Rizzardini Milena, Lupi Monica, Mangolini Alessandra, Babetto Elisabetta, Ubezio Paolo, Cantoni Lavinia
Laboratory of Molecular Pathology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
Brain Res Bull. 2006 Apr 28;69(4):465-74. doi: 10.1016/j.brainresbull.2006.02.013. Epub 2006 Mar 10.
G93A Cu/Zn superoxide dismutase (SOD1), a human mutant SOD1 associated with familial amyotrophic lateral sclerosis, increased the toxicity of the mitochondrial toxin rotenone in the NSC-34 motoneuronal cell line. G93ASOD1 cells died more than untransfected and wild-type SOD1 cells after 6 and 24h exposure to 12.5 microM rotenone. Biparametric flow cytometry showed that rotenone induced rapid hyperpolarization of mitochondrial membrane potential (deltapsi(m)) in all the cell lines, followed by depolarization, and then by cell death. However, G93ASOD1 mitochondria were significantly more likely to shift from a hyperpolarized to a depolarized condition, and within the still viable cell population there was a higher proportion with depolarized mitochondria, a condition that can be envisaged as a commitment to cell death. ATP, which is needed to prevent loss of deltapsi(m), decreased more rapidly and to a greater extent in rotenone-treated G93ASOD1 cells than in the untransfected and wtSOD1cells. In all the cell lines, 1h after rotenone exposure, mitochondrial hyperpolarization was accompanied by the formation of a comparable amount of reactive oxygen species. However, G93ASOD1 cells reached the highest reactive oxygen species level since their basal level was higher than in untransfected and wild-type SOD1 cells. Our findings indicate that the mutant protein G93ASOD1 enhances the vulnerability of motor neurons to rotenone by mechanism(s) involving oxidative stress and perturbed mitochondrial homeostasis. This suggests that motor neurons from individuals carrying the mutant G93ASOD1 are at greater risk of death after inhibition of the electron transport chain.
G93A 铜/锌超氧化物歧化酶（SOD1）是一种与家族性肌萎缩侧索硬化相关的人类突变型 SOD1，它增加了线粒体毒素鱼藤酮对 NSC - 34 运动神经元细胞系的毒性。在暴露于 12.5 微摩尔鱼藤酮 6 小时和 24 小时后，G93A SOD1 细胞比未转染及野生型 SOD1 细胞死亡更多。双参数流式细胞术显示，鱼藤酮在所有细胞系中均诱导线粒体膜电位（Δψm）迅速超极化，随后去极化，进而导致细胞死亡。然而，G93A SOD1 线粒体显著更易从超极化状态转变为去极化状态，并且在仍存活的细胞群体中，线粒体去极化的比例更高，这种状态可被视为细胞死亡的前奏。用于防止 Δψm 丧失的三磷酸腺苷（ATP）在鱼藤酮处理的 G93A SOD1 细胞中比未转染及野生型 SOD1 细胞更快且更大量地减少。在所有细胞系中，鱼藤酮暴露 1 小时后，线粒体超极化伴随着相当数量活性氧的形成。然而，G93A SOD1 细胞达到的活性氧水平最高，因为其基础水平高于未转染及野生型 SOD1 细胞。我们的研究结果表明，突变蛋白 G93A SOD1 通过涉及氧化应激和线粒体稳态紊乱的机制增强了运动神经元对鱼藤酮的易损性。这表明携带突变型 G93A SOD1 的个体中的运动神经元在电子传递链受到抑制后死亡风险更高。
