DISSECTING THE INTERACTION BETWEEN THE ABERRANT PHASE SEPARATION OF ALPHA-SYNUCLEIN AND ORGANELLE MISLOCALIZATION

Lewy bodies (LBs), the pathological hallmarks of Parkinson’s disease, exhibit a distinctive core-shell architecture in which α-synuclein forms a dense shell surrounding a core enriched in proteins. Recent ultrastructural analyses show that LBs accumulate membrane-bound organelles. Despite extensive research, the mechanisms driving LB formation remain poorly understood. Here, we aim to uncover the cellular and molecular processes underlying LB biogenesis by reconstituting LBs in a minimal cellular model. To this end, we co-expressed α-synuclein and synphilin-1 to induce condensate formation via phase separation and used live-cell confocal imaging combined with immunofluorescence to monitor condensate assembly, maturation, and interactions with membrane-bound organelles. Visualizing mitochondria, lysosomes, and other organelles revealed their enrichment at the periphery of these inclusions, indicating that membrane-bound organelles are actively engaged during the maturation process. Ultrastructural analyses using X-ray tomography indicate that mitochondria are fragmented in the vicinity of aberrant α-synuclein inclusions. Overall, this minimal system provides a platform to explore how aberrant phase separation and organelle interactions of α-synuclein contribute to the pathogenesis of Parkinson’s disease. In the future, this system could be adapted for high-throughput compound screening to identify therapeutic candidates that modulate α-synuclein phase behavior and inclusion maturation in Parkinson’s disease.