Functional Analysis of Mrgpr Family in Itch Sensation

描述（由申请人提供）：我们的研究目标是了解组胺不依赖型瘙痒的细胞和分子机制。背根神经节（dordorroot ganglia， DRG）的初级感觉神经元通过其位于皮肤的外周轴突检测瘙痒刺激，并通过其中央轴突将信号发送到脊髓，在瘙痒产生中发挥重要作用。最典型的瘙痒介质是组胺。然而，抗组胺药物在大多数瘙痒条件下无效，提示涉及组胺非依赖性途径。了解组胺非依赖性瘙痒的主要障碍是缺乏标记DRG中瘙痒敏感神经元和细胞表面受体的分子标记，这些标记直接被瘙痒刺激而不是组胺激活。最近，我们已经证明Mrgprs是一个G蛋白偶联受体家族，在DRG神经元的一小部分中特异性表达，作为抗疟疾药物氯喹的受体，并且是氯喹引起的瘙痒所必需的。除了氯喹，Mrgprs和表达mrgpr的DRG神经元也对其他几种瘙痒诱导化合物有反应，这表明Mrgprs是一种新的瘙痒受体，可以直接感知这些化合物，表达这些受体的神经元可以传导瘙痒信号。在本研究中，我们将从分子、遗传、行为和电生理等方面分析Mrgprs的功能和表达mrgpr的DRG神经元在瘙痒中的特性。我们的初步数据表明，Mrgprs介导由蛋白酶激活受体2 （PAR2）的肽激动剂SLIGRL诱导的瘙痒。这一令人惊讶的结果挑战了PAR2在DRG中作为瘙痒受体介导蛋白酶诱导的瘙痒的传统观念。目的一是验证Mrgprs作为瘙痒信号中蛋白酶产生的PAR2的裂解n端受体的假设。我们最近在异源细胞中发现了一种小分子化合物，可以特异性地抑制几种瘙痒诱导化合物（包括氯喹和SLIGRL）对人和小鼠Mrgpr的激活。在Aim II中，我们将确定拮抗剂治疗是否可以阻断氯喹和sligrl诱导的小鼠神经元和行为反应。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Aim III中，我们将使用我们新生成的转基因小鼠系，其中表达mrgpr的DRG神经元被GFP-Cre标记，以研究这些神经元在外周和脊髓的电生理特性和轴突投射。Mrgprs和表达mrgpr的神经元的功能分析将为了解瘙痒的关键机制提供洞见，并为开发新的瘙痒疗法打开大门。

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. PUBLIC HEALTH RELEVANCE: Chronic itch interferes with normal daily activity and can have serious clinical consequences. Our studies suggest Mrgprs are novel itch receptors that directly detect itch-inducing compounds and that sensory neurons expressing these receptors transduce itch signals. Therefore, functional analysis of Mrgprs and Mrgpr-expressing neurons will not only provide a mechanistic understanding of itch but also open new avenues to develop novel itch therapeutics.