Reduction of α1GABA_A receptor mediated by tyrosine kinase C (PKC) phosphorylation in a mouse model of fragile X syndrome

Abstract

Fragile X syndrome (FXS) caused by lack of fragile X mental retardation protein (Fmr1) is the most common cause of inherited intellectual disability and characterized by many cognitive disturbances like attention deficit, autistic behavior, and audiogenic seizure and have region-specific altered expression of some gamma-aminobutyric acid (GABA_A) receptor subunits. Quantitative real-time polymerase chain reaction and western blot experiments were performed in the cultured cortical neurons and forebrain obtained from wild-type (WT) and Fmr1 KO mice demonstrate the reduction in the expression of α1 gamma-aminobutyric acid (α1GABA_A) receptor, phospho-α1GABA_A receptor, PKC and phosphor-PKC in Fmr1 KO mice comparing with WT mice, both in vivo and in vitro. Furthermore, we found that the phosphorylation of the α1GABA_A receptor was mediated by PKC. Our results elucidate that the lower phosphorylation of the α1GABA_A receptor mediated by PKC neutralizes the seizure-promoting effects in Fmr1 KO mice and point to the potential therapeutic targets of α1GABA_A agonists for the treatment of fragile X syndrome.

Introduction

Fragile X syndrome (FXS), the most common single-gene cause of inherited intellectual disability, is caused by epigenetic silencing of the fragile X mental retardation gene (Fmr1) and eventually lack of fragile X mental retardation protein (FMRP), which leads to decreased inhibition of translation of many synaptic proteins [1]. As a selective RNA-binding protein, FMRP mostly located at the synapse in neurons regulates RNA transportation, stabilization, and translation. There are 5-44 CGG repeats on the Fmr1 gene located on the X chromosome and this trinucleotide repeat length can expand to an unstable repeat length [2]. The absence of expression of FMRP caused by a dynamic mutation of more than 200 CGG trinucleotide repeats in the 5’ untranslated region on the Fmr1 gene results FXS [3].

Fmr1 KO mice with absence of FMRP expression were radically susceptibility to audiogenic seizures when compared WT mice [4]. Audiogenic seizures are a major form of rodent neurological disorder that can be genetically mediated and can also be readily induced in both young and mature animals [5]. Previous work has demonstrated reduce expression of gamma-aminobutyric acid A (GABA_A) receptors in subjects with fragile X syndrome [6]. Less is known about levels for GABA_A receptor subunit α1 expression in brains of subjects with audiogenic seizures. Here we show that the depression of α1GABA_A receptor, phospho-α1GABA_A receptor, PKC and phospho-PKC and the higher audiogenic seizures susceptibility in Fmr1 KO mice. Furthermore, we found the PKC was involved phosphorylation of α1GABA_A receptor in mouse cortical neurons. These findings suggest that the lower phosphorylation level of α1GABA_A receptor mediated by PKC is a potential signaling relating to increase of audiogenic seizures susceptibility in Fmr1 KO mice. Our results also suggest the α1GABA_A agonists may be a potential therapy method for the treatment of fragile X syndrome.

Materials and methods

Animals

All animal experiments were carried out in accordance with the guidelines set out by the XX University Animal Care and Use Committee. Fmr1 KO mice on the FVB background were purchased from the Jackson Laboratory (stock number: 004624) and bred at the University of XX. All possible efforts were made to minimize the number of animals used in experiments and their discomfort. All experimental animals were maintained in a temperature/humidity-controlled room on a 12 h/12 h light/dark cycle with free access to food and water.

Genotyping

Fmr1 genotyping was based on the presence or absence of the wild-type or knockout Fmr1 allele. For the wild-type allele, primer S1 (5’ GTG GTT AGC TAA AGT GAG GAT GAT 3’) and S2 (5’ CAG GTT TGT TGG GAT TAA CAG ATC 3’) and the knockout allele using primer M2 (5’ ATC TAG TCA TGC TAT GGA TAT CAG C 3’) and N2 (5’ GTG GGC TCT ATG GCT TCT GAG G 3’). The following PCR conditions were used: 95°C for 5 min; 34 PCR cycles were performed composed of 30 sec at 95°C, 30 sec at 61°C, and 1 min at 72°C. KO and WT PCR reactions were run separately; the reaction products were then combined and electrophoresed on a 1.5% agarose gel [WT: 465 BP (S1/S2); KO: 800 BP (M2/N2)].

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from mouse forebrain or cortical neurons using the RNeasy kit (Qiagen) following the manufacturer’s protocol. Two micrograms of total RNA was reverse-transcribed with random nonomers (Sigma) using the Superscript II Reverse Transcriptase (Invitrogen) as described by the manufacturer. The cDNA samples were amplified using the DyNAmo Flash SYBR Green quantitative PCR kit (Thermo Scientific) and detected via the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Primers for α1GABA_A receptor, PKC and β-actin quantification were designed with Express v2.0 software (Applied Biosystems) or manually and contained an intronic sequence in between. The primers used were: α1GABA_A receptor, sense 5-TTA TGG GTG CAT GGA TCA CTC C-3, antisense 5-CCC ACG TAA GGT CAT CAT GGA T-3; PKC sense 5-GGA TCC AAG TGT AAT TGT TC-3, antisense 5-GTA GTA CTA TGA ACG GTA TC-3; β-actin, sense 5-TGG AAT CCT GTG GCA TCC ATG AAA C-3, antisense 5-TAA AAC GCA GCT CAG TAA CAG TCC G-3.

Preparation and phospholabeling of cortical neurons

Primary cortical neurons isolated from embryonic day 18-19 WT and Fmr1 KO mice were plated at 175,000 cells/mL in Neurobasal Media (Invitrogen) supplemented with 2% B-27 (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 0.25% 100X glutamine and 0.1% of 10 mM glutamate (Invitrogen). After 7 days in culture, neurons were washed in phosphate-free medium (Life Technologies, Inc. Ltd; 37°C, 10% CO₂) and incubated for 4 h in phosphate-free medium containing 0.5 mCi/ml [³²P] orthophosphoric acid (Amersham Pharmacia Biotech). Approximately 10⁶ neurons were used per treatment. PDBu (100 nM, activator of PKC) and Calphostin C (2.5 mM, inhibitor of PKC) were added for 5-30 min during or at the end of the labeling period. α1GABA_A receptor was then immunoprecipitated using polyclonal antisera against the receptor α1GABA_A receptor coupled to protein A-Sepharose as described below.

Immunoprecipitation

Cortical neurons transiently expressing GABA_A receptors were solubilized in a buffer containing 1% Triton X-100, 0.5% deoxycholate, 10 mM sodium pyrophosphate, 20 mM sodium phosphate, 40 mM Tris, pH 7.4, 50 mM sodium fluoride, 150 mM sodium chloride, 1 mM sodium vanadate, 5 mM EGTA, 5 mM EDTA, 5 mM phenylmethylsulfonyl fluoride, 20 mg/ml antipain, leupeptin, pepstatin, and 10 units/ml aprotinin. After microcentrifugation at 14,000 rpm, the detergent-soluble cell lysates were collected. The GABA_A receptor was then immunoprecipitated using a polyclonal antisera against the receptor α1/6 or α1 subunits coupled to protein A-Sepharose as described previously [7]. Precipitated material was resolved by SDS-PAGE, visualized by autoradiography, and quantified using a phosphorimager (Bio-Rad). For the experiment in Figure 2, a precipitated material was subjected to Western blotting with against the α1GABA_A receptor.

Figure 2.

The α1GABA_A receptor is phosphorylated in Fmr1 KO and WT mouse cortical neurons and this phosphorylation was mediated by a PKC-dependent mechanism. A. Cortical neurons were lysed and GABA_A receptor α subunits were immunoprecipitated with antibodies against the indicated subunits. Precipitated material was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a α1-specific antibody. IN represents 25% of the input material used for the immunoprecipitation. B. Cortical neurons were prelabeled for 4 h with [³²P] orthophosphate and lysed, and receptors were immunoprecipitated with α1/6 antisera or control IgG. Precipitated material was then separated by SDS-PAGE and visualized by autoradiography. C. Cortical neurons were prelabeled as described in 2. 30 min prior to lysis, cells were treated with specific kinase inhibitors as indicated. Lane 2, PDBu (100 mM, activator of PKC); lane 3, Calphostin C (2.5 mM, an inhibitor of PKC). UT, untreated control cells. Receptors were immunoprecipitated with α1/6 antisera, and precipitated material was then separated by SDS-PAGE and visualized by autoradiography. D. Cortical neurons were labeled with [³²P] orthophosphoric acid and were then treated for various time periods with PDBu (100 nM). GABA_A receptors were then immunoprecipitated with anti-α1/6 antisera, resolved by SDS-PAGE and visualized with autoradiography. E. The level of GABA_A receptor α1 subunit phosphorylation was quantified using a phosphorimager and plotted against time. Counts are in relation to the value at the zero time point. F. Samples of cortical neurons were treated with PDBu (100 nM) or treated with vehicle alone for 15 min, and the protein level of the α1 subunit was then evaluated with antisera specific for the α1 subunit by Western blotting. Data shown are representative of three independent experiments.

Western blotting

Adult WT and Fmr1 KO mice were euthanized with an overdose of ketamine/xylazine. Proteins were extracted from the whole forebrain or cortical neurons were homogenized in a RIPA lysis buffer (Sigma) containing protease inhibitors (Roche) and the total protein was extracted and detected and placed on ice. Twenty micrograms protein per sample was separated by SDS-polyacrylamide gel electrophoresis, with samples being loaded onto each lane of 12% polyacrylamide gels. The protein was transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 250 mA. The membranes were blocked with 5% nonfat milk in TRIS buffered saline containing 0.1% Tween 20 at room temperature and then probed with the following primary antibodies at 4°C overnight: rabbit anti-α1GABA_A receptor (1:1000; ab33299, Abcam), rabbit anti-phospho-α1GABA_A receptor (1:1000, phosphorylated S408/409 epitope, gift from XXX), goat anti-α6GABA_A receptor (1:1000; ab117100, Abcam), mouse anti-PKC (1:2000; ab31, Abcam), rabbit anti-PKC (phospho T497) (1:1000; ab59411, Abcam) and rabbit anti-β-actin (1:5000; ab179467, Abcam). Following by an overnight incubation in primary antibodies at 4°C, the protein was transferred onto a polyvinylidene difluoride (PVDF) membrane for 120 min at 250 mA. The membranes were then incubated with second antibodies for 60 min at room temperature. After incubation with secondary antibodies diluted in 5% nonfat milk in TRIS buffered saline containing 0.1% Tween 20, the membranes were subjected to enhanced chemiluminescence (ECL detection system; sc-2048, Santa Cruz Biotechnology).

Audiogenic seizure susceptibility

Audiogenic seizure sensitivity was determined by placing mice individually to into the empty plastic box (28×17×14 cm) with a sound-absorbent tile lid under which the siren was mounted with a 125 db at 0.25 m siren (modified personal alarm, Radioshack model 49-1010, powered from a DC converter) for 2 minutes prior to stimulus onset. Mice were tested between 03:00 p.m. and 05:00 p.m. for possible circadian variation. Seizures were scored using a seizure severity score as follows: wild running = 1; clonic seizure = 2; tonic seizure = 3; status epilepticus/respiratory arrest/death = 4 [8].

For drug injection studies, an intraperitoneal injection of drug or vehicle (0.1 ml/10 g body weight) was administered 30 or 45 min before seizure testing. The drug doses and vehicles are as follows: 1.0 mg/kg zolpidem (α1GABA_A agonist) [9] (Sigma) in distilled water (85%); 1.0 mg/kg β-Carboline-3-carboxylate-t-butyl ester (β-CCt) 1.0 mg/kg (GABA_A antagonist) [9] (Sigma) in propylene glycol (14%). Fisher’s exact test was used for statistical analysis of seizure susceptibility data.

Statistical analyses

All data were analyzed using analysis of variance (ANOVA) with Graphpad Prism 4 software. We use t-tests for post hoc analyses and significance determined as P<0.05 across the two genotypes (WT and Fmr1 KO). All experiments were performed a minimum of three times.

Results

The mRNA and/or protein expression patterns of PKC, phosphorylated PKC, α1GABA_A receptor and phosphorylated α1GABA_A receptor

The mRNA and/or protein expression patterns of PKC, phosphorylated PKC, α1GABA_A receptor and phosphorylated α1GABA_A receptor in cortical neurons and whole forebrain were analyzed q-PCR and western blotting in vivo and in vitro, comparing WT and Fmr1 KO mice. q-PCR assays indicated that mRNA expression of PKC and α1GABA_A receptor decrease significantly in cortical neurons and whole forebrain (Figure 1A; Table 1, P<0.05). Western blotting analyses revealed that PKC, phosphorylated PKC, α1GABA_A receptor and phosphorylated α1GABA_A receptor protein levels in cortical neurons and brains of Fmr1 KO mice were significantly lower by half compared to WT mice (Figure 1B; P<0.05).

Figure 1.

Level of PKC, phosphorylated PKC, α1GABA_A receptor and phosphorylated α1GABA_A receptor. A. Expression of PKC and α1GABA_A receptor in cortical neurons and whole forebrain of WT versus Fmr1 KO mice. B. Proteins from cortical neurons and whole forebrain of WT and Fmr1 KO mice were homogenized in RIPA buffer and analyzed for PKC, phosphorylated PKC, α1GABA_A receptor, phosphorylated α1GABA_A receptor β-actin by western blotting. These protein levels were normalized to β-actin. Histogram bars as mean ± s.e., *P<0.05.

Table 1.

qRT-PCR results for PKC and α1GABA_A receptor in cortical neurons

Lateral cerebellum		Schizophrenia
Gene	ANOVA	Fold change	P *
GABRQ	0.046	0.64	0.016
GABRR2	0.017	1.33	0.11
GRM5	0.034	0.49	0.039
FMR1	0.099	0.65	0.16

Abbreviations: ANOVA, analysis of variance; PKC, protein kinase C; qRT-PCR, quantitative real-time polymerase chain;

^*P<0.05.

The α1GABA_A receptor is basally phosphorylated in cortical neurons

Immunoprecipitation was used to examine the phosphorylation of α1GABA_A receptor in cultured cortical neurons, both WT and Fmr1 KO mice. Detergent-solubilized extracts of cultured cortical neurons were immunoprecipitated with α1/6 and anti-α1 antibodies or with control IgG. This procedure resulted in the immunoprecipitation of a major band of 51 kDa that was recognized by antisera specific for the α1GABA_A receptor by Western blotting (Figure 2A). Material precipitated with anti-α1/6 was also probed with antisera specific for the α6 subunit [10]. No signal was obtained with this antisera suggesting that only low amounts of α6 subunit are expressed in cultured cortical neurons. A molecular mass of 51 kDa has previously been reported for the α1 subunit in HEK293 cells [10]. Immunoprecipitation with anti-α1/6 receptor from cortical neurons that had been prelabeled with [³²P] orthophosphoric acid specifically precipitated a major phosphoprotein of 51 kDa, demonstrating that the α1 subunit is basally phosphorylated in cortical neurons (Figure 2B).

Phosphorylation of the α1GABA_A receptor in cortical neurons is mediated by PKC Activity

GABA_A receptors subunits contain phosphorylation sites and could be phosphorylated by a number of second messenger-dependent protein kinases in recombinant preparations, including PKA, PKC, CamKII, and cGMP-dependent kinase. Phosphorylation can alter receptor function directly by changing its conformation and/or indirectly by altering receptor expression [11]. PKC could phosphorylate α1GABA_A receptor in rat cerebral cortical neurons [12]. To confirm this result in mouse, specific kinase inhibitors were utilized. Activator of PKC (PDBu) and inhibitor of PKC (Calphostin C) had little effect on the basal phosphorylation (less than 10% in three separate experiments) of the b3 subunit (Figure 2C). Inhibition of PKC activity and with the specific PKC inhibitor calphostin C had a dramatic effect on phosphorylation of the α1 subunit. Quantitation of inhibition indicated that calphostin C produced a 3-fold decrease (0.3±0.1 of control, n = 3) in α1 subunit phosphorylation (Table 2). To further analyze the role of PKC activity in controlling the phosphorylation of the α1 subunit in cortical neurons, the effect of phorbol esters was tested. Treatment of cortical cultures with 100 nM PDBu, an activator of PKC, caused a time-dependent increase in the phosphorylation of α1 as measured by immunoprecipitation after [³²P] orthophosphate labeling (Figure 2D and 2E). This increase was evident after 5 min and reached steady state after 15 min (Figure 2D and 2E). Therefore, all further experiments were performed after 15 min of exposure to the kinase activator. PDBu on average produced a 2.4-fold increase in b3 subunit phosphorylation (Table 2). PKC activity did not appear to be associated with receptor degradation as illustrated by Western blotting with α1-specific antisera which revealed identical protein levels in the presence and absence of PDBu treatment (Figure 2F). Together, these observations strongly suggest that a PKC-dependent pathway is responsible for the high basal phosphorylation of the α1 subunit in cortical neurons.

Table 2.

Effect of PKC activators and inhibitors on the phosphorylation of the α1 subunit

Treatment	Basal	+ PDBu	+ calphostin C
β3 phosphorylation	1.0	2.4±0.4α	0.34±0.1α

^αSignificantly different using the Student’s test (P<0.05).

Phosphorylation of the α1 subunit was compared in cortical neurons that had been treated with either 100 nM PDBu or 2.5 mM calphostin C to modulate the activity of PKC. The α1 subunit was immunoprecipitated from cortical neurons prelabeled with [³²P] orthophosphoric acid after treatment as indicated. The level of phosphorylation was quantified using a phosphorimager. Values for cultures treated with PDBu or calphostin C were then compared to basal conditions, which was given an arbitrary value of 1. N = 3 for each case.

Increase of audiogenic seizures susceptibility in Fmr1 KO mice and zolpidem reduces this phynotype

It has been reported that Fmr1 KO mice are more sensitive to seizures induced by auditory stimulus than WT animals [4]. To confirm this phenotype, audiogenic seizure susceptibility were performed in Fmr1 KO mice comparing with WT mice. Mice were exposed to a 125 db alarm for 2 min, and seizure susceptibility was evaluated as described previously [8]. In total, 53% of Fmr1 KO mice exhibited sound induced seizures compared with 4% of wild-type animals (Figure 3A, P<0.001). Our result found this to be the case again and Fmr1 KO mice were radically more prone to audiogenic seizures. To test the role of GABA_A receptors in audiogenic seizure susceptibility, Fmr1 KO mice were treated with the GABA_A agonist zolpidem (1.0 or 2.0 mg/kg i.p.) administered 45 min before seizure testing. Treatment with 1.0 and 2.0 mg/kg zolpidem produced a 67% (P<0.05) and 79% (P<0.01) decrease, respectively, in seizure incidence (Figure 3B) compared with vehicle controls. These results showed that stimulus mediated by α1GABA_A signaling rescues seizures in Fmr1 KO mice.

Figure 3.

Increase of susceptibility of Fmr1 KO mice to audiogenic seizures and α1GABA_A agonist zolpidem can reduce this phenotype. A. Mice were tested for audiogenic seizures at postnatal day 27 to 30 and Fmr1 KO mice were more susceptible to audiogenic seizures than WT mice (***, P<0.001). B. Fmr1 KO mice (27-30 days old) were treated with 1 or 2 mg/kg zolpidem. zolpidem significantly reduced audiogenic seizure incidence compared with vehicle controls (*, P<0.05; **, P<0.01).

Discussion

Here, we have shown that both levels of total α1GABA_A receptor and PKC and levels of phosphorylated α1GABA_A receptor and phosphorylated PKC decrease in cortical neurons and forebrain in Fmr1 KO mice versus WT mice. Since Fmr1 KO mice show increased audiogenic seizures susceptibility versus WT animals [4], the lower phosphorylation of α1GABA_A receptor in Fmr1 KO mice may relate with the higher audiogenic seizures susceptibility. The GABAergic system was first implicated in the pathogenesis of FXS based on studies of GABA_A receptor expression in Fmr1 KO mice. Glutamate and gamma-aminobutyric acid (GABA) are major excitatory and inhibitory (E-I) neurotransmitters and the E-I balance plays an important role in brain functions [11]. The GABA system mediating most fast excitatory and inhibitory transmission in the brain contributes to the forms of synaptic plasticity thought to underlie memory acquisition [12]. The neurotransmitter GABA binds to these receptors, changing their conformation state and thereby opening the pore to allow chloride ions (Cl^-) to pass down an electrochemical gradient. The flux of chloride ions hyperpolarizes the membrane leading to neuronal inhibition [13].

There are GABA_A receptor and GABAB receptor. GABA_A receptors are a family of chloride ion channels that allows the flow of chloride ions across the membrane, which predominately mediate rapid inhibitory neurotransmission by hyperpolarizing the neuron’s postsynaptic membrane and minimizes the effect of any coincident synaptic input [14]. Functional GABA_A receptors are heteropentamers, and subunit composition is an important determinant of inhibitory transmission, dictating receptor characteristics like response kinetics, subcellular localization, and sensitivity to a number of clinically important compounds [15]. Molecular cloning has revealed multiple GABA_A receptor subunits that can be divided by homology into subunit classes with several members: α (1-6), β (1-3), γ (1-3), δ, ε, θ, π, and ρ (1-3) [16]. GABA_A receptor subunits contain phosphorylation sites for many protein kinases including PKC, PKA, and fyn kinase involving in trafficking of GABA_A receptors. Phosphorylation can alter receptor function by changing its conformation and/or by altering receptor expression [14]. Our results identified that the phosphorylation of phosphorylation of the α1GABA_A receptor in mouse cortical neurons was mediated by PKC activity. It is possible that attenuated α1GABA_A receptor signaling mediated by PKC in neurons may explain the observed increase in susceptibility to audiogenic seizures in Fmr1 KO mice when compared with WT mice.

The GABA_A receptor α1 subunit is the most abundant α subunit in adult brain, highly expressed throughout most brain regions and is a component of ~50% of all GABA_A receptors [17]. PKA was involved in the phosphorylation of α1GABA_A receptor in cerebellar granule cells [15]. PKCγ co-immunoprecipitates with α1-and α4-GABA_A receptors in the cerebral cortex [18]. PKC was involved in the phosphorylation of α1GABA_A receptor in rat cerebral cortical neurons [19]. Phosphorylation of GABA_A receptor subunits can modify GABA binding to its receptor channel conductance, and possibly internalization [20]. Since GABA_A receptor α1 subunits are the most abundant subunit in the cerebral cortex, the reduction in the expression of this subunit is likely to have significant functional consequences. In support of a role for a protective effect of GABA signaling in the prevention of fragile X audiogenic seizures, we observed reduced audiogenic seizures in Fmr1 KO mice after treatment with the α1GABA_A agonist-zolpidem. Mounting evidence suggests that in the auditory pathways involved in audiogenic seizure induction and progression, auditory signals are balanced by trafficking of α1GABA_A receptor through PKC signaling.

FMRP can bind subunit the mRNAs encoding eight different GABA_A receptor subunits (α1, α3, α4, β1, β2, γ1, γ2, and &) and significantly reduce the expression of these receptors in the brains of Fmr1 KO mice in the behaviorally relevant brain regions such as cortex and hippocampus [21].

There are downregulation of PKC expression and decrease in phosphorylation level of PKC in fibroblastic cell lines derived from Fmr1 KO embryo [22]. Consequences of the reduced GABA_A receptor expression in FXS likely include oversensitivity to sensory stimuli, audiogenic seizures, and anxiety. Our results have confirm this phenotype again and further clarified that the lower phosphorylated level of α1GABA_A receptor mediated by PKC is a potential signaling relating to increase of seizure susceptibility in Fmr1 KO mice. Taken together, it appears that PKC mediated phosphorylation of α1GABA_A receptor plays an important role in Fme1 KO mice and point to the potential therapeutic targets of GABA_A agonists for the treatment of fragile X syndrome.

Disclosure of conflict of interest

None.