α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson's disease.

A compelling link is emerging between the posttranslational modification O-GlcNAc and protein aggregation. A prime example is α-synuclein, which forms toxic aggregates that are associated with neurodegeneration in Parkinson's and related diseases. α-Synuclein has been shown to be O-GlcNAcylated at nine different positions in in vivo proteomics experiments from mouse and human tissues. This raises the possibility that O-GlcNAc may alter the aggregation of this protein and could be both an important biological mediator of neurodegeneration and also a therapeutic target. Here, we expand upon our previous research in this area through the chemical synthesis of six site-specifically O-GlcNAcylated variants of α-synuclein. We then use a variety of biochemical experiments to show that O-GlcNAc in general inhibits the aggregation of α-synuclein but can also alter the structure of α-synuclein aggregates in site-specific ways. Additionally, an α-synuclein protein bearing three O-GlcNAc modifications can inhibit the aggregation of unmodified protein. Primary cell culture experiments also show that several of the O-GlcNAc sites inhibit the toxicity of extracellular α-synuclein fibers that are likely culprits in the spread of Parkinson's disease. We also demonstrate that O-GlcNAcylation can inhibit the aggregation of an aggressive mutant of α-synuclein, indicating that therapies currently in development that increase this modification might be applied in animal models that rely on this mutant. Finally, we also show that the pan-selective antibody for O-GlcNAc does not generally recognize this modification on α-synuclein, potentially explaining why it remains understudied. These results support further development of O-GlcNAcylation tools and therapeutic strategies in neurodegenerative diseases.