Neuroprotection by a Nuclear Carbonic Anhydrase in C. elegans

线虫核碳酸酐酶的神经保护作用

基本信息

批准号：
8131788
负责人：
Keith Nehrke
金额：
$ 32.8万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8131788
关键词：
Ablation Address Anabolism Animal Model Area Attenuated Automobile Driving Behavioral Assay Bicarbonates Biochemical Biological Assay Biology Biosensor Brain Buffers Caenorhabditis elegans Candidate Disease Gene Carbon Dioxide Catalytic Domain Cell Culture Techniques Cell Death Cell Nucleus Cell Survival Cell physiology Cells Cellular Morphology Cellular Stress Cerebrum Cytoplasm Data Disease Electrolytes Environment Equilibrium Exposure to Fatty acid glycerol esters Functional disorder Gene Expression Regulation Genetic Genetic Models Glucose Goals Homeostasis Human Hypoxia Hypoxia Inducible Factor Immunologic Techniques Impaired cognition Injury Ischemia Label Lesion Life Ligation Mammals Measures Mediating Memory Minerals Modeling Molecular Mus Nematoda Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neurons Nuclear Organism Orthologous Gene Output Oxidation-Reduction Oxidative Stress Oxygen Phenotype Physiological Physiological Processes Process Proteins Protons Recombinants Regulation Reporter Respiration Role Signal Transduction Slice Source Staining method Stains Stress Stroke Techniques Testing Tissues Transgenes Transgenic Organisms Translating Tumor Markers Variant Work bone carbonate dehydratase cellular imaging computerized data processing deprivation design improved loss of function man middle cerebral artery mouse model mutant neuron loss neuroprotection novel overexpression pH Homeostasis preconditioning promoter protective effect public health relevance research study response stress management sugar synaptic function therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Carbonic anhydrase (CA) catalyzes the conversion of CO2 to a proton (H+) and bicarbonate (HCO3-) and is involved in many physiologic and pathophysiologic processes. Our preliminary data demonstrate that the nematode C. elegans expresses a CA that selectively localizes to the cell nucleus, is induced by hypoxia, and when lost results in neurodegeneration (ie dysfunction followed by cell death). This is the first example of a classic a-CA that is targeted to the nucleus in any organism. Our central hypothesis is that nuclear CA (NCA) activity protects neurons from hypoxic stress by buffering nuclear pH. We have developed a set of tools that will allow us to study neurodegeneration in a stain that is deficient in NCA and to assess whether its activity is significant for physiologic responses to hypoxia. We will also study signaling processes that are relevant to NCA expression. Finally, using novel genetically-encoded biosensors we will test whether nuclear CA can buffer pH in neuronal nuclei and what effect this has on nuclear redox status (or oxidative stress, which follows hypoxia). All of these approaches will utilize integrative physiologic techniques in live worms. These experiments are geared toward defining the mechanism whereby nuclear CA promotes cell function and viability. A second goal of this proposal is to test whether nuclear CA activity protects mammalian neurons during ischemia. Preliminary evidence demonstrates that transgenic expression of the nematode nuclear CA in mammalian cortical neurons is protective during oxygen-glucose deprivation and hypoxia. In addition to pursuing these studies, the cortical cell culture model will be used as a tissue source for a biochemical approach to identifying a predicted endogenous mammalian NCA. There are currently two candidate genes that are induced by hypoxia and cell stress, respectively, that will be examined using immunologic techniques and recombinant expression assays. In addition, a mouse middle cerebral artery ligation stroke model will be used to determine whether endogenous NCA activity is regulated by hypoxia in an intact brain, with a focus on our two candidate gene products. We hypothesize that worms require a dedicated nuclear CA because of their constant exposure to the environment, but that mammals express or target a CA to the nucleus only under stress conditions. The experiments proposed in this application are focused on defining a novel neuroprotective mechanism that involves pH and electrolyte homeostasis in the cell nucleus mediated by nuclear CA, and as such bridges two relatively diverse, but extremely significant, areas of biology. PUBLIC HEALTH RELEVANCE: We have identified an a-carbonic anhydrase in the genetic model organism C. elegans that regulates neuronal cell death decisions that occur in response to stress, and in particular hypoxia, by buffering the pH of the nucleus. Nuclear pH regulation is a novel mechanism for protecting neurons against ischemia and/or oxidative stress such as occurs in neurodegenerative disease. This application is focused on understanding the mechanism behind nuclear CAs protective effects in nematodes and translating these findings to mammals as a way of targeting cognitive impairment.

描述（由申请人提供）：碳酸酐酶（CA）催化CO2转化为质子（H+）和碳酸氢盐（HCO3-），并参与许多生理和病理生理过程。我们的初步数据表明，线虫秀丽隐杆线虫表达一种选择性定位于细胞核的CA，由缺氧诱导，当丢失时会导致神经变性（即功能障碍，随后细胞死亡）。这是针对任何生物体细胞核的经典 a-CA 的第一个例子。我们的中心假设是核 CA (NCA) 活性通过缓冲核 pH 值来保护神经元免受缺氧应激。我们开发了一套工具，使我们能够研究缺乏 NCA 的染色剂中的神经变性，并评估其活性是否对缺氧的生理反应具有重要意义。我们还将研究与 NCA 表达相关的信号传导过程。最后，使用新型基因编码生物传感器，我们将测试核 CA 是否可以缓冲神经元核中的 pH 值，以及这对核氧化还原状态（或缺氧后的氧化应激）有何影响。所有这些方法都将利用活体蠕虫的综合生理技术。这些实验旨在确定核 CA 促进细胞功能和活力的机制。该提案的第二个目标是测试核 CA 活性是否在缺血期间保护哺乳动物神经元。初步证据表明，哺乳动物皮质神经元中线虫核 CA 的转基因表达在氧糖剥夺和缺氧期间具有保护作用。除了进行这些研究之外，皮质细胞培养模型还将用作生化方法的组织来源，以鉴定预测的内源性哺乳动物 NCA。目前有两个候选基因分别由缺氧和细胞应激诱导，将使用免疫技术和重组表达测定法对其进行检查。此外，小鼠大脑中动脉结扎中风模型将用于确定完整大脑中内源性NCA活性是否受到缺氧的调节，重点是我们的两个候选基因产物。我们假设蠕虫需要专门的核 CA，因为它们经常暴露在环境中，但哺乳动物仅在应激条件下表达或将 CA 靶向细胞核。本申请中提出的实验重点是定义一种新颖的神经保护机制，该机制涉及由核CA介导的细胞核中的pH和电解质稳态，并因此弥合了两个相对不同但极其重要的生物学领域。公共健康相关性：我们在遗传模型生物体秀丽隐杆线虫中发现了一种α-碳酸酐酶，它通过缓冲细胞核的 pH 值来调节因应激（特别是缺氧）而发生的神经细胞死亡决策。核pH调节是一种保护神经元免受缺血和/或氧化应激（例如神经退行性疾病中发生的）的新机制。该应用的重点是了解核 CA 对线虫的保护作用背后的机制，并将这些发现转化为哺乳动物，作为针对认知障碍的一种方式。