Arizona College of Graduate Studies, Midwestern University, Glendale, AZ, USA.
Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ, USA.
Methods Mol Biol. 2021;2276:113-127. doi: 10.1007/978-1-0716-1266-8_8.
Disruptions in mitochondrial redox activity are implicated in maladies ranging from those in which cells degenerate to those in which cell division is unregulated. This is not surprising given the pivotal role of mitochondria as ATP producers, reactive oxygen species (ROS) generators, and gatekeepers of apoptosis. While increased ROS are implicated in such a wide variety of disorders, pinpointing the cause of their hyperproduction is challenging. Elevated levels of ROS can result from increases in their production and/or decreases in their turnover. Disruptions in and/or hyperactivity of NADH-ubiquinone oxidoreductase or ubiquinone-cytochrome c oxidoreductase can cause excessive ROS generation. Alternatively, if respiration is functioning in a homeostatic manner, decreases in levels or activity of antioxidants like glutathione, CuZn- and Mn-superoxide dismutase, and catalase could result in excessive ROS. Because of the diversity of disorders in which oxidative damage occurs, the most effective therapeutic strategies may be those that address the putatively diverse causes of increased ROS. Strategies for determining antioxidant activity typically involve semiquantitative measurement of relative protein levels using immunochemistry and mass spectrometry. These methods can be applied to a variety of samples, but they do not lend themselves to detection of cell-specific analyses within tissue like brain.Because we are interested in elucidating the cause of oxidative stress in selectively vulnerable brain neurons, we have taken advantage of the easily manipulatable genetics and high fecundity of the fly. Using a cell type-targeting approach, we have driven redox sensitive green fluorescent proteins (roGFP2 ) into the mitochondria of tyrosine hydroxylase-producing (dopaminergic) neurons. In oxidizing conditions, the fluorophore's maximal excitation wavelength reversibly shifts. Therefore, the relative amount of mitochondrial protein oxidation can be determined by taking the ratio of fluorescence excited with two different lasers. In addition, these GFPs have been independently fused to human glutaredoxin-1 (mito-roGFP2-Grx1) and yeast oxidant receptor peroxidase (mito-roGFP2-Orp1), facilitating measurements of relative mitochondrial glutathione redox potential and HO levels, respectively. In order to obtain a more comprehensive observation of redox states, we capture 3D images of roGFP2 excited by two different lasers. Mito- and cytoplasmic-roGFP2 -Grx1 and -Orp1 expression can be driven by hundreds of genetic drivers in Drosophila , facilitating fixed or living whole organism or tissue- and cell-specific redox measurements.
线粒体氧化还原活性的紊乱与从细胞退化到细胞分裂不受调节的各种疾病有关。鉴于线粒体作为 ATP 产生者、活性氧 (ROS) 产生者和细胞凋亡的守门员的关键作用，这并不奇怪。虽然 ROS 的增加与如此广泛的多种疾病有关，但确定其过度产生的原因具有挑战性。ROS 水平的升高可能是由于其产生的增加和/或其周转率的降低。NADH-泛醌氧化还原酶或泛醌-细胞色素 c 氧化还原酶的紊乱和/或过度活跃会导致过多的 ROS 产生。或者，如果呼吸以动态平衡的方式发挥作用，谷胱甘肽、CuZn-和 Mn-超氧化物歧化酶和过氧化氢酶等抗氧化剂的水平或活性降低，可能会导致过多的 ROS。由于氧化损伤发生在多种疾病中，因此最有效的治疗策略可能是针对增加 ROS 的推定不同原因的策略。确定抗氧化活性的策略通常涉及使用免疫化学和质谱法对半定量测量相对蛋白质水平。这些方法可应用于各种样品，但不适合检测组织（如大脑）中的细胞特异性分析。由于我们有兴趣阐明选择性脆弱的大脑神经元中氧化应激的原因，我们利用了果蝇易于操作的遗传学和高繁殖力。使用细胞类型靶向方法，我们将氧化还原敏感的绿色荧光蛋白 (roGFP2) 驱动到产生酪氨酸羟化酶（多巴胺能）神经元的线粒体中。在氧化条件下，荧光团的最大激发波长可逆地移动。因此，可以通过用两个不同的激光激发荧光来确定线粒体蛋白氧化的相对量。此外，这些 GFP 已分别与人类谷胱甘肽还原酶 1（mito-roGFP2-Grx1）和酵母氧化剂受体过氧化物酶（mito-roGFP2-Orp1）融合，分别促进相对线粒体谷胱甘肽氧化还原电势和 HO 水平的测量。为了更全面地观察氧化还原状态，我们用两个不同的激光激发 roGFP2 捕获 3D 图像。mito-和细胞质-roGFP2-Grx1 和 -Orp1 的表达可以由果蝇中的数百个遗传驱动来驱动，这有助于进行固定或活体整个生物体或组织和细胞特异性的氧化还原测量。
