Background: Ischemic stroke leads to severe cerebral ischemia/reperfusion (I/R) injury, resulting in neuronal death and neurological deficits. The N 6 -methyladenosine (m 6 A) methyltransferase METTL16 has emerged as a key regulator of RNA metabolism, but its specific role and mechanism in ischemic stroke remain unclear. Methods: A transient middle cerebral artery occlusion (MCAO) model was established in adult male C57BL/6J mice. METTL16 was knocked down via intracerebroventricular injection of shRNA-expressing lentivirus at 6 hours post-reperfusion. Cerebral infarct volume (TTC staining), neurological function (mNSS, adhesive removal, corner-turning, and rotarod tests), histopathology (H&E, TUNEL, Nissl, NeuN), and apoptosis-related protein expression were evaluated. Primary cortical neurons and astrocytes were subjected to oxygen-glucose deprivation (OGD). Cell apoptosis, LDH release, inflammatory cytokines (TNF-α, IL-1β), and synaptic protein (Synapsin-1, PSD-95) expression were assessed. The METTL16-TIPARP interaction and m 6 A modification were analyzed by RIP and m 6 A-RIP-qPCR. Protein stability was determined using cycloheximide chase assay. Rescue experiments were performed by overexpressing TIPARP in METTL16-depleted cells. Results: METTL16 knockdown significantly reduced infarct volume, attenuated neuronal apoptosis and inflammation, and improved sensorimotor and coordination functions after MCAO. In vitro, METTL16 depletion protected neurons and astrocytes from OGD-induced apoptosis, cytokine release, and synaptic damage. Mechanistically, METTL16 directly bound to TIPARP mRNA and enhanced its m 6 A modification, thereby stabilizing TIPARP transcripts and upregulating TIPARP protein expression. Critically, TIPARP overexpression completely abolished the protective effects of METTL16 knockdown against OGD-induced apoptosis and synaptic impairment. Conclusion: METTL16 promotes cerebral I/R injury by stabilizing TIPARP mRNA via m 6 A methylation, leading to elevated TIPARP expression. The METTL16-m 6 A-TIPARP axis represents a novel pathogenic mechanism and a potential therapeutic target for ischemic stroke. Keywords: stroke, neurons, astrocytes, METTL16, RNA methylation, TIPARP
Xiang et al. (Wed,) studied this question.