This study investigates the impact of O-GlcNAc modification on the amyloid formation and pathology of α-synuclein, a protein implicated in Parkinson's disease. The researchers found that O-GlcNAc modification at serine 87 (S87) of α-synuclein results in the formation of amyloid fibrils with a distinct core structure, as revealed by cryogenic electron microscopy. These modified fibrils exhibit diminished seeding activity in neuronal and rodent models of Parkinson's disease. Mechanistically, heat shock proteins interact with these O-GlcNAc-modified fibrils, inhibiting their seeding activity. The study highlights that post-translational modifications, such as O-GlcNAc, play a crucial role in determining the amyloid strain and pathogenicity of α-synuclein. The findings suggest that O-GlcNAc may alter the interactome of α-synuclein fibrils, leading to reduced seeding activity in vivo. This work provides new insights into the mechanisms underlying the formation of different amyloid strains and their impact on neurodegenerative diseases.This study investigates the impact of O-GlcNAc modification on the amyloid formation and pathology of α-synuclein, a protein implicated in Parkinson's disease. The researchers found that O-GlcNAc modification at serine 87 (S87) of α-synuclein results in the formation of amyloid fibrils with a distinct core structure, as revealed by cryogenic electron microscopy. These modified fibrils exhibit diminished seeding activity in neuronal and rodent models of Parkinson's disease. Mechanistically, heat shock proteins interact with these O-GlcNAc-modified fibrils, inhibiting their seeding activity. The study highlights that post-translational modifications, such as O-GlcNAc, play a crucial role in determining the amyloid strain and pathogenicity of α-synuclein. The findings suggest that O-GlcNAc may alter the interactome of α-synuclein fibrils, leading to reduced seeding activity in vivo. This work provides new insights into the mechanisms underlying the formation of different amyloid strains and their impact on neurodegenerative diseases.