Single-vesicle tracking of α-synuclein oligomers reveals pore formation by a three-stage model modulated by charge, curvature, lipids and ligands

Neurodegenerative disorders like Parkinson’s disease (PD) pose significant health challenges. A major hallmark is the aggregation of α-synuclein into toxic oligomers (αSO) and fibrils. While many efforts focus on slowing disease progression, the molecular origins and mechanisms of αSO toxicity remain poorly understood, particularly its proposed link to membrane disruption. To address this, we have developed a single-vesicle analysis platform for direct and real-time measurements of αSO and membrane interaction. This allows us to show real-time translocation of dyes through αSO pores with single-particle resolution and single-channel electrical recordings for analyzing pore formation in planar lipid bilayers. Across methods, our data reveal evidence for a three-stage model for αSO and membrane interactions with initial membrane recruitment followed by partial pore insertion and subsequent full pore formation. Strikingly, while αSO recruitment was found to favor curved membranes, pore formation occurred more efficiently in less curved membranes, hence recruitment is divorced from a membrane charge-promoted reorientation and pore integration. Single αSO pore formations are undergoing multiple translocation steps hence pore formation is highly dynamic cycling back and forth from partial insertion to full pore formation. The dynamic nature of pore formation can be modulated by lipid charge, lipid headgroup class, and ligand binding. Our findings suggest that increased dynamic pore formation could implies increased membrane toxicity. Evidence for the three-stage model is important for future targeting strategies for blocking αSO mitigated PD-related cellular dysfunction and we envision the single-vesicle assay enables screening for ligands modulating the pore formation.