Functional Analysis of Mrgpr Family in itch sensation

项目摘要 我们研究的目的是了解慢性瘙痒的细胞和分子机制， 干扰正常的日常活动，并可能产生严重的临床后果。许多病理条件 可导致慢性瘙痒，如局部皮肤病或更全身性的疾病，如胆汁淤积， 肾衰竭瘙痒也可能是许多治疗药物的副作用。目前的治疗方法包括 抗组胺药在大多数慢性瘙痒症中无效 独立的道路。了解组胺非依赖性瘙痒的一个主要障碍是缺乏知识 非组胺刺激直接激活的受体。背根初级感觉神经元 神经节（DRG）在检测瘙痒中起重要作用。我们的实验室已经鉴定出一种G蛋白偶联的 受体（GPCR）Mrgpr家族，其在DRG中特异性表达并作为瘙痒受体起作用。最近 我们发现小鼠中的MrgprA 1及其人类同源物MRGPRX 4作为新的瘙痒受体发挥功能， 检测胆红素和胆汁酸（BA）。更重要的是，我们证明了MrgprA 1/X4在细胞内起着重要的作用。 介导胆汁淤积性瘙痒（瘙痒）的作用，这是一种由于胆管炎导致胆红素和BA升高的疾病 堵塞。我们的初步数据表明，MrgprA 1是各种治疗药物的新型受体， 介导药物引起的瘙痒副作用。初步数据显示，MrgprA 1也有助于过敏性接触 皮炎（ACD）。此外，以前的研究和我们的初步数据表明，BA也是 尿毒症瘙痒症。在本提案中，我们将采取多学科方法来检验以下假设： MrgprA 1/X4是瘙痒受体并介导由不同疾病状况（包括药物）引起的瘙痒 引起瘙痒副作用、ACD和肾脏疾病，严重影响患者的生活质量， 潜在的机制仍不清楚。在目的I中，我们将测试一些具有瘙痒侧的药物的假设， 作用是通过使用MrgprA 1直接激活刺激感受（瘙痒感测）神经元， 小鼠遗传学、药理学、体外Ca 2+成像、电生理学和体内DRG成像。在Aim II中， 我们将测试感觉神经中的MrgprA 1及其肽激动剂NPFF从附近释放的假设， 皮肤中的肥大细胞有助于使用不同的ACD小鼠模型结合小鼠 遗传学和药理学方法。此外，我们还将确定是否有免疫感受神经元， 在ACD条件下激活，如果是这样，是否MrgprA 1和NPFF介导激活使用体内 DRG GCaMP成像。在目的III中，我们将使用两种尿毒症瘙痒小鼠模型，腺嘌呤诱导的或腺嘌呤诱导的。 尿毒症瘙痒患者血浆注射模型，以检验BA通过激活 MRGPRX 4在小鼠DRG神经元中表达。此外，我们将确定其他瘙痒介质存在于 因肾衰竭接受血液透析的患者血浆。该项目的成果将提供 深入了解瘙痒的关键机制，并为开发新的瘙痒治疗方法打开大门。

Project Summary The goal of our research is to understand the cellular and molecular mechanisms of chronic itch, a disease that interferes with normal daily activity and can have serious clinical consequences. Many pathological conditions can lead to chronic itch such as localized skin diseases or more systemic conditions like cholestasis and kidney failure. Itch can also be a side effect of many therapeutic drugs. Current therapies including antihistamines are ineffective in most chronic itch conditions suggesting the involvement of histamine independent pathways. A major hurdle in understanding histamine-independent itch is the lack of knowledge about the receptors directly activated by non-histaminergic stimuli. Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in detecting itch. Our lab has identified members of a G protein-coupled receptor (GPCR) Mrgpr family that are specifically expressed in DRG and function as itch receptors. Recently we showed that MrgprA1 in mice and its human homologue MRGPRX4 function as novel itch receptors, detecting bilirubin and bile acids (BAs). More importantly, we demonstrated that MrgprA1/X4 play an essential role for mediating cholestatic pruritus (itch), a condition resulting in elevated bilirubin and BAs due to bile duct blockage. Our preliminary data suggest that MrgprA1 are novel receptors for various therapeutic drugs and mediates drug-induced itch side effect. Preliminary data show that MrgprA1 also contributes to allergic contact dermatitis (ACD). Furthermore, previous studies and our preliminary data suggest BAs are also mediators for uremic pruritus. In this proposal, we will take a multidisciplinary approach to test the hypothesis that MrgprA1/X4 are itch receptors and mediate itch caused by different disease conditions including medication caused itch side effect, ACD, and kidney disease which dramatically impact the quality of patient's life and the underlying mechanisms remain unclear. In Aim I, we will test the hypothesis that some of drugs with itch side effect are pruritogens themselves by directly activating pruriceptive (itch sensing) neurons via MrgprA1 using mouse genetics, pharmacology, in vitro Ca2+ imaging, electrophysiology, and in vivo DRG imaging. In Aim II, we will test the hypothesis that MrgprA1 in sensory nerves and its peptide agonist NPFF released from nearby mast cells in the skin contributes to ACD itch using different ACD mouse models combined with mouse genetics and pharmacological approaches. In addition, we will determine whether pruriceptive neurons are activated under ACD conditions and if so whether MrgprA1 and NPFF mediate the activation using in vivo DRG GCaMP imaging. In Aim III, we will employ two uremic itch mouse models, an adenine-induced or a uremic itch patient plasma injection model to test the hypothesis that BAs mediate uremic itch by activating MRGPRX4 expressed in mouse DRG neurons. Moreover, we will identify other itch mediators present in patient plasma who are receiving hemodialysis due to kidney failure. The results of this project will provide insight into key itch mechanisms and open the door for the development of novel itch therapeutics.