NSUN4-mediated m5C RNA methylation protects retinal cells against excitotoxic injury via the SHH signaling pathway

Glaucoma, a leading global cause of blindness, is characterized by progressive retinal neuronal loss. NOP2/Sun RNA methyltransferase 4 (NSUN4), a writer of 5-methylcytosine (m5C) RNA modifications, has established roles in methylation and mitoribosome assembly, yet its function in retinal cell survival remains unexplored. In this study, integrated methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq analysis in an NMDA-induced retinal injury model revealed widespread mRNA hypomethylation enriched in the Sonic Hedgehog (SHH) signaling pathway, accompanied by significant downregulation of Nsun4. To investigate the underlying mechanisms, we utilized the R28 retinal cell line, a widely accepted model for studying retinal neuroprotection. In glutamate-stimulated R28 cells, NSUN4 overexpression mitigated excitotoxic injury, attenuating Ca² ⁺ overload, mitochondrial dysfunction, and apoptosis. Mechanistically, NSUN4 enhanced m5C methylation on key SHH pathway transcripts (Shh, Gli1, and Gli2). Crucially, the neuroprotective effect of NSUN4 was abolished upon pharmacological inhibition of the SHH pathway using Vismodegib, confirming that pathway activation is essential for NSUN4-mediated protection. Clinically, NSUN4 levels were significantly reduced in the aqueous humor of patients with primary open-angle glaucoma compared to controls. Together, these findings establish NSUN4 as an m5C-dependent activator of the SHH pathway that protects retinal cells against excitotoxic injury, nominating it as a novel candidate for glaucoma neuroprotection.