Cholesterol modulates vesicle clustering mediated by alpha-synuclein in a nonlinear fashion

α-Synuclein (α-Syn) is a presynaptic protein primarily associated with Parkinson's disease and other neurodegenerative diseases. The cholesterol content in SV membranes regulates α-Syn binding to synaptic vesicles, changing its function and modifying its aggregation. Using single-vesicle imaging, we show that low concentrations of cholesterol reduce vesicle clustering, and high concentrations enhance vesicle clustering mediated by α-Syn. Furthermore, using all-atom molecular dynamics simulation, we investigate the role of cholesterol in synaptic-like vesicle clustering mediated by α-Syn. In particular, we found cholesterol reduces hydrogen bonds and interaction energies in low concentrations, while high concentrations of cholesterol increase hydrogen bonds and interaction energies. Moreover, cholesterol also regulates lipid packing defects, and the condensation of cholesterol leads to the suppression of shallow packing defects, and enhancement of large defects with increasing cholesterol concentration. We revealed that cholesterol promoted vesicle clustering is due to the electrostatic interaction between cholesterol in the membrane and the N-terminal region of α-Syn. Moreover, this increased electrostatic interaction arises from a change in packing defect distribution of the protein-membrane interface induced by cholesterol condensation. This work highlights the complex interplay between α-Syn and cholesterol, emphasizing the importance of cholesterol levels in membranes and their impact on α-Syn function.