Project Summary/Abstract α-Synuclein is a small, soluble neuronal protein that is the primary component of the Lewy body aggregates that are the hallmark of Parkinson's disease. While the mechanistic details are not yet well-understood, emerging evidence suggests that cell-to-cell transmission of toxic forms of α-Synuclein is the basis of disease propagation. Our lab has recently identified complex N-linked glycans as mediators of cellular internalization of both monomer and aggregate forms of α-Synuclein bearing an N-terminal acetyl group, a physiological modification of the protein. We specifically identified the neuronal glycoprotein neurexin 1β as capable of driving internalization of α-Synuclein in a glycan-dependent manner. The goal of our proposed research is to characterize the structural basis of α-Synuclein binding to neurexin 1β, the role of both N-terminal acetylation and glycosylation in conferring specificity in this interaction, and determine the molecular mechanisms resulting cellular internalization of α-Synuclein following binding neurexin 1β. Our hypothesis is that cell-to-cell transmission of αS is dependent on interactions with neurexin 1β and that the selectivity in these interactions is dependent on transient structural changes in α-Synuclein conferred by the N-terminal acetyl group. To investigate this hypothesis, we have developed three specific aims with the following goals: determine the structural features of α-Synuclein bound to neurexin 1β, including defining a minimal α-Synuclein construct required for binding (Aim 1); determine the mechanisms by which binding to neurexin 1β results in cellular internalization of α-Synuclein (Aim 2); and understand the functional impact of α-Synuclein binding to neurexin 1β (Aim 3). To achieve these goals, we will carry out in vitro coarse grain and high resolution structural characterization of α-Synuclein:neurexin 1β complexes and use live-cell imaging to quantify internalization of α-Synuclein and the ability of internalized α-Synuclein to seed aggregation of endogenous α-Synuclein. We will contrast WT monomer, PD-associated point mutants and fibrillar forms of α-Synuclein. Through this research we expect to characterize key interactions involved in propagation of α-Synuclein pathology in Parkinson's disease, as well as to gain insight into the structural features of α-Synuclein:neurexin 1β complexes. Ultimately, the characterization of α-Synuclein: neurexin 1β interactions carried out through our studies may provide a new target for Parkinson's disease treatment and serve as the basis identifying novel small molecule therapeutics.