Orai1 is a crucial downstream partner of group I metabotropic glutamate receptor signaling in dorsal horn neurons.

ABSTRACT: Group I metabotropic glutamate receptors (group I mGluRs) have been implicated in several central nervous system diseases including chronic pain. It is known that activation of group I mGluRs results in the production of inositol triphosphate (IP3) and diacylglycerol that leads to activation of extracellular signal-regulated kinases (ERKs) and an increase in neuronal excitability, but how group I mGluRs mediate this process remains unclear. We previously reported that Orai1 is responsible for store-operated calcium entry and plays a key role in central sensitization. However, how Orai1 is activated under physiological conditions is unknown. Here, we tested the hypothesis that group I mGluRs recruit Orai1 as part of its downstream signaling pathway in dorsal horn neurons. We demonstrate that neurotransmitter glutamate induces STIM1 puncta formation, which is not mediated by N-Methyl-D-aspartate (NMDA) or α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Glutamate-induced Ca2+ entry in the presence of NMDA or AMPA receptor antagonists is eliminated in Orai1-deficient neurons. Dihydroxyphenylglycine (DHPG) (an agonist of group I mGluRs)-induced Ca2+ entry is abolished by Orai1 deficiency, but not affected by knocking down of transient receptor potential cation channel 1 (TRPC1) or TRPC3. Dihydroxyphenylglycine-induced activation of ERKs and modulation of neuronal excitability are abolished in cultured Orai1-deficient neurons. Moreover, DHPG-induced nociceptive behavior is markedly reduced in Orai1-deficient mice. Our findings reveal previously unknown functional coupling between Orai1 and group I mGluRs and shed light on the mechanism underlying group I mGluRs-mediated neuronal plasticity.

Orai1 Plays a Crucial Role in Central Sensitization by Modulating Neuronal Excitability.

Pathological pain is a common and debilitating condition that is often poorly managed. Central sensitization is an important mechanism underlying pathological pain. However, candidate molecules involved in central sensitization remain unclear. Store-operated calcium channels (SOCs) mediate important calcium signals in nonexcitable and excitable cells. SOCs have been implicated in a wide variety of human pathophysiological conditions, including immunodeficiency, occlusive vascular diseases, and cancer. However, the role of SOCs in CNS disorders has been relatively unexplored. Orai1, a key component of SOCs, is expressed in the human and rodent spinal cord dorsal horn, but its functional significance in dorsal horn neurons is poorly understood. Here we sought to explore a potential role of Orai1 in the modulation of neuronal excitability and A-type potassium channels involved in pain plasticity. Using both male and female Orai1 knock-out mice, we found that activation of Orai1 increased neuronal excitability and reduced A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway in dorsal horn neurons. Orai1 deficiency significantly decreased acute pain induced by noxious stimuli, nearly eliminated the second phase of formalin-induced nociceptive response, markedly attenuated carrageenan-induced ipsilateral pain hypersensitivity and abolished carrageenan-induced contralateral mechanical allodynia. Consistently, carrageenan-induced increase in neuronal excitability was abolished in the dorsal horn from Orai1 mutant mice. These findings uncover a novel signaling pathway involved in the pain process and central sensitization. Our study also reveals a novel link among Orai1, ERK, A-type potassium channels, and neuronal excitability.SIGNIFICANCE STATEMENT Orai1 is a key component of store-operated calcium channels (SOCs) in many cell types. It has been implicated in such pathological conditions as immunodeficiency, autoimmunity, and cancer. However, the role of Orai1 in CNS disorders remains poorly understood. The functional significance of Orai1 in neurons is elusive. Here we demonstrate that activation of Orai1 modulates neuronal excitability and Kv4-containing A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway. Genetic knock-out of Orai1 nearly eliminates the second phase of formalin-induced pain and markedly attenuates carrageenan-induced pain hypersensitivity and neuronal excitability. These findings reveal a novel link between Orai1 and neuronal excitability and advance our understanding of central sensitization.

Neuronal P2X7 receptor-induced reactive oxygen species production contributes to nociceptive behavior in mice.

ATP can activate a variety of pathways through P2 purinoreceptors, leading to neuroprotection and pathology in the CNS. Among all P2X receptors, the P2X7 receptor (P2X7R) is a well-defined therapeutic target for inflammatory and neuropathic pain. Activation of P2X7R can generate reactive oxygen species (ROS) in macrophages and microglia. However, the role of ROS in P2X7R-induced pain remains unexplored. Here, we investigated the downstream effects of neuronal P2X7R activation in the spinal cord. We found that ATP induces ROS production in spinal cord dorsal horn neurons, an effect eliminated by ROS scavenger N-tert-butyl-α-phenylnitrone (PBN) and P2X7R antagonist A438079. A similar effect was observed with a P2X7R agonist, BzATP, and was attenuated by a NADPH oxidase inhibitor apocynin. Intrathecal administration of BzATP resulted in ROS production in the spinal cord and oxidative DNA damage in dorsal horn neurons. BzATP also induced robust biphasic spontaneous nociceptive behavior. Pre-treatment with A438079 abolished all BzATP-induced nociceptive behaviors, while ROS scavengers dose-dependently attenuated the secondary response. Here, we provide evidence that neuronal P2X7R activation leads to ROS production and subsequent nociceptive pain in mice. Together, the data indicate that P2X7R-induced ROS play a critical role in the P2X7R signaling pathway of the CNS.

Circulating microRNA Signatures in Rodent Models of Pain.

MicroRNAs (miRNAs) remain stable in circulation and have been identified as potential biomarkers for a variety of conditions. We report miRNA changes in blood from multiple rodent models of pain, including spinal nerve ligation and spared nerve injury models of neuropathic pain; a complete Freund's adjuvant (CFA) model of inflammatory pain; and a chemotherapy-induced model of pain using the histone deacetylase inhibitor JNJ-26481585. The effect of celecoxib, a cyclooxygenase-2-selective nonsteroidal anti-inflammatory drug, was investigated in the CFA model as proof of principle for assessing the utility of circulating miRNAs as biomarkers in determining treatment response. Each study resulted in a unique miRNA expression profile. Despite differences in miRNAs identified from various models, computational target prediction and functional enrichment have identified biological pathways common among different models. The Wnt signaling pathway was affected in all models, suggesting a crucial role for this pathway in the pathogenesis of pain. Our studies demonstrate the utility of circulating miRNAs as pain biomarkers and suggest the potential for rigorous forward and reverse translational approaches. Evaluating alterations in miRNA fingerprints under different pain conditions and after administering therapeutic agents may be beneficial in evaluating clinical trial outcomes, predicting treatment response, and developing correlational outcomes between preclinical and human studies.

Effect of histone deacetylase inhibitor JNJ-26481585 in pain.

Recent studies have shown that histone deacetylase (HDAC) inhibitors can alleviate inflammatory and neuropathic pain. We investigated the effects of JNJ-26481585, a pan-HDAC inhibitor on basal mechanical sensitivity. Unlike previous reports for HDAC inhibitors, JNJ-26481585 induced mechanical hypersensitivity in mice. This effect was reversible with gabapentin. Voltage-dependent calcium channel subunit alpha-2/delta-1, one of the putative targets for gabapentin, was upregulated in the spinal cord from JNJ-26481585-treated mice. Transcriptional profiling of spinal cord from JNJ-26481585-treated mice showed significant alterations in pathways involved in axon guidance, suggesting overlap in mechanisms underlying neurotoxicity caused by other known chemotherapeutic agents. To investigate the mechanisms underlying the development of pain, RAW 264.7 mouse macrophage cells were treated with JNJ-26481585. There was a dose- and time-dependent activation of nuclear factor-kappaB and interleukin-1ß increase. Thus, alterations in the axon guidance pathway, increase in voltage-dependent calcium channel alpha(2)delta-1 subunit, and the induction of proinflammatory mediators by JNJ-26481585 could all contribute to increased mechanical sensitivity. Our data indicate that the effect of HDAC inhibitors may be unique to the compound studied and highlights the potential to develop chemotherapy-induced peripheral neuropathy with the use of a pan-HDAC inhibitor for cancer treatment, and this pain may be alleviated by gabapentin.

Functional significance of macrophage-derived exosomes in inflammation and pain.

Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary, depending on the source and the physiological conditions of cells, and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome-mediated information transfer in vitro, in a rodent model of inflammatory pain, and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from lipopolysaccharide-stimulated cells were sufficient to cause nuclear factor-κB activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage-derived exosomes. Macrophage-derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain, and should be explored for their therapeutic utility.

Potent analgesic effects of a store-operated calcium channel inhibitor.

Chronic pain often accompanies immune responses and immune cells are known to be involved in chronic pain. Store-operated calcium (SOC) channels are calcium-selective cation channels and play an important role in the immune system. YM-58483, a potent SOC channel inhibitor, has been shown to inhibit cytokine production from immune cells and attenuate antigen-induced hypersensitivity reactions. Here, we report that YM-58483 has analgesic actions in chronic pain and produces antinociceptive effects in acute pain and prevents the development of chronic pain in mice. Oral administration of 10mg/kg or 30 mg/kg YM-58483 dramatically attenuated complete Freund adjuvant (CFA)-induced thermal hyperalgesia and prevented the development of thermal and mechanical hypersensitivity in a dose-dependent manner. Analgesic effects were observed when YM-58483 was administered systemically, intrathecally and intraplantarly. YM-58483 decreased spared nerve injury (SNI)-induced thermal and mechanical hypersensitivity and prevented the development of SNI-induced pain hypersensitivity. Pretreatment with YM-58483 strongly reduced both the first and second phases of formalin-induced spontaneous nocifensive behavior in a dose-dependent manner. YM-58483 produced antinociception in acute pain induced by heat or chemical or mechanical stimuli at a dose of 30 mg/kg. YM-58483 diminished CFA-induced paw edema, and reduced production of TNF-α, IL-1ß and PGE2 in the CFA-injected paw. In vitro, SOC entry in nociceptors was more robust than in nonnociceptors, and the inhibition of SOC entry by YM-58483 in nociceptors was much greater than in nonnociceptors. Our findings indicate that YM-58483 is a potent analgesic and suggest that SOC channel inhibitors may represent a novel class of therapeutics for pain.

Central mechanisms of menthol-induced analgesia.

Menthol is one of the most commonly used chemicals in our daily life, not only because of its fresh flavor and cooling feeling but also because of its medical benefit. Previous studies have suggested that menthol produces analgesic action in acute and neuropathic pain through peripheral mechanisms. However, the central actions and mechanisms of menthol remain unclear. Here, we report that menthol has direct effects on the spinal cord. Menthol decreased both ipsilateral and contralateral pain hypersensitivity induced by complete Freund's adjuvant in a dose-dependent manner. Menthol also reduced both first and second phases of formalin-induced spontaneous nocifensive behavior. We then identified the potential central mechanisms underlying the analgesic effect of menthol. In cultured dorsal horn neurons, menthol induced inward and outward currents in a dose-dependent manner. The menthol-activated current was mediated by Cl(-) and blocked by bicuculline, suggesting that menthol activates γ-aminobutyric acid type A receptors. In addition, menthol blocked voltage-gated sodium channels and voltage-gated calcium channels in a voltage-, state-, and use-dependent manner. Furthermore, menthol reduced repetitive firing and action potential amplitude, decreased neuronal excitability, and blocked spontaneous synaptic transmission of cultured superficial dorsal horn neurons. Liquid chromatography/tandem mass spectrometry analysis of brain menthol levels indicated that menthol was rapidly concentrated in the brain when administered systemically. Our results indicate that menthol produces its central analgesic action on inflammatory pain probably via the blockage of voltage-gated Na(+) and Ca(2+) channels. These data provide molecular and cellular mechanisms by which menthol decreases neuronal excitability, therefore contributing to menthol-induced central analgesia.

Group II metabotropic glutamate receptor agonist ameliorates MK801-induced dysfunction of NMDA receptors via the Akt/GSK-3ß pathway in adult rat prefrontal cortex.

Pharmacological intervention targeting mGluRs has emerged as a potential treatment for schizophrenia, whereas the mechanisms involved remain elusive. We explored the antipsychotic effects of an mGluR2/3 agonist in the MK-801 model of schizophrenia in the rat prefrontal cortex. We found that the mGluR2/3 agonist LY379268 effectively recovered the disrupted expression of NMDA receptors induced by MK-801 administration. This effect was attributable to the direct regulatory action of LY379268 on NMDA receptors via activation of the Akt/GSK-3ß signaling pathway. As occurs with the antipsychotic drug clozapine, acute treatment with LY379268 significantly increased the expression and phosphorylation of NMDA receptors, as well as Akt and GSK-3ß. Physiologically, LY379268 significantly enhanced NMDA-induced current in prefrontal neurons and a GSK-3ß inhibitor occluded this effect. In contrast to the widely proposed mechanism of modulating presynaptic glutamate release, our results strongly argue that mGluR2/3 agonists modulate the function of NMDA receptors through postsynaptic actions and reverse the MK-801-induced NMDA dysfunction via the Akt/GSK-3ß pathway. This study provides novel evidence for postsynaptic mechanisms of mGluR2/3 in regulation of NMDA receptors and presents useful insights into the mechanistic actions of mGluR2/3 agonists as potential antipsychotic agents for treating schizophrenia.