Mettl3-Mediated N6-Methyladenosine Modification Mitigates Ganglion Cell Loss and Retinal Dysfunction in Retinal Ischemia–Reperfusion Injury by Inhibiting FoxO1-Mediated Autophagy

Purpose: N6-methyladenosine (m6A) modification has been implicated in ischemia–reperfusion injury in various systems and in several neurodegenerative diseases. Glaucoma is characterized by degeneration of retinal ganglion cells (RGCs) and shares similar pathologic injury characteristics with retinal ischemia–reperfusion (RIR) injury. However, the specific role of m6A modification in RIR injury is unclear, and the involvement of autophagy in RIR injury also remains controversial. Therefore, our study explored the role of m6A modification and autophagy in RIR injury. Methods: Male wild-type C57BL/6J mice (6–8 weeks old) were used to induce RIR injury. Retinal flat-mount immunofluorescence was performed to assess RGC survival rate. Electroretinogram and optomotor response were conducted to evaluate the retinal electrophysiologic function and visual acuity. Autophagy level was reflected by Western blot and transmission electron microscope images. M6A modification levels were determined via m6A dot blot. Methyltransferase-like protein 3 (Mettl3) and forkhead box O1 (FoxO1) protein expressions were tested by Western blot. Methylated RNA immunoprecipitation–quantitative PCR was conducted to examine m6A modification level on FoxO1 mRNA. We also employed 3-methyladenine and rapamycin to regulate autophagy level in RIR injury. Results: Inhibiting autophagy ameliorated RGC loss and preserved retinal electrophysiologic function in RIR injury. Additionally, a decrease in Mettl3-mediated m6A modification was observed in RIR injury mice. By overexpressing Mettl3 via intravitreal injection of type 2 recombinant adeno-associated virus before RIR injury, we established that Mettl3 overexpression can also ameliorate RGC loss and retinal electrophysiologic dysfunction induced by RIR injury. Furthermore, Mettl3 overexpression inhibited autophagy and reduced FoxO1 expression by upregulating m6A modifications on FoxO1 mRNA. Conclusions: Mettl3-mediated m6A modification mitigates RGC loss and retinal electrophysiologic dysfunction by inhibiting FoxO1-mediated autophagy in RIR injury.