Sensory specializations drive octopus and squid behaviour.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Structural basis of sensory receptor evolution in octopus.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via 'taste by touch'1. CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that the channel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour.

Sensory Neurotransmitter Calcitonin Gene-Related Peptide Modulates Tumor Growth and Lymphocyte Infiltration in Oral Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma are highly innervated by peripheral sensory neurons. Local neurotransmitter release (e.g., calcitonin gene-related peptide (CGRP)) from sensory neurons innervating cancer is linked to tumorigenesis. CGRP-immunoreactive nerve presence comprised 9.53±1.9% of total nerve area across 11 HNSCC patients. A syngeneic tongue tumor transplant mouse model of oral cancer and a global Calca knockout mouse (CGRPKO ) are used to investigate the impact of CGRP signaling on tumor growth and the associated immune response in vivo. In tumor-bearing CGRPKO mice, there is a significant reduction in tumor size over time compared to wildtype mice using two different mouse oral cancer cell lines. Furthermore, tumor tissue from CGRPKO mice had a significant increase in tumor-infiltrating CD4+ T cells, cytotoxic CD8+ T cells, and NK1.1+ NK cells compared to wildtype. Fluorescent-activated cell sorting and real-time qPCR are used to confirm that CD4+ T cells are isolated from tumor-bearing wildtype mice containing a high expression of Ramp1 compared to sham mice. These data suggest that sensory neurotransmitter CGRP may modulate oral cancer progression via tumor immunosurveillance. Understanding the relationship between sensory neurons and cancer will aid in repurposing clinically available nervous system drugs for the treatment of cancer.

The impact of SBF2 on taxane-induced peripheral neuropathy.

Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients. In addition to its impact on quality of life, this toxicity may lead to dose reductions or treatment discontinuation, adversely impacting survival outcomes and leading to health disparities in African Americans (AA). Our lab has previously identified deleterious mutations in SET-Binding Factor 2 (SBF2) that significantly associated with severe TIPN in AA patients. Here, we demonstrate the impact of SBF2 on taxane-induced neuronal damage using an ex vivo model of SBF2 knockdown of induced pluripotent stem cell-derived sensory neurons. Knockdown of SBF2 exacerbated paclitaxel changes to cell viability and neurite outgrowth while attenuating paclitaxel-induced sodium current inhibition. Our studies identified paclitaxel-induced expression changes specific to mature sensory neurons and revealed candidate genes involved in the exacerbation of paclitaxel-induced phenotypes accompanying SBF2 knockdown. Overall, these findings provide ex vivo support for the impact of SBF2 on the development of TIPN and shed light on the potential pathways involved.

Combining chitin biological conduits with small autogenous nerves and platelet-rich plasma for the repair of sciatic nerve defects in rats.

AIMS: Peripheral nerve defects are often difficult to recover from, and there is no optimal repair method. Therefore, it is important to explore new methods of repairing peripheral nerve defects. This study explored the efficacy of nerve grafts constructed from chitin biological conduits combined with small autogenous nerves (SANs) and platelet-rich plasma (PRP) for repairing 10-mm sciatic nerve defects in rats. METHODS: To prepare 10-mm sciatic nerve defects, SANs were first harvested and PRP was extracted. The nerve grafts consisted of chitin biological conduits combined with SAN and PRP, and were used to repair rat sciatic nerve defects. These examinations, including measurements of axon growth efficiency, a gait analysis, electrophysiological tests, counts of regenerated myelinated fibers and observations of their morphology, histological evaluation of the gastrocnemius muscle, retrograde tracing with Fluor-Gold (FG), and motor endplates (MEPs) distribution analysis, were conducted to evaluate the repair status. RESULTS: Two weeks after nerve transplantation, the rate and number of regenerated axons in the PRP-SAN group improved compared with those in the PRP, SAN, and Hollow groups. The PRP-SAN group exhibited better recovery in terms of the sciatic functional index value, composite action potential intensity, myelinated nerve fiber density, myelin sheath thickness, and gastrectomy tissue at 12 weeks after transplantation, compared with the PRP and SAN groups. The results of FG retrograde tracing and MEPs analyses showed that numbers of FG-positive sensory neurons and motor neurons as well as MEPs distribution density were higher in the PRP-SAN group than in the PRP or SAN group. CONCLUSIONS: Nerve grafts comprising chitin biological conduits combined with SANs and PRP significantly improved the repair of 10-mm sciatic nerve defects in rats and may have therapeutic potential for repairing peripheral nerve defects in future applications.

Somatosensory innervation of healthy human oral tissues.

The oral somatosensory system relays essential information about mechanical stimuli to enable oral functions such as feeding and speech. The neurochemical and anatomical diversity of sensory neurons across oral cavity sites have not been systematically compared. To address this gap, we analyzed healthy human tongue and hard-palate innervation. Biopsies were collected from 12 volunteers and underwent fluorescent immunohistochemistry (≥2 specimens per marker/structure). Afferents were analyzed for markers of neurons (ßIII tubulin), myelinated afferents (neurofilament heavy, NFH), and Merkel cells and taste cells (keratin 20, K20). Hard-palate innervation included Meissner corpuscles, glomerular endings, Merkel cell-neurite complexes, and free nerve endings. The organization of these somatosensory endings is reminiscent of fingertips, suggesting that the hard palate is equipped with a rich repertoire of sensory neurons for pressure sensing and spatial localization of mechanical inputs, which are essential for speech production and feeding. Likewise, the tongue is innervated by afferents that impart it with exquisite acuity and detection of moving stimuli that support flavor construction and speech. Filiform papillae contained end bulbs of Krause, as well as endings that have not been previously reported, including subepithelial neuronal densities, and NFH+ neurons innervating basal epithelia. Fungiform papillae had Meissner corpuscles and densities of NFH+ intraepithelial neurons surrounding taste buds. The differing compositions of sensory endings within filiform and fungiform papillae suggest that these structures have distinct roles in mechanosensation. Collectively, this study has identified previously undescribed neuronal endings in human oral tissues and provides an anatomical framework for understanding oral mechanosensory functions.

Neuropeptide expression in the box jellyfish Tripedalia cystophora-New insights into the complexity of a "simple" nervous system.

Box jellyfish have an elaborate visual system and perform advanced visually guided behaviors. However, the rhopalial nervous system (RNS), believed to be the main visual processing center, only has 1000 neurons in each of the four eye carrying rhopalia. We have examined the detailed structure of the RNS of the box jellyfish Tripedalia cystophora, using immunolabeling with antibodies raised against four putative neuropeptides (T. cystophora RFamide, VWamide, RAamide, and FRamide). In the RNS, T. cystophora RF-, VW-, and RAamide antibodies stain sensory neurons, the pit eyes, the neuropil, and peptide-specific subpopulations of stalk-associated neurons and giant neurons. Furthermore, RFamide ir+ neurites are seen in the epidermal stalk nerve, whereas VWamide antibodies stain the gastrodermal stalk nerve. RFamide has the most widespread expression including in the ring and radial nerves, the pedalium nerve plexus, and the tentacular nerve net. RAamide is the putative neurotransmitter in the motor neurons of the subumbrellar nerve net, and VWamide is a potential marker for neuronal differentiation as it is found in subpopulations of undifferentiated cells both in the rhopalia and in the bell. The results from the FRamide antibodies were not included as only few cells were stained, and in an unreproducible way. Our studies show hitherto-unseen details of the nervous system of T. cystophora and allowed us to identify specific functional groups of neurons. This identification is important for understanding visual processing in the RNS and enables experimental work, directly addressing the role of the different neuropeptides in vision.

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

The 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.

A proteomic view on the differential phenotype of Schwann cells derived from mouse sensory and motor nerves.

Schwann cells (SCs) are myelin-forming glial cells of the peripheral nervous system. Recent studies suggested that SCs comprise two phenotypes: sensory SCs and motor SCs, which are associated with the modality-specific promotion of sensory and motor axon growth during peripheral neuronal regeneration. However, the molecular basis of the two phenotypic SCs is unclear. We established a workflow to obtain highly purified SCs derived from sensory nerve (SNdSCs) and motor nerve (MNdSCs) from B6; D2-Tg(s100B-EGFP)1Wjt/J mice. Subsequently, a quantitative proteomic analysis based on iTRAQ labeling was performed to compare the proteome of SNdSCs and MNdSCs. A total of 6,567 proteins were identified, of which 63 and 11 proteins were overexpressed in SNdSCs and MNdSCs, respectively. Three of the overexpressed proteins were further validated by western blot and immunocytochemistry: GMFB and CNPase, which were overexpressed in sensory SNdSCs, and histone H4, which was overexpressed in MNdSCs. The expression pattern of the three proteins was also validated in the dorsal roots and ventral roots. Bioinformatics analysis indicated that proteins highly expressed in SNdSCs are mainly involved in RNA processing and protein synthesis, while those overexpressed in MNdSCs are related to cell proliferation. Real-time cell analysis confirmed that the proliferation activity of MNdSCs is higher than that of SNdSCs. This study is the first to provide a proteomic view of the differential phenotype of mouse SNdSCs and MNdSCs. The data may serve as a valuable source for the study of the biological characteristics of these two SC phenotypes and their roles in nerve-specific regeneration.

Evidence of chitin in the ampullae of Lorenzini of chondrichthyan fishes.

We previously reported that the polysaccharide chitin, a key component of arthropod exoskeletons and fungal cell walls, is endogenously produced by fishes and amphibians in spite of the widely held view that it was not synthesized by vertebrates [1]. Genes encoding chitin synthase enzymes were found in the genomes of a number of fishes and amphibians and shown to be correspondingly expressed at the sites where chitin was localized [1,2]. In this report, we present evidence suggesting that chitin is prevalent within the specialized electrosensory organs of cartilaginous fishes (Chondrichthyes). These organs, the Ampullae of Lorenzini (AoL), are widely distributed and comprise a series of gel-filled canals emanating from pores in the skin (Figure 1A). The canals extend into bulbous structures called alveoli that contain sensory cells capable of detecting subtle changes in electric fields (Figure 1B) [3,4]. The findings described here extend the number of vertebrate taxa where endogenous chitin production has been detected and raise questions regarding chitin's potential function in chondrichthyan fishes and other aquatic vertebrates.

Inhibition of PTEN activity promotes IB4-positive sensory neuronal axon growth.

Traumatic nerve injuries have become a common clinical problem, and axon regeneration is a critical process in the successful functional recovery of the injured nervous system. In this study, we found that peripheral axotomy reduces PTEN expression in adult sensory neurons; however, it did not alter the expression level of PTEN in IB4-positive sensory neurons. Additionally, our results indicate that the artificial inhibition of PTEN markedly promotes adult sensory axon regeneration, including IB4-positive neuronal axon growth. Thus, our results provide strong evidence that PTEN is a prominent repressor of adult sensory axon regeneration, especially in IB4-positive neurons.

Sensory cutaneous papillae in the sea lamprey (Petromyzon marinus L.): I. Neuroanatomy and physiology.

Molecules present in an animal's environment can indicate the presence of predators, food, or sexual partners and consequently, induce migratory, reproductive, foraging, or escape behaviors. Three sensory systems, the olfactory, gustatory, and solitary chemosensory cell (SCC) systems detect chemical stimuli in vertebrates. While a great deal of research has focused on the olfactory and gustatory system over the years, it is only recently that significant attention has been devoted to the SCC system. The SCCs are microvillous cells that were first discovered on the skin of fish, and later in amphibians, reptiles, and mammals. Lampreys also possess SCCs that are particularly numerous on cutaneous papillae. However, little is known regarding their precise distribution, innervation, and function. Here, we show that sea lampreys (Petromyzon marinus L.) have cutaneous papillae located around the oral disk, nostril, gill pores, and on the dorsal fins and that SCCs are particularly numerous on these papillae. Tract-tracing experiments demonstrated that the oral and nasal papillae are innervated by the trigeminal nerve, the gill pore papillae are innervated by branchial nerves, and the dorsal fin papillae are innervated by spinal nerves. We also characterized the response profile of gill pore papillae to some chemicals and showed that trout-derived chemicals, amino acids, and a bile acid produced potent responses. Together with a companion study (Suntres et al., Journal of Comparative Neurology, this issue), our results provide new insights on the function and evolution of the SCC system in vertebrates.

Cutaneous inflammation differentially regulates the expression and function of Angiotensin-II types 1 and 2 receptors in rat primary sensory neurons.

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

Deep Sequencing of Somatosensory Neurons Reveals Molecular Determinants of Intrinsic Physiological Properties.

Dorsal root ganglion (DRG) sensory neuron subtypes defined by their in vivo properties display distinct intrinsic electrical properties. We used bulk RNA sequencing of genetically labeled neurons and electrophysiological analyses to define ion channel contributions to the intrinsic electrical properties of DRG neuron subtypes. The transcriptome profiles of eight DRG neuron subtypes revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by these data, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties. The distinct ensembles of voltage-gated ion channels predicted to underlie the unique intrinsic physiological properties of eight DRG neuron subtypes are presented.

DNA methylation patterns in human iPSC-derived sensory neuronal differentiation.

Sensory neurons of the peripheral nervous system are critical in health and disease. Sensory neurons derived from induced pluripotent stem (iPS) cells are now being used increasingly for in vitro models of neuropathy, pain, and neurotoxicity. DNA methylation is critical for neurodevelopment and has been implicated in many neuronal diseases, but has not been examined in iPS-derived sensory neurons. In order to better characterize the iPS-derived sensory neuron model, we have undertaken a genome-wide DNA methylation study on the cells from human iPS to iPS-derived sensory neurons during differentiation through reduced representation and bisulfite sequencing. We report decreasing DNA methylation with iPS-derived sensory neuronal differentiation that is reflected in increasing numbers and proportions of hypomethylated individual CpGs and regions, as well as lowered DNMT3b expression. Furthermore, genes with changes in DNA methylation near their TSS suggest key pathways that may be involved in iPS-derived sensory neuronal differentiation. These findings provide insights into sensory neuronal differentiation and can be used for further in vitro modelling of disease states.

Reduced level of docosahexaenoic acid shifts GPCR neuroreceptors to less ordered membrane regions.

G protein-coupled receptors (GPCRs) control cellular signaling and responses. Many of these GPCRs are modulated by cholesterol and polyunsaturated fatty acids (PUFAs) which have been shown to co-exist with saturated lipids in ordered membrane domains. However, the lipid compositions of such domains extracted from the brain cortex tissue of individuals suffering from GPCR-associated neurological disorders show drastically lowered levels of PUFAs. Here, using free energy techniques and multiscale simulations of numerous membrane proteins, we show that the presence of the PUFA DHA helps helical multi-pass proteins such as GPCRs partition into ordered membrane domains. The mechanism is based on hybrid lipids, whose PUFA chains coat the rough protein surface, while the saturated chains face the raft environment, thus minimizing perturbations therein. Our findings suggest that the reduction of GPCR partitioning to their native ordered environments due to PUFA depletion might affect the function of these receptors in numerous neurodegenerative diseases, where the membrane PUFA levels in the brain are decreased. We hope that this work inspires experimental studies on the connection between membrane PUFA levels and GPCR signaling.

Production, purification and characterization of an elastin-like polypeptide containing the Ile-Lys-Val-Ala-Val (IKVAV) peptide for tissue engineering applications.

Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.

Characterization of perinatally born glutamatergic neurons of the mouse olfactory bulb based on NeuroD6 expression reveals their resistance to sensory deprivation.

During postnatal olfactory bulb (OB) neurogenesis, predetermined stem cells residing in the ventricular-subventricular zone continuously generate progenitors that migrate in the rostral migratory stream and integrate into the OB. Although the vast majority of these postnatally generated interneurons are inhibitory, a sub-fraction represents glutamatergic neurons that integrate into the superficial glomerular layer. In the present work, we demonstrate that the bHLH transcription factor NeuroD6 is specifically and transitorily expressed in the dorsal neurogenic lineage that generates glutamatergic juxtaglomerular cells (JGCs) for the OB. Using lineage tracing combined with whole brain clearing, we provide new insight into timing of generation, morphology, and connectivity of glutamatergic JGCs. Specifically, we show that all glutamatergic JGCs send complex axons with varying projection patterns into different layers of the OB. Moreover, we find that, contrary to GABAergic OB interneurons, glutamatergic JGCs survive under sensory deprivation, indicating that inhibitory and excitatory populations are differentially susceptible to environmental stimulation.

MBD1 Contributes to the Genesis of Acute Pain and Neuropathic Pain by Epigenetic Silencing of Oprm1 and Kcna2 Genes in Primary Sensory Neurons.

The transmission of normal sensory and/or acute noxious information requires intact expression of pain-associated genes within the pain pathways of nervous system. Expressional changes of these genes after peripheral nerve injury are also critical for neuropathic pain induction and maintenance. Methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, regulates gene transcriptional activity. We report here that MBD1 in the primary sensory neurons of DRG is critical for the genesis of acute pain and neuropathic pain as DRG MBD1-deficient mice exhibit the reduced responses to acute mechanical, heat, cold, and capsaicin stimuli and the blunted nerve injury-induced pain hypersensitivities. Furthermore, DRG overexpression of MBD1 leads to spontaneous pain and evoked pain hypersensitivities in the WT mice and restores acute pain sensitivities in the MBD1-deficient mice. Mechanistically, MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 is likely a key player under the conditions of acute pain and neuropathic pain.SIGNIFICANCE STATEMENT In the present study, we revealed that the mice with deficiency of methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, in the DRG displayed the reduced responses to acute noxious stimuli and the blunted neuropathic pain. We also showed that DRG overexpression of MBD1 produced the hypersensitivities to noxious stimuli in the WT mice and rescued acute pain sensitivities in the MBD1-deficient mice. We have also provided the evidence that MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 may participate in the genesis of acute pain and neuropathic pain likely through regulating DNMT3a-controlled Oprm1 and Kcna2 gene expression in the DRG neurons.

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.