PROJECT SUMMARY Parkinson's Disease (PD) is a complex and progressive neurodegenerative disorder that culminates in the deposition of alpha synuclein (a-syn) containing protein aggregates, named Lewy bodies, and the degeneration of nigrostriatal dopamine neurons. Evidence suggests that neuroinflammation plays a causative role in the pathogenesis of PD. Post-mortem- and longitudinal PET imaging studies of the PD brain reveal an increase in the number of activated microglia that occur early in the disease process and remain elevated throughout the course of the disease. Activated microglia can convert astrocytes to a subtype of toxic “A1- astrocyte” and these reactive astrocytes are observed in the PD brain. A1 astrocytes coordinate selective neurodegeneration by releasing components of the complement system. The complement system is a division of the innate immune system that coordinates the removal of pathogens and cell debris. However, the complement system, and specifically complement component 3 (C3), is also used by the central nervous system to tag vulnerable synapses and neurons for phagocytic clearance. Within the PD brain, toxic A1- astrocytes dramatically increase the expression of C3. Taken together, we hypothesize that following an initial immunogenic signal (pathological a-syn misfolding) C3 derived from A1 astrocytes is used to tag inclusion-containing neurons for destruction by microglia. Supporting this idea, both midbrain dopamine neurons and Lewy bodies label with activated C3 in the PD brain. Neuroinflammation has been reported with virtually every model of PD. However the majority of PD models fail to accurately recapitulate the defining features of PD pathology: The progressive aggregation of endogenous a-syn and subsequent degeneration of nigrostriatal neurons. In contrast, intrastriatal injection of recombinant a- syn pre-formed fibrils (PFFs) is able to accurately model these hallmarks of PD pathology, and a neuroinflammatory response that mimics the human disease. We have extensively characterized this model and found that injection of a-syn PFFs into the rat striatum results in peak a-syn aggregate formation within the substantia nigra pars compacta (SNc) at 2 months post-injection, followed by significant nigral degeneration by 6 months. During the peak of a-syn aggregation we observe peak microglial activation as well as robust astrogliosis. Genetic expression and morphological profiling of reactive astrocytes is consistent with the presence of toxic A1-astrocytes. As such we hypothesize that C3 derived from A1 reactive astrocytes is used to tag inclusion-containing nigral neurons for destruction. Supporting this idea, we observe a large increase in the expression of C3 during this time point, and have localized C3 to reactive astrocytes. Importantly, the presence of toxic A1-astrocytes and the increase in C3 expression occurs months prior to overt nigral degeneration, suggesting astrocytic C3 may play a role in the deg...