Journal Club – 2022.09.29

Patch-seq of mouse DRG neurons reveals candidate genes for specific mechanosensory functions

Thibaud Parpaite, Lucie Brosse, Nina Se´ journe´ , Amandine Laur, Yasmine Mechioukhi, Patrick Delmas, Bertrand Coste

Abstract

A variety of mechanosensory neurons are involved in touch, proprioception, and pain. Many molecular components of the mechanotransduction machinery subserving these sensory modalities remain to be discovered. Here, we combine recordings of mechanosensitive (MS) currents in mechanosensory neurons with single-cell RNA sequencing. Transcriptional profiles are mapped onto previously identified sensory neuron types to identify cell-type correlates between datasets. Correlation of current signatures with single-cell transcriptomes provides a one-to-one correspondence between mechanoelectric properties and transcriptomically defined neuronal populations. Moreover, a gene-expression differential comparison provides a set of candidate genes for mechanotransduction complexes. Piezo2 is expectedly found to be enriched in rapidly adapting MS current-expressing neurons, whereas Tmem120a and Tmem150c, thought to mediate slowtype MS currents, are uniformly expressed in all mechanosensory neuron subtypes. Further knockdown experiments disqualify them as mediating MS currents in sensory neurons. This dataset constitutes an open resource to explore further the cell-type-specific determinants of mechanosensory properties.

Presenter: Hye In Kim

Journal Club-2022.09.23

miRNA-203b-3p induces acute and chronic pruritus via 5-HTR2B and TRPV4

Francesco De Logu, Roberto Maglie, Mustafa Titiz, Giulio Poli, Lorenzo Landini, Matilde Marini, Daniel Souza Monteiro de Araujo, Gaetano De Siena, Marco Montini, Daniela Almeida Cabrini, Michel Fleith Otuki, Priscila Lúcia Pawloski, Emiliano Antiga, Tiziano Tuccinardi, João Batista Calixto, Pierangelo Geppetti, Romina Nassini, Eunice André

Abstract

Growing evidence indicates that transient receptor potential (TRP) channels contribute to different forms of pruritus. However, the endogenous mediators that cause itch via TRP channels signaling are poorly understood. Herein, we show that genetic deletion or pharmacological antagonism of TRP vanilloid 4 (TRPV4) attenuated itch in a mouse model of psoriasis induced by topical application of imiquimod. Human psoriatic lesions showed increased expression of several miRNAs, including the miR-203b-3p, which induced a Ca2+ response in rodent dorsal root ganglion neurons and scratching behavior in mice via serotonin receptor 2B (5-HTR2B) activation and the protein kinase C-dependent phosphorylation of TRPV4. Computer simulation revealed that the miR-203b-3p core sequence (GUUAAGAA) that causes 5-HTR2B/TRPV4-dependent itch, targets the extracellular side of 5-HTR2B by interacting with a portion of the receptor pocket consistent with its activation. Overall, we reveal the unconventional pathophysiological role of an extracellular miRNA that can behave as an itch promoter via 5-HTR2B and TRPV4.

Keywords: itch; miRNA; neurons; scratching behavior; skin diseases.

Journal Club-2022.09.16

Scutellarein attenuates atopic dermatitis by selectively inhibiting transient receptor potential vanilloid 3 channels

Yujing Wang1 | Liaoxi Tan1 Shan Jiang1 | Younan Ren1 Khalid N. M. Abdelazeem4 | Zhengyu Cao1

| Kejun Jiao1 | Chu Xue1 | Qinglian Tang1 | | Hao Chen2 | Tarek Mohamed Abd El-Aziz3 |

Ye Yu5 | Fang Zhao1 | Michael X. Zhu6 |
1State Key Laboratory of Natural Medicines and Department of TCM Pharmacology, School of Traditional Chinese Pharmacy, China Pharmaceutical University,

Nanjing, China
2Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing, China
3Zoology Department, Faculty of Science, Minia University, El-Minia, Egypt
4Radiation Biology Research Department, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt
5Department of Basic Medicine, School of Basic Medicine and Clinic Pharmacy, China Pharmaceutical University, Nanjing, China
6Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas, USA

Background and Purpose: Atopic dermatitis (AD) is one of the most common chronic inflammatory cutaneous diseases with unmet clinical needs. As a common ingredient found in several medicinal herbs with efficacy on cutaneous inflammatory diseases, Scutellarein (Scu) has been shown to possess anti-inflammatory and anti-proliferative activities. We aimed to evaluate the therapeutic efficacy of Scu against AD and its underlying molecular mechanism.

Experimental Approach: Efficacy of Scu on AD was evaluated in 2,4-dinitrofluorobenzene (DNFB) and carvacrol-induced dermatitis mouse models. Cytokine mRNA and serum IgE levels were examined using qPCR and ELISA, respec- tively. Voltage clamp recordings were used to measure currents mediated by tran- sient receptor potential (TRP) channels. In silico docking, site-direct mutagenesis, and covalent modification were used to explore the binding pocket of Scu on TRPV3.

Key Results: Subcutaneous administration of Scu efficaciously suppresses DNFB and carvacrol-induced pruritus, epidermal hyperplasia and skin inflammation in wild type mice but has no additional benefit in Trpv3 knockout mice in the carvacrol model. Scu is a potent and selective TRPV3 channel allosteric negative modulator with an appar- ent affinity of 1.18 μM. Molecular docking coupled with site-direct mutagenesis and covalent modification of incorporated cysteine residues demonstrate that Scu targets the cavity formed between the pore helix and transmembrane helix S6. Moreover, Scu attenuates endogenous TRPV3 activity in human keratinocytes and inhibits carvacrol-induced proliferative and proinflammatory responses.

Conclusion and Implications: Collectively, these data demonstrate that Scu amelio- rates carvacrol-induced skin inflammation by directly inhibiting TRPV3, and TRPV3 represents a viable therapeutic target for AD treatment.

KEYWORDS

atopic dermatitis, keratinocytes, scutellarein, TRPV3page2image61528160

Journal Club-2022.09.02

PIEZO1 transduces mechanical itch in mice

Rose Z Hill 1Meaghan C Loud 1Adrienne E Dubin 2Brooke Peet 1Ardem Patapoutian 3

1Howard Hughes Medical Institute, Department of Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA, USA.

2Department of Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA, USA.

3Howard Hughes Medical Institute, Department of Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA, USA. ardem@scripps.edu.

Abstract

Itch triggers scratching, a behavioural defence mechanism that aids in the removal of harmful irritants and parasites1. Chemical itch is triggered by many endogenous and exogenous cues, such as pro-inflammatory histamine, which is released during an allergic reaction1. Mechanical itch can be triggered by light sensations such as wool fibres or a crawling insect2. In contrast to chemical itch pathways, which have been extensively studied, the mechanisms that underlie the transduction of mechanical itch are largely unknown. Here we show that the mechanically activated ion channel PIEZO1 (ref. 3) is selectively expressed by itch-specific sensory neurons and is required for their mechanically activated currents. Loss of PIEZO1 function in peripheral neurons greatly reduces mechanically evoked scratching behaviours and both acute and chronic itch-evoked sensitization. Finally, mice expressing a gain-of-function Piezo1 allele4 exhibit enhanced mechanical itch behaviours. Our studies reveal the polymodal nature of itch sensory neurons and identify a role for PIEZO1 in the sensation of itch.

https://www.nature.com/articles/s41586-022-04860-5#additional-information

Presenter: Hyein Kim

Journal Club – 22.08.26

TRPC Channels Mediate a Muscarinic Receptor-Induced Afterdepolarization in Cerebral Cortex

Hai-Dun Yan, Claudio Villalobos, and Rodrigo Andrade
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48230

Abstract

Activation of muscarinic cholinergic receptors on pyramidal cells of the cerebral cortex induces the appearance of a slow afterdepolarization that can sustain autonomous spiking after a brief excitatory stimulus. Accordingly, this phenomenon has been hypothesized to allow for the transient storage of memory traces in neuronal networks. Here we investigated the molecular basis underlying the muscarinic receptor-induced afterdepolarization using molecular biological and electrophysiological strategies. We find that the ability of muscarinic receptors to induce the inward aftercurrent underlying the slow afterdepolarization is inhibited by expression of a Gq-11 dominant negative and is also markedly reduced in a phospholipase C 1 (PLC1) knock-out mouse. Furthermore, we show, using a genetically encoded biosensor,that activation ofmuscarinic receptorinducesthe breakdown of phosphatidylinositol 4,5-bisphosphatein pyramidal cells. These results indicate that the Gq-11 /PLC1 cascade plays a key role in the ability of muscarinic receptors to signal the inward aftercurrent. We have shown previously that the muscarinic afterdepolarization is mediated by a calcium-activated nonselective cation current, suggestingthe possible involvement of TRPC channels.Wefindthat expression of a TRPC dominant negative inhibits, and overexpression of wild-type TRPC5 or TRPC6 enhances, the amplitude of the muscarinic receptor-induced inward aftercurrent. Furthermore, we find that coexpression of TRPC5 and T-type calcium channels is sufficient to reconstitute a muscarinic receptor-activated inward aftercurrent in human embryonic kidney HEK-293 cells. These results indicate that TRPC channels mediate the muscarinic receptor-induced slow afterdepolarization seen in pyramidal cells of the cerebral cortex and suggest a possible role for TRPC channels in mnemonic processes.

Journal Club – 22.08.19

Modified Proteinase-Activated Receptor-1 and -2 Derived Peptides Inhibit Proteinase-Activated Receptor-2 Activation by Trypsin

BAHJAT AL-ANI, MAHMOUD SAIFEDDINE, SURANGA J. WIJESURIYA and MORLEY D. HOLLENBERG

ABSTRACT
Trypsin activates proteinase-activated receptor-2 (PAR2) by a mechanism that involves the release of a tethered receptor activating sequence. We have identified two peptides, FSLLRY-NH2 (FSY-NH2) and LSIGRL-NH2 (LS-NH2) that block the ability of trypsin to activate PAR2 either in PAR2-expressing Kirsten virus-transformed kidney (KNRK) cell lines or in a rat aorta ring preparation. The reverse PAR2 peptide, LRGILS-NH2(LRG-NH2) did not do so and FSY-NH2 failed to block thrombin activation of PAR1 in the aorta ring or in PAR1-expressing human embryonic kidney cells. Half-maximal inhibition (IC50) by FSY-NH2 and LS-NH2 of the activation of PAR2 by trypsin in a PAR2 KNRK calcium-signaling assay was observed at about 50 and 200 M, respectively. In contrast, the activation of PAR2 by the PAR2-activating peptide, SLIGRL-NH2 (SL-NH2) was not inhibited by FSY-NH2, LS-NH2, or LRG-NH2. In a casein proteolysis assay, neither FSY-NH2 nor LS-NH2 inhibited the proteolytic action of trypsin on its substrate. In addition, FSY-NH2
and LS-NH2 were unable to prevent trypsin from hydrolyzing a 20-amino acid peptide, GPNSKGR/SLIGRLDTPYGGC representing the trypsin cleavage/activation site of rat PAR2. Similarly, FSY-NH2 and LS-NH2 failed to block the ability of trypsin to release the PAR2 N-terminal epitope that is cleaved from the receptor upon proteolytic activation of receptor-expressing KNRK cells. We conclude that the peptides FSY-NH2 and LSNH2 block the ability of trypsin to activate PAR2 by a mechanism that does not involve a simple inhibition of trypsin proteolytic activity, but possibly by interacting with a tethered ligand receptor-docking site.

Presenter: Gi Baek Lee

2022.08.12

IL-31 levels correlate with pruritus in patients with cholestatic and metabolic liver diseases and is farnesoid X receptor responsive in NASH

Jun Xu Ya Wang Mina Khoshdeli Matt Peach Jen-Chieh Chuang Julie Lin Wen-Wei Tsai Sangeetha Mahadevan Wesley Minto Lauri Diehl Ruchi Gupta Michael Trauner Keyur Patel Mazen Noureddin Kris V Kowdley Aliya Gulamhusein Christopher L Bowlus Ryan S Huss Robert P Myers Chuhan Chung Andrew N Billin 

Abstract

Background and aims: Pruritus is associated with multiple liver diseases, particularly those with cholestasis, but the mechanism remains incompletely understood. Our aim was to evaluate serum IL-31 as a putative biomarker of pruritus in clinical trials of an farnesoid X receptor (FXR) agonist, cilofexor, in patients with NASH, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC).

Approach and results: Serum IL-31 was measured in clinical studies of cilofexor in NASH, PSC, and PBC. In patients with PSC or PBC, baseline IL-31 was elevated compared to patients with NASH and healthy volunteers (HVs). IL-31 correlated with serum bile acids among patients with NASH, PBC, and PSC. Baseline IL-31 levels in PSC and PBC were positively correlated with Visual Analog Scale for pruritus and 5-D itch scores. In patients with NASH, cilofexor dose-dependently increased IL-31 from Week (W)1 to W24. In patients with NASH receiving cilofexor 100 mg, IL-31 was higher in those with Grade 2-3 pruritus adverse events (AEs) than those with Grade 0-1 pruritus AEs. IL-31 weakly correlated with C4 at baseline in patients with NASH, and among those receiving cilofexor 100 mg, changes in IL-31 and C4 from baseline to W24 were negatively correlated. IL-31 messenger RNA (mRNA) was elevated in hepatocytes from patients with PSC and NASH compared to HVs. In a humanized liver murine model, obeticholic acid increased IL-31 mRNA expression in human hepatocytes and serum levels of human IL-31.

Conclusions: IL-31 levels correlate with pruritus in patients with cholestatic disease and NASH, with FXR agonist therapy resulting in higher serum levels in the latter group. IL-31 appears to derive in part from increased hepatocyte expression. These findings have therapeutic implications for patients with liver disease and pruritus.

Journal Club – 22.07.15

GPR15L is an epithelial inflammation-derived pruritogen

Pang-Yen Tseng and Mark A. Hoon*

Molecular Genetics Section, National Institute of Dental and Craniofacial Research/ NIH, 35 Convent Drive, Bethesda, MD 20892, USA.
*Corresponding author. Email: mark.hoon@nih.gov

Itch is an unpleasant sensation that often accompanies chronic dermatological conditions. Although many of the itch receptors and the neural pathways underlying this sensation are known, the identity of endogenous ligands is still not fully appreciated. Using an unbiased bioinformatic approach, we identified GPR15L as a candidate pruritogen whose expression is robustly up-regulated in psoriasis and atopic dermatitis. Although GPR15L was previously shown to be a cognate ligand of the receptor GPR15, expressed in dermal T cells, here we show that it also contributes to pruritogenesis by activating Mas-related G protein–coupled receptors (MRGPRs). GPR15L can selectively stimulate mouse dorsal root ganglion neurons that express Mrgpra3 and evokes intense itch re- sponses. GPR15L causes mast cell degranulation through stimulation of MRGPRX2 and Mrgprb2. Genetic dis- ruption of GPR15L expression attenuates scratch responses in a mouse model of psoriasis. Our study reveals unrecognized features of GRP15L, showing that it is a potent itch-inducing agent.

Journal Club – 22.07.08

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

Journal Club – 22.07.01

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