H2O2-dependent redox homeostasis in a zebrafish model of endocrine disease

氧化还原反应是生物学中的基本反应。必须严格控制这些反应产生的活性氧（ROS），以防止对核酸、蛋白质和脂质造成损害。过多的ROS产生是许多疾病的特征，包括内分泌和代谢疾病。然而，ROS在生物体中也具有信号传导或调节功能，特别是通过蛋白质半胱氨酸残基中硫醇的可逆修饰。有充分的证据表明，这种可逆修饰改变了蛋白质的构象、定位和活性。然而，到目前为止，只有少数这些硫醇开关的调节被很好地表征。此外，关于ROS在多细胞生物细胞和组织中的时空分布的知识是有限的。我们需要这些信息来绘制硫醇开关正常工作的位置和过程，并使我们能够更好地掌握ROS稳态变化与（病理）生理过程之间的因果关系。在这里，我们希望使用斑马鱼模型系统在胚胎发育和病理条件下模拟内分泌和代谢疾病的体内氧化还原事件成像。为此，我们将使用表达工程硫醇开关生物传感器的斑马鱼转基因系，如roGFP2-Orp1和最近开发的更敏感的roGFP2-Tsa2deltaCR，用于监测胚胎组织发育过程中细胞质和线粒体中的H2O2动态。初步数据表明，胚胎中存在迁移细胞，持续数分钟至数小时的H2O2水平增加。通过将传感器系与携带细胞特异性荧光标记的系相结合，我们的目标是识别这些细胞的性质。对显示不同氧化还原信号水平的细胞进行FACS，然后对其转录组进行下一代测序分析，将为与闪光相关的调节和功能变化提供线索，这将通过化学处理或基因操作来操纵氧化还原水平进一步研究。为了检查内分泌疾病模型中的氧化还原变化，我们将传感器系引入rx3强突变体，这是一种肾上腺功能不全的斑马鱼模型，我们之前已经描述了许多糖皮质激素依赖性的转录和代谢动力学变化。我们还将检查用糖皮质激素处理的胚胎是否产生过量的糖皮质激素信号，正如在库欣综合征中观察到的那样。这些数据将揭示糖皮质激素缺乏和过量对各种组织中ROS水平和动态的影响。鉴于关于糖皮质激素系统紊乱患者氧化还原变化的报道稀缺，这一信息将特别有价值。重要的是，该项目将为模型生物的氧化还原生物学研究提供工具和概念，该模型生物非常适合体内药物筛选方法。

Redox reactions are fundamental reactions in biology. Reactive oxygen species (ROS) arising from these reactions must be tightly controlled to prevent damage to nucleic acids, proteins and lipids. Excessive ROS production is a feature of a number of diseases, including endocrine and metabolic diseases. However, ROS also serve signaling or regulatory functions in the organism, notably via reversible modifications of thiols in cysteine residues of proteins. It is well documented that such reversible modifications change protein conformation, localization and activity. Nevertheless, regulation of only a few of these thiol switches has been well characterized so far. In addition, knowledge on the spatiotemporal distribution of ROS in cells and tissues of multicellular organisms is limited. This information is needed to map sites and processes where thiol switches normally operate and to allow us a better grasp of causal relationships between shifts in ROS homeostasis and (patho)physiological processes.Here, we wish to use the zebrafish model system for the imaging of in vivo redox events across embryonic development and under pathological conditions mimicking endocrine and metabolic diseases. To this end, we will use zebrafish transgenic lines expressing engineered thiol-switch biosensors, such as roGFP2-Orp1 and the recently developed more sensitive roGFP2-Tsa2deltaCR, for the monitoring of H2O2 dynamics across cytosol and mitochondria of embryonic tissues during development. Preliminary data indicate the presence of migrating cells in the embryo that show flashes of increased H2O2 levels lasting for minutes to hours. By combining the sensor lines with lines carrying cell specific fluorescent markers, we aim to identify the nature of these cells. FACS of cells showing different redox signal levels followed by next generation sequencing analysis of their transcriptomes will give hints as to the regulatory and functional changes linked with the flashes, which will be further studied by manipulating redox levels via chemical treatment or genetic manipulation.To examine redox changes in an endocrine disease model, we will introduce the sensor lines into rx3 strong mutants, a zebrafish model of adrenal insufficiency in which we have previously described numerous glucocorticoid dependent changes in transcriptional and metabolic dynamics. We will also examine embryos treated with glucocorticoids to create an excess of glucocorticoid signalling, as observed in Cushings syndrome. The data will reveal the consequences of both a lack and an excess of glucocorticoids on ROS levels and dynamics in various tissues. This information will be particularly valuable given the scarcity of reports on redox changes in human patients suffering from disorders of the glucocorticoid system. Importantly, the project will provide tools and concepts for the study of redox biology in a model organism highly amenable to in vivo drug screening approaches.