Defective heme transport in the development of congenital hydrocephalus

项目摘要/摘要 先天性脑积水(CH)是一种具有复杂遗传和环境因素的衰弱神经系统疾病 输入，特征是脑脊液(CSF)过度积累和脑室扩大。新兴 研究表明，神经前体细胞(NPC)增殖/分化紊乱，大脑异常 血管生成和缺氧可能参与了脑出血的发病机制。尽管最近取得了这些进展，但仍然存在 由于缺乏信息模型，我们对疾病病因学的知识存在严重差距。我们开发了一种小鼠 增生性血管病变和遗传性先天性脑积水模型的建立 通过血红素转运蛋白Flvcr2的突变。与人类相似，血管中Flvcr2基因缺失的小鼠 内皮细胞(ECs)出现异常脑血管、组织缺氧、鼻咽癌分化受阻以及 陈氏在初步研究中，我们还发现神经细胞产生和输出大量的血红素，即npc。 强烈表达血红素输出蛋白Flvcr1a，鼻咽癌特异性的Flvcr1a缺失会导致脑积水 表型与Flvcr2突变小鼠相似。总而言之，这项工作将异常血管生成与中断联系在一起 大脑发育和CH，并揭示了血红素在这些病理中的核心作用。在这份提案中， 我们研究了血红素，一种在体内运送氧气的重要分子，是如何参与发病机制的。 PVHH。我们假设从鼻咽癌释放的血红素调节脑血管生成和鼻咽癌 微环境，并且扰乱了血红素的运输导致脑血管形成减少，组织 缺氧和下游脑积水。我们将在三个不同但相互关联的目标上测试这一假设： 在目标1中，我们将确定血红素是如何在大脑中被交易的。使用创新的血红素记者和新的 蛋白质组学方法，我们将确定血红素的主要细胞来源，机制 运输/贩运，以及大脑中与血红素相互作用的蛋白质。在目标2中，我们将重点介绍亚铁血红素是如何 调节PVHH的脑血管生成。我们的初步数据表明，血红素直接调节DLL4-Notch 信号，一种已知的抑制血管新生萌发和减少血管生长的途径。使用药理学 治疗和基因扰动，我们将在体外和体内调节血红素和DLL4-Notch信号，以及 确定DLL4-Notch对于产生PVHH表型是否充分和必要。在《目标3》中，我们将 确定低氧和HIF-VEGF信号在PVHH中的具体作用。在我们的PVHH模型中，我们观察到 严重低氧，NPC中低氧信号因子HIF2A的强烈上调，以及与之相关的 HIF靶基因，血管内皮生长因子。在患有脑积水的人类中发现缺氧和血管内皮生长因子升高，并靶向 脑出血小鼠模型中的血管内皮生长因子可减少脑积水。在这里，我们将使用Genetic来阻止HIF-VEGF信号 和药理学方法，然后确定对PVHH表型的影响。这三个目标加在一起， 将探索血红素在PVHH发展中的新作用，更广泛的目标是了解和 为其他形式的CH确定新的治疗靶点。

PROJECT SUMMARY/ABSTRACT Congenital hydrocephalus (CH) is a debilitating neurologic condition with complex genetic and environmental inputs, characterized by excessive accumulation of cerebro-spinal fluid (CSF) and enlarged ventricles. Emerging research suggests that disrupted neuroprogenitor cell (NPC) proliferation/differentiation, abnormal brain angiogenesis and hypoxia may be involved in CH pathogenesis. Despite these recent advances there remain critical gaps in our knowledge of disease etiology due to the lack of informative models. We developed a mouse model for Proliferative Vasculopathy and Hydranencephaly Hydrocephalus (PVHH), a genetic form of CH caused by mutation in the heme transporter, Flvcr2. Similar to humans, mice with genetic deletion of Flvcr2 in vascular endothelial cells (ECs) develop abnormal brain blood vessels, tissue hypoxia, disrupted NPC differentiation, and CH. In preliminary studies, we also found that neural cells produce and export large amounts of heme, that NPCs strongly express the heme exporter, Flvcr1a, and that NPC-specific deletion of Flvcr1a causes a hydrocephalus phenotype similar to Flvcr2 mutant mice. Together, this work links abnormal angiogenesis to disrupted brain development and CH, and uncovers a central role for heme in these pathologies. In this proposal, we investigate how heme, a molecule important for carrying oxygen in the body, is involved in the pathogenesis of PVHH. We hypothesize that heme released from NPCs regulates brain angiogenesis and the NPC micro-environment, and that disrupted heme transport causes reduced brain vascularization, tissue hypoxia and downstream hydrocephalus. We will test this hypothesis in three distinct but interrelated aims: In Aim 1, we will determine how heme is trafficked in the brain. Using innovative heme reporters and new proteomics approaches, we will determine the primary cellular source of heme, mechanisms of heme transport/trafficking, and the proteins interacting with heme in the brain. In Aim 2, we will focus on how heme regulates brain angiogenesis in PVHH. Our preliminary data indicate that heme directly regulates Dll4-Notch signaling, a pathway known to suppress angiogenic sprouting and reduce vascular growth. Using pharmacologic treatments and gene perturbations, we will modulate heme and Dll4-Notch signaling in vitro and in vivo, and determine whether Dll4-Notch is sufficient and necessary to produce the PVHH phenotype. In Aim 3, we will determine the specific role of hypoxia and HIF-VEGF signaling in PVHH. In our PVHH models, we observe severe hypoxia, strong upregulation of hypoxic signaling factor HIF2a in NPCs, and associated increase in the HIF target gene, VEGF. Hypoxia and increased VEGF is found in humans with hydrocephalus, and targeting VEGF in mouse models of CH reduces hydrocephalus. Here, we will block HIF-VEGF signaling using genetic and pharmacologic approaches, then determine the impact on the PVHH phenotype. Together, these three aims will explore a new role for heme in the development of PVHH, with the broader goal of understanding and identifying new treatment targets for other forms of CH.