Abstracts

Seizure frequency in women often correlates with catamenial changes in gonadal steroid hormone levels during the menstrual cycle. Moreover, progesterone and its metabolite, allopregnanolone (5[alpha]-pregnan-3[alpha]-ol-20-one, ALLO), have anticonvulsant properties in several model systems. We hypothesized that neurosteroid regulation of GABA[sub]A[/sub] receptor (GABAR) subunit expression could contribute to catamenial changes in seizure susceptibility. In our prior [italic]in vitro[/italic] studies of GABAR subunit expression in NT2-N neuronal cells using RT-PCR, exposure to peak ALLO concentration for 2 days increased mRNA levels of the GABAR [italic][epsilon] [/italic]subunit; withdrawal from ALLO further increased [alpha]2 and [alpha]4 subunit mRNA expression. Unlike progesterone, ALLO has low affinity at the nuclear progesterone receptor, hence the mechanism of GABAR subunit transcriptional regulation by ALLO is unclear. To identify genes that may contribute to ALLO regulation of GABAR subunits, we have used focused microarrays to identify signaling pathways activated by ALLO withdrawal. NT2-N cells 11 weeks after onset of retinoic acid treatment were exposed to ALLO (1 [mu]M) or vehicle control for 2 days and then withdrawn from ALLO for 1 day prior to RNA harvesting. Biotin-labeled cRNA was hybridized to 60-mer cDNA probes corresponding to established signal transduction elements from multiple pathways using the human Signal Transduction Pathfinder Array (OHS-014, Superarray Bioscience). Chemiluminescence was captured on x-ray film and densities were analyzed and normalized to housekeeping gene densities. Genes with more than two-fold increase or decrease from control were considered significant. Gene transcription was upregulated for MMP7, BRCA1, NRIP1 (RIP140), Wnt 1, and IL-4R. Several genes were downregulated, including Wnt 2, c-Fos, RBBP8, TRIM25, and PKC[alpha]. These changes suggest possible involvement of the estrogen, NF[kappa]B and other signaling pathways. Regulation of GABAR subunit expression by ALLO exposure and withdrawal may involve multiple signaling pathways, particularly genes regulated in the estrogen receptor pathway, though these findings must be confirmed by RT-PCR and immunochemistry. Further studies of genes regulated by ALLO and their interactions with GABAR subunit promoters may elucidate the mechanisms of GABAR regulation by this important neurosteroid, and lead to better understanding of catamenial epilepsy and perimenstrual dysphoric disorder. (Supported by Myoclonus Research Foundation Grant (LJG), Epilepsy Foundation Predoctoral Fellowship (RCP).)