Notch3 signaling in small-artery-diseases

描述（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，这是开发治疗方法的先决条件。我们提出的研究重点是Notch3信号通路。Notch信号是一种进化保守的信号通路，在大多数脊椎动物器官的发育和成熟中起着核心作用。我们发现Notch3是CADASIL的致病基因，CADASIL是一种越来越被认为是常染色体显性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Notch3细胞外结构域内的半胱氨酸残基数量为奇数；2¿)Notch3的表达主要局限于小动脉和血管平滑肌细胞（vSMC）， 3¿)表达以vSMC为靶点的典型CADASIL突变（R90C）的小鼠发展出CADASIL动脉病变的特征。我们的具体假设是，适当水平的Notch3活性通过调节RBP-JK依赖、Hes/HEY独立的途径，对小动脉的结构和功能完整性至关重要。我们最近的研究结果支持了这一假设：1)在成年Notch3缺失小鼠中，小动脉表现出结构性缺陷，脑血管反应性严重缺陷。值得注意的是，Notch3 null和CADASIL表型差异很大；2¿)Notch3缺失小鼠动脉中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Notch3依赖性小血管疾病和Notch3信号传导，并解决一个主要的未解决的问题：CADASIL突变在多大程度上损害了Notch3活性。我们将构建和分析Notch3功能获得突变小鼠，以确定增加小动脉中Notch3活性的影响（目的1）。我们将确定Notch3缺失和功能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Notch3的直接靶基因（Aim # 2）。我们将使用我们的Notch3R90C小鼠、Notch3缺失小鼠作为“拯救”系统和转基因RBP-JK报告小鼠（Aim # 3）来研究原型R90C CADASIL突变对体内Notch3野生型活性的影响。

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).