m6A Methylation‐Induced Autophagy Impairment by TFEB Regulation in SOD1 ‐ G93A ALS Cell Model

ABSTRACT We investigate the role of m6A RNA methylation in regulating transcription factor EB (TFEB) and its contribution to mitochondrial autophagy (mitophagy) dysfunction in amyotrophic lateral sclerosis (ALS). ALS cell models were used to analyse mitophagy markers and TFEB expression under METTL3 and TFEB modulation, using RT‐qPCR, Western blot, MeRIP, RIP, and immunofluorescence. Elevated m6A methylation and reduced TFEB expression were observed in hSOD1‐G93A models. METTL3 overexpression suppressed TFEB expression, leading to impaired mitophagy, while METTL3 knockdown alleviated these effects. MeRIP assays confirmed increased m6A modifications on TFEB mRNA, and RIP assays demonstrated direct interaction between METTL3 and TFEB mRNA. Notably, TFEB overexpression rescued mitophagy dysfunction, whereas TFEB knockdown exacerbated the impairment. METTL3‐mediated m6A methylation inhibits mitophagy by downregulating TFEB expression, revealing the m6A‐TFEB pathway as a promising therapeutic target for ALS.