Pyruvate and oxaloacetate limit zinc-induced oxidative HT-22 neuronal cell injury

Abstract During CNS ischaemia, accumulating evidence suggests that raised intracellular Zn 2+ levels may play a significant role in inducing neuronal cell death. Several mechanisms mediating Zn 2+ -induced cell death have been suggested, however the precise molecular mechanisms remain uncertain. Employing the HT-22 murine hippocampal neuronal cell line, we have evaluated possible mechanisms of cytotoxic extracellular Zn 2+ insults. Increased extracellular Zn 2+ levels was found to induce concentration-dependent cytotoxicity. When tested at 200 μM, Zn 2+ increased intracellular Zn 2+ levels (determined via FluoZin-3 fluorescence) and rapidly induced cell death. However, neither L-type (nimodipine) nor T-type (mibefradil) voltage-activated Ca 2+ channel inhibitors limited Zn 2+ -induced cytotoxicity. Furthermore, and in contrast with staurosporine, Zn 2+ cytotoxic insults failed to induce significant caspase-3 activation and were insensitive to the poly-caspase inhibitor, zVAD-fmk. Antioxidant co-application (Trolox and N , N ′-diphenyl-1,4-phenylenediamine (DPPD)) was neuroprotective versus 6 h Zn 2+ insults. Additionally, despite inducing significant mitochondrial membrane potential loss, Zn 2+ failed to induce detectable increased superoxide production. However, both pyruvate and oxaloacetate were found to afford significant neuroprotection versus Zn 2+ cytotoxic insults, without significantly influencing intracellular Zn 2+ accumulation. We conclude that cultured HT-22 neurones are vulnerable to Zn 2+ cytotoxic insults via a non-caspase-3 mediated mechanism, which involves glycolytic inhibition.