Testing cell autonomy of AD phenotypes using human IPS cells

描述（由申请人提供）：家族性和散发性阿尔茨海默病（FAD和SAD）疗法的开发需要对疾病的发生和进展有更深入的了解。一个关键的未回答的问题是，所有FAD和SAD神经元的不良行为是否仅仅是由分泌的淀粉样前体蛋白（APP）片段如A？和sAPP（因此细胞非自主）引发或增强的过程的结果，或者是否是由APP蛋白水解产物驱动的A？-独立的细胞内过程，例如，C-末端片段（CTF）（因此可能是细胞自主的）过程是主要的贡献者。具体而言，AD起始和进展的主要假设是淀粉样蛋白级联假说，其提出人APP的Ass肽片段对于起始和驱动AD进展的所有典型下游病理（包括突触缺陷）是必要且充分的[1]。替代的想法包括由早老素或APP突变体/片段引起的细胞内缺陷，其在内体或溶酶体途径、轴突运输、神经营养信号传导、转录控制、细胞周期重新启动、氧化缺陷等中产生缺陷[2-7]。也有两次打击模型，其中A？是与细胞内损伤组合的起始或增强损伤的一部分[8-11]。这三种类型的模型（自主的、非自主的、两次击中的）对使用源自人诱导多能干细胞（hIPSC）的神经元的混合细胞培养实验做出不同的实验预测。例如，（非自主）淀粉样蛋白级联假说和基于APP（或其他分子）的分泌片段的毒性的相关非自主假说预测，由于患病神经元分泌毒性产物，患病和未患病神经元的混合物应在未患病神经元中引起疾病表型。或者，在不抑制毒性产物分泌的AD的细胞自主模型中，患病和未患病神经元的混合物不应导致未患病神经元中的疾病表型。最后，在两种命中模型中，细胞自主过程和细胞非自主过程可能联合收割机组合，使得表型的细胞自主起始可能被A？或其他分泌的毒性介质增强。我们建议开始测试这些想法，进一步开发一个新的平台hIPSC人类神经元模型的AD。这些调查，如果成功的话，可以揭示新的自主和非自主的过程和两次击中模型的相对贡献。我们将使用由来自非痴呆对照患者（NDC）、FAD APPDp患者和SAD患者（称为SAD 2）的hIPSC系制成的神经元。我们有两个具体的实验目的：目的1）测试在纯化的神经元中观察到的一些或所有缺陷的假设，所述纯化的神经元含有FAD APP重复或来自患有SAD的个体的基因组，称为SAD 2。目的2）验证携带不同ApoE等位基因的星形胶质细胞在纯化神经元中增强或抑制AD表型的假说。

DESCRIPTION (provided by applicant): Development of familial and sporadic Alzheimer's Disease (FAD and SAD) therapies requires deeper understanding of disease initiation and progression. A key unanswered question is whether all FAD and SAD neuronal misbehavior is solely the result of processes initiated or enhanced by secreted Amyloid Precursor Protein (APP) fragments such as A¿ and sAPP (and therefore cell non-autonomous), or whether A¿- independent intracellular processes driven by APP proteolytic products, e.g., the C-terminal fragment (CTF) (and therefore potentially cell autonomous) processes are a major contributor. Specifically, the major hypothesis for AD initiation and progression is the amyloid cascade hypothesis, which proposes that Ass peptide fragments of human APP are necessary and sufficient to initiate and to drive all downstream pathologies typical of AD progression including synaptic defects [1]. Alternative ideas include intracellular defects caused by presenilin or APP mutants/fragments that generate defects in endosomal or lysosomal pathways, axonal transport, neurotrophic signaling, transcriptional control, cell cycle reinitiation, oxidative defets, etc. [2-7]. There are also two-hit models in which A¿ is part of an initiating or enhancing insult n combination with intracellular insults [8-11]. These three types of models (autonomous, non-autonomous, two- hit) make distinct experimental predictions for mixed cell culture experiments using neurons derived from human induced pluripotent stem cells (hIPSC). For example, the (non-autonomous) amyloid cascade hypothesis, and related non-autonomous hypotheses based on toxicity of secreted fragments of APP (or other molecules) predict that mixtures of diseased and non-diseased neurons should cause disease phenotypes in the non-diseased neurons owing to secretion of toxic products by diseased neurons. Alternatively, in cell autonomous models of AD that do not posit secretion of toxic products, mixtures of diseased and non-diseased neurons should not lead to disease phenotypes in non-diseased neurons. Finally in two hit models, cell autonomous processes and cell nonautonomous processes might combine such that cell autonomous initiation of phenotypes might be enhanced by A¿ or other secreted toxic mediators. We propose to begin testing these ideas by further developing a new platform hIPSC human neuronal model of AD. These investigations, if successful, can shed new light on the relative contributions of autonomous and non-autonomous processes and two-hit models. We will use neurons made from hIPSC lines derived from a non-demented control patient (NDC), an FAD APPDp patient, and an SAD patient (called SAD2). We have two specific experimental aims: Aim 1) To test the hypothesis that some or all defects observed in purified neurons containing either an FAD APP duplication or a genome from an individual with SAD called SAD2 are cell-autonomous. Aim 2) To test the hypothesis that astrocytes carrying different ApoE alleles enhance or suppress AD phenotypes in purified neurons.