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

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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.