Mechanisms Underlying the Suppression of Transcytosis at the Blood Brain Barrier

抑制血脑屏障转胞吞作用的机制

• 批准号: 8830634
• 负责人: Benjamin Joseph Andreone
• 金额: $ 3.48万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8830634
• 关键词: Ablation; Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Biochemical; Biological Assay; Blood - brain barrier anatomy; Blood Vessels; Blood capillaries; Brain; Brain Neoplasms; Caveolae; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Central Nervous System Diseases; Cerebrum; Chemicals; Clathrin; Clathrin-Coated Vesicles; Co-Immunoprecipitations; Complex; Cytoplasm; Data; Drug Delivery Systems; Endothelial Cells; Endothelium; Ensure; Environment; Event; Extravasation; Family; Functional disorder; Genetic; Goals; Homeostasis; In Vitro; Ions; Label; Mediating; Molecular; Multiple Sclerosis; Mus; Mutate; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Nutrient; Pathway interactions; Peripheral; Permeability; Pharmaceutical Preparations; Phenotype; Physiological; Process; Property; Proteins; Research; Solid; Structure; Synaptic Transmission; System; Testing; Therapeutic; Therapeutic Agents; Tight Junctions; Tracer; Vesicle; Work; base; capillary; density; immunoreactivity; in vitro Assay; in vivo; insight; member; monolayer; mutant; nervous system disorder; novel; overexpression; polarized cell; prevent; public health relevance; repaired; research study; seal; tool; trafficking; transcytosis

DESCRIPTION (provided by applicant): The tightly controlled chemical environment of the CNS required for proper synaptic transmission is maintained by the 'blood brain barrier' (BBB), which is composed of highly specialized blood vessels whose endothelial cells act as a physiological barrier to seal the CNS and control substance influx and efflux. Two unique features of CNS endothelial cells determine BBB integrity, namely specialized tight junctions between the endothelial cells lining the CNS capillaries and extremely low rates of vesicular trafficking between the luminal and abluminal plasma membranes, a process termed transcytosis. A solid understanding of how BBB function is regulated to ensure brain homeostasis is missing in the field, as there are little to no molecular insights into how CNS endothelial cells acquire and maintain their specialized properties. Furthermore, the most promising strategies for delivering therapeutic agents across the BBB involve the manipulation of transcytotic pathways, an avenue of research which requires mechanistic understanding of how this process is normally regulated. Previous work in our lab has identified MFSD2A as the first molecule to maintain BBB integrity specifically by suppressing transcytosis. MFSD2A is exclusively expressed in CNS endothelial cells, and genetic ablation of Mfsd2a results in both extravasation of exogenous tracer from the vessel lumen to brain parenchyma and an increased density of vesicles within CNS endothelial cells, while intercellular tight junctions remain normal The goal of this study is to understand the molecular mechanism whereby MFSD2A suppresses transcytosis. My preliminary data show that MFSD2A is localized to the luminal plasma membrane of CNS endothelial cells and that loss of Mfsd2a results in increased immunoreactivity of Cav-1. Therefore, I hypothesize that MFSD2A acts as a suppressor of caveolae-mediated transcytosis in CNS endothelial cells, possibly by interacting with transcytotic machinery to inhibit caveolae dynamics at the luminal plasma membrane. To this end, I have developed a rigorous experimental plan with three aims: I will (1) use in vivo tools and an in vitro cell-based system I have developed to determine which specific transcytotic pathway is suppressed by MFSD2A in CNS endothelial cells, (2) perform structure-function experiments to dissect how structural domains of MFSD2A contribute to its function in suppressing transcytosis, and (3) test the hypothesis that MFSD2A interacts with well-known molecular determinants of caveolae-mediated transcytosis to elicit its function. I expect that these experiments will provide novel insight into how BBB function is regulated, leading to new pathways for BBB manipulation for therapeutic purposes.

描述（由申请人提供）：正确突触传递所需的中枢神经系统严格控制的化学环境由“血脑屏障”（BBB）维持，该屏障由高度特化的血管组成，其内皮细胞充当生理屏障密封中枢神经系统并控制物质的流入和流出。 CNS 内皮细胞的两个独特特征决定了 BBB 的完整性，即 CNS 毛细血管内壁内皮细胞之间的专门紧密连接，以及腔内和腔外质膜之间极低的囊泡运输率，这一过程称为转胞吞作用。该领域缺乏对血脑屏障功能如何调节以确保大脑稳态的深入了解，因为对中枢神经系统内皮细胞如何获得和维持其特殊特性几乎没有分子见解。此外，跨血脑屏障递送治疗剂的最有前途的策略涉及转胞吞途径的操纵，这是一种需要从机制上理解该过程通常如何调节的研究途径。我们实验室之前的工作已确定 MFSD2A 是第一个通过抑制转胞吞作用来维持 BBB 完整性的分子。 MFSD2A仅在中枢神经系统内皮细胞中表达，Mfsd2a的基因消融导致外源示踪剂从血管腔外渗至脑实质，并导致中枢神经系统内皮细胞内囊泡密度增加，而细胞间紧密连接保持正常。目的是了解 MFSD2A 抑制转胞吞作用的分子机制。我的初步数据表明，MFSD2A 定位于 CNS 内皮细胞的管腔质膜，Mfsd2a 的缺失会导致 Cav-1 的免疫反应性增加。因此，我假设 MFSD2A 作为 CNS 内皮细胞中小凹介导的转胞吞作用的抑制剂，可能是通过与转胞吞机制相互作用来抑制管腔质膜上的小凹动力学。为此，我制定了一个严格的实验计划，具有三个目标：我将（1）使用我开发的体内工具和体外基于细胞的系统来确定中枢神经系统内皮细胞中哪种特定的转胞吞途径被 MFSD2A 抑制， (2) 进行结构-功能实验来剖析 MFSD2A 的结构域如何促进其抑制转胞吞作用的功能，以及 (3) 检验 MFSD2A 与众所周知的分子决定因素相互作用的假设。小凹介导的转胞吞作用以引发其功能。我期望这些实验将为 BBB 功能如何调节提供新的见解，从而为治疗目的的 BBB 操纵提供新的途径。