Sensory Schwann cells set perceptual thresholds for touch and selectively regulate mechanical nociception.

Previous work identified nociceptive Schwann cells that can initiate pain. Consistent with the existence of inherently mechanosensitive sensory Schwann cells, we found that in mice, the mechanosensory function of almost all nociceptors, including those signaling fast pain, were dependent on sensory Schwann cells. In polymodal nociceptors, sensory Schwann cells signal mechanical, but not cold or heat pain. Terminal Schwann cells also surround mechanoreceptor nerve-endings within the Meissner's corpuscle and in hair follicle lanceolate endings that both signal vibrotactile touch. Within Meissner´s corpuscles, two molecularly and functionally distinct sensory Schwann cells positive for Sox10 and Sox2 differentially modulate rapidly adapting mechanoreceptor function. Using optogenetics we show that Meissner's corpuscle Schwann cells are necessary for the perception of low threshold vibrotactile stimuli. These results show that sensory Schwann cells within diverse glio-neural mechanosensory end-organs are sensors for mechanical pain as well as necessary for touch perception.

Touch receptor end-organ innervation and function require sensory neuron expression of the transcription factor Meis2.

Touch sensation is primarily encoded by mechanoreceptors, called low-threshold mechanoreceptors (LTMRs), with their cell bodies in the dorsal root ganglia. Because of their great diversity in terms of molecular signature, terminal endings morphology, and electrophysiological properties, mirroring the complexity of tactile experience, LTMRs are a model of choice to study the molecular cues differentially controlling neuronal diversification. While the transcriptional codes that define different LTMR subtypes have been extensively studied, the molecular players that participate in their late maturation and in particular in the striking diversity of their end-organ morphological specialization are largely unknown. Here we identified the TALE homeodomain transcription factor Meis2 as a key regulator of LTMRs target-field innervation in mice. Meis2 is specifically expressed in cutaneous LTMRs, and its expression depends on target-derived signals. While LTMRs lacking Meis2 survived and are normally specified, their end-organ innervations, electrophysiological properties, and transcriptome are differentially and markedly affected, resulting in impaired sensory-evoked behavioral responses. These data establish Meis2 as a major transcriptional regulator controlling the orderly formation of sensory neurons innervating peripheral end organs required for light touch.

Touch sensation requires the mechanically gated ion channel ELKIN1.

Touch perception is enabled by mechanically activated ion channels, the opening of which excites cutaneous sensory endings to initiate sensation. In this study, we identify ELKIN1 as an ion channel likely gated by mechanical force, necessary for normal touch sensitivity in mice. Touch insensitivity in Elkin1-/- mice was caused by a loss of mechanically activated currents (MA currents) in around half of all sensory neurons activated by light touch (low-threshold mechanoreceptors). Reintroduction of Elkin1 into sensory neurons from Elkin1-/- mice restored MA currents. Additionally, small interfering RNA-mediated knockdown of ELKIN1 from induced human sensory neurons substantially reduced indentation-induced MA currents, supporting a conserved role for ELKIN1 in human touch. Our data identify ELKIN1 as a core component of touch transduction in mice and potentially in humans.

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.

Role of TMEM100 in mechanically insensitive nociceptor un-silencing.

Mechanically silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors in mice. Moreover, we show that mice lacking TMEM100 do not develop secondary mechanical hypersensitivity-i.e., pain hypersensitivity that spreads beyond the site of inflammation-during knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce mechanical hypersensitivity in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary mechanical hypersensitivity during inflammation.

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.

The Somatosensory World of the African Naked Mole-Rat.

The naked mole-rat (Heterocephalus glaber) is famous for its longevity and unusual physiology. This eusocial species that lives in highly ordered and hierarchical colonies with a single breeding queen, also discovered secrets enabling somewhat pain-free living around 20 million years ago. Unlike most mammals, naked mole-rats do not feel the burn of chili pepper's active ingredient, capsaicin, nor the sting of acid. Indeed, by accumulating mutations in genes encoding proteins that are only now being exploited as targets for new pain therapies (the nerve growth factor receptor TrkA and voltage-gated sodium channel, NaV1.7), this species mastered the art of analgesia before humans evolved. Recently, we have identified pain-insensitivity as a trait shared by several closely related African mole-rat species. In this chapter we will show how African mole-rats have evolved pain insensitivity as well as discussing what the proximate factors may have been that led to the evolution of pain-free traits.

African Naked Mole-Rats Demonstrate Extreme Tolerance to Hypoxia and Hypercapnia.

Naked mole-rats are extremely tolerant to low concentrations of oxygen (hypoxia) and high concentrations of carbon dioxide (hypercapnia), which is consistent with the environment that they inhabit. Naked mole-rats combine subterranean living with living in very densely populated colonies where oxygen becomes depleted and carbon dioxide accumulates. In the laboratory, naked mole-rats fully recover from 5 h exposure to 5% O2 and 5 h exposure to 80% CO2, whereas both conditions are rapidly lethal to similarly sized laboratory mice. During anoxia (0% O2) naked mole-rats enter a suspended animation-like state and switch from aerobic metabolism of glucose to anaerobic metabolism of fructose. Additional fascinating characteristics include that naked mole-rats show intrinsic brain tolerance to anoxia; a complete lack of hypoxia-induced and CO2-induced pulmonary edema; and reduced aversion to high concentrations of CO2 and acidic fumes. Here we outline a constellation of physiological and molecular adaptations that correlate with the naked mole-rat's hypoxic/hypercapnic tolerance and which offer potential targets for ameliorating pathological conditions in humans, such as the damage caused during cerebral ischemia.

Hearing and Vocalizations in the Naked Mole-Rat.

Since their discovery, naked mole-rats have been speaking to us. Early field studies noted their extensive vocalizations, and scientists who are fortunate enough to spend time with these creatures in the laboratory setting cannot help but notice their constant peeping, chirruping and grunting (Hill et al., Proc Zool Soc Lond 128:455-514, 1957). Yet, few dwell on the function of these chirps and peeps, being instead drawn to the many other extraordinary aspects of naked mole-rat physiology detailed throughout this book. Still, no biology is complete without a description of how an organism communicates. While the field of naked mole-rat bioacoustics and acoustic communication has been largely silent for many years, we highlight recent progress in understanding how and what Heterocephalus glaber hears and which vocalizations it uses. These efforts are essential for a complete understanding of naked mole-rat cooperation, society and even culture.

A Sweet Story of Metabolic Innovation in the Naked Mole-Rat.

The naked mole-rat's (Heterocephalus glaber) social and subterranean lifestyle imposes several evolutionary pressures which have shaped its physiology. One example is low oxygen availability in a crowded burrow system which the naked mole-rat has adapted to via several mechanisms. Here we describe a metabolic rewiring which enables the naked mole-rat to switch substrates in glycolysis from glucose to fructose thereby circumventing feedback inhibition at phosphofructokinase (PFK1) to allow unrestrained glycolytic flux and ATP supply under hypoxia. Preferential shift to fructose metabolism occurs in other species and biological systems as a means to provide fuel, water or like in the naked mole-rat, protection in a low oxygen environment. We review fructose metabolism through an ecological lens and suggest that the metabolic adaptation to utilize fructose in the naked mole-rat may have evolved to simultaneously combat multiple challenges posed by its hostile environment.

A Role for STOML3 in Olfactory Sensory Transduction.

Stomatin-like protein-3 (STOML3) is an integral membrane protein expressed in the cilia of olfactory sensory neurons (OSNs), but its functional role in this cell type has never been addressed. STOML3 is also expressed in dorsal root ganglia neurons, where it has been shown to be required for normal touch sensation. Here, we extended previous results indicating that STOML3 is mainly expressed in the knob and proximal cilia of OSNs. We additionally showed that mice lacking STOML3 have a morphologically normal olfactory epithelium. Because of its presence in the cilia, together with known olfactory transduction components, we hypothesized that STOML3 could be involved in modulating odorant responses in OSNs. To investigate the functional role of STOML3, we performed loose patch recordings from wild-type (WT) and Stoml3 knock-out (KO) OSNs. We found that spontaneous mean firing activity was lower with additional shift in interspike intervals (ISIs) distributions in Stoml3 KOs compared with WT neurons. Moreover, the firing activity in response to stimuli was reduced both in spike number and duration in neurons lacking STOML3 compared with WT neurons. Control experiments suggested that the primary deficit in neurons lacking STOML3 was at the level of transduction and not at the level of action potential generation. We conclude that STOML3 has a physiological role in olfaction, being required for normal sensory encoding by OSNs.

Cultural transmission of vocal dialect in the naked mole-rat.

Naked mole-rats (Heterocephalus glaber) form some of the most cooperative groups in the animal kingdom, living in multigenerational colonies under the control of a single breeding queen. Yet how they maintain this highly organized social structure is unknown. Here we show that the most common naked mole-rat vocalization, the soft chirp, is used to transmit information about group membership, creating distinctive colony dialects. Audio playback experiments demonstrate that individuals make preferential vocal responses to home colony dialects. Pups fostered in foreign colonies in early postnatal life learn the vocal dialect of their adoptive colonies, which suggests vertical transmission and flexibility of vocal signatures. Dialect integrity is partly controlled by the queen: Dialect cohesiveness decreases with queen loss and remerges only with the ascendance of a new queen.

USH2A is a Meissner's corpuscle protein necessary for normal vibration sensing in mice and humans.

Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner's corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in terminal Schwann cells within Meissner's corpuscles. Pathogenic USH2A mutations cause Usher's syndrome, associated with hearing loss and visual impairment. We show that patients with biallelic pathogenic USH2A mutations also have clear and specific impairments in vibrotactile touch perception, as do mutant mice lacking USH2A. Forepaw rapidly adapting mechanoreceptors innervating Meissner's corpuscles, recorded from Ush2a-/- mice, showed large reductions in vibration sensitivity. However, the USH2A protein was not found in sensory neurons. Thus, loss of USH2A in corpuscular end-organs reduced mechanoreceptor sensitivity as well as vibration perception. Thus, a tether-like protein is required to facilitate detection of small-amplitude vibrations essential for the perception of fine-grained tactile surfaces.

SUMOylation of Enzymes and Ion Channels in Sensory Neurons Protects against Metabolic Dysfunction, Neuropathy, and Sensory Loss in Diabetes.

Diabetic peripheral neuropathy (DPN) is a highly frequent and debilitating clinical complication of diabetes that lacks therapies. Cellular oxidative stress regulates post-translational modifications, including SUMOylation. Here, using unbiased screens, we identified key enzymes in metabolic pathways and ion channels as novel molecular targets of SUMOylation that critically regulated their activity. Sensory neurons of diabetic patients and diabetic mice demonstrated changes in the SUMOylation status of metabolic enzymes and ion channels. In support of this, profound metabolic dysfunction, accelerated neuropathology, and sensory loss were observed in diabetic gene-targeted mice selectively lacking the ability to SUMOylate proteins in peripheral sensory neurons. TRPV1 function was impaired by diabetes-induced de-SUMOylation as well as by metabolic imbalance elicited by de-SUMOylation of metabolic enzymes, facilitating diabetic sensory loss. Our results unexpectedly uncover an endogenous post-translational mechanism regulating diabetic neuropathy in patients and mouse models that protects against metabolic dysfunction, nerve damage, and altered sensory perception.

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.

Independent evolution of pain insensitivity in African mole-rats: origins and mechanisms.

The naked mole-rat (Heterocephalus glaber) is famous for its longevity and unusual physiology. This eusocial species that lives in highly ordered and hierarchical colonies with a single breeding queen, also discovered secrets enabling somewhat pain-free living around 20 million years ago. Unlike most mammals, naked mole-rats do not feel the burn of chili pepper's active ingredient, capsaicin, nor the sting of acid. Indeed, by accumulating mutations in genes encoding proteins that are only now being exploited as targets for new pain therapies (the nerve growth factor receptor TrkA and voltage-gated sodium channel, NaV1.7), this species mastered the art of analgesia before humans evolved. Recently, we have identified pain insensitivity as a trait shared by several closely related African mole-rat species. One of these African mole-rats, the Highveld mole-rat (Cryptomys hottentotus pretoriae), is uniquely completely impervious and pain free when confronted with electrophilic compounds that activate the TRPA1 ion channel. The Highveld mole-rat has evolved a biophysical mechanism to shut down the activation of sensory neurons that drive pain. In this review, we will show how mole-rats have evolved pain insensitivity as well as discussing what the proximate factors may have been that led to the evolution of pain-free traits.