Characterization of Alzheimer's Mutations in ADAM10.

ADAM10 中阿尔茨海默病突变的表征。

基本信息

批准号：
8438141
负责人：
RUDOLPH Emile TANZI
金额：
$ 34.59万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8438141
关键词：
Age of Onset Alzheimer&apos s Disease Amyloid Amyloid beta-Protein Precursor Animal Model Attenuated Biochemical Biological Assay Biotinylation Brain Catalytic Domain Cell membrane Cerebrum Clinical Cognitive Collection Data Density Gradient Centrifugation Development Disease Dominant-Negative Mutation Electrophysiology (science)Etiology Exhibits Family Foundations Gene Expression Genes Genetic Genotype In Vitro Knock-in Mouse Late Onset Alzheimer Disease Linkage Disequilibrium Long-Term Potentiation Memory Metalloproteases Missense Mutation Molecular Molecular Chaperones Mus Mutation National Institute of Mental Health Pathogenesis Pathogenicity Peptide Hydrolases Phenotype Play Prevention Production Property Proteins Regulation Reporting Role Sampling Senile Plaques Single Nucleotide Polymorphism Staining method Stains Structure Surface System Testing Tg2576 Transgenic Mice Transgenic Organisms Validation Variant Yeasts amyloid precursor protein processing base cognitive function disease phenotype enzyme activity familial Alzheimer disease genome wide association study in vivo mouse model mutant neuropathology novel overexpression proband protein folding secretase segregation trafficking

项目摘要

DESCRIPTION (provided by applicant): Abundant clinical, genetic, and biochemical data support the hypothesis that abnormal processing of the amyloid precursor protein (APP) and the cerebral accumulation of its metabolite, A¿, play key roles in the etiology and pathogenesis of Alzheimer's disease (AD). A¿ is generated via serial cleavage of APP by ¿- and ¿- secretase. In contrast, cleavage of APP by ¿-secretase precludes A¿ production; the major ¿-secretase in the brain is ADAM10. We recently reported two rare missense mutations in ADAM10 that strongly co- segregates with late-onset AD (LOAD). Both are prodomain mutations that were found in 7 of 1000 NIMH and NIA LOAD families tested (average age of onset, ~70 years). We have also previously reported that both mutations significantly attenuated ¿-secretase activity and elevated A¿ levels in vitro. To further validate and expand upon these novel findings, we propose to 1. Search for additional novel familial LOAD mutations in ADAM10; 2. Validate the pathogenicity of these two missense mutations in vivo; and 3. Determine the molecular mechanism by which these two mutations impair ¿-secretase activity. For Aim 1, we will search for additional novel familial LOAD mutations in ADAM10, through targeted re-sequencing (and genotyping) of 2454 additional AD families (N=6516 subjects) from the NIMH and NIA AD family collections. Our family-based GWAS on >900 NIMH and NIA AD families suggest the existence of additional AD-associated SNPs in ADAM10. With regard to Aim 2, we will attempt to validate the pathogenicity of these two mutations in vivo in transgenic mice (already generated) overexpressing either wild-type (WT) or mutant (Q170H, R181G, artificial dominant-negative) forms of ADAM10. These mice have already been crossed with Tg2576 (APPswe) AD mice so that we can also test for effects of these mutations on neuropathological and cognitive phenotypes of AD. Our preliminary in vivo results reveal that the ADAM10 prodomain mutations attenuate non-amyloidogenic processing of APP, and that senile plaque counts and A¿ levels were significantly increased in the brains of APPswe/ADAM10 double transgenic mice expressing mutant forms of ADAM10, as compared to those expressing WT forms. Moreover, to test the impact of the prodomain mutations under more physiologically relevant conditions, we have added a plan to generate and characterize ADAM10 mutant knock-in mice. Finally, in Aim 3, we will investigate the molecular mechanism by which the prodomain mutations down- regulate ADAM10 ¿-secretase activity. Briefly, we will test for effects of these mutations on three prodomain functions: regulation of enzyme activity, intracellular trafficking, and intramolecular chaperoning. At the completion of this project, we hope to provide genetic, biochemical, and mechanistic evidence validating the pathogenicity of late-onset familial AD mutations in ADAM10. Moreover, the data emerging from the proposed study would serve as a firm foundation for the discovery and development of new therapies targeting ADAM10 for the treatment and prevention of this devastating disease. PUBLIC HEALTH RELEVANCE: We will attempt to establish in vivo validation for the pathogenicity and to elucidate the molecular mechanism of two ADAM10 prodomain mutations, which we have already confirmed in several late-onset Alzheimer's disease (AD) families. We will also search for additional AD mutations in the ADAM10 gene.

描述（由申请人提供）：大量的临床、遗传和生化数据支持以下假设：淀粉样前体蛋白 (APP) 的异常加工及其代谢物 A¿ ，在阿尔茨海默病（AD）的病因学和发病机制中发挥关键作用。是通过 ¿ 连续切割 APP 生成的- 和 - 相比之下，APP 被 ¿ 切割。 -分泌酶排除A¿生产；主要 ¿我们最近报道了 ADAM10 中的两种罕见错义突变，它们与迟发性 AD (LOAD) 强烈共分离，这两种突变均在测试的 1000 个 NIMH 和 NIA LOAD 家族中的 7 个中发现。发病年龄，约 70 岁）我们之前也报道过这两种突变均显着减弱 ¿ -分泌酶活性和升高的A¿为了进一步验证和扩展这些新发现，我们建议 1. 寻找 ADAM10 中其他新的家族 LOAD 突变；2. 验证这两种错义突变的体内致病性；3. 通过以下方法确定分子机制：这两种突变会损害 ¿对于目标 1，我们将通过对来自 NIMH 和 NIA AD 家族收藏的 2454 个其他 AD 家族（N=6516 名受试者）进行有针对性的重测序（和基因分型）来寻找 ADAM10 中的其他新型家族 LOAD 突变。对超过 900 个 NIMH 和 NIA AD 家族进行的基于家族的 GWAS 表明，ADAM10 中存在其他与 AD 相关的 SNP。如图 2 所示，我们将尝试在过表达 ADAM10 野生型 (WT) 或突变型（Q170H、R181G、人工显性失活）形式的转基因小鼠（已生成）体内验证这两种突变的致病性。与 Tg2576 (APPswe) AD 小鼠杂交，以便我们还可以测试这些突变对 AD 神经病理学和认知表型的影响。 ADAM10 前结构域突变会减弱 APP 的非淀粉样蛋白形成过程，并且老年斑计数和 A¿与表达 WT 形式的小鼠相比，表达 ADAM10 突变形式的 APPswe/ADAM10 双转基因小鼠的大脑中的水平显着增加。此外，为了测试前结构域突变在更生理相关的条件下的影响，我们添加了一项计划生成并表征 ADAM10 突变敲入小鼠最后，在目标 3 中，我们将研究前结构域突变下调 ADAM10 的分子机制。 -简单地说，我们将测试这些突变对三个的影响。前结构域功能：酶活性、细胞内运输和分子内伴侣的调节在该项目完成时，我们希望提供遗传、生化和机制证据来验证 ADAM10 中晚发家族性 AD 突变的致病性。拟议研究的结果将为发现和开发针对 ADAM10 的新疗法奠定坚实的基础，以治疗和预防这种毁灭性疾病。公共健康相关性：我们将尝试建立致病性的体内验证并阐明两个 ADAM10 前结构域突变的分子机制，我们已经在几个迟发性阿尔茨海默病 (AD) 家族中证实了这一点。我们还将寻找其他突变。 ADAM10 基因发生 AD 突变。