Cellular Mechanisms Regulating Blood-Brain Barrier Permeability

调节血脑屏障通透性的细胞机制

• 批准号: RGPIN-2016-06025
• 负责人: Miller, Donald
• 金额: $ 2.4万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710100
• 关键词: Cellular; Mechanisms; Regulating; Blood; Brain

The brain microvessel endothelial cells that form the blood-brain barrier (BBB) have an important role in maintaining the proper extracellular environment within the brain required for central nervous system function. Morphologically, the brain microvessel endothelial cells form tight junctions that restrict the passage of solutes between the endothelial cells. In addition, the brain microvessel endothelial cells express a variety of transporters that move solutes and nutrients into and out of the brain. While often considered a static barrier, it is now understood that BBB permeability can be influenced by both neuronal and humoral signals that can alter tight junction and transporter function. However, the cellular mechanisms by which such alterations in BBB permeability can be produced remain to be fully characterized. Much emphasis has been placed on the tight junction complex that forms between adjacent brain microvessel endothelial cells and regulation of BBB permeability. However, it is increasingly recognized that there are plasma membrane proteins, in close proximity to the tight junction complex, that may also influence both paracellular and transcellular passage of solutes, macromolecules and even immune cells. The present Discovery grant application examines the role of two of these plasma membrane proteins in regulation of BBB permeability at the cellular and molecular level. The studies will proceed down two separate and independent research tracks. The first involves understanding the contribution of the adherens junction in regulating both paracellular diffusion and intracellular routes of BBB penetration involving solute carriers and vesicular transport. In particular, the role of the junction adhesion molecule, cadherin (CAD), will be systematically examined from the cellular to whole animal level. The hypothesis is that CAD not only regulates paracellular diffusion across brain endothelial cells but also influences intracellular transport through interactions with scaffold proteins involved in solute and macromolecules transport. The second research theme evaluates the role of connexins (Cxs) in solute and macromolecule permeability. For both CAD and Csx, approaches will first involve examination of these processes in various cell culture models of the BBB using a combination of specifically targeted peptide based inhibitors of these proteins along with siRNA knock-down and gene transfection methods. In vivo studies correlating BBB permeability changes with alterations in CAD and Csx function will also be performed providing research opportunities to trainees (2 grad and 1 undergrad) incorporating functional as well as cellular and molecular biology. Studies will provide key knowledge of cellular processes involved in regulating BBB permeability as well as information on cellular junction formation important in a variety of barrier cells.

形成血脑屏障（BBB）的脑微血管内皮细胞在维持中枢神经系统功能所需的脑内适当的细胞外环境中具有重要作用。 在形态学上，脑微血管内皮细胞形成紧密连接，限制内皮细胞之间的溶质通过。 此外，脑微血管内皮细胞表达多种转运蛋白，将溶质和营养物质移入和移出脑。 虽然经常被认为是静态屏障，但现在理解的是，BBB通透性可以受到神经元和体液信号的影响，这些信号可以改变紧密连接和转运蛋白功能。 然而，这种BBB通透性改变的细胞机制仍有待充分表征。 许多重点放在紧密连接复合物，形成相邻的脑微血管内皮细胞和BBB通透性的调节。 然而，越来越多的人认识到，存在紧密连接复合物附近的质膜蛋白，其也可能影响溶质、大分子甚至免疫细胞的细胞旁和跨细胞通过。本发明的发现资助申请研究了这些质膜蛋白中的两种在细胞和分子水平上调节BBB通透性的作用。这些研究将沿着两条独立的研究轨道进行。 第一个涉及到了解的adherens连接在调节两个细胞旁扩散和细胞内的BBB渗透涉及溶质载体和囊泡运输的途径的贡献。 特别是，连接粘附分子，钙粘蛋白（CAD）的作用，将系统地检查从细胞到整个动物的水平。 假设CAD不仅调节跨脑内皮细胞的细胞旁扩散，而且通过与溶质和大分子转运中涉及的支架蛋白的相互作用影响细胞内转运。 第二个研究主题是评估连接蛋白（Cxs）在溶质和大分子渗透性中的作用。 对于CAD和Csx，方法将首先涉及使用这些蛋白质的基于特异性靶向肽的抑制剂沿着siRNA敲低和基因转染方法的组合在BBB的各种细胞培养模型中检查这些过程。 还将进行体内研究，将BBB通透性变化与CAD和Csx功能的改变相关联，为学员（2名格拉德和1名本科生）提供研究机会，将功能以及细胞和分子生物学结合起来。研究将提供参与调节血脑屏障通透性的细胞过程的关键知识，以及在各种屏障细胞中重要的细胞连接形成的信息。