Tuan Trang

P2X4-receptor mediated synthesis and release of brain-derived neurotrophic factor in microglia is dependent on calcium and p38-mitogen-activated protein kinase activation

Microglia in the dorsal horn of the spinal cord are increasingly recognized as being crucial in the pathogenesis of pain hypersensitivity after injury to a peripheral nerve. It is known that P2X4 purinoceptors (P2X4Rs) cause the release of brain-derived neurotrophic factor (BDNF) from microglia, which is necessary for maintaining pain hypersensitivity after nerve injury. However, there is a critical gap in understanding how activation of microglial P2X4Rs leads to the release of BDNF. Here, we show that stimulating P2X4Rs with ATP evokes a biphasic release of BDNF from microglia: an early phase occurs within 5 min, whereas a late phase peaks 60 min after ATP stimulation. Concomitant with the late phase of release is an increased level of BDNF within the microglia. Both phases of BDNF release and the accumulation within the microglia are dependent on extracellular Ca2+. The late phase of BDNF release and accumulation, but not the early phase of release, are suppressed by inhibiting transcription and translation, indicating that activation of P2X4R causes an initial release of a pre-existing pool of BDNF followed by an increase in de novo synthesis of BDNF. The release of BDNF is abolished by inhibiting SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated exocytosis. Furthermore, we find that the P2X4R-evoked release and synthesis of BDNF are dependent on activation of p38-mitogen-activated protein kinase (MAPK). Together, our findings provide a unifying mechanism for pain hypersensitivity after peripheral nerve injury through P2X4R-evoked increase in Ca2+ and activation of p38-MAPK leading to the synthesis and exocytotic release of BDNF from microglia.

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury–induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.

Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl- homeostasis

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We found that hyperalgesia-inducing treatment with morphine resulted in downregulation of the K+-Cl− co-transporter KCC2, impairing Cl− homeostasis in rat spinal lamina l neurons. Restoring the anion equilibrium potential reversed the morphine-induced hyperalgesia without affecting tolerance. The hyperalgesia was also reversed by ablating spinal microglia. Morphine hyperalgesia, but not tolerance, required μ opioid receptor–dependent expression of P2X4 receptors (P2X4Rs) in microglia and μ-independent gating of the release of brain-derived neurotrophic factor (BDNF) by P2X4Rs. Blocking BDNF-TrkB signaling preserved Cl− homeostasis and reversed the hyperalgesia. Gene-targeted mice in which Bdnf was deleted from microglia did not develop hyperalgesia to morphine. However, neither morphine antinociception nor tolerance was affected in these mice. Our findings dissociate morphine-induced hyperalgesia from tolerance and suggest the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target for preventing hyperalgesia without affecting morphine analgesia.

P2X4R+ microglia drive neuropathic pain

Neuropathic pain, the most debilitating of all clinical pain syndromes, may be a consequence of trauma, infection or pathology from diseases that affect peripheral nerves. Here we provide a framework for understanding the spinal mechanisms of neuropathic pain as distinct from those of acute pain or inflammatory pain. Recent work suggests that a specific microglia response phenotype characterized by de novo expression of the purinergic receptor P2X4 is critical for the pathogenesis of pain hypersensitivity caused by injury to peripheral nerves. Stimulating P2X4 receptors initiates a core pain signaling pathway mediated by release of brain-derived neurotrophic factor, which produces a disinhibitory increase in intracellular chloride in nociceptive (pain-transmitting) neurons in the spinal dorsal horn. The changes caused by signaling from P2X4R+ microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans, and they point to specific interventions to alleviate this debilitating condition.

Brain-derived neurotrophic factor from microglia: a molecular substrate for neuropathic pain.

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 expresses 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 signaling 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.

Calcium-permeable ion channels in pain signaling.

The detection and processing of painful stimuli in afferent sensory neurons is critically dependent on a wide range of different types of voltage- and ligand-gated ion channels, including sodium, calcium, and TRP channels, to name a few. The functions of these channels include the detection of mechanical and chemical insults, the generation of action potentials and regulation of neuronal firing patterns, the initiation of neurotransmitter release at dorsal horn synapses, and the ensuing activation of spinal cord neurons that project to pain centers in the brain. Long-term changes in ion channel expression and function are thought to contribute to chronic pain states. Many of the channels involved in the afferent pain pathway are permeable to calcium ions, suggesting a role in cell signaling beyond the mere generation of electrical activity. In this article, we provide a broad overview of different calcium-permeable ion channels in the afferent pain pathway and their role in pain pathophysiology.

Blocking microglial pannexin-1 channels alleviates morphine withdrawal in rodents

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (10panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia.

P2X4 purinoceptor signaling in chronic pain

ATP, acting via P2 purinergic receptors, is a known mediator of inflammatory and neuropathic pain. There is increasing evidence that the ATP-gated P2X4 receptor (P2X4R) subtype is a locus through which activity of spinal microglia and peripheral macrophages instigate pain hypersensitivity caused by inflammation or by injury to a peripheral nerve. The present article highlights the recent advances in our understanding of microglia–neuron interactions in neuropathic pain by focusing on the signaling and regulation of the P2X4R. We will also develop a framework for understanding converging lines of evidence for involvement of P2X4Rs expressed on macrophages in peripheral inflammatory pain.

Animal models of chronic pain: Advances and challenges for clinical translation

Chronic pain is a global problem that has reached epidemic proportions. An estimated 20% of adults suffer from pain, and another 10% are diagnosed with chronic pain each year (Goldberg and McGee, ). Despite the high prevalence of chronic pain (an estimated 1.5 billion people are afflicted worldwide), much remains to be understood about the underlying causes of this condition, and there is an urgent requirement for better pain therapies. The discovery of novel targets and the development of better analgesics rely on an assortment of preclinical animal models; however, there are major challenges to translating discoveries made in animal models to realized pain therapies in humans. This review discusses common animal models used to recapitulate clinical chronic pain conditions (such as neuropathic, inflammatory, and visceral pain) and the methods for assessing the sensory and affective components of pain in animals. We also discuss the advantages and limitations of modeling chronic pain in animals as well as highlighting strategies for improving the predictive validity of preclinical pain studies.

Microglial P2X4R-evoked pain hypersensitivity is sexually dimorphic in rats

Abstract Microglia–neuron signalling in the spinal cord is a key mediator of mechanical allodynia caused by peripheral nerve injury. We recently reported sex differences in microglia in pain signalling in mice: spinal mechanisms underlying nerve injury–induced allodynia are microglial dependent in male but not female mice. Whether this sex difference in pain hypersensitivity mechanisms is conserved in other species is unknown. Here, we show that in rats, the spinal mechanisms of nerve injury–induced hypersensitivity in males differ from those in females, with microglial P2X4 receptors (P2X4Rs) being a key point of divergence. In rats, nerve injury produced comparable allodynia and reactive microgliosis in both sexes. However, inhibiting microglia in the spinal cord reversed allodynia in male rats but not female rats. In addition, pharmacological blockade of P2X4Rs, by an intrathecally administered antagonist, attenuated pain hypersensitivity in male rats only. Consistent with the behavioural findings, nerve injury increased cell surface expression and function of P2X4Rs in acutely isolated spinal microglia from male rats but not from female rats. Moreover, in microglia cultured from male rats, but not in those from female rats, stimulating P2X4Rs drove intracellular signalling through p38 mitogen-activated protein kinase. Furthermore, chromatin immunoprecipitation–qPCR revealed that the transcription factor IRF5 differentially binds to the P2rx4 promoter region in female rats vs male rats. Finally, mechanical allodynia was produced in otherwise naive rats by intrathecally administering P2X4R-stimulated microglia from male rats but not those from female rats. Together, our findings demonstrate the existence of sexually dimorphic pain signalling in rats, suggesting that this sex difference is evolutionarily conserved, at least across rodent species.