Neuroprotection by a Nuclear Carbonic Anhydrase in C. elegans

描述（由申请人提供）：碳酸酐酶（CA）催化二氧化碳转化为质子（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，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，当失去时导致神经退行性变（即功能障碍后细胞死亡）。这是第一个经典的a- ca在任何生物体中靶向细胞核的例子。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。我们已经开发了一套工具，使我们能够在缺乏NCA的染色中研究神经变性，并评估其活性是否对缺氧的生理反应有重要意义。我们还将研究与NCA表达相关的信号传导过程。最后，我们将使用新的基因编码生物传感器来测试核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）有什么影响。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。这个提议的第二个目标是测试核CA活性是否在缺血时保护哺乳动物神经元。初步证据表明，线虫核CA在哺乳动物皮质神经元中的转基因表达在缺氧和缺氧时具有保护作用。除了进行这些研究之外，皮质细胞培养模型将被用作生物化学方法的组织来源，以确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，将通过免疫技术和重组表达试验进行检测。此外，将使用小鼠大脑中动脉结扎卒中模型来确定内源性NCA活性是否受缺氧在完整大脑中的调节，重点关注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，因为它们经常暴露在环境中，但哺乳动物只在应激条件下表达或将CA靶向到细胞核。本应用中提出的实验重点是定义一种新的神经保护机制，该机制涉及核CA介导的细胞核pH和电解质稳态，并因此连接两个相对多样化但极其重要的生物学领域。

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.