Signaling In Sensory Neurons Research Articles

Regulation of voltage-gated sodium channels by TNF-α during herpes simplex virus latency establishment.

During lytic or latent infection of sensory neurons with herpes simplex virus type 1 (HSV-1) there are significant changes in the expression of voltage-gated Na+ channels, which may disrupt the transmission of pain information. HSV-1 infection can also evoke the secretion of various pro-inflammatory cytokines, including TNF-α and IL-6. In this work, we hypothesized that TNF-α regulates the expression of Na+ channels during HSV-1 latency establishment in ND7/23 sensory-like neurons. Latency establishment was mimicked by culturing HSV-1 infected ND7/23 cells in the presence of acyclovir (ACV) for 3 days. Changes in the functional expression of voltage-gated Na+ channels were assessed by whole-cell recordings. Our results demonstrate that infection of ND7/23 cells with the HSV-1 strain McKrae with GFP expression (M-GFP) causes a significant decrease in sodium currents during latency establishment. Exposure of ND7/23 cells to TNF-α during latency establishment reverses the effect of HSV-1, resulting in a significant increase in sodium current density. However, Na+ currents were not restored by 3day-treatment with IL-6. There were no changes in the pharmacological and biophysical properties of sodium currents promoted by TNF-α, including sensitivity to tetrodotoxin and the current-voltage relationship. TNF-α stimulation of ND7/23 cells increases p38 signaling. Inhibition of p38 signaling with SB203580 or SB202190 eliminates the stimulatory effect of TNF-α on sodium currents. These results indicate that TNF-α signaling in sensory neurons during latency establishment upregulates the expression of voltage-gated Na+ channels in order to maintain the transmission of pain information.

Interleukin-22 receptor 1-mediated stimulation of T-type Ca2+ channels enhances sensory neuronal excitability through the tyrosine-protein kinase Lyn-dependent PKA pathway

BackgroundInterleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24’s role in peripheral nociception remain unclear.MethodsUsing patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels).ResultsIL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2.ConclusionOur findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.

Effects of abrocitinib on pruritus and eczema symptoms and tolerance in patients with moderate‑to‑severe atopic dermatitis in randomized, double‑blind and placebo‑controlled trials: A systematic review and a meta‑analysis.

Abrocitinib is a highly selective Janus kinase 1 (JAK1) inhibitor that can block a multitude of inflammatory signaling pathways that underlie atopic dermatitis (AD). In addition, abrocitinib inhibits JAK1 signaling in sensory neurons to alleviate acute and chronic pruritus during AD. However, substantial variations in efficacy and safety risks remain due to variations in doses applied in clinical use. Therefore for the present study, differences in the efficacy and tolerability of 100 and 200 mg abrocitinib for treating pruritus and eczema symptoms in patients with moderate-to-severe AD were evaluated compared with placebo. Specifically, randomized controlled trials (RCTs) of abrocitinib compared with placebo for the treatment of moderate-to-severe AD were searched on Pubmed, E.B. Stephens Company, China National Knowledge Infrastructure, Wanfang Medical network, Web of Science and related Clinical Trials Registry up to November 2023. In total, two researchers evaluated the quality of the included literature according to the Cochrane Handbook of Systematic Reviews. RevMan 5.3 software was used to conduct a meta-analysis of the efficacy and safety indicators in a cross-comparison of the effects exerted by placebo and 100 and 200 mg abrocitinib. A total of 1,825 patients with moderate-to-severe AD were included across five double-blind, placebo RCTs. Compared with the placebo group, during the double-blind trial period, significant improvements were observed in the investigator's global assessment score, response rate of eczema area and severity index (EASI)-50, EASI-75, EASI-90 and pruritus numerical rating scale (P-NRS) in the 100 and 200 mg abrocitinib groups (P<0.05). However, pairwise control analysis of the 100 and 200 mg group yielded significant differences (P<0.05) in all of the aforementioned therapeutic indicators except for the P-NRS score. In terms of safety, compared with the placebo group, there were significantly higher incidence of nausea, upper respiratory tract viral infection, infections and infestations in the 100 mg abrocitinib group (P<0.05). In addition, there were significantly higher incidence of nausea, gastrointestinal disorder, headache and dizziness in the 200 mg group (P<0.05). There were also significant differences in the incidence of nausea, gastrointestinal disorder and dizziness between the 100 and 200 mg groups (P<0.05). For patients with moderate-to-severe AD, oral administration of 100 or 200 mg abrocitinib once/day was concluded to ameliorate skin pruritus and eczema symptoms to varying degrees, with the efficacy significantly superior at the 200 mg dose. However, the risk of a number of adverse reactions, such as headache, dizziness, nausea and gastrointestinal dysfunction, is also significantly increased. Therefore, patients should be made aware of the risk of adverse drug effects prior to the administration of long-term high abrocitinib doses. Furthermore, large-scale, multi-center, rigorous clinical trials remain necessary to validate the findings from the present study.

Pathophysiological Roles of Ion Channels in Epidermal Cells, Immune Cells, and Sensory Neurons in Psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by the rapid abnormal growth of skin cells in the epidermis, driven by an overactive immune system. Consequently, a complex interplay among epidermal cells, immune cells, and sensory neurons contributes to the development and progression of psoriasis. In these cellular contexts, various ion channels, such as acetylcholine receptors, TRP channels, Ca2+ release-activated channels, chloride channels, and potassium channels, each serve specific functions to maintain the homeostasis of the skin. The dysregulation of ion channels plays a major role in the pathophysiology of psoriasis, affecting various aspects of epidermal cells, immune responses, and sensory neuron signaling. Impaired function of ion channels can lead to altered calcium signaling, inflammation, proliferation, and sensory signaling, all of which are central features of psoriasis. This overview summarizes the pathophysiological roles of ion channels in epidermal cells, immune cells, and sensory neurons during early and late psoriatic processes, thereby contributing to a deeper understanding of ion channel involvement in the interplay of psoriasis and making a crucial advance toward more precise and personalized approaches for psoriasis treatment.

A note on estimating absolute cytosolic Ca2+ concentration in sensory neurons using a single wavelength Ca2+ indicator.

Ca2+ imaging is frequently used in the investigation of sensory neuronal function and nociception. In vitro imaging of acutely dissociated sensory neurons using membrane-permeant fluorescent Ca2+ indicators remains the most common approach to study Ca2+ signalling in sensory neurons. Fluo4 is a popular choice of single-wavelength indicator due to its brightness, high affinity for Ca2+ and ease of use. However, unlike ratiometric indicators, the emission intensity from single-wavelength indicators can be affected by indicator concentration, optical path length, excitation intensity and detector efficiency. As such, without careful calibration, it can be difficult to draw inferences from differences in the magnitude of Ca2+ transients recorded using Fluo4. Here, we show that a method scarcely used in sensory neurophysiology - first proposed by Maravall and colleagues (2000) - can provide reliable estimates of absolute cytosolic Ca2+ concentration ([Ca2+]cyt) in acutely dissociated sensory neurons using Fluo4. This method is straightforward to implement; is applicable to any high-affinity single-wavelength Ca2+ indicator with a large dynamic range; and provides estimates of [Ca2+]cyt in line with other methods, including ratiometric imaging. Use of this method will improve the granularity of sensory neuron Ca2+ imaging data obtained with Fluo4.

IL-33 potentiates histaminergic itch

Itch is a common symptom that can greatly diminish quality of life. Histamine is a potent endogenous pruritogen, and while antihistamines are often the first-line treatment for itch, in conditions like chronic spontaneous urticaria (CSU), many patients remain symptomatic while receiving maximal doses. Mechanisms that drive resistance to antihistamines are poorly defined. Signaling of the alarmin cytokine IL-33 in sensory neurons is postulated to drive chronic itch by inducing neuronal sensitization to pruritogens. Thus, we sought to determine if IL-33 can augment histamine-induced (histaminergic) itch. Itch behavior was assessed in response to histamine after IL-33 or saline administration. Various stimuli and conditional and global knockout mice were utilized to dissect cellular mechanisms. Multiple existing transcriptomic data sets were evaluated, including single-cell RNA sequencing of human and mouse skin, microarrays of isolated mouse mast cells at steady state and after stimulation with IL-33, and microarrays of skin biopsy samples from subjects with CSU and healthy controls. IL-33 amplifies histaminergic itch independent of IL-33 signaling in sensory neurons. Mast cells are the top expressors of the IL-33 receptor in both human and mouse skin. When stimulated by IL-33, mouse mast cells significantly increase IL-13 levels. Enhancement of histaminergic itch by IL-33 relies on a mast cell- and IL-13-dependent mechanism. IL-33 receptor expression is increased in lesional skin of subjects with CSU compared to healthy controls. Our findings suggest that IL-33 signaling may be a key driver of histaminergic itch in mast cell-associated pruritic conditions such as CSU.

Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice

BackgroundAdipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown.MethodsUsing electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons.ResultsAdiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCβ1). Blocking PKCβ1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCβ1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2.ConclusionOur study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCβ1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.

Trace amine-associated receptor 1 regulation of Kv1.4 channels in trigeminal ganglion neurons contributes to nociceptive behaviors

BackgroundTrace amines, such as tyramine, are endogenous amino acid metabolites that have been hypothesized to promote headache. However, the underlying cellular and molecular mechanisms remain unknown.MethodsUsing patch-clamp recording, immunostaining, molecular biological approaches and behaviour tests, we elucidated a critically functional role of tyramine in regulating membrane excitability and pain sensitivity by manipulating Kv1.4 channels in trigeminal ganglion (TG) neurons.ResultsApplication of tyramine to TG neurons decreased the A-type K+ current (IA) in a manner dependent on trace amine-associated receptor 1 (TAAR1). Either siRNA knockdown of Gαo or chemical inhibition of βγ subunit (Gβγ) signaling abrogated the response to tyramine. Antagonism of protein kinase C (PKC) prevented the tyramine-induced IA response, while inhibition of conventional PKC isoforms or protein kinase A elicited no such effect. Tyramine increased the membrane abundance of PKCθ in TG neurons, and either pharmacological or genetic inhibition of PKCθ blocked the TAAR1-mediated IA decrease. Furthermore, PKCθ-dependent IA suppression was mediated by Kv1.4 channels. Knockdown of Kv1.4 abrogated the TAAR1-induced IA decrease, neuronal hyperexcitability, and pain hypersensitivity. In a mouse model of migraine induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus, blockade of TAAR1 signaling attenuated mechanical allodynia; this effect was occluded by lentiviral overexpression of Kv1.4 in TG neurons.ConclusionThese results suggest that tyramine induces Kv1.4-mediated IA suppression through stimulation of TAAR1 coupled to the Gβγ-dependent PKCθ signaling cascade, thereby enhancing TG neuronal excitability and mechanical pain sensitivity. Insight into TAAR1 signaling in sensory neurons provides attractive targets for the treatment of headache disorders such as migraine.

Pathogen recognition by sensory neurons: hypotheses on the specificity of sensory neuron signaling.

Sensory neurons cooperate with barrier tissues and resident immune cells to form a significant aspect of defensive strategies in concert with the immune system. This assembly of neuroimmune cellular units is exemplified across evolution from early metazoans to mammalian life. As such, sensory neurons possess the capability to detect pathogenic infiltrates at barrier surfaces. This capacity relies on mechanisms that unleash specific cell signaling, trafficking and defensive reflexes. These pathways exploit mechanisms to amplify and enhance the alerting response should pathogenic infiltration seep into other tissue compartments and/or systemic circulation. Here we explore two hypotheses: 1) that sensory neurons' potential cellular signaling pathways require the interaction of pathogen recognition receptors and ion channels specific to sensory neurons and; 2) mechanisms which amplify these sensing pathways require activation of multiple sensory neuron sites. Where possible, we provide references to other apt reviews which provide the reader more detail on specific aspects of the perspectives provided here.

APP Genetic Deficiency Alters Intracellular Ca2+ Homeostasis and Delays Axonal Degeneration in Dorsal Root Ganglion Sensory Neurons.

The activation of self-destructive cellular programs helps sculpt the nervous system during development, but the molecular mechanisms used are not fully understood. Prior studies have investigated the role of the APP in the developmental degeneration of sensory neurons with contradictory results. In this work, we sought to elucidate the impact of APP deletion in the development of the sensory nervous system in vivo and in vitro. Our in vivo data show an increase in the number of sciatic nerve axons in adult male and female APP-null mice, consistent with the hypothesis that APP plays a pro-degenerative role in the development of peripheral axons. In vitro, we show that genetic deletion of APP delays axonal degeneration triggered by nerve growth factor deprivation, indicating that APP does play a pro-degenerative role. Interestingly, APP depletion does not affect caspase-3 levels but significantly attenuates the rise of axoplasmic Ca2+ that occurs during degeneration. We examined intracellular Ca2+ mechanisms that could be involved and found that APP-null DRG neurons had increased Ca2+ levels within the endoplasmic reticulum and enhanced store-operated Ca2+ entry. We also observed that DRG axons lacking APP have more mitochondria than their WT counterparts, but these display a lower mitochondrial membrane potential. Finally, we present evidence that APP deficiency causes an increase in mitochondrial Ca2+ buffering capacity. Our results support the hypothesis that APP plays a pro-degenerative role in the developmental degeneration of DRG sensory neurons, and unveil the importance of APP in the regulation of calcium signaling in sensory neurons.SIGNIFICANCE STATEMENT The nervous system goes through a phase of pruning and programmed neuronal cell death during development to reach maturity. In such context, the role played by the APP in the peripheral nervous system has been controversial, ranging from pro-survival to pro-degenerative. Here we present evidence in vivo and in vitro supporting the pro-degenerative role of APP, demonstrating the ability of APP to alter intracellular Ca2+ homeostasis and mitochondria, critical players of programmed cell death. This work provides a better understanding of the physiological function of APP and its implication in developmental neuronal death in the nervous system.

LB982 IL-33 enhancement of histaminergic itch is dependent on IL-13

Histamine is a critical mediator if itch in urticaria, however, many patients with chronic spontaneous urticaria (CSU) itch in spite of treatment with antihistamines. Further, it is widely known that in the CSU, patients over time can lose clinical itch responses to antihistamines as well. We hypothesized that the limitation of these therapies is partially due to other immune mediators amplifying the histaminergic itch pathway. We focused specifically on IL-33, an alarmin cytokine that we and others have recently shown is critical for the development of chronic itch in different mouse models through its direct signaling in sensory neurons.

Task-induced neural covariability as a signature of approximate Bayesian learning and inference.

Perception is often characterized computationally as an inference process in which uncertain or ambiguous sensory inputs are combined with prior expectations. Although behavioral studies have shown that observers can change their prior expectations in the context of a task, robust neural signatures of task-specific priors have been elusive. Here, we analytically derive such signatures under the general assumption that the responses of sensory neurons encode posterior beliefs that combine sensory inputs with task-specific expectations. Specifically, we derive predictions for the task-dependence of correlated neural variability and decision-related signals in sensory neurons. The qualitative aspects of our results are parameter-free and specific to the statistics of each task. The predictions for correlated variability also differ from predictions of classic feedforward models of sensory processing and are therefore a strong test of theories of hierarchical Bayesian inference in the brain. Importantly, we find that Bayesian learning predicts an increase in so-called "differential correlations" as the observer's internal model learns the stimulus distribution, and the observer's behavioral performance improves. This stands in contrast to classic feedforward encoding/decoding models of sensory processing, since such correlations are fundamentally information-limiting. We find support for our predictions in data from existing neurophysiological studies across a variety of tasks and brain areas. Finally, we show in simulation how measurements of sensory neural responses can reveal information about a subject's internal beliefs about the task. Taken together, our results reinterpret task-dependent sources of neural covariability as signatures of Bayesian inference and provide new insights into their cause and their function.

IL-33 signaling in sensory neurons promotes dry skin itch

Chronic pruritus, or itch, is common and debilitating, but the neuroimmune mechanisms that drive chronic itch are only starting to be elucidated. Recent studies demonstrate that the IL-33 receptor (IL-33R) is expressed by sensory neurons. However, whether sensory neuron-restricted activity of IL-33 is necessary for chronic itch remains poorly understood. We sought to determine if IL-33 signaling in sensory neurons is critical for the development of chronic itch in 2 divergent pruritic disease models. Plasma levels of IL-33 were assessed in patients with atopic dermatitis (AD) and chronic pruritus of unknown origin (CPUO). Mice were generated to conditionally delete IL-33R from sensory neurons. The contribution of neuronal IL-33R signaling to chronic itch development was tested in mouse models that recapitulate key pathologic features of AD and CPUO, respectively. IL-33 was elevated in both AD and CPUO as well as their respective mouse models. While neuron-restricted IL-33R signaling was dispensable for itch in AD-like disease, it was required for the development of dry skin itch in a mouse model that mirrors key aspects of CPUO pathology. These data highlight how IL-33 may be a predominant mediator of itch in certain contexts, depending on the tissue microenvironment. Further, this study provides insight into future therapeutic strategies targeting the IL-33 pathway for chronic itch.

The inflammatory regulation of TRPA1 expression in human A549 lung epithelial cells

Transient receptor potential ankyrin-1 (TRPA1) is an ion channel mediating pain and cough signals in sensory neurons. We and others have shown that TRPA1 is also expressed in some non-neuronal cells and supports inflammatory responses. To address the pathogenesis and to uncover potential targets for pharmacotherapy in inflammatory lung diseases, we set out to study the expression of TRPA1 in human A549 lung epithelial cells under inflammatory conditions. TRPA1 expression was determined by RT-qPCR and Western blotting at a mRNA and protein level, respectively and its function was studied by Fluo 3-AM intracellular Ca2+ measurement in A549 lung epithelial cells. TRPA1 promoter activity was assessed by reporter gene assay.TRPA1 expression was very low in A549 cells in the absence of inflammatory stimuli. Tumor necrosis factor-α (TNF-α) significantly increased TRPA1 expression and a synergy was found between TNF-α, interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Reporter gene experiments indicate that the combination of TNF-α and IL-1β increases TRPA1 promoter activity while the effect of IFN-γ seems to be non-transcriptional. Interestingly, the glucocorticoid dexamethasone downregulated TRPA1 expression in A549 cells by reducing TRPA1 mRNA stability in a transcription-dependent manner. Furthermore, pharmacological blockade of TRPA1 reduced the production of the pro-inflammatory cytokine IL-8.In conclusion, TRPA1 was found to be expressed and functional in human A549 lung epithelial cells under inflammatory conditions. The anti-inflammatory steroid dexamethasone reduced TRPA1 expression through post-transcriptional mechanisms. The results reveal TRPA1 as a potential mediator and drug target in inflammatory lung conditions.

OROFACIAL ALLODYNIA INDUCED BY HIGH OMEGA-6 DIET

Chronic orofacial pain is estimated to be experienced by up to 26% of the adult population, which greatly decreases life quality. A common complaint of orofacial pain is from mechanical stimuli, although spontaneous pain also occurs in several orofacial pain conditions. Health care providers recommend dietary interventions for the management of cardiovascular disease and diabetes. However, it is possible that diet may also contribute to chronic peripheral pain conditions, as omega-6 fatty acids such as linoleic acid (LA) and arachidonic acid (AA), which are essential polyunsaturated fatty acids (PUFA), are regulated by dietary intake. However, there is a large gap in knowledge on the role of diet as a risk factor or potential therapy for chronic orofacial pain. The current study characterized changes in sensory neuron signaling after a high omega-6 diet and its contribution to increases in mechanical nociception within the dorsal root ganglia (DRG) and trigeminal ganglia (TG) system. To measure the effect of a high omega-6 diet (H6D) on orofacial nociception, male and female C57 mice were fed standardized diets containing either a high 10% omega-6 diet (Dyets, Inc) or a low 0.5% omega-6 diet (Dyets, Inc) for 8 weeks with weekly monitoring of mechanical nociceptive withdrawal thresholds (n=10/group) using von Frey filaments by a blinded observer. 2-way ANOVA analyzed data. The results demonstrate after 8 weeks of H6D, there is a decrease in mechanical nociceptive thresholds seen in the orofacial region. These findings support the conclusion that a H6D triggers mechanical allodynia in both the DRG and TG systems. Therefore, dietary intervention may represent novel therapeutic approaches for treating chronic pain conditions. NIDCR T-32 DE14318 and R01 NS110948.

106 IL-33 signaling in sensory neurons promotes dry skin itch

106 IL-33 signaling in sensory neurons promotes dry skin itch

Junctophilin-4 facilitates inflammatory signalling at plasma membrane-endoplasmic reticulum junctions in sensory neurons.

Rat somatosensory neurons express a junctional protein, junctophilin-4 (JPH4) JPH4 is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the junctions between plasma membrane and endoplasmic reticulum in these neurons. Knockdown of JPH4 impairs endoplasmic reticulum Ca2+ store refill and junctional Ca2+ signalling in sensory neurons. In vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly attenuated experimentally induced inflammatory pain in rats. Junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms. Junctions of endoplasmic reticulum and plasma membrane (ER-PM junctions) form signalling nanodomains in eukaryotic cells. ER-PM junctions are present in peripheral sensory neurons and are important for the fidelity of G protein coupled receptor (GPCR) signalling. Yet little is known about the assembly, maintenance and physiological role of these junctions in somatosensory transduction. Using fluorescence imaging, proximity ligation, super-resolution microscopy, in vitro and in vivo gene knockdown we demonstrate that a member of the junctophilin protein family, junctophilin-4 (JPH4), is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the ER-PM junctions in rat somatosensory neurons. Thus we show that JPH4 localises to the ER-PM junctional areas and co-clusters with SOCE proteins STIM1 and Orai1 upon ER Ca2+ store depletion. Knockdown of JPH4 impairs SOCE and ER Ca2+ store refill in sensory neurons. Furthermore, we demonstrate a key role of the JPH4 and junctional nanodomain Ca2+ signalling in the pain-like response induced by the inflammatory mediator bradykinin. Indeed, an in vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly shortened the duration of nocifensive behaviour induced by hindpaw injection of bradykinin in rats. Since the ER supplies Ca2+ for the excitatory action of multiple inflammatory mediators, we suggest that junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

Regulation of Mitochondrial Function by Epac2 Contributes to Acute Inflammatory Hyperalgesia.

Gαs-coupled receptors signaling through cAMP provide a key mechanism for the sensitization of nociceptive sensory neurons, and the cAMP effector Epac has been implicated in the transition from acute to chronic pain. Epac exerts its effects through Rap1 and protein kinase C (PKC). To identify targets of Epac-PKC signaling in sensory neurons of the mouse dorsal root ganglion (DRG), we profiled PKC substrate proteins phosphorylated in response to the activation of Epac with the proinflammatory prostaglandin E2 (PGE2). A prominent Epac-dependent phospho-protein band induced by PGE2 was identified by mass spectrometry as the mitochondrial enzyme pyruvate dehydrogenase (Pdha1). In dissociated DRG from both males and females, the recruitment of Pdha1 to phospho-protein fractions was rapidly induced by PGE2 and prevented by selective inhibition of Epac2. Epac activation increased mitochondrial respiration, consistent with an increase in Pdha1 function mediated by Epac2. Hindpaw injection of PGE2 induced heat hyperalgesia in males and females, but Pdha1 phosphorylation occurred only in males. Hyperalgesia was attenuated in males but not in females by systemic inhibition of Epac2, and also by a mitochondrial membrane potential uncoupler, dinitrophenol, supporting a role for mitochondrial regulation in acute hyperalgesia. These findings identify a mechanism for the regulation of mitochondrial function by Epac2 that contributes to acute inflammatory hyperalgesia in male mice. Systemic administration of the cyclooxygenase 2 inhibitor celecoxib suppressed both PGE2-induced heat hyperalgesia and Pdha1 phosphorylation in DRG of males but not females, suggesting that prostaglandin synthesis within the DRG mediates the phosphorylation of Pdha1 in response to hindpaw insult.SIGNIFICANCE STATEMENT There has been extensive investigation of mitochondrial dysfunction as a causative factor in neuropathic pain disorders. In contrast, results reported here implicate enhanced mitochondrial function as a contributing factor in the development of acute inflammatory hyperalgesia. We describe a mechanism in which Epac2 activation by prostaglandin receptors leads to phosphorylation of pyruvate dehydrogenase and an increase in mitochondrial respiration in peripheral sensory neurons. Although Epac2 activation leads to Pdha1 (pyruvate dehydrogenase) phosphorylation in dissociated neurons from mice of both sexes, induction of this pathway in vivo by hindpaw insult is restricted to males and appears to require intraganglionic prostaglandin synthesis. These findings support a model in which Gs-coupled receptor modulation of mitochondrial function promotes acute nociceptive signaling and inflammatory hyperalgesia.

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

SummaryMany 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.

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 which regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hindpaw 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 only found 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.