Negatively charged α-synuclein condensate modulates partitioning of molecules.

α-Synuclein (αSyn) aggregation via liquid-liquid phase separation (LLPS) has recently emerged as a crucial mechanism underlying amyloid fibril formation implicated in Parkinson's disease. However, comprehensive investigation of the physicochemical properties of αSyn condensate remains incomplete. Here, we demonstrate that αSyn condensates exhibit a highly negative electrostatic potential, revealed by preferential enrichment of positively charged fluorophores or fluorophore-labeled αSyn. We further demonstrate that αSyn LLPS is regulated by a balance between self-association and electrostatic repulsion, with excess negative charge inhibiting phase separation. To study αSyn LLPS in a cellular context, we generated a stably transfected SH-SY5Y cell line that forms gel-like, nontoxic αSyn condensates upon differentiation. Most importantly, these intracellular condensates maintain a negative electrostatic potential, as evidenced by mEGFP probes systematically engineered with increasing positive charge. Our findings underscore the central role of electrostatic potential in modulating molecular partitioning within αSyn condensates and suggest that similar electrostatic principles may apply to other biomolecular condensates.