ATP as a Neuromediator at the Brain-Immune Interface

The purinergic signaling system, also known as a ‘purinome’, represents extracellular signaling cascades, which are mediated by Adenosine Triphosphate (ATP) and its extracellular breakdown product, adenosine. This system consists of receptors, enzymes and transporters responsible for their release, actions and extracellular inactivation (Figure 1). ATP is well-known as the universal ‘energy currency’ of living cells. The idea that in addition to this role, purines might also influence the activity of other cells stems from 1929 by Albert Szent-Györgyi, the Nobel laureate biochemist, when he reported that adenine compounds, including adenosine, have profound effect on heart rate and cardiovascular function [1]. This idea was reanimated by Geoffrey Burnstock in the early seventies who proposed the ‘purinergic nerve hypothesis’, suggesting that ATP is a specific neurotransmitter in the nervous system [2].

In the past three decades this concept has gained solid experimental proof in a number of synapses of the peripheral and central nervous system [3, 4]. Moreover, it also turned out that ATP has a more versatile function in the neuronal information processing than a classical neurotransmitter, participating in pre- and post-synaptic neuromodulation. The most recent recognized function of ATP is that it mediates the glia and neuron cell communication as well as between glial cells, and is one of the most important gliotransmitters [5]. Finally, taken into account that ATP is released to the extracellular space upon pathological conditions such as cell death, and participates in disease pathophysiology, ATP also functions as a ‘danger signal’ in the brain.

It has now also become clear that ATP-mediated signaling is not restricted within the border of the nervous system. Purinergic receptors are expressed in essentially all cells and tissues, including the cells of the immune system. Therefore, ATP is now regarded as a universal signaling substance, rather than a pure neurotransmitter or neuromodulator, playing a major role in the cross talk between different cell types, and at the interface between the nervous system and the immune system.

Figure 1. Purinergic signaling in the nervous system. The ‘purinome’ consists of receptors, enzymes and transporters responsible for the release (1), extracellular inactivation (2) and actions (3) of nucleotides and nucleosides.

Synthesis, utilization and storage of ATP in the nervous system

It is well-known that all metabolically active cells including neurons and glia are able to synthesize ATP. The majority of ATP under normal metabolic conditions is formed from ADP by oxidative phosphorylation in the mitochondria. In addition, ATP is also generated in a minor amount in the glycolytic pathway and in the tricarboxylic acid cycle. ATP formation in the mitochondria is governed by the actual metabolic demand; this is the so-called ‘respiratory control’, as the mitochondrial ATP production is coupled to the respiratory chain and is driven by the actual ADP concentration of the cell.  The adenine ring of the ATP molecule is synthesized during the multiple steps of de novo purine biosynthesis from phosphoribosyl pyrophosphate resulting in inosine monophosphate (IMP) production.   IMP is transaminated to AMP then directly phosphorylated to ADP and serves as a substrate for mitochondrial oxidative phosphorylation.  However, the de novo purine biosynthesis is an energy consuming process; therefore nerve terminals utilize purine salvage mechanisms rather than de novo purine biosynthesis. Hence, they take up adenosine via the nucleoside transport systems and the adenosine kinase enzyme converts adenosine to AMP, which then enters into the reactions detailed above. Taken together, the activity of all these reactions result in approx 10 mM ATP concentration in the cytoplasm under normal metabolic conditions.

The majority of ATP produced by the nerve terminal is utilized for fuel energy by cellular functions and among them the most important are: (i) the maintenance of resting membrane potential by the Na+/K+ pump, (ii) the function of the energy requiring transport mechanisms, such as the Ca2+ pumps of the plasma membrane and the mitochondria (iii) the synthesis of neurotransmitters, receptors, ion channels, transporters, and other signaling proteins and molecules, like G proteins, protein kinases, GTP etc. (iv) the build-up of the vesicular proton gradient by the vacuolar H+ATPase and (v) the steps of the exocytosis itself.  Nevertheless, cytoplasmic ATP is also available for intra- and extracellular signaling process and ATP is also taken up by synaptic vesicles [6, 7]. The uptake of ATP into secretory and probably into synaptic vesicles is mediated by the recently identified vesicular nucleotide transporter (VNUT) [8], and is a Cl- -dependent process, similar to the vesicular uptake of glutamate.

In cholinergic and noradrenergic vesicles, the amount of ATP is usually outnumbered by its co-transmitter mate. Thus, storage ratios of 3:1 to 50:1 were established, depending upon the type of vesicles, corresponding to about 1-200 mM concentration of ATP inside the vesicle. However, ATP content may differ in individual vesicles and can change under various conditions, e.g. upon different patterns of neuronal activity. In addition to ATP other nucleotides which are thought to play a role as signaling substances are also stored in synaptic vesicles, i.e. ADP, AMP, UTP, Ap4A, Ap5A and guanine nucleotides,. Although their concentration is less than that of ATP, it is still relatively high, i.e. in millimolar range, and enough to serve as a pool for their release.

The release and extracellular inactivation of ATP

Extracellular purine availability in the nervous system is determined by the balance of release, removal by enzymatic degradation and uptake. An important prerequisite of the participation of ATP in extracellular signaling is that it should be released to the extracellular space in response to physiological and/or pathological stimuli, which could lead to purine levels sufficiently high to activate nucleotide receptors expressed on the surface of pre- and postsynaptic membranes. Indeed, a wide variety of stimuli is known to release ATP such as: (i) electrical or chemical depolarization of nerve terminals (ii) activation of cell surface receptors (iii) mechanical stimuli (iv)  hypoxia/hypoglycemia/ischemia and the consequent cellular energy deprivation (v) hypoosmotic challenge (vi) inflammatory stimuli (vii) cellular damage. The release of ATP upon neuronal activity was demonstrated for the first time by Holton [9] using antidromic stimulation of sensory nerves. Since then the stimulation-dependent release of endogenous ATP has been reported from many sites of the nervous system, using electrical field stimulation, direct stimulation of specific neuronal pathways or chemical depolarization [6, 7]. More recently, the enzyme based microelectrode biosensor method was developed [10] to detect  ATP and adenosine release on a real time scale in vitro and in vivo. For example, using this technique, stimulation-dependent physiological ATP release was demonstrated during the hypoxic ventilatory response from the carotid body, in response to the elevation of blood pCO2 from the chemosensitive region of the medulla oblongata  [11-13] and during locomotor activity from spinal networks [10].

Given its ubiquitous nature however, ATP could be released not only from neurons but also from non-neuronal cells in the nervous system. Therefore one of the most intriguing questions is to identify the source of ATP involved in the regulation of neuronal functions. Several neuronal pathways have been identified as a source of ATP release during neuronal activity, which include the septohabenular projection, [14-16], the ventral noradrenergic bundle [17], the Shaffer collateral pathway providing the main excitatory input to the hippocampus [18], the afferent nerve bundle projecting to the rat superior cervical ganglion [19] and sympathetic nerves innervating the vas deferens  [20, 21]. ATP is shown to be released from these pathways in a vesicular fashion, i.e. upon sodium channel activity and subsequent Ca2+ dependent exocytosis, alone or together with its co-transmitter mate. In other studies no evidence was found for the neuronal origin of released ATP, which implicates that ATP may be also secreted via autocrine-paracrine pathways from non-neuronal cells [22-24].

Besides neurons other potential sources of extracellular ATP are glial cells, postsynaptic target cells, blood vessel endothelium and the resident immune cells of the neural tissue. The release of ATP from glia has been repeatedly demonstrated in response to various stimuli [25-27]. An additional mechanism, whereby ATP could be released in the nervous system, is a receptor operated retrograde release from the postsynaptic target cells, which seems to be significant mechanism in the periphery, at the autonomic [21, 28, 29] and skeletal  neuromuscular junction  [30, 31]. Postsynaptic ATP could contribute to neuronal ATP release and serve as an amplifying mechanism of the pre- and postsynaptic actions of extracellular ATP and its degradation product, adenosine.

Although the stimulation-dependent release of ATP upon conventional [15, 32] and high frequency [18] neuronal activity is well documented, these stimuli probably result in a spatially restricted, localized increase in extracellular purine levels, which serve the fast synaptic transmission and its modulation within the synaptic cleft. However, ATP-metabolizing ectoenzymes, present on the nerve terminal membrane, and glial cells may strongly limit nucleotide availability under these conditions.

On the other hand, pathological events such as mechanical or metabolic stress, inflammation, cellular injury or changes in the ionic environment are also known to elicit purine release. This might result in a purine-rich extracellular milieu leading to a more widespread activation of receptors reaching also the extrasynaptic receptors on the neighboring nerve terminals or distant cells such as astrocytes.  Pathological stimuli that lead to the release of ATP also include hypoxia/hypoglycemia and the consequent energy deprivation [23, 33] and inflammatory stimuli, such as bacterial lipopolysaccharide [16, 34] and the proinflammatory cytokine IL-1beta [35]. It is important to note, however, that the source of purines released by physiological and pathlogical stimuli are not identical. For example, efflux of ATP and adenosine, released by in vitro ischemic-like conditions is [Ca2+]o– independent and probably has cytoplasmic origin [36].

ATP is a highly polarized molecule and cannot pass freely through the cell membrane. The release mechanism for which ATP enters the extracellular space is also the subject of extensive investigation. These include (1) vesicular exocytosis (2) transporter mediated release (3) release through channels and membrane pores (4) and cytolytic release.

(1) Vesicular exocytosis is a prototype mechanism for neurotransmitters to enter the extracellular space and is expected to be a [Ca2+]o-dependent process. Since ATP is a constituent of synaptic vesicles, it is reasonable to assume that exocytosis is accompanied by the release of ATP to the extracellular space. Indeed, Ca2+-dependent vesicular release has been demonstrated in a number of central synapses by both electrophysiological and neurochemical detection [7, 37]. Vesicular exocytosis of ATP has been demonstrated in astrocytes [26, 38]. Moreover, lysosome exocytosis [39] has also been identified as a mechanism of ATP efflux from astrocytes.

(2) Although specific transporters capable for the transmembrane movement of ATP have yet to be molecularly identified in neurons, some data suggest that ATP could also be released in a carrier-mediated manner in the nervous system. In non-neuronal cells, ABC (ATP binding cassette) proteins have been implicated as an ATP transporter [40-42]. These transporters are also expressed in glial cells [43]  and mediate ATP release upon hypoosmotic challenge [44, 45]. Moreover, nucleotides and nucleosides by themselves may promote further release of purines, by a homo- or heteroexchange mechanism if they reach relatively high concentration in the extracellular space [46].

(3) Other alternative routes of ATP release are channels and pores able to conduit the release of ATP.  Thus, connexin and pannexin hemichannels [47, 48] volume sensitive anion channels [45, 49], and P2X7 receptor channels [25, 50], are described as mediators of the release of ATP. These latter mechanisms have primarily been identified in non-neuronal cells such as cultured astrocytes, and less information is available whether these pathways also serve as a conduit of ATP release for purines involved in the modulation of synaptic signaling. Nevertheless, as hypothesized in a recent review,  channel-mediated ATP release seems to be a more effective and less energetically costly mechanism for the release of larger quantities of ATP compared to exocytosis, which may gain significance under certain circumstances such as development [51].

(4) Although only scarcely supported by direct experimental proof [52], the general assumption is that any kind of cellular injury could result in high local ATP concentrations in the extracellular space. In this case, the millimolar cytoplasmic ATP is expected to flow out to the extracellular space by the damage caused to the cell membrane.  Cytolytic ATP release could occur under a wide variety of pathological situations in the CNS, e.g. in traumatic brain or spinal cord injury, ischemic neurodegeneration Alzheimer’s disease, sclerosis multiplex and in the generation of chronic pain.

Apart from release, the other major factor determining extracellular nucleotide availability is their removal from the extracellular space by enzymatic degradation and transporters. Several enzyme families are responsible for the extracellular degradation of ATP in the nervous system [53]. The first step of the inactivation of ATP is mediated by the family of ectonucleoside triphosphate diphospho-hydrolases (E-NTPDases, EC 3.6.1.5, also known as ectoATPase or apyrase), which are able to hydrolyse ATP into ADP and AMP. Among these enzymes, E-NTPDase 1,2,3, and 8 are present in the brain [54, 55] and have low micromolar Km for ATP and ADP, which gives rise to rapid and highly effective hydrolysis of ATP in almost all neuronal tissues. In addition to the E-NTPDase family, ATP and other nucleotides can also be dephosphorylated by ecto-nucleotide pyrophosphatases (E-NPPs) and by alkaline phosphatases, both having broader substrate specificity, but also widespread tissue distribution [53].

The final step of extracellular inactivation is the hydrolysis of AMP by the ecto5`nucleotidase (EC 3.1.3.5) enzyme, which is the rate-limiting step giving rise to the formation of adenosine, which is a new extracellular signal, acting on its own receptors. Nevertheless, the short half-life of ATP in the extracellular space could still be long enough for the activation of ionotropic ATP receptors, which act on a millisecond time scale, and probably also for metabotropic ATP receptors, which act on a slower, secondary time scale. In addition to nucleotide catabolizing pathways, enzymes capable to rephosphorylate nucleotides (e.g. adenylate kinase) also exist in the extracellular space in the nervous system [56, 57].

Structure and properties of ATP receptors

ATP exerts its biological actions in the nervous system through diverse families of P2 nucleotide receptors (Table 1).

Table 1. Classification of identified purine/pyrimidine receptors

P2 receptors can be subdivided into two families, ionotropic (P2X) and metabotropic (P2Y) receptors [58]. Ionotropic P2X receptors are 379-595 amino acid long ligand gated cation channels, having two transmembrane domains (TM1 and TM2) and a large extracellular loop [59, 60]. The ligand binding domain is located within a cysteine rich extracellular loop between the lysine resides of positions 69 and 71, and binding sites for different antagonists have also been identified on the extracellular domain of the receptor protein. P2X receptors are non selective cationic channels having permeability to both monovalent (Na+, K+) and divalent (Ca2+) cations. Moreover, upon prolonged or repetitive agonist application they might also undergo pore dilation which makes the channel permeable to high molecular weight cations up to 800 Da. This effect eventually leads to cell death, but depends on the cell type. Thus, pore formation is typically observed on peripheral immune cells in response to P2X7 receptor activation [61]. The molecular mechanism of pore formation is subject to current debate, i.e.  whether P2X7 receptors by themselves form the pore or they gate pannexin hemichannels  [62].

Seven individual subunits of P2X receptor family have been identified numbered from P2X1 to P2X7, and have individual kinetics and pharmacological phenotypes (Table 1) [63]. These receptor proteins, however, do not function as individual receptors, but co-assemble into various homo- or heterooligomeric assemblies to form functional receptors. Among possible combinations so far are 16 variations that have been proved to be functional [64]. These are all of the homooligomeric receptors, except P2X6, which does not function in homooligomeric form, and the rest are heterooligomers, formed from P2X1-P2X6 subunits. On the other hand, the P2X7 receptor functions primarily in homooligomeric form and does not co-assemble with other known P2X receptor subunits, except P2X4 [65]. As for the stoichiometry of the subunits, chemical cross-linking [66] and atomic force microscopic  studies [67] indicates that functional receptors are formed as trimers.   More recent investigations identifying the crystal structure of P2X4 receptors essentially reaffirms this assumption [68].

P2X receptors are sensitive to ATP and ADP but not to AMP and adenosine and the ligand binding profile of homomeric P2X receptors are well established (for further information, see [63]). The affinity of ATP to homomeric P2X receptors is between 1-10 microM, while the P2X7 receptor is between 100 microM-1 mM. On the other hand, less is known about the pharmacology of heteromeric receptors; among them, the pharmacological characterized profiles of P2X2/3, P2x2/6, P2X1/2, P2x1/4, p2x1/5 and P2X4/6.

P2Y receptors all belong to G protein coupled receptors, having seven hydrophobic transmembrane domains, and possess their ATP binding sites on the external side of the TM3 and TM7 domains [69-73].  P2Y receptor family has 8 individual members, numbered  P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 (Table 1). These receptors are subdivided into two subgroups, those which primarily activate the Gq/G11/PLC/IP3 pathway (P2Y1, P2Y2, P2Y4, P2y6, P2y11)  and those which are coupled to Gi/o and inhibits adenylcyclase and ion channel activity (P2y12, p2y13, p2Y14). P2Y receptors are activated by adenine and uridine nucleotides, such as ATP, ADP, UDP and UTP, but not by nucleosides and according to their pharmacological profile, are classified as either adenine nucleotide or uridine nucleotide preferring receptors. P2Y1, P2Y11, P2Y12, P2Y13 receptors belong to the former, whereas P2Y2, P2Y4, P2Y6 receptors belong to the latter subgroup, while P2Y14 receptors are activated by UDP-sugar moieties, such as UDP-glucose. In addition, certain P2Y receptors, such as P2y12 are also activated by cysteinyl-leukotrienes (CysLTE4). Similar to other G protein coupled receptors, P2Y1 receptors can heteromerize with other P2Y receptors [74] and with A1 adenosine receptors [75].

Expression of P2 receptors in the nervous system

All seven P2X receptor subunits are widely expressed in the nervous system, however, their expression patterns are different and show region- and cell-type specific distinct distribution. Among the P2X receptors, P2X2, P2X4 and P2X6 seem to be most abundantly expressed in the brain, whereas other subunits show more restricted localization. The typical localization of P2X2 receptor is on nerve terminals of the brain and the periphery [76, 77] but also appears postsynaptically [78]. P2X1 receptors, initially suggested to be exclusively expressed on smooth muscle membrane, consistent in its role of mediating fast synaptic transmission at the autonomic neuroeffector junction, are also present on central and peripheral neurons [79, 80]. The same holds true for P2X3 receptors, which are primarily associated to sensory pathways, but functional studies indicate that they are also expressed in other brain regions and autonomic pathways [81-83]. P2X4 receptor shows heavy expression in several brain areas such as the cerebral cortex, hippocampus, thalamus and brainstem [84] and associated with postsynaptic specialization of synaptic contacts [78]. P2X5 subunits display the most restricted localization in the brain, although it shows strong representation in certain areas, e.g. nucleus tractus solitarii (NTS) [85]. Finally, mRNA encoding  P2X7 receptors are expressed in reactive microglia and astroglia in the brain [86], while immunocytochemical studies revealed  widespread presynaptic expression of P2X7 receptors in several brain regions, spinal cord, and the skeletal neuromuscular junction [87-89]. More recent studies indicate that several splice variants of P2X7 receptors exist and they partially retain their functionality in the knockouts [90, 91]. Splice variants might be responsible for the pseudo-immunoreactivity found in p2X7 receptor knockout animals [92, 93].

mRNA encoding all known P2Y receptors, i.e. P2Y1, P2y2, P2Y4, p2Y6, P2y11, P2Y12, P2Y13, and P2Y14 are present in the brain. Although our knowledge on their cell-specific localization at the protein level is still incomplete, it appears that a number of receptors, such as P2Y1, P2Y2, and P2Y6 are expressed both on neurons and astrocytes.  Whereas others are  predominantly but not exclusively localized  to astrocytes (P2Y13: [94] P2Y14: [95]), oligodendrocytes (P2Y12: [96]) or microglia (P2Y12: [97]). However, immuncytochemical data should be handled with caution due to the lack of the verification of the specificity of many of the available antibodies. For detailed information on the distribution of individual P2Y receptor mRNAs and proteins we refer to recent reviews on this particular topic [98, 99].

The fast transmitter action of ATP

The principal function attributed to extracellular ATP is when activating postsynaptic P2X receptors; it acts as a fast excitatory neurotransmitter in neuro-neuronal and neuro-effector synapses. P2X receptor gated synaptic currents were identified in a number of synapses in the CNS and PNS. The first brain area where the fast transmitter function of ATP has been proven was the medial habenula [100]. Followed by the demonstration of ATP-mediated synaptic currents in other neuro-neuronal synapses between the submucosal  and celiac neurons [101, 102], enteric neurons [103], in the locus coeruleus nucleus of the brainstem [104, 105], lateral hypothalamus [106], rat trigeminal mesencephalic neurons [107], dorsal horn of the spinal cord [108], CA1 and CA3 region of the hippocampus [109] and the somatosensory cortex [110].

Nevertheless, it should be noted that almost two decades after the first demonstration of a purinergic synapse in the brain, the number of identified purinergic currents is still relatively limited. Moreover, purinergic currents could be recorded usually only in a proportion of cells. In general, a relatively strong stimulation paradigm and simultaneous blockade of the action of other excitatory and inhibitory neurotransmitters i.e. glutamate, GABA, acetylcholine and serotonin are also necessary conditions to observe purinergic currents [59, 111]. It is also unclear whether ATP acts in these synapses as a genuine co-transmitter released from common vesicles with its co-transmitter mate or an individual transmitter.

In contrast, the co-transmitter role of ATP is well established in neuroeffector synapses as it acts as a co-transmitter with noradrenaline at the sympathetic neuroeffector junction, which has been demonstrated in a number of tissue preparations, including the vas deferens, blood vessels, cutaneous microcirculation etc. [112]. ATP also acts as a co-transmitter with acetylcholine in certain transmission sites of the parasympathetic nerves, e.g. in the urinary bladder [113].

ATP as a neuromodulator

The presynaptic nerve terminal is an important regulatory site where the efficacy of synaptic transmission can be locally and efficiently controlled. Accordingly, axon terminals in the central nervous system and in the periphery are equipped with a wide variety of auto- and heteroceptors [114, 115]. Whereas presynaptic metabotropic receptors convey negative feedback regulation of transmitter release, presynaptic ionotropic receptors can amplify synaptic transmission. Co-activation of different presynaptic receptors therefore provides delicate fine-tuning mechanisms whereby different neurotransmitters and modulators could influence each other’s and their own actions. Moreover, presynaptic and extrasynaptic receptors controlling transmitter release also offer attracting target sites for existing and future pharmacotherapy, as they can modify the normal and pathological synaptic information processing without all-or-none actions [116].

Since ATP and its related nucleotides are ubiquitous signaling molecules, it is not surprising that their receptors, i.e. ionotropic P2X and metabotropic P2Y receptors participate both in the negative and positive feedback modulation of neurotransmitter release. Although originally it was suggested that ATP-sensitive P2 receptors are located exclusively on postsynaptic sites, presynaptic P2 receptors have already been identified in the early ‘90’s on cholinergic nerve terminals of the guinea-pig [117]. Later, P2X receptors were identified to be responsible for the facilitation of acetylcholine release [118].

Since P2X receptors have relatively high Ca2+ permeability [119, 120] their activation could also directly elicit transmitter release without preceding action potential and subsequent activation of voltage sensitive Ca2+ channels by Ca2+ influx through the receptor ion channel complex, provided that they are located nearby the release sites.  Activation of P2X receptors in this way elicits noradrenaline release from sympathetic nerve terminals [121-123] and from central noradrenergic varicosities innervating the hippocampus [82]. In addition, P2 receptors enhance the release of serotonin  and dopamine from  different parts of the brain  [124], [125], [126] in vivo, although the latter effects are thought to be mediated by P2Y receptors.

The release of other transmitters, including the main excitatory and inhibitory transmitters of the brain, is also subject to facilitation by presynaptic P2X receptors.  Activation of P2X receptors elicits glutamate release in the spinal cord [127], brainstem [128] and hippocampus [89, 129]. As for the underlying receptor subunits involved in these effects, P2X2, P2X7, as well as P2X1, P2X3 and P2X2/3 receptors were identified in different synapses. On the other hand, the regulation of GABA release by P2X receptors seems to be more restricted, with the exception of spinal cord  [130] and the brainstem, where the excitatory and inhibitory synaptic transmission is subtype specifically facilitated, via P2X3 and P2X1 receptors, respectively [131]. Finally, the release of another inhibitory transmitter, glycin, is augmented by P2X receptor activation in the dorsal horn [132] and in the brainstem trigeminal nucleus [133].

In addition to facilitatory modulation, P2 receptors are also involved in the inhibitory modulation of the release of various transmitters and metabotropic P2Y receptors, which are engineered to act on a longer time scale and play a major role in these actions.  Hence, in the CNS,  ATP inhibits the release of acetylcholine [134], noradrenaline [135, 136], serotonin [137], dopamine [138], and glutamate [83] release, whereas the release of GABA is not subject to inhibitory neuromodulation by P2 receptors. Convincing evidence is also available on similar modulation of acetylcholine and noradrenaline release in the periphery (for further references, see [139].

The role of ATP in glia-neuron and  glia-glia signaling

In addition to its role as a fast transmitter and as a presynaptic modulator, rapidly emerging data indicate that ATP is an important signaling molecule in the communication between glia and neurons and within glial networks. Although glial cells were traditionally regarded as a simple support for the neuronal networks, it has now become clear that they are more active players in synaptic transmission [140, 141].  Thus, there is a bidirectional communication between neurons and glial cells, and  not only the glia responds to signals originating from neurons, but it could also release transmitters, which then act on cell surface receptors present on the neuronal membrane and modulate synaptic activity pre- and postsynaptically. Among these gliotransmitters, ATP seems to be one of the most important, in addition to glutamate and other amino acids [142].

Mechanical or non-NMDA receptor-mediated stimulation of astrocytes leads to the generation of Ca2+ waves in astrocytes, which spread by the release of ATP and subsequent activation of P2 receptors and lead to the depression of excitatory synaptic transmission by P2Y and  A1 adenosine receptors, respectively [143, 144], [145]. On the other hand, astrocytic ATP is also involved in the modulation of inhibitory transmission, by the excitation of inhibitory interneurons via P2Y1 receptors, which leads to an increased synaptic inhibition within intact hippocampal synaptic networks [146] and to long term synaptic plasticity events [147]. In addition to presynaptic modulation, glial derived ATP also causes enduring changes in postsynaptic efficacy:  in the hypothalamic paraventricular nucleus, noradrenaline, acting on α1-adronoceptors, releases ATP from astrocytes which then act on P2X7 receptors enhancing excitatory transmission postsynaptically by the activation of phosphatidylinositol 3-kinase and subsequent insertion of AMPA receptors to the cell membrane [148].

In addition to its mediator role between glial cells and neurons, ATP is also the primary mediator of the extracellular communication between astrocytes [149]. Astrocyte populations coordinate their functions via Ca2+ waves and the spread of the Ca2+ signal is implemented in two ways: an intercellular pathway mediated by gap junctions, and an extracellular pathway mediated by ATP and P2 receptors [149]. Astrocytes communicate by calcium-mediated signaling not only with each other but also with neighboring cells including neurons (see above, [143]) and microglia. Thus, astrocyte-derived ATP activates P2X7 receptors on microglial cells and elicits Ca2+ signals in the microglia, which eventually leads to cytolysis of this cell type [27].

Purinergic signaling at the brain-immune interface: modulation of the microglia response by P2 receptors (Figure 2)

Figure 2. The role of purinergic mechanisms in microglia activation ATP is released to the extracellular space in response to a wide variety of signals, including neuronal firing, mechanical stimuli, ischemia/energy deprivation, bacterial endotoxin and cellular damage from nerve terminals, astrocytes, and the microglia itself. ATP acts on ionotropic P2X7-, P2X4-, and on metabotopic P2Y receptors, all present on the surface of activated microglia. Whilst the activation of P2X receptors triggers an inward cationic current and depolarizes the microglial membrane, the activation of P2Y receptors is coupled to the G protein-phospholipase C (PLC) signal transduction pathway, both resulting in the elevation of intracellular Ca2+. P2X receptors are involved in the expression, posttranslational processing and secretion of various factors, i.e. IL-1β, IL-6, IL-18, TNF-alpha, reactive oxygen intermediates (ROI), plasminogen, 2-arachydonoyl glycerate (2-AG), and microglial response factor-1 (MRF-1) and thereby shaping both the proinflammatory and antiinflammatory aspects of microglial activation. In addition, P2X7 receptors eventually also mediate apoptosis by caspase 1 activation or necrosis. The stimulation of P2Y receptors hyperpolarizes the microglia, via an outward K+ conductance and inhibits the production of proinflammatory mediators.

Microglial cells originate from monocyte/macrophage precursors and are regarded as the major immunocompetent cell type of the nervous system, constituting about 10% of all cells in the brain. The immune response of the brain is spatially segregated from the peripheral immune response by the blood brain barrier and together with astroglial cells and infiltrating peripheral immune cells, is predominantly executed by microglial cells.

This cell type is rapidly activated in response to pathological signals such as ischemia or bacterial endotoxin and responds with morphological changes transforming the resting ramified microglia to an amoeboid form with phagocytic activity, proliferation and the production of a wide array of inflammatory mediators. Although microglial activation is a highly complex process consisting of a number of interrelated extra- and intracellular pathways, it is a rather uniform response, triggered by any environmental challenge, which affects the functional integrity of the nervous tissue. Therefore, microglial activation is heavily implicated in the pathogenesis of virtually all CNS diseases and the following repair process, including brain and spinal cord injury, stroke, Alzheimer’s (AD) and Parkinson’s disease (PD), sclerosis multiplex (SM), amyotrophic lateral sclerosis (ALS) and sensory neuropathies. Besides cytokines, growth factors and other bioactive substances, nucleotides are crucial mediators, which regulate and orchestrate various aspects of microglial activation by the interaction of different subtypes of P2 receptors.

It has been well known for a long time that microglial cells respond with both ionotropic and metabotropic actions to ATP application [150]. Whereas the activation of ligand-gated P2X receptors produces depolarization and Ca2+ influx through the receptor-ion channel complex, the stimulation of the G protein coupled P2Y receptors elicits hyperpolarization via phospholipase C (PLC) and IP3 mediated mobilization of Ca2+ from intracellular stores and subsequent opening of K+ channels [151]. Data confirms that all members of the P2 receptor family are expressed on resting and activated microglial cells at the mRNA and/or protein level [152, 153].

Among various subtypes of the ionotropic P2 receptors, the role of P2X7 receptors in the microglial response is especially well delineated (Figure 2). Because P2X7 receptors are expressed predominantly on antigen-presenting immune cells and epithelia, they have been early postulated to function as immunomodulatory receptors [86, 154]. Supporting this concept, a wealth of data confirms that P2X7 receptors regulate many aspects of immune response in the periphery [155] but this holds true for microglial activation as well [156].

The microglia respond to P2X7 receptor activation  with an inward current, membrane depolarization, a sustained increase in intracellular free Ca2+ and the uptake of high molecular weight fluorescent dyes. Moreover, the central role of P2X7 receptors of the posttranslational processing of IL-1beta in microglial cells has been repeatedly proven [157-159]. The P2X7 receptor  activation provides the necessary co-stimulus for the cleavage of pro-IL-1beta and subsequent release of mature IL-1beta following  LPS challenge [159]. This mechanism appears to participate not only in the exogenous but also in the endogenous activation of P2X7 receptors, since LPS releases ATP from microglial cells, and the P2X7 receptor selective antagonist oxiATP prevents the LPS-induced IL-1beta release [34].

In addition to IL-1beta, the synthesis and release of other cytokines are also stimulated by P2X7 receptor activation in the microglia. Hence, ATP is a full stimulus (i.e. without the requirement of priming by LPS) to induce TNF-alpha production via a Ca2+ dependent, extracellular signal regulated protein kinase (ERK)/JNK/p38 signaling pathway [160, 161]. P2X7 receptors might also be involved in the regulation of the production of the antiinflammatory cytokine, interleukin-6 (IL-6) [162-164]. Moreover, P2X7 receptors play a role in the distinct modulation of cytokine secretory pathways not only after LPS, but also upon amyloid beta peptide (Abeta) pre-activation [165]. In contrast, when the production of IL-1beta, IL-1alpha, TNF-alpha and IL-18 was increased,  IL-6, the anti-inflammatory cytokine, was attenuated under these conditions implicating the involvement of P2X7 receptors in the pathogenesis of Alzheimer’s disease [165].

High concentrations of ATP induce  or enhance inducible nitric oxide synthase (iNOS) mRNA expression and increase nitric oxide (NO) production from rat [166] and murine [167] microglia, an effect potentially mediated by P2X7 receptors.  Activation of P2X7 receptors also promotes the generation of reactive oxygen intermediates (ROI), in particular superoxide, in a way depending on extracellular Ca2+ and the p38 mitogen activated protein kinase (MAPK) pathway [168]. The stimulation of P2X7 receptors elicits a pronounced increase in the secretion of the endocannabinoid, 2-arachydonoylglycerate (2-AG) in primary microglial cell cultures by the activation of diacylglycerol lipase and the simultaneous inhibition of monoacylycerol lipase, the enzyme responsible for endocannabinoid degradation [169]. This mechanism is noteworthy because endocannabinoids are also recognized as key regulators of microglial activation [170]. Finally, activation of P2X7 receptors stimulates the release of the neuroprotective mediator plasminogen from cultured microglia [171]. Therefore, regulation of the production of putatively protective (plasminogen, TNF-alpha, 2-AG) and harmful (IL-1beta, NO) mediators by P2X7 receptors appears to follow a strict time- and concentration-dependent pattern [163].

In addition to its role to regulate the production of inflammatory mediators, the activation of P2X7 receptors elicits changes in microglia at the transcriptional level; it rapidly activates the transcription factor nuclear factor of activated T cells (NFAT) in a [Ca2+]o dependent manner [172] as well as causes the nuclear translocation of NF-κB via ROIs and caspase activation leading to the transcription of a subset of NF-κB target genes. The expression of other transcription factors such as MRF-1, are also regulated time-dependently upon P2X7 receptor activation [173, 174]. This mechanism plays a role in the neuron-microglia cross-talk during microglial activation in response to apoptotic signals [174].

According to its pore-forming property, the sustained activation of P2X7 receptors leads to cytolysis in an apoptotic fashion in the microglia [175]. The P2X7 receptor-mediated apoptosis involves the activation of the proteolytic pathway of the caspase activation, which leads to nuclear DNA damage, but is not an absolute requirement for the membrane damage and cytolysis, i.e. if caspases are inhibited, cell death proceeds through the necrotic pathway [172]. Nevertheless, the P2X7 receptor activated IL-1beta secretion and cell death, although both processes involve caspases and both are eliminated in P2X7-/- mice, appear to be independent from each other [157].

Finally, P2X7 receptor activation also plays an important role in Ca2+ signaling between astrocytes and microglial cells [27]. Thus astrocyte-derived ATP activates P2X7 receptors on microglial cells and elicits Ca2+ signals in the microglia, a process which eventually leads to cytolysis of this cell type [27]. It is interesting to note that P2X7 receptor-activation in astrocytes usually does not lead to cytolysis, while the same effect might cause cellular death in microglia. The reason for the different resistance of astrocytes and microglia against the P2X7 receptor mediated cytolysis is unknown.

The widespread and profound effects of P2X7 receptor activation on different aspects of microglial activation implicate the role of these receptors in the pathology of CNS and PNS diseases. Rapidly emerging knowledge supports such a role, in particular the demonstration of (1) the activity–dependent expression of P2X7 receptors under pathological conditions, and (2) the protective role of P2X7 receptor ligands in animal disease models. The diseases, in which microglial P2X7 receptors may play either a harmful or protective function, include neurodegenerative and neuroinflammatory diseases, such as ischemia-reperfusion and traumatic injury, Alzheimer’s disease, sclerosis multiplex, neuro- and retinopathies. In fact, the upregulation of microglial P2X7 receptors has been observed in several pathological models, including in vivo ischemia [176, 177]; around amyloid plaques in a transgenic model of Alzheimer’s disease [168], as well as in human tissue samples obtained from patients suffering in proliferative vitreoretinopathy [178], and sensory nerve injury [162].These data indicate that the expression of P2X7 receptors is strongly activity-dependent during pathological situations.

Nonetheless, whether the change in their expression pattern and functional responsiveness is a simple adaptive change or plays a more active pathogenetic role warrants further investigation. It is also a largely open question whether the final outcome of versatile actions mediated by microglial P2X7 receptors is protective or harmful. As an example, P2X7 receptor deficient mice show increased susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model of sclerosis multiplex [179], which is potentially due to the decreased microglial secretion of the protective endocannabinoid 2-AG [169]. On the other hand, chronic inflammatory and neuropathic pain is almost completely abolished in that same murine line, which is attributed partly to disrupted microglial production of the inflammatory cytokine IL-1beta [162]. A decreased vulnerability upon treatment with specific P2X7 receptor antagonists are also reported in acute spinal cord injury [180] and Huntington’s disease [181].  However, specific P2X7 receptor mediated cellular actions underlying these latter actions remains to be investigated.

In addition to P2X7 receptors, other subtypes of the P2 receptor family, responding to lower concentration of ATP, are also involved in different aspects of microglial activation [182]. Thus, microglial Ca2+ influx could be also initiated by lower agonist concentrations, effects which are associated to a non-P2X7 ionotropic, probably P2X4 receptor activation, but there is also a metabotropic, P2Y receptor mediated response [182]. The expression of P2X4 receptors is enhanced in the spinal cord microglia after peripheral nerve injury and this receptor and the subsequent activation of the p38 MAP kinase pathway have been shown to be involved in the generation of inflammatory and neuropathic pain [183, 184].

On the other hand, P2Y1 receptors mediate the inhibition of the LPS induced IL-1beta and TNF-alpha production [185]. The level of proinflammatory cytokines therefore seems to be differentially and oppositely regulated by ionotropic and metabotropic P2 receptors. The production of the anti-inflammatory cytokine IL-6 upon LPS challenge is inhibited by P2Y receptors [185] although the involvement of pertussin toxin insensitive P2Y receptors in the induction of IL-6 production has also been reported [186]. In addition, stimulation of a Gi protein coupled P2Y12-like receptor induces membrane ruffling and chemotaxis in cultured primary microglia [187]. Finally, the activation of P2Y but not P2X receptors induces cyclooxigenase-2 (COX-2) expression in the human microglia [188].

Conclusions

It is now clear that ATP is an important signaling molecule in the nervous system and is involved in a wide variety of neuronal functions including synaptic transmission, neuromodulation, glia-neuron interactions and neuroimmunomodulation. Therefore, the initial idea of a ‘purinergic nervous system’ has developed into a more diverse concept of a ‘purinergic signaling system’.  Nevertheless, there are a number of aspects which need further investigation. Despite the wealth of data on ATP mediated signaling at the molecular and cellular level, present knowledge is still limited at the network level.  This holds true to any aspects of purinergic mechanisms including the release and inactivation mechanisms and ATP-receptor mediated responses at the brain-immune interface. A new and interesting extension of current research is expected on the behavioral translation of ATP-mediated responses, including those which convey the influence of immune system on cognition and mood.  Progress on this may lead to the better therapeutic utilization of a purinergic signaling system, which offers a number of potential target sites for pharmacological intervention.

Acknowledgments

This study was supported by grants from the Hungarian Research and Development Fund (NN79957) and the Hungarian Medical Research Council (ETT 05-102).

Nonstandard Abbreviations: (2-AG), 2-arachydonoylglycerate; (AD), Alzheimer’s disease; (ALS), amyotrophic lateral sclerosis; (ABC), ATP binding cassette; (COX-2), cyclooxigenase-2; (CysLTE4), cysteinyl-leukotrienes; (E-NTPDase), ectonucleoside triphosphate diphospho-hydrolase; (E-NPP), ecto-nucleotide pyrophosphatase; (EAE), experimental autoimmune encephalomyelitis; (ERK), extracellular signal regulated protein kinase; (GFAP), glial fibrillary acidic protein; (iNOS), inducible nitric oxide synthase; (IMP), inosine monophosphate; (IL), interleukin; (MAPK), mitogen activated protein kinase; (NO), nitric oxide; (NFAT), nuclear factor of activated T cells; (NTS), nucleus tractus solitarii; (PD), Parkinson’s disease; (PLC), phospholipase C; (ROI), reactive oxygen intermediates; (SM), sclerosis multiplex; (VNUT), vesicular nucleotide transporter

Author(s) Affiliation

B Sperlágh – Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary
C Csölle – Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary

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