Alzheimer's Disease and Impaired APP Proteolysis

阿尔茨海默病和 APP 蛋白水解受损

• 批准号: 7140287
• 负责人: Warren C LADIGES
• 金额: $ 19.37万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140287
• 关键词: Alzheimer' s disease; amyloid proteins; aspartic endopeptidases; enzyme activity; family genetics; gene expression; gene mutation; genetic models; genetic screening; genetically modified animals; histology; laboratory mouse; model design /development; proteolysis; reporter genes

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.

描述（由申请人提供）：家族性阿尔茨海默病（FAD）是一种遗传性显性疾病。导致 p-淀粉样前体蛋白 (APR) 中单个氨基酸取代的特定错义突变与 FAD 相关。 APP 中的 FAD 突变聚集在两个区域周围，即邻近 α 和 (3- 分泌酶位点的胞外结构域，以及邻近已知 y 分泌酶裂解位点的膜内区域。本提案的目的是研究 FAD 突变对完整小鼠大脑中 y 分泌酶介导的 APP 裂解的影响。该方法涉及培育转基因小鼠 该模型采用先前开发并在体外验证的 y-分泌酶活性报告系统。在细胞培养系统中使用此筛选，我们证明 FAD 突变会导致 APP 裂解导致 APP 羧基末端的释放减少。虽然这些数据令人信服，但 y-分泌酶介导的 APP 裂解的减少是否是完整大脑中 FAD 突变的常见影响仍有待测试。该项目将测试 假设FAD突变减少了生理条件下γ-分泌酶介导的APP羧基末端片段从脑膜的蛋白水解释放。具体目标是： 1) 开发转基因报告小鼠模型来测定体内γ-分泌酶活性。将开发在延伸因子1α(EF1a)启动子控制下表达APP-Gal 4VP16激活剂的转基因小鼠系，并与Gal4-荧光素酶EGFP转基因小鼠杂交。基因筛查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检测方法进行验证。 2) 使用转基因报告小鼠模型比较 y-分泌酶介导的野生型和 FAD 突变体 APP-Gal4VP16 的切割。 FAD突变体APP-Gal4VP16转基因系将与Gal4-荧光素酶EGFP报告小鼠杂交，并且野生型和FAD突变体的报告输出的比较分析将用于确定每组转基因系中的相对γ分泌酶活性。