PROJECT SUMMARY Growing evidence suggest that central nervous system (CNS) inflammation is a major pathophysiological process in Parkinson’s disease (PD). The inflammation is characterized by an increase in the number of microglia and in the levels of proinflammatory cytokines and chemokines. The proinflammatory cytokines, which are released when microglia are activated, could exacerbate α-synuclein (α-syn) aggregation and neuronal death ultimately driving the chronic progression of PD. Dying or dead dopaminergic neurons released α-syn aggregates can be taken up into the microglia, which leads to microglia activation. This in turn contributes to the accelerated progression of neurodegenerative processes. Therefore, identification of molecular targets in microglia that can be modulated to inhibit their inflammatory response may provide new strategy for the treatment of PD. However, there is a lack of knowledge about the cellular mechanisms underlying microglia-mediated neuroinflammatory events following stimulation with α-syn aggregates. To gain a comprehensive understanding of the cellular mechanisms of α-syn aggregates induced microglial activation, we conducted RNAseq analysis using α-syn preformed fibrils (α-syn PFF) activated microglia. From the screening, we identified NOD2/RIPK2 complex as a key modulator in neuroinflammatory responses due to α-syn aggregate. In our preliminary studies, we found that α-syn aggregates activate NOD2/RIPK2 mediated inflammatory responses in microglia to exacerbates neuronal death through a series of signaling events, which included M1 microglial activation and toxic A1 type astrocytes conversion by secreting IL-1α, TNFα and C1q. On the other hand, we found that the depletion of NOD2/RIPK2 complex protects neuronal death via blocking microglia-mediated A1 astrocyte conversion in vitro and in vivo. To further confirm and expand our novel preliminary findings, we will systematically pursue the following specific aims: (i) to define the levels of NOD2, RIPK2 mRNAs and proteins and the levels of phosphorylation of RIPK2 in microglia in mouse models of α-syn induced degeneration and PD postmortem brains, ii) to characterize the interaction between NOD2 and α-syn aggregates in microglia and define how α-syn PFF binding to NOD2 affects NOD2/RIPK2 dependent signaling pathways including RIPK2 activation/ubiquitination and MAPKs and NF-kB activation in microglia, iii) determine the depletion effect of NOD2/RIPK2 signaling in microglia-induced A1 astrocytes formation due to α-syn aggregates both in vitro and in vivo, and (iv) to determine the effects of blocking of NOD2/RIPK2 signaling in α-syn PFF induced model of PD through genetic ablation of NOD2 and RIPK2 and pharmacological inhibition of RIPK2. Cellular, neurochemical, neuropathological and behavioral approache...