Exploring the Role of Aging in Cerebral Ischemic Small Vessel Disease Using Notch3 Mutant Mice

脑小血管病（SVD）是老年人最常见的神经系统疾病之一， 中风和认知障碍的主要原因。在本申请中，我们提出研究 CADASIL（脑常染色体显性遗传性动脉病）小鼠模型中SVD发展中的衰老 伴有皮质下梗死和白质脑病），这是脑SVD最常见的遗传形式。CADASIL 是由显性突变引起的，涉及Notch 3胞外结构域中的半胱氨酸残基， 受体，其特征在于小血管中的进行性变性平滑肌细胞（SMC） 脑穿动脉和血管壁中颗粒状嗜锇物质（GOM）的积聚。 CADASIL患者在三四十岁时开始遭受复发性缺血性中风， 从五六十岁开始出现损伤和痴呆。 基于以下观察结果：1）Notch 3突变功能丧失的患者没有明显的血管病变， 发育异常，2）杂合子患者发生SVD的时间比纯合子患者晚得多 在老年患者中，我们假设老年人对Notch 3信号传导的需求高于老年人， 发展或年轻时。因此，衰老似乎会影响实验结果 与Notch 3信号操纵有关，老年小鼠将是研究分子和 Notch 3信号传导与SVD的细胞机制为了研究这一点，我们建议测试假设 操纵Notch 3信号对小鼠中SVD相关表型的影响将随着年龄的变化而变化。 为了检验我们的假设，我们将利用我们在先前的实验中产生和表征的小鼠， 出版物（Arboleda-Velasquez等人，2008; Arboleda-Velasquez等人，2011年），包括Notch 3淘汰赛 以及两种允许野生型人Notch 3或人Notch 3的诱导型表达的敲入模型 携带C455 R CADASIL突变，其位于Notch 3配体结合结构域（LBD）（Arboleda- Velasquez等人，2002年）。我们对这些模型小鼠的分析表明，C455 R突变损害了细胞内的信号传导。 在体外和体内，导致一个强大的SMC病理，和（像其他已发表的CADASIL突变位于 LBD，C428 S）显示显性负性。 在项目的UH 2阶段，我们计划：1）繁殖携带Notch 3突变的小鼠，并将其年龄延长至24岁 2）通过检查Notch 3转基因确定可行性 在老年小鼠血管中的表达。在项目UH 3阶段，我们提出以下建议 实验目标：1）通过以下方式研究年龄如何影响操纵Notch 3信号对SVD的影响： 监测老年人和年轻人SVD表型的进展和SMC基因表达的变化 野生型和Notch 3突变小鼠;和2）监测诱导型野生型人Notch 3突变小鼠的能力， 转基因以随着小鼠年龄的增长挽救SVD表型。最终，这些研究旨在确定 携带Notch 3突变的老年小鼠是研究SVD的更合适模型， 在小鼠中操纵SVD相关表型上的Notch 3信号传导将随着年龄而改变。

Cerebral small vessel disease (SVD) is one of the most prevalent neurological conditions of old age, and a leading cause of stroke and cognitive impairment. In this application, we propose to investigate the role of aging in the development of SVD in a mouse model of CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), the most common genetic form of cerebral SVD. CADASIL is caused by dominant mutations involving cysteine residues in the extracellular domain of the Notch 3 receptor, and is characterized by progressive degeneration smooth muscle cells (SMCs) in the small penetrating arteries of the brain and accumulation of granular osmiophilic material (GOM) in vessel walls. CADASIL patients start suffering recurrent ischemic strokes in their thirties or forties, and progressive cognitive impairment and dementia starting in their fifties or sixties. Based on the observations that: 1) patients with loss-of-function Notch 3 mutations do not have overt vascular developmental abnormalities, and 2) heterozygous patients develop SVD much later in life than homozygous patients, we hypothesize that the requirements for Notch 3 signaling are higher in old age than during development or at younger ages. Therefore, it seems likely that aging will influence experimental outcomes related to Notch 3 signaling manipulation, and that older mice will be better models to study the molecular and cellular mechanisms linking Notch 3 signaling to SVD. In order to study this, we propose to test the hypothesis that the impact of manipulating Notch 3 signaling on SVD-relevant phenotypes in mice will change with age. In order to test our hypothesis, we will utilize mice that we generated and characterized in previous publications (Arboleda-Velasquez et al., 2008; Arboleda-Velasquez et al., 2011), including a Notch 3 knockout and two knockin models that allow inducible expression of either wild type human Notch 3 or human Notch 3 carrying the C455R CADASIL mutation, which lies in the Notch 3 ligand binding domain (LBD) (Arboleda- Velasquez et al., 2002). Our analyses of these model mice show that the C455R mutation impairs signaling in vitro and in vivo, leads to a robust SMC pathology, and (like other published CADASIL mutations located in the LBD, C428S) displays dominant-negative properties. In the UH2 phase of the project, we plan to: 1) breed mice carrying Notch 3 mutations and age them out to 24 months for use in the UH3 phase of the project and 2) determine feasibility by examining Notch 3 transgene expression in vessels of aged mice. During the UH3 phase of the project, we propose the following experimental goals: 1) to examine how age affects the impact of manipulating Notch 3 signaling on SVD by monitoring the progression of SVD phenotypes and changes in SMC gene expression in aged and young wildtype and Notch 3 mutant mice; and 2) to monitor the ability of an inducible wildtype human Notch 3 transgene to rescue SVD phenotypes as mice age. Ultimately, these studies are designed to establish whether old mice carrying mutations in Notch 3 are a more appropriate model to study SVD and whether the impact of manipulating Notch 3 signaling on SVD-relevant phenotypes in mice will change with age.