In vitro Phase Separation of α-Synuclein: A Model System to Investigate Molecular Pathology of Parkinson’s Disease

Abstract

Objectives Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterised by the presence of Lewy bodies in the post-mortem brain histology. Lewy bodies are mostly made up of insoluble aggregated α-synuclein protein, which has been recently suggested to function through phase separation. Phase separation, while contributing to function, also increases the probability of aggregate formation, as it is one of the key cellular mechanisms to increase the local concentration of biomolecules. The conversion of soluble α-synuclein to insoluble aggregate is not well understood, due to a lack of good model systems with physiologically relevant conditions. In this study, we develop a model system based on PEG-dextran to study phase separation of α-synuclein in the presence or absence of other biomolecules like ATP, RNA, and DNA at physiological salt and buffer conditions. Material and Methods Phase separation reactions were optimised by screening a variety of polymeric crowding agents. Recombinant α-synuclein-GFP protein was purified and phase separated under physiological conditions in vitro . The effect of biomolecules like ATP, RNA, and ssDNA was investigated using epifluorescence microscopy. Microscopy images were quantified using ImageJ. Results 4% PEG and 10% dextran showed optimal phase separation at pH 7.4 at physiological conditions within 5 min of reaction set-up. In these conditions, α-synuclein phase separated between 10-70 µM concentration that is similar to neuronal concentration of the protein. RNA increased the phase separation of α-synuclein in a broad range of concentration through likely a sequence-specific electrostatic interaction, while ssDNA did not show a remarkable change in phase separation. Conclusion α-Synuclein can phase separate at concentrations of 10-70 µM at physiological salinity and pH; these conditions are closer to physiological conditions than some of the previous reports. Both ATP and RNA modulate the phase separation of α-synuclein, ATP within a very narrow range (peaking at 60 mM), promotes phase separation of α-synuclein, while RNA increases the extent of phase separation over a broad range. ssDNA does not influence the phase separation of α-synuclein.