Induction of antiviral interferon-stimulated genes by neuronal STING promotes the resolution of pain in mice.

Inflammation and pain are intertwined responses to injury, infection, or chronic diseases. While acute inflammation is essential in determining pain resolution and opioid analgesia, maladaptive processes occurring during resolution can lead to the transition to chronic pain. Here we found that inflammation activates the cytosolic DNA-sensing protein stimulator of IFN genes (STING) in dorsal root ganglion nociceptors. Neuronal activation of STING promotes signaling through TANK-binding kinase 1 (TBK1) and triggers an IFN-ß response that mediates pain resolution. Notably, we found that mice expressing a nociceptor-specific gain-of-function mutation in STING exhibited an IFN gene signature that reduced nociceptor excitability and inflammatory hyperalgesia through a KChIP1-Kv4.3 regulation. Our findings reveal a role of IFN-regulated genes and KChIP1 downstream of STING in the resolution of inflammatory pain.

The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain.

The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non-small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.

Gut-innervating TRPV1+ Neurons Drive Chronic Visceral Pain via Microglial P2Y12 Receptor.

BACKGROUND & AIMS: Chronic abdominal pain is a common symptom of inflammatory bowel diseases (IBDs). Peripheral and central mechanisms contribute to the transition from acute to chronic pain during active disease and clinical remission. Lower mechanical threshold and hyperexcitability of visceral afferents induce gliosis in central pain circuits, leading to persistent visceral hypersensitivity (VHS). In the spinal cord, microglia, the immune sentinels of the central nervous system, undergo activation in multiple models of VHS. Here, we investigated the mechanisms of microglia activation to identify centrally acting analgesics for chronic IBD pain. METHODS: Using Designer Receptors Exclusively Activated by Designer Drugs (DREADD) expressed in transient receptor potential vanilloid member 1-expressing visceral neurons that sense colonic inflammation, we tested whether neuronal activity was indispensable to control microglia activation and VHS. We then investigated the neuron-microglia signaling system involved in visceral pain chronification. RESULTS: We found that chemogenetic inhibition of transient receptor potential vanilloid member 1+ visceral afferents prevents microglial activation in the spinal cord and subsequent VHS in colitis mice. In contrast, chemogenetic activation, in the absence of colitis, enhanced microglial activation associated with VHS. We identified a purinergic signaling mechanism mediated by neuronal adenosine triphosphate (ATP) and microglial P2Y12 receptor, triggering VHS in colitis. Inhibition of P2RY12 prevented microglial reactivity and chronic VHS post-colitis. CONCLUSIONS: Overall, these data provide novel insights into the central mechanisms of chronic visceral pain and suggest that targeting microglial P2RY12 signaling could be harnessed to relieve pain in patients with IBD who are in remission.

TRPV1 Activation Promotes ß-arrestin2 Interaction with the Ribosomal Biogenesis Machinery in the Nucleolus:Implications for p53 Regulation and Neurite Outgrowth.

Transient receptor potential vanilloids (TRPV1) are non-selective cation channels that sense and transduce inflammatory pain signals. We previously reported that activation of TRPV1 induced the translocation of ß-arrestin2 (ARRB2) from the cytoplasm to the nucleus, raising questions about the functional role of ARRB2 in the nucleus. Here, we determined the ARRB2 nuclear signalosome by conducting a quantitative proteomic analysis of the nucleus-sequestered L395Q ARRB2 mutant, compared to the cytosolic wild-type ARRB2 (WT ARRB2), in a heterologous expression system. We identified clusters of proteins that localize to the nucleolus and are involved in ribosomal biogenesis. Accordingly, L395Q ARRB2 or WT ARRB2 after capsaicin treatment were found to co-localize and interact with the nucleolar marker nucleophosmin (NPM1), treacle protein (TCOF1) and RNA polymerase I (POL I). We further investigated the role of nuclear ARRB2 signaling in regulating neuroplasticity. Using neuroblastoma (neuro2a) cells and dorsal root ganglia (DRG) neurons, we found that L395Q ARRB2 mutant increased POL I activity, inhibited the tumor suppressorp53 (p53) level and caused a decrease in the outgrowth of neurites. Together, our results suggest that the activation of TRPV1 promotes the ARRB2-mediated regulation of ribosomal biogenesis in the nucleolus. The ARRB2-TCOF1-p53 checkpoint signaling pathway might be involved in regulating neurite outgrowth associated with pathological pain conditions.

TACAN Is an Ion Channel Involved in Sensing Mechanical Pain.

Mechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying essential physiological functions such as touch and pain sensing, hearing, and proprioception. Although the mechanisms for some of these functions have been identified, the molecules essential to the sense of pain have remained elusive. Here we report identification of TACAN (Tmem120A), an ion channel involved in sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, a nociceptor-specific inducible knockout of TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to painful mechanical stimuli but not to thermal or touch stimuli. We propose that TACAN is an ion channel that contributes to sensing mechanical pain.

TRPV1 promotes opioid analgesia during inflammation.

Pain and inflammation are inherently linked responses to injury, infection, or chronic diseases. Given that acute inflammation in humans or mice enhances the analgesic properties of opioids, there is much interest in determining the inflammatory transducers that prime opioid receptor signaling in primary afferent nociceptors. Here, we found that activation of the transient receptor potential vanilloid type 1 (TRPV1) channel stimulated a mitogen-activated protein kinase (MAPK) signaling pathway that was accompanied by the shuttling of the scaffold protein ß-arrestin2 to the nucleus. The nuclear translocation of ß-arrestin2 in turn prevented its recruitment to the µ-opioid receptor (MOR), the subsequent internalization of agonist-bound MOR, and the suppression of MOR activity that occurs upon receptor desensitization. Using the complete Freund's adjuvant (CFA) inflammatory pain model to examine the role of TRPV1 in regulating endogenous opioid analgesia in mice, we found that naloxone methiodide (Nal-M), a peripherally restricted, nonselective, and competitive opioid receptor antagonist, slowed the recovery from CFA-induced hypersensitivity in wild-type, but not TRPV1-deficient, mice. Furthermore, we showed that inflammation prolonged morphine-induced antinociception in a mouse model of opioid receptor desensitization, a process that depended on TRPV1. Together, our data reveal a TRPV1-mediated signaling pathway that serves as an endogenous pain-resolution mechanism by promoting the nuclear translocation of ß-arrestin2 to minimize MOR desensitization. This previously uncharacterized mechanism may underlie the peripheral opioid control of inflammatory pain. Dysregulation of the TRPV1-ß-arrestin2 axis may thus contribute to the transition from acute to chronic pain.

Inhibition of Cav 3.2 calcium channels: A new target for colonic hypersensitivity associated with low-grade inflammation.

BACKGROUND AND PURPOSE: Abdominal pain associated with low-grade inflammation is frequently encountered in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) during remission. Current treatments are not very effective and new therapeutic approaches are needed. The role of CaV 3.2 channels, which are important in other chronic pain contexts, was investigated in a murine model of colonic hypersensitivity (CHS) associated with low-grade inflammation. EXPERIMENTAL APPROACH: Low doses of dextran sulfate sodium (DSS; 0.5%) were chronically administered to C57BL/6j mice in drinking water. Their inflammatory state was assessed by systemic and local measures of IL-6, myeloperoxidase, and lipocalin-2 using elisa. Colonic sensitivity was evaluated by the visceromotor responses to colorectal distension. Functional involvement of CaV 3.2 channels was assessed with different pharmacological (TTA-A2, ABT-639, and ethosuximide) and genetic tools. KEY RESULTS: DSS induced low-grade inflammation associated with CHS in mice. Genetic or pharmacological inhibition of CaV 3.2 channels reduced CHS. Cav3.2 channel deletion in primary nociceptive neurons in dorsal root ganglia (CaV 3.2Nav1.8 KO mice) suppressed CHS. Spinal, but not systemic, administration of ABT-639, a peripherally acting T-type channel blocker, reduced CHS. ABT-639 given intrathecally to CaV 3.2Nav1.8 KO mice had no effect, demonstrating involvement of CaV 3.2 channels located presynaptically in afferent fibre terminals. Finally, ethosuximide, which is a T-type channel blocker used clinically, reduced CHS. CONCLUSIONS AND IMPLICATIONS: These results suggest that ethosuximide represents a promising drug reposition strategy and that inhibition of CaV 3.2 channels is an attractive therapeutic approach for relieving CHS in IBS or IBD.

A simple strategy for culturing morphologically-conserved rat hypothalamic tanycytes.

Hypothalamic tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle. Given their strategic location, tanycytes are believed to play several key functions including being a selective barrier and controlling the amount of hypothalamic-derived factors reaching the anterior pituitary. The in vitro culture of these cells has proved to be difficult. Here, we report an improved method for the generation of primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day-old rats and cultured in a chemically defined medium containing DMEM:F12, serum albumin, insulin, transferrin and the antibiotic gentamycin. After 7 days in vitro, ∼30% of the cultured cells exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. Tanycyte-like cells were strongly immuno-reactive for vimentin and dopamine-cAMP-regulated phospho-protein (DARPP-32) and weakly immune-reactive for glial fibrillary acidic protein. Tanycyte-like cells displayed a stable negative resting plasma membrane potential and failed to show spiking properties in response to current injections. When exposed to fluorescent beads in the culture medium, tanycyte-like cells exhibited a robust endocytosis. Thus, the present method effectively yields cultures containing tanycyte-like cells that resemble in vivo tanycytes in terms of morphologic features and molecular markers as well as electrical and endocytic activity. To our knowledge, this is the first protocol that allows the culturing of tanycyte-like cells that can be individually identified and that conserve the morphology of tanycytes in their natural physiological environment.

Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons.

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G protein-coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the voltage-gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and agonist-dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.

Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.