Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the β-Subunit of Acid Ceramidase

Yasuhiro Teranishi 1,†,‡, Hiroshi Kuwahara 1,†,§, Masaru Ueda 1, Tadashi Takemura 1, Masanori Kusumoto 1,§, Keiji Nakamura 1, Jun Sakai 1, Toru Kimura 1, Yasuji Furutani 1, Makoto Kawashima 2, Genji Imokawa 3,* and Mari Nogami-Itoh 4,*

Abstract: A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the
dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that
sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield
their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of
ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency.
Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains
unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities
of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates
by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively.
Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung >
heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and
Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent
molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS
using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid
sequence to be determined and identified as the β-subunit of aCDase, which consists of α- and
β-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with
2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies
to the α-subunit at ~56 kDa and ~13 kDa and the β-subunit at ~43 kDa, the purified SM deacylase was
detectable only by the antibody to the β-subunit at ~43 kDa. Breaking the S-S bond of recombinant
human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading β-subunit that evokes the ceramide deficiency in AD skin.

Keywords: atopic dermatitis; ceramide; ceramide deficiency; barrier function; water reservoir faction;
stratum corneum; sphingomyelin deacylase; sphingosylphosphorylcholine; acid ceramidase

Functional Interaction between Transient Receptor Potential V4 Channel and Neuronal Calcium Sensor 1 and the Effects of Paclitaxel

Julio C. Sanchez and Barbara E. Ehrlich

ABSTRACT
Neuronal calcium sensor 1 (NCS1), a calcium-binding protein, and transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, are fundamental in the regulation of calcium homeostasis. The interactions of these proteins and their regulation by paclitaxel (PTX) were investigated using biochemical, pharmacological, and electrophysiological approaches in both a breast cancer epithelial cell model and a neuronal model. TRPV4 and NCS1 reciprocally immunoprecipitated each other, suggesting that they make up a signaling complex. The functional consequence of this physical association was that TRPV4 currents increased with increased NCS1 expression. Calcium fluxes through TRPV4 correlated with the magnitude of TRPV4 currents, and these calcium fluxes depended on NCS1
expression levels. Exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes but decreased the expression of NCS1. These findings augment the understanding of the properties of TRPV4, the role of NCS1 in the regulation of TRPV4, and the cellular mechanisms of PTX-induced neuropathy.

Presenter: Ki Baek Lee

TRPV4 inhibition prevents paclitaxel-induced neurotoxicity in preclinicalmodels

Wolfgang Boehmerlea,b,c,⁎,1, Petra Huehnchena,b,c,1, Sabrina Lin Lin Leeb,d, Christoph Harmsa,b,c,d,1, Matthias Endresa,b,c,d,e,f,1

Paclitaxel is a cytotoxic drug which frequently causes sensory peripheral neuropathy in patients. Increasing
evidence suggests that altered intracellular calcium (Ca2+) signals play an important role in the pathogenesis of
this condition. In the present study, we examined the interplay between Ca2+ release channels in the endoplasmic reticulum (ER) and Ca2+ permeable channels in the plasma membrane in the context of paclitaxel
mediated neurotoxicity. We observed that in small to medium size dorsal root ganglia neurons (DRGN) the
inositol-trisphosphate receptor (InsP3R) type 1 was often concentrated in the periphery of cells, which is in
contrast to homogenous ER distribution. G protein-coupled designer receptors were used to further elucidate
phosphoinositide mediated Ca2+ signaling: This approach showed strong InsP3 mediated Ca2+ signals close to
the plasma membrane, which can be amplified by Ca2+ entry through TRPV4 channels. In addition, our results
support a physical interaction and partial colocalization of InsP3R1 and TRPV4 channels. In the context of
paclitaxel-induced neurotoxicity, blocking Ca2+ influx through TRPV4 channels reduced cell death in cultured
DRGN. Pretreatment of mice with the pharmacological TRPV4 inhibitor HC067047 prior to paclitaxel injections
prevented electrophysiological and behavioral changes associated with paclitaxel-induced neuropathy.
In summary, these results underline the relevance of TRPV4 signaling for the pathogenesis of paclitaxelinduced neuropathy and suggest novel preventive strategies

Keywords: Paclitaxel, Neuropathy, Calcium, TRPV4, Inositol-trisphosphate receptor

Presenter: Hye In Kim

A re‐innervated in vitro skin model of non‐histaminergic itch and skin neurogenic inflammation: PAR2‐, TRPV1‐ and TRPA1‐agonist induced functionality

N. Lebonvallet, J. W. Fluhr, C. Le Gall‐Ianotto, R. Leschiera, M. Talagas, J.‐L. Carré, A. Reux, L. Misery, A. Bataille, C. Brun3, T. Oddos3, J.‐P. Pennec4

• Published in:- Skin Health and Disease

Abstract

Background: Skin, and epidermis, is innervated by sensory nerve fibres. Interactions between them and signal transduction are only partially eluci- dated in physiological/pathological conditions, especially in pruritus.

Objectives: To study the mechanisms involved in pruritus in vitro, we developed a skin explant model re‐innervated by sensory neurons.

Methods: This model is based on the co‐culture of human skin explants and sensory neurons from dorsal root ganglia of rats. Innervation and the expression of protease activated receptor 2 (PAR2), transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin one (TRPA1) was analysed by immunostaining. The response of the model to TRPV1, PAR2 and TRPA1 agonists was analysed by patch‐clamp, qPCR and enzyme‐linked immunosorbent assay.

Results: After 5 days of re‐innervating nerve fibres was evidenced in the epidermis. Re‐innervation was correlated with decrease of epidermal thickness and the number of apoptotic cells in the tissue. The major actors of non‐histaminergic itch (PAR‐2, thymic stromal lymphopoietin [TSLP], TSLP‐R, TRPA1 and TRPV1) were expressed in neurons and/or epidermal cells of skin explants. After topical exposure of TRPV1‐(Capsaicin), TRPA1‐ (Polygodial) and PAR2‐agonist (SLIGKV‐NH2) activation of reinnervating neurons could be shown in patch‐clamp analysis. The release of TSLP was increased with capsaicin or SLIGKV but decreased with polygodial. Release of CGRP was increased by capsaicin and polygodial but decreased with SLIGKV. Activation by SLIGKV showed a decrease of VEGF; polygodial induced an increase of TSLP, Tumour necrosis factor (TNF) and nerve growth factor and capsaicin lead to a decrease of sema3 and TNF expression.

Conclusion: The present model is suitable for studying itch and neurogenic inflammation pathways in vitro. We observed that activation of TRPV1, TRPA1 and PAR‐2 leads to different response profiles in re‐innervated skin explants.

The role of Na _v 1.7 and methylglyoxal-mediated activation of TRPA1 in itch and hypoalgesia in a murine model of type 1 diabetes

Ruo-Xiao Cheng, Yu Feng, Di Liu, Zhi-Hong Wang, Jiang-Tao Zhang, Li-Hua Chen, Cun-Jin Su, Bing Wang, Ya Huang, Ru-Rong Ji, Ji Hu, Tong Liu

Abstract

Methylglyoxal (MGO), an endogenous reactive carbonyl compound, plays a key role in the pathogenesis of diabetic neuropathy. The aim of this study is to investigate the role of MGO in diabetic itch and hypoalgesia, two common symptoms associated with diabetic neuropathy. 

Methods: Scratching behavior, mechanical itch (alloknesis), and thermal hypoalgesia were quantified after intradermal (i.d.) injection of MGO in naïve mice or in diabetic mice induced by intraperitoneal (i.p.) injection of streptozotocin (STZ). Behavioral testing, patch-clamp recording, transgenic mice, and gene expression analysis were used to investigate the mechanisms underlying diabetic itch and hypoalgesia in mice. 

Results: I.d. injection of MGO evoked dose-dependent scratching in normal mice. Addition of MGO directly activated transient receptor potential ankyrin 1 (TRPA1) to induce inward currents and calcium influx in dorsal root ganglia (DRG) neurons or in TRPA1-expressing HEK293 cells. Mechanical itch, but not spontaneous itch was developed in STZ-induced diabetic mice. Genetic ablation of Trpa1 (Trpa1^-/- ), pharmacological blockade of TRPA1 and Na_v1.7, antioxidants, and mitogen-activated protein kinase kinase enzyme (MEK) inhibitor U0126 abrogated itch induced by MGO or in STZ-induced diabetic mice. Thermal hypoalgesia was induced by intrathecal (i.t.) injection of MGO or in STZ-induced diabetic mice, which was abolished by MGO scavengers, intrathecal injection of TRPA1 blockers, and in Trpa1^-/- mice. 

Conclusion: This study revealed that Na_v1.7 and MGO-mediated activation of TRPA1 play key roles in itch and hypoalgesia in a murine model of type 1 diabetes. Thereby, we provide a novel potential therapeutic strategy for the treatment of itch and hypoalgesia induced by diabetic neuropathy.

Keywords: Diabetes; Hypoalgesia; Itch; Methylglyoxal; TRPA1.

The Role of Transient Receptor Potential A1 and G Protein-Coupled Receptor 39 in Zinc-Mediated Acute and Chronic Itch in Mice

Yue Hu^1†, Qing-Yue Fu^1†, Dan-Ni Fu^1†, Xue-Long Wang^2†, Zhi-Hong Wang¹, Jiang-Tao Zhang³, Wen-Jing Xu³, Guo-Kun Zhou³, Li-Hua Chen^4* and Tong Liu^3,5,6*

• ¹Jiangsu Key Laboratory of Neuropsychiatric Diseases and Institute of Neuroscience, Soochow University, Suzhou, China
• ²Department of Thoracic Surgery, Capital Medical University Electric Power Teaching Hospital Beijing, Beijing, China
• ³Institute of Pain Medicine and Special Environmental Medicine, Nantong University, Nantong, China
• ⁴Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong, China
• ⁵College of Life Sciences, Yanan University, Yan’an, China
• ⁶Suzhou Key Laboratory of Intelligent Medicine and Equipment, Soochow University, Suzhou, China

Itching is a common symptom of many skin or systemic diseases and has a negative impact on the quality of life. Zinc, one of the most important trace elements in an organism, plays an important role in the regulation of pain. Whether and how zinc regulates itching is largely unclear. Herein, we explored the role of Zn²⁺ in the regulation of acute and chronic itch in mice. It is found that intradermal injection (i.d.) of Zn²⁺ dose-dependently induced acute itch and transient receptor potential A1 (TRPA1) participated in Zn²⁺-induced acute itch in mice. Moreover, the pharmacological analysis showed the involvement of histamine, mast cells, opioid receptors, and capsaicin-sensitive C-fibers in Zn²⁺-induced acute itch in mice. Systemic administration of Zn²⁺ chelators, such as N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), pyrithione, and clioquinol were able to attenuate both acute itch and dry skin-induced chronic itch in mice. Quantitative polymerase chain reaction (Q-PCR) analysis showed that the messenger RNA (mRNA) expression levels of zinc transporters (ZIPs and ZnTs) significantly changed in the dorsal root ganglia (DRG) under dry skin-induced chronic itch condition in mice. Activation of extracellular signal-regulated kinase (ERK) pathway was induced in the DRG and skin by the administration of zinc or under dry skin condition, which was inhibited by systemic administration of Zn²⁺ chelators. Finally, we found that the expression of GPR39 (a zinc-sensing GPCR) was significantly upregulated in the dry skin mice model and involved in the pathogenesis of chronic itch. Together, these results indicated that the TRPA1/GPR39/ERK axis mediated the zinc-induced itch and, thus, targeting zinc signaling may be a promising strategy for anti-itch therapy.

Keywords: GPR39; Itch; TRPA1; Zn2+; p-ERK.

Sensory neuron expressed TRPC3 mediates acute and chronic itch.

Yan Liu , Yutong Liu , Nathachit Limjunyawong , Claire Narang , Hanna Jamaldeen, Shimeng Yu , Shivanie Patiram , Hong Nie , Michael J. Caterina , Xinzhong Dong , Lintao Qu

Abstract

Chronic pruritus is a prominent symptom of allergic contact dermatitis(ACD) and represent a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. Yet, the role of TRPC3 in acute and chronic itch is still not well defined. Here, we show that, among mouse trigeminal ganglion (TG) neurons, Trpc3 mRNA is predominantly expressed in nonpeptidergic small diameter TG neurons of mice. Moreover, Trpc3 mRNA signal was present in the majority of presumptively itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch- and pain-like behaviors in naïve mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of contact hypersensitivity (CHS), Trpc3 mRNA expression level and function were upregulated in the TG following CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch via a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.

Keywords: TRPC3, itch, pain, primary sensory neurons, dermatitis

Neuronal pentraxin 2 is required for facilitating excitatory synaptic inputs onto spinal neurons involved in pruriceptive transmission in a model of chronic itch

Kensho Kanehisa, Keisuke Koga, Sho Maejima, Yuto Shiraishi, Konatsu Asai, Miho Shiratori-Hayashi, Mei-Fang Xiao, Hirotaka Sakamoto, Paul F. Worley & Makoto Tsuda

Abstract

An excitatory neuron subset in the spinal dorsal horn (SDH) that expresses gastrin-releasing peptide receptors (GRPR) is critical for pruriceptive transmission. Here, we show that glutamatergic excitatory inputs onto GRPR⁺ neurons are facilitated in mouse models of chronic itch. In these models, neuronal pentraxin 2 (NPTX2), an activity-dependent immediate early gene product, is upregulated in the dorsal root ganglion (DRG) neurons. Electron microscopy reveals that NPTX2 is present at presynaptic terminals connected onto postsynaptic GRPR⁺ neurons. NPTX2-knockout prevents the facilitation of synaptic inputs to GRPR⁺ neurons, and repetitive scratching behavior. DRG-specific NPTX2 expression rescues the impaired behavioral phenotype in NPTX2-knockout mice. Moreover, ectopic expression of a dominant-negative form of NPTX2 in DRG neurons reduces chronic itch-like behavior in mice. Our findings indicate that the upregulation of NPTX2 expression in DRG neurons contributes to the facilitation of glutamatergic inputs onto GRPR⁺ neurons under chronic itch-like conditions, providing a potential therapeutic target.

Presenter: Lee Ki Baek

Molecular and neural basis of pleasant touch sensation

Benlong Liu, Lina Qiao, Kun Liu1, Juan Liu, Tyler J. Piccinni-Ash, Zhou-Feng Chen

DOI: 10.1126/science.abn2479

Abstract

Pleasant touch provides emotional and psychological support that helps mitigate social isolation and stress. However, the underlying mechanisms remain poorly understood. Using a pleasant touch-conditioned place preference (PT-CPP) test, we show that genetic ablation of spinal excitatory interneurons expressing prokineticin receptor 2 (PROKR2), or its ligand PROK2 in sensory neurons, abolishes PT-CPP without impairing pain and itch behaviors in mice. Mutant mice display profound impairments in stress response and prosocial behaviors. Moreover, PROKR2 neurons respond most vigorously to gentle stroking and encode reward value. Collectively, we identify PROK2 as a long-sought neuropeptide that encodes and transmits pleasant touch to spinal PROKR2 neurons. These findings may have important implications for elucidating mechanisms by which pleasant touch deprivation contributes to social avoidance behavior and mental disorders.

Presenter: Hyein Kim

The role of PTEN in primary sensory neurons in processing itch and thermal information in mice

Published in :- Cell reports ; I.F.:- 9.423

Abstract

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Dele- tion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-CreER: PTENflox/flox (PTEN CKO) mice with oral administration of tamoxifen, and CKO mice develop pathological itch and elevated itch responses on exposure to various pruritogens. PTEN deletion leads to ectopic expression of TRPV1 and MrgprA3 in IB4+ non-peptidergic DRG neurons, and the TRPV1 is responsive to capsaicin. Importantly, the elevated itch re- sponses are no longer present in Drg11-CreER: PTENflox/flox: TRPV1flox/flox (PTEN: TRPV1 dCKO) mice. In addi- tion, thermal stimulation is enhanced in PTEN CKO mice but blunted in dCKO mice. PTEN-involved regulation of itch-related gene expression in DRG neurons provides insights for understanding molecular mechanism of itch and thermal sensation at the spinal level.