Cellular and Molecular Role of CXCR4 signaling in Painful Diabetic Neuropathy

摘要痛性糖尿病神经病变(PDN)是糖尿病最常见、最难治的症状之一。 糖尿病，影响25%的糖尿病患者1-4。PDN的特点是神经病理性疼痛和细小纤维。 变性，表现为背根神经节(DRG)伤害性轴突5，6。神经病理性疼痛 在没有生理上合适的刺激的情况下与伤害性感受器的过度兴奋有关7-9。然而， 导致PDN背根节伤害性感受器过度兴奋和神经病理性疼痛的分子机制是 尚不清楚，导致小纤维退化的机制也是如此。我们知识中的这一根本鸿沟 是发展PDN新的治疗方法的关键障碍。确实是当前的 可用于治疗疼痛的药物并未被证明对治疗PDN3、24和长期有效 特别是阿片类药物的影响是非常有问题的3。因此，它将具有很好的治疗作用 如果能够开发出治疗PDN相关性疼痛的新药，那么基于明确的 了解导致与高血压相关的症状的分子因素 疾病。此外，鉴于G蛋白偶联受体(GPCRs)已被证明是特别的 “可用药”靶标，与发育相关的GPCRs(如CXCR4受体) PDN症状的研究将是开发新的、有效的、 非阿片类药物，也不是PDN的辅助治疗药物。 我们拨款提案的一个主要目的是“CXCR4信号在痛性糖尿病中的细胞和分子作用” 神经病“是使用一种无偏见的方法来识别其表达特定的基因 与PDN的自然历史有关。自赠款资助期开始以来，我们已经启动了 已经进行了一些非常有趣的观察，提出了一些新的治疗靶点 与疾病的发展有关。尤其是背根神经节的转录分析 从高脂饮食(HFD)喂养的小鼠中提取的(DRG)神经元表达PDN10的主要症状， 显示一组基因的变化，这些基因都与特定的DRG神经元亚型相关 表达与Mas相关的G蛋白偶联受体D(MRgprD)，该受体先前被认为与 神经病理性疼痛11，12。因此，我们将这组基因命名为“MRGPRD相关簇”(MAC)。 本应用程序的目标是严格验证MRGPrD信号在 PDN伤害性感受器兴奋性、神经病理性疼痛和小纤维变性的发病机制。先生是一位 具有显著的结构性活性的兴奋性受体13，表明具有相反作用的小分子 该受体的激动剂活性可能在PDN的治疗中特别有用。我们的中心假设是 MrgprD信号驱动伤害性感受器过度兴奋，导致神经病理性疼痛和小纤维变性 在PDN中。我们将通过以下具体目标来检验这一中心假设： AIM1.确定pDN自然发展过程中mrgprD及相关MAC基因的调控 在老鼠身上。我们的初步结果是，确定了与表达DRG群体的MRgprD相关的MAC基因， 将对照组小鼠与HFD治疗10周后完全建立PDN的小鼠进行比较。 我们希望绘制出这些基因在整个过程中表达的详细模式 疾病，在HFD治疗开始后立即开始。这些数据很重要，因为它们会准确地告诉我们 当mrgprD和其他MAC基因可能成为发挥最大作用的靶点时。此外，针对 对分离的MRgprD表达群体的转录分析将被预测为允许更详细的 分析与PDN发生发展相关的基因转录变化。 AIM2.确定操纵mrgprD信号对PDN症状的影响。为了 提出一个令人信服的理由将mrgprD受体作为治疗PDN相关疼痛的靶点，这是至关重要的 证明操纵这些受体可以减少PDN的症状。我们将研究 这种可能性使用了几种实验方法。首先，我们将测试激活抑制的效果 DREADD受体靶向于DRG神经元中表达MRgprD的群体。第二，我们将研究 利用条件性基因敲除小鼠基因表达降低mRgprD受体表达的效果 基于CRISPR的方法。基于CRISPR的方法也可以用来检查后果 操纵任何与MAC相关的基因。检查的终点将包括疼痛的发展 典型的与PDN相关的过敏行为和皮肤感觉神经的消退。 AIM3.验证MRGPrD作为治疗PDN的潜在靶点。因为mrgprd是一个gpr，展示了 一个显著程度的结构性活动，预测其过度表达，即使在没有 激活配体，会产生神经元的超兴奋性，从而导致疼痛过敏行为。如果 在这种情况下，mrgprd反向激动剂应该被证明在治疗PDN方面是有效的。 伴随的疼痛。我们将通过测试具有这些特征的小分子来检查这种可能性 在体给药后DRG神经元的兴奋性和PDN的症状。 MRGPrD受体作为治疗PDN相关疼痛的新靶点的研究 是我们以前发表的解决这个问题的方法与我们目前的 不偏不倚的转录方法，因此构成了非常耐人寻味的铅。

ABSTRACT Painful diabetic neuropathy (PDN) is one of the most common and intractable symptoms of diabetes, affecting 25% of diabetic patients1-4. The hallmarks of PDN are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (DRG) nociceptor axons5,6. Neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli7-9. However, the molecular mechanisms leading to the hyper-excitability of DRG nociceptors and neuropathic pain in PDN are unknown, as are the mechanisms leading to small fiber degeneration. This fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for PDN. Indeed current drugs available for treating pain have not proved to be particularly effective for treating PDN3,24 and the long term effects of drugs like opioids, in particular, are highly problematic3. It would therefore be of great therapeutic significance if novel drugs could be developed for treating PDN associated pain that are based on a clear understanding of the molecular factors that are responsible for producing the symptoms associated with the disease. Moreover, given the fact that G-protein coupled receptors (GPCRs) have been shown to be particularly “druggable” targets, GPCRs (such as the CXCR4 receptor) that are specifically associated with the development of the symptoms of PDN would represent particularly interesting targets for the development of new, effective, non-opioid, and not additive therapeutics for PDN. A major aim of our grant proposal entitled “Cellular and Molecular Role of CXCR4 signaling in Painful Diabetic Neuropathy” was to use an unbiased approach for identifying genes whose expression was specifically associated with the natural history of PDN. Since the start of the grant funding period we have initiated these studies and have already made some extremely interesting observations suggesting novel therapeutic targets associated with the development of the disease. In particular transcriptomal analysis of dorsal root ganglion (DRG) neurons taken from high fat diet (HFD) fed mice which express the major symptoms of PDN10, demonstrate changes in a set of genes which all cluster in association with a specific DRG neuronal subtype expressing the Mas-related G protein-coupled receptor D (MrgprD) that has previously been implicated in neuropathic pain11,12. We have therefore, designated this set of genes the “MrgprD Associated Cluster” (MAC). The objective of this application is to rigorously validate the functional role of MrgprD signaling in the pathogenesis of nociceptor excitability, neuropathic pain and small fiber degeneration in PDN. MrgprD is an excitatory receptor that has significant constitutive activity13, suggesting that small molecules that have inverse agonist activity at this receptor might be of particular use in the treatment of PDN. Our central hypothesis is that MrgprD signaling is driving nociceptor hyper-excitability leading to neuropathic pain and small fiber degeneration in PDN. We will test this central hypothesis through the following specific aims: AIM1. Determine the regulation of MrgprD and associated MAC genes during the natural history of PDN in mice. Our initial results, which identified MAC genes associated with the MrgprD expressing DRG population, compared control mice with those in which PDN was completely established after 10 weeks of HFD treatment. We wish to map the detailed pattern of the expression of these genes over the complete time course of the disease, starting right after the initiation of HFD treatment. These data are important as they will tell us precisely when MrgprD and other MAC genes might be targeted for maximal effect. Moreover, specific targeting of transcriptomal analysis to the isolated MrgprD expressing population would be predicted to allow more detailed analysis of changes in gene transcription associated with the development of PDN. AIM2. Determine of the effects of manipulating MrgprD signaling on the symptoms of PDN. In order to make a compelling case for MrgprD receptors as a target for the treatment of PDN associated pain, it is vital to demonstrate that manipulation of these receptors produces a reduction in the symptoms of PDN. We will examine this possibility using several experimental approaches. First, we will test the effects of activating inhibitory DREADD receptors targeted to the MrgprD expressing population of DRG neurons. Secondly, we will examine the effects of reducing the expression of MrgprD receptors using conditional knockout mouse genetics and CRISPR based approaches. The CRISPR based approach can also be used to examine the consequences manipulating any of the MAC associated genes. Endpoints to be examined will include the development of pain hypersensitivity behavior and the dying back of cutaneous sensory innervation typically associated with PDN. AIM3. Validate MrgprD as potential target for the treatment of PDN. Because MrgprD is a GPCR that exhibits a significant degree of constitutive activity, it is predicted that its overexpression, even in the absence of an activating ligand, would produce neuronal hyperexcitability that would lead to pain hypersensitivity behavior. If this is the case then an MrgprD inverse agonist should prove to be therapeutically useful in the treatment of PDN associated pain. We will examine this possibility by testing small molecules with these characteristics on the excitability of DRG neurons and the symptoms of PDN following their administration in vivo. The identification of MrgprD receptors as a promising novel target for the treatment PDN associated pain is the result of the confluence of our previously published approach to this problem together with our current unbiased transcriptomal approach and therefore constitutes and extremely intriguing lead.