Gap junctions and hemichannels composed of connexins: potential therapeutic targets for neurodegenerative diseases

Microglia are macrophage-like resident immune cells that contribute to the maintenance of homeostasis in the central nervous system (CNS). Abnormal activation of microglia can cause damage in the CNS, and accumulation of activated microglia is a characteristic pathological observation in neurologic conditions such as trauma, stroke, inflammation, epilepsy, and neurodegenerative diseases. Activated microglia secrete high levels of glutamate, which damages CNS cells and has been implicated as a major cause of neurodegeneration in these conditions. Glutamate-receptor blockers and microglia inhibitors (e.g., minocycline) have been examined as therapeutic candidates for several neurodegenerative diseases; however, these compounds exerted little therapeutic benefit because they either perturbed physiological glutamate signals or suppressed the actions of protective microglia. The ideal therapeutic approach would hamper the deleterious roles of activated microglia without diminishing their protective effects. We recently found that abnormally activated microglia secrete glutamate via gap-junction hemichannels on the cell surface. Moreover, administration of gap-junction inhibitors significantly suppressed excessive microglial glutamate release and improved disease symptoms in animal models of neurologic conditions such as stroke, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease. Recent evidence also suggests that neuronal and glial communication via gap junctions amplifies neuroinflammation and neurodegeneration. Elucidation of the precise pathologic roles of gap junctions and hemichannels may lead to a novel therapeutic strategies that can slow and halt the progression of neurodegenerative diseases.


INTRODUCTION
Microglia are macrophage-like immune cells that reside in the central nervous system (CNS), where they play multiple roles: presenting antigen to initiate immunological reactions, directing attack against non-self antigens, debris clearance, support of neuronal circuit development (Kreutzberg, 1996;Kempermann and Neumann, 2003;Block et al., 2007;Takeuchi, 2010;Boche et al., 2013), and so on. Microglia contribute to maintenance of CNS homeostasis, but abnormal activation of these cells often causes damage to surrounding cells and tissues. Microgliosis, the accumulation of activated microglia, is a characteristic pathological feature in many neurologic conditions such as trauma, stroke, inflammation, epilepsy, and neurodegenerative diseases (Cagnin et al., 2001;Eikelenboom et al., 2002;McGeer and McGeer, 2002;Nelson et al., 2002;Orr et al., 2002;Bruijn et al., 2004;Pavese et al., 2006). Activated microglia release massive amounts of glutamate, at much higher levels than astrocytes and neurons (mM vs. μM), and destroy neural cells; these processes have been implicated as a major cause of neuronal damage in neurologic diseases (Piani et al., 1992;Barger and Basile, 2001;Schwartz et al., 2003;Ye et al., 2003;Kipnis et al., 2004;Takeuchi et al., 2005Takeuchi et al., , 2008bHerman and Jahr, 2007;Liang et al., 2008;Yawata et al., 2008). Therefore, blockade of glutamate signaling and inhibition of microglial activation have been explored as therapeutic candidates for several neurodegenerative diseases. However, glutamate receptor blockers also perturb physiological glutamate signals and cause severe adverse side effects (Parsons et al., 2007). Tetracycline and two of its derivatives (doxycycline and minocycline) have been used as inhibitors of microglial activation, but these compounds exerted little therapeutic benefit, because activated microglia also exert neuroprotective effects such as production of neurotrophic factors and sequestration of neurotoxic substances (Zietlow et al., 1999;Kempermann and Neumann, 2003;Kipnis et al., 2004;Koenigsknecht and Landreth, 2004;Schwab and Schluesener, 2004). Thus, the optimal therapeutic strategy would inhibit the deleterious effects of activated microglia without diminishing their protective roles (Takeuchi, 2010). We recently found that neurotoxic activated microglia secrete glutamate through gap-junction hemichannels. Recent evidence also suggests that neuronal and glial communication by gap junctions amplifies neuroinflammation and neurodegeneration. Therefore, elucidation of the pathologic roles of gap junctions and hemichannels may provide us with new therapeutic strategies against many neurologic diseases.

MICROGLIA AS THE "ENEMY WITHIN"
Microglia, which originate from bone marrow-derived myeloid cells, account for approximately 10% of cells in the human CNS (Del Rio-Hortega, 1932). Microglia are predominantly observed in gray matter, especially in the olfactory bulb, hippocampus, basal ganglia, and substantia nigra (Lawson et al., 1990). Under healthy physiological conditions, microglia persist in a quiescent state with ramified morphology (resting microglia) and survey the environment of the CNS (Davalos et al., 2005;Nimmerjahn et al., 2005). Under pathological conditions, microglia dramatically change their morphology and adopt an amoeboid appearance in the activated state. Activated microglia express surface molecules such as Fc receptor, CD11b, CD11c, CD14, major histocompatibility complex (MHC) molecules, Toll-like receptors (TLRs), scavenger receptors, and cytokine/chemokine receptors, and they can act as both antigen-presenting cells and immunological effector cells (Suzumura et al., 1987;Rock et al., 2004). In addition to innate immunity, activated microglia also play other beneficial roles, such as neuroprotection mediated by release of neurotrophic factors (Zietlow et al., 1999;Bessis et al., 2007;Liang et al., 2010), maintenance of CNS homeostasis by clearance of cellular debris and toxic substances (Upender and Naegele, 1999;Marin-Teva et al., 2004;Iribarren et al., 2005;Simard et al., 2006;Richard et al., 2008), and guidance of stem-cell migration in neuronal repair and neurogenesis (Aarum et al., 2003;Ziv et al., 2006a,b).

GLUTAMATE AS A MAJOR NEUROTOXIC FACTOR FROM MICROGLIA
Glutamate is the most potently neurotoxic factor released from activated microglia. Excessive glutamate induces severe neuronal damage via excitotoxicity (Piani et al., 1992;Barger and Basile, 2001;Takeuchi et al., 2005Takeuchi et al., , 2006. A common misconception is that inflammatory cytokines produced by activated microglia directly induce neuronal damage. In fact, these cytokines have little direct neurotoxic effect Takeuchi, 2010). Although tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) are considered to be the most deleterious inflammatory cytokines produced by activated microglia, these cytokines have only weak direct neurotoxic effects because they also enhance neuroprotective cascades involving mitogen-activated protein kinase (MAPK) and expression of nuclear factor κB (NF-κB) (Ghezzi and Mennini, 2001;Kamata et al., 2005). In general, inflammatory cytokines induce neurotoxicity indirectly by stimulating microglia in an autocrine/paracrine manner. These stimuli induce microglia to release high levels of glutamate, resulting in neuronal damage via excitotoxicity. Moreover, a recent paper showed that activated microglial glutamate suppresses astrocytic glutamate transporters, which play a pivotal role in maintenance of the physiological extracellular glutamate level (Takaki et al., 2012); this suppression probably worsens excitotoxic neuronal damage. Although microglia also express glutamate transporters, they seem much less effective at maintaining extracellular glutamate homeostasis than astrocytic glutamate transporters .
One of the earliest pathologic features of excitotoxicity is formation of neuritic beading, i.e., focal bead-like swelling in dendrites and axons (Takeuchi et al., 2005;Mizuno et al., 2008). Neuritic beading is a common neuropathological hallmark of many neurologic conditions such as ischemia, epilepsy, mechanical pressure, brain tumor, aging, neuroinflammatory diseases, and neurodegenerative diseases such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) (Delisle and Carpenter, 1984;Hori and Carpenter, 1994;Takahashi et al., 1997;Trapp et al., 1998;Dickson et al., 1999;Mattila et al., 1999;Swann et al., 2000;Goel et al., 2003;Pavlidis et al., 2003;Saito et al., 2003;Dutta and Trapp, 2007). Recent studies elucidated the detailed role of microglial glutamate in formation of neuritic beading and subsequent neuronal death. Glutamate produced by activated microglia activates neuronal N-methyl-D-aspartate (NMDA) receptor signaling, which promotes Ca 2+ influx and activates Ca 2+ /calmodulin-dependent protein kinase (CaMK). CaMK activates neuronal nitric oxide synthase (nNOS) and increases the intracellular concentration of nitric oxide (NO). NO in turn inhibits mitochondrial respiratory chain complex IV, resulting in a rapid reduction in intracellular ATP levels. Ultimately, the loss of intracellular energy pools suppresses dendritic and axonal transport, leading to bead-like accumulation of cytoskeletal and motor proteins along neurites and the formation of neuritic beading. Thus, a low-energy state results in neuronal dysfunction. Persistence of this neuronal dysfunction eventually causes neuronal death [i.e., excitotoxic neuronal death or non-cell-autonomous neuronal death (Lobsiger and Cleveland, 2007)].
Recent studies have revealed the precise mechanism of glutamate production by activated microglia (Takeuchi et al., 2005 (Figure 1). Two pathways are involved in cellular glutamate synthesis (Newsholme and Newsholme, 1989;Newsholme and Calder, 1997;Yudkoff, 1997;Nissim, 1999). One of these pathways is mediated by glutamate dehydrogenase, which converts α-ketoglutarate to glutamate. Most cells use this pathway to maintain cellular homeostasis of glutamate levels. The other pathway is mediated by glutaminase, which produces glutamate from extracellular glutamine brought into the cell via glutamine transporters. Resting microglia maintain their physiological glutamate level via the glutamate dehydrogenase pathway, as in other cell types, and secrete very little glutamate into the extracellular space (Figure 1). By contrast, activated microglia produce excessive amounts of glutamate as a result of upregulation of glutaminase, but not glutamate dehydrogenase. Subsequently, activated microglia release massive amounts of glutamate via gapjunction hemichannels. Inflammatory cytokines such as TNF-α and IFN-γ enhance not only glutaminase expression level but also cell-surface localization of hemichannels in microglia (Eugenin et al., 2001;Takeuchi et al., 2006). These two phenomena may act synergistically to release excess glutamate, leading to excitotoxic neuronal damage (Figure 1). Moreover, the extracellular glutamine level is critical for microglial glutamate production . In the CNS, glutamine from astrocytes is essential for glutamate production in neurons (Tsacopoulos and Magistretti, 1996), suggesting that it also plays an important role in microglial glutamate production.

GAP JUNCTIONS IN CNS CELLS
Gap junctions contribute to formation of intercellular channels that directly connect the cytoplasmic compartments of neighboring cells (Yeager and Harris, 2007). These channels pass various small molecules (∼1000 Da) and ions, although the charges and shapes of these molecules may affect the rate of transfer through gap junctions (Goldberg et al., 2004). Each gap junction is composed of a pair of hemichannels docked in a head-to-head configuration. Hemichannels are organized as hexagonal cylinders with central pores, and each hemichannel consists of a hexameric cluster of protein subunits called connexins (in vertebrates) or innexins (in invertebrates). Connexins are encoded by a conserved family of genes with at least 21 members in mammals. There are 21 connexin genes in the human genome and 20 connexin genes in the mouse genome; 19 of these proteins have orthologs in both humans and mice (Willecke et al., 2002;Laird, 2006). The connexin isoforms structurally interact in multiple ways. Homomeric hemichannels consist of a single connexin isoform, whereas heteromeric hemichannels contain two or more different connexin isoforms. Likewise, a homotypic gap junction channel is composed of two identical hemichannels, whereas a heterotypic gap junction channel contains two different hemichannels. Thus, the compositions of gap junctions can be classified into four types: homomeric and homotypic; heteromeric and homotypic; homomeric and heterotypic; and heteromeric and heterotypic FIGURE 1 | Mechanism of glutamate production and release by activated microglia. Like other types of cells, resting microglia use glutamate dehydrogenase to synthesize glutamate from intracellular α-ketoglutarate in order to maintain a physiologically normal level of glutamate. Under resting conditions, microglia release very little glutamate into the extracellular space. By contrast, under pathological conditions, glutaminase and gap-junction hemichannels are upregulated in activated microglia (e.g., in response to stimulation by TNF-α). Glutaminase synthesizes excess glutamate from extracellular glutamine, which is brought into the cell via glutamine transporters. Subsequently, high levels of glutamate are secreted through gap-junction hemichannels, resulting in eventual neuronal damage. FIGURE 2 | The composition of gap junctions and hemichannels. Each colored column (orange, blue, green, or purple) represents a different connexin isoform. Hemichannels may be homomeric (composed of one connexin isoform) or heteromeric (composed of more than one connexin isoform). Gap-junction channels may be homotypic (formed by identical hemichannels) or heterotypic (formed by different hemichannels).
( Figure 2). This heterogeneity of connexin configurations confers complexity to the gap junction/hemichannel system.
Whereas vertebrate cells use connexins to form gap junctions and hemichannels, invertebrate cells use innexins, which lack sequence homology to connexins. A search of the human genome identified three innexin-related genes (Barbe et al., 2006). Because of the occurrence of homologous genes in both vertebrates and invertebrates, the corresponding proteins were termed pannexins: pannexin1 (Panx1), pannexin2 (Panx2), and pannexin3 (Panx3). Pannexins have the same transmembrane topology as connexins, with four transmembrane domains and cytoplasmic amino-terminal and carboxyl-terminal domains. Recent evidence indicates that pannexins also form uncoupled hemichannels in mammalian cells; however, it is not clear whether they can form functional gap junctions (Dahl and Locovei, 2006).
The evidence described above might give the false impression that CNS cells express only a narrow range of combinations of homomeric hemichannels and gap junctions. However, the precise configuration of these hemichannels [i.e., homomeric and homotypic; heteromeric and homotypic; homomeric and heterotypic; and heteromeric and heterotypic (Figure 2)] has yet to be elucidated. In addition, our recent reverse transcription-PCR analysis using mouse primary cultures indicated that gap junctions/hemichannels in neurons and glial cells may consist of a wider range of combinations of connexins than expected ( Table 1) : neurons predominantly express

GAP JUNCTIONS COMPOSED OF CONNEXINS AS A NOVEL THERAPEUTIC TARGET FOR NEUROLOGIC DISEASES
As mentioned above, glial gap junctions play an important role in maintenance of homeostasis in the CNS under the physiological conditions. These structures, however, also contribute to the initiation and propagation of pathologic conditions (Orellana et al., 2009). Stroke and trauma provide examples that illustrate this mechanism. Ischemia or contusion leads to a rapid decrease in intracellular oxygen levels and subsequent reduction in ATP synthesis, resulting in eventual cell death (Kalogeris et al., 2012). Injured cells contain toxic ions and molecules at high concentrations (e.g., Ca 2+ , K + , ROS, and NO). These toxic molecules are propagated from injured cells to healthier cells through gap junctions. Ischemic conditions also induce uncoupled hemichannels to open, leading to paracrine transfer of toxic molecules (Thompson et al., 2006;De Vuyst et al., 2007). These waves of death signals activate astrocytes and microglia, inducing the release of toxic molecules including glutamate, ROS, NO, and pro-inflammatory cytokines and chemokines. This vicious amplification spiral of signaling could worsen neuroinflammation by recruiting leukocytes and increasing the lesion area (Orellana et al., 2009) (Figure 3). Moreover, gap junction and hemichannel blockers have exerted therapeutic effects in experimental models of stroke and spinal cord injury (Rawanduzy et al., 1997;Frantseva et al., 2002;De Pina-Benabou et al., 2005;Takeuchi et al., 2008a;Tamura et al., 2011;Huang et al., 2012;Umebayashi et al., 2014). Abnormal expression of glial connexins has been observed in the inflamed lesions in multiple sclerosis (MS) and an animal model of this disease, experimental autoimmune encephalomyelitis (EAE). In particular, downregulation of oligodendrocytic Cx32 and Cx47 and astrocytic Cx43 have been observed in the active lesions of MS patients and EAE mice (Brand-Schieber et al., 2005;Eugenin et al., 2012;Markoullis et al., 2012). Expression levels of Cx47 and Cx32 were upregulated during remyelination, but downregulated in the relapsing phase, and Cx32 deletion resulted in exacerbates symptoms in EAE, specifically increased demyelination and axonal loss (Markoullis et al., 2012). Whereas mice lacking astrocytic expression of Cx43/Cx30 exhibited white-matter vacuolation and hypomyelination, the severity of EAE in these animals was similar to that in wild-type mice (Lutz et al., 2012). Therefore, oligodendrocytic expression levels of Cx32 and Cx47 appear to be associated with the degree of damage and remyelination, whereas astrocytic expression levels of Cx43 do not. However, recent studies showed that a loss of Cx43 in astrocytes precedes demyelination in the MS-related disorders neuromyelitis optica and Balo's disease (Matsushita et al., 2011;Masaki et al., 2012), suggesting that the temporal expressional pattern of astrocytic Cx43 plays a significant role in the disease process.
Accumulating evidence has also implicated neuroinflammation, including gliosis by activated astrocytes and microglia, in the pathogenesis of such neurodegenerative diseases as HIV encephalopathy, AD, PD, and ALS (Glass et al., 2010;Valcour et al., 2011). Microglial activation followed by astrocytic activation is the earliest pathologic feature in the pre-symptomatic phases of these diseases. Our recent studies have shown that activated microglia release excess glutamate through Cx32 hemichannels, resulting in excitotoxic neuronal death , 2008a; Yawata et al., 2008). Furthermore, microglia-derived glutamate and pro-inflammatory cytokines induce dysfunction of gap junctions and hemichannels in astrocytes (Kielian, 2008), thereby potentially disrupting CNS homeostasis. On the other hand, reactive astrocytes neighboring amyloid β (Aβ) plaques in the brains of AD patients expressed elevated levels of Cx43 and Cx30 (Koulakoff et al., 2012). Aβ peptide induces the release of glutamate and ATP via uncoupled hemichannels in microglia and astrocytes, leading to neuronal death (Orellana et al., 2011). Corroborating this observation, blockade of gap junctions/hemichannels improved memory impairment in a mouse model of AD (Takeuchi et al., 2011). Recent studies also revealed that astrocytic gap junctions/hemichannels are involved in the disease progression of HIV encephalopathy (Eugenin and Berman, 2013;Orellana et al., 2014). PD animal models (MTPTtreated mice and rotenone-treated rats) exhibited upregulation of astrocytic Cx43 expression in affected areas (Rufer et al., 1996;Kawasaki et al., 2009), and a gap junction/hemichannel blocker ameliorated the disease symptoms of a PD mouse model . A recent report revealed that α-synuclein directly binds Cx32, and that overexpression of α-synuclein suppresses the activity of Cx32 in the SH-SY5Y dopaminergic neuroblastoma cell line (Sung et al., 2007). Other studies have shown that microglia and astrocytes are determinants of disease progression in ALS (the non-autonomous neuronal death hypothesis) (Boillee et al., 2006;Yamanaka et al., 2008). Activation of microglia and astrocytes is associated with elevated expression of gap junctions and hemichannels (Cui et al., 2014). In fact, treatment with a gap junction/hemichannel blocker ameliorated disease progression in a mouse model of ALS (Takeuchi et al., 2011). Juvenile neuronal ceroid lipofuscinosis (JNCL) also shows the activation of microglia and astrocytes preceding neuronal loss (Pontikis et al., 2005;Xiong and Kielian, 2013), and treatment with a gap junction/hemichannel blocker attenuated the disease symptoms of a JNCL mouse model (Burkovetskaya et al., 2014). Few reports, however, have focused on the expression profiles and functions of connexins in these diseases. Further studies are needed to elucidate the precise role of glial connexins in the pathogenesis of these diseases.

CONCLUSIONS
A growing body of evidence has demonstrated the pathologic roles of gap junctions and hemichannels in various neurologic diseases. For example, dysfunction and dysregulation of gap junctions and hemichannels in glial cells contribute to neuroinflammation in the CNS, which results in neuronal damage (a situation in which glial cells are "bad neighbors" of neurons) (Block et al., 2007). Despite recent progress in elucidating the pathological roles of gap junctions and hemichannels, many challenges remain, due in part to technical limitations. For instance, few high-quality antibodies against each connexin are available for immunostaining and immunoblotting. Moreover, reagents that are commonly used to block connexin channels are not specific for those channels. In fact, connexin channel blockers such as glycyrrhetinic acid, its derivative carbenoxolone, niflumic acid, and octanol also block pannexin channels. Although the most specific gap junction and hemichannel blockers currently available are mimetic peptides with sequences very similar to that of the extracellular loop of connexins, recent studies showed that mimetic peptides specific for Cx32 ( 32 gap 24 and 32 gap 27), Cx43 ( 43 gap 27), or Panx1 ( 10 panx1) non-specifically block both connexins and pannexins (Wang et al., 2007). Although aptamers and siRNA may be used as blockers for specific connexins (Knieps et al., 2007;Xu et al., 2014), they still have a problem of the blood-brain barrier penetration. The heterogeneity of gap-junction and hemichannel configurations (Figure 2) and the ability of various connexins to compensate for the loss of other isoforms (e.g., in connexin-knockout studies) also complicate analysis of this system. Although EGFP-tagged connexins have facilitated live-cell imaging, tagging and/or overexpression of connexins in cultured cells often produce abnormally large gap-junction plaques (Lopez et al., 2001;Gaietta et al., 2002;Hunter et al., 2003). Moreover, tagging the amino-termini of connexins results in non-functional channels, whereas tagging the carboxyl-termini alters the properties of the channels (Bukauskas et al., 2000;Contreras et al., 2003). Therefore, future investigations should attempt to elucidate the spatiotemporal expression profiles of connexin isoforms under pathological conditions in the CNS; this work will require development of specific blockers and tracers for each connexin isoform, hemichannel, and gap junction. Understanding the precise pathologic roles of gap junctions and hemichannels may lead to new therapeutic strategies against multiple chronic neurodegenerative diseases.