Functional Analysis of Mrgpr Family in itch sensation

项目摘要 我们研究的目标是了解慢性瘙痒的细胞和分子机制，这种疾病 干扰正常的日常活动，并可能产生严重的临床后果。许多病理情况 可导致慢性瘙痒，如局限性皮肤病或更多全身疾病，如胆汁淤积和 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疾病中无效，这表明组胺参与了这一过程 独立的小路。理解组胺非依赖性瘙痒的一个主要障碍是缺乏知识 关于非组胺能刺激直接激活的受体。背根内的初级感觉神经元 神经节(DRG)在检测瘙痒中起着至关重要的作用。我们的实验室已经鉴定出G蛋白偶联的成员 受体(Gpr)：mrgpr家族，在DRG中特异表达，作为瘙痒受体发挥作用。最近 我们发现在小鼠体内的MRGPRA1及其人类同源物MRGPRX4作为新的瘙痒受体发挥功能， 检测胆红素和胆汁酸(BAs)。更重要的是，我们证明了Mr gprA1/X4在 调节胆汁淤积性瘙痒(瘙痒)的作用，瘙痒是一种由胆管引起的胆红素和BAS升高的情况 堵塞。我们的初步数据表明，MrgprA1是多种治疗药物的新型受体，并且 调节药物引起的瘙痒副作用。初步数据显示，mrgprA1也与过敏接触有关 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。在这项提案中，我们将采用多学科方法来检验以下假设 MRGPRA1/X4是瘙痒受体，介导包括药物在内的不同疾病条件下引起的瘙痒 引起瘙痒副作用、ACD和肾脏疾病，极大地影响患者的生活质量和 潜在的机制仍不清楚。在Aim I中，我们将检验这样一个假设，即一些有瘙痒副作用的药物 其作用是瘙痒物质本身，通过使用 小鼠遗传学、药理学、体外钙离子成像、电生理学和体内DRG成像。在AIM II中， 我们将检验感觉神经中的mrgprA1及其多肽激动剂NPFf从附近释放的假设 不同ACD小鼠模型结合小鼠皮肤肥大细胞对ACD瘙痒的影响 遗传学和药理学方法。此外，我们将确定瘙痒感觉神经元是否 在ACD条件下被激活，如果是这样的话，是否MRgprA1和NPFF在体内介导激活 DRG GCaMP成像。在AIM 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 尿毒症瘙痒患者血浆注射模型验证bas通过激活介导尿毒症瘙痒的假说 MRGPRX4在小鼠背根神经节神经元中表达。此外，我们还将确定在场的其他瘙痒调解人 因肾功能衰竭而接受血液透析的患者血浆。该项目的成果将提供 深入了解关键的瘙痒机制，为新的瘙痒疗法的开发打开大门。

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.