Do seizures damage the brain? New human and evolving experimental data support seizure-induced programmed cell death in the brain but the significance of gene-directed neuronal cell death in human epileptics and following experimental seizures is largely unknown. As these data have substantial relevance for protection of brain during seizures, the broad, long-term objective of this project is to characterize the involvement of gene-controlled cell death following seizures and the therapeutic potential of its modulation. The hypotheses to be tested are: (A) Cell death following human or experimental seizures involves the expression of genes that control programmed cell death; (B) The caspase-3 pathway is integral to cell death following seizures; and (C) Specific nuclear (DNA damage) and non-nuclear (mitochondrial) processes are involved in the execution of such cell death.
The Specific Aims are to: (1) Characterize the transcriptional and post-transcriptional expression of the programmed cell death-regulating genes Bcl2, Bax, BCI-XL, and ICE using brain tissue from an animal model of focal limbic seizures induced by intra-amygdaloid kainic acid (KA), from Bc1-2 knockout mice, and from the brains of patients who have undergone temporal lobe surgery for intractable epilepsy. (2) demonstrate the involvement of mitochondrial-associated processes in the pathway to caspase-3 activation, including caspase-8, bid, and cytochrome C, in the in vitro seizure model and in Bid and Bax knockout mice. (3) Demonstrate the role of caspase-3 in the seizure model by determining transcriptional and post-transcriptional expression, ex viva activity, the effects of in vivo inhibition, and the activation (cleavage) and function (in viva inhibition) of the specific caspase-3 substrate, poly (ADP-ribose) polymerase (PARP) in the rat seizure model and in PARP knockout mice. (4) Determine the nature and profile of DNA damage following experimental seizures, by examining the temporal and spatial appearance of single and double stranded DNA breaks, the response of the DNA damage-inducible gene GADD45, and the role of the caspase activated deoxyribonuclease (CAD) and its inhibitor ICAD. The characterization of death modulatory gene regulation resulting from seizures, proposed in this application, has the potential to change our understanding of the benignity of seizures and offers strategies of neuroprotective therapy for epileptic patients.
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