Neuroprotective effects of novel EAAT2 allosteric modulators in excitotoxic and ischemic injury models

Excitatory amino acid transporters (EAATs) regulate extracellular glutamate levels in the central nervous system, and impairment of their function contributes to excitotoxic neuronal injury in ischemic stroke and other neurological disorders. We investigated the neuroprotective properties of novel EAAT2 allosteric modulators in primary neuron-glia cultures subjected to glutamate-induced excitotoxicity or oxygen-glucose deprivation (OGD), an in vitro model of ischemic injury. The EAAT2-selective positive allosteric modulator (PAM) NA-014 increased neuronal survival in both injury paradigms and remained effective when administered several hours after insult. In contrast, the EAAT2-selective negative allosteric modulator (NAM) NA-010 reduced neuronal survival, whereas an inactive analog had no effect. Neuroprotection by NA-014 was abolished by the EAAT2 inhibitor WAY-213613, supporting an EAAT2-dependent mechanism. Calcium imaging demonstrated that co-application of NA-014 attenuated glutamate-evoked intracellular Ca²⁺ responses, while morphological analyses indicated partial preservation of neuronal structure. In contrast to the EAAT2-selective PAM, the non-selective EAAT1/2/3 PAM did not confer neuroprotection, suggesting that transporter selectivity or differences in the mechanisms of modulation may influence neuroprotective efficacy. Together, these findings identify NA-014 as a promising neuroprotective candidate and support selective EAAT2 modulation as a potential therapeutic strategy for excitotoxic and ischemic brain injury.