Retrograde Signaling in Alcohol-Induced Mitochondrial Stress and Biogenesis.

描述（由申请人提供）：人们普遍认为长期酒精暴露会导致线粒体损伤。线粒体稳态的一个未探索的方面是线粒体应激反应（MSR），其调节基质伴侣蛋白的产生（例如，HSP 60和10）和响应于线粒体应激的线粒体生物发生（例如，蛋白质错误折叠或氧化修饰），如酒精所引起的。MSR和生物发生反应主要通过核基因的上调介导。我们已经开始探索这些反应的上游传感器和触发器，称为线粒体逆行信号传导，我们的初步结果已经确定了一个关键的转录辅激活因子（TORC 3），它位于线粒体基质中，似乎在线粒体损伤后易位到细胞核。因此，本申请的目的是：1）确认TORC 3在线粒体中的定位并与TORC 2的定位进行比较。TORC 2和TORC 3是肝脏中的两个转录共激活因子，其上调Hsp 60和PGC-1a。假设存在TORC 2（胞质溶胶）和TORC 3的单独区室化，（线粒体基质）将使用差速或密度梯度离心、线粒体和高分辨率共聚焦成像进行仔细测试：2）在细胞培养模型中检查TORC 3在MSR和线粒体生物发生中的作用（HepG 2细胞和原代小鼠肝细胞）的线粒体损伤，使用鱼藤酮，抗霉素A，或Cyp 2 e1在线粒体中的表达，目的是（a）表征TORC 3从线粒体释放和核转位与MSR和生物发生基因表达相关的详细时程，对线粒体氧化应激的反应（B）比较TORC 2与TORC 3向Hsp 60启动子的核转位，以及沉默TORC 2和/或TORC 3对MSR和线粒体生物发生以及对致死性线粒体损伤的易感性的影响;（c）确定CHOP和伴随的ER应激相对于TORC 2和TORC 3对线粒体应激和生物发生反应的作用;这些研究将利用实时PCR、蛋白质印迹、ChIP测定和siRNA; 3）确定吡唑诱导的Cyp 2 e1对体内MSR和线粒体生物发生的作用;初步结果证实吡唑诱导线粒体和ER Cyp 2 e1，这导致MSR;因此，吡唑治疗可以提供用于选择性线粒体损伤的方便的体内模型; 4）描述慢性灌胃酒精小鼠MSR和线粒体生物合成基因的表达模式。本申请的结果应该令人信服地表征了一种新的直接逆行信号通路从线粒体到细胞核，涉及TORC 3转录辅激活因子，定义其相对于其他信号通路的作用，并开始建立其相关性酒精损伤体内。

DESCRIPTION (provided by applicant): There is widespread agreement that chronic alcohol exposure leads to mitochondrial damage. An unexplored aspect of mitochondrial homeostasis is the mitochondrial stress response (MSR) which regulates matrix chaperone production (e.g., Hsp60 and 10) and mitochondrial biogenesis in response to stress to mitochondria (e.g., protein malfolding or oxidative modifications) as would occur from alcohol. The MSR and biogenesis responses are mediated mainly by up-regulation of nuclear genes. We have begun to explore the upstream sensor and trigger of these responses, referred to as mitochondrial retrograde signaling and our Preliminary Results have identified a key transcriptional co-activator (TORC3) which resides in the mitochondrial matrix and appears to translocate to the nucleus after mitochondrial damage. Therefore, the aims of the current application are to: 1) Confirm localization of TORC3 in mitochondria and compare with localization of TORC2. TORC 2 and 3 are two transcriptional co-activators in liver which up-regulate Hsp60 and PGC-1a. The hypothesis that there is separate compartmentation of TORC2 (cytosol) and TORC3 (mitochondria matrix) will be carefully tested defined using differential or density gradient centrifugation, mitoplasts and high resolution confocal imaging: 2) Examine the role of TORC3 in MSR and mitochondrial biogenesis in cell culture models (HepG2 cells and primary mouse hepatocytes) of mitochondrial damage using rotenone, antimycin A, or expression of Cyp2e1 in mitochondria with the goals of (a) characterizing the detailed time course of TORC3 release from mitochondria and nuclear translocation in relation to MSR and biogenesis gene expression in response to mitochondrial oxidative stress (b) comparing nuclear translocation of TORC2 versus TORC3 to Hsp60 promoter and the effect of silencing TORC2 and or TORC3 on MSR and mitochondrial biogenesis as well as susceptibility to lethal mitochondrial injury; (c) determining the role of CHOP and concomitant ER stress in relation to TORC2 and TORC3 on mitochondrial stress and biogenesis responses; these studies will utilize real-time PCR, Western blots, ChIP assays and siRNA; 3) Define the effect of pyrazole-induced Cyp2e1 on MSR and mitochondrial biogenesis in vivo; preliminary results confirm pyrazole induces mitochondrial and ER Cyp2e1 and this causes MSR; therefore, pyrazole treatment may provide a convenient in vivo model for selective mitochondrial damage; 4) Describe the expression pattern of MSR and mitochondrial biogenesis genes in chronic intragastric alcohol fed mice. The results of this application should convincingly characterize a novel direct retrograde signaling pathway from mitochondria to nucleus involving TORC3 transcription co-activator, define its role relative to other signaling pathways, and begin to establish its relevance to alcohol injury in vivo.