Neurotrophins and Neuropeptides in Colon and Bladder Hypersensitivity

描述(申请人提供)：炎症性肠病患者经常经历膀胱感觉反应性增强，反映神经源性膀胱过度活动。这种内脏症状的重叠涉及到背根节(DRG)的神经元交叉激活，在那里膀胱传入神经元是敏感的。该项目的长期目标是了解结肠和膀胱感觉通路之间的复杂神经元相互作用，并找出调节结肠炎引起的膀胱感觉过敏的介质。我们的文献和初步数据表明，脑源性神经营养因子(BDNF)/TrkB系统在膀胱活动的调节中具有重要作用。在这一新的应用中，我们假设磷脂酶C-γ(PLC？)-钙(Ca~(2+))通路位于内源性BDNF/TrkB增加的膀胱传入神经元的下游，并通过在结肠炎过程中依赖于Ca~(2+)对神经活性物质的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。针对这一假说，提出了三个相互关联的特定目标，以研究结肠炎前和结肠炎期间膀胱传入神经元中PLC-钙通路的调控机制和靶点，包括钙/钙调素依赖的蛋白激酶(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(Cbln1)。在AIM 1中，我们将研究一系列调节钙动员的成分(磷脂酶C？，InsP3R-1，电压门控钙通道，主要是N型通道Cav2.2)和钙依赖性神经元激活(CaMKII和CREB)在结肠炎7天和21天时的表达谱。这将在分子(mRNA和蛋白质)和功能(细胞内钙记录和电生理)水平上完成。在AIM 2中，我们将研究在结肠炎过程中内源性BDNF激活膀胱传入神经元PLC？-钙通路的调节机制。为此，我们将使用一种新开发的但特性良好的BDNF/-鼠品系。在AIM 3中，我们将结合分子生物学、药理学、神经化学和行为测试来研究BDNF-Ca~(2+)轴在结肠炎期间膀胱过度活动中的功能作用。我们将描述钙依赖通路如何参与CGRP和Cbln1的表达，以及它们如何调节结肠炎时膀胱传入神经元的过度活动和调节膀胱排尿参数。对于上述建议的研究，我们将利用各种体内(转基因动物、鞘内给药、行为研究和体外/体外(DRG外植体、分离的DRG神经元培养和转染法)系统)。三硝基苯磺酸(TNBS)结肠内滴注可诱导大鼠结肠局灶性炎症。由于几种钙通道的小分子拮抗剂正在进行临床试验，用于治疗其他疼痛症状，我们预计目前的系统研究将为形成治疗内脏过敏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.