OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2.

PKA is a downstream effector of many inflammatory mediators that induce pain hypersensitivity by increasing the mechanosensitivity of nociceptive sensory afferent. Here, we examine the molecular mechanism underlying PKA-dependent modulation of the mechanically activated ion channel PIEZO2, which confers mechanosensitivity to many nociceptors. Using phosphorylation site prediction algorithms, we identified multiple putative and highly conserved PKA phosphorylation sites located on intracellular intrinsically disordered regions of PIEZO2. Site-directed mutagenesis and patch-clamp recordings showed that substitution of one or multiple putative PKA sites within a single intracellular domain does not alter PKA-induced PIEZO2 sensitization, whereas mutation of a combination of nine putative sites located on four different intracellular regions completely abolishes PKA-dependent PIEZO2 modulation, though it remains unclear whether all or just some of these nine sites are required. By demonstrating that PIEZO1 is not modulated by PKA, our data also reveal a previously unrecognized functional difference between PIEZO1 and PIEZO2. Moreover, by demonstrating that PKA only modulates PIEZO2 currents evoked by focal mechanical indentation of the cell, but not currents evoked by pressure-induced membrane stretch, we provide evidence suggesting that PIEZO2 is a polymodal mechanosensor that engages different protein domains for detecting different types of mechanical stimuli.

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.

Neuropathic pain caused by miswiring and abnormal end organ targeting.

Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together1-3. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury4-7-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients.

Intrinsically disordered intracellular domains control key features of the mechanically-gated ion channel PIEZO2.

A central question in mechanobiology is how mechanical forces acting in or on cells are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we examined the role of the intracellular domains of PIEZO2, which account for 25% of the channel, and demonstrate that these domains fine-tune properties such as poking and stretch-sensitivity, velocity coding and single channel conductance. Moreover, we show that the intrinsically disordered linker between the transmembrane helices twelve and thirteen (IDR5) is required for the activation of PIEZO2 by cytoskeleton-transmitted forces. The deletion of IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, while it only partially affected its sensitivity to cell indentation and does not alter its stretch sensitivity. Thus, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO function.

Identification of a population of peripheral sensory neurons that regulates blood pressure.

The vasculature is innervated by a network of peripheral afferents that sense and regulate blood flow. Here, we describe a system of non-peptidergic sensory neurons with cell bodies in the spinal ganglia that regulate vascular tone in the distal arteries. We identify a population of mechanosensitive neurons, marked by tropomyosin receptor kinase C (TrkC) and tyrosine hydroxylase in the dorsal root ganglia, which projects to blood vessels. Local stimulation of TrkC neurons decreases vessel diameter and blood flow, whereas systemic activation increases systolic blood pressure and heart rate variability via the sympathetic nervous system. Ablation of the neurons provokes variability in local blood flow, leading to a reduction in systolic blood pressure, increased heart rate variability, and ultimately lethality within 48 h. Thus, a population of TrkC+ sensory neurons forms part of a sensory-feedback mechanism that maintains cardiovascular homeostasis through the autonomic nervous system.

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.

TTX-Resistant Sodium Channels Functionally Separate Silent From Polymodal C-nociceptors.

Pronounced activity-dependent slowing of conduction has been used to characterize mechano-insensitive, "silent" nociceptors and might be due to high expression of NaV1.8 and could, therefore, be characterized by their tetrodotoxin-resistance (TTX-r). Nociceptor-class specific differences in action potential characteristics were studied by: (i) in vitro calcium imaging in single porcine nerve growth factor (NGF)-responsive neurites; (ii) in vivo extracellular recordings in functionally identified porcine silent nociceptors; and (iii) in vitro patch-clamp recordings from murine silent nociceptors, genetically defined by nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) expression. Porcine TTX-r neurites (n = 26) in vitro had more than twice as high calcium transients per action potential as compared to TTX-s neurites (n = 18). In pig skin, silent nociceptors (n = 14) characterized by pronounced activity-dependent slowing of conduction were found to be TTX-r, whereas polymodal nociceptors were TTX-s (n = 12) and had only moderate slowing. Mechano-insensitive cold nociceptors were also TTX-r but showed less activity-dependent slowing than polymodal nociceptors. Action potentials in murine silent nociceptors differed from putative polymodal nociceptors by longer duration and higher peak amplitudes. Longer duration AP in silent murine nociceptors linked to increased sodium load would be compatible with a pronounced activity-dependent slowing in pig silent nociceptors and longer AP durations could be in line with increased calcium transients per action potential observed in vitro in TTX-resistant NGF responsive porcine neurites. Even though there is no direct link between slowing and TTX-resistant channels, the results indicate that axons of silent nociceptors not only differ in their receptive but also in their axonal properties.

Structure-guided examination of the mechanogating mechanism of PIEZO2.

Piezo channels are mechanically activated ion channels that confer mechanosensitivity to a variety of different cell types. Piezos oligomerize as propeller-shaped homotrimers that are thought to locally curve the membrane into spherical domes that project into the cell. While several studies have identified domains and amino acids that control important properties such as ion permeability and selectivity as well as inactivation kinetics and voltage sensitivity, only little is known about intraprotein interactions that govern mechanosensitivity-the most unique feature of PIEZOs. Here we used site-directed mutagenesis and patch-clamp recordings to investigate the mechanogating mechanism of PIEZO2. We demonstrate that charged amino acids at the interface between the beam domain-i.e., a long α-helix that protrudes from the intracellular side of the "propeller" blade toward the inner vestibule of the channel-and the C-terminal domain (CTD) as well as hydrophobic interactions between the highly conserved Y2807 of the CTD and pore-lining helices are required to ensure normal mechanosensitivity of PIEZO2. Moreover, single-channel recordings indicate that a previously unrecognized intrinsically disordered domain located adjacent to the beam acts as a cytosolic plug that limits ion permeation possibly by clogging the inner vestibule of both PIEZO1 and PIEZO2. Thus, we have identified several intraprotein domain interfaces that control the mechanical activation of PIEZO1 and PIEZO2 and which might thus serve as promising targets for drugs that modulate the mechanosensitivity of Piezo channels.

Differential modulation of voltage-gated sodium channels by nerve growth factor in three major subsets of TrkA-expressing nociceptors.

Nerve growth factor is an inflammatory mediator that induces long-lasting hyperalgesia, which can partially be attributed to nerve growth factor-induced sensitization of primary afferent nociceptors. It was shown that nerve growth factor increases the excitability of polymodal C-fibre nociceptors by modulating tetrodotoxin-sensitive and tetrodotoxin-resistant voltage-gated sodium channels, but hitherto only little is known about the effects of nerve growth factor on sodium currents in other nociceptor subtypes that express the nerve growth factor receptor TrkA. We previously characterized two reporter mouse lines that allow the unequivocal identification of two important subclasses of TrkA-expressing nociceptors - i.e. neuropeptide Y receptor type 2 (NPY2R+ ) Aδ-fibre nociceptors that mediate pinprick pain and nicotinic acetylcholine receptor alpha-3 subunit (CHRNA3+ ) silent nociceptors, which are the most abundant TrkA+ nociceptors in visceral organs and deep somatic tissues. Here, we utilized these mouse lines to investigate the expression patterns and the possible nerve growth factor-dependent modulation of sodium channels in these neurons using whole-cell patch-clamp recordings and quantitative real-time polymerase chain reaction. We demonstrate that NPY2R+ nociceptors, CHRNA3+ 'silent' nociceptors and polymodal C-fibre nociceptors express different combinations of sodium channel α- and ß-subunits and accordingly exhibit functionally different sodium currents. Moreover, we demonstrate that nerve growth factor produces robust hyperpolarizing shifts in the half-activation voltage of tetrodotoxin-resistant currents in NPY2R+ nociceptors and polymodal C-fibre nociceptors and also shifts the half-activation of tetrodotoxin-sensitive currents in polymodal C-fibre nociceptors. In silent nociceptors, however, nerve growth factor solely increases the current density of the tetrodotoxin-resistant current but does not alter other sodium channel properties. Considering the different peripheral target tissues and the previously reported roles in different forms of pain of the nociceptor subpopulations that were examined here, our results suggest that nerve growth factor differentially contributes to the development visceral and cutaneous pain hypersensitivity and highlights the importance of developing different therapeutic strategies for different forms of pain.

Control of mechanical pain hypersensitivity in mice through ligand-targeted photoablation of TrkB-positive sensory neurons.

Mechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both necessary and sufficient for producing pain from light touch after nerve injury in mice. Mice in which TrkB-Cre-expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB-positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.

Myotubularin related protein-2 and its phospholipid substrate PIP2 control Piezo2-mediated mechanotransduction in peripheral sensory neurons.

Piezo2 ion channels are critical determinants of the sense of light touch in vertebrates. Yet, their regulation is only incompletely understood. We recently identified myotubularin related protein-2 (Mtmr2), a phosphoinositide (PI) phosphatase, in the native Piezo2 interactome of murine dorsal root ganglia (DRG). Here, we demonstrate that Mtmr2 attenuates Piezo2-mediated rapidly adapting mechanically activated (RA-MA) currents. Interestingly, heterologous Piezo1 and other known MA current subtypes in DRG appeared largely unaffected by Mtmr2. Experiments with catalytically inactive Mtmr2, pharmacological blockers of PI(3,5)P2 synthesis, and osmotic stress suggest that Mtmr2-dependent Piezo2 inhibition involves depletion of PI(3,5)P2. Further, we identified a PI(3,5)P2 binding region in Piezo2, but not Piezo1, that confers sensitivity to Mtmr2 as indicated by functional analysis of a domain-swapped Piezo2 mutant. Altogether, our results propose local PI(3,5)P2 modulation via Mtmr2 in the vicinity of Piezo2 as a novel mechanism to dynamically control Piezo2-dependent mechanotransduction in peripheral sensory neurons.

Functional and Molecular Characterization of Mechanoinsensitive "Silent" Nociceptors.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation.

Touch Receptor-Derived Sensory Information Alleviates Acute Pain Signaling and Fine-Tunes Nociceptive Reflex Coordination.

Painful mechanical stimuli activate multiple peripheral sensory afferent subtypes simultaneously, including nociceptors and low-threshold mechanoreceptors (LTMRs). Using an optogenetic approach, we demonstrate that LTMRs do not solely serve as touch receptors but also play an important role in acute pain signaling. We show that selective activation of neuropeptide Y receptor-2-expressing (Npy2r) myelinated A-fiber nociceptors evokes abnormally exacerbated pain, which is alleviated by concurrent activation of LTMRs in a frequency-dependent manner. We further show that spatial summation of single action potentials from multiple NPY2R-positive afferents is sufficient to trigger nocifensive paw withdrawal, but additional simultaneous sensory input from LTMRs is required for normal well-coordinated execution of this reflex. Thus, our results show that combinatorial coding of noxious and tactile sensory input is required for normal acute mechanical pain signaling. Additionally, we established a causal link between precisely defined neural activity in functionally identified sensory neuron subpopulations and nocifensive behavior and pain.

PIEZO2 is required for mechanotransduction in human stem cell-derived touch receptors.

Human sensory neurons are inaccessible for functional examination, and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro, we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals, their most salient characteristic in vivo. Strikingly, mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors, which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity.

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.

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.