Lactylation-mediated degradation of ANKS1B mitigates ischemic excitotoxicity by impairing GluN2B trafficking

Ischemic stroke triggers a cascade of metabolic shifts and excitotoxicity, partially mediated by GluN2B-containing N-methyl-d-aspartate receptors (NMDARs). While protein lactylation has emerged as a key metabolic regulator, its role in modulating neurotoxicity remains underexplored. In this study, we integrated proteomics datasets from middle cerebral artery occlusion (MCAO) mouse models and identified ANKS1B as a unique protein that is both differentially lactylated and downregulated in ischemic tissue. Using an oxygen-glucose deprivation/reperfusion (OGD/R) model in SH-SY5Y cells, we demonstrated that ischemia induces ANKS1B lactylation at the conserved K1222 site. This modification targets ANKS1B for ubiquitin-proteasome-mediated degradation. Functionally, ANKS1B regulates the endoplasmic reticulum (ER) export of GluN2B. Lactylation-driven ANKS1B loss leads to GluN2B retention in the ER. Notably, expressing a lactylation-resistant mutant (ANKS1B-K1222R) prevented this degradation, resulting in restored GluN2B surface trafficking. However, this preservation of ANKS1B proved detrimental, as it exacerbated intracellular Ca²⁺ overload and increased neuronal death following OGD/R. These results indicate that ischemia-induced ANKS1B lactylation acts as an adaptive, neuroprotective feedback mechanism to limit excitotoxicity by downregulating surface NMDARs. This study reveals a novel link between metabolic reprogramming and synaptic protein trafficking, providing new insights into stroke pathophysiology and potential therapeutic targets.