TMC6 functions as a GPCR-like receptor to sense noxious heat via Gαq signaling.

Thermosensation is vital for the survival, propagation, and adaption of all organisms, but its mechanism is not fully understood yet. Here, we find that TMC6, a membrane protein of unknown function, is highly expressed in dorsal root ganglion (DRG) neurons and functions as a Gαq-coupled G protein-coupled receptor (GPCR)-like receptor to sense noxious heat. TMC6-deficient mice display a substantial impairment in noxious heat sensation while maintaining normal perception of cold, warmth, touch, and mechanical pain. Further studies show that TMC6 interacts with Gαq via its intracellular C-terminal region spanning Ser780 to Pro810. Specifically disrupting such interaction using polypeptide in DRG neurons, genetically ablating Gαq, or pharmacologically blocking Gαq-coupled GPCR signaling can replicate the phenotype of TMC6 deficient mice regarding noxious heat sensation. Noxious heat stimulation triggers intracellular calcium release from the endoplasmic reticulum (ER) of TMC6- but not control vector-transfected HEK293T cell, which can be significantly inhibited by blocking PLC or IP3R. Consistently, noxious heat-induced intracellular Ca2+ release from ER and action potentials of DRG neurons largely reduced when ablating TMC6 or blocking Gαq/PLC/IP3R signaling pathway as well. In summary, our findings indicate that TMC6 can directly function as a Gαq-coupled GPCR-like receptor sensing noxious heat.

Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome.

Expansion of the hexanucleotide repeat GGGGCC in the C9orf72 gene is the most common genetic factor in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Poly-Gly-Ala (poly-GA), one form of dipeptide repeat proteins (DPRs) produced from GGGGCC repeats, tends to form neurotoxic protein aggregates. The C9orf72 GGGGCC repeats and microglial receptor TREM2 are both associated with risk for ALS/FTD. The role and regulation of TREM2 in C9orf72-ALS/FTD remain unclear. Here, we found that poly-GA proteins activate the microglial NLRP3 inflammasome to produce interleukin-1ß (IL-1ß), which promotes ADAM10-mediated TREM2 cleavage and inhibits phagocytosis of poly-GA. The inhibitor of the NLRP3 inflammasome, MCC950, reduces the TREM2 cleavage and poly-GA aggregates, resulting in the alleviation of motor deficits in poly-GA mice. Our study identifies a crosstalk between NLRP3 and TREM2 signaling, suggesting that targeting the NLRP3 inflammasome to sustain TREM2 is an approach to treat C9orf72-ALS/FTD.

Structure-guided peptide engineering of a positive allosteric modulator targeting the outer pore of TRPV1 for long-lasting analgesia.

Transient receptor potential vanilloid 1 (TRPV1) ion channel is a classic analgesic target, but antagonists of TRPV1 failed in clinical trials due to their side effects like hyperthermia. Here we rationally engineer a peptide s-RhTx as a positive allosteric modulator (PAM) of TRPV1. Patch-clamp recordings demonstrate s-RhTx selectively potentiated TRPV1 activation. s-RhTx also slows down capsaicin-induced desensitization of TRPV1 in the presence of calcium to cause more calcium influx in TRPV1-expressing cells. In addition, our thermodynamic mutant cycle analysis shows that E652 in TRPV1 outer pore specifically interacts with R12 and K22 in s-RhTx. Furthermore, we demonstrate in vivo that s-RhTx exhibits long-lasting analgesic effects in noxious heat hyperalgesia and CFA-induced chronic inflammatory pain by promoting the reversible degeneration of intra-epidermal nerve fiber (IENF) expressing TRPV1 channels in mice, while their body temperature remains unaffected. Our results suggest s-RhTx is an analgesic agent as a PAM of TRPV1.

mTOR-neuropeptide Y signaling sensitizes nociceptors to drive neuropathic pain.

Neuropathic pain is a refractory condition that involves de novo protein synthesis in the nociceptive pathway. The mTOR is a master regulator of protein translation; however, mechanisms underlying its role in neuropathic pain remain elusive. Using the spared nerve injury-induced neuropathic pain model, we found that mTOR was preferentially activated in large-diameter dorsal root ganglion (DRG) neurons and spinal microglia. However, selective ablation of mTOR in DRG neurons, rather than microglia, alleviated acute neuropathic pain in mice. We show that injury-induced mTOR activation promoted the transcriptional induction of neuropeptide Y (Npy), likely via signal transducer and activator of transcription 3 phosphorylation. NPY further acted primarily on Y2 receptors (Y2R) to enhance neuronal excitability. Peripheral replenishment of NPY reversed pain alleviation upon mTOR removal, whereas Y2R antagonists prevented pain restoration. Our findings reveal an unexpected link between mTOR and NPY/Y2R in promoting nociceptor sensitization and neuropathic pain.

GPR177 in A-fiber sensory neurons drives diabetic neuropathic pain via WNT-mediated TRPV1 activation.

Diabetic neuropathic pain (DNP) is a common and devastating complication in patients with diabetes. The mechanisms mediating DNP are not completely elucidated, and effective treatments are lacking. A-fiber sensory neurons have been shown to mediate the development of mechanical allodynia in neuropathic pain, yet the molecular basis underlying the contribution of A-fiber neurons is still unclear. Here, we report that the orphan G protein-coupled receptor 177 (GPR177) in A-fiber neurons drives DNP via WNT5a-mediated activation of transient receptor potential vanilloid receptor-1 (TRPV1) ion channel. GPR177 is mainly expressed in large-diameter A-fiber dorsal root ganglion (DRG) neurons and required for the development of DNP in mice. Mechanistically, we found that GPR177 mediated the secretion of WNT5a from A-fiber DRG neurons into cerebrospinal fluid (CSF), which was necessary for the maintenance of DNP. Extracellular perfusion of WNT5a induced rapid currents in both TRPV1-expressing heterologous cells and nociceptive DRG neurons. Computer simulations revealed that WNT5a has the potential to bind the residues at the extracellular S5-S6 loop of TRPV1. Using a peptide able to disrupt the predicted WNT5a/TRPV1 interaction suppressed DNP- and WNT5a-induced neuropathic pain symptoms in rodents. We confirmed GPR177/WNT5A coexpression in human DRG neurons and WNT5A secretion in CSF from patients with DNP. Thus, our results reveal a role for WNT5a as an endogenous and potent TRPV1 agonist, and the GPR177-WNT5a-TRPV1 axis as a driver of DNP pathogenesis in rodents. Our findings identified a potential analgesic target that might relieve neuropathic pain in patients with diabetes.

Rational Design of a Modality-Specific Inhibitor of TRPM8 Channel against Oxaliplatin-Induced Cold Allodynia.

Platinum-based compounds in chemotherapy such as oxaliplatin often induce peripheral neuropathy and neuropathic pain such as cold allodynia in patients. Transient Receptor Potential Melastatin 8 (TRPM8) ion channel is a nociceptor critically involved in such pathological processes. Direct blockade of TRPM8 exhibits significant analgesic effects but also incurs severe side effects such as hypothermia. To selectively target TRPM8 channels against cold allodynia, a cyclic peptide DeC-1.2 is de novo designed with the optimized hot-spot centric approach. DeC-1.2 modality specifically inhibited the ligand activation of TRPM8 but not the cold activation as measured in single-channel patch clamp recordings. It is further demonstrated that DeC-1.2 abolishes cold allodynia in oxaliplatin treated mice without altering body temperature, indicating DeC-1.2 has the potential for further development as a novel analgesic against oxaliplatin-induced neuropathic pain.

GPR151 in nociceptors modulates neuropathic pain via regulating P2X3 function and microglial activation.

Neuropathic pain is a major health problem that affects up to 7-10% of the population worldwide. Currently, neuropathic pain is difficult to treat because of its elusive mechanisms. Here we report that orphan G protein-coupled receptor 151 (GPR151) in nociceptive sensory neurons controls neuropathic pain induced by nerve injury. GPR151 was mainly expressed in non-peptidergic C-fibre dorsal root ganglion neurons and highly upregulated after nerve injury. Importantly, conditional knockout of Gpr151 in adult nociceptive sensory neurons significantly alleviated chronic constriction injury-induced neuropathic pain-like behaviour but did not affect basal nociception. Moreover, GPR151 in DRG neurons was required for chronic constriction injury-induced neuronal hyperexcitability and upregulation of colony-stimulating factor 1 (CSF1), which is necessary for microglial activation in the spinal cord after nerve injury. Mechanistically, GPR151 coupled with P2X3 ion channels and promoted their functional activities in neuropathic pain-like hypersensitivity. Knockout of Gpr151 suppressed P2X3-mediated calcium elevation and spontaneous pain behaviour in chronic constriction injury mice. Conversely, overexpression of Gpr151 significantly enhanced P2X3-mediated calcium elevation and dorsal root ganglion neuronal excitability. Furthermore, knockdown of P2X3 in dorsal root ganglia reversed chronic constriction injury-induced CSF1 upregulation, spinal microglial activation and neuropathic pain-like behaviour. Finally, the coexpression of GPR151 and P2X3 was confirmed in small-diameter human dorsal root ganglion neurons, indicating the clinical relevance of our findings. Together, our results indicate that GPR151 in nociceptive dorsal root ganglion neurons plays a key role in the pathogenesis of neuropathic pain and could be a potential target for treating neuropathic pain.

Basal forebrain mediates prosocial behavior via disinhibition of midbrain dopamine neurons.

Sociability is fundamental for our daily life and is compromised in major neuropsychiatric disorders. However, the neuronal circuit mechanisms underlying prosocial behavior are still elusive. Here we identify a causal role of the basal forebrain (BF) in the control of prosocial behavior via inhibitory projections that disinhibit the midbrain ventral tegmental area (VTA) dopamine (DA) neurons. Specifically, BF somatostatin-positive (SST) inhibitory neurons were robustly activated during social interaction. Optogenetic inhibition of these neurons in BF or their axon terminals in the VTA largely abolished social preference. Electrophysiological examinations further revealed that SST neurons predominantly targeted VTA GABA neurons rather than DA neurons. Consistently, optical inhibition of SST neuron axon terminals in the VTA decreased DA release in the nucleus accumbens during social interaction, confirming a disinhibitory action. These data reveal a previously unappreciated function of the BF in prosocial behavior through a disinhibitory circuitry connected to the brain's reward system.

Interleukin-17 Regulates Neuron-Glial Communications, Synaptic Transmission, and Neuropathic Pain after Chemotherapy.

The proinflammatory cytokine interleukin-17 (IL-17) is implicated in pain regulation. However, the synaptic mechanisms by which IL-17 regulates pain transmission are unknown. Here, we report that glia-produced IL-17 suppresses inhibitory synaptic transmission in the spinal cord pain circuit and drives chemotherapy-induced neuropathic pain. We find that IL-17 not only enhances excitatory postsynaptic currents (EPSCs) but also suppresses inhibitory postsynaptic synaptic currents (IPSCs) and GABA-induced currents in lamina IIo somatostatin-expressing neurons in mouse spinal cord slices. IL-17 mainly expresses in spinal cord astrocytes, and its receptor IL-17R is detected in somatostatin-expressing neurons. Selective knockdown of IL-17R in spinal somatostatin-expressing interneurons reduces paclitaxel-induced hypersensitivity. Overexpression of IL-17 in spinal astrocytes is sufficient to induce mechanical allodynia in naive animals. In dorsal root ganglia, IL-17R expression in nociceptive sensory neurons is sufficient and required for inducing neuronal hyperexcitability after paclitaxel. Together, our data show that IL-17/IL-17R mediate neuron-glial interactions and neuronal hyperexcitability in chemotherapy-induced peripheral neuropathy.

Is Optogenetic Activation of Vglut1-Positive Aß Low-Threshold Mechanoreceptors Sufficient to Induce Tactile Allodynia in Mice after Nerve Injury?

Mechanical allodynia is a cardinal feature of pathological pain. Recent work has demonstrated the necessity of Aß-low-threshold mechanoreceptors (Aß-LTMRs) for mechanical allodynia-like behaviors in mice, but it remains unclear whether these neurons are sufficient to produce pain under pathological conditions. We generated a transgenic mouse in which channelrhodopsin-2 (ChR2) is conditionally expressed in vesicular glutamate transporter 1 (Vglut1) sensory neurons (Vglut1-ChR2), which is a heterogeneous population of large-sized sensory neurons with features consistent with Aß-LTMRs. In naive male Vglut1-ChR2 mice, transdermal hindpaw photostimulation evoked withdrawal behaviors in an intensity- and frequency-dependent manner, which were abolished by local anesthetic and selective A-fiber blockade. Surprisingly, male Vglut1-ChR2 mice did not show significant differences in light-evoked behaviors or real-time aversion after nerve injury despite marked hypersensitivity to punctate mechanical stimuli. We conclude that optogenetic activation of cutaneous Vglut1-ChR2 neurons alone is not sufficient to produce pain-like behaviors in neuropathic mice.SIGNIFICANCE STATEMENT Mechanical allodynia, in which innocuous touch is perceived as pain, is a common feature of pathological pain. To test the contribution of low-threshold mechanoreceptors (LTMRs) to nerve-injury-induced mechanical allodynia, we generated and characterized a new transgenic mouse (Vglut1-ChR2) to optogenetically activate cutaneous vesicular glutamate transporter 1 (Vglut1)-positive LTMRs. Using this mouse, we found that light-evoked behaviors were unchanged by nerve injury, which suggests that activation of Vglut1-positive LTMRs alone is not sufficient to produce pain. The Vglut1-ChR2 mouse will be broadly useful for the study of touch, pain, and itch.

GPR37 regulates macrophage phagocytosis and resolution of inflammatory pain.

The mechanisms of pain induction by inflammation have been extensively studied. However, the mechanisms of pain resolution are not fully understood. Here, we report that GPR37, expressed by macrophages (MΦs) but not microglia, contributes to the resolution of inflammatory pain. Neuroprotectin D1 (NPD1) and prosaptide TX14 increase intracellular Ca2+ (iCa2+) levels in GPR37-transfected HEK293 cells. NPD1 and TX14 also bind to GPR37 and cause GPR37-dependent iCa2+ increases in peritoneal MΦs. Activation of GPR37 by NPD1 and TX14 triggers MΦ phagocytosis of zymosan particles via calcium signaling. Hind paw injection of pH-sensitive zymosan particles not only induces inflammatory pain and infiltration of neutrophils and MΦs, but also causes GPR37 upregulation in MΦs, phagocytosis of zymosan particles and neutrophils by MΦs in inflamed paws, and resolution of inflammatory pain in WT mice. Mice lacking Gpr37 display deficits in MΦ phagocytic activity and delayed resolution of inflammatory pain. Gpr37-deficient MΦs also show dysregulations of proinflammatory and antiinflammatory cytokines. MΦ depletion delays the resolution of inflammatory pain. Adoptive transfer of WT but not Gpr37-deficient MΦs promotes the resolution of inflammatory pain. Our findings reveal a previously unrecognized role of GPR37 in regulating MΦ phagocytosis and inflammatory pain resolution.

Distinct Analgesic Actions of DHA and DHA-Derived Specialized Pro-Resolving Mediators on Post-operative Pain After Bone Fracture in Mice.

Mechanisms of pain resolution are largely unclear. Increasing evidence suggests that specialized pro-resolving mediators (SPMs), derived from fish oil docosahexaenoic acid (DHA), promote the resolution of acute inflammation and potently inhibit inflammatory and neuropathic pain. In this study, we examined the analgesic impact of DHA and DHA-derived SPMs in a mouse model of post-operative pain induced by tibial bone fracture (fPOP). Intravenous perioperative treatment with DHA (500 µg), resolvin D1 (RvD1, 500 ng) and maresin 1 (MaR1, 500 ng), 10 min and 24 h after the surgery, delayed the development of fPOP (mechanical allodynia and cold allodynia). In contrast, post-operative intrathecal (IT) administration of DHA (500 µg) 2 weeks after the surgery had no effects on established mechanical and cold allodynia. However, by direct comparison, IT post-operative treatment (500 ng) with neuroprotectin D1 (NPD1), MaR1, and D-resolvins, RvD1 and RvD5, but not RvD3 and RvD4, effectively reduced mechanical and cold allodynia. ELISA analysis showed that perioperative DHA treatment increased RvD1 levels in serum and spinal cord samples after bone fracture. Interestingly, sham surgery resulted in transient allodynia and increased RvD1 levels, suggesting a correlation of enhanced SPM levels with acute pain resolution after sham surgery. Our findings suggest that (1) perioperative treatment with DHA is effective in preventing and delaying the development of fPOP and (2) post-treatment with some SPMs can attenuate established fPOP. Our data also indicate that orthopedic surgery impairs SPM production. Thus, DHA and DHA-derived SPMs should be differentially supplemented for treating fPOP and improving recovery.

Interferon alpha inhibits spinal cord synaptic and nociceptive transmission via neuronal-glial interactions.

It is well known that interferons (IFNs), such as type-I IFN (IFN-α) and type-II IFN (IFN-γ) are produced by immune cells to elicit antiviral effects. IFNs are also produced by glial cells in the CNS to regulate brain functions. As a proinflammatory cytokine, IFN-γ drives neuropathic pain by inducing microglial activation in the spinal cord. However, little is known about the role of IFN-α in regulating pain sensitivity and synaptic transmission. Strikingly, we found that IFN-α/ß receptor (type-I IFN receptor) was expressed by primary afferent terminals in the superficial dorsal horn that co-expressed the neuropeptide CGRP. In the spinal cord IFN-α was primarily expressed by astrocytes. Perfusion of spinal cord slices with IFN-α suppressed excitatory synaptic transmission by reducing the frequency of spontaneous excitatory postsynaptic current (sEPSCs). IFN-α also inhibited nociceptive transmission by reducing capsaicin-induced internalization of NK-1 and phosphorylation of extracellular signal-regulated kinase (ERK) in superficial dorsal horn neurons. Finally, spinal (intrathecal) administration of IFN-α reduced inflammatory pain and increased pain threshold in naïve rats, whereas removal of endogenous IFN-α by a neutralizing antibody induced hyperalgesia. Our findings suggest a new form of neuronal-glial interaction by which IFN-α, produced by astrocytes, inhibits nociceptive transmission in the spinal cord.

ß-arrestin-2 regulates NMDA receptor function in spinal lamina II neurons and duration of persistent pain.

Mechanisms of acute pain transition to chronic pain are not fully understood. Here we demonstrate an active role of ß-arrestin 2 (Arrb2) in regulating spinal cord NMDA receptor (NMDAR) function and the duration of pain. Intrathecal injection of the mu-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin produces paradoxical behavioural responses: early-phase analgesia and late-phase mechanical allodynia which requires NMDAR; both phases are prolonged in Arrb2 knockout (KO) mice. Spinal administration of NMDA induces GluN2B-dependent mechanical allodynia, which is prolonged in Arrb2-KO mice and conditional KO mice lacking Arrb2 in presynaptic terminals expressing Nav1.8. Loss of Arrb2 also results in prolongation of inflammatory pain and neuropathic pain and enhancement of GluN2B-mediated NMDA currents in spinal lamina IIo not lamina I neurons. Finally, spinal over-expression of Arrb2 reverses chronic neuropathic pain after nerve injury. Thus, spinal Arrb2 may serve as an intracellular gate for acute to chronic pain transition via desensitization of NMDAR.

Inhibition of mechanical allodynia in neuropathic pain by TLR5-mediated A-fiber blockade.

Mechanical allodynia, induced by normally innocuous low-threshold mechanical stimulation, represents a cardinal feature of neuropathic pain. Blockade or ablation of high-threshold, small-diameter unmyelinated group C nerve fibers (C-fibers) has limited effects on mechanical allodynia. Although large, myelinated group A fibers, in particular Aß-fibers, have previously been implicated in mechanical allodynia, an A-fiber-selective pharmacological blocker is still lacking. Here we report a new method for targeted silencing of A-fibers in neuropathic pain. We found that Toll-like receptor 5 (TLR5) is co-expressed with neurofilament-200 in large-diameter A-fiber neurons in the dorsal root ganglion (DRG). Activation of TLR5 with its ligand flagellin results in neuronal entry of the membrane-impermeable lidocaine derivative QX-314, leading to TLR5-dependent blockade of sodium currents, predominantly in A-fiber neurons of mouse DRGs. Intraplantar co-application of flagellin and QX-314 (flagellin/QX-314) dose-dependently suppresses mechanical allodynia after chemotherapy, nerve injury, and diabetic neuropathy, but this blockade is abrogated in Tlr5-deficient mice. In vivo electrophysiology demonstrated that co-application of flagellin/QX-314 selectively suppressed Aß-fiber conduction in naive and chemotherapy-treated mice. TLR5-mediated Aß-fiber blockade, but not capsaicin-mediated C-fiber blockade, also reduced chemotherapy-induced ongoing pain without impairing motor function. Finally, flagellin/QX-314 co-application suppressed sodium currents in large-diameter human DRG neurons. Thus, our findings provide a new tool for targeted silencing of Aß-fibers and neuropathic pain treatment.

Emerging targets in neuroinflammation-driven chronic pain.

Current analgesics predominately modulate pain transduction and transmission in neurons and have limited success in controlling disease progression. Accumulating evidence suggests that neuroinflammation, which is characterized by infiltration of immune cells, activation of glial cells and production of inflammatory mediators in the peripheral and central nervous system, has an important role in the induction and maintenance of chronic pain. This Review focuses on emerging targets - such as chemokines, proteases and the WNT pathway - that promote spinal cord neuroinflammation and chronic pain. It also highlights the anti-inflammatory and pro-resolution lipid mediators that act on immune cells, glial cells and neurons to resolve neuroinflammation, synaptic plasticity and pain. Targeting excessive neuroinflammation could offer new therapeutic opportunities for chronic pain and related neurological and psychiatric disorders.