GLIAL PLASTICITY IN DEVELOPMENT AND REGENERATION

这是一项相互竞争的延续拨款，旨在寻求五年的支持。这个 目前赠款期间的目标是调查以下方面的影响 可溶性营养因子和胶质细胞表面分子在调节血管内皮生长中的作用 下丘脑神经元的存活和发育。在下一个项目中 期间，我们将扩展和扩大这项工作，以发挥神经/神经胶质的作用 塑造神经发育和再生的相互作用 系统。我们已经确定了一组1型星形胶质细胞(命名为 对神经细胞的黏附和生长有强烈的抑制作用 与绝大多数“扁平”星形胶质细胞相比， 神经元黏附和生长。洛基星形细胞胞外 基质(ECM)含有高水平的肌腱蛋白和硫酸软骨素-6- 蛋白多糖(CS-6-PG)，与边界相关的分子 在大脑中形成。对某些细胞因子的反应，扁平星形细胞 肌腱蛋白和CS-6-PG的合成及其对神经元的不利影响 生长，类似于岩石状的星形细胞。我们建议，在开发过程中， 在损伤后，细胞因子对星形胶质细胞有显著的作用 改变细胞表面分子的表达，进而改变神经元 迁移和神经元生长。我们有三个相关的具体目标。目标1 将检验细胞因子改变允许的扁平星形胶质细胞的假设 以抑制神经元生长和再生的方式。 培养的大鼠大脑皮层星形胶质细胞将暴露于伽马-干扰素， 肿瘤坏死因子-α和转化生长因子-β1。我们将评估组成的变化 用Western blotting和免疫印迹法检测星形胶质细胞单层的细胞外基质 免疫细胞化学技术，并评估黏附和生长 分离的胚胎神经元与这些细胞和细胞外基质的关系 这些细胞的准备工作。神经元生长锥体的动态运动 在这些单层上，将使用延时视频增强进行评估 显微镜和激光扫描共聚焦显微镜。特定目标2将 检测人Tenascin在大鼠星形胶质细胞中的表达 转基因)可直接抑制体外培养的神经元生长。 神经元的黏附和生长将在不同类型的细胞上进行评估 Tenascin的表达水平。我们还将测试以下假设： 允许和不允许的细胞外基质分子在细胞外基质上的表达平衡 细胞调节神经元的黏附和生长。最后，我们将评估 人交错剪接变异体表达的影响 Tenascin。具体目标3将测试阻断Tenascin和 用CS-6-PG和抗体来评估这些ECM 各组分对神经元黏附有协同抑制作用， 轴突生长。我们还将评估糖或蛋白质 蛋白多糖的部分参与了神经元的调节 黏附/生长。我们还将测试阻断Tenascin的假设 而CS-6-PG会降低或消除生长锥的感知能力 允许和不允许的星形胶质细胞之间的边界。这个 拟议的实验为解决以下主要问题提供了统一的方法 神经/神经胶质细胞在发育过程中的相互作用，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.