Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics Alteration of Epileptogenesis Genes Amy R. Brooks-Kayal,*† Yogendra H. Raol,* and Shelley J. Russek‡ *Division of Neurology, Department of Pediatrics, University of Colorado Denver School of Medicine, †The Children’s Hospital, Aurora, Colorado 80045, and ‡Laboratory of Translational Epilepsy, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118 Summary: Retrospective studies suggest that precipitating expression and function have been reported in adult animals events such as prolonged seizures, stroke, or head trauma in- beginning immediately after prolonged seizures (status epilepticus crease the risk of developing epilepsy later in life. The process [SE]) and continue as animals become chronically epileptic. Pre- of epilepsy development, known as epileptogenesis, is associ- vention of GABAA receptor subunit changes after SE using viral ated with changes in the expression of a myriad of genes. One gene transfer inhibits development of epilepsy in an animal model, of the major challenges for the epilepsy research community suggesting that these changes directly contribute to epileptogen- has been to determine which of these changes contributes to esis. The mechanisms that regulate differential expression of epileptogenesis, which may be compensatory, and which may GABAA receptor subunits in hippocampus after SE have recently be noncontributory. Establishing this for any given gene is been identified, and include the CREB-ICER, JAK-STAT, BDNF, essential if it is to be considered a therapeutic target for the and Egr3 signaling pathways. Targeting signaling pathways that prevention or treatment of epilepsy. Our laboratories have ex- alter the expression of genes involved in epileptogenesis may amined alterations in gene expression related to inhibitory neu- provide novel therapeutic approaches for preventing or inhibiting rotransmission that have been proposed as contributing factors the development of epilepsy after a precipitating insult. Key in epileptogenesis. The GABAA receptor mediates most fast Words: GABA receptor subunits, epilepsy, epileptogenesis, hip- synaptic inhibition, and changes in GABAA receptor subunit pocampus, gene transfer, transcriptional regulation. INTRODUCTION used as antiepileptic drugs. Also, drugs that block GABAergic inhibition can induce seizures in animals, It is clear from the results of research using various further supporting the potential importance of alterations animal models and from human retrospective studies that in GABAergic transmission in epileptogenesis. an initial precipitating event such as status epilepticus (SE) can increase the risk of later epilepsy development (epileptogenesis). The process of epileptogenesis is ROLE OF GABAA RECEPTORS IN EPILEPSY likely to be complex and multifactorial. Determining whether changes in gene regulation that accompany the Three types of GABA receptors are found in the epileptic condition are consequential to or causative of mature central nervous system: GABAA, GABAB, and disease etiology is the major difficulty of research in this GABAC. Both GABAA and GABAC are ionotropic re- area. Many laboratories, including our own, have fo- ceptors; GABAB is a metabotropic receptor. Most fast cused on the role of gene regulation in determining synaptic inhibition in the mature brain is mediated by changes in GABA receptor plasticity that occur during GABAA receptors, whereas slow inhibition is mediated the latent period after brain insult and prior to the devel- by GABAB receptors. GABAA receptors are composed opment of the epileptic state. GABA is the major inhib- of multiple subunit subtypes (␣1–6, ␤1–3, ␥1–3, ␦, ␧, ␲, itory neurotransmitter in the mature brain, and various ␪, and ␳1–3) that form a pentameric anion-selective 1,2 drugs that enhance GABAergic inhibition are commonly channel. The most common in vivo subunit composi- tion is two ␣, two ␤, and one ␥ subunit. There is remark- able receptor heterogeneity, with subtype combinations Address correspondence and reprint requests to: Amy R. Brooks- varying in different brain regions and cell types, and Kayal, M.D., Department of Pediatrics, Division of Neurology, Uni- during different times in ontogeny.3–6 Different subunit versity of Colorado Denver, 12700 E, 19th Avenue, Rm P15-4122A, Mail Stop 8605, Aurora, CO 80045. E-mail: brooks-kayal.amy@ subtypes confer distinct functional and pharmacological tchden.org. properties to the receptors.3 312 Vol. 6, 312–318, April 2009 © The American Society for Experimental NeuroTherapeutics, Inc. ALTERATION OF EPILEPTOGENESIS GENES 313 Prolonged seizures (i.e., SE) result in alterations in the by 2 weeks after SE compared to the control groups. Rats expression and membrane localization of several GABAA were continuously video-EEG monitored to determine receptor subunits (␣1, ␣4, ␥2, and ␦) in hippocampal the latency for development of spontaneous seizures. dentate granule neurons.7–9 These alterations, which are AAV-␣1 injection resulted in a threefold increase in the associated with changes in phasic and tonic GABAA mean time to the first spontaneous seizure after SE, and ␣ receptor-mediated inhibition, and in GABAA receptor only 39% of AAV- 1 injected rats were observed to modulation by benzodiazepines, neurosteroids, and zinc, develop spontaneous seizures in the first 4 weeks after begin soon after SE and continue as animals become SE, compared with 100% of rats receiving sham injec- epileptic.7–10 tions. Because all groups of rats experienced similar SE Several laboratories have documented changes in after pilocarpine injection, these findings provide the first GABAA receptor subunit composition in human tem- direct evidence that increasing the levels of a single poral lobe epilepsy (TLE) and in animal models of GABAA receptor subunit in the DG can inhibit the de- TLE.7,9,11,12 In the pilocarpine model of SE in adult velopment of spontaneous seizures after SE. Together, ␣ ␣ rodents, GABAA receptor 1-subunit mRNA expression these data support a role for GABAA receptor -subunit decreases, ␣4-subunit mRNA expression increases in changes in the process of epileptogenesis. dentate granule cells (DGCs) of the hippocampus, and animals uniformly go on to develop the recurrent spon- MECHANISMS REGULATING GABA taneous seizures that define epilepsy.7 The change in A RECEPTOR SUBUNIT EXPRESSION subunit expression correlates with a decreased sensitivity to zolpidem augmentation and increased sensitivity to ␣1 Subunit regulation 7 zinc inhibition of GABAA receptor responses. Similar Although viral gene transfer is a promising therapeutic functional and subunit expression changes have been avenue for modifying aberrant gene expression associ- observed in DGCs isolated from surgically resected hip- ated with epileptogenesis, producing the optimal level of pocampus from patients with intractable TLE.12 The expression over a prolonged period can be challenging. changes in GABAA receptor subunit expression and Another possible approach is to modify the mechanisms function in DGCs of epileptic animals precede the de- regulating gene expression. Recent work in our labora- velopment of epilepsy, suggesting that these changes tories has established cAMP response element binding contribute to the epileptogenesis process. In contrast, protein (CREB) and inducible cAMP early repressor neonatal SE (postnatal day 10) in rats results in increased (ICER) as critical mediators of the GABAA receptor ␣ ␣ GABAA receptor 1-subunit expression and does not 1-subunit mRNA decreases that occur after SE in the subsequently lead to development of epilepsy.13 DG. The CREB is a stimulus-induced bZIP transcription These studies suggest that GABAA receptor subunit factor that is activated by phosphorylation at its Ser 133 alterations may be an important component of epilepto- site. Phosphorylated CREB (pCREB) dimerizes and binds genesis. To more directly determine if GABAA receptor to cAMP response element (CRE) motifs on promoters that changes are critical contributors to epilepsy develop- contain the consensus sequence TGACGTCA.16 Along ment, we used gene transfer to prevent these GABAA with its chromatin regulator, the CREB binding protein receptor subunit changes. To directly test the hypothesis (CBP), pCREB upregulates transcription of target genes. that the expression of higher ␣1-subunit levels inhibits Transcriptional regulation through CREB has been im- development of epilepsy after SE, an adeno-associated plicated in mechanisms of cell survival, plasticity, and virus (AAV) gene transfer vector (AAV2), serotype 5, learning and memory paradigms.17 Target genes of was designed to express a bicistronic RNA that codes for pCREB include CREB family members encoding cAMP ␣ both the GABAA receptor 1-subunit and the reporter response element modulator (CREM), ICER, and acti- (enhanced yellow fluorescence protein [eYFP]).14 Ex- vating transcription factors. These para- and homologs of pression of this RNA was placed under control of the CREB also bind CRE elements to modulate the tran- ␣ GABAA receptor 4-subunit gene (GABRA4) core pro- scription of particular genes. The CREM gene produces moter region, because this was previously shown to be many spliced isoforms that can act as transcriptional markedly activated in dentate gyrus (DG) after SE15: repressors or activators. One of these repressor forms is activity of the ␣4 promoter is upregulated