Introducing Cellular Aging in Human iPS Cells to Investigate Alzheimer Pathogenesis

摘要 虽然年龄增长是阿尔茨海默病（AD）的主要危险因素，但目前尚不清楚AD的发病率为何 会随着年龄的增长而急剧上升这一提议将开发出一种强大的人类诱导多能干细胞 (iPS)细胞资源，以促进我们对衰老对AD细胞发展的影响的理解 病理学;特别是细胞衰老，淀粉样前体蛋白（APP）和三体21之间的相互作用。 我们的研究将利用现有的来自唐氏综合征（DS）的iPS细胞等基因组 在一些实施方案中，本发明的细胞将包括非DS AD患者来源的iPS细胞，但也将包括非DS AD患者来源的iPS细胞。早发性AD痴呆几乎不可避免 在患有DS的个体中，他们也明显表现出过早衰老。基本上所有患有DS的人都有 淀粉样蛋白脑病变早在青春期就开始了。我们的初步结果和其他出版的 有证据表明，从DS患者来源的iPS细胞分化的神经元表现出AD的多种标志 细胞病理学，并提供了一个强大的人类细胞模型来研究AD发病机制。劳伦斯实验室 创建了一个充分表征的、全等基因的DS患者源性细胞系组，其中一些是工程化的 通过XIST RNA的诱导表达沉默一个Chr21，而其他的是由于自发的 失去一个Chr21。为了在此基础上研究衰老的影响，长期从事这项研究的张实验室 研究细胞衰老和衰老，将与劳伦斯实验室联手，在多个细胞中诱导细胞衰老。 通过两种不同的方法建立AD iPS细胞系。使用CRISPR/Cas9技术，我们将针对两个重要的 细胞衰老的调节因子端粒酶和Klotho几种AD细胞病理（A β 42分泌、淀粉样蛋白 聚集体、tau磷酸化、4R-tau产生、内体增大和细胞损失）将在 iPS细胞在3D类器官培养物中分化为皮质神经元、神经胶质和其他神经细胞。除了DS， 将与Tracy Young-Pearse合作研究多种家族性或散发性AD iPS细胞系 （哈佛医学院）。目的1是以端粒酶为靶点，诱导iPS细胞， 缩短，这是衰老细胞的特征。这些细胞为细胞衰老做好准备，但只有在 大量的增殖，使我们能够解决这种形式的细胞老化在分化的神经细胞中的影响。 细胞目的2将操纵Klotho的表达，Klotho的缺乏与衰老密切相关， 在小鼠神经退行性变的小鼠模型中，其增加的表达减轻了认知衰退。 ad.此外，我们将测试使用Klotho蛋白的概念，该蛋白具有减轻对AD的衰老影响的潜力。 发病机制这项研究具有很高的潜力，可以产生广泛有价值的细胞资源， 解决几个具体的假设，这将促进我们对衰老和 AD发病机制

ABSTRACT While advancing age is a major risk factor of Alzheimer’s disease (AD), it is unclear why the incidence of AD rises dramatically as we grow older. This proposal will develop a powerful human induced pluripotent stem (iPS) cell resource to advance our understanding of the impact of aging on the development of AD cell pathology; particularly on the interplay between cellular aging, amyloid precursor protein (APP) and trisomy 21. Our studies will capitalize on an existing isogenic panel of iPS cells derived from a Down Syndrome (DS) patient, but will also include non-DS AD patient-derived iPS cells. Early-onset AD dementia is almost inevitable in individuals with DS, who notably also exhibit premature aging. Essentially all individuals with DS have amyloid brain pathology beginning as early as adolescence. Our preliminary results and other published evidence indicate that neurons differentiated from DS patient-derived iPS cells exhibit multiple hallmarks of AD cell pathology and provide a robust human cell model to study AD pathogenesis. The Lawrence lab has created a well characterized, all-isogenic panel of DS patient-derived cell lines, some of which are engineered to silence one Chr21 by inducible expression of XIST RNA, and others are normal disomic due to spontaneous loss of one Chr21. To build on this foundation to study the impact of aging, the Zhang lab, which has long studied cellular senescence and aging, will join forces with the Lawrence lab to induce cellular aging in multiple AD iPS cell lines by two different approaches. Using the CRISPR/Cas9 technology, we will target two important regulators of cellular aging, telomerase and Klotho. Several AD cell pathologies (Aβ42 secretion, amyloid aggregates, tau phosphorylation, 4R-tau production, endosome enlargement and cell loss) will be examined in iPS cells differentiated to cortical neurons, glia and other neural cells in 3D organoid cultures. In addition to DS, multiple familial or sporadic AD iPS cell lines will be studied through collaboration with Tracy Young-Pearse (Harvard Medical School). Aim 1 will target telomerase to produce iPS cells which undergo telomere shortening, a characteristic of aging cells. These cells become poised for cellular senescence but only after substantial proliferation, allowing us to address the impact of this form of cellular aging in differentiated neural cells. Aim 2 will manipulate the expression of Klotho, deficiency of which is strongly linked to aging and neurodegeneration in mice, and increased expression of which mitigates cognitive decline in a mouse model of AD. Further, we will test a concept of using Klotho protein with potential to mitigate aging effects on AD pathogenesis. This research has high potential to produce broadly valuable cellular resources as well as address several specific hypotheses that will advance our understanding of the interplay between aging and AD pathogenesis.