GABAA receptors are associated with retinal ganglion cell death 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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