Cellular Mechanisms Regulating Blood-Brain Barrier Permeability

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起着重要作用。在形态学上，脑微血管内皮细胞形成紧密连接，限制了溶质在内皮细胞之间的通过。此外，脑微血管内皮细胞表达多种转运蛋白，将溶质和营养物质运进和运出大脑。虽然血脑屏障通常被认为是一种静态屏障，但现在人们了解到血脑屏障的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的功能。然而，产生血脑屏障通透性改变的细胞机制仍有待充分研究。许多重点放在紧密连接复合体，形成相邻的脑微血管内皮细胞和血脑屏障的通透性调节。然而，人们越来越认识到，在紧密连接复合体附近存在质膜蛋白，它们也可能影响溶质、大分子甚至免疫细胞的细胞旁和细胞外通道。目前的发现基金申请研究了这两种质膜蛋白在细胞和分子水平上调节血脑屏障通透性的作用。这些研究将沿着两条独立的研究轨道进行。首先要了解粘附体连接在调节血脑屏障渗透的细胞旁扩散和细胞内途径（包括溶质载体和囊泡运输）中的作用。特别是，连接粘附分子钙粘蛋白（CAD）的作用，将从细胞到整个动物水平进行系统研究。假设CAD不仅调节脑内皮细胞的细胞旁扩散，还通过与参与溶质和大分子运输的支架蛋白的相互作用影响细胞内运输。第二个研究主题是评价连接蛋白（Cxs）在溶质和大分子渗透性中的作用。对于CAD和Csx，方法将首先涉及在血脑屏障的各种细胞培养模型中使用这些蛋白质的特异性靶向肽抑制剂以及siRNA敲除和基因转染方法的组合来检查这些过程。血脑屏障通透性变化与CAD和Csx功能变化之间的体内研究也将进行，为学员（2名研究生和1名本科生）提供研究机会，包括功能以及细胞和分子生物学。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。

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.