The TREK-1 potassium channel is involved in both the analgesic and anti-proliferative effects of riluzole in bone cancer pain.

BACKGROUND: The metastasis of tumors into bone tissue typically leads to intractable pain that is both very disabling and particularly difficult to manage. We investigated here whether riluzole could have beneficial effects for the treatment of prostate cancer-induced bone pain and how it could influence the development of bone metastasis. METHODS: We used a bone pain model induced by intratibial injection of human PC3 prostate cancer cells into male SCID mice treated or not with riluzole administered in drinking water. We also used riluzole in vitro to assess its possible effect on PC3 cell viability and functionality, using patch-clamp. RESULTS: Riluzole had a significant preventive effect on both evoked and spontaneous pain involving the TREK-1 potassium channel. Riluzole did not interfere with PC3-induced bone loss or bone remodeling in vivo. It also significantly decreased PC3 cell viability in vitro. The antiproliferative effect of riluzole is correlated with a TREK-1-dependent membrane hyperpolarization in these cells. CONCLUSION: The present data suggest that riluzole could be very useful to manage evoked and spontaneous hypersensitivity in cancer-induced bone pain and has no significant adverse effect on cancer progression.

Pasteurized akkermansia muciniphila improves irritable bowel syndrome-like symptoms and related behavioral disorders in mice.

Gut - brain communications disorders in irritable bowel syndrome (IBS) are associated with intestinal microbiota composition, increased gut permeability, and psychosocial disturbances. Symptoms of IBS are difficult to medicate, and hence much research is being made into alternative approaches. This study assesses the potential of a treatment with pasteurized Akkermansia muciniphila for alleviating IBS-like symptoms in two mouse models of IBS with different etiologies. Two clinically relevant animal models were used to mimic IBS-like symptoms in C57BL6/J mice: the neonatal maternal separation (NMS) paradigm and the Citrobacter rodentium infection model. In both models, gut permeability, colonic sensitivity, fecal microbiota composition and colonic IL-22 expression were evaluated. The cognitive performance and emotional state of the animals were also assessed by several tests in the C. rodentium infection model. The neuromodulation ability of pasteurized A. muciniphila was assessed on primary neuronal cells from mice dorsal root ganglia using a ratiometric calcium imaging approach. The administration of pasteurized A. muciniphila significantly reduced colonic hypersensitivity in both IBS mouse models, accompanied by a reinforcement of the intestinal barrier function. Beneficial effects of pasteurized A. muciniphila treatment have also been observed on anxiety-like behavior and memory defects in the C. rodentium infection model. Finally, a neuroinhibitory effect exerted by pasteurized A. muciniphila was observed on neuronal cells stimulated with two algogenic substances such as capsaicin and inflammatory soup. Our findings demonstrate novel anti-hyperalgesic and neuroinhibitory properties of pasteurized A. muciniphila, which therefore may have beneficial effects in relieving pain and anxiety in subjects with IBS.

Antihyperalgesic properties of gut microbiota: Parabacteroides distasonis as a new probiotic strategy to alleviate chronic abdominal pain.

ABSTRACT: The potential role of gut microbiota in pain modulation is arousing an emerging interest since recent years. This study investigated neuromodulatory properties of gut microbiota to identify next-generation probiotics to propose alternative therapies for visceral pain management. Neuromodulation ability of 10 bacterial strains isolated from a healthy donor was assessed both on ND7/23 immortalized cell line and primary neuronal cells from rat dorsal root ganglia. This screening highlighted the neuroinhibitory property of Parabacteroides distasonis (F1-2) strain, supported both by its intracellular content and membrane fraction, which was further investigated in visceral pain mouse models. Oral administration of F1-2 resulted in a significant decrease of colonic hypersensitivity (CHS) in dextran sulfate sodium (0.5%) model associated with low-grade inflammation and a significant decrease of CHS in Citrobacter rodentium postinfectious models. No effect of F1-2 oral administration on CHS was observed in a neonatal maternal separation stress model. Antihyperalgesic effect unlikely involved modulation of inflammatory processes or restoration of intestinal barrier. Exploration of direct dialogue mechanisms between this strain and nervous system, assessed by calcium imaging experiments, revealed that F1-2 interacts directly with nociceptors by reducing activation level on capsaicin, inflammatory soup, and bradykinin stimulations. Our study provides new insights about bacteria-host interaction and places P distasonis as a potential therapeutic strategy in the treatment of visceral pain observed in leaky gut-associated pathologies.

Epigenetics Involvement in Oxaliplatin-Induced Potassium Channel Transcriptional Downregulation and Hypersensitivity.

Peripheral neuropathy is the most frequent dose-limiting adverse effect of oxaliplatin. Acute pain symptoms that are induced or exacerbated by cold occur in almost all patients immediately following the first infusions. Evidence has shown that oxaliplatin causes ion channel expression modulations in dorsal root ganglia neurons, which are thought to contribute to peripheral hypersensitivity. Most dysregulated genes encode ion channels involved in cold and mechanical perception, noteworthy members of a sub-group of potassium channels of the K2P family, TREK and TRAAK. Downregulation of these K2P channels has been identified as an important tuner of acute oxaliplatin-induced hypersensitivity. We investigated the molecular mechanisms underlying this peripheral dysregulation in a murine model of neuropathic pain triggered by a single oxaliplatin administration. We found that oxaliplatin-mediated TREK-TRAAK downregulation, as well as downregulation of other K+ channels of the K2P and Kv families, involves a transcription factor known as the neuron-restrictive silencer factor (NRSF) and its epigenetic co-repressors histone deacetylases (HDACs). NRSF knockdown was able to prevent most of these K+ channel mRNA downregulation in mice dorsal root ganglion neurons as well as oxaliplatin-induced acute cold and mechanical hypersensitivity. Interestingly, pharmacological inhibition of class I HDAC reproduces the antinociceptive effects of NRSF knockdown and leads to an increased K+ channel expression in oxaliplatin-treated mice.

TREK1 channel activation as a new analgesic strategy devoid of opioid adverse effects.

BACKGROUND AND PURPOSE: Opioids are effective painkillers. However, their risk-benefit ratio is dampened by numerous adverse effects and opioid misuse has led to a public health crisis. Safer alternatives are required, but isolating the antinociceptive effect of opioids from their adverse effects is a pharmacological challenge because activation of the µ opioid receptor triggers both the antinociceptive and adverse effects of opioids. EXPERIMENTAL APPROACH: The TREK1 potassium channel is activated downstream of µ receptor and involved in the antinociceptive activity of morphine but not in its adverse effects. Bypassing the µ opioid receptor to directly activate TREK1 could therefore be a safer analgesic strategy. KEY RESULTS: We developed a selective TREK1 activator, RNE28, with antinociceptive activity in naive rodents and in models of inflammatory and neuropathic pain. This activity was lost in TREK1 knockout mice or wild-type mice treated with the TREK1 blocker spadin, showing that TREK1 is required for the antinociceptive activity of RNE28. RNE28 did not induce respiratory depression, constipation, rewarding effects, or sedation at the analgesic doses tested. CONCLUSION AND IMPLICATIONS: This proof-of-concept study shows that TREK1 activators could constitute a novel class of painkillers, inspired by the mechanism of action of opioids but devoid of their adverse effects.

Testosterone-androgen receptor: The steroid link inhibiting TRPM8-mediated cold sensitivity.

Recent studies have revealed gender differences in cold perception, and pointed to a possible direct action of testosterone (TST) on the cold-activated TRPM8 (Transient Receptor Potential Melastatin Member 8) channel. However, the mechanisms by which TST influences TRPM8-mediated sensory functions remain elusive. Here, we show that TST inhibits TRPM8-mediated mild-cold perception through the noncanonical engagement of the Androgen Receptor (AR). Castration of both male rats and mice increases sensitivity to mild cold, and this effect depends on the presence of intact TRPM8 and AR. TST in nanomolar concentrations suppresses whole-cell TRPM8-mediated currents and single-channel activity in native dorsal root ganglion (DRG) neurons and HEK293 cells co-expressing recombinant TRPM8 and AR, but not TRPM8 alone. AR cloned from rat DRGs shows no difference from standard AR. However, biochemical assays and confocal imaging reveal the presence of AR on the cell surface and its interaction with TRPM8 in response to TST, leading to an inhibition of channel activity.

Replacement of current opioid drugs focusing on MOR-related strategies.

The scarcity and limited risk/benefit ratio of painkillers available on the market, in addition to the opioid crisis, warrant reflection on new innovation strategies. The pharmacopoeia of analgesics is based on products that are often old and derived from clinical empiricism, with limited efficacy or spectrum of action, or resulting in an unsatisfactory tolerability profile. Although they are reference analgesics for nociceptive pain, opioids are subject to the same criticism. The use of opium as an analgesic is historical. Morphine was synthesized at the beginning of the 19th century. The efficacy of opioids is limited in certain painful contexts and these drugs can induce potentially serious and fatal adverse effects. The current North American opioid crisis, with an ever-rising number of deaths by opioid overdose, is a tragic illustration of this. It is therefore legitimate to develop research into molecules likely to maintain or increase opioid efficacy while improving their tolerability. Several avenues are being explored including targeting of the mu opioid receptor (MOR) splice variants, developing biased agonists or targeting of other receptors such as heteromers with MOR. Ion channels acting as MOR effectors, are also targeted in order to offer compounds without MOR-dependent adverse effects. Another route is to develop opioid analgesics with peripheral action or limited central nervous system (CNS) access. Finally, endogenous opioids used as drugs or compounds that modify the metabolism of endogenous opioids (Dual ENKephalinase Inhibitors) are being developed. The aim of the present review is to present these various targets/strategies with reference to current indications for opioids, concerns about their widespread use, particularly in chronic non-cancer pains, and ways of limiting the risk of opioid abuse and misuse.

Targeting the TREK-1 potassium channel via riluzole to eliminate the neuropathic and depressive-like effects of oxaliplatin.

Neurotoxicity remains the most common adverse effect of oxaliplatin, limiting its clinical use. In the present study, we developed a mouse model of chronic oxaliplatin-induced neuropathy, which mimics both sensory and motor deficits observed in patients, in a clinically relevant time course. Repeated oxaliplatin administration in mice induced both cephalic and extracephalic long lasting mechanical and cold hypersensitivity after the first injection as well as delayed sensorimotor deficits and a depression-like phenotype. Using this model, we report that riluzole prevents both sensory and motor deficits induced by oxaliplatin as well as the depression-like phenotype induced by cumulative chemotherapeutic drug doses. All the beneficial effects are due to riluzole action on the TREK-1 potassium channel, which plays a central role in its therapeutic action. Riluzole has no negative effect on oxaliplatin antiproliferative capacity in human colorectal cancer cells and on its anticancer effect in a mouse model of colorectal cancer. Moreover, riluzole decreases human colorectal cancer cell line viability in vitro and inhibits polyp development in vivo. The present data in mice may support the need to clinically test riluzole in oxaliplatin-treated cancer patients and state for the important role of the TREK-1 channel in pain perception.

Inhibition of somatosensory mechanotransduction by annexin A6.

Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron-derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein. Annexin A6-deficient mice showed increased sensitivity to mechanical stimuli. Sensory neurons from these mice showed increased activity of the cation channel Piezo2, which mediates a rapidly adapting mechano-gated current linked to proprioception and touch, and a decrease in mechanically activated, slowly adapting currents. Conversely, overexpression of annexin A6 in sensory neurons inhibited rapidly adapting currents that were partially mediated by Piezo2. Furthermore, overexpression of annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis, a disease in which mechanically evoked pain is particularly problematic. These data suggest that annexin A6 can be exploited to inhibit chronic mechanical pain.

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice.

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope-tagged NaV1.7 mouse that showed normal pain behaviours to identify channel-interacting proteins. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo The sodium channel ß3 (Scn3b), rather than the ß1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing-response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane-trafficking protein synaptotagmin-2 (Syt2), L-type amino acid transporter 1 (Lat1) and transmembrane P24-trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co-immunoprecipitation (Co-IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein-regulated inducer of neurite outgrowth (Gprin1), an opioid receptor-binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope-tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel.

Development of the First Two-Pore Domain Potassium Channel TWIK-Related K+ Channel 1-Selective Agonist Possessing in Vivo Antinociceptive Activity.

The TWIK-related K+ channel, TREK-1, has recently emerged as an attractive therapeutic target for the development of a novel class of analgesic drugs, suggesting that activation of TREK-1 could result in pain inhibition. Here, we report the synthesis of a series of substituted acrylic acids (1-54) based on our previous work with caffeate esters. The analogues were evaluated for their ability to modulate TREK-1 channel by electrophysiology and for their in vivo antinociceptive activity (acetic acid-induced writhing and hot plate assays), leading to the identification of a series of novel molecules able to activate TREK-1 and displaying potent antinociceptive activity in vivo. Furyl analogue 36 is the most promising of the series.

New Insight in Cold Pain: Role of Ion Channels, Modulation, and Clinical Perspectives.

Cold temperature detection involves the process of sensory transduction in cutaneous primary sensory nerve terminals, which converts thermal stimuli into depolarizations of the membrane. This transformation into electrical signals is followed by the subsequent propagation of action potentials in cold-sensitive afferent nerve fibers. A large array of ion channels shapes this process; however, the precise contribution of specific ion channel subtypes to cold perception and cold pain remains elusive. This review aims at giving an update on our current understanding of the role played by TRPs, leak K+ and voltage-gated Na+ and K+ channels in the transduction of cold by nociceptors and in cold-induced pain.

Endogenous opioids contribute to insensitivity to pain in humans and mice lacking sodium channel Nav1.7.

Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.

Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia.

The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

The Nav1.9 channel is a key determinant of cold pain sensation and cold allodynia.

Cold-triggered pain is essential to avoid prolonged exposure to harmfully low temperatures. However, the molecular basis of noxious cold sensing in mammals is still not completely understood. Here, we show that the voltage-gated Nav1.9 sodium channel is important for the perception of pain in response to noxious cold. Nav1.9 activity is upregulated in a subpopulation of damage-sensing sensory neurons responding to cooling, which allows the channel to amplify subthreshold depolarizations generated by the activation of cold transducers. Consequently, cold-triggered firing is impaired in Nav1.9(-/-) neurons, and Nav1.9 null mice and knockdown rats show increased cold pain thresholds. Disrupting Nav1.9 expression in rodents also alleviates cold pain hypersensitivity induced by the antineoplastic agent oxaliplatin. We conclude that Nav1.9 acts as a subthreshold amplifier in cold-sensitive nociceptive neurons and is required for the perception of cold pain under normal and pathological conditions.

Mechanical allodynia.

Mechanical allodynia (other pain) is a painful sensation caused by innocuous stimuli like light touch. Unlike inflammatory hyperalgesia that has a protective role, allodynia has no obvious biological utility. Allodynia is associated with nerve damage in conditions such as diabetes, and is likely to become an increasing clinical problem. Unfortunately, the mechanistic basis of this enhanced sensitivity is incompletely understood. In this review, we describe evidence for the involvement of candidate mechanosensitive channels such as Piezo2 and their role in allodynia, as well as the peripheral and central nervous system mechanisms that have also been implicated in this form of pain. Specific treatments that block allodynia could be very useful if the cell and molecular basis of the condition could be determined. There are many potential mechanisms underlying this condition ranging from alterations in mechanotransduction and sensory neuron excitability to the actions of inflammatory mediators and wiring changes in the CNS. As with other pain conditions, it is likely that the range of redundant mechanisms that cause allodynia will make therapeutic intervention problematic.

Botulinum toxin-A treatment reduces human mechanical pain sensitivity and mechanotransduction.

The mechanisms underlying the analgesic effects of botulinum toxin serotype A (BoNT-A) are not well understood. We have tested the hypothesis that BoNT-A can block nociceptor transduction. Intradermal administration of BoNT-A to healthy volunteers produced a marked and specific decrease in noxious mechanical pain sensitivity, whereas sensitivity to low-threshold mechanical and thermal stimuli was unchanged. BoNT-A did not affect cutaneous innervation. In cultured rodent primary sensory neurons, BoNT-A decreased the proportion of neurons expressing slowly adapting mechanically gated currents linked to mechanical pain transduction. Inhibition of mechanotransduction provides a novel locus of action of BoNT-A, further understanding of which may extend its use as an analgesic agent.

Activation of TREK-1 by morphine results in analgesia without adverse side effects.

Morphine is the gold-standard pain reliever for severe acute or chronic pain but it also produces adverse side effects that can alter the quality of life of patients and, in some rare cases, jeopardize the vital prognosis. Morphine elicits both therapeutic and adverse effects primarily through the same µ opioid receptor subtype, which makes it difficult to separate the two types of effects. Here we show that beneficial and deleterious effects of morphine are mediated through different signalling pathways downstream from µ opioid receptor. We demonstrate that the TREK-1 K(+) channel is a crucial contributor of morphine-induced analgesia in mice, while it is not involved in morphine-induced constipation, respiratory depression and dependence-three main adverse effects of opioid analgesic therapy. These observations suggest that direct activation of the TREK-1 K(+) channel, acting downstream from the µ opioid receptor, might have strong analgesic effects without opioid-like adverse effects.

Nav1.9 channel contributes to mechanical and heat pain hypersensitivity induced by subacute and chronic inflammation.

Inflammation is known to be responsible for the sensitization of peripheral sensory neurons, leading to spontaneous pain and invalidating pain hypersensitivity. Given its role in regulating neuronal excitability, the voltage-gated Nav1.9 channel is a potential target for the treatment of pathological pain, but its implication in inflammatory pain is yet not fully described. In the present study, we examined the role of the Nav1.9 channel in acute, subacute and chronic inflammatory pain using Nav1.9-null mice and Nav1.9 knock-down rats. In mice we found that, although the Nav1.9 channel does not contribute to basal pain thresholds, it plays an important role in heat pain hypersensitivity induced by subacute paw inflammation (intraplantar carrageenan) and chronic ankle inflammation (complete Freund's adjuvant-induced monoarthritis). We showed for the first time that Nav1.9 also contributes to mechanical hypersensitivity in both models, as assessed using von Frey and dynamic weight bearing tests. Consistently, antisense-based Nav1.9 gene silencing in rats reduced carrageenan-induced heat and mechanical pain hypersensitivity. While no changes in Nav1.9 mRNA levels were detected in dorsal root ganglia (DRGs) during subacute and chronic inflammation, a significant increase in Nav1.9 immunoreactivity was observed in ipsilateral DRGs 24 hours following carrageenan injection. This was correlated with an increase in Nav1.9 immunolabeling in nerve fibers surrounding the inflamed area. No change in Nav1.9 current density could be detected in the soma of retrolabeled DRG neurons innervating inflamed tissues, suggesting that newly produced channels may be non-functional at this level and rather contribute to the observed increase in axonal transport. Our results provide evidence that Nav1.9 plays a crucial role in the generation of heat and mechanical pain hypersensitivity, both in subacute and chronic inflammatory pain models, and bring new elements for the understanding of its regulation in those models.