Suchitra Joshi

Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABAR β2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency.

Receptors with low affinity for neurosteroids and GABA contribute to tonic inhibition of granule cells in epileptic animals

Neurosteroid sensitivity of GABA(A) receptor mediated inhibition of the hippocampal dentate granule cells (DGCs) is reduced in animal models of temporal lobe epilepsy. However, the properties and subunit composition of GABA(A) receptors mediating tonic inhibition in DGCs of epileptic animals have not been described. In the DGCs of epileptic animals, allopregnanolone and L-655708 sensitivity of holding current was diminished and δ subunit was retained in the endoplasmic reticulum and its surface expression was decreased the in the hippocampus. Ro15-4513 and lanthanum had distinct effects on holding current recorded from DGCs of control and epileptic animals suggesting that the pharmacological properties of GABA(A) receptors maintaining tonic inhibition in DGCs of epileptic animals were similar to those containing the α4βxγ2 subunits. Furthermore, surface expression of the α4 subunit increased and a larger fraction of the subunit co-immunoprecipitated with theγ2 subunit in hippocampi of epileptic animals. Together, these studies revealed that functional α4βxδ and α5βxγ2 receptors were reduced in the hippocampi of epileptic animals and that novel α4bxγ2 receptors contributed to the maintenance of tonic inhibition. The presence of α4βxγ2 receptors resulted in low GABA affinity and neurosteroid sensitivity of tonic currents in the DGCs of epileptic animals that could potentially increase seizure vulnerability. These receptors may represent a novel therapeutic target for anticonvulsant drugs without sedative actions.

Slow intracellular accumulation of GABAA receptor δ subunit is modulated by brain-derived neurotrophic factor

GABA(A) receptors composed of the gamma2 and delta subunits have distinct properties, functions and subcellular localization, and pathological conditions differentially modulate their surface expression. Recent studies demonstrate that acute seizure activity accelerated trafficking of the gamma2 and beta2/3 subunits but not that of the delta subunit. The trafficking of the gamma2 and beta2/3 subunits is relatively well understood but that of the delta subunit has not been studied. We compared intracellular accumulation of the delta and gamma2 subunits in cultured hippocampal neurons using an antibody feeding technique. Intracellular accumulation of the delta subunit peaked between 3 and 6 h, whereas, maximum internalization of the gamma2 subunit took 30 min. In the organotypic hippocampal slice cultures internalization of the delta subunit studied using a biotinylation assay revealed highest accumulation between 3 and 5 h and that of the gamma2 subunit between 15 and 45 min. The surface half-life of the delta subunit was 171 min in cultured hippocampal neurons and 102 min in the organotypic hippocampal slice cultures. In the subsequent studies, internalization of the delta subunit was found to be dependent on network activity but independent of ligand-binding. Brain-derived neurotrophic factor (BDNF) reduced buildup of the delta subunit in the cytoplasmic compartments and increased its surface expression, and this BDNF effect was independent of network activity. BDNF effect was mediated by activation of TrkB receptors, PLCgamma and PKC. Increase in the basal PKC activity augmented cell surface stability of the delta subunit. These results suggest that rate of intracellular accumulation of the delta subunit was distinct and modulated by BDNF.

Receptor trafficking hypothesis revisited: Plasticity of AMPA receptors during established status epilepticus

Status epilepticus (SE) is associated with a dynamic plasticity of postsynaptic neurotransmitter receptors. The plasticity of AMPA receptor (AMPAR)–mediated glutamatergic transmission during established SE (ESE), after development of benzodiazepine resistance, was evaluated. There was increased frequency and inward rectification of AMPAR‐mediated excitatory postsynaptic currents at Schaffer collateral ‐ CA1 pyramidal neuron synapses during ESE. Surface expression of the GluA1 subunit increased, and this was a consequence of N‐methyl‐d‐aspartate receptor activation. Further, diminishing glutamate release by activation of somatostatin receptors prevented SE. These studies suggest that AMPAR‐mediated glutamatergic transmission is strengthened during ESE.

GABAergic transmission in temporal lobe epilepsy: the role of neurosteroids.

Modification of GABAergic inhibition is an intensely investigated hypothesis guiding research into mechanisms underlying temporal lobe epilepsy (TLE). Seizures can be initiated by blocking γ amino butyric acid type A (GABAA receptors, GABARs), which mediate fast synaptic inhibition in the brain, and controlled by drugs that enhance their function. Derivatives of steroid hormones called neurosteroids are natural substances that physiologically enhance GABAR function and suppress seizures. GABAR structure, function, expression, assembly, and pharmacological properties are changed in the hippocampus of epileptic animals. These alterations render GABARs less sensitive to neurosteroid modulation, which may contribute to seizure susceptibility. Plasticity of GABARs could play a role in periodic exacerbation of seizures experienced by women with epilepsy, commonly referred to as catamenial epilepsy.

Impact of receptor changes on treatment of status epilepticus.

Neurotransmitter receptors play a key role in the pathogenesis and current treatment strategies for status epilepticus (SE). Benzodiazepines are commonly used to treat SE exert their anticonvulsant action by stimulating γ-aminobutyric type A receptors (GABAA receptors). These drugs are quite effective in terminating SE in approximately 55–66% of patients, but there is a residual group of patients with seizures resistant to benzodiazepeines. The mechanisms underlying initiation and maintenance of prolonged SE were explored.

GABAA receptor plasticity in status epilepticus

Seizures become self‐sustaining during status epilepticus (SE) in part due to altered inhibitory neurotransmission mediated by γ‐aminobutyric acid (GABAA) receptors (GABAARs). This chapter reviews mechanisms of altered GABAAR function during SE. For an expanded treatment of this topic see Jasper’s Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado‐Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at http://www.ncbi.nlm.nih.gov/books).

GABAA receptor membrane insertion rates are specified by their subunit composition.

γ Amino-butyric acid type-A receptors (GABARs) containing γ2 or δ subunits form separate pools of receptors in vivo, with distinct localization and function. We determined the rate of surface membrane insertion of native and recombinant γ2 and δ subunit-containing GABARs (γ2-GABARs and δ-GABARs). Insertion of the α-bungarotoxin binding site (BBS) tagged γ2 subunit (t-γ2)-containing GABARs in the surface membrane of HEK293 cells occurred within minutes and reached a peak by 30 min. In contrast, insertion of the BBS-tagged δ subunit (t-δ)-containing receptors required longer incubation and peaked in 120 min. Insertion of the t-γ2 subunit-containing receptors was not influenced by assembling α1 or α4 subunits. In contrast, insertion of the α4β3t-δ subunit-containing receptors was faster than those containing α1β3t-δ subunits. The rate of insertion of native GABARs in the surface membrane of cultured hippocampal neurons, determined by an antibody saturation assay, was similar to that of the recombinant receptors expressed in HEK293 cells. Insertion of the γ2-GABARs was rapid and new γ2-GABARs were detected on the surface membrane of cell soma and dendrites within minutes. In contrast, insertion of the δ-GABARs was slow and newly inserted receptors were initially present only in the surface membrane of cell soma and later also appeared over the dendrites. Thus the rate of insertion of GABARs was dependent on their subunit composition.

Enhanced AMPA receptor-mediated neurotransmission on CA1 pyramidal neurons during status epilepticus

Status epilepticus (SE) is a common neurological emergency that results from the failure of the mechanisms responsible for seizure termination or the initiation of mechanisms that lead to abnormally prolonged seizures. Although the failure of inhibitory mechanisms during SE is well understood, the seizure-initiating mechanisms are poorly understood. We tested whether hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated transmission was enhanced during SE and assessed the underlying molecular mechanism. In animals in self-sustaining limbic SE the amplitudes of the miniature, spontaneous, and AMPA-evoked excitatory currents recorded from the CA1 pyramidal neurons were larger than those recorded in the controls. The evoked EPSCs rectified inwardly. In these animals, the surface expression of GluA1 subunit-containing AMPARs was increased in the CA1 pyramidal neurons. The phosphorylation of the GluA1 subunit on S831 and S845 residues was reduced in animals in SE. In contrast, the GluA1 subunit surface expression and AMPAR-mediated neurotransmission of dentate granule cells (DGCs) was not altered. Treating animals in SE with the NMDAR antagonist MK-801 or with diazaepam blocked the increased surface expression of the GluA1 subunits. NMDAR blockade also prevented the dephosphorylation of the S845 residue but not that of S831. Targeting NMDARs and AMPARs may provide novel strategies to treat benzodiazepine-refractory SE.

A Mouse Monoclonal Antibody Against the γ2 Subunit of GABAA Receptors

A mouse monoclonal antibody directed against the N terminal extracellular epitope of rat γ amino butyric acid (GABA) type-A (GABA(A)R) receptor γ2 subunit was generated. This antibody identified a protein of approximately 42 kDa in Western blot assays using rat and mouse hippocampal proteins. The antibody also detected the expression of γ2 subunit by immunohistochemistry and could immunoprecipitate the γ2 subunit.