Characterization of Alzheimer's Mutations in ADAM10.

描述（申请人提供）：大量的临床、遗传学和生化数据支持淀粉样蛋白前体蛋白（APP）的异常加工及其代谢物A¿的大脑积累在阿尔茨海默病（AD）的病因和发病机制中起关键作用的假设。A¿是通过-和-分泌酶对APP的连续裂解产生的。相反，分泌酶对APP的裂解阻止了A -的产生；大脑中主要的分泌酶是ADAM10。我们最近报道了ADAM10中两个罕见的错义突变，它们与迟发性AD （LOAD）强烈共分离。这两种突变都是在1000个NIMH和NIA LOAD家族中发现的前域突变（平均发病年龄约70岁）。我们之前也报道过这两种突变在体外显著降低了分泌酶活性并升高了A¿水平。为了进一步验证和扩展这些新发现，我们建议：1。在ADAM10中寻找新的家族性LOAD突变2. 在体内验证这两种错义突变的致病性；和3。确定这两种突变损害分泌酶活性的分子机制。在Aim 1中，我们将通过对NIMH和NIA AD家族收集的2454个AD家族（N=6516名受试者）进行有针对性的重测序（和基因分型），寻找ADAM10中其他新的家族LOAD突变。我们基于bbbb900 NIMH和NIA AD家族的GWAS表明，在ADAM10中存在额外的AD相关snp。关于Aim 2，我们将尝试在体内验证这两种突变在转基因小鼠（已经产生）中过表达野生型（WT）或突变型（Q170H, R181G，人工显性阴性）ADAM10形式的致病性。这些小鼠已经与Tg2576 (APPswe) AD小鼠杂交，因此我们也可以测试这些突变对AD神经病理和认知表型的影响。我们的初步体内实验结果显示，ADAM10原结构域突变减弱了APP的非淀粉样变性过程，与表达WT型的ADAM10突变型相比，表达ADAM10突变型的APPswe/ADAM10双转基因小鼠的大脑中老年斑计数和A¿水平显著增加。此外，为了测试前结构域突变在更多生理相关条件下的影响，我们增加了生成和表征ADAM10突变敲入小鼠的计划。最后，在Aim 3中，我们将研究原结构域突变下调ADAM10 -分泌酶活性的分子机制。简而言之，我们将测试这些突变对三个人的影响

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.