Amelioration of Mitochondrial Dysfunction by Thioredoxin in Hyperoxia

 描述（由申请人提供）：氧气治疗是一种常见的临床必需品，但它具有显著的负面副作用。具体来说，它产生的高氧条件产生了许多活性氧，包括导致线粒体功能障碍的超氧阴离子。虽然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），一种细胞质氧化还原蛋白，可以减少氧化应激和再生氧化已失活的酶。最近我们发表了硫氧还蛋白表达增加对高氧下Trx-Tg小鼠的肺损伤具有保护作用并增加其存活率，但低表达Trx的小鼠对高氧更敏感并遭受显著的死亡率。然而，高水平硫氧还蛋白保护肺损伤的机制仍然未知。我们的初步数据表明，细胞质Trx 1易位线粒体在高氧，但这种运动不会发生在dnTrx-Tg小鼠。这些发现促使我们假设Trx通过UCP-2依赖性解偶联降低氧化应激来保护线粒体免受高氧，并且此外通过保护高氧中的线粒体功能障碍来保护线粒体，因为线粒体中的超氧阴离子产生是肺氧毒性的关键机制。因此，在目标1中，我们将确定是否以及如何易位胞质Trx 1保护线粒体功能障碍，高氧。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（Drp 1）的激活，从而防止线粒体断裂和功能障碍。在目的3中，我们将确定Trx-Tg小鼠中增加的PGC-1α向细胞核的易位是否可以保护免受高氧引起的线粒体功能障碍。使用最先进的技术，包括线粒体通量分析，EPR光谱，生化酶测定和尖端的分子方法，我们将剖析Trx 1在高氧保护功能失调的线粒体中发挥的作用。我们还将创建一种新的条件性Trx敲除小鼠，一种具有增加或减少的Trx表达的PGC 1a敲除小鼠，以了解高水平Trx对高氧线粒体功能障碍的体内作用。该项目预计将提供一个清楚的了解胞质Trx 1影响线粒体功能在常氧和高氧。使用转基因小鼠（和来自他们的细胞）在体内和体外的机制研究，我们希望发现线粒体机制，在高氧期间由Trx 1调制。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.