Tatjana I. Kichko

Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Nav1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Nav1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Nav1.8 knockout (Scn10−/−) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway.

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO–TRPA1–CGRP pathway. We propose that this neuroendocrine HNO–TRPA1–CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage

The Sox10 transcription factor is required to maintain as well as specify glial identity, adding new causes for the neuropathies associated with SOX10 mutations.

TRPA1 and Substance P Mediate Colitis in Mice

BACKGROUND & AIMS The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P. METHODS We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice. RESULTS TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis. CONCLUSIONS Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

Sox10 is required for Schwann-cell homeostasis and myelin maintenance in the adult peripheral nerve.

The transcription factor Sox10 functions during multiple consecutive stages of Schwann‐cell development in the peripheral nervous system (PNS). Although Sox10 continues to be expressed in mature Schwann cells of the adult peripheral nerve, it is currently unclear whether it is still functional. Here, we used a genetic strategy to selectively delete Sox10 in glia of adult mice in a tamoxifen‐dependent manner. The tamoxifen‐treated mice developed a severe peripheral neuropathy that was associated with dramatic alterations in peripheral nerve structure and function. Demyelination and axonal degeneration were as much evident as signs of neuroinflammation. Compound action potentials exhibited pathophysiological alterations. Sox10‐deleted Schwann cells persisted in the peripheral nerve, but did not exhibit a mature, myelinating phenotype arguing that Sox10 is rather required for differentiation and maintenance of the differentiated state than for survival. Our report is the first evidence that Sox10 is still essentially required for Schwann‐cell function in the adult PNS and establishes a useful model in which to study human peripheral neuropathies.

Human TRPA1 is a heat sensor displaying intrinsic U-shaped thermosensitivity.

Thermosensitive Transient Receptor Potential (TRP) channels are believed to respond to either cold or heat. In the case of TRP subtype A1 (TRPA1), there seems to be a species-dependent divergence in temperature sensation as non-mammalian TRPA1 is heat-sensitive whereas mammalian TRPA1 is sensitive to cold. It has been speculated but never experimentally proven that TRPA1 and other temperature-sensitive ion channels have the inherent capability of responding to both cold and heat. Here we show that redox modification and ligands affect human TRPA1 (hTRPA1) cold and heat sensing properties in lipid bilayer and whole-cell patch-clamp recordings as well as heat-evoked TRPA1-dependent calcitonin gene-related peptide (CGRP) release from mouse trachea. Studies of purified hTRPA1 intrinsic tryptophan fluorescence, in the absence of lipid bilayer, consolidate hTRPA1 as an intrinsic bidirectional thermosensor that is modified by the redox state and ligands. Thus, the heat sensing property of TRPA1 is conserved in mammalians, in which TRPA1 may contribute to sensing warmth and uncomfortable heat in addition to noxious cold.

Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release

Significance The cation channel TRPM3 is highly expressed in the sensory system, where it plays a key role in the detection of noxious heat and the development of inflammatory heat hypersensitivity. Our understanding of the physiological role of TRPM3 in the sensory system and other tissues is hampered by the lack of potent pharmacologic tools, however. This study describes CIM0216, a small-molecule TRPM3 agonist. Our results indicate that CIM0216 is much more potent than established TRPM3 agonists, particularly owing to its ability to open two distinct cation-permeable pores in TRPM3. Using CIM0216 as a pharmacologic tool, we reveal that activation of TRPM3 evokes the release of calcitonin gene-related peptide from sensory nerve terminals and of insulin from pancreatic islets. Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation.

TRPV1 controls acid- and heat-induced calcitonin gene-related peptide release and sensitization by bradykinin in the isolated mouse trachea

Chronic cough derives from inflammatory hypersensitivity of tracheobronchial nerve endings, most of which express the polymodal capsaicin receptor‐channel transient receptor potential vanilloid (TRPV) type 1 and the secretory neuropeptide calcitonin gene‐related peptide (CGRP). An isolated mouse trachea preparation was established to measure chemically and thermally stimulated CGRP release as an index for sensory transduction of potential cough‐inducing stimuli. TRPV1 knockout mice were employed to assess the TRPV1 contribution to tracheal responsiveness and sensitization. Graded heat‐induced CGRP release depended entirely on extracellular calcium and partly on TRPV1; knockout mice showed 60% less CGRP release at 45°C (for 5 min) than wild‐types. This heat response was facilitated by the TRPV1 agonist ethanol and the TRPV1–3 agonist 2‐aminoethoxydiphenyl borate, effects that were reduced or absent in TRPV1−/−, respectively. The TRPV1 antagonists ruthenium red and N‐(4‐t‐butylphenyl)‐4‐(3‐chloropyridin‐2‐yl) tetrahydropyrazine‐1(2H)‐carboxamide were ineffective on the basal heat response. A step increase of temperature from 22 to 40°C caused a TRPV1‐independent CGRP release that was doubled by bradykinin in wild‐types but not TRPV1−/−. Proton stimulation resulted in a bell‐shaped concentration–response curve with threshold at pH 6.7 and a maximum at pH 5.7; responses were greatly reduced but not abolished in TRPV1−/−. Coadministration of amiloride (30 μm), the blocker of acid‐sensing ion channels, was ineffective in both TRPV1 genotypes. The data suggest that tracheal acid sensing mainly involves TRPV1 but not acid‐sensing ion channels, whereas noxious heat responsiveness partly depends and (inflammatory) sensitization to heat largely depends on the capsaicin receptor in tracheal nerve endings. Lowering of their heat threshold to near body temperature may sustain hypersensitivity and neurogenic inflammation of the upper airways.

Bimodal concentration-response of nicotine involves the nicotinic acetylcholine receptor, transient receptor potential vanilloid type 1, and transient receptor potential ankyrin 1 channels in mouse trachea and sensory neurons.

High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 µM (−)-nicotine, a maximum at 100 µM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pHe-dependent, – five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 µM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.

Establishment of myelinating schwann cells and barrier integrity between central and peripheral nervous systems depend on Sox10

The transcription factor Sox10 is expressed throughout Schwann cell development and has already been shown to be essential for specification and for the identity and further development of immature Schwann cells. Here, we show that Sox10 is also required in Schwann cells for establishing the myelinating state. This is concluded from the fact that a peripheral neuropathy develops in mice in which Sox10 is deleted by a Cre recombinase whose expression is under control of Krox20 regulatory elements. This neuropathy is characterized by altered marker gene expression along the peripheral nerve, decreased conductivity, and severe persistent hypomyelination. As the Cre recombinase is additionally active in boundary cap cells, we also analyzed the role of Sox10 during embryogenesis in establishment and maintenance of the boundary between central and peripheral nervous systems. Sox10 deletion did not affect establishment or survival of boundary cap cells but appeared to compromise barrier function as cells expressing oligodendrocyte and astrocyte markers were no longer restricted to the central nervous system, and instead found in peripheral nerves. We infer that in addition to its many roles in Schwann cells, Sox10 is also important for the integrity of the boundary between central and peripheral nervous systems.