Clptm1, a new target in suppressing epileptic seizure by regulating GABAAR-mediated inhibitory synaptic transmission in a PTZ-induced epilepsy model

Abstract

Disruption of gamma-amino butyric acid type A receptors (GABA_ARs) synaptic clustering and a decrease in the number of GABA_ARs in the plasma membrane are thought to contribute to alteration of the balance between excitatory and inhibitory neurotransmission, which promotes seizure induction and propagation. The multipass transmembrane protein cleft lip and palate transmembrane protein 1 (Clptm1) controls the forward trafficking of GABA_AR, thus decaying miniature inhibitory postsynaptic current (mIPSC) of inhibitory synapses. In this study, using a pentylenetetrazol (PTZ)-induced epilepsy rat model, we found that Clptm1 regulates epileptic seizures by modulating GABA_AR-mediated inhibitory synaptic transmission. First, we showed that Clptm1 expression was elevated in the PTZ-induced epileptic rats. Subsequently, we found that downregulation of Clptm1 expression protected against PTZ-induced seizures, which was attributed to an increase in the number of GABA_ARγ2s in the plasma membrane and the amplitude of mIPSC. Taken together, our findings identify a new anti-seizure target that provides a theoretical basis for the development of novel strategies for the prevention and treatment of epilepsy.

INTRODUCTION

Epilepsy is a chronic brain disease that currently affects an estimated 50 million people worldwide.¹ Abnormal neuronal discharges in patients with epilepsy are due to failure of inhibitory and/or excitatory neurotransmission. Receptors of gamma-amino butyric acid (GABA) are involved in seizure induction and propagation.² GABA type A receptors (GABA_ARs) are considered the most important GABA receptors in epilepsy,³ and persistent activation of GABA_ARs can suppress neuronal voltage responses to incoming excitatory potentials.⁴ Thus, it is important to investigate the molecular mechanisms underlying GABA_AR-mediated inhibitory synaptic transmission in epilepsy.

Clptm1, a multipass transmembrane protein, was identified due to the association of a chromosomal translocation with a cleft lip and palate.⁵ It modulates the postsynaptic assembly of GABA_ARs. Knocking down Clptm1 increases the number of GABA_ARγ2s in the plasma membrane and the amplitude of the miniature inhibitory postsynaptic current (mIPSC) in neurons.⁶ However, it has not been established whether Clptm1 regulates epileptic seizures by modulating GABA_AR-mediated inhibitory synaptic transmission.

In this study, we examined the role of Clptm1 in epilepsy. We demonstrated that Clptm1 expression is increased in pentylenetetrazol (PTZ)-induced epileptic rats. Then, we showed that downregulation of Clptm1 expression attenuates epileptic seizures and that this change is associated with increases in the number of GABA_ARγ2s in the cell membrane and the amplitude of mIPSC in neurons. These results show the significant role of Clptm1 in GABA_AR-mediated inhibitory synaptic transmission and its involvement in epileptic seizures. Therefore, Clptm1 is a promising target for the treatment of epilepsy.

MATERIALS AND METHODS Ethical statement

All animal protocols were approved by the Commission of Chongqing Medical University for Ethics of Experiments on Animals and were performed in accordance with the principles of Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines.

Rat model of seizure or epilepsy

All animals were obtained from the Laboratory Animal Centre of Chongqing Medical University. We used the PTZ-induced epilepsy rat model. PTZ (35 mg/kg, intraperitoneal injection (i.p.), Sigma) were given daily for 30 days in the chronic model, while 70 mg/kg in the acute model, and EEG recordings were collected.Behaviors were scored as described in the previous studies.^7–9 For Clptm1-shRNA + PTX + PTZ administration, rats were intrahippocampal injection of Clptm1-shRNA 2 weeks before treatment with picrotoxin (PTX; 1 mg/kg, i.p. 5 min prior to receiving PTZ) and then with PTZ.

Immunofluorescence labeling

Immunofluorescence staining was operated as described in our previous study.⁹ In brief, brain tissues were permeabilized with 0.4% Triton X-100 for 15 min, and blocked with donkey serum (1:100, Proteintech, Wuhan, China) for 1.5 h. The brain tissues were incubated overnight with mixed antibodies at 4°C, and then incubated with secondary antibodies for 30 min. The following antibodies were used: mouse anti-Clptm1 (1:50, sc-374,619, Santa Cruz, CA, USA), rabbit anti-GABA_ARγ2 (1:100, 222,837, US Biological, GA, USA), chicken anti- Microtubule-associated protein 2 (MAP2; 1:200, AB5543, Millipore, MA, USA) and rabbit anti-glial fibrillary acidic protein (GFAP; 1:150, 16825-1-AP, Proteintech).

Western blot and co-immunoprecipitation

According to instructions of the plasma membrane protein extraction kit (Abcam, Cambridge, UK) and total protein extraction kit (Sangon Biotech, Shanghai, China), we extracted plasma membrane-associated proteins and total proteins. Then, a bicinchoninic acid protein assay was used to determine the concentration of pure protein. The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Next, the PVDF membrane was incubated with a mouse anti-Clptm1 (1:150, sc-374,619, Santa Cruz) or a mouse anti-GABA_ARγ2 (1:500, MABN875, Millipore) antibody. The next day, the membrane was incubated with secondary antibody for 1.5 h at 37°C. Co-immunoprecipitation was conducted as described in the previous study.⁹ The following antibodies were used: mouse anti-Clptm1 (1:50, sc-374,619, Santa Cruz) and mouse anti-GABA_ARγ2 (1:50, MABN875, Millipore). Immunoblot assays were performed with mouse anti-GABA_ARγ2 (1:500, MABN875, Millipore) and mouse anti-Clptm1 (1:150, sc-374,619, Santa Cruz) antibodies.

Downregulation of Clptm1 expression and electrophysiological assessments

A lentivirus vector expressing Clptm1-shRNA and scramble-shRNA were constructed by GeneChem company (GeneChem Co., Ltd. Shanghai, China). Clptm1-shRNA or scramble-shRNA were microinjected into the CA1 region of the hippocampus in each rat. Electrophysiological assessments were performed as described in the previous publication.⁹ For the electrophysiological assessments, rats brain slices (300 μm thick) were cut and immersed in an ice-cold buffer. Then brain sections were immediately placed in artificial cerebrospinal fluid for 1.5 h at 34°C. Cells in the CA1 region of the hippocampus were recorded under an inverted phase contrast microscope (Nikon, Japan). When stable mIPSCs were obtained, recordings were sustained for at least 3 min. Then, PTX was added to the brain slices to confirm that mIPSC was conducted via GABA_AR.

Statistics

All data are shown as the mean ± SEM. The normality of the data was measured using the Shapiro–Wilk test. Student's t-test or Mann–Whitney U test was performed to compare means between groups, and one-way analysis of variance was performed to compare means among multiple groups. All testes were two-sided, and p < 0.05 was considered statistically significant.

RESULTS Clptm1 is localized in neurons and interacts with GABA_ARγ2 in PTZ-induced epileptic rat

We first determined whether Clptm1 is localized in neurons, as epilepsy is known to be characterized by neuronal hyperexcitability. Confocal microscopy analyses revealed that Clptm1 colocalized with the neuron-specific marker MAP2 in the cortex and hippocampus of PTZ-induced epileptic rat. However, it did not colocalize with the astrocyte-specific marker GFAP (Figure 1A,B). Additionally, co-immunoprecipitation showed that Clptm1 interacted with GABA_ARγ2 in the cortex and hippocampus of PTZ-induced epileptic rat (Figure 1C,D). These findings suggest that Clptm1 is localized in neurons and interacts with GABA_ARγ2 in the epileptic brain.

FIGURE 1. Clptm1 is localized in neurons and interacts with GABAARγ2 in the cortex and hippocampus of PTZ-induced epileptic rat. (A,B) The localization of Clptm1 in the epileptic brain. Immunofluorescence showing that Clptm1 (red) was colocalized with MAP2 (green) in the cortex and hippocampus of PTZ-induced epileptic rat. Clptm1 (red) was not colocalized with GFAP (green). Additionally, Clptm1 (red) was colocalized with GABAARγ2 (green) in the cortex and hippocampus of PTZ-induced epileptic rat. Scale bar = 20 μm. (C,D) The interaction between Clptm1 and GABAARγ2 in the epileptic brain. Co-immunoprecipitation showed that Clptm1 interacted with GABAARγ2 in the cortex and hippocampus of PTZ-induced epileptic rat

Clptm1 expression is increased in the cortex and hippocampus of PTZ-induced epileptic rat

To observe whether Clptm1 plays a role in epilepsy, we first measured Clptm1 expression in the PTZ-induced epilepsy model. Immunoblotting showed that Clptm1 expression was increased in the hippocampal and cortical tissues of PTZ-induced epileptic rat (Figure 2A,B). These findings suggest that Clptm1 may be involved in epileptic seizures.

FIGURE 2. Clptm1 and GABAARγ2 expression are altered in the cortex and hippocampus of PTZ-induced epileptic rat. (A,B) Clptm1 expression in the PTZ-induced epileptic rats and control rats. Clptm1 expression was increased in the cortex and hippocampus of PTZ-induced epilepsy rat model (n = 3 rats in each group). (C,D) The protein expression level of GABAARγ2 in the cell membrane of PTZ-induced epileptic rats and control rats. The plasma membrane expression of GABAARγ2 was significantly decreased in the cortex and hippocampus of PTZ-induced epilepsy rat model (n = 3 rats in each group). ***p [less than] 0.001 versus the control groups. The error bars denote the SEM

GABA_ARγ2 expression is decreased in the cortex and hippocampus of PTZ-induced epileptic rat

Disruption of GABA_AR-mediated inhibitory synaptic transmission is related to a postsynaptic mechanism involved in epilepsy. Clptm1 negatively regulates the number of GABA_ARγ2s in the cell membrane.⁶ Hence, we measured GABA_ARγ2 levels in the epileptic brain. As shown in Figure 2C,D, the protein expression level of GABA_ARγ2 in the cell membrane was significantly decreased in the cortex and hippocampus of rat with chronic epilepsy compared to that in the control group. These changes may disrupt GABA_AR-mediated inhibitory synaptic transmission in epilepsy.

Downregulation of Clptm1 expression attenuates PTZ-induced seizures

Subsequently, to identify the relationship between the increase in Clptm1 expression and epilepsy, we used Clptm1-shRNA to downregulate Clptm1 protein expression and then assessed the effects of Clptm1 on epileptic seizures. Two weeks after intrahippocampal injection of Clptm1-shRNA, green fluorescent protein (GFP)-positive cells were present in the hippocampus (Figure 3A). Furthermore, immunoblotting revealed that Clptm1 expression in the hippocampus was gradually decreased after intrahippocampal injection of Clptm1-shRNA (Figure 3B). These findings suggest that Clptm1-shRNA was successfully transfected into the rat hippocampus. Subsequently, we observed epileptic seizures after PTZ injection (Figure 3C). We found that the latency to the first seizure was markedly increased and the number of generalized tonic–clonic seizure (GTCS) per rat was significantly decreased in the Clptm1-shRNA + PTZ group compared to the scramble-shRNA + PTZ group; however, the antiseizure effects of Clptm1 downregulation were blocked by PTX (an antagonist of GABA_AR; Figure 3C). Taken together, these findings indicate that Clptm1 regulates epileptic seizures and that downregulation of Clptm1 expression exerts antiseizure effects.

FIGURE 3. Clptm1 regulates PTZ-induced seizures. (A) The presence of GFP-positive cells in the hippocampus suggested that Clptm1-shRNA was successfully transfected into the hippocampus of rats. Scale bar = 200 μm. (B) Clptm1 protein expression after Clptm1-shRNA or scramble-shRNA intrahippocampal injection. Western blot analysis demonstrated that Clptm1 expression was decreased in the hippocampus at 7 d and 14 d after Clptm1-shRNA microinjection compared to the scramble-shRNA group (n = 3 rats in each group). **p [less than] 0.01 and ***p [less than] 0.001 versus the scramble-shRNA group. (C) Schematic overview of behavior evaluation after PTZ injection. The latency to the first seizure was markedly increased and the number of GTCS per rat was significantly decreased in the Clptm1-shRNA + PTZ group compared to the scramble-shRNA + PTZ group; however, the antiseizure effects of Clptm1 downregulation were blocked by PTX (n = 10 rats in each group). *p [less than] 0.05 and **p [less than] 0.01 versus the scramble-shRNA + PTZ group, #p [less than] 0.05 and ##p [less than] 0.01 versus the Clptm1-shRNA + PTZ group. The error bars denote the SEM

Clptm1 modulates GABA_AR-mediated inhibitory synaptic transmission in PTZ-induced epileptic rat

Since Clptm1 inhibits GABA_AR forward trafficking and alters the protein expression of GABA_ARγ2 in the cell membrane,⁶ we investigated whether Clptm1 regulates GABA_ARγ2 expression after Clptm1-shRNA transfection. Membrane proteins of hippocampus were extracted, and the results showed that GABA_ARγ2 expression in the cell membrane was increased in the Clptm1-shRNA group (Figure 4A). Furthermore, to determine whether downregulation of Clptm1 is involved in GABA_AR-mediated inhibitory synaptic transmission, we recorded mIPSC from hippocampal brain slices of rats in each group. It is well known that GABA_AR are predominantly located at postsynaptic areas, and mIPSC amplitude in patch clamp experiments usually reflects postsynaptic function; whereas, mIPSC frequency usually reflects presynaptic function.⁹ As shown in Figure 4B, mIPSC amplitude was increased in the Clptm1-shRNA + PTZ group compared to the scramble-shRNA + PTZ group, but the mIPSC frequency was not significantly changed. Consistent with a previous study, Clptm1 had no effect on mIPSC frequency, overexpression of Clptm1 significantly decreased mIPSC amplitude, while knockdown of Clptm1 increased mIPSC amplitude.⁶ Taken together, these findings indicate that Clptm1 regulates GABA_AR-mediated inhibitory synaptic transmission in the PTZ-induced epilepsy model.

FIGURE 4. Clptm1 modulates GABAAR-mediated inhibitory synaptic transmission in PTZ-induced epileptic rat. (A) The plasma membrane expression of GABAARγ2. Western blotting analysis demonstrated that the protein expression level of GABAARγ2 in the cell membrane was significantly increased in the Clptm1-shRNA group compared to that in the scramble-shRNA group (n = 3 rats in each group). ***p [less than] 0.001 versus the scramble-shRNA group. (B) Representative hippocampal mIPSC recordings in each group. The mIPSC amplitude was increased in the Clptm1-shRNA + PTZ group compare to the scramble-shRNA + PTZ group (n = 3 rats in each group). No significant difference in the mIPSC frequency was observed in each group. **p [less than] 0.01 and ***p [less than] 0.001 versus the control group, #p [less than] 0.05 versus the scramble-shRNA + PTZ group. The error bars denote the SEM

DISCUSSION

In this study, we found that Clptm1 regulates epileptic seizures by modulating the number of GABA_ARγ2 in the plasma membrane and inhibitory synaptic transmission (Figure 5). Controlling GABA_AR-mediated inhibitory synaptic transmission has been the target in some antiepileptic drugs in clinical trials. There is extensive evidence linking epilepsy and dysregulation of GABA_AR-mediated inhibitory synaptic transmission.^10–14 A reduction in the number of GABA_ARs present in the cell membrane increases excitability in animal models of epilepsy.^15–17 Epileptic rodents that experience spontaneous seizures also show failure of inhibitory synaptic transmission in the chronic stage.¹⁸ The interactions between GABA_AR and its regulatory proteins mediates synaptic transmission and plasticity. GABA_AR exits the endoplasmic reticulum and is trafficked to the plasma membrane. GABA_AR trafficking and accumulation are regulated by interactions between GABA_AR and several binding partners. These interactions lead to the formation of mature receptor complexes, which enables their proper localization and functions in the cell membrane and in synapses.^19,20

FIGURE 5. Schematic representation of Clptm1 modulates epileptic seizures. Clptm1 expression is upregulated in the cortex and hippocampus of PTZ-induced epileptic rat, leading to the reduction of GABAARγ2 in the plasma membrane. Reduced membrane expression of GABAARγ2 impairs inhibitory synaptic transmission, thereby promoting epileptic seizures

A functional association between Clptm1 and GABA_ARγ2 has been reported in neuron. Ge et al.⁶ confirmed that Clptm1, a negative regulator that traps GABA_ARs within intracellular compartments, can limit GABA_ARs expression in the cell membrane. Clptm1 was originally reported in a mutational study of a family affected with cleft lip and palate.⁵ Previous studies have indicated that Clptm1 plays a role in intrathymic T-cell development and oral squamous cell carcinoma.^21,22 However, the role of clptm1 in central nervous system diseases is not fully understood. A few studies have shown that Clptm1 is associated with Alzheimer's disease and low-density lipoprotein cholesterol levels.^23,24 In the present study, we used the PTZ-induced epilepsy rat model to explore the role of Clptm1 in GABA_AR function and epileptic seizures. The PTZ-induced epilepsy model is considered a suitable animal model for preclinical epilepsy research.^9,25,26 PTZ can interact with the benzodiazepine recognition sites of GABA_AR.²⁶ GABA_AR function was impaired by single or repeated PTZ administration.^9,27 The CA1 region of the hippocampus is a critical area for epileptic lesions in patients with epilepsy and animal models of epilepsy.^26,28 Additionally, the CA1 region of the hippocampus has always been the center of synaptic morphological alteration and epileptogenic network origin.²⁸ Immunofluorescence results showed that Clptm1 was expressed in the CA1 region of the hippocampus of PTZ-induced epileptic rat. This indicates that Clptm1 may be involved in synaptic morphological and functional changes in epilepsy. Then, we demonstrated that Clptm1 expression was upregulated in the PTZ-induced epilepsy model, and that downregulation of Clptm1 expression increased the number of GABA_ARγ2s in the plasma membrane and the amplitude of mIPSC. Taken together, our results show that downregulation of Clptm1 expression may be an approach for the treatment of epilepsy.

It is generally considered that GABA_ARγ2 plays an important role in GABA_AR-mediated inhibitory synaptic transmission. The γ2 subunit is distributed throughout the central nervous system and is near-ubiquitous at synaptic GABA_AR.^29,30 Thus, mutations in this receptor subunit fundamentally change GABA_AR-mediated synaptic inhibition. γ2 subunit mutations have been implicated in severe myoclonic epilepsy in infancy, Dravet syndrome, genetic epilepsy syndromes, simple febrile seizures, and childhood absence epilepsy.³¹ γ2 mutations disrupt inhibitory synaptic transmission by decreasing IPSC decay rates.^32–37 Our results showed that GABA_ARγ2 membrane expression was decreased in PTZ-induced epileptic rats and that GABA_ARγ2 membrane expression was restored after downregulation of Clptm1 expression. This suggests that Clptm1 involves in GABA_AR-mediated inhibitory synaptic transmission by regulating GABA_ARγ2 membrane expression.

In summary, GABA_AR determine the efficacy of GABA-targeting drugs in epilepsy patients.^4,38–40 Understandably, elucidating the cellular mechanisms underlying the modulation of GABA_AR assembly in the cell membrane is of considerable interest. This study shows that Clptm1 modulates GABA_AR-mediated inhibitory synaptic transmission by regulating the number of GABA_ARγ2 in the plasma membrane in the context of epilepsy. Nevertheless, it is necessary to further elucidate the molecular association networks involving Clptm1 and GABA_ARs.

CONFLICT OF INTEREST

All authors declare no conflict of interest.

Abbreviations List
• Clptm1
• cleft lip and palate transmembrane protein 1
• GABA
• gamma-amino butyric acid
• GABA_ARs
• gamma-amino butyric acid type A receptors; GFP, green fluorescent protein
• GTCS
• generalized tonic–clonic seizure
• mIPSC
• miniature inhibitory postsynaptic current
• PTX
• picrotoxin
• PTZ
• pentylenetetrazol
• shRNA
• short hairpin RNA