Apolipoprotein E regulates the integrity of tight junctions in an isoform-dependent manner in an in vitro blood-brain barrier model

Abstract

Apolipoprotein E (apoE) is a major apolipoprotein in the brain. The ε4 allele of apoE is a major risk factor for Alzheimer disease, and apoE deficiency in mice leads to blood-brain barrier (BBB) leakage. However, the effect of apoE isoforms on BBB properties are as yet unknown. Here, using an in vitro BBB model consisting of brain endothelial cells and pericytes prepared from wild-type (WT) mice, and primary astrocytes prepared from human apoE3- and apoE4-knock-in mice, we show that the barrier function of tight junctions (TJs) was impaired when the BBB was reconstituted with primary astrocytes from apoE4-knock-in mice (apoE4-BBB model). The phosphorylation of occludin at Thr residues and the activation of protein kinase C (PKC)η in mBECs were attenuated in the apoE4-BBB model compared with those in the apoE3-BBB model. The differential effects of apoE isoforms on the activation of PKCη, the phosphorylation of occludin at Thr residues, and TJ integrity were abolished following the treatment with an anti-low density lipoprotein receptor-related protein 1 (LRP1) antibody or a LRP1 antagonist receptor-associated protein. Consistent with the results of in vitro studies, BBB permeability was higher in apoE4-knock-in mice than in apoE3-knock-in mice. Our studies provide evidence that TJ integrity in BBB is regulated by apoE in an isoform-dependent manner.

FIGURE 1.

TEER in apoE3-BBB, apoE4-BBB, WT-BBB, and apoE-KO-BBB models. A, triple co-culture BBB models were prepared by using primary pericytes and mBECs from WT mouse brains and primary astrocytes from human apoE3- or apoE4-knock-in mice and were cultured for 7 days. TEER was measured on the indicated culture days and presented as Ω × cm². The data presented are means ± S.D. (error bars) (n = 3). *, p < 0.001 compared with the values of apoE3-BBB models on day 7 or day 5 (unpaired Student's t test). B, triple co-culture BBB models were prepared by using primary pericytes and mBECs from WT mouse brains and primary astrocytes from human apoE3- or apoE4-knock-in mice, WT mice, or apoE-KO mice and were cultured for 7 days. To determine the effect of the apoE-containing conditioned media, double co-culture model using primary pericytes and mBECs in the absence of astrocytes was sued. The conditioned media (48 h) from apoE3-expressing astrocytes (apoE3-CM) or apoE4-expressing astrocytes (apoE4-CM) were added only to the luminal side of double co-culture models and maintained for 7 days. Then, TEER was measured, and the values are presented as Ω × cm². The data presented are means ± S.D. (n = 3). *, p < 0.05 versus apoE3-BBB model.

FIGURE 2.

Phosphorylation of occludin and PKCη in mBECs of apoE3-BBB, apoE4-BBB, WT-BBB, and apoE-KO-BBB models. A, triple co-culture BBB models were prepared by using primary pericytes and mBECs from WT mouse brains and primary astrocytes from human apoE3 or apoE4 knock-in mice, WT mice, or apoE-KO mice and were cultured for 7 days. Total RNA of mBECs was collected. Relative real-time PCR analysis was performed to compare the expression levels of occludin. Results are shown as relative ratios to actin. The data presented are means ± S.D. (error bars) (n = 3). B, triple co-cultured models were cultured for 7 days. On day 7, mBECs were harvested, and the obtained cell lysates were subjected to immunoprecipitation and Western blotting. For the detection of occludin phosphorylated at its Thr residues, immunoprecipitation with the anti-Thr(P) antibody followed by immunoblotting with the anti-occludin antibody was performed. For the detection of total occludin, cell lysates were subjected to SDS-PAGE followed by probing with the anti-occludin antibody. The anti-β actin antibody was used as the loading control. The graph shows the levels of phosphorylated occludin at Thr residues and total occludin. The data presented are means ± S.D. (n = 3). *, p < 0.001 versus apoE3-BBB model. C, triple co-cultured models were cultured for 7 days. On day 7, mBECs were harvested, and the obtained cell lysates were subjected to Western blotting with the anti-phosphorylated PKCη and anti-PKCη antibodies. The graph shows the levels of phosphorylated and total pPKCη. The data presented are means ± S.D. (n = 3). *, p < 0.001 versus apoE3-BBB model.

FIGURE 3.

LRP1 is involved in the regulation of TJ integrity in the apoE3-BBB model. A–D, apoE3- and apoE4-BBB models were cultured for 7 days. On day 7, the in vitro BBB models were treated with RAP (A) (1 μm) or the anti-LRP1 (B), anti-LDLR (C), or anti-VLDLR antibody (D) (25 μg/ml) for 4 h at 37 °C. After treatment, mBECs were harvested using a cell scraper, and the obtained cell lysates were subjected to Western blotting using anti-phosphorylated PKCη and anti-PKCη antibodies. E, apoE3- and apoE4-BBB models were cultured for 7 days. On day 7, the in vitro BBB models were treated with the anti-LRP1 antibody or isotype IgG (25 μg/ml) for 4 h at 37 °C. After treatment, mBEC lysates were immunoprecipitated with the polyclonal anti-Thr(P) antibody, and the immunoprecipitates were subjected to Western blotting using the anti-occludin antibody or anti-phosphooccludin antibody. F, apoE3- and apoE4-BBB models were cultured for 7 days and then treated with the anti-LRP1 antibody or isotype IgG (25 μg/ml) for 4 h at 37 °C. After treatment, TEER was measured, and the values are presented as Ω × cm². The data presented are means ± S.D. (error bars) (n = 3). *, p < 0.005 compared with the values of apoE3-BBB models treated with control IgG (Dunnett's test).

FIGURE 4.

BBB integrity is impaired in apoE4-knock-in mice. The BBB integrity in apoE3-knock-in mice, apoE4-knock-in mice, and apoE-KO mice was evaluated using an Evans blue dye technique. Two hundred microliters of 20% mannitol was injected through the tail vein. Thirty minutes after injection, 200 μl of 2% Evans blue dye was injected intraperitoneally. The distribution of the dye was confirmed by a visible change in the color of the skin within 1 h after injection. Three hours after the injection, the mice were killed, and their brains were removed. The cerebrum and cerebellum were immediately collected, weighed, and incubated in 500 μl of formamide for 72 h at room temperature in the dark. The absorbance of the extracted dye was measured at 630 nm. The data presented are means ± S.D. (error bars) (n = 3). *, p < 0.05.