Analyses of AD relevant phenotypes in neural cells derived from human iPSCs

描述（由申请人提供）：干细胞生物学的最新进展为研究人员提供了一个独特的机会，可以研究来自受影响受试者细胞的活神经元中神经系统发育疾病的分子机制。世界各地的几个实验室正在从数百名神经系统疾病患者身上产生诱导多能干细胞（iPS）细胞系。在某些情况下，受试者有已知的因果基因突变，而另一些人表达遗传风险等位基因，还有一些人没有已知的遗传易感性。虽然iPS细胞技术为研究感兴趣的细胞中遗传改变的影响提供了一个令人兴奋的进展，但确定使用该技术创建模型系统来研究遗传变异的可行性是很重要的。在这里，我们的目标是建立检测方法来研究强烈的、显性遗传的家族性阿尔茨海默病（fAD）突变和APOE等位基因变异增加AD风险的机制后果。我们建议利用干细胞的多能性，引导这些细胞向特定的神经命运分化。在Aim 1中，iPS细胞将被定向分化为AD中受影响最大的细胞类型，即海马和大脑皮层的兴奋性锥体神经元。我们将探讨AD发病机制的一些关键特征，即A¿的产生和分泌、Tau蛋白的磷酸化和神经的完整性。除了研究具有不同遗传背景的神经元细胞中这些表型的内在编程表现外，我们还将研究这些细胞对外源添加的从死后人类AD大脑中纯化的A¿的反应。在第二个目标中，我们将引导细胞走向神经胶质命运，并检查APOE基因型对这些细胞清除a¿的影响。在第三个目标中，iPS细胞将被定向到多巴胺能和运动神经元，它们在阿尔茨海默病中不那么脆弱。与Aim 1类似，将分析具有这些命运的细胞的A¿生成和清除，Tau磷酸化和神经炎完整性。通过比较具有相同遗传背景的不同细胞类型的结果，我们的目标是开始解决AD大脑中选择性易感性的机制。

DESCRIPTION (provided by applicant): Recent advances in stem cell biology provide a unique opportunity for researchers to investigate the molecular mechanisms underlying developmental diseases of the nervous system in living neurons derived from the cells of the affected subject. Several laboratories around the world are generating induced pluripotent stem (iPS) cell lines from hundreds of individuals with neurological disease. In some cases the subject has a known causal genetic mutation, while others express genetic risk alleles, and still others have no known genetic predisposition. While iPS cell technology provides an exciting inroad to study the effects of genetic alterations in the cells of interest, it is important to determine the feasibility of creating model systems to study genetic variation using this technology. Here, we aim to establish assays to investigate the mechanistic consequences of both a strong, dominantly inherited familial Alzheimer's disease (fAD) mutation and an allelic variant in APOE that increases risk for AD. We propose to take advantage of the pluripotency of stem cells, and direct the differentiation of these cells to specific neural fates. In Aim 1, iPS clls will be directed to differentiate to the cell type most affected in AD, excitatory pyramidal neuron of the hippocampus and cerebral cortex. Certain features key to AD pathogenesis will be examined, namely, generation and secretion of A¿, phosphorylation of Tau, and neuritic integrity. In addition to examining the intrinsically programmed presentation of these phenotypes in neuronal cells with varied genetic background, we also will examine the responses of these cells to exogenously added A¿ purified from postmortem human AD brain. In the second aim, we will direct the cells to a glial fate, and examine the effects of APOE genotype on clearance of A¿ by these cells. In the third aim, iPS cells will be directed to dopaminergic and motor neuron fates, which are less vulnerable in AD. Similar to Aim 1, cells with these fates will be analyzed for A¿ generation and clearance, Tau phosphorylation, and neuritic integrity. By comparing the results in different cell types with the same genetic background, we aim to begin to address the mechanisms of selective vulnerability in the AD brain. PUBLIC HEALTH RELEVANCE: Understanding how genetic alterations lead to molecular changes that ultimately lead to disease is key for the identification of novel therapeutic targets. These studies aim to utilize stem cells to establish a human neuronal model to study key features of Alzheimer's disease pathogenesis.