A30P and A30G Mutations in α-Synuclein Promote Metastable Long-Lived Aggregates with Distinct Structural Responses to...

Synucleinopathies are neurodegenerative disorders marked by the accumulation of misfolded α-synuclein and its familial mutants in brain cells. The mechanistic understanding of how α-synuclein mutations exacerbate disease remains unsolved. Here, the in vitro aggregation kinetics of α-synuclein and its mutants revealed that A30P and a recently discovered A30G variant displayed aggregation kinetics uncharacteristically slower than those of wild-type (WT) and other mutants. We delineated the amyloidogenesis pathway of these variants by characterizing different intermediates through time-dependent CD and AFM analysis. AFM-Raman spectroscopy and proteinase-K digestion further distinguished structural features of these species. We infer that WT and the A30 variants aggregate through a common pathway, albeit with variant-specific rates, yielding kinetically metastable aggregates with structural differences in the order A30P < A30G < WT. These metastable aggregates underwent further rearrangements to form stable fibrils upon vigorous agitation, incubation with osmolytes, or seeding with fibrillar seeds. Addition of the amyloid-modifying compound EGCG to the metastable and the final fibrillar states converted them to β-sheet-rich small fibrillar and prefibrillar structures, respectively. However, EGCG directed monomers toward amorphous aggregation. We present a viewpoint that A30 mutations that are located outside the canonical amyloid core of α-synuclein could augment its toxic gain of function by producing kinetically long-lived prefibrillar species rather than by promoting thermodynamically alternative conformations. These kinetically metastable conformers of varying hierarchy were differentially sensitive to EGCG. Our findings provide significant insights into how α-synuclein mutations contribute to synucleinopathies and how amyloid modulators differentially act on intermediates versus mature fibrils.