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）的全球患病率上升，提高了开发有效的AD治疗方法的紧迫性。尽管我们付出了巨大的努力，但通过药物治疗来遏制阿尔茨海默病的进展或发病，我们还不够成功。要做到这一点，我们需要对阿尔茨海默病病理生物学的基本要素有更深入的了解。阿尔茨海默病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来了解疾病机制，从而有效地靶向治疗。最近的进展揭示了一些因素，如早老素功能的部分丧失和非细胞自主相互作用可能有助于AD的发病机制。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ad样神经病理和临床特征，包括神经变性。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PSEN2 N141I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PSEN2突变，该突变导致c端早老素2 （PS2）蛋白减少，进一步支持了PSEN2功能丧失导致AD的假设。这些发现，加上迄今为止伽马分泌酶抑制剂在临床试验中缺乏成功，以及最近关于PSEN1功能部分丧失与AD相关的报道，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，AD的发病还涉及哪些其他机制？目前正在研究a42生产的动态和意义；然而，CNS A清除率降低本身与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缺陷中观察到的那样，小胶质细胞行为的改变可能在阿尔茨海默病的发病机制中起着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，PSEN2 fAD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导致疾病。我们实验室的目标

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.