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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616058
• 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）的大脑微血管内皮细胞在维持中枢神经系统功能所需的大脑内适当的细胞外环境方面发挥着重要作用。在形态上，脑微血管内皮细胞形成紧密连接，限制溶质在内皮细胞之间的通过。此外，大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大脑。虽然通常被认为是静态屏障，但现在人们了解到，BBB 通透性可能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，产生这种 BBB 通透性改变的细胞机制仍有待充分表征。人们非常重视相邻脑微血管内皮细胞之间形成的紧密连接复合物以及血脑屏障通透性的调节。然而，人们越来越认识到，紧邻紧密连接复合物的质膜蛋白也可能影响溶质、大分子甚至免疫细胞的细胞旁和跨细胞通道。目前的发现资助申请研究了其中两种质膜蛋白在细胞和分子水平调节血脑屏障通透性中的作用。这些研究将沿着两个独立的研究轨道进行。第一个涉及了解粘附连接在调节涉及溶质载体和囊泡运输的 BBB 渗透的细胞旁扩散和细胞内途径中的贡献。特别是，将从细胞到整个动物水平系统地检查连接粘附分子钙粘蛋白（CAD）的作用。该假设认为，CAD 不仅调节脑内皮细胞的旁细胞扩散，而且还通过与参与溶质和大分子运输的支架蛋白相互作用来影响细胞内运输。第二个研究主题评估连接蛋白 (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以及 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。还将进行将 BBB 通透性变化与 CAD 和 Csx 功能改变相关的体内研究，为学员（2 名研究生和 1 名本科生）提供结合功能以及细胞和分子生物学的研究机会。研究将提供参与调节 BBB 通透性的细胞过程的关键知识，以及有关各种屏障细胞中重要的细胞连接形成的信息。