GABAA receptors are associated with retinal ganglion cell death induced by oxidative stress

https://doi.org/10.1016/j.exer.2008.01.019Get rights and content

Abstract

A previously proteomic study from our laboratory showed that the expression of the protein of the GABAA receptor β1 subunit was significantly increased in the retina of EAAC1-deficient (EAAC1−/−) mice, a mouse model of glaucoma. The purpose of this study was to investigate the role played by GABAA receptor β1 subunit in the death of retinal ganglion cells (RGCs) caused by oxidative stress. The retinal sites and expression pattern of GABAA receptor β1 subunit were determined by immunohistochemistry in the retina of ICR mice. A RGC line, RGC-5, was exposed to GABAA receptor agonist and antagonist, and the cell viability was determined by the MTS assay. The effect of GABAA receptor antagonist on the death of RGCs, and the activation of oxidative stress signaling induced by hydrogen peroxide was investigated. Our results showed that the GABAA receptor β1 subunit was expressed on the RGCs of ICR mice. GABAA receptor agonist induced RGCs death, and this death was inhibited by bicuculline, a GABAA receptor antagonist. The hydrogen peroxide-induced death of RGCs was reduced by GABAA receptor antagonist, and the oxidative stress signaling activated by hydrogen peroxide was also inhibited. These results indicate that GABAA receptors are expressed on RGCs and may play a role in the death of RGCs induced by oxidative stress.

Introduction

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter, is expressed on neurons in the central nervous system of humans and other mammals (Bowery and Smart, 2006). GABA is synthesized from glutamate by glutamic acid decarboxylase in neurons (Wiessner et al., 2002), and GABA-dependent signaling plays an important role in the retina (Wässle and Boycott, 1991).

GABA receptors are categorized into three types: GABAA, GABAB, and GABAC receptors. GABAA receptors are associated with Cl ion channels (Bormann et al., 1987), and consist of several subunits, viz., α1–6, β1–4, γ1–4, δ, ε, θ, π, and ρ1–3 (Rudolph and Möhler, 2006). Muscimol is an agonist of GABAA receptors, and has little influence on GABAC receptors. Bicuculline is a specific antagonist of GABAA receptors and has no effect on the other receptors. GABAA receptors are expressed on all types of neurons in the vertebrate retina (Yang, 2004). Retinal precursor cells also possess these receptors (Sun et al., 2002).

Excitatory amino acid carrier1 (EAAC1), one of the glutamate transporters (Sepkuty et al., 2002), is expressed on different types of cells but especially on neurons (Rothstein et al., 1994). EAAC1 functions as an anti-apoptotic agent, and can rescue motor neurons after injury (Kiryu-Seo et al., 2006). EAAC1 is expressed on horizontal cells, amacrine cells, and retinal ganglion cells (RGCs) in the retina (Barnett and Grozdanic, 2004).

The RGCs of EAAC1-deficient (EAAC1−/−) mice die in response to oxidative stress without an increase of the intraocular pressure (Harada et al., 2007). Proteomic screenings studies have been performed in our laboratory to identify the proteins that are up- or down-regulated in EAAC1−/− mice (Okumichi et al., 2007). Thirteen proteins were identified, and the GABAA receptor β1 subunit was one of the identified proteins. The volume of the GABAA receptor β1 subunit protein was significantly increased on two-dimensional gels.

Based on these results, we hypothesized that GABAA receptor-dependent signaling plays a role in the RGC death induced by oxidative stress.

Section snippets

Animals and cells

All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology's statement on the use of animals in ophthalmic research. These experiments were approved by the Animal Use Committee of Hiroshima University.

EAAC1−/− mice (on ICR mice background; Peghini et al., 1997) were kindly provided by Dr. Kohichi Tanaka (Tokyo Medical and Dental University), and wild type ICR mice were purchased from CLEA Japan, Inc (Tokyo, Japan). All animals were maintained in

Up-regulation of expression of GABAA receptor β1 subunit protein in retina of EAAC1−/− mice

To confirm our proteomics results (Okumichi et al., 2007), western blot analysis was performed to determine the level of expression of the protein of GABAA receptor β1 subunit. The expression of GABAA receptor β1 subunit protein was higher in the EAAC1−/− retina than in the retina of ICR mice (Fig. 1, lanes 1 and 2). The specificity of the anti-GABA-A receptor beta-1 antibody was high as shown by the overexpressed HA-tagged GABA-A receptor beta-1 proteins that were blotted with anti-HA antibody

Discussion

Our immunohistochemical results showed that the GABAA receptor β1 subunit was present on the RGCs of both EAAC1−/− and ICR mice (Fig. 2A). However, it could not be determined whether the quantity of GABAA receptor β1 expression in the retina of EAAC1−/− was significantly different from that of ICR mice. Western blot analysis showed clearly that GABAA receptor β1 in EAAC1−/− was expressed more strongly than in ICR mice (Fig. 1). These results suggested that the GABAA receptor β1 in the retina

Acknowledgments

We thank Prof. Neeraj Agawal for providing the RGC-5 cells and Professor Trevor G. Smart for the modified vector with GABAA receptor β1.

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