Runx1 Determines Nociceptive Sensory Neuron Phenotype and Is Required for Thermal and Neuropathic Pain

Chih-Li Chen, Daniel C Broom, Yang Liu, Joriene C de Nooij, Zhe Li, Chuan Cen, Omar Abdel Samad, Thomas M Jessell, Clifford J Woolf, Qiufu Ma

Abstract

In mammals, the perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by nociceptive sensory neurons. The molecular mechanisms responsible for the specification of distinct sensory modality are, however, largely unknown. We show here that Runx1, a Runt domain transcription factor, is expressed in most nociceptors during embryonic development but in adult mice, becomes restricted to nociceptors marked by expression of the neurotrophin receptor Ret. In these neurons, Runx1 regulates the expression of many ion channels and receptors, including TRP class thermal receptors, Na+-gated, ATP-gated, and H+-gated channels, the opioid receptor MOR, and Mrgpr class G protein coupled receptors. Runx1 also controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. Thus, Runx1 coordinates the phenotype of a large cohort of nociceptors, a finding with implications for pain therapy.

Epithelia-sensory neuron crosstalk underlies cholestatic itch induced by lysophosphatidylcholine

Yong Chen, Zi-Long Wang, Michele Yeo, Qiao-Juan Zhang, Ana E. López- Romero, Hui-Ping Ding, Xin Zhang, Qian Zeng, Sara L. Morales-Lázaro, Carlene Moore, Ying-Ai Jin, Huang-He Yang, Johannes Morstein, Andrey Bortsov, Marcin Krawczyk, Frank Lammert, Manal Abdelmalek, Anna Mae Diehl, Piotr Milkiewicz, Andreas E. Kremer, Jennifer Y. Zhang, Andrea Nackley, Tony E. Reeves, Mei- Chuan Ko, Ru-Rong Ji, Tamara Rosenbaum, Wolfgang Liedtke

BACKGROUND & AIMS

Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of anti-pruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus.

METHODS

Pruritogenicity of LPC, LPA’s precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC’s pruritogenicity involving keratinocyte-TRPV4 was studied using genetic and pharmacological approaches, cultured keratinocytes, ion channel physiology and structural-computational modeling. Activation of pruriceptor-sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in-vitro and ex-vivo Ca²⁺-imaging assays. Sera from primary biliary cholangitis (PBC) patients were used for measuring the levels of LPC and miR-146a.

RESULTS

LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. 3D-structural modeling, site-directed mutagenesis and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4-activation by LPC induced extracellular release of miR-146a, which activated TRPV1⁺-sensory neurons to cause itch. Both LPC and miR-146a levels were elevated in sera of PBC patients with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates.

CONCLUSIONS

We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron crosstalk, whereby it directly activates skin keratinocyte-TRPV4, which rapidly release miR-146a to activate skin-innervating TRPV1⁺-pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons and central neural pathways supporting pruriception.

Spinal Inhibitory Ptf1a-Derived Neurons Prevent Self-Generated Itch

Augusto Escalante , Rüdiger Klein

Abstract

Chronic itch represents an incapacitating burden on patients suffering from a spectrum of diseases. Despite recent advances in our understanding of the cells and circuits implicated in the processing of itch information, chronic itch often presents itself without an apparent cause. Here, we identify a spinal subpopulation of inhibitory neurons defined by the expression of Ptf1a, involved in gating mechanosensory information self-generated during movement. These neurons receive tactile and motor input and establish presynaptic inhibitory contacts on mechanosensory afferents. Loss of Ptf1a neurons leads to increased hairy skin sensitivity and chronic itch, partially mediated by the classic itch pathway involving gastrin-releasing peptide receptor (GRPR) spinal neurons. Conversely, chemogenetic activation of GRPR neurons elicits itch, which is suppressed by concomitant activation of Ptf1a neurons. These findings shed light on the circuit mechanisms implicated in chronic itch and open novel targets for therapy developments.

Keywords: DREADD receptors; GRPR; Ptf1a; dorsal horn interneurons; intersectional genetics; itch; spinal cord.

Low-Threshold Mechanosensitive VGLUT3-Lineage Sensory Neurons Mediate Spinal Inhibition of Itch by Touch

Kent Sakai, Kristen M. Sanders, Shing-Hong Lin, Darya Pavlenko, Hideki Funahashi, Taisa Lozada, Shuanglin Hao, Chih-Cheng Chen and Tasuku Akiyama

Abstract

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre/NpHR-EYFPmice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.

Significance Statement 

Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.

Central opioid receptors mediate morphine-induced itch and chronic itch via disinhibition

Zilong Wang, Changyu Jiang, Hongyu Yao, Ouyang Chen, Sreya Rahman, Yun Gu, Junli Zhao, Yul Huh, Ru-Rong Ji 

Abstract

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data have also provided mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.

Keywords: inhibitory interneurons; itch; lymphoma; opioid; spinal cord.

TACAN Is an Ion Channel Involved in Sensing Mechanical Pain

Lou Beaulieu-Laroche ¹, Marine Christin ¹, Annmarie Donoghue ², Francina Agosti ³, Noosha Yousefpour ⁴, Hugues Petitjean ¹, Albena Davidova ¹, Craig Stanton ¹, Uzair Khan ⁵, Connor Dietz ⁵, Elise Faure ⁶, Tarheen Fatima ¹, Amanda MacPherson ⁵, Stephanie Mouchbahani-Constance ¹, Daniel G Bisson ⁷, Lisbet Haglund ⁷, Jean A Ouellet ⁸, Laura S Stone ⁹, Jonathan Samson ¹⁰, Mary-Jo Smith ¹¹, Kjetil Ask ¹², Alfredo Ribeiro-da-Silva ⁴, Rikard Blunck ¹³, Kate Poole ¹⁴, Emmanuel Bourinet ³, Reza Sharif-Naeini ¹⁵

Abstract

Mechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying essential physiological functions such as touch and pain sensing, hearing, and proprioception. Although the mechanisms for some of these functions have been identified, the molecules essential to the sense of pain have remained elusive. Here we report identification of TACAN (Tmem120A), an ion channel involved in sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, a nociceptor-specific inducible knockout of TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to painful mechanical stimuli but not to thermal or touch stimuli. We propose that TACAN is an ion channel that contributes to sensing mechanical pain.

Keywords: TACAN; bilayer; ion channel; mechanosensitive; mechanotransduction; nociceptor; pain; patch clamp; pillar.

Allantoin induces pruritus by activating MrgprD in chronic kidney disease 

Yan Yang, Yulin Sun, Donglang Guan, Dan Chen, Dijun Wang, Tongtong Liu, Meixiao Sheng, Tao Jing, Shi Jun, Chan Zhu, Guang Yu, Xinzhong Dong, Zongxiang Tang

Abstract

Chronic kidney disease is a disease with decreased, irreversible renal function. Pruritus is the most common skin symptom in patients with chronic kidney disease, especially in end-stage renal disease (AKA chronic kidney disease-associated pruritus [CKD-aP]); however, the underlying molecular and neural mechanism of the CKD-aP in patients remains obscure. Our data show that the level of allantoin increases in the serum of CKD-aP and CKD model mice. Allantoin could induce scratching behavior in mice and active DRG neurons; the calcium influx and the action potential were significantly reduced in DRG neurons of MrgprD KO or TRPV1 KO mice. U73122, an antagonist of PLC, could also block calcium influx in DRG neurons induced by allantoin. Thus, our results concluded that allantoin plays an important role in CKD-aP, mediated by MrgprD and TrpV1, in CKD patients.

A spinal neural circuitry for converting touch to itch sensation

Sihan Chen, Xiao-Fei Gao, Yuxi Zhou, Ben-Long Liu, Xian-Yu Liu, Yufen Zhang, Devin M Barry, Kun Liu, Yingfu Jiao, Rita Bardoni, Weifeng Yu , Zhou-Feng Chen

Abstract

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2^Cre) receive direct Aβ low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2^Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.

Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors

Devin M Barry ^1 2, Xue-Ting Liu ^1 2 3, Benlong Liu ^1 2, Xian-Yu Liu ^1 2, Fang Gao ^1 2, Xiansi Zeng ^1 2 4, Juan Liu ^1 2, Qianyi Yang ^1 2, Steven Wilhelm ^1 2, Jun Yin ^1 2, Ailin Tao ^1 3, Zhou-Feng Chen ^5 6 7 

Abstract

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission.

Irritant-evoked activation and calcium modulation of the TRPA1 receptor

Jianhua Zhao, John V. Lin King, Candice E. Paulsen, Yifan Cheng & David Julius

Abstract

The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, including small volatile environmental toxicants and endogenous algogenic lipids¹. TRPA1 is also a ‘receptor-operated’ channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies². However, the mechanisms by which TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers remain unknown. Here we use strutural studies and electrophysiology to show that electrophiles act through a two-step process in which modification of a highly reactive cysteine residue (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium-binding pocket that is highly conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.

https://www.nature.com/articles/s41586-020-2480-9.pdf