Alzheimer's Disease and Impaired APP Proteolysis

描述（由申请人提供）：家族性阿尔茨海默病（FAD）是一种遗传遗传性显性疾病。导致p-淀粉样蛋白前体蛋白（APR）单氨基酸取代的特异性错义突变与FAD有关。APP中的FAD突变集中在两个区域周围，靠近a-和3-分泌酶位点的细胞外区域，以及靠近已知y-分泌酶切割位点的膜内区域。本研究的目的是研究FAD突变对完整小鼠大脑中y分泌酶介导的APP裂解的影响。该方法涉及到利用先前在体外开发并验证的y分泌酶活性报告系统开发一种先天性小鼠模型。在细胞培养系统中使用该筛选，我们已经证明FAD突变导致APP切割导致APP羧基末端释放减少。虽然这一数据令人信服，但y分泌酶介导的APP切割减少是否是完整脑内FAD突变的常见影响仍有待检验。本项目将验证在生理条件下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释放的假设。具体目的是：1)建立转基因报告小鼠模型，测定体内y分泌酶活性。将开发表达APP-Ga！在延伸因子1 α (EF1a)启动子的控制下，将4VP16激活子与Gal4- luciferaseEGFP转基因小鼠杂交。基因筛选系统的成功部署，以及报告基因输出的y分泌酶依赖性，将通过组织学和报告基因测定方法进行验证。2)利用转基因报告小鼠模型比较y分泌酶介导的野生型和FAD突变型APP-Gal4VP16的裂解。将FAD突变体APP-Gal4VP16转基因系与Gal4- luciferaseEGFP报告小鼠杂交，通过对比分析野生型和FAD突变体的报告输出，确定每组转基因系中y-分泌酶的相对活性。

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.