Abstracts

RATIONALE: Prolonged continuous seizure activity (status epilepticus, SE) results in neuronal degeneration in selected brain regions. We have previously documented that in these affected brain regions, biochemical hallmarks of apoptosis, namely internucleosomal DNA fragmentation and caspase-3 activation, appear in the aftermath of SE. The mechanism by which caspase-3 induces DNA fragmentation is via the dissociation of a heterodimer containing [dsquote]caspase-activated DNase[dsquote] (CAD, DFF40). This dissociation is accomplished by the caspase-3 mediated cleavage of [dsquote]ICAD[dsquote], an inhibitory component of the heterodimer, thereby releasing active CAD. Accordingly, the present study set out to test the hypothesis that cleavage of ICAD will be associated with SE-induced DNA fragmentation.
METHODS: SE was induced in adult male Sprague-Dawley rats by kainic acid injection (12 mg/kg i.p.) and seizures were terminated with diazepam after 2 hr. At 24, 48, or 72 hr after SE termination, protein levels of CAD and its inhibitor (ICAD) were measured in hippocampus and rhinal cortex by Western blotting (after SDS-PAGE) using specific antibodies.
RESULTS: At 48 and 72 hr after SE termination ICAD levels significantly decreased (by more than 60%) as compared to animals not experiencing seizures. This effect was observed in both hippocampus and rhinal cortex and was inversely related to the extent of DNA fragmentation, i.e., the decrease in ICAD was accompanied by a sharp increase in DNA fragmentation. This is consistent with the role of ICAD as an inhibitor of caspase-activated DNase. No changes were detected in total levels of SDS-dissociated CAD protein, confirming the specificity of the loss of ICAD. The decrease in ICAD at 48 and 72 hr post SE may trigger an increase in DNase activity of CAD (i.e. increased proportion of active CAD), leading to DNA fragmentation.
CONCLUSIONS: Our results indicate that CAD may participate in caspase-3 mediated internucleosomal DNA fragmentation, thus promoting SE-induced apoptotic cell death in both hippocampus and rhinal cortex. Moreover, our data suggests that the disappearance of ICAD protein may serve as a quantitative index of the extent of apoptotic cell death. Accordingly, we are now investigating whether pre-exposure to brief noninjurious electroshock seizures, which protects against SE-induced DNA fragmentation (Mol Brain Res, in press), will also prevent the decrease in ICAD. The present findings are consistent with our previous observations (Mol Brain Res 75: 216-224, 2000) of a neuroprotective action of caspase-3 inhibitors against apoptotic neuronal cell death following SE.
Support: HHS grants NS36035 and MH02040.