Study the Protective Roles of ApoE2 in Alzheimer's Disease Using Reprogrammed Isogenic Cells

使用重编程同基因细胞研究 ApoE2 在阿尔茨海默病中的保护作用

• 批准号: 10615690
• 负责人: YADONG HUANG
• 金额: $ 72.43万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615690
• 关键词: Aging; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease patient; Alzheimer' s disease risk; Animal Model; Apolipoprotein E; Astrocytes; CRISPR/Cas technology; Cell Aging; Cell Differentiation process; Cell Line; Cells; Complement; Disease; Disease model; Elements; Epigenetic Process; Fibroblasts; Generations; Genes; Genotype; Goals; Human; Late Onset Alzheimer Disease; Late-Onset Disorder; Mediating; Modeling; Modification; Molecular; Mus; Mutation; Nature; Neurons; Outcome; Pathogenesis; Pathologic; Pathology; Phenotype; Process; Property; Protein Isoforms; Regenerative Medicine; Reporting; Research; Role; Signal Pathway; Source; Technology; Transgenic Mice; Variant; age related; apolipoprotein E-3; apolipoprotein E-4; cell type; drug development; drug discovery; genetic risk factor; human disease; induced pluripotent stem cell; induced pluripotent stem cell technology; mouse model; nerve stem cell; novel; prevent; species difference; stem cell biology; therapy development

The complexity and multifactorial nature of Alzheimer’s disease (AD) pose unique challenges for mechanistic studies and developing therapies. Although many transgenic mouse models have been generated for AD research and these models are important for our understanding of the pathological basis of the disease, none of them has captured the entire spectrum of the disease pathologies. This is likely due to significant species differences between mouse and human neural cells. Therefore, there is an urgent need to establish human disease modeling platforms to complement studies in animal models for AD research. Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, human iPSCs (hiPSCs) have been widely used for disease modeling and drug discovery. However, given the relative immaturity of cells differentiated from hiPSCs, it is challenging to use them for modeling late-onset diseases, such as AD, for which cellular aging is important in disease pathologies. Direct reprogramming is an alternative cellular reprogramming technology, which allows direct conversion of one type of cells, such as fibroblasts, into another type of cells, such as neural stem cells (NSCs) or neurons. It has been shown that direct reprogramming enables generation of human neurons that possess key elements of cellular aging, because this reprogramming process does not go through the iPSC stage involving extensive epigenetic modifications. While the strongest risk factor for AD is aging, the strongest genetic risk factor of AD is apolipoprotein E4 (apoE4). Among the three isoforms of human apoE (apoE2, apoE3, and apoE4), apoE4 increases AD risk, apoE3 is neutral, and apoE2 is protective. Although the roles of apoE4 in AD pathogenesis have been extensively studied, the protective roles of apoE2 in AD have been surprisingly understudied. Clearly, better understanding of the molecular and cellular mechanisms underlying apoE2’s protective roles in AD will likely provide novel targets or signaling pathways for anti-AD drug development, especially for late-onset AD. The objectives of this proposal are to develop aging-relevant human neuron models of late-onset AD, using direct reprogramming technology in combination with CRISPR/Cas9-mediated gene editing, and to dissect the underlying mechanisms of apoE2’s protective roles in AD. We propose three complementary aims to accomplish the goals. Aim 1: To generate isogenic human fibroblast lines with an apoE2/2 or apoE3/3 genotype from the parental human fibroblast lines with an apoE4/4 genotype as cell sources for direct reprogramming. Aim 2: To dissect the protective roles of apoE2 and their underlying mechanisms using directly reprogrammed human neurons and astrocytes. Aim 3: To dissect the protective roles of apoE2 and their underlying mechanisms using directly reprogrammed NSC-derived neurons and astrocytes. The outcomes of the proposed studies will promote our understanding of the molecular and cellular mechanisms underlying apoE2’s protective roles in AD, and will likely identify novel targets for anti-AD drug development.

阿尔茨海默氏病（AD）的复杂性和多因素性质为机械带来了独特的挑战 研究和开发疗法。尽管已经为AD生成了许多转基因小鼠模型 研究和这些模型对于我们对疾病的病理基础很重要，没有 其中，捕获了疾病病理的整个范围。这可能是由于重要物种 小鼠和人神经细胞之间的差异。因此，迫切需要建立人类 疾病建模平台，用于在动物模型中完成AD研究的完成研究。自十年前诱导多能干细胞（IPSC）技术的推进以来，人IPSC（HIPSC）已被广泛用于疾病建模和药物发现。但是，鉴于与HIPSC分化的细胞的相对不成熟，将它们用于建模较晚发作的疾病（例如AD）对细胞衰老在疾病病理中很重要的AD。直接重编程是一种替代性细胞重编程技术，它允许将一种类型的细胞（例如成纤维细胞）直接转换为另一种类型的细胞，例如神经元（NSC）或神经元。已经表明，直接重编程使能够产生具有细胞衰老关键要素的人类神经元，因为此重编程过程并未经过涉及广泛表观遗传修饰的IPSC阶段。尽管AD的强大风险因素是衰老，但AD的强遗传风险因素是载脂蛋白E4 （APOE4）。在人类APOE（APOE2，APOE3和APOE4）的三种同工型中，APOE4增加了AD风险， APOE3是中性的，并且APOE2受到保护。尽管APOE4在AD发病机理中的作用已经 广泛研究了APOE2在AD中的保护作用令人惊讶地理解。显然，更好 了解APOE2在AD中的保护作用的分子和细胞机制可能会 为抗AD药物开发提供新的靶标或信号通路，尤其是对于晚期AD。 该提案的目标是开发与衰老相关的人类神经元模型，使用 直接重新编程技术与CRISPR/CAS9介导的基因编辑结合，并剖析 APOE2在AD中受保护角色的基本机制。我们提出了三个完整的目标 目标1：用APOE2/2或APOE3/3生成等生的人成纤维细胞系 来自亲本人类成纤维细胞系的基因型，具有APOE4/4基因型作为直接的细胞来源 重新编程。目标2：使用使用APOE2的保护作用及其基本机制 直接重编程的人类神经元和星形胶质细胞。目标3：剖析APOE2和 它们使用直接重编程的NSC衍生神经元和星形胶质细胞的基本机制。 拟议研究的结果将促进我们对分子和细胞机制的理解 APOE2在AD中的保护作用，可能会确定抗AD药物开发的新目标。