Notch3 signaling in small-artery-diseases

小血管疾病是导致血管性痴呆的主要原因。我们的长期目标是澄清 控制小动脉结构和功能完整性的信号通路，并了解 这些途径与小血管疾病的发展之间的联系，作为 治疗学的发展。我们提出的研究重点是NotchS信号通路。凹槽 信号是一种进化保守的途径，在发育和成熟过程中起着核心作用。 大多数脊椎动物的器官。我们确定NotchS是CADASIL的致病基因，CADASIL是一种日益增长的 公认的常染色体显性形式的全身性小血管疾病，导致中风和痴呆。我们 以前显示CADASIL患者携带高度刻板印象的错义突变，导致奇怪的 NotchS胞外区的半胱氨酸残基数量；2)NotchS的表达很大程度上 仅限于小动脉和血管平滑肌细胞(VSMC)和3？)小鼠表达原型 以vSMC为靶点的CADASIL突变(R90C)发展为CADASIL动脉病的特征。我们的特定 假说认为，适当水平的NotchS活性对小脑的结构和功能完整性至关重要 通过调节RBP-JK依赖的、Hes/Hey不依赖的通路来调节动脉。这一假设得到了支持 根据我们最近的发现：1)在成年NotchS基因缺失小鼠中，小动脉表现出结构性缺陷和 脑血管反应性有很大缺陷。值得注意的是，NotchS NULL和CADASIL表型非常 NotchS基因缺失小鼠动脉中RBP-JK依赖活性被取消，但其表达水平 HES/嘿基因不受影响。在这里，我们提出三个具体目标，以加深我们对NotchS的理解- 依赖性小血管疾病和NotchS信号转导并解决一个主要的悬而未决的问题： 哪些CADASIL突变会削弱NotchS的活性。我们将构造和分析NotchS函数增益 突变小鼠以确定增加小动脉中NotchS活性的效果(目标1)。我们将决定 NotchS功能缺失和功能获得等位基因的基因表达特征及直接靶基因的筛选 通过对分离的小动脉进行微阵列分析来研究NotchS(目标2)。我们将调查这一措施的影响 Notch3R90C小鼠NotchS体内原型R90C CADASIL突变对野生型活性的影响 空白小鼠作为“拯救”系统和转基因RBP-JK报告小鼠(目标3)。

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 NotchS 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 NotchS 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 NotchS; 2¿) NotchS 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 NotchS 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 NotchS null mice, small arteries exhibit structural defects and cerebrovascular reactivity is strongly defective. Notably, NotchS null and CADASIL phenotypes are very different; 2¿) RBP-JK dependent activity is abolished in arteries of NotchS null mice but expression level of Hes/HEY genes is unaffected. Here we propose three specific aims to further our understanding of NotchS- dependent small-vessel-diseases and NotchS signaling and address a major unresolved issue: the extent to which CADASIL mutations impair NotchS activity. We will construct and analyze NotchS gain-of-function mutant mice to determine effect of increasing NotchS activity in small arteries (Aim #1). We will determine the gene expression signature of loss and gain-of-function alleles of NotchS and identify direct target genes of NotchS by microarray analysis on isolated small arteries (Aim # 2). We will investigate effect of the archetypal R90C CADASIL mutation on NotchS wildtype activity in vivo using our Notch3R90C mice, NotchS null mice as a "rescue" system and transgenic RBP-JK reporter mice (Aim # 3).