The goal of this project is to examine the role of Rab27 in synucleinopathies, including Parkinson's disease and Dementia with Lewy Bodies. Misfolding and cell-to-cell propagation of αsyn are hypothesized to play a major role in the pathogenesis of Parkinson's Disease and Dementia with Lewy Bodies. Key steps implicated in α-syn spread include active release, uptake, and misfolding, yet the key mechanisms that regulate the spread of α-syn are poorly understood. Rab proteins are small GTPase proteins that interact with Rab effector proteins to control protein trafficking and degradation and have been implicated in αsyn pathogenesis. We recently identified a potential role for Rab27b in synucleinopathies. Rab27b, which is highly enriched in neurons, plays a role in protein secretion in multiple cell types including neurons, but has also been linked to autophagy and endocytosis. Alterations in Rab27b are associated with Alzheimer's disease, Dementia with Lewy Bodies, and X-linked dystonia parkinsonism syndrome. We recently found a dramatic increase in Rab27b in human PD postmortem brains and in an inducible αsyn cell line (isyn). Rab 27b knockdown (KD) in isyn cells reduces αsyn release and impairs autophagic clearance, consequently promoting αsyn toxicity. In contrast, Rab27a/b double knockout (DKO) leads to a dramatic reduction in αsyn aggregation in neurons exposed to extracellular αsyn protofibrils (PFFs) in vitro and in vivo, most likely due to reduced uptake. Based on these data, we hypothesize that Rab27b plays two independent roles that affect αsyn handling: 1) it regulates entry of extracellular αsyn via endocytosis; and 2) it promotes clearance of intracellular αsyn via secretion and autophagy. In disease, fibrillar αsyn co-opts cell entry pathways via Rab27b and thus indirectly disrupts Rab27b's clearance of intracellular αsyn. We propose that differential effects of Rab27b on autophagy, secretion, and uptake are mediated by different Rab27 effector pathways. In Aim 1, we will test the ability of Rab27b to mediate αsyn entry into neurons and the relevant Rab27 effectors that mediate uptake. In Aim 2, we will focus on the impact of Rab27b on clearance of intracellular α-syn by active secretion and autophagy, determine the relevant Rab27 effectors that mediate these effects on autophagy and secretion, and test whether fibrillar αsyn induces a redistribution of Rab27b away from endolysosomal structures to interfere with clearance activities. In Aim 3, we will test the consequences of Rab27b and relevant effectors on cognitive function, αsyn inclusion formation, and neuronal loss in the in vivo α-syn PFF model. Further understanding of the mechanisms by which Rab27b affects αsyn trafficking is critical to understand its role in disease and its therapeutic potential.