New tools for understanding the blood brain barrier

描述（由申请人提供）：中枢神经系统（CNS）需要一个严格控制的无毒素和病原体的环境，为突触传递提供适当的化学条件。这种环境由“血脑屏障”（BBB）维持，血脑屏障由高度特化的血管组成，其内皮显示特化的紧密连接和异常低的跨细胞囊泡转运（胞吞转运）速率。虽然BBB破坏最近与各种神经系统疾病有关，但完整的BBB也对药物递送至CNS构成主要障碍。制药公司花费数十亿美元开发可以穿透血脑屏障治疗疾病的药物。然而，由于在理解BBB功能如何调节和识别控制其过程的基本分子成分方面存在显著的知识缺口，因此在操纵BBB方面几乎没有取得进展。这种有限的理解也阻碍了我们在治疗上操纵BBB的能力。了解血脑屏障的主要障碍是确定其基本组成部分，并阐明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，其已经包含阻止其用于重构研究的独特性质。类似地，研究BBB的主要技术是EM，然而其静态快照不提供关于主动和动态囊泡运输、方向性或其特定途径的信息，以允许研究者询问调节BBB完整性的关键分子机制。最近，随着我们在神经和血管系统发育共同布线的研究背景， 我们使用传统的发育方法，首先绘制BBB形成的精确时间，然后 确定了诱导CNS内皮获得BBB特性的神经线索，以及可能具有BBB特性的分子。 从CNS和外周内皮细胞之间的简单转录组比较中可以看出， 令人惊讶的是，我们还发现，与其是结构上重要的紧密结构的物理积累或破坏， 正如以前认为的那样，转胞吞调节似乎更有可能是主要的机制。 基本的血脑屏障完整性在描述这些发育特性时，我意识到这些发现是 这只是冰山一角，真正根本的问题仍然是确定核心途径， 了解它们如何调节BBB功能。因此，需要新的工具来理解BBB。这里我们 建议首先开发一种新的基于干细胞的系统，以允许在体外重建功能性BBB，并且 然后开发一种遗传光学系统，用于监测体内BBB的功能完整性， 真实的时间的亚细胞分辨率。这一综合办法将解决有关 这将导致更有效的治疗策略和针对BBB的特异性靶点 修复和操纵。

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.