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肽激素，通常激活G蛋白偶联物， 受体（GPCR），并参与了大量的生理和心理过程中， 大脑和周围。我们通过计算确定的一种神经肽被称为NPQ， 被另一个小组称为Spexin，他们用不同的方法独立鉴定了同一种分子， 计算方法衍生自preproNPQ/Spexin的肽存在于NPQ/Spexin的非常局部的区域中。 大脑，我们已经确定，他们参与疼痛反应，以及有肾脏和 心血管作用。最近，发现NPQ/spexin属于甘丙肽家族， 结合并激活甘丙肽受体3（GALR 3）。事实上，正如我们已经证实的，NPQ激活GALR 3 比甘丙肽激活受体的浓度低得多基于这一发现，我们 假设NPQ而不是甘丙肽是介导GALR 3作用的主要神经肽， 通过GALR 3，NPQ在急性和慢性疼痛中发挥作用，并且是作为非靶点的潜在新靶点。 成瘾性止痛药为了验证这些假设，具体目标1将使用一系列体外试验来检查 NPQ肽与GALR 3在体外的相互作用。含有GALR 3的CHO细胞 将使用所产生的样品来检查NPQ和NPQ 53-70的结合和功能活性。Aim 1还将 使用膜片钳电生理学检查NPQ阻断GABA释放的能力， 腹外侧PAG，具有高水平NPQ和GALR 3的区域，并且已知参与疼痛感知。 Specific Aim 2将使用体内测定直接检查NPQ和GALR 3在疼痛中的参与， 急性热和炎性疼痛以及慢性神经性和炎性疼痛的体内模型。具体 AIM 3将筛选组合文库以产生hit/lead，从而鉴定新的小分子激动剂 并开始新疗法开发的先导鉴定过程。具体目标4将 通过检查来源于以下的肽的免疫组织化学定位来进一步验证该靶点： proNPQ，以及激素原（proNPQ）本身。这将与GALR 3的本地化进行比较， 甘丙肽为了确定NPQ和GALR 3如何参与慢性神经性和炎性疼痛，NPQ 肽和GALR 3免疫反应性，以及mRNA水平，将在幼稚的，脊髓神经结扎， 和CFA处理的小鼠。将通过使用GALR 3来验证GALR 3在NPQ行动中的参与 敲除小鼠这些实验将验证NPQ激活GALR 3的观察结果，在体外和体内使用， 研究NPQ/GALR 3系统如何参与疼痛的体内方法，并启动筛选程序， 通过新GPCR靶点鉴定小分子激动剂作为潜在的非成瘾性镇痛剂。

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.