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

Abstract

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.

α-Synuclein oligomers form by secondary nucleation

“…The presence of secondary processes at neutral pH was hinted at by data from Buell et al, and our detailed investigation we has now confirmed that the process is secondary nucleation and not fragmentation [21]. Moreover, several additional studies have reported seed-concentration dependent aggregation kinetics at neutral pH, suggesting that secondary nucleation is a general feature of α-synuclein aggregation [35,36,37,34,38].…”

Optimization of microfluidic synthesis of silver nanoparticles: A generic approach using machine learning

“…The proposed strategy, which used optimal and flexible experiment designs based on uncertainty analysis, can provide more flexibility compared to existing approaches such as design of experiments [33], since the exact values of the initial parameters are not fixed and the training features could be parameters which have been calculated based on physical parameters, such as nucleation and growth constants. The nucleation and growth constants were derived from an independent set of experiments carried out in a beaker using the Finke-Watzky (F-W) two-step mechanism [34] which has been applied previously to describe well a wide range of kinetic processes such as protein aggregation [35], [36] and metal nanoparticle formation including silver [37], gold [38], palladium [39] and rhodium [40]. Only one set of chemicals was used in this study providing several pairs of nucleation and growth constants.…”

Liquid-liquid Phase Separation of Alpha-synuclein Increases the Structural Variability of Fibrils Formed during Amyloid Aggregation

“…To solve this problem, two different approaches were applied. First, we induced amorphous aggregate disassembly while retaining the α-syn fibrils using sample denaturation by low concentrations of guanidinium hydrochloride (GuHCl) 52 . The LLPS and non-LLPS (positive control) condition aggregates were resuspended into a range of GuHCl concentrations (Figure 2A), and the effect of denaturation was monitored by scanning sample optical densities (OD 600 ).…”