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

阿尔茨海默病（AD）的复杂性和多因素性对机制提出了独特的挑战。 研究和开发疗法。尽管已经产生了许多AD的转基因小鼠模型， 研究和这些模型对于我们理解疾病的病理基础很重要， 已经涵盖了整个疾病的病理。这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，迫切需要建立一个人 疾病建模平台，以补充AD研究的动物模型研究。自从十年前诱导多能干细胞（iPSC）技术出现以来，人类iPSC（hiPSC）已被广泛用于疾病建模和药物发现。然而，考虑到从hiPSC分化的细胞的相对不成熟，使用它们来建模迟发性疾病（例如AD）是具有挑战性的，其中细胞衰老在疾病病理学中是重要的。直接重编程是一种替代的细胞重编程技术，其允许将一种类型的细胞（例如成纤维细胞）直接转化为另一种类型的细胞（例如神经干细胞（NSC）或神经元）。已经表明，直接重编程能够产生具有细胞老化关键要素的人类神经元，因为这种重编程过程不经历涉及广泛表观遗传修饰的iPSC阶段。虽然AD最强的危险因素是衰老，但AD最强的遗传危险因素是载脂蛋白E4 （apoE4）。在人apoE的三种亚型（apoE 2、apoE 3和apoE 4）中，apoE 4增加AD风险， apoE 3是中性的，apoE 2是保护性的。虽然apoE 4在AD发病机制中的作用已经被证实， 尽管已经进行了广泛的研究，但令人惊讶的是，apoE 2在AD中的保护作用研究不足。很明显， 了解apoE 2在AD中保护作用的分子和细胞机制， 为抗AD药物的开发提供了新的靶点或信号通路，特别是对于晚发型AD。 本提案的目标是开发晚发性AD的衰老相关人类神经元模型，使用 直接重编程技术与CRISPR/Cas9介导的基因编辑相结合， apoE 2在AD中保护作用的潜在机制。我们提出三个互补的目标， 实现目标。目的1：建立具有apoE 2/2或apoE 3/3基因的人成纤维细胞系 来自具有apoE 4/4基因型的亲本人成纤维细胞系的基因型作为用于直接免疫的细胞来源。 重新编程目的2：探讨apoE 2在脑缺血再灌注损伤中的保护作用及其机制。 直接重编程人类神经元和星形胶质细胞。目的3：探讨apoE 2和apoE 3对心肌细胞的保护作用。 它们的潜在机制使用直接重编程的NSC衍生的神经元和星形胶质细胞。的 这些研究结果将促进我们对分子和细胞机制的理解 潜在的apoE 2在AD中的保护作用，并可能为抗AD药物开发确定新的靶点。

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.