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: 10926473
• 负责人: Michael Gottesman
• 金额: $ 85.21万
• 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/10926473
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Adult; Astrocytes; Biological Assay; Biological Models; Blood; Blood - brain barrier anatomy; Brain; Caring; Categories; Cells; Cerebrovascular system; Collaborations; Consumption; Data; Drug Delivery Systems; Drug or chemical Tissue Distribution; Drug resistance; Firefly Luciferases; Fishes; Generations; Glial Fibrillary Acidic Protein; Goals; Homologous Gene; Human; In Situ Hybridization; Kidney; Knockout Mice; Light; Liver; Luciferases; Malignant Neoplasms; Measures; Modeling; Monitor; Mus; National Center for Advancing Translational Sciences; Nature; Orthologous Gene; Penetration; Permeability; Play; Promega; Property; Reaction; Resistance; Role; Shrimp; Signal Transduction; Site; Spinal Cord; Study models; Substrate Specificity; System; Testing; Time; Transgenic Organisms; Variant; Water; Zebrafish; blood-brain barrier crossing; blood-brain barrier penetration; blood-brain barrier permeabilization; brain tissue; coelenterazine; cytotoxicity; deep ocean; high throughput analysis; high throughput screening; improved; inhibitor; luciferin; model organism; 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. 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.

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。我们开发并鉴定了带有纳米荧光素酶的转基因斑马鱼品系，纳米荧光素酶是由 Promega 从深海虾中衍生而来，受 GFAP 启动子控制。在此模型中，NanoLuc 在发育中的斑马鱼大脑和脊髓中表达。腔肠素是 NanoLuc 的底物之一，由 ABCB1 和 ABCG2 转运。 Furimazine 是一种腔肠素衍生物，具有非常高的光产量，是 ABCG2 底物。因此，该模型可用于研究转运蛋白在血脑屏障中的作用，但也可用于筛选可能增加屏障通透性的化合物，无论其机制如何。我们已经证明，向含有斑马鱼幼虫的水中添加底物（例如呋喃嗪）和 Abcg2 抑制剂会产生与呋喃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，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。我们对这些斑马鱼 ABCG2 直向同源物进行了功能分类，并确定了斑马鱼 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 细胞中稳定表达每种转运蛋白，并使用已知的 ABCG2 底物进行细胞毒性和荧光流出测定。我们发现 Abcg2a 与 ABCG2 具有最大的底物重叠，而 Abcg2d 似乎在功能上最不相似。使用 RNAscope 原位杂交，我们根据 abcg2a 在claudin-5阳性脑血管系统中的定位，确定 abcg2a 是在成体和幼体斑马鱼 BBB 上表达的唯一同源物。这些结果证明了斑马鱼 Abcg2a 的保守功能，并表明斑马鱼可能是研究 ABCG2 在 BBB 中的作用的合适模型生物。在确定斑马鱼 Abcb4 和 Abcg2a 为斑马鱼 BBB 上的同源转运蛋白后，我们表征了斑马鱼和人类 BBB 转运蛋白转运 NanoLuc 底物的能力。我们检查了几种腔肠素衍生物以及一些腔肠素衍生物，发现腔肠素是测试的最亮的 NanoLuc 底物，并且由人 ABCG2 和斑马鱼 Abcg2a 转运。腔肠素 h 是最亮的腔肠素衍生物，也由人类 ABCG2 和斑马鱼 Abcg2a 转运。因此，这些化合物可用于研究 ABCG2 在 BBB 中的作用。我们还收到了更多来自 Promega Corporation 的呋喃嗪衍生物，在他们的研究中未发现这些衍生物能够穿透血脑屏障，希望找到可能由斑马鱼中的 Abcb4 或 Abcg2a 转运的其他 NanoLuc 底物。

项目成果

Abcg2a is the functional homolog of human ABCG2 expressed at the zebrafish

Abcg2a 是斑马鱼表达的人类 ABCG2 的功能同源物

• 发表时间: 2024-09-14
• 作者: Joanna R. Thomas;W. J. Frye;Robert W. Robey;Andrew C. Warner;Donna;Butcher;Jennifer L. Matta;Tamara C. Morgan;E. Edmondson;M. Michael;Gottesman
• 通讯作者: Gottesman