EAAT2 dysfunction mediates acrylamide-induced excitotoxicity and neuronal damage in a SH-SY5Y/U251 co-culture model.

Acrylamide (ACR) is a pervasive environmental and workplace contaminant with established neurotoxic effects but unclear pathogenic mechanisms. In this study, we screened for potential ACR binding targets associated with neurotoxicity and identified the astrocytic glutamate transporter EAAT2. Molecular docking and dynamics simulations revealed that ACR interacts stably with the glutamate-binding pocket of EAAT2, potentially impairing transport function. After exposing SH-SY5Y human neuroblastoma cells to ACR (0-500 μg/mL) for 1, 3, or 5 days, a significant decrease in EAAT2 expression was indeed observed. Concurrently, it induced significant time- and dose-dependent reductions in viable cell numbers, increases in Tau phosphorylation (AT8, pS396, pS262), and the accumulation of insoluble Tau oligomers, as well as the downregulation of neurotrophic signaling factors BDNF and TrkB. Moreover, in transwell co-cultures of mature SY5Y cells and U251 astrocytes, ACR administration (111 μg/mL, 72 h) resulted in reactive transformation of astrocytes, extracellular glutamate accumulation and enhanced neuronal calcium influx via extrasynaptic NMDA receptors. This resulted in downstream neurotoxic responses including BDNF/TrkB suppression, caspase-3 activation, Tau hyperphosphorylation and secondary neuronal injury. Astrocytic overexpression of SLC1A2 (EAAT2) significantly reversed all of these pathogenic responses. Taken together, these findings suggest that ACR induces neuronal excitotoxicity by interfering with astrocytic EAAT2-mediated regulation of extracellular glutamate, leading to extrasynaptic NMDAR overactivation, intracellular calcium overload, and Tau-related neurodegeneration. The EAAT2 is a potential therapeutic target for mitigating ACR-induced neurotoxicity and associated sequelae such as cognitive impairment.