Abstracts

Humans with temporal lobe epilepsy (TLE) and models of TLE, including pilocarpine treated rats, display [gamma]-aminobutyric acid type A receptors (GABA-A-Rs) with altered functional properties in dentate granule cells (DGCs). Altered receptor function is accompanied by upregulation in certain GABA-A-R subunit mRNAs, including [alpha]4. GABA-A-Rs containing [alpha]4 have unique pharmacological properties. Our previous work suggests that protein kinase C (PKC) and early growth response factor 3 (Egr3) upregulate [alpha]4 levels in DGCs from pilocarpine-treated rats and in primary hippocampal cultures. The following studies were undertaken to identify potential endogenous signals responsible for seizure-induced upregulation of [alpha]4 gene (GABRA4) expression. Because mRNAs and protein specific to brain derived neurotrophin factor (BDNF) are elevated in TLE patients and in models of TLE, we examined whether BDNF through its receptor (TrkB) may regulate levels of Egr3 and [alpha]4., Gene expression was monitored by quantitative realtime PCR. Western blot analysis was used to assess protein levels after BDNF treatment. Promoter activity was examined using luciferase reporter assays and transcription factor association was monitored by chromatin immunoprecipitation., BDNF mRNA levels are elevated 14 fold in DGCs during the latent period (24 hours following status epilepticus (SE)). Elevation of BDNF after SE is accompanied by increases in levels of both Egr3 and [alpha]4 subunit mRNAs. To investigate whether there is a relationship between increased BDNF and expression of Egr3 and/or [alpha]4 subunits, molecular studies were performed in BDNF treated primary hippocampal neurons. Levels of Egr3 mRNAs and protein are elevated in hippocampal neurons following BDNF treatment and are dependent upon activation by PKC and mitogen-activated protein kinase (MAPK). Like treatment with phorbol myristate acetate, BDNF elevates [alpha]4 levels in cultured hippocampal neurons after 6 or 12 hours of treatment. Enhancement of GABRA4 promoter activity by BDNF suggests that BDNF induced elevation of endogenous [alpha]4 subunit levels may be due to a transcriptional mechanism. Moreover, blockade of TrK activity by K-252a suggests that at least 50 % of GABRA4 activity in developing hippocampal neurons is dependent upon presence of neurotrophins., Our findings support a role for BDNF in Egr3-mediated [alpha]4 subunit gene regulation that may be relevant to our understanding of the molecular determinants of TLE and the development of novel therapeutics., (Supported by R01NS050393.)