Characterization of Alzheimer's Mutations in ADAM10.

ADAM10 中阿尔茨海默病突变的表征。

• 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
• 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' 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

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）：丰富的临床、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产物A？的脑内蓄积在阿尔茨海默病（AD）的病因学和发病机制中起关键作用。A是通过APP被<$-和<$-分泌酶连续切割而产生的。相反，APP被<$-分泌酶切割排除了A <$的产生;大脑中主要的<$-分泌酶是ADAM 10。我们最近报道了两个罕见的错义突变的ADAM 10强烈共分离与迟发性AD（LOAD）。两者都是在1000个NIMH和NIA LOAD家族中的7个中发现的前结构域突变（平均发病年龄，约70岁）。我们以前也报道过，这两种突变在体外显著减弱了β-分泌酶活性和升高了A β水平。为了进一步验证和扩展这些新的发现，我们建议1。在ADAM 10中寻找额外的新家族性LOAD突变; 2.探讨这两种错义突变在体内的致病性; 3.确定这两个突变损害β-分泌酶活性的分子机制。对于目标1，我们将通过对NIMH和NIA AD家族收集的2454个额外AD家族（N=6516例受试者）进行靶向重新测序（和基因分型），在ADAM 10中寻找额外的新型家族性LOAD突变。我们对>900个NIMH和NIA AD家族进行的基于家族的GWAS表明ADAM 10中存在额外的AD相关SNP。关于目标2，我们将尝试在过表达野生型（WT）或突变型（Q170 H，R181 G，人工显性阴性）形式的ADAM 10的转基因小鼠（已生成）中验证这两种突变的体内致病性。这些小鼠已经与Tg 2576（APPswe）AD小鼠杂交，因此我们也可以测试这些突变对AD的神经病理学和认知表型的影响。我们的初步体内研究结果表明，ADAM 10前结构域突变减弱APP的非淀粉样蛋白形成过程，与表达WT形式的小鼠相比，表达突变形式ADAM 10的APPswe/ADAM 10双转基因小鼠脑中老年斑计数和A？水平显著增加。此外，为了测试前结构域突变在更生理相关条件下的影响，我们增加了一个计划来产生和表征ADAM 10突变体敲入小鼠。最后，在目标3中，我们将研究前结构域突变下调ADAM 10 <$-分泌酶活性的分子机制。简单地说，我们将测试这些突变对三个人的影响。 前结构域功能：调节酶活性、细胞内运输和分子内伴侣。在这个项目完成后，我们希望提供遗传，生化和机制的证据验证ADAM 10中迟发性家族性AD突变的致病性。此外，拟议研究中出现的数据将为发现和开发针对ADAM 10的新疗法以治疗和预防这种毁灭性疾病奠定坚实的基础。 公共卫生关系：我们将试图建立在体内验证的致病性和阐明的分子机制，我们已经证实了在几个迟发性阿尔茨海默病（AD）的家庭两个ADAM 10前结构域突变。我们还将寻找ADAM 10基因中的其他AD突变。