Co‐existence of Two Different α‐Synuclein Oligomers with Different Core Structures Determined by Hydrogen/Deuterium Exchange Mass Spectrometry

Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson’s disease involves the protein α‐synuclein (αSN). Cytotoxicity is mainly associated with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild‐type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co‐existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D₂O until they dissociate into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39–A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.     

Cytotoxic Oligomers and Fibrils Trapped in a Gel‐like State of α‐Synuclein Assemblies

α‐Synuclein (α‐Syn) aggregation is associated with Parkinson's disease (PD) pathogenesis. In PD, the role of oligomers versus fibrils in neuronal cell death is debatable, but recent studies suggest oligomers are a proximate neurotoxin. Herein, we show that soluble α‐Syn monomers undergo a transformation from a solution to a gel state on incubation at high concentration. Detailed characterization of the gel showed the coexistence of monomers, oligomers, and short fibrils. In vitro, the gel was highly cytotoxic to human neuroblastoma cells. The individual constituents of the gel are short‐lived species but toxic to the cells. They comprise a structurally heterogeneous population of α‐helical and β‐sheet‐rich oligomers and short fibrils with the cross‐β motif. Given the recent evidence of the gel‐like state of the protein associated with neurodegenerative diseases, the gel state of α‐Syn in this study represents a mechanistic and structural model for the in vivo toxicity of α‐Syn in PD.     

PAMAM Dendrimers as Potential Agents against Fibrillation of α‐Synuclein,a Parkinson's Disease‐Related Protein

The effect of PAMAM dendrimers (generations G3, G4 and G5) on the fibrillation of α‐synuclein was examined by fluorescence and CD spectroscopy, TEM and SANS. PAMAM dendrimers inhibited fibrillation of α‐synuclein and this effect increased both with generation number and PAMAM concentration. SANS showed structural changes in the formed aggregates of α‐synuclein – from cylindrical to dense three‐dimensional ones – as the PAMAM concentration increased, on account of the inhibitory effect. PAMAM also effectively promoted the breaking down of pre‐existing fibrils of α‐synuclein. In both processes – that is, inhibition and disassociation of fibrils – PAMAM redirected α‐synuclein to an amorphous aggregation pathway.