In this proposal, we will determine if we can use microglial extracellular vesicles (EV) that have been shed into the cerebral spinal fluid (CSF) and serum to predict the microglial activation state in the Parkinson’s brain (PD). Currently, a major obstacle in the field is that there are no established and validated methods to detect brain inflammation in response to neurodegeneration during life. Microglia, the resident immune cell of the CNS constantly patrol the brain, looking for signs of infection or inflammation caused by a host of immune stimulants. The role of microglia is to clear potential threats to the CNS, but their chemical signatures based on their presence are continuously released (cytokines). These cytokines activate neighboring microglia initiating a cascade of events that are believed to drive disease pathogenesis. Although cytokines are generally considered to function as soluble molecules, recent efforts have shown that cytokines are encapsulated in EVs. These EVs contain a host of inflammatory-associated mRNAs that encode cytokine-associated genes among other signaling molecules that are known to reflect the physiological state of the parent cell. Unlike microglia, EVs can cross the blood-brain barrier under leaky and inflamed conditions, both of which are known physiological processes in PD. To determine if we can identify microglial EVs in the periphery we will characterize EVs isolated from disease-associated microglia (DAM) using RNAseq and Liquid chromatography-mass spectrometry (LC/MS). This novel data will be used to generate targets for discovery work in the periphery. This proposal aims to address two unmet needs 1) the possibility to detect brain inflammation in the living and 2) the ability to distinguish EVs released from microglia from those released from peripheral blood mononuclear cells. We hypothesize that microglial EVs extracted from the serum/CSF will reflect EV profiles from DAM. To test our hypothesis, we propose to 1) Analyze membrane-bound proteins and EV cargo in disease-associated microglia (DAM), 2) analyze membrane-bound proteins and EV cargo in peripheral EVs isolated from PD peripheral blood mononuclear cells, 3) identify and validate unique microglial-specific EV membrane antigens for antibody discovery work and 4) use these validated antibodies to pull down microglial-specific EVs from CSF and serum. All the tissues proposed in the application were collected from premortem PD patients who have since passed and have pathological confirmation of disease without comorbidities. We believe that each of these individual aims is high impact, and novel on their own, but collectively could provide us the tools necessary to predict brain inflammation.