Neuroprotection by a Nuclear Carbonic Anhydrase in C. elegans

线虫核碳酸酐酶的神经保护作用

• 批准号: 8461188
• 负责人: Keith Nehrke
• 金额: $ 31.96万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 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/8461188
• 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

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.

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（HCO 3-），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C。线虫表达的CA选择性地定位于细胞核，由缺氧诱导，当失去时导致神经变性（即功能障碍，随后细胞死亡）。这是任何生物体中靶向细胞核的经典a-CA的第一个例子。我们的中心假设是，核CA（NCA）的活动保护神经元缺氧应激缓冲核pH值。我们已经开发了一套工具，使我们能够研究神经变性的染色是缺乏NCA和评估其活动是否是显着的生理反应缺氧。我们还将研究与NCA表达相关的信号传导过程。最后，使用新的遗传编码的生物传感器，我们将测试核CA是否可以缓冲神经元细胞核中的pH值，以及这对核氧化还原状态（或缺氧后的氧化应激）有什么影响。所有这些方法都将利用活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。 该提议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，线虫核CA在哺乳动物皮层神经元中的转基因表达在氧-葡萄糖剥夺和缺氧期间具有保护作用。除了进行这些研究外，皮质细胞培养模型将用作生物化学方法的组织来源，以鉴定预测的内源性哺乳动物NCA。目前有两个候选基因，诱导缺氧和细胞应激，分别将使用免疫学技术和重组表达测定。此外，小鼠大脑中动脉结扎中风模型将用于确定内源性NCA活性是否受完整脑中缺氧的调节，重点是我们的两个候选基因产物。我们假设，蠕虫需要一个专用的核CA，因为他们不断暴露于环境中，但哺乳动物表达或目标CA的细胞核，只有在压力条件下。本申请中提出的实验集中于定义一种新的神经保护机制，其涉及由核CA介导的细胞核中的pH和电解质稳态，并且因此桥接了两个相对不同但极其重要的生物学领域。