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

ABC转运蛋白不仅负责癌症的耐药性，而且是血脑屏障（BBB）和血胎盘屏障的主要组成部分。血脑屏障中最重要的三种转运蛋白是ABCB 1、ABCC 1和ABCG 2。我们以前开发了一种用于分析ABCG 2在血脑屏障上表达的小鼠模型，该模型是基于以下事实，即，白蛋白是ABCG 2底物，并且其进入大脑被转运蛋白表达阻止。在该模型中，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。当给小鼠注射蓝蛋白时，由于ABCG 2阻止蓝蛋白穿过血脑屏障，因此没有检测到来自大脑的光信号。然而，当将ABCG 2抑制剂与BACG蛋白共同给药时，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以产生光信号。由于小鼠血脑屏障的研究是耗时和昂贵的，我们正在开发斑马鱼的同源模型，因为斑马鱼血脑屏障的组成部分似乎非常相似的哺乳动物血脑屏障。已经开发了两种转基因斑马鱼系，其具有GFAP启动子控制下的萤火虫荧光素酶或nanoLuc荧光素酶。荧光素是萤火虫荧光素酶的底物，由ABCG 2转运，而腔肠素是nanoLuc的底物，由ABCB 1和ABCG 2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。如果斑马鱼被认为是一个合适的模型，用于研究转运蛋白在血脑屏障，斑马鱼同系物的人类转运蛋白必须仔细的特点。斑马鱼没有人类ABCB 1的直接同源物，而是有两个类似的变体-Abcb 4和Abcb 5。这些转运蛋白在异源系统中的表达使其能够详细表征和抑制特性。与NCATS的Matthew Hall合作，我们发现斑马鱼Abcb 4与人类ABCB 1几乎相同，对近100种已知的ABCB 1底物具有抗性。斑马鱼Abcb 4定位于BBB和斑马鱼的其他屏障和排泄部位。斑马鱼也有人类ABCG 2的4个同源物-Abcg 2a、Abcg 2b、Abcg 2c和Abcg 2d。我们最近将Abcg 2a定位于斑马鱼血脑屏障，并且正在对转运蛋白的底物特异性进行详细表征。转染细胞中的初步数据表明，Abcg 2a与人ABCG 2具有最相似的底物特异性，但它们并不相同。

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.