A Novel Approach to Identify the Mediators in Chronic Itch

描述(申请人提供)：慢性瘙痒是许多与皮肤、免疫和神经系统相关的疾病的症状。慢性瘙痒在很大程度上对传统的抗组胺药物具有耐药性，而且几乎没有有效的治疗方法。为了开发治疗慢性瘙痒的新药物，迫切需要鉴定相关的ITC特异性信号分子。我们最近在脊髓中发现了一小部分瘙痒特异性神经元，它们表达胃泌素释放肽受体(GRPR)，一种瘙痒受体。GRPR+神经元是瘙痒感觉所必需的，但不是痛觉传递所必需的，这表明它们含有无数的瘙痒特异性基因。为了便于在GRPR+神经元中识别新的瘙痒基因，我们设计了一种新的策略，将全基因组GRPR+神经元基因表达谱与功能筛选相结合，包括行为测试和钙成像。比较了有正常GRPR+神经元和不有GRPR+神经元的浅层基因表达谱。这一策略使我们能够寻找在GRPR+神经元中表达的基因，这些基因在功能上是介导非组胺能瘙痒所必需的。将进行三组实验来检验关键假设，即脊髓中存在组胺能和非组胺能瘙痒的不同信号特征。首先，将通过微阵列、qRT-PCR和原位杂交研究GRPR+神经元的全基因组基因表达谱。在GRPR+神经元中表达的候选基因将被确定。其次，将使用siRNA敲除方法评估候选基因在急性和慢性瘙痒中的参与程度，并确定在急性或慢性瘙痒中介导非组胺能瘙痒的关键基因。第三，候选基因和GRPR之间的关系将通过检测该基因的敲除是否会损害鞘内GRP引起的抓挠以及是否会进一步影响GRPR突变小鼠的抓挠行为来确定。此外，候选基因是否参与GRPR依赖的钙信号将在异源系统中使用siRNA敲除和钙成像进行检测。拟议的研究具有很高的翻译性，因为瘙痒介体直接从慢性瘙痒模型中识别出来，并且可能是瘙痒特有的。我们提出的研究将极大地扩大未来药物发现计划可能探索的靶点。

DESCRIPTION (provided by applicant): Chronic itch is a presenting sign in numerous diseases associated with the skin, the immune, and the nervous systems. Chronic itch is largely resistant to conventional antihistamines, and few effective therapies are available. In order to develop novel therapeutics against chronic itch, there is a pressing need for identification of itc-specific signaling molecules involved. We have recently uncovered a small subset of itch-specific neurons which express gastrin-releasing peptide receptor (GRPR), an itch receptor, in the spinal cord. GRPR+ neurons are required for pruritoceptive but not for nociceptive transmission, indicating that they contain a myriad of itch-specific genes. To facilitate the identification of novel itch genes in GRPR+ neurons, we design a novel strategy that combines a genome-wide profiling of gene expression in GRPR+ neurons with functional screening, including behavioral tests and Ca2+ imaging. Gene expression profiles between the superficial laminae with normal GRPR+ neurons and without are compared. This strategy enables us to search for the genes that are expressed in GRPR+ neurons and are functionally required for mediating nonhistaminergic itch. Three sets of experiments will be performed to test the key hypothesis that there are distinct signaling signatures for histaminergic and nonhistaminergic itch in the spinal cord. First, genome-wide profiling of gene expression in GRPR+ neurons by microarray, qRT-PCR and in situ hybridization studies will be performed. The candidate genes expressed in GRPR+ neurons will be identified. Second, the involvement of the candidate genes in acute and chronic itch will be evaluated using a siRNA knockdown approach, and the key genes for mediating nonhistaminergic itch in acute or chronic itch will be identified. Third, the relationship between the candidate genes and GRPR will be determined by examining whether the knockdown of the gene would impair intrathecal GRP-evoked scratching and would further impact scratching behavior of GRPR mutant mice. In addition, whether the candidate genes are involved in GRPR-dependent Ca2+ signaling will be examined in a heterologous system using siRNA knockdown and Ca2+ imaging. The proposed studies are highly translational because the itch mediators are identified directly from chronic itch models, and likely to be itch-specific. Our proposed studies will significantly expand the pool of the targets that may be explored for future drug discovery program.