Heterogeneous GABAA receptor subunit expression in pediatric epilepsy patients

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Abstract

The γ-amino-butyric acid type A receptors (GABAAR) are a heteropentameric receptor complex, composed of 16 possible subunits in various combinations, forming a ligand-gated ion channel. Subunit composition is the primary determinant of GABAAR physiology and pharmacology. Here we have measured mRNA levels for 16 GABAAR subunits in isolated dentate granule neurons (DGN) from eight pediatric patients undergoing resective surgery for intractable epilepsy. We found tightly correlated expression of a subset of GABAAR subunit mRNAs within a single DGN (α1, γ1, and γ2; α4, α5, and β2; α4 and β3). Analysis of inter-patient variability (ANOVA) of eleven highly expressed GABAAR subunit mRNAs found seven of the subunits varied between patients, as did whole cell GABAAR currents. Due to inter-patient differences, there is heterogeneity in DGN GABAAR subunit mRNA and physiology within pediatric epilepsy patients. Patient-specific GABAAR expression might contribute to variability in anti-epileptic drug efficacy, side-effect profiles, and seizure susceptibility.

Introduction

γ-Amino-butyric acid receptors are the major inhibitory neurotransmitter receptors expressed in the central nervous system. They are divided into 3 families, A, B, and C, with GABAA receptors (GABAAR) being the primary fast ligand-gated anion channel. There are at least 16 different GABAAR subunits grouped together based on their sequence homology, α (1–6), β (1–3), γ (1–3), δ, ε, θ, and π (Ticku and Mehta, 1999, Whiting, 1999). GABAARs are heteromeric receptors formed from five subunits, and while a large number of theoretical combinations could form a pentameric receptor, animal studies suggest only subsets of compositions are actually present (Barnard et al., 1998). The most common formulation contains two α, one or two β, and one or two γ subunits, with potential substitution of ε or δ for the γ subunit.

Specific GABAAR subunit compositions have been associated with certain phenotypes such as anxiety, aggression, learning, and memory deficits (Collinson et al., 2002, Delaney and Sah, 1999, Jorgensen and Miczek, 2003, Rudolph et al., 1999). Recombinant and gene knockout studies suggest that altering subunit composition changes receptor kinetics, pharmacology, and physiology, which impacts behavior. GABAAR subunit composition also directs sub-cellular localization to synaptic and extrasynaptic regions, and thus contributes to fast and tonic forms of GABA-mediated inhibition, respectively (Kay et al., 2001, Mody and Nusser, 2002). Phosphorylation sites are found on the β subunits allowing for modulation of GABAAR by a diverse cascade of signals (McDonald et al., 1998, Poisbeau et al., 1999). Endogenous modulators of GABAAR functions such as neurosteroids, dopamine receptors, zinc, and protons have different efficacy depending on GABAAR subunit composition (Harrison et al., 1999, Hosie et al., 2003, Liu et al., 2000, Wegelius et al., 2000). Finally, there are a variety of exogenous drugs, benzodiazepines, ethanol, steroids, and barbiturates, which depending on subunit composition modulate a subset of GABAAR (Sieghart, 2000). Thus, slight variations in GABAAR subunit composition could contribute to both normal behavior as well as pathologic conditions.

Findings in animal models suggest that GABAAR subunit expressions vary across cell types, structural regions, and over the course of brain development (Laurie et al., 1992a, Laurie et al., 1992b, Wisden et al., 1992). A subset of GABAAR subunits is primarily synaptic (α1, α2, α3, and γ2) and another group that is preferentially extra-synaptic (α4, α5, and δ) (Brunig et al., 2002, Wei et al., 2003). The regulation of GABAAR subunit expression appears to be highly controlled with transcription being one level of regulation (Holt et al., 1997, McLean et al., 2000, Mu and Burt, 1999, Russek et al., 2000). Cell type and sub-cellular-specific expression patterns are also controlled by protein sequences that guide translation and subcellular localization (Connolly et al., 1996, Nusser et al., 1998, Nymann-Andersen et al., 2002).

Altered GABAAR-mediated inhibitory synaptic transmission has been implicated in multiple types of epilepsy. Mutations in certain subunits (γ1, γ2, α1) have been reported in familial forms of generalized epilepsy (Baulac et al., 2001, Cossette et al., 2002, Harkin et al., 2002, Marini et al., 2003). Animal studies suggest that there are dramatic changes in the composition, expression levels, and physiology of GABAAR during and following status epilepticus that may contribute to the development of focal epilepsy such as temporal lobe epilepsy (TLE) (Brooks-Kayal et al., 1998a, Brooks-Kayal et al., 1998b, Buckmaster and Dudek, 1997a, Buckmaster and Dudek, 1997b, Kapur and Macdonald, 1997, Rikhter et al., 1997). Findings in animal models suggest that changes in inhibitory neurotransmission in epilepsy occur at multiple levels, with inhibitory cell loss, increased and decreased GABAAR expression, and alterations in GABAAR subunit composition (Buckmaster and Dudek, 1997a, Brooks-Kayal et al., 1998b, Sloviter, 1983, Titulaer et al., 1995). Immunohistochemical studies of GABAAR subunit expression in patients with TLE found differences in GABAAR subunits in specific cell types of the hippocampus as compared to autopsy controls. The dentate gyrus, the major excitatory input region into the hippocampus, had increased expression of all 3 β isoforms, as well as, α1, α2, and γ2 subunits (Loup et al., 2000, Pirker et al., 2003). Our prior study in adults with intractable TLE found coordinated regulation of some GABAAR subunit mRNAs within hippocampal dentate granule neurons (DGN) (Brooks-Kayal et al., 1999). There also was heterogeneity of GABAAR subunit expression in DGN with at least two populations of DGN expressing varied GABAAR subunit combinations. Our prior study was limited to five adults with TLE and wished to expand our focus to include GABAAR subunit expression during normal development and in pediatric epilepsy models. Here we have studied pediatric epilepsy patients to determine if they have a unique pattern of DGN GABAAR subunit expression (Brooks-Kayal and Pritchett, 1993, Brooks-Kayal et al., 1998a). We describe DGN GABAAR subunit expression and whole cell GABA electrophysiology from 8 children and adolescents that underwent resections including mesial temporal structures for treatment of their medically intractable epilepsy.

Section snippets

Methods

This study was carried out at the Children's Hospital of Philadelphia with the approval of the Institutional Review Board on Human Studies.

Results

Fifty-two DGNs from 8 children and adolescents undergoing hippocampal resections for intractable epilepsy underwent RNA amplification to measure the relative expression of the 16 GABAAR subunits. They are a diverse group of patients reflecting a spectrum of pediatric epilepsy disorders (see Table 1). Out of the 16 measured GABAAR subunits, 11 had mean expression levels felt to be consistently above our cutoff for background non-specific hybridization (see Fig. 1 and Methods).

Correlation

Discussion

Our data support coordinated RNA expression levels of a subset of GABAAR subunits, suggesting transcriptional co-regulation. α1, γ1, and γ2 are the most tightly correlated and the most highly expressed subunits in these human DGN. Several studies suggest that these subunits are predominantly synaptic, form an allosteric interaction, and at their interface form the benzodiazepine-binding pocket (Nusser et al., 1998, Rudolph et al., 1999). The second group of highly correlated subunit mRNAs, α4,

Acknowledgment

This work was funded by the NINDS; R01 NS38595-01 to ABK, K08 NS 044869 to BEP. We wish to thank Drs. Jeffery Golden and Alexander Judkins for neuropathology assistance.

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