1. Introduction
In a healthy brain, microglia exhibit a resting surveillance state associated with active exploration of their environment for exogenous or endogenous signals representing a threat to the homeostasis [1-5]. When physiological balance is impaired in the central nervous system (CNS), resting phenotype of microglia shift to a reactive phenotype with different degrees of activation according to the nature of the stimuli and the context. During intense CNS inflammation, rather than show a repair-orientated activity profile, reactive microglia constitute a source of toxic factors and participate in the recruitment of non-resident brain cells involved in the innate immune response, which worsen brain damage. The brain performs exceptionally complex and dynamic tasks that depend on the coordinated interaction of glial cells, therefore it is conceivable that impairment of intercellular signaling and coordination among microglia could play an important role on several CNS disorders. In vertebrate cells, this synchronization is in part mediated by gap junctions [6-10]. They are aggregates of intercellular channels termed gap junction channels that allow direct, but selective, cytoplasmic continuity between contacting cells, promoting the exchange of ions (allowing eletrical coupling), metabolites (e.g., ADP, glucose, glutamate and glutathione) and second messengers (e.g., cAMP and IP3)[11-16]. Whereas a gap junction channel is formed by the serial docking of two hemichannels each one contributed by one of two adjacent cells, each hemichannel is composed by six protein subunits termed connexins (Fig. 1). The latter belong to a highly conserved protein family encoded by 21 genes in human and 20 in mouse with orthologs in other vertebrate species [17-19]. Connexins are abundantly expressed in cells of the CNS, and they are named after their predicted molecular mass expressed in kDa, so that connexin43 (Cx43) has a molecular mass of ~43 kDa.
For a long time the main function attributed to connexin hemichannels was the formation of gap junction channels. Nevertheless, in the last decade, the presence of functional connexin hemichannels in nonjunctional membranes has been demonstrated by several experimental approaches [20-24]. These channels serve like aqueous pores permeable to ions and small molecules that permit diffusional exchange between the intra and extracellular compartments, allowing cellular release of relevant quantities of autocrine/paracrine signaling molecules (e.g., ATP, glutamate, NAD+ and PGE2) to the extracellular milieu [25-30], as well as uptake of small molecules (e.g., glucose) [31]. One decade ago, a new gene family of gap junction proteins composed by three members was discovered in chordates [32, 33]. These proteins are the chordate homologs of innexins (the gap junction proteins of non chordates), and were denominated pannexins (panx1, 2 and 3) because apparently they are present in all eumetazoans except echinoderms [34] (Fig. 1). It has been suggested that gap junctional intercellular communication occur via Panx3 in osteoblasts [35], whereas other studies have shown that overexpression of exogenous Panx1 could form gap junctions
Current knowledge regarding brain hemichannels state that, under physiological conditions, they have a low activity, but enough to ensure the release of paracrine substances necessary for diverse functions of the CNS, including ischemic tolerance [39, 40], establishment of adhesive interactions [41]; fear memory consolidation [42], glucosensing [30], chemoreception [43], blood-brain barrier permeability [44], neuronal migration [45, 46] and metabolic autocrine regulation [47]. Nevertheless, under acute or chronic neurodegeneration dysregulation of hemichannel properties could be critical on the beginning and maintenance of homeostatic imbalances observed in diverse brain diseases [48-50]. Pioneering findings from Paul and colleagues showed that Xenopus oocytes transfected with Cx46 mRNA exhibited non-selective cation currents associated to depolarization and cell lysis within 24 h [51]. From then on, several studies supported the idea that dysregulated opening of hemichannels is incompatible with normal cell life. In the CNS, the first convincing evidence of hemichannel opening was provided by Contreras and colleagues, whose work showed that opening of Cx43 hemichannels accelerate astroglial cell death induced by ischemia-like conditions [52]. Such increased hemichannel activity induced by ischemia-like conditions has been observed in neurons [40, 53-55], oligodendrocytes [56], and also in brain cells subjected to other pro-inflammatory conditions [48]. Up to now, it is believed that sustained hemichannel opening contributes to increased intracellular Ca2+ concentration ([Ca2+]i), which in turn may favor even more the hemichannel activity (De Vuyst et al., 2007, Schalper et al., 2008), inducing Ca2+ and Na+ intracellular overload (Fig. 2).
Under these conditions, ionic (or electrolyte) imbalance leads to an osmotic imbalance that results in cell swelling and plasma membrane breakdown. Calcium overload induced in part by hemichannel opening may also activate phospholipase A2, with the subsequent generation of arachidonic acid and activation of cyclooxygenase/lipoxygenase pathways leading to increased free radicals, lipid peroxidation and further plasma membrane damage. Possibly, exacerbated or uncontrolled hemichannel opening could lead to cellular damage by several ways: 1) High increase of [Ca2+]i by Ca2+ entry through hemichannels, 2) cellular swelling by increased entry of Na2+ and Cl- through hemichannels, 3) release of metabolic products essential to cell viability as glucose, NAD+ or glutathione via hemichannels and 4) alternatively, spread of toxic molecules released by hemichannels (e.g., glutamate) could affect the viability of healthy neighboring cells.
Taking into account that hemichannels participate in the paracrine signaling among brain cells, the current chapter attempts to review and discuss the role of gap junction channels and hemichannels in microglia on normal and inflamed brain.
2. Gap junction channels in microglia
In a resting surveillance state, microglia express almost undetectable levels of Cx43 and Cx36 [59-65]. Nevertheless, when microglia are subjected to pro-inflammatory conditions, they exhibit expression of Cx43 and are able to form gap junction channels among them, as evaluated by dye-coupling experiments. In fact, Cx43 expression and gap junctional communication is induced in microglia by LPS, TNF-α plus IFN-γ [61], calcium ionophore plus PMA [66], or Staphylococcus aureus-derived peptidoglycan [64]. Despite the above, cultured human or mouse microglia treated with LPS, granulocyte-macrophage colony-stimulating factor, INF-γ or TNF-α do not exhibit modifications in connexin expression [60, 63]. Recently has been showed that resting microglia exhibit detectable levels of surface and total Cx43, whereas upon treatment with amyloid-β peptide (Aβ) a high increase in Cx43 expression is observed (Orellana 2011a). The discrepancy in the above mentioned studies may be related to different types of animal used to obtain brain tissue, dissimilar methods to take out cells and different culture conditions.
The ability to establish gap junctional communication among microglia, requires a rise in [Ca2+]i [66], while cAMP, cGMP or activation of PKC have been ruled out as possible inductors gap junction-mediated coupling [66]. In this regard, different degrees of microglial activation may trigger intracellular pathways that further result in a specific pattern of expression of gap juntion proteins. Communication via gap junctions may allow to activated microglia to recruit resting microglia at the site of injury, resulting in more damage or repair depending on the circumstances. Interestingly, microglia stimulated with cytokines or LPS exhibit reduced levels of Cx43 expression and gap junctional communication in astrocytes when both cell types are in co-culture or when conditioned media from activated microglia is used [31, 55, 59, 60, 67, 68]. Interestingly, gap junctions among dendritic cells ensure sharing of antigenic peptides [69-74], suggesting the possibility that these channels in microglia also could coordinate the CNS immune response. Importantly, recently it has been shown that the release of TNF-α and IL-1β by microglia depend on the activity of gap junction channels, because secretion of those cytokines was partially blocked by a gap junction blocker, α-glicirretinic acid [75]. Thus, it was proposed that gap junction channels play a key role into coordinate the microglial mediated inflammation.
3. Hemichannels in microglia
Up to now only few studies have documented the expression of functional hemichannels in microglia. Contrary to the expectations regarding as Cx43 the most possible protein to form hemichannels in microglia, TNF-α treatment was shown to induce release of glutamate through a pathway inhibited by a Cx32 (32Gap27), but not Cx43 (43Gap27) mimetic peptide [76]. Moreover, surface levels of Cx32 were increased in microglia treated with TNF-α. Noteworthy, the increased neuronal death associated with the release of glutamate was inhibited completely with the 32Gap27 mimetic peptide [76]. Later, the same group of authors proposed that glutamate released via Cx32 hemichannels play a key role in neuronal damage originated by brain ischemia [77] and experimental autoimmune encephalomyelitis [78]. 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 32Gap24, two Cx32 hemichannel mimetic peptides [79]. It is relevant to kept in mind that these and other mimetic peptides are homologous to extracellular domains of the respective connexin sequences, but their effects on hemichannel activity have not been documented, thereby some studies have questioned their specificity [80-82]. The use of cell cultures derived from connexin null mice and/or performing knockdown of the respective connexin, along the appropriate use of mimetic peptides could ensure the involvement of Cx32 hemichannels in these studies.
Almost two years ago, the opening of Cx43 and Panx1 hemichannels, evaluated by dye uptake and macroscopic cell membrane currents, were shown to be increased in microglia by Aβ25-35 exposure (Orellana et al. 2012a). 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) in the presence of external divalent cations, suggesting that opening of microglial hemichannels may occur in Alzheimer's disease (AD). Importantly, ATP and glutamate released from microglia treated with Aβ25-35 trigger hemichannel opening in neurons causing deleterious effects on them [83]. Supporting the idea of hemichannels as possible regulators in damage observed in AD, a novel putative hemichannel blocker (INI-0602) that crosses the blood brain barrier was recently shown to inhibit
4. Conclusions
Microglial cells are known to play a relevant role in neuronal survival [3]. In pathological situations, dysregulation of connexin- and pannexin-based channels expressed by microglia, contribute importantly to determine the neuronal fate [48, 50]. Microgliosis and brain inflammation are associated with most, if not all, brain injuries and pathologies. Hemichannel activation in microglia could play a crucial role in the reinforcement of the neuronal death, due to their capacity to release glutamate and ATP (Fig. 3) [55, 76, 83, 85]. Opening of Cx43, Cx32 and Panx1 hemichannels could increase [Ca2+]i in microglia, which further propagate Ca2+ waves via gap junction channels to neighbor cells (Fig. 3). Moreover, in distant microglia, Ca2+ waves can activate hemichannels, as demonstrated previously [57, 58, 86]. 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 [87] (Fig. 3). Thus, the prevention of hemichannel activation under pro-inflammatory conditions may represent an unexplored strategy to prevent neuronal damage and death. Altogether these observations strengthen the emerging concept that unregulated membrane permeability through enhanced hemichannel permeability and dysfunctional gap junction channels may contribute to the development of CNS pathologies and connexins as well as pannexins might represent potential and alternative targets for therapeutic intervention in neuroinflammatory diseases.
Acknowledgments
This work was partially supported by CONICYT 79090028 and FONDECYT 11121133 (to JAO) grants.References
- 1.
Kettenmann H et al Physiology of microglia. Physiol Rev,2011 461 553 - 2.
andBlock M. L J. S Hong Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog Neurobiol,2005 77 98 - 3.
Block M. L andL Zecca J. S Hong Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci,2007 57 69 - 4.
Hanisch U. K Microglia as a source and target of cytokines. Glia,2002 140 155 - 5.
Streit W. J Microglia as neuroprotective, immunocompetent cells of the CNS. Glia,2002 133 139 - 6.
Saez J. C et al Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev,2003 1359 1400 - 7.
andSohl G K Willecke Gap junctions and the connexin protein family. Cardiovasc Res,2004 228 232 - 8.
Laird D. W The gap junction proteome and its relationship to disease. Trends Cell Biol,2010 92 101 - 9.
andGoodenough D. A D. L Paul Gap junctions. Cold Spring Harb Perspect Biol,2009 a002576 - 10.
Evans W. H andE De Vuyst L Leybaert The gap junction cellular internet: connexin hemichannels enter the signalling limelight. Biochem J,2006 1 14 - 11.
Saez J. C et al cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture. Am J Physiol,1989 Pt 1):C1 11 - 12.
andConnors B. W M. A Long Electrical synapses in the mammalian brain. Annu Rev Neurosci,2004 393 418 - 13.
Goldberg G. S andP. D Lampe B. J Nicholson Selective transfer of endogenous metabolites through gap junctions composed of different connexins. Nat Cell Biol,1999 457 459 - 14.
Kam Y et al Transfer of second messengers through gap junction connexin 43 channels reconstituted in liposomes. Biochim Biophys Acta,1998 384 388 - 15.
Lawrence T. S andW. H Beers N. B Gilula Transmission of hormonal stimulation by cell-to-cell communication. Nature,1978 501 506 - 16.
Niessen H et al Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate. J Cell Sci,2000 Pt 8):1365 1372 - 17.
andCruciani V S. O Mikalsen The connexin gene family in mammals. Biol Chem,2005 325 332 - 18.
andAbascal F R Zardoya Evolutionary analyses of gap junction protein families. Biochim Biophys Acta,2012 - 19.
Willecke K et al Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem,2002 725 737 - 20.
andGoodenough D. A D. L Paul Beyond the gap: functions of unpaired connexon channels. Nat Rev Mol Cell Biol,2003 285 294 - 21.
Stout C andD. A Goodenough D. L Paul Connexins: functions without junctions. Curr Opin Cell Biol,2004 507 512 - 22.
Saez J. C et al Connexin-based gap junction hemichannels: gating mechanisms. Biochim Biophys Acta,2005 215 224 - 23.
Schalper K. A et al Currently used methods for identification and characterization of hemichannels. Cell Commun Adhes,2008 207 218 - 24.
Saez J. C et al Cell membrane permeabilization via connexin hemichannels in living and dying cells. Exp Cell Res,2010 2377 2389 - 25.
Bruzzone S et al Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells. FASEB J,2001 10 12 - 26.
Ye Z. C et al Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci,2003 3588 3596 - 27.
Cherian P. P et al Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin. Mol Biol Cell,2005 3100 3106 - 28.
Stout C. E et al Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem,2002 10482 10488 - 29.
Braet K et al Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells. J Cell Physiol,2003 205 213 - 30.
Orellana J. A et al Glucose increases intracellular free Ca(2+) in tanycytes via ATP released through connexin 43 hemichannels. Glia,2012 53 68 - 31.
Retamal M. A et al Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia. J Neurosci,2007 13781 13792 - 32.
Panchin Y et al A ubiquitous family of putative gap junction molecules. Curr Biol,2000 R473 R474 - 33.
Bruzzone R et al Pannexins, a family of gap junction proteins expressed in brain. Proc Natl Acad Sci U S A,2003 13644 13649 - 34.
andShestopalov V. I Y Panchin Pannexins and gap junction protein diversity. Cell Mol Life Sci,2008 376 394 - 35.
Ishikawa M et al Pannexin 3 functions as an ER Ca(2+) channel, hemichannel, and gap junction to promote osteoblast differentiation. J Cell Biol,2011 1257 1274 - 36.
Lai C. P et al Tumor-suppressive effects of pannexin 1 in C6 glioma cells. Cancer Res,2007 1545 1554 - 37.
Vanden Abeele F., et al.,Functional implications of calcium permeability of the channel formed by pannexin 1. J Cell Biol,2006 535 546 - 38.
MacVicar B.A. and R.J. Thompson,Non-junction functions of pannexin-1 channels. Trends Neurosci,2010 93 102 - 39.
Lin J. H et al A central role of connexin 43 in hypoxic preconditioning. J Neurosci,2008 681 695 - 40.
Schock S. C et al ATP release by way of connexin 36 hemichannels mediates ischemic tolerance in vitro. Biochem Biophys Res Commun,2008 138 144 - 41.
Cotrina M. L andJ. H Lin M Nedergaard Adhesive properties of connexin hemichannels. Glia,2008 1791 1798 - 42.
Stehberg J et al Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala. FASEB J,2012 - 43.
Wenker I. C et al Regulation of ventral surface CO2/H+-sensitive neurons by purinergic signalling. J Physiol,2012 Pt 9):2137 2150 - 44.
De Bock M et al Connexin channels provide a target to manipulate brain endothelial calcium dynamics and blood-brain barrier permeability. J Cereb Blood Flow Metab,2011 1942 1957 - 45.
Liu X et al Gap junctions/hemichannels modulate interkinetic nuclear migration in the forebrain precursors. J Neurosci,2010 4197 4209 - 46.
Liu X et al Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex. Proc Natl Acad Sci U S A,2012 8280 8285 - 47.
Masino,Kawamura M Jr D. N Ruskin andS. A Metabolic autocrine regulation of neurons involves cooperation among pannexin hemichannels, adenosine receptors, and KATP channels. J Neurosci,2010 3886 3895 - 48.
Orellana J. A et al Modulation of brain hemichannels and gap junction channels by pro-inflammatory agents and their possible role in neurodegeneration. Antioxid Redox Signal,2009 369 399 - 49.
Orellana J. A et al Hemichannels in the neurovascular unit and white matter under normal and inflamed conditions. CNS Neurol Disord Drug Targets,2011 404 414 - 50.
Orellana J. A et al Glial hemichannels and their involvement in aging and neurodegenerative diseases. Rev Neurosci,2012 163 177 - 51.
Paul D. L et al Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol,1991 1077 1089 - 52.
Contreras J. E et al Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proc Natl Acad Sci U S A,2002 495 500 - 53.
and B.A. MacVicar,Thompson R. J N Zhou Ischemia opens neuronal gap junction hemichannels. Science,2006 924 927 - 54.
Thompson R. J et al Activation of pannexin-1 hemichannels augments aberrant bursting in the hippocampus. Science,2008 1555 1559 - 55.
Orellana J. A et al ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels. J Neurochem,2011 826 840 - 56.
Domercq M et al 2X7 receptorsmediate ischemic damage to oligodendrocytes . Glia,2010 p. 730-40. - 57.
Schalper K. A et al Connexin 43 hemichannels mediate the Ca2+ influx induced by extracellular alkalinization. Am J Physiol Cell Physiol,2010 C1504 C1515 - 58.
De Vuyst E et al Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor. Mol Biol Cell,2007 34 46 - 59.
Rouach N et al Brain macrophages inhibit gap junctional communication and downregulate connexin 43 expression in cultured astrocytes. Eur J Neurosci,2002 403 407 - 60.
Meme W et al Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by beta-amyloid. FASEB J,2006 494 496 - 61.
Eugenin E. A et al Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-gamma and tumor necrosis factor-alpha. Proc Natl Acad Sci U S A,2001 4190 4195 - 62.
Parenti R et al Immunocytochemical and RT-PCR analysis of connexin36 in cultures of mammalian glial cells. Arch Ital Biol,2002 101 108 - 63.
Dobrenis K et al Human and mouse microglia express connexin36, and functional gap junctions are formed between rodent microglia and neurons. J Neurosci Res,2005 306 315 - 64.
Syed, and T. Kielian,Garg S M Md Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia. J Neurochem,2005 475 483 - 65.
Lee I. H et al Glial and neuronal connexin expression patterns in the rat spinal cord during development and following injury. J Comp Neurol,2005 1 10 - 66.
Martinez A. D et al Identification of second messengers that induce expression of functional gap junctions in microglia cultured from newborn rats. Brain Res,2002 191 201 - 67.
Faustmann P. M et al Microglia activation influences dye coupling and Cx43 expression of the astrocytic network. Glia,2003 101 108 - 68.
Hinkerohe D et al Effects of cytokines on microglial phenotypes and astroglial coupling in an inflammatory coculture model. Glia,2005 85 97 - 69.
Neijssen J et al Cross-presentation by intercellular peptide transfer through gap junctions. Nature,2005 83 88 - 70.
Matsue H et al Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs. J Immunol,2006 181 190 - 71.
Corvalan L. A et al Injury of skeletal muscle and specific cytokines induce the expression of gap junction channels in mouse dendritic cells. J Cell Physiol,2007 649 660 - 72.
Handel A et al Gap junction-mediated antigen transport in immune responses. Trends Immunol,2007 463 466 - 73.
Mendoza-naranjo A et al Functional gap junctions facilitate melanoma antigen ransfer and cross-presentation between human dendritic cells. J Immunol,2007 6949 6957 - 74.
Pang B et al Direct antigen presentation and gap junction mediated cross-presentation during apoptosis. J Immunol,2009 1083 1090 - 75.
Eugenin E. A et al The Role of Gap Junction Channels During Physiologic and Pathologic Conditions of the Human Central Nervous System. J Neuroimmune Pharmacol,2012 - 76.
Takeuchi H et al Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner. J Biol Chem,2006 21362 21368 - 77.
Takeuchi H et al Blockade of microglial glutamate release protects against ischemic brain injury. Exp Neurol,2008 144 146 - 78.
Shijie J et al Blockade of glutamate release from microglia attenuates experimental autoimmune encephalomyelitis in mice. Tohoku J Exp Med,2009 87 92 - 79.
Maezawa I et al Rett syndrome astrocytes are abnormal and spread MeCP2 deficiency through gap junctions. J Neurosci,2009 5051 5061 - 80.
Wang J et al Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters. Am J Physiol Cell Physiol,2007 C1112 C1119 - 81.
Dahl G Gap junction-mimetic peptides do work, but in unexpected ways. Cell Commun Adhes,2007 259 264 - 82.
andEvans W. H L Leybaert Mimetic peptides as blockers of connexin channel-facilitated intercellular communication. Cell Commun Adhes,2007 265 273 - 83.
Orellana J. A et al Amyloid beta-induced death in neurons involves glial and neuronal hemichannels. J Neurosci,2011 4962 4977 - 84.
Takeuchi H et al Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer’s disease. PLoS One,2011 e21108 - 85.
Kang J et al Connexin 43 hemichannels are permeable to ATP. J Neurosci,2008 4702 4711 - 86.
Sanchez H. A et al Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells. Am J Physiol Cell Physiol,2009 C665 C678 - 87.
andSzydlowska K M Tymianski Calcium, ischemia and excitotoxicity. Cell Calcium,2010 122 129