Inhibition of Mettl3-mediated m6A RNA modification of HMGCS1 protects retinal ganglion cells from glutamate excitotoxicity-induced ferroptosis in a rat model of glaucoma.

BACKGROUND: Glaucoma, a leading cause of irreversible blindness worldwide, is characterized by progressive retinal ganglion cells (RGCs) degeneration. The accumulation of glutamate in the retina is a common mechanism underlying the of RGCs death in various forms of glaucoma. Neuroprotective strategies for RGCs remain an unmet need, urging exploration of novel molecular mechanisms beyond intraocular pressure (IOP) control. MATERIALS AND METHODS: Retinal m6A methylation sequencing, RT-qPCR, and dot blot techniques were performed to identify m6A methylation levels change in an N-methyl-D-aspartate (NMDA)-induced glaucoma model of rats and find potential targets. The effect of methyltransferase-like 3 (Mettl3) inhibition on ferroptosis of R28 and RGCs glutamate excitotoxicity model was detected by using Mettl3 inhibitor STM2457, Cell Counting Kit-8 (CCK-8), FerroOrange, Liperfluo, malondialdehyde (MDA) assay, glutathione (GSH) assay, immunofluorescence staining, Hematoxylin-Eosin (H&E) staining and flash visual evoked potential (f-VEP) were used to study the ferroptosis in R28 and RGCs. Using si-RNA and oe-RNA to knockdown YTHN6-methyladenosine RNA binding protein 2 (YTHDF2) or Hydroxyl-3-methylglutaryl-Coenzyme A synthase 1 (HMGCS1) in the R28. Lentiviral vectors were applied to overexpress the HMGCS1 in rat retina. MeRIP-qPCR and Western Blot to study the mechanism of how Mettl3 regulates m6A methylation and expression of downstream targets. RESULTS: Our findings demonstrate that NMDA-induced excitotoxicity significantly elevated retinal m6A methylation levels. HMGCS1 m6A methylation was significantly increased while its expression significantly decreased in the NMDA group. In R28 cells, inhibition of Mettl3 significantly alleviated glutamate excitotoxicity-induced R28 and RGCs ferroptosis and restored the visual function of rats. Knockdown HMGCS1 significantly reduced the protective effect of Mettl3 inhibition on the R28 cells and overexpress HMGCS1 protected R28 cell and RGCs from NMDA-induced glutamate excitotoxicity. YTHDF2 reverses this protective effect by recognizing and degrading m6A-modified HMGCS1 mRNA, thereby promoting ferroptosis. CONCLUSION: Our study investigates the inhibition of Mettl3-mediated m6A RNA modification of HMGCS1 as a critical regulator of RGC ferroptosis in glaucoma, providing a potential therapeutic target for glaucoma.