Probing SORL1 Risk Factors with Human Induced Pluripotent Stem Cell Technology

项目1：摘要。 了解和最终治疗阿尔茨海默病（AD）的一个关键问题是我们的不完整的 了解遗传风险因素在迟发性、散发性AD（SAD）中的作用。虽然没有明确的 单一遗传损伤导致SAD，观察到的高遗传力表明个体遗传 背景起着重要的作用。在这里，我们提出了独特的应用，人类诱导多能 干细胞（hIPSC）技术，以剖析如何个人遗传背景和确定的风险因素 在人类神经元中易患SAD生化表型，并将该信息与以下临床数据联系起来： 个体患者和死后病理学。我们的工作是基于我们最近的发现， hIPSC技术测试了R单倍型导致SORL 1普遍减少的假设 导致淀粉样β（A？）肽增加的表达和随后发生SAD的风险[3，7- 10]。使用hIPSC技术，我们发现R单倍型损害了SORL 1基因的信号输入。 具体而言，P单倍型通过诱导SORL 1表达对BDNF作出反应，而R单倍型则不。 基础表达水平与R或P单倍型无相关性。因此，SORL 1基因的贡献 SAD可能是由活的人类神经元中复杂的调节变化引起的。我们现在建议测试 我们的工作模型，SORL 1单倍型如何有助于SAD神经元表型，从而SAD的风险， 在体外的人类和在人类患者体内。我们建议测试：1）BDNF诱导的假设， SORL 1表达调节APP和下游SAD相关的淀粉样蛋白形成过程 生化变化;以及2）SORL 1单倍型对神经元表型影响的假设 在SAD患者的临床数据中，特别是BDNF和SORL 1的相对量， 蛋白质在脑脊液（CSF）和死后神经病理表型。在相关目标中，我们 将研究从我们收集的患者hIPSC系中纯化的神经元是否与 个别患者的临床表现。目前，我们的hIPSC细胞系太少，无法明确建立 相关度严格，但鉴于存在的线，我们将开始收集比较 数据作为一种有助于未来研究的方式。总之，这些实验将提供有关 SORL 1变体对具有不同遗传学特征的人类神经元SAD表型的贡献的详细信息 背景，并可能导致药物发现和临床试验分层的新途径 基于遗传背景。我们的具体目标是：1.检验这个假设， 具有SORL 1风险变异单倍型的纯化人神经元的诱导反应增强SORL 1- 依赖于下游生化SAD表型。2.为了验证一个假设， SORL 1基因座的患者对CSF中测量的临床表型标志物有显著影响， 尸检病理学鉴定

PROJECT 1: ABSTRACT. A key problem in understanding and eventually treating Alzheimer's disease (AD) is our incomplete understanding of the role of genetic risk factors in late-onset, sporadic AD (SAD). While there is no clear single genetic lesion that causes SAD, the observed high heritability suggests that individual genetic background plays a significant role. Here we propose unique applications of human induced pluripotent stem cell (hIPSC) technology to dissect how individual genetic background and identified risk factors predispose to SAD biochemical phenotypes in human neurons and to link that information to clinical data on individual patients and to post-mortem pathology. We are basing our work on our recent finding using hIPSC technology that tested the hypothesis that R haplotypes cause general reduction of SORL1 expression leading to increased amyloid beta (A¿) peptides and consequent risk of developing SAD [3, 7- 10]. Using hIPSC technology we found that R haplotypes impair a signaling input to the SORL1 gene. Specifically, P haplotypes respond to BDNF by inducing SORL1 expression, while R haplotypes do not. Basal expression levels show no correlation with R or P haplotypes. Thus, the SORL1 genetic contribution to SAD may be caused by complex regulatory variation in living human neurons. We now propose to test our working model for how SORL1 haplotypes contribute to SAD neuronal phenotypes and thus SAD risk in humans in vitro, and in vivo in human patients. We propose to test: 1) the hypothesis that BDNF-induced SORL1 expression modulates amyloidogenic processing of APP and downstream SAD-associated biochemical changes; and 2) the hypothesis that effects of SORL1 haplotype on neuronal phenotypes in vitro are mirrored in clinical data on SAD patients, specifically the relative amounts of BDNF and SORL1 proteins in cerebrospinal fluid (CSF) and post-mortem neuropathological phenotypes. In a related goal, we will investigate whether purified neurons made from our collection of patient hIPSC lines correlates with clinical behavior of individual patients. At present, we have too few hIPSC lines to definitively establish the degree of correlation rigorously, but given the existence of the lines, we will begin to collect comparative data as a way of contributing to future studies. Together, these experiments will provide new data about the details of SORL1 variant contributions to SAD phenotypes in human neurons with differing genetic backgrounds and potentially lead to new pathways for drug discovery and stratification of clinical trials based on genetic background. Our specific aims are to: 1. Test the hypothesis that the reduced BDNF induction response of purified human neurons with SORL1 risk variant haplotypes enhances SORL1- dependent downstream biochemical SAD phenotypes. 2. To test the hypothesis that the genetic status of patients at the SORL1 locus has a significant influence on clinical phenotypic markers measured in CSF or by post-mortem pathology.