Mitochondrial (MT) dysfunction is a hallmark of Alzheimer’s Disease (AD), but the scope and severity of these specific deficits across forms of AD are not well characterized. We designed a high-throughput, longitudinal, phenotypic assay to track MT dynamics and bioenergetics in glutamatergic iPSC-derived human neurons possessing mutations in presenilin 1 (PSEN1), presenilin 2 (PSEN2) and the amyloid beta precursor protein (APP). Each gene set was comprised of iPSC-derived neurons from an AD patient as well as two to three engineered mutations with appropriate isogenic and age matched controls. These iPSC-derived neurons were imaged every other day beginning at DIV10 to assess how MT length and content change over a ten day time course using a mitochondrially targeted reporter. A second cytosolic reporter allowed for visualization of neurites. Bioenergetics assays, focusing on MT respiration and individual electron transport chain (ETC) complexes, were also surveyed over this time course. Mutations in all three genes altered MT function measured by basal, ATP-linked, and maximal oxygen consumption rate; and spare respiratory capacity, with PSEN1/PSEN2 alleles being more severe than APP mutations. Electron flow through Complexes I-IV was decreased in PSEN1/PSEN2 mutations but; in contrast, APP alleles had only modest impairments of Complexes I and II. We measured aspects of MT dynamics, including fragmentation, and neurite degeneration, both of which were dramatic in PSEN1/PSEN2 alleles, but essentially absent in APP alleles. The marked differences in MT pathology may occur from the distinct ways amyloids are processed into amyloid beta peptides (Aβ) and may correlate with the disease severity.

中文翻译：

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阿尔茨海默病诱导的多能干细胞衍生神经元的线粒体动力学和生物能量学


线粒体 （MT） 功能障碍是阿尔茨海默病 （AD） 的标志，但不同形式的 AD 的这些特定缺陷的范围和严重程度尚未得到很好的表征。我们设计了一种高通量、纵向、表型测定法来跟踪具有早老素 1 （PSEN1）、早老素 2 （PSEN2） 和淀粉样蛋白 β 前体蛋白 （APP） 突变的谷氨酸能 iPSC 衍生的人类神经元中的 MT 动力学和生物能量学。每个基因集由来自 AD 患者的 iPSC 衍生神经元以及 2 到 3 个具有适当同基因和年龄匹配对照的工程突变组成。从 DIV10 开始，每隔一天对这些 iPSC 衍生的神经元进行成像，以使用线粒体靶向报告基因评估 MT 长度和内容在 10 天的时间过程中如何变化。第二个胞质报告基因允许神经突可视化。在此期间，还调查了专注于 MT 呼吸和单个电子传递链 （ETC） 复合物的生物能量学分析。所有三个基因的突变都改变了通过基础、ATP 相关和最大耗氧率测量的 MT 功能;和备用呼吸能力，其中 PSEN1/PSEN2 等位基因比 APP 突变更严重。在 PSEN1/PSEN2 突变中，通过复合物 I-IV 的电子流减少，但是;相比之下，APP 等位基因对复合物 I 和 II 只有适度的损害。我们测量了 MT 动力学的各个方面，包括断裂和神经突变性，这两者都在 PSEN1/PSEN2 等位基因中是显着的，但在 APP 等位基因中基本上不存在。MT 病理学的显着差异可能是由于淀粉样蛋白加工成淀粉样蛋白 β 肽 （Aβ） 的不同方式造成的，并且可能与疾病的严重程度有关。