Single-vesicle Tracking of α-Synuclein Oligomers Reveals Pore Formation by a Three-Stage Model

Neurodegenerative disorders, such as Parkinson's disease (PD) pose significant health challenges. A major hallmark of PD 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 regarding 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 platform allows us to demonstrate real-time translocation of dyes through αSO pores with single-particle resolution and use single-channel electrical recordings to analyze pore formation in planar lipid bilayers. Across methods, our data provide evidence for a three-stage model of αSO and membrane interactions, comprising initial membrane recruitment followed by partial pore insertion and subsequent full pore formation. Notably, while αSO recruitment was found to favor curved membranes, pore formation occurred more efficiently in less curved membranes, hence, recruitment is decoupled from a membrane charge-promoted reorientation and pore integration. Single αSO pore formations undergo multiple translocation steps making pore formation highly dynamic, cycling back and forth between partial insertion and 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 imply increased membrane toxicity. Evidence for the three-stage model is important for developing future targeting strategies to block αSO-mediated PD-related cellular dysfunction. We envision that the single-vesicle assay will enable screening of ligands modulating the pore formation.