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。一个尚未解答的关键问题是，所有 FAD 和 SAD 神经元不当行为是否仅仅是由分泌的淀粉样前体蛋白 (APP) 片段（例如 A¿ 和 sAPP）（因此细胞非自主）启动或增强的过程的结果，或者由 APP 蛋白水解产物驱动的 A 独立细胞内过程（例如 C 末端片段 (CTF)（因此可能是细胞自主的）过程）是否是主要贡献者。具体而言，AD 起始和进展的主要假说是淀粉样蛋白级联假说，该假说提出，人类 APP 的 Ass 肽片段对于起始和驱动 AD 进展的所有下游病理（包括突触缺陷）是必要且充分的[1]。其他想法包括由早老素或 APP 突变体/片段引起的细胞内缺陷，这些缺陷会在内体或溶酶体途径、轴突运输、神经营养信号、转录控制、细胞周期重新启动、氧化缺陷等方面产生缺陷。 [2-7]。还有二次打击模型，其中 A¿ 是与细胞内损伤相结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、非自主、两次打击）对使用源自人类诱导多能干细胞（hIPSC）的神经元的混合细胞培养实验做出了不同的实验预测。例如，（非自主）淀粉样蛋白级联假说和基于 APP（或其他分子）分泌片段毒性的相关非自主假说预测，由于患病神经元分泌有毒产物，患病和非患病神经元的混合物应在非患病神经元中引起疾病​​表型。或者，在不分泌有毒产物的 AD 细胞自主模型中，患病和非患病神经元的混合物不应导致非患病神经元出现疾病表型。最后，在两个命中模型中，细胞自主过程和细胞非自主过程可能会结合起来，使得表型的细胞自主启动可能会被 A 或其他分泌的毒性介质增强。我们建议通过进一步开发 AD 的新平台 hIPSC 人类神经元模型来开始测试这些想法。这些研究如果成功，可以为自主和非自主过程以及两次打击模型的相对贡献提供新的线索。我们将使用来自非痴呆对照患者 (NDC)、FAD APPDp 患者和 SAD 患者（称为 SAD2）的 hIPSC 系制成的神经元。我们有两个具体的实验目标： 目标 1) 检验以下假设：在含有 FAD APP 重复或来自 SAD 个体（称为 SAD2）的基因组的纯化神经元中观察到的部分或所有缺陷是细胞自主的。目标 2) 检验携带不同 ApoE 等位基因的星形胶质细胞增强或抑制纯化神经元中 AD 表型的假设。

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.