Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, United States.
Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, United States.
ACS Sens. 2022 Mar 25;7(3):722-729. doi: 10.1021/acssensors.1c01381. Epub 2022 Feb 28.
Oxidative stress is important for the etiology and pathogenesis of Alzheimer's disease (AD). Research tools that can conveniently evaluate oxidative stress in AD models are expected to catalyze and accelerate research on AD. This study explored the use of genetically encoded fluorescent indicators (GEFIs) to detect mitochondrial oxidative stress in organotypic brain slices and AD mouse models. To enable ratiometric normalization and avoid tissue autofluorescence, we genetically fused a green fluorescent hydrogen peroxide (HO) indicator, HyPer7, with each of two selected, bright red fluorescent proteins (RFPs), mScarlet-I and tdTomato. The resultant indicators, namely, HyPerGR and HyPerGR, were tagged with mitochondrial targeting sequences and examined for localization and function in cultured HeLa cells and primary mouse neurons. We further utilized HyPerGR, which is a genetic fusion of HyPer7 with tdTomato, to monitor mitochondrial HO in response to the human β-amyloid 1-42 isoform (Aβ) in cultured brain slices and an AD mouse model. Owing to the high sensitivity and low autofluorescence interference resulting from HyPerGR, we successfully detected Aβ-mediated mitochondrial HO in these AD models. The results suggest that HyPerGR is a valuable tool for studying mitochondrial oxidative stress in tissues and animals.
氧化应激对阿尔茨海默病（AD）的病因和发病机制很重要。人们希望能够方便地评估 AD 模型中氧化应激的研究工具，这将有助于推动 AD 研究。本研究探索了使用遗传编码荧光指示剂（GEFI）检测脑器官型切片和 AD 小鼠模型中线粒体氧化应激的方法。为了实现比率归一化并避免组织自发荧光，我们将绿色荧光过氧化氢（HO）指示剂 HyPer7 与两种选定的明亮红色荧光蛋白（RFP）mScarlet-I 和 tdTomato 分别进行基因融合。所得的指示剂分别命名为 HyPerGR 和 HyPerGR，它们带有线粒体靶向序列，并在培养的 HeLa 细胞和原代小鼠神经元中检测其定位和功能。我们进一步利用遗传融合了 tdTomato 的 HyPer7 的 HyPerGR 来监测培养脑切片和 AD 小鼠模型中人类β-淀粉样蛋白 1-42 异构体（Aβ）诱导的线粒体 HO。由于 HyPerGR 具有高灵敏度和低自发荧光干扰的特点，我们成功地在这些 AD 模型中检测到 Aβ介导的线粒体 HO。结果表明，HyPerGR 是研究组织和动物中线粒体氧化应激的一种有价值的工具。
