Elucidating AD genotype-phenotype relationships using genetics of human IPS cells

描述（由申请人提供）：阿尔茨海默病（AD）是常见的，毁灭性的，并造成巨大的社会和经济负担。目前，没有有效的疾病修饰AD治疗是可用的或即将到来的，部分原因是我们缺乏对疾病期间人类神经元和神经胶质细胞失败的细胞机制和途径的完整理解，部分原因是我们没有充分了解常见的遗传变异如何改变人类神经元和神经胶质细胞表型。在这里，我们建议测试APP和PS突变是否在人类诱导多能干细胞（hIPSC）的共同遗传背景中产生的产生相同的早期神经元表型，然后调查在何种程度上GWAS研究确定的候选基因组产生可比表型时，减少，增加，或改变自然发生的变异。为了解决这两个问题，我们提出了hIPSC技术的独特应用：1）剖析FAD突变如何改变关键通路，然后2）测试个体遗传背景和已识别的风险因素如何使人类神经元和星形胶质细胞中的SAD生化表型易感。在可能的情况下，我们将把体外信息与个体患者的临床数据和UCSD ADRC的尸检病理学联系起来。对SAD患者的hIPSC系的分析对于探索常见遗传风险因素如何在神经元和星形胶质细胞中起作用至关重要，并且还将初步估计SAD患者和对照中导致体外分化的神经细胞中相关SAD表型的基因组频率。这种频率估计将有助于解决hIPSC系是否可用于预测给定个体将发展SAD的可能性的重要长期问题，即，以产生SAD的预测性基因组/hIPSC诊断。该提议利用了我们和其他人先前的工作，这些工作分析了来自携带APP重复（APPDp）或21三体的患者的hIPSC系。这两种情况似乎都是通过在整倍体基因组中增加APP表达50%来引起FAD。由这些hIPSC系制成的神经元表现出典型的AD生物化学改变，包括升高的A β、升高的GSK 3活化和升高的tau磷酸化。我们还发现APPV 717 F而不是PS1 dE 9突变导致p-tau水平升高。因此，APP和早老素突变的早期神经元表型可能是不同的，这提高了可能存在可以使用hIPSC技术研究的多种早期致病途径的可能性。我们还发现hIPSC研究可以阐明一种常见的遗传风险因子SORL 1如何在人类神经元中起作用。因此，我们提出了三个具体的目标：1）测试的假设，APP，PS1，和？-分泌酶突变触发相同的早期事件在人类神经元和星形胶质细胞导致下游生化病理典型的AD。2)检验GWAS研究中确定为风险因素的基因在过度表达或表达不足时产生AD表型和改变的内吞作用、运输或转运的假设。3)测试GWAS研究中鉴定的常见遗传变异通过改变神经元或星形胶质细胞中的基因表达起作用的假设。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is common, devastating, and creates enormous social and financial burdens. At present, no effective disease-modifying AD treatment is available or imminent, in part because we lack a complete understanding of the cellular mechanisms and pathways that fail in human neurons and glial cells during disease, and in part because we don't adequately understand how common genetic variants alter human neuronal and glial phenotypes. Here we propose to test whether APP and PS mutations generated in common genetic backgrounds in human induced pluripotent stem cells (hIPSC) generate the same early neuronal phenotypes and then to investigate the extent to which a candidate set of genes identified by GWAS studies generate comparable phenotypes when reduced, increased, or altered by naturally occurring variants. To tackle both problems, we propose unique applications of hIPSC technology to 1) dissect how FAD mutations alter key pathways and then 2) to test how individual genetic background and identified risk factors predispose to SAD biochemical phenotypes in human neurons and astrocytes. Where possible, we will link the in vitro information to clinical data on individual patients and to post-mortem pathology from the UCSD ADRC. The analysis of hIPSC lines from SAD patients will be crucial to probe how common genetic risk factors act in neurons and astrocytes and will also give an initial estimate of the frequency of genomes in SAD patients and controls that cause relevant SAD phenotypes in neural cells differentiated in vitro. This frequency estimate will help address the important long-term question of whether hIPSC lines can be used to predict the likelihood that a given individual will develop SAD, i.e., to generate a predictive genomic/hIPSC diagnostic for SAD. This proposal capitalizes upon previous work from us and others that analyzed hIPSC lines from patients carrying an APP duplication (APPDp) or trisomy 21. Both situations appear to cause FAD by increasing APP expression by 50% in an otherwise euploid genome. Neurons made from these hIPSC lines exhibit typical AD biochemical alterations including elevated A¿, elevated activation of GSK3, and elevated phosphorylation of tau at a proposed pathological site. We also found that APPV717F but not PS1dE9 mutations cause elevated p-tau levels. Thus, early neuronal phenotypes of APP and presenilin mutations might be different raising the possibility that there may be multiple early pathogenic pathways that can be studied using hIPSC technology. We also found that hIPSC studies can elucidate how one common genetic risk factor, SORL1, acts in human neurons. We thus propose three specific aims: 1) Test the hypothesis that APP, PS1, and ¿-secretase mutations trigger the same early events in human neurons and astrocytes leading to downstream biochemical pathology typical of AD. 2) Test the hypothesis that genes identified as risk factors in GWAS studies generate AD phenotypes and altered endocytosis, trafficking, or transport when over or underexpressed. 3) Test the hypothesis that common genetic variants identified in GWAS studies act by altering gene expression in neurons or astrocytes.