Mechanisms of endocytosis and transcytosis into endothelial cells of the blood-brain barrier

概述：本课程的总体目标是提供一个非常需要的关于身体如何与大脑沟通的理解。更具体地说，我们将破译血液中循环的生物活性分子的内吞作用和细胞内/细胞间运输的机制，并与位于血脑屏障（BBB）的转运体/受体相互作用。背景：目前尚不清楚内源性血源性底物，如转铁蛋白，在多大程度上经历内吞作用，聚集在核内体和溶酶体中，或通过血脑屏障进入大脑（胞吞作用）。有一件事是肯定的，循环化合物必须首先与脑微血管内皮细胞（BMECs）管腔侧表达的受体相互作用，形成血脑屏障。然而，随后的细胞途径和分子机制仍然难以捉摸。最近的进展：在过去的几年中，我们的团队开发了一个研究平台，通过结合体外和体内方法与高分辨率技术，该平台具有准确和定量监测bmec和其他脑细胞内化合物的所有工具。特异性目的：血管内皮生长因子受体2 （VEGFR2）、胰岛素样生长因子2受体（IGF2R）和ephrin - a型受体2 （ePHA2）的内源性（底物）或外源性（单克隆抗体）配体在与培养的BMEC受体结合后的亚细胞命运将被确定。机制将利用温度的变化和内吞途径的各种抑制剂来探索。技术方法将结合结合研究和免疫荧光。细胞和亚细胞分布和共定位分析将进行超分辨率STED荧光显微镜和扫描电子显微镜。将采用定量和定性方法在体内、小鼠脑原位灌注和静脉注射进行验证性实验。新颖性：我们一般方法的新颖性源于使用体外和体内研究以及高分辨率技术的定量和定性方法的结合。来自不同受体介导系统的一致结果，以及互补的方法，将相互加强，以破译内吞分子途径。结论:目前的资助申请的主要成果将是：(i)培养具有血脑屏障细胞运输机制相关技术培训的研究生，这在世界上只有少数实验室可以获得；（ii）巩固血脑屏障内化配体的研究平台；（iii）更好地了解血脑屏障内化配体与受体结合时触发的血脑屏障内吞噬机制及其对血脑屏障功能和大脑稳态的影响。

OVERVIEW: The overall aim of the present program is to provide a much needed understanding of how the body communicates with the brain. More specifically, we will decipher the mechanisms underlying the endocytosis and intra-/intercellular trafficking of bioactive molecules circulating in the bloodstream and interacting with transporters/receptors located at the blood-brain barrier (BBB). BACKGROUND: It remains unclear to which extent large endogenous blood-borne substrates, like transferrin, undergo endocytosis, amass in endosomes and lysosomes, or are ferried across the BBB into the brain (transcytosis). One thing that is certain is that circulating compounds have to interact first with receptors expressed on the luminal side of brain microvessel endothelial cells (BMECs), forming the BBB. However, subsequent cellular routes and molecular mechanisms involved remain elusive. RECENT PROGRESS: In the last years, our group has developed a research platform with all the tools to accurately and quantitatively monitor compounds inside BMECs and in other brain cells, by combining in vitro and in vivo approaches with high-resolution technology. SPECIFIC OBJECTIVES: The subcellular fate of endogenous (substrates) or exogenous (monoclonal antibodies) ligands of vascular endothelial growth factor receptor 2 (VEGFR2), insulin-like growth factor 2 receptor (IGF2R) and ephrin type-A receptor 2 (ePHA2) will be determined after binding to their receptor in cultured BMEC. Mechanisms will be probed using changes in temperature and various inhibitors of endocytic pathways. Technical approaches will combine binding studies and immunofluorescence. Cellular and subcellular distribution and colocalization analyses will be performed with super-resolution STED fluorescence microscopy and scanning electron microscopy. Confirmatory experiments using quantitative and qualitative methods will be performed in vivo, using in situ cerebral perfusion in mice and intravenous injections. NOVELTY: The novelty of our general approach stems from the combination of quantitative and qualitative methodologies using both in vitro and in vivo studies and high-resolution technology. Consistent results from different receptor-mediated systems, with complementary methods, will strengthen each other to decipher endocytic molecular pathways. CONCLUSION: The main deliverables of the present grant application will be (i) the formation of graduate students with training on techniques pertaining to cellular transport mechanisms in the BBB, which are available in just a few laboratories in the world, (ii) the consolidation of a research platform for BMEC-internalizing ligands, (iii) a better understanding of endocytic mechanisms within BMECs triggered upon ligand binding to receptors and their impact on BBB function and brain homeostasis.