iPSC for Neurodegenerative Diseases

描述（由申请人提供）： 我们建议在阿尔茨海默病（AD）患者来源的诱导多能干细胞（iPSC）分化的人类神经元中建立细胞生物标志物，以预测特定的药物反应。这些人细胞系将用作离体细胞模型，以表征驱动AD发病机制的关键蛋白质，淀粉样蛋白（A）和Tau，并与来自同一供体的死后脑病理学相关。我们还将从同一个捐赠者那里收集死后的大脑和脑脊液样本。我们将量化A？单体和寡聚体，pTau和Tau水平，并验证这些生物标志物在细胞和脑组织中的内在特性。我们将测试具有较高A或pTau蛋白的神经元对治疗剂的反应不同的假设。通过验证这一假设，我们将解决AD治疗开发中的一个关键问题，即特征性A？/Tau生物标志物是否可用于预测散发性AD（SAD）患者的治疗疗效。目前，已经开发出由脑淀粉样蛋白成像和脑脊液（CSF）A？和pTau/Tau蛋白组成的生物标志物，并用于临床试验的患者选择。在不进行脑淀粉样蛋白成像和CSF收集的情况下，没有测试AD生物标志物以预测个体的脑病理学和CSF特征的体外方法。 我们在开展这些研究方面具有独特的优势。我们在AD研究的细胞建模方面有近二十年的经验，并且有一个良好表征的患者群体，为离体建模和体内确认提供血细胞和死后组织。在我的实验室工作中，使用哺乳动物细胞培养模型来研究早老素在淀粉样前体蛋白（APP）加工和A？生成中的功能作用。使用广泛的生物化学测定和高灵敏度的ELISA，我们已经量化了培养细胞，动物和人体组织中的A？和Tau蛋白的各种亚型。在将这一专业知识应用于人类iPSC的过程中，我们将直接评估AD衍生的人类神经元中的A？和Tau蛋白特征，并使用该特征来预测对治疗药物的反应。 我们的机构审查委员会（IRB）申请已获得批准，29名受试者已同意参与。所有参与者都同意通过代理进行脑尸检以确认AD的诊断。我们将从生活在AD临终关怀病房的患者中获得iPSC，其中预期寿命为6个月或更短。 用于iPSC制备。将从捐献血细胞用于iPSC制备的相同个体收集脑室CSF和死后脑组织。临床诊断将通过详细的神经病理学评价和表征来证实。通过创建源自AD患者的iPSC，这项工作将产生iPSC衍生的人类神经元细胞，这些细胞携带不同的A？/Tau蛋白特征。为了避免课程中引入的变化， 为了研究iPSC的转化和分化，我们将比较在相同的神经元细胞系中，有或没有药物治疗的情况下A？/Tau的变化，并预测对不同治疗剂的反应。具体而言，我们会达致以下目标。目标1.为了确定A是否iPSC衍生的人神经元中的/Tau蛋白特征与来自同一供体的死后脑组织中的一致。已建立的方法将用于将血细胞转化为iPSC，然后分化为神经元细胞。我们将定量A？单体和寡聚体蛋白、总蛋白和pTau蛋白。我们将确定培养的神经元中A？和Tau/pTau的水平是否固有地反映在来自原始细胞供体的死后CSF和脑组织中，从而验证用于治疗测试的细胞生物标志物。目标2.确定A？和pTau水平是否预测细胞对分泌酶或激酶抑制剂的反应。将用β-分泌酶抑制剂处理具有不同水平的A β/Tau的人神经元，并且将量化对A β 42和A β寡聚体的影响。GSK 3抑制剂将用于减少Tau磷酸化。我们将确定培养的神经元中高水平的A？或pTau是否分别预测对分泌酶和激酶抑制剂的更好反应。

DESCRIPTION (provided by applicant): We propose to establish cellular biomarkers in human neurons differentiated from induced pluripotent stem cells (iPSC) of Alzheimer's disease (AD) patient origin to predict specific drug responses. These human cell lines will be used as an ex vivo cellular model to characterize key proteins driving AD pathogenesis, amyloid ¿ protein (A¿) and Tau, and to correlate to postmortem brain pathology from the same donor. We will also collect postmortem brain and CSF samples from the same donor. We will quantify A¿ monomers and oligomers, pTau and Tau levels and validate intrinsic properties of these biomarkers in cells and brain tissues. We will test the hypotheses that neurons having higher A¿ or pTau proteins respond to therapeutic agents differently. By testing this hypothesis, we will address a critical question in AD therapeutic development, whether characteristic A¿/Tau biomarkers can be used to predict therapeutic efficacy in sporadic AD (SAD) patients. Currently, biomarkers consisting of a combination of brain amyloid imaging and cerebrospinal fluid (CSF) A¿ and pTau/Tau proteins have been developed and are used in patient selection for clinical trials. There is no in vitro method of testing AD biomarkers to predict individual's brain pathology and CSF profile without performing brain amyloid imaging and CSF collection. We are uniquely positioned to carry out these studies. We have almost two-decades of experience in cellular modeling for AD research and a well-characterized patient population contributing blood cells and postmortem tissue for ex vivo modeling and in vivo confirmation. Work in my laboratory has used the mammalian cell culture model to study the functional role of presenilin in amyloid precursor protein (APP) processing and A¿ generation. Using extensive biochemical assays and highly sensitive ELISA, we have quantified various isoforms of A¿ and Tau proteins in cultured cells, animals, and human tissues. In bringing this expertise to bear on human iPSC, we will directly assess the A¿ and Tau protein signatures in AD-derived human neurons and use this signature to predict the responses to therapeutics. Our Institutional Review Board (IRB) application has been approved, and 29 subjects have been consented for participation. All participants have agreed, by proxy, to brain autopsy to confirm the diagnosis of AD. We will derive iPSC from patients living at an inpatient AD hospice unit, where life expectancy is 6 months or less. Blood cells will be used for iPSC preparation. Ventricular CSF and postmortem brain tissues will be collected from the same individuals who have donated blood cells for iPSC preparation. Clinical diagnosis will be confirmed by detailed neuropathological evaluation and characterization. By creating iPSC originating from AD patients, this work will generate iPSC-derived human neuronal cells that carry different signatures of A¿/Tau proteins. To avoid the variation introduced in the course of iPSC conversion and differentiation, we will compare the changes of A¿/Tau in same neuronal cell lines with or without pharmacologic treatment and predict responses to different therapeutic agents. Specifically, we will achieve the following aims. Aim 1. To determine whether the A¿/Tau protein signature in iPSC-derived human neurons is consistent with that in postmortem brain tissue from the same donor. Established methods will be applied to convert blood cells to iPSC, followed by differentiation into neuronal cells. We will quantify the A¿ monomer and oligomer proteins, total and pTau proteins. We will determine whether levels of A¿ and Tau/pTau in cultured neurons are inherently reflected in postmortem CSF and brain tissue from the original cell donors, validating cellular biomarkers for therapeutic tests. Aim 2. o determine whether levels of A¿ and pTau predict cellular responses to secretase or kinase inhibitors. Human neurons with different levels of A¿/Tau will be treated with a ¿-secretase inhibitor, and the effect on A¿42 and A¿ oligomers will be quantified. GSK3¿ inhibitor will be used to reduce Tau phosphorylation. We will determine whether high levels of A¿ or pTau in cultured neurons predict better responses to secretase and kinase inhibitors, respectively.