Mechanisms of enhanced aggregation and fibril formation of Parkinson's disease-related variants of α-synuclein.

Aggregation of α-synuclein (α-syn) into amyloid fibrils is closely associated with Parkinson's disease (PD). Familial mutations or posttranslational truncations in α-syn are known as risk factor for PD. Here, we examined the effects of the PD-related A30P or A53T point mutation and C-terminal 123-140 or 104-140 truncation on the aggregating property of α-syn based on the kinetic and thermodynamic analyses. Thioflavin T fluorescence measurements indicated that A53T, Δ123‒140, and Δ104-140 variants aggregated faster than WT α-syn, in which the A53T mutation markedly increases nucleation rate whereas the Δ123‒140 or Δ104‒140 truncation significantly increases both nucleation and fibril elongation rates. Ultracentrifugation and western blotting analyses demonstrated that these mutations or truncations promote the conversion of monomer to aggregated forms of α-syn. Analysis of the dependence of aggregation reaction of α-syn variants on the monomer concentration suggested that the A53T mutation enhances conversion of monomers to amyloid nuclei whereas the C-terminal truncations, especially the Δ104-140, enhance autocatalytic aggregation on existing fibrils. In addition, thermodynamic analysis of the kinetics of nucleation and fibril elongation of α-syn variants indicated that both nucleation and fibril elongation of WT α-syn are enthalpically and entropically unfavorable. Interestingly, the unfavorable activation enthalpy of nucleation greatly decreases for the A53T and becomes reversed in sign for the C-terminally truncated variants. Taken together, our results indicate that the A53T mutation and the C-terminal truncation enhance α-syn aggregation by reducing unfavorable activation enthalpy of nucleation, and the C-terminal truncation further triggers the autocatalytic fibril elongation on the fibril surfaces.