Altered Kir and gap junction channels in temporal lobe epilepsy

Neurochemistry International 63 (2013) 682–687

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Review

Altered Kir and gap junction channels in temporal lobe epilepsy Peter Bedner, Christian Steinhäuser ⇑ Institute of Cellular Neurosciences, University of Bonn, Bonn, Germany

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Article history: Available online 26 January 2013 Keywords: Epilepsy Astrocyte Kir4.1 Cx43 Cx30 Dye coupling

a b s t r a c t Since astrocytes may sense and respond to neuronal activity these cells are now considered important players in brain signaling. Astrocytes form large gap junction coupled syncytia allowing them to clear the extracellular space from K+ and neurotransmitters accumulating during neuronal activity, and redistribute it to sites of lower extracellular concentrations. Increasing evidence suggests a crucial role for dysfunctional astrocytes in the etiology of epilepsy. Notably, alterations in expression, localization and function of astroglial K+ channels as well as impaired K+ buffering was observed in specimens from patients with pharmacoresistant temporal lobe epilepsy and in chronic epilepsy models. Altered astroglial gap junction coupling has also been reported in epileptic tissue which, however, seems to play a dual role: (i) junctional coupling counteracts hyperactivity by facilitating clearance of elevated extracellular K+ and glutamate while (ii) it also provides a pathway for energetic substrates and fuels neuronal activity. Dysfunctional astrocytes should be considered promising targets for new therapeutic strategies. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Epilepsy is a condition of the brain characterized by the unpredictable occurrence of seizures, and despite medication about one third of patients have poor seizure control and become medically refractory. Many antiepileptic drugs (AEDs) must be taken chronically for seizure suppression, which often leads to unwanted side effects. Thus, there is an urgent need for the development of more specific AEDs. Recent critical reviews state the little progress AED development has made over the past years, and call for new concepts and fresh thinking to identify new targets for improved treatment of epilepsy (Löscher and Schmidt, 2011; Simonato et al., 2012). Still, epilepsy research is neuron-centered, which might be part of the dilemma because many of the alterations neurons undergo in the course of epilepsy seem to be secondary rather than causative for the epileptic condition. Astrocytes have now been recognized as active partners in neural information processing. These cells express a plethora of ion channels and transmitter receptors (Verkhratsky and Steinhäuser, 2000) which allow them to sense neuronal activity and feed back to neurons to modulate CNS signaling (Halassa and Haydon, 2010; Perea and Araque, 2010). While the pathways enabling activation of these cells under physiological conditions are still matter of investigation, evidence is emerging suggesting a critical role of

⇑ Corresponding author. Address: Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Sigmund Freud Str. 25, 53105 Bonn, Germany. Tel.: +49 228 287 14669; fax: +49 228 287 19121. E-mail addresses: [email protected] (P. Bedner), [email protected] (C. Steinhäuser). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.01.011

astrocyte dysfunction in the pathogenesis of neurological disorders (Seifert et al., 2006), including epilepsy (Seifert et al., 2010). In this review, we will focus on astroglial alterations in inwardly rectifying K+ (Kir) channels and gap junction coupling in temporal lobe epilepsy. The published data suggest that these changes might be causative for hyperexcitation, neurotoxicity and the generation or spread of seizure activity, and that astrocytes might represent promising new targets in the search for improved antiepileptic therapies. 2. Kir channels and temporal lobe epilepsy 2.1. K+ uptake and K+ spatial buffering Prolonged neuronal activity causes transient accumulation of extracellular K+ ([K+]o) which, if uncompensated, would lead to neuronal depolarization and consequently to hyperexcitability. During epileptiform activity, [K+]o can increase from the resting concentration of 3 mM to a ceiling level of 10–12 mM (Heinemann and Lux, 1977). Higher [K+]o concentrations (up to 60 mM) are observed under conditions such as hypoxia/ischemia and during spreading depression (Hansen, 1985; Somjen, 2001). Since even small changes in [K+]o can influence neuronal excitability, K+ homeostasis in the brain needs to be tightly regulated. Key players in this regulation are astrocytes whose membranes are highly permeable for K+ at resting potential and which are connected to each other via gap junction (GJ) channels to form a functional syncytium. During neuronal activity K+ buffering has been proposed to be realized by two different mechanisms: K+ uptake and spatial K+ buffering (for review see (Kofuji and Newman, 2004). Net

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uptake of K+ is mainly accomplished by glial and neuronal Na,KATPase and Na–K–Cl cotransporters (D’Ambrosio et al., 2002; Kofuji and Newman, 2004; Ransom et al., 2000). On the other hand, glial Kir channels in concert with Cl channels might contribute to K+ clearance through its net uptake. Kir channels have a high open probability at resting potential and their conductance increases at high [K+]o (Ransom and Sontheimer, 1995). The membrane conductance of astrocytes is dominated by Kir4.1 channels (Seifert et al., 2009) and accordingly, astrocyte-specific deletion of Kir4.1 caused defective K+ clearance and seizures (see below), emphasizing their crucial role in the regulation of [K+]o (Djukic et al., 2007; Haj-Yasein et al., 2011). However, the relative contribution of Kir channels vs. Na–K pumps in net K+ uptake is still under debate (Kofuji and Newman, 2004; Olsen and Sontheimer, 2008; Walz, 2000). The model of spatial buffering, first described by (Orkand et al., 1966), implies that K+ taken up by astrocytes at sides of high neuronal activity is transported through the GJ connected astrocytic network to distal regions of lower [K+]o. This mechanism is energy-independent, driven by the local K+ equilibrium potential and the glial syncytium membrane potential (Kofuji and Newman, 2004; Orkand et al., 1966; Walz, 2000), and more efficient than diffusion through the extracellular space (Gardner-Medwin, 1983). To evaluate the importance of the glial syncytium for efficient buffering of K+, Wallraff et al. (2006) used K+-sensitive electrodes and assessed changes in [K+]o in hippocampal slices obtained from mice devoid of astrocytic GJ proteins (Cx30/; Cx43fl/fl hGFAP-Cre mice; termed dko mice). They found impaired spatial buffering in the stratum lacunosum-moleculare but, unexpectedly, preserved buffering in the stratum radiatum (Wallraff et al., 2006). Obviously GJ-independent mechanisms contribute to K+ buffering in the radiatum. In this context the concept of ‘indirect coupling’ has been suggested, which implies that the K+ released by one cell can be taken up by more distally located, non-coupled neighboring cells through K+ channels (Newman, 1995; Ransom, 1996). It is thought that in such a case, spatial buffer current may flow within single elongated astrocytes from sites of elevated [K+]o towards sites of lower [K+]o (Ransom, 1996; Wallraff et al., 2006). Such a process, termed ‘K+ siphoning’, has earlier been described within retinal Müller cells which transfer excess K+ from the inner plexiform layer to the vitreous humor and blood vessels (Newman et al., 1984). Besides K+ clearance, K+ siphoning was hypothesized to mediate neurovascular coupling (Paulson and Newman, 1987) which, however, could not be confirmed later on (Metea et al., 2007). In all forms of spatial buffering, the weakly-rectifying Kir4.1 channels which allow bidirectional diffusion of K+ across the membrane, may mediate not only uptake of K+ but also its release. 2.2. Kir4.1channels and K+ buffering in epilepsy To investigate the role of Kir4.1 channels in K+ buffering the effect of Ba2+, which at sub-mM concentrations selectively blocks Kir channels, on stimulus-induced rises in [K+]o or iontophoretically applied K+ was analyzed in sclerotic and non-sclerotic human hippocampal slices as well as in a rat model of epilepsy. It could be demonstrated that Ba2+ enhances [K+]o accumulation in non-sclerotic human and control rat hippocampus, but had no effect in sclerotic human and rat tissue. These results not only emphasized the importance of Kir4.1 channels in K+ buffering in the non-sclerotic hippocampus, but also provided evidence for the disturbance of Ba2+-sensitive K+-uptake in sclerosis (Heinemann et al., 2000; Jauch et al., 2002; Kivi et al., 2000). Consistent with this idea are the results from patch clamp studies performed in the human sclerotic CA1 region, demonstrating significantly reduced (Hinterkeuser et al., 2000; Schröder et al., 2000) or even complete loss (Bordey and Sontheimer, 1998) of Kir currents. In line with these reports,

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Western blot analysis by (Das et al., 2012) revealed a 50% reduction of Kir4.1 protein levels in sclerotic human hippocampus compared to post-mortem control tissue. Taken together, these data strongly indicate that in TLE with hippocampal sclerosis, impaired K+ clearance and increased seizure susceptibility result from reduced expression of Kir channels. Whether these events represent the cause or the consequence of the disease is still an open question. (Takahashi et al., 2010) found no changes in astrocytic Kir currents 7–16 days following systemic injection of kainic acid in rats, which would indicate that Kir downregulation is a consequence of epilepsy. In contrast in an albumin model of epilepsy, downregulation of Kir4.1 was found to occur already before the onset of epileptic activity (David et al., 2009). Since blood–brain barrier lesion and the resulting extravasation of albumin are considered primary events in human epileptogenesis (Heinemann et al., 2012), the results by David et al. (2009) support a causative role for Kir channel downregulation in epilepsy. Genetic inactivation of the Kir4.1 encoding gene, KCNJ10, substantiated an essential role for Kir4.1 in glial K+ buffering (Djukic et al., 2007; Kofuji et al., 2000). General knock-out of Kir4.1 resulted in a severe pathology and lethality between postnatal days 8 and 24 (Neusch et al., 2001). Similarly, conditional knock-out mice with glia-specific deletion of Kir4.1 displayed a severe phenotype, including ataxia, seizures and early lethality (Djukic et al., 2007). In addition, the deletion caused substantial depolarization of gray matter astrocytes which not only impaired K+ buffering but also glutamate uptake. As a consequence, decreased spontaneous neuronal activity and enhanced synaptic potentiation was found in these mice (Djukic et al., 2007). The crucial role of Kir4.1 in K+ buffering has been confirmed in follow-up studies demonstrating that disruption of Kir4.1 expression causes an epileptic phenotype (Chever et al., 2010; Haj-Yasein et al., 2011). 2.3. KCNJ10 gene mutations cause epilepsy KCNJ10 is considered a potential causative candidate for increased seizure susceptibility (Ferraro et al., 2004). In children with seizures, ataxia, sensorineural deafness, mental retardation and electrolytic imbalance (SeSAME or EAST syndrome; (Bockenhauer et al., 2009; Scholl et al., 2009) sequencing of the affected KCNJ10 gene revealed various missense and nonsense mutations. These loss-of-function mutations of KCNJ10 were homozygous or compound heterozygous (Bockenhauer et al., 2009; Scholl et al., 2009). Heterologous expression confirmed that the mutations also affected Kir4.1 channel function, e.g. its Ba2+-sensitivity (Bockenhauer et al., 2009; Reichold et al., 2010; Williams et al., 2010). EAST/SeSAME syndrome-related mutations caused a decrease in K+ conductance, and the mutated Kir4.1 had a dominant negative effect when co-expressed with wildtype Kir4.1. Another group of epilepsy patients carried gain-of-function mutations of KCNJ10, and the afflicted patients presented with seizures, autism spectrum disorders and intellectual disability (Sicca et al., 2011). Single nucleotide variations in the KCNJ10 gene were detected in the DNA of TLE patients presenting with hippocampal sclerosis and antecedent febrile seizures. It was concluded that variations in AQP4 (encoding a water channel) and the KCNJ10/KCNJ9 region are likely to be associated with TLE and febrile seizures (Heuser et al., 2010). 3. GJ channels and temporal lobe epilepsy 3.1. Potential roles of GJs in epilepsy In the adult brain, hippocampal astrocytes express the GJ proteins connexin43 (Cx43) and connexin30 (Cx30) (Nagy and Rash, 2000). Consistently, mice lacking these two proteins were devoid

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of tracer coupling (Wallraff et al., 2006). The major astrocytic GJ protein in the hippocampus seems to be Cx43, since only a 22% reduction of interastrocytic coupling was observed in Cx30-deficient mice (Gosejacob et al., 2011). The role of the astroglial network in the pathophysiology of epilepsy is still controversial. A potential antiepileptic function is based on its ability to decrease neuronal excitability by clearance and redistribution of extracellular ions and neurotransmitter (Fig. 1). Disturbance of this function may cause accumulation of K+ and glutamate in the extracellular space (ECS) and, consequently, epileptic seizures. Moreover, disruption of interastrocytic communication was found to cause a reduction of the ECS volume due to astrocytic swelling, further increasing extracellular ion concentrations (Pannasch et al., 2011). Accordingly, disturbed K+ and glutamate clearance, astrocytic swelling, increased synaptic transmission as well as spontaneous epileptiform activity and a decreased threshold for evoking seizure activity have been described in mice with coupling-deficient astrocytes (Pannasch et al., 2011; Wallraff et al., 2006). While these results provide strong evidence for an anticonvulsive role of glial networks, a potential pro-epileptic function arose from findings of Rouach et al. (2008). These authors elegantly demonstrated that GJs are needed for the intercellular trafficking of glucose and its metabolites from

blood vessels through the astrocytic syncytium to distal neurons in an activity-dependent manner (Fig. 1). They proposed that this process is important to sustain synaptic activity under pathological conditions such as epilepsy (Rouach et al., 2008). Thus, reduction of astrocytic coupling seems to exert opposite effects on neuronal excitability: a rapid, seizure-promoting effect due to impaired K+ and glutamate redistribution, but a delayed, anticonvulsive effect because of insufficient energy supply. One could speculate that in this scenario, increased extracellular ion and neurotransmitter levels are responsible for seizure initiation while the disruption of neuronal energy supply terminates hyperactivity. In addition to interastrocytic coupling, panglial communication between astrocyte and oligodendrocytes through heterotypic GJs has been proposed to be involved in the regulation of K+ homeostasis. In this concept, K+ ions released during axonal activity are taken up by oligodendrocytes and transferred through reflexive GJs, which connect adjacent myelin layers of the same cell, to the cell body, and from there through heterotypic junctions into the astrocytic syncytium (Menichella et al., 2006; Rash, 2010). Disruption of this mechanism may result in inadequate K+ clearance and seizures. In accordance with this concept, mice lacking oligodendrocytic connexins (Cx32/ Cx47/ double KO) as well as mice in which panglial coupling is diminished (Cx30/ Cx47/ and

Fig. 1. Dual role of the astroglial syncytium in epilepsy. 1. Spatial K+ buffering. Neuronal activity causes transient elevations of [K+]o. The difference between the membrane potential and the K+ equilibrium potential causes K+ influx into nearby astrocytes. Intracellularly K+ propagates along the electrochemical gradient through the astroglial network and leaves at sites of lower [K+]o. 2. Spatial glutamate buffering. Glutamate released during synaptic activity is taken up astrocytes through the glutamate transporters GLAST and GLT-1. In astrocytes, glutamate is converted into glutamine by the enzyme glutamine synthetase (GS). Glutamine is then released into the ECS, taken up by neurons and reused for glutamate production. During high neuronal activity, dissipation of glutamate is facilitated by its diffusion through the gap junction connected astroglial syncytium. 3. Diffusion of energetic metabolites through the astroglial network sustains synaptic transmission. Glucose is taken up from the blood by astrocytes via the GLUT1 glucose transporter, metabolized to lactate and released into the ECS for neuronal use. Astrocytic gap junctional coupling mediates activity-dependent trafficking of glucose from blood vessels to the sites of high energy demand.

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Cx32/ Cx43/ double KO) display epileptic seizures (Maglione et al., 2010; Menichella et al., 2003, 2006; Tress et al., 2012). Thus, intercellular communication between astrocytes and oligodendrocytes and/or oligodendrocyte reflexive coupling may possess antiepileptic function. 3.2. GJ expression and communication in epileptic tissue Many studies have explored changes in connexin expression using a variety of animal models of seizures and human tissue, with conflicting results. In animals, increased, unchanged and decreased Cx mRNA and/or protein levels have been reported (Condorelli et al., 2002; Elisevich et al., 1997a, 1998; Gajda et al., 2003; Khurgel and Ivy, 1996; Li et al., 2001; Samoilova et al., 2003; Söhl et al., 2000; Szente et al., 2002; Takahashi et al., 2010; Xu et al., 2009). Differences between animal models and in seizure duration might account for this inconsistency. In human epileptic tissue resected from patients with intractable epilepsy, unchanged or elevated Cx43 mRNA and/or proteins levels were described (Aronica et al., 2001; Collignon et al., 2006; Das et al., 2012; Elisevich et al., 1997b; Fonseca et al., 2002; Naus et al., 1991). However, studies on human specimens have to be interpreted with caution because the tissue is usually obtained from patients with pharmacoresistant chronic epilepsy, raising the possibility that it is affected by the long-term treatment with AEDs. Furthermore, human studies suffer from the lack of appropriate controls. Generally, tumor or autopsy specimens were used as controls and it is possible that apparent increases in Cx levels in epileptic tissue are actually caused by decreased levels in controls (Nemani and Binder, 2005). In addition, Cx RNA or protein levels and the extent of functional coupling do not necessarily correlate, since posttranslational modifications, such as phosphorylation, may change gating properties, assembly or subcellular localization of the GJ channels. Functional analysis may therefore provide more accurate results. Increased interastrocytic communication has been described in a post-SE rat model of epilepsy (Takahashi et al., 2010) and in hippocampal slice cultures chronically exposed to bicuculline (Samoilova et al., 2003). Opposite results were reported by Xu et al. (2009), who observed reduced coupling in a mouse model of tuberous sclerosis complex. To our knowledge no functional coupling analyses have been performed in acute human epileptic slices so far. Using fluorescence recovery after photobleaching increased GJ coupling was found in astrocytes cultured from human epileptic specimens when compared with cultures from non-epileptic tissue (Lee et al., 1995). Another strategy to investigate the role of the astrocytic syncytium in epilepsy is pharmacological disruption of GJ communication using inhibitors (such as carbenoxolone, halothan and octanol), substances producing intracellular acidification (sodium propionate, carbon dioxide) or Cx mimetic peptides (small peptides mimicking a sequence of the connexin subunit, inhibiting GJ and Cx hemichannels by binding to their extracellular domain) (Bostanci and Bagirici, 2006, 2007; Gajda et al., 2003; Gigout et al., 2006; Jahromi et al., 2002; Kohling et al., 2001; Medina-Ceja et al., 2008; Perez-Velazquez et al., 1994; Ross et al., 2000; Samoilova et al., 2003, 2008; Szente et al., 2002; Voss et al., 2009). Most of these studies reported antiepileptic effects of GJ blockade although proepileptic effects have also been observed (Voss et al., 2009). In neocortical slices from patients with TLE or focal cortical dysplasia, GJ blockers inhibited spontaneous and evoked epileptiform activity, pointing to a proepileptic role of GJ coupling (Gigout et al., 2006). Here one has to consider that GJ blockers have usually dramatic side effects and barely discriminate between neuronal and glial GJs. In addition, pharmacological blockade may not only inhibit intercellular channels but also Cx and pannexin hemichannels (HCs) which possibly play a differential role in excitability.

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3.3. Role of connexin hemichannels and pannexin channels in epilepsy There is abundant evidence that astrocytic connexins in addition to intercellular GJ channels also form functional membranespanning HCs (for review see (Saez et al., 2003; Theis and Giaume, 2012). These channels possess a low open probability under physiological conditions which, however, increases upon depolarization, altered Ca2+ concentrations, metabolic inhibition or cytokine activation (Giaume et al., 2010; Saez et al., 2005). Opened HCs are permeable for glutamate, ATP, glucose and glutathione (Cotrina et al., 2000; Rana and Dringen, 2007; Retamal et al., 2007; Ye et al., 2003). Elevated open probability of HCs may promote seizures due to excessive release of ATP and glutamate which in turn may increase synaptic excitability. Moreover, ATP release may facilitate the propagation of glial Ca2+ waves, leading to hypersynchronization and spread of ictal activity (Gomez-Gonzalo et al., 2010). It is also reasonable to assume that the role of HCs in epilepsy differs from that of GJ channels. Thus, inflammatory cytokines, which are overproduced in epileptic tissue (Avignone et al., 2008; Vezzani et al., 2012), decrease GJ coupling but increase the open probability of HCs (Meme et al., 2006; Retamal et al., 2007). Further support for this assumption came from the analysis of patients with oculodentodigital dysplasia (ODDD), a rare genetic disease which is caused by mutations in the gene encoding Cx43. Among other neurological symptoms these patients also developed seizures (Loddenkemper et al., 2002). In cell culture, some of the ODDD-associated mutations of Cx43 caused loss of GJ coupling, but increased HC activity (Dobrowolski et al., 2007). Moreover, (Yoon et al., 2010) showed that selective blockade of HCs by low doses of mimetic peptides had protective effects on seizure spread while inhibition of both HCs and GJs by high concentrations of the peptide exacerbated the lesions. Several recent studies questioned the functional significance of Cx HCs and suggest instead that another family of proteins, the pannexins (Panx), form functional non-junctional plasma membrane channels under physiological conditions (Scemes et al., 2009; Scemes and Spray, 2012). These channels are opened by increases in [K+]o, depolarization, cytosolic Ca2+ elevation, mechanical stress and P2X7 receptor activation and permeable for large molecules, such as ATP (Scemes and Spray, 2012; Suadicani et al., 2012). Since high neuronal activity entails increases in [K+]o it was suggested that the resulting opening of Panx might contribute to seizures, through release of ATP. Indeed, pharmacological blockade or deletion of Panx1 in transgenic mice attenuated seizure activity during kainic acid-induced status epilepticus (Santiago et al., 2011). In line with these findings, (Mylvaganam et al., 2010) reported increased Panx transcript levels in an in vitro seizure model.

4. Concluding remarks The new view of brain function considers astrocytes as communication partners of neurons. Accordingly, it is not surprising that increasing evidence suggests a role for these cells in epilepsy. Several laboratories reported dysfunction and dysregulation of astroglial Kir channels and gap junction networks in human and experimental epilepsy. These alterations entail impaired removal and redistribution of K+ and glutamate which are also key features of human TLE with sclerosis. Although in many cases it is still unclear whether the reported astroglial alterations are causative or a consequence of the condition, glial cells, and astrocytes in particular, emerge as new, promising targets for the development of improved AEDs. Future work will clarify whether TLE is a glial rather than a neuronal disorder and hopefully facilitate more specific therapeutic approaches to treat this condition.

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Acknowledgement Work of the authors is supported by Deutsche Forschungsgemeinschaft (SFB/TR3 C1, C9) and European Commission (FP7202167 NeuroGLIA).

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