Kainic acid induces selective mitochondrial oxidative phosphorylation enzyme dysfunction in cerebellar granule neurons: protective effects of melatonin and GSH ethyl ester.

Kainic acid (KA), a potent central excitotoxin, may elicit neuronal death via generation of reactive oxygen species (ROS). The present study was undertaken to further characterize KA neurotoxicity and its relationship to ROS production and mitochondrial dysfunction. Exposure of rat cerebellar granule neurons at 14 days in vitro to 0.5 mM KA for 30 min resulted in the death of 53% of cells 24 h later. ROS production, evaluated by 2',7'-dichlorofluorescein diacetate, increased in KA-treated granule neurons. Resolution of mitochondrial oxidative phosphorylation enzymes by blue native polyacrylamide gel electrophoresis, followed by histochemical staining, showed that KA induced a strong decrease (–40%, P<0.01) in succinate dehydrogenase (SDH) activity of complex II. Western analysis revealed a marked reduction in quantity of the catalytic portion of complex II enzyme in KA-treated cells. No significant changes were observed in the activities of other mitochondrial complex enzymes. The actions of KA at the mitochondrial level, as well as on ROS generation and cell viability, were prevented by the KA receptor-selective antagonist 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione. Pretreatment of granule neurons with melatonin, a direct scavenger of ROS, or with the reduced glutathione (GSH) delivery agent GSH ethyl ester, before KA challenge prevented both the decrease in cell viability and complex II damage. The last result supports a link between KA-induced mitochondrial oxidative enzyme dysfunction and ROS generation. Together the results suggest mitochondria to be a critical target in KA injury to neurons.