Identifying Oligomers and Lipid Vesicles Effects during α-Synuclein Fibril Formation through a Solid-State Nanopore

Abstract

α-Synuclein (α-Syn) is a small intrinsically disordered 140 amino acids (14 kDa) protein expressed abundantly in human brain, regulating the release of neurotransmitters, vesicle recycling, and synaptic integrity. Adequate α-Syn monomers in solution undergo a nucleation-dependent mechanism growing into oligomers, which are able to elongate to form profibers through monomers addition and finally mature fibers. It is well acknowledged that the abnormal aggregation process of α-Syn monomers is associated with pathophysiology of Parkinson's disease. Identifying different oligomer types and tracking their aggregation dynamic process in lipid free or lipid co-incubated system, which not only present the significant step during fibrillation but also play a key role in pathogenesis of Parkinson's disease, is hard to know due to the inherent challenges of studying heterogeneous and transient system by using traditional biotechnology. In this study, we use solid-state nanopores to study the aggregation progress of α-Syn only sample and lipid-small unilamellar vesicles co-incubated α-Syn sample in a single-molecule approach. A tween 20 surface coating method was developed to enable continuous and smooth α-Syn translocation through SiN nanopores. Through analysis of translocation events for α-Syn incubated at different time, we identified four types of oligomers forming during aggregation process. These four types oligomers inferred as intermediates show a time-dependent quantity fraction change, suggesting that the formation of large oligomers is based on the consumption of small oligomers. Furthermore, we investigated the effect of lipid small unilamellar vesicles on α-Syn aggregation process. The experimental results indicate that it is the presence of 20% 1,2-dioleoyl-sn- glycero-3- [phospho-L-serine] (DOPS) dramatically enhances the aggregation rate of α-Syn, while the aggregation pathway is similar to α-Syn only samples.