Functional Analysis of Mrgpr Family in Itch Sensation

描述（由申请人提供）：我们研究的目标是了解组胺非依赖性瘙痒的细胞和分子机制。背根神经节（DRG）中的初级感觉神经元通过其外周轴突检测皮肤中的瘙痒刺激并通过其中央轴突将信号发送到脊髓，在产生瘙痒中起重要作用。最具特征的瘙痒介质是组胺。然而，抗组胺药物在大多数瘙痒病症中无效，表明涉及组胺非依赖性途径。理解组胺非依赖性瘙痒的主要障碍是缺乏标记DRG中的瘙痒敏感神经元和由组胺以外的瘙痒刺激直接激活的细胞表面受体的分子标记物。最近，我们已经表明，Mrkl 2，一个家族的G蛋白偶联受体特异性地表达在DRG神经元的一个小的子集，作为受体的抗疟疾药物氯喹的功能和氯喹诱导的瘙痒所需的。除氯喹外，Mrlactone和表达Mrgpr的DRG神经元还对其他几种瘙痒诱导化合物产生反应，这表明Mrlactone是通过直接感受这些化合物的新型瘙痒受体，并且表达这些受体的神经元传递瘙痒信号。在本研究中，我们将从分子、遗传、行为和电生理学的角度来研究Mrgpr在瘙痒中的功能和表达Mrgpr的DRG神经元的特性。我们的初步数据表明，Mrabel介导由SLIGRL（蛋白酶激活受体2（PAR 2）的肽激动剂）诱导的瘙痒。这一令人惊讶的结果挑战了传统观念，即PAR 2作为DRG中的瘙痒受体介导蛋白酶诱导的瘙痒。目的I是检验MrP 2作为由瘙痒信号传导中的蛋白酶产生的PAR 2的切割的N-末端的受体起作用的假设。我们最近已经确定了一种小分子化合物，可以特异性地抑制人类和小鼠MrGPR激活的几种瘙痒诱导化合物，包括氯喹和SLIGRL在异源细胞。在目的II中，我们将确定是否与拮抗剂治疗可以阻断氯喹和SLIGRL诱导的神经元和行为反应的小鼠。我们的细胞分析表明，表达Mrgpr的神经元是瘙痒敏感神经元。在目的III中，我们将使用我们新生成的转基因小鼠品系，其中Mrgpr表达DRG神经元被GFP-Cre标记，以研究这些神经元在外周和脊髓中的电生理特性和轴突投射。Mrgpr和Mrgpr表达神经元的功能分析将为瘙痒的关键机制提供深入了解，并为开发新的瘙痒治疗方法打开大门。

DESCRIPTION (provided by applicant): The goal of our research is to understand the cellular and molecular mechanisms of histamine-independent itch. Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in generating itch by detecting itch stimuli through their peripheral axons in the skin and sending the signals to the spinal cord via their central axons. The best characterized itch mediator is histamine. However, anti-histamine drugs are ineffective in most itch conditions suggesting the involvement of histamine- independent pathways. The major hurdle in understanding histamine-independent itch is the dearth of molecular markers that label itch-sensitive neurons in DRG and cell surface receptors directly activated by itch stimuli other than histamine. Recently, we have shown that Mrgprs, a family of G protein-coupled receptors specifically expressed in a small subset of DRG neurons, function as receptors for the anti- malaria drug chloroquine and are required for chloroquine-induced itch. Besides chloroquine, Mrgprs and Mrgpr-expressing DRG neurons also respond to several other itch-inducing compounds suggesting that Mrgprs are novel itch receptors by directly sensing these compounds and that neurons expressing these receptors transduce itch signals. In this proposal, we will take molecular, genetic, behavioral, and electrophysiological approaches to dissect the functions of Mrgprs and properties of Mrgpr-expressing DRG neurons in itch. Our preliminary data show that Mrgprs mediate itch induced by SLIGRL, a peptide agonist for protease-activated receptor 2 (PAR2). This surprising result challenges the traditional notion that PAR2 functions as the itch receptor in DRG to mediate protease-induced itch. Aim I is to test the hypothesis that Mrgprs function as receptors for the cleaved N-terminus of PAR2 generated by proteases in itch signaling. We have recently identified a small molecule compound that can specifically inhibit human and mouse Mrgpr activation by several itch-inducing compounds including chloroquine and SLIGRL in heterologous cells. In Aim II, we will determine whether treatment with the antagonist can block chloroquine- and SLIGRL-induced neuronal and behavioral responses in mice. Our cellular analyses suggest that Mrgpr-expressing neurons are itch-sensitive neurons. In Aim III, we will use our newly generated transgenic mouse lines in which Mrgpr-expressing DRG neurons are labeled by GFP-Cre to study the electrophysiological properties and axonal projections of these neurons in the periphery and the spinal cord. Functional analysis of Mrgprs and Mrgpr-expressing neurons will provide insight into key mechanisms of itch as well as open the door for the development of novel itch therapeutics.