The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson’s disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.

It is well-established that ⍺-synuclein aggregation may proceed through an initial lipid-dependent aggregate formation and, if at acidic pH, a subsequent aggregate-dependent proliferation. It has also been recently reported that the aggregation of ⍺-synuclein may also take place through an alternative pathway, which takes place within dense liquid condensates produced through liquid-liquid phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we developed a fluorescence-based assay to perform a kinetic analysis of the aggregation process of ⍺-synuclein within liquid condensates, and applied it to determine the corresponding mechanism of aggregation. Our analysis shows that at pH 7.4 the aggregation process of ⍺-synuclein within dense condensates starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Taken together, these results reveal a highly efficient pathway for the appearance and proliferation of ⍺-synuclein aggregates at physiological pH.