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: 9265372
• 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/9265372
• 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; Epigenetic Process; Family; Fibroblasts; Functional disorder; Funding; Genes; Genetic; Goals; Health; Inflammatory; Inflammatory Response; Inherited; Laboratories; Lead; Measures; Mediator of activation protein; Microglia; Molecular; Mutation; Nature; Nerve Degeneration; Neuroglia; Neuronal Injury; Neurons; Pathogenesis; Pathogenicity; 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; Treatment Efficacy; 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; public health relevance; 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（由申请人提供）：阿尔茨海默病（AD）全球患病率的上升增加了开发有效AD治疗剂的紧迫性。尽管付出了巨大的努力，但我们通过药物治疗来抑制AD的进展或启动并不成功。要做到这一点，我们需要更好地了解AD病理生物学的基本要素。AD发病机制的多因素性质正变得越来越清楚，因此我们可以从广泛的方法中受益，以了解疾病机制，以实现有效的治疗靶向。最近的研究发现，早老素功能的部分丧失和非细胞自主相互作用等因素可能导致AD的发病机制。例如，其中两种早老素基因在前脑神经元中均不存在的鼠模型发展出AD样神经病理学和临床特征，包括神经变性。我们已经证明早老素2（PSEN 2）缺乏与小胶质细胞中过度的促炎反应相关，并且fAD相关的PSEN 2 N141 I突变导致小胶质细胞中γ-分泌酶活性降低。我们还报道了一种新的AD相关PSEN 2突变，导致C-末端早老素2（PS2）蛋白减少，进一步支持PSEN 2功能丧失导致AD的假设。这些发现与迄今为止γ分泌酶抑制剂在临床试验中缺乏成功以及最近关于与AD相关的PSEN 1功能部分丧失的报道一起提出了关于AD发病机制的关键问题。除了神经元A β 42的产生，AD的发病机制还涉及哪些其他机制？A42产生的动力学和意义正在研究中;然而，CNS A42清除率下降本身与AD有关。小胶质细胞是A β清除的关键介质。因此，改变小胶质细胞的行为，我们观察到与PS2缺陷，可能发挥关键的非细胞自主的作用，在AD的发病机制。考虑到所有最近可用的数据，我们假设AD发病机制涉及多种细胞类型的组合功能障碍，PSEN 2 fAD突变除了先前描述的毒性获得功能外，还通过毒性丧失功能导致疾病。我们实验室的目标 目的是研究AD神经元损伤的细胞自主和非细胞自主机制。我们正在进行一项R 01资助的项目，研究PSEN 2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。为了支持R 01项目的重要性和人类疾病相关性，我们正在我们的研究计划中开发其他技术，这些技术具有解决这些假设的令人兴奋的潜力。使用患者来源的诱导多能干细胞（iPSC）是检查特定突变的分子表型及其细胞类型特异性效应的有利方法。在华盛顿大学（UW），我们有独特的优势，通过使用几个关键资源来解决上述问题。首先，UW阿尔茨海默病研究中心（ADRC）已经从充分表征的fAD队列中储存了成纤维细胞。其次，我们可以使用已建立的设施来衍生和表征诱导多能干细胞（iPSC）系。我们已经创建了多个iPSC系，这些iPSC系正在分子、表观遗传学和其他关键iPSC要求中的畸胎瘤形成能力方面进行充分表征。尚未报道fAD突变对iPSC衍生的神经胶质细胞的影响以及特异性PSEN 2突变对任何神经细胞类型的生物学的影响。在这个K 02提案中，我的目标是与该领域的iPSC先驱合作，具有双重目的：1）为我的职业发展开发一套新的技能，2）贡献有关PSEN 2 fAD突变的独特信息，并确定神经元和神经胶质细胞过程可能相互作用的潜在途径，导致神经变性。因此，我们提出以下实验方案。我们将研究导致fAD的两种不同PSEN 2突变的细胞类型特异性效应。我们假设AD相关的PSEN 2突变导致PSEN 2功能的部分丧失，这将改变神经元和小胶质细胞的行为。A）在来自含有PS2突变的患者成纤维细胞的iPSC系中产生、表征和评估APP加工活性。B）将含有PSEN 2 N141 I或PSEN 2缺失突变（PS2 del）的iPSC分化成神经元。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分泌酶活性的影响。C）将1B中使用的iPSC系分化成小胶质细胞，并评估促炎细胞因子释放、吞噬作用和炎症途径信号传导。接下来，我们将研究fAD PS2突变对神经元和胶质细胞之间相互作用的非细胞自主影响。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而导致AD。通过采用神经元-胶质细胞共培养，我们将研究在携带PS2 fAD突变的小胶质细胞存在下的神经元过程。我们将：A）当与野生型或PS2 fAD小胶质细胞共培养时，在不存在和存在A42的情况下测量野生型神经元突触生理学。B）评估在野生型或PS2 fAD小胶质细胞存在下神经元对神经毒性的易感性。