NPQ/Spexin the Endogenous Ligand for the Galanin Receptor 3

项目摘要 神经肽是一类神经递质或多肽激素，通常激活G蛋白偶联 受体(GPCRs)和参与了大量的生理和心理过程。 大脑和外围设备。我们通过计算确定了一种这样的神经肽，我们称之为NPQ和 被另一个小组称为Spexin，该小组独立地通过不同的 计算方法。来自前ProNPQ/Spexin的多肽在非常局部化的区域发现 大脑，我们已经确定它们参与疼痛反应，以及肾脏和 心血管活动。最近，人们发现NPQ/spexin属于甘丙肽家族，并且 结合并激活Galanin受体3(GALR3)。事实上，正如我们已经验证的那样，NPQ激活了GALR3 在比甘丙素低得多的浓度下激活这一受体。基于这一发现，我们有 假设NPQ而不是甘丙肽是介导GALR3活动的主要神经肽， 通过GALR3，NPQ在急性和慢性疼痛中发挥作用，是AS非 令人上瘾的止痛药。为了验证这些假设，《特定目标1》将使用一系列体外测试来检验 NPQ多肽与GALR3的体外相互作用含有GALR3的CHO细胞 产生的产物将用于检测NPQ和NPQ 53-70的结合和功能活性。Aim 1也将 用膜片钳电生理学检测NPQ阻断脑片GABA释放的能力 腹外侧部PAG是一个NPQ和GALR3水平较高的区域，已知参与痛觉。 SPICAL AIM 2将使用体内试验直接检测NPQ和GALR3在疼痛中的作用 急性热痛和炎症性疼痛以及慢性神经病理性和炎症性疼痛的活体模型。特定的 AIM 3将筛选Hit/Lead生成的组合库以寻找新的小分子激动剂 并开始为开发新的治疗药物进行铅识别的过程。具体目标4将 进一步验证这一目标，通过检查来自 ProNPQ，以及前激素(ProNPQ)本身。这将与GALR3和GALR3的本地化进行比较 甘丙素。为了确定NPQ和GALR3如何参与慢性神经病理性和炎症性疼痛，NPQ 多肽和GALR3免疫反应性以及mRNA水平将在幼稚、脊神经结扎、 和CFA处理的小鼠。GALR3在NPQ行动中的参与将通过使用GALR3进行验证 基因敲除老鼠。这些实验将验证NPQ激活GALR3的观察结果，在体外和在 活体方法研究NPQ/GALR3系统如何参与疼痛，并启动筛查程序以 通过一个新的GPCR靶点确定小分子激动剂作为潜在的非成瘾性止痛药。

Project Summary Neuropeptides are a class of neurotransmitters or peptide hormones that generally activate G-Protein Coupled Receptors (GPCRs) and are involved in a large number of physiological and psychological processes in the brain and in the periphery. One such neuropeptide that we have identified computationally we called NPQ and was called Spexin by another group that independently identified the same molecule by a different computational method. Peptides derived from preproNPQ/Spexin are found in very localized regions of the brain, and we have determined that they are involved in pain response as well as having renal and cardiovascular actions. Very recently, it was discovered that NPQ/spexin is in the galanin peptide family and binds to and activates the Galanin Receptor 3 (GALR3). In fact, as we have validated, NPQ activates GALR3 at a far lower concentration than galanin activates this receptor. Based upon this discovery, we have hypothesized that NPQ, rather than galanin, is the primary neuropeptide that mediates the actions of GALR3, and through GALR3, NPQ has a role in acute and chronic pain and is a potential new target for as non- addicting analgesic. To test these hypotheses, Specific Aim 1 will use a series of in vitro assays to examine the interaction between NPQ peptides and GALR3 in vitro. GALR3 containing CHO cells that we have produced will be used to examine the binding and functional activity of NPQ and NPQ 53-70. Aim 1 will also use patch clamp electrophysiology to examine the ability of NPQ to block GABA release in slices from the ventrolateral PAG, a region with high levels of NPQ and GALR3, and known to be involved in pain perception. Specific Aim 2 will use in vivo assays to directly examine the involvement of NPQ and GALR3 in pain using in vivo models of acute thermal and inflammatory pain, and chronic neuropathic and inflammatory pain. Specific Aim 3 will screen combinatorial libraries for hit/lead generationto identify new small molecule agonists andbegin the process of lead identification for the development of novel therapeutics. Specific Aim 4 will further validate this target by examining the immunohistochemical localization of peptides derived from proNPQ, as well as the prohormone (proNPQ) itself. This will be compared with the localization of GALR3 and galanin. To determine how NPQ and GALR3 are involved in chronic neuropathic and inflammatory pain, NPQ peptides and GALR3 immunoreactivity, as well as mRNA levels, will compared in naïve, spinal nerve ligated, and CFA treated mice. The involvement of GALR3 in the actions of NPQ will be verified by the use of GALR3 knockout mice. These experiments will verify the observation that NPQ activates GALR3, use in vitro and in vivo methods to examine how the NPQ/GALR3 system is involved in pain, and initiate a screening program to identify small molecule agonists as potential non-addicting analgesics through a novel GPCR target.