Astrocyte dystroglycan complexes in brain development

描述(申请人摘要)：营养不良蛋白-糖蛋白复合体(DGC) 是一个特征良好的细胞质、跨膜和 细胞外基质蛋白形成关键的连接之间的联系 细胞骨架和横纹肌的基板。在中环内 神经系统(CNS)，与基底板类似的营养不良糖链是 存在于星形胶质细胞形成的两个界面：(1)足突与 脑血管和(2)形成胶质细胞界限的足突。 大脑的软膜表面。前一种界面对地层至关重要 和维持血脑屏障，而后者很可能发挥作用 在神经元迁移过程中锚定放射状胶质细胞的重要作用。基本的 已在某些形式的脑胶质瘤中发现了神经胶质细胞限制层的异常。 人类先天性肌营养不良症(例如福山肌营养不良症)和 神经胶质细胞限制区基底膜断裂导致中枢神经系统发育异常 在动物模型中。在这份提案中，我们将重点关注中央 蛋白质在星形胶质细胞-基底膜的连接中，营养不良。我们的具体目标 建议确定星形胶质细胞-营养不良多糖复合体的蛋白质成分， 阐明蛋白质在复合体中的相互作用，并证明 星形胶质细胞营养不良多糖复合体在中枢神经系统发育中的重要性。 通过使用Cre-lox方法学，我们计划创建一种新的小鼠模型 中枢神经系统发育障碍。这个项目是一个跨学科的项目 具有临床神经病理学专业知识的研究人员之间的合作 基础神经科学、分子生物学、细胞生物学和膜生理学 他们处于开展拟议研究的独特位置。目标1：定义 星形胶质细胞-营养不良多糖复合体(ES)的组成，我们将测试 星形胶质细胞中存在一个或多个营养不良多糖复合体的假说 使用生化和免疫组织化学相结合的方法。这些 研究将利用野生型的组织切片和培养的星形胶质细胞 小鼠和带有自然发生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成分。目标2： 选择性地建立中枢神经系统发育异常的新模型 破坏星形胶质细胞-营养不良多糖复合体。营养不良多糖+/-，营养不良多糖 LOX/LOX，以及GFAP-CRE小鼠将被培育成产生GFAP-CRE/DYSTROGLAN LOX/- 和GFAP-Cre/dystroglan lox/lox小鼠。这一战略应该会扰乱 星形胶质细胞DGC开始于胚胎发育的后半期。我们相信 这一策略将产生具有神经元迁移和脑血管功能的小鼠 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.