Connexins in Ischemia-Induced Neuronal Death

描述(由申请人提供)：在心脏骤停和心脏手术期间观察到的短暂但严重的全局性缺血，或在动物实验中诱导的，导致选择性和延迟性神经变性。CA1区锥体细胞最为敏感，CA3区和齿状回颗粒细胞对缺血损伤有抵抗作用，CA1区GABA能中间神经元也存活。这种神经元死亡模式背后的分子机制还不是很清楚。这项拟议的研究旨在研究缝隙连接在全脑缺血后神经元损伤“成熟”的几天中所起的作用。本实验室的最新发现表明，在神经元死亡开始之前，全脑缺血在脆弱的CA1区的GABA能中间神经元中触发了Cx36(和Cx32)蛋白的选择性上调，这与这些神经元的存活所起的作用是一致的。此外，Cx32(Y/-)小鼠的CA1神经元对全脑缺血诱导的神经元死亡表现出更强的脆弱性。这些数据表明，通过抑制中间神经元的同步化来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。濒临死亡的细胞可以通过神经胶质“兄弟会”(旁观者死亡)杀死耐药的邻近神经胶质细胞，从而将损伤传播到邻近区域。另一方面，星形胶质细胞的缝隙连接偶联介导了它们之间的代谢合作，并减轻了氧化应激模型中神经元的死亡。这一提议的基本假设是，缝隙连接在决定神经元在全脑缺血后的死亡和存活中发挥着重要作用。未来五年的研究计划重点是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性质的变化。具体目标是1.表征缺血诱导的大鼠和小鼠脆弱的CA1和耐药的CA3和齿状回中连接蛋白表达和缝隙连接特性的变化。实验将通过免疫细胞化学和Western blotting检测全脑缺血诱导的抑制性中间神经元偶联和连接蛋白表达的变化，通过原位杂交检测连接蛋白mRNAs的表达。实验将确定特定连接蛋白被反义寡核苷酸急性敲除对神经元脆弱性的影响，并将检测Cx32(Y/-)小鼠、Cx36(-/-)小鼠和星形胶质细胞Cx43缺陷小鼠的神经元易损性。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片培养的影响。用电生理学方法和图像分析方法检测急性脑片和器官型海马片培养中缺血诱导的缝隙连接特性的变化。这项拟议的研究预计将对干预全球缺血的新治疗策略的开发产生影响。全球缺血是一种与人类心脏骤停相关的虚弱且往往是致命的创伤。此外，这项研究对其他神经退行性疾病的研究也有重要意义，包括局灶性脑缺血、癫痫、艾滋病脑病和阿尔茨海默病。

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.