Astrocyte Development and Reactive Gliosis

描述（由申请人提供）：本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。如果得到资助，这一提议将确定两个因素的作用，骨形态发生蛋白7 （BMP7）和音hedgehog (SHH)，在增加一种特别有害的细胞状态的发展中所起的作用，这种状态被称为反应性胶质瘤。反应性胶质细胞存在于许多眼病中，包括青光眼、年龄相关性黄斑变性、视网膜色素变性、糖尿病性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视神经区域，在正常的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起着重要的支持作用。然而，在受伤或患病的眼睛中，这些细胞变得具有反应性，因此它们开始表达不利于视神经再生的分子。本研究的重点是确定BMP7和SHH调节PAX2表达的机制，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。该应用提出了一种新的机制，其中SHH和BMP7通过改变Pax2表达抑制剂的关联来调节发育过程中Pax2的表达。这项应用将测试这种新机制是否在体内和体外驱动反应性星形胶质细胞的发展。本申请中提出的研究采用多学科方法，包括1)体外实验确定BMP和SHH通路成员与其他调节dna结合蛋白的相互作用，如共免疫沉淀、染色质免疫沉淀和荧光素酶测定；2)体外和体内操作视神经损伤后BMP通路，确定BMP7在反应性胶质瘤中的作用；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；4)利用免疫组织化学、Western blots、原位杂交、定量聚合酶链反应和图像分析表征视神经星形胶质细胞在体外和体内的表型特性。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的必要机制的理解，也可能为减少视神经损伤和疾病的反应性胶质增生提供治疗靶点。公共卫生相关性：本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正常和反应性星形胶质细胞发育所需基因调控中的作用，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、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.