Connexins in Ischemia-Induced Neuronal Death

在心脏骤停和心脏手术期间观察到的短暂但严重的全身性缺血 在动物实验中诱导，诱导选择性和延迟性神经变性。中的锥体细胞 CA1是最敏感的；CA3和齿状回颗粒细胞(DG)对缺血耐受 损伤后，CA1内的GABA能中间神经元仍存活。这背后的分子机制 神经元死亡的模式还不是很清楚。拟议的研究旨在研究GAP的作用 全脑缺血后神经元损伤“成熟”的几天内的连接。最新发现 来自该实验室的研究表明，全球缺血触发了Cx36(和Cx32)蛋白的选择性上调 易损性CA1区GABA能中间神经元在神经元死亡前的表达 与这些神经元的存活所起的作用相一致。此外，Cx32(Y/-)小鼠的CA1神经元表现出 增强了对全球缺血诱导的神经元死亡的易感性。这些数据表明， 通过抑制中间神经元的同步化抑制锥体细胞可能具有神经保护作用。差距 星形胶质细胞之间的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“杀伤性”(旁观者死亡)杀死具有抵抗力的邻近胶质细胞，从而将伤害传播到 邻近地区。另一方面，星形胶质细胞的缝隙连接偶联调节新陈代谢 它们之间的合作，并减轻氧化应激模型中的神经元死亡。潜在的 这一提议的假设是，缝隙连接在决定神经元死亡方面起着重要作用。 以及全脑缺血后的存活率。未来五年的研究计划侧重于以下方面的变化 脑缝隙连接的丰度、分布及其分子和生物物理特性 神经性侮辱。具体目标是：1.表征缺血诱导的连接蛋白改变 易感CA1、抗性CA3和齿状回的表达和缝隙连接特性 老鼠和老鼠。实验将检测全脑缺血诱导的抑制偶联变化 免疫细胞化学和免疫印迹法检测中间神经元和连接蛋白的表达 连接蛋白mRNAs的原位杂交和。实验将确定急性击倒的效果 反义寡核苷酸特异性连接蛋白对神经细胞易损性的影响 Cx32(Y/-)小鼠、Cx36(-/-)小鼠和星形胶质细胞Cx43缺陷小鼠的脆弱性。2.检查以下措施的效果 原位免疫细胞化学方法研究缺氧缺糖对海马片培养的影响 杂交和电生理方法。观察缺血引起的缝隙连接的变化 电生理方法在急性脑片和器官型海马片培养中的特性 图像分析。这项拟议的研究预计将对新疗法的开发产生影响 全球缺血的干预策略，这是一种与心脏相关的衰弱且往往是致命的创伤 在人类身上被逮捕。此外，本研究对其他神经退行性疾病的研究也具有重要意义。 疾病包括局灶性脑缺血、癫痫、艾滋病脑病和阿尔茨海默病。

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 postischemic 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.