Pain. 2024 Feb 28.  doi: 10.1097/j.pain.0000000000003189.

ATF4 inhibits TRPV4 function and controls itch perception in rodents and nonhuman primate

Man-Xiu Xie ¹, Jun-Hua Rao ², Xiao-Yu Tian ³, Jin-Kun Liu ^3 4, Xiao Li ⁵, Zi-Yi Chen ⁶, Yan Cao ⁵, An-Nan Chen ⁶, Hai-Hua Shu ⁷, Xiao-Long Zhang 

• PMID: 38422489
• DOI: 10.1097/j.pain.0000000000003189

Abstract

Acute and chronic itch are prevalent and incapacitating, yet the neural mechanisms underlying both acute and chronic itch are just starting to be unraveled. Activated transcription factor 4 (ATF4) belongs to the ATF/CREB transcription factor family and primarily participates in the regulation of gene transcription. Our previous study has demonstrated that ATF4 is expressed in sensory neurons. Nevertheless, the role of ATF4 in itch sensation remains poorly understood. Here, we demonstrate that ATF4 plays a significant role in regulating itch sensation. The absence of ATF4 in dorsal root ganglion (DRG) neurons enhances the itch sensitivity of mice. Overexpression of ATF4 in sensory neurons significantly alleviates the acute and chronic pruritus in mice. Furthermore, ATF4 interacts with the transient receptor potential cation channel subfamily V member 4 (TRPV4) and inhibits its function without altering the expression or membrane trafficking of TRPV4 in sensory neurons. In addition, interference with ATF4 increases the itch sensitivity in nonhuman primates and enhances TRPV4 currents in nonhuman primates DRG neurons; ATF4 and TRPV4 also co-expresses in human sensory neurons. Our data demonstrate that ATF4 controls pruritus by regulating TRPV4 signaling through a nontranscriptional mechanism and identifies a potential new strategy for the treatment of pathological pruritus.

Cav3.2 T-type calcium channel mediates acute itch and contributes to chronic itch and inflammation in experimental atopic dermatitis

Ji-Woong Ahn ¹, Song-Ee Kim ², Do-Young Kim ³, Inhye Jeong ², Sohyun Kim ¹, Seungsoo Chung ⁴, Sang Eun Lee ⁵

• PMID: 37863387
• DOI: 10.1016/j.jid.2023.07.029

Abstract

Voltage-gated calcium channels regulate neuronal excitability. The Cav3.2 isoform of the T-type voltage-activated calcium channel is expressed in sensory neurons and is implicated in pain transmission. However, its role in itch remains unclear. Herein, we demonstrated that Cav3.2 is expressed by mechanosensory and peptidergic subsets of mouse dorsal root ganglion (DRG) neurons and colocalized with TRPV1 and receptors for type 2 cytokines. Cav3.2-positive neurons innervate human skin. A deficiency of Cav3.2 reduces histamine, IL-4/IL-13, and thymic stromal lymphopoietin-induced itch in mice. Cav3.2 channels were upregulated in the DRGs of an atopic dermatitis (AD)-like mouse model and mediated neuronal excitability. Genetic knockout of Cav3.2 or T-type calcium channel blocker mibefradil treatment reduced spontaneous and mechanically induced scratching behaviors and skin inflammation in an AD-like mouse model. Substance P and vasoactive intestinal polypeptide levels were increased in the trigeminal ganglia (TG) from AD-like mouse model, and genetic ablation or pharmacological inhibition of Cav3.2 reduced their gene expression. Cav3.2 knockout also attenuated the pathologic changes in ex vivo skin explants co-cultured with TG neurons from AD-induced mice. Our study identifies the role of Cav3.2 in both histaminergic and non-histaminergic acute itch. Cav3.2 channel also contributes to AD-related chronic itch and neuroinflammation.

Keywords: Cav3.2; T-type voltage-activated calcium channel; atopic dermatitis; itch; neuroinflammation; substance P; vasoactive intestinal polypeptide.

Nat Commun. 2023 Apr 29;14(1):2478. doi: 10.1038/s41467-023-38209-x.

Yuanbo Jia ^# 1 2, Jiahui Hu ^# 2 3, Keli An ^1 2, Qiang Zhao ^2 3, Yang Dang ^2 3, Hao Liu ^1 2, Zhao Wei ^1 2, Songmei Geng ⁴, Feng Xu ^5 6

• PMID: 37120459
• PMCID: PMC10148840
• DOI: 10.1038/s41467-023-38209-x

Abstract

Atopic dermatitis (AD) is a chronic skin disease caused by skin immune dyshomeostasis and accompanied by severe pruritus. Although oxidative stress and mechanical scratching can aggravate AD inflammation, treatment targeting scratching is often overlooked, and the efficiency of mechano-chemically synergistic therapy remains unclear. Here, we find that enhanced phosphorylation of focal adhesion kinase (FAK) is associated with scratch-exacerbated AD. We then develop a multifunctional hydrogel dressing that integrates oxidative stress modulation with FAK inhibition to synergistically treat AD. We show that the adhesive, self-healing and antimicrobial hydrogel is suitable for the unique scratching and bacterial environment of AD skin. We demonstrate that it can scavenge intracellular reactive oxygen species and reduce mechanically induced intercellular junction deficiency and inflammation. Furthermore, in mouse AD models with controlled scratching, we find that the hydrogel alleviates AD symptoms, rebuilds the skin barrier, and inhibits inflammation. These results suggest that the hydrogel integrating reactive oxygen species scavenging and FAK inhibition could serve as a promising skin dressing for synergistic AD treatment.

Br J Pharmacol. 2023 Mar 16. doi: 10.1111/bph.16072. Online ahead of print.

Molecular mechanisms of MrgprA3-independent activation of the transient receptor potential ion channels TRPA1 and TRPV1 by chloroquine

Tabea C Fricke ¹, Sebastian Pantke ¹, Bjarne Lüttmann ¹, Frank G Echtermeyer ¹, Christine Herzog ¹, Mirjam J Eberhardt ¹, Andreas Leffler ¹

• PMID: 36928865
• DOI: 10.1111/bph.16072

Abstract

Background and purpose: Itch associates several pathologies and is a common drug-induced side effect. Chloroquine (CQ) was reported to induce itch by activating the Mas-related G protein-coupled receptor MrgprA3 and subsequently TRPA1. In this study we demonstrate that CQ employs at least two MrgprA3-independent mechanisms to activate or sensitize TRPA1 and TRPV1.

Experimental approach: Patch Clamp and calcium-imaging were utilized to examine effects of CQ on TRPA1 and TRPV1 expressed in HEK-293T cells.

Key results: In calcium-imaging, CQ induces a concentration-dependent but MrgprA3-independent activation of TRPA1 and TRPV1. While CQ itself inhibits TRPA1 and TRPV1 in patch clamp recordings, co-application of CQ and UVA-light evokes membrane currents through both channels. This effect is inhibited by the reducing agent dithiothreitol (DTT) and reduced on mutants lacking cysteine residues accounting for reactive oxygen species (ROS)-sensitivity. The combination of CQ and UVA-light triggers an accumulation of intracellular ROS, removes fast inactivation of voltage-gated sodium currents and activates TRPV2. On the other hand, CQ is a weak base and induces intracellular alkalosis. Intracellular alkalosis can activate TRPA1 and TRPV1, and CQ applied at alkaline pH-values indeed activates both channels.

Conclusion and implications: Our data reveal novel pharmacological properties of CQ allowing activation of TRPA1 and TRPV1 via photosensitization as well as intracellular alkalosis. These findings add complexity to the commonly accepted dogma that CQ-induced itch is specifically mediated by MrgprA3 coupling to TRPA1.

Keywords: chloroquine; histamine; itch; oxidative stress; sensory neuron.

Microglia–neuron interactions promote chronic itch via the NLRP3-IL-1β-GRPR axis

Allergy 2023 Mar 6. doi: 10.1111/all.15699. Online ahead of print.

Xueting Liu ¹, Yanmei Wang ¹, Yueling Zeng ¹, De Wang ¹, Yuhuan Wen ¹, Limin Fan ¹, Ying He ¹, Junyan Zhang ¹, Weimin Sun ¹, Yongping Liu ¹, Ailin Tao ¹

• PMID: 36876522
• DOI: 10.1111/all.15699

Abstract

Background: Spinal astrocytes contribute to chronic itch via sensitization of itch-specific neurons expressing gastrin-releasing peptide receptor (GRPR). However, whether microglia-neuron interactions contribute to itch remains unclear. In this study, we aimed to explore how microglia interact with GRPR⁺neurons and promote chronic itch.

Methods: RNA sequencing, quantitative real-time PCR, western blot, immunohistochemistry, RNAscope ISH, pharmacologic and genetic approaches were performed to examine the roles of spinal NLRP3 (The NOD-like receptor family, pyrin-containing domain 3) inflammasome activation and IL-1β-IL1R1 signaling in chronic itch. Grpr-eGFP and Grpr KO mice were used to investigate microglia-GRPR⁺neuron interactions.

Results: We observed NLRP3 inflammasome activation and IL-1β production in spinal microglia under chronic itch conditions. Blockade of microglial activation and the NLRP3/caspase-1/IL-1β axis attenuated chronic itch and neuronal activation. Type 1 IL-1 receptor (IL-1R1) was expressed in GRPR⁺neurons, which are essential for the development of chronic itch. Our studies also find that IL-1β⁺microglia are localized in close proximity to GRPR⁺ neurons. Consistently, intrathecal injection of IL1R1 antagonist or exogenous IL-1β indicate that the IL-1β-IL-1R1 signaling pathway enhanced the activation of GRPR⁺ neurons. Furthermore, our results demonstrate that the microglial NLRP3/caspase-1/IL-1β axis contributes to several different chronic itches triggered by small molecules and protein allergens from the environment and drugs.

Conclusion: Our findings reveal a previously unknown mechanism in which microglia enhances the activation of GRPR⁺ neurons through the NLRP3/caspase-1/IL-1β/IL1R1 axis. These results will provide new insights into the pathophysiology of pruritus and novel therapeutic strategies for patients with chronic itch.

Keywords: IL-1β; NLRP3 inflammasome; chronic itch; gastrin-releasing peptide receptor; microglia.

© 2023 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.

J Med Chem. 2022 Feb 24;65(4):3218-3228. doi: 10.1021/acs.jmedchem.1c01709.Epub 2022 Feb 4.

Thieno[2,3- d]pyrimidine-Based Positive Allosteric Modulators of Human Mas-Related G Protein-Coupled Receptor X1 (MRGPRX1)

Ilyas Berhane, Niyada Hin, Ajit G Thomas, Qian Huang, Chi Zhang, Vijayabhaskar Veeravalli, Ying Wu, Justin Ng, Jesse Alt, Camilo Rojas, Hiroe Hihara ¹, Mika Aoki ¹, Kyoko Yoshizawa ¹, Tomoki Nishioka ¹, Shuichi Suzuki ¹, Shao-Qiu He, Qi Peng, Yun Guan, Xinzhong Dong, Srinivasa N Raja, Barbara S Slusher, Rana Rais, Takashi Tsukamoto

• PMID: 35119273
• DOI: 10.1021/acs.jmedchem.1c01709

Abstract

Mas-related G protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and potential target for the treatment of pain. Positive allosteric modulators (PAMs) of MRGPRX1 have the potential to preferentially activate the receptors at the central terminals of primary sensory neurons and minimize itch side effects caused by peripheral activation. Using a high-throughput screening (HTS) hit, a series of thieno[2,3-d]pyrimidine-based molecules were synthesized and evaluated as human MRGPRX1 PAMs in HEK293 cells stably transfected with human MrgprX1 gene. An iterative process to improve potency and metabolic stability led to the discovery of orally available 6-(tert-butyl)-5-(3,4-dichlorophenyl)-4-(2-(trifluoromethoxy)phenoxy)thieno[2,3-d]pyrimidine (1t), which can be distributed to the spinal cord, the presumed site of action, following oral administration. In a neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI), compound 1t(100 mg/kg, po) reduced behavioral heat hypersensitivity in humanized MRGPRX1 mice, demonstrating the therapeutic potential of MRGPRX1 PAMs in treating neuropathic pain.