Abstracts

Nuclear-factor kappaB (NF-[kappa]B) has been identified as a potential regulator of gene responses in epilepsy. The specific genes targeted by NF-[kappa]B in this context are incompletely understood. Kainate-induced status epilepticus (SE) stimulates the expression of numerous genes that may underlie changes in hippocampal organization and contribute to epilepsy. To further define the role of gene regulation by NF-[kappa]B in epilepsy, we investigated whether hippocampal NF-[kappa]B activation couples to specific genes early following SE and when the animals developed chronic epilepsy. I[kappa]B[alpha] and BDNF are among the candidate NF-[kappa]B gene targets. I[kappa]B[alpha] gene expression is regulated by NF-[kappa]B. BDNF is an immediate-early gene activated a few hours following kainate injection. We evaluated NF-[kappa]B activation in hippocampus following kainate (IP) administration in adult rats. Hippocampal NF-[kappa]B activation was determined using an antibody against the p65 NF-[kappa]B subunit when phosphorylated at Ser-536. RT-PCR was used to determine hippocampal exon-specific BDNF and I[kappa]B[alpha] gene expression after SE (3 hr post-kainate injection) and epilepsy (3 to 6 mon post-SE). We investigated whether NF-[kappa]B activation coupled to hippocampal BDNF gene changes following SE and epilepsy. Chromatin immunoprecipitation (ChIP) assays were used to measure levels of NF-[kappa]B (p65) at the BDNF promoters in hippocampus after SE and epilepsy. Using the NF-[kappa]B inhibitor, diethyldithiocarbamate (DDTC) we assessed the role of hippocampal NF-[kappa]B-mediated gene regulation after SE and epilepsy. Immunoblotting analysis revealed significant increases in Ser-536 p65 phosphorylation in area CA3 of hippocampus (p [lt] 0.05) with no significant change in phosphorylated Ser-536 p65 levels in area CA1 and dentate gyrus after SE. Phosphorylated Ser-536 p65 levels were increased in areas CA3 (p [lt] 0.01) and CA1 (p [lt] 0.05) and dentate gyrus (p [lt] 0.05) from epileptic animals. RT-PCR revealed an increase in I[kappa]B[alpha] mRNA levels in CA3, CA1, and dentate gyrus (p [lt] 0.05), while there was no change in epileptic animals. Together, these results suggest that changes in Ser-536 p65 phosphorylation in hippocampus do not correlate with NF-[kappa]B-mediated I[kappa]B[alpha] gene regulation after SE and in epilepsy. Hippocampal BDNF (exons 1-3) mRNA levels were significantly modulated in all subfields after SE (p [lt] 0.01), but not in epileptic animals. Preliminary ChIP analysis indicated that p65 NF-[kappa]B was recruited to BDNF promoters 2 and 4 in hippocampus. Pilot inhibitor studies using DDTC suggest that NF-[kappa]B activation regulates BDNF gene expression in area CA3 after SE. Additional DDTC studies are underway for assessment of BDNF gene expression in hippocampus following SE. In the present studies, we show biochemical evidence for NF-[kappa]B activation acutely following SE and in chronic epilepsy. However, our molecular studies suggest that NF-[kappa]B is transcriptionally active in SE but not in chronic epilepsy. (Supported by NIH/NINDS, EF and SFN Fellowships.)