Renewal Request for R01-NS064934 “Mechanisms of LRRK2 Neurotoxicity” Parkinson's disease (PD) and related Lewy body diseases represent common causes of Alzheimer's disease related dementias (ADRDs). According to the National Alzheimer's Project Act, Lewy body diseases affect more than one-million Americans, with onset of disease typically earlier in life than dementia caused by Alzheimer's disease. Tens of thousands of Americans harbor LRRK2 missense variants that can cause PD. Longitudinal studies show LRRK2 mutation carriers largely follow a typical course of motor and cognitive impairment over time, mirroring idiopathic disease. Post-mortem studies show that LRRK2 mutations are a potent genetic risk factor for the aggregation of tau and α-synuclein. LRRK2 variants associate with susceptibility to Crohn's disease and mycobacteria infection, highlighting LRRK2 function in immune cells. In the past project period, we observed a dual-activation phenomenon with intrinsic LRRK2 modifying α-synuclein aggregation in neurons, as well as high LRRK2 expression in myeloid cells recruited to the brain in disease models. In a novel enzymatic cycle that we intend to closely examine, LRRK2 mutations may increase cis LRRK2 kinase activity in autophosphorylation as well as trans phosphorylation of a small subset of Rab proteins. In our continued efforts to understand LRRK2 function in disease, we focus our attention to LRRK2- Rab substrate interactions. We propose a series of quantitative proximity labeling approaches to probe the inner catalytic core of LRRK2-Rab enzymatic cycling in disease-relevant cells. These experiments will be anchored through structure-function relationships identified by cryo-electron microscopy analysis of relevant protein complexes. We predict these experiments will shed light on how disparate pathogenic mutations in LRRK2 share a common final output in Rab hyper-phosphorylation. Further, we will explore how LRRK2 variants linked to protection from disease might affect the LRRK2-Rab catalytic cycle. Both LRRK2 and its Rab substrate constituency demonstrate some of the highest expression in subsets of myeloid cells in the immune system. In complement to our biochemical approach, we will dissect the role of LRRK2 in pro-inflammatory macrophages recruited to the brain in an AAV-α-synuclein mouse model. We anticipate mutant LRRK2 expression in peripheral immune cells will exacerbate deleterious neuroinflammation that correlates to dopaminergic neurodegeneration. We further predict that LRRK2 knockout in peripheral immune cells will attenuate pro-inflammatory responses in the brain and α-synuclein-induced loss of dopamine neurons. As several exploratory therapies that target LRRK2 have recently advanced through safety trials in the clinic, with project success, we predict our studies will further emphasize LRRK2-Rab signaling and peripheral immune responses in LRRK2-linked disease.