Abstracts

Functional changes in the properties of [gamma]-aminobutyric acid type A receptors (GABA-ARs) occurring in the dentate gyrus after pilocarpine-induced status epilepticus (SE) are accompanied by changes in the mRNA levels of certain GABA-AR subunits, including those specific to [alpha]4. Because receptors containing [alpha]4 are believed to make up the majority of extrasynaptic GABA-ARs, it has been suggested that an increase in their expression could alter the relationship between tonic and phasic inhibition. Similar changes in GABA-AR function and gene expression are also seen in temporal lobe epilepsy (TLE) patients suggesting that altered GABA-AR subunit composition may at least in part contribute to the disease process. In order to understand the changes responsible for altered GABA-AR function, we have studied the binding property and transcriptional activity of the [alpha]4 gene (GABRA4) both in primary cultured neurons and in vivo. Bioinformatics was first used to identify potential DNA binding sites by comparing GABRs whose activity is upregulated in response to SE to those downregulated. Chromatin immunoprecipitation (ChIP) was then used to determine occupancy of GABRA4 consensus DNA binding sites in vivo using slices of adult rat dentate granule tissue isolated from pilocarpine-treated rats and promoter function was examined using reporter assays. Endogenous GABRA4 is bound by early growth response factor 3 (Egr3) 24 hours after induction of SE. Increased binding is accompanied by increases in Egr3 proteins and [alpha]4 and Egr3 mRNAs suggesting increased synthesis of Egr3 contributes at least in part to sustained response of GABRA4 to SE. To investigate a potential mechanism for Egr3 up-regulation in vivo, hippocampal neurons were exposed to phorbol myristate acetate (PMA) in the presence and absence of specific antagonists (U0126 and calphostin C). Changes in GABRA4 mRNAs were monitored using real-time PCR. ChIP performed in parallel demonstrates that increases in [alpha]4 mRNA levels after MAPK/PKC activation are accompanied by increased binding of Egr3 to endogenous GABRA4. Functional studies in hippocampal neurons show that over-expression of Egr3 up-regulates promoter activity through the Egr3 binding site and that Egr3 dominant negatives prevent increased activity in response to PMA. Egr3 knock-out mice also display about 50 % less GABRA4 mRNAs (with no change in [alpha]1). Our findings support a role for Egr3 as a major regulator of GABRA4 in neurons and provide the foundation for future studies aimed at determining the impact of the Egr3 signaling pathway to the etiology and treatment of temporal lobe epilepsy. (Supported by NIH NS42363-01 to ABK and SJR.)