Advanced Review
P2X receptors in neuroglia Alexei Verkhratsky,1,2,3∗ Yuri Pankratov,4 Ulyana Lalo1 and Maiken Nedergaard5 Different types of ionotropic P2X purinoceptors are expressed in all major types of neuroglia, where they mediate a variety of physiological and pathological signaling. Cortical astrocytes express specific P2X1/5 heteromeric receptors that are activated by ongoing synaptic transmission and can trigger fast local signaling through elevation in cytoplasmic Ca2+ and Na+ concentrations. Oligodendrocytes express several types of P2X receptors that may control their development and mediate axonal–glial interactions. In microglia, P2X4 and P2X7 receptors regulate numerous events associated with microglial activation, motility, and release of proinflammatory factors. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. How to cite this article:
WIREs Membr Transp Signal 2012, 1:151–161. doi: 10.1002/wmts.12
INTRODUCTION: ATP AS A NEUROTRANSMITTER
A
TP, its derivatives, and adenosine are ubiquitous and ancient intercellular messengers that mediate cell-to-cell signaling in variety of tissues.1–3 In the central nervous system (CNS), ATP acts as homocellular (neuronal–neuronal and glial–glial) and heterocellular (neuronal–glial and glial–neuronal) neurotransmitter.4,5 ATP is physiologically released from many cellular elements of the CNS including neuronal terminals, axons, and astroglia. ATP release pathways include concentration-gradient driven diffusion through plasmalemmal channels with large permeability (as cytosolic concentration of ATP approaches 5–10 mM and extracellular is set at a low nanomolar range, the resulting concentration gradient is arguably one of the highest existing in biological systems); release through ATP-binding cassette transporters and secretion by exocytosis.4,6,7 The diffusion of ATP can occur through several sets of ∗
Correspondence to: [email protected]
1
Faculty of Medical and Human Sciences, The University of Manchester, Manchester, UK 2 IKERBASQUE,
Basque Foundation for Science, Bilbao, Spain
3 Department
of Neurosciences, University of the Basque Country UPV/EHU, Leioa, Spain
4 School
of Life Sciences, University of Warwick, Coventry, UK
5 Division
of Glial Disease and Therapeutics, Center for Translational Neuromedicine, Department of Neurosurgery, University of Rochester Medical Center, Rochester, NY, USA
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plasmalemmal channels such as unpaired connexins/ hemichannels8 or volume-sensitive anion channels.9 The latter mechanism was demonstrated to mediate physiological release of ATP associated with axonal firing9 ; although physiological relevance of diffusional ATP release in other CNS structures remains unclear. The most widespread pathway for ATP release in the context of signaling in neural networks is represented by Ca2+ -regulated exocytosis, which was identified in neuronal terminals6,7 and in astrocytes.10–12 Massive release of ATP also accompanies cellular death and destruction; hence ATP acts as a universal ‘damage’ signal and is involved in wide variety of pathological reactions in the nervous system.
P2X RECEPTORS IN ASTROGLIA Functional properties of P2X receptors (e.g., their agonist sensitivity, desensitization, or Ca2+ permeability) are determined by the subunit composition (Table 1). The P2X receptors can be formed through homo- or heteromeric assembly of P2X1 to P2X7 subunits (so far homomeric composition was shown for P2X1–5,7 subunits, whereas P2X6 subunits apparently cannot oligomerize); heteromeric compositions are represented by P2X1/2 , P2X1/4 , P2X1/5 , P2X2/3 , P2X2/6 , and P2X4/6 channels.13,14 On a transcriptional level, all seven P2X subunits were identified in astrocytes in vitro and in situ.15 Specific mRNA for P2X1–5 and P2X7 receptors was detected in primary cultured rat cortical
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TABLE 1 General Pharmacology of P2X Receptors Receptor P2X1
Kd/EC50
Preferred Substrates
References
Kd(ATP) = 2–3 nM
No specific agonists for P2X1 only
102
EC50 (ATP) = 0.07 μM
BzATP > 2meSATP > ATP > αβmeATP
103
BzATP > ATP = 2meSATP αβmeATP
103
2meSATP ATP > αβmeATP
104
BzATP > 2meSATP > ATP > αβmeATP
103
BzATP > ATP = 2meSATP αβmeATP
103
EC50 (BzATP) = 0.003 μM P2X2
EC50 (ATP) = 1.2 μM EC50 (BzATP) = 0.75 μM
P2X3 EC50 (ATP) = 0.5 μM EC50 (BzATP) = 0.08 μM P2X4
EC50 (ATP) = 10 μM
P2X5
EC50 (ATP) = 10 μM
ATP = 2meSATP αβmeATP > BzATP
103
P2X6 (hetero)
EC50 (ATP) = 12 μM
2meSATP ≥ ATP αβmeATP
103
BzATP > ATP = 2meSATP αβmeATP
103
EC50 (BzATP) = 7 μM
EC50 (2meSATP) = 9 μM P2X7
EC50 (ATP) = 100 μM EC50 (BzATP) = 20 μM
astrocytes.16,17 In tissue extracts from rat nucleus accumbens, expression of mRNA for all seven P2X subunits was revealed RT-PCR.18 Specific mRNA for P2X3 , P2X4 , and P2X5 subunits was identified in ¨ freshly isolated rat retinal Muller cells,19 the P2X7 receptor-specific mRNA was identified in human ¨ Muller glia.20 In acutely isolated mouse cortical astrocytes, only P2X1 - and P2X5 -specific mRNAs were found.21 Expression of P2X subunits in astroglia was also characterized by immunohistochemistry. In nucleus accumbens, immunofluorescence revealed P2X2–4 receptors co-localized with GFAP-labeled astroglial profiles.18 Mechanical lesion of the nucleus accumbens resulted in upregulation of P2X1–4 and P2X7 immunofluorescence in astrocytes.18 Immunoreactivity for P2X1 and P2X2 receptors was detected in astroglial cells in the cerebellum,22,23 P2X2 receptors were visualized in spinal cord astrocytes,24 and P2X4 receptors were identified in the processes and somatas of the astrocytes from the brainstem.25 In hippocampal astrocytes, immunostaining revealed expression of P2X1–4 , P2X6 , and P2X7 subunits.26 At a functional level, however, P2X receptorsmediated responses are not ubiquitous property of astroglia. ATP triggered membrane currents, depolarization, and Ca2+ signaling in cultured astrocytes,27,28 although the subunit composition of underlying receptors was not investigated. In hippocampal astrocytes in slices or after acute isolation, no P2X-mediated currents were identified.29 Similarly, ATP-induced currents were not observed in Bergmann glial cells 152
in acute cerebellar slices.30 Nonetheless, absence of ATP-induced currents in the in situ experiments mentioned above cannot be considered conclusive, because complex geometry of glial cells, diffusional barriers, and rapid degradation of ATP in slice tissue may hinder the detection of membrane responses.
P2X1/5 Receptors in Cortical Astrocytes Astrocytes in cortex express functional P2X1/5 heteromeric receptors, which upon activation produce idiosyncratic membrane responses (Figure 1 and Ref 21). Astroglial P2X1/5 receptor is extraordinary sensitive to ATP (KD ∼ 50 nM), has distinct pharmacology (complete inhibition by 30 μM PPADS and almost complete inhibition by 1 μM TNP-ATP) and biophysical properties (absence of desensitization and activation of ‘rebound’ tail current in response to the washout of the agonist). The P2X1/5 receptors contribute to fast ion currents triggered in astrocytes in response to stimulation of neuronal afferents in cortical slices.31,32 The same receptors are involved in generation of spontaneous ‘miniature’ postsynaptic currents measured from astrocytes in cortical slices.31,33 Astroglial P2X1/5 receptors have intermediate Ca2+ permeability (PCa /Pmonovalent ∼ 2.2) and their activation by exogenous agonists as well as by synaptically released ATP triggers astroglial Ca2+ signals (Figure 2 and Ref 32). Expression of P2X1/5 receptors in astrocytes is age-dependent,33 being maximal in 3–6 months old mice and substantially declining with an advanced age.
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P2X receptors in neuroglia
(a)
(b)
(c)
FIGURE 1 | P2X1/5 receptor-mediated currents in acutely isolated cortical astrocytes. (a) The family of ATP currents evoked by repetitive applications of the agonist. The currents show no apparent desensitization. Current traces have a complex kinetics comprising the peak, the steady-state component, and the ‘rebound’ inward current recorded upon ATP washout as indicated on the graph. (b) Concentration dependence of ATP-induced currents in cortical astrocytes. Membrane currents recorded from a single cell in response to different ATP concentrations are shown on the left. The right panel shows the concentration response curves constructed from nine similar experiments; current amplitudes were measured at the initial peak and at the end of the current, as indicated on the graph. (c) Inhibition of ATP-induced currents by 2 ,3 -O -(2,4,6-trinitrophenyl) adenosine 5 -triphosphate (TNP-ATP). The graph shows ATP-induced currents measured from the cortical astrocyte in control conditions, in the presence of different concentrations of TNP-ATP and after the washout. Application of TNP-ATP started 2 min before the application of ATP. All recordings were made at holding potential of −80 mV. (Reprinted with permission from Ref 21. Copyright 2008 Society for Neuroscience)
Astroglial P2X7 Receptors The role and functional expression of the cytolitic, pore-forming P2X7 receptors14,34 in astroglia remains controversial. Both P2X7 -specific mRNA and P2X7 Volume 1, March/April 2012
receptor protein have been detected in cultured astroglia (reviewed in Refs 15 and 35). Similarly, stimulation of P2X7 receptors in cultured astrocytes often triggered [Ca2+ ]i transients, mimicked by
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(a)
(b)
FIGURE 2 | P2X1/5 receptor-mediated Ca2+ -signaling in cortical astrocytes. (a) Acutely isolated astrocytes were loaded with Fluo-4 via a patch
pipette. Fluorescent images were recorded simultaneously with transmembrane currents evoked by application of 100 μM ATP or 10 μM α,β-methyleneATP. [Ca2+ ]i -transients are represented as the F /F0 ratio averaged over the cell soma (green traces). Holding potential was −80 mV. (b) Synaptically induced ionotropic Ca2+ signals in protoplasmic cortical astrocytes in situ. Cortical layer II astrocytes were loaded with Fura-2 in situ via patch pipette. Fluorescent images were recorded simultaneously with glial currents evoked by neuronal afferent stimulation in the presence of CNQX (control) and after application of 10 nM of NF-449, selective antagonist of P2X receptors. [Ca2+ ]i -transients (green traces) are expressed as F340 /F380 ratio. (Reprinted with permission from Ref 32. Copyright 2010 Elsevier)
P2X7 receptor agonist 2 ,3 -O-(benzoyl-4-benzoyl)ATP (BzATP) and inhibited by antagonist oxidized ATP (oxATP).17,36,37 Characteristic P2X7 receptor currents activated by 1–5 mM ATP or by 0.1 mM Bz-ATP were recorded from cultured astrocytes.38,39 Likewise, P2X7 receptor-mediated currents were ¨ identified in freshly isolated human retinal Muller glial cells.20 It has to be remembered, however, that Bz-ATP can activate other P2X receptors13 and can be degraded to Bz-adenosine, which stimulates P1 receptors.40 Similarly, the oxATP also inhibits P2X1 and P2X2 receptors.13 Stimulation of P2X7 receptors in astrocytes in vitro affects numerous biochemical process, 154
for example, synthesis of endocannabinoid 2arachidonoylglycerol, stimulation of production of NO and of lipid mediators of inflammation cysteinyl leukotrienes, regulation of NF-κB signaling, release of gliotransmitters and TNF-α, etc.41–45 Furthermore, stimulation of P2X7 receptors in cultured astrocytes induces secretion of various transmitter molecules (‘glio’ transmitters) including glutamate, ATP, and GABA.38,46,47 Recently, the P2X7 currents were found in cortical astrocytes in situ, in acutely isolated slices,48 and P2X7 receptor-mediated Ca2+ signals were identified in astrocytes from acutely isolated optic nerve.49 The role of P2X7 receptors in astroglial physiology remains unknown, as indeed activation of
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the receptor requires very high (in excess of 1 mM) ATP concentrations. It was, however, suggested that low ATP concentrations may activate P2X7 receptor without opening the channel pore; this ‘low-level’ stimulation can be relevant for regulation of a variety of trophic/metabolic processes.50 At the same time, astroglial expression of P2X7 receptors is usually increased in neuropathology (see Ref 15 for review), which may indicate their role in regulation of pathologically relevant astroglial reactions.
P2X RECEPTORS IN OLIGODENDROGLIA Expression of P2X1,2,3,4,7 proteins was found in oligodendroglial precursor cells (OPCs) in purified postnatal cultures,51,52 although P2X-mediated ion currents were not detected in cultured OPCs and mature oligodendrocytes.53 In the isolated optic nerve, application of a broad agonist of P2X receptors α,β-methylene ATP induced relatively small [Ca2+ ]i elevation, thus suggesting possible involvement of P2X receptors.54 In oligodendrocytes in situ in corpus callosum slices, ATP failed to activate transmembrane currents.53 Some evidence indicating expression of functional P2X7 receptors in cells of oligodendroglial lineage was gathered recently. In cultured OPCs, application of Bz-ATP triggered large Ca2+ transients, which were effectively inhibited by oxATP thereby suggesting Ca2+ entry though P2X7 receptors.51 Immunoreactivity for P2X7 receptors was detected in oligodendrocytes from the optic nerve and the spinal cord.55,56 In cultured oligodendrocytes from optic nerve, high concentrations of ATP (EC50 ∼ 8.8 mM) and much lower concentrations of BzATP (EC50 ∼ 0.5 mM) triggered sustained inward currents. These currents were potentiated in divalentcations-free extracellular solutions and were inhibited by oxATP. In addition, high concentrations of ATP and BzATP induced a rapid increase in [Ca2+ ]i , which was almost exclusively dependent on transmembrane Ca2+ entry.56 Finally, over-stimulation of P2X7 receptors (with 1 mM ATP or BzATP for 15 min) induced significant oligodendroglial death in culture and in situ in the optic nerve.56 The P2X7 -mediated death of oligodendrocytes may be relevant for variety of demyelinating diseases including multiple sclerosis (MS). Treatment of animals suffering from experimental autoimmune encephalitis (EAE, which is generally considered as a model for MS), with P2X7 antagonists oxATP or brilliant blue G, inhibited demyelination and restored axon conduction velocity.55,56 Furthermore, Volume 1, March/April 2012
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expression of P2X7 receptors increased in white matter of MS patients.56
NG2 GLIA Very little is known about expression of P2X receptors in the NG2-glia, which was recently identified as a separate class of neuroglia. The NG2 glial cells (identifiable by the expression of NG2 chondroitin sulfate proteoglycan57 ) are present throughout the developing and adult brain and may serve as pluripotent stem cells, OPCs or being a specific elements of neural circuitry.58,59 Recently, it was reported that ATP released from axons and astrocytes in electrically stimulated optic nerve triggers [Ca2+ ]i transients in NG2-glia, which at least in part can be mediated by P2X7 receptors.60
P2X RECEPTORS CONTROL ACTIVATION OF MICROGLIA Purinergic signaling system is particularly developed in microglia. Numerous experiments in vitro and in vivo have demonstrated that ATP and its analogues trigger rapid functional responses of microglial cells, which include induction of microglial motility (both of the processes and the soma), the outgrowth of microglial processes, membrane raffling, and the release of cytokines and inflammatory proteins (see Ref 61 for comprehensive review). Effects of purines/pyrimidines on microglia are mediated through all types of purinoceptors (P1 and P2) abundantly expressed in microglial cells throughout the nervous system.61 Expression of P2X receptor subunits in microglia is developmentally regulated. At embryonic day 16, majority of microglial cells expressed mRNA specific for P2X1 and P2X4 subunits, whereas only 30% of these cells expressed P2X7 receptor transcripts. From postnatal day 7, the P2X4 -expressing microglia concentrated around blood vessels. Finally, at postnatal day 30, microglial cells expressed only P2X7 and P2X4 mRNAs; the P2X7 -positive cells were distributed evenly through the forebrain, whereas cells bearing P2X4 receptors outlined blood vessels and subarachnoid space.62 Expression of different types of purinoceptors in cultured microglia undergoes substantial remodeling while progressing through activation.63 Likewise, expression of purinoceptors changes in various neurological diseases. For example, activation of hippocampal microglia following epileptic seizures (triggered by injections of kainate) resulted in the upregulation of the expression of mRNA specific for
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P2X1,4,7 and P2Y6,12,13 receptors. This upregulation led to an increase in ATP-induced membrane currents and ATP-induced microglial motility.64 The upregulation of microglial P2X4 , P2X7 , and P2Y6 receptors were also observed in the experimental model of amyotrophic lateral sclerosis, in mice expressing mutant superoxide dismutase 1.65 The P2X-mediated currents were identified in microglial cultures prepared from several tissues including mouse embryos, newborn rat, and human brains.66–68 In all these preparations, application of 100 μM ATP triggered desensitizing cationic currents and induced [Ca2+ ]i elevation produced by Ca2+ entry through ionotropic purinoceptors. Similar ATP-induced cationic currents were also detected in microglial cells in acute slices from an adult mouse brain.69
P2X4 Receptors in Pathologically Remodeled Spinal Cord Microglia Peripheral nerve injury triggers rapid and substantial activation of microglia in the spinal cord, this process being fundamentally important in pathogenesis of neuropathic pain.70–73 Development of tactile symptoms associated with neuropathic pain (e.g., tactile allodynia) in the rat spinal nerve injury model could be reversed by inhibition of P2X4 receptors, suggesting their role in neuropathic pain.74 This initial observation for further corroborated when it was shown that intraspinal injection of P2X4 antisense oligodeoxynucleotide inhibited P2X4 receptors expression and reduced tactile allodynia following nerve injury. The intrathecal injection of activated cultured microglia expressing P2X4 receptors triggered allodynia without peripheral nerve lesion.74 The lesion to the peripheral nerve resulted in a significant upregulation of P2X4 receptor expression in spinal cord microglia74 ; the P2X4 protein levels began to increase 1 day after surgery and reached maximal levels after 14 days; this time course matched the development of tactile allodynia. Similarly P2X4 expression was upregulated following formalin injection into the spinal cord (a common inflammatory pain model) or intraperitoneal injection of LPS75,76 or mechanical lesion to the spinal cord.77 An increase in P2X4 expression was also observed in rats with experimental autoimmune neuritis.78
P2X7 Receptors in Microglia Functional P2X7 receptors were identified in microglial cells in situ, in amoeboid microglia collected from the surface of corpus callosum slices,79 and in vitro in freshly isolated mouse microglia.80 In 156
cultured microglia, ATP (in millimolar concentrations) and Bz-ATP evoked Ca2+ influx and uptake of ethidium bromide or Lucifer Yellow both being indicative of the formation of P2X7 receptor associated pore. These effects were blocked by oxATP.80,81 In the amoeboid microglia from corpus callosum slices of 5–7 days old mice, 1 mM ATP induced classical P2X7 mediated currents (Figure 3). Sensitivity of receptor was greatly enhanced following removal of extracellular divalent cations.79 The P2X7 receptor-mediated currents were also detected in ramified microglia in acute slices from adult (6–8 weeks old) mice.69 The P2X7 receptor seem to be constitutively expressed in resting microglia; in the healthy brain P2X7 -positive microglial cells are homogeneously distributed throughout all brain regions.82 Different forms of insults to the brain trigger rapid and substantial increase in the expression of microglial P2X7 receptors.35,83,84 Expression of P2X7 receptors is upregulated after acute ischemic and traumatic attacks85,86 as well as chronic neuropathologies such as MS87 and Alzheimer’s disease.88,89 A significant increase in expression of P2X7 -specific mRNA and in P2X7 immunoreactivity was also detected in reactive microglial cells surrounding senile plaques in AD transgenic model.90 Microglial expression of P2X7 receptors was also increased following exposure of cultured microglia to β-amyloid or after intrahippocampal injection of the latter.88 An increase in expression of P2X7 receptors was also reported in microglia from rat model of epilepsy91 and from brains of scrapie-infected mice.92 Pharmacological manipulations with P2X7 receptors were neuroprotective in ischemia86 ; it also reduced microglial expression and release of proinflammatory factors.93 Activation of P2X7 receptors regulates various functions associated with microglial activation. The P2X7 receptors, for example, were obligatory for triggering activation of microglia and inetrleukin-1 production in response to an intrahippocampal injection of Aβ protein; both activation and IL-1 accumulation were absent in P2X7 knockout mice.94 Overexpression of P2X7 receptors in cultured microglia on its own right triggered microglial activation that could be blocked by oxATP.95–100 This microglial activation required formation of P2X7 receptor-associated pore; when microglial cells were transfected with a point mutant P2X7 RG345Y that does not allow pore formation, microglial activation was suppressed.95 Microglial P2X7 receptors also control release of multiple proinflammatory factors. Activation of P2X7 initiates release of cytokines IL-1α and IL-1β 96,97 that may involve plasmalemmal vesicle formation and shedding.98 The P2X7 receptors also control
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(a)
(c)
(e)
microglial synthesis and release of CC-chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1α 99 ; release of chemokine CXCL2,100 plasminogen,101 and many other proinflammatory factors (see Ref 61 for comprehensive review).
CONCLUSIONS P2X receptors are expressed in all types of neuroglial cells. The full regional mapping of glial presence of P2X receptors is far from being complete;
P2X receptors in neuroglia
(b)
(d)
FIGURE 3 | ATP triggers P2X7 receptor-mediated currents in amoeboid microglial cells collected from the surface of corpus callosum slices of young (P5–P7) mice. (a–d) Method of patch-voltage clamping of single amoeboid microglial cells from the surface of corpus callosum slice. Combinations of images taken by means of infrared video microscopy (left panels) and schematic drawings showing the method of cell patch-clamping. Video images were captured with an intensified CCD camera (Hamamatsu, Japan) and digitized by a frame grabber connected to the PC. (a) Initial position of amoeboid microglial cells situated on the surface of a corpus callosum slice. (b) A single microglial cell was approached with a micropipette and a whole-cell patch clamp configuration was established. (c) After 2–3 min, the cell partially spread over the pipette, intensifying the cell-to-pipette contact. (d) Finally, the cell was lifted for 200 μm over the slice surface. Scale bar in (d) = 10 μm. (e) P2X7 -mediated membrane currents measured from amoeboid microglial cells in control conditions and Ca2+ /Mg2+ -free bath solution. (Reprinted with permission from Ref 79. Copyright 1996 Elsevier)
however, we already may suggest their roles in various types of signaling in neuronal–glial circuits. In cortical astroglia, P2X1/5 receptors participate in rapid synaptic-associated signaling producing local [Na+ ]i and [Ca2+ ]i transients in glial perisynaptic processes. In oligodendroglia, activation of P2X7 receptors is somehow involved in pathogenesis of demyelinating diseases. In microglia, P2X4 and P2X7 receptors control various aspects of microglial activation and secretion of proinflammatory factors.
ACKNOWLEDGMENTS This research was supported by Alzheimer’s Research Trust (UK) Programme Grant (ART/PG2004A/1) to A.V.
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27. Magoski NS, Walz W. Ionic dependence of a P2purinoceptor mediated depolarization of cultured astrocytes. J Neurosci Res 1992, 32:530–538. 28. Walz W, Gimpl G, Ohlemeyer C, Kettenmann H. Extracellular ATP-induced currents in astrocytes: involvement of a cation channel. J Neurosci Res 1994, 38:12–18. 29. Jabs R, Matthias K, Grote A, Grauer M, Seifert G, Steinhauser C. Lack of P2X receptor mediated currents in astrocytes and GluR type glial cells of the hippocampal CA1 region. Glia 2007, 55:1648–1655. 30. Kirischuk S, Moller T, Voitenko N, Kettenmann H, Verkhratsky A. ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells. J Neurosci 1995, 15:7861–7871. 31. Lalo U, Pankratov Y, Parpura V, Verkhratsky A. Ionotropic receptors in neuronal-astroglial signalling: what is the role of ‘‘excitable’’ molecules in nonexcitable cells. Biochim Biophys Acta 2011, 1813: 992–1002. 32. Palygin O, Lalo U, Verkhratsky A, Pankratov Y. Ionotropic NMDA and P2X1/5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes. Cell Calcium 2010, 48:225–231. 33. Lalo U, Palygin O, North RA, Verkhratsky A, Pankratov Y. Age-dependent remodelling of ionotropic signalling in cortical astroglia. Aging Cell 2011, 10: 392–402. 34. Surprenant A, Rassendren F, Kawashima E, North RA, Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7 ). Science 1996, 272:735–738. 35. Sperlagh B, Vizi ES, Wirkner K, Illes P. P2X7 receptors in the nervous system. Prog Neurobiol 2006, 78: 327–346.
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36. Ballerini P, Rathbone MP, Di Iorio P, Renzetti A, Giuliani P, D’Alimonte I, Trubiani O, Caciagli F, Ciccarelli R. Rat astroglial P2Z (P2X7 ) receptors regulate intracellular calcium and purine release. Neuroreport 1996, 7:2533–2537. 37. Nobile M, Monaldi I, Alloisio S, Cugnoli C, Ferroni S. ATP-induced, sustained calcium signalling in cultured rat cortical astrocytes: evidence for a non-capacitative, P2X7-like-mediated calcium entry. FEBS Lett 2003, 538:71–76. 38. Duan S, Anderson CM, Keung EC, Chen Y, Swanson RA. P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J Neurosci 2003, 23: 1320–1328. 39. Norenberg W, Schunk J, Fischer W, Sobottka H, Riedel T, Oliveira JF, Franke H, Illes P. Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia. Br J Pharmacol 2010, 160: 1941–1952. 40. Kukley M, Stausberg P, Adelmann G, Chessell IP, Dietrich D. Ecto-nucleotidases and nucleoside transporters mediate activation of adenosine receptors on hippocampal mossy fibers by P2X7 receptor agonist 2 -3 -O-(4-benzoylbenzoyl)-ATP. J Neurosci 2004, 24:7128–7139. 41. Kucher BM, Neary JT. Bi-functional effects of ATP/P2 receptor activation on tumor necrosis factor-alpha release in lipopolysaccharide-stimulated astrocytes. J Neurochem 2005, 92:525–535. 42. Walter L, Dinh T, Stella N. ATP induces a rapid and pronounced increase in 2-arachidonoylglycerol production by astrocytes, a response limited by monoacylglycerol lipase. J Neurosci 2004, 24:8068–8074. 43. Duan S, Neary JT. P2X7 receptors: properties and relevance to CNS function. Glia 2006, 54:738–746. 44. Ballerini P, Ciccarelli R, Caciagli F, Rathbone MP, Werstiuk ES, Traversa U, Buccella S, Giuliani P, Jang S, Nargi E, Visini D, et al. P2X7 receptor activation in rat brain cultured astrocytes increases the biosynthetic release of cysteinyl leukotrienes. Int J Immunopathol Pharmacol 2005, 18:417–430. 45. Gendron FP, Neary JT, Theiss PM, Sun GY, Gonzalez FA, Weisman GA. Mechanisms of P2X7 receptormediated ERK1/2 phosphorylation in human astrocytoma cells. Am J Physiol Cell Physiol 2003, 284:C571–C581. 46. Suadicani SO, Brosnan CF, Scemes E. P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling. J Neurosci 2006, 26: 1378–1385. 47. Fellin T, Pozzan T, Carmignoto G. Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes. J Biol Chem 2006, 281: 4274–4284. 48. Oliveira JF, Riedel T, Leichsenring A, Heine C, Franke H, Krugel U, Norenberg W, Illes P. Rodent
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cortical astroglia express in situ functional P2X7 receptors sensing pathologically high ATP concentrations. Cereb Cortex 2010, 21:806–820. 49. Hamilton N, Vayro S, Kirchhoff F, Verkhratsky A, Robbins J, Gorecki DC, Butt AM. Mechanisms of ATP- and glutamate-mediated calcium signaling in white matter astrocytes. Glia 2008, 56:734–749. 50. Di Virgilio F, Ferrari D, Adinolfi E. P2X7 : a growthpromoting receptor-implications for cancer. Purinergic Signal 2009, 5:251–256. 51. Agresti C, Meomartini ME, Amadio S, Ambrosini E, Volonte C, Aloisi F, Visentin S. ATP regulates oligodendrocyte progenitor migration, proliferation, and differentiation: involvement of metabotropic P2 receptors. Brain Res Brain Res Rev 2005, 48:157–165. 52. Meomartini ME, Amadio S, Visentin S, Franchini L, Aloisi F, Volonta` C, Visentin S. Expression and functional analysis of P2 receptors in oligodendrocytes. Glia 2003, 43, suppl 2:59. 53. Kirischuk S, Scherer J, Kettenmann H, Verkhratsky A. Activation of P2-purinoreceptors triggered Ca2+ release from InsP3-sensitive internal stores in mammalian oligodendrocytes. J Physiol 1995, 483 (Pt 1):41–57. 54. James G, Butt AM. P2X and P2Y purinoreceptors mediate ATP-evoked calcium signalling in optic nerve glia in situ. Cell Calcium 2001, 30:251–259. 55. Matute C. P2X7 receptors in oligodendrocytes: a novel target for neuroprotection. Mol Neurobiol 2008, 38:123–128. 56. Matute C, Torre I, Perez-Cerda F, Perez-Samartin A, Alberdi E, Etxebarria E, Arranz AM, Ravid R, Rodriguez-Antiguedad A, Sanchez-Gomez M, Domercq M. P2X7 receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis. J Neurosci 2007, 27:9525–9533. 57. Levine JM, Beasley L, Stallcup WB. Localization of a neurectoderm-associated cell surface antigen in the developing and adult rat. Brain Res 1986, 392:211–222. 58. Butt AM, Hamilton N, Hubbard P, Pugh M, Ibrahim M. Synantocytes: the fifth element. J Anat 2005, 207:695–706. 59. Nishiyama A, Komitova M, Suzuki R, Zhu X. Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity. Nat Rev Neurosci 2009, 10:9–22. 60. Hamilton N, Vayro S, Wigley R, Butt AM. Axons and astrocytes release ATP and glutamate to evoke calcium signals in NG2-glia. Glia 2010, 58:66–79. 61. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. Physiology of microglia. Physiol Rev 2010, 91: 461–553. 62. Xiang Z, Burnstock G. Expression of P2X receptors on rat microglial cells during early development. Glia 2005, 52:119–126.
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63. Moller T, Kann O, Verkhratsky A, Kettenmann H. Activation of mouse microglial cells affects P2 receptor signaling. Brain Res 2000, 853:49–59. 64. Avignone E, Ulmann L, Levavasseur F, Rassendren F, Audinat E. Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic signaling. J Neurosci 2008, 28:9133–9144. 65. D’Ambrosi N, Finocchi P, Apolloni S, Cozzolino M, Ferri A, Padovano V, Pietrini G, Carri MT, Volonte C. The proinflammatory action of microglial P2 receptors is enhanced in SOD1 models for amyotrophic lateral sclerosis. J Immunol 2009, 183:4648–4656. 66. Walz W, Ilschner S, Ohlemeyer C, Banati R, Kettenmann H. Extracellular ATP activates a cation conductance and a K+ conductance in cultured microglial cells from mouse brain. J Neurosci 1993, 13:4403–4411. 67. Norenberg W, Langosch JM, Gebicke-Haerter PJ, Illes P. Characterization and possible function of adenosine 5 -triphosphate receptors in activated rat microglia. Br J Pharmacol 1994, 111:942–950. 68. McLarnon JG, Zhang L, Goghari V, Lee YB, Walz W, Krieger C, Kim SU. Effects of ATP and elevated K+ on K+ currents and intracellular Ca2+ in human microglia. Neuroscience 1999, 91:343–352. 69. Boucsein C, Zacharias R, Farber K, Pavlovic S, Hanisch UK, Kettenmann H. Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro. Eur J Neurosci 2003, 17:2267–2276. 70. Inoue K. ATP receptors of microglia involved in pain. Novartis Found Symp 2006, 276:263–272; discussion 273–281. 71. Inoue K, Tsuda M. Microglia and neuropathic pain. Glia 2009, 57:1469–1479. 72. Inoue K, Tsuda M, Koizumi S. Chronic pain and microglia: the role of ATP. Novartis Found Symp 2004, 261:55–64; discussion 64–57, 149–154. 73. Inoue K, Tsuda M, Koizumi S. ATP receptors in pain sensation: Involvement of spinal microglia and P2X4 receptors. Purinergic Signal 2005, 1:95–100. 74. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 2003, 424:778–783. 75. Guo LH, Trautmann K, Schluesener HJ. Expression of P2X4 receptor by lesional activated microglia during formalin-induced inflammatory pain. J Neuroimmunol 2005, 163:120–127. 76. Guo LH, Schluesener HJ. Acute but not chronic stimulation of glial cells in rat spinal cord by systemic injection of lipopolysaccharide is associated with hyperalgesia. Acta Neuropathol 2006, 112:703–713. 77. Schwab JM, Guo L, Schluesener HJ. Spinal cord injury induces early and persistent lesional P2X4 receptor expression. J Neuroimmunol 2005, 163:185–189.
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78. Zhang Z, Zhang ZY, Fauser U, Schluesener HJ. Mechanical allodynia and spinal up-regulation of P2X4 receptor in experimental autoimmune neuritis rats. Neuroscience 2008, 152:495–501. 79. Haas S, Brockhaus J, Verkhratsky A, Kettenmann H. ATP-induced membrane currents in ameboid microglia acutely isolated from mouse brain slices. Neuroscience 1996, 75:257–261. 80. Ferrari D, Villalba M, Chiozzi P, Falzoni S, RicciardiCastagnoli P, Di Virgilio F. Mouse microglial cells express a plasma membrane pore gated by extracellular ATP. J Immunol 1996, 156:1531–1539. 81. Di Virgilio F, Sanz JM, Chiozzi P, Falzoni S. The P2Z / P2X7 receptor of microglial cells: a novel immunomodulatory receptor. Prog Brain Res 1999, 120:355–368. 82. Yu Y, Ugawa S, Ueda T, Ishida Y, Inoue K, Kyaw Nyunt A, Umemura A, Mase M, Yamada K, Shimada S. Cellular localization of P2X7 receptor mRNA in the rat brain. Brain Res 2008, 1194:45–55. 83. Collo G, Neidhart S, Kawashima E, Kosco-Vilbois M, North RA, Buell G. Tissue distribution of the P2X7 receptor. Neuropharmacology 1997, 36:1277–1283. 84. Sperlagh B, Illes P. Purinergic modulation of microglial cell activation. Purinergic Signal 2007, 3: 117–127. 85. Franke H, Gunther A, Grosche J, Schmidt R, Rossner S, Reinhardt R, Faber-Zuschratter H, Schneider D, Illes P. P2X7 receptor expression after ischemia in the cerebral cortex of rats. J Neuropathol Exp Neurol 2004, 63:686–699. 86. Melani A, Amadio S, Gianfriddo M, Vannucchi MG, Volonte C, Bernardi G, Pedata F, Sancesario G. P2X7 receptor modulation on microglial cells and reduction of brain infarct caused by middle cerebral artery occlusion in rat. J Cereb Blood Flow Metab 2006, 26: 974–982. 87. Yiangou Y, Facer P, Durrenberger P, Chessell IP, Naylor A, Bountra C, Banati RR, Anand P. COX-2, CB2 and P2X7 -immunoreactivities are increased in activated micro-glial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord. BMC Neurol 2006, 6:12. 88. McLarnon JG, Ryu JK, Walker DG, Choi HB. Upregulated expression of purinergic P2X7 receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus. J Neuropathol Exp Neurol 2006, 65:1090–1097. 89. Takenouchi T, Sekiyama K, Sekigawa A, Fujita M, Waragai M, Sugama S, Iwamaru Y, Kitani H, Hashimoto M. P2X7 receptor signaling pathway as a therapeutic target for neurodegenerative diseases. Arch Immunol Ther Exp (Warsz) 2010, 58:91–96. 90. Parvathenani LK, Tertyshnikova S, Greco CR, Roberts SB, Robertson B, Posmantur R. P2X7 mediates
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superoxide production in primary microglia and is upregulated in a transgenic mouse model of Alzheimer’s disease. J Biol Chem 2003, 278:13309–13317.
evidence for a role of the P2X7 receptor in the release of IL-1 beta in the murine brain. Brain Behav Immunol 2008, 22:234–244.
91. Rappold PM, Lynd-Balta E, Joseph SA. P2X7 receptor immunoreactive profile confined to resting and activated microglia in the epileptic brain. Brain Res 2006, 1089:171–178.
98. Bianco F, Pravettoni E, Colombo A, Schenk U, Moller T, Matteoli M, Verderio C. Astrocyte-derived ATP induces vesicle shedding and IL-1 β release from microglia. J Immunol 2005, 174:7268–7277.
92. Takenouchi T, Iwamaru Y, Imamura M, Kato N, Sugama S, Fujita M, Hashimoto M, Sato M, Okada H, Yokoyama T, Mohri S, et al. Prion infection correlates with hypersensitivity of P2X7 nucleotide receptor in a mouse microglial cell line. FEBS Lett 2007, 581:3019–3026. 93. Choi HB, Ryu JK, Kim SU, McLarnon JG. Modulation of the purinergic P2X7 receptor attenuates lipopolysaccharide-mediated microglial activation and neuronal damage in inflamed brain. J Neurosci 2007, 27:4957–4968. 94. Sanz JM, Chiozzi P, Ferrari D, Colaianna M, Idzko M, Falzoni S, Fellin R, Trabace L, Di Virgilio F. Activation of microglia by amyloid β requires P2X7 receptor expression. J Immunol 2009, 182:4378–4385. 95. Monif M, Reid CA, Powell KL, Smart ML, Williams DA. The P2X7 receptor drives microglial activation and proliferation: a trophic role for P2X7 R pore. J Neurosci 2009, 29:3781–3791. 96. Ferrari D, Chiozzi P, Falzoni S, Hanau S, Di Virgilio F. Purinergic modulation of interleukin-1 β release from microglial cells stimulated with bacterial endotoxin. J Exp Med 1997, 185:579–582. 97. Mingam R, De Smedt V, Amedee T, Bluthe RM, Kelley KW, Dantzer R, Laye S. In vitro and in vivo
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99. Kataoka A, Tozaki-Saitoh H, Koga Y, Tsuda M, Inoue K. Activation of P2X7 receptors induces CCL3 production in microglial cells through transcription factor NFAT. J Neurochem 2009, 108:115–125. 100. Shiratori M, Tozaki-Saitoh H, Yoshitake M, Tsuda M, Inoue K. P2X7 receptor activation induces CXCL2 production in microglia through NFAT and PKC/MAPK pathways. J Neurochem 2010, 114: 810–819. 101. Inoue K, Nakajima K, Morimoto T, Kikuchi Y, Koizumi S, Illes P, Kohsaka S. ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia. Br J Pharmacol 1998, 123:1304–1310. 102. Bo X, Sexton A, Xiang Z, Nori SL, Burnstock G. Pharmacological and histochemical evidence for P2X receptors in human umbilical vessels. Eur J Pharmacol 1998, 353:59–65. 103. Jarvis MF, Khakh BS. ATP-gated P2X cationchannels. Neuropharmacology 2009, 56:208–215. 104. Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, Wood JN. A P2X purinoceptor expressed by a subset of sensory neurons. Nature 1995, 377:428–431.
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P2X receptors in neuroglia Alexei Verkhratsky,1,2,3∗ Yuri Pankratov,4 Ulyana Lalo1 and Maiken Nedergaard5 Different types of ionotropic P2X purinoceptors are expressed in all major types of neuroglia, where they mediate a variety of physiological and pathological signaling. Cortical astrocytes express specific P2X1/5 heteromeric receptors that are activated by ongoing synaptic transmission and can trigger fast local signaling through elevation in cytoplasmic Ca2+ and Na+ concentrations. Oligodendrocytes express several types of P2X receptors that may control their development and mediate axonal–glial interactions. In microglia, P2X4 and P2X7 receptors regulate numerous events associated with microglial activation, motility, and release of proinflammatory factors. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. How to cite this article:
WIREs Membr Transp Signal 2012, 1:151–161. doi: 10.1002/wmts.12
INTRODUCTION: ATP AS A NEUROTRANSMITTER
A
TP, its derivatives, and adenosine are ubiquitous and ancient intercellular messengers that mediate cell-to-cell signaling in variety of tissues.1–3 In the central nervous system (CNS), ATP acts as homocellular (neuronal–neuronal and glial–glial) and heterocellular (neuronal–glial and glial–neuronal) neurotransmitter.4,5 ATP is physiologically released from many cellular elements of the CNS including neuronal terminals, axons, and astroglia. ATP release pathways include concentration-gradient driven diffusion through plasmalemmal channels with large permeability (as cytosolic concentration of ATP approaches 5–10 mM and extracellular is set at a low nanomolar range, the resulting concentration gradient is arguably one of the highest existing in biological systems); release through ATP-binding cassette transporters and secretion by exocytosis.4,6,7 The diffusion of ATP can occur through several sets of ∗
Correspondence to: [email protected]
1
Faculty of Medical and Human Sciences, The University of Manchester, Manchester, UK 2 IKERBASQUE,
Basque Foundation for Science, Bilbao, Spain
3 Department
of Neurosciences, University of the Basque Country UPV/EHU, Leioa, Spain
4 School
of Life Sciences, University of Warwick, Coventry, UK
5 Division
of Glial Disease and Therapeutics, Center for Translational Neuromedicine, Department of Neurosurgery, University of Rochester Medical Center, Rochester, NY, USA
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plasmalemmal channels such as unpaired connexins/ hemichannels8 or volume-sensitive anion channels.9 The latter mechanism was demonstrated to mediate physiological release of ATP associated with axonal firing9 ; although physiological relevance of diffusional ATP release in other CNS structures remains unclear. The most widespread pathway for ATP release in the context of signaling in neural networks is represented by Ca2+ -regulated exocytosis, which was identified in neuronal terminals6,7 and in astrocytes.10–12 Massive release of ATP also accompanies cellular death and destruction; hence ATP acts as a universal ‘damage’ signal and is involved in wide variety of pathological reactions in the nervous system.
P2X RECEPTORS IN ASTROGLIA Functional properties of P2X receptors (e.g., their agonist sensitivity, desensitization, or Ca2+ permeability) are determined by the subunit composition (Table 1). The P2X receptors can be formed through homo- or heteromeric assembly of P2X1 to P2X7 subunits (so far homomeric composition was shown for P2X1–5,7 subunits, whereas P2X6 subunits apparently cannot oligomerize); heteromeric compositions are represented by P2X1/2 , P2X1/4 , P2X1/5 , P2X2/3 , P2X2/6 , and P2X4/6 channels.13,14 On a transcriptional level, all seven P2X subunits were identified in astrocytes in vitro and in situ.15 Specific mRNA for P2X1–5 and P2X7 receptors was detected in primary cultured rat cortical
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TABLE 1 General Pharmacology of P2X Receptors Receptor P2X1
Kd/EC50
Preferred Substrates
References
Kd(ATP) = 2–3 nM
No specific agonists for P2X1 only
102
EC50 (ATP) = 0.07 μM
BzATP > 2meSATP > ATP > αβmeATP
103
BzATP > ATP = 2meSATP αβmeATP
103
2meSATP ATP > αβmeATP
104
BzATP > 2meSATP > ATP > αβmeATP
103
BzATP > ATP = 2meSATP αβmeATP
103
EC50 (BzATP) = 0.003 μM P2X2
EC50 (ATP) = 1.2 μM EC50 (BzATP) = 0.75 μM
P2X3 EC50 (ATP) = 0.5 μM EC50 (BzATP) = 0.08 μM P2X4
EC50 (ATP) = 10 μM
P2X5
EC50 (ATP) = 10 μM
ATP = 2meSATP αβmeATP > BzATP
103
P2X6 (hetero)
EC50 (ATP) = 12 μM
2meSATP ≥ ATP αβmeATP
103
BzATP > ATP = 2meSATP αβmeATP
103
EC50 (BzATP) = 7 μM
EC50 (2meSATP) = 9 μM P2X7
EC50 (ATP) = 100 μM EC50 (BzATP) = 20 μM
astrocytes.16,17 In tissue extracts from rat nucleus accumbens, expression of mRNA for all seven P2X subunits was revealed RT-PCR.18 Specific mRNA for P2X3 , P2X4 , and P2X5 subunits was identified in ¨ freshly isolated rat retinal Muller cells,19 the P2X7 receptor-specific mRNA was identified in human ¨ Muller glia.20 In acutely isolated mouse cortical astrocytes, only P2X1 - and P2X5 -specific mRNAs were found.21 Expression of P2X subunits in astroglia was also characterized by immunohistochemistry. In nucleus accumbens, immunofluorescence revealed P2X2–4 receptors co-localized with GFAP-labeled astroglial profiles.18 Mechanical lesion of the nucleus accumbens resulted in upregulation of P2X1–4 and P2X7 immunofluorescence in astrocytes.18 Immunoreactivity for P2X1 and P2X2 receptors was detected in astroglial cells in the cerebellum,22,23 P2X2 receptors were visualized in spinal cord astrocytes,24 and P2X4 receptors were identified in the processes and somatas of the astrocytes from the brainstem.25 In hippocampal astrocytes, immunostaining revealed expression of P2X1–4 , P2X6 , and P2X7 subunits.26 At a functional level, however, P2X receptorsmediated responses are not ubiquitous property of astroglia. ATP triggered membrane currents, depolarization, and Ca2+ signaling in cultured astrocytes,27,28 although the subunit composition of underlying receptors was not investigated. In hippocampal astrocytes in slices or after acute isolation, no P2X-mediated currents were identified.29 Similarly, ATP-induced currents were not observed in Bergmann glial cells 152
in acute cerebellar slices.30 Nonetheless, absence of ATP-induced currents in the in situ experiments mentioned above cannot be considered conclusive, because complex geometry of glial cells, diffusional barriers, and rapid degradation of ATP in slice tissue may hinder the detection of membrane responses.
P2X1/5 Receptors in Cortical Astrocytes Astrocytes in cortex express functional P2X1/5 heteromeric receptors, which upon activation produce idiosyncratic membrane responses (Figure 1 and Ref 21). Astroglial P2X1/5 receptor is extraordinary sensitive to ATP (KD ∼ 50 nM), has distinct pharmacology (complete inhibition by 30 μM PPADS and almost complete inhibition by 1 μM TNP-ATP) and biophysical properties (absence of desensitization and activation of ‘rebound’ tail current in response to the washout of the agonist). The P2X1/5 receptors contribute to fast ion currents triggered in astrocytes in response to stimulation of neuronal afferents in cortical slices.31,32 The same receptors are involved in generation of spontaneous ‘miniature’ postsynaptic currents measured from astrocytes in cortical slices.31,33 Astroglial P2X1/5 receptors have intermediate Ca2+ permeability (PCa /Pmonovalent ∼ 2.2) and their activation by exogenous agonists as well as by synaptically released ATP triggers astroglial Ca2+ signals (Figure 2 and Ref 32). Expression of P2X1/5 receptors in astrocytes is age-dependent,33 being maximal in 3–6 months old mice and substantially declining with an advanced age.
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(a)
(b)
(c)
FIGURE 1 | P2X1/5 receptor-mediated currents in acutely isolated cortical astrocytes. (a) The family of ATP currents evoked by repetitive applications of the agonist. The currents show no apparent desensitization. Current traces have a complex kinetics comprising the peak, the steady-state component, and the ‘rebound’ inward current recorded upon ATP washout as indicated on the graph. (b) Concentration dependence of ATP-induced currents in cortical astrocytes. Membrane currents recorded from a single cell in response to different ATP concentrations are shown on the left. The right panel shows the concentration response curves constructed from nine similar experiments; current amplitudes were measured at the initial peak and at the end of the current, as indicated on the graph. (c) Inhibition of ATP-induced currents by 2 ,3 -O -(2,4,6-trinitrophenyl) adenosine 5 -triphosphate (TNP-ATP). The graph shows ATP-induced currents measured from the cortical astrocyte in control conditions, in the presence of different concentrations of TNP-ATP and after the washout. Application of TNP-ATP started 2 min before the application of ATP. All recordings were made at holding potential of −80 mV. (Reprinted with permission from Ref 21. Copyright 2008 Society for Neuroscience)
Astroglial P2X7 Receptors The role and functional expression of the cytolitic, pore-forming P2X7 receptors14,34 in astroglia remains controversial. Both P2X7 -specific mRNA and P2X7 Volume 1, March/April 2012
receptor protein have been detected in cultured astroglia (reviewed in Refs 15 and 35). Similarly, stimulation of P2X7 receptors in cultured astrocytes often triggered [Ca2+ ]i transients, mimicked by
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(a)
(b)
FIGURE 2 | P2X1/5 receptor-mediated Ca2+ -signaling in cortical astrocytes. (a) Acutely isolated astrocytes were loaded with Fluo-4 via a patch
pipette. Fluorescent images were recorded simultaneously with transmembrane currents evoked by application of 100 μM ATP or 10 μM α,β-methyleneATP. [Ca2+ ]i -transients are represented as the F /F0 ratio averaged over the cell soma (green traces). Holding potential was −80 mV. (b) Synaptically induced ionotropic Ca2+ signals in protoplasmic cortical astrocytes in situ. Cortical layer II astrocytes were loaded with Fura-2 in situ via patch pipette. Fluorescent images were recorded simultaneously with glial currents evoked by neuronal afferent stimulation in the presence of CNQX (control) and after application of 10 nM of NF-449, selective antagonist of P2X receptors. [Ca2+ ]i -transients (green traces) are expressed as F340 /F380 ratio. (Reprinted with permission from Ref 32. Copyright 2010 Elsevier)
P2X7 receptor agonist 2 ,3 -O-(benzoyl-4-benzoyl)ATP (BzATP) and inhibited by antagonist oxidized ATP (oxATP).17,36,37 Characteristic P2X7 receptor currents activated by 1–5 mM ATP or by 0.1 mM Bz-ATP were recorded from cultured astrocytes.38,39 Likewise, P2X7 receptor-mediated currents were ¨ identified in freshly isolated human retinal Muller glial cells.20 It has to be remembered, however, that Bz-ATP can activate other P2X receptors13 and can be degraded to Bz-adenosine, which stimulates P1 receptors.40 Similarly, the oxATP also inhibits P2X1 and P2X2 receptors.13 Stimulation of P2X7 receptors in astrocytes in vitro affects numerous biochemical process, 154
for example, synthesis of endocannabinoid 2arachidonoylglycerol, stimulation of production of NO and of lipid mediators of inflammation cysteinyl leukotrienes, regulation of NF-κB signaling, release of gliotransmitters and TNF-α, etc.41–45 Furthermore, stimulation of P2X7 receptors in cultured astrocytes induces secretion of various transmitter molecules (‘glio’ transmitters) including glutamate, ATP, and GABA.38,46,47 Recently, the P2X7 currents were found in cortical astrocytes in situ, in acutely isolated slices,48 and P2X7 receptor-mediated Ca2+ signals were identified in astrocytes from acutely isolated optic nerve.49 The role of P2X7 receptors in astroglial physiology remains unknown, as indeed activation of
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the receptor requires very high (in excess of 1 mM) ATP concentrations. It was, however, suggested that low ATP concentrations may activate P2X7 receptor without opening the channel pore; this ‘low-level’ stimulation can be relevant for regulation of a variety of trophic/metabolic processes.50 At the same time, astroglial expression of P2X7 receptors is usually increased in neuropathology (see Ref 15 for review), which may indicate their role in regulation of pathologically relevant astroglial reactions.
P2X RECEPTORS IN OLIGODENDROGLIA Expression of P2X1,2,3,4,7 proteins was found in oligodendroglial precursor cells (OPCs) in purified postnatal cultures,51,52 although P2X-mediated ion currents were not detected in cultured OPCs and mature oligodendrocytes.53 In the isolated optic nerve, application of a broad agonist of P2X receptors α,β-methylene ATP induced relatively small [Ca2+ ]i elevation, thus suggesting possible involvement of P2X receptors.54 In oligodendrocytes in situ in corpus callosum slices, ATP failed to activate transmembrane currents.53 Some evidence indicating expression of functional P2X7 receptors in cells of oligodendroglial lineage was gathered recently. In cultured OPCs, application of Bz-ATP triggered large Ca2+ transients, which were effectively inhibited by oxATP thereby suggesting Ca2+ entry though P2X7 receptors.51 Immunoreactivity for P2X7 receptors was detected in oligodendrocytes from the optic nerve and the spinal cord.55,56 In cultured oligodendrocytes from optic nerve, high concentrations of ATP (EC50 ∼ 8.8 mM) and much lower concentrations of BzATP (EC50 ∼ 0.5 mM) triggered sustained inward currents. These currents were potentiated in divalentcations-free extracellular solutions and were inhibited by oxATP. In addition, high concentrations of ATP and BzATP induced a rapid increase in [Ca2+ ]i , which was almost exclusively dependent on transmembrane Ca2+ entry.56 Finally, over-stimulation of P2X7 receptors (with 1 mM ATP or BzATP for 15 min) induced significant oligodendroglial death in culture and in situ in the optic nerve.56 The P2X7 -mediated death of oligodendrocytes may be relevant for variety of demyelinating diseases including multiple sclerosis (MS). Treatment of animals suffering from experimental autoimmune encephalitis (EAE, which is generally considered as a model for MS), with P2X7 antagonists oxATP or brilliant blue G, inhibited demyelination and restored axon conduction velocity.55,56 Furthermore, Volume 1, March/April 2012
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expression of P2X7 receptors increased in white matter of MS patients.56
NG2 GLIA Very little is known about expression of P2X receptors in the NG2-glia, which was recently identified as a separate class of neuroglia. The NG2 glial cells (identifiable by the expression of NG2 chondroitin sulfate proteoglycan57 ) are present throughout the developing and adult brain and may serve as pluripotent stem cells, OPCs or being a specific elements of neural circuitry.58,59 Recently, it was reported that ATP released from axons and astrocytes in electrically stimulated optic nerve triggers [Ca2+ ]i transients in NG2-glia, which at least in part can be mediated by P2X7 receptors.60
P2X RECEPTORS CONTROL ACTIVATION OF MICROGLIA Purinergic signaling system is particularly developed in microglia. Numerous experiments in vitro and in vivo have demonstrated that ATP and its analogues trigger rapid functional responses of microglial cells, which include induction of microglial motility (both of the processes and the soma), the outgrowth of microglial processes, membrane raffling, and the release of cytokines and inflammatory proteins (see Ref 61 for comprehensive review). Effects of purines/pyrimidines on microglia are mediated through all types of purinoceptors (P1 and P2) abundantly expressed in microglial cells throughout the nervous system.61 Expression of P2X receptor subunits in microglia is developmentally regulated. At embryonic day 16, majority of microglial cells expressed mRNA specific for P2X1 and P2X4 subunits, whereas only 30% of these cells expressed P2X7 receptor transcripts. From postnatal day 7, the P2X4 -expressing microglia concentrated around blood vessels. Finally, at postnatal day 30, microglial cells expressed only P2X7 and P2X4 mRNAs; the P2X7 -positive cells were distributed evenly through the forebrain, whereas cells bearing P2X4 receptors outlined blood vessels and subarachnoid space.62 Expression of different types of purinoceptors in cultured microglia undergoes substantial remodeling while progressing through activation.63 Likewise, expression of purinoceptors changes in various neurological diseases. For example, activation of hippocampal microglia following epileptic seizures (triggered by injections of kainate) resulted in the upregulation of the expression of mRNA specific for
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P2X1,4,7 and P2Y6,12,13 receptors. This upregulation led to an increase in ATP-induced membrane currents and ATP-induced microglial motility.64 The upregulation of microglial P2X4 , P2X7 , and P2Y6 receptors were also observed in the experimental model of amyotrophic lateral sclerosis, in mice expressing mutant superoxide dismutase 1.65 The P2X-mediated currents were identified in microglial cultures prepared from several tissues including mouse embryos, newborn rat, and human brains.66–68 In all these preparations, application of 100 μM ATP triggered desensitizing cationic currents and induced [Ca2+ ]i elevation produced by Ca2+ entry through ionotropic purinoceptors. Similar ATP-induced cationic currents were also detected in microglial cells in acute slices from an adult mouse brain.69
P2X4 Receptors in Pathologically Remodeled Spinal Cord Microglia Peripheral nerve injury triggers rapid and substantial activation of microglia in the spinal cord, this process being fundamentally important in pathogenesis of neuropathic pain.70–73 Development of tactile symptoms associated with neuropathic pain (e.g., tactile allodynia) in the rat spinal nerve injury model could be reversed by inhibition of P2X4 receptors, suggesting their role in neuropathic pain.74 This initial observation for further corroborated when it was shown that intraspinal injection of P2X4 antisense oligodeoxynucleotide inhibited P2X4 receptors expression and reduced tactile allodynia following nerve injury. The intrathecal injection of activated cultured microglia expressing P2X4 receptors triggered allodynia without peripheral nerve lesion.74 The lesion to the peripheral nerve resulted in a significant upregulation of P2X4 receptor expression in spinal cord microglia74 ; the P2X4 protein levels began to increase 1 day after surgery and reached maximal levels after 14 days; this time course matched the development of tactile allodynia. Similarly P2X4 expression was upregulated following formalin injection into the spinal cord (a common inflammatory pain model) or intraperitoneal injection of LPS75,76 or mechanical lesion to the spinal cord.77 An increase in P2X4 expression was also observed in rats with experimental autoimmune neuritis.78
P2X7 Receptors in Microglia Functional P2X7 receptors were identified in microglial cells in situ, in amoeboid microglia collected from the surface of corpus callosum slices,79 and in vitro in freshly isolated mouse microglia.80 In 156
cultured microglia, ATP (in millimolar concentrations) and Bz-ATP evoked Ca2+ influx and uptake of ethidium bromide or Lucifer Yellow both being indicative of the formation of P2X7 receptor associated pore. These effects were blocked by oxATP.80,81 In the amoeboid microglia from corpus callosum slices of 5–7 days old mice, 1 mM ATP induced classical P2X7 mediated currents (Figure 3). Sensitivity of receptor was greatly enhanced following removal of extracellular divalent cations.79 The P2X7 receptor-mediated currents were also detected in ramified microglia in acute slices from adult (6–8 weeks old) mice.69 The P2X7 receptor seem to be constitutively expressed in resting microglia; in the healthy brain P2X7 -positive microglial cells are homogeneously distributed throughout all brain regions.82 Different forms of insults to the brain trigger rapid and substantial increase in the expression of microglial P2X7 receptors.35,83,84 Expression of P2X7 receptors is upregulated after acute ischemic and traumatic attacks85,86 as well as chronic neuropathologies such as MS87 and Alzheimer’s disease.88,89 A significant increase in expression of P2X7 -specific mRNA and in P2X7 immunoreactivity was also detected in reactive microglial cells surrounding senile plaques in AD transgenic model.90 Microglial expression of P2X7 receptors was also increased following exposure of cultured microglia to β-amyloid or after intrahippocampal injection of the latter.88 An increase in expression of P2X7 receptors was also reported in microglia from rat model of epilepsy91 and from brains of scrapie-infected mice.92 Pharmacological manipulations with P2X7 receptors were neuroprotective in ischemia86 ; it also reduced microglial expression and release of proinflammatory factors.93 Activation of P2X7 receptors regulates various functions associated with microglial activation. The P2X7 receptors, for example, were obligatory for triggering activation of microglia and inetrleukin-1 production in response to an intrahippocampal injection of Aβ protein; both activation and IL-1 accumulation were absent in P2X7 knockout mice.94 Overexpression of P2X7 receptors in cultured microglia on its own right triggered microglial activation that could be blocked by oxATP.95–100 This microglial activation required formation of P2X7 receptor-associated pore; when microglial cells were transfected with a point mutant P2X7 RG345Y that does not allow pore formation, microglial activation was suppressed.95 Microglial P2X7 receptors also control release of multiple proinflammatory factors. Activation of P2X7 initiates release of cytokines IL-1α and IL-1β 96,97 that may involve plasmalemmal vesicle formation and shedding.98 The P2X7 receptors also control
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(a)
(c)
(e)
microglial synthesis and release of CC-chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1α 99 ; release of chemokine CXCL2,100 plasminogen,101 and many other proinflammatory factors (see Ref 61 for comprehensive review).
CONCLUSIONS P2X receptors are expressed in all types of neuroglial cells. The full regional mapping of glial presence of P2X receptors is far from being complete;
P2X receptors in neuroglia
(b)
(d)
FIGURE 3 | ATP triggers P2X7 receptor-mediated currents in amoeboid microglial cells collected from the surface of corpus callosum slices of young (P5–P7) mice. (a–d) Method of patch-voltage clamping of single amoeboid microglial cells from the surface of corpus callosum slice. Combinations of images taken by means of infrared video microscopy (left panels) and schematic drawings showing the method of cell patch-clamping. Video images were captured with an intensified CCD camera (Hamamatsu, Japan) and digitized by a frame grabber connected to the PC. (a) Initial position of amoeboid microglial cells situated on the surface of a corpus callosum slice. (b) A single microglial cell was approached with a micropipette and a whole-cell patch clamp configuration was established. (c) After 2–3 min, the cell partially spread over the pipette, intensifying the cell-to-pipette contact. (d) Finally, the cell was lifted for 200 μm over the slice surface. Scale bar in (d) = 10 μm. (e) P2X7 -mediated membrane currents measured from amoeboid microglial cells in control conditions and Ca2+ /Mg2+ -free bath solution. (Reprinted with permission from Ref 79. Copyright 1996 Elsevier)
however, we already may suggest their roles in various types of signaling in neuronal–glial circuits. In cortical astroglia, P2X1/5 receptors participate in rapid synaptic-associated signaling producing local [Na+ ]i and [Ca2+ ]i transients in glial perisynaptic processes. In oligodendroglia, activation of P2X7 receptors is somehow involved in pathogenesis of demyelinating diseases. In microglia, P2X4 and P2X7 receptors control various aspects of microglial activation and secretion of proinflammatory factors.
ACKNOWLEDGMENTS This research was supported by Alzheimer’s Research Trust (UK) Programme Grant (ART/PG2004A/1) to A.V.
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