Abstracts

Prolonged seizures increase oxidative stress in the brain, leading to cellular distress and DNA damage. DNA damage can reach a threshold where it initiates programmed cell death pathways in the brain, but the extent to which DNA repair mechanisms can compensate for this damage remains unknown. We hypothesized that the regulation of DNA double-strand break (DSB) repair may be a determinant of vulnerability to seizure-induced cell death. In previous studies, we found evidence for a downregulation of DSB repair mechanisms at 48 and 72hr after seizure termination. In the present study, we examined earlier time points to see if upregulation preferentially occurs during a period preceeding cell death execution. As an indicator of DNA repair, protein levels of Ku70, a regulatory component of DSB repair machinery, were measured as a function of seizure duration. Adult male Sprague-Dawley rats were treated with kainic acid (12.5 mg/kg, i.p.) or saline (controls without seizures) and given diazepam (30 mg/kg, i.p.) at defined durations. Nuclear and cytosolic protein levels of Ku70 were determined in specific brain regions by Western blotting at 4, 8, and 20hr after seizure termination. In rhinal cortex, a region especially vulnerable to seizure-evoked injury, seizures lasting 30 or 120min resulted in an upregulation of Ku 70 protein that was detected at 4 and/or 8hr following seizure termination. The cellular distribution of the increase was as follows: [table1]In addition, there was a trend toward a downregulation of Ku70 protein at 20hr following both 30 and 120min of seizures; this is consistent with our previously reported observation of downregulation at 48 and 72hr following seizure termination. The pattern of changes in Ku70 will be compared with that for histone H2AX phosphorylation (a measure of the extent of DSB). Our results indicate that the DNA repair component, Ku70, is rapidly and transiently upregulated in response to prolonged seizure activity. Furthermore, the pattern of this regulation is influenced by the duration of seizure activity. Seizures lasting 30min were sufficient to induce what appears to be a maximal effect. This raises the possibility that the components of the DSB DNA repair mechanisms may serve as a target for neuroprotective strategies. (Supported by NIH predoctoral fellowship NS 046199, NIH grants NS 20576, MH 02040, NS 041231)