Mechanisms of Endosomal Signaling of Itch

项目摘要 瘙痒是一个复杂的生理过程，包括皮肤感觉神经元对刺激物的检测。 这会激活脊髓中间神经元，并最终激活皮质投射神经元来产生反应。G 蛋白偶联受体(GPCRs)在瘙痒感觉和传递的各个层面上发挥着不可或缺的作用。 尽管传统上认为细胞表面受体是脱敏和内化的 配基结合，新的证据证实了GPCRs从内体发出信号的能力。然而，几乎没有 已知调节GPCRs内体信号的机制，但对此一无所知 内体GPCRs信号在瘙痒中的作用或内体GPCRs是否是可行的治疗靶点 止痒。本研究假设：1.胃泌素释放肽受体(GRPR)和神经激肽1 受体(NK1R)是脊髓瘙痒传递的两个关键受体，可以募集和组装多个受体。 来自内体隔室的促进内体信号转导和中介的蛋白质复合体 脊髓中间神经元长时间的过度兴奋。GRPR和NK1R发出信号的能力 内体导致脊髓瘙痒传递和阻断GRPR和NK1R的内吞抑制物 内体信号可以抑制小鼠的抓挠行为。2.靶向GRPR和GRPR的内体信号传导 使用纳米颗粒的NK1R在抑制瘙痒方面比靶向细胞表面受体更有效。 GRPR和NK1R的内体信号将在模型细胞系、脊髓中间神经元和 调节瘙痒的传播，并在完好的动物身上。将使用药物和遗传方法来 抑制GRPR和NK1R的内体转运。目标1将描述内吞体的重要性 GRPR和NK1R在脊髓中间神经元中的运输和信号转导。的作用 瘙痒的内体信号将通过电生理学和完整的瘙痒行为分析来解决。 动物。目标2将表征GRPR运输内小体和组装多蛋白的能力 导致亚细胞特异性信号事件的复合体。这些信号复合体将使用 具有高时空分辨率的先进生物物理和成像方法。AIM 3将使用高级 化学生物学、纳米工程和纳米颗粒包裹技术用于传递GRPR和NK1R拮抗剂 对于内体，探讨GRPR和NK1R内体信号在瘙痒传递和修复中的重要性。 以确定内体GPCRs是否是可行的治疗靶点。

Project Summary Itch is a complex physiological process that incorporates detection of irritants by sensory neurons in the skin which activate spinal interneurons and ultimately, cortical projection neurons to generate a response. G protein-coupled receptors (GPCRs) play an integral role at each level of itch sensation and transmission. Although conventionally considered cell surface receptors that are desensitized and internalized following ligand binding, new evidence has established the ability of GPCRs to signal from endosomes. However, little is known about the mechanisms that regulate endosomal signaling of GPCRs and nothing is known about the role of endosomal GPCRs signaling in itch or whether endosomal GPCRs are a viable therapeutic target for itch. This proposal hypothesizes that: 1. Gastrin releasing peptide receptor (GRPR) and neurokinin 1 receptor (NK1R), two key receptors in itch transmission in the spinal cord, can recruit and assemble multi- protein complexes from the endosomal compartment that facilitate endosomal signaling and mediate prolonged hyperexcitability of spinal interneurons. That the ability of GRPR and NK1R to signaling from endosomes leads to itch transmission in the spinal cord and endocytic inhibitors that block GRPR and NK1R endosomal signaling can inhibit scratching behavior in mice. 2. Targeting endosomal signaling of GRPR and NK1R using nanoparticles is a more effective strategy in inhibiting itch than targeting cell surface receptors. Endosomal signaling of GRPR and NK1R will be characterized in model cell lines, spinal interneurons that mediate itch transmission, and in intact animals. Pharmaceutical and genetic approaches will be used to inhibit endosomal trafficking of GRPR and NK1R. Aim 1 will characterize the importance of endosomal trafficking and signaling of GRPR and NK1R in spinal interneurons for the transmission of itch. The role of endosomal signaling in itch will be addressed by electrophysiology, and by itch behavioral assays in intact animals. Aim 2 will characterize the ability of GRPR to traffic to endosomes and assemble the multi-protein complexes that result in subcellular specific signaling events. These signaling complexes will be studied using advanced biophysical and imaging approaches with high spatiotemporal resolution. Aim 3 will use advanced chemical biology, nanoengineering and nanoparticle encapsulation to deliver GRPR and NK1R antagonists to endosomes, probing the importance of endosomal signaling of GRPR and NK1R in itch transmission and to determine whether endosomal GPCRs are a viable therapeutic target.