Neurotrophins and Neuropeptides in Colon and Bladder Hypersensitivity

描述（由申请人提供）：炎症性肠病患者通常会出现膀胱感觉反应性增加，反映神经源性膀胱过度活动。这种内脏症状重叠涉及背根神经节（DRG）中的神经元交叉激活，其中膀胱传入神经元被敏化。该项目的长期目标是了解结肠和膀胱感觉通路之间复杂的神经元相互作用，并确定调节结肠炎引起的膀胱感觉超敏反应的介质。我们的出版物和初步数据表明，脑源性神经营养因子（BDNF）/TrkB系统在膀胱活动的调节中具有突出的作用。在本更新申请中，我们假设磷脂酶C-γ（PLC？）-钙（Ca 2+）途径是膀胱传入神经元中增加的内源性BDNF/TrkB的独特下游，并且在结肠炎期间通过神经活性化合物的Ca 2+依赖性转录和翻译后调节而在膀胱传入激活中发挥不可或缺的作用。为了解决这一假设，三个相互关联的具体目标，提出了审查的监管机制和目标的PLC？在结肠炎之前和期间，膀胱传入神经元中的Ca 2+通路包括Ca 2 +/钙调素依赖性蛋白激酶（CaMK）II、cAMP反应元件结合蛋白（CREB）、降钙素基因相关肽（CGRP）和小脑蛋白1前体（Cbln 1）。在AIM 1中，我们将描述一系列调节Ca 2+动员的组分（磷脂酶C？，InsP 3R-1、电压门控Ca 2+通道（主要是N型通道Cav2.2）和Ca 2+依赖性神经元激活（CaMK II和CREB）在结肠炎7天和21天的膀胱传入神经元中。这将在分子（mRNA和蛋白质）和功能（细胞内Ca 2+记录和电生理学）水平上完成。在AIM 2中，我们将研究PLC的监管机制。结肠炎时膀胱传入神经元内内源性BDNF激活Ca 2+通路为此，我们将使用一种新开发的但特征良好的BDNF+/-大鼠品系。在AIM 3中，我们将结合联合收割机分子生物学、药理学、神经化学和行为学测试来检查BDNF-Ca 2+轴在结肠炎期间膀胱过度活动中的功能作用。我们将描述Ca 2+依赖性途径如何参与CGRP和Cbln 1表达，以及它们如何调节结肠炎期间膀胱传入神经元的过度活跃和调节膀胱排尿参数。对于以上提出的研究，我们将利用各种体内（转基因动物、鞘内药物递送、行为研究和离体/体外（DRG外植体、分离的DRG神经元培养和转染）系统。三硝基苯磺酸（TNBS）可引起大鼠结肠局部炎症反应。由于几种Ca 2+通路的小分子拮抗剂正在进行治疗其他疼痛症状的临床试验，我们预计目前的系统研究将为形成治疗内脏高敏感性的治疗策略提供见解。

DESCRIPTION (provided by applicant): Patients with inflammatory bowel disease often experience increased sensory responsiveness in the urinary bladder reflecting neurogenic bladder overactivity. This visceral symptom overlap involves neuronal cross-activation in the dorsal root ganglia (DRG) where the bladder afferent neurons are sensitized. The long- term goal of this project has been to understand the complex neuronal interaction between colonic and bladder sensory pathways, and to identify mediators that regulate bladder sensory hypersensitivity as a result of colitis. Our publications and preliminary data have shown that the brain-derived neurotrophic factor (BDNF)/TrkB system has a prominent role in the regulation of bladder activity. In this renewal application, we hypothesize that the phospholipase C-gamma (PLC?)-calcium (Ca2+) pathways are unique downstream of the increased endogenous BDNF/TrkB in bladder afferent neurons, and play an integral role in bladder afferent activation by Ca2+-dependent transcriptional and posttranslational regulation of neuroactive compounds during colitis. To address this hypothesis, three interrelated Specific Aims are proposed to examine the regulatory mechanism and the targets of the PLC?-Ca2+ pathways including Ca2+/calmodulin-dependent protein kinase (CaMK)II, cAMP-response element binding protein (CREB), calcitonin gene-related peptide (CGRP), and cerebellin 1 precursor (Cbln1) in bladder afferent neurons before and during colitis. In AIM 1, we will characterize the expression profiles of a series of components regulating Ca2+ mobilization (phospholipase C?, InsP3R-1, voltage-gated Ca2+ channels predominantly the N-type channel Cav2.2) and Ca2+-dependent neuronal activation (CaMKII and CREB) in bladder afferent neurons at 7 days and 21 days of colitis. This will be done at the molecular (mRNA and protein) and functional (intracellular Ca2+ recording and electrophysiology) levels. In AIM 2, we will examine the regulatory mechanism by which the PLC?-Ca2+ pathways are activated by endogenous BDNF in bladder afferent neurons during colitis. For this purpose, we will use a newly developed yet well-characterized BDNF+/- rat strain. In AIM 3, we will combine molecular biological, pharmacological, neurochemical, and behavioral tests to examine the functional role of the BDNF-Ca2+ axis in bladder hyperactivity during colitis. We will characterize how the Ca2+-dependent pathways are involved in CGRP and Cbln1 expression, and how they regulate bladder afferent neuronal hyperactivity and modulate bladder micturition parameters during colitis. For studies proposed above, we will utilize a variety of in vivo (transgenic animals, intrathecal delivery of drugs, behavioral studies and ex vivo/in vitro (DRG explants, isolated DRG neuron culture and transfection) systems. The localized colonic inflammation will be induced by intracolonic instillation of tri-nitrobenzene sulfonic acid (TNBS) in rat. As several small molecule antagonists of the Ca2+ pathways are under clinical trials in treatment of other pain symptoms, we anticipate that the current systematic studies will provide insights into forming therapeutic strategies in the treatment of visceral hypersensitivity.