MtDNA repair: An isolated pharmacologic target in acute lung injury

描述(申请人提供)：许多研究探索抗氧化剂作为急性肺损伤(ALI)的治疗剂，但其临床试验结果令人失望。这一失败的一个解释是，ALI中产生的氧化剂不仅具有细胞毒性，而且还在肺细胞增殖和分化所需的途径中发挥第二信使的作用。因此，非选择性抗氧化剂可能会干扰细胞生存和恢复所需的信号转导。一种相关的可能性是，整合ROS对肺血管系统作用的关键分子(S)尚未确定；抗氧化剂可能无法保护这些可能控制细胞死亡程序激活的前哨分子(S)免受损害。基于从细胞培养研究中获得的一系列令人信服的证据，在此，我们建议检验线粒体(Mt)DNA是决定氧化应激反应中肺细胞命运的前哨分子，以及mtDNA修复途径是ALI干预的靶点的概念。具体目标1将开始将细胞培养的结果转化为实际应用，测试使用新型融合蛋白结构增加mtDNA糖基酶活性的假说，即在患有铜绿假单胞菌或呼吸机诱导的ALI的啮齿动物中，增加mtDNA糖基酶活性可以预防和逆转ALI。这些实验构成了我们朝着开发mtDNA修复融合蛋白作为治疗ALI的平台药物分子的长期目标迈出的重要一步。尽管线粒体DNA修复的增强抑制了ROS介导的细胞死亡，但这种有益作用的机制仍然不清楚。传统观念认为，加速线粒体dna修复可以防止氧化mtdna损伤的积累，从而减弱线粒体产生的促凋亡的ROS，以及随之而来的线粒体驱动的细胞死亡的激活，但并不是目前所有可用的数据。 支持这一模型，一些研究表明线粒体DNA修复本身并不重要。因此，特异性目标2将使用野生型和突变型Ogg1蛋白，它们修复mtDNA的能力不同，但不能结合mtDNA，以确定是否需要修复RS诱导的mtDNA损伤，以防止RS诱导的EC细胞毒性、屏障功能障碍，并形成损伤传播的mtDNA片段的细胞外释放。

DESCRIPTION (provided by applicant): Numerous studies have explored anti-oxidants as therapeutic agents in acute lung injury (ALI), but their outcome in clinical trials has been disappointing. One explanation for this failure is that oxidants generated in ALI are not only cytotoxic, but they also function as second messengers in pathways required for lung cell proliferation and differentiation. Thus, non-selective antioxidants may disrupt signaling required for cell survival and recovery. A related possibility is that key molecule(s) integrating ROS actions on the pulmonary vasculature have yet to be identified; antioxidants may fail to protect against damage to these putative sentinel molecule(s) governing activation of cell death programs. Based on multiple lines of compelling evidence obtained from cell culture studies, herein we propose to test the concept that mitochondrial (mt) DNA is a sentinel molecule dictating lung cell fate in response to oxidant stress and that the mtDNA repair pathway is a target for intervention in ALI. Specific Aim 1 will begin translating findings from cell culture ino practical application by testing the hypothesis that increasing mtDNA glycosylase activity using novel fusion protein constructs protects against, and reverses, ALI in rodents with either Pseudomonas aeruginosa- or ventilator- induced ALI. These experiments comprise an important step towards our longer term goal of developing mtDNA repair fusion proteins as platform drug molecules for treating ALI. Even though enhancement of mtDNA repair suppresses ROS-mediated cell death, the mechanism of this salutary effect has remained elusive. Traditional concepts hold that accelerated mtDNA repair prevents accumulation of oxidative mtDNA damage, thereby attenuating mitochondrial generation of pro-apoptotic ROS and attendant activation of mitochondrially-driven cell death, but not all currently available data support this model and some studies suggest that mtDNA repair, per se, is unimportant. Accordingly, Specific Aim 2 will use wild type and mutant Ogg1 proteins differing in their capacities to repair - but not to bind - mtDNA to determine whether repair of RS-induced mtDNA damage is required for prevention of RS-induced EC cytotoxicity, barrier dysfunction, and formation an extracellular release of injury-propagating mtDNA fragments.