Defective heme transport in the development of congenital hydrocephalus

项目总结/摘要 先天性脑积水（CH）是一种具有复杂遗传和环境因素的神经衰弱性疾病 输入，其特征为脑脊液（CSF）过度积聚和脑室扩大。新兴 研究表明，破坏神经祖细胞（NPC）增殖/分化，异常的大脑 血管生成和缺氧可能参与CH的发病机制。尽管最近取得了这些进展， 由于缺乏信息模型，我们对疾病病因学的认识存在重大差距。我们发明了一种老鼠 脑积水（PVHH），一种遗传形式的CH引起的 血红素转运蛋白Flvcr 2突变与人类相似，血管中Flvcr 2基因缺失的小鼠， 内皮细胞（EC）发展出异常的脑血管、组织缺氧、NPC分化中断，以及 CH.在初步研究中，我们还发现神经细胞产生并输出大量血红素， 强表达血红素输出蛋白Flvcr 1a，且NPC特异性Flvcr 1a缺失导致脑积水 表型与Flvcr 2突变小鼠相似。总之，这项工作将异常血管生成与破坏 脑发育和CH，并揭示了血红素在这些病理中的核心作用。在这一提议中， 我们研究了血红素，一种在体内携带氧气的重要分子，是如何参与发病机制的。 的PVHH。我们推测，从NPC释放的血红素调节脑血管生成和NPC 微环境，血红素运输中断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中检验这一假设： 在目标1中，我们将确定血红素在大脑中的运输方式。使用创新的血红素报告和新的 蛋白质组学方法，我们将确定血红素的主要细胞来源，血红素的机制， 运输/贩运，以及与脑中血红素相互作用的蛋白质。在目标2中，我们将重点关注血红素如何 调节PVHH中的脑血管生成。我们的初步数据表明，血红素直接调节Dll 4-Notch 信号传导，一种已知抑制血管生成发芽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Dll 4-Notch信号传导， 确定D114-Notch是否是产生PVHH表型的充分和必要条件。在目标3中，我们 确定缺氧和HIF-VEGF信号传导在PVHH中的具体作用。在PVHH模型中，我们观察到 严重缺氧，NPC中缺氧信号传导因子HIF 2a的强烈上调，以及相关的 HIF靶基因VEGF。在脑积水患者中发现缺氧和VEGF增加， CH小鼠模型中的VEGF减少脑积水。在这里，我们将使用基因工程技术阻断HIF-VEGF信号传导。 和药理学方法，然后确定对PVHH表型的影响。总之，这三个目标 将探索血红素在PVHH发展中的新作用，其更广泛的目标是了解和 为其他形式的CH确定新的治疗目标。

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.