Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research, University of Maryland School of Medicine, Baltimore, MD 21201, USA; Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Exp Neurol. 2020 Jun;328:113282. doi: 10.1016/j.expneurol.2020.113282. Epub 2020 Mar 9.
Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that "map" locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.
基于细胞的呼吸仪，如 Seahorse 细胞外通量分析仪，是评估贴壁神经元以及其他细胞类型中线粒体功能的有用工具。Mito Stress Test 是最常采用的药物添加方案，用于评估线粒体生物能量功能。细胞依次暴露于 ATP 合酶抑制剂（如寡霉素）和解偶联剂（如 FCCP）会引起耗氧量的变化，从而可以估计细胞的效率和线粒体 ATP 合成能力。虽然这是评估实验处理或遗传操作是否影响线粒体能量学的有用的第一步，但 Mito Stress Test 并不能确定呼吸链功能改变的特定部位。本文讨论了 Mito Stress Test 的局限性，提出了一种用于比较细胞群体的改良方案，该方案需要在多个细胞密度下进行独立的药物滴定，并描述了一系列基于呼吸测定的逐步分析，“映射”电子传递缺陷的位置。这些策略包括测试细胞色素 c 释放、探测完整或通透细胞中特定电子传递链复合物的功能以及测量 NADH 氧化的偶联活性复合物 I、III 和 IV。为了说明其效用，我们表明，尽管 UK5099 和 ABT-737 都降低了皮质神经元的备用呼吸能力，但逐步分析揭示了不同的潜在机制，与它们既定的药物靶点一致：由线粒体丙酮酸载体抑制剂 UK5099 诱导的复合物 I 底物供应不足和由抗凋亡 BCL-2 家族蛋白抑制剂 ABT-737 诱导的细胞色素 c 释放。
