Abstracts

Regulated expression of type A GABA receptor (GABA-AR) subunit genes plays a critical role in neuronal maturation, synaptogenesis, and has been implicated in a variety of neurological diseases. Changes in expression of [alpha] subunit genes (GABRA) have been observed in models of TLE. The kind of [alpha] subunit influences sensitivity of GABA-AR to GABA and to allosteric modulators such as Zinc, barbiturates, and benzodiazepines. Understanding genetic regulation behind such changes will lead to a better understanding of the mechanisms controlling GABA-AR function and have the promise of providing potential new therapeutic tools for the prevention or cure of a variety of neurological disorders. We have focused on developing an understanding of GABRA1 regulation whose promoter activity decreases in animal models of disease, including pilocarpine-induced status epilepticus (SE) and progesterone withdrawal. We have used transient transfection of primary hippocampal neurons to determine functional properties of DNA binding sites in GABRA1 and their specific interactions with the cAMP response element-binding protein (CREB). Chromatin immunoprecipitation (ChIP) was used to relate findings to the occupation of endogenous GABRA1 by activator and repressor proteins both in cultured neurons and in vivo in the pilocarpine model of TLE. PMA stimulation of primary cultured neurons increases GABA-AR [alpha]1 subunit mRNA levels and GABRA1 promoter activity through a PKC dependent and MAPK independent pathway. Increased GABA-AR [alpha]1 subunit mRNA levels are accompanied by increased binding of phospho-CREB to endogenous GABRA1. Overexpression of wildtype CREB increases GABRA1 activity while overexpression of a mutant form (M-CREB) is without effect. A 2bp mutation in the CRE site blocks the response to PMA and to CREB-mediated increases in activity. A mutant form of CREB (K-CREB) that has lost its ability to bind fails to increase activity. Overexpression of CREB related isoforms ATF4 and ICER markedly inhibit GABRA1. These observations are the first to identify a transcriptional mechanism that could contribute to downregulation of GABRA1 gene expression in response to cell signals. Pilocarpine-induced SE increases CREB and p-CREB association with the endogenous GABRA1 promoter in dentate gyrus 24 hours after SE. Our results demonstrate that the CREB binding site in human GABRA1 is functional and that it can bind CREB family members. Repression of transcription through heterodimers of CREB/ICER and homodimers of ATF4 suggest a new mechanism of GABRA1 gene expression that may be operative in neurological diseases such as temporal lobe epilepsy. (Supported by NIH NS42363-01 to ABK and SJR.)