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 Aβ nociceptor, 2 Aδ, 2 Aβ, 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.

Migraine is the second leading cause for disability worldwide and the most common neurological disorder. It is also three times more common in women; reasons for this sex difference are not known. Using preclinical behavioral models of migraine, we show that application of calcitonin gene-related peptide (CGRP) to the rat dura mater produces cutaneous periorbital hypersensitivity. Surprisingly, this response was observed only in females; dural CGRP at doses from 1 pg to 3.8 μg produce no responses in males. In females, dural CGRP causes priming to a pH 7.0 solution after animals recover from the initial CGRP-induced allodynia. Dural application of interleukin-6 causes acute responses in males and females but only causes priming to subthreshold dural CGRP (0.1 pg) in females. Intracisternal application of BDNF also causes similar acute hypersensitivity responses in males and females but only priming to subthreshold dural CGRP (0.1 pg) in females. Females were additionally primed to a subthreshold dose of the NO-donor sodium nitroprusside (0.1 mg/kg) following dural CGRP. Finally, the sexually dimorphic responses to dural CGRP were not specific to rats as similar female-specific hypersensitivity responses were seen in mice, where increased grimace responses were also observed. These data are the first to demonstrate that CGRP-induced headache-like behavioral responses at doses up to 3.8 μg are female-specific both acutely and following central and peripheral priming. These data further implicate dural CGRP signaling in the pathophysiology of migraine and propose a model where dural CGRP-based mechanisms contribute to the sexual disparity of this female-biased disorder.SIGNIFICANCE STATEMENTCalcitonin gene-related peptide (CGRP) has long been implicated in the pathophysiology of migraine, and CGRP-based therapeutics are efficacious for the treatment of migraine in humans. However, the location of action for CGRP in migraine remains unclear. We show here that application of CGRP to the cranial meninges causes behavioral responses consistent with headache in preclinical rodent models. Surprisingly, however, these responses are only observed in females. Acute responses to meningeal CGRP are female-specific and sensitization to CGRP after two distinct stimuli are also female-specific. These data implicate the dura mater as a primary location of action for CGRP in migraine and suggest that female-specific mechanisms downstream of CGRP receptor activation contribute to the higher prevalence of migraine in women.

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.

Chronic pain, encompassing conditions, such as low back pain, arthritis, persistent post-surgical pain, fibromyalgia, and neuropathic pain disorders, is highly prevalent but remains poorly treated. The vast majority of therapeutics are directed solely at neurons, despite the fact that signaling between immune cells, glia, and neurons is now recognized as indispensable for the initiation and maintenance of chronic pain. This review highlights recent advances in understanding fundamental neuroimmune signaling mechanisms and novel therapeutic targets in rodent models of chronic pain. We further discuss new technological developments to study, diagnose, and quantify neuroimmune contributions to chronic pain in patient populations.

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 inScn10a-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 STATEMENTNeuropathic 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.