Abstract DP091: Enhanced Lactylation of Methyltransferase-Like 3 (METTL3) Drives Maladaptive Autophagy and Ineffective Reperfusion in Ischemic Stroke

Objectives: Reperfusion remains the cornerstone of ischemic stroke treatment, but a significant proportion of patients experience ineffective reperfusion, where vessel recanalization fails to salvage tissue. The molecular basis of this paradox is unclear. We investigated whether lactate-driven histone and protein lactylation contribute to maladaptive autophagy via METTL3, thereby promoting ineffective reperfusion injury. Methods: Rat middle cerebral artery occlusion (MCAO,2 h vs 6 h) and SH-SY5Y oxygen–glucose deprivation/reoxygenation (OGD/R) models were used to study epigenetic-metabolic regulation of autophagy. Infarct size (TTC), neurological function (mNSS), and ultrastructure (TEM) were assessed. Autophagy flux was quantified by LC3-II/I ratio and p62. Multi-omics (RNA-seq, proteomics) and mass spectrometry identified lactylation targets. Chromatin immunoprecipitation (ChIP-qPCR) verified promoter occupancy. Interventions included lactate supplementation, lactylation inhibition (C646, oxamate), and AAV-mediated delivery of METTL3 K215R mutants. Results: Ineffective reperfusion determined by delayed reperfusion (after 6 h MCAO) led to larger infarcts (p<0.01), worse neurological deficits (p<0.05), and abnormal accumulation of autophagosomes (p<0.01). Lactate levels increased significantly (p<0.001), driving H3K18 lactylation at the Mettl3 promoter (p<0.01), which enhanced transcription. METTL3 underwent direct lactylation at K215 (p<0.001), shifted from nuclear to cytoplasmic localization (p<0.001), and increased TFEB mRNA binding and m6A modification (P<0.01). Cytoplasmic METTL3 facilitated TFEB translation, activating maladaptive autophagy (LC3-II/I, p62, p<0.05). In contrast, effective reperfusion (after shorter MCAO with 2 h) showed lower lactate, reduced H3K18la, and balanced autophagy. Functional blockade by K215R mutation or pharmacological lactylation inhibition restored nuclear METTL3, reduced LC3-II/I (P<0.01), increased p62 (p<0.05), improved neuronal viability (p<0.01), and decreased apoptosis (p<0.01). These interventions reduced infarct volume (p<0.01) and improved neurological scores (p<0.05). Conclusions: We identify a pathogenic lactate–H3K18la–METTL3-K215la–TFEB signaling axis and TFEB-driven maladaptive autophagy, as a central mechanism of ineffective reperfusion. Interventions that disrupt METTL3 lactylation or its downstream signaling restore autophagy balance and enhance the protective efficacy of reperfusion therapies.