Abstracts

Alterations in transcriptional regulation and subsequent gene regulation are candidate mechanisms for long-term changes associated with epilepsy. The specific transcription factors involved in epilepsy are not well-defined. Furthermore, little is known about the signaling pathways that couple to transcriptional regulation in epilepsy. In these studies we characterized activation of two transcription factors, NF-[kappa]B and CREB, in the KA-seizure model [italic]in vivo [/italic]and assayed KA-induced transcriptional changes [italic]in vitro[/italic]. We also investigated whether KA-induced NF-[kappa]B and CREB activation couple to mitogen-activated protein kinase (MAPK) activation, specifically the extracellular signal-regulated kinase (ERK subfamily), in hippocampus.
For these studies, we evaluated NF-[kappa]B, CREB, and ERK-MAPK activation in hippocampus [italic]in vivo[/italic] and [italic]in vitro [/italic]following KA administration. KA-mediated NF-[kappa]B and CREB DNA binding activity was investigated by gel-shift analysis. Immunostaining was also performed to evaluate localization of KA-mediated transcriptional activation in hippocampus. In addition, we evaluated ERK-MAPK activation by western blotting using phospho-selective antibodies.
Immunoblot analysis of hippocampal area CA3 nuclear extracts from animals with kainate-induced status epilepticus showed increased levels of phospho-NF-[kappa]B (p [le] 0.05) and increased ERK-MAPK (p [le] 0.05) activation. Phospho-CREB levels showed no change in CA3 nuclear fractions by western blotting; however immunostaining show increases in phospho-CREB levels in CA3 pyramidal neurons. We further characterized KA-mediated transcription factor modulation in hippocampal slices [italic]in vitro.[/italic] Interestingly, we found a time-dependent increase in NF-[kappa]B and CREB phosphorylation levels within 15 minutes of kainate treatment in nuclear fractions compared to control levels [p-NF-[kappa]B (p[lt]0.01) and p-CREB (p[lt]0.05)]. In addition, we found a time-dependent increase (15 min) in phosphorylated ERK (p [le] 0.01) levels following KA bath application. Preliminary inhibitor studies suggest that ERK-MAPK activation may be necessary for the kainate-induced activation of NF-[kappa]B and CREB in hippocampal slices.
In summary, we have shown that kainate modulates activation of several transcription factors in hippocampus suggesting potential mechanisms for long-term changes that are seen in the kainate epilepsy model. Our findings also suggest that this kainate effect may be coupled to ERK-MAPK in hippocampus.
[Supported by: NIH/NINDS ]