Neurodegenerative diseases are a primary cause of debilitation in the aging population, yet no disease-modifying treatments are available to slow the progression of disease. α-Synuclein accumulates in pathological aggregates in the brains of patients with Parkinson’s disease, dementia with Lewy bodies and ~50% of Alzheimer’s disease. α-Synuclein is also mutated in familial forms of the disease. Yet, little is known about the pathogenesis pathway underlying synucleinopathies, leading to a paucity of therapeutic targets. The improved understanding provided by systematic analysis of the molecular components of this pathway will provide targets that may be exploited using hypothesis-driven clinical interventions. The experiments proposed here will take advantage of an established cell model that recapitulates major features of synucleinopathies, in addition to novel technologies to dissect the molecular components of a recently identified molecular pathway in which microtubule-affinity regulating kinases (MARKs) are important modulators of α-synuclein pathogenesis. MARKs are phosphorylated at an activating residue and accreted in Lewy bodies of cell and animal models of synucleinopathy as well as human brains. Inhibition of MARKs leads to an exacerbation of α-synuclein pathology, suggesting that MARKs may protect against α-synuclein-induced pathogenesis. The long-term objective of this proposal is to explore the hypothesis that MARKs, their effectors and substrates are components of an α-synuclein pathogenesis pathway that can be exploited through therapeutic targeting of the proteins involved. The hypothesis will be tested through two specific aims. In Aim 1, a CRISPRi/a platform will be utilized to selectively increase or decrease expression of MARK effectors and substrates to determine how these proteins affect α-synuclein pathology. Together with manipulation of MARK kinase activity this assay will be used to define protein components of the α-synuclein pathogenesis cascade. In Aim 2, the link between MARK activity and the targeting of misfolded α-synuclein for autophagy will be explored using a live imaging paradigm in parallel with immunocytochemistry to determine the effect MARKs have on the axonal transport and toxicity of α-synuclein pathology. The proposed work will offer valuable insight into how α-synuclein pathology is formed and identify proteins that control this process with the goal of developing those proteins for therapeutic treatment of PD and related synucleinopathies.