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患者或动物实验诱导的短暂但严重的全身缺血，可诱导选择性和延迟性神经变性。CA1中的锥体细胞最敏感；CA3和齿状回（DG）颗粒细胞对缺血性损伤具有抵抗性，CA1中的gaba能中间神经元也能存活。这种神经元死亡模式的分子机制尚不清楚。本研究旨在研究间隙连接在全脑缺血后神经元损伤数天“成熟”过程中的作用。该实验室最近的研究结果表明，在神经元死亡之前，全局缺血会触发易感CA1的gaba能中间神经元中Cx36（和Cx32）蛋白表达的选择性上调，这与这些神经元存活的作用一致。此外，Cx32 （Y/-）小鼠的CA1神经元对全局缺血诱导的神经元死亡表现出增强的易感性。这些数据表明，通过抑制中间神经元的同步增加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间隙连接也被认为在缺血后神经元死亡中起作用。垂死的细胞可以通过神经胶质“自相残杀”（旁观者死亡）杀死具有抗性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，星形胶质细胞的间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的神经元死亡。这一提议的基本假设是，间隙连接在决定全脑缺血后神经元的死亡和存活中起重要作用。未来五年的研究计划集中在神经损伤后脑间隙连接的丰度、分布、分子和生物物理特性的变化。具体目标：表征缺血诱导的大鼠和小鼠易损CA1和抗性CA3和齿状回连接蛋白表达和间隙连接特性的改变。实验将通过免疫细胞化学和Western blotting检测抑制中间神经元偶联和连接蛋白表达的全球缺血诱导的变化，并通过原位杂交和Western blotting检测连接蛋白mrna的表达。实验将确定反义寡核苷酸急性敲除特定连接蛋白对神经元易感性的影响，并将检测Cx32（Y/-）小鼠、Cx36（-/-）小鼠和星形胶质细胞Cx43缺乏小鼠的神经元易感性。2. 通过免疫细胞化学、原位杂交和电生理方法检测缺氧/葡萄糖剥夺对海马切片培养的影响。采用电生理方法和图像分析方法，观察急性海马切片和器官型海马切片缺血后间隙连接特性的变化。拟议的研究预计将影响全球缺血干预新治疗策略的发展，全球缺血是一种与人类心脏骤停相关的使人衰弱且通常致命的创伤。此外，该研究对局灶性缺血、癫痫、艾滋病脑病和阿尔茨海默病等其他神经退行性疾病的研究也具有重要意义。

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.