Employing Familial AD Induced Pluripotent Stem Cells to Study Neurodegeneration

利用家族性 AD 诱导多能干细胞研究神经退行性疾病

• 批准号: 9061554
• 负责人: SUMAN JAYADEV
• 金额: $ 14.14万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9061554
• 关键词: Address; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Behavior; Biology; Cell Line; Cell physiology; Cells; Characteristics; Clinical; Clinical Trials; Coculture Techniques; Collaborations; Data; Deletion Mutation; Derivation procedure; Development; Disease; Electrophysiology (science); Elements; Family; Fibroblasts; Functional disorder; Funding; Genes; Goals; Health; Inflammatory; Inflammatory Response; Inherited; Laboratories; Lead; Measures; Mediator of activation protein; Medical Genetics; Microglia; Mutation; Nature; Nerve Degeneration; Neuroglia; Neuronal Injury; Neurons; Pathogenesis; Pathway interactions; Patients; Peptides; Phagocytosis; Pharmacotherapy; Physiology; Play; Positioning Attribute; Predisposition; Prevalence; Process; Production; Property; Prosencephalon; Reporting; Research; Resources; Role; Sampling; Signal Pathway; Synapses; Techniques; Teratoma; Therapeutic; Universities; Washington; abeta accumulation; amyloid precursor protein processing; career development; cell type; cohort; combinatorial; cytokine; familial Alzheimer disease; gain of function; gamma secretase; global health; human disease; induced pluripotent stem cell; inhibitor/antagonist; loss of function; molecular phenotype; mouse model; mutant; neuroinflammation; neurotoxicity; novel; presenilin; presenilin-1; presenilin-2; programs; secretase; skills; success; therapeutic target

DESCRIPTION (provided by applicant): The rising global prevalence of Alzheimer disease (AD) has heightened the urgency to develop effective AD therapeutics. Despite extraordinary efforts, we have been less than successful to curb either the progression or initiation of AD through drug therapy. To do so, we need a stronger understanding of the fundamental elements of AD pathobiology. The multifactorial nature of AD pathogenesis is becoming increasing clear and thus we can benefit from a broad approach to understanding disease mechanisms for effective therapeutic targeting. Recent advances have revealed factors such as partial loss of presenilin function and non-cell autonomous interactions which may contribute to AD pathogenesis. For example, murine models in which both presenilin genes are absent in forebrain neurons develop AD-like neuropathological and clinical features including neurodegeneration. We have demonstrated that presenilin 2 (PSEN2) deficiency is associated with an exaggerated pro-inflammatory response in microglia and that the fAD associated PSEN2 N141I mutation leads to decreased gamma- secretase activity in microglia. We have also reported a novel AD associated PSEN2 mutation that leads to decreased c-terminus Presenilin 2 (PS2) protein, further supporting the hypothesis that PSEN2 loss of function contributes to AD. These findings in conjunction with the lack of success thus far of gamma secretase inhibitors in clinical trials and recent reports on partial loss of PSEN1 function associated with AD raise a critical question regarding the pathogenesis of AD. In addition to neuronal A�42 production what additional mechanisms are involved in AD pathogenesis? The dynamics and significance of A�42 production are being investigated; however, decreased CNS A� clearance itself has been implicated in AD. Microglia, are key mediators of A� clearance. Therefore altered microglia behavior, as we observed with PS2 deficiency, may play a critical non-cell autonomous role in AD pathogenesis. Taking all recently available data into consideration, we hypothesize that AD pathogenesis involves combinatorial dysfunction in multiple cell types and that PSEN2 fAD mutations contribute to disease through toxic-loss-of-function in addition to the previously described toxic-gain-of-function. The goal of our laboratory is to study cell autonomous and non-cell autonomous mechanisms of neuronal injury in AD. We are pursuing an R01 funded project examining the impact of PSEN2 mutations on microglia and neuroinflammation as it relates to non-cell autonomous neurodegeneration in AD. To bolster the significance and human disease relevance of the R01 project, we are developing additional techniques in our research program with exciting potential to address these hypotheses. The use of patient derived induced pluripotent stem cells (iPSCs) is an expedient approach to examine the molecular phenotype of specific mutations as well as their cell type specific effects. At the University of Washington (UW), we are uniquely positioned to address the questions posed above by employing several key resources. First, the UW Alzheimer Disease Research Center (ADRC) has banked fibroblasts from well-characterized fAD cohorts. Second, we have access to established facilities for the derivation and characterization of induced pluripotent stem cell (iPSC) lines. We have created multiple iPSC lines which are being fully characterized molecularly, epigenetically and for capacity for teratoma formation among other crucial iPSC requirements. The impact of fAD mutations on iPSC derived glial cells and the effect of specific PSEN2 mutations on the biology of any neural cell type has not been reported. In this K02 proposal, I aim to collaborate with iPSC pioneers in the field with dual purpose to 1) develop a new skill set for my career development and 2) contribute unique information about PSEN2 fAD mutations and identify potential pathways where neuronal and glial cell processes may interact, leading to neurodegeneration. Thus, we propose the following experimental plan. We will investigate the cell type specific effects of two different PSEN2 mutations that cause fAD. We hypothesize that AD associated PSEN2 mutations lead to partial loss of PSEN2 function that will alter the behavior of neurons and microglia. To address this hypothesis we will: A) Generate, characterize and assess APP processing activity in iPSC lines from patient fibroblasts containing PS2 mutations. B) Differentiate iPSCs containing PSEN2 N141I or PSEN2 deletion mutation (PS2del) into neurons. Determine the effects of PS2 deletion on intrinsic electrophysiological properties, synaptic physiology and gamma secretase activity of these neurons. C) Differentiate the iPSC lines used in 1B into microglia and evaluate for pro-inflammatory cytokine release, phagocytosis and inflammatory pathway signaling. Next, we will study the non-cell autonomous impact of fAD PS2 mutations on the interaction between neurons and glia. We hypothesize that these two PS2 mutations contribute to AD through consequences of glial dysfunction leading to neuronal injury. By employing neuronal-glial co-cultures we will study neuronal processes in the presence of PS2 fAD mutation carrying microglia. We will: A) Measure wildtype neuronal synaptic physiology in the absence and presence of A�42 when cocultured with wildtype or PS2 fAD microglia. B) Assess neuronal susceptibility to neurotoxicity in the presence of wildtype or PS2 fAD microglia.

描述（由适用提供）：阿尔茨海默氏病（AD）的全球患病率上升，提高了开发有效的AD疗法的紧迫性。尽管做出了巨大的努力，但我们无法通过药物治疗来遏制AD的发展或主动性的成功。为此，我们需要对AD病理生物学的基本要素有更深入的了解。 AD发病机理的多因素性质变得越来越清晰，因此我们可以从了解有效治疗靶向的疾病机制中受益。最近的进步揭示了可能导致AD发病机理的部分丧失表现素功能和非细胞自主相互作用的因素。例如，在前脑神经元中不存在两个呈现蛋白基因的鼠模型，形成了广告样神经病理学和临床特征，包括神经变性。我们已经证明，presenilin 2（PSEN2）缺乏与小胶质细胞中夸大的促炎反应有关，而FAD相关的PSEN2 N141I突变会导致小胶质细胞中的γ-神经酶活性降低。我们还报道了一种新型的AD相关的PSEN2突变，导致C-末端presenilin 2（PS2）蛋白降低，进一步支持PSEN2功能丧失有助于AD的假设。这些发现与迄今为止在临床试验中缺乏成功的伽马分泌酶抑制剂以及有关与AD相关的PSEN1功能的部分丧失的报告引发了有关AD发病机理的关键问题。除了神经元A 42产生AD发病机理还涉及哪些其他机制？正在研究42生产的动态和意义；但是，在AD中隐含了CNS降低的通量本身。小胶质细胞是清除率的关键介体。因此，正如我们在PS2缺乏症中观察到的那样，小胶质细胞行为的改变可能在AD发病机理中起关键的非细胞自主作用。考虑到所有最近可用的数据，我们假设AD发病机理涉及多种细胞类型中的组合功能障碍，并且PSEN2 FAD突变除了先前描述的毒性获得功能外，还通过功能毒性造成的功能促进了疾病。我们实验室的目标 是研究AD中神经元损伤的细胞自主和非细胞自主机制。我们正在追求一个由R01资助的项目，研究PSEN2突变对小胶质细胞和神经炎症的影响，因为它与AD中的非细胞自主神经变性有关。为了增强R01项目的重要性和人类疾病相关性，我们正在研究计划中开发其他技术，具有令人兴奋的潜力来解决这些假设。使用患者衍生的诱导多能干细胞（IPSC）的使用是一种方便方法，用于检查特定突变的分子表型及其细胞类型特异性效应。在华盛顿大学（UW），我们唯一可以通过使用多个关键资源来解决上述问题。首先，阿尔茨海默氏大学疾病研究中心（ADRC）已从特征良好的时尚同胞组成了成纤维细胞。其次，我们可以访问已建立的设施，用于诱导多能干细胞（IPSC）线的推导和表征。我们创建了多个IPSC线，这些线路在分子，表观遗传上充分表征，并在其他至关重要的IPSC要求中为畸胎瘤形成的能力。尚未报道FAD突变对IPSC衍生细胞的影响以及特定PSEN2突变对任何神经细胞类型的生物学的影响。在这项K02提案中，我的目标是与该领域的IPSC先驱，双重目的达到1）为我的职业发展开发新技能，2）为PSEN2 FAD突变提供了独特的信息，并确定神经元和神经胶质细胞过程可能会相互作用的潜在途径，从而导致神经变性。那，我们提出以下实验计划。我们将研究引起FAD的两个不同PSEN2突变的细胞类型特异性效应。我们假设与AD相关的PSEN2突变导致PSEN2功能的部分丧失，这将改变神经元和小胶质细胞的行为。为了解决这一假设，我们将：a）从含有PS2突变的患者成纤维细胞中生成，表征和评估IPSC系列的应用程序处理活动。 b）将含有PSEN2 N141I或PSEN2缺失突变（PS2DEL）的IPSC区分为神经元。确定PS2缺失对这些神经元的内在电生理特性，突触生理和γ泌尿酶活性的影响。 c）将1B中使用的IPSC线区分为小胶质细胞，并评估促炎性细胞因子释放，吞噬作用和炎症途径信号传导。接下来，我们将研究FAD PS2突变对神经元与神经胶质之间相互作用的非电池自主影响。我们假设这两个PS2突变通过神经胶质功能障碍导致神经元损伤的后果有助于AD。通过采用神经元的共培养，我们将在携带小胶质细胞的PS2 FAD突变存在下研究神经元过程。我们将：a）在与野生型或PS2 FAD小胶质细胞共培养时，在不存在和存在42的情况下测量野生型神经元突触生理。 b）在存在野生型或PS2 FAD小胶质细胞的情况下，评估神经元易感性。