Evolutionarily Conserved Roles for Blood-Brain Barrier Xenobiotic Transporters in Endogenous Steroid Partitioning and Behavior

Abstract

Central nervous system (CNS) chemical protection depends upon discrete control of small-molecule access by the blood-brain barrier (BBB). Curiously, some drugs cause CNS side-effects despite negligible transit past the BBB. To investigate this phenomenon, we asked whether the highly BBB-enriched drug efflux transporter MDR1 has dual functions in controlling drug and endogenous molecule CNS homeostasis. If this is true, then brain-impermeable drugs could induce behavioral changes by affecting brain levels of endogenous molecules. Using computational, genetic, and pharmacologic approaches across diverse organisms, we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice). Furthermore, we show that MDR1-interacting drugs are associated with anxiety-related behaviors in humans. We propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates and implicates the BBB in behavioral regulation.

Keywords: behavior; blood brain barrier; central nervous system; drug side effect mechanisms; drug transporters; endobiotics; steroid hormones; toxicology.

Figure 1. The Drosophila steroid 20-hydroxyecdysone is a predicted Mdr65 substrate

(A) Diagram of the invertebrate and vertebrate blood-brain barrier. The invertebrate BBB is a compound structure, formed by subperineurial glia (SPG), outer perineurial glia, and a basement membrane; only the SPG are shown for simplicity. The vertebrate BBB is primarily formed by the brain vascular endothelial cells (VE), and its functions are supported by the surrounding pericytes (P) within the basement membrane (BM), and the end feet of the astrocyte glia (AG). These cellular and non-cellular layers form a compound barrier structure: the neurovascular unit (NVU). Both the vertebrate and invertebrate BBBs express junctional proteins that form the diffusion barrier (DB), as well as ATP-binding Cassette (ABC) transporters that form the transport barrier and protect the brain from xenobiotics. CG; cortex glia. (B) Substrate predictions for the fly Mdr65 and mouse Mdr1 efflux transporters from substrate docking computer modeling. (C) Homology model of Mdr65 (yellow) overlaid on the mouse Mdr1 template PDB ID: 3G60 (green). Original template ligand, QZ59-RRR, is shown in pink. The enlarged window shows the top-scored pose of ecdysone docked in the Mdr65 model. Also shown are some of the residues predicted to be involved in hydrogen bonding and hydrophobic interactions with the ligand. (D) Competitive in vivo efflux transport assay using Rhodamine B and 20E. Fluorescence readings are normalized to wild type (WT) brains injected with Rho B and vehicle. At least 4 biological replicates were performed for each condition. Error bars represent SEM. ANOVA *, p<0.05; ***, p≤0.001.

Figure 2. Drosophila Mdr65 mutants show altered blood-brain barrier partitioning of 20-hydroxyecdysone

(A) Western blot time-course of EcRLBD>Stinger GFP fly heads following injection of 20E or vehicle. β-actin was used as a loading control. N=2 technical replicates. (B) Confocal images of wild type (i–v) and Mdr65 null mutant (vi–x) brains expressing EcRLBD>Stinger GFP after hemolymph injection of vehicle or 20E. Co-injected 70kDa Texas Red Dextran marks the BBB. Images are shown at the BBB layer (Brain surface; i, ii, vi & vii), below the BBB layer (Internal brain space; iii & viii) and at the optic lobe cross section (Cross section; iv, v, ix & x). Regions from the cross sections were enlarged for clarity (v & x). N> 9 biological replicates. Scale bars, 20 μm (i–iii, vi–viii), 10 μm (iv & ix) and 5 μm (v & x). (C) Quantification of the total number of GFP-positive cells/z-stack section for the initial 15% depth into the optic lobes of EcRLBD>Stinger GFP wild type and Mdr65 null flies hemolymph-injected with 20E. Data were normalized to the number of GFP-positive cells present on the optic lobe surface. N > 5 biological replicates. (D & E) QPCR analysis of E74B (D) and Cyp18a1 (E) transcript levels in whole brains from wild type and Mdr65 null flies (without the EcRLBD>Stinger GFP reporter). Error bars represent SEM. T test **, p<0.005; ***, p≤0.001.

Figure 3. Endogenous aldosterone levels are increased in Mdr1 mutant mice

(A and B) HPLC-MSMS analysis of endogenous steroids from whole brains (A) and sera (B) of adult male Mdr1 control and mutant mice. N= 18 control and 7 Mdr1 mutants. ND, no data. (C) HPLC-MSMS analysis of endogenous aldosterone from whole brains of adult male BCRP control and mutant mice. N=18 Controls and 9 BCRP mutants. (D) HPLC-MSMS analysis of aldosterone from whole brains of wild type mice injected first with either vehicle (n=4) or Cyclosporin A (CsA) (n=4) then followed by aldosterone. Error bars represent SEM. Mann-Whitney test * p<0.05.

Figure 4. Drosophila Mdr65 can modulate behavior

(A–C) The cumulative percentage of adult flies hatching of wild type and Mdr65 null mutants (A), and following knock-down of Mdr65 in all glia (including the BBB) using the driver repo-Gal4 (B; Glia Gal4>Mdr65RNAi) or in the BBB using 9-137-Gal4 (C; BBB Gal4>Mdr65RNAi) compared to the heterozygote transgene controls. *, p<0.05; **, p<0.005. (D) The length of sleep bouts (min/30 minutes) for wild type and Mdr65 null mutants during the daytime and nighttime. (E) Total amount of sleep (hours/12 hour period) and average bout length during the daytime and nighttime. Error bars represent SEM. T test *, p<0.05; ****, p≤0.0005.

Figure 5. Anxiety-like behaviors are elevated in Mdr1 mutant mice

(A) Amount of time spent in the Open and Closed sections of the elevated zero maze (EZM) apparatus. (B) Amount of movement and ambulation by adult littermate male Mdr1 control and mutant mice during the EZM test. N= 16 controls and 9 Mdr1 mutants. Error bars represent SEM. Mann-Whitney * p<0.05.

Figure 6. The adverse drug reaction Anxiety is linked with Mdr1-associated drugs

(A) Model diagram for target-ADR enrichment analysis. Enrichment factors were calculated from a unique set of 441 FDA-approved drugs, 10,098 adverse drug reactions (ADRs), and 631 CHEMBL targets after filtering (see Methods). Together, these analyses generated 1,018,208 EFs, of which only 2171 (0.2%) were statistically significant. (B & C) All target-ADR pairs significantly associated with the adverse drug reaction “Anxiety” or “Emotional Distress”. Targets are ordered by enrichment factor (EF) values, with a greater EF value being indicative of an increased number of observed target-ADR associations relative to the expected number of observations. Q-values indicate degree of confidence that the EF for each target-ADR association is not a false positive.