Alzheimer's Disease and Impaired APP Proteolysis

描述(申请人提供)：家族性阿尔茨海默病(FAD)是一种可遗传的显性疾病。导致β-淀粉样前体蛋白(APR)中单一氨基酸替换的特定错义突变与FAD有关。APP的FAD突变集中在两个区域，胞外区与a-和(3-分泌酶)位点相邻，膜内区与已知的y-分泌酶裂解位点相邻。这项建议的目的是研究FAD突变对正常小鼠大脑中y-分泌酶介导的APP切割的影响。该方法包括利用先前开发的并在体外验证的y-分泌酶活性报告系统来开发转基因小鼠模型。在细胞培养系统中使用这一屏幕，我们已经证明了FAD突变导致APP羧基末端的释放减少，这是由APP切割引起的。虽然这一数据令人信服，但y-分泌酶介导的APP裂解减少是否是完整大脑中FAD突变的常见结果仍有待测试。该项目将验证这样一种假设，即在生理条件下，FAD突变会减少y-分泌酶介导的APP羧基末端片段从脑膜上的蛋白水解性释放。其具体目的是：1)建立转基因报告小鼠模型，用于体内γ-分泌酶活性的检测。在延伸因子1α(EF1a)启动子的控制下，建立表达APP-GA4VP16激活子的转基因小鼠系，并与Gal4-荧光素酶EGFP转基因小鼠杂交。基因筛选系统的成功部署，以及报告输出对y-分泌酶的依赖性，将通过组织学和报告分析方法进行验证。2)利用转基因报告小鼠模型比较了γ-分泌酶对野生型和FAD突变型APP-Gal4VP16的切割作用。将FAD突变体APP-Gal4VP16转基因系与Gal4-LuciferaseEGFP报告小鼠杂交，对野生型和Fad突变体的报告输出进行比较分析，以确定每组转基因系中的相对γ-分泌酶活性。

DESCRIPTION (provided by applicant): Familial Alzheimer's Disease (FAD) is a genetically heritable dominant disorder. Specific missense mutations that result in a single amino acid substitution in p-amyloid precursor protein (APR) have been linked with FAD. FAD mutations in APP cluster around two regions, the extracellular domain adjacent to the a- and (3-secretase sites, and within the intramembranous region adjacent to known y-secretase cleavage sites. The goal of this proposal is to study the effects of FAD mutations upon y-secretase mediated cleavage of APP in the brains of intact mice. The approach involves the development of a trarisgenic mouse model employing a y-secretase activity reporter system previously developed and validated in vitro. Using this screen in cell culture systems, we have demonstrated that FAD mutations result in a decrement in liberation of the APP carboxy-terminus resulting from APP cleavage. While this data is compelling, it remains to be tested whether decrements in y-secretase-mediated APP cleavage are a common effect of FAD mutations within intact brain. This project will test the hypothesis that FAD mutations decrease y-secretase- mediated proteolytic liberation of the carboxy-terminal fragment of APP from the membrane in brain under physiological conditions. The Specific Aims are: 1) Develop a transgenic reporter mouse model to assay y-secretase activity in vivo. Transgenic mouse lines will be developed expressing the APP-Ga!4VP16 activator under the control of the elongation factor 1 alpha (EF1a) promoter, and crossed with Gal4- luciferaseEGFP transgenic mice. Successful deployment of the genetic screening system, and the y-secretase dependency of the reporter output, will be validated by histological and reporter assay methods. 2) Compare y-secretase-mediated cleavage of wild-type and FAD mutant APP-Gal4VP16 using the transgenic reporter mouse model. FAD mutant APP-Gal4VP16 transgenic lines will be crossed with Gal4- luciferaseEGFP reporter mice, and comparative analysis of the reporter output of the wild-type and FAD mutants will be used to determine relative y-secretase activity in each set of transgenic: lines.