Abstracts

RATIONALE: Nuclear factor- kappa B (NF-[kappa]B) is activated by a wide range of stimuli and operates in several different signaling pathways. A number of these signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade, modulate transcriptional activation through phosphorylation of transcription factors. Members of the MAPK family, extracellular-signal regulated kinase (ERK), p38 MAPK, and c-jun N-terminal kinase (JNK), have been shown to activate NF-[kappa]B in a variety of cell types. Previous studies in the kainate (KA) model of epilepsy have demonstrated activation of the MAPK signaling cascade in hippocampus following seizures. However, the downstream transcriptional effectors of MAPK in the KA model of epilepsy are not well understood. In these studies we evaluated KA-mediated activation of the transcription factor, NF-[kappa]B, in rat hippocampus. We also investigated whether KA-induced NF-[kappa]B activation couples to MAPK activation in hippocampus.
METHODS: We first investigated phospho-NF-[kappa]B levels and phosphorylation and degradation of total levels of I[kappa]B[alpha], the NF-[kappa]B inhibitor in KA-treated hippocampal slices by western blotting. ERK, p38 MAPK and JNK activation in KA-treated hippocampal slices were evaluated by western blotting using phospho-selective antibodies. Additionally, we sought to correlate findings from KA studies in hippocampal slices, in vitro with studies in the KA model, in vivo. For these studies, we evaluated ERK, p38 MAPK, JNK and NF-[kappa]B activation in hippocampus following KA-induced status epilepticus.
RESULTS: Immunoblot analysis revealed a dose-dependent increase in hippocampal NF-[kappa]B phosphorylation (p [lte] 0.05) following KA treatment compared to controls in hippocampal slices. Similarly, we found a dose-dependent increase in phosphorylated ERK (p [lte] 0.01) and p38 MAPK (p [lte] 0.05) levels, but not phospho-JNK levels, following KA. Preliminary inhibitor studies are underway to evaluate MAPK coupling to KA-induced activation of NF-[kappa]B in hippocampal slices. Interestingly, we found increased levels of phospho-NF-[kappa]B (p [lte] 0.0001) in hippocampus following KA-induced status epilepticus.
CONCLUSIONS: We have shown that KA modulates activation of NF-[kappa]B in hippocampus, suggesting a potential mechanism for changes in gene-expression that contribute to the long-term changes seen in the KA epilepsy model. We are investigating whether this KA effect is coupled to MAPK in hippocampus. These studies provide insights into the role of KA-mediated transcriptional activation.
[Supported by: NINDS]