Astrocyte dystroglycan complexes in brain development

描述（申请人摘要）：肌营养不良蛋白-糖蛋白复合物（DGC） 是细胞质，跨膜， 细胞外基质蛋白，形成了一个关键的连接之间的 细胞骨架和横纹肌的基底层。在中央 神经系统（CNS），与基底板层类似的肌营养不良聚糖连接是 存在于星形胶质细胞形成的两个界面：（1）足突邻接 脑血管和（2）足突，形成胶质界膜， 大脑的软脑膜表面前一个界面是形成的关键 和维持血脑屏障，而后者可能发挥作用， 在神经元迁移过程中锚定放射状胶质细胞的重要作用。基底 在胶质界膜的层异常已经在某些形式的 人类先天性肌营养不良症（例如福山肌营养不良症）和 神经胶质界膜的基底层破坏导致异常的CNS发育 在动物模型中。在这份建议中，我们将重点放在中央 蛋白质在星形胶质细胞-基底层连接，肌营养不良聚糖。我们的具体目标 提出鉴定星形胶质细胞-肌营养不良蛋白聚糖复合物的蛋白元件， 阐明复合物内的蛋白质相互作用，并证明 星形胶质细胞抗肌萎缩蛋白聚糖复合物在中枢神经系统发育过程中的重要性。 通过使用Cre-lox方法，我们计划建立一种新的小鼠模型 中枢神经系统发育障碍这个项目是一个交叉学科 具有临床神经病理学专业知识的研究者之间的合作， 在基础神经科学、分子生物学、细胞生物学和膜生理学方面 他们是唯一能够进行拟议研究的人。目标1：界定 星形胶质细胞-肌营养不良蛋白聚糖复合物的组成，我们将测试 一种或多种肌营养不良蛋白聚糖复合物存在于星形胶质细胞中的假设 使用生物化学和免疫组织化学方法的组合。这些 研究将利用来自野生型的组织切片和培养的星形胶质细胞 小鼠和具有天然存在的或基因工程突变的小鼠， 已知在星形胶质细胞中表达的一种或多种DGC组分。目标二： 通过选择性地建立一种新的中枢神经系统发育异常模型， 破坏星形胶质细胞-肌营养不良蛋白聚糖复合物。肌营养不良聚糖+/-，肌营养不良聚糖 lox/lox和GFAP-Cre小鼠将被饲养以产生GFAP-Cre/肌营养不良聚糖lox/- 和GFAP-Cre/肌营养不良聚糖lox/lox小鼠。这一战略将打破 星形胶质细胞DGC开始于胚胎发育的后半期。我们认为 这种策略将产生具有神经元迁移和脑血管疾病的小鼠， 缺陷

DESCRIPTION (Applicant's abstract): The dystrophin-glycoprotein complex (DGC) is a well characterized array of cytoplasmic, membrane spanning, and extracellular matrix proteins that form a critical linkage between the cytoskeleton and the basal lamina of striated muscle. Within the central nervous system (CNS), similar dystroglycan linkages to basal laminae are present at two interfaces formed by astrocytes: (1) foot processes abutting on cerebral blood vessels and (2) foot processes that form the glia limitans at the pial surface of the brain. The former interface is critical for formation and maintenance of the blood-brain barrier, while the latter is likely to play important roles in anchoring radial glia during neuronal migration. Basal lamina abnormalities at the glia limitans have been identified in some forms of congenital muscular dystrophy in humans (e.g. Fukuyama muscular dystrophy) and basal lamina disruption at the glia limitans leads to abnormal CNS development in animal models. In this proposal, we will focus attention on the central protein in the astrocyte-basal lamina linkage, dystroglycan. Our Specific Aims propose to identify protein elements of the astrocyte-dystroglycan complex, elucidate protein interactions within the complex, and demonstrate the importance of the astrocyte dystroglycan complex during CNS development. Through the use of Cre-lox methodology, we plan to create a novel murine model of CNS developmental disorders. This project is a cross-discipline collaboration among investigators with expertise in clinical neuropathology and in basic neuroscience, molecular biology, cell biology, and membrane physiology who are uniquely situated to carry out the proposed studies. Aim 1: To define the composition of the astrocyte-dystroglycan complex(es), we will test the hypothesis that one-or-more dystroglycan complexes are present in astrocytes using a combination of biochemical and immunohistochemical methods. These studies will utilize tissue sections and cultured astrocytes from wild type mice and mice with naturally occurring or genetically engineered mutations of one or more of the DGC components known to be expressed in astrocytes. Aim 2: To create a new model of CNS developmental abnormalities by selectively disrupting the astrocyte-dystroglycan complex. Dystroglycan +/-, dystroglycan lox/lox, and GFAP-Cre mice will be bred to produce GFAP-Cre/dystroglycan lox/- and GFAP-Cre/dystroglycan lox/lox mice. This strategy should disrupt the astrocyte DGC beginning in the latter half of embryonic development. We believe this strategy will produce mice with neuronal migration and cerebrovascular defects.