model systems established...