Peripheral Nociceptive Neurons Research Articles

125 Contribution of Sensory Neuron-associated Macrophages to Neuropathic Pain via Up-regulation of Calcium Channel in C Fiber Sensory Neurons in Dorsal Root Ganglion: An Animal Model Study

INTRODUCTION: Animal models have demonstrated that sensory neuron-associated macrophages (sNAMs) play a key role in the initiation and persistence of neuropathic pain. The Cav3.2 calcium channel (Cav3.2) is also known to be involved in modulating neuropathic pain in peripheral sensory neurons. METHODS: Male Sprague-Dawley rats underwent unilateral tight ligation of the lumbar 5 spinal nerve and were divided into three groups: a control group (SHAM), a group that underwent ligation without, and with minocycline (SNL and SNL+Mino). The mechanical pain threshold of the hind paw was assessed with von-Frey filament application. Immunohistochemical staining techniques were used to evaluate the levels of sNAM, Cav3.2, and C-fiber sensory neurons with anti-Iba1, anti-Cav3.2, and IB4 antibodies, respectively. Cytokine levels were analyzed using cytokine arrays and western blotting. RESULTS: The SNL group had a significantly lower pain threshold compared to the control group (p < 0.001). In the DRG, neuropathic pain led to an accumulation of macrophages and increased their ability to attach to Cav 3.2. The expression of Cav 3.2 then triggered the activation of IB4 cells in the DRG. Additionally, cytokine array and western blot analyses revealed a higher expression rate of TNF-α in the SNL group compared to the control group (p = 0.048). Treatment with minocycline resulted in increased pain threshold, decreased number of macrophages in the DRG, and interruption of the attachment between macrophages and Cav 3.2, leading to lower expression of TNF-α. CONCLUSIONS: In conclusion, the results of this study suggest that sNAMs may play a role in the development of neuropathic pain by increasing the expression of Cav3.2 through the upregulation of TNF-α release in peripheral sensory nociceptive neurons.

Protease-Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota Is Associated With Visceral and Somatic Hypersensitivity

Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally-housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. In vivo and in vitro assays of visceral sensation were performed on mice treated with the non-absorbable antibiotic vancomycin (50 μg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition. Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro, and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared to controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naïve mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nM) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 μM) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2. Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect.

Opioid-Induced Hyperalgesia and Tolerance Are Driven by HCN Ion Channels.

Prolonged exposure to opioids causes an enhanced sensitivity to painful stimuli (opioid-induced hyperalgesia, OIH) and a need for increased opioid doses to maintain analgesia (opioid-induced tolerance, OIT), but the mechanisms underlying both processes remain obscure. We found that pharmacological block or genetic deletion of HCN2 ion channels in primary nociceptive neurons of male mice completely abolished OIH but had no effect on OIT. Conversely, pharmacological inhibition of central HCN channels alleviated OIT but had no effect on OIH. Expression of C-FOS, a marker of neuronal activity, was increased in second-order neurons of the dorsal spinal cord by induction of OIH, and the increase was prevented by peripheral block or genetic deletion of HCN2, but block of OIT by spinal block of HCN channels had no impact on C-FOS expression in dorsal horn neurons. Collectively, these observations show that OIH is driven by HCN2 ion channels in peripheral nociceptors, while OIT is driven by a member of the HCN family located in the CNS. Induction of OIH increased cAMP in nociceptive neurons, and a consequent shift in the activation curve of HCN2 caused an increase in nociceptor firing. The shift in HCN2 was caused by expression of a constitutively active μ-opioid receptor (MOR) and was reversed by MOR antagonists. We identified the opioid-induced MOR as a six-transmembrane splice variant, and we show that it increases cAMP by coupling constitutively to Gs HCN2 ion channels therefore drive OIH, and likely OIT, and may be a novel therapeutic target for the treatment of addiction.

Central and peripheral mechanism of MOTS-c attenuates pain hypersensitivity in a mice model of inflammatory pain

ABSTRACT Background Inflammatory pain is caused by damaged tissue or noxious stimuli, accompanied by the release of inflammatory mediators that often leads to severe hyperalgesia and allodynia with limited therapy options. Recently, a novel mitochondrial-derived peptide (named MOTS-c) was reported to regulate obesity, metabolic homeostasis and inflammatory response. The aim of this study was to investigate the effects of MOTS-c and its related regulatory mechanisms involved in inflammatory pain. Methods Male Kunming mice (8–10 weeks-old) were intraplantar injected with formalin, capsaicin, λ-Carrageenan and complete Freund adjuvant (CFA) to establish acute and chronic inflammatory pain. The effects of MOTS-c on the above inflammatory pain mice and its underlying mechanisms were examined by behavioral tests, quantitative polymerase chain reaction (qPCR), western blotting, enzyme linked immunosorbent assay (ELISA), immunohistochemistry (IHC) and immunofluorescence (IF). Results Behavioral experiments investigated the potential beneficial effects of MOTS-c on multiple acute and chronic inflammatory pain in mice. The results showed that MOTS-c treatment produced potent anti-allodynic effects in formalin-induced acute inflammatory pain, capsaicin-induced nocifensive behaviors and λ-Carrageenan/CFA-induced chronic inflammatory pain model. Further mechanistic studies revealed that central MOTS-c treatment significantly ameliorated CFA-evoked the release of inflammatory factors and activation of glial cells and neurons in the spinal dorsal horn. Moreover, peripheral MOTS-c treatment reduced CFA-evoked inflammatory responses in the surface structure of hindpaw skin, accompanied by inhibiting excitation of peripheral calcitonin gene-related peptide (CGRP) and P2X3 nociceptive neurons. Conclusions The present study indicates that MOTS-c may serve as a promising therapeutic target for inflammatory pain.

Descending GABAergic pathway links brain sugar-sensing to peripheral nociceptive gating in Drosophila

Although painful stimuli elicit defensive responses including escape behavior for survival, starved animals often prioritize feeding over escape even in a noxious environment. This behavioral priority is typically mediated by suppression of noxious inputs through descending control in the brain, yet underlying molecular and cellular mechanisms are incompletely understood. Here we identify a cluster of GABAergic neurons in Drosophila larval brain, designated as SEZ-localized Descending GABAergic neurons (SDGs), that project descending axons onto the axon terminals of the peripheral nociceptive neurons and prevent presynaptic activity through GABAB receptors. Remarkably, glucose feeding to starved larvae causes sustained activation of SDGs through glucose-sensing neurons and subsequent insulin signaling in SDGs, which attenuates nociception and thereby suppresses escape behavior in response to multiple noxious stimuli. These findings illustrate a neural mechanism by which sugar sensing neurons in the brain engages descending GABAergic neurons in nociceptive gating to achieve hierarchical interaction between feeding and escape behavior.

Selective Inhibition of NaV1.8 with VX-548 for Acute Pain

BackgroundThe NaV1.8 voltage-gated sodium channel, expressed in peripheral nociceptive neurons, plays a role in transmitting nociceptive signals. The effect of VX-548, an oral, highly selective inhibitor of NaV1.8, on control of acute pain is being studied.MethodsAfter establishing the selectivity of VX-548 for NaV1.8 inhibition in vitro, we conducted two phase 2 trials involving participants with acute pain after abdominoplasty or bunionectomy. In the abdominoplasty trial, participants were randomly assigned in a 1:1:1:1 ratio to receive one of the following over a 48-hour period: a 100-mg oral loading dose of VX-548, followed by a 50-mg maintenance dose every 12 hours (the high-dose group); a 60-mg loading dose of VX-548, followed by a 30-mg maintenance dose every 12 hours (the middle-dose group); hydrocodone bitartrate–acetaminophen (5 mg of hydrocodone bitartrate and 325 mg of acetaminophen every 6 hours); or oral placebo every 6 hours. In the bunionectomy trial, participants were randomly assigned in a 2:2:1:2:2 ratio to receive one of the following over a 48-hour treatment period: oral high-dose VX-548; middle-dose VX-548; low-dose VX-548 (a 20-mg loading dose, followed by a 10-mg maintenance dose every 12 hours); oral hydrocodone bitartrate–acetaminophen (5 mg of hydrocodone bitartrate and 325 mg of acetaminophen every 6 hours); or oral placebo every 6 hours. The primary end point was the time-weighted sum of the pain-intensity difference (SPID) over the 48-hour period (SPID48), a measure derived from the score on the Numeric Pain Rating Scale (range, 0 to 10; higher scores indicate greater pain) at 19 time points after the first dose of VX-548 or placebo. The main analysis compared each dose of VX-548 with placebo.ResultsA total of 303 participants were enrolled in the abdominoplasty trial and 274 in the bunionectomy trial. The least-squares mean difference between the high-dose VX-548 and placebo groups in the time-weighted SPID48 was 37.8 (95% confidence interval [CI], 9.2 to 66.4) after abdominoplasty and 36.8 (95% CI, 4.6 to 69.0) after bunionectomy. In both trials, participants who received lower doses of VX-548 had results similar to those with placebo. Headache and constipation were common adverse events with VX-548.ConclusionsAs compared with placebo, VX-548 at the highest dose, but not at lower doses, reduced acute pain over a period of 48 hours after abdominoplasty or bunionectomy. VX-548 was associated with adverse events that were mild to moderate in severity. (Funded by Vertex Pharmaceuticals; VX21-548-101 and VX21-548-102 ClinicalTrials.gov numbers, NCT04977336 and NCT05034952.)

Deficient AMPK activity contributes to hyperexcitability in peripheral nociceptive sensory neurons and thermal hyperalgesia in lupus mice.

Patients with systemic lupus erythematosus (SLE) often suffer from chronic pain. Little is known about the peripheral mechanisms underlying the genesis of chronic pain induced by SLE. The aim of this study was to investigate whether and how membrane properties in nociceptive neurons in the dorsal root ganglions (DRGs) are altered by SLE. We found elevation of resting membrane potentials, smaller capacitances, lower action potential thresholds and rheobases in nociceptive neurons in the DRGs from MRL/lpr mice (an SLE mouse model) with thermal hyperalgesia. DRGs from MRL/lpr mice had increased protein expressions in TNFα, IL-1β, and phosphorylated ERK but suppressed AMPK activity, and no changes in sodium channel 1.7 protein expression. We showed that intraplantar injection of Compound C (an AMPK inhibitor) induced thermal hyperalgesia in normal mice while intraplantar injection of AICAR (an AMPK activator) reduced thermal hyperalgesia in MRL/Lpr mice. Upon inhibition of AMPK membrane properties in nociceptive neurons from normal control mice could be rapidly switched to those found in SLE mice with thermal hyperalgesia. Our study indicates that increased excitability in peripheral nociceptive sensory neurons contributes to the genesis of thermal hyperalgesia in mice with SLE, and AMPK regulates membrane properties in nociceptive sensory neurons as well as thermal hyperalgesia in mice with SLE. Our study provides a basis for targeting signaling pathways regulating membrane properties of peripheral nociceptive neurons as a means for conquering chronic pain caused by SLE.

Elucidating the toxic effect and disease mechanisms associated with Lyso-Gb3 in Fabry disease.

Fabry disease stems from a deficiency of alpha-galactosidase and results in the accumulation of globotriaosylceramide (Gb3). However, the production of its deacylated form globotriaosylsphingosine (lyso-Gb3) is also observed and its plasma levels have closer association with disease severity. Studies have shown that lyso-Gb3 directly affects podocytes and causes sensitisation of peripheral nociceptive neurons. However, little is understood of the mechanisms of this cytotoxicity. To study the effect on neuronal cells, we incubated SH-Sy5y cells with lyso-Gb3 at low (20ng/mL) and high (200ng/mL) levels, to mimic mild and classical FD serum levels. We used glucosylsphingosine as a positive control to determine specific effects of lyso-Gb3. Proteomic analyses revealed that cellular systems affected by lyso-Gb3 included cell signalling particularly protein ubiquitination and protein translation. To confirm ER/proteasome perturbations, we performed an immune enrichment of ubiquitinated proteins and demonstrated specific increased protein ubiquitination at both doses. The most ubiquitinated proteins observed included the chaperone/heat shock proteins, cytoskeletal proteins and synthesis/translation proteins. To detect proteins that interact directly with lyso-Gb3, we immobilised lyso-lipids, then incubated them with neuronal cellular extracts and identified bound proteins using mass spectrometry. Proteins that specifically bound were chaperones and included HSP90, HSP60 and the TRiC complex. In conclusion, lyso-Gb3 exposure affects pathways involved in protein translation and folding. This response is observed as increased ubiquitination and changes in signalling proteins which may explain the multiple biological processes, particularly cellular remodelling, often associated with FD.

The Cytidine N-Acetyltransferase NAT10 Participates in Peripheral Nerve Injury-Induced Neuropathic Pain by Stabilizing SYT9 Expression in Primary Sensory Neurons.

RNA N4-acetylcytidine (ac4C) modification is increasingly recognized as an important layer of gene regulation; however, the involvement of ac4C in pain regulation has not been studied. Here, we report that N-acetyltransferase 10 protein (NAT10; the only known ac4C "writer") contributes to the induction and development of neuropathic pain in an ac4C-dependent manner. Peripheral nerve injury increases the levels of NAT10 expression and overall ac4C in injured dorsal root ganglia (DRGs). This upregulation is triggered by the activation of upstream transcription factor 1 (USF1), a transcription factor that binds to the Nat10 promoter. Knock-down or genetic deletion of NAT10 in the DRG abolishes the gain of ac4C sites in Syt9 mRNA and the augmentation of SYT9 protein, resulting in a marked antinociceptive effect in nerve-injured male mice. Conversely, mimicking NAT10 upregulation in the absence of injury evokes the elevation of Syt9 ac4C and SYT9 protein and induces the genesis of neuropathic-pain-like behaviors. These findings demonstrate that USF1-governed NAT10 regulates neuropathic pain by targeting Syt9 ac4C in peripheral nociceptive sensory neurons. Our findings establish NAT10 as a critical endogenous initiator of nociceptive behavior and a promising new target for treating neuropathic pain.SIGNIFICANCE STATEMENT The cytidine N4-acetylcytidine (ac4C), a new epigenetic RNA modification, is crucial for the translation and stability of mRNA, but its role for chronic pain remains unclear. Here, we demonstrate that N-acetyltransferase 10 (NAT10) acts as ac4C N-acetyltransferase and plays an important role in the development and maintenance of neuropathic pain. NAT10 was upregulated via the activation of the transcription factor upstream transcription factor 1 (USF1) in the injured dorsal root ganglion (DRG) after peripheral nerve injury. Since pharmacological or genetic deleting NAT10 in the DRG attenuated the nerve injury-induced nociceptive hypersensitivities partially through suppressing Syt9 mRNA ac4C and stabilizing SYT9 protein level, NAT10 may serve as an effective and novel therapeutic target for neuropathic pain.

Encoding of inflammatory hyperalgesia in mouse spinal cord.

Inflammation modifies the input-output properties of peripheral nociceptive neurons such that the same stimulus produces enhanced nociceptive firing. This increased nociceptive output enters the superficial dorsal spinal cord (SDH), an intricate neuronal network composed largely of excitatory and inhibitory interneurons and a small percentage of projection neurons. The SDH network comprises the first central nervous system network integrating noxious information. Using in vivo calcium imaging and a computational approach, we characterized the responsiveness of the SDH network in mice to noxious stimuli in normal conditions and investigated the changes in SDH response patterns after acute burn injury-induced inflammation. We show that the application of noxious heat stimuli to the hind paw of naïve mice results in an overall increase in SDH network activity. Single-cell response analysis reveals that 70% of recorded neurons increase or suppress their activity, while ∼30% of neurons remain nonresponsive. After acute burn injury and the development of inflammatory hyperalgesia, application of the same noxious heat stimuli leads to the activation of previously nonresponding neurons and desuppression of suppressed neurons. We further demonstrate that an increase in afferent activity mimics the response of the SDH network to noxious heat stimuli under inflammatory conditions. Using a computational model of the SDH network, we predict that the changes in SDH network activity result in overall increased activity of excitatory neurons, amplifying the output from SDH to higher brain centers. We suggest that during acute local peripheral inflammation, the SDH network undergoes dynamic changes promoting hyperalgesia.

Protease‐Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota is Associated with Visceral Hypersensitivity

Abdominal pain is a major symptom of diseases associated with microbial dysbiosis. Disruption of the gut microbiota with antibiotics increases visceral pain, and germ‐free mice are more prone to pain than conventionally‐raised mice. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. Patch clamp electrophysiological recordings of dorsal root ganglion (DRG) neuron excitability were obtained from control mice and mice treated with the non‐absorbable antibiotic vancomycin (50 µg/ml in drinking water) for one week. Ten days prior to recording visceromotor response (VMR) telemetric transmitters were placed into the abdominal cavity of the mice and allowed to recover. VMR was measured by insertion of balloon catheter into the rectum under light anesthetization in both control and vancomycin treated mice, then distended to 80 mmHg and VMR recorded. Bacterial dysbiosis was verified by metagenomic analysis of stool microbial composition. Mice treated with vancomycin were more sensitive to colorectal distension in vivo (VMR increased by 70% at 80 mmHg compared to control), and DRG neurons from vancomycin‐treated mice were hyperexcitable in vitro compared to water‐treated controls (rheobase decreased by 30% relative to control). Interestingly, hyperexcitability of DRG neurons was not restricted to gut projecting neurons, suggesting a widespread effect of gut dysbiosis on pain pathways. Incubation of DRG neurons from naïve mice in serum from vancomycin‐treated mice increased neuron excitability (rheobase decreased by 30% relative to control), suggesting that microbial dysbiosis alters circulating mediators that influence nociception. Multiplex ELISA measurements did not detect any significant changes in serum cytokines or chemokines between vancomycin‐treated and control mice. The cysteine protease inhibitor E64 (30 nM) and the protease‐activated receptor 2 (PAR2) antagonist GB‐83 (10 µM) each blocked the increase in DRG neuron excitability in response to serum from vancomycin‐treated mice. Naïve DRG neurons incubated with fecal supernatants from vancomycin‐treated mice also exhibited increased excitability (rheobase decreased by 40% relative to control), but supernatants derived from colonic tissue failed to cause hyperexcitability. Overall, this data suggests that microbial dysbiosis within the gut alters pain sensitivity. This effect is not caused by inflammation or host derived factors, rather bacterially‐derived cysteine proteases activating PAR2 on DRG neurons.

The role of neuroimmune interactions in musculoskeletal pain

Abstract Interactions of the immune system and the nociceptive system play an important role in the generation and maintenance of pain in musculoskeletal diseases and in disease development. In inflamed tissue peripheral nociceptive neurons are rendered hyperexcitable by proinflammatory cytokines, antigen/antibody complexes and other immune mediators. Spinal nociceptive neurons are rendered hyperexcitable with the support of microglial cells, the immune cells of the central nervous system. The so-elicited sensitization of pain pathways has a strong impact on pain processing in the brain. On the other hand, immune processes are regulated by the nervous system. Sensory neurons, by releasing neuropeptides, and efferent neurons of the sympathetic nervous system support immune processes which promote disease development.

Nuclear Factor κB-COX2 Pathway Activation in Non-myelinating Schwann Cells Is Necessary for the Maintenance of Neuropathic Pain in vivo.

Chronic neuropathic pain leads to long-term changes in the sensitivity of both peripheral and central nociceptive neurons. Glial fibrillary acidic protein (GFAP)-positive glial cells are closely associated with the nociceptive neurons including astrocytes in the central nervous system (CNS), satellite glial cells (SGCs) in the sensory ganglia, and non-myelinating Schwann cells (NMSCs) in the peripheral nerves. Central and peripheral GFAP-positive cells are involved in the maintenance of chronic pain through a host of inflammatory cytokines, many of which are under control of the transcription factor nuclear factor κB (NFκB) and the enzyme cyclooxygenase 2 (COX2). To test the hypothesis that inhibiting GFAP-positive glial signaling alleviates chronic pain, we used (1) a conditional knockout (cKO) mouse expressing Cre recombinase under the hGFAP promoter and a floxed COX2 gene to inactivate the COX2 gene specifically in GFAP-positive cells; and (2) a tet-Off tetracycline transactivator system to suppress NFκB activation in GFAP-positive cells. We found that neuropathic pain behavior following spared nerve injury (SNI) significantly decreased in COX2 cKO mice as well as in mice with decreased glial NFκB signaling. Additionally, experiments were performed to determine whether central or peripheral glial NFκB signaling contributes to the maintenance of chronic pain behavior following nerve injury. Oxytetracycline (Oxy), a blood-brain barrier impermeable analog of doxycycline was employed to restrict transgene expression to CNS glia only, leaving peripheral glial signaling intact. Signaling inactivation in central GFAP-positive glia alone failed to exhibit the same analgesic effects as previously observed in animals with both central and peripheral glial signaling inhibition. These data suggest that the NFκB-COX2 signaling pathway in NMSCs is necessary for the maintenance of neuropathic pain in vivo.

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation.

Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release.

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.

A spotlight on underlying the mechanism of AMPK in diabetes complications.

Type 2 diabetes (T2D) is one of the centenarian metabolic disorders and is considered as a stellar and leading health issue worldwide. According to the International Diabetes Federation (IDF) Diabetes Atlas and National Diabetes Statistics, the number of diabetic patients will increase at an exponential rate from 463 to 700 million by the year 2045. Thus, there is a great need for therapies targeting functions that can help in maintaining the homeostasis of glucose levels and improving insulin sensitivity. 5' adenosine monophosphate-activated protein kinase (AMPK) activation, by various direct and indirect factors, might help to overcome the hurdles (like insulin resistance) associated with the conventional approach. A thorough review and analysis was conducted using various database including MEDLINE and EMBASE databases, with Google scholar using various keywords. This extensive review concluded that various drugs (plant-based, synthetic indirect/direct activators) are available, showing tremendous potential in maintaining the homeostasis of glucose and lipid metabolism, without causing insulin resistance, and improving insulin sensitivity. Moreover, these drugs have an effect against diabetes and are therapeutically beneficial in the treatment of diabetes-associated complications (neuropathy and nephropathy) via mechanism involving inhibition of nuclear translocation of SMAD4 (SMAD family member) expression and association with peripheral nociceptive neurons mediated by AMPK. From the available information, it may be concluded that various indirect/direct activators show tremendous potential in maintaining the homeostasis of glucose and lipid metabolism, without resulting in insulin resistance, and may improve insulin sensitivity, as well. Therefore, in a nut shell, it may be concluded that the regulation of APMK functions by various direct/indirect activators may bring promising results. These activators may emerge as a novel therapy in diabetes and its associated complications.

Inhibition of Nav1.7 channel by a novel blocker QLS-81 for alleviation of neuropathic pain.

Voltage-gated sodium channel Nav1.7 robustly expressed in peripheral nociceptive neurons has been considered as a therapeutic target for chronic pain, but there is no selective Nav1.7 inhibitor available for therapy of chronic pain. Ralfinamide has shown anti-nociceptive activity in animal models of inflammatory and neuropathic pain and is currently under phase III clinical trial for neuropathic pain. Based on ralfinamide, a novel small molecule (S)-2-((3-(4-((2-fluorobenzyl) oxy) phenyl) propyl) amino) propanamide (QLS-81) was synthesized. Here, we report the electrophysiological and pharmacodynamic characterization of QLS-81 as a Nav1.7 channel inhibitor with promising anti-nociceptive activity. In whole-cell recordings of HEK293 cells stably expressing Nav1.7, QLS-81(IC50 at 3.5 ± 1.5 μM) was ten-fold more potent than its parent compound ralfinamide (37.1 ± 2.9 μM) in inhibiting Nav1.7 current. QLS-81 inhibition on Nav1.7 current was use-dependent. Application of QLS-81 (10 μM) caused a hyperpolarizing shift of the fast and slow inactivation of Nav1.7 channel about 7.9 mV and 26.6 mV, respectively, and also slowed down the channel fast and slow inactivation recovery. In dissociated mouse DRG neurons, QLS-81 (10 μM) inhibited native Nav current and suppressed depolarizing current pulse-elicited neuronal firing. Administration of QLS-81 (2, 5, 10 mg· kg-1· d-1, i.p.) in mice for 10 days dose-dependently alleviated spinal nerve injury-induced neuropathic pain and formalin-induced inflammatory pain. In addition, QLS-81 (10 μM) did not significantly affect ECG in guinea pig heart ex vivo; and administration of QLS-81 (10, 20 mg/kg, i.p.) in mice had no significant effect on spontaneous locomotor activity. Taken together, our results demonstrate that QLS-81, as a novel Nav1.7 inhibitor, is efficacious on chronic pain in mice, and it may hold developmental potential for pain therapy.

The T-type calcium channel antagonist, Z944, reduces spinal excitability and pain hypersensitivity.

Background and PurposeT‐type voltage‐gated calcium channels are an emerging therapeutic target for neurological disorders including epilepsy and pain. Inhibition of T‐type channels reduces the excitability of peripheral nociceptive sensory neurons and reverses pain hypersensitivity in male rodent pain models. However, administration of peripherally restricted T‐type antagonists failed to show efficacy in multiple clinical and preclinical pain trials, suggesting that inhibition of peripheral T‐type channels alone may be insufficient for pain relief.Experimental ApproachWe utilized the selective and CNS‐penetrant T‐type channel antagonist, Z944, in electrophysiological, calcium imaging and behavioural paradigms to determine its effect on lamina I neuron excitability and inflammatory pain behaviours.Key ResultsVoltage‐clamp recordings from lamina I spinal neurons of adult rats revealed that approximately 80% of neurons possess a low threshold T‐type current, which was blocked by Z944. Due to this highly prevalent T‐type current, Z944 potently blocked action‐potential evoked somatic and dendritic calcium transients in lamina I neurons. Moreover, application of Z944 to spinal cord slices attenuated action potential firing rates in over half of laminae I/II neurons. Finally, we found that intraperitoneal injection of Z944 (1–10 mg·kg−1) dose‐dependently reversed mechanical allodynia in the complete Freund's adjuvant model of persistent inflammatory pain, with a similar magnitude and time course of analgesic effects between male and female rats.Conclusion and ImplicationsT‐type calcium channels critically shape the excitability of lamina I pain processing neurons and inhibition of these channels by the clinical stage antagonist Z944 potently reverses pain hypersensitivity across sexes.

Derivation of Peripheral Nociceptive, Mechanoreceptive, and Proprioceptive Sensory Neurons from the same Culture of Human Pluripotent Stem Cells.

SummaryThe three peripheral sensory neuron (SN) subtypes, nociceptors, mechanoreceptors, and proprioceptors, localize to dorsal root ganglia and convey sensations such as pain, temperature, pressure, and limb movement/position. Despite previous reports, to date no protocol is available allowing the generation of all three SN subtypes at high efficiency and purity from human pluripotent stem cells (hPSCs). We describe a chemically defined differentiation protocol that generates all three SN subtypes from the same starting population, as well as methods to enrich for each individual subtype. The protocol yields high efficiency and purity cultures that are electrically active and respond to specific stimuli. We describe their molecular character and maturity stage and provide evidence for their use as an axotomy model; we show disease phenotypes in hPSCs derived from patients with familial dysautonomia. Our protocol will allow the modeling of human disorders affecting SNs, the search for treatments, and the study of human development.

STING controls nociception via type I interferon signalling in sensory neurons.

The innate immune regulator STING is a critical sensor of self- and pathogen-derived DNA. DNA sensing by STING leads to the induction of type-I interferons (IFN-I) and other cytokines, which promote immune-cell-mediated eradication of pathogens and neoplastic cells1,2. STING is also a robust driver of antitumour immunity, which has led to the development of STING activators and small-molecule agonists as adjuvants for cancer immunotherapy3. Pain, transmitted by peripheral nociceptive sensory neurons (nociceptors), also aids in host defence by alerting organisms to the presence of potentially damaging stimuli, including pathogens and cancer cells4,5. Here we demonstrate that STING is a critical regulator of nociception through IFN-I signalling in peripheral nociceptors. We show that mice lacking STING or IFN-I signalling exhibit hypersensitivity to nociceptive stimuli and heightened nociceptor excitability. Conversely, intrathecal activation of STING produces robust antinociception in mice and non-human primates. STING-mediated antinociception is governed by IFN-Is, which rapidly suppress excitability of mouse, monkey and human nociceptors. Our findings establish the STING-IFN-I signalling axis as a critical regulator of physiological nociception and a promising new target for treating chronic pain.