Employing Familial AD Induced Pluripotent Stem Cells to Study Neurodegeneration

描述(由申请人提供)：阿尔茨海默病(AD)全球发病率的上升加剧了开发有效的AD疗法的紧迫性。尽管付出了非凡的努力，但无论是通过药物治疗来抑制AD的进展还是开始，我们都不太成功。要做到这一点，我们需要对AD病理生物学的基本要素有更深入的了解。AD发病机制的多因素性质正变得越来越清楚，因此我们可以从理解疾病机制的广泛方法中受益，从而有效地进行靶向治疗。最近的进展揭示了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参与了AD的发病。例如，前脑神经元中两种早老素基因缺失的小鼠模型会出现类似AD的神经病理和临床特征，包括神经退行性变。我们已经证明，早老素2(PSEN2)缺乏与小胶质细胞中过度的促炎反应有关，并且FAD相关的PSEN2N141I突变导致小胶质细胞中伽玛分泌酶活性降低。我们还报道了一个与AD相关的新的PSEN2突变，该突变导致c末端早老素2(PS2)蛋白减少，进一步支持了PSEN2功能丧失与AD有关的假说。这些发现，再加上迄今为止伽玛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PSEN1功能部分丧失的报道，提出了一个关于AD发病机制的关键问题。除了神经元A�42的产生外，还有什么其他机制参与AD的发病？A�42产生的动力学和意义正在研究中；然而，中枢神经系统A�清除减少本身已被认为与AD有关。小胶质细胞是A�清除的关键介质。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的发病机制中发挥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的发病机制涉及多种细胞类型的组合功能障碍，并且PSEN2FAD突变除了先前描述的毒性功能获得外，还通过毒性功能丧失而导致疾病。我们实验室的目标是 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的非细胞自主神经变性有关。为了支持R01项目的重要性和与人类疾病的相关性，我们正在研究计划中开发更多的技术，这些技术具有解决这些假设的令人兴奋的潜力。使用患者诱导的多能干细胞(IPSCs)是检测特定突变的分子表型以及它们的细胞类型特异性效应的一种便捷方法。在华盛顿大学(UW)，我们通过使用几个关键资源，在解决上述问题方面处于独特的地位。首先，威斯康星州阿尔茨海默病研究中心(ADRC)已经储存了具有良好特征的时尚队列中的成纤维细胞。其次，我们可以利用现有的设施来衍生和鉴定诱导多能干细胞(IPSC)系。我们已经创建了多个IPSC系，这些系正在分子、表观遗传学和畸胎瘤形成能力以及其他关键的IPSC要求方面得到充分表征。FAD突变对IPSC来源的神经胶质细胞的影响以及特定的PSEN2突变对任何类型神经细胞生物学的影响尚未见报道。在这份K02提案中，我的目标是与IPSC领域的先驱合作，具有双重目的：1)为我的职业发展开发一套新的技能；2)提供有关PSEN2 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导致神经变性的潜在途径。因此，我们提出了以下实验方案。我们将研究两种不同的PSEN2突变对细胞类型的特定影响，这些突变会导致FAD。我们假设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产生、表征和评估IPSC系中的APP处理活性。B)将含有PSEN2N141I或PSEN2缺失突变(PS2del)的IPSCs分化为神经元。确定PS2缺失对这些神经元的内在电生理特性、突触生理学和伽马分泌酶活性的影响。C)将1B中使用的IPSC系分化为小胶质细胞，并评估促炎症细胞因子的释放、吞噬作用和炎症途径信号。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突变通过导致神经损伤的神经胶质功能障碍的后果而导致AD。通过采用神经元-神经胶质细胞共培养，我们将研究携带小胶质细胞的PS2FAD突变存在的神经元过程。我们将：a)在没有和存在A�42的情况下，当与野生型或PS2FAD小胶质细胞共同培养时，测量野生型神经元突触生理学。B)在野生型或PS2 FAD小胶质细胞存在的情况下，评估神经元对神经毒性的敏感性。

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.