Human apolipoprotein E ɛ4 expression impairs cerebral vascularization and blood-brain barrier function in mice

Abstract

Human apolipoprotein E (APOE) exists in three isoforms ɛ2, ɛ3, and ɛ4, of which APOE4 is the main genetic risk factor of Alzheimer's disease (AD). As cerebrovascular defects are associated with AD, we tested whether APOE genotype has an impact on the integrity and function of the blood-brain barrier (BBB) in human APOE-targeted replacement mice. Using the quantitative in situ brain perfusion technique, we first found lower (13.0% and 17.0%) brain transport coefficient (Clup) of [(3)H]-diazepam in APOE4 mice at 4 and 12 months, compared with APOE2 and APOE3 mice, reflecting a decrease in cerebral vascularization. Accordingly, results from immunohistofluorescence experiments revealed a structurally reduced cerebral vascularization (26% and 38%) and thinner basement membranes (30% and 35%) in 12-month-old APOE4 mice compared with APOE2 and APOE3 mice, suggesting vascular atrophy. In addition, APOE4 mice displayed a 29% reduction in [(3)H]-d-glucose transport through the BBB compared with APOE2 mice without significant changes in the expression of its transporter GLUT1 in brain capillaries. However, an increase of 41.3% of receptor for advanced glycation end products (RAGE) was found in brain capillaries of 12-month-old APOE4 mice. In conclusion, profound divergences were observed between APOE genotypes at the cerebrovascular interface, suggesting that APOE4-induced BBB anomalies may contribute to AD development.

Figure 1

The brain transport coefficient (Clup, μL/g s) of [³H]-diazepam was decreased in APOE4 mice. Clup was determined in APOE2, APOE3, and APOE4 mice at (A) 2, (B) 4, and (C) 12 months of age using the in situ brain perfusion technique. Data are shown as mean± standard error of the mean (s.e.m.). Statistical analyses: one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test *P<0.05 (n=6 to 12).

Figure 2

Relative vessel density in the hippocampus was reduced in APOE4 mice. Area occupied by vessels was determined by immunohistofluorescence using anti-collagen IV antibody in 12-month-old APOE2, APOE3, and APOE4 mice. Image analyses were performed in the CA2-CA3 region of hippocampal slices located between bregma −1.46 and bregma −1.70. Data are shown as mean±standard error of the mean (s.e.m.). Statistical analyses: one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P<0.05; ***P<0.001 (n=5 to 6).

Figure 3

The apparent thickness of the basement membrane of brain capillaries (μm) was reduced in APOE4 mice, as assessed using immunohistofluorescence with an anti-collagen IV antibody in 12-month-old APOE2, APOE3, and APOE4 mice. Data are shown as mean thickness±standard error of the mean (s.e.m.). Statistical analyses: one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P<0.01; ***P<0.001 (n=5 to 6).

Figure 4

The calculated vascular volume (V_vasc, μL/g) was not different between the groups. V_vasc was determined in APOE2, APOE3, and APOE4 mice at (A) 2, (B) 4, and (C) 12 months by in situ brain perfusion of the vascular marker [¹⁴C]-sucrose. The biodistribution of human immunoglobulins (hIgG) was comparable in (D) the plasma and (E) tissue homogenates of 5-month-old APOE2 and APOE4 mice after three injections of 30 mg hIgG, 96, 24, and 1 hour(s) before kiling. Western blot quantification of (F) occludin and (G) claudin-5 in brain capillary fractions did not evidence any difference between the three APOE genotypes at 12 months of age. All data represent mean±standard error of the mean (s.e.m.). The number of mice in each group is indicated in the bars of the histograms. Statistical analyses: one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (A, B, C, F, and G) or Student's unpaired t test (D and E).

Figure 5

The brain transport coefficient (Clup, μL/g s) of [³H]d-glucose was decreased in 12-month-old APOE4 mice. Clup was determined in APOE2, APOE3, and APOE4 mice at (A) 4 and (B) 12 months of age by in situ brain perfusion technique. Data are shown as mean±standard error of the mean (s.e.m.). Statistical analyses: one-way analysis of variance followed by Dunnett's multiple comparison test. ***P<0.001 (n=4 to 12). (C) GLUT1 expression was not significantly different between the groups. GLUT1 expression was measured by western blot in 12-month-old mice. Data are shown as mean±s.e.m. (n=8 to 9 mice).

Figure 6

Brain capillary concentration of receptor for advanced glycation end products (RAGE) was increased in APOE4 mice. (A) RAGE, (B) lipoprotein receptor-related protein-1 (LRP1), (C) ApoE, and (D) LR11 were measured by western blot in the brain capillaries of 12-month-old mice. Data are shown as mean± standard error of the mean (s.e.m.). Statistical analyses: one-way ANOVA followed by Dunnett's multiple comparison test. *P<0.05; **P<0.01; (n=6 to 9 mice).