Genetic Analysis of Neuronal Hypoxia Resistance

项目总结 缺氧(O2剥夺)在包括缺血性中风、心肌梗死在内的多种人类疾病中起着核心作用 脑梗塞、肺动脉高压、脑瘫、新冠肺炎和癌症。后生动物对缺氧的反应是 采用保守的低氧反应途径。该途径通过一种脯氨酸羟基酶来感知O2 (PHD)酶，它使用O2来羟化缺氧诱导因子α上的特定Pro侧链 (HIFα)。一旦羟化，缺氧诱导因子α被冯·希佩尔-林道(VHL)泛素连接酶泛素化，导致 它的蛋白质分解作用。当氧气充足时，HIFα是不稳定的。当缺氧接踵而至时，PHD酶缺乏氧气来 羟化缺氧诱导因子α，导致缺氧诱导因子α稳定和多个靶基因的转录调节 帮助有机体存活下来。在某些情况下(例如实体瘤、干细胞壁龛)，HIFα被激活 尽管氧气水平足够(即沃堡效应)，但在有氧条件下反应如何不同 不清楚。虽然缺氧诱导因子α通路在组织培养中已经得到了很好的研究，但对它是如何 在体内的特定组织(特别是神经元)中运作以提供量身定制的反应是必要的。 这一提议利用了遗传学和一种完整的、同基因的模式生物(秀丽线虫) 可以在低氧下茁壮成长，其环境和遗传可以通过保真度和 再现性。线虫只有PHD(EGL-9)、VHL(VHL-1)和HIFα(HIF-1)基因。 这一建议的总体前提是低氧反应通路对低氧具有保护作用。 损伤通过(1)通过有丝分裂去除线粒体，从而消除ROS的来源，以及(2) 动员抗氧化剂代谢，在缺氧和复氧时对ROS进行解毒。更好的 了解这一途径的反应将为与缺氧相关的疾病提供治疗靶点。 初步的芯片序列、RNA序列和代谢组学表明，HIF-1促进糖异生， 磷酸戊糖途径，和抗氧化剂的产生。我们假设HIF-1促进这种代谢 通过结合增强子序列和激活PEP羧基激酶Pck-的表达来重新编程- 1，一种通过糖异生来移动代谢产物的关键酶。Aim 1通过使用以下命令来验证这一假设 CRISPR/Cas9编辑以移除此增强子，然后测试对HIF-1结合、PCK-1和全局的影响 基因表达、新陈代谢、抗氧化性、神经退行性变和低氧生存。 用基因编码的荧光报告基因检测有丝分裂的初步细胞生物学方法 提示HIF-1促进有丝分裂吞噬。我们假设HIF-1通过结合促进有丝分裂 增强子序列和激活有丝分裂受体fndc-1和dct-1的表达。AIM 2测试 这一假设是通过使用CRISPR/Cas9编辑来移除这些增强子，然后测试对HIF的影响- 1结合，全球基因表达，有丝分裂和大量自噬，代谢，氧化应激抵抗， 神经退行性变，低氧生存。

PROJECT SUMMARY Hypoxia (O2 deprivation) plays a central role in diverse human diseases, including ischemic stroke, myocardial infarction, pulmonary hypertension, Cerebral Palsy, COVID-19, and cancer. Metazoans respond to hypoxia by employing the conserved hypoxia response pathway. The pathway senses O2 through a prolyl hydroxylase (PHD) enzyme, which uses O2 to hydroxylate specific proline side chains on the Hypoxia Inducible Factor α (HIFα). Once hydroxylated, HIFα is ubiquitinated by the Von Hippel-Lindau (VHL) ubiquitin ligase, resulting in its proteolysis. When O2 is abundant, HIFα is unstable. When hypoxia ensues, PHD enzymes lack O2 to hydroxylate HIFα, resulting in HIFα stabilization and the transcriptional regulation of multiple target genes that help the organism survive. Under some circumstances (e.g., solid tumors, stem cell niches), HIFα is activated despite adequate O2 levels (i.e., the Warburg effect), but how the response differs under aerobic conditions is unclear. While the HIFα pathway has been well studied in tissue culture, a full understanding of how it operates in specific tissues (particularly neurons) in vivo to provide tailored responses is needed. This proposal takes advantage of genetics and an intact, isogenic model organism (C. elegans) that can thrive under hypoxia, and whose environment and genetics can be controlled with fidelity and reproducibility. C. elegans possess single genes for the PHD (EGL-9), the VHL (VHL-1), and the HIFα (HIF-1). The overall premise of this proposal is that the hypoxia response pathway pathway protects against hypoxic damage by (1) removing mitochondria through mitophagy, which eliminates a source of ROS, and by (2) mobilizing antioxidant metabolism, which detoxifies ROS during hypoxia and reoxygenation. A better understanding of the pathway response will provide therapeutic targets for diseases associated with hypoxia. Preliminary ChIP-seq, RNA-seq, and metabolomics suggest that HIF-1 promotes gluconeogenesis, the pentose phosphate pathway, and antioxidant generation. We hypothesize that HIF-1 promotes this metabolic reprograming by binding an enhancer sequence and activating the expression of the PEP carboxykinase pck- 1, a key enzyme for moving metabolites through gluconeogenesis. Aim 1 tests this hypothesis by using CRISPR/Cas9 editing to remove this enhancer, then testing for the effects on HIF-1 binding, pck-1 and global gene expression, metabolism, oxidative stress resistance, neurodegeneration, and hypoxia survival. Preliminary cell biological approaches with a genetically encoded fluorescent reporter for mitophagy suggest that HIF-1 promotes mitophagy. We hypothesize that HIF-1 promotes mitophagy by binding enhancer sequences and activating the expression of the mitophagy receptors fndc-1 and dct-1. Aim 2 tests this hypothesis by using CRISPR/Cas9 editing to remove these enhancers, then testing for the effects on HIF- 1 binding, global gene expression, mitophagy and bulk autophagy, metabolism, oxidative stress resistance, neurodegeneration, and hypoxia survival.