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.

Sex-stratified genome-wide association study of multisite chronic pain in UK Biobank.

Chronic pain is highly prevalent worldwide and imparts a significant socioeconomic and public health burden. Factors influencing susceptibility to, and mechanisms of, chronic pain development, are not fully understood, but sex is thought to play a significant role, and chronic pain is more prevalent in women than in men. To investigate sex differences in chronic pain, we carried out a sex-stratified genome-wide association study of Multisite Chronic Pain (MCP), a derived chronic pain phenotype, in UK Biobank on 178,556 men and 209,093 women, as well as investigating sex-specific genetic correlations with a range of psychiatric, autoimmune and anthropometric phenotypes and the relationship between sex-specific polygenic risk scores for MCP and chronic widespread pain. We also assessed whether MCP-associated genes showed expression pattern enrichment across tissues. A total of 123 SNPs at five independent loci were significantly associated with MCP in men. In women, a total of 286 genome-wide significant SNPs at ten independent loci were discovered. Meta-analysis of sex-stratified GWAS outputs revealed a further 87 independent associated SNPs. Gene-level analyses revealed sex-specific MCP associations, with 31 genes significantly associated in females, 37 genes associated in males, and a single gene, DCC, associated in both sexes. We found evidence for sex-specific pleiotropy and risk for MCP was found to be associated with chronic widespread pain in a sex-differential manner. Male and female MCP were highly genetically correlated, but at an rg of significantly less than 1 (0.92). All 37 male MCP-associated genes and all but one of 31 female MCP-associated genes were found to be expressed in the dorsal root ganglion, and there was a degree of enrichment for expression in sex-specific tissues. Overall, the findings indicate that sex differences in chronic pain exist at the SNP, gene and transcript abundance level, and highlight possible sex-specific pleiotropy for MCP. Results support the proposition of a strong central nervous-system component to chronic pain in both sexes, additionally highlighting a potential role for the DRG and nociception.

Intercellular Arc Signaling Regulates Vasodilation.

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

Studying human nociceptors: from fundamentals to clinic.

Chronic pain affects one in five of the general population and is the third most important cause of disability-adjusted life-years globally. Unfortunately, treatment remains inadequate due to poor efficacy and tolerability. There has been a failure in translating promising preclinical drug targets into clinic use. This reflects challenges across the whole drug development pathway, from preclinical models to trial design. Nociceptors remain an attractive therapeutic target: their sensitization makes an important contribution to many chronic pain states, they are located outside the blood-brain barrier, and they are relatively specific. The past decade has seen significant advances in the techniques available to study human nociceptors, including: the use of corneal confocal microscopy and biopsy samples to observe nociceptor morphology, the culture of human nociceptors (either from surgical or post-mortem tissue or using human induced pluripotent stem cell derived nociceptors), the application of high throughput technologies such as transcriptomics, the in vitro and in vivo electrophysiological characterization through microneurography, and the correlation with pain percepts provided by quantitative sensory testing. Genome editing in human induced pluripotent stem cell-derived nociceptors enables the interrogation of the causal role of genes in the regulation of nociceptor function. Both human and rodent nociceptors are more heterogeneous at a molecular level than previously appreciated, and while we find that there are broad similarities between human and rodent nociceptors there are also important differences involving ion channel function, expression, and cellular excitability. These technological advances have emphasized the maladaptive plastic changes occurring in human nociceptors following injury that contribute to chronic pain. Studying human nociceptors has revealed new therapeutic targets for the suppression of chronic pain and enhanced repair. Cellular models of human nociceptors have enabled the screening of small molecule and gene therapy approaches on nociceptor function, and in some cases have enabled correlation with clinical outcomes. Undoubtedly, challenges remain. Many of these techniques are difficult to implement at scale, current induced pluripotent stem cell differentiation protocols do not generate the full diversity of nociceptor populations, and we still have a relatively poor understanding of inter-individual variation in nociceptors due to factors such as age, sex, or ethnicity. We hope our ability to directly investigate human nociceptors will not only aid our understanding of the fundamental neurobiology underlying acute and chronic pain but also help bridge the translational gap.

Differences between Dorsal Root and Trigeminal Ganglion Nociceptors in Mice Revealed by Translational Profiling.

Nociceptors located in the trigeminal ganglion (TG) and DRG are the primary sensors of damaging or potentially damaging stimuli for the head and body, respectively, and are key drivers of chronic pain states. While nociceptors in these two tissues show a high degree of functional similarity, there are important differences in their development lineages, their functional connections to the CNS, and recent genome-wide analyses of gene expression suggest that they possess some unique genomic signatures. Here, we used translating ribosome affinity purification to comprehensively characterize and compare mRNA translation in Scn10a-positive nociceptors in the TG and DRG of male and female mice. This unbiased method independently confirms several findings of differences between TG and DRG nociceptors described in the literature but also suggests preferential utilization of key signaling pathways. Most prominently, we provide evidence that translational efficiency in mechanistic target of rapamycin (mTOR)-related genes is higher in the TG compared with DRG, whereas several genes associated with the negative regulator of mTOR, AMP-activated protein kinase, have higher translational efficiency in DRG nociceptors. Using capsaicin as a sensitizing stimulus, we show that behavioral responses are greater in the TG region and this effect is completely reversible with mTOR inhibition. These findings have implications for the relative capacity of these nociceptors to be sensitized upon injury. Together, our data provide a comprehensive, comparative view of transcriptome and translatome activity in TG and DRG nociceptors that enhances our understanding of nociceptor biology.SIGNIFICANCE STATEMENT The DRG and trigeminal ganglion (TG) provide sensory information from the body and head, respectively. Nociceptors in these tissues are critical first neurons in the pain pathway. Injury to peripheral neurons in these tissues can cause chronic pain. Interestingly, clinical and preclinical findings support the conclusion that injury to TG neurons is more likely to cause chronic pain and chronic pain in the TG area is more intense and more difficult to treat. We used translating ribosome affinity purification technology to gain new insight into potential differences in the translatomes of DRG and TG neurons. Our findings demonstrate previously unrecognized differences between TG and DRG nociceptors that provide new insight into how injury may differentially drive plasticity states in nociceptors in these two tissues.

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.

A pharmacological interactome between COVID-19 patient samples and human sensory neurons reveals potential drivers of neurogenic pulmonary dysfunction.

The SARS-CoV-2 virus infects cells of the airway and lungs in humans causing the disease COVID-19. This disease is characterized by cough, shortness of breath, and in severe cases causes pneumonia and acute respiratory distress syndrome (ARDS) which can be fatal. Bronchial alveolar lavage fluid (BALF) and plasma from mild and severe cases of COVID-19 have been profiled using protein measurements and bulk and single cell RNA sequencing. Onset of pneumonia and ARDS can be rapid in COVID-19, suggesting a potential neuronal involvement in pathology and mortality. We hypothesized that SARS-CoV-2 infection drives changes in immune cell-derived factors that then interact with receptors expressed by the sensory neuronal innervation of the lung to further promote important aspects of disease severity, including ARDS. We sought to quantify how immune cells might interact with sensory innervation of the lung in COVID-19 using published data from patients, existing RNA sequencing datasets from human dorsal root ganglion neurons and other sources, and a genome-wide ligand-receptor pair database curated for pharmacological interactions relevant for neuro-immune interactions. Our findings reveal a landscape of ligand-receptor interactions in the lung caused by SARS-CoV-2 viral infection and point to potential interventions to reduce the burden of neurogenic inflammation in COVID-19 pulmonary disease. In particular, our work highlights opportunities for clinical trials with existing or under development rheumatoid arthritis and other (e.g. CCL2, CCR5 or EGFR inhibitors) drugs to treat high risk or severe COVID-19 cases.

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.

RNA sequencing on muscle biopsy from a 5-week bed rest study reveals the effect of exercise and potential interactions with dorsal root ganglion neurons.

Sedentary lifestyle, chronic disease, or microgravity can cause muscle deconditioning that then has an impact on other physiological systems. An example is the nervous system, which is adversely affected by decreased physical activity resulting in increased incidence of neurological problems such as chronic pain. We sought to better understand how this might occur by conducting RNA sequencing experiments on muscle biopsies from human volunteers in a 5-week bed-rest study with an exercise intervention arm. We also used a computational method for examining ligand-receptor interactions between muscle and human dorsal root ganglion (DRG) neurons, the latter of which play a key role in nociception and are generators of signals responsible for chronic pain. We identified 1352 differentially expressed genes (DEGs) in bed rest subjects without an exercise intervention but only 132 DEGs in subjects with the intervention. Among 591 upregulated muscle genes in the no intervention arm, 26 of these were ligands that have receptors that are expressed by human DRG neurons. We detected a specific splice variant of one of these ligands, placental growth factor (PGF), in deconditioned muscle that binds to neuropilin 1, a receptor that is highly expressed in DRG neurons and known to promote neuropathic pain. We conclude that exercise intervention protects muscle from deconditioning transcriptomic changes, and prevents changes in the expression of ligands that might sensitize DRG neurons, or act on other cell types throughout the body. Our work creates a set of actionable hypotheses to better understand how deconditioned muscle may influence the function of sensory neurons that innervate the entire body.

Sex Differences in Nociceptor Translatomes Contribute to Divergent Prostaglandin Signaling in Male and Female Mice.

BACKGROUND: There are clinically relevant sex differences in acute and chronic pain mechanisms, but we are only beginning to understand their mechanistic basis. Transcriptome analyses of rodent whole dorsal root ganglion (DRG) have revealed sex differences, mostly in immune cells. We examined the transcriptome and translatome of the mouse DRG with the goal of identifying sex differences. METHODS: We used translating ribosome affinity purification sequencing and behavioral pharmacology to test the hypothesis that in Nav1.8-positive neurons, most of which are nociceptors, translatomes would differ by sex. RESULTS: We found 80 genes with sex differential expression in the whole DRG transcriptome and 66 genes whose messenger RNAs were sex differentially actively translated (translatome). We also identified different motifs in the 3' untranslated region of messenger RNAs that were sex differentially translated. In further validation studies, we focused on Ptgds, which was increased in the translatome of female mice. The messenger RNA encodes the prostaglandin PGD2 synthesizing enzyme. We observed increased PTGDS protein and PGD2 in female mouse DRG. The PTGDS inhibitor AT-56 caused intense pain behaviors in male mice but was only effective at high doses in female mice. Conversely, female mice responded more robustly to another major prostaglandin, PGE2, than did male mice. PTGDS protein expression was also higher in female cortical neurons, suggesting that DRG findings may be generalizable to other nervous system structures. CONCLUSIONS: Our results demonstrate sex differences in nociceptor-enriched translatomes and reveal unexpected sex differences in one of the oldest known nociceptive signaling molecule families, the prostaglandins.

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.

Diversity of Receptor Expression in Central and Peripheral Mouse Neurons Estimated from Single Cell RNA Sequencing.

Because somatosensory PNS neurons, in particular nociceptors, are specially tuned to be able to detect a wide variety of both exogenous and endogenous signals, one might assume that these neurons express a greater variety of receptor genes. This assumption has not been formally tested. Because cells detect such signals via cell surface receptors, we sought to formally test the hypothesis that PNS neurons might express a broader array of cell surface receptors than CNS neurons using existing single cell RNA sequencing resources from mouse. We focused our analysis on ion channels, G-protein coupled receptors (GPCRS), receptor tyrosine kinase and cytokine family receptors. In partial support of our hypothesis, we found that mouse PNS somatosensory, sympathetic and enteric neurons and CNS neurons have similar receptor expression diversity in families of receptors examined, with the exception of GPCRs and cytokine receptors which showed greater diversity in the PNS. Surprisingly, these differences were mostly driven by enteric and sympathetic neurons, not by somatosensory neurons or nociceptors. Secondary analysis revealed many receptors that are very specifically expressed in subsets of PNS neurons, including some that are unique among neurons for nociceptors. Finally, we sought to examine specific ligand-receptor interactions between T cells and PNS and CNS neurons. Again, we noted that most interactions between these cells are shared by CNS and PNS neurons despite the fact that T cells only enter the CNS under rare circumstances. Our findings demonstrate that both PNS and CNS neurons express an astonishing array of cell surface receptors and suggest that most neurons are tuned to receive signals from other cells types, in particular immune cells.

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.

Nasal administration of mitochondria reverses chemotherapy-induced cognitive deficits.

Up to seventy-five percent of patients treated for cancer suffer from cognitive deficits which can persist for months to decades, severely impairing quality of life. Although the number of cancer survivors is increasing tremendously, no efficacious interventions exist. Cisplatin, most commonly employed for solid tumors, leads to cognitive impairment including deficits in memory and executive functioning. We recently proposed deficient neuronal mitochondrial function as its underlying mechanism. We hypothesized nasal administration of mitochondria isolated from human mesenchymal stem cells to mice, can reverse cisplatin-induced cognitive deficits. Methods: Puzzle box, novel object place recognition and Y-maze tests were used to assess the cognitive function of mice. Immunofluorescence and high-resolution confocal microscopy were employed to trace the nasally delivered mitochondria and evaluate their effect on synaptic loss. Black Gold II immunostaining was used to determine myelin integrity. Transmission electron microscopy helped determine mitochondrial and membrane integrity of brain synaptosomes. RNA-sequencing was performed to analyse the hippocampal transcriptome. Results: Two nasal administrations of mitochondria isolated from human mesenchymal stem cells to mice, restored executive functioning, working and spatial memory. Confocal imaging revealed nasally delivered mitochondria rapidly arrived in the meninges where they were readily internalized by macrophages. The administered mitochondria also accessed the rostral migratory stream and various other brain regions including the hippocampus where they colocalized with GFAP+ cells. The restoration of cognitive function was associated with structural repair of myelin in the cingulate cortex and synaptic loss in the hippocampus. Nasal mitochondrial donation also reversed the underlying synaptosomal mitochondrial defects. Moreover, transcriptome analysis by RNA-sequencing showed reversal of cisplatin-induced changes in the expression of about seven hundred genes in the hippocampus. Pathway analysis identified Nrf2-mediated response as the top canonical pathway. Conclusion: Our results provide key evidence on the therapeutic potential of isolated mitochondria - restoring both brain structure and function, their capability to enter brain meninges and parenchyma upon nasal delivery and undergo rapid cellular internalization and alter the hippocampal transcriptome. Our data identify nasal administration of mitochondria as an effective strategy for reversing chemotherapy-induced cognitive deficits and restoring brain health, providing promise for the growing population of both adult and pediatric cancer survivors.

A peptide encoded within a 5' untranslated region promotes pain sensitization in mice.

ABSTRACT: Translational regulation permeates neuronal function. Nociceptors are sensory neurons responsible for the detection of harmful stimuli. Changes in their activity, termed plasticity, are intimately linked to the persistence of pain. Although inhibitors of protein synthesis robustly attenuate pain-associated behavior, the underlying targets that support plasticity are largely unknown. Here, we examine the contribution of protein synthesis in regions of RNA annotated as noncoding. Based on analyses of previously reported ribosome profiling data, we provide evidence for widespread translation in noncoding transcripts and regulatory regions of mRNAs. We identify an increase in ribosome occupancy in the 5' untranslated regions of the calcitonin gene-related peptide (CGRP/Calca). We validate the existence of an upstream open reading frame (uORF) using a series of reporter assays. Fusion of the uORF to a luciferase reporter revealed active translation in dorsal root ganglion neurons after nucleofection. Injection of the peptide corresponding to the calcitonin gene-related peptide-encoded uORF resulted in pain-associated behavioral responses in vivo and nociceptor sensitization in vitro. An inhibitor of heterotrimeric G protein signaling blocks both effects. Collectively, the data suggest pervasive translation in regions of the transcriptome annotated as noncoding in dorsal root ganglion neurons and identify a specific uORF-encoded peptide that promotes pain sensitization through GPCR signaling.

A Role for Protease Activated Receptor Type 3 (PAR3) in Nociception Demonstrated Through Development of a Novel Peptide Agonist.

The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca2+ response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3-/- mice. We characterized other nociceptive phenotypes in PAR3-/- mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3-/- mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. PERSPECTIVE: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.

ACE2 and SCARF expression in human dorsal root ganglion nociceptors: implications for SARS-CoV-2 virus neurological effects.

SARS-CoV-2 has created a global crisis. COVID-19, the disease caused by the virus, is characterized by pneumonia, respiratory distress, and hypercoagulation and can be fatal. An early sign of infection is loss of smell, taste, and chemesthesis-loss of chemical sensation. Other neurological effects of the disease have been described, but not explained. It is now apparent that many of these neurological effects (for instance joint pain and headache) can persist for at least months after infection, suggesting a sensory neuronal involvement in persistent disease. We show that human dorsal root ganglion (DRG) neurons express the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 at the RNA and protein level. We also demonstrate that SARS-CoV-2 and coronavirus-associated factors and receptors are broadly expressed in human DRG at the lumbar and thoracic level as assessed by bulk RNA sequencing. ACE2 mRNA is expressed by a subset of nociceptors that express MRGPRD mRNA, suggesting that SARS-CoV-2 may gain access to the nervous system through entry into neurons that form free nerve endings at the outermost layers of skin and luminal organs. Therefore, DRG sensory neurons are a potential target for SARS-CoV-2 invasion of the peripheral nervous system, and viral infection of human nociceptors may cause some of the persistent neurological effects seen in COVID-19.

Pharmacological target-focused transcriptomic analysis of native vs cultured human and mouse dorsal root ganglia.

Dorsal root ganglion (DRG) neurons detect sensory inputs and are crucial for pain processing. They are often studied in vitro as dissociated cell cultures with the assumption that this reasonably represents in vivo conditions. However, to the best of our knowledge, no study has directly compared genome-wide transcriptomes of DRG tissue in vivo versus in vitro or between laboratories and culturing protocols. Comparing RNA sequencing-based transcriptomes of native to cultured (4 days in vitro) human or mouse DRG, we found that the overall expression levels of many ion channels and G-protein-coupled receptors specifically expressed in neurons are markedly lower although still expressed in culture. This suggests that most pharmacological targets expressed in vivo are present under the condition of dissociated cell culture, but with changes in expression levels. The reduced relative expression for neuronal genes in human DRG cultures is likely accounted for by increased expression of genes in fibroblast-like and other proliferating cells, consistent with their mitotic status in these cultures. We found that the expression of a subset of genes typically expressed in neurons increased in human and mouse DRG cultures relative to the intact ganglion, including genes associated with nerve injury or inflammation in preclinical models such as BDNF, MMP9, GAL, and ATF3. We also found a striking upregulation of a number of inflammation-associated genes in DRG cultures, although many were different between mouse and human. Our findings suggest an injury-like phenotype in DRG cultures that has important implications for the use of this model system for pain drug discovery.

The cellular basis of protease-activated receptor 2-evoked mechanical and affective pain.

Protease-activated receptor 2 (PAR2) has long been implicated in inflammatory and visceral pain, but the cellular basis of PAR2-evoked pain has not been delineated. Although PAR2-evoked pain has been attributed to sensory neuron expression, RNA-sequencing experiments show ambiguous F2rl1 mRNA detection. Moreover, many pharmacological tools for PAR2 are nonspecific, acting also on the Mas-related GPCR family (Mrg) that are highly enriched in sensory neurons. We sought to clarify the cellular basis of PAR2-evoked pain. We developed a PAR2-conditional knockout mouse and specifically deleted PAR2 in all sensory neurons using the PirtCre mouse line. Our behavioral findings show that PAR2 agonist-evoked mechanical hyperalgesia and facial grimacing, but not thermal hyperalgesia, are dependent on PAR2 expression in sensory neurons that project to the hind paw in male and female mice. F2rl1 mRNA is expressed in a discrete population (~4%) of mostly small-diameter sensory neurons that coexpress the Nppb and IL31ra genes. This cell population has been implicated in itch, but our work shows that PAR2 activation in these cells causes clear pain-related behaviors from the skin. Our findings show that a discrete population of DRG sensory neurons mediate PAR2-evoked pain.

A pharmacological interactome between COVID-19 patient samples and human sensory neurons reveals potential drivers of neurogenic pulmonary dysfunction.

The SARS-CoV-2 virus infects cells of the airway and lungs in humans causing the disease COVID-19. This disease is characterized by cough, shortness of breath, and in severe cases causes pneumonia and acute respiratory distress syndrome (ARDS) which can be fatal. Bronchial alveolar lavage fluid (BALF) and plasma from mild and severe cases of COVID-19 have been profiled using protein measurements and bulk and single cell RNA sequencing. Onset of pneumonia and ARDS can be rapid in COVID-19, suggesting a potential neuronal involvement in pathology and mortality. We sought to quantify how immune cells might interact with sensory innervation of the lung in COVID-19 using published data from patients, existing RNA sequencing datasets from human dorsal root ganglion neurons and other sources, and a genome-wide ligand-receptor pair database curated for pharmacological interactions relevant for neuro-immune interactions. Our findings reveal a landscape of ligand-receptor interactions in the lung caused by SARS-CoV-2 viral infection and point to potential interventions to reduce the burden of neurogenic inflammation in COVID-19 disease. In particular, our work highlights opportunities for clinical trials with existing or under development rheumatoid arthritis and other (e.g. CCL2, CCR5 or EGFR inhibitors) drugs to treat high risk or severe COVID-19 cases.