Targeting gap junction in epilepsy: Perspectives and challenges

Biomedicine & Pharmacotherapy 109 (2019) 57–65

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Review

Targeting gap junction in epilepsy: Perspectives and challenges Qin Li a b

a,b

, Qiu-Qi Li

a,b

, Ji-Ning Jia

a,b

, Zhao-Qian Liu

a,b

, Hong-Hao Zhou

a,b

, Xiao-Yuan Mao

a,b,⁎

T

Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, China Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha 410078, Hunan, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Epilepsy Gap junction Connexin Neurons Astrocytes

Gap junctions (GJs) are multiple cellular intercellular connections that allow ions to pass directly into the cytoplasm of neighboring cells. Electrical coupling mediated by GJs plays a role in the generation of highly synchronous electrical activity. Accumulative investigations show that GJs in the brain are involved in the generation, synchronization and maintenance of seizure events. At the same time, GJ blockers exert potent curative potential on epilepsy in vivo or in vitro. This review aims to shed light on the role of GJs in epileptogenesis. Targeting GJs is likely to be served as a novel therapeutic approach on epileptic patients.

1. Introduction Epilepsy, a widespread neurological disorder that has been documented for thousands of years [1], is characterized by sudden, repetitive over-discharge of the brain neurons in the central nervous system (CNS) [2]. Nowadays, it is estimated that nearly 22 million people suffer from epileptic seizures around the world [3,4]. Several syndromes including cognitive decline and depression [5–7], have a detrimental effect on the life quality of patients. According to the previous studies, approximately 6 percent to 52.6 percent of epileptic patients suffered from depression [8–12]. So far, three major medications including surgery, drug therapy and dietary supplementations are employed to ameliorate epileptic seizures, among which pharmacotherapy remains the dominant one [13,14]. In spite of the great progress in the development of new anti-epileptic drugs, there are still about 30% to 40% of patients that are not sensitive to the current drugs [13,15–17]. As early as 2014, it was reported that nearly 100 patients with epilepsy were not well treated with traditional anti-epileptic drugs including valproic acid, levetiracetam, carbamazepine, lamotrigine, topiramate and gabapentin in 218 patients [18]. In addition, several adverse effects such as hepatic toxicity [19], cognitive deficits [20,21] and skin epidermal necrosis (mainly Stevens-Johnson syndrome) [22] often appear after treatment with these antiepileptic drugs [23–25]. Therefore, it is of desperate need to probe the etiology of epileptogenesis and develop the novel drugs for suppressing epilepsy and/or decreasing adverse effects following epileptogenesis.

2. Cell communication serves as a great contributor of epileptogenesis The occurrence of epilepsy can be influenced by a variety of factors, such as cell communication, oxidative stress [26–28] and others [29]. There is a general acceptance supporting that epilepsy is triggered by the altered cell communication. Sanae Hasegawa et al. reported that the neuronal communication was remarkably increased in the CA1 region of mice hippocampus following kainic acid-induced neurotoxicity [30]. In general, cell communication contains chemical transmission (indirect communications) and electrical synapses (direct communications). Traditionally, chemical transmission contributes to the generation of epilepsy. It was previously assumed that an imbalance between glutamate-mediated excitatory neurotransmission and γ-aminobutyric acid (GABA)-associated synaptic inhibition resulted in epileptogenesis [31–34]. Substantial investigations depicted that excessive generation of glutamate triggered neuronal hyperexcitability in epileptic patients [35–38]. In addition, it was also found that the level of extracellular glutamate was increased in the focal pilocarpine model of limbic seizures [36]. With further research on the pathogenesis of epilepsy, there is more and more studies illustrating that direct cell-to-cell communication including gap junction (GJ) and mitochondrial transfer [39–41] also serve as a critical contributor of epileptogenesis [42]. It was previously reported that neuronal GJs could modulate the largescale simultaneous firing of neurons during seizures [43], implying that enhanced GJ is likely to be a basic mechanism in the occurrence of seizures [44,45]. Furthermore, increased electrotonic coupling was reported to facilitate the synchronization of neuronal firing and human

⁎ Corresponding author: Dr Xiao-Yuan Mao, Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, China and Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha 410078, Hunan, China. E-mail address: [email protected] (X.-Y. Mao).

https://doi.org/10.1016/j.biopha.2018.10.068 Received 13 July 2018; Received in revised form 8 October 2018; Accepted 12 October 2018 0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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4. Alterations of GJs in epilepsy

primary astrocytes isolated from patients with intractable epilepsy showed increased gap junctional coupling [46]. Besides, in the pilocarpine-induced epileptic model, the expression of connexin36 (Cx36, a common molecule of gap junction) was significantly reduced at 1 h, 4 h and 1 week after status epilepticus, which implied that Cx36 was negatively correlated with the onset of epileptogenesis in the early stage [47]. Similarly, Cx36 knockout was also found to decrease the threshold of pentylenetetrazol (PTZ)-induced seizures in mice [43]. These findings implicate that the GJ-associated molecule Cx36 may serve as a novel target for the treatment of epilepsy.

4.1. Neuron-neuron GJs in epilepsy GJs between neurons are often thought as “connections” between epilepsy and GJs. It is generally believed that GJs form direct intercellular connections between neurons, promoting hypersynchrony neuronal activity associated with seizures [68–72]. Early studies indicated that the interconnection between neurons via GJs played a key role in neuronal synchronization and epileptogenesis [73]. High-frequency oscillations (HFOs), a type of brain activity, are recorded from brain regions that can generate seizures [74]. It has been considered as an indication of a network mechanism of epileptic seizures in the early stages of seizures [75,76]. An increasing number of evidences indicates that GJs interconnecting neurons are vital in generating these synchronous oscillations. In the pilocarpine-induced temporal lobe epilepsy rats model, GJ blockers carbenoxolone and quinine decreased the average amount of fast ripples and the average amount of oscillation cycles per fast ripple event in the hippocampus [77]. Coincidently, Maier N et al found that spontaneous sharp waves and ripple oscillations were less frequently in slices from mice lacking Cx36 than in littermate controls [78]. In addition, with the study of the neuron-neuron GJs, it was found that the Cx was also closely related to the occurrence of epilepsy [72,79]. Cx36 is the major but not the only neuronal connexin [80,81]. The critical role of Cx36 exemplified from the report that Cx36 knockout in mice resulted in approximately 95% loss of neuronal GJ coupling [82]. The Cx36 GJ-coupled GABAergic populations play an irreplaceable role in setting basal inhibitory stress in several brain regions and maintaining physiological resistance to epileptiform activity [83]. A previous investigation demonstrated that inhibition or deficiency Cx36 could exert anti-epileptogenic, pro-epileptogenic, and bystander effects [84]. In terms of its anti-epileptogenic effects of Cx36, suppression of the Cx36 disrupted the inhibitory delivery of GABAergic interneurons in the CA1 region of hippocampus in amygdala kindling model, and subsequently accelerate seizure severity and epileptogenesis [85,86]. Likewise, Cx36 knockout mice exhibit higher susceptibility to seizures induced by the PTZ-induced epilepsy model compared to wildtype mice [43]. Nevertheless, some other findings are controversial. A previous study indicated that the hippocampal inhibitory circuit was mainly composed of Cx36 gap junctions in CA1. Any disruption in this pathway can trigger hippocampus hyperexcitability and promote epileptogenesis [87]. Similarly, quinine, a blocker of neuronal Cx36mediated GJs increased both the frequency and amplitude of lowmagnesium-induced seizure-like events in rat cortical slices [88]. The alterations of Cx36 are differentiated expressed in various animal models of epilepsy. For details, in a pilocarpine model of epilepsy, the expression level of Cx36 had no significant difference between the epileptic model and matched control rats at different time points [89]. And in the kindling model of epilepsy, both mRNA and protein level of Cx36 was recovered to a basal level after obtaining a secondary generalized seizure [90]. In parallel, mefloquine, a gap junctions’ blocker, was nearly 75-fold more potent at blocking Cx36 and Cx50 gap junctions than quinine. It was surprising that the mefloquine failed to elicit any change in seizure-like event amplitude, frequency, or duration in low-magnesium and aconitine-treated neocortical slice preparations between wild type mice and Cx36 knockout mice [91,92]. Taken together, although the role of Cx36 in the development of epilepsy is diverse. A lot of reports indicate that gap junctions between neurons can still act as the main regulators of epileptogenesis.

3. GJs in the brain 3.1. Structure of GJs GJs are the crucial membrane channel in cells that can exchange metabolites and information directly [48,49]. There is a wide range of GJs among various cells of the CNS, which is the structural basis for direct intercellular communication between cells [48,50–52]. GJs show a layered composition, which are formed by the apposition of connexons from adjacent cells [52]. Each connexon is formed by six connexin proteins. The main structural component is the membrane protein connexin, which is organized as the basic unit of the structure [53]. The connexon protein is a hexameric structure having a ring-like appearance in the negative staining preparation. At present, 20 different connexin genes have been identified in mice and 21 in humans [54–57]. A single connexon from one cell docks or associates with a homologous connexon on an adjacent cell to form a GJ channel. The channel allows the passive diffusion of molecules with a molecular weight less than 1 KDa or a diameter less than 1.5 nm. These small molecules include intracellular metabolites, second messengers (cAMP, Ca2+, IP3) and other regulatory substances involved in cell growth and differentiation [58–60].

3.2. Cxs act as the basic unit of GJ As the primary component of GJs, Cx is a large class of membrane proteins and each Cx consists of four membrane-spanning domains, two extracellular loops, three cytoplasmic components, one amino- and carboxy-terminal region and a cytoplasmic loop [45,52]. The cytoplastic loop consists of about 50% of the amino acid sequences of each family member. The amino (N-terminal) and carboxyl (C-terminal) ends of Cx are located in the cytoplasm. Internally, the amino terminus is relatively conserved, while the carboxyl terminus is more diverse. The level of phosphorylation of the amino-terminal filament/threonine and tyrosine residues influences the functional status of GJ [48,61]. So far, 20–21 Cxs subunits have been identified in mammals, 9 of which have been differentially expressed in the CNS, including Cx26, Cx32, Cx33, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx46 [62]. The results of in situ immunolabeling studies in the brain showed that the distribution of Cx types in the mammal brain was different and had certain specificity. Cx32, Cx36 and Cx26 are the major GJ proteins in neurons while Cx43, Cx30, Cx45, Cx40, and Cx32 are mainly distributed in astrocytes. Additionally, Cx32 and Cx45 are mainly present in the oligodendrocytes. Cx43, the dominant Cx subunit, is not only expressed in astrocytes but also expressed in meningeal endothelial cells and ependymal cells. The second most predominant connexin in the brain is Cx32, which is mainly expressed in oligodendrocytes and also expressed in pyramidal cells, granulosa cells, midbrain and basal ganglia neurons in the cerebral cortex and hippocampus [63–66]. GJs not only exist between the same type of cells but also between different types of cells. There are at least three different sorts of GJs in the CNS according to cell types, including neuron-neuron, astrocyte-astrocyte and neuron-astrocyte GJs [67] (Table 1).

4.2. Astrocyte-Astrocyte GJs in epilepsy Like neurons, astrocytes are an active factor in neuronal signaling processing and numerous GJs are expressed in astrocytes. GJs are formed by the major Cx proteins Cx43 and Cx30 in astrocytes, which allow long-range intercellular exchange of ions, metabolites, amino 58

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Table 1 The distribution and expression of connexin subtypes in different epileptic models. Connexin

Distribution

Expression

Epileptic model

References

Cx26 Cx30

Neuron, Astrocyte Astrocyte

Up-regulated No change Slightly down-regulated

[158] [159] [160]

Cx32

Neuron, Oligodendrocyte

Cx36

Neuron, Oligodendrocyte, Microglia

No change Down-regulated Down-regulated Up-regulated 6 h after KA kindling then down-regulated in 3 d and 7d after KA kindling Up-regulated during focal seizures, then back to basal levels after onset of generalized seizures Up-regulated Up-regulated Up-regulated No change Up-regulated Up-regulated

4-aminopyridine induced epilepsy model In vivo kindling model Kainate-treated animal models of human temporal lobe epilepsy In vivo kindling model In mesial temporal lobe epilepsy in humans Pilocarpine-induced status epilepticus Kainic acid kindling induced epilepsy Adult Wistar partially-kindled rat model

[90]

Temporal lobe epilepsy Pilocarpine-induced epilepsy model Pentylenetetrazole (PTZ) induced kindling model Adult Wistar partially-kindled rat model Temporal lobe epilepsy Intractable epilepsy

[123] [89] [162] [90] [163] [96]

Cx43

Neuron, Astrocyte, Microglia

[159] [95] [47] [161]

Fig. 1. The relationship between gap junctions and epileptic seizures. Gap junctions (GJs) are formed by the apposition of connexons from adjacent cells. Each connexon is formed by six connexin proteins; GJ channels are regulated by diverse signaling molecules in neurons and astrocytes. Protein kinase signaling pathways intensified by N-methyl-D-aspartate (NMDA) receptors are potentially important ways to increase neuronal coupling. Astrocytes respond to receptors for neurotransmitters via Ca2+ intracellular oscillations and intercellular Ca2+ waves. Ca2+ waves can be transmitted through gap junctions between adjacent astrocytes, thereby activating several astrocytes at the same time, they can also trigger the release of glutamate, ATP, cyclic adenosine monophosphate (cAMP) and so on. These molecules can activate adjacent astrocytes and participate in the transmission of intracellular Ca2+ waves, and activate receptors including NMDA, and purinergic receptors. ATP released from astrocyte hemichannels through the extracellular space activates metabotropic P2Y receptors and ionotropic P2X receptors lead to the closure of astrocytes GJ channels. Accumulation of extracellular glutamate as a result of astrocytic uncoupling is expected to generate and sustain spontaneous epileptiform activity. 59

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[169] [170] [171] [172] [173]

[167] [168]

40 mg/kg of mefloquine inhibit the generalized tonic-clonic seizures induced by PTZ. Heptanol attenuated the burst amplitude and duration of epileptiform activity at low concentration (0.2 mM), and blocked bursting at higher concentration (0.5 mM). 1-heptanol markedly depressed all the epileptiform markers of the evoked responses.

acids and nucleotides, to maintain synaptic transmission [93]. Cx43 is the most expressed GJ protein in the mammalian CNS and is primarily expressed in astrocytes [94]. A lot of studies have found that Cx43 was up-regulated with the development of epilepsy, and the intensity of Cx43 expression was affected by the time of epileptic seizure [95–97]. These findings implicate that Cx43 may contribute to the development of epilepsy. Previous investigations also depicted that Cx43 was altered dynamically at different time points after kainic acid-induced epileptic seizures in rats. The results showed that the expression of Cx43 was increased with the prolongation of seizures in the cerebral cortex and hippocampus of rats after kainic acid-induced seizures. The relevant mechanism may be that the increased expression of Cx43 promotes the formation of GJs, increases the number of new electrical synapses and electrical conductivity, and leads to the expansion of synchronized firing of neurons and causes epilepsy [98]. Rouach et al. indicated that GJ between astrocytes for delivery of glucose or lactic acid to astrocytes was required for maintenance of excitatory synaptic transmission and epileptiform activity through double knockdown of glial Cx30 and Cx43 [99]. Meanwhile, Carissa G Fonseca et al. reported that Cx43 was also increasingly regulated in astrocytes in temporal lobe epilepsy patients via double immunohistochemical labeling of Cx43 and GFAP (the astrocytes marker) and up-regulation of Cx43 in astrocytes further exacerbated comprehensive seizures [100]. According to reports, the high expression of Cx43 in low-grade tumors and in the peritumoral reactive astrocytes suggested that they may contribute to tumor-associated seizures [101–103]. In addition, studies have suggested that extensive GJs make astrocytes as a functional syncytium, and when seizures occur, a large amount of Ca2+ enters into neurons, leading to a decrease in extracellular Ca2+. The reduction of extracellular Ca2+ plays an important role in the synchronization of epileptiform discharges and the onset of epileptic discharges. Excitatory Ca2+ influx from astrocytes can be disseminated in this functional syncytium, known as Ca2+ wave, which is important for neuromodulation [104]. The recent studies have also shown that the inflammation-induced interference of astrocytic GJ coupling and K+ clearance is a key event in the etiology of temporal lobe epilepsy [105]. In summary, there is increasing evidence suggesting that GJs between astrocytes play a key role in the pathogenesis of epilepsy [106].

GJ and Cx hemichannels

GJ and Cx hemichannels

GJ and Cx hemichannels

GJ and Cx hemichannels

Heptanol

Octanol

Meclofenamic acid

Flufenamic acid

4.3. Astrocyte-neuron GJs in epilepsy Ca2+ imaging techniques and electrophysiological revealed a similar pattern of ion channels and transmitter receptors in astrocytes and neurons, suggesting that astrocytes can sense and respond to neuronal activity [107]. Interestingly, neurons and astrocytes are found to be coupled as demonstrated by the presence of GJ proteins and both electrical and dye transfer between the two cell types. It is confirmed the view that astrocytes are the direct communication target of neurons [108]. As mentioned before, any stimulation that initiated the conversion of astroglia pathophysiology to reactive astrocytes could cause seizure delay [109]. Except as the long-term changes in physiology of astrocytes, physiologically active astrocyte-neuron GJ in the hippocampus may also lead to the generation or exacerbation of epileptic activity [110,111]. Astrocyte-neuron GJ involves a variety of signaling mechanisms, most of which are dependent on astrocyte Ca2+ signaling [112,113]. Similar to previous reports in Alzheimer's disease [114], the distribution of resting [Ca2+] in astrocytes may be affected by epileptic activity. However, Ca2+-dependent release of astrocytes may not induce epileptic activity by itself, it can promote synchronization of neuronal activity and thus reduce the threshold of epileptic activity [115], implicating that the released Ca2+ from astrocytes can influence Ca2+-dependent astrocyte-neuron communication and consequently maintain altered network excitability. On the other hand, in astrocytedeficient mice models, impaired K+ clearance led to spontaneous epileptiform activity, and subsequently reduced the threshold of seizure activity [116]. Considering that astrocytic GJs play an important role in

GJ: Gap junction.

GJ, Cx and Panx hemichannels Mefloquine

High K+–low Ca2+ induction of evoked and spontaneous epileptiform field potentials Cs-induced field bursts Penicillin-induced epilepsy Focal cortical epilepsy Amygdaloid kindling and hippocampus rapid kindling models 4-aminopyridine/Mg2+ induced epileptiform activity

Cs-induced field bursts were markedly suppressed by octanol. Octanol significantly decreased epileptiform activity. Meclofenamic acid reduced percentage seizure time to a significant extent. Meclofenamic acid blocked limbic epileptogenesis in both two kindling models. Flufenamic acid simultaneously decreased glutamatergic excitatory synaptic activity and reduced neuronal excitability.

[166] [135] Quinine may dose-dependently decrease the severity of seizure. Mefloquine significantly decreased the amplitude and duration of seizure-like activity.

[141]

[165] GJ and Cx hemichannels Quinine

Low-Ca2+ or high-K+ artificial cerebrospinal fluid induced epileptiform activity Pentylenetetrazole (PTZ)-kindled rats epilepsy model Co-administration of 250 μM 4-AP and 5 μM bicuculline induced drugresistant seizure-like activity PTZ-inducced tonic-clonic seizures Zero-Ca2+ model of epileptiform activity

[164] [149]

Carbenoxolone inhibited astroglial synchronization, seizure-like events generation in slices. Carbenoxolone significantly inhibited hyperexcitability of the hippocampal neuronal network and persistent seizure discharge. Quinine could selectively suppress the generation of ictal activity. GJ and Cx hemichannels Carbenoxolone

Low-Mg2+ epilepsy model Kainic acid-kindled rats epilepsy model

References Results Models Mechanisms Drugs

Table 2 Effects of some gap junction blockers and openers in epileptic model.

Q. Li et al.

60

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mediating the local uptake and spatial buffering of K+ in the extracellular space, the researchers conducted a series of simulations using the single astrocyte-neuron network models and the coupled astrocyteneuron module network. The results showed that changes in Kir4.1 channel conductance and GJ strength induced spontaneous epileptic activity in the absence of external stimuli [117]. These studies indicate that the communication between astrocytes and neurons regulates the occurrence and development of epilepsy through multiple pathways. This also suggests that GJ between astrocytes and neurons may serve as an effective target for epilepsy treatment. As the same time, GJ-mediated astrocytic coupling was lost in epilepsy [105]. So, it is indispensable to understand the molecular modifications that lead to uncoupling of GJs. Current research has determined that changed of Cx43 phosphorylation was a possible mechanism leading to uncoupling. It was known that at least five serine and two tyrosine residues located in the Cx43-C-terminus (CT) were phosphorylated in vivo, resulting in down-regulation (Y247, S255, S262, Y265, S279/282, S368) of gap junction intercellular communication (GJIC) [118,119]. Serine phosphorylation, which were mediated by PKC (S368) [120], mitogen activated protein kinase (MAPK) (S255, S262, and S279/S282) [121], and the cell-cycle dependent kinase CDC2 [122], could reduce GJIC. Deshpande T et al. found that phosphorylation by MAPK at S255 decreased coupling in mesial temporal lobe epilepsy with hippocampal sclerosis [123]. Besides, a large number of structural and cytoplasmic regulatory proteins were known to interact with Cx43 mainly at its C-terminus [118,124]. One of these regulatory proteins is a member of the membrane-bound guanylate kinase family called zonula occludens-1 (ZO-1), which directly interacts with Cx43 and other connexins through its PDZ-2 domain [118]. Recently, studies have shown that the Cx43 connexons were bound to ZO1 when they docked with connexons from an adjacent cell near the gap junction plaques. ZO-1 binding down-regulated the rate of channels that added to the GJ plaques [125]. According to the report, proinflammatory cytokines also induced uncoupling of astrocytes [105]. And cytokines like IL-1β and TNF-α were upregulated and released in epilepsy [126]. In agreement, it was reported that IL-1β produced a strong decrease in astroglia coupling when combined with TNF-α [127]. Recently, Alyu and Dikmen demonstrated that inflammatory processes participated to epileptogenesis in several ways such as affecting fibroblast growth factor-2 (FGF-2) [128] and targeting of ATP-gated purinergic P2 receptors [129]. And FGF-2 could cause a reduction of Cx43protein, -mRNA, and intercellular communication revealed by dye spreading [130]. Nucleotides such as adenosine-5′-triphosphate (ATP) was also elevated by 300% during spontaneous seizures in the pilocarpine-induced temporal lobe epilepsy [129]. Moreover, ATP, via P2X and P2Y receptor activation, reduced hemichannel activity in several cell types including astrocytes and neurons [131]. Other than that, glutamate is a key neurotransmitter that plays a key role in chemical and electrical synapses. And metabotropic glutamate (mGlu) receptors are known to regulate spike and wave discharges (SWDs), the electroclinical hallmarks of absence seizures [132]. At the same time, previous study indicated that activation of group II metabotropic glutamate receptors (mGluRs) enhanced electrotonic and increased the protein levels of Cx36 [133] (Fig. 1). All of these findings suggest that uncoupling of astrocytes has profound implications for epileptogenesis. Prevention of uncoupling has potential therapeutic benefits. Therefore, determining the exact molecular pathway that leads to uncoupling at different stages of epilepsy is worthy.

junction blockers and openers can regulate the open or closed state of the GJs, thereby affecting the occurrence of epilepsy. The role of blockade agents such as carbenoxolone, quinine, mefloquine, quinidine, and tonabersat in epilepsy has been reported in epileptic animal models [134–146]. And a lot of reports that studied the relationship between GJ blockers/openers and epileptiform activity were carried out in hippocampal slices or thalamocortical slices. As was reported by scholars in early years, carbenoxolone (a broad-spectrum GJ blocker) and mefloquine decreased the amplitude and duration of seizure-like activity in thalamocortical slices [134,135]. Carbenoxolone could also depress the frequency of spontaneous epileptiform activity in rat hippocampal slices [136]. Interestingly, quinine also observed the similar effects with carbenoxolone in the neocortex in patients with temporal lobe epilepsy [137,138]. Besides, spontaneous synchronous events generated by application of 4-aminopyridine in neocortical slices from temporal lobe epileptic patients was also decreased by carbenoxolone [137]. Also, antiepileptic effects of other GJ blockers have been reported. Quinine, as a potent gap junction blocker, depressed GABAergic ictal-like events in rat hippocampal slices exposed to GABA(B) receptor antagonists [139]. And quinidine abolished the ictal-like activities in rat thalamocortical slices induced by 4-aminopyridine [140]. 1-heptanol and 1-octanol had been reported to significantly depress all the epileptiform markers of the evoked responses induced in CA3 area of rat hippocampal slices by a high K+-low Ca2+ perfusion fluid [141]. Tonabersat, a novel cis benzopyran derivative, is used for the treatment of migraine and epilepsy [142–144]. A previous study reported that tonabersat could elevate the threshold for PTZ-induced tonic forelimb extension seizures [145]. Meanwhile, tonabersat was observed to prevent inflammatory damage via blocking Cx43 hemichannels in vitro ischemia-reperfusion model [146]. Therefore, it is possible that tonabersat may exert anti-epileptic effect by suppressing Cx43 hemichannels. On the contrary, trimethylamine, a GJ opener, increased secondary and tertiary discharges induced by Mg2+-free solution [147]. As well as, trimethylamine significantly enhanced seizure-like activity induced by 4-AP and bicuculline [135]. Similarly, ammonium chloride increases the frequency and duration of spontaneous field activity in the Ca2+-free model of epilepsy [148]. In addition to the studies in the in vitro model, increasing evidence from in vivo experimental studies also indicates that GJs are involved in the epileptiform activity. In the kainic acid-kindled rats, carbenoxolone, quinine, and quinidine reversed the overexpression of MAP-2 and SYP and led to the inhibition of propagation of seizure activity [149]. According to the report, fast ripples are considered to be potential biomarkers for epilepsy [150,151]. Carbenoxolone reduced the average number of oscillation cycles per fast ripples event in the hippocampus of rats with spontaneous seizures induced by pilocarpine [151]. Carbenoxolone could significantly decrease the spectral power and the amplitude of the epileptiform activity induced by PTZ [152]. Similarly, in the PTZ-induced epilepsy model, quinine could attenuate seizure severity and the mean seizure stages at different doses compared with the control [151]. In addition, quinine could inhibit epileptiform activity by decreasing the amplitude and frequency of epileptiform spikes [153] (Table 2). Cx mimetic peptides, short synthetic peptides corresponding to intracellular amino acid sequences of diverse Cx have better specificity compared to traditional GJ blockers and openers. In particular, it was reported that Cx mimetic peptides reversibly inhibited GJ channel function in a concentration and time-dependent manner [154]. Therefore, it can reasonably speculate that Cx mimetic peptides may contribute to the development of epilepsy. As reported in previous study, after treatment with Cx43 mimetic peptides more than 10 h, the spontaneous recurrent epileptiform activity was reduced through selectively inhibition of Cx43 GJ in rat hippocampal slices [155]. In vivo, pilocarpine-induced seizures were suppressed by TAT-Gap 19 (an inhibitory Cx mimetic peptide for Cx43 hemichannels) [156]. Besides, in the model of fetal sheep ischemia, the specific Cx43 mimetic peptide

5. The role of GJ blockers and openers in epilepsy As previously known, GJ-mediated electrical coupling plays a role in the generation of highly synchronous electrical activity. The hypersynchronous neuronal activity is a significant feature of convulsive events, so it can be speculated that gap junctional communication may be a potential mechanism for epileptogenesis and maintenance. The gap 61

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markedly reduced the duration of seizures by blocking the Cx43 hemichannels after ischemia [157]. Currently, it is a promising pharmacology tool for studying the relationship between specific connexin proteins and epilepsy therapy. In agreement with previous studies, it can be proposed that GJ blockers attenuate seizures and GJ openers can promote the occurrence of epilepsy both in vivo and in vitro models. More and more researchers are concerned about the role of GJ blockers/openers in the treatment of epilepsy. Therefore, GJ blockers/openers can provide new strategies for the development of new antiepileptic drugs.

[10]

[11] [12]

[13] [14]

6. Concluding remarks and perspectives

[15]

Currently, a large number of in vitro and in vivo experiments have shown that GJs could predictably affect the epileptogenesis: blockers reduce epileptic seizures and openers increase seizure severity. Further investigations are indispensible to explore the potential molecular mechanisms on how Cxs-mediated GJs manipulate generation of epileptic seizures. In terms of the diversity of GJs and the corresponding constituent units (Cxs), identifying the role of specific GJ and Cx in the development of epilepsy will help to develop the novel therapeutic target for treating patients who are subject to epilepsy. In spite of current experimental data showing the suppressive effects of GJ blockers on epilepsy, non-specificity still exists and various side effects appear, which limits their therapeutic potential in clinical practices. In the future, further screening of specific GJ blockers is necessary and the effects of these GJ blockers on epileptic patients are validated in multicenter clinical trials. Anyway, Cxs-mediated GJs are critical to epileptogenesis and the development and synthesis of anti-epileptic drugs based on Cxs-associated GJs will provide new therapeutic avenues for the treatment of epilepsy.

[16]

Conflict of interest

[25]

[17]

[18] [19]

[20]

[21]

[22]

[23] [24]

The authors declare that they have no potential conflict of interest

[26] [27]

Acknowledgments This work is partially financially supported by the National Natural Science Foundation of China (Nos. 81671293 and 81302750), Natural Science Foundation of Hunan Province (No. 2017JJ3479), and the Fundamental Research Funds for the Central Universities of Central South University (2018zzts900).

[28]

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