GABA blocks pathological but not acute TRPV1 pain signals.

Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.

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.

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.

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.

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.

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.

Collagen Organization Within the Cartilage of Trpv4-/- Mice Studied with Two-Photon Microscopy and Polarized Second Harmonic Generation.

The polymodal channel TRPV4 has been shown to regulate development and maintenance of cartilage. Here we investigate whether TRPV4 activity regulates the early deposition and structure of collagen matrix in the femoral head cartilage by comparing the 3D morphology and the sub-micrometer organization of the collagen matrix between wild type and Trpv4 -/- mice pups four to five days old. Two-photon microscopy can be used to conduct label-free imaging of cartilage, as collagen generates a second harmonic signal (second harmonic generation [SHG]) under pulsed infrared excitation. In one set of measurements, we use circularly polarized laser light to reconstruct the 3D morphology of the femoral head cartilage and to measure the tissue thickness. Second, by rotating the direction of the linearly polarized light and using polarized SHG detection, we investigate the sub-micrometer orientation of collagen fibers in the cartilage. At this developmental stage, we cannot detect statistically significant differences between the two mice strains, although a tendency toward a more random orientation of collagen fibers and a higher thickness of the whole cartilage seems to characterize the Trpv4 -/- mice. We discuss possible reasons for these observations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

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.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

Specialized mechanoreceptor systems in rodent glabrous skin.

KEY POINTS: An ex vivo preparation was developed to record from single sensory fibres innervating the glabrous skin of the mouse forepaw. The density of mechanoreceptor innervation of the forepaw glabrous skin was found to be three times higher than that of hindpaw glabrous skin. Rapidly adapting mechanoreceptors that innervate Meissner's corpuscles were severalfold more responsive to slowly moving stimuli in the forepaw compared to those innervating hindpaw skin. We found a distinct group of small hairs in the centre of the mouse hindpaw glabrous skin that were exclusively innervated by directionally sensitive D-hair receptors. The directional sensitivity, but not the end-organ anatomy, were the opposite to D-hair receptors in the hairy skin. Glabrous skin hairs in the hindpaw are not ubiquitous in rodents, but occur in African and North American species that diverged more than 65 million years ago. ABSTRACT: Rodents use their forepaws to actively interact with their tactile environment. Studies on the physiology and anatomy of glabrous skin that makes up the majority of the forepaw are almost non-existent in the mouse. Here we developed a preparation to record from single sensory fibres of the forepaw and compared anatomical and physiological receptor properties to those of the hindpaw glabrous and hairy skin. We found that the mouse forepaw skin is equipped with a very high density of mechanoreceptors; >3 times more than hindpaw glabrous skin. In addition, rapidly adapting mechanoreceptors that innervate Meissner's corpuscles of the forepaw were severalfold more sensitive to slowly moving mechanical stimuli compared to their counterparts in the hindpaw glabrous skin. All other mechanoreceptor types as well as myelinated nociceptors had physiological properties that were invariant regardless of which skin area they occupied. We discovered a novel D-hair receptor innervating a small group of hairs in the middle of the hindpaw glabrous skin in mice. These glabrous skin D-hair receptors were direction sensitive albeit with an orientation sensitivity opposite to that described for hairy skin D-hair receptors. Glabrous skin hairs do not occur in all rodents, but are present in North American and African rodent species that diverged more than 65 million years ago. The function of these specialized hairs is unknown, but they are nevertheless evolutionarily very ancient. Our study reveals novel physiological specializations of mechanoreceptors in the glabrous skin that likely evolved to facilitate tactile exploration.

Mechanoelectrical transduction in chondrocytes.

Cartilage tissue lines the joints of mammals, helping to lubricate joint movement and distribute mechanical loads. This tissue is comprised of isolated cells known as chondrocytes which are embedded in an extracellular matrix. Chondrocytes produce and maintain the cartilage by sensing and responding to changing mechanical loads. Mechanosensitive ion channels have been implicated in chondrocyte mechanotransduction and recent studies have shown that both PIEZO1 and TRPV4 can be activated by mechanical stimuli in these cells. The 2 channels mediate separate but overlapping mechanoelectrical transduction pathways, PIEZO1 in response to stretch and substrate deflections and TRPV4 in response to substrate deflections alone. These distinct pathways of mechanoelectrical transduction suggest a mechanism by which chondrocytes can distinguish between different stimuli that arise in their complex mechanical environment.

A stomatin dimer modulates the activity of acid-sensing ion channels.

Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved stomatin domain of mouse stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.

Sensory mechanotransduction at membrane-matrix interfaces.

Sensory cells specialized to detect extremely small mechanical changes are common to the auditory and somatosensory systems. It is widely accepted that mechanosensitive channels form the core of the mechanoelectrical transduction in hair cells as well as the somatic sensory neurons that underlie the sense of touch and mechanical pain. Here, we will review how the activation of such channels can be measured in a meaningful physiological context. In particular, we will discuss the idea that mechanosensitive channels normally occur in transmembrane complexes that are anchored to extracellular matrix components (ECM) both in vitro and in vivo. One component of such complexes in sensory neurons is the integral membrane scaffold protein STOML3 which is a robust physiological regulator of native mechanosensitive currents. In order to better characterize such channels in transmembrane complexes, we developed a new electrophysiological method that enables the quantification of mechanosensitive current amplitude and kinetics when activated by a defined matrix movement in cultured cells. The results of such studies strongly support the idea that ion channels in transmembrane complexes are highly tuned to detect movement of the cell membrane in relation to the ECM.

A genetic basis for mechanosensory traits in humans.

In all vertebrates hearing and touch represent two distinct sensory systems that both rely on the transformation of mechanical force into electrical signals. There is an extensive literature describing single gene mutations in humans that cause hearing impairment, but there are essentially none for touch. Here we first asked if touch sensitivity is a heritable trait and second whether there are common genes that influence different mechanosensory senses like hearing and touch in humans. Using a classical twin study design we demonstrate that touch sensitivity and touch acuity are highly heritable traits. Quantitative phenotypic measures of different mechanosensory systems revealed significant correlations between touch and hearing acuity in a healthy human population. Thus mutations in genes causing deafness genes could conceivably negatively influence touch sensitivity. In agreement with this hypothesis we found that a proportion of a cohort of congenitally deaf young adults display significantly impaired measures of touch sensitivity compared to controls. In contrast, blind individuals showed enhanced, not diminished touch acuity. Finally, by examining a cohort of patients with Usher syndrome, a genetically well-characterized deaf-blindness syndrome, we could show that recessive pathogenic mutations in the USH2A gene influence touch acuity. Control Usher syndrome cohorts lacking demonstrable pathogenic USH2A mutations showed no impairment in touch acuity. Our study thus provides comprehensive evidence that there are common genetic elements that contribute to touch and hearing and has identified one of these genes as USH2A.

Subunit-specific inhibition of acid sensing ion channels by stomatin-like protein 1.

There are five mammalian stomatin-domain genes, all of which encode peripheral membrane proteins that can modulate ion channel function. Here we examined the ability of stomatin-like protein 1 (STOML1) to modulate the proton-sensitive members of the acid-sensing ion channel (ASIC) family. STOML1 profoundly inhibits ASIC1a, but has no effect on the splice variant ASIC1b. The inactivation time constant of ASIC3 is also accelerated by STOML1. We examined STOML1 null mutant mice with a ß-galactosidase-neomycin cassette gene-trap reporter driven from the STOML1 gene locus, which indicated that STOML1 is expressed in at least 50% of dorsal root ganglion (DRG) neurones. Patch clamp recordings from mouse DRG neurones identified a trend for larger proton-gated currents in neurones lacking STOML1, which was due to a contribution of effects upon both transient and sustained currents, at different pH, a finding consistent with an endogenous inhibitory function for STOML1.

Hairy sensation.

The hairs of the skin not only function to prevent heat loss but also have important sensory functions. Recent work has now established that each hair of the skin is innervated by one or more of three types of mechanoreceptor ending. Each of these three mechanoreceptor types possesses distinct molecular features and detects distinctive information about skin touch, which is relayed to specific brain locations in a somatotopic fashion.

Pro-neurotrophins, sortilin, and nociception.

Nerve growth factor (NGF) signaling is important in the development and functional maintenance of nociceptors, but it also plays a central role in initiating and sustaining heat and mechanical hyperalgesia following inflammation. NGF signaling in pain has traditionally been thought of as primarily engaging the classic high-affinity receptor tyrosine kinase receptor TrkA to initiate sensitization events. However, the discovery that secreted proforms of nerve NGF have biological functions distinct from the processed mature factors raised the possibility that these proneurotrophins (proNTs) may have distinct function in painful conditions. ProNTs engage a novel receptor system that is distinct from that of mature neurotrophins, consisting of sortilin, a type I membrane protein belonging to the VPS10p family, and its co-receptor, the classic low-affinity neurotrophin receptor p75NTR. Here, we review how this new receptor system may itself function with or independently of the classic TrkA system in regulating inflammatory or neuropathic pain.

Evidence for a protein tether involved in somatic touch.

The gating of ion channels by mechanical force underlies the sense of touch and pain. The mode of gating of mechanosensitive ion channels in vertebrate touch receptors is unknown. Here we show that the presence of a protein link is necessary for the gating of mechanosensitive currents in all low-threshold mechanoreceptors and some nociceptors of the dorsal root ganglia (DRG). Using TEM, we demonstrate that a protein filament with of length approximately 100 nm is synthesized by sensory neurons and may link mechanosensitive ion channels in sensory neurons to the extracellular matrix. Brief treatment of sensory neurons with non-specific and site-specific endopeptidases destroys the protein tether and abolishes mechanosensitive currents in sensory neurons without affecting electrical excitability. Protease-sensitive tethers are also required for touch-receptor function in vivo. Thus, unlike the majority of nociceptors, cutaneous mechanoreceptors require a distinct protein tether to transduce mechanical stimuli.

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.