α-Synuclein oligomers form by secondary nucleation

Abstract

Oligomeric species arising during aggregation of α-synuclein are proposed to be a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, their mechanism of formation and role in aggregation are largely unresolved. Here we first show that, at physiological pH, α-synuclein aggregates by secondary nucleation, rather than fragmentation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under physiologically relevant conditions.

Molecular mechanism of α-synuclein aggregation on lipid membranes revealed

“…This tells us that in this lipid-induced assay, secondary nucleation or fragmentation, if present at all, only has a relatively minor effect on the overall aggregation kinetics. This finding is in line with previous work showing that a different s et of conditions w as generally required to enable significant secondary nucleation[20, 22, 24].…”

“…The above considerations highlight the fibril as the most suitable target for small molecules to inhibit this process and maximise chances of translatability. By contrast, given the negligible effect on the kinetics in this particular in vitro assay of any secondary processes that may be present, their inhibition will have little effect on the aggregation speed, and instead different conditions should be used to investigate this process[20, 22, 24].…”

“…Moreover, the air water interface or plate interfaces are found to be sites for primary nucleation across different proteins and experimental setups[23, 51], so our finding of interface-catalysed nucleation may be equally general. Finally, secondary nucleation appears to be only dominant under specific conditions in the aggregation of pure α -synuclein[20, 22, 24], so it would not be surprising if it is found to also be insignificant with lipids other than DMPS.…”

“…At neutral pH, purified α -synuclein is surprisingly resistant to aggregation, generally requiring vigorous agitation or the introduction of preformed seed fibrils to trigger the aggregation process[20, 22]. This resistance of α -synuclein to aggregation is likely due to the low rate of primary nucleation.…”

Toward a molecular mechanism for the interaction of ATP with alpha-synuclein

“…[21][22][23][24] ATP inhibits the N-terminally-driven interaction between aS monomers and brils Since secondary nucleation is a major driver of aS aggregation and is electrostatically-driven by the N-terminal residues of aS monomers binding to brils, we hypothesized that the inhibition of late-stage aS bril formation by ATP may involve the suppression of bril elongation and/or secondary nucleation due to the N-terminal binding of aS monomers by ATP. 9,26,[34][35][36][37] To test this hypothesis, we measured residue-specic transverse 15 N aS R 2 amide relaxation rates in the presence of late-stage WT aS amyloid brils in both the absence and presence of ATP (Fig. 6a).…”