Genetic Analysis of Neuronal Hypoxia Resistance

项目摘要 缺氧（O2剥夺）在多种人类疾病中起着核心作用，包括缺血性中风、心肌梗塞、心肌缺血性中风等。 梗死、肺动脉高压、脑瘫、COVID-19和癌症。后生动物对缺氧的反应是 采用保守的缺氧反应途径。该途径通过脯氨酰羟化酶感知O2 (PHD)一种利用O2使缺氧诱导因子α上的特定脯氨酸侧链羟基化的酶 (HIFα）。一旦羟基化，HIFα被Von Hippel-Lindau（VHL）泛素连接酶泛素化，导致 它的蛋白水解。当氧气充足时，HIFα不稳定。当缺氧加剧时，PHD酶缺乏O2， 羟基化HIFα，导致HIFα稳定和多个靶基因的转录调节， 帮助有机体生存。在某些情况下（例如，实体瘤，干细胞龛），HIFα被激活 尽管有足够的O2水平（即，瓦尔堡效应），但在有氧条件下的反应如何不同， 不清楚虽然HIFα通路在组织培养中已经得到了很好的研究，但对HIFα通路如何作用的充分理解， 需要在体内特定组织（特别是神经元）中操作以提供定制的响应。 这个提议利用了遗传学和一个完整的、等基因的模式生物（C。elegans）， 可以在缺氧条件下茁壮成长，其环境和遗传可以被忠实地控制， 再现性C.线虫具有PHD（EGL-9）、VHL（VHL-1）和HIFα（HIF-1）的单一基因。 该提案的总体前提是缺氧反应途径途径保护免受缺氧 （1）通过线粒体自噬去除线粒体，这消除了ROS的来源，以及（2） 动员抗氧化剂代谢，在缺氧和复氧期间解毒ROS。更好的 对该通路反应的理解将为与缺氧相关的疾病提供治疗靶点。 初步的ChIP-seq、RNA-seq和代谢组学研究表明，HIF-1促进了肿瘤的发生， 戊糖磷酸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并激活PEP羧激酶pck的表达进行重编程， 1，一种通过细菌异生作用移动代谢物的关键酶。Aim 1通过使用 CRISPR/Cas9编辑以去除这种增强子，然后测试对HIF-1结合，pck-1和全局的影响。 基因表达、代谢、抗氧化应激、神经变性和缺氧存活。 用遗传编码的荧光报告基因进行线粒体自噬的初步细胞生物学方法 提示HIF-1促进线粒体自噬。我们推测HIF-1通过结合 增强子序列并激活线粒体自噬受体FNDC-1和DCT-1的表达。Aim 2测试 通过使用CRISPR/Cas9编辑来去除这些增强子，然后测试对HIF的影响， 1结合，整体基因表达，线粒体自噬和大量自噬，代谢，抗氧化应激， 神经变性和缺氧存活。

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.