Genetic Analysis of Neuronal Hypoxia Resistance

神经元耐缺氧的遗传分析

• 批准号: 10297456
• 负责人: Christopher G Rongo
• 金额: $ 41.2万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 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/10297456
• 关键词: Aerobic; Anaerobic Bacteria; Animal Model; Antioxidants; Autophagocytosis; Binding; Biological; Biological Models; COVID-19; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Cerebral Palsy; ChIP-seq; Disease; Enhancers; Environment; Enzymes; Etiology; Excision; Gene Expression; Generations; Genes; Genetic; Genetic Enhancer Element; Genetic Models; Genetic Transcription; Gluconeogenesis; 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; Spinal cord injury; Testing; Tissues; Transcriptional Regulation; Traumatic Brain Injury; Tumor Stem Cells; Warburg Effect; combat; deprivation; disorder prevention; experimental study; factor A; genetic analysis; human disease; hypoxia inducible factor 1; in vivo; metabolomics; mutant; normoxia; novel; promoter; receptor; response; therapeutic target; tissue culture; transcriptome; transcriptome sequencing; ubiquitin ligase

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.

项目摘要 缺氧（O2剥夺）在潜水人类疾病中起着核心作用，包括缺血性中风，心肌 梗塞，肺动脉高压，大脑麻痹，Covid-19和癌症。后生动物对缺氧的反应 采用保守的缺氧反应途径。该途径通过羟基羟化酶感测O2 （博士学位）酶，该酶使用O2在缺氧诱导因子α上羟基特异性脯氨酸侧链α （HIFα）。一旦羟基化，HIFα就被Von Hippel-Lindau（VHL）泛素连接酶泛素化，导致 它的蛋白水解。当O2丰富时，HIFα不稳定。当发生缺氧时，博士酶缺乏O2 羟基HIFα，导致HIFα稳定和多个靶基因的转录调节 帮助生物生存。在某些情况下（例如实体瘤，干细胞壁ni），HIFα被激活 尽管有足够的O2水平（即Warburg效应），但是有氧条件下的反应差异是如何 不清楚。虽然HIFα途径在组织培养中已经很好地研究了 需要在特定的组织（部分神经元）中进行体内进行量身定制的反应。 该提议利用了遗传学和完整的同基因模型生物（秀丽隐杆线虫） 可以在缺氧下壮成长，并且可以通过忠诚和遗传学来控制其环境和遗传学 可重复性。秀丽隐杆线虫具有PHD（EGL-9），VHL（VHL-1）和HIFα（HIF-1）的单个基因。 该提议的总体前提是缺氧反应途径途径可预防缺氧 通过（1）通过线粒体去除线粒体的损​​坏，这消除了ROS的来源，以及（2） 动员抗氧化剂代谢，该代谢在缺氧和氧化过程中排毒ROS。更好 对途径反应的理解将为与缺氧相关的疾病提供治疗靶标。 初步的CHIP-SEQ，RNA-SEQ和代谢组学表明，HIF-1促进糖异生，即 戊糖磷酸盐途径和抗氧化剂产生。我们假设HIF-1促进了这种代谢 通过结合增强子序列并激活Pep羧基酶PCK-的表达来重新编程 1，用于通过糖异生的代谢产物的关键酶。 AIM 1通过使用 CRISPR/CAS9编辑以删除此增强器，然后测试对HIF-1绑定，PCK-1和Global的影响 基因表达，代谢，氧化应激抗性，神经退行性和缺氧存活。 初步的细胞生物学方法，采用一般编码的线粒体编码的荧光记者 建议HIF-1促进线粒体。我们假设HIF-1通过结合促进线粒体 增强子序列并激活线粒体接收器FNDC-1和DCT-1的表达。 AIM 2测试 该假设通过使用CRISPR/CAS9编辑来删除这些增强剂，然后测试对HIF的影响 1结合，整体基因表达，线粒体和散装自噬，代谢，氧化应激性， 神经变性和缺氧存活。