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

脑部小血管疾病(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。在本应用程序中，我们建议调查 衰老在CADASIL(常染色体显性遗传性脑动脉病)小鼠SVD发展中的作用 大脑皮质下梗塞和白质脑病)，这是脑SVD最常见的遗传形式。CADASIL 是由Notch 3胞外区半胱氨酸残基的显性突变引起的 受体，以小鼠进行性变性的平滑肌细胞为特征。 穿透大脑动脉并在血管壁上堆积颗粒嗜奥斯性物质(GOM)。 CADASIL患者在30多岁或40多岁开始遭受反复的缺血性中风，并进行认知 从50多岁或60多岁开始出现损害和痴呆。 基于以下观察：1)功能丧失Notch 3突变的患者没有明显的血管 发育异常，2)杂合子患者比纯合子患者患SVD的时间要晚得多 患者，我们假设对Notch 3信号的需求在老年比在 在发育或更年轻的时候。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信号与SVD相关的细胞机制。为了研究这一点，我们建议检验这一假设 操纵Notch 3信号对小鼠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，我们将使用我们在前面的 出版物(Arboleda-Velasquez等人，2008年；Arboleda-Velasquez等人，2011年)，包括Notch 3击倒 和两个敲打模型，允许野生型人类Notch 3或人类Notch 3的诱导表达 携带C455R CADASIL突变，位于Notch 3配体结合域(LBD)(Arboleda- 贝拉斯克斯等人，2002年)。我们对这些模型鼠的分析表明，C455R突变会损害信号转导。 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、C428S)表现出显性-负性。 在该项目的UH2阶段，我们计划：1)培育携带Notch 3突变的小鼠，并将它们老化到24岁 在项目的UH3阶段使用数月，以及2)通过检查Notch 3转基因来确定可行性 在衰老小鼠血管中的表达。在项目的UH3阶段，我们提出以下建议 实验目标：1)检测年龄如何影响操纵Notch 3信号对SVD的影响 监测老年人和年轻人SVD表型进展和SMC基因表达的变化 野生型和Notch 3突变小鼠；以及2)监测可诱导的野生型人类Notch 3的能力 随着小鼠年龄的增长，转基因拯救SVD表型。归根结底，这些研究旨在确定 携带Notch 3基因突变的老年小鼠是研究SVD的更合适的模型，以及 操纵Notch 3信号在小鼠SVD相关表型上的作用将随着年龄的增长而改变。

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.