Notch3 signaling in small-artery-diseases

描述（由申请人提供）：小血管疾病是血管性痴呆的主要原因。我们的长期目标是阐明控制小动脉结构和功能完整性的信号通路，并了解这些通路与小血管疾病发展之间的联系，作为开发治疗方法的先决条件。我们建议的研究重点是Notch 3信号通路。Notch信号通路是一种进化上保守的通路，在大多数脊椎动物器官的发育和成熟中起着核心作用。我们确定Notch 3是CADASIL的致病基因，CADASIL是一种日益被认可的常染色体显性形式的系统性小血管疾病，可导致中风和痴呆。我们之前的研究表明：1）CADASIL患者携带高度定型的错义突变，导致Notch 3细胞外结构域内的半胱氨酸残基数量为奇数; 2）Notch 3表达主要局限于小动脉和血管平滑肌细胞（vSMC）; 3）表达靶向vSMC的原型CADASIL突变（R90 C）的小鼠出现CADASIL动脉病的特征。我们的具体假设是适当水平的Notch 3活性通过调节RBP-JK依赖性、Hes/HEY非依赖性途径对小动脉的结构和功能完整性至关重要。我们最近的发现支持了这一假设：1）在成年Notch 3基因敲除小鼠中，小动脉表现出结构缺陷，脑血管反应性严重缺陷。值得注意的是，Notch 3 null和CADASIL表型非常不同; 2）在Notch 3 null小鼠的动脉中，RBP-JK依赖性活性被消除，但Hes/HEY基因的表达水平不受影响。在这里，我们提出了三个具体的目标，以进一步了解Notch 3依赖性小血管疾病和Notch 3信号转导，并解决一个主要的悬而未决的问题：CADASIL突变损害Notch 3活性的程度。我们将构建并分析Notch 3功能获得性突变小鼠，以确定增加小动脉中Notch 3活性的效果（目标#1）。我们将确定Notch 3的功能丧失和获得等位基因的基因表达特征，并通过微阵列分析在分离的小动脉上鉴定Notch 3的直接靶基因（目标#2）。我们将使用我们的Notch 3R 90 C小鼠、作为“拯救”系统的Notch 3缺失小鼠和转基因RBP-JK报告小鼠研究原型R90 C CADASIL突变对Notch 3野生型活性的体内影响（目标#3）。

DESCRIPTION (provided by applicant): Small-vessel-diseases are a leading cause of Vascular Dementia. Our long-term goal is to elucidate the signaling pathways that control the structural and functional integrity of small arteries and understand the connections between these pathways and the development of small-vessel-diseases, as a prerequisite to the development of therapeutics. The research we propose is focused on the Notch3 signaling pathway. Notch signaling is an evolutionary conserved pathway that plays a central role in the development and maturation of most vertebrate organs. We identified Notch3 as the causative gene of CADASIL, an increasingly recognized autosomal dominant form of systemic small-vessel-disease causing stroke and dementia. We previously showed that 1¿) CADASIL patients carry highly stereotyped missense mutations leading to an odd number of cysteine residues within the extracellular domain of Notch3; 2¿) Notch3 expression is largely confined to small arteries and vascular Smooth Muscle Cells (vSMC) and 3¿) Mice expressing an archetypal CADASIL mutation (R90C) targeted in vSMC develop features of the CADASIL arteriopathy. Our specific hypothesis is that appropriate level of Notch3 activity is critical for structural and functional integrity of small arteries by modulating an RBP-JK dependent, Hes/HEY independent pathway. That hypothesis is supported by our recent findings: 1¿) In adult Notch3 null mice, small arteries exhibit structural defects and cerebrovascular reactivity is strongly defective. Notably, Notch3 null and CADASIL phenotypes are very different; 2¿) RBP-JK dependent activity is abolished in arteries of Notch3 null mice but expression level of Hes/HEY genes is unaffected. Here we propose three specific aims to further our understanding of Notch3-dependent small-vessel-diseases and Notch3 signaling and address a major unresolved issue: the extent to which CADASIL mutations impair Notch3 activity. We will construct and analyze Notch3 gain-of-function mutant mice to determine effect of increasing Notch3 activity in small arteries (Aim #1). We will determine the gene expression signature of loss and gain-of-function alleles of Notch3 and identify direct target genes of Notch3 by microarray analysis on isolated small arteries (Aim # 2). We will investigate effect of the archetypal R90C CADASIL mutation on Notch3 wildtype activity in vivo using our Notch3R90C mice, Notch3 null mice as a "rescue" system and transgenic RBP-JK reporter mice (Aim # 3).