Astrocyte Development and Reactive Gliosis

描述（由申请人提供）：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。这项提案如果得到资助，将确定骨形态发生蛋白7（BMP 7）和音速刺猬（SHH）这两个因素在增加一种称为反应性神经胶质增生的特别有害的细胞状态的发展中的作用。反应性胶质细胞存在于许多眼部疾病中，包括青光眼、年龄相关性黄斑变性、色素性视网膜炎、糖尿病性视网膜病变和早产儿视网膜病变。转化为反应性神经胶质的细胞来自成为视神经的区域，并且在正常未受伤或未患病的眼睛中，星形胶质细胞对视网膜的神经节细胞神经元执行必要的支持功能。然而，一旦损伤患病眼睛或患病眼睛中，这些细胞变得具有反应性，于是它们开始表达不利于视神经再生的分子。该建议集中于确定BMP 7和SHH调节PAX 2表达的机制，PAX 2是一种对视神经星形胶质细胞发育至关重要的DNA结合蛋白。本申请提出了一种新的机制，其中SHH和BMP 7通过改变PAX 2表达抑制剂的结合来调节发育期间Pax 2的表达。该应用程序将测试这种新的机制是否驱动体外和体内反应性星形胶质细胞的发展。本申请中提出的研究使用多学科方法，包括1）体外测定以确定BMP和SHH途径成员与其它调节性DNA结合蛋白的相互作用，例如免疫共沉淀、染色质免疫沉淀和荧光素酶测定，2）视神经损伤后BMP途径的体外和体内操作以确定BMP 7在反应性神经胶质增生中的作用，3）体内和体外反义技术以测试BMP 7作为减少反应性星形胶质细胞的潜在靶标，和4）使用免疫组织化学、蛋白质印迹、原位杂交、定量聚合酶链反应和图像分析在体外和体内表征视神经星形胶质细胞的表型性质。从这些研究中获得的信息将进一步我们的反应性星形胶质细胞的发展所需的机制的理解，也可能提供治疗目标，减少视神经损伤和疾病的反应性胶质增生。公共卫生相关性：该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。通过研究生长因子在调节正常和反应性星形胶质细胞发育所必需的基因中的作用，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、年龄相关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.