Genetic Analysis of Neuronal Hypoxia Resistance

神经元耐缺氧的遗传分析

基本信息

批准号：
10683094
负责人：
Christopher G Rongo
金额：
$ 33.73万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10683094
关键词：
Aerobic Antioxidants Autophagocytosis Binding Biological Biological Models COVID-19 CRISPR/Cas technology Caenorhabditis elegans Cells Cerebral Palsy ChIP-seq Disease Drug Metabolic Detoxication Enhancers Environment Enzymes Etiology Excision Gene Expression Generations Genes Genetic Genetic Enhancer Element Genetic Models Genetic Transcription Gluconeogenesis Hydroxylation Hypoxia Hypoxia Inducible Factor Impairment Individual Ischemic Stroke Malignant Neoplasms Metabolic Metabolism Mitochondria Muscle Mutation Myocardial Infarction NADP Nerve Degeneration Neuronal Hypoxia Neurons Organism Orthologous Gene Oxidative Phosphorylation Oxidative Stress Pathway interactions Pentosephosphate Pathway Phase Play Procollagen-Proline Dioxygenase Production Proline Proteins Proteolysis Pulmonary Hypertension Reactive Oxygen Species Reduced Glutathione Regulation Reporter Reproducibility Resistance Role Side Solid Neoplasm Source Testing Tissues Transcriptional Regulation Traumatic Brain Injury Warburg Effect combat deprivation disorder prevention experimental study genetic analysis human disease hypoxia inducible factor 1 in vivo metabolomics model organism mutant normoxia novel promoter protective pathway receptor response spinal cord and brain injury stem cell niche therapeutic target tissue culture transcriptome transcriptome sequencing ubiquitin ligase

项目摘要

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.

项目概要缺氧（缺氧）在多种人类疾病中发挥着核心作用，包括缺血性中风、心肌病梗塞、肺动脉高压、脑瘫、COVID-19 和后生动物对缺氧的反应。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) 酶，使用 O2 羟基化缺氧诱导因子 α 上的特定脯氨酸侧链 (HIFα) 一旦羟基化，HIFα 就会被 Von Hippel-Lindau (VHL) 泛素连接酶泛素化，从而产生当氧气充足时，HIFα 不稳定；当缺氧时，PHD 酶缺乏氧气。羟基化 HIFα，导致 HIFα 稳定和多个靶基因的转录调节在某些情况下（例如实体瘤、干细胞生态位），HIFα 被激活。尽管有足够的氧气水平（即瓦尔堡效应），但在有氧条件下反应有何不同是虽然 HIFα 通路已在组织培养中得到了充分研究，但仍不清楚它是如何发挥作用的。需要在体内特定组织（特别是神经元）中运作以提供定制的反应。该提案利用了遗传学和完整的等基因模型生物（秀丽隐杆线虫），可以在缺氧条件下茁壮成长，并且其环境和遗传可以精确控制线虫拥有 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可防止缺氧通过 (1) 通过线粒体自噬去除线粒体，从而消除 ROS 来源，以及 (2) 动员抗氧化代谢，在缺氧和复氧过程中更好地解毒活性氧。了解通路反应将为缺氧相关疾病提供治疗靶点。初步 ChIP-seq、RNA-seq 和代谢组学表明 HIF-1 促进糖异生，我们认为 HIF-1 可以促进这种代谢。通过结合增强子序列并激活 PEP 羧激酶 pck- 的表达来重新编程 1，一种通过糖异生移动代谢物的关键酶，目的 1 通过使用来测试这一假设。 CRISPR/Cas9 编辑删除该增强子，然后测试对 HIF-1 结合、pck-1 和全局的影响基因表达、代谢、氧化应激抵抗、神经变性和缺氧生存。使用基因编码荧光报告基因进行线粒体自噬的初步细胞生物学方法表明 HIF-1 促进线粒体自噬我们发现 HIF-1 通过结合促进线粒体自噬。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。这个假设是通过使用 CRISPR/Cas9 编辑去除这些增强子，然后测试对 HIF 的影响 1 结合、全局基因表达、线粒体自噬和大量自噬、代谢、氧化应激抵抗、神经退行性变和缺氧生存。