Glutamate-induced apoptosis is related with the damage of mitochondria that results in cytochrome C release into cytosol in cultured hippocampal neurons

AIM: To set up a glutamate-induced cell damage model in cultured hippocampal neurons, and to determine whether glutamate-induced neuronal apoptosis changes and whether this process is mediated by mitochondrial signal transduction pathways involving the release of cytochrome C. METHODS: Hippocampal neurons, isolated and cultured from new born Wistar rats, were exposed to various concentrations of glutamate. Extent of cell death was assessed by measuring the release of lactate dehydrogenase (LDH) in the culture media. Based on these data, an appropriate concentration of glutamate was selected, and all subsequent experiments were carried out under the concentration. Kinetics of glutamate-induced both apoptotic and necrotic cell death after exposure to glutamate for various times(3-24 h) were determined by flow cytometry and LDH release. The caspase-3 protein levels and cytochrome C release from mitochondria into cytosol in hippocampal neurons were determined by Western blotting. RESULTS: Glutamate treatment induced hippocampal neurons death in dose-dependent and time-dependent manners. A significant increase in LDH release (18.4%) was induced in the cells treated with 50 μmol/L glutamate, compared to control untreated cells(P<0.05). A significant increases in LDH release and apoptosis were observed at 6 h after glutamate treatment (P<0.05). The significant increase in the level of caspase-3 protein occurred at 3 h after glutamate exposure, which preceded neuronal cell apoptosis, and reached maximum levels at 6 h(62.4%). Treatment with glutamate induced a rapid release of cytochrome C into cytosol. Cytosolic cytochrome C showed a significant increase (P<0.05) as early as 30 min after glutamate treatment, which preceded the increase in caspase-3 level, and after 3 h, the level of cytochrome C was higher in the cytosol compared to the mitochondria. Concomitant with these changes in cytosol, the mitochondrial levels of cytochrome C decreased significantly. CONCLUSION: Exposure to 50 μmol/L glutamate induces apoptosis in cultured hippocampal neurons. The glutamate-induced apoptosis may be via the damage of mitochondria that results in cytochrome C release into cytosol, which activates caspase cascade and induces apoptotic cell death.