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。血脑屏障上三个最重要的转运蛋白是 ABCB1、ABCC1 和 ABCG2。我们之前开发了一种小鼠模型，用于分析血脑屏障处的 ABCG2 表达，基于以下事实：荧光素是 ABCG2 底物，并且转运蛋白表达会阻止其进入大脑。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。当给小鼠注射荧光素时，由于 ABCG2 阻止荧光素穿过血脑屏障，因此检测不到来自大脑的光信号。然而，当荧光素与 ABCG2 抑制剂共同给药时，它可以穿过血脑屏障并与星形胶质细胞中表达的荧光素酶反应，产生可定量测量的光。由于对小鼠 BBB 的研究既耗时又昂贵，因此我们正在斑马鱼中开发同源模型，因为斑马鱼 BBB 的成分似乎与哺乳动物 BBB 的成分非常相似。在 GFAP 启动子的控制下，使用萤火虫荧光素酶或 nanoLuc 开发了两种转基因斑马鱼品系。荧光素是萤火虫荧光素酶的底物，由 ABCG2 运输，而腔肠素是 nanoLuc 的底物之一，由 ABCB1 和 ABCG2 运输。因此，这两种模型都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，无论其机制如何。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 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其具有详细的表征和抑制特性。通过与 NCATS 的 Matthew Hall 合作，我们发现斑马鱼 Abcb4 在对 90 种已知 ABCB1 底物具有抗性方面与人类 ABCB1 几乎相同。 Abcb5 也是一种功能性转运蛋白，对许多 ABCB1 底物具有抗性，但底物特异性稍窄。虽然斑马鱼 Abcb4 是定位于 BBB 的唯一同源物，但 Abcb4 和 Abcb5 在斑马鱼的其他屏障和排泄部位（例如肠道、肝脏和肾脏）表达。斑马鱼还具有人类 ABCG2 的 4 个同源物——Abcg2a、Abcg2b、Abcg2c 和 Abcg2d。 我们已经确定 Abcg2a 是在斑马鱼 BBB 上表达的唯一 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，但它们并不相同。由于nanoLuc的光信号明显比萤火虫荧光素酶的光信号亮，因此我们最初关注nanoLuc转基因鱼。天然腔肠素及其几种衍生物与 nanoLuc 系统兼容，我们确定腔肠素和腔肠素 h 是最亮的。研究发现，腔肠素 h 和腔肠素 h 均由斑马鱼 Abcg2a 转运，但不由 Abcb4 转运。当胚胎鱼在 ABCG2 抑制剂 Ko143 存在的情况下与腔肠素 h 一起孵育时，我们注意到与单独用腔肠素 h 孵育的鱼相比，其发光水平更高，这是概念验证的结果。进一步的工作将包括测试其他 nanoLuc 底物以及其他已知的抑制剂。