Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

Abstract

Capillary endothelial cells of the human blood–brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.

Zebrafish behavioral response to ivermectin: insights into potential neurological risk

“…Our interpretation of behavioral responses to ivermectin in zebrafish is also in line with literature focused on neurotoxicity risk dependent on the absorption-extrusion activity of the drug from the gastrointestinal tract and blood brain-barrier, which is regulated by P-glycoprotein (P-gp), environment-susceptible genes coding for Pgp, such as ABCB1 (also known as multi-drug resistant express the ABCB1 gene, they do express the homologs ABCB4 and ABCB5, and ABCB4 has indeed been demonstrated to phenocopy P-gp function in zebrafish [36].) Genetic mutations in this context may therefore further increase the risk of penetration of ivermectin across the blood brain barrier, e.g.…”

Generation and characterization of a zebrafish knockout model of abcb4, a homolog of the human multidrug efflux transporter P-glycoprotein

“…Generation and validation of a knockout model of the zebra sh homolog of the human ABCB1 Two orthologs of human ABCB1, abcb4 and abcb5, are found in the zebra sh, of which abcb4 is functionally similar to human ABCB1 [16,17]. To elucidate the in vivo roles of zebra sh abcb4 in more details, we generated an abcb4-mutated zebra sh line using CRISPR/Cas9 genome editing technology.…”

“…In zebra sh, Abcb4 and Abcb5 are both associated with e ux transport activities. However, zebra sh Abcb4 protein has a highly overlapping substrate speci city pro le with human Pgp [17]. In addition, previous studies based on morpholino knockdown found that zebra sh Abcb4 transports several uorescent Pgp substrates in embryos [16].…”

“…Zebra sh has a structurally similar endothelial membrane system to higher vertebrates, including humans, in the BBB and the intestinal tract [13][14][15]. Although synteny analysis found that zebra sh has two Pgp orthologs, abcb4, and abcb5 [16], high-throughput screening of human Pgp substrates characterized Abcb4 as functionally phenocopied to human Pgp [17]. It has been reported that the C219 antibody that recognizes human Pgp cross-reacts with zebra sh Abcb4 and Abcb5 [15].…”

“…It has been reported that the C219 antibody that recognizes human Pgp cross-reacts with zebra sh Abcb4 and Abcb5 [15]. Therefore, coupling antibody staining with RNAscope techniques was required to observe Abcb4 localization in zebra sh [17]. However, the precise characterization of Abcb4 protein expression in zebra sh using immunohistochemistry was still not feasible due to the lack of antibodies speci c to zebra sh Abcb4.…”