Clptm1 Downregulation Exerts Antiepileptic Activity by Regulating GABAAR-mediated Inhibitory Synaptic Transmission in PTZ-induced Epileptic Model

Background: Disruption of GABA A R synaptic clustering and a decrease number in their cell surface are thought to contribute to the alteration in the balance between excitatory and inhibitory neurotransmission, which contributes to seizure induction and propagation. Cleft lip and palate transmembrane protein 1 (Clptm1), a multi-pass transmembrane protein, has been showed that it is an intracellular molecule that controls forward tracking of GABA A R. Clptm1 downregulating increased miniature inhibitory postsynaptic current (mIPSC) in vivo. Thus, Clptm1 controls phasic and tonic inhibitory transmission in brain. In this study, we hypothesized that Clptm1 may be involved in epileptic seizure by regulating GABA A R-mediated inhibitory synaptic transmission in epileptic model. Methods and Results: In PTZ-induced epileptic model, we found that Clptm1 was increased in temporal lobe epilepsy (TLE) patients as well as in epileptic model. Then, we showed that Clptm1 downregulation exerted antiepileptic activities in epileptic model, which was associated to the increased surface GABA A Rγ2 expression and mIPSCs amplitudes. Conclusions: Clptm1 downregulation exerted antiepileptic activities in epileptic model, thus, it may be a promising target for antiepileptic treatments.


Introduction
TLE is the most prevalent form of focal epilepsy. It is characterized as a form of adult focal onset epilepsy and is highly associated with refractory epilepsy (1). Abnormal neuronal discharges in patients with TLE is due to failure of inhibitory and/or excitatory neurotransmission. Gamma-amino butyric acid (GABA) receptors (GABA A R), the main inhibitory neurotransmitter, may be involved in seizure induction and propagation (2). Of all of the GABAR, the GABA A receptors (GABA A Rs) are considered as the most important in TLE induction and propagation (3), which persistently activated GABA A Rs can suppress neuronal voltage responses towards incoming excitation (4). Thus, it is important to investigate the molecular mechanisms underlying the effects of GABA A R-mediated inhibitory synaptic transmission on epileptic seizure. Clptm1 is identi ed by association of a chromosomal translocation with cleft lip and palate (5). It modulates postsynaptic assembly of GABA A Rs. Knocking down Clptm1 increased the number of GABA A Rγ2 in cell surface and mIPSC amplitudes in neurons (6). However, whether Clptm1 regulates epileptic seizure by regulating GABA A R-mediated inhibitory synaptic transmission has not been established.
In this study, we examined the role of Clptm1 in epilepsy. We demonstrated that Clptm1 is increased in TLE patients as well as in epileptic models. Then, we showed that Clptm1 downregulation attenuated seizure susceptibility and severity in PTZ mouse and that these changes are associated with the increase of the number of GABA A Rγ2 in cell surface and mIPSCs amplitudes. These results show the signi cance role of Clptm1 in GABA A R-mediated inhibitory synaptic transmission and involved in epileptic seizure. Therefore, Clptm1 is a promising target for antiepileptic treatments.

Ethical statement
This study was conducted following the guidelines for the National Institutes of Health of China and the Committee on Human Research at Chongqing Medical University. Written informed consent for human brain tissues were collected. All protocols for the performing animal experiments were admitted by the Commission of Chongqing Medical University for Ethics in Animal Experiments (Approval number:0002648).

Human samples
Ten samples of TLE patients and ten normal samples who experienced head trauma were obtained as our previous publication (7). Clinical characteristics of patients are shown in Table1.

Mouse model of seizure or epilepsy
All animal were obtained from the Laboratory Animal Center of Chongqing Medical University. We established PTZ-induced epileptic model, which is considered an appropriate animal model for preclinical research on the roles of GABA A Rs in epilepsy (8-10). Acute and chronic epileptic models were established as our previous publication (7).

Behavioural tests
The behaviours were scored as previous study (11). For the shRNA-clptm1+ PTX intervention, mice were administered with shRNA-clptm1 or LV-GFP two weeks before inducing PTZ and thereafter with PTX (1 mg/kg. Ip. 5 min before PTZ inducing).

Double immuno uorescence labelling
Immuno uorescence (IF) staining was operated as previously described (7). The antibodies were mouse

Establishment of the Lentiviral Vector and Stereotactic Injection
LV vectors with Clptm1 constructs shRNA-Clptm1 were constructed by GeneChem company (GeneChem Co., Ltd. Shanghai, China). We microinjected Clptm1-shRNA or scramble-shRNA into the bilateral CA1 region of every mouse.

Electrophysiological Assessments
Mouse cortical brain slices (300 μm thick) were cut and immersed in an ice-cold buffer. Then brain sections were immediately placed in arti cial cerebrospinal uid (ACSF) for 1.5 h at 34°C. Cells in the CA1 region of the hippocampus were recordings under an inverted phase contrast microscope (Nikon, Japan).
When stable mIPSCs were obtained, recordings were sustained for at least 3 min. Then, GABA (100 or 500 μM, Sigma, USA) was added to the brain slices to assess whether the mIPSCs required GABA activity.

Statistics
All data are shown as mean ± SEM. Student's t test was conducted for between group comparisons and LSD tests if three or four groups were compared. ANOVA repeated measures were used for comparisons of sequential measurements to a single control measurement followed by Dennett's test. P < 0.05 was the cut-off for signi cance.

Results
Clptm1 is localized in neuron and interacted with GABA A Rγ2 in epileptic brain We rst detected whether Clptm1 protein is localized in neuron, which epilepsy is known to be characterized by neuronal hyperexcitability. Confocal microscopy analyses revealed that Clptm1 immunoreactivity is located at cortical neurons of TLE patients (Fig. 1A-1C), and we showed that Clptm1 is located at the cortex as well as hippocampal of the epileptic mouse. Clptm1 co-localize with the speci c neural marker MAP2. However, it did not co-localize with the speci c astrocyte marker GFAP.
Additionally, we found Clptm1 co-localizes with GABA A Rβ 2/3 in epileptic brain, and coimmunoprecipitation showed that Clptm1 is interacted with GABA A Rγ2 in TLE patients, and the hippocampus and cortex of the epileptic mouse are also (Fig. 1D-1F). These ndings suggest that Clptm1 localized in neuron and interacted with GABA A Rγ2 in epileptic brain. GABA A Rγ2 expression is decreased in epileptic brain Disruption of GABA A R-mediated inhibitory synaptic transmission is related to a post synaptic mechanism in epilepsy, thus, we rst detected GABA A Rγ2 levels in the epileptic brain. As shown in gures 2A, 2B and 2C, the immunoreactivity of GABA A Rγ2 in the cortex of patients with TLE and in chronic epileptic model showed a signi cant decrease compared to the control. These changes may underlie the disrupted GABAergic transmission in epilepsy.

Clptm1 expression is increased in epileptic brain
To observe whether Clptm1 plays a role in epilepsy, we rst detected the Clptm1 expression in the brains of the TLE patients and epileptic mouse model. Immunoblotting showed that Clptm1 expression was increased in TLE patients compared to the control (Fig. 2D), and in hippocampal and cortical tissue of the epileptic mouse model were also increased ( Fig. 2E-2F). These ndings suggested that Clptm1 upregulation may be involved in epileptic seizure.

Clptm1 downregulation attenuates PTZ-induced Seizure
Subsequently, to identify the relation between the increasing of Clptm1 expression and epilepsy, we used Clptm1-shRNA to downregulate Clptm1 proteins and assessed the effects of Clptm1 on PTZ-induced seizures. Two weeks post-administration of the exogenous recombinant lentivirus vector (LV), we found green uorescent protein (LV-GFP)-positive cells in the hippocampus (Fig. 3A). Moreover, immunoblotting revealed Clptm1 expression is decreased after initial administering (Fig. 3B). These ndings suggesting that Clptm1-shRNA was transfected successfully in epileptic model. Subsequently, we observed behavioural alterations after PTZ-induced seizures (Fig. 3C), we found seizure susceptibility and severity were markedly decreased in Clptm1-shRNA group compared to the control (Fig. 3D). Taken together, we found Clptm1 regulates epileptic seizures and that Clptm1 downregulation exerts antiepileptic activities in epileptic model.

Clptm1 modulates GABA A R-mediated inhibitory synaptic transmission in epileptic mouse model
Since Clptm1 limits GABA A R forward tra cking and synaptic inhibition by altering surface GABA A Rγ2 expression (6), we studied weather this mechanism is involved in epilepsy. Biochemical measurements of surface-expressed receptors showed the surface GABA A Rγ2 expression is increased in the Clptm1-shRNA group compared to the control (Fig. 4A). Moreover, to determine whether these effects of Clptm1 downregulation on surface GABA A Rγ2 numbers were involved in the e cacy of synaptic suppression, mIPSCs were recorded from mouse treated with either PTZ alone or Clptm1-shRNA. As shown in Fig. 4B, the mIPSC amplitudes were increased in Clptm1-shRNA group compared to the control, but mIPSC frequencies were not changed. Taken together, these ndings indicate that Clptm1 regulate GABA A Rmediated inhibitory synaptic transmission in epileptic model.

Discussion
Epilepsy frequently dues to an imbalance in inhibition and excitation, which is a failure of neurotransmission. The evidence between epilepsy and dysfunctional GABA A R-mediated inhibitory synaptic transmission is substantial and has been extensively reported (12)(13)(14)(15)(16). A decrease in the number of GABA A Rs present on the cell surface increased excitability in animal models of TLE (17)(18)(19)(20). The epileptic animals which experienced spontaneous seizures also show failed inhibitory neurotransmission in the chronic stage (21). Association between GABA A R and regulatory proteins regulate synaptic transmission and plasticity via multiple pathways. Firstly, GABA A R exits the endoplasmic reticulum (ER) and is tra c to the plasma membrane. Receptor tra cking and accumulation is regulated by associations between GABA A R and several binding partners. These associations lead to the formation of mature receptor complexes, which enables their proper localization and functions at cell surfaces and in synapses (22,23). Recently, Ge et al con rmed that Clptm1, a negative regulator that traps GABA A Rs within intracellular compartments, could limit their expression on the cell surface (6). In this study, we examined the role of clptm1 function and dysregulation of GABAergic neurotransmission in epilepsy.
Clptm1 was originally reported in a study of gene mutations in a cleft lip and palate patient family and has a role in intrathymic T-cell development (5,24,25). Very recently, Clptm1 has been shown to modulates forward tra cking of GABA A R and inhibitory transmission in neuron (5). However, the function of clptm1 is not fully clear in central nervous system diseases. Few studies showed that Clptm1 is associated with Alzheimer disease (AD) and low-density lipoprotein cholesterol levels (26,27). In this study, we con rmed that Clptm1 regulates epileptic seizures by modulating the number of GABA A Rγ2 in the plasma membrane and e cacy of inhibitory synaptic. We demonstrated that Clptm1 was upregulated in chronic epileptic animal models and in TLE patients. Then we observed that Clptm1 downregulation increased the number of GABA A Rγ2 in the plasma membrane and mIPSCs amplitudes. Taken together, we showed that Clptm1 downregulation may be a molecular target for treatment of epilepsy.
In summary, GABA A receptors determine the e cacy of GABA-targeting drugs in TLE patients (37 -40). Understandably, elucidating the cellular mechanisms that modulates their assembly on the cell surface is of considerable interest. This study showed that Clptm1 is likely to modulate GABA A Rmediated inhibitory synaptic transmission throughout the brain by regulating the number of GABA A Rs at the plasma membrane in epilepsy. Nevertheless, elucidation of molecular association networks involving Clptm1 and GABA A Rs is necessary. All patients provided written informed consents. Chongqing Medical University admitted this study.

Consents for publication
Not applicable.

Availability of data and materials
The datasets generated during the current study are available from the corresponding author on reasonable request. Clptm1 is localized in neuron and interacted with GABA A Rγ2 in epileptic brain A, B, C: The location of Clptm1 in epileptic brain. Immuno uorescent labelling showed that Clptm1 (red) co-localized with MAP2 (green) in TLE patients and both at cortex or hippocampus of epileptic mouse, but Clptm1 (red) are not co-localized with GFAP (green). Additionally, Clptm1 (red) co-localized with GABA A Rγ2 (green) in epileptic brain. Scale bar = 50 µm (400×).
D, E, F: The interaction of Clptm1 and GABA A Rγ2 in epileptic brain. Coimmunoprecipitation showed that Clptm1 is interacted with GABA A Rγ2 in TLE patients, and the cortex as well as hippocampal neuron of the epileptic mouse are also.