Abnormalities in SOD Isoenzyme Patterns and DNA Fragmentation During Development of the Epileptic Mutant EL Mouse
Authors: Murashima Yoshiya L. , Suzuki Jiro 
Published: August 2009

Purpose: Several lines of evidence suggest that free radicals and their scavenger enzyme superoxide dismutase (SOD) are involved in long‐term potentiation (LTP) and epileptogenesis in the CA1 field of the hippocampus. In rats with kainate‐induced epilepsy and tottering mice (a model for petit ma1 seizures), “apoptosis” has been observed in neurons. Free radicals in the brain have been suggested to facilitate the apoptosis. The EL mouse, a mutant model of epilepsy, shows secondarily generalized seizures. In the mutant mice, the susceptibility to seizures gradually increases during development and through the experience of repetitive seizures. The parietal cortex in EL mice plays an important role in the seizure initiation, and the hippocampus shows a high electrical excitability during generalization of the seizures. In this study, we examined SOD isoenzyme activities and protein contents in the parietal cortex and hippocampus of EL mice during development. To investigate the region‐specific targets of free radicals in EL mice, the spatial specificity of DNA fragmentation also was studied. Methods: SOD activities were determined by using the method of Fridovich (1991). Total SOD activity included cyanide‐insensitive Mn‐SOD and cyanide‐sensitive Cu, Zn‐SOD. After coincubation with 1 mM cyanide, the activities of Mn‐SOD were assayed. Cu, Zn‐SOD activities were obtained from total SOD activities by subtracting Mn‐SOD activities. Mn‐SOD and Cu, Zn‐SOD contents were assayed with a commercially available enzyme‐linked immunosorbent assay (ELISA) kit (Boehringer). DNA fragmentation was detected in EL mice by using in situ terminal transferase‐mediated deoxyuridine triphosphate (dUTP) nick‐labeling detected with alkaline phosphatase and peroxidase. Results: EL mice which had experienced frequent seizures and established epileptogenicity, EL[s], revealed abnormal SOD isoenzyme activity patterns characterized by a remarkable decrease in the hippocampus. Once epileptogenicity was established, SOD activities, especially cyanide‐sensitive Cu, Zn‐SOD activities, were maintained at low levels. Despite the conspicuous decrease in Cu, Zn‐SOD activities, the protein contents of this isoenzyme were increased in the hippocampus. Cyanide‐insensitive Mn‐SOD activities were not changed. On the other hand, EL mice that had not experienced seizure‐provoking stimulations and exhibited no seizures, EL[ns], showed a moderate decrease in their SOD isoenzyme activities. In EL[s] mice, there was a fragmentation of DNA in the CAI field of the hippocampus and the parietal cortex, whereas in EL[ns], the DNA fragmentation was restricted to the parietal cortex and was much less obvious. Conclusions: The results revealed a region‐specific decrease in the Cu, Zn‐SOD activity and an increase in the regional free radical concentration in the EL brain during epileptogenesis. The discrepancy between enzyme activity and protein content might indicate the induction of an inactive Cu, Zn‐SOD isoenzyme during development. We speculated that region‐specific abnormalities in the activity of Cu, Zn‐SOD isoenzyme, presumably due to a disorder of Cu metabolism, produce spatially specific free radicals, which in turn could cause DNA fragmentation. This process might contribute to the common pathway for epileptogenesis.