Connexins in Ischemia-Induced Neuronal Death

描述（由申请方提供）：在心脏骤停和心脏手术期间的患者中观察到或在动物中实验诱导的短暂但严重的全脑缺血，诱导选择性和延迟性神经变性。CA 1区的锥体细胞对缺血性损伤最为敏感，CA 3区和齿状回颗粒细胞对缺血性损伤具有抵抗力，CA 1区的GABA能中间神经元也存活。这种神经元死亡模式的分子机制还没有很好的理解。这项研究的目的是研究缝隙连接在全脑缺血后神经元损伤的几天“成熟”过程中的作用。该实验室的最新研究结果表明，在神经元死亡开始之前，全脑缺血触发了脆弱的CA 1的GABA能中间神经元中Cx 36（和Cx 32）蛋白表达的选择性上调，这与这些神经元的存活作用一致。此外，Cx 32（Y/-）小鼠中的CA 1神经元表现出对全脑缺血诱导的神经元死亡的增强的脆弱性。这些数据表明，通过抑制性中间神经元的同步化增加对锥体细胞的抑制可能具有神经保护作用。星形胶质细胞之间的缝隙连接也被认为在缺血后神经元死亡中起作用。死亡细胞可以通过胶质细胞“自相残杀”（旁观者死亡）杀死耐药的相邻胶质细胞，从而将损伤传播到相邻区域。另一方面，星形胶质细胞的间隙连接偶联介导它们之间的代谢合作，并减弱氧化应激模型中的神经元死亡。这个建议的基本假设是，缝隙连接在决定神经元死亡和生存全脑缺血后发挥重要作用。未来五年的研究计划集中在神经损伤后大脑缝隙连接的丰度，分布以及分子和生物物理特性的变化。具体目标是1。表征缺血诱导的大鼠和小鼠易损CA 1和抗性CA 3以及齿状回中连接蛋白表达和间隙连接特性的变化。实验将研究全球缺血诱导的变化，抑制性中间神经元和连接蛋白的表达的耦合免疫细胞化学和蛋白质印迹和连接蛋白的mRNA的原位杂交和。实验将确定通过反义寡核苷酸急性敲除特定连接蛋白对神经元易损性的影响，并将检查Cx 32（Y/-）小鼠、Cx 36（-/-）小鼠和星形胶质细胞Cx43缺陷小鼠中的神经元易损性。2.用免疫细胞化学、原位杂交和电生理方法检测缺氧缺糖对海马脑片培养的影响。通过电生理学方法和图像分析检测急性脑片和器官型海马脑片培养中缺血诱导的缝隙连接特性的变化。拟议的研究预计将影响新的治疗策略的发展，用于干预全球缺血，这是一种与人类心脏骤停相关的衰弱且往往致命的创伤。此外，这项研究对其他神经退行性疾病，包括局灶性缺血，癫痫，艾滋病脑病和阿尔茨海默病的研究有重要意义。

DESCRIPTION (provided by applicant): Transient, but severe global ischemia, observed in patients during cardiac arrest and cardiac surgery or induced experimentally in animals, induces selective and delayed neurodegeneration. Pyramidal cells in CA1 are the most sensitive; CA3 and granule cells of the dentate gyrus (DG) are resistant to ischemic damage, and GABAergic interneurons in CA1 also survive. The molecular mechanisms underlying this pattern of neuronal death are not well understood. The proposed research aims to study the role of gap junctions during the several days of "maturation" of neuronal injury after global ischemia. Recent findings from this laboratory indicate that global ischemia triggers a selective upregulation of Cx36 (and Cx32) protein expression in GABAergic interneurons of the vulnerable CA1 at times prior to the onset of neuronal death, consistent with a role in the survival of these neurons. Moreover, CA1 neurons in Cx32 (Y/-) mice exhibit enhanced vulnerability to global ischemia-induced neuronal death. These data suggest that increased inhibition of pyramidal cells through synchronization of inhibitory interneurons may be neuroprotective. Gap junctions between astrocytes are also thought to have a role in post-ischemic neuronal death. Dying cells can kill resistant neighboring glial cells via glial "fratricide" (bystander death) and thereby propagate injury to neighboring regions. On the other hand, gap junctional coupling of astrocytes mediates metabolic cooperation among them and attenuates neuronal death in models of oxidative stress. The underlying hypothesis of this proposal is that gap junctions play important roles in determining neuronal death and survival following global ischemia. The research plan for the next five years focuses on changes in the abundance, distribution and molecular and biophysical properties of brain gap junctions following neurological insult. Specific Aims are 1. Characterize ischemia-induced alterations in connexin expression and gap junction properties in the vulnerable CA1 and resistant CA3 and dentate gyrus of rats and mice. Experiments will examine global ischemia-induced changes in coupling of inhibitory interneurons and expression of connexin proteins by immunocytochemistry and Western blotting and of connexin mRNAs by in situ hybridization and. Experiments will determine the effects of acute knockdown of specific connexins by antisense oligonucleotides on neuronal vulnerability and will examine neuronal vulnerability in Cx32(Y/-) mice, Cx36(-/-) mice and mice deficient in astrocyte Cx43. 2. Examine effects of oxygen/glucose deprivation on hippocampal slice cultures by immunocytochemistry, in situ hybridization and electrophysiological methods. To examine ischemia-induced changes in gap junction properties in acute slices and organotypic hippocampal slice cultures by electrophysiological methods and image analysis. The proposed research is expected to impact on the development of new treatment strategies for intervention in global ischemia, a debilitating and often fatal trauma associated with cardiac arrest in humans. Moreover, this study has important implications for research on other neurodegenerative disorders including focal ischemia, epilepsy, AIDS encephalopathy, and Alzheimer's disease.