Retrograde Signaling in Alcohol-Induced Mitochondrial Stress and Biogenesis.

描述（由申请人提供）：人们普遍认为长期饮酒会导致线粒体损伤。线粒体稳态的一个未被探索的方面是线粒体应激反应（MSR），它调节基质伴侣蛋白的产生（例如，Hsp60和10）和线粒体生物发生，以响应线粒体应激（例如，蛋白质折叠不良或氧化修饰），如酒精引起的。MSR和生物发生反应主要是由核基因上调介导的。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，我们的初步结果已经确定了一个关键的转录共激活因子（TORC3），它位于线粒体基质中，在线粒体损伤后似乎会转移到细胞核。因此，本应用的目的是：1)确认TORC3在线粒体中的定位，并与TORC2的定位进行比较。TORC 2和3是肝脏中的两种转录共激活因子，可上调Hsp60和PGC-1a。假设TORC2（细胞质）和TORC3（线粒体基质）存在独立的区隔，将通过差速或密度梯度离心、有丝分裂体和高分辨率共聚焦成像进行仔细测试。2)在鱼藤酮、抗霉素A、线粒体损伤的细胞培养模型（HepG2细胞和原代小鼠肝细胞）中，研究TORC3在MSR和线粒体生物发生中的作用。目的是(a)表征线粒体氧化应激下TORC3从线粒体释放和核易位与MSR和生物发生基因表达相关的详细时间过程(b)比较TORC2与TORC3对Hsp60启动子的核易位以及沉默TORC2和/或TORC3对MSR和线粒体生物发生以及致死性线粒体损伤易感的影响；(c)确定CHOP和伴随的内质网应激与TORC2和TORC3在线粒体应激和生物发生反应中的作用；这些研究将利用实时PCR、Western blots、ChIP分析和siRNA；3)确定吡唑诱导的Cyp2e1对体内MSR和线粒体生物发生的影响；初步结果证实吡唑诱导线粒体和内质网Cyp2e1，引起MSR；因此，吡唑治疗可能为选择性线粒体损伤提供一种方便的体内模型；4)描述慢性灌胃酒精喂养小鼠中MSR和线粒体生物发生基因的表达模式。这一应用的结果将令人信服地描述一种涉及TORC3转录共激活子的从线粒体到细胞核的新型直接逆行信号通路，确定其相对于其他信号通路的作用，并开始确定其与体内酒精损伤的相关性。

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.