GAP JUNCTIONS AND ASTROCYTES

本项目的目标是分析GAP的生理作用。 星形胶质细胞形成的连接。压倒一切的假设是这种差距 连接在关键的星形细胞功能中很重要， 耦合的中断将对这些功能产生不利影响。我们会 应用缝隙连接通信的标准调制方法， 所有这些都至少部分是在该计划中开发的，以及新方法 包括同源重组和反义基因敲除 战略。另一个重要的进一步方法将是恢复耦合 通过瞬时转染法或野生型Cx43病毒转导法， 与突变体Cx43或与另一种在功能上不同于 Cx43。具体目的1.鉴定S表达的缝隙连接蛋白 星形胶质细胞。星形胶质细胞的主要连接蛋白是Cx43；我们有很强的 有证据表明Cx40也有表达，尽管程度较小。 Cx43基因敲除小鼠的星形胶质细胞表现出罕见的弱电信号 和染料耦合；连接通道属性不同于 Cx43等位基因。具体目标2.检验缝隙连接的假设 星形胶质细胞间的偶联介导钙波的传播 促进神经营养物质的分泌和摄取 神经递质。依赖于缝隙连接通讯的阻滞剂 在连接蛋白核苷酸或蛋白质序列上将允许高度特异性 接合联轴器的块。Cx43基因敲除小鼠的星形胶质细胞 还提供了一种偶联程度较低的制剂。具体目标3.测试 在培养中，假设神经元和其他细胞分泌的因子 细胞类型调节星形胶质细胞间隙的水平、分布和性质 交汇点。星形胶质细胞间缝隙连接的发生率发生变化 体内创伤和偶联可通过以下方式调节 体外神经递质。我们将尝试在体外复制 反应性胶质细胞增生症中星形胶质细胞连接蛋白的变化。特定的 目的4.表征反应性星形胶质细胞偶联的变化 神经胶质增多症。我们将确定联轴器是否有变化 与Cx43分布和免疫反应性的变化有关。 我们将确定胶质细胞增多症的其他变化，如向上- 对GFAP的调节，受偶联修饰的影响。具体目标5. 检验星形胶质细胞间缝隙连接减少的假设 对致痫刺激的敏感性。我们将使用脑片在 培养和测定修饰对惊厥阈值的影响 结合式耦合。这项基础广泛的研究应该会大大增加我们的 了解星形胶质细胞的作用，并提供 针对创伤的神经保护的治疗策略 预防神经退行性变和改善疾病 流程。

The goal of this project is to analyze the physiological roles of gap junctions formed by astrocytes. The overriding hypothesis is that gap junctions are important in critical astrocytic functions and that disruption of coupling will impact adversely on these functions. We will apply standard methods of modulation of gap junctional communication, all at least partially developed in this Program, and new methods including gene knockout by homologous recombination and antisense strategies. An important further approach will be to restore coupling by transient transfection or viral transduction with wild type Cx43, with mutant Cx43 or with another connexin that differs functionally from Cx43. Specific Aim 1. Characterize the connexin(s) expressed by astrocytes. The principal connexin in astrocytes is Cx43; we have strong evidence that Cx40 is also expressed, although to a lesser degree. Astrocytes from a Cx43 knockout mouse exhibit infrequent weak electrical and dye coupling; the junctional channel properties are distinct from those of Cx43. Specific Aim 2. Test the hypothesis that gap junctional coupling between astrocytes mediates propagation of Ca2+ waves and facilitates secretion of neurotrophic substances and uptake of neurotransmitters. Blockers of gap junctional communication that depend on connexin nucleotide or protein sequences will allow highly specific block of junctional coupling. Astrocytes from the Cx43 knockout mouse also provide a preparation with reduced coupling. Specific Aim 3. Test in culture the hypothesis that factors secreted by neurons and other cell types modulate levels, distribution and properties of astrocyte gap junctions. The incidence of gap junctions between astrocytes changes following trauma in vivo and coupling can be modulated by neurotransmitters in vitro. We will attempt to reproduce in vitro the changes in astrocyte connexins that occur in reactive gliosis. Specific Aim 4. Characterize the changes in coupling of astrocytes in reactive gliosis. We will determine whether there are alterations in coupling associated with the changes in Cx43 distribution and immunoreactivity. We will determine whether other changes in gliosis, such as up- regulation of GFAP, are affected by modifying coupling. Specific Aim 5. Test the hypothesis that gap junctions between astrocytes decrease susceptibility to epileptogenic stimuli. We will use brain slices in culture and determine effects on seizure thresholds of modifying junctional coupling. This broad based study should greatly increase our understanding of the roles of astrocytes and provide possibilities of therapeutic strategies for neural protection against trauma, for prevention of neurodegeneration and for amelioration of disease processes.