Prox2 and Runx3 vagal sensory neurons regulate esophageal motility.

Vagal sensory neurons monitor mechanical and chemical stimuli in the gastrointestinal tract. Major efforts are underway to assign physiological functions to the many distinct subtypes of vagal sensory neurons. Here, we use genetically guided anatomical tracing, optogenetics, and electrophysiology to identify and characterize vagal sensory neuron subtypes expressing Prox2 and Runx3 in mice. We show that three of these neuronal subtypes innervate the esophagus and stomach in regionalized patterns, where they form intraganglionic laminar endings. Electrophysiological analysis revealed that they are low-threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis in freely behaving mice. Our work defines the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders.

Lack of evidence for participation of TMEM150C in sensory mechanotransduction.

The membrane protein TMEM150C has been proposed to form a mechanosensitive ion channel that is required for normal proprioceptor function. Here, we examined whether expression of TMEM150C in neuroblastoma cells lacking Piezo1 is associated with the appearance of mechanosensitive currents. Using three different modes of mechanical stimuli, indentation, membrane stretch, and substrate deflection, we could not evoke mechanosensitive currents in cells expressing TMEM150C. We next asked if TMEM150C is necessary for the normal mechanosensitivity of cutaneous sensory neurons. We used an available mouse model in which the Tmem150c locus was disrupted through the insertion of a LacZ cassette with a splice acceptor that should lead to transcript truncation. Analysis of these mice indicated that ablation of the Tmem150c gene was not complete in sensory neurons of the dorsal root ganglia (DRG). Using a CRISPR/Cas9 strategy, we made a second mouse model in which a large part of the Tmem150c gene was deleted and established that these Tmem150c-/- mice completely lack TMEM150C protein in the DRGs. We used an ex vivo skin nerve preparation to characterize the mechanosenstivity of mechanoreceptors and nociceptors in the glabrous skin of the Tmem150c-/- mice. We found no quantitative alterations in the physiological properties of any type of cutaneous sensory fiber in Tmem150c-/- mice. Since it has been claimed that TMEM150C is required for normal proprioceptor function, we made a quantitative analysis of locomotion in Tmem150c-/- mice. Here again, we found no indication that there was altered gait in Tmem150c-/- mice compared to wild-type controls. In summary, we conclude that existing mouse models that have been used to investigate TMEM150C function in vivo are problematic. Furthermore, we could find no evidence that TMEM150C forms a mechanosensitive channel or that it is necessary for the normal mechanosensitivity of cutaneous sensory neurons.

Immune competence and spleen size scale with colony status in the naked mole-rat.

Naked mole-rats (NM-R; Heterocephalus glaber) live in multi-generational colonies with a social hierarchy, and show low cancer incidence and long life-spans. Here we asked if an immune component might underlie such extreme physiology. The largest lymphoid organ is the spleen, which plays an essential role in responding to immunological insults and may participate in combating cancer and slowing ageing. We investigated the anatomy, molecular composition and function of the NM-R spleen using RNA-sequencing and histological analysis in healthy NM-Rs. Spleen size in healthy NM-Rs showed considerable inter-individual variability, with some animals displaying enlarged spleens. In all healthy NM-Rs, the spleen is a major site of adult haematopoiesis under normal physiological conditions. However, myeloid-to-lymphoid cell ratio is increased and splenic marginal zone showed markedly altered morphology when compared to other rodents. Healthy NM-Rs with enlarged spleens showed potentially better anti-microbial profiles and were much more likely to have a high rank within the colony. We propose that the anatomical plasticity of the spleen might be regulated by social interaction and gives immunological advantage to increase the lifespan of higher-ranked animals.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

TMEM87a/Elkin1, a component of a novel mechanoelectrical transduction pathway, modulates melanoma adhesion and migration.

Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are converted into electro-chemical signals by mechanically activated ion channels. We describe here the presence of mechanically activated currents in melanoma cells that are dependent on TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 exhibit defective mechanoelectrical transduction, decreased motility and increased dissociation from organotypic spheroids. By analysing cell adhesion properties, we demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular adhesions and regulates melanoma cell migration and cell-cell interactions.

When cells receive signals about their surrounding environment, this initiates a chain of signals which generate a response. Some of these signalling pathways allow cells to sense physical and mechanical forces via a process called mechanotransduction. There are different types of mechanotransduction. In one pathway, mechanical forces open up specialized channels on the cell surface which allow charged particles to move across the membrane and create an electrical current. Mechanoelectrical transduction plays an important role in the spread of cancer: as cancer cells move away from a tumour they use these signalling pathways to find their way between cells and move into other parts of the body. Understanding these pathways could reveal ways to stop cancer from spreading, making it easier to treat. However, it remains unclear which molecules regulate mechanoelectrical transduction in cancer cells. Now, Patkunarajah, Stear et al. have studied whether mechanoelectrical transduction is involved in the migration of skin cancer cells. To study mechanoelectrical transduction, a fine mechanical input was applied to the skin cancer cells whilst measuring the flow of charged molecules moving across the membrane. This experiment revealed that a previously unknown protein named Elkin1 is required to convert mechanical forces into electrical currents. Deleting this newly found protein caused skin cancer cells to move more slowly and dissociate more easily from tumour-like clusters of cells. These findings suggest that Elkin1 is part of a newly identified mechanotransduction pathway that allows cells to sense mechanical forces from their surrounding environment. More work is needed to determine what role Elkin1 plays in mechanoelectrical transduction and whether other proteins are also involved. This could lead to new approaches that prevent cancer cells from dissociating from tumours and spreading to other body parts.

Maf links Neuregulin1 signaling to cholesterol synthesis in myelinating Schwann cells.

Cholesterol is a major constituent of myelin membranes, which insulate axons and allow saltatory conduction. Therefore, Schwann cells, the myelinating glia of the peripheral nervous system, need to produce large amounts of cholesterol. Here, we define a crucial role of the transcription factor Maf in myelination and cholesterol biosynthesis and show that Maf acts downstream from Neuregulin1 (Nrg1). Maf expression is induced when Schwann cells begin myelination. Genetic ablation of Maf resulted in hypomyelination that resembled mice with defective Nrg1 signaling. Importantly, loss of Maf or Nrg1 signaling resulted in a down-regulation of the cholesterol synthesis program, and Maf directly binds to enhancers of cholesterol synthesis genes. Furthermore, we identified the molecular mechanisms by which Nrg1 signaling regulates Maf levels. Transcription of Maf depends on calmodulin-dependent kinases downstream from Nrg1, whereas Nrg1-MAPK signaling stabilizes Maf protein. Our results delineate a novel signaling cascade regulating cholesterol synthesis in myelinating Schwann cells.

Fxyd2 regulates Aδ- and C-fiber mechanosensitivity and is required for the maintenance of neuropathic pain.

Identification of the molecular mechanisms governing sensory neuron subtype excitability is a key requisite for the development of treatments for somatic sensory disorders. Here, we show that the Na,K-ATPase modulator Fxyd2 is specifically required for setting the mechanosensitivity of Aδ-fiber low-threshold mechanoreceptors and sub-populations of C-fiber nociceptors, a role consistent with its restricted expression profile in the spinal somatosensory system. We also establish using the spared nerve injury model of neuropathic pain, that loss of Fxyd2 function, either constitutively in Fxyd2-/- mice or acutely in neuropathic rats, efficiently alleviates mechanical hypersensitivity induced by peripheral nerve lesions. The role of Fxyd2 in modulating Aδ- and C-fibers mechanosensitivity likely accounts for the anti-allodynic effect of Fxyd2 knockdown. Finally, we uncover the evolutionarily conserved restricted expression pattern of FXYD2 in human dorsal root ganglia, thus identifying this molecule as a potentially promising therapeutic target for peripheral neuropathic pain management.

Hypofunctional TrkA Accounts for the Absence of Pain Sensitization in the African Naked Mole-Rat.

The naked mole-rat is a subterranean rodent lacking several pain behaviors found in humans, rats, and mice. For example, nerve growth factor (NGF), an important mediator of pain sensitization, fails to produce thermal hyperalgesia in naked mole-rats. The sensitization of capsaicin-sensitive TRPV1 ion channels is necessary for NGF-induced hyperalgesia, but naked mole-rats have fully functional TRPV1 channels. We show that exposing isolated naked mole-rat nociceptors to NGF does not sensitize TRPV1. However, the naked mole-rat NGF receptor TrkA displays a reduced ability to engage signal transduction pathways that sensitize TRPV1. Between one- and three-amino-acid substitutions in the kinase domain of the naked mole-rat TrkA are sufficient to render the receptor hypofunctional, and this is associated with the absence of heat hyperalgesia. Our data suggest that evolution has selected for a TrkA variant that abolishes a robust nociceptive behavior in this species but is still compatible with species fitness.

A Molecular Signature of Myalgia in Myotonic Dystrophy 2.

BACKGROUND: Chronic muscle pain affects close to 20% of the population and is a major health burden. The underlying mechanisms of muscle pain are difficult to investigate as pain presents in patients with very diverse histories. Treatment options are therefore limited and not tailored to underlying mechanisms. To gain insight into the pathophysiology of myalgia we investigated a homogeneous group of patients suffering from myotonic dystrophy type 2 (DM2), a monogenic disorder presenting with myalgia in at least 50% of affected patients. METHODS: After IRB approval we performed an observational cross-sectional cohort study and recruited 42 patients with genetically confirmed DM2 plus 20 healthy age and gender matched control subjects. All participants were subjected to an extensive sensory-testing protocol. In addition, RNA sequencing was performed from 12 muscle biopsy specimens obtained from DM2 patients. FINDINGS: Clinical sensory testing as well as RNA sequencing clearly separated DM2 myalgic from non-myalgia patients and also from healthy controls. In particular pressure pain thresholds were significantly lowered for all muscles tested in myalgic DM2 patients but were not significantly different between non-myalgic patients and healthy controls. The expression of fourteen muscle expressed genes in myalgic patients was significantly up or down-regulated in myalgic compared to non-myalgic DM2 patients. INTERPRETATION: Our data support the idea that molecular changes in the muscles of DM2 patients are associated with muscle pain. Further studies should address whether muscle-specific molecular pathways play a significant role in myalgia in order to facilitate the development of mechanism-based therapeutic strategies to treat musculoskeletal pain. FUNDING: This study was funded by the German Research Society (DFG, GK1631), KAP programme of Charité Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine.

Inhibition of c-Kit signaling is associated with reduced heat and cold pain sensitivity in humans.

The tyrosine kinase receptor c-Kit is critically involved in the modulation of nociceptive sensitivity in mice. Ablation of the c-Kit gene results in hyposensitivity to thermal pain, whereas activation of c-Kit produces hypersensitivity to noxious heat, without altering sensitivity to innocuous mechanical stimuli. In this study, we investigated the role of c-Kit signaling in human pain perception. We hypothesized that subjects treated with Imatinib or Nilotinib, potent inhibitors of tyrosine kinases including c-Kit but also Abl1, PDFGFRα, and PDFGFRß, that are used to treat chronic myeloid leukemia (CML), would experience changes in thermal pain sensitivity. We examined 31 asymptomatic CML patients (14 male and 17 female) receiving Imatinib/Nilotinib treatment and compared them to 39 age- and sex-matched healthy controls (12 male and 27 female). We used cutaneous heat and cold stimulation to test normal and noxious thermal sensitivity, and a grating orientation task to assess tactile acuity. Thermal pain thresholds were significantly increased in the Imatinib/Nilotinib-treated group, whereas innocuous thermal and tactile thresholds were unchanged compared to those in the control group. In conclusion, our findings suggest that the biological effects of c-Kit inhibition are comparable in mice and humans in that c-Kit activity is required to regulate thermal pain sensitivity but does not affect innocuous thermal and mechanical sensation. The effect on experimental heat pain observed in our study is comparable to those of several common analgesics; thus modulation of the c-Kit pathway can be used to specifically modulate noxious heat and cold sensitivity in humans.

Nerve growth factor and nociception: from experimental embryology to new analgesic therapy.

Nerve growth factor (NGF) is central to the development and functional regulation of sensory neurons that signal the first events that lead to pain. These sensory neurons, called nociceptors, require NGF in the early embryo to survive and also for their functional maturation. The long road from the discovery of NGF and its roles during development to the realization that NGF plays a major role in the pathophysiology of inflammatory pain will be reviewed. In particular, we will discuss the various signaling events initiated by NGF that lead to long-lasting thermal and mechanical hyperalgesia in animals and in man. It has been realized relatively recently that humanized function blocking antibodies directed against NGF show remarkably analgesic potency in human clinical trials for painful conditions as varied as osteoarthritis, lower back pain, and interstitial cystitis. Thus, anti-NGF medication has the potential to make a major impact on day-to-day chronic pain treatment in the near future. It is therefore all the more important to understand the precise pathways and mechanisms that are controlled by NGF to both initiate and sustain mechanical and thermal hyperalgesia. Recent work suggests that NGF-dependent regulation of the mechanosensory properties of sensory neurons that signal mechanical pain may open new mechanistic avenues to refine and exploit relevant molecular targets for novel analgesics.

Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination.

Myelination depends on the synthesis of large amounts of myelin transcripts and proteins and is controlled by Nrg1/ErbB/Shp2 signaling. We developed a novel pulse labeling strategy based on stable isotope labeling with amino acids in cell culture (SILAC) to measure the dynamics of myelin protein production in mice. We found that protein synthesis is dampened in the maturing postnatal peripheral nervous system, and myelination then slows down. Remarkably, sustained activation of MAPK signaling by expression of the Mek1DD allele in mice overcomes the signals that end myelination, resulting in continuous myelin growth. MAPK activation leads to minor changes in transcript levels but massively up-regulates protein production. Pharmacological interference in vivo demonstrates that the effects of activated MAPK signaling on translation are mediated by mTOR-independent mechanisms but in part also by mTOR-dependent mechanisms. Previous work demonstrated that loss of ErbB3/Shp2 signaling impairs Schwann cell development and disrupts the myelination program. We found that activated MAPK signaling strikingly compensates for the absence of ErbB3 or Shp2 during Schwann cell development and myelination.

Neural precursor cells induce cell death of high-grade astrocytomas through stimulation of TRPV1.

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics.

The transcription factor c-Maf controls touch receptor development and function.

The sense of touch relies on detection of mechanical stimuli by specialized mechanosensory neurons. The scarcity of molecular data has made it difficult to analyze development of mechanoreceptors and to define the basis of their diversity and function. We show that the transcription factor c-Maf/c-MAF is crucial for mechanosensory function in mice and humans. The development and function of several rapidly adapting mechanoreceptor types are disrupted in c-Maf mutant mice. In particular, Pacinian corpuscles, a type of mechanoreceptor specialized to detect high-frequency vibrations, are severely atrophied. In line with this, sensitivity to high-frequency vibration is reduced in humans carrying a dominant mutation in the c-MAF gene. Thus, our work identifies a key transcription factor specifying development and function of mechanoreceptors and their end organs.

Peripheral sensitisation of nociceptors via G-protein-dependent potentiation of mechanotransduction currents.

Mechanical stimuli impinging on the skin are converted into electrical signals by mechanically gated ion channels located at the peripheral nerve endings of dorsal root ganglion (DRG) neurons. Under inflammatory conditions sensory neurons are commonly sensitised to mechanical stimuli; a putative mechanism that may contribute to such sensitisation of sensory neurons is enhanced responsiveness of mechanotransduction ion channels. Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones. Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y(2) nucleotide receptor. Moreover, using the in vitro skin nerve technique, we show that UTP also increases action potential firing rates in response to mechanical stimuli in a subpopulation of skin C-fibre nociceptors. Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

Nociceptive tuning by stem cell factor/c-Kit signaling.

The molecular mechanisms regulating the sensitivity of sensory circuits to environmental stimuli are poorly understood. We demonstrate here a central role for stem cell factor (SCF) and its receptor, c-Kit, in tuning the responsiveness of sensory neurons to natural stimuli. Mice lacking SCF/c-Kit signaling displayed profound thermal hypoalgesia, attributable to a marked elevation in the thermal threshold and reduction in spiking rate of heat-sensitive nociceptors. Acute activation of c-Kit by its ligand, SCF, resulted in a reduced thermal threshold and potentiation of heat-activated currents in isolated small-diameter neurons and thermal hyperalgesia in mice. SCF-induced thermal hyperalgesia required the TRP family cation channel TRPV1. Lack of c-Kit signaling during development resulted in hypersensitivity of discrete mechanoreceptive neuronal subtypes. Thus, c-Kit can now be grouped with a small family of receptor tyrosine kinases, including c-Ret and TrkA, that control the transduction properties of sensory neurons.

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons.

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.

GFR alpha2/neurturin signalling regulates noxious heat transduction in isolectin B4-binding mouse sensory neurons.

The GFR alpha2 receptor is the cognate co-receptor for the neurotrophic factor neurturin and GFR alpha2 is selectively expressed by isolectin B(4) (IB(4))-binding nociceptive sensory neurons. Here, we used two physiological approaches in combination with mice that have a targeted deletion of the GFR alpha2 gene (GFR alpha2 -/- mice) in order to determine whether GFR alpha2/neurturin signalling regulates the functional properties or the survival of IB(4)-binding nociceptors. Because 50 % of IB(4)-binding neurons respond to noxious heat and because patch clamp recordings of isolated dorsal root ganglion sensory neurons allow one to neurochemically identify subpopulations of neurons, we analysed the noxious heat responsiveness of IB(4)-positive and -negative small-diameter neurons isolated from adult GFR alpha2 -/- and littermate control mice. The percentage of IB(4)-positive neurons that had large (> 100 pA) heat-evoked inward currents was severely reduced in GFR alpha2 -/- mice (12 %) compared to wild-type littermates (47 %), and this loss in large-magnitude heat currents was accounted for by an increase in neurons with very small (< 100 pA) heat-evoked currents as well as an increase in neurons with no detectable heat current. Counts of IB(4)-positive and -negative neurons, as well as counts of unmyelinated axons in the saphenous nerve, confirmed that the loss in neurons with large-amplitude heat currents was due to a deficit in heat transduction and not a decrease in cell survival. The effect was modality specific for heat because mechanical transduction of all fibre types, including IB(4)-positive C fibres, was normal. Our data are the first to indicate a transduction-function role for GFR alpha2/neurturin signalling in a specific class of sensory neurons.