O -GlcNAcylation effect on Tau in modulating its seeding and cellular transmission in Alzheimer’s Disease

Post-translational modifications critically regulate neurodegenerative disease progression. In Alzheimer’s disease (AD) and tauopathies, Tau hyperphosphorylation promotes aggregation and pathological spreading, whereas O- GlcNAcylation has emerged as a protective modification alongside its interplay with phosphorylation. However, the role of in vitro site-specific and global O - G lcNAcylation in Tau proteinopathy remains elusive. Here, using full-length Tau-441 (2N4R), we showed that O -GlcNAcylated Tau aggregated slowly, formed distinct aggregate morphology and exhibited reduced seeding capacity compared to wild-type (WT) Tau. Under phase-separated conditions, O -GlcNAcylated Tau formed oligomer like condensates. Mutation of the key O -GlcNAc sites reduced O -GlcNAc-transferase (OGT) mediated O -GlcNAcylation, cellular transmission and affected cross-talk with phosphorylation relative to WT. OGT overexpression alleviated WT-Tau toxicity in cells, and O-Tau fibrils were less toxic to primary cortical neurons compared to WT Tau fibrils. Finally, an in-house novel site-specific S422 O -GlcNAc-Tau antibody revealed reduced S422 O -GlcNAcylation in AD brain tissues, highlighting its protective role in AD pathogenesis.