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

ABC转运蛋白不仅对癌症的耐药性负责，而且是血脑屏障(BBB)和血胎盘屏障的主要组成部分。血脑屏障上最突出的三种转运蛋白是ABCB1、ABCC1和ABCG2。我们之前开发了一个小鼠模型来分析ABCG2在血脑屏障的表达，这是基于荧光素是ABCG2的底物，它通过转运蛋白的表达阻止其进入大脑的事实。在这个模型中，萤火虫荧光素酶在GFAP启动子的表达下，导致其在星形胶质细胞中表达。当小鼠注射荧光素时，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抑制剂联合使用时，它能够穿过血脑屏障，并与星形胶质细胞中表达的荧光素酶反应，产生光信号。由于对小鼠血脑屏障的研究既耗时又昂贵，我们正在斑马鱼中开发类似的模型，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，获得了两个转基因斑马鱼株系，分别含有萤火虫荧光素酶和纳米Luc荧光素酶。荧光素是萤火虫荧光素酶的底物，由ABCG2转运，而腔肠净是纳米Luc的底物，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血脑屏障通透性的化合物。如果斑马鱼被认为是研究血脑屏障转运蛋白的合适模型，必须仔细描述人类转运蛋白的斑马鱼同源物。斑马鱼没有人类ABCB1的直接同源物，而是有两个类似的变体-Abcb4和ABCB5。这些转运蛋白在异源系统中的表达使其具有详细的表征和抑制特性。在与NCATS的Matthew Hall合作中，我们发现斑马鱼Abcb4在对近100种已知ABCB1底物产生抗性方面与人类ABCB1几乎相同。斑马鱼Abcb4定位于斑马鱼的血脑屏障和其他屏障和排泄物部位。斑马鱼也有人类ABCG2的4个同源物-Abcg2a、Abcg2b、Abcg2c和Abcg2d。我们最近已经将Abcg2a定位于斑马鱼血脑屏障，转运蛋白底物特异性的详细表征正在进行中。在转基因细胞中的初步数据表明，Abcg2a与人ABCG2具有最相似的底物特异性，但它们并不完全相同。

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 is able to cross the blood-brain barrier and react with luciferase expressed in the astrocytes to yield a light signal. 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 luciferase under the control of the GFAP promoter. Luciferin is the substrate for firefly luciferase and is transported by ABCG2, while coelenterazine is the substrate 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. 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 nearly 100 known ABCB1 substrates. Zebrafish Abcb4 localizes to the BBB and other barrier and excretory sites in zebrafish. Zebrafish also have 4 homologs of human ABCG2-Abcg2a, Abcg2b, Abcg2c and Abcg2d. We have recently localized Abcg2a to the zebrafish blood-brain barrier 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.