REDOX-regulation of mitochondrial transcription by mitochondrial DNA topoisomerase I

线粒体拓扑异构酶I （TOP1MT）可去除线粒体DNA （mtDNA）中的负超旋，已被确定为人类线粒体疾病的候选基因。然而，尽管负mtDNA超卷曲明显增加，TOP1MT-/-小鼠并未表现出明显的线粒体功能障碍。因此，TOP1MT被认为是负mtDNA超卷曲的调节因子，但该功能在线粒体稳态中的作用尚不清楚。我们已经证明，TOP1MT是mtdna转录的直接负调控因子。在分离的线粒体中，这种功能可以通过刺激内源性ROS的产生而被抑制。当表达抗氧化TOP1MT突变体时，这种效应就消失了。我们的观察结果支持双负环模型，其中TOP1MT负调控mtdna转录，并反过来被氧化负调控。我们的目标是提供推测的调节回路的因果证据（目标1），描述所涉及的成分和分子相互作用（目标2），并解决该机制在氧化应激适应和年龄相关的线粒体功能障碍中的作用（目标3）。为此，我们将在分离的线粒体中证明(i)氧化还原酶对mtDNA转录的调节在TOP1MT耗尽或缺失时丢失，并且不会被无活性或抗氧化的TOP1MT突变体补充；（ii）线粒体ROS的急剧增加减少了重链和轻链启动子对TOP1MT的招募，减少了TOP1MT的类核关联，增加了负mtDNA超缠绕。并且这些反应在TOP1MT0/0细胞中由野生型TOP1MT补充，但不是失活或抗氧化的TOP1MT突变体。然后，我们将通过将上述互补模型置于急性和/或慢性间歇性UVA暴露下，进一步确定线粒体应激反应中top1mt依赖的机制，并监测UVA对ROS水平、mtDNA转录、mtDNA拷贝数、核有丝分裂生物发生、核和mtDNA编码呼吸复合物平衡以及呼吸功能的影响。我们将最终验证top1mt依赖的适应过程的生理学相关性，通过研究它们在从暴露和防晒的老年和年轻人类供体皮肤中分离的原代成纤维细胞中的状态。最终，我们的目标是通过诱导小鼠的TOP1MT敲除来验证我们的结果，但这些实验超出了本应用程序的范围。我们期望发现一种提供mtDNA转录局部氧化还原调控的机制。我们希望阐明这一机制是否能调节线粒体基质内氧化还原状态的呼吸能力，并在线粒体生物遗传应激反应中微调线粒体核通讯。我们可能会证实，在人类真皮成纤维细胞中，uva诱导的线粒体功能障碍和与年龄相关的线粒体功能障碍与推测的调节机制的破坏有关。

Mitochondrial topoisomerase I (TOP1MT) removes negative supercoils from mitochondrial DNA (mtDNA) and has been identified as mitochondrial disease candidate gene in humans. However, TOP1MT-/- mice do not exhibit overt mitochondrial dysfunction despite a marked increase in negative mtDNA supercoiling. TOP1MT is thus indicated a regulator of negative mtDNA supercoiling, but the role of that function in mitochondrial homeostasis remains unclear.We have demonstrated that TOP1MT acts as direct negative regulator of mtDNA-transcription. This function can be inhibited in isolated mitochondria by stimulating endogenous ROS production. Upon expression of oxidation-resistant TOP1MT mutants the effect is abolished. Our observations support the model of a double negative loop, in which TOP1MT negatively regulates mtDNA-transcription and is in turn negatively regulated by oxidation. We aim to provide causal proof of the surmised regulatory loop (objective 1), characterise the components and molecular interactions involved (objective 2), and address the role of this mechanism in oxidative stress adaptation and age-associated mitochondrial dysfunction (objective 3). For that pupose, we will demonstrate in isolated mitochondria (i) that REDOX-modulation of mtDNA transcription is lost upon depletion or deletion of TOP1MT and not complemented by inactive or oxidation-resistant TOP1MT mutants, and (ii) that acute increases in mitochondrial ROS diminish the recruitment of TOP1MT to heavy and light strand promoters, decrease nucleoid association of TOP1MT and increase negative mtDNA supercoiling, and that these responses are complemented in TOP1MT0/0 cells by wild type TOP1MT but not inactive or oxidation-resistant TOP1MT mutants. We will then move on to identify TOP1MT-dependent mechanisms in mitochondrial stress responses by subjecting the above complementation models to acute and/or chronic intermittent UVA exposure and monitor the UVA-impact on ROS levels, mtDNA-transcription, mtDNA copy number, nuclear mito-biogenesis, the balance of nuclear and mtDNA-encoded respiratory complexes, and respiratory function. We will finally validate the physiological relevance of TOP1MT-dependent adaptation processes by addressing their state in primary fibroblasts isolated form sun-exposed and sun-protected skin of old and young human donors. Ultimately, we aim at validating our results by inducible TOP1MT knock out in mice, but these experiments are outside the purview of this application.We expect to uncover a mechanism that provides local REDOX-regulation of mtDNA transcription. We expect to elucidate whether this mechanism adjusts respiratory capacity to the REDOX state inside the mitochondrial matrix and serves to fine tune mito-nuclear communication in mito-biogenetic stress responses. We will possibly confirm that disruption of the surmised regulatory mechanism is involved in UVA-induced and age-related mitochondrial dysfunction in human dermal fibroblasts.