Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death

Abstract

Elevated levels of extracellular glutamate are neurotoxic. The cytotoxic property of extracellular glutamate is known to mediate two primary mechanisms, excitotoxicity and excitotoxicity-independent processes. The excitotoxicity-independent pathway was investigated in the current study in a mouse hippocampal-derived HT4 cell line. Exposure of HT4 cells to glutamate for 12 h induced loss of cell viability preceded by rapid loss of intracellular reduced glutathione followed by accumulation of intracellular reactive oxygen species, elevation of intracellular Ca 2+, progressive loss of mitochondrial membrane potential swelling and loss of mitochondrial outer membrane integrity. Glutamate-induced loss of DNA integrity has been detected. The antioxidants α-tocopherol and trolox, mitochondrial calcium uniporter inhibitor Ruthenium Red and protein synthesis inhibitor cycloheximide all showed protection against glutamate-induced toxicity. None of the protective agents except for α-tocopherol controlled the glutamate-induced reactive oxygen species build-up. However, these cell death regulators prevented the glutamate-induced mitochondrial damage and regulated glutamate-induced increase in intracellular Ca 2+. Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, a mitochondrial uncoupler, partially protected against glutamate-induced cell death and mitochondrial damage, while the mitochondrial ribosomal inhibitor chloramphenicol and extracellular Ca 2+ chelator ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid did not protect the cells against glutamate treatment. The results of this study demonstrated that mitochondrial dysfunction was a key event in the excitotoxicity-independent component of neuronal cell death. Reactive oxygen species accumulation and glutathione depletion were prominent in glutamate-treated cells; however, these events were not direct mediators of cell death.