Secondary Processes Dominate the Quiescent, Spontaneous Aggregation of α-Synuclein at Physiological pH with Sodium Salts

Abstract

The accurate recapitulation in an in vitro assay of the aggregation process of α-synuclein in Parkinson's disease has been a significant challenge. As α-synuclein does not aggregate spontaneously in most currently used in vitro assays, primary nucleation is triggered by the presence of surfaces such as lipid membranes or interfaces created by shaking, to achieve aggregation on accessible time scales. In addition, secondary nucleation is typically only observed by lowering the pH below 5.8. Here we investigated assay conditions that enables spontaneous primary nucleation and secondary nucleation at pH 7.4. Using 400 mM sodium phosphate, we observed quiescent spontaneous aggregation of α-synuclein and established that this aggregation is dominated by secondary processes. Furthermore, the presence of potassium ions enhanced the reproducibility of quiescent αsynuclein aggregation. This work provides a framework for the study of spontaneous α-synuclein aggregation at physiological pH.

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.…”

“…Introduction of preformed aggregates increases the speed of aggregation considerably, further supporting the idea that a slow primary nucleation step is the main reason for the aggregation resistance of α -synuclein[20]. Lowering the pH [20, 21] or using high salt concentrations [24] are other ways to induce aggregation, likely by reducing electrostatic repulsion between the aggregating proteins[25, 26].…”

Molecular mechanism of α-synuclein aggregation on lipid membranes revealed

“…23 Introduction of preformed aggregates increases the speed of aggregation considerably, further supporting the idea that a slow primary nucleation step is the main reason for the aggregation resistance of α-synuclein. 20 Lowering the pH 20,21 or using high salt concentrations 24 are other ways to induce aggregation, likely by reducing electrostatic repulsion between the aggregating proteins. 25,26…”