Investigating the impact of Cu2+ on α-synuclein aggregation: A single-molecule approach

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by the abnormal aggregation of α-synuclein. This study investigated the impact of Cu²⁺ ions on α-synuclein aggregation in oligomer distribution using two single-molecule techniques. The aggregation of α-synuclein monomers with and without Cu²⁺ revealed that Cu²⁺ accelerated the formation of ThT-positive β-sheet structured aggregates. Nanopipettes of varying diameters from to 7 to 134 nm were employed to characterize the oligomers formed during the lag phase, demonstrating that Cu²⁺ generated a wider range of oligomers from 10 nm³ to 20,000 nm³ over time. Confocal fluorescence spectroscopy analysis of ThT-positive fibrils in the plateau phase showed that Cu²⁺ induces larger oligomers and fewer in number. The introduction of preformed seeds to the control and Cu²⁺-containing samples further accelerated the aggregation. The combination of seeds and Cu²⁺ produced structurally distinct oligomers, with seeds catalyzing the formation of small oligomers that detach from the parent fibers and Cu²⁺, promoting the formation of larger oligomers. These results reveals that seeds and Cu²⁺ act synergistically, with two different aggregation pathways coexisting in the early phase, leading to an intermediate composition of fibers and clusters at the end of aggregation.