OGT regulates RNA-binding protein nucleocytoplasmic partitioning, but this fails in amyotrophic lateral sclerosis

O-linked N-acetylglucosamine transferase (OGT) catalyzes the O-linked β-N-acetylglucosamine (O-GlcNAc) modification to proteins and has been implicated in a range of diseases, including amyotrophic lateral sclerosis (ALS), a rapidly progressive and currently incurable neurodegenerative disorder. However, the precise mechanisms by which OGT influences ALS pathogenesis remain largely unresolved. In this study, we used human-induced pluripotent stem cell (hiPSC)-derived spinal motor neurons (hiPSC-MNs), the principal cell type that undergoes degeneration in ALS, to systematically reveal that OGT-related mechanisms regulate RNA-binding protein (RBP) nucleocytoplasmic compartmentalization. Using a combination of antisense oligonucleotide (ASO) technology and small molecular pharmacology, we reveal two distinct regulatory mechanisms of RBP compartmentalization, including by intron-retaining transcript-mediated sequestration and O-linked N-acetylglucosaminylation (O-GlcNAcylation)-mediated nuclear transport. We finally examine the tractability of manipulating OGT activity in the ALS context but demonstrate that the OGT-related mechanisms governing RBP localization in healthy hiPSC-MNs are impaired in the context of ALS-causing VCP and TARDBP mutations.