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-)，参与许多生理和病理生理过程。我们的初步数据表明，线虫表达一种选择性地定位于细胞核的CA，由低氧诱导，当丢失时会导致神经退化(即功能障碍随后细胞死亡)。这是第一个以生物体细胞核为靶点的经典a-CA的例子。我们的中心假设是，核CA(NCA)活性通过缓冲核pH来保护神经元免受低氧应激。我们已经开发了一套工具，使我们能够研究NCA缺乏的染色中的神经退化，并评估其活性是否对低氧的生理反应有重要意义。我们还将研究与NCA表达相关的信号传递过程。最后，使用新型的遗传编码生物传感器，我们将测试核CA是否可以缓冲神经元核中的pH，以及这对核的氧化还原状态(或低氧后的氧化应激)有什么影响。所有这些方法都将利用活蠕虫的综合生理学技术。这些实验旨在确定核CA促进细胞功能和活性的机制。这项提议的第二个目标是测试核CA活动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在哺乳动物皮质神经元中的转基因表达对缺氧缺糖和缺氧具有保护作用。除了进行这些研究外，皮质细胞培养模型还将被用作组织来源，用于生物化学方法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，将使用免疫学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中风模型来确定内源性NCA活性是否受完整大脑中缺氧的调节，重点放在我们的两个候选基因产物上。我们假设，蠕虫需要一个专门的核CA，因为它们经常暴露在环境中，但哺乳动物只有在应激条件下才表达或靶向细胞核的CA。本申请中提出的实验集中于定义一种新的神经保护机制，涉及由核CA介导的细胞核内pH和电解质的动态平衡，并因此连接两个相对不同但极其重要的生物学领域。 与公共健康相关：我们已经在遗传模式生物秀丽线虫中发现了一种a-碳酸氢酶，它通过缓冲细胞核的pH来调节应激，特别是低氧时发生的神经细胞死亡决定。核pH调节是一种新的保护神经元免受缺血和/或氧化应激的机制，如在神经退行性疾病中发生的。这项应用的重点是了解线虫核CA保护作用背后的机制，并将这些发现翻译到哺乳动物身上，作为一种靶向认知障碍的方式。

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.