Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice

Abstract

Folate deficiency has been associated with many adverse clinical manifestations. The blood-brain barrier (BBB), formed by brain capillary endothelial cells, protects the brain from exposure to neurotoxicants. The function of BBB is modulated by multiple ABC transporters, particularly P-glycoprotein. A proton-coupled folate transporter (PCFT)-deficient mouse has been previously described as a model for systemic folate deficiency. Herein, we demonstrate that exposing mouse brain capillaries to the antiepileptic drug, valproic acid (VPA; 5 μM), significantly increased P-glycoprotein transport function in the wild-type animals. A ligand to the aryl hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produced a similar induction of P-glycoprotein, which tightened the BBB, thereby increasing the neuroprotection. However, VPA- or TCDD-induced P-glycoprotein transport was blocked in the PCFT-nullizygous mice, indicating that multiple neuroprotective mechanisms are compromised under folate-deficient conditions. Brain capillaries from S-folinic acid (SFA; 40 mg/kg)-treated PCFT-nullizygous mice exhibited increased P-glycoprotein transport following VPA exposure. This suggests that SFA supplementation restored the normal BBB function. In addition, we show that tight-junction proteins are disintegrated in the PCFT mutant mice. Taken together, these findings strongly suggest that folate deficiency disrupts the BBB function by targeting the transporter and tight junctions, which may contribute to the development of neurological disorders.

Figure 1.

Localization of PCFT in the BBB. A) Mouse brain capillaries were freshly isolated from PCFT wild-type mouse brains and immunostained with anti-PCFT antibody. Confocal imaging showed the luminal location (arrow) of PCFT in the BBB. Negative control, no primary antibody added. Isotype control, rabbit IgG used as primary antibody. B) Freshly isolated mouse brain capillaries were dual-labeled with anti-PCFT (red) and anti-PGP (green) antibodies by immunocytochemistry. Image (xy section) is a single confocal slice from the center of Z-stack serial images. The xz sections show cross-sections of the capillaries reconstructed from the slices where the arrow points to. Confocal imaging showed PCFT and PGP were localized along the luminal membrane in the BBB. Scale bars = 5 μm.

Figure 2.

VPA-induced PGP transport in the BBB. A) Representative confocal images showing luminal accumulation of NBD-CSA, a fluorescent substrate for PGP, in freshly isolated mouse brain capillaries. Luminal fluorescence was reduced by 5 μM PSC 833, a specific inhibitor of PGP, and was increased following 3 h of exposure to 10 μM VPA. Scale bar = 5 μm. B) Exposing mouse brain capillaries to VPA increased luminal accumulation of NBD-CSA in a concentration-dependent manner. Values are expressed as means ± se for 8-12 capillaries from a single preparation (each containing pooled brain tissue from 3-5 mice). ***P < 0.001 vs. control.

Figure 3.

PGP transport blocked by transcription or translation inhibitors. Mouse brain capillaries were freshly isolated from PCFT wild-type mouse brains. Inhibiting transcription by 1 μM actinomycin D (ActD; A) or translation by 100 μg/ml cycloheximide (CHX; B) abolished the effect of 5 μM VPA on PGP transport activity. ***P < 0.001 vs. control.

Figure 4.

PGP transport induction abolished in nullizygous mice. A) Representative images and fluorescence quantitation show that luminal fluorescence accumulation was not altered in freshly isolated brain capillaries of PCFT-nullizygous mice following VPA exposure. Scale bar = 5 μm. B) TCDD (0.1 nM) induced PGP transport in freshly isolated brain capillaries of wild-type mice (left panel), but it was blocked in PCFT-nullizygous mice (right panel). ***P < 0.001 vs. control.

Figure 5.

PGP transport function recovered in SFA-dosed PCFT-nullizygous mice. PCFT-nullizygous mice (4 wk old) were given 40 mg/kg SFA by subcutaneous injection 2×/wk for 2 wk. Brain capillaries were freshly isolated and treated with VPA (5 μM). Luminal NBD-CSA accumulation was analyzed for PGP transport function. SFA supplementation restored PGP transport function in the BBB. ***P < 0.001 vs. control.

Figure 6.

Western blot of transporter proteins and tight-junction protein in wild type and PCFT-nullizygous mice. A) Antibodies against ABC transporters PGP, Mrp2, BCRP, and tight-junction protein ZO1 were used to probe the membranes for protein expression. Mouse brain capillary membranes were isolated from wild-type (WT) and PCFT-nullizygous (null) mice and analyzed by Western blots. β-Actin was used as a loading control. B) Mouse brain capillaries were isolated from wild-type mice and exposed to VPA (10 μM). Mouse brain capillary membranes were isolated for PGP expression. Representative lots, and quantified band intensities normalized to actin, involving measurement from 3 separate experiments are shown.

Figure 7.

Expression of tight-junction proteins in wild-type and PCFT-nullizygous mice by immunocytochemistry. Brain capillaries were freshly isolated from mouse brains from wild-type or PCFT-nullizygous mice and immunostained with anti-ZO1, anti-claudin-1, or anti-occludin antibody. Tight junctional proteins were significantly impaired in the BBB of PCFT-nullizygous mice compared to control animals.