Glial hemichannels and their involvement in aging and neurodegenerative diseases
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(2) Rev. Neurosci., Vol. 23(2): 163–177, 2012 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/revneuro-2011-0065. Glial hemichannels and their involvement in aging and neurodegenerative diseases. Juan A. Orellana1,*, Rommy von Bernhardi1, Christian Giaume2–4 and Juan C. Sáez5,6 1 Facultad de Medicina, Departamento de Neurología, Pontificia Universidad Católica de Chile, Santiago, Chile 2 Center for Interdisciplinary Research in Biology (CIRB), CNRS UMR 7241/INSERM U1050, Collège de France, 75005 Paris, France 3 University Pierre et Marie Curie, ED, Nº 158, 75005 Paris, France 4 MEMOLIFE Laboratory of Excellence and Paris Science Lettre, Collège de France, 75005 Paris, France 5 Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago, Chile 6 Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Valparaíso, Chile. * Corresponding author e-mail: [email protected]. Abstract During the last two decades, it became increasingly evident that glial cells accomplish a more important role in brain function than previously thought. Glial cells express pannexins and connexins, which are member subunits of two protein families that form membrane channels termed hemichannels. These channels communicate intra- and extracellular compartments and allow the release of autocrine/paracrine signaling molecules [e.g., adenosine triphosphate (ATP), glutamate, nicotinamide adenine dinucleotide, and prostaglandin E2] to the extracellular milieu, as well as the uptake of small molecules (e.g., glucose). An increasing body of evidence has situated glial hemichannels as potential regulators of the beginning and maintenance of homeostatic imbalances observed in diverse brain diseases. Here, we review and discuss the current evidence about the possible role of glial hemichannels on neurodegenerative diseases. A subthreshold pathological threatening condition leads to microglial activation, which keeps active defense and restores the normal function of the central nervous system. However, if the stimulus is deleterious, microglial cells and the endothelium become overactivated, both releasing bioactive molecules (e.g., glutamate, cytokines, prostaglandins, and ATP), which increase the activity of glial hemichannels, reducing the astroglial neuroprotective functions, and further reducing neuronal viability. Because ATP and glutamate are released via glial hemichannels in neurodegenerative conditions, it is expected that they contribute to neurotoxicity. More importantly, toxic molecules released via glial hemichannels. could increase the Ca2+ entry in neurons also via neuronal hemichannels, leading to neuronal death. Therefore, blockade of hemichannels expressed by glial cells and/or neurons during neuroinflammation might prevent neurodegeneration. Keywords: astrocytes; brain; connexins; inflammation; microglia; pannexins.. Introduction In the central nervous system (CNS), the two main cellular components are glial cells and neurons. The former constitute ∼90% of CNS cells, and the latter represent just ∼10%. However, both cellular types contribute in equal form (50% and 50%) to the total cell mass of the brain (Verkhratsky and Toescu, 2006). Glial cells are divided into macroglia (oligodendrocytes, astrocytes, and ependymoglial cells) and microglia, which are from neuroectodermal and mesenchymal origin, respectively. Whereas neurons and macroglial cells are endogenous cells of the brain, microglia invade the CNS early during embryonic development (Carson et al., 2006). For a long time, glial cells were considered as part of the brain connective tissue that provides support to neurons. Nevertheless, during the last two decades, it became evident that glial cells have more significant roles in brain function than previously thought. The brain performs exceptionally complex and dynamic tasks that depend on the coordinated interaction of endothelial cells, macroglial cells, microglia, and neurons. For instance, hundreds of astrocytes can be connected with each other (Giaume et al., 2010). Such astrocyte-to-astrocyte intercellular communication is attained by sharing cytoplasmic content through membrane specializations termed gap junctions. These cell junctions are aggregates of few tens to thousands of intercellular conduits termed gap junction channels (GJCs) that allow direct but selective cytoplasmic continuity between contacting cells. Through GJCs, the intercellular exchange of metabolites (e.g., ADP, glucose, glutamate, and glutathione), second messengers (e.g., cyclic adenosine monophosphate and inositol triphosphate), and the intercellular spread of electrotonic potentials in excitable and non-excitable tissues (Sáez et al., 2003; Sohl and Willecke, 2004; Evans et al., 2006) are possible. Whereas a GJC is formed by the serial docking of two hemichannels (each one contributed by one of two adjacent cells), each hemichannel is composed of six protein subunits termed connexins (Cxs). The latter belong to a highly conserved protein family encoded by 21 genes in humans and 20 in mice with orthologs in other vertebrate species (Cruciani and Mikalsen, 2005).. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. Cato. de Chile) Authenticated | 172.16.1.226 Download Date | 4/5/12 4:47 PM.
(3) 164. J.A. Orellana et al.. Although for a long time the main function attributed to Cx hemichannels, also called connexons, was the formation of GJCs, in the last decade, the presence of functional hemichannels in nonjunctional membranes has been demonstrated by several experimental approaches (Sáez et al., 2010). Up until now, it is broadly recognized that hemichannels allow cellular release of relevant quantities of autocrine/paracrine signaling molecules [e.g., ATP, glutamate, nicotinamide adenine dinucleotide (NAD+), and prostaglandin E2 (PGE2)] to the extracellular milieu (Bruzzone et al., 2001; Stout et al., 2002; Cherian et al., 2005), as well as uptake of small molecules (e.g., glucose) (Retamal et al., 2007). Recently, another gene family encoding a set of three membrane proteins, named pannexins (Panx1–3), has been demonstrated to form hemichannels (Panchin et al., 2000; Bruzzone et al., 2003). In addition, gap junctional communication via Panx3 has been recently demonstrated to occur between osteoblasts (Ishikawa et al., 2011). However, the absence of ultrastructural evidences for gap junction formation in mammalian cells and demonstration of functional communication mediated by other endogenously expressed Panxs has led to propose that the main function of Panx-based channels is paracrine/ autocrine communication, acting as hemichannels (MacVicar and Thompson, 2010). Cxs and Panxs share similar membrane topology, with four α-helical transmembrane domains connected by two extracellular loops, where both N- and C-termini are intracellular. However, comparison of the primary structure between members of these two protein families does not reveal relevant homology (<16% overall identity) (Figure 1). Recently, the involvement of hemichannels in several biological functions and responses has been shown. For instance, a relevant role in cellular proliferation and tissue remodeling has been attributed to Cx hemichannels (Schalper et al., 2008b; Burra and Jiang, 2009), while in the CNS, they have been proposed to mediate ischemic tolerance (Lin et al., 2008; Schock et al., 2008) and establish adhesive interactions (Cotrina et al., 2008). So far, most studies suggest that under normal brain conditions, Cx hemichannels allow the release of molecules relevant for intercellular signaling, including ATP and NAD+ required for the propagation of intercellular Ca2+ waves (Orellana et al., 2011a). However, an increasing body of evidence has situated Cx and Panx hemichannels as potential regulators of the beginning and maintenance of homeostatic imbalances present in diverse brain diseases (Orellana et al., 2009). A constant increase of intracellular free Ca2+ concentration ([Ca2+]i) due to Ca2+ entry through Cx hemichannels, which are permeable to Ca2+ (Schalper et al., 2010), and deficient or insufficient Ca2+ handling by injured cells could lead to cell death. Here, we review and discuss the current evidence on the possible role of glial and neuronal hemichannels on neurodegenerative diseases.. Glia: enemy, victim, or patient ally in neurodegenerative diseases? Neuroinflammation and microglial cell activation have been extensively studied in neurodegenerative diseases. However,. there is some debate whether microglia serve beneficial or deleterious functions and whether inflammation and microglial accumulation are causative of the disease or represent attempts of the diseased brain to fight the underlying disease cause. The emergence of the concept of dysfunctional microglia (von Bernhardi et al., 2007), secondary to aging, chronic inflammation, or epigenetic changes, together with the high plasticity of microglia, leads to new thoughts for disease mechanisms as well as therapies based on modulation of their function, instead of their outright elimination. Neuroinflammation and microglial cell activation. Microglia are the immunological effectors of the CNS that continuously survey brain parenchyma (Hanisch and Kettenmann, 2007). Under unstimulated conditions, microglia are in a quiescent state. However, the immune response is rapidly triggered by injury or proinflammatory stimuli. Activation is defined by the integration of activating and inhibitory signals that ultimately determine the kinetic, magnitude, and/or length of the response. Overwhelming reactions due to increased activation or lack of inhibitory signals can destroy not only abnormal but also healthy tissues. An excess of negative signals may lead to responses insufficient for clearing abnormal tissue components. Microglia present a complex pattern rather than simplistic on and off states (activated and resting phenotypes). Similarly, neuroinflammation is recognized as a polymorphic and finely tuned response of the CNS to various stimuli such as pathogens, modified molecules/cells, or injuries. Disturbance of brain homeostasis leads to what is referred to as the ‘activated state’ of microglia, which translates in a reversible sequence of morphological changes, acquisition of distinct expression pattern of diverse proteins including K+ channels and neurotransmitter receptors, migratory and proliferative behavior, release of various reactive oxygen species, reactive nitrogen species, cytokines, growth factors, and phagocytic activity. The inflammatory response of microglia presents many faces depending on the stimuli. These faces can be ascribed to variations of the M1 and M2 profiles initially defined as the inflammatory response of macrophages to interferon-γ and lipopolysaccharide (LPS) and their anti-inflammatory response to interleukin-4 (IL-4), respectively (Mantovani et al., 2004). The exacerbated inflammatory response of microglia reflects the M1-like inflammatory, poorly phagocytic, and cytotoxic profile. At the other extreme of a continuum of activation profiles associated with different functions, the M2-like anti-inflammatory, neuroprotective, phagocytic profile can be found (Mosser and Edwards, 2008; Ransohoff and Cardona, 2010; Vereyken et al., 2011). Among the various cytokines that constitute the repertoire of neuroinflammation, transforming growth factor β (TGF-β) is a key molecule involved in the modulation of microglia activation through its receptor-activated Smad signaling (Liu and Feng, 2010). It induces an M2-like profile of microglia activation and prevents microglial response to inflammatory signals (Paglinawan et al., 2003). TGF-β also exerts neuroprotective functions apparently mediated through Smad-independent. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. Cato. de Chile) Authenticated | 172.16.1.226 Download Date | 4/5/12 4:47 PM.
(4) Glial hemichannels in neurodegeneration. 165. Figure 1 Diagram illustrating basic structures of Cx and Panx undocked hemichannels present at the cell surface. Cxs and Panxs share similar membrane topology, with four α-helical transmembrane domains (M1-M4) connected by two extracellular loops (E1 and E2), and one cytoplasmic loop (CL) where both amino (NH2)- and carboxy (COOH)-termini are intracellular. Top and bottom center show hemichannels formed by six Cx or Panx subunits each. The middle center shows an aggregate of Cx GJCs, a section through a gap junction ‘plaque,’ at a close contact between cells 1 and 2 as shown in the left. A hemichannel is formed by six Cxs or Panxs that oligomerize laterally, leaving a central pore in the activated state (open). Under resting conditions, hemichannels remain preferentially closed, but they can be activated by diverse physiological and pathological conditions, offering a diffusional transmembrane route between the intra- and extracellular milieu.. phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) pathways (Zhu et al., 2002, 2004). Both IL-1β and TGF-β1 inhibit nitric oxide (NO) production by microglia through modulation of ERK (Saud et al., 2005). The elevated production of TGF-β observed in normal brain aging or with AD (Harry et al., 2000; Bye et al., 2001; Rota et al., 2006; Motta et al., 2007; Sierra et al., 2007), together with impairment of its signaling (Colangelo et al., 2002; Lee et al., 2006), can lead to a dysregulation of microglial function and regulation.. Regulation of microglia functions by astrocytes. Astrocytes are key regulators of microglia function, in which TGF-β released by astrocytes plays a key role. Astrocytes also release gliotransmitters which comprise purines and glutamate, which most likely contribute to regulate microglial activity to the same extent as do neurons (Volterra and Meldolesi, 2005). ATP-mediated calcium signaling between astrocytes and microglia can even induce microglial cell death, which might be a way of silencing and regulating the. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. Cato. de Chile) Authenticated | 172.16.1.226 Download Date | 4/5/12 4:47 PM.
(5) 166. J.A. Orellana et al.. number of activated microglia in pathological conditions (Verderio and Matteoli, 2001). The microglial reactivity to amylod β peptide (Aβ) (Smits et al., 2001; von Bernhardi and Ramírez, 2001) and their phagocytic activity (DeWitt et al., 1998) can be attenuated by astrocytes. However, glial cell activation by Aβ is markedly amplified by their inflammatory priming, while its attenuation by astrocytes is lost (von Bernhardi and Eugenin, 2004) and neurotoxicity is enhanced (Ramírez et al., 2008). The fact that astrocytes appear to have both beneficial (reduction of cytotoxicity) and deleterious (inhibition of clearance) effects on microglia is quite remarkable. TGF-β1 can be one of the modulating factors of cytotoxicity. Microglial modulation by astrocytes is abolished by immunoneutralization with anti-TGF-β1 specific antibody (Herrera-Molina and von Bernhardi, 2005; Ramírez et al., 2005). Those observations suggest that impairment of astrocyte regulation in the agingassociated inflammatory environment could further enhance microglial cell activation. In addition, aging has also been associated with increased neurotoxicity. Microinjection of Aβ in the cortex of Rhesus monkey showed Aβ-induced neuronal loss, tau phosphorylation, and microglial proliferation only in aged but not in young monkeys. These results suggest that Aβ neurotoxicity is a pathological product of the aging brain (Geula et al., 1998). Microglia appear to be primed in senescence in such a way that several additional stimuli can result in a persistent inflammatory response that might induce neuronal damage (Block et al., 2007). For example, cortical plaques in non-demented elders are mostly diffuse, but can be abundant and show a similar distribution with those of AD brains (Knopman et al., 2003). The difference is that plaques of non-demented elders lack glia and inflammatory reactivity. Evolution toward the mature senile plaques observed in AD is associated with microglial cell activation (Sheng et al., 1997; Wegiel et al., 2000). In neurodegenerative diseases, there is loss of the control of neurons and astrocytes on microglial cell activation. TGF-β-mediated regulation of cytotoxicity and promotion of phagocytosis is impaired under conditions of chronic inflammation because of changes in its signal transduction pathways. Chronically activated microglia release chemokines and cytokines such as IL-1, IL-6, and tumor necrosis factor α (TNF-α) (Akiyama et al., 2000) which are highly neurotoxic. Microglia and astrocytes express several scavenger receptors (Alarcón et al., 2005) which bind Aβ, thereby amplifying the production of neurotoxic cytokines and NO (Li et al., 2003). Cytotoxic activation may even be potentiated by infiltrated macrophages that phagocyte Aβ more efficiently than microglia (Malm et al., 2005, 2008; Simard and Rivest, 2006; Simard et al. 2006). Therapeutic intervention on microglia. Although much is known about AD and other neurodegenerative conditions, most attempted therapies have been symptomatic (Mangialasche et al., 2010). The failure of current therapies is a clear call for the design of novel or complementary therapies. An appropriate immunomodulation of the. activation state of microglia (von Bernhardi et al., 2011) may facilitate restoration of adequate microglia inflammatory profile which would lead to neuroprotection and efficient clearing of abnormal proteins, and elimination of dying neurons. The role of microglia as therapeutic targets is still under discussion. Their relevance in neurodegenerative diseases have been challenged by studies that selectively ablate them (Carmen et al., 2006; Maysinger et al., 2007; Grathwohl et al., 2009). Existing evidence shows that inhibition of microglia can be harmful, as it has been observed in regeneration models (Keilhoff et al., 2007; Potter et al., 2009) and demyelinating lesions (Glezer et al., 2007). The beneficial or deleterious effect might depend in part on the type of activation undergone by microglia and the stage of disease progression. The ideal therapeutic approach should involve attenuation of deleterious activation, but preservation of their beneficial effects. Microglial cell-mediated immune response in aging seems to be shifted to an adenomatous polyposis coli (APC) phenotype and Th1 cytokine profile, meaning that phagocytic activity and neuroprotective effects are reduced, whereas cytotoxicity is increased. Then, microglial cell activation toward an innate immune Th2 response, promoting anti-inflammatory activity with decreased oxidative damage and promotion of neuronal protection (Shie et al., 2009), could be more effective. In animal models, the blockade of CD40L has shown to decrease the APC phenotype, decreasing levels of proinflammatory cytokines and increasing levels of anti-inflammatory cytokines in the brain (Townsend et al., 2005; Obregon et al., 2008). Agonists and antagonists of various metabotropic glutamate receptors (mGluRs) appear to ameliorate microglia neurotoxicity in experimental autoimmune encephalomyelitis (Pinteaux-Jones et al., 2008) and other neurodegenerative diseases (Byrnes et al., 2009). Clinical trials of immunotherapy are underway. The underlying mechanisms for immunotherapy-mediated microglial activation are not completely elucidated, but the existence of multiple mechanisms is likely (von Bernhardi et al., 2010). One possibility is that immunization improves microglial cell uptake and degradation of Aβ (Wilcock et al., 2003). An alternative mechanism could be modulation of microglia cytotoxic activation. Mediators produced by the innate immune system can stimulate (Simard and Rivest, 2006) and induce a beneficial activation of microglia (Morgan et al., 2005). Immunotherapy also reduces cytokine levels (Rakover et al., 2007), shifting microglial cells toward a phenotype less cytotoxic and with increasing effectiveness in clearing Aβ deposits (Morgan, 2006). Reports also show that Aβ-specific Th2 cells improve cognitive function and reduce plaque load without inducing measurable anti-Aβ antibodies (Dodart et al., 2002; Ethell et al., 2006). Cognitive improvement can also depend on the suppression of neurotoxic activation of microglia, because cytokines influence synaptic transmission, and inflammation is involved in disruption of synaptic function in pathological conditions and induction of neurodegeneration (Rogers et al., 2002; von Bernhardi and Eugenín, 2004; Ramírez et al., 2005; von Bernhardi et al., 2007).. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. 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(6) Glial hemichannels in neurodegeneration. Functional expression pattern of brain hemichannels Depending on the developmental stage, cell type, and brain region, the expression of diverse Cxs and Panxs differs (Dermietzel et al., 1989; Nadarajah et al., 1997; Leung et al., 2002; Bruzzone et al., 2003; Ray et al., 2005; Zappala et al., 2006, 2007; Cina et al., 2007). Consequently, the CNS expresses different hemichannel types (Figure 2), and here, we describe the expression pattern of these cell membrane channels in brain cells. Neurons. Despite the detection of Cxs 26, 30.2, 31.1, 32, 37, 40, 43, 47, 57 and Panxs 1 and 2 in different neuronal types at different developmental stages (Nadarajah et al., 1997; Bruzzone et al., 2003; Zappala et al., 2006, 2007; Cina et al., 2007), so far, only functional hemichannels composed of Panx1 or Cx36 have been identified in neurons (Figure 2). Pioneering findings by Thompson et al. (2006) showed for the first time the presence of functional hemichannels in neurons. They recorded the rapid activation of a large inward current in acutely isolated rat hippocampal neurons subjected to oxygen-glucose deprivation (OGD). Moreover, membrane currents and efflux and influx of calcein and sulforhodamine, respectively, were inhibited by 100 μm carbenoxolone (CBX) – a concentration that blocks both Cx and Panx hemichannels (Thompson et al., 2006). In addition, the OGD-induced currents were also blocked with La3+, which does not block Panx1 hemichannels but blocks Cx hemichannels (Schalper et al., 2008a) and transient receptor potential channels (Halaszovich et al., 2000). Nonetheless, the authors proposed Panx1 hemichannels as the main current pathway because single-channel recordings. 167. showed the expected unitary conductance for Panx1 hemichannels (∼550 pS) recorded in Xenopus oocytes used as an exogenous expression system (Bao et al., 2004). Moreover, due to the large size of Panx1 unitary hemichannel currents recorded at positive holding potentials, it was suggested that they likely contribute substantially to anoxic depolarization during ischemia (Thompson et al., 2006). However, lack of experiments in Panx1-/- neurons or inducing Panx1 knockdown leaves open the possible involvement of other hemichannel types. In fact, Madry et al. (2010) showed in situ that neuronal Panx1 hemichannels do not contribute considerably to the generation of anoxic depolarization and do not mediate neuronal dye uptake at later times in ischemia. The disparity between these two studies could be explained by the use of different preparations; the former used acute isolated pyramidal neurons (Thompson et al., 2006), whereas the latter utilized pyramidal neurons from acute brain slices (Madry et al., 2010). Other groups have reported that ischemia-like conditions could modulate the activity of neuronal hemichannels. Indeed, hippocampal neurons exposed to OGD or metabolic inhibition exhibit increased hemichannel activity measured as calcein leakage, which was inhibited by CBX (Zhang et al., 2008). Interestingly, the NO donor S-nitrosoglutathione induced rapid calcein leakage from neurons, suggesting that NO per se and/or its metabolites could change the activity of neuronal hemichannels (Zhang et al., 2008). However, these studies were only based on the use of CBX, a nonselective hemichannel blocker that also blocks P2X7 receptors (Suadicani et al., 2006) and volume-regulated anion channels (Ye et al., 2009). Thus, further studies are needed to support the above interpretation. Recently, Kawamura et al. (2010) proposed that the metabolic state of neurons could modulate the functional state of Panx1 hemichannels. They demonstrated. Figure 2 Cellular distribution of hemichannels in brain cells. This figure includes only the available information obtained under in vivo and/or in vitro studies using more than one experimental approach. Hemichannels are indicated in neurons, astrocytes, oligodendrocytes, and microglia. At the right, the respective unitary conductance in pS of each hemichannel type so far characterized is indicated. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. Cato. de Chile) Authenticated | 172.16.1.226 Download Date | 4/5/12 4:47 PM.
(7) 168. J.A. Orellana et al.. that in conditions of sufficient intracellular ATP, the reduction of extracellular glucose promotes ATP release via Panx1 hemichannels in CA3 neurons (Kawamura et al., 2010). More importantly, as it occurs with the ATP release via astroglial Cx43 hemichannels during hypoxic preconditioning (Lin et al., 2008), ATP released through neuronal Panx1 hemichannels is dephosphorylated to adenosine, which activates neuronal adenosine A1 receptors and then hyperpolarizes neurons via ATP-sensitive K+ channels (Kawamura et al., 2010). In this scenario, the release of ATP could also result in activation of Panx1 hemichannels indirectly via P2X7 receptors as it has been demonstrated in macrophages (Locovei et al., 2006) or directly as it has been recently suggested to occur in vasopressin neurons isolated from the rat supraoptic nucleus (Ohbuchi et al., 2011). Under physiological conditions, it is expected that ATP concentrations over 100 μm generate direct inhibition of Panx1 hemichannels (Qiu and Dahl, 2009), avoiding a vicious cycle of Panx1 hemichannel opening, which could lead to activation of the inflammasome and induce neuronal death (Silverman et al., 2009). A few years ago, it was demonstrated in pyramidal neurons that a secondary current (I2nd) and calcein efflux induced by N-methyl-d-aspartate (NMDA) are blocked by the 10Panx1 mimetic peptide (homologous to a domain of extracellular loop 1 of Panx1) or by down-regulation of Panx1 with siRNA (Thompson et al., 2008). Moreover, recordings of extracellular field potentials in hippocampal brain slices show that the frequency of interictal bursts and mean amplitude of spikes induced by activation of NMDA receptors (NMDARs) are decreased by 10Panx1 (Thompson et al., 2008). Therefore, the authors propose that Ca2+ deregulation in hippocampal pyramidal neurons and establishment of acquired epilepsy could be related to this NMDAR-evoked I2nd mostly due to activation of Panx1 hemichannels. Accordingly, glutamate released from activated astrocytes increase the Panx1 hemichannel activity in neurons via activation of neuronal NMDARs (Orellana et al., 2011b). More importantly, blockade of NMDARs and/ or Panx1 hemichannels reduced neuronal death induced by astrocytes subjected to proinflammatory conditions (Orellana et al., 2011b). Altogether, the above-mentioned data reveal that neurons exhibit functional Panx1 hemichannels. Indeed, a unified patch clamp protocol for the characterization of Panx1 hemichannels has been described for isolated neurons and acute brain slices (Gründken et al., 2011). A recent report support the idea that Panx1 hemichannels linked to P2X7 receptors reduce the muscarinic acetylcholine 1 receptor-mediated seizure activity in vivo. In this work, CBX, probenecid, and knockdown of Panx1 increased the convulsive activity in various seizure models generated in P2X7-/- mice (Kim and Kang, 2011). Although oocytes injected with Cx36 mRNA do not exhibit functional hemichannels (Al-Ubaidi et al., 2000), a recent report showed that Cx36 forms functional hemichannels in neurons. Accordingly, down-regulation of Cx36 with siRNA impairs the ATP release and high K+-induced ischemic tolerance to potassium cyanide in cerebellar cortical and granule neurons (Schock et al., 2008). On the same line, treatment with the neurotoxic fragment of Aβ, 25–35 (Aβ25–35),. increases hemichannel opening in neurons as monitored by single-channel and dye uptake recordings (Orellana et al., 2011c). The hemichannel forming proteins responsible for these responses were Panx1 and, possibly, Cx36 (Orellana et al., 2011c). Accordingly, single-channel events with unitary conductances characteristic of Panx1 and Cx36 hemichannels were recorded in neurons treated with Aβ25–35 (Orellana et al., 2011c). Moreover, both ATP and glutamate present in conditioned medium by either microglia or astrocytes treated with Aβ25–35 increased neuronal ethidium (Etd) uptake and mortality, which was prevented by inhibitors of P2X/NMDA receptors and Panx1 hemichannels (Orellana et al., 2011c). Astrocytes. Astrocytes constitute the cell population with the highest level of Cx expression in the CNS, being Cx43 and Cx30 the most abundant astroglial Cxs in adult brain (Giaume and Theis, 2009). The presence of hemichannels in astrocytes was first suggested by Hofer and Dermietzel (1998), who applied an antibody that interferes with external loop domains of Cx43 and reduced the Lucifer yellow uptake induced by low extracellular Ca2+ concentration ([Ca2+]e). However, definitive demonstration was provided by Contreras et al. (2002) using astrocytes of constitutive Cx43 knockout mice or astrocytes with cell-specific inactivation of the Cx43 gene; they demonstrated that astroglial death induced by ischemialike conditions is accelerated by enhanced opening of Cx43 hemichannels. The same year, Stout et al. (2002) showed that astrocytes exhibit low [Ca2+]e-activated whole-cell currents consistent with hemichannel currents that were inhibited by flufenamic acid (FFA), a blocker of Cx more than Panx1 hemichannels. Importantly, mechanically stimulated astrocytes were shown to release ATP via hemichannels, which was potentiated by low [Ca2+]e and inhibited by FFA or Gd3+, suggesting the involvement of Cx43 more than Panx1 hemichannels (Stout et al., 2002). These findings support the notion proposing astroglial Cx hemichannels as conduit for the release of small bioactive molecules. In addition, a further study demonstrated that astrocytes exposed to low [Ca2+]e release glutamate and taurine via Cx hemichannels, as the release of these molecules was inhibited by octanol and heptanol – two Cx hemichannel blockers (Ye et al., 2003). All the above along with further studies performed in primary cultures or in brain slices, established that astroglial Cx43 hemichannels can release glutamate, taurine, ATP, and glutathione in low [Ca2+]e and/or [Mg2+]e conditions (Stout and Charles, 2003; Rana and Dringen, 2007; Stridh et al., 2008, 2010), but release of these molecules can also occur in the presence of physiological concentrations of divalent cations under physiological (Kang et al., 2008; Garré et al., 2010) or pathophysiological conditions (Jiang et al., 2011; Orellana et al., 2011a,b,c). Because astrocytes provide metabolic and structural support to neurons and control the extracellular concentration of glutamate, K+, and H+, astroglial damage associated to hemichannel opening has been proposed to increase neuronal susceptibility to proinflammatory conditions (Contreras et al., 2004; Orellana et al., 2009). Moreover,. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. Cato. de Chile) Authenticated | 172.16.1.226 Download Date | 4/5/12 4:47 PM.
(8) Glial hemichannels in neurodegeneration. in cultured astrocytes and brain slices, IL-1β reversed the inhibitory effect on hemichannel activity induced by epidermal growth factor and basic fibroblast growth factor (b-FGF) (Morita et al., 2007). In the same line, astrocytes treated with conditioned medium from LPS-activated microglia or treated with TNF-α or IL-1β show increased Cx43 hemichannel activity through a p38 MAP kinase-dependent pathway (Retamal et al., 2007). Moreover, these cytokines increase dye uptake mediated by hemichannels as early as 30 min after their addition. This effect was blocked by La3+, and does not occur in astrocytes from Cx43 knockout mice (Retamal et al., 2007). However, it has long been known that hyperglycemia worsens the outcome of acute brain ischemia acting as a proinflammatory condition in animals and in humans (Kagansky et al., 2001). Recently, high extracellular glucose level was shown in astrocytes to enhance the increased hemichannel activity and reduction in gap junction permeability induced by hypoxia (Orellana et al., 2010). These changes were transient after 3 h of hypoxia in high glucose. However, they were more prominent after 6 h of hypoxia and last for over 3 h of reoxygenation, and were followed by death of numerous astrocytes (Orellana et al., 2010). Contrary to the above findings, a few years ago, Panx1 was proposed as the molecular substrate of astroglial hemichannels (Iglesias et al., 2009). Under physiological extracellular concentrations of Ca2+ and Mg2+, either cell membrane depolarization or activation of P2X7 receptors with 3′-O(4-benzoyl)benzoyl ATP increased Panx1-like currents and dye uptake in astrocytes from WT or Cx43-/- mice – a response that was greatly attenuated in Panx1-/- astrocytes, suggesting Panx1 hemichannels as the main contributors in this response (Iglesias et al., 2009). Although Panxs 1 and 2 are clearly detected in neurons of the CNS (Ray et al., 2005; Vogt et al., 2005), up to now, Panx expression in glial cells in vivo has been detected only in few cases (Zappala et al., 2007; Karpuk et al., 2011). Moreover, cultured astrocytes exposed to hypoxia-reoxygenation or treated with proinflammatory cytokines or Aβ peptide show hemichannel activity mediated by Cx43, but not Panx1 (Retamal et al., 2007; Orellana et al., 2010, 2011a,b). However, two recent studies, one using spinal cord astrocytes treated with FGF-1 (Garré et al., 2010) and the other using brain slices from mice harboring a brain abscess (Karpuk et al., 2011), demonstrated that both Cx43 or Panx1 form functional hemichannels. Consequently, the respective contribution of Panxs and Cxs on astroglial hemichannel activity may depend on specific conditions not yet identified that trigger their activation – an issue that requires further investigation. Oligodendrocytes. Despite that oligodendrocytes express Cxs 29, 32, 36, 45, and 47 (Nagy et al., 2004), only functional hemichannels composed of Panx1 have been detected so far (Domercq et al., 2010) (Figure 2). In this study, 1 h of OGD increased the ATP release and cell death, which were potentiated by the ecto-ATPase inhibitor ARL67156, but not by ARL67156 plus inhibitors of P2X7 receptors (brilliant blue G and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate), and hemichannel. 169. blockers (CBX and FFA) (Domercq et al., 2010). Therefore, OGD induces opening of Panx1-based hemichannels and ATP release which kills oligodendrocytes due to a Ca2+ overload via open P2X7 receptors and, possibly, Panx1 hemichannels. In the same way, it is likely that Panx1 hemichannels could explain the ATP release in autoimmune encephalomyelitis where ATP excitotoxicity via P2X receptors occurs in oligodendrocytes (Matute et al., 2007). Further studies are needed to elucidate whether other Panxs or Cxs could form functional hemichannels under physiological or pathophysiological conditions including trauma and multiple sclerosis where oligodendrocytes degeneration is prominent. Microglia. Under resting conditions, microglia in culture or in vivo exhibit low levels of Cxs, but in activation in vitro by TNF-α plus INF-γ or in vivo by stab wounds, they exhibit a diffuse intracellular Cx43 immunoreactivity (Eugenín et al., 2001). Moreover, Staphylococcus aureus-derived peptidoglycan triggers Cx43 mRNA and protein expression associated with a low but significant gap junction coupling between microglia, as assessed by dye coupling assay (Garg et al., 2005). Up to now, only few studies have documented the expression of functional hemichannels in microglia. Contrary to the expectations, TNF-α treatment was shown to induce release of glutamate through a pathway inhibited by a Cx32 (32Gap27), but not Cx43 (43Gap27) mimetic peptide (Takeuchi et al., 2006). Moreover, levels of cell surface Cx32 were increased in microglia treated with TNF-α. Noteworthy, the increased neuronal death associated with release of glutamate was inhibited completely with the 32Gap27 mimetic peptide (Takeuchi et al., 2006). Accordingly, microglial cells from Mecp2 null mice, a model of a neurodevelopmental disorder known as Rett syndrome, promote neuronal death through glutamate release via a cell membrane pathway inhibited by 32Gap27 and 32 Gap24 – two Cx32 hemichannel mimetic peptides (Maezawa et al., 2009). It is relevant to keep in mind that these and other mimetic peptides are homologous to extracellular domains of the respective Cx sequences, but their effects on hemichannel activity have not been documented; therefore, some studies have questioned their specificity (Dahl, 2007; Wang et al., 2007). The use of cell cultures derived from Cx null mice and/or performing knockdown of the respective Cxs along the appropriate use of mimetic peptides could ensure definitely the involvement of Cx32 hemichannels in these studies. Recently, Cx43 and Panx1 hemichannel activities, evaluated by dye uptake and macroscopic cell membrane currents, were shown to be increased by Aβ25–35 exposure (Orellana et al., 2011c). These observations were confirmed by using microglial cultures from Cx43 KO mice and Panx1 mimetic peptides. These currents were recorded at negative holding potential (-60 mV) and in the presence of external divalent cations, suggesting that opening of microglial hemichannel may occur in AD. Importantly, ATP and glutamate present in extracellular medium conditioned by microglia treated with Aβ25–35 were shown to trigger hemichannel opening in neurons, causing deleterious effects in them (Orellana et al.,. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. Cato. de Chile) Authenticated | 172.16.1.226 Download Date | 4/5/12 4:47 PM.
(9) 170. J.A. Orellana et al.. 2011c). Interestingly, a novel putative hemichannel blocker (INI-0602) that crosses the blood-brain barrier was recently shown to inhibit in vivo the LPS-induced glutamate release from microglia and to improve memory deficits in APP/PS1 mice (Takeuchi et al., 2011). Due to the pharmacological sensitivity, these effects were proposed to be mediated by Cx32 hemichannels. However, the possible involvement of other hemichannel forming proteins or even other channels was not ruled out and studies on the specificity of INI-0602 requires further demonstration, using, e.g., in vivo experiments with Cx32-/- microglia or knockdown of Cx32. To further demonstrate the hemichannel involvement in this pathology, the next step could be the analysis of the functional state of microglial hemichannels in brain slices from AD model mice (APP/PS1) using patch clamp and membrane permeability assays.. Possible neurotoxic molecules released via glial hemichannels Glial hemichannels participate in the coordination of several cell processes relevant to intercellular signaling through the release of paracrine/autocrine signaling molecules (Orellana et al., 2011a). Several gliotransmitters including d-serine (Yang et al., 2003; Wu et al., 2004; Henneberger et al., 2010), glutamate (Noda et al., 1999; Malarkey and Parpura, 2008), and ATP (Ferrari et al., 1997; Gourine et al., 2010) have been demonstrated to be released by glial cells and most of them have been documented to be released via hemichannels in microglia (Takeuchi et al., 2006; Orellana et al., 2011c) and astrocytes (Stout et al., 2002; Ye et al., 2003; Kang et al., 2008; Iglesias et al., 2009; Orellana et al., 2011a,b). Although it is expected that gliotransmitters released via hemichannels possibly modulate presynaptic efficacy and postsynaptic responses at the synapse (Orellana et al., 2011a), several studies indicate that proinflammatory conditions increase the release of gliotransmitter via hemichannels affecting neuronal viability. Accordingly, microglia stimulated with TNF-α (Takeuchi et al., 2006), LPS (Takeuchi et al., 2006), or Aβ25–35 (Orellana et al., 2011c) release glutamate via hemichannels in concentrations toxic for hippocampal or cortical neurons. In the same way, astrocytes pre-treated with conditioned media from Aβ25–35-treated microglia release glutamate and ATP via hemichannels when subjected to hypoxia in high glucose (Orellana et al., 2011b). The release of both glutamate and ATP via astroglial Cx43 hemichannels results in increased incidence of cortical neuronal death mediated by activation of Panx1 hemichannels (Orellana et al., 2011b,c). Moreover, neuronal hemichannel activity induced by inflamed astrocytes was prevented either by NMDA or P2X receptor antagonists in neurons (Orellana et al., 2011b,c). Similarly, hippocampal brain slices treated with Aβ25–35 resulted in Etd uptake in astrocytes, identified by their hGFAP eGFP expression, and in pyramidal neurons as well (Orellana et al., 2011c). In astrocytes, the membrane ‘permeabilization’ to Etd was mediated by Cx43 hemichannels as suggested by their pharmacological sensitivity and confirmed by the lack of Aβ25–35-induced Etd uptake in brain slices obtained from Cx43fl/flcreGFAP mouse;. whereas in pyramidal neurons, Etd uptake was essentially due to opening of Panx1 hemichannels identified by their sensitivity to different specific blockers. Importantly, neuronal death detected in Aβ25–35-treated slices was prevented either by blocking astroglial or neuronal hemichannels or by blocking neuronal NMDA and P2X receptors. These results confirm that short-term treatment with the Aβ25–35 induces a cascade of hemichannel activation in glia and neurons leading to neuronal death. Further studies will help to determine how the same gliotransmitters released through hemichannels promote physiological and pathophysiological responses. Different tissue conditions and concentrations of paracrine molecules (e.g., ATP and glutamate) are likely to be the explanation of the different responses.. Cannabinoids prevent glial hemichannelmediated neurotoxicity It is well established that glial cells are targets for synthetic as well as endogenous cannabinoids (CBs) (Stella, 2009). Interestingly, CBs have a potential therapeutic application through an anti-inflammatory action (Croxford, 2003; Cabral and Griffin-Thomas, 2008), and astrocytes as well as microglial cells are one of their targets in the nervous system (Stella, 2004). Indeed, increasing number of studies have revealed that the anti-inflammatory properties of CBs are exerted both in peripheral and in central levels (for reviews, see Klein et al., 2003; Zurier, 2003; Walter and Stella, 2004; Klein, 2005; Pacher et al., 2006). For example, the active plantderived CB compound Δ9-tetrahydrocannabinol significantly reduces the symptoms generated in an animal model of multiple sclerosis (Lyman et al., 1989) through modulation of the neuroinflammation (Baker et al., 2000; Croxford et al., 2008). Moreover, CBs block the release of proinflammatory products from activated microglia (Coffey et al., 1996; Waksman et al., 1999), switching their phenotype toward an anti-inflammatory phenotype (Stella, 2009). In addition, the synthetic CB agonist WIN 55,212-2 (WIN) reduces (1) the release of TNF-α from LPS-activated microglia (Facchinetti et al., 2003) and (2) the release of inflammatory mediators (iNOS and NO) from IL-1β-treated human astrocytes (Sheng et al., 2005); TNF-α and IL-1β also affect Cx-based channels in astrocytes (Meme et al., 2006; Retamal et al., 2007). Glial cells express CB receptors in both healthy and diseased brain. Indeed, while it was shown that microglia express both CB1 and CB2 receptors (Waksman et al., 1999; Facchinetti et al., 2003), CB2 receptors are primarily expressed by activated microglia in a variety of inflammatory situations and brain diseases (Stella, 2004), like in AD (Benito et al., 2003). There are also reports stating that CB1 receptors but not CB2 receptors are expressed by astrocytes (Molina-Holgado et al., 2002; Salio et al., 2002; Walter and Stella, 2004). The understanding of cellular and molecular events linking their respective glial activation status is essential to dissect the mechanisms triggering neurotoxicity. Accordingly, anti-inflammatory agents, such as CBs, might interfere with changes in the functional status of glial cells evoked. Brought to you by | Pontificia Univ. Cato. de Chile (Pontificia Univ. Cato. de Chile) Authenticated | 172.16.1.226 Download Date | 4/5/12 4:47 PM.
(10) Glial hemichannels in neurodegeneration. by proinflammatory treatments. Thus, increased astroglial hemichannel activity induced by cytokines or LPS-treated microglia (Retamal et al., 2007) represents a target for antiinflammatory action of CBs. Such working hypothesis was recently addressed by using either (1) astrocytes/microglia co-cultures treated with LPS or pure cultures of astrocytes treated with TNF-α and IL-1β (Froger et al., 2009). In these studies, the glial actions of endogenous (Meth, methanandamide a non-hydrolyzable analogue of anandamide and 2-AG, 2-arachidonylglycerol) and synthetic (WIN and CP [CP 55,940]) agonists of CBs were investigated at two levels – the release of the proinflammatory cytokines from LPStreated microglia and the activity of Cx-based channels in astrocytes. Cannabinoids inhibit the LPS-induced TNF-α and IL-1β secretion from cultured microglia. Co-incubation with LPS and WIN, CP, Meth, or 2AG reduced the production of TNF-α and IL-1β by microglia (Froger et al., 2009). These observations are in agreement with a study performed in rat microglia showing that endogenous and synthetic agonists of CBs abolish the LPS-induced TNF-α release (Facchinetti et al., 2003). In addition, they complete a previous report indicating that treatment with Δ9-tetrahydrocannabinol, Meth, or CP inhibits the LPS-induced increase at mRNA levels of these two cytokines (Puffenbarger et al., 2000). Interestingly, the amounts of IL-1β and TNF-α measured under LPS treatment are in the same range to the lowest concentrations required to inhibit gap junctional communication in astrocytes, i.e., 2 and 20 ng/ml, respectively (Meme et al., 2006). Moreover, when LPS-activated microglia are co-treated with CB agonists, the cytokine levels become ineffective on astroglial GJCs, as demonstrated by the application of the harvested conditioned medium from microglia. These data demonstrate that in astrocyte-microglia co-cultures, the lack of effect of LPS on astroglial Cx43 gap junctions is in part due to the low level of IL-1β and TNF-α secretion that is maintained under threshold by CB stimulation. Cannabinoid inhibition of cytokine-induced Cx43 hemichannel activity in astrocytes is neuroprotective. To determine the effects of CBs on Cx43-based channel activity in astrocytes, two proinflammatory treatments have been applied – either conditioned medium harvested from LPSactivated microglia, or the mixture of IL-1β and TNF-α. In both situations, Meth, WIN, and CP were shown to partially prevent the inhibition of GJCs and the activation of hemichannels induced by the two treatments (Froger et al., 2009). These observations demonstrate that astrocytes are also a target for CBs. However, the pharmacological profile of Meth and synthetic CBs was distinct and their effects were additive, indicating that they likely act on different receptors. Inflammation induces Cx43 hemichannel activation in astrocytes which has been proposed to be involved in neuroglial interactions (Orellana et al., 2009). Their contribution to NMDA-induced excitotoxicity in neuron/astrocyte. 171. co-cultures was tested after treatment with TNF-α and IL-β. Interestingly, NMDA treatment induced a higher amount of neurotoxicity in cytokine-treated co-cultures than in untreated ones, whereas this extent of neurotoxicity was absent in neurons co-cultured with Cx43 knockout astrocytes or in the presence of Cx43 hemichannel blockers (Froger et al., 2010). Altogether, these observations lead to suggest that inflammation-induced astroglial hemichannel activation plays a critical role in neuronal death and point out a potential neuroprotective role of Cx43 hemichannel blockade. As discussed above, CBs prevent hemichannel activity in astrocytes; this rose the possibility that CB treatment could have a neuroprotective effect in excitotoxic situations. Recently, this was addressed by demonstrating that the synthetic CB WIN abolishes the increase in NMDA neurotoxicity produced by proinflammatory treatment (Froger et al., 2010). As indicated above, activated microglia release TNF-α and IL-1β, which trigger Cx43 hemichannel opening in astrocytes via activation of NOS and p38 MAP kinase pathways (Retamal et al., 2007). Importantly, these effects are inhibited by CBs, known to inhibit NO production (Ortega-Gutierrez et al., 2005), and the activation of p38 MAP kinase in astrocytes (Sheng et al., 2009). It is noteworthy that the inhibition of NO production is mediated by CB1 receptor, while action on p38 MAP kinase is preferentially driven by CB2 receptors. Accordingly, these properties could explain the CB1/CB2 pharmacology of synthetic and endogenous CBs in astrocytes.. Concluding remarks Glial cells are known to play a relevant role in neuronal survival (Kirchhoff et al., 2001). In pathological situations, molecules released via Cx- and Panx-based channels expressed by glial cells, contribute importantly to determine the neuronal fate (Nakase and Naus, 2004; Orellana et al., 2009). However, their involvement in neuroprotection and/or neurotoxicity is still a matter of debate (Nakase and Naus, 2004; Farahani et al., 2005), possibly because up-to-recently, the hemichannel function of Cxs and Panxs in glial cells was not fully taken into account in this context (but see Orellana et al., 2009). Reactive gliosis and brain inflammation are associated with most, if not all, brain injuries and pathologies. Hemichannel activation in astrocytes and microglia could play a crucial role in the reinforcement of the neuronal death, due to their capacity to release glutamate and ATP. Then, opening of neuronal Panx1 hemichannels could be triggered by the rise in [Ca2+]i via activation of NMDA and P2X receptors by glutamate and ATP, respectively. Panx1 hemichannels are likely to contribute to the intracellular Ca2+ overload that activates neurotoxic intracellular cascades during excitotoxicity (Szydlowska and Tymianski, 2010). Thus, the prevention of hemichannel activation under proinflammatory conditions may represent an unexplored strategy to prevent neuronal damage and death. Relevant to this point, a growing amount of evidence suggest that CBs may be neuroprotective in CNS inflammatory conditions. CBs act on the two main actors of reactive gliosis (i.e.,. 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(11) 172. J.A. Orellana et al.. microglia and astrocytes) and prevent the opposite regulation of Cx43-based channels in astrocytes. Accordingly, the determination of astroglial CB receptors subtypes that are activated differentially by synthetic and endogenous CBs may contribute to define therapeutic targets to prevent a deleterious function of Cx43, i.e., hemichannel activity, while maintaining the other, i.e., gap junctional communication, and, thus, counteract deleterious aspects induced by reactive gliosis in neuroglial interaction. Altogether, these observations strengthen the emerging concept that unregulated membrane permeability through enhanced hemichannel permeability may contribute to the development of CNS pathologies, and Cx as well as Panx1 hemichannels might represent potential and alternative targets for therapeutic intervention in neuroinflammatory diseases.. Acknowledgments This work was partially supported by CONICYT 79090028 (to Juan A. Orellana), CRPCEN (to Christian Giaume), INSERM (to Christian Giaume), FONDEF DO7I1086 (to Juan C. Sáez), FONDECYT 1090353 (to Rommy von Bernhardi), ANILLO ACT-71 (to Juan C. Sáez), and ECOS/CONICYT C10S01 (to Christian Giaume and Juan C. Sáez) grants.. References Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., et al. (2000). Inflammation and Alzheimer ’s disease. Neurobiol. Aging 21, 383–421. Al-Ubaidi, M.R., White, T.W., Ripps, H., Poras, I., Avner, P., Gomes, D., and Bruzzone, R. (2000). Functional properties, developmental regulation, and chromosomal localization of murine connexin36, a gap-junctional protein expressed preferentially in retina and brain. J. Neurosci. Res. 59, 813–826. Alarcón, R., Fuenzalida, C., Santibanez, M., and von Bernhardi, R. (2005). Expression of scavenger receptors in glial cells. 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