Brain-Derived Neurotrophic Factor from Microglia: A Molecular Substrate for Neuropathic Pain

Abstract

One of the most significant advances in pain research is the realization that neurons are not the only cell type involved in the etiology of chronic pain. This realization has caused a radical shift from the previous dogma that neuronal dysfunction alone accounts for pain pathologies, to the current framework of thinking that takes into account all cell types within the central nervous system (CNS). This shift in thinking stems from growing evidence that glia can modulate the function and directly shape the cellular architecture of nociceptive networks in the CNS. Microglia, in particular, are increasingly recognized as active principal players that respond to changes in physiological homeostasis by extending their processes toward the site of neural damage, and by releasing specific factors that have profound consequences on neuronal function and that contribute to CNS pathologies caused by disease or injury. A key molecule that modulates microglia activity is ATP, an endogenous ligand of the P2 receptor family. Microglia express several P2 receptor subtypes, and of these the P2X4 receptor subtype has emerged as a core microglia-neuron signaling pathway: activation of this receptor drives the release of brain-derived neurotrophic factor (BDNF), a cellular substrate that causes disinhibition of pain-transmitting spinal lamina I neurons. Converging evidence points to BDNF from spinal microglia as being a critical microglia-neuron signalling molecule that gates aberrant nociceptive processing in the spinal cord. The present review highlights recent advances in our understanding of P2X4 receptor-mediated signaling and regulation of BDNF in microglia, as well as the implications for microglia-neuron interactions in the pathobiology of neuropathic pain.

INTRODUCTION

Acute nociceptive pain is instructive and warns against imminent or existing tissue damage, whereas chronic pain has no known defensive or beneficial function and remains one of the most vexing challenges in medicine. In the United States alone, the direct economic costs of acute and chronic pain conditions are estimated to cost $560–635 billion annually (Institute of Medicine Report, 2011). Although major advances have been made towards understanding the fundamental processes that underlie acute pain, little is known about the cellular and molecular processes initiated by acute pain that lead to the progression to chronic pain, which is characterized by pain persisting long after the tissue damage has healed. Paradoxically, the degree of injury is not necessarily predictive of the severity or chronicity of the pain, and the development of chronic pain following injury is highly variable between individuals. Considerable evidence suggests that transition from acute to chronic pain is a consequence of alterations in the cellular, molecular, and anatomical organization of nociceptive neural networks in the spinal dorsal horn and brain (Latremoliere and Woolf, 2009;Scholz and Woolf, 2002;Voscopoulos and Lema, 2010;Woolf and Salter, 2000). Thus, an acute injury can have profound effects on the nociceptive neural circuitry, transforming the CNS from conveying normally protective acute nociceptive signals into a pathologically altered system in which pain can occur spontaneously and responses to innocuous and noxious stimuli are amplified.

Among the most severe and debilitating types of chronic pain conditions is neuropathic pain which can arise following lesions to the somatosensory nervous system caused by trauma, infection, or pathology (Scholz and Woolf, 2002;Zimmermann, 2001;Gwak and Hulsebosch, 2009). Such damage to a nerve can instigate a series of cellular and molecular changes that directly affect neuronal plasticity, leading ultimately to altered synaptic connectivity and the reorganization of peripheral and central nociceptive circuitry (Scholz and Woolf, 2002;Woolf and Salter, 2000;Latremoliere and Woolf, 2009). These changes have profound effects on pain transmission by suppressing mechanisms that inhibit pain and by enhancing mechanisms that facilitate pain in the central nervous system (CNS). The consequent shift in inhibitory and excitatory control can cause a pathological amplification that alters the modality of sensory input and output from the spinal cord to elicit exaggerated pain responses that typifies chronic pain conditions (Costigan et al., 2009).

A principal locus of this pathologically altered activity arises from the nociceptive neurons in lamina I of the spinal dorsal horn. The action potential discharge of these neurons is normally evoked only in response to noxious peripheral stimulation (Keller et al., 2007). However, after peripheral nerve injury the output of lamina I neurons is transformed such that innocuous stimulus can evoke action potential discharges, and response to noxious stimulation is greatly exaggerated. In addition, after peripheral nerve injury lamina I neurons exhibit spontaneous bursting in the absence of overt stimulation whereas in uninjured animals these neurons are normally silent. These changes in the output of lamina I neurons provide a neural basis for the three cardinal signs of neuropathic pain in humans (Woolf and Salter, 2000): mechanical allodynia (discharge in response to innocuous stimulation), hyperalgesia (exaggerated response to noxious stimulation), and spontaneous pain (spontaneous bursting in the absence of an overt stimulus). The symptoms of neuropathic pain are often resistant to the current available treatments, which have typically been directed against cellular targets in neurons. Failure of the current cadre of drugs in treating the sequelae of neuropathic pain has intensified the search for new molecular players and cellular substrates that can be targeted for development of novel pharmacological therapies. This search has generated a rapidly growing body of evidence that indicates interactions between neurons and glia are critical in establishing and maintaining neuropathic pain (Beggs and Salter, 2010;Grace et al., 2011;Inoue and Tsuda, 2006;Milligan and Watkins, 2009;Gwak and Hulsebosch, 2010). Microglia in particular, have emerged as key players in the initiation and in the expression of neuropathic pain (Inoue and Tsuda, 2006;Trang et al., 2011;Tsuda et al., 2003;Tsuda et al., 2005;Watkins et al., 2001;Watkins and Maier, 2003).

Spinal microglia respond to peripheral nerve injury

Microglia originate from embryonic macrophages derived from the yolk sac during late prenatal development (Ginhoux et al., 2010). In the adult CNS, microglia comprise 5–10% of the total glial population (Kreutzberg, 1996;Lawson et al., 1990;Nakajima and Kohsaka, 2001). Likened to the electricians of the CNS, microglia are able to modulate the electrical activity within neuronal circuits (Graeber, 2010). In the surveillance mode of activity, microglia possess small soma bearing thin ramified processes that cover large non-overlapping territories throughout the brain and spinal cord (Bushong et al., 2002;Kreutzberg, 1996). In response to stimuli that potentially disrupt homeostasis, microglia rapidly extend their processes toward the site of neural damage, forming a barrier between healthy and injured cells that limits damage in the CNS (Davalos et al., 2005;Nimmerjahn et al., 2005). The rapid microglia responsiveness precedes a more slowly developing series of changes in morphology, gene expression, function, and proliferation (Hanisch and Kettenmann, 2007;Kettenmann et al., 2011;Kreutzberg, 1996;Nakajima and Kohsaka, 2001). These changes are observed in spinal microglia in rodent models of peripheral nerve injury caused by compression, ligation, or transaction (Calvo and Bennett, 2011;Calvo et al., 2011;Echeverry et al., 2008;Liu et al., 1995;Tsuda et al., 2003;Zhang and De Koninck, 2006). The stereotypical microglial response also entails the upregulation of surface marker proteins belonging to the complement cascade: complement receptor 3 (CR3), Toll-like receptor 4 (TLR4), CD14, CD4, and major histocompatibility complex (MHC) class I and II (Coyle, 1998;Liu et al., 1995;Sweitzer et al., 2002;Tanga et al., 2004;Tsuda et al., 2003). Upregulation of these surface proteins concomitant with distinct anatomical changes in morphology – from ramified to ameboid – are stereotypical of microglia in the ‘enhanced response state’. Although microglia in this state have been reported to release of a myriad of factors that signal to neurons in the spinal cord to alter neuronal excitability, in pain neuroplasticity after peripheral nerve injury a specific core pathway has been implicated through P2X4 receptor-stimulated release of BDNF (Beggs and Salter, 2010;Trang et al., 2011).

Spinal microglia express P2 receptors

A critical molecular substrate for microglia-neuron communication is ATP, an endogenous ligand of the P2 receptor family comprising of P2Y metabotropic and P2X ionotropic receptors (Coull et al., 2005;Di, 2006;Jarvis, 2010;Maeda et al., 2010). Microglia express a variety of P2 receptors: of the metabotropic P2Y receptors, microglia express P2Y1, 2, 4, 6, and 12 receptors (Boucsein et al., 2003;Farber and Kettenmann, 2005;Inoue and Tsuda, 2006;Sasaki et al., 2003), whereas microglial expression of the ionotropic P2X receptor is restricted to the P2X4 and P2X7R subtypes (Inoue and Tsuda, 2006;Collo et al., 1997;Ferrari et al., 1996;Tsuda et al., 2003). Despite the diverse repertoire of P2Y receptors expressed on microglia, only P2Y12 receptors have been demonstrably implicated in the development of tactile allodynia associated with peripheral nerve injury (Kobayashi et al., 2008;Tozaki-Saitoh et al., 2008). By contrast, both P2X4 and P2X7 receptors are causally involved in expressing nerve injury-induced pain behaviours (Chessell et al., 2005;Coull et al., 2005;Kobayashi et al., 2008;Tozaki-Saitoh et al., 2008;Tsuda et al., 2003;Tsuda et al., 2009a;Tsuda et al., 2009c;Ulmann et al., 2008).

A compelling argument has been made for involvement of P2X7 receptors in inflammatory pain and in neuropathic pain on the basis of reduced pain sensitivity in P2X7 receptor-deficient mice (Chessell et al., 2005) and after pharmacological blockade of the receptor (Broom et al., 2008;Dell’Antonio et al., 2002;Honore et al., 2006;Honore et al., 2009;McGaraughty et al., 2007;Perez-Medrano et al., 2009). Recent evidence has also established a linked between genetic variations in the P2X7 receptor that affect its function and variability in chronic pain sensitivity in mice and in humans (Sorge et al., In Press). The specific cell type(s) responsible for the P2X7 receptor mediated pain phenotypes has been difficult to precisely pinpoint because, in addition to being expressed on microglia, P2X7 receptors are localized on macrophages, neurons, and astrocytes (Donnelly-Roberts et al., 2008;Donnelly-Roberts and Jarvis, 2007;Skaper et al., 2010). Thus, conclusions about the specific role microglial P2X7Rs play in inflammatory and neuropathic pain is confounded by expression of this receptor on a wide variety of cell types. By contrast, much of our conceptual understanding of the role microglial P2 receptors play in neuropathic pain stems from elucidating the fundamental mechanisms that regulate P2X4 receptor expression, as well as the identification of signaling pathways downstream from this receptor.

Microglial P2X4 receptors are critically required for neuropathic pain

The first compelling clues that identified P2X4 receptors as a critical molecular component in neuropathic pain were the discoveries that blocking P2X4 receptor function pharmacologically or suppressing its expression with antisense RNA transiently reverses mechanical allodynia (Tsuda et al., 2003). The development of mechanical allodynia was found to correlate temporally with an increase in spinal P2X4 receptor expression, and unexpectedly this increase was confined to microglia in the ipsilateral spinal dorsal horn. These observations were subsequently confirmed in CX3CR1^+/GFP mice, in which induction of P2X4 receptors resulting from peripheral nerve lesion is restricted to activated eGFP expressing spinal microglia (Ulmann et al., 2008), and in mice lacking the P2X4 receptor, which do not develop mechanical allodynia after peripheral nerve injury (Tsuda et al., 2009a;Ulmann et al., 2008). Although neuropathic pain behaviours in the P2X4 receptor deficient mice are absent, the microglial proliferative response and the alterations in microglia morphology induced by peripheral nerve injury were not affected (Tsuda et al., 2003; Ulmann et al., 2008), suggesting that while tonic P2X4 receptor activation is required for maintaining peripheral nerve injury-induced allodynia, the proliferation and upregulation of microglial P2X4 receptors in the spinal cord are mediated by distinct intracellular mechanisms. Direct evidence that stimulation of P2X4 receptors expressed on microglia is sufficient to elicit pain hypersensitivity comes from the finding that injection of P2X4 receptor-stimulated cultured microglia into the spinal cords of naïve animals elicits robust mechanical allodynia that is blocked by 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) (Coull et al., 2005;Tsuda et al., 2003;Tsuda et al., 2008b). Taken together, the pharmacological, genetic, and behavioral findings indicate that activity of P2X4 receptors expressed on spinal microglia is critically involved in the functional alterations in the spinal dorsal horn that maintain ongoing pain following peripheral nerve injury.

Regulation of P2X4 receptor expression in microglia

A major question arising from the observation that development of mechanical hypersensitivity is correlated with a progressive increase in spinal P2X4 receptor expression is how peripheral nerve injury initiates signalling in the spinal dorsal horn to specifically cause an increase in P2X4 receptor expression in microglia. The answer to this question appears to involve the release of several signalling elements including: CCL21, a chemokine released from injured neurons that functions as an upstream activator of P2X4 receptor (Biber et al., 2011;de Jong et al., 2005), interferon γ, a cytokine that transforms resting spinal microglia into an activated state (Tsuda et al., 2009b), and tryptase, a protease released from mast cells that activates proteinase-activated receptor 2 in microglia (Yuan et al., 2010). Also critical for upregulating expression of P2X4 receptors is the extracellular matrix molecule fibronectin, which through activity of Lyn kinase and downstream activation of intracellular signalling pathways involving phosphatidylinositol 3-kinase (PI3K)-Akt and mitogen-activated protein kinase kinase (MAPK kinase, MEK)-extracellular signal-regulated kinase (ERK), modulates the transcriptional and post-transcriptional levels of P2X4 receptor expression in microglia (Nasu-Tada et al., 2006;Tsuda et al., 2008a;Tsuda et al., 2008b;Tsuda et al., 2009c). Thus, several elements of the molecular machinery required for upregulation of P2X4 receptors in microglia following peripheral nerve injury have recently been identified (Figure 1). The implications of this diverse modulation and whether they are causally connected through a convergent common pathway that controls P2X4 receptor expression is not known.

Figure 1.

Dynamic regulation of P2X4 receptors in microglia. Microglia in the physiological CNS actively monitor their surrounding environment for potential stimuli that threaten homeostasis. In response to peripheral nerve injury spinal microglia upregulate expression of P2X4 receptors, which normally are expressed at low levels in the ‘resting/surveillance state.’ Upregulation of P2X4 receptors is a critical mechanistic step through which spinal microglia signal to neurons in the spinal dorsal horn to cause neuropathic pain. Activation of P2X4 receptors initiates the p38 MAPK-BDNF-KCC2 signalling cascade to cause aberrant nociceptive output that underlies pain hypersensitivity characterized by hyperalgesia, allodynia, and spontaneous pain. Molecules released from injured neurons, such as the chemokines CCL2 and CCL21, as well as the cytokine IFN-γ, increase P2X4 receptor expression in microglia. The fibronectin-Lyn kinase signalling cascade and tryptase released from mast cells have also been found to upregulate P2X4 receptors in microglia. Thus, the P2X4 receptor and the components of its signalling pathway have emerged as a core mechanism through which spinal microglia contribute to neuropathic pain.

Significant inroads have also been made in understanding the intracellular trafficking of P2X4 receptors. It is now known that the proportion of P2X4 receptors on the cell surface is regulated by rapid constitutive internalizationand reinsertion into the plasma membrane (Bobanovic et al., 2002;Fujii et al., 2011;Royle et al., 2002;Toulme et al., 2006). Internalization is controlled by the C-terminus of the P2X4 receptor (Fujii et al., 2011;Qureshi et al., 2007;Royle et al., 2002), a region also important for agonist-induced desensitization (Fountain and North, 2006) and phosphoinositide PIP₂ modulation of P2X4 receptor function (Bernier et al., 2008). In microglia, internalized P2X4 receptors are targeted to lysosomes which contain a large pool of P2X4 receptors. Mobilization of P2X4 receptors contained within these lysosomes to, as well as the retrieval of these receptors from, the plasma membrane regulates the proportion of P2X4 receptors expressed on the cell surface (Qureshi et al., 2007). Dynamic targeting of P2X4 receptor containing lysosomes in microglia can be induced by the chemokine CCL2, acting via the CCR2 chemokine receptor (Toyomitsu et al., 2012). Moreover, a rise in intracellular Ca²⁺ is sufficient to trigger lysosome exocytosis that leads to accumulation of P2X4 receptors on the cell surface and enhancement of P2X4 receptor-mediated currents (Qureshi et al., 2007).

Activation of cell surface P2X4 receptors by ATP causes structural changes that result in two distinct conformations – a prototypical cation channel or a macropore structure. In the presence of extracellular Ca²⁺, ATP stimulation induces the P2X4 receptor to transiently open a non-selective cation permeable channel, but in the absence of extracellular Ca²⁺ the receptor forms a macropore that allows passage of large molecules (Shinozaki et al., 2009). P2X4 receptors expressed in microglia possess this ability to function both as a cation channel and as a macropore (Bernier et al., 2010;Seil et al., 2010); however, the implication of this dual mode of functioning in the context of neuropathic pain remains an intriguingly open question. The discovery of two distinct P2X4 receptor modes of function, in addition to identification of the P2X4 receptor crystal structure, have illuminated many atomic details about its extracellular domain structure, the putative ATP binding site, transmembrane regions, and ion permeation pathway (Kawate et al., 2009).

P2X4R activation drives release of brain-derived neurotrophic factor

Another key question arising from the discovery that microglial P2X4 receptors are critically involved in neuropathic pain is – how does P2X4R signaling in microglia affect nociceptive processing in the spinal dorsal horn? It was reasoned that P2X4 receptors in microglia must initiate signalling that is communicated to neurons in the spinal nociceptive network which then relay the information to the brain (Tsuda et al., 2003;Tsuda et al., 2005). It was predicted that such signaling could occur through the release of one or more diffusible chemical messengers from microglia upon stimulation of P2X4 receptors. The components of the microglia-neuron signalling pathway was elucidated by the discovery that activation of P2X4 receptors in microglia evokes the release of brain-derived neurotrophic factor (BDNF) which fundamentally alters the output of spinal lamina I neurons to the brain (Coull et al., 2005;De Koninck, 2007;Keller et al., 2007). BDNF was found to down-regulate expression of the K⁺-Cl⁻ co-transporter KCC2, the main Cl⁻ transporter in spinal lamina I neurons, causing a rise in intracellular [Cl⁻] within these cells (Coull et al., 2003;Coull et al., 2005). With the increase in [Cl⁻], opening of GABA_A or glycine channels was less effective in producing inhibition, and in approximately one-third of lamina I neurons GABA-evoked responses were converted from hyperpolarizing to depolarizing (Coull et al., 2005).

Several key lines of evidence support BDNF as being the critical microglia-neuron signalling molecule: 1) disrupting BDNF-TrkB signalling with TrkB antibody, or sequestering BDNF with TrkB-Fc fusion protein, prevents mechanical allodynia evoked by administering P2X4 receptor-stimulated microglia; 2) siRNA knockdown of BDNF prevents the effects of intrathecally administered microglia on lamina I neurons and on the microglia-elicited pain behaviours; and 3) ATP activation of P2X4 receptors causes BDNF release from microglia in culture, a response prevented by TNP-ATP or by knocking down expression of BDNF with siRNA (Coull et al., 2005). Taken together, the most parsimonious explanation for these findings is that P2X4 receptor stimulated microglia signal to spinal lamina I nociceptive neurons causing aberrant spinal nociceptive processing, and that the critical microglia-neuron signaling molecule is BDNF. The requirement for P2X4 receptors in the release of BDNF is consistent with observations that P2X4 receptor-deficient mice have impaired microglial BDNF release, possess altered BDNF signaling in the spinal cord, and they are protected from developing mechanical allodynia following peripheral nerve injury (Ulmann et al., 2008). Indeed, there is overwhelming evidence for BDNF involvement in the initiation of central sensitization associated with neuropathic pain (Biggs et al., 2010;Lever et al., 2003;Lu et al., 2007;Obata et al., 2011). However, the previous conclusions that BDNF derived from primary afferent neurons is entirely responsible for spinal nociceptive hypersensitivity has been brought into question by evidence indicating that there is a lack of primary afferent evoked BDNF release in the spinal cord after nerve injury (Lever et al., 2003), and that eliminating BDNF from primary afferents suppresses inflammatory pain but has no effect on nerve injury-induced mechanical allodynia (Zhao et al., 2006). Together, these findings raise the possibility that neuronal-derived and microglial-derived BDNF have distinct roles – BDNF from neurons may be required for inflammatory pain, whereas BDNF from microglia may mediate neuropathic pain.

Microglial P2X4 receptors signal through Ca²⁺ and p38-MAPK

ATP stimulation of P2X4 receptors instigates a series of conformational changes that allow cations, such as Ca²⁺ and Na⁺, entry into the cell through a non-selective channel (Burnstock, 2006a;Burnstock, 2006b;North, 2002). In primary microglia culture, influx of extracellular Ca²⁺ is necessary for P2X4 receptor-stimulated release of pre-existing and newly synthesized BDNF (Trang et al., 2009;Ulmann et al., 2008). Ca²⁺-dependency is a hallmark of release by exocytosis, and in microglia, P2X4 receptor-stimulated release of BDNF involves a Ca²⁺- and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicular exocytotic pathway (Trang et al., 2009). This release mechanism is consistent with evidence that SNARE proteins SNAP-23, syntaxin-1, and cellubrevin are expressed in microglial cells (Hepp et al., 1999).

Ca²⁺-dependent release of BDNF is well-characterized in neurons and occurs from both pre-synaptic terminals and post-synaptic dendrites (Balkowiec and Katz, 2002;Kolarow et al., 2007). In nerve terminals, the release of BDNF is triggered by depolarization that depends upon Ca²⁺ influx through voltage-gated Ca²⁺ channels, and upon release of Ca²⁺ from intracellular stores (Buldyrev et al., 2006;Lever et al., 2003). Release of BDNF from post-synaptic dendrites is also mediated by influx of Ca²⁺, initiated by opening NMDA receptors or voltage-gated Ca²⁺ channels (Kolarow et al., 2007). By contrast, BDNF release from microglia appears to be independent of intracellular Ca²⁺ stores (Trang et al., 2009).

In addition to causing release of BDNF, P2X4 receptor-stimulated Ca²⁺ influx drives the transcription and translation of BDNF which results in accumulation of the neurotrophin in the microglia (Trang et al., 2009). BDNF was originally identified in neurons as an immediate-early gene (Lauterborn et al., 1996;West et al., 2001), transcription of which is initiated by Ca²⁺-influx through L-type voltage-gated Ca²⁺ channels or via NMDA receptors (Ghosh et al., 1994;Kolarow et al., 2007;Tao et al., 1998). Thus, Ca²⁺-dependent stimulation of BDNF transcription is common to neurons and microglia. In neurons, stimulus-evoked increase in Ca²⁺ activates key transcription factors that regulate the BDNF gene, such as cAMP response element binding protein (Tao et al., 1998), nuclear factor kappaB (Lipsky et al., 2001;Marini et al., 2004), methyl-CpG-binding protein 2 (Chen et al., 2003;Zhou et al., 2006), and calcium-responsive transcription factor (Tao et al., 1998). Whether the P2X4 receptor-stimulated increase in BDNF transcription in microglia involves these transcription factors, or others, is as yet undetermined.

In neurons, expression of BDNF is mediated by the extracellular signal-regulated protein kinase (ERK) and p38-MAPK signaling pathways (Ji and Woolf, 2001;Obata et al., 2004;Rao et al., 2007). Activation of these pathways in spinal microglia contributes to the development of neuropathic pain behaviors in nerve-injured rats (Jin et al., 2003;Tsuda et al., 2004). However, in primary microglia cultures isolated from the rat brain, pharmacological inhibitors of ERK have no effect on the release or the accumulation of BDNF, indicating ERK is not required in the P2X4 receptor-BDNF signaling pathway in microglia. By contrast, inhibiting activity of p38-MAPK prevents both the P2X4 receptor-evoked accumulation and release of BDNF (Trang et al., 2009). This finding suggests that activation of p38-MAPK by ATP is necessary for the BDNF response, and together with the Ca²⁺-dependence of p38-MAPK activation, the simplest explanation is that influx of Ca²⁺ through the P2X4 receptor is a critical step linking stimulation of these receptors to the p38-MAPK signalling pathway. By demonstrating that p38-MAPK is a cellular intermediary in the BDNF response, a unifying mechanism in microglia can account for ongoing expression of neuropathic pain behaviors as requiring activity of both P2X4 receptor and p38-MAPK.

Ca²⁺-dependent activation of p38-MAPK has been reported in a number of cell types (Blanquet, 2000;Dehez et al., 2001), including peripheral macrophages (Ulmann et al., 2010). Like microglia, macrophages are immune cells that constitutively express functional P2X4 receptors (Brone et al., 2007;Qureshi et al., 2007;Ulmann et al., 2010). A recent study has uncovered that stimulating P2X4 receptors expressed in macrophages triggers Ca²⁺ influx and p38-MAPK activation leading to the production and release of prostaglandin E2 (Ulmann et al., 2010), a principal substrate for peripheral inflammation (Portanova et al., 1996;Samad et al., 2002). p38-MAPK signalling therefore appears to gate the release of distinct signaling molecules from microglia (BDNF) and peripheral macrophages (prostaglandin E2) that have distinct roles in the pathoetiology of neuropathic pain and inflammatory pain, respectively.

Convergence of P2X7 and P2Y12 receptor activation on p38-MAPK signalling

In addition to P2X4 receptors, P2X7 and P2Y12 receptors expressed on microglia are increasingly implicated in neuropathic pain (Clark et al., 2007;Clark et al., 2010;Kobayashi et al., 2008;Tozaki-Saitoh et al., 2008). Activation of P2X7 receptors can cause release of interleukin-1β and cathepsin S, molecules which in the spinal cord may contribute to mechanical hypersensitivity following injury to a peripheral nerve (Clark et al., 2007;Clark et al., 2010). Analogous to P2X4 receptor-mediated release of BDNF, the release of interleukin-1β and cathepsin S from microglia requires P2X7 receptor signaling to p38-MAPK (Clark et al., 2010). Likewise, involvement of microglial P2Y12 receptors in neuropathic pain critically depends on p38-MAPK activation (Kobayashi et al., 2008); however, the signaling events downstream from P2Y12 receptor activation of p38-MAPK have yet to be elucidated. Collectively, these findings suggest that p38-MAPK is a cellular intermediary and its activation is a key point of convergence for P2X4, P2X7, and P2Y12 receptor signaling in neuropathic pain. The next major challenges are to determine the significance of this convergence in signalling for microglia function, and how the interplay of upstream and downstream components in these signalling pathways contributes to neuropathic pain.

Conclusions

Conventional neurocentric bias has focused almost entirely on neuron-to-neuron signalling as being the fundamental basis of neuropathic pain. However, this view fails to taken into account the diversity of cell types and the intricacies of cell-cell interactions in the CNS. In this respect, there has been a seismic shift towards understanding pathologies in the CNS as being a consequence of the complex interplay between diverse cell types involving multiple convergent and divergent signalling pathways. There is now a canon of literature detailing a plethora of molecular links between neurons and glia and their involvement in the pathogenesis of pain hypersensitivity arising from peripheral nerve injury. Microglia, in particular, have emerged as key cellular targets for treating neuropathic pain (Beggs and Salter, 2010;Calvo and Bennett, 2011;Scholz and Woolf, 2002;Trang et al., 2006). In the search for drugs directed against microglia, caution must be heeded in developing ‘microglia inhibitors’ with non-specific glial modulating properties given that microglia may play both a protective and a pathological role (Rivest et al., 2009; Milligan and Watkins, 2009). Rather than attempting to globally inhibit microglia, an approach might be to specifically target key microglia-neuron signalling pathways. As described above, the P2X4 receptor is a cellular hub through which microglia transform the nociceptive output of the spinal dorsal horn. Therefore, strategies directed against the P2X4 receptor signalling pathway is a high value target for the potential treatment of neuropathic pain caused by peripheral nerve injury. Moreover, elucidation of the core P2X4 receptor signalling pathway opens the possibility that this pathway, or its components, may be a common mechanism underlying other pathological pain states in addition to neuropathic pain.

Major advances have been made in identifying the essential molecular components that regulate P2X4 receptor expression and the cellular machinery that drives release of microglial BDNF, a critical substrate for neuropathic pain (Figure 2). These discoveries have built a complete framework for understanding microglia-to-neuron signaling in neuropathic pain, positioning the P2X4 receptor as a core signaling pathway necessary for ongoing expression of tactile allodynia following nerve injury.

Figure 2.

ATP activation of P2X4 receptors causes the synthesis and release of BDNF from microglia through a Ca²⁺ and p38-MAPK dependent signalling cascade. Activation of P2X4 receptors expressed on the cell surface instigates a series of conformational changes that allow cations, such as Ca²⁺ and Na⁺, entry into the cell through a non-selective channel. In a recent study, we reported ATP stimulation causes a biphasic release of BDNF and a concomitant increase in BDNF protein level within primary cultured microglia (Trang et al., 2009). Although drug treatment affected both BDNF accumulation and release, points 1–4 only illustrate the effects on the peak BDNF release response at 60 min post ATP stimulation: 1) In the presence of extracellular Ca²⁺, activating P2X4 receptors with ATP causes the release of BDNF; 2) Treatment with the p38-MAPK inhibitor SB203580 prevents BDNF release; 3) Inhibiting transcription or translation with actinomycin-D or cycloheximide, respectively, prevents BDNF release; and 4) Microglia treated with TAT-NSF, which interferes with NSF hexamerization necessary for SNARE-dependent exocytosis, abolishes BDNF release. The current model is that influx of Ca²⁺ through P2X4 receptors drives the activation of p38-MAPK that leads to increase in the SNARE-dependent release per se of BDNF and to an increase in BDNF synthesis in microglia. Modified from Trang et al., 2009.

A key cellular intermediary in the P2X4 receptor signalling pathway is p38-MAPK. Activation of this kinase is a point of convergence for involvement of microglial P2X7 and P2Y12 receptors in neuropathic pain, and for macrophage P2X4 receptor-mediate inflammatory pain. However, in contrast to the P2X4 receptor signalling pathway which has been systematically dissected, the upstream and downstream components of the P2X7 and P2Y12 receptor pathways have yet to be uncovered. Thus, the next major challenges are to determine the significance of this convergence in signaling for microglia function and how the components of P2X7 and P2Y12 receptor signalling contribute to aberrant spinal nociceptive processing in neuropathic pain.