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，产生可以定量测量的光。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。我们已经建立并鉴定了一个转基因斑马鱼系，该转基因斑马鱼系由Promega从深海对虾中提取的纳米荧光素酶，受GFAP启动子的控制。在这个模型中，NanoLuc在发育中的斑马鱼大脑和脊髓中表达。舒喘宁是纳米Luc的底物之一，由ABCB1和ABCG2转运。呋喃西嗪是ABCG2的底物，是一种具有很高光产率的腔肠嗪衍生物。因此，这个模型可以用来研究转运蛋白在血脑屏障中的作用，也可以用来筛选可能增加血脑屏障通透性的化合物，而不考虑其机制。我们已经证明，在含有斑马鱼幼体的水中添加底物，如呋喃咪嗪和ABCG2抑制剂，可以产生与呋喃咪嗪穿过血脑屏障一致的光产生。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90种已知ABCB1底物产生抗性方面与人类ABCB1几乎相同。ABCB5也是一种功能转运蛋白，对许多ABCB1底物具有抗性，但底物特异性略低。虽然斑马鱼Abcb4是唯一定位于BBB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的4个同源物-Abcg2a、Abcg2b、Abcg2c和Abcg2d。我们对斑马鱼ABCG2同源物进行了功能分类，并测定了斑马鱼ABCG2同源物的脑组织分布。为了确定转运蛋白的底物，我们在HEK-293细胞中稳定地表达了每一种转运蛋白，并用已知的ABCG2底物进行了细胞毒性和荧光外排试验。我们发现Abcg2a与ABCG2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，根据abcg2a定位于claudin-5阳性脑血管系统，我们确定abcg2a是斑马鱼成体和幼体BBB中唯一表达的同源物。这些结果证明了斑马鱼Abcg2a的保守功能，并提示斑马鱼可能是研究ABCG2在血脑屏障中作用的合适的模式生物。在确定斑马鱼Abcb4和Abcg2a为斑马鱼BBB的同源转运体后，我们表征了纳米Luc底物被斑马鱼和人类BBB转运体转运的能力。我们考察了几种呋喃西嗪类化合物和几种室温净类化合物，发现呋喃类药物是最亮的纳米Luc底物，可以被人ABCG2和斑马鱼Abcg2a转运。Coelenterazine h是最明亮的coelenterazine衍生物，也可被人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西嗪衍生物，在他们的研究中没有发现它们能穿透BBB，希望找到其他可能由Abcb4或Abcg2a在斑马鱼体内转运的NanoLuc底物。

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.