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）治疗的发展需要对疾病的发生和进展有更深入的了解。一个关键的悬而未决的问题是，所有FAD和SAD神经元的不当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（因此是细胞非自主的）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¿独立的细胞内过程，例如c端片段（CTF）（因此是潜在的细胞自主的）过程是主要的因素。具体来说，阿尔茨海默病发生和进展的主要假说是淀粉样蛋白级联假说，该假说认为，人类APP的Ass肽片段是必要的，足以启动和驱动阿尔茨海默病进展的所有典型下游病理，包括突触缺陷[1]。其他观点包括早老素或APP突变体/片段引起的细胞内缺陷，这些缺陷在内体或溶酶体途径、轴突运输、神经营养信号、转录控制、细胞周期再启动、氧化缺陷等方面产生缺陷[2-7]。也有双击模型，其中A¿是启动或增强侮辱与细胞内侮辱结合的一部分[8-11]。这三种类型的模型（自主、非自主、双命中）对使用人类诱导多能干细胞（hIPSC）衍生的神经元进行混合细胞培养实验做出了不同的实验预测。例如，（非自主的）淀粉样蛋白级联假说以及基于APP（或其他分子）分泌片段毒性的相关非自主假说预测，由于病变神经元分泌有毒产物，病变和非病变神经元的混合物应在非病变神经元中引起疾病表型。另外，在不假定分泌有毒产物的AD细胞自主模型中，病变和非病变神经元的混合物不应导致非病变神经元的疾病表型。最后，在两个hit模型中，细胞自主过程和细胞非自主过程可能结合在一起，从而细胞自主启动表型可能被A¿或其他分泌的有毒介质增强。我们建议通过进一步开发一个新的平台hIPSC人类AD神经元模型来开始测试这些想法。这些调查，如果成功，可以揭示自主和非自主过程和双击模型的相对贡献。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细胞系制成的神经元。我们有两个特定的实验目的：目的1)验证在含有FAD APP复制或来自SAD个体的基因组（称为SAD2）的纯化神经元中观察到的部分或所有缺陷是细胞自主的假设。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.