Regulation of the expression of GABAA receptor subunits by an antiepileptic drug QYS

Neuroscience Letters 392 (2006) 145–149

Regulation of the expression of GABAA receptor subunits by an antiepileptic drug QYS Xianchun Li a , Qingxiong Yang b , Yinghe Hu a,∗ a

Key Lab of Brain Functional Genomics, MOE & STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, Shanghai 200062, China b Yunnan Yunyao Laboratory Co., Ltd., Kunming 650106, China Received 27 July 2005; received in revised form 4 September 2005; accepted 6 September 2005

Abstract It has been reported that the antiepileptic drug qingyangshenylycosides (QYS) modulated the function of GABAergic system. However, little is known about the effects of QYS on the gene expression of GABA receptors in the central nervous system (CNS). In the present study, we examined the effects of QYS on the expression of GABAA receptor subunits in different regions of the mouse brain. The results showed that treatment of QYS significantly increased the expressions of Gabra1, Gabra2 and Gabr4 and decreased the expression of Gabrg2 in inferior colliculus. Moreover, Gabrb2 expression was up-regulated and Gabra5 was down-regulated in hippocampus, while the expressions of Gabra1 and Gabrb2 were induced in cortex after QYS treatment. These data indicated that QYS had different effects on the expression of GABAA receptor subunits in different brain regions. These results may help to reveal the molecular mechanism of anticonvulsant action of QYS. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: GABAA receptor; Qingyangshenylycosides; Gene expression; Central nervous system (CNS)

Epilepsy is one of the most common brain diseases in humans. About 1% of the population is diagnosed with the disease [27]. Epileptic seizures result from excessive discharge in a population of hyperexcitable neurons. Most epileptic seizures are due to discharges generated in cortical and hippocampal structures, but subcortical structures are also involved in some seizure types [1]. It is well known that inhibitory GABA systems in the mammalian central nervous system play an essential role in the control of neural activity and the suppression of epileptiform discharge [41]. Failure of GABAergic inhibition is a widely proposed mechanism for the generation of epilepsy [28]. For example, repeated intra-amygdala application of the selective GABA–A receptor antagonist bicuculline would produce epileptic response. This effect could be blocked by coinjection with GABA agonists, such as muscimol [42]. Other evidence demonstrated that several kinds of human epilepsy, such as idiopathic generalized epilepsy (IGE), febrile seizure, and juvenile myoclonic epilepsy (JME), were associated with mutations of the GABAA receptor ␣1 [5], ␥2 [2,44] and ␦ [26]

∗

Corresponding author. Tel.: +86 21 62232789; fax: +86 21 62601953. E-mail address: [email protected] (Y. Hu).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.09.011

and GABABR1 subunits [3,31]. These mutations could affect GABAA receptor gating, expression, and trafficking of the receptor to the cell surface. Much evidence indicated that qingyangshenylycosides (QYS) had a significant anticonvulsant action on kindling effect [32], rat audiogenic seizures [32], and electroshock seizure [16]. We have found that QYS could prolong the latency of audiogenic seizure (AGS) and reduce the severity of AGS in DBA mice [21]. Furthermore, QYS has been used clinically for the treatment of chronic human epilepsy [30], but the mechanism of action of the drug is not clear. In the present study we focused on the gene expression of GABAA receptor subunits in different brain regions. Our results suggested that alteration of GABAA receptor subunits expression might contribute to the antiepileptic effects of QYS. Qingyangshenylycosides (QYS) were the gift from Yunnan Baiyao Group Co., Ltd. The drug was dissolved in 1.5% Tween80 saline, filtered and stored at 4 ◦ C before use. Twelve 3- to 4-week-old DBA/2J mice were used in this study (from the Experimental Center of Chinese Academic of Sciences, Shanghai, China). Animals were housed in an environment of 21 ± 0.5 ◦ C with a relative humidity of 50 ± 10%. Every cage had a complete exchange of air 15–18 times per hour

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Table 1 The primers of GABAA receptor subunits and GAPDH Access ion number

Subunit

Forward primer

Reverse primer

NM 010250 NM 008066 AK013727 AF090374 NM 008070 NM 008071 NM 008073 M32599

alpha1 alpha2 alpha4 alpha5 beta2 beta3 gamma2 GAPDH

5-GCAGATTGGATATTGGGAAGCA-3 5-CGGCCACGACTCCTCAAG-3 5-GAGAGGCTGGAAACGTGAACA-3 5-CAGGATCTTGGACGGACTCTT-3 5-AAACCGTATGATTCGATTGC-3 5-AAGACAGCCAAGGCCAAGAA-3 5-TTTCTGCTTTGGTGGAGTATGG-3 5-AGGAGCGAGACCCCACTAACAT-3

5-GGTCCAGGCCCAAAGATAGTC-3 5-AGAGACTGCAGCAGCCTAAAGAC-3 5-CTGTAACAGGACCCCCAAATC-3 5-AATGGACTTCTTGCCGTTGTG-3 5-ACGATGGAGAACTGAGGAAGC-3 5- GCCTGCAACCTCATTCATTTC-3 5-TCGGCAATCTTCAAAACAGCAG-3 5-GTGATGGCATGGACTGTGGT-3

and a 12-h light/12-h dark cycle with no twilight. Water and food were continuously available. The mice received intraperitoneal injections of QYS at 200 mg/kg every 24 h [16], and the control mice received the same amount of saline. After six treatments of QYS, the inferior colliculus, hippocampus, and cortex were dissected quickly and total RNA was extracted by TRIZOL solutions (Invitrogen, USA). Seven GABAA receptor subunit genes were investigated by fluorescence real-time quantitative PCR (QPCR). The primers (see Table 1) were designed using Primerexpress software and synthesized by Sangon. GAPDH was used as a reference gene. The experiment was performed on Opticon 2 (MJ research, USA). Each reaction was repeated four times and relative gene expression levels were calculated and presented with 2−∆Ct and 2−∆∆Ct method [22]. Our previous experiments using high-density oligomicroarray to analyze gene expression profiling in inferior colliculus (IC) demonstrated that expression levels of 671 genes were altered after QYS treatment, including a number of GABAA subunits. For example, GABAA alpha1 (Gabra1), alpha2 (Gabra2), and alpha4 (Gabra4) subunit genes were upregulated two- to threefold (Table 2), while GABAA receptor gamma2 (Gabrg2) subunit gene was down-regulated significantly (Table 2). However, the expression of GABAA alpha5 (Gabra5), beta2 (Gabrb2) and beta3 (Gabrb3) subunit genes remained the same after the administration of QYS (Table 2). We further confirmed the microarray data using fluorescence realtime quantitative PCR. Our results have shown that the expression of Gabra1, Gabra2, and Gabr4 genes were up-regulated in the inferior colliculus after QYS treatment, which was consistent with the microarray data. Similarly, Gabrg2 gene expression was

also decreased in our quantitative PCR experiment (Fig. 1). The expression levels of other GABAA subunits including Gabra5, Gabrb2, and Gabrb3, did not show significant changes. Inferior colliculus is an important brain region involved in AGS. Since hippocampus and cortex have been shown to regulate other forms of epilepsy, we examined the differential expressions of GABAA receptor subunits in these brain regions after QYS treatment. Interestingly, we found that the expression level of Gabra1 was increased about fivefold in cortex (Fig. 2A), but it was not altered in hippocampus after QYS treatment (Fig. 2B). On the other hand, Gabra5 expression did not change in cortex but significantly down-regulated in hippocampus after administration of QYS (Fig. 2A and B). Although Gabrb2 gene expression was not altered in IC after QYS treatment, it has been significantly up-regulated in both hippocampus and cortex (Fig. 2A and B). Furthermore, QYS reduced the expression of Gabrg2 in IC, but the same treatment had no effect on its expression in both hippocampus and cortex (Fig. 2A and B). The gene expression analysis demonstrated that administration of QYS had distinct effects on the expression of GABAA receptor subunits in different brain regions. These results may help to explain the molecular mechanism of anticonvulsant activity of QYS in different forms of epilepsy. Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain, producing inhibitory postsynaptic potentials (IPSPs) in both feedforward and feedback circuits. GABA-mediated inhibition plays a critical role in the antiepileptic process by contributing to the termination of the discharge and limiting the spread of hyperex-

Table 2 The fold change (FC) of GABAA receptor subunits after QYS treatment Gene name

Fold change (FC)

GABAA receptor, subunit alpha 1 (Gabra1) GABAA receptor, subunit alpha 2 (Gabra2) GABAA receptor, subunit alpha 4(Gabra4) GABAA receptor, subunit alpha 5 (Gabra5) GABAA receptor, subunit beta 2 (Gabrb2) GABAA receptor, subunit beta 3 (Gabrb3) GABAA receptor, subunit gamma 2 (Gabrg2)

3.02 2.32 2.03 NC NC NC −1.67

Fold change (FC): the relative expression level after QYS treatment compared to the untreated control; NC: no significant change of gene expression. FC is calculated from ratio values, which equals to the ratio of Cy5-processed signal to Cy3-processed signal.

Fig. 1. Expression of GABAA receptor subunits in the inferior colliculus. Fold changes (2−∆∆Ct value) were shown in terms of means of relative gene expression (2−∆Ct value) and the standard error of mean in the control mice (solid) and QYS-treated mice (white). Paired Student t-test, c P < 0.01; d P < 0.001 vs. Con.

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Fig. 2. Expression of GABAA receptor subunits in the hippocampus and cortex: (A) hippocampus and (B) cortex. Fold changes (2−∆∆Ct value) were shown in terms of means of relative gene expression (2−∆Ct value) and the standard error of mean in the control mice (solid) and QYS-treated mice (white). Paired Student’s t-test, b P < 0.05; c P < 0.01; d P < 0.001 vs. Con.

citability [4]. GABA plays essential physiological roles in the brain through ligand-operated GABAA receptors and G-proteincoupled metabotropic GABAB receptors [39]. It has been shown that impairment of GABAergic transmission by genetic mutations or application of GABA receptor antagonists induced epileptic seizures, whereas drugs augmenting GABAergic transmission have been developed for antiepileptic therapy [13]. Extensive studies demonstrated that IC was involved in the initiation and propagation of generalized tonic–clonic audiogenic seizures (AGS). Inhibition of GABAA receptors in IC could produce AGS susceptibility in normal animals [8,35]. However, hippocampus and certain regions of cortex were involved into other forms of epilepsy [3,28]. It has been demonstrated that QYS had a significant anticonvulsant activity on audiogenic seizure induced by high-intensity noise [19,21,30]. Furthermore, Lang et al. [20] reported that oral administration of QYS could increase the release of GABA in the brain. However, it is not clear whether QYS could regulate the gene expression of GABAA receptor subunits. In the present study, we have demonstrated that administration of QYS could enhance the expression of Gabra1, Gabra2, and Gabra4 in the inferior colliculus (Table 2, Fig. 1). In addition, Gabrb2 expression in the hippocampus (Fig. 2A) and Gabra1 and Gabrb2 expressions in the cortex were also induced by QYS (Fig. 2B). Therefore, our results suggested that one possible mechanism of antiepileptic action of QYS was related to the modulating the expression of GABAA receptor subunits in the central nervous system. Numerous strategies for developing antiepileptic drugs that act by increasing GABAergic neurotransmission have been used, including the identification of GABAA agonists, GABA-transaminase inhibitors, GABA uptake blockers, GABA prodrugs, and allosteric modulators of the GABAA receptor. For example, the GABAA receptor agonist gaboxadol and GABA prodrug progabide exhibited potent anticonvulsant activity in animal seizure models [9,23]. Tiagabine, a neuronal and glial GABA uptake blocker, had potent anticonvulsant effect in hippocampal-kindled and other epileptic animal models [29,38]. This drug was also effective in human complex partial seizures [37]. The irreversible GABAtransaminase inhibitor vigabatrin exhibited robust anticonvulsant activity in a variety of animal seizure models [25,36] and

displayed good therapeutic effects in the treatment of complex partial epilepsy in human. Some allosteric modulators of the GABAA receptor-Cl− ionophore, such as diazepam, have been used primarily for status epilepticus [33]. It has been reported that barbiturates exerted positive allosteric effects on GABAA neurotransmission by directly activating the Cl− ion channel, and are utilized in the treatment of generalized tonic–clonic and partial seizures [7]. In addition, many other antiepileptic drugs regulated the GABA receptor expression or GABA-mediated current, such as phenytoin (PHT) [10], carbamazepine (CBZ) [18,43], and valproic acid (VPA) [24]. Many antiepileptic drugs are targeting multiple molecular targets. For example, topiramate (TPM) was clinically effective for simple or complex partial seizures and also used for the treatment of generalized tonic–clonic seizures. Several mechanisms have been proposed for antiepileptic effect of TPM, such as (1) activity-dependent attenuation of voltage-dependent sodium currents, possibly by stabilizing sodium channels in their inactivated state [40]; (2) potentiation of GABAA-mediated currents [45] and enhancement of the expression of several subtypes of GABAA receptors [18]; (3) inhibition of AMPA/kainate receptors [11,34]; (4) negative modulation of calcium channels [17,46]; and (5) regulation of the expression of potassium channel [6]. Our previous gene expression profiling data showed that more than 100 gene expressions were altered after QYS treatment, including sodium channel, calcium channel, many kinds of metabolic enzymes (such as Glutamine synthetase and acetylcholinesterase) and cytokines [21]. Guo et al. reported that QYS had significant effects on kainic acid induced epilepsy. Intraperitoneal injection of QYS and diphenylhydantoin sodium (DPH) reduced the expression of c-fos in the hippocampus during acute seizures but not the chronic seizures [12]. Furthermore, Kuang et al. showed that oral administration of QYS significantly increased the acetylcholine [14] and 5-hydroxytryptomine (5HT) concentrations [15] in the cerebral hemispheres and brain stem. Therefore, the anticonvulsant mechanism of QYS, like that of many other antiepileptic drugs, may regulate multiple signal pathways in the brain. Further molecular and pharmacological studies are necessary to identify the effective therapeutic targets for QYS.

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Acknowledgements This work was supported by the grants from Ministry of Science and Technology of China (2003AA221061, 2003CB716601), and Yunnan Commission for Science and Technology. We thank Yunnan Baiyao Group and Dr. Qingxiong Yang for their support and Dr. Paul Gregor for critically reading of the manuscript.

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