Astrocyte Development and Reactive Gliosis

描述(申请人提供)：该实验室的长期目标是增强人类神经系统，特别是视网膜和/或视神经的再生能力。如果这项提议得到资助，将确定骨形态发生蛋白7(BMP7)和声波刺猬(SHH)这两个因素在增加一种特别有害的细胞状态-反应性胶质增生-发展中的作用。反应性胶质细胞存在于许多眼科疾病中，包括青光眼、老年性黄斑变性、视网膜色素变性、糖尿病视网膜病变和早产儿视网膜病变。转化为反应性胶质细胞的细胞来自成为视神经的区域，在正常的未损伤或未患病的眼睛中，星形胶质细胞为视网膜的神经节细胞神经元发挥必要的支持功能。然而，一旦眼睛受伤或患病，这些细胞就会变得有反应，于是它们开始表达不利于视神经再生的分子。这项建议侧重于确定骨形态发生蛋白7和SHH调节PAX2表达的机制(S)，PAX2是一种DNA结合蛋白，对视神经星形胶质细胞的发育至关重要。本申请提出了一种新的机制，其中SHH和BMP7都通过改变PAX2表达抑制物的关联来调节发育过程中Pax2的表达。这项应用将测试这一新机制是否会在体外和体内驱动反应性星形胶质细胞的发育。本申请中提出的研究采用多学科方法，包括1)体外检测BMP和SHH通路成员与其他调节DNA结合蛋白的相互作用，如免疫共沉淀、染色质免疫沉淀和荧光素酶检测；2)体外和体内对视神经损伤后BMP通路的操纵，以确定BMP7在反应性胶质增生中的作用；3)体内和体外反义技术检测BMP7作为减少反应性星形胶质细胞的潜在靶点；以及4)利用免疫组织化学、Western blotts、原位杂交、定量聚合酶链式反应和图像分析来表征视神经星形胶质细胞的表型特性。从这些研究中获得的信息将进一步加深我们对反应性星形胶质细胞发育所必需的机制的理解，也可能为减少视神经损伤和疾病中的反应性胶质增生提供治疗靶点。公共卫生相关性：这项提案的重点是研究视神经中的星形胶质细胞及其对损伤的反应。通过研究生长因子对正常和反应性星形胶质细胞发育所需基因的调控作用，我们希望增加青光眼、糖尿病视网膜病变、早产儿视网膜病变、老年性黄斑变性和视网膜色素变性等疾病的治疗靶点。

DESCRIPTION (provided by applicant): The long term goal of this laboratory is to enhance the ability of the human nervous system, particularly the retina and/or optic nerve, to regenerate. This proposal, if funded, would determine the role of two factors, bone morphogenetic protein 7 (BMP7) and sonic hedgehog (SHH), in increasing the development of a particularly detrimental cell state known as reactive gliosis. Reactive glia are present in many eye diseases, including glaucoma, age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy and retinopathy of prematurity. The cells that transform into reactive glia arise from the region that becomes the optic nerve, and in the normal uninjured or non-diseased eye, the astrocytes perform essential supportive functions for the ganglion cell neurons of the retina. However, upon injury to or in the diseased eye, these cells become reactive, whereupon they begin expressing molecules that are unfavorable to the regeneration of the optic nerve. This proposal focuses on determining the mechanism(s) whereby BMP7 and SHH regulate the expression of PAX2, a DNA- binding protein that is essential for the development of optic nerve astrocytes. This application proposes a novel mechanism in which both SHH and BMP7 modulate the expression of Pax2 during development by altering the association of an inhibitor of PAX2 expression. This application will test if this novel mechanism drives the development of reactive astrocytes in vitro and in vivo. The studies proposed in this application use a multi-disciplinary approach, including 1) in vitro 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 following optic nerve injury to determine the role of BMP7 in reactive gliosis, 3) in vivo and in vitro antisense technology to test BMP7 as potential target for reducing reactive astrocytes, and 4) 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 reactive astrocytes and may also provide therapeutic targets for decreasing reactive gliosis in optic nerve injury and disease. PUBLIC HEALTH RELEVANCE: The focus of this proposal is the study of the astrocytes in the optic nerve and their response to injury. By studying the role of growth factors in the regulation of genes that are necessary for the development of normal and reactive astrocytes, we hope to increase the therapeutic targets for diseases such as glaucoma, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and retinitis pigmentosa.