GLIAL PLASTICITY IN DEVELOPMENT AND REGENERATION

这是一个竞争性的连续赠款，寻求五年的支持。 的 本补助期的目标是调查 可溶性营养因子和胶质细胞表面分子在调节 下丘脑神经元的存活和发育。在下一个项目中 在此期间，我们将扩展和放大这项工作的作用，神经/神经胶质细胞 在塑造神经系统的发育和再生中的相互作用 系统我们已经确定了一个人口的1型星形胶质细胞（命名为 “rocky”），其强烈抑制神经元粘附和生长， 与绝大多数“扁平”星形胶质细胞相反， 神经元粘附和生长。细胞外的岩石状星形胶质细胞 基质（ECM）含有高水平的腱生蛋白和硫酸软骨素-6-。 蛋白聚糖（CS-6-PG），与边界相关的分子， 形成大脑。 扁平星形胶质细胞对某些细胞因子的反应 合成腱生蛋白和CS-6-PG，并变得对神经元不利 生长，类似岩石星形胶质细胞。我们建议，在开发过程中， 损伤后，细胞因子对星形胶质细胞有显著作用， 改变细胞表面分子的表达， 迁移和神经元生长。我们有三个相关的具体目标。要求1 将检验细胞因子改变允许的扁平星形胶质细胞的假设， 抑制神经元的生长和再生。 将培养的大鼠大脑皮质星形胶质细胞暴露于γ-IFN， TNF-α和TGF-β 1。我们将评估组成的变化， 这些星形胶质细胞单层的ECM使用蛋白质印迹， 免疫细胞化学技术，并评估粘附和生长 分离的胚胎神经元与这些细胞以及ECM 这些细胞的制备。神经生长锥的动态运动 将使用延时视频增强技术对这些单层进行评估。 显微镜和激光扫描共聚焦显微镜。具体目标2将 检测人腱生蛋白在大鼠星形胶质细胞（来自 转基因）直接导致体外神经元生长的抑制。 神经元粘附和生长将在具有不同细胞粘附的细胞上进行评价。 tenascin表达水平。我们还将测试假设， 允许性与非允许性ECM分子的表达的平衡 细胞调节神经元的粘附和生长。最后，我们将评估 人的可变剪接变异体的表达的影响， 腱生蛋白特异性目标3将测试阻断生腱蛋白的效果， CS-6-PG与抗体，以评估这些ECM 组分对神经元粘附具有协同抑制作用， 神经突生长我们还将评估糖或蛋白质 蛋白聚糖的部分参与神经元的调节， 粘附/生长。我们还将检验阻断腱生蛋白 而CS-6-PG会降低或消除生长锥感知 允许性和非允许性星形胶质细胞之间的界限。的 提出的实验提供了一个统一的方法来解决一个主要问题， 神经/神经胶质细胞的相互作用在发展过程中，也可能适用于 受伤后缺乏再生。

This is a competing continuation grant seeking five years of support. The objectives of the present grant period were to investigate the effects of soluble trophic factors and glial cell surface molecules in regulating the survival and development of hypothalamic neurons. In the next project period we will extend and amplify this work to the role of neural/glial interactions in shaping the development and regeneration of the nervous system. We have identified a population of type 1 astrocytes (named "rocky") which are strongly inhibitory to neuronal adhesion and growth, in contrast to the vast majority of "flat" astrocytes which are permissive to both neuronal adhesion and growth. The rocky astrocyte extracellular matrix (ECM) contains high levels of tenascin and chondroitin sulfate-6- proteoglycan (CS-6-PG), molecules that have been associated with boundary formation in the brain. In response to certain cytokines, flat astrocytes synthesize tenascin and CS-6-PG and become unfavorable for neuronal growth, resembling rocky astrocytes. We propose that, during development and following injury, cytokines have significant actions on astrocytes to alter their expression of cell surface molecules that then alter neuronal migration and neuronal growth. We have three related Specific Aims. Aim 1 will test the hypothesis that cytokines alter permissive flat astrocytes in ways that make them inhibitory to neuronal growth and regeneration. Cultured rat cerebral cortical astrocytes will be exposed to gamma-IFN, TNF-alpha, and TGF-beta1. We will evaluate changes in the composition of the ECM of these astrocyte monolayers using Western blotting and immunocytochemical techniques and also evaluate the adhesion and outgrowth of dissociated embryonic neurons to these cells as well as to ECM preparations of these cells. The dynamic movement of neuronal growth cones on these monolayers will be evaluated using time-lapse video enhanced microscopy and laser scanning confocal microscopy. Specific Aim 2 will test whether the expression of human tenascin in rat astrocytes (from a transgene) leads directly to inhibition of neuronal growth in vitro. Neuronal adhesion and growth will be evaluated on cells with different levels of tenascin expression. We will also test the hypothesis that the balance of expression of permissive vs. non-permissive ECM molecules on cells regulates neuronal adhesion and growth. Finally, we will evaluate the effects of the expression of the alternately spliced variants of human tenascin. Specific Aim 3 will test the effects of blocking tenascin and CS-6-PG with antibodies in order to evaluate the hypothesis that these ECM components have a synergistic, inhibitory effect on neuronal adhesion and neurite outgrowth. We will also evaluate whether the sugar or the protein moieties of the proteoglycans are involved in modulation of neuronal adhesion/growth. We will also test the hypothesis that blocking tenascin and CS-6-PG will reduce or eliminate the ability of growth cones to sense boundaries between permissive and non-permissive astrocytes. The experiments proposed provide a unified approach to a major problem of neural/glial interactions during development and may also apply to the lack of regeneration following injury.