Gap Junction-Mediated Neuroprotection

C H A P T E R

14 Gap Junction-Mediated Neuroprotection Michael G. Kozoriz, Christian C. Naus Department of Cellular & Physiological Sciences, The Life Sciences Institute, University of British Columbia, Vancouver, Canada

O U T L I N E Introduction

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Are Gap Junctions Protective or Destructive? Gap Junction Proteins and Cellular Protection Gap Junction Proteins and Cellular Destruction

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Channel Mechanisms of Neuroprotection or Neuronal Injury Intercellular Communication: Gap Junction Channels Connexin Hemichannels Pannexins

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INTRODUCTION One of the principal communication pathways in the central nervous system (CNS) involves the unique intercellular channels formed by gap junctions. With the expression of 11 connexin (Giaume, 2010) and two pannexin (Baranova et al., 2004) genes in the CNS, it is not surprising to find that there are diverse functions attributed to these channel proteins. In addition to the electrical synapses between neurons, the majority of gap junctional channels reside in the non-neuronal compartment of the CNS, namely the glial cells (astrocytes, oligodendrocytes, microglia) and endothelial cells. One longheld view of the role of astrocytic gap junctions has been to provide a mechanism by which the extracellular environment of neurons can be carefully regulated, particularly in times of excitation when high levels of potassium and glutamate are released by neurons. Gap junctions have provided a key feature in the “spatial buffering” hypothesis to provide neuroprotection (Orkand et al., 1966). However, while there is significant support for this role of gap junctions, this is contested in

E. Dere (Ed): Gap Junctions in the Brain. ISBN 978-0-12-415901-3. http://dx.doi.org/10.1016/B978-0-12-415901-3.00014-1

Non-Channel Mechanisms Connexin Interacting Proteins Mitochondria

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Cellular Mechanisms Gliosis Microglia

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Conclusion Acknowledgments

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a number of situations. Furthermore, it is apparent that the simple view of gap junction proteins only forming intercellular channels falls short of explaining the diverse functions of connexins and pannexins.

ARE GAP JUNCTIONS PROTECTIVE OR DESTRUCTIVE? In cellular injury, the role of gap junctions has more recently been described as “controversial” because several studies have shown that gap junction proteins are either protective or destructive. Understanding how gap junctions participate in either of these roles in neuroprotection is complicated by the fact that different cell types express different (and usually several) connexins and pannexins. Thus, any manipulation of gap junction channels (e.g. blockers) or the expression of various connexins or pannexins (e.g. overexpression, gene knockouts or mutations) is likely to affect multiple cell types. In addition, changes in expression of connexins have been shown to affect the expression of many other

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genes (Iacobas et al., 2003). The complexity of these issues along with various cellular interactions must be kept in mind. The main gap junction protein in astrocytes is connexin43 (Cx43) (Dermietzel et al., 1989, 1991; Giaume et al., 1991; Naus et al., 1990; Yamamoto et al., 1990). This particular protein has been shown to be protective in a number of in vivo and in vitro models (Blanc et al., 1998; Boengler et al., 2007; Giardina et al., 2007; Goubaeva et al., 2007; Heinzel et al., 2005; Lin et al., 2003, 2008; Nakase et al., 2004; Oguro et al., 2001; Ozog et al., 2002; Rodriguez-Sinovas et al., 2006; Siushansian et al., 2001; Theis et al., 2003). Blockade of gap junctions in vitro enhances neuronal death in an astrocyte neuron coculture system (Ozog et al., 2002). In vivo studies have shown that decreased Cx43, both in heterozygote knockout (KO) mice (Nakase et al., 2003a; Siushansian et al., 2001) and in conditional deletion of Cx43 from astrocytes (Nakase et al., 2003a, b, 2004), increases cerebral infarct volume, apoptosis and inflammation following ischemic injury. In these studies gap junction intercellular communication may mediate neuroprotection during stroke by allowing metabolites [e.g. antioxidants, adenosine triphosphate (ATP), glucose] to move into areas of high energy demand, while also buffering cytotoxic levels of excitatory amino acids and ions through the astrocytic syncytium. In contrast, several studies have reported that gap junction proteins promote cellular injury (de PinaBenabou et al., 2005; Frantseva et al., 2002a; Lin et al., 1998; Nodin et al., 2005; Perez Velazquez et al., 2006; Rami et al., 2001; Rawanduzy et al., 1997; Thompson et al., 2006; Warner et al., 1995), possibly through intercellular propagation of cytotoxic substances, spreading depression-like depolarizations or by opening of neuronal hemichannels or pannexin channels. Gap junctions are thought to remain open during simulated ischemic conditions (e.g. calcium overload, oxidative stress or metabolic inhibition) (Cotrina et al., 1998; Lin et al., 1998); however, it is not clearly understood when propagation of harmful or protective molecules takes precedence. That being said, differences in the experimental design and the type of connexins or pannexins under study may explain why gap junction proteins could have opposing roles. Adding to the complexity of determining a precise role of gap junction proteins in cellular injury is the fact that connexins have many roles beyond astrocyteeastrocyte junctional communication. Connexins are not limited to glial cells, and thus neurons also express connexins and pannexins. Some studies have also shown that microglia and oligodendrocytes, as well as endothelial cells, express connexins as well. Furthermore, hemichannels, which allow for direct communication with the extracellular space, have been shown to play an important role

in cellular injury (Orellana et al., 2010, 2011a, b). In addition, mitochondrial Cx43 is known to influence mitochondrial function (Boengler et al., 2011; Kozoriz et al., 2010b; Rottlaender et al., 2010, 2012), which may have downstream consequences in cellular viability. Lastly, connexins are appreciated to be more than a passive channel. They have been shown to interact with many molecules that play important roles in the functioning of this protein (Laird, 2010). This chapter explores the role of gap junction proteins in neuronal injury and highlights the numerous roles that these proteins play.

Gap Junction Proteins and Cellular Protection Many different approaches have been used to examine the role of gap junctions, connexins and pannexins in CNS injury, and several different subtypes of gap junction protein have been explored. One underlying hypothesis for gap junction-based neuroprotection is that gap junction-coupled astrocytes provide a conduit to buffer harmful ions or molecules and provide a route for helpful molecules to reach an area of injury (Contreras et al., 2004; Perez Velazquez et al., 2003; Talhouk et al., 2008). In several studies, protection has been shown using a permanent middle cerebral artery occlusion (MCAO) focal stroke model, in which infarct volume was usually assessed 4 days after occlusion. When wild-type (Cx43þ/þ) and heterozygote (Cx43þ/) mice were subjected to MCAO, infarct volume was approximately 50% greater in the Cx43þ/ mice (Siushansian et al., 2001). In a separate study it was again found that infarct volume was greater in Cx43þ/ mice following MCAO, and apoptosis was greater in Cx43þ/ mice than in Cx43þ/þ mice at 4 days; however, at 1 day no differences in apoptosis were observed (Nakase et al., 2003a). In a more definitive study, in order to specifically target the effect of Cx43 expression in astrocytes and avoid complications that might arise during development owing to globally reduced levels of Cx43, a glial fibrillary acidic protein (GFAP) promoter cre-lox recombinase system was used which selectively deletes Cx43 from astrocytes (Cx43fl/fl/hGFAP-cre) (Nakase et al., 2003b). The advantage of this system is that the role of Cx43 in astrocytes can be assessed without altering Cx43 expression in most other cell types (note that neuronal progenitor cells may also have Cx43 deleted in these mutants). It was found that infarct volume was approximately 65% larger in Cx43fl/fl/hGFAP-cre transgenic mice. Cx43fl/fl/hGFAP-cre mice were also shown to have greater apoptosis, reduced gliosis and increased inflammatory cell invasion following stroke (Nakase et al., 2004). These studies show that in the absence of Cx43, damage due to focal stroke is greater, suggesting a role for endogenous

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astrocytic Cx43 in neuronal protection. These results are also supported by a recent study using carbenoxolone to block gap junctions during ischemic injury (Tamura et al., 2011). The authors reported that gap junction blockage accelerated the initiation and propagation of cortical spreading depression as well as increasing the injury from focal cerebral ischemia. However, most of these studies do not specifically address channel versus non-channel functions of gap junction proteins in neuroprotection (see “Channel Mechanisms of Neuroprotection or Neuronal Injury” and “Non-Channel Mechanisms”, below). One limitation of these studies is that mutant mice may have altered expression of many genes (Iacobas et al., 2003), which may be a confounding variable. It is also not clear from these studies how astrocytic Cx43 could be acting to protect neurons. One proposed mechanism focuses on the spatial buffering role that astrocytic gap junctions may play in the CNS (Orkand et al., 1966). Although gap junction coupling is often thought of as a potential route for the delivery of protective molecules, recently it has been shown that hemichannels may play a role, as shown in a hypoxic preconditioning study. Preconditioning involves sublethal exposure to a substance (or injury), which confers protection against subsequent insults (or injuries) when a larger dose (or injury) is given (Murry et al., 1986). Exposure to a sublethal hypoxic stimulus increases ATP release via hemichannels (Lin et al., 2008). ATP is then converted to a neuroprotective molecule, adenosine, which was found to exert a protective effect following MCAO (Lin et al., 2008). The MCAO experiments described at the start of this section were performed on non-preconditioned mice, so it is possible that hemichannel contribution to extracellular adenosine may not be a factor in those studies. However, the study by Lin et al. (2008) did compare the effect of MCAO in non-preconditioned mice with a double deletion of astrocytic Cx43 and Cx30 (Cx43fl/fl/hGFAP-cre/Cx30/). Consistent with other findings, infarct volume was larger in the double mutant lacking both Cx30 and Cx43 in comparison to wild-type. Some studies have suggested that neuronal gap junction coupling is a protective mechanism. Although not tested explicitly, Cx32 and Cx36 are thought to be protective in the hippocampus following global ischemia because these connexins were reported to be expressed in interneurons and may play a role in synchronizing g-aminobutyric acid (GABA) release (Oguro et al., 2001). In this study, Cx32 KO mice were found to have more hippocampal CA1 neuron cell death, lending support to this theory. This could also be attributed to a loss of oligodendrocytic Cx32, which could be involved in a spatial buffering role as well (Menichella et al., 2006; Nagy and Rash, 2003). Menichella and

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colleagues provide evidence that oligodendrocyte connexins and Kir4.1 function in a common pathway, implicating oligodendrocyte gap junctions as having a critical role in the buffering of Kþ released during neuronal activity. Several cell culture studies have also shown cellular protection during application of injurious molecules. Using a neuroneastrocyte coculture model, neuronal death is enhanced upon application of glutamate in the presence of gap junction blockers (Ozog et al., 2002). Glutamate was applied uniformly across the culture, so gradients for protective molecules to pass along to areas of demand, as in a focal stroke model, are unlikely to exist. However, it is possible that protection could be explained by an action of adenosine, as described previously (Lin et al., 2008), or by direct coupling between neurons and astrocytes, which has been shown to occur in cell culture (Froes et al., 1999). Similarly, in a coculture system, it has been shown that blockade of gap junctions increases neuronal death when exposed to oxidative stress (Blanc et al., 1998). Although in this study neuroneastrocyte coupling was minimal, potentially other factors were at play (e.g. release of protective molecules or buffering of harmful molecules through hemichannels). The final cell culture study to be discussed is one in which Cx43, Cx32 or Cx40 overexpression in C6 glioma cells conferred resistance to cell death when exposed to a variety of nonischemic insults (Lin et al., 2003). Although it may be difficult to compare studies using glioma cell lines to those with primary astrocytes, one interesting caveat to this work is that cellular protection was independent of gap junctional communication, as isolated cells or cells expressing constructs which do not form gap junctions also displayed resistance to cell death.

Gap Junction Proteins and Cellular Destruction In contrast to the above, numerous studies have suggested that gap junctions are destructive. One theory of cellular destruction is the propagation of harmful molecules through gap junctions and thus increased cellular injury. One of the first studies suggesting that gap junctions are destructive used intraperitoneal injection with octanol (a gap junction blocker) followed by MCAO in the rat (Rawanduzy et al., 1997). Infarct volume was assessed 24 h later. As gap junctions play a role in spreading depression (Nedergaard et al., 1995), it was suggested that gap junction blockade would reduce the expansion of infarct volume and block spreading depression-like depolarizations through gap junctions. Indeed, infarct volume was reduced and experimentally induced spreading depression was inhibited by octanol treatment. However, these results are not consistent with

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a recent study using carbenoxolone to block gap junctions during ischemic injury (Tamura et al., 2011). These authors occluded two adjacent cortical veins in the rat using Rose Bengal dye and fiberoptic illumination after intraventricular injection of carbenoxolone, and assessed infarct volume 7 days later. They found a doubling of the infarct volume in the rats treated with carbenoxolone. The earlier results of Rawanduzy et al. (1997) also contrast with experiments where infarct volume increased following MCAO in mice with an astrocyte-targeted deletion of Cx43 (Nakase et al., 2003b, 2004). In addition, experimentally induced spreading depression velocity is increased when Cx43 is deleted from astrocytes (Theis et al. 2003). Therefore, it is likely that the Rawanduzy study may have revealed an astrocyte Cx43-independent pathway for protection that may or may not involve gap junctions. Since octanol was delivered intraperitoneally (rather than intraventricularly as in Tamura et al., 2011), it is not known whether this compound reached the brain at high enough concentrations to block gap junctions or whether there were indirect actions of octanol on other organs (e.g. the heart). Octanol is also not specific for gap junctions; in fact, octanol and many other anesthetics may exert neuroprotective properties though action on various ion channels or neurotransmitter release (Matchett et al., 2009; Narahashi et al., 1998). It is now known that neuronal pannexin channels open in response to ischemia (Thompson et al., 2006), and potentially octanol was acting on neuronal connexins or pannexins to reduce cell damage rather than acting on astrocytic gap junctions. Intraperitoneal injection of octanol has also been performed in a transient global ischemia model where both carotid arteries were occluded (Rami et al., 2001). Hippocampal cell death was reduced in the octanol treatment group. Similar results have been found in a four-vessel occlusion model with application of the gap junction blocker carbenoxolone via cannulae implanted into the hippocampus (Perez Velazquez et al., 2006). In addition to the points raised for the Rawanduzy study, these studies used global ischemia, and thus are unlike the studies noted above that demonstrate protection in a focal ischemia model. Differences in stroke models are known to exist (Hossmann, 2008; Traystman, 2003), and may contribute to the differences observed in these studies. Whereas focal occlusion is known to create a necrotic core and penumbral region, global ischemia tends to cause damage in brain regions susceptible to cell death, and by increasing the length of ischemia the number of affected regions increases. A global ischemia model may not be ideal to assess a beneficial role for gap junctions as there may not be the same gradients of healthy and non-healthy cells as created in stroke models with a penumbral region.

However, global ischemic studies may have relevance to the events that occur in the necrotic core of a focal stroke, where gradients of healthy and unhealthy cells are unlikely to exist. Exposure of hippocampal slices to oxygeneglucose deprivation with gap junction blockers reduced cell death at 48 h (Frantseva et al., 2002b). As oxygenegluose deprivation is applied uniformly to the hippocampal brain slice preparation, this model may reflect states where blood supply is uniformly disrupted, for example systemic hypoperfusion. In this study an antisense RNA approach was used to reduce levels of Cx26 and Cx32, both reported as neuronal connexins; this resulted in decreased cell death, suggesting that neuronal connexins are important in mediating this process. This also fits with the experiments above describing a reduction in cell death with application of gap junction blockers (e.g. octanol) that may be acting on neuronal connexins (or pannexins). These authors also reduced Cx43 expression, which led to a reduction in cell death. The authors noted that hippocampal neurons may express Cx43, which could play a role in cellular destruction (Simburger et al., 1997). A similar experiment was performed in slice cultures, where it was found that gap junction blockade also decreased cell death at time points up to 24 h (de Pina-Benabou et al., 2005). This study further examined the effect of gap junction blockade on intrauterine hypoxiaeischemia, essentially finding the same result as in the slice cultures. These results do not dispute findings that astrocyte Cx43 plays a role in cellular protection in a focal ischemic model. Rather, they highlight the potential importance of neuronal connexins in cell death and suggest that the mechanisms at play in global ischemia are likely to be different. Culture models have also been used to study cell death. In one study, cultured hippocampal astrocytes were treated with iodoacetate to deplete ATP levels (Nodin et al., 2005). It was found that gap junction blockade reduced cell death. Again, this could potentially be explained by the uniformly applied treatment. Another commonly cited paper which argues that gap junctions are destructive is a study that used a glioma cell line with overexpression of the human proto-oncogene bcl2 (Lin et al., 1998). The bcl2-expressing cells are more resistant to cell death; however, when coupled with cells that do not express this gene and then exposed to several types of insult (none of which was ischemic), the more resistant cells would die. The interpretation is that gap junction-coupled cells that are susceptible to cell death can cause “bystander” cell death by propagating harmful molecules through gap junctions to cells that are normally protected from chemical insults. In this study the ratio of healthy to non-healthy cells was important. When the number of resistant cells was increased, the bystander cell death effect was abolished, indicating that too many

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injured cells may tip the balance toward cellular destruction. Based on the above studies it is obvious that gap junction proteins play a diverse role in cellular injury. It is overly simplistic to state that these proteins as a group are either protective or destructive as it appears that protection or destruction depends on the nature of the insult and the type of connexin or pannexin involved. Different in vitro model systems (i.e. cell lines, primary cultures of neurons and/or astrocytes, brain slices) may also give different results, and sensitivity to ischemia also varies with different strains of mice or rats (Carmichael, 2005; Liu and McCullough, 2011). The following sections will explore how gap junction proteins may serve this dual role.

CHANNEL MECHANISMS OF NEUROPROTECTION OR NEURONAL INJURY Connexin proteins, such as Cx43, are synthesized in the endoplasmic reticulum and trafficked to the Golgi apparatus where they undergo oligomerization into a connexon hexamer (Musil and Goodenough, 1993). After microtubule-facilitated transport to the cell surface, connexons can dock with connexon counterparts located in the plasma membrane of neighboring cells to form gap junction channels. In addition, substantial evidence suggests that connexons can also form hemichannels in the cell membrane to exchange small molecules with the extracellular environment (Evans et al., 2006). There is some controversy regarding the existence of connexin hemichannels since some of their observed characteristics can be accounted for by other channels (Spray et al., 2006), and in some situations, by the presence of pannexins (Iglesias et al., 2009). Both gap junction channels and hemichannels have been implicated to play a role in neuronal protection and/or injury (Chew et al., 2010).

Intercellular Communication: Gap Junction Channels The initial channel-dependent mechanism invoked for neuroprotection has focused on the gap junction intercellular channel, which establishes cytoplasmic continuity between coupled cells, i.e. establishing a syncytial cellular network. First proposed by Orkand et al. (1966), gap junctional coupling between astrocytes has long been hypothesized to underlie the potassium siphoning essential for models of spatial buffering. If the glial syncytium is disrupted, either by blocking gap junctions or by decreasing the amount of connexins

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present, a decrease in neuroprotection would be predicted. In fact, this is the case for the genetic mouse models with reduced Cx43 which exhibit enhanced neuronal injury in the MCAO stroke model (see earlier discussion). The specific effect of connexin deletion on potassium buffering has been characterized by Wallraff et al. (2006), who demonstrated a significant attenuation of potassium buffering in hippocampal slices from mice with both Cx43 and Cx30 deletions. The additional importance of the glial syncytium in glucose sharing via astrocytes to neurons has been shown to be dependent on astrocytic gap junctional coupling (Rouach et al., 2008). These authors demonstrated that Cx43 and Cx30 were needed for the gap junctional intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking was shown to be regulated by glutamatergic synaptic activity mediated by a-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons. Alternatively, the gap junction intercellular channel has been proposed to function as a conduit for the passage of neurotoxic substances to propagate cell injury, effectively spreading damage through a “bystander effect” (Lin et al., 1998; Perez Velazquez et al., 2003; Rossi et al., 2007; Talhouk et al., 2008). Based on the premise that gap junctions are detrimental during CNS injury, several approaches have been used to interfere with their function as a potential therapy. These include pharmacological blockers of gap junctions (i.e. octanol, carbenoxolone), many of which lack specificity. These have already been discussed above. To influence gap junctions more directly, several reports have demonstrated effective use of connexin messenger RNA (mRNA) antisense approaches (Cronin et al., 2008; Danesh-Meyer et al., 2008; Qiu et al., 2003; Yoon et al., 2010b) and gap junction mimetic peptides (Evans and Leybaert, 2007; Herve and Dhein, 2010; O’Carroll et al., 2008; Yoon et al., 2010a). With all of these approaches, however, it is not possible to distinguish between effects on gap junction channels or hemichannels. Correlating Cx43 expression with beneficial or detrimental effects on neuroprotection does not address the specific state of the channel, i.e. as a gap junction intercellular channel or as a hemichannel. In order to determine distinct effects attributed to the status of the channel, various approaches have been used.

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Connexin Hemichannels While there is substantial evidence supporting the existence of connexin hemichannels, this is still an area of debate (Spray et al., 2006). Further insight into the role of hemichannels has been obtained with the discovery of another family of gap junction proteins, the pannexins (see below). Based on the evidence supporting the existence of connexin hemichannels, strategies to specifically block or target these have been used. A recent study by Decrock et al. (2009) provided further evidence for gap junctions mediating a detrimental bystander effect, but in this case via hemichannels. The authors used in situ electroporation with cytochrome c to induce apoptosis in C6 glioma cells stably transfected with Cx43 and found that surrounding cells underwent apoptosis. They concluded that Cx43 hemichannels, as well as gap junction channels, play a role in propagating cytochrome c-induced apoptotic cell death messages to surrounding cells. Froger et al. (2010) investigated the contribution of Cx43 to N-methyl-D-aspartate (NMDA)-induced excitotoxicity in neuron/astrocyte cocultures, after treatment with a proinflammatory cytokine mixture containing tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b), which stimulated astroglial Cx43 hemichannel activity. NMDA treatment induced a higher amount of neurotoxicity in cytokine-treated cocultures than in untreated ones, whereas this extent of neurotoxicity was absent in enriched neuron cultures or in cocultures with Cx43 KO astrocytes; application of Cx43 hemichannel blockers or a synthetic cannabinoid prevented the cytokine-induced potentiated NMDA neurotoxicity. These findings demonstrate that inflammation-induced astroglial hemichannel activation plays a critical role in neuronal death and suggest a neuroprotective role of Cx43 hemichannel blockade. Several additional studies support the role of hemichannels in neuronal injury. Under proinflammatory conditions involving microglia activation, astrocyte gap junctions close while hemichannels open (Retamal et al., 2007a). As shown by Orellana et al. (2011b), signaling between microgliaeastrocytes and neurons can have important consequences for neuronal death. Application of b-amyloid leads to glial release of glutamate and ATP via hemichannels that results in neuronal death by triggering the opening of neuronal hemichannels (Orellana et al., 2011b). Similarly, astrocyte ATP and glutamate release from Cx43 hemichannels also mediates cell death by opening neuronal pannexin channels (Orellana et al., 2011a). It is also appreciated that inhibiting astrocyte Cx43 hemichannel activity serves a protective role (Froger et al., 2010). A recent report outlines an in vivo study using this strategy in a fetal

sheep model, where the authors infused a Cx43 mimetic peptide designed to specifically block hemichannels (Davidson et al., 2012).

Pannexins A more recently described family of gap junction proteins, the pannexins (Panx), presents candidates to consider in the context of these channel proteins and neuroprotection. Pannexins were first discovered owing to their homology to the invertebrate gap junction proteins, innexins (Bruzzone et al., 2003; Panchin, 2005). Two of these, Panx1 and Panx2, are present in the CNS and have been considered in different CNS injury models. The evidence for pannexin channels not forming intercellular gap junction channels, but rather forming the equivalent of a connexin “hemichannel”, has recently been addressed (Sosinsky et al., 2011). Thompson et al. (2006) identified the activation of large-conductance channels (500 pS) following neuronal excitotoxicity during oxygeneglucose deprivation, leading to swelling and calcium dysregulation (Thompson et al., 2006). They showed that these channel openings could be blocked by inhibitors of hemichannels. They concluded that Panx1 channel opening leads to ionic dysregulation during ischemic conditions contributing to neuronal death. Panx1 has been shown to be permeable to ATP, and its association with P2X7 receptor has been shown to be necessary for ATPinduced ATP release (Chekeni et al., 2010; Iglesias et al., 2009; MacVicar and Thompson, 2010). The most definitive study to date on the role of pannexins in neuronal injury has been provided by Bargiotas et al. (2011), who examined Panx1, Panx2 and double Panx1/2 KO mice in an ischemic injury model. They found that channel function in astrocytes and cortical spreading depolarization were not altered in these mice, indicating that, in contrast to previous concepts, these processes occur normally in the absence of pannexin channels. However, ischemia-induced dye release from cortical neurons in the double knockout was lower, indicating that channel function in Panx1/2 KO neurons was impaired. Furthermore, Panx1/2 KO mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. They concluded that Panx1 and Panx2 underlie channel function in neurons and contribute to ischemic brain damage. Complementary to these findings, the role of Panx1 was examined in a mouse seizure model (Santiago et al., 2011). With the use of both pharmacological blockers and transgenic mice that do not express Panx1, the severity of seizures was attenuated, thus having a likely impact on subsequent neurodegenerative effects.

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Panx2 has also been examined specifically in this context. Zappala` et al. (2007) examined the localization of Panx2 in the hippocampus, showing that it was normally present in neurons. After bilateral transient carotid artery occlusion for 20 min, followed by different times of reperfusion, they noted an intense astrogliosis in the hippocampus, with most of the astrocytes now transiently expressing Panx2. Similarly, in primary cocultures of hippocampal neurons and astrocytes submitted to a transient ischemia-like injury, it was shown that Panx2 was again transiently expressed. They concluded that expression of Panx2 in astrocytes may be induced either from injured neurons or by biochemical pathways internal to the astrocyte itself. Their results showed the transient expression of Panx2 in reactive astrocytes occurring in the hippocampus following injury. They hypothesized the involvement of Panx2 in the formation of channels for the release of signaling molecules devoted to influencing the cellular metabolism and the redox status of the surrounding environment.

NON-CHANNEL MECHANISMS Clearly, the channel aspects of gap junction proteins are the most obvious considerations when examining the effects of connexins in neuronal injury or protection models. However, in addition to the channel-mediated roles of gap junction proteins, there has been substantial characterization of the functions that connexins may play apart from channel formation per se.

Connexin Interacting Proteins While some interactions of connexins with other proteins have been studied in the context of channel function (i.e. gating effects) (Herve et al., 2004), the expanding repertoire of interacting proteins (Herve et al., 2012; Laird, 2010) has led to novel considerations of connexin functions in various cellular processes, including cell protection. It is recognized that connexins interact with other proteins. The study of the connexin proteome is in its infancy but to date over 40 proteins have been found to interact directly or indirectly with connexins (reviewed in Laird, 2010). These interacting proteins include kinases, phosphatases, proteins for proteasome-based degradation, and proteins involved in scaffolding, trafficking, cytoskeletal interactions and growth regulation. Many of these interactions are known to occur at the C-terminal domain of connexins, specifically Cx43. The cytoplasmic C-terminal of connexins is a region of diversity among the connexin family. Cx43 has several known interaction sites of importance, and has

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been shown to bind proteins such as a-tubulin, b-tubulin, CCN3, c-src and zonula occludens-1 (ZO-1), and has phosphorylation sites for protein kinase A (PKA), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) (Fu et al., 2004; Giepmans and Moolenaar, 1998; Giepmans et al., 2001a, b; Herve et al., 2007). These interactions are believed to play a role in a variety of gap junction-related processes, including assembly, activity and gating (Duffy et al., 2002; Herve et al., 2007; Kim et al., 1999; Lampe and Lau, 2000; Lampe et al., 2000; Liu et al., 1993; Martinez et al., 2003; Musil et al., 1990; Solan and Lampe, 2009). Although the significance of many of these interactions is not fully understood, one area of particular interest is the role of the C-terminal region interactions and channel gating. The C-terminal region has many sites for phosphorylation. Dephosphorylating agents are known to decrease gap junction coupling (Duthe et al., 2000), while stimulation of kinases (PKC, PKA or MAPK) increases gap junction coupling (Kwak and Jongsma, 1996; Kwak et al., 1995; Zhang et al., 1999). However, it is important to note that Cx43 phosphorylation is complicated and in some instances agents that phosphorylate or dephosphorylate may have the opposite effect to that described above (Lampe and Lau, 2000; Marquez-Rosado et al., 2012). This is likely to be due to the specific amino acid residues that are being acted upon in the context of numerous other interacting molecules. In addition to gating by phosphorylation, Cx43 is known to be gated by a decline in pH. The C-terminal region of Cx43 is known to be a pH sensor and is important in channel gating (Liu et al., 1993; Stergiopoulos et al., 1999). Because so many molecules interact at the C-terminal domain, several studies have investigated changes that occur when this region is truncated. Truncation of the C-terminal region prolongs the mean open time of the Cx43 channel and increases channel conductance (Fishman et al., 1991; Moreno et al., 2002). A mouse with a truncated Cx43 C-terminal region (Cx43DCT) has been generated by mutating the adenine base at codon 258 to thymine, resulting in a translational stop codon (Maass et al., 2004). Mice with both alleles truncated (Cx43DCT/DCT) die shortly after birth owing to an epidermal barrier deficit (Maass et al., 2004), but mice with one truncated allele (either Cx43DCT/þ or Cx43DCT/ mice) survive (Maass et al., 2007). It has been shown that cardiomyocytes in mice with one truncated allele and one knockout allele (Cx43DCT/) couple (Maass et al., 2007, 2009) and have channel properties similar to studies where truncated Cx43 has been transfected into cells (Fishman et al., 1991; Moreno et al., 2002). One notable change with the Cx43DCT mutation is a reduction in the total number of gap junction plaques, yet these plaques are larger in size (Maass et al.,

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2007). This could be explained by the possibility that the C-terminal region may be involved in channel degradation (Li et al., 2008) and a lack of this region increases the channel half-life (Maass et al., 2004). The hearts of these mutant mice have been subjected to ischemic conditions by occluding the left descending coronary artery (Maass et al., 2009). In this study it was found that Cx43DCT/ mice had increased infarct size compared to control mice, and a reduced sensitivity to acid-induced uncoupling. Similarly, in the brain, mice with the Cx43DCT mutation have increased stroke damage 4 days following MCAO (Kozoriz et al., 2010a). This indicates that disruption of the C-terminal region alters the ability of astrocytes to protect the brain during stroke. One hypothesis of the involvement of the C-terminal region in cellular injury involves Cx43 gating by pH. Gap junctions close with a reduction in pH, a process that involves the C-terminal region (Duffy et al., 2002, 2004; Liu et al., 1993; Morley et al., 1996). During stroke it can be reasoned that full-length Cx43 would be gated once pH declines, an effect that may be beneficial to reduce passage of cytotoxic molecules to neighboring cells (Lin et al., 1998). Such channel gating is impaired in Cx43DCT channels, potentially allowing for increased passage of harmful molecules. The above results do not contradict the previously mentioned studies where reduced expression of Cx43 leads to increased infarct volume during MCAO (Nakase et al., 2003b, 2004; Siushansian et al., 2001). It is possible that in the early stages of stroke, gap junction coupling allows for passage of protective molecules and buffering of toxic molecules to reduce ischemic damage, while in the latter stages, where pH declines, a lack of C-terminal gating would allow for passage of harmful molecules that would otherwise be gated by a functional C-terminal region. Lastly, it is unclear how the numerous protein and phosphorylation interactions with the Cx43 C-terminal region contribute during stroke pathology. Future studies may shed light on the potential complex multitude of pathways that underlie connexin regulation in ischemic states.

Mitochondria Some studies have found that protection from cellular injury by connexins is independent of gap junction intercellular communication (Lin et al., 2003, 2008), suggesting that other non-coupling-related mechanisms may be involved. The presence of Cx43 in mitochondria was originally suggested in studies using human umbilical vein endothelial cells (H. Li et al., 2002) and has now been shown more conclusively in cardiomyocytes

(Boengler et al., 2005; Goubaeva et al., 2007; Halestrap, 2006). Several techniques have been used to demonstrate the presence of Cx43 in heart mitochondria, including flow cytometry, confocal immunohistochemistry, Western blot of mitochondrial membrane fractions and electron microscopy with immunogold labeled antibodies (Boengler et al., 2005, 2009; Goubaeva et al., 2007; Halestrap, 2006; Miro-Casas et al., 2009; RodriguezSinovas et al., 2006). In these studies several antibodies have been used to confirm the presence of Cx43 in mitochondria, and an absence of staining has been shown in a knockout (Cx43Cre-ER(tetracycline)/fl) system (Boengler et al., 2005). Rodriguez-Sinovas et al. (2006) studied how Cx43 reaches the mitochondrial membrane. It appears that Cx43 is imported via translocase of the outer membrane/ translocase of the inner membrane (TOM/TIM) mitochondrial protein import system, as Cx43 has been shown to associate by coimmunoprecipitation with members of this system, such as TOM20 and heat-shock protein 90 (Rodriguez-Sinovas et al., 2006). Furthermore, an inhibitor of protein import through the TOM/TIM system, geldanamycin, halts the import of Cx43 into mitochondria (Rodriguez-Sinovas et al., 2006). The function and localization of mitochondrial Cx43 are not entirely clear. As mitochondria contain an inner and outer membrane, it is attractive to think of connexins as connectors of these two layers, much like between two adjacent plasma membranes. One group has found that Cx43 localizes primarily to the inner mitochondrial membrane (Miro-Casas et al., 2009; Rodriguez-Sinovas et al., 2006), while another has determined its presence to be primarily on the outer membrane (Goubaeva et al., 2007). The reason for the difference has been suggested to be due to different subfractionation protocols (Ruiz-Meana et al., 2008). Gap junction-like structures appear to form between the outer mitochondrial membrane and endoplasmic reticulum in intrafusal muscle fibers (Ovalle, 1971), although this observation remains unexplored. Recently, it has been shown that Cx43 can form hemichannels on the inner mitochondrial membrane that are involved in Kþ uptake (Miro-Casas et al., 2009). The existence of hemichannels may also be of interest as plasma membrane Cx43 has been suggested to release amino acids, ATP, glutamate and glutathione (Kang et al., 2008; Stridh et al., 2008; Ye et al., 2003), molecules that are also present in mitochondria. Given the importance of maintaining ion gradients for mitochondrial function, the presence of Cx43 hemichannels may be of concern since ion gradients could be dissipated by unregulated hemichannel openings. However, extrapolating from what is known about plasma membrane hemichannel opening, it is possible that mitochondrial Cx43 could be regulated by phosphorylation state, voltage, Ca2þ and pH (Contreras

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CELLULAR MECHANISMS

et al., 2003a; Retamal et al., 2007b; Saez et al., 2005; Thompson et al., 2006), so that openings occur only in selected states. Mitochondrial Cx43 is thought to play a role in ischemic preconditioning in cardiomyocytes. Preconditioning was first demonstrated by Murry et al. (1986), where brief ischemic episodes led to subsequent cardioprotection after exposure to a stimulus that would normally be lethal. Nearly any substance that causes cellular injury can cause preconditioning when given at a sublethal dose (Dirnagl et al., 2003). Several lines of evidence have pointed to mitochondria as a site important in preconditioning (Murphy and Steenbergen, 2008), but the specific mechanisms involved remain unclear. Cellular stress by homocysteine led to profound increases in mitochondrial Cx43 in human umbilical vein endothelial cells (H. Li et al., 2002). Such increases in mitochondrial Cx43 were also observed in cardiomyocytes after preconditioning (Boengler et al., 2005). Gap junction blockers have been shown to inhibit the effects of preconditioning in the heart (G. Li et al., 2002) and preconditioning is not observed in the heart of Cx43þ/ mice (Schwanke et al., 2002, 2003). Although it is difficult to dissociate the role of plasma membrane Cx43 and mitochondrial Cx43 in these studies, it is important to note that cell survival has been reported to occur independently of gap junction intercellular communication (Lin et al., 2003, 2008; Plotkin and Bellido, 2001). Furthermore, mitochondrial reactive oxygen species (ROS) generation has been shown to play a role in preconditioning. When ROS generation was tested using diazoxide-induced preconditioning, it was found that Cx43þ/ cardiomyocytes had reduced ROS generation and were not protected during reperfusion injury (Heinzel et al., 2005). Also, when mitochondrial Cx43 levels were reduced by application of geldanamycin, an inhibitor of the TOM/TIM pathway, protective effects of mitoKATP opening by diazoxide on ischemiaereperfusion were reduced (Rodriguez-Sinovas et al., 2006). The specific connection between mitochondrial Cx43 and ROS preconditioning pathways is not well defined (Halestrap et al., 2007; Rodriguez-Sinovas et al., 2007). However, it may involve the Cx43-based mitochondrial Kþ uptake, as other mitochondrial Kþ channels are known to generate ROS and lead to preconditioning (Pain et al., 2000). Cx43 is known to play a role in mitochondrial Kþ uptake in astrocytes (Kozoriz et al., 2010b) and a signaling link has been proposed between Cx43 and mitoKATP channels (Rottlaender et al., 2010). In further studies it was shown that glycogen synthase kinase-3a, an important molecule in cytoprotective signaling, can act through mitochondrial Cx43 to influence mitoKATP channels (Rottlaender et al., 2012). Mitochondrial Cx43 has been shown to affect the

mitochondrial respiratory complex I (Boengler et al., 2011), which may play an important role in cellular energetics during ischemic conditions. In non-preconditioned heart mitochondria, Kþ uptake occurs through mitochondrial Cx43 hemichannels. Mitochondrial Kþ uptake has been shown to be protective (Hansson et al., 2010; Kowaltowski et al., 2001), and as mentioned mitochondria in astrocytes take up Kþ via a Cx43-dependent mechanism (Kozoriz et al., 2010b).

CELLULAR MECHANISMS A variety of cellular mechanisms come into play following cellular stress, including a number associated with neurological insults. Two of the major responses of the CNS to injury include gliosis and increases in immune cell reactions (Czlonkowska and KurkowskaJastrzebska, 2011). There are emerging roles for gap junctions in these responses. While many other responses occur, they are beyond the scope of this review.

Gliosis One of the major responses in the CNS following injury is reactive gliosis (Pekny and Nilsson, 2005; Ridet et al., 1997; Sofroniew, 2009; Sofroniew and Vinters, 2010). Gliosis itself is an active area or research and it has been shown to serve both beneficial and detrimental roles (Sofroniew, 2009; Zhang et al., 2010). For example, forming a gliotic scar can serve to limit damage by walling off an area of injury; however, this can exclude access by restorative neuronal elements across the gliotic area. Since Cx43 is highly expressed in astrocytes, it is not surprising that increased Cx43 immunoreactivity is associated with gliosis (Haupt et al., 2007; Nakase et al., 2003a, 2004). In the case of brain ischemia, Cx43 expression is elevated in the peri-infarct region (Nakase et al., 2006), and has been shown to be up regulated in reactive and proliferating astrocytes following stroke (Haupt et al., 2007). In vitro reactivity of astrocytes is impaired when Cx43 is depleted (Homkajorn et al., 2010). The precise role of Cx43 in gliosis is not fully understood, but could involve coordinated scar formation through gap junctions or through interaction with purinergic receptors that are important for cell migration and proliferation (Haupt et al., 2007; Scemes, 2008; Scemes et al., 2003). In experiments using Cx43-deleted mice, astrogliosis was reduced in the peri-infarct region of the stroke (Nakase et al., 2003a, 2004). This may be explained in part by reduced expression of P2Y1 receptors known to occur in Cx43/ mice (Scemes et al., 2003). P2Y1 receptors are thought to interact with the C-terminal region of Cx43 (Scemes, 2008). Therefore, it

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is not surprising that reduced gliosis was also found in Cx43DCT mutant mice following MCAO (Kozoriz et al., 2010a).

Microglia Inflammation and microglial activation are hallmarks of CNS injury (Amor et al., 2010; Thiel and Heiss, 2011; Yenari et al., 2010). Under certain conditions, such as a stab injury to the brain or application of Staphylococcus aureus-derived peptidoglycan, microglia have been shown to express Cx43 (Eugenin et al., 2001; Garg et al., 2005). Cx36 is also expressed in microglial

cultures, with some coupling between neurons and microglia demonstrated (Dobrenis et al., 2005). Furthermore, Cx32 has been demonstrated to release glutamate from microglia, which contributes to neurotoxic damage (Takeuchi et al., 2006). The presence of these connexins raises the question of their role in cellular injury. It is appreciated that microglia and astrocytes communicate with each other through several mediators (Liu et al., 2011). Proinflammatory cytokines such as TNF-a, IL-1b, IL-6 and interferon-g have been shown to reduce gap junction coupling (Hinkerohe et al., 2005; Orellana et al., 2009). Another cytokine, ciliary neurotrophic factor, is known to increase gap junction

(D)

(F) (C) (B)

(G) (E)

(A)

(I) (H)

FIGURE 14.1 Summary of the multiple roles that gap junction proteins may play in cellular injury. During focal ischemic injury healthy astrocytes may communicate with injured penumbral astrocytes. (a) It is proposed that harmful substances such as glutamate, Kþ or other injurious factors can be buffered away from the ischemic region. However, if the extent of damage is large the balance of protection could tip toward propagation of cellular injury to neighboring cells. (b) In this system protective molecules such as glucose, ATP and antioxidants can pass through gap junctions to offer support to dying cells. (c) It is known that astrocyte hemichannels can release ATP which is converted to adenosine in the extracellular space. This acts on neuronal adenosine receptors, which serves a protective role. (d) During ischemia it is appreciated that neuronal pannexin channels open, leading to cell death. (e) Cx43 is known to interact with at least 40 different molecules. Disruption of the Cterminal region of Cx43 increases infarct size following stroke. Understanding these interactions may unlock key therapeutic targets in the treatment of stroke. (f,g) Astrocytes buffer rises in extracellular Kþ via a mechanism involving mitochondrial Cx43. Mitochondrial Cx43 has also been shown to interact with cytoprotective molecules such as glycogen synthase kinase-3b and with mitoKATP channel (dashed lines), which may play an important role in cellular injury. (h) Cellular elements, such as microglia, also contribute to the milieu of the ischemic brain. Released cytokines are known to open astrocyte hemichannels and reduce gap junction coupling. Their exact role in focal ischemia is not fully determined; however, microglia recruitment is increased in Cx43 KO models following stroke. (i) Lastly, reactive gliosis occurs during brain injury. It is known that gap junction proteins are upregulated during injury and quite possibly gap junctions play an important role in scar formation. In summary, there are multiple mechanisms involving gap junction proteins at play during ischemia which can have both protective and destructive roles. This figure is reproduced in color in the color plate section.

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CONCLUSION

coupling and play a protective role (Ozog et al., 2004, 2008). Activated microglia are also known to reduce astrocyte gap junction intercellular communication and enhance astrocyte hemichannel activity (Kielian and Esen, 2004; Meme et al., 2006; Orellana et al., 2009; Retamal et al., 2007a). Furthermore, it is known that application of non-specific hemichannel blockers reduces microglia activity in the setting of traumatic brain tissue injury (Davalos et al., 2005), the mechanism of which is thought to involve ATP signaling through hemichannels. In support of this, innexin hemichannels in glial cells in the leech have been shown to release ATP following nerve injury, resulting in migration of microglial cells to the injury site (Samuels et al., 2010). It is hypothesized that microglia activation leads to potential harm by opening astrocyte hemichannels and reducing astrocyte gap junction functioning (Orellana et al., 2009). This would place them in a situation where they would be unable to offer support to neurons, resulting in cell injury. The role of direct microgliaemicroglia coupling or astrocyteemicroglia coupling during ischemic damage is not known. It seems reasonable that such communication could play an important role in coordinating cellular signals during injurious states. Lastly, microglial responses were increased in Cx43DCT mutant mice and in Cx43fl/fl/hGFAP-cre mice following MCAO (Kozoriz et al., 2010a; Nakase et al., 2004). This could be related to altered communication in either of these mutants leading to a failure to control microglial recruitment and aggravation of brain injury. Cx43DCT mice also have increased neutrophil recruitment and inflammation in a lung injury paradigm (Sarieddine et al., 2009).

CONCLUSION The potential pathways contributing to cellular protection and injury are summarized in Figure 14.1. In addition to their traditional role of providing an intercellular conduit for passage of electrical activity and molecules, connexins are now recognized to have several other cellular functions. Connexins form hemichannels, which allow for direct communication with the extracellular space (Contreras et al., 2003b; Spray et al., 2006). Based on studies of tumor cells, connexins are found to be involved in cell growth, proliferation and invasion (Bates et al., 2007; Fu et al., 2004; Zhang et al., 2003), and although the pathways of this action are not entirely clear, it is possible that connexins act in the nucleus to regulate cell growth (Dang et al., 2003). Cx43 is expressed in mitochondria (Boengler et al., 2005, 2007, 2009; Goubaeva et al., 2007; H. Li et al., 2002; Rodriguez-Sinovas et al., 2006), where it

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has been shown to play a role in Kþ uptake (Kozoriz et al., 2010b; Miro-Casas et al., 2009). Connexins are known to interact either directly or indirectly with at least 40 proteins (Laird, 2010). These interactions may serve to alter basic functions of connexins such as channel localization, activity and degradation, and may form a platform for cellular signaling. The study of connexins is further complicated by the fact that pharmacological agents that block or enhance specific connexin actions are generally not available. However, newly developed peptide compounds show promise in blocking hemichannel activity and thus provide valuable new tools to investigate the role of gap junction in various situations. Because of the range of functions attributed to connexins, it is not surprising that a clear understanding of the role of connexins in cellular injury has remained elusive. Many papers state that the role of connexins in cellular injury is controversial, yet to the authors’ knowledge, no two published studies have used a similar design to study the role of connexins in cellular injury. Given the range of roles for connexins it is quite possible that they are involved in both cellular protection and cellular destruction, and it is the experimental context that dictates cellular fate. Differences in results may be due to the type of connexin(s) or pannexin(s) expressed, the type of cells used (e.g. neuronal, glial, mixed or cell line), the experimental design or the ratio of healthy to non-healthy cells. Some general patterns emerge in the connexin literature. In studies of focal ischemia (Lin et al., 2008; Nakase et al., 2003a, b, 2004; Siushansian et al., 2001), where an ischemic core and penumbra are created, gap junctions tend to be protective, potentially because harmful compounds can be buffered away from the penumbra and protective molecules can move to the site of injury. Astrocyte hemichannels may also play a role in the release of protective molecules and may buffer toxic substances (Lin et al., 2008). The ratio of healthy to non-healthy cells may also be more favorable in this situation. In contrast, in global ischemia, or if a brain slice is uniformly exposed to an ischemic insult, gap junctions tend to increase cell death, potentially through spreading of toxic molecules and through the opening of neuronal hemichannels (de Pina-Benabou et al., 2005; Frantseva et al., 2002b; Perez Velazquez et al., 2006; Rami et al., 2001; Simburger et al., 1997). An exception to this situation is if connexins in interneurons are disrupted, which may affect protective GABA release (Oguro et al., 2001). In addition, because Panx1 channels can be blocked by most traditional connexin blockers, previous studies must be reinterpreted, taking into account the fact that neuronal Panx1 channels open during ischemic conditions (Thompson et al., 2006), as well as the presence of pannexins in astrocytes (Iglesias

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et al., 2009). This may explain why “gap junctions” were found to be destructive in these studies. One of the main limitations in gap junction studies is the lack of selective blockers or enhancers for specific connexin or pannexin subtypes, and the lack of selectivity between hemichannels and gap junctions. It is also difficult to study the role of connexins in cellular damage because connexin functions are not limited to cellecell communication and hemichannel activity. As mentioned earlier, connexins also interact with at least 40 different proteins, which may have importance in signaling events that occur in response to cellular injury.

Acknowledgments The work referenced from the authors’ laboratory was supported in part by grants from the Heart & Stroke Foundation of BC & Yukon, and the Canadian Institutes of Health Research. MK was supported by scholarships from the Natural Sciences & Engineering Research Council, the Michael Smith Foundation for Health Research, the Canadian Institutes of Health Research Vancouver Coastal Health Research Institute UBC MD/PhD Studentship Award and the Dorothy May Ladner Memorial Fellowship. CCN holds a Canada Research Chair.

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V. GAP JUNCTIONS AND NEUROLOGICAL AND NEUROPSYCHIATRIC DISEASES