Mechanisms of Astrocyte Development

描述（由申请人提供）：中枢神经系统含有一组专门的神经胶质细胞，称为星形胶质细胞。成熟的视神经和视网膜星形胶质细胞对视网膜神经节细胞起代谢支持作用，并维持血-视网膜屏障。这些细胞也可以转化为神经系统的破坏性成分，称为反应性星形胶质细胞，形成轴突再生的永久屏障。发育中的视神经星形胶质细胞（星形胶质细胞前体）发挥着许多眼睛所特有的作用。首先，星形胶质细胞前体作为神经节细胞轴突的引诱剂，因为它们离开眼睛向中枢神经系统其余部分的其他靶细胞生长。其次，星形胶质细胞前体细胞的亚群迁移到眼睛中，在那里它们吸引来自眼睛外部的内皮细胞进入并充当内皮细胞在其上形成原代视网膜血管系统的基质。我的实验室的长期目标是了解某些生长和模式化因子，如骨形态发生蛋白（BMP）和音刺猬（SHH），在视杯和神经中正常和反应性星形胶质细胞中直接分化和基因表达的机制。目前的建议的目标是确定的分子机制，其中Pax 2，在星形胶质细胞分化的关键基因，可能是由BMP 7和SHH在发展过程中进行调节。 本申请将检验SHH和BMP 7通过调节抑制蛋白TLX的活性在原代视网膜星形胶质细胞中协同调节Pax 2的假设。本申请中提出的研究涉及多学科方法，包括1）测定BMP和SHH途径成员与其它调节性DNA结合蛋白的相互作用的体外和体内测定，例如免疫共沉淀、染色质免疫沉淀和荧光素酶测定，2）BMP途径的体外和体内操作，3）采用免疫组化、Western印迹、原位杂交、定量聚合酶链反应和图像分析等方法对体外和体内视神经星形胶质细胞的表型特征进行了表征。从这些研究中获得的信息将进一步我们的视网膜和视神经星形胶质细胞的发展所必需的机制的理解，也可能增加我们的理解反应性星形胶质细胞如何发展后损伤或疾病的视网膜。 公共卫生相关性：视柄中发育中的星形胶质细胞对于视网膜神经节细胞离开眼睛时的路径发现和视网膜血管系统的发育至关重要。其中一个转录因子Pax 2是星形胶质细胞分化所必需的。我们的实验室提出了一种新的机制，即在发育中的视柄，骨形态发生蛋白7和音刺猬，在阻遏蛋白TLX在Pax 2启动子的调制中合作的两个因素的细胞内信号通路。该应用的资助将导致1）增加对视网膜星形胶质细胞发育机制的了解，2）将增强我们对这些因素如何在眼睛其他部分发挥作用的理解，3）增强我们对反应性星形胶质细胞如何在疾病中形成的理解，以及神经系统损伤后，并可能提供治疗反应性星形胶质细胞的治疗靶点。

DESCRIPTION (provided by applicant): The central nervous system contains a specialized group of glial cells, known as astrocytes. Mature optic nerve and retinal astrocytes play a metabolically supportive role for retinal ganglion cells, as well as maintain the blood-retinal barrier. These cells can also transform into a destructive component of the nervous system, known as reactive astrocytes, which form permanent barriers to axonal regeneration. Developing optic nerve astrocytes (astrocyte precursors) play a number of roles that are unique to the eye. First, astrocyte precursors act as an attractant for ganglion cell axons as they leave the eye to grow towards other target cells in the rest of the central nervous system. Second, a subpopulation of the astrocyte precursor cells migrates into the eye where they beckon to endothelial cells from outside the eye to enter and act as the substrate upon which the endothelial cells form the primary retinal vasculature. The long term goal of my laboratory is to understand the mechanisms by which certain growth and patterning factors, such as bone morphogenetic proteins (BMPs) and sonic hedgehog (SHH), direct differentiation and gene expression in normal and reactive astrocytes in the optic cup and nerve. The goal of the current proposal is to determine the molecular mechanisms by which Pax2, a gene critical in astrocyte differentiation, may be regulated by BMP7 and SHH during development. This application will test the hypothesis that SHH and BMP7 cooperate in the regulation of Pax2 in primary retinal astrocytes by moderating the activity of the suppressor protein TLX. The studies proposed in this application involve a multi-disciplinary approach, including 1) in vitro and in vivo assays to determine interactions of BMP and SHH pathway members with other regulatory DNA-binding proteins such as co-immunoprecipitation, chromatin immunoprecipitation, and luciferase assays, 2) in vitro and in vivo manipulations of BMP pathways, 3) characterization of the phenotypic properties of optic nerve astrocytes in vitro and in vivo using immunohistochemistry, Western blots, in situ hybridization, quantitative polymerase chain reaction and image analysis. Information derived from these studies will further our understanding of the mechanisms necessary for the development of retinal and optic nerve astrocytes and may also increase our understanding of how reactive astrocytes develop following injury or disease to the retina. PUBLIC HEALTH RELEVANCE: Developing astrocytes in the optic stalk are critical for the path finding of retina ganglion cells as they leave the eye and the development of retinal vasculature. One transcription factor, Pax2, is necessary and sufficient for the differentiation of astrocytes. Our lab has proposed a novel mechanism whereby the intracellular signaling pathways of two factors found in the developing optic stalk, bone morphogenetic protein 7 and sonic hedgehog, cooperate in the modulation of repressor protein TLX at the Pax2 promoter. Funding of this application would result in 1) increased knowledge of the mechanisms involved in the development of retinal astrocytes and 2) will enhance our understanding of how these factors may work in the rest of the eye, and 3) augment our understanding of how reactive astrocytes may form in disease and following injury of the nervous system and perhaps provide therapeutic targets to treat reactive astrocytes.