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方面起着重要作用。从形态上看，脑微血管内皮细胞形成紧密的连接，限制了内皮细胞之间的溶质通道。此外，脑微血管内皮细胞表达多种转运体，将溶质和营养物质运入和运出大脑。虽然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，导致血脑屏障通透性改变的细胞机制仍未完全确定。研究的重点是相邻脑微血管内皮细胞之间形成的紧密连接复合体和血脑屏障通透性的调节。然而，人们越来越认识到，紧密连接复合体附近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。目前的发现金申请研究了其中两种质膜蛋白在细胞和分子水平上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。第一个涉及理解粘着连接在调节BBB穿透的细胞旁扩散和细胞内途径中的作用，涉及溶质载体和囊泡运输。特别是，连接黏附分子钙粘附素(CAD)的作用将从细胞到整个动物水平进行系统的研究。该假说认为，CAD不仅调节细胞间跨内皮细胞的扩散，而且通过与参与溶质和大分子运输的支架蛋白的相互作用来影响细胞内的运输。第二个研究主题评估了连接蛋白(CXS)在溶质和大分子通透性中的作用。对于CAD和CSX，方法将首先涉及在各种血脑屏障细胞培养模型中检查这些过程，使用基于这些蛋白质的特定靶向多肽抑制剂的组合，以及siRNA敲除和基因转染法。还将进行将血脑屏障通透性变化与CAD和CSX功能变化相关联的活体研究，为结合功能生物学以及细胞和分子生物学的受训者(2名本科和1名本科生)提供研究机会。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.