1α,25-Dihydroxyvitamin D3 reduces cerebral amyloid-β accumulation and improves cognition in mouse models of Alzheimer's disease

Abstract

We demonstrate a role of the vitamin D receptor (VDR) in reducing cerebral soluble and insoluble amyloid-β (Aβ) peptides. Short-term treatment of two human amyloid precursor protein-expressing models, Tg2576 and TgCRND8 mice, with 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], the endogenous active ligand of VDR, resulted in higher brain P-glycoprotein (P-gp) and lower soluble Aβ levels, effects negated with coadministration of elacridar, a P-gp inhibitor. Long-term treatment of TgCRND8 mice with 1,25(OH)2D3 during the period of plaque formation reduced soluble and insoluble plaque-associated Aβ, particularly in the hippocampus in which the VDR is abundant and P-gp induction is greatest after 1,25(OH)2D3 treatment, and this led to improved conditioned fear memory. In mice fed a vitamin D-deficient diet, lower cerebral P-gp expression was observed, but levels were restored on replenishment with VDR ligands. The composite data suggest that the VDR is an important therapeutic target in the prevention and treatment of Alzheimer's disease.

Keywords: Alzheimer's; P-glycoprotein; amyloid beta; blood-brain barrier; vitamin D receptor.

Figure 1.

Distribution of Vdr and P-gp in 8-week-old C57BL/6 mouse brains and effects of 1,25(OH)₂D₃ treatment on P-gp levels in brain regions. a, P-gp (green), along with Vdr (red), was expressed in brain capillaries within the cerebral cortex. Scale bars, 100 μm. Vdr levels were highest in the hippocampus and cortex (b), and P-gp levels were elevated mostly in the hippocampal and cortical regions after 1,25(OH)₂D₃ treatment (c) (for striatum, n = 4; for other regions, n = 6). Data are mean ± SEM. Differences among groups were compared using one-way ANOVA, and p values were determined by Bonferroni's multiple comparisons test. PFC, Prefrontal cortex; STR, striatum; HC, hippocampus; CER, cerebellum; OLB, olfactory bulb.

Figure 2.

Changes in 1,25(OH)₂D₃ levels and Vdr target gene expression during the 1,25(OH)₂D₃ treatment period in control and 1,25(OH)₂D₃-treated 8-week-old C57BL/6 mice. a, After treatment with repeated doses of 1,25(OH)₂D₃, brain levels (black symbols) of 1,25(OH)₂D₃ rose and fell in unison with those in plasma [gray symbols; from a previous publication (Chow et al., 2013)]; open and filled symbols represent levels in brains of untreated and treated mice, respectively. The lines connect the mean levels. b, Basal mRNA levels of Vdr target genes, Cyp24a1 and Mdr1a, were relatively unaltered in vehicle-treated animals but were increased after treatment. Cyp24a1 levels rose and fell sharply, but Mdr1a mRNA levels were sustained during the dosing period (n = 2–4 for each time point).

Figure 3.

Vitamin D (Vit D) deficiency lowers plasma 1,25(OH)₂D₃ and cerebral P-gp expression. a, C57BL/6 mice (8 weeks old) that were fed a vitamin D-deficient diet exhibited significantly lower (75%) plasma 1,25(OH)₂D₃ levels after 6 weeks of the diet (n = 4 for samples at 2, 4, and 6 weeks). Replenishment of the vitamin D-deficient mice after week 6 with dietary vitamin D or 1,25(OH)₂D₃ elevated plasma 1,25(OH)₂D₃ levels, albeit not to basal levels (n ≥ 6). b, Mdr1a mRNA expression and P-gp protein levels were fully restored on replenishment with 1,25(OH)₂D₃. For vitamin D-deficient and vitamin D-sufficient mice, n = 6. Data are mean ± SEM; a one-way ANOVA was used to evaluate differences between groups, with p values determined by Bonferroni's multiple comparisons test.

Figure 4.

1,25(OH)₂D₃ treatment induces P-gp in 10-week-old Tg2576 mice (a) and reduces soluble hAβ_1–40 and hAβ_1–42 in the brain (b). N/D denotes that levels were below detection limits. For Tg2576 mice, n = 5; for non-Tg mice, n = 6. A one-way ANOVA was used to evaluate differences in Mdr1a/P-gp between groups, with p values determined by Bonferroni's multiple comparisons test. To compare Aβ levels between treated and control Tg2576 mice, p values were determined by Student's two-tailed t test.

Figure 5.

Relative brain Mdr1a mRNA and P-gp protein levels (a) and brain concentrations of soluble and insoluble hAβ_1–40 and hAβ_1–42 (b) in non-Tg and TgCRND8 mice (at 4 months) after treatment with 1,25(OH)₂D₃ or vehicle for 8 weeks. Significant reduction of soluble hAβ_1–42 and insoluble hAβ_1–40 and hAβ_1–42 was observed after 1,25(OH)₂D₃ treatment. N/D denotes that levels were below detection limits. For TgCRND8 mice, n ≥ 7; for non-Tg mice, n ≥ 4. Data are mean ± SEM; a one-way ANOVA was used to evaluate differences in Mdr1a/P-gp between groups, with p values determined by Bonferroni's multiple comparisons test. To compare Aβ levels between treated and control TgCRND8 mice, p values were determined by Student's two-tailed t test.

Figure 6.

1,25(OH)₂D₃ treatment decreases dense and diffuse amyloid plaques in TgCRND8 mice. a, Representative images of brains of vehicle-treated and 1,25(OH)₂D₃-treated TgCRND8 mice after long-term treatment and significant decreases in dense and diffuse amyloid plaques in cortex and hippocampus. For each group, n = 7 mice. Every fifth section of 25 sections from each brain were stained and quantified. Data are mean ± SEM; p values were determined by Student's two-tailed t test. Scale bars, 1 mm. b, Resorufin staining in the cerebral cortex. bi, bii, Patterns of resorufin binding observed in cerebral arteries of TgCRND8 mice at 17 weeks of age. Exterior surface of cerebral artery (bi) and cross-section of cerebral artery demonstrating interspersed laminar stripes of resorufin distribution on abluminal surface (bii). biii–bv, Resorufin staining seen in cortex of vehicle- treated animals; bvi–bviii, resorufin staining observed in cortex of 1,25(OH)₂D₃-treated animals. Although statistically significant differences in resorufin staining were not observed in cerebral vasculature, a diminution of resorufin-positive neuritic plaques was noted in 1,25(OH)₂D₃-treated animals (arrowheads). Scale bars (in iii–viii), 100 mm. For each group, n = 5 mice. For 105 sections, the first four of every 30 sections from each brain was stained and quantified. Data are mean ± SEM; p values were determined by Student's two-tailed t test.

Figure 7.

Fear conditioning studies: frequency of freezing behavior is partially restored in TgCRND8 mice that received 1,25(OH)₂D₃ treatment. Data are mean ± SEM percentage of time mice exhibited freezing behavior before and after the CS. Vehicle-treated non-Tg mice, n = 10; 1,25(OH)₂D₃-treated non-Tg mice, n = 8; vehicle-treated TgCRND8 mice, n = 8; and 1,25(OH)₂D₃-treated TgCRND8 mice, n = 10. A one-way ANOVA was used to evaluate differences between groups, with p values determined by Bonferroni's multiple comparisons test.

Figure 8.

1,25(OH)₂D₃ treatment increases P-gp in 20-week-old TgCRND8 mice (a) and reduces soluble hAβ_1–40 and hAβ_1–42 in the brain (b). Data are mean ± SEM. For all groups, n = 4. A one-way ANOVA was used to evaluate differences between groups, with p values determined by Bonferroni's multiple comparisons test.