New tools for understanding the blood brain barrier

描述(申请人提供)：中枢神经系统(CNS)需要一个严密控制的没有毒素和病原体的环境，为突触传递提供适当的化学条件。这种环境是由血脑屏障(BBB)维持的，血脑屏障由高度专门化的血管组成，其内皮细胞显示出特殊的紧密连接和异常低的跨细胞囊泡运输(跨细胞)率。虽然血脑屏障的破坏最近与各种神经疾病有关，但完整的血脑屏障也对药物输送到中枢神经系统构成了主要障碍。制药公司花费数十亿美元开发能够穿透血脑屏障治疗疾病的药物。然而，由于在了解BBB功能是如何调节的以及确定控制其过程的基本分子成分方面存在显著的知识差距，在操纵BBB方面进展甚微。这种有限的理解也阻碍了我们在治疗上操纵血脑屏障的能力。理解血脑屏障的主要障碍是确定其基本成分，并解开这些关键调节因子控制血脑屏障功能的机制。然而，目前的体外模型依赖于完全分化的内皮细胞。 细胞，这些细胞已经包含独特的属性，使其无法用于重建研究。同样，研究血脑屏障的主要技术一直是EM，然而它的静态快照不提供关于主动和动态的囊泡运输、方向性或其特定路线的信息，从而使研究人员能够询问调节血脑屏障完整性的关键分子机制。最近，以我们在神经系统和血管系统发育共同连接方面的研究背景， 我们使用传统的发育方法，首先绘制出血脑屏障形成的准确时间，然后 识别了诱导中枢神经系统内皮细胞获得血脑屏障特性的神经线索，以及可能具有 中枢神经系统和外周内皮细胞的简单转录组比较在血脑屏障功能中的作用。 令人惊讶的是，我们还发现，紧凑的结构不是物理上的积聚或破坏 正如之前所认为的，跨细胞调节似乎更有可能是主要的机制。 潜在的BBB完整性。在描述这些发育特性时，我意识到这些发现 这只是冰山一角，真正根本的问题仍然是确定核心途径和 了解它们是如何调节血脑屏障功能的。因此，需要新的工具来理解BBB。在这里我们 首先建议开发一种新的基于干细胞的系统，以允许在体外重建具有功能的血脑屏障，以及 然后开发一种遗传光学系统，用于监测体内血脑屏障的功能完整性 实时亚细胞分辨率。这一综合方法将解决有关 调节血脑屏障，这将导致更有效的治疗策略和针对血脑屏障的特定靶点 恢复和操纵。

DESCRIPTION (provided by applicant): The central nervous system (CNS) requires a tightly controlled environment free of toxins and pathogens to provide the proper chemical conditions for synaptic transmission. This environment is maintained by the 'blood brain barrier' (BBB), which is composed of highly specialized blood vessels whose endothelium display specialized tight junctions and unusually low rates of transcellular vesicular transport (transcytosis). While BBB breakdown has recently been associated with various neurological disorders, an intact BBB also poses a major obstacle for drug delivery to the CNS. Pharmaceutical companies spend billions of dollars to develop drugs that can penetrate the BBB to treat disease. However, little progress has been made on manipulating the BBB due to a significant knowledge gap in understanding how BBB function is regulated and identifying the essential molecular constituents governing its processes. This limited understanding has also thwarted our ability to therapeutically manipulate the BBB. The major impediment to understanding the BBB is identifying its essential constituent and unraveling the mechanism by which these key regulators control BBB function. However, the current in vitro models rely on fully differentiated endothelial cells, which already contain unique properties that prevent their use in reconstitution studies. Similarly, the main technique to study the BBB has been EM, however its static snapshots do not provide information on active and dynamic vesicular transport, directionality, or their specifi routes to allow investigators to interrogate the key molecular mechanisms that regulate BBB integrity. Recently, with our research background in developmental co-wiring of nervous and vascular systems, we used the traditional developmental approach, to first mapped the precise timing of BBB formation and then identified neural cues that induce CNS endothelium to acquire BBB properties, and molecules with possible roles in BBB function from simple transcriptome comparisons between CNS and peripheral endothelial cells. Surprisingly, we also found that instead of a physical buildup or disruption of structurally important tight junctions as previously thought, transcytosis regulation seems to be the more likely the major mechanism underlying BBB integrity. In characterizing these developmental properties, I realized that these findings are just the tip of the iceberg and that truly fundamental questions remain in identifying the core pathway and understand how they regulate BBB function. New tools thus are needed for understanding the BBB. Here we propose first to develop a new stem cell-based system to allow reconstitution of a functional BBB in vitro, and then to develop a genetic-optical system for monitoring the functional integrity of the BBB in vivo at subcellular resolution in real time. This integrated approach will address fundamental questions about the regulation of the BBB, which will then lead to more effective therapeutic strategies and specific targets for BBB restoration and manipulation.