An iPS Disease-in-a-Dish Model of Familial Alzheimers

描述(申请人提供)：阿尔茨海默病(AD)是最常见的痴呆症，其特点是淀粉样多肽的沉积，即‘老年性’-淀粉样斑块，由异常磷酸化的tau蛋白组成的神经纤维缠结，以及神经元功能障碍和丢失。目前可用的AD治疗在数量上对疾病的影响很小，对改善患有这种衰弱疾病的患者的生活质量或持续时间几乎没有作用，这种疾病的临床特征是丧失肺脏和更高的皮质功能。开发既有效又安全的阿尔茨海默病治疗方法的一个关键关键是能够忠实地概括人类综合症的模型系统。在这方面，携带导致早发性家族性阿尔茨海默病(FAD)的一个或多个基因突变的转基因小鼠在询问潜在的治疗靶点和了解疾病的病理机制方面都有巨大的帮助。然而，由于它们的物种，这些模型必然是有限的，对于小鼠是否能够忠实地模拟人类AD的神经病理学，仍然是一个悬而未决的问题。我们R21拨款申请的中心主题是使用一种在模拟人类疾病方面前景广阔的新兴技术：人类诱导多能干细胞(HIPS)。基本步骤包括培养携带导致FAD的基因突变的个体的皮肤成纤维细胞，或者来自年龄匹配的没有疾病的对照亲属的皮肤成纤维细胞，并将它们重新编程为髋关节细胞，这些细胞随后分化为前脑谷氨酸能神经元。一旦分化，这些前脑神经元将被功能询问，以专门评估人类AD的病理特征。我们建议分两部分进行这项工作。具体目标1的重点是从4个FAD突变型和4个相关的对照成纤维细胞系建立iPS细胞系。我们将提取成纤维细胞 来自携带FAD突变的个体和年龄匹配的对照亲属的~175个系，由科里尔医学研究所的NIH/NIA衰老细胞培养库维护。具体目标2的主要目标是询问从重新编程的iPS细胞分化出的FAD突变和对照前脑神经元中的阿尔茨海默病表型。在次目标2a中，我们假设阿尔茨海默病的表型将会发生，并通过实验诱导FAD突变型与非突变型HIPS衍生的前脑神经元的兴奋毒性而加剧。次目标2b将测试概念验证，以确定当前治疗AD的标准药物美金刚是否至少部分挽救了分化的FAD突变前脑神经元的阿尔茨海默病表型(S)。这项探索性工作的完成有望带来一种人类时尚的“盘中病”模型。这样的模型可以为理解这种疾病的基本病理机制以及潜在的治疗方法铺平道路。 与公共卫生相关：目前有300多万美国人患有阿尔茨海默病，预计到2050年，这一数字将增加到900万，突显出一场迅速发展的公共卫生危机。我们建议利用尖端的人类诱导多能干细胞(HIPS)技术在培养的神经元中模拟这种毁灭性的疾病。如果成功，这种令人兴奋的碟中病模型可能会允许对治疗方法进行临床前测试。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common dementia, and is hallmarked by deposition of amyloid-¿ peptides as 'senile' ¿-amyloid plaques, neurofibrillary tangles comprised of abnormally phosphorylated tau protein, and neuronal dysfunction and loss. Currently available AD treatments have a quantitatively minor impact on the disease, doing little to improve the quality or duration of life of patients suffering from thi debilitating illness, which is clinically characterized by loss of pneumonic and higher cortical functions. A critical lynchpin for the development of an AD treatment that is both effective and safe is model systems that faithfully recapitulate the human syndrome. In this regard, transgenic mice harboring mutations in one or more genes that cause early-onset familial AD (fAD) have been enormously helpful, both in terms of interrogating potential therapeutic targets and also for understanding pathological mechanisms of disease. Yet, these models are necessarily limited due to their species, and it remains an open question as to whether the mouse will ever be able to faithfully model AD neuropathology as it occurs in the human. The central theme of our R21 grant application is to use an emerging technology with great promise for modeling human diseases: human induced pluripotent stem (hiPS) cells. The basic steps involve culturing skin fibroblasts from individuals bearing mutations in genes that cause fAD or from age-matched control relatives lacking disease, and reprogramming them into hiPS cells that are later differentiated into forebrain glutamatergic neurons. Once differentiated, these forebrain neurons will be functionally interrogated to specifically assess pathologic hallmarks of human AD. We propose to carry out this work in two parts. The focus of Specific Aim 1 is to establish iPS cell lines from four fAD mutant and four related control fibroblast cell lines. We will draw fibroblasts from ~175 lines derived from individuals bearing fAD mutations and age-matched control relatives, maintained through the NIH/NIA Aging Cell Culture Repository at the Coriell Institute for Medical Research. The main goal of Specific Aim 2 is to interrogate Alzheimer phenotypes in fAD mutant vs. control forebrain neurons differentiated from reprogrammed iPS cells. In Sub-Aim 2a, we hypothesize that Alzheimer disease phenotypes will occur and be exacerbated by experimental induction of excitotoxicity in fAD mutant vs. non-mutant hiPS-derived forebrain neurons. Sub-Aim 2b will test proof-of-concept for whether the current standard of care AD drug, memantine, will at least partially rescue Alzheimer phenotype(s) in differentiated fAD mutant forebrain neurons. Completion of this exploratory work is expected to lead to a 'disease-in-a-dish' model of human fAD. Such a model could pave the way toward understanding both basic pathologic mechanisms of the disease as well as potential therapeutic approaches. PUBLIC HEALTH RELEVANCE: There are now over 3 million Americans afflicted with Alzheimer's disease, a figure that is projected to increase to 9 million by 2050, underscoring a rapidly developing public health crisis. We propose to utilize cutting-edge human induced pluripotent stem (hiPS) cell technology to model this devastating disease in cultured neurons. If successful, this exciting disease-in-a-dish model could allow pre-clinical testing of therapeutic approaches.