Cellular and Molecular Role of CXCR4 signaling in Painful Diabetic Neuropathy

CXCR4 信号传导在疼痛性糖尿病神经病变中的细胞和分子作用

• 批准号: 9816498
• 负责人: Daniela M Menichella
• 金额: $ 37.21万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9816498
• 关键词: Affect; Agonist; Applications Grants; Automobile Driving; Back; Behavior; CXCR4 Receptors; CXCR4 gene; Cells; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Cutaneous; Data; Development; Diabetes Mellitus; Diabetic Neuralgia; Disease; Exhibits; Fiber; Funding; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Gene Cluster; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Grant; High Fat Diet; Hypersensitivity; Knockout Mice; Knowledge; Lead; Ligands; Long-Term Effects; Maps; Mission; Molecular; Mus; Natural History; Neurons; Neuropathy; Nociceptors; Opioid; Pain; Pain management; Pathogenesis; Pharmaceutical Preparations; Physiological; Population; Public Health; Publishing; Regulation; Research; Role; Sensory; Signal Transduction; Spinal Ganglia; Symptoms; Testing; Therapeutic; Time; United States National Institutes of Health; associated symptom; base; designer receptors exclusively activated by designer drugs; diabetic; druggable target; human disease; in vivo; nerve supply; new therapeutic target; non-opioid analgesic; novel; novel therapeutic intervention; novel therapeutics; overexpression; pain symptom; painful neuropathy; receptor; small molecule

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.

抽象疼痛的糖尿病神经病（PDN）是最常见和棘手的符号之一 糖尿病，影响25％的糖尿病患者1-4。 PDN的标志是神经性疼痛和小纤维 退化，表现为背根神经节（DRG）伤害感受器轴突5,6。神经性疼痛是 在没有物理上适当的刺激7-9的情况下，与伤害感受器的高脱位性有关。然而， 导致DRG伤害感受器和PDN神经性疼痛的分子机制是 未知，导致小纤维变性的机制也是如此。我们所知的这个根本差距 代表了开发PDN新型治疗方法进展的关键障碍。确实是当前的 可用于治疗疼痛的药物尚未证明对治疗PDN3,24特别有效 尤其是阿片类药物等药物的作用是高度问题3。因此，这将是出色的疗法 如果可以开发出新的药物来治疗PDN相关疼痛，这是基于清晰的疼痛的意义 了解负责产生与该符号的分子因素 疾病。此外，鉴于G蛋白偶联受体（GPCR）已被证明特别是 与发育特别相关的“可药”目标，GPCR（例如CXCR4受体） PDN的症状将代表开发新的，有效， 非阿片类药物，而不是PDN的添加剂疗法。 我们的赠款提案的主要目的为“ CXCR4信号在疼痛糖尿病中的细胞和分子作用 神经病”是使用一种无​​偏的方法来识别其表达的基因 与PDN的自然史有关。自赠款期开始以来，我们已经启动了这些 研究并已经做出了一些非常有趣的观察结果，暗示了新的治疗靶标 与疾病的发展有关。特别是背根神经的转录组分析 （DRG）从高脂饮食（HFD）喂养的小鼠中取的神经元，该小鼠表达PDN10的主要符号， 展示了一组基因的变化，这些基因都与特定的DRG神经元亚型相关联 表达与MAS相关的G蛋白偶联受体D（MRGPRD）以前与 神经性疼痛11,12。因此，我们设计了这组基因“ MRGPRD相关群集”（MAC）。 该应用的目的是严格验证MRGPRD信号在 PDN中伤害感受器兴奋性，神经性疼痛和小纤维变性的发病机理。 MRGPRD是一个 具有显着构成活性的兴奋性接收器13，表明具有逆的小分子 该受体的激动剂活性在PDN的治疗中可能特别使用。我们的中心假设是 MRGPRD信号传导正在驱动伤害感受器高脱位性，导致神经性疼痛和小纤维变性 在PDN。我们将通过以下特定目的检验该中心假设： AIM1。在PDN的自然史上确定MRGPRD和相关MAC基因的调节 在老鼠中。我们的最初结果确定了与表达DRG人群的MRGPRD相关的MAC基因 将对照小鼠与HFD治疗10周后完全确定的对照小鼠进行了比较。 我们希望在整个时间过程中绘制这些基因表达的详细模式 疾病，在HFD治疗倡议之后开始。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基因可能被靶向以获得最大效果时。此外，特定的定位 向孤立的MRGPRD表达人群的转录组分析将被预测为允许更多细节 分析与PDN发展相关的基因转录变化。 AIM2。确定操纵MRGPRD信号传导对PDN症状的影响。为了 为MRGPRD接收器做一个令人信服的案例，作为治疗PDN相关疼痛的目标，对 证明对这些受体的操作会减少PDN症状。我们将检查 使用几种实验方法的这种可能性。首先，我们将测试激活抑制的影响 Dreadd接收器针对MRGPRD表示DRG神经元的人群。其次，我们将检查 使用条件基因敲除小鼠遗传学和 基于CRISPR的方法。基于CRISPR的方法也可以用于检查后果 操纵任何MAC相关的基因。要检查的终点将包括疼痛的发展 超敏反应行为和皮肤感觉神经的死亡通常与PDN相关。 AIM3。验证MRGPRD是治疗PDN的潜在靶标。因为MRGPRD是一个展示的GPCR 具有很大程度的构成活性，可以预测其过表达，即使没有 激活配体会产生神经元过度兴奋性，从而导致疼痛过敏行为。如果 这种情况是这种情况，MRGPRD逆激动剂应证明在PDN的治疗中具有热有用 相关的疼痛。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 DRG神经元的兴奋性和PDN在体内给药后的症状。 将MRGPRD接收器鉴定为治疗PDN相关疼痛的有望的新目标 是我们先前发表的该问题方法以及当前的融合的结果 无偏的转录组方法，因此构成并极为有趣。