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了解身体如何与大脑沟通。更具体地说，我们将破译内吞和细胞内/细胞间运输的生物活性分子在血流中循环，并与位于血脑屏障（BBB）的转运蛋白/受体相互作用的机制。 背景技术背景：目前尚不清楚大的内源性血源性底物，如转铁蛋白，在何种程度上经历内吞作用，积聚在内体和溶酶体中，或穿过BBB进入大脑（转胞吞作用）。有一件事是肯定的是，循环化合物必须首先与脑微血管内皮细胞（BMEC）腔侧表达的受体相互作用，形成BBB。然而，随后的细胞途径和分子机制仍然难以捉摸。 最新进展：在过去的几年里，我们的团队开发了一个研究平台，通过将体外和体内方法与高分辨率技术相结合，利用所有工具准确定量地监测BMEC和其他脑细胞内的化合物。 具体说明：将在培养的BMEC中与其受体结合后，测定血管内皮生长因子受体2（VEGFR 2）、胰岛素样生长因子2受体（IGF 2 R）和肝配蛋白A型受体2（ePHA 2）的内源性（底物）或外源性（单克隆抗体）配体的亚细胞命运。将使用温度变化和内吞途径的各种抑制剂来探索机制。技术方法将结合研究和免疫荧光联合收割机。将使用超分辨率STED荧光显微镜和扫描电子显微镜进行细胞和亚细胞分布和共定位分析。使用定量和定性方法进行体内验证实验，使用小鼠原位脑灌注和静脉注射。 新奇：我们的一般方法的新奇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组合。来自不同受体介导系统的一致结果，加上互补的方法，将相互加强，以破译内吞分子途径。 结论：本资助申请的主要成果将是（i）培养研究生，培训有关BBB细胞转运机制的技术，这些技术在世界上只有少数实验室可用，（ii）巩固BMEC内化配体的研究平台，（iii）更好地理解在配体与受体结合后触发的BMEC内的内吞机制及其对BBB功能和脑内稳态的影响。

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.