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: 10702837
• 负责人: Michael Gottesman
• 金额: $ 60.75万
• 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/10702837
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Astrocytes; Biological Assay; Biological Models; Blood; Blood - brain barrier anatomy; Brain; Caring; Cells; Collaborations; Consumption; Data; Drug Delivery Systems; Drug resistance; Embryo; Firefly Luciferases; Fishes; Glial Fibrillary Acidic Protein; Goals; Homologous Gene; Human; Incubated; Kidney; Knockout Mice; Light; Liver; Luciferases; Malignant Neoplasms; Measures; Modeling; Monitor; Mus; National Center for Advancing Translational Sciences; Nature; Permeability; Play; Property; Resistance; Role; Signal Transduction; Site; Study models; Substrate Specificity; System; Testing; Time; Transgenic Organisms; Variant; Work; Zebrafish; base; blood-brain barrier crossing; blood-brain barrier permeabilization; coelenterazine; high throughput analysis; high throughput screening; improved; inhibitor; luciferin; luminescence; 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. Two transgenic zebrafish lines have been developed with either firefly luciferase or nanoLuc under the control of the GFAP promoter. Luciferin is the substrate for firefly luciferase and is transported by ABCG2, while coelenterazine is one of the substrates for nanoLuc and is transported by both ABCB1 and ABCG2. Thus, either model could potentially be used to study the role of transporters at the blood-brain barrier, but they could also be used to screen compounds that might increase permeability of the barrier irregardless of the mechanism. 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 determined that Abcg2a is the only ABCG2 homolog expressed at the zebrafish BBB and a detailed characterization of the substrate specificity of the transporters is underway. Preliminary data in transfected cells suggest that Abcg2a has the most similar substrate specificity to human ABCG2, but they are not identical. As the light signal from nanoLuc is significantly brighter than that of firefly luciferase, we initially focused on the nanoLuc transgenic fish. Native coelenterazine and several of its derivatives are compatible with the nanoLuc system and we identified furimazine and coelenterazine h as the brightest. Both furimazine and coelenterazine h were found to be transported by zebrafish Abcg2a but not Abcb4. When embryonic fish were incubated with coelenterazine h in the presence of the ABCG2 inhibitor Ko143, we noted higher levels of luminescence compared to fish incubated with coelenterazine h alone, a proof-of-concept result. Further work will include testing other nanoLuc substrates as well as other known inhibitors.

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。在GFAP启动子的控制下，用萤火虫荧光素酶或nanoLuc开发了两种转基因斑马鱼系。萤光素是萤火虫萤光素酶的底物，由ABCG2转运，而coelenterazine是nanoLuc的底物之一，由ABCB1和ABCG2转运。因此，这两种模型都有可能用于研究转运蛋白在血脑屏障中的作用，但它们也可以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。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。我们已经确定Abcg2a是唯一在斑马鱼血脑屏障上表达的ABCG2同源物，并且正在对转运蛋白的底物特异性进行详细表征。转染细胞的初步数据表明，Abcg2a与人ABCG2具有最相似的底物特异性，但它们并不完全相同。由于nanoLuc的光信号比萤火虫荧光素酶的光信号要亮得多，所以我们首先关注了nanoLuc转基因鱼。天然coelenterazine及其衍生物与纳米oluc体系相容，其中furimazine和coelenterazine h最亮。furimazine和coelenterazine h均可通过斑马鱼Abcg2a转运，但Abcb4不转运。当胚胎鱼在ABCG2抑制剂Ko143存在的情况下与coelenterazine h孵育时，我们注意到与单独使用coelenterazine h孵育的鱼相比，鱼的发光水平更高，这是一个概念验证结果。进一步的工作将包括测试其他纳米uc底物以及其他已知的抑制剂。