Zebrafish model of blood-brain barrier to improve drug delivery to the brain

血脑屏障斑马鱼模型可改善药物向大脑的输送

• 批准号: 10926473
• 负责人: Michael Gottesman
• 金额: $ 85.21万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926473
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Adult; Astrocytes; Biological Assay; Biological Models; Blood; Blood - brain barrier anatomy; Brain; Caring; Categories; Cells; Cerebrovascular system; Collaborations; Consumption; Data; Drug Delivery Systems; Drug or chemical Tissue Distribution; Drug resistance; Firefly Luciferases; Fishes; Generations; Glial Fibrillary Acidic Protein; Goals; Homologous Gene; Human; In Situ Hybridization; Kidney; Knockout Mice; Light; Liver; Luciferases; Malignant Neoplasms; Measures; Modeling; Monitor; Mus; National Center for Advancing Translational Sciences; Nature; Orthologous Gene; Penetration; Permeability; Play; Promega; Property; Reaction; Resistance; Role; Shrimp; Signal Transduction; Site; Spinal Cord; Study models; Substrate Specificity; System; Testing; Time; Transgenic Organisms; Variant; Water; Zebrafish; blood-brain barrier crossing; blood-brain barrier penetration; blood-brain barrier permeabilization; brain tissue; coelenterazine; cytotoxicity; deep ocean; high throughput analysis; high throughput screening; improved; inhibitor; luciferin; model organism; mouse model; nanoluciferase; novel; prevent; promoter

Not only are ABC transporters responsible for drug resistance in cancer, but they are a major component of the blood-brain barrier (BBB) and blood-placental barrier. The three most prominent transporters at the blood-brain barrier are ABCB1, ABCC1, and ABCG2. We previously developed a murine model for analysis of ABCG2 expression at the blood-brain barrier based on the fact that luciferin is an ABCG2 substrate and its entry into the brain is prevented by transporter expression. In this model, firefly luciferase is under the expression of the GFAP promoter, leading to its expression in the astrocytes. When mice are injected with luciferin, no light signal from the brain is detected due to ABCG2 preventing luciferin from crossing the blood-brain barrier. However, when luciferin is coadministered with an ABCG2 inhibitor, it can cross the blood-brain barrier and react with luciferase expressed in the astrocytes to produce light which can be quantitatively measured. Because studies of the BBB in mice are time-consuming and expensive, we are developing homologous models in the zebrafish, as components of the zebrafish BBB appear to be very similar to those of the mammalian BBB. We have developed and characterized a transgenic zebrafish line with NanoLuciferase, derived by Promega from a deep sea shrimp, under the control of the GFAP promoter. In this model, NanoLuc is expressed in the developing zebrafish brain and spinal cord. Coelenterazine is one of the substrates for NanoLuc and is transported by both ABCB1 and ABCG2. Furimazine, a coelenterazine derivative with very high yield of light, is an ABCG2 substrate. Thus, this model can be used to study the role of transporters at the blood-brain barrier, but could also be used to screen compounds that might increase permeability of the barrier irregardless of the mechanism. We have shown that addition of a substrate, such as furimazine, and an Abcg2 inhibitor to the water containing larval zebrafish result in light generation consistent with penetration of furimazine across the BBB. If zebrafish are to be considered an appropriate model for study of transporters at the blood-brain barrier, the zebrafish homologs of human transporters must be carefully characterized. Zebrafish do not have a direct homolog of human ABCB1 but instead have 2 similar variants-Abcb4 and Abcb5. Expression of these transporters in heterologous systems has enabled their detailed characterization and inhibition properties. In collaboration with Matthew Hall at NCATS, we have found that zebrafish Abcb4 is nearly identical to human ABCB1 in conferring resistance to 90 known ABCB1 substrates. Abcb5 is also a functional transporter and confers resistance to many ABCB1 substrates but has a slightly narrower substrate specificity. While zebrafish Abcb4 is the only homolog that localizes to the BBB, Abcb4 and Abcb5 are expressed at other barrier and excretory sites in zebrafish, such as the gut, liver and kidneys. Zebrafish also have 4 homologs of human ABCG2-Abcg2a, Abcg2b, Abcg2c and Abcg2d. We have functionally categorized these zebrafish ABCG2 orthologs and determined the brain tissue distribution of zebrafish ABCG2 homologs. To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. Using RNAscope in situ hybridization we identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for the studying the role of ABCG2 at the BBB. Having identified zebrafish Abcb4 and Abcg2a as the homologous transporters at the zebrafish BBB, we characterized the ability of NanoLuc substrates to be transported by zebrafish and human transporters at the BBB. We examined several coelenterazine derivatives as well as some furimazine derivatives and found that furimazine was the brightest NanoLuc substrate tested and was transported by human ABCG2 and zebrafish Abcg2a. Coelenterazine h was the brightest coelenterazine derivative and was also transported by human ABCG2 and zebrafish Abcg2a. Thus, these compounds could be used to study the role of ABCG2 at the BBB. We also received more furimazine derivatives from Promega Corporation that were not found to penetrate the BBB in their studies in hopes of finding other NanoLuc substrates that might be transported by Abcb4 or Abcg2a in the zebrafish.

ABC转运蛋白不仅与癌症的耐药性有关，而且还是血脑屏障（BBB）和血胎盘屏障的主要组成部分。血脑屏障中三种最重要的转运蛋白是ABCB1、ABCC1和ABCG2。基于荧光素是ABCG2底物且转运蛋白表达阻止其进入大脑这一事实，我们之前开发了一种小鼠模型来分析血脑屏障中ABCG2的表达。在本模型中，萤火虫荧光素酶在GFAP启动子的作用下表达，导致其在星形胶质细胞中表达。当给小鼠注射荧光素时，由于ABCG2阻止荧光素穿过血脑屏障，因此没有检测到来自大脑的光信号。然而，当荧光素与ABCG2抑制剂共给药时，它可以穿过血脑屏障，与星形胶质细胞中表达的荧光素酶反应，产生可定量测量的光。由于研究小鼠血脑屏障耗时且昂贵，我们正在开发斑马鱼血脑屏障的同源模型，因为斑马鱼血脑屏障的成分似乎与哺乳动物的血脑屏障非常相似。我们在GFAP启动子的控制下，开发并鉴定了一种含有纳米荧光素酶的转基因斑马鱼系，该系由Promega从深海虾中提取。在这个模型中，NanoLuc在发育中的斑马鱼大脑和脊髓中表达。Coelenterazine是NanoLuc的底物之一，可通过ABCB1和ABCG2进行转运。Furimazine是一种具有很高光收率的coelenterazine衍生物，是ABCG2底物。因此，该模型可用于研究转运蛋白在血脑屏障中的作用，但也可用于筛选可能增加屏障通透性的化合物，而不管其机制如何。我们已经证明，在含有斑马鱼幼虫的水中添加一种底物，如furimazine和一种Abcg2抑制剂，可以产生与furimazine穿过血脑屏障一致的光。如果将斑马鱼视为研究血脑屏障转运蛋白的合适模型，则必须仔细表征斑马鱼的人类转运蛋白同源物。斑马鱼没有人类ABCB1的直接同源基因，但有2个相似的变体——abcb4和Abcb5。这些转运蛋白在异源系统中的表达使得它们的详细表征和抑制特性成为可能。通过与NCATS的Matthew Hall合作，我们发现斑马鱼的Abcb4对90种已知ABCB1底物的抗性几乎与人类的ABCB1相同。Abcb5也是一种功能性转运蛋白，对许多ABCB1底物具有抗性，但对底物的特异性略窄。虽然Abcb4是斑马鱼唯一定位于血脑屏障的同源物，但Abcb4和Abcb5在斑马鱼的其他屏障和排泄部位表达，如肠道、肝脏和肾脏。斑马鱼也有人类ABCG2-Abcg2a、Abcg2b、Abcg2c和Abcg2d的4个同源物。我们对这些斑马鱼ABCG2同源物进行了功能分类，并确定了斑马鱼ABCG2同源物的脑组织分布。为了确定这些转运蛋白的底物，我们在HEK-293细胞中稳定地表达了这些转运蛋白，并用已知的ABCG2底物进行了细胞毒性和荧光外排实验。我们发现Abcg2a与ABCG2具有最大的底物重叠，而Abcg2d似乎在功能上最不相似。利用RNAscope原位杂交技术，我们发现abcg2a是斑马鱼成年和幼虫血脑屏障中唯一表达的同源基因，基于其定位于cladin -5阳性的脑血管系统。这些结果证明了斑马鱼Abcg2a的保守功能，提示斑马鱼可能是研究ABCG2在血脑屏障中的作用的合适模式生物。在鉴定了斑马鱼血脑屏障上的同源转运蛋白Abcb4和Abcg2a后，我们表征了NanoLuc底物被斑马鱼和人类血脑屏障转运蛋白转运的能力。我们检测了几种coelenterazine衍生物和一些furimazine衍生物，发现furimazine是测试的最亮的NanoLuc底物，并通过人ABCG2和斑马鱼Abcg2a运输。Coelenterazine h是最亮的Coelenterazine衍生物，也被人ABCG2和斑马鱼Abcg2a转运。因此，这些化合物可用于研究ABCG2在血脑屏障中的作用。我们还从Promega公司获得了更多的呋喃嘧啶衍生物，这些衍生物在他们的研究中没有发现穿透血脑屏障，希望找到其他可能通过Abcb4或Abcg2a在斑马鱼中运输的NanoLuc底物。