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. 2013 Apr;54(4):616-24.
doi: 10.1111/epi.12063. Epub 2013 Jan 7.

Down-regulation of gephyrin and GABAA receptor subunits during epileptogenesis in the CA1 region of hippocampus

Marco I González  1 , Yasmin Cruz Del Angel, Amy Brooks-Kayal
Affiliations

Down-regulation of gephyrin and GABAA receptor subunits during epileptogenesis in the CA1 region of hippocampus

Marco I González et al. Epilepsia. 2013 Apr.

Abstract

Purpose: Epileptogenesis is the process by which a brain becomes hyperexcitable and capable of generating recurrent spontaneous seizures. In humans, it has been hypothesized that following a brain insult there are a number of molecular and cellular changes that underlie the development of spontaneous seizures. Studies in animal models have shown that an injured brain may develop epileptiform activity before appearance of epileptic seizures and that the pathophysiology accompanying spontaneous seizures is associated with a dysfunction of γ-aminobutyric acid (GABA)ergic neurotransmission. Here, we analyzed the effects of status epilepticus on the expression of GABAA receptors (GABAA Rs) and scaffolding proteins involved in the regulation of GABAA R trafficking and anchoring.

Methods: Western blot analysis was used to determine the levels of proteins involved in GABAA R trafficking and anchoring in adult rats subjected to pilocarpine-induced status epilepticus (SE) and controls. Cell surface biotinylation using a cell membrane-impermeable reagent was used to assay for changes in the expression of receptors at the plasma membrane. Finally, immunoprecipitation experiments were used to evaluate the composition of GABAA Rs. We examined for a correlation between total GABAA R subunit expression, plasma membrane expression, and receptor composition.

Key findings: Analysis of tissue samples from the CA1 region of hippocampus show that SE promotes a loss of GABAA R subunits and of the scaffolding proteins associated with them. We also found a decrease in the levels of receptors located at the plasma membrane and alterations in GABAA R composition.

Significance: The changes in protein expression of GABAA Rs and scaffolding proteins detected in these studies provide a potential mechanism to explain the deficits in GABAergic neurotransmission observed during the epileptogenic period. Our current observations represent an additional step toward the elucidation of the molecular mechanisms underlying GABAA R dysfunction during epileptogenesis.

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Conflict of interest statement

DISCLOSURE

None of the authors has any conflict of interest to disclose. The authors have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Figures

Figure 1
Figure 1. Expression of scaffolding proteins in CA1 during the epileptogenic period
Hippocampal slices (600 µm) were obtained from control (Ctl, day 0) and SE animals (1, 4 or 8 days after SE induction) to microdissect the CA1 region and prepare tissue lysates for Western blot analysis. (A) Representative blots of samples from the CA1 region probed with antibodies for gephyrin, GRIP, NSF, GABARAP or actin. (B) Densitometric analysis of the immunoreactivity for each scaffolding protein normalized to actin immunoreactivity. Data was obtained from four independent experiments and it is presented as the mean ± SEM (*p < 0.05 or **p < 0.01 compared to control by ANOVA followed by Bonferroni post hoc test). The overall values for the ANOVA analysis were: Gephyrin (F = 4.604, p < 0.0229), GRIP (F = 5.845, p < 0.0106), and GABARAP (F = 2.140, p < 0.1484).
Figure 2
Figure 2. Detection of gephyrin distribution in the CA1 region of hippocampus
Brain sections (40 µM) were collected from control (Ctl) and SE animals at 4 days post-SE to qualitatively evaluate the distribution of neuronal profiles and gephyrin staining. (A) Representative images of hippocampal sections of control and SE animals stained with the neuronal marker, NeuN. Low magnification images (4X) of the hippocampal structure do not show an evident alteration in the neuronal layers. Images of the CA1 region at a higher magnification (20X) show a similar distribution of neuronal profiles in control and SE tissue. (B) Double labeled immunofluorescence of brain sections with anti-gephyrin (green) and anti-VGAT (red) antibodies were used to qualitatively assess the distribution of gephyrin in the CA1 region of hippocampus. Confocal images show that gephyrin staining (green) at the cell bodies is surrounded by the VGAT staining (red) that delineate the cell bodies, some colocalization (yellow) is observed at both the cell bodies and stratum radiatum. Both gephyrin and VGAT staining appears to be more disperse in tissue obtained from SE animals. Images are representative of four independent experiments. Scale bar represents 20 µM.
Figure 3
Figure 3. Expression of GABAAR subunits in CA1 during the epileptogenic period
Tissue samples of CA1 region were obtained at 0, 1, 4 or 8 days after SE and analyzed by western blot. (A) Representative blots for the expression of α1, α4, β2/3, γ2 and actin in CA1 samples. (B) Summary of the immunoreactivity detected for each GABAAR subunit normalized to actin immunoreactivity. Data was obtained from four independent experiments and is presented as the mean ± SEM. (*p < 0.05, **p < 0.01 or ***p < 0.01 compared to control by ANOVA followed by Bonferroni post hoc test). The overall values for the ANOVA analysis were: α1 (F = 1.575, p < 0.2469), α4 (F = 18.361, p < 0.000), β2/3 (F = 8.396, p < 0.0028), and γ2 (F = 10.098, p < 0.0013).
Figure 4
Figure 4. Cell surface expression of GABAAR subunits during the epileptogenic period
Freshly prepared hippocampal slices (400 µM) from control (Ctl, day 0) or SE animals (at 1, 4 or 8 after SE) were labeled using a cell impermeable biotinylation reagent (sulfo-NHS-LC-biotin). All data are presented as mean ± SEM of four to five independent experiments. (A) Representative blots showing the immunoreactivity of α1, α4, β2/3, γ2 or actin in total lysates and biotinylated fraction. (B) Densitometric analysis for each GABAAR subunit in the biotinylated fraction, immunoreactivity in the biotinylated fraction of SE samples was compared to immunoreactivity in control samples by ANOVA with a Bonferroni post hoc test (*p < 0.05, **p < 0.01 or ***p < 0.001 compared to control). The overall values for the ANOVA analysis were: α1 (F = 6.307, p < 0.0056), α4 (F = 6.597, p < 0.0046), β2/3 (F = 3.471, p < 0.0430), and γ2 (F = 20.422, p < 0.0001). (C) Immunoreactivity for GABAAR subunits in the biotinylated fraction (plasma membrane) was normalized to immunoreactivity in the lysate (total protein expression) and compared control values to determine the fraction of each receptor subunit located at the plasma membrane. The overall values for the ANOVA analysis were: α1 (F = 0.9445, p < 0.4439), α4 (F = 1.424, p < 0.2750), β2/3 (F = 3.079, p < 0.0596), and γ2 (F = 4.801, p < 0.0154). Bonferroni post hoc test *p < 0.05 compared to control).
Figure 5
Figure 5. GABAAR subunit composition following SE induction
GABAAR were immunoprecipitated from samples obtained 1 day after SE induction. Pre-cleared lysates (300–350 µg of protein) of whole tissue obtained from control (Ctl) and SE animals were incubated with 5 µg of mouse monoclonal antibodies directed to β2/3 or α1 subunits or with non-immune mouse IgG (used as control). Immune complexes were separated by SDS-PAGE and probed with α4, γ2 or α1 antibodies to determine the immunoreactivity associated to ββ2/3 or α1 subunits. (A) Representative blots of the α4, γ2 and α1 immunoreactivity detected in β2/3 immunoprecipitates. (B) Analysis of α4, γ2 and α1 immunoreactivity associated to β2/3 subunits. Data is presented as mean ± SEM of four to five independent experiments. *p < 0.05 compared with immunoreactivity in control samples by paired students t-test. The values for the t-test were: α4 (t = 2.846, p < 0.0466), α1 (t = 3.899, p < 0.0176), and γ2 (t = 4.007, p < 0.0279). (C) Representative blots showing the α4, γ2, α1 immunoreactivity detected in immunoprecipitates prepared using an α1 antibody. (D) Analysis of α4 and γ2 immunoreactivity detected in α1 immunoprecipitations. Data is presented as mean ± SEM of five independent experiments. *p < 0.05 compared with immunoreactivity in control samples by students t-test. The values for the t-test were: α4 (t = 3.027, p < 0.0389) and γ2 (t = 0.7874, p < 0.4751). (◄) Points to the signal produced by the heavy chains of the control IgG or antibodies for β2/3 or α1 subunits. (formula image) Points to the immunoreactivity of α4, γ2 or α1 subunits associated with β2/3 or α1 subunits.
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