Shelley J. Russek

Enhancing GABAA Receptor α1 Subunit Levels in Hippocampal Dentate Gyrus Inhibits Epilepsy Development in an Animal Model of Temporal Lobe Epilepsy

Differential expression of GABAA receptor (GABR) subunits has been demonstrated in hippocampus from patients and animals with temporal lobe epilepsy (TLE), but whether these changes are important for epileptogenesis remains unknown. Previous studies in the adult rat pilocarpine model of TLE found reduced expression of GABR α1 subunits and increased expression of α4 subunits in dentate gyrus (DG) of epileptic rats compared with controls. To investigate whether this altered subunit expression is a critical determinant of spontaneous seizure development, we used adeno-associated virus type 2 containing the α4 subunit gene (GABRA4) promoter to drive transgene expression in DG after status epilepticus (SE). This novel use of a condition-dependent promoter upregulated after SE successfully increased expression of GABR α1 subunit mRNA and protein in DG at 1–2 weeks after SE. Enhanced α1 expression in DG resulted in a threefold increase in mean seizure-free time after SE and a 60% decrease in the number of rats developing epilepsy (recurrent spontaneous seizures) in the first 4 weeks after SE. These findings provide the first direct evidence that altering GABR subunit expression can affect the development of epilepsy and suggest that α1 subunit levels are important determinants of inhibitory function in hippocampus.

Molecular Identification of the Human GABABR2: Cell Surface Expression and Coupling to Adenylyl Cyclase in the Absence of GABABR1

We have identified a gene encoding a GABAB receptor, the human GABABR2, located on chromosome 9q22.1, that is distinct from the recently reported rat GABABR1. GABABR2 structurally resembles GABABR1 (35% identity), having seven transmembrane domains and a large extracellular region, but differs in having a longer carboxy-terminal tail. GABABR2 is localized to the cell surface in transfected COS cells, and negatively couples to adenylyl cyclase in response to GABA, baclofen, and 3-aminopropyl(methyl)phosphinic acid in CHO cells lacking GABABR1. Baclofen action is inhibited by the GABABR antagonist, 2-hydroxysaclofen. The human GABABR2 and GABABR1 genes are differentially expressed in the nervous system, with the greatest difference being detected in the striatum in which GABABR1 but not GABABR2 mRNA transcripts are detected. GABABR2 and GABABR1 mRNAs are also coexpressed in various brain regions such as the Purkinje cell layer of the cerebellum. Identification of a functional homomeric GABABR2 coupled to adenylyl cyclase suggests that the complexity of GABAB pharmacological data is at least in part due to the presence of more than one receptor and opens avenues for future research leading to an understanding of metabotropic GABA receptor signal transduction mechanisms.

BDNF and the diseased nervous system : a delicate balance between adaptive and pathological processes of gene regulation

It is clear that brain‐derived neurotrophic factor (BDNF) plays a crucial role in organizing the response of the genome to dynamic changes in the extracellular environment that enable brain plasticity. BDNF has emerged as one of the most important signaling molecules for the developing nervous system as well as the impaired nervous system, and multiple diseases, such as Alzheimer’s, Parkinson’s, Huntington’s, epilepsy, Rett’s syndrome, and psychiatric depression, are linked by their association with potential dysregulation of BDNF‐driven signal transduction programs. These programs are responsible for controlling the amount of activated transcription factors, such as cAMP response element binding protein, that coordinate the expression of multiple brain proteins, like ion channels and early growth response factors, whose job is to maintain the balance of excitation and inhibition in the nervous system. In this review, we will explore the evidence for BDNFs role in gene regulation side by side with its potential role in the etiology of neurological diseases. It is hoped that by bringing the datasets together in these diverse fields we can help develop the foundation for future studies aimed at understanding basic principles of gene regulation in the nervous system and how they can be harnessed to develop new therapeutic opportunities.

cAMP Response Element-Binding Protein, Activating Transcription Factor-4, and Upstream Stimulatory Factor Differentially Control Hippocampal GABABR1a and GABABR1b Subunit Gene Expression through Alternative Promoters

Expression of metabotropic GABAB receptors is essential for slow inhibitory synaptic transmission in the CNS, and disruption of GABAB receptor-mediated responses has been associated with several disorders, including neuropathic pain and epilepsy. The location of GABAB receptors in neurons determines their specific role in synaptic transmission, and it is believed that sorting of subunit isoforms, GABABR1a and GABABR1b, to presynaptic or postsynaptic membranes helps to determine this role. GABABR1a and GABABR1b are thought to arise by alternative splicing of heteronuclear RNA. We now demonstrate that alternative promoters, rather than alternative splicing, produce GABABR1a and GABABR1b isoforms. Our data further show that subunit gene expression in hippocampal neurons is mediated by the cAMP response element-binding protein (CREB) by binding to unique cAMP response elements in the alternative promoter regions. Double-stranded oligonucleotide decoys selectively alter levels of endogenous GABABR1a and GABABR1b in primary hippocampal neurons, and CREB knock-out mice show changes in levels of GABABR1a and GABABR1b transcripts, consistent with decoy competition experiments. These results demonstrate a critical role of CREB in transcriptional mechanisms that control GABABR1 subunit levels in vivo. In addition, the CREB-related factor activating transcription factor-4 (ATF4) has been shown to interact directly with GABABR1 in neurons, and we show that ATF4 differentially regulates GABABR1a and GABABR1b promoter activity. These results, together with our finding that the depolarization-sensitive upstream stimulatory factor (USF) binds to a composite CREB/ATF4/USF regulatory element only in the absence of CREB binding, indicate that selective control of alternative GABABR1 promoters by CREB, ATF4, and USF may dynamically regulate expression of their gene products in the nervous system.

Brain-derived Neurotrophic Factor (BDNF)-induced Synthesis of Early Growth Response Factor 3 (Egr3) Controls the Levels of Type A GABA Receptorα4 Subunits in Hippocampal Neurons

Altered function of γ-aminobutyric acid type A receptors (GABAARs) in dentate granule cells of the hippocampus has been associated with temporal lobe epilepsy (TLE) in humans and in animal models of TLE. Such altered receptor function (including increased inhibition by zinc and lack of modulation by benzodiazepines) is related, in part, to changes in the mRNA levels of certain GABAAR subunits, including α4, and may play a role in epileptogenesis. The majority of GABAARs that contain α4 subunits are extra-synaptic due to lack of the γ2 subunit and presence of δ. However, it has been hypothesized that seizure activity may result in expression of synaptic receptors with altered properties driven by an increased pool of α4 subunits. Results of our previous work suggests that signaling via protein kinase C (PKC) and early growth response factor 3 (Egr3) is the plasticity trigger for aberrant α4 subunit gene (GABRA4) expression after status epilepticus. We now report that brain derived neurotrophic factor (BDNF) is the endogenous signal that induces Egr3 expression via a PKC/MAPK-dependent pathway. Taken together with the fact that blockade of tyrosine kinase (Trk) neurotrophin receptors reduces basal GABRA4 promoter activity by 50%, our findings support a role for BDNF as the mediator of Egr3-induced GABRA4 regulation in developing neurons and epilepsy and, moreover, suggest that BDNF may alter inhibitory processing in the brain by regulating the balance between phasic and tonic inhibition.

BDNF Selectively Regulates GABAA Receptor Transcription by Activation of the JAK/STAT Pathway

BDNF regulates a GABA receptor subunit through the repressor ICER. Exciting Changes in Inhibitory Receptors In adult rats, prolonged seizures lead to independent changes in the expression of different subunits of the γ-aminobutyric acid (GABA) type A receptor (GABAAR), which is the main inhibitory neurotransmitter receptor in the brain, in the dentate gyrus of the hippocampus. These changes in subunit expression, which are accompanied by functional changes in GABAAR-mediated synaptic inhibition, are associated with the development of epilepsy. Seizures increase the abundance of brain-derived neurotrophic factor (BDNF), and Lund et al. now report that both prolonged seizures and BDNF activate the JAK/STAT signaling pathway to decrease transcription of the GABAAR α1 subunit by means of the transcription factors CREB and ICER. Previous research has shown that BDNF increases the abundance of the GABAAR α4 subunit independently of JAK/STAT signaling, suggesting that BDNF regulates GABAergic synaptic transmission through at least two distinct signaling pathways. The γ-aminobutyric acid (GABA) type A receptor (GABAAR) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABAAR subunit expression remain poorly understood. We report that the seizure-induced decrease in GABAAR α1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway regulated by brain-derived neurotrophic factor (BDNF). BDNF- and seizure-dependent phosphorylation of STAT3 cause the adenosine 3′,5′-monophosphate (cAMP) response element–binding protein (CREB) family member ICER (inducible cAMP early repressor) to bind with phosphorylated CREB at the Gabra1:CRE site. JAK/STAT pathway inhibition prevents the seizure-induced decrease in GABAAR α1 abundance in vivo and, given that BDNF is known to increase the abundance of GABAAR α4 in a JAK/STAT-independent manner, indicates that BDNF acts through at least two distinct pathways to influence GABAAR-dependent synaptic inhibition.

Sulfated steroids as endogenous neuromodulators.

Central nervous system function is critically dependent upon an exquisitely tuned balance between excitatory synaptic transmission, mediated primarily by glutamate, and inhibitory synaptic transmission, mediated primarily by GABA. Modulation of either excitation or inhibition would be expected to result in altered functionality of finely tuned synaptic pathways and global neural systems, leading to altered nervous system function. Administration of positive or negative modulators of ligand-gated ion channels has been used extensively and successfully in CNS therapeutics, particularly for the induction of sedation and treatment of anxiety, seizures, insomnia, and pain. Excessive activation of excitatory glutamate receptors, such as in cerebral ischemia, can result in neuronal damage via excitotoxic mechanisms. The discovery that neuroactive steroids exert rapid, direct effects upon the function of both excitatory and inhibitory neurotransmitter receptors has raised the possibility that endogenous neurosteroids may play a regulatory role in synaptic transmission by modulating the balance between excitatory and inhibitory neurotransmission. The sites to which neuroactive steroids bind may also serve as targets for the discovery of therapeutic neuromodulators.

Up-regulation of NMDAR1 subunit gene expression in cortical neurons via a PKA-dependent pathway

Transcription mediated by protein kinase A and the cAMP response element binding protein (CREB) has been linked to the establishment of long‐term memory and cell survival. However, all of the major targets for activated CREB have yet to be identified. Given the fact that CREB‐mediated transcription is intimately involved in cellular processes of learning and memory and that CREB activity can be regulated by synaptic N‐methyl‐d‐aspartate receptors (NMDARs) and metabotropic GABA receptors, we have studied the role of the cAMP‐dependent signaling pathway in the regulation of the NMDA receptor subunit 1 (NMDAR1), a subunit required for functional receptor formation. We now report that levels of NMDAR1 subunit protein in primary neocortical cultures are increased 66% in response to forskolin, an activator of adenylyl cyclase. Up‐regulation of NMDAR1 is paralleled by a twofold increase in mRNA levels and an 83% increase in NMDAR1 promoter/luciferase reporter activity that is dependent on protein kinase A. Three cAMP regulatory elements (CREs) in the rat NMDAR1 promoter (− 228, − 67, and − 39) bind CREB in vitro and forskolin increases binding to two of the sites (− 228 and – 67). Chromatin immunoprecipitation of neuronal rat genomic DNA reveals that CREB is bound in vivo to the endogenous NMDAR1 gene. Increased presence of the activated Ser133 phosphorylated form is dependent on the length of exposure to forskolin. Taken together with the results of mutational analysis, the findings strongly suggest that transcription of NMDAR1 is regulated by the c‐AMP signaling pathway, most likely through the binding of CREB and its activation by signal‐dependent phosphorylation.

Neurosteroid modulation of recombinant ionotropic glutamate receptors

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1(100)+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1(100)+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1(100)+NR2A cRNAs as compared with NR1(100) cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.

Evolution of GABAA receptor diversity in the human genome

Nowhere is the record of receptor evolution more accessible than in the organization of the 19 vertebrate genes coding for subunits of the major inhibitory neurotransmitter receptor in the central nervous system, the gamma-aminobutyric acid receptor (GABAAR). Co-expression of alpha, beta, and gamma subunit genes is necessary for the formation of a GABAAR that is potentiated by widely used anxiolytics, anticonvulsants, and hypnotics. The identification of alpha, beta, and gamma genes on chromosomes 4, 5, and 15 suggests that co-localization of a gamma gene with an alpha and beta may be important for brain function. We have now directly examined the organization of GABAAR subunit genes on human chromosomes. Estimates of physical distance using in situ hybridization to cells in interphase, and gene localization using hybridization to cells in metaphase demonstrate the existence of beta-alpha-alpha-gamma gene clusters in cytogenetic bands on chromosomes 4(p12) and 5(q34). Sequencing of PAC clones establishes intercluster conservation of a unique head-to-head configuration for alpha and beta genes on chromosomes 4, 5, and 15. Remarkably, phylogenetic tree analysis predicts the existence of a beta-alpha-gamma ancestral gene cluster in which internal duplication of an ancestral alpha was followed by cluster duplication, resulting in the relative chromosomal positions of modern GABAAR subunit genes in the human genome.