Abstract Metal exposure has increasingly been recognized as a potential environmental contributor to chronic neurodegnerative diseases including Parkinson's disease (PD) and Alzheimer's disease (AD). Chronic manganese (Mn) exposure has been implicated in Parkinson's-like neurological conditions in humans. Protein aggregation and its prion-like propagation are now considered the central pathophysiological mechanisms of many neurodegenerative diseases collectively known as proteinopathies. However, the role of metals in protein aggregation and the neurotoxicological mechanisms that drive degenerative processes are not well understood. α-Synuclein (αSyn) protein aggregation has been implicated in PD, and this protein features multiple divalent metal-binding motifs that have been suggested to play a role in αSyn's fibrillization and neurotoxicity. Since Mn shows affinity to the metal binding sites in αSyn, we have examined the effect of Mn on αSyn in neuronal models. Interestingly, we found that αSyn protected against Mn-induced neurotoxicity during early stages of Mn exposure, but prolonged Mn exposure promoted αSyn aggregation. In agreement with the emerging concept that aggregated proteins propagate cell-to-cell via exosomal release, we also observed enhanced release of exosomes containing αSyn into the extracelluar environment during Mn exposure. Thus, our exciting finding of Mn-induced αSyn aggregation and release of exosomes with αSyn cargo prompts us to further characterize the cellular and molecular mechanisms of neurodegenerative processes in Mn neurotoxicity. Our proposal will test the novel hypothesis that Mn exposure promotes αSyn misfolding and impairs intracellular αSyn trafficking, thereby increasing the formation and release of exosomes containing αSyn protein aggregates, which subsequently trigger microglial activation through the NLRP3 inflammasome pathway in a PKCδ-dependent manner. Sustained activation of the PKCδ-dependent NLRP3 inflammasome pathway contributes to Mn neurotoxicity. Specific objectives of the proposal are i) to characterize the cellular mechanism of Mn-induced impairment in endosomal trafficking, retromer dysfunction and exosome release in cell culture and animal models of Mn neurotoxicity, ii) to determine NLRP2/3 inflammasome neuroinflammatory signaling in microglia and astrocytes triggered by Mn-induced exosomal αSyn aggregates and to characterize the proinflammatory regulatory function of PKCδ in NLRP2/3 inflammasome activation in Mn neurotoxicity, and iii) to examine the role of PKCδ in mediating the exosomal αSyn aggregate-induced proinflammatory response in animal models of Mn neurotoxicity and to confirm the presence of αSyn protein aggregation in Mn-exposed human brain tissues. Our integrated cellular and molecular approach to unraveling the formation and release of exosomal αSyn aggregates and their functional consequences on neuroinflammatory signaling in manganese metal neurotoxicity will provide novel...