Modulating alpha-Synuclein Liquid-Liquid Phase Separation

Liquid-liquid phase separation (LLPS) is a crucial phenomenon for the formation of functional membraneless organelles. However, LLPS is also responsible for protein aggregation in various neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease (PD). Recently, several reports, including ours, have shown that alpha-synuclein (alpha-Syn) undergoes LLPS and a subsequent liquid-to-solid phase transition, which leads to amyloid fibril formation. However, how the environmental (and experimental) parameters modulate the alpha-Syn LLPS remains elusive. Here, we show that in vitro alpha-Syn LLPS is strongly dependent on the presence of salts, which allows charge neutralization at both terminal segments of protein and therefore promotes hydrophobic interactions supportive for LLPS. Using various purification methods and experimental conditions, we showed, depending upon conditions, alpha-Syn undergoes either spontaneous (instantaneous) or delayed LLPS. Furthermore, we delineate that the kinetics of liquid droplet formation (i.e., the critical concentration and critical time) is relative and can be modulated by the salt/counterion concentration, pH, presence of surface, PD-associated multivalent cations, and N-terminal acetylation, which are all known to regulate alpha-Syn aggregation in vitro. Together, our observations suggest that alpha-Syn LLPS and subsequent liquid-to-solid phase transition could be pathological, which can be triggered only under disease-associated conditions (high critical concentration and/or conditions promoting alpha-Syn self-assembly). This study will significantly improve our understanding of the molecular mechanisms of alpha-Syn LLPS and the liquid-to-solid transition.