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

阿尔茨海默病(AD)的复杂性和多因素特性对机械学提出了独特的挑战 研究和开发治疗方法。虽然已经为AD建立了许多转基因小鼠模型 研究和这些模型对于我们理解疾病的病理基础是重要的，没有一个 它们中的一个已经捕捉到了疾病病理的全部光谱。这很可能是由于重要的物种。 小鼠和人类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，为AD研究补充动物模型中的研究。自十年前出现诱导多能干细胞(IPSC)技术以来，人类IPSCs(HiPSCs)已被广泛应用于疾病建模和药物开发。然而，由于从HiPSCs分化出的细胞相对不成熟，用它们来模拟迟发性疾病，如AD，是具有挑战性的，对这些疾病来说，细胞老化在疾病病理中是重要的。直接重编程是一种替代的细胞重编程技术，它允许将一种类型的细胞(如成纤维细胞)直接转换为另一种类型的细胞，如神经干细胞(NSCs)或神经元。已有研究表明，直接重编程能够产生具有细胞衰老关键成分的人类神经元，因为这种重编程过程不会经历涉及广泛表观遗传修饰的IPSC阶段。虽然AD的最强危险因素是年龄，但AD的最强遗传风险因素是载脂蛋白E4 (ApoE4)。在人类apoE的三种亚型(apo2、apoE3和apoE4)中，apoE4增加了AD的风险， APOE3是中性的，APOE2是保护性的。尽管载脂蛋白E4在AD发病机制中的作用已被 在广泛的研究中，APOE2在AD中的保护作用却出人意料地被研究得不够。显然，更好 对载脂蛋白2 S在阿尔茨海默病中保护作用的分子和细胞机制的理解将可能 为抗AD药物开发提供新的靶点或信号通路，尤其是针对晚发性AD。 这项提议的目标是开发与衰老相关的迟发性AD的人类神经元模型，使用 直接重编程技术结合CRISPR/Cas9介导的基因编辑，并剖析了 载脂蛋白2的S在AD中保护作用的机制我们提出三个相辅相成的目标 完成目标。目的1：建立含apoE2/2或apoE3/3基因的人成纤维细胞系 以apoE4/4基因为细胞来源的亲本人成纤维细胞系的基因 重新编程。目的2：分析APOE2的保护作用及其潜在的机制 直接对人类神经元和星形胶质细胞重新编程。目的3：分析APOE2和APOE2的保护作用 其潜在机制使用直接重新编程的神经干细胞来源的神经元和星形胶质细胞。这个 拟议的研究结果将促进我们对分子和细胞机制的理解。 潜在的载脂蛋白2的S在AD中的保护作用，并可能确定抗AD药物开发的新靶点。

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.