Characterization of the BMP signaling pathways that produce nociceptor sensitization in Drosophila

 描述(由申请者提供)：正常疼痛通过警告我们潜在的组织损伤来促进健康，但异常疼痛损害了世界各地数百万人的生活质量。据估计，痛苦对美国经济的影响超过6000亿美元。疼痛敏感化是组织损伤后疼痛信号的增加，也通过在愈合过程中防止再次受伤来促进健康。某些类型的异常疼痛，包括慢性疼痛类型，是由疼痛敏感化系统的失调引起的。现有的治疗慢性疼痛的方法是不够的，部分原因是我们最好的止痛药阿片类药物的有害副作用随着使用时间的延长而加剧。迫切需要对异常疼痛进行更好的治疗。我们建议通过开发果蝇模型的强大遗传工具包来揭示止痛药的新靶点。当苍蝇幼虫的表皮被控制剂量的紫外线(UV)破坏时，苍蝇会表现出超敏疼痛，这是一种伤害性敏感化。这意味着受损的苍蝇将对大多数未受损的苍蝇认为无害的热刺激做出反应，并以明确无误的开塞钻滚转的形式做出明示的回避行为。这种损伤诱导的敏化模式以前被用来证明伤害性感受器神经元需要通过骨形态发生蛋白(BMP)途径发出信号才能产生痛觉异常。果蝇体内的许多BMP信号成分与它们的哺乳动物同源基因非常相似。我们的初步结果表明，苍蝇体内已知的三种BMP之一DPP及其受体Put是紫外线损伤后痛觉过敏的伤害性感受器神经元所必需的。目的1将研究其他BMP配体GBB和SCW以及其他BMP受体TKV、SAX、WIT和BABO的必要性。这将通过Gal4/UAS系统将每个基因的RNAi沉默等位基因特异地定位到伤害性感受器神经元来实现。AIM 2将再次使用Gal4/UAS和RNAi工具检查细胞内中继组件MAD、MED、Smox、R-SMAD、Shn和BRK。目的3将通过磁珠辅助细胞分选和RNA测序描述伤害性感受器神经元中BMP信号通路的转录输出。这一过程将识别伤害性感受器神经元中受该通路调控的靶基因。最后，将通过分析紫外线敏化范式中的RNAi突变体来测试这些目标基因表达的必要性。这项拟议的研究将确定BMP信号通路中与痛觉异常有关的成分。由于苍蝇和哺乳动物BMP之间的高度功能保守，这些实验确定的成分代表了治疗人类异常疼痛的新型药物的靶点。

 DESCRIPTION (provided by applicant): Normal pain promotes health by warning us of potential tissue damage, but abnormal pain compromises the quality of life for millions around the world. The impact of pain on the US economy has been estimated at over $600B. Pain sensitization, an increase in pain signaling after tissue damage, also promotes health by preventing re-injury during healing. Some types of abnormal pain, including types of chronic pain, result from dysregulation of the pain sensitization system. Available treatment for chronic pain is inadequate, in part because the deleterious side effects of our best analgesics, the opioids, are accentuated with longer use. Better treatments for abnormal pain are badly needed. We propose to reveal novel targets for pain medications by exploiting the powerful genetic toolkit of the Drosophila model. When the epidermis of the fly larva is damaged by a controlled dose of ultraviolet (UV) light, the fly exhibits allodynia, a type of nociceptive sensitization. Ths means that the damaged fly will react, with a nocifensive avoidance behavior in the form of an unmistakable corkscrew roll, to a thermal stimulus that most undamaged flies find innocuous. This damage induced sensitization paradigm has previously been used to demonstrate that the nociceptor neuron requires signaling by the Bone Morphogenetic Proteins (BMP) pathway in order to produce allodynia. Many BMP signaling components in the fly are very similar to their mammalian orthologs. Our preliminary results show that one of the three known BMPs in the fly, dpp, and its receptor put are necessary in the nociceptor neurons for allodynia following UV damage. Aim 1 will examine the necessity of the other BMP ligands gbb and scw, and the other BMP receptors tkv, sax, wit and babo. This will be accomplished by targeting an RNAi silencing allele of each gene specifically to the nociceptor neurons by the Gal4/UAS system. Aim 2 will examine the intracellular relay components mad, med, smox, R-SMAD, shn and brk, again using Gal4/UAS and RNAi tools. Aim 3 will describe the transcriptional output of the BMP signaling pathway in the nociceptor neuron by magnetic bead- assisted cell sorting and RNA sequencing. This procedure will identify the target genes regulated by the pathway in the nociceptor neurons. Finally, the necessity for the expression of these target genes will be tested by analyzing RNAi mutants in the UV sensitization paradigm. The proposed studies will identify the components of the BMP signaling pathway that are required for allodynia. Because of the high degree of functional conservation between fly and mammalian BMPs, components identified by these experiments represent targets for novel medications for the treatment of abnormal pain in humans.