Reducing human apolipoprotein E levels attenuates age-dependent Aβ accumulation in mutant human amyloid precursor protein transgenic mice

Abstract

Apolipoprotein E4 (apoE4) plays a major role in the pathogenesis of Alzheimer's disease. Brain amyloid-β (Aβ) accumulation depends on age and apoE isoforms (apoE4 > apoE3) both in humans and in transgenic mouse models. Brain apoE levels are also isoform dependent, but in the opposite direction (apoE4 < apoE3). Thus, one prevailing hypothesis is to increase brain apoE expression to reduce Aβ levels. To test this hypothesis, we generated mutant human amyloid precursor protein transgenic mice expressing one or two copies of the human APOE3 or APOE4 gene that was knocked in and flanked by LoxP sites. We report that reducing apoE3 or apoE4 expression by 50% in 6-month-old mice results in efficient Aβ clearance and does not increase Aβ accumulation. However, 12-month-old mice with one copy of the human APOE gene had significantly reduced Aβ levels and plaque loads compared with mice with two copies, regardless of which human apoE isoform was expressed, suggesting a gene dose-dependent effect of apoE on Aβ accumulation in aged mice. Additionally, 12-month-old mice expressing one or two copies of the human APOE4 gene had significantly higher levels of Aβ accumulation and plaque loads than age-matched mice expressing one or two copies of the human APOE3 gene, suggesting an isoform-dependent effect of apoE on Aβ accumulation in aged mice. Moreover, Cre-mediated APOE4 gene excision in hippocampal astrocytes significantly reduced insoluble Aβ in adult mice. Thus, reducing, rather than increasing, apoE expression is an attractive approach to lowering brain Aβ levels.

Figure 1.

Generation and characterization of floxed human apoE knock-in mice. A, Targeting vector containing human apoE exons 2–4 (represented by black rectangles) flanked by loxP sites with endogenous mouse exon 1 and mouse upstream and downstream elements used for homologous recombination (mouse exons represented by gray rectangles). B, C, E, F, Representative immunofluorescence images for apoE (green) and GFAP (red) in the hippocampal subfield highlighting apoE-producing astrocytes in APOE-fKI mice (B, C) and APOE-KI mice (E, F). D, G, Western blot and densitometric quantification of apoE protein levels in the hippocampus of homozygous APOE-fKI mice (D) and homozygous APOE-KI mice (G). N = 3–4 mice per genotype. Values are mean ± SEM. *p < 0.05 by two-tailed, unpaired t test. Scale bar, 10 μm.

Figure 2.

Reducing apoE levels by half does not increase Aβ accumulation in young mice. A–C, Levels of Aβ_1-x were measured by sandwich ELISA in combined soluble and insoluble hippocampal lysates (A), low-detergent-soluble lysates (B), and insoluble lysates (C) for E3^+/−/hAPP, E3^+/+/hAPP, E4^+/−/hAPP, E4^+/+/hAPP, and E^−/−/hAPP mice at 6–8 months of age. D–F, Levels of Aβ₄₂ in combined soluble and insoluble lysates (D), low-detergent-soluble lysates (E), and insoluble lysates (F). G–K, Representative sections from an Aβ immunostain of the hippocampal subfield of E3^+/−/hAPP (G), E3^+/+/hAPP (H), E4^+/−/hAPP (J), E4^+/+/hAPP (K), and E^−/−/hAPP (I) mice. L, Quantification by densitometry of the percentage area covered by Aβ deposition. N = 7–12 mice per genotype, three sections per mouse. Values are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus all other groups by one-way ANOVA with Bonferroni's post hoc test. Scale bar, 250 μm.

Figure 3.

Human apoE expression levels are reduced in young hemizygous apoE mice without altering APP expression levels. A, B, Western blotting was performed on low-detergent-soluble (A) and insoluble (B) hippocampal lysates and probed for apoE, tubulin, and human full-length APP protein levels in E3^+/−/hAPP, E3^+/+/hAPP, E4^+/−/hAPP, E4^+/+/hAPP, and E^−/−/hAPP mice at 6–8 months of age. C–F, Quantification of Western blots by densitometry for total apoE levels (C), low-detergent-soluble apoE (D), insoluble apoE (E), and ratios of soluble to insoluble pools of apoE (F). G, H, Western blot quantification of total full-length APP levels (G) and total tubulin levels as loading controls (H). Values are mean ± SEM. N = 3–4 mice per genotype. *p < 0.05, ***p < 0.001, by two-tailed, unpaired t test.

Figure 4.

Halving the levels of apoE significantly attenuates Aβ accumulation in aged mice. A–F, Levels of Aβ_1-x (A–C) and Aβ₄₂ (D–F) were measured by sandwich ELISA in both soluble and insoluble lysates (A, D), low-detergent-soluble lysates (B, E), and insoluble lysates (C, F) in the hippocampus of E3^+/−/hAPP, E3^+/+/hAPP, E4^+/−/hAPP, E4^+/+/hAPP, and E^−/−/hAPP mice at 12 months of age. Values are mean ± SEM. N = 6–13 mice per genotype. *p < 0.05, **p < 0.01, and ***p < 0.001, by two-tailed, unpaired t test.

Figure 5.

Human apoE expression levels are reduced in aged hemizygous apoE mice without altering APP expression levels. A, B, Western blotting was performed on low-detergent-soluble (A) and insoluble (B) hippocampal lysates, and probed for apoE, tubulin, and human full-length APP protein levels in E3^+/−/hAPP, E3^+/+/hAPP, E4^+/−/hAPP, E4^+/+/hAPP, and E^−/−/hAPP mice at 12 months of age. C–F, Quantification of Western blots by densitometry for total apoE levels (C), low-detergent-soluble apoE (D), insoluble apoE (E), and ratios of soluble to insoluble pools of apoE (F). G, H, Western blot quantification of total full-length APP levels (G) and total tubulin levels as loading controls (H). Values are mean ± SEM. N = 3–4 mice per genotype.

Figure 6.

Halving the levels of apoE significantly attenuates Aβ deposition in aged mice. A–E, Representative sections from 12-month-old aged E3^+/−/hAPP (A), E3^+/+/hAPP (B), E4^+/−/hAPP (C), E4^+/+/hAPP (D), and E^−/−/APP (E) mice immunostained for Aβ. F, Quantification of Aβ immunostain by densitometry for the percentage area of Aβ deposition in the five genotypes of mice. G–K, Representative sections from 12-month-old E3^+/−/hAPP (G), E3^+/+/hAPP (H), E4^+/−/hAPP (I), E4^+/+/hAPP (J), and E^−/−/hAPP (K) mice stained with thioflavin S dye to fluorescently label fibrillar/amyloid plaques. L, Quantification of percentage area positive for thioflavin S by densitometry. Values are mean ± SEM. N = 6–13 mice per genotype, three sections per mouse. *p < 0.05, by two-tailed, unpaired t test; **p < 0.01 versus all other groups by one-way ANOVA with Bonferroni's post hoc test. Scale bars, 250 μm.

Figure 7.

Gene dose- and isoform-dependent codistribution of apoE with Aβ plaques in aged mice. A–J, Representative fluorescent immunostaining for apoE (green) and Aβ (red) in various subregions of the hippocampus in E3^+/−/hAPP (A, B), E3^+/+/hAPP (C, D), E4^+/−/hAPP (E, F), E4^+/+/hAPP (G, H), and E^−/−/hAPP (I, J) mice at 12 months of age. K, Quantification of Aβ deposits and codistribution with apoE. Mice with two copies of apoE3 or apoE4 have a greater percentage of plaques containing apoE. Values are mean ± SEM. N = 3–4 mice per genotype, two sections per mouse. *p < 0.05, **p < 0.01, by two-tailed, unpaired t test. Scale bar, 25 μm.

Figure 8.

Cre-mediated APOE gene excision in hippocampal astrocytes decreases Aβ levels in young adult mice. A, B, Representative confocal images of brain sections from E4^+/+/hAPP mice at 6–7 months of age stained for GFAP (blue) from the hippocampus injected with adenovirus expressing Cre (Ad-Cre) and GFP (green) (A) or saline (B). Brains were collected 1 month after the Ad-Cre virus or saline injection. C–F, Representative confocal images of brain sections from E4^+/+/hAPP mice stained for GFP (green; C, D, F), GFAP (blue; C, E, F), and apoE (red; D–F) from the hippocampus injected with Ad-Cre and expressing GFP (green). G, H, Aβ levels in Ad-Cre-injected and saline-injected hippocampi were quantified 1 month after injection. Levels of Aβ_1-x (G) in both soluble and insoluble hippocampal lysates were similar in control compared with Ad-Cre-injected hemibrains, whereas levels of Aβ₄₂ (H) showed a specific difference in the insoluble lysates. Values are mean ± SEM. N = 8 mice. *p < 0.05 by two-tailed, paired t test. Scale bars: A, B, 250 μm; C–F, 20 μm.