alpha-Ketoglutarate dehydrogenase contributes to production of reactive oxygen species in glutamate-stimulated hippocampal neurons in situ

The alpha-ketoglutarate dehydrogenase complex (KGDHC) which catalyzes the conversion of alpha-ketoglutarate to succinyl-CoA and NADH in mitochondria, is known to generate O(2).- in vitro. To find out if KGDHC contributes to neuronal Reactive Oxygen Species (ROS) increase in situ, we investigated whether the specific inhibitors of cellular KGDHC, succinyl phosphonate (SP) and the SP triethyl ester (TESP), might affect the glutamate-induced ROS production in cultured hippocampal neurons from rats. The concentration-dependent decrease in the mitochondrial potential of the glutamate-overstimulated neurons in the presence of SP or TESP indicated that under the conditions inducing neuronal ROS generation, the inhibitors are delivered to mitochondria, and their subsequent inhibition of KGDHC decreases the mitochondrial potential. The production of O(2).- was detected by reaction with hydroethidine. The distribution of the resulting fluorescence of DNA-ethidium coincided with that of the mitochondrial marker Mitotracker, pointing to the mitochondrial origin of the hydroethidine-detected ROS in response to glutamate (100 microM). At 200 microM, both TESP and SP administered together with glutamate, inhibited the glutamate-induced ROS production by about 20%, with the inhibition increasing to 44% at 500 microM TESP. The decrease in neuronal ROS by specific inhibitors of KGDHC demonstrates that KGDHC is a source of ROS in cultured neurons responding to glutamate. However, increasing the concentration of the strongest KGDHC inhibitor SP to 500 microM even increased the ROS production compared with glutamate alone, presumably due to secondary effects arising upon the strong KGDHC inhibition. Our work extends the current understanding of the glutamate-induced ROS generation in neurons, shedding light on the pathological mechanisms of the KGDHC involvement in glutamate neurotoxicity. In conclusion, potent KGDHC inhibitors are promising diagnostic tools for in situ study of neurodegenerative mechanisms.