Modulation of α-Synuclein Aggregation Process and Fibril Stability by Co-solvents

Amyloid fibrils are involved in several amyloid-related pathologies such as Parkinson's disease, Alzheimer's disease and type II diabetes. As a result, scientific community is nowadays addressing considerable efforts towards the comprehension of fibrillation mechanisms, particularly focusing on how they are affected by environmental conditions, small molecules and/or membrane presence. In this scenario, alpha-synuclein (aSN), a small protein involved in Parkinson's disease, represents a challenging model system for studying aggregation phenomena, and understanding the pathogenesis at molecular level. Indeed, it is poorly understood how fibril formation is linked to the progressive neurodegeneration. Here we report an in vitro experimental study on the wild type aSN aggregation process in physiological conditions and in the presence of Trifluoroethanol (TFE). TFE is known to induce structural changes in protein structures, and it is often used in aqueous solution to mimic extracellular or cytosolic environment. Aggregation kinetics were monitored by means of Rayleigh Scattering and Thioflavin-T fluorescence. Moreover, Circular Dichroism (CD) and two photon excitation fluorescence microscopy were used to characterize secondary structure and morphology of the aggregates. Our data clearly show that, both the initial aSN conformation and aggregation process are strongly affected by TFE. Disaggregation is clearly observed if TFE concentration is decreased by dilution procedure. Different environments critically alter aggregate structure, size and morphology as revealed by CD and fluorescence microscopy experiments. From a broader perspective, our results strongly suggest that experimental factors, mimicking a cellular environment, are important to take into account in in vitro studies of amyloid aggregation.