Protease-Activated Receptor 2 (PAR2) Expressed in Sensory Neurons Contributes to Signs of Pain and Neuropathy in Paclitaxel Treated Mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common, dose-limiting side effect of cancer therapy. Protease-activated receptor 2 (PAR2) is implicated in a variety of pathologies, including CIPN. In this study, we demonstrate the role of PAR2 expressed in sensory neurons in a paclitaxel (PTX)-induced model of CIPN in mice. PAR2 knockout/wildtype (WT) mice and mice with PAR2 ablated in sensory neurons were treated with PTX administered via intraperitoneal injection. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. We then examined immunohistochemical staining of dorsal root ganglion (DRG) and hind paw skin samples from CIPN mice to measure satellite cell gliosis and intra-epidermal nerve fiber (IENF) density. The pharmacological reversal of CIPN pain was tested with the PAR2 antagonist C781. Mechanical allodynia caused by PTX treatment was alleviated in PAR2 knockout mice of both sexes. In the PAR2 sensory neuronal conditional knockout (cKO) mice, both mechanical allodynia and facial grimacing were attenuated in mice of both sexes. In the DRG of the PTX-treated PAR2 cKO mice, satellite glial cell activation was reduced compared to control mice. IENF density analysis of the skin showed that the PTX-treated control mice had a reduction in nerve fiber density while the PAR2 cKO mice had a comparable skin innervation as the vehicle-treated animals. Similar results were seen with satellite cell gliosis in the DRG, where gliosis induced by PTX was absent in PAR cKO mice. Finally, C781 was able to transiently reverse established PTX-evoked mechanical allodynia. PERSPECTIVE: Our work demonstrates that PAR2 expressed in sensory neurons plays a key role in PTX-induced mechanical allodynia, spontaneous pain, and signs of neuropathy, suggesting PAR2 as a possible therapeutic target in multiple aspects of PTX CIPN.

Efficacy of dual enkephalinase inhibition in a preclinical migraine model is mediated by activation of peripheral delta opioid receptors.

OBJECTIVE: The aim of this study was to evaluate whether elevating levels of enkephalin by inhibiting their degradation can attenuate stress-induced migraine-like behaviors in mice. BACKGROUND: Previous studies in animals have suggested the delta opioid receptor (DOR) as a novel migraine target. The primary endogenous ligands for DOR are enkephalins and their levels can be increased by pharmacological inhibition of enkephalinases; however, it is not clear whether enkephalinase inhibition can be efficacious in preclinical migraine models through activation of DOR or whether other opioid receptors might be involved. Further, it is not clear whether opioid receptors in the central nervous system are necessary for these effects. METHODS: This study used a model of repetitive restraint stress in mice that induces periorbital hypersensitivity and priming to the nitric oxide donor sodium nitroprusside (SNP; 0.1 mg/kg). Von Frey filaments were used to measure periorbital mechanical thresholds and grimace scores were evaluated by observing mouse facial features. Animals were treated with the dual enkephalinase inhibitor (DENKI) PL37. RESULTS: On day two post-stress, PL37 given to mice via either intravenous injection (10 mg/kg) or oral gavage (20 mg/kg) significantly attenuated stress-induced periorbital hypersensitivity and facial grimace responses. Additionally, both intravenous (10 mg/kg) and oral gavage (20 mg/kg) of PL37 prior to SNP (0.1 mg/kg) administration on day 14 post-stress significantly reduced SNP-induced facial hypersensitivity. Injection of the DOR antagonist naltrindole (0.1 mg/kg) but not the mu-opioid receptor antagonist CTAP (1 mg/kg) prior to PL37 treatment blocked the effects. Finally, pretreatment of mice with the peripherally restricted opioid receptor antagonist naloxone methiodide (5 mg/kg) blocked the effects of PL37. CONCLUSIONS: These data demonstrate that inhibiting enkephalinases, and thus protecting enkephalins from degradation, attenuates stress-induced migraine-like behavior via activation of peripheral DOR. Peripheral targeting of endogenous opioid signaling may be an effective therapeutic strategy for migraine.

Prolactin in headache and migraine: A systematic review of preclinical studies.

OBJECTIVE: To systemically review preclinical studies investigating the implication of prolactin signaling in headache and migraine pathophysiology. BACKGROUND: The features of migraine attacks, including characteristics, duration, frequency, and prevalence, are sex-dependent with variability across a lifetime, indicating the involvement of the hypothalamus-pituitary-gonadal axis. Prolactin is a key regulator of this axis, and a new line of evidence implicates prolactin signaling in sex-related differences in pain perception. METHODS: In this systematic review, we searched PubMed and EMBASE for the terms prolactin, hyperprolactinemia, macroprolactinemia, hypoprolactinemia, migraine, headache, head pain, and trigeminal pain pathway to find preclinical studies investigating prolactin signaling in headache and migraine. Two reviewers independently screened 841 articles for population, intervention, comparison, outcome, and study design. Studies were restricted to the English language and were excluded if they had a nonexperimental methodology. RESULTS: Of a total of 15 preclinical articles selected, 11 were both ex vivo and in vivo, 3 were ex vivo, and 1 was an in vivo study. The main findings were that prolactin receptors are distributed in the trigeminal pain pathway, and prolactin induced migraine-like behavior in rodents. Moreover, prolactin signaling has a crucial role in calcitonin gene-related peptide (CGRP) release, a key molecule in migraine pathogenesis, and prolactin gene deletion attenuated CGRP-induced migraine-like behavior. CONCLUSION: Preclinical data indicate a key role of prolactin and its receptors in mechanisms causing migraine. Further randomized and placebo-controlled clinical studies targeting prolactin signaling are needed to further clarify the influences of prolactin in migraine-attack initiation.

Prolactin in headache and migraine: A systematic review of clinical studies.

OBJECTIVE: To systemically review clinical studies investigating the role of prolactin and its receptors in headache and migraine. BACKGROUND: Migraine prevalence is more common in women compared to men. As prolactin is a crucial regulator of the hypothalamus-pituitary-gonadal axis, prolactin and its receptors might contribute to signaling mechanisms underlying migraine. METHODS: In this systematic review, we searched PubMed and EMBASE with the terms: prolactin, hyperprolactinemia, macroprolactinemia, hypoprolactinemia, migraine, headache, head pain and trigeminal pain pathway for clinical studies investigating prolactin signaling in headache and migraine. Two reviewers independently screened 841 articles for population, intervention, comparison, outcome, and study design. Studies were restricted to the English language and were excluded if they had a nonexperimental methodology. RESULTS: Nineteen clinical studies met the inclusion criteria and were included in the qualitative and quantitative analysis. The main findings were that serum prolactin levels were found to be higher in individuals with migraine compared to healthy controls, and prolactinomas (prolactin-secreting pituitary adenomas) were correlated with higher incidence of headache in otherwise healthy individuals and migraine attacks in individuals with migraine. CONCLUSION: Considerable evidence suggests a key role of prolactin and its receptors in migraine pathophysiology. Further randomized and placebo-controlled clinical studies targeting prolactin signaling are needed to further clarify influences of prolactin in migraine attack initiation.

MNK1/2 contributes to periorbital hypersensitivity and hyperalgesic priming in preclinical migraine models.

Migraine is thought to involve sensitization of the trigeminal nociceptive system. In preclinical pain models, activation of MNK-eIF4E signalling contributes to nociceptor sensitization and the development of persistent pain. Despite these observations, the role of MNK signalling in migraine remains unclear. Here, we investigate whether activation of MNK contributes to hypersensitivity in two rodent models of migraine. Female and male wild-type (WT) and MNK1 knock-out mice were subjected to repeated restraint stress or a dural injection of interleukin-6 (IL-6) and tested for periorbital hypersensitivity and grimacing. Upon returning to baseline thresholds, stressed mice were administered a low dose of the nitric oxide donor sodium nitroprusside and mice previously injected with IL-6 were given a second dural injection of pH 7.0 to test for hyperalgesic priming. MNK1 knock-out mice were significantly less hypersensitive than the WT following dural IL-6 and did not prime to pH 7.0 or sodium nitroprusside. Furthermore, treatment with the selective MNK inhibitor, eFT508, in WT mice prevented hypersensitivity caused by dural IL-6 or pH 7.0. Together, these results implicate MNK-eIF4E signalling in the development of pain originating from the dura and strongly suggest that targeting MNK inhibition may have significant therapeutic potential as a treatment for migraine.

RNA profiling of human dorsal root ganglia reveals sex differences in mechanisms promoting neuropathic pain.

Neuropathic pain is a leading cause of high-impact pain, is often disabling and is poorly managed by current therapeutics. Here we focused on a unique group of neuropathic pain patients undergoing thoracic vertebrectomy where the dorsal root ganglia is removed as part of the surgery allowing for molecular characterization and identification of mechanistic drivers of neuropathic pain independently of preclinical models. Our goal was to quantify whole transcriptome RNA abundances using RNA-seq in pain-associated human dorsal root ganglia from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain-associated dorsal root ganglia. We sequenced 70 human dorsal root ganglia, and among these 50 met inclusion criteria for sufficient neuronal mRNA signal for downstream analysis. Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14 and OSM in male and CCL1, CCL21, PENK and TLR3 in female dorsal root ganglia associated with neuropathic pain. Coexpression modules revealed enrichment in members of JUN-FOS signalling in males and centromere protein coding genes in females. Neuro-immune signalling pathways revealed distinct cytokine signalling pathways associated with neuropathic pain in males (OSM, LIF, SOCS1) and females (CCL1, CCL19, CCL21). We validated cellular expression profiles of a subset of these findings using RNAscope in situ hybridization. Our findings give direct support for sex differences in underlying mechanisms of neuropathic pain in patient populations.

Resveratrol increases tear production and ocular pain after corneal abrasion in male, but not female, rats using a photorefractive keratectomy model.

Photorefractive keratectomy (PRK) is an alternative to LASIK and can cause intense acute pain that is often not relieved by standard treatments. To assess potential therapeutics for this type of acute pain, appropriate preclinical models are needed. We describe a preclinical corneal abrasion rat model that simulates the initial stages of PRK surgery and demonstrates similar pain and tear dysfunction as seen clinically. We used both behavioral and homeostatic assays to determine the therapeutic potential of resveratrol on pain and tear production. Studies were conducted in male and female Sprague-Dawley rats. Heptanol was applied to one eye and the superficial corneal epithelium was removed, mimicking the abrasion used in PRK. Spontaneous pain was assessed with orbital tightening (OT) scores for 7 days. Topical resveratrol increased OT scores sex-specifically in abraded males, but not females, at 72 h and 1 week after abrasion. Resveratrol increased tear production in abraded males, with no effect in abraded females. There was no correlation between OT score at 1 week and tear production measurements, demonstrating no relationship between spontaneous ocular pain and tear dysfunction in this model. These findings demonstrate the usefulness of our corneal abrasion preclinical PRK model for the assessment of ocular pain therapeutics and indicate that topical resveratrol may not be useful for managing PRK-induced pain.

Spatial transcriptomics of dorsal root ganglia identifies molecular signatures of human nociceptors.

Nociceptors are specialized sensory neurons that detect damaging or potentially damaging stimuli and are found in the dorsal root ganglia (DRG) and trigeminal ganglia. These neurons are critical for the generation of neuronal signals that ultimately create the perception of pain. Nociceptors are also primary targets for treating acute and chronic pain. Single-cell transcriptomics on mouse nociceptors has transformed our understanding of pain mechanisms. We sought to generate equivalent information for human nociceptors with the goal of identifying transcriptomic signatures of nociceptors, identifying species differences and potential drug targets. We used spatial transcriptomics to molecularly characterize transcriptomes of single DRG neurons from eight organ donors. We identified 12 clusters of human sensory neurons, 5 of which are C nociceptors, as well as 1 C low-threshold mechanoreceptors (LTMRs), 1 Aß nociceptor, 2 Aδ, 2 Aß, and 1 proprioceptor subtypes. By focusing on expression profiles for ion channels, G protein-coupled receptors (GPCRs), and other pharmacological targets, we provided a rich map of potential drug targets in the human DRG with direct comparison to mouse sensory neuron transcriptomes. We also compared human DRG neuronal subtypes to nonhuman primates showing conserved patterns of gene expression among many cell types but divergence among specific nociceptor subsets. Last, we identified sex differences in human DRG subpopulation transcriptomes, including a marked increase in calcitonin-related polypeptide alpha (CALCA) expression in female pruritogen receptor-enriched nociceptors. This comprehensive spatial characterization of human nociceptors might open the door to development of better treatments for acute and chronic pain disorders.

Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.

Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-/-) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR-/- line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2-/- grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.

Interleukin-6 induces spatially dependent whole-body hypersensitivity in rats: implications for extracephalic hypersensitivity in migraine.

BACKGROUND: Migraine is a complex neurological disorder that is characterized by throbbing head pain, increased sensitivity to light, sound, and touch, as well as nausea and fatigue. It is one of the most common and most disabling disorders globally but mechanisms causing migraine are poorly understood. While head pain is a typical feature of attacks, they also often present with cutaneous hypersensitivity in the rest of the body. In contrast, primary pain conditions in the lower parts of the body are less commonly associated with cephalic hypersensitivity. Previous studies indicate that application of stimuli to the meninges of rodents causes cutaneous facial as well as hindpaw hypersensitivity. In the present study, we asked whether widespread hypersensitivity is a unique feature of dural stimulation or whether body-wide responses occur similarly when the same stimulus is given in other locations. METHODS: Rats were given the same dose of IL-6 either via dural, intraplantar, subcutaneous, intramuscular, intracisternal, or intrathecal injection. Cutaneous facial and hindpaw allodynia was assessed using Von Frey following injection into each location. RESULTS: Hindpaw allodynia was observed following dural and intraplantar injection of IL-6 in both males and females. Hindpaw allodynia was only observed in females following intracisternal and intrathecal IL-6 injections. In contrast, facial allodynia was only observed in either sex following dural and intracisternal injections, which would activate meningeal afferents and the trigeminal nucleus caudalis (TNC), respectively. CONCLUSIONS: Here we show that while stimulation of upper body regions with IL-6 including the meninges and brainstem can cause widespread hypersensitivity spreading to the paws, similar stimulation of the lower body does not cause the spread of hypersensitivity into the head. These data are consistent with the observations that whole body hypersensitivity is specific to conditions such as migraine where pain is present in the head and they may provide insight into co-morbid pain states associated with migraine.

A ligand-receptor interactome platform for discovery of pain mechanisms and therapeutic targets.

In the peripheral nervous system, ligand-receptor interactions between cells and neurons shape sensory experience, including pain. We set out to identify the potential interactions between sensory neurons and peripheral cell types implicated in disease-associated pain. Using mouse and human RNA sequencing datasets and computational analysis, we created interactome maps between dorsal root ganglion (DRG) sensory neurons and an array of normal cell types, as well as colitis-associated glial cells, rheumatoid arthritis-associated synovial macrophages, and pancreatic tumor tissue. These maps revealed a common correlation between the abundance of heparin-binding EGF-like growth factor (HBEGF) in peripheral cells with that of its receptor EGFR (a member of the ErbB family of receptors) in DRG neurons. Subsequently, we confirmed that increased abundance of HBEGF enhanced nociception in mice, likely acting on DRG neurons through ErbB family receptors. Collectively, these interactomes highlight ligand-receptor interactions that may lead to treatments for disease-associated pain and, furthermore, reflect the complexity of cell-to-neuron signaling in chronic pain states.

De novo protein synthesis is necessary for priming in preclinical models of migraine.

BACKGROUND: Migraine attacks are often triggered by normally innocuous stimuli, suggesting that sensitization within the nervous system is present. One mechanism that may contribute to neuronal sensitization in this context is translation regulation of new protein synthesis. The goal of this study was to determine whether protein synthesis contributes to behavioral responses and priming in preclinical models of migraine. METHODS: Mice received a dural injection of interleukin-6 in the absence or presence of the protein synthesis inhibitor anisomycin or the translation initiation inhibitor 4EGI-1 and were tested for facial hypersensitivity. Upon returning to baseline, mice were given a second, non-noxious dural injection of pH 7.0 to test for priming. Additionally, eIF4ES209Amice lacking phosphorylation of mRNA cap-binding protein eIF4E received dural interleukin-6 or were subjected to repeated restraint stress and then tested for facial hypersensitivity. After returning to baseline, mice were given either dural pH 7.0 or a systemic sub-threshold dose of the nitric oxide donor sodium nitroprusside and tested for priming. RESULTS: Dural injection of interleukin-6 in the presence of anisomycin or 4EGI-1 or in eIF4ES209Amice resulted in the partial attenuation of acute facial hypersensitivity and complete block of hyperalgesic priming. Additionally, hyperalgesic priming following repeated restraint stress was blocked in eIF4ES209Amice. CONCLUSIONS: These studies show that de novo protein synthesis regulated by activity-dependent translation is critical to the development of priming in two preclinical models of migraine. This suggests that targeting the regulation of protein synthesis may be a novel approach for new migraine treatment strategies.

Neuroendocrine Mechanisms Governing Sex Differences in Hyperalgesic Priming Involve Prolactin Receptor Sensory Neuron Signaling.

Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL-6) priming and PGE2 as a second stimulus as a model for pain chronicity. Intraplantar IL-6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE2 hypersensitivity was more persistent in females. This difference in PGE2 response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using ΔPRL, a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female, but not male, mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling.SIGNIFICANCE STATEMENT Females are more likely to experience chronic pain than males, but the mechanisms that underlie this sex difference are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice, where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that prolactin receptor expression in Nav1.8+ neurons was necessary for hyperalgesic priming in female, but not male, mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor.

IL-6 induced upregulation of T-type Ca2+ currents and sensitization of DRG nociceptors is attenuated by MNK inhibition.

Phosphorylation of the 5' cap-binding protein eIF4E by MAPK-interacting kinases (MNK1/2) is important for nociceptor sensitization and the development of chronic pain. IL-6-induced dorsal root ganglion (DRG) nociceptor excitability is attenuated in mice lacking eIF4E phosphorylation, in MNK1/2-/- mice, and by the nonselective MNK1/2 inhibitor cercosporamide. Here, we sought to better understand the neurophysiological mechanisms underlying how IL-6 causes nociceptor excitability via MNK-eIF4E signaling using the highly selective MNK inhibitor eFT508. DRG neurons were cultured from male and female ICR mice, 4-7 wk old. DRG cultures were treated with vehicle, IL-6, eFT508 (pretreat) followed by IL-6, or eFT508 alone. Whole cell patch-clamp recordings were done on small-diameter neurons (20-30 pF) to measure membrane excitability in response to ramp depolarization. IL-6 treatment (1 h) resulted in increased action potential firing compared with vehicle at all ramp intensities, an effect that was blocked by pretreatment with eFT508. Basic membrane properties, including resting membrane potential, input resistance, and rheobase, were similar across groups. Latency to the first action potential in the ramp protocol was lower in the IL-6 group and rescued by eFT508 pretreatment. We also found that the amplitudes of T-type voltage-gated calcium channels (VGCCs) were increased in the DRG following IL-6 treatment, but not in the eFT508 cotreatment group. Our findings are consistent with a model wherein MNK-eIF4E signaling controls the translation of signaling factors that regulate T-type VGCCs in response to IL-6 treatment. Inhibition of MNK with eFT508 disrupts these events, thereby preventing nociceptor hyperexcitability.NEW & NOTEWORTHY In this study, we show that the MNK inhibitor and anti-tumor agent eFT508 (tomivosertib) is effective in attenuating IL-6 induced sensitization of dorsal root ganglion (DRG) nociceptors. Pretreatment with eFT508 in DRG cultures from mice helps mitigate the development of hyperexcitability in response to IL-6. Furthermore, our data reveal that the upregulation of T-type voltage-gated calcium channels following IL-6 application can be blocked by eFT508, implicating the MNK-eIF4E signaling pathway in membrane trafficking of ion channels.

Mycobacterium tuberculosis Sulfolipid-1 Activates Nociceptive Neurons and Induces Cough.

Pulmonary tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), manifests with a persistent cough as both a primary symptom and mechanism of transmission. The cough reflex can be triggered by nociceptive neurons innervating the lungs, and some bacteria produce neuron-targeting molecules. However, how pulmonary Mtb infection causes cough remains undefined, and whether Mtb produces a neuron-activating, cough-inducing molecule is unknown. Here, we show that an Mtb organic extract activates nociceptive neurons in vitro and identify the Mtb glycolipid sulfolipid-1 (SL-1) as the nociceptive molecule. Mtb organic extracts from mutants lacking SL-1 synthesis cannot activate neurons in vitro or induce cough in a guinea pig model. Finally, Mtb-infected guinea pigs cough in a manner dependent on SL-1 synthesis. Thus, we demonstrate a heretofore unknown molecular mechanism for cough induction by a virulent human pathogen via its production of a complex lipid.

Prolactin Regulates Pain Responses via a Female-Selective Nociceptor-Specific Mechanism.

Many clinical and preclinical studies report an increased prevalence and severity of chronic pain among females. Here, we identify a sex-hormone-controlled target and mechanism that regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hind paw or spinal cord substantially reduced hyperalgesia in inflammatory models. This effect was mimicked by sensory neuronal ablation of Prlr. Although Prlr mRNA is expressed equally in female and male peptidergic nociceptors and central terminals, Prlr protein was found only in females and PRL-induced excitability was detected only in female DRG neurons. PRL-induced excitability was reproduced in male Prlr+ neurons after prolonged treatment with estradiol but was prevented with addition of a translation inhibitor. We propose a novel mechanism for female-selective regulation of pain responses, which is mediated by Prlr signaling in sensory neurons via sex-dependent control of Prlr mRNA translation.

Temporal and sex differences in the role of BDNF/TrkB signaling in hyperalgesic priming in mice and rats.

Brain-derived neurotrophic factor (BDNF) signaling through its cognate receptor, TrkB, is a well-known promoter of synaptic plasticity at nociceptive synapses in the dorsal horn of the spinal cord. Existing evidence suggests that BDNF/TrkB signaling in neuropathic pain is sex dependent. We tested the hypothesis that the effects of BDNF/TrkB signaling in hyperalgesic priming might also be sexually dimorphic. Using the incision postsurgical pain model in male mice, we show that BDNF sequestration with TrkB-Fc administered at the time of surgery blocks the initiation and maintenance of hyperalgesic priming. However, when BDNF signaling was blocked prior to the precipitation of hyperalgesic priming with prostaglandin E2 (PGE2), priming was not reversed. This result is in contrast to our findings in male mice with interleukin-6 (IL6) as the priming stimulus where TrkB-Fc was effective in reversing the maintenance of hyperalgesic priming. Furthermore, in IL6-induced hyperalgesic priming, the BDNF sequestering agent, TrkB-fc, was effective in reversing the maintenance of hyperalgesic priming in male mice; however, when this experiment was conducted in female mice, we did not observe any effect of TrkB-fc. This markedly sexual dimorphic effect in mice is consistent with recent studies showing a similar effect in neuropathic pain models. We tested whether the sexual dimorphic role for BDNF was consistent across species. Importantly, we find that this sexual dimorphism does not occur in rats where TrkB-fc reverses hyperalgesic priming fully in both sexes. Finally, to determine the source of BDNF in hyperalgesic priming in mice, we used transgenic mice (Cx3cr1CreER  × Bdnfflx/flx mice) with BDNF eliminated from microglia. From these experiments we conclude that BDNF from microglia does not contribute to hyperalgesic priming and that the key source of BDNF for hyperalgesic priming is likely nociceptors in the dorsal root ganglion. These experiments demonstrate the importance of testing mechanistic hypotheses in both sexes in multiple species to gain insight into complex biology underlying chronic pain.

Nociceptor Translational Profiling Reveals the Ragulator-Rag GTPase Complex as a Critical Generator of Neuropathic Pain.

Nociceptors, sensory neurons in the DRG that detect damaging or potentially damaging stimuli, are key drivers of neuropathic pain. Injury to these neurons causes activation of translation regulation signaling, including the mechanistic target of rapamycin complex 1 (mTORC1) and mitogen-activated protein kinase interacting kinase (MNK) eukaryotic initiation factor (eIF) 4E pathways. This is a mechanism driving changes in excitability of nociceptors that is critical for the generation of chronic pain states; however, the mRNAs that are translated to lead to this plasticity have not been elucidated. To address this gap in knowledge, we used translating ribosome affinity purification in male and female mice to comprehensively characterize mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain (CIPN) caused by paclitaxel treatment. This unbiased method creates a new resource for the field, confirms many findings in the CIPN literature and also find extensive evidence for new target mechanisms that may cause CIPN. We provide evidence that an underlying mechanism of CIPN is sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling. This demonstrates a novel translation regulation signaling circuit wherein MNK1-eIF4E activity drives mTORC1 via control of RagA translation. CIPN and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We identify a novel translational circuit for the genesis of neuropathic pain caused by chemotherapy with important implications for therapeutics.SIGNIFICANCE STATEMENT Neuropathic pain affects up to 10% of the population, but its underlying mechanisms are incompletely understood, leading to poor treatment outcomes. We used translating ribosome affinity purification technology to create a comprehensive translational profile of DRG nociceptors in naive mice and at the peak of neuropathic pain induced by paclitaxel treatment. We reveal new insight into how mechanistic target of rapamycin complex 1 is activated in neuropathic pain pointing to a key role of MNK1-eIF4E-mediated translation of a complex of mRNAs that control mechanistic target of rapamycin complex 1 signaling at the surface of the lysosome. We validate this finding using genetic and pharmacological techniques. Our work strongly suggests that MNK1-eIF4E signaling drives CIPN and that a drug in human clinical trials, eFT508, may be a new therapeutic for neuropathic pain.

Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain.

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Adult mouse sensory neurons on microelectrode arrays exhibit increased spontaneous and stimulus-evoked activity in the presence of interleukin-6.

Following inflammation or injury, sensory neurons located in the dorsal root ganglia (DRG) may exhibit increased spontaneous and/or stimulus-evoked activity, contributing to chronic pain. Current treatment options for peripherally mediated chronic pain are highly limited, driving the development of cell- or tissue-based phenotypic (function-based) screening assays for peripheral analgesic and mechanistic lead discovery. Extant assays are often limited by throughput, content, use of tumorigenic cell lines, or tissue sources from immature developmental stages (i.e., embryonic or postnatal). Here, we describe a protocol for culturing adult mouse DRG neurons on substrate-integrated multiwell microelectrode arrays (MEAs). This approach enables multiplexed measurements of spontaneous as well as stimulus-evoked extracellular action potentials from large populations of cells. The DRG cultures exhibit stable spontaneous activity from 9 to 21 days in vitro. Activity is readily evoked by known chemical and physical agonists of sensory neuron activity such as capsaicin, bradykinin, PGE2, heat, and electrical field stimulation. Most importantly, we demonstrate that both spontaneous and stimulus-evoked activity may be potentiated by incubation with the inflammatory cytokine interleukin-6 (IL-6). Acute responsiveness to IL-6 is inhibited by treatment with a MAPK-interacting kinase 1/2 inhibitor, cercosporamide. In total, these findings suggest that adult mouse DRG neurons on multiwell MEAs are applicable to ongoing efforts to discover peripheral analgesic and their mechanisms of action. NEW & NOTEWORTHY This work describes methodologies for culturing spontaneously active adult mouse dorsal root ganglia (DRG) sensory neurons on microelectrode arrays. We characterize spontaneous and stimulus-evoked adult DRG activity over durations consistent with pharmacological interventions. Furthermore, persistent hyperexcitability could be induced by incubation with inflammatory cytokine IL-6 and attenuated with cercosporamide, an inhibitor of the IL-6 sensitization pathway. This constitutes a more physiologically relevant, moderate-throughput in vitro model for peripheral analgesic screening as well as mechanistic lead discovery.