The Effect of Small Molecules on Early and Late Events of Alpha Synuclein Aggregation in Solution and Inside Living Cells

Abstract

Parkinson disease (PD) is the most common motor-neurodegenerative disease, affecting 1-2% of the population older than 65 years and 4-5% of those aged over 85 years. PD is a movement disorder, associated with different motor and nonmotor syndromes, arising from degeneration of dopaminergic neurons of substantia nigra. The hallmark of PD is the observation of intracellular protein aggregates, Lewy Bodies (LB). Alpha-synuclein is one of the major components of LB. The heterogeneity of aggregation process and the presence of large number of triggering mechanisms results in the difficulty to devise therapeutic strategies against these toxic inclusions formation. There have been continuing efforts to search for small molecules against alpha synuclein aggregation.With the help of fluorescence correlation spectroscopy (FCS), confocal imaging, FRAP and other biophysical experiments we have studied the early and late events of alpha-synuclein aggregation. Subsequently, we have explored the possibilities of identifying possible small molecules, which would prevent early oligomer formation. For the FCS experiments, we have labeled a single cysteine mutant of alpha synuclein (G132C) using Alexa488Maleimide. Our FCS data have shown significant changes in the conformation of the protein in the presence of an inhibiting small molecule. To obtain additional insights into these molecules, we have studied their effects inside two mammalian model cell lines, namely HeLa and SH-SY5Y. For this purpose, we have transiently transfected the cells using a tetra-cysteine tagged alpha-synuclein, following which the cells have been stained using Flash-EDT2 dye for fluorescence imaging and FRAP studies. The results show that the electrostatic differences between the protein and the small molecules contribute significantly towards the ability of the small molecules to modulate the early and late stages of aggregation.