Amelioration of Mitochondrial Dysfunction by Thioredoxin in Hyperoxia

 描述(申请人提供)：氧疗是一种常见的临床必需品，但它伴随着显著的副作用。具体地说，它产生的高氧状态会产生许多活性氧物种，包括导致线粒体功能障碍的超氧阴离子。尽管这种毒性是氧疗应用中的一个关键因素，但人们对高氧如何影响线粒体能量产生，或能够提供保护作用的蛋白质再生机制知之甚少。我们的研究项目集中在硫氧还蛋白(TRX)上，这是一种细胞质氧化还原蛋白，可以减少氧化应激并再生氧化失活的酶。最近，我们发表了硫氧还蛋白表达增加对高氧下Trx-TG小鼠的肺损伤和存活的保护作用，但Trx低表达的小鼠对高氧更敏感，并出现显著的死亡率。然而，高水平硫氧还蛋白保护肺损伤的机制仍不清楚。我们的初步数据表明，在高氧期间，细胞质Trx1移位到线粒体，但这种移动在dnTrx-TG小鼠中没有发生。这些发现促使我们假设，TRX通过UCP-2依赖的解偶联来减少氧化应激，进而保护线粒体在高氧中的功能障碍，从而保护线粒体免受高氧的影响，因为线粒体中超氧阴离子的产生是肺氧中毒的关键机制。因此，在目标1中，我们将确定移位的细胞质Trx1是否以及如何保护高氧下的线粒体功能障碍。在目标2中，我们将发现高水平的Trx是否可以阻止动力蛋白相关蛋白(Drp1)的激活，从而保护线粒体免受断裂和功能障碍的影响。在目标3中，我们将确定在Trx-TG小鼠中增加pGC-1α到细胞核的易位是否可以保护高氧引起的线粒体功能障碍。利用最先进的技术，包括线粒体通量分析、EPR光谱分析、生化酶分析和尖端分子方法，我们将剖析Trx1在高氧中保护功能障碍的线粒体中所起的作用。我们还将创建一种新型的条件性TRX基因敲除小鼠，一种TRX表达增加或减少的PGC1a基因敲除小鼠，以在体内了解高水平TRX在高氧下线粒体功能障碍中的作用。该项目有望为了解胞质Trx1在常氧和高氧期间影响线粒体功能的方式提供清晰的理解。利用转基因小鼠(及其衍生的细胞)进行体内和体外机制研究，我们希望揭示在高氧期间受Trx1调控的线粒体机制。我们相信，该项目产生的结果将激发新的干预策略，以保护患者免受氧疗引起的肺部毒性。

 DESCRIPTION (provided by applicant): Oxygen therapy is a common clinical necessity, but it comes with significant negative side effects. Specifically, the hyperoxic condition it produces generates a number of reactive oxygen species, including superoxide anions that cause mitochondrial dysfunction. Although this toxicity is a key factor in the application of oxygen therapy, little is known about how hyperoxia impacts mitochondrial energy production, or the protein regeneration mechanisms that can offer protection from its effects. Our research program focuses on thioredoxin (Trx), a cytoplasmic redox protein that can reduce oxidative stress and regenerate enzymes that oxidation has inactivated. Recently we published that increased expression of Thioredoxin protects the lung injury and increased survival of Trx-Tg mice in hyperoxia, but mice with lower expression of Trx were more sensitive to hyperoxia and suffered significant mortality. However, the mechanism by which high levels of Thioredoxin protects against lung injury remains unknown. Our preliminary data establish that cytoplasmic Trx1 translocates to mitochondria during hyperoxia, but this movement does not occur in dnTrx-Tg mice. These findings propel us to hypothesize that Trx protects mitochondria from hyperoxia by reducing oxidative stress through UCP-2-dependent uncoupling, and furthermore by protecting mitochondrial dysfunction in hyperoxia as superoxide anion generation in the mitochondria is a key mechanism of pulmonary oxygen toxicity. Accordingly, in Aim 1 we will determine whether and how translocated cytoplasmic Trx1 protects against mitochondrial dysfunction in hyperoxia. In Aim 2 we will find if high levels of Trx prevents dynamin-related protein (Drp1) activation and thereby protects against mitochondrial fragmentation and dysfunction. In Aim 3 we will determine if increased translocation of PGC-1α to the nucleus in Trx-Tg mice can protect against the mitochondrial dysfunction caused by hyperoxia. Using state-of-the-art techniques that include mitochondrial flux analysis, EPR spectroscopy, biochemical enzymatic assay, and cutting-edge molecular approaches, we will dissect the role Trx1 plays in protecting the dysfunctional mitochondria in hyperoxia. We will also create a novel conditional Trx knockout mouse, a PGC1a-knockout mouse with increased or decreased expression of Trx to understand in vivo role of high levels of Trx on mitochondrial dysfunction in hyperoxia. The project is expected to provide a clear understanding of the way cytosolic Trx1 affects mitochondrial function during normoxia and hyperoxia. Using the transgenic mice (and cells derived from them) for in vivo and in vitro mechanistic studies, we expect to uncover mitochondrial mechanisms that are modulated by Trx1 during hyperoxia. We believe results produced by the project will incite novel intervention strategies to protect patients against pulmonary toxicity resulting from oxygen therapy.