Mitoflash和蛋白组研究揭示线粒体在线虫衰老中的重要作用
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摘要
生物衰老的时钟理论已经被提出几十年1,暗示线粒体是调节衰老的核心因素,但是直到目前为止,仍缺少支持证据。在本研究中,通过活体线虫的单个线粒体水平观察超氧炫来研究线粒体与生物衰老理论。线粒体超氧炫作为读码器,可以反映单个线粒体的活性氧自由基的产生和能量代谢状态2。本实验室的早期研究已经有很多发现,例如,在线虫成年第三天和第九天的咽部肌肉细胞中会分别出现两个非常强的超氧炫信号峰,第一个可能与线虫的产卵有关,第二个可能与线虫的死亡有关。
我们发现电子传递链的活性与超氧炫的发生密切相关,而且,饥饿或者激光诱导荧光蛋白所产生的超氧化合物也能够分别抑制或者增加超氧炫活性,表明超氧炫与线粒体的氧化状态和代谢活性有密切联系。此外,我采用RNAi沉默靶基因的方法使线虫寿命延长或缩短,发现两个超氧炫信号峰的位置在长寿或者短寿的线虫中确实会出现明显移动,短寿线虫的信号峰前移,长寿线虫的信号峰后移,该结果揭示了线粒体超氧炫与衰老之间有密切关系。用24种环境因素影响线虫寿命和相应的超氧炫信号,发现这些成虫第三天的超氧炫信号强度与它们的寿命呈负相关。除环境因素外,也验证了随机因素对寿命和超氧炫信号的影响,分别在长寿age-1和短寿elo-5线虫的单个个体中进行比较,发现超氧炫信号与单个线虫的寿命也呈负相关,该结果与环境因素所引起的变化非常相似。最后,研究胰岛素信号通路调节的衰老与超氧炫的关系。我们发现线粒体复合物Ⅱ和乙醛酸循环活性在长寿线虫daf-2(e1370)中上调,推测可能与daf-2突变株的长寿有关。通过研究异柠檬酸裂解酶ICL-1与寿命和超氧炫信号的关系,我们发现该酶贡献了daf-2的长寿和使超氧炫信号降低。总之,遗传因素、环境因素和随机因素是决定生物体寿命的主要因素,线粒体在这些因素引起的寿命变化中起着重要的作用。而且成虫第三天的超氧炫信号能够指示寿命。这些结果与衰老时钟理论非常吻合,暗示着在相当大程度上线虫的衰老速度在成年早期已经被决定了。
抑制胰岛素信号通路(IIS)可以延长线虫,果蝇和小鼠的寿命3。考虑到线粒体在生物衰老过程中起着重要作用4,5,我们结合定量线粒体蛋白组方法,分析线粒体在IIS中的作用。DAF-2是胰岛素受体,DAF-16是IIS下游的负调控转录因子。我们纯化了野生型线虫N2、突变体线虫daf-2和daf-2; daf-16的线粒体,通过定量蛋白组的方法分析了线粒体蛋白的丰度,鉴定了1824个线粒体蛋白。其中,有83个蛋白的表达在daf-2突变体中被上调,60个蛋白的表达被下调。通过Gene Ontology和KEGG对所有定量蛋白进行分析,发现参与线虫寿命、过氧化物清除和线粒体脂肪酸、乙醛酸、丙酸和支链氨基酸代谢的相关过程都上调。代谢网络的构建表明daf-2突变体的线粒体代谢状态处于一种和谐的上调模式。通过对该模式的上调因子进行研究,我们发现它们对daf-2突变体的长寿起着重要作用。
It has been theorized for decades that mitochondria act as the biological clock of ageing1, but direct evidence is lacking. Here, we investigated the mitochondrial theory of ageing by in vivo visualization of the mitochondrial flash (mitoflash), a frequency-coded optical readout reflecting free-radical production and energy metabolism at the single-mitochondrion level2. In the previous study, the rate of occurrence of spontaneous mitoflashes in C. elegans pharyngeal muscles peaked on day3during activere production and on day9when animals started to die off. Inhibition of electron transport chain (ETC) strongly decreases mitoflash. Moreover, photo-induced free radical production clearly activates the singal of mitoflash. RNAi of age-1or elo-5caused longer or shorter lifespan, respectively. In the animals with longevity, peaks of mitoflash shifted backward, contrastly, peaks of mitoflash shifted forward in short-lived animals. A plethora of24environmental factors inversely modified the lifespan and the day-3mitoflash frequency. Effects of stochastic factors on individual lifespan and mitoflash was also investigated in animals with longer and shorter lifespan, the result is very similar to that in environmental treatment. Furthermore, we showed that reduced mitochondrial superoxide production in early adulthood due to coordinately enhanced complex II activity and the glyoxylate cycle contributed to the longevity of daf-2(e1370) mutant animals. RNAi of icl-1, an isocitrate lyase, could shorten the lifespan of daf-2mutant and meantime increase mitoflash.
Collectively, these results provide compelling evidence bolstering the central role of mitochondria in lifespan regulation by genetic, environmental, and stochastic factors.That day-3mitoflash frequency is a unifying predictor of C. elegans lifespan supports the notion that the rate of aging, although adjustable in later life, has been set to a considerable degree before reproduction ceases.
The insulin/insulin-like signaling pathway (IIS) is an evolutionarily conserved mechanism that regulates aging3. Given the importance of mitochondria in the aging process, we decided to investigate IIS-mediated the mitochondrial functional fluctuations by quantitative proteomics approach4,5. DAF-2acts as insulin receptor, and DAF-16is a main downstream target negatively regulated by IIS. We performed quantitative proteomic analysis of mitochondria purified from N2(wild type, WT), a long-lived daf-2(e1370) mutant, and a slightly short-lived daf-2(e1370); daf-16(mu86) double mutant.1824potential mitochondrial proteins were identified, of which83were up-regulated and60were down-regulated in daf-2compared to WT and daf-2; daf-16. Gene Ontology and KEGG analysis of all the quantitative proteins showed that aging-related processes like adult lifespan and radical oxygen species (ROS) defense were in up-regulatioin, as well as mitochondrial intermediary metabolims including fatty acid degradation, proproate catabolism, glyoxylate and branch chain amino acid metabolism were also up-regulated. Interconnect network of these metabolims was constructed and suggested a coherently modulated module. Inactivation of some candidates involving in these metabolisms by RNA intereference (RNAi) revealed that the coordinated mediated catabolism exerted a potential effect on the healthy longevity in daf-2mutant.
我们发现电子传递链的活性与超氧炫的发生密切相关,而且,饥饿或者激光诱导荧光蛋白所产生的超氧化合物也能够分别抑制或者增加超氧炫活性,表明超氧炫与线粒体的氧化状态和代谢活性有密切联系。此外,我采用RNAi沉默靶基因的方法使线虫寿命延长或缩短,发现两个超氧炫信号峰的位置在长寿或者短寿的线虫中确实会出现明显移动,短寿线虫的信号峰前移,长寿线虫的信号峰后移,该结果揭示了线粒体超氧炫与衰老之间有密切关系。用24种环境因素影响线虫寿命和相应的超氧炫信号,发现这些成虫第三天的超氧炫信号强度与它们的寿命呈负相关。除环境因素外,也验证了随机因素对寿命和超氧炫信号的影响,分别在长寿age-1和短寿elo-5线虫的单个个体中进行比较,发现超氧炫信号与单个线虫的寿命也呈负相关,该结果与环境因素所引起的变化非常相似。最后,研究胰岛素信号通路调节的衰老与超氧炫的关系。我们发现线粒体复合物Ⅱ和乙醛酸循环活性在长寿线虫daf-2(e1370)中上调,推测可能与daf-2突变株的长寿有关。通过研究异柠檬酸裂解酶ICL-1与寿命和超氧炫信号的关系,我们发现该酶贡献了daf-2的长寿和使超氧炫信号降低。总之,遗传因素、环境因素和随机因素是决定生物体寿命的主要因素,线粒体在这些因素引起的寿命变化中起着重要的作用。而且成虫第三天的超氧炫信号能够指示寿命。这些结果与衰老时钟理论非常吻合,暗示着在相当大程度上线虫的衰老速度在成年早期已经被决定了。
抑制胰岛素信号通路(IIS)可以延长线虫,果蝇和小鼠的寿命3。考虑到线粒体在生物衰老过程中起着重要作用4,5,我们结合定量线粒体蛋白组方法,分析线粒体在IIS中的作用。DAF-2是胰岛素受体,DAF-16是IIS下游的负调控转录因子。我们纯化了野生型线虫N2、突变体线虫daf-2和daf-2; daf-16的线粒体,通过定量蛋白组的方法分析了线粒体蛋白的丰度,鉴定了1824个线粒体蛋白。其中,有83个蛋白的表达在daf-2突变体中被上调,60个蛋白的表达被下调。通过Gene Ontology和KEGG对所有定量蛋白进行分析,发现参与线虫寿命、过氧化物清除和线粒体脂肪酸、乙醛酸、丙酸和支链氨基酸代谢的相关过程都上调。代谢网络的构建表明daf-2突变体的线粒体代谢状态处于一种和谐的上调模式。通过对该模式的上调因子进行研究,我们发现它们对daf-2突变体的长寿起着重要作用。
It has been theorized for decades that mitochondria act as the biological clock of ageing1, but direct evidence is lacking. Here, we investigated the mitochondrial theory of ageing by in vivo visualization of the mitochondrial flash (mitoflash), a frequency-coded optical readout reflecting free-radical production and energy metabolism at the single-mitochondrion level2. In the previous study, the rate of occurrence of spontaneous mitoflashes in C. elegans pharyngeal muscles peaked on day3during activere production and on day9when animals started to die off. Inhibition of electron transport chain (ETC) strongly decreases mitoflash. Moreover, photo-induced free radical production clearly activates the singal of mitoflash. RNAi of age-1or elo-5caused longer or shorter lifespan, respectively. In the animals with longevity, peaks of mitoflash shifted backward, contrastly, peaks of mitoflash shifted forward in short-lived animals. A plethora of24environmental factors inversely modified the lifespan and the day-3mitoflash frequency. Effects of stochastic factors on individual lifespan and mitoflash was also investigated in animals with longer and shorter lifespan, the result is very similar to that in environmental treatment. Furthermore, we showed that reduced mitochondrial superoxide production in early adulthood due to coordinately enhanced complex II activity and the glyoxylate cycle contributed to the longevity of daf-2(e1370) mutant animals. RNAi of icl-1, an isocitrate lyase, could shorten the lifespan of daf-2mutant and meantime increase mitoflash.
Collectively, these results provide compelling evidence bolstering the central role of mitochondria in lifespan regulation by genetic, environmental, and stochastic factors.That day-3mitoflash frequency is a unifying predictor of C. elegans lifespan supports the notion that the rate of aging, although adjustable in later life, has been set to a considerable degree before reproduction ceases.
The insulin/insulin-like signaling pathway (IIS) is an evolutionarily conserved mechanism that regulates aging3. Given the importance of mitochondria in the aging process, we decided to investigate IIS-mediated the mitochondrial functional fluctuations by quantitative proteomics approach4,5. DAF-2acts as insulin receptor, and DAF-16is a main downstream target negatively regulated by IIS. We performed quantitative proteomic analysis of mitochondria purified from N2(wild type, WT), a long-lived daf-2(e1370) mutant, and a slightly short-lived daf-2(e1370); daf-16(mu86) double mutant.1824potential mitochondrial proteins were identified, of which83were up-regulated and60were down-regulated in daf-2compared to WT and daf-2; daf-16. Gene Ontology and KEGG analysis of all the quantitative proteins showed that aging-related processes like adult lifespan and radical oxygen species (ROS) defense were in up-regulatioin, as well as mitochondrial intermediary metabolims including fatty acid degradation, proproate catabolism, glyoxylate and branch chain amino acid metabolism were also up-regulated. Interconnect network of these metabolims was constructed and suggested a coherently modulated module. Inactivation of some candidates involving in these metabolisms by RNA intereference (RNAi) revealed that the coordinated mediated catabolism exerted a potential effect on the healthy longevity in daf-2mutant.
引文
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