权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Genetic Analysis of Neuronal Hypoxia Resistance

神经元耐缺氧的遗传分析

基本信息

批准号：
10835277
负责人：
Christopher G Rongo
金额：
$ 10.44万
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/10835277
关键词：
Address Administrative Supplement Antioxidants Autophagocytosis Binding Biological COVID-19 CRISPR/Cas technology Caenorhabditis elegans Cells Cerebral Palsy Disease Drug Metabolic Detoxication Enhancers Environment Enzymes Equipment Equipment Failure Etiology Free Radicals Funding Genes Genetic Genetic Enhancer Element Gluconeogenesis Hydroxylation Hypoxia Hypoxia Inducible Factor Ischemic Stroke Life Malignant Neoplasms Metabolic Metabolism Mitochondria Myocardial Infarction Nerve Degeneration Neuronal Hypoxia Neurons Organism Oxidative Stress Pathway interactions Play Procollagen-Proline Dioxygenase Production Pulmonary Hypertension Reporter Reproducibility Resistance Role Source Stress Testing Tissues Transcriptional Regulation combat deprivation disorder prevention experimental study genetic analysis human disease hypoxia inducible factor 1 in vivo model organism novel protective pathway receptor response therapeutic target tissue culture transcription factor transcriptome

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 negatively regulates the transcription factor Hypoxia Inducible Factor α (HIFα). 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. 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. 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 oxidative stress, and by (2) mobilizing antioxidant metabolism, which detoxifies free radicals during hypoxia and reoxygenation. 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. Recently, our media sterilizer and automated Petri dish pourer reached the end of its life. This large equipment failure is inhibiting our progress towards both aims, as this fundamental equipment is essential for every experiment we conduct. An administrative supplement is requested to replace this equipment and restore our progress towards understanding how the hypoxia response pathway operates. A better understanding of the pathway will provide therapeutic targets for diseases associated with hypoxia.

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) 酶，负向调节转录因子缺氧诱导因子 α (HIFα)。什么时候当缺氧发生时，PHD 酶缺乏 O2 来羟基化 HIFα，从而导致 HIFα 稳定和帮助生物体生存的多个靶基因的转录调控。虽然 HIFα 通路有在组织培养中得到了充分的研究，充分了解它如何在特定组织（特别是神经元）在体内提供定制的反应是必要的。该提议利用了遗传学和完整的等基因模式生物（秀丽隐杆线虫），可以在缺氧下茁壮成长，其环境和可以以保真度和再现性控制遗传学。该提案的总体前提是缺氧反应途径通过（1）去除线粒体来防止缺氧损伤线粒体自噬，消除氧化应激的来源，并通过（2）调动抗氧化代谢，在缺氧和复氧过程中解毒自由基。我们假设 HIF-1 通过结合增强子序列来促进这种代谢重编程并激活 PEP 羧激酶 pck-1 的表达，这是一种将代谢物转移到体内的关键酶糖异生。目标 1 通过使用 CRISPR/Cas9 编辑删除该增强子来测试该假设，然后测试对 HIF-1 结合、pck-1 和整体基因表达、代谢、氧化应激的影响抵抗力、神经退行性变和缺氧生存。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。我们假设 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。目标 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。最近，我们的培养基灭菌器和自动培养皿倾倒器达到了其使用寿命。这个大设备故障阻碍了我们实现这两个目标的进展，因为这种基本设备对于我们进行的每一个实验。需要行政补充来更换该设备，并且恢复我们在理解缺氧反应途径如何运作方面的进展。更好的了解该途径将为缺氧相关疾病提供治疗靶点。