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

描述（由申请人提供）：阿尔茨海默病（AD）是最常见的痴呆，其特征在于淀粉样蛋白肽沉积为“老年性”淀粉样蛋白斑、由异常磷酸化的tau蛋白组成的神经元缠结以及神经元功能障碍和损失。目前可用的AD治疗对该疾病的影响在数量上很小，对改善患有这种衰弱性疾病的患者的生活质量或持续时间几乎没有作用，这种衰弱性疾病的临床特征是肺部和高级皮质功能的丧失。 开发既有效又安全的AD治疗的关键关键是忠实地再现人类综合征的模型系统。在这方面，在一个或多个导致早发性家族性AD（fAD）的基因中携带突变的转基因小鼠在询问潜在的治疗靶点和理解疾病的病理机制方面都有很大的帮助。然而，这些模型由于其物种而必然受到限制，并且关于小鼠是否能够忠实地模拟AD神经病理学，因为它发生在人类中，这仍然是一个悬而未决的问题。 我们的R21资助申请的中心主题是使用一种新兴技术，这种技术在模拟人类疾病方面具有很大的前景：人类诱导多能干细胞（hiPS）。基本步骤包括从携带导致fAD的基因突变的个体或从年龄匹配的对照亲属中培养皮肤成纤维细胞，并将其重新编程为hiPS细胞，这些细胞随后分化为前脑神经元。一旦分化，这些前脑神经元将进行功能性询问，以具体评估人类AD的病理标志。 我们建议分两部分进行这项工作。具体目标1的重点是从四种fAD突变体和四种相关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约175个品系，通过Coriell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fAD突变体与从重编程iPS细胞分化的对照前脑神经元中的阿尔茨海默病表型。在子目标2a中，我们假设在fAD突变型与非突变型hiPS衍生的前脑神经元中实验诱导兴奋性毒性将发生阿尔茨海默病表型并加重。子目标2b将测试当前标准治疗AD药物美金刚是否将至少部分挽救分化的fAD突变前脑神经元中的阿尔茨海默病表型的概念验证。这项探索性工作的完成预计将导致人类fAD的“盘中病”模型。这种模型可以为理解疾病的基本病理机制以及潜在的治疗方法铺平道路。 公共卫生相关性：现在有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，这突出表明公共卫生危机正在迅速发展。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。如果成功的话，这种令人兴奋的盘中疾病模型可以允许治疗方法的临床前测试。

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.