Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease

Abstract

Apolipoprotein E (apoE) alleles determine the age-adjusted relative risk (epsilon4 > epsilon3) for Alzheimer's disease (AD). ApoE may affect AD pathogenesis by promoting deposition of the amyloid-beta (Abeta) peptide and its conversion to a fibrillar form. To determine the effect of apoE on Abeta deposition and AD pathology, we compared APP(V717F) transgenic (TG) mice expressing mouse, human, or no apoE (apoE(-/-)). A severe, plaque-associated neuritic dystrophy developed in APP(V717F) TG mice expressing mouse or human apoE. Though significant levels of Abeta deposition also occurred in APP(V717F) TG, apoE(-/-) mice, neuritic degeneration was virtually absent. Expression of apoE3 and apoE4 in APP(V717F) TG, apoE(-/-) mice resulted in fibrillar Abeta deposits and neuritic plaques by 15 months of age and substantially (>10-fold) more fibrillar deposits were observed in apoE4-expressing APP(V717F) TG mice. Our data demonstrate a critical and isoform-specific role for apoE in neuritic plaque formation, a pathological hallmark of AD.

Figure 1

Neuritic plaques in APP^V717F TG mice are thioflavine-S-positive. A section containing the hippocampus from a 12-month-old APP^{V717F +/−} TG mouse [low power (A and B); high power (C and D)] and the temporal neocortex of an AD brain [high power (E and F)] were stained with the de Olmos silver degeneration method (A, C, and E), and the same sections were counterstained with thioflavine-S and visualized via epifluorescence microscopy with a UV filter (B, D, and F). Most of the neuritic plaques stained with this method had a thioflavine-S-positive core. [Bar in B = 100 μm (A and B); bar in F = 17 μm (C–F).]

Figure 2

Aβ deposition and neuritic plaques in APP^V717F TG mice in the presence and absence of apoE. Sections of the hippocampus from APP^{V717F +/−}, apoE^+/+ (A, C, and E) and APP^{V717F +/−}, apoE^−/− mice (B, D, and E) at 12 months of age were examined for Aβ IR (A and B) and the de Olmos silver degeneration method (C–F). Aβ-IR deposits were observed in the hippocampus of APP^V717F TG mice in the presence (A) and absence (B) of apoE; however, the distribution of Aβ deposits within the hippocampus was different. The area between arrowheads in A and B is the molecular layer of the dentate gyrus. Both low- (C and D) and higher-power (E and F) photomicrographs within the hippocampus reveal the presence of numerous neuritic plaques only in the presence (C and E) as opposed to in the absence (D and F) of apoE. Arrows in C point to neuritic plaques. Large arrow in E points to a large, distended, dystrophic neurite in a plaque, and small arrow in E points to fine dystrophic neurites. [Bar in D = 180 μm (A–D); bar in F = 30 μm (E and F).]

Figure 3

Quantitation of neuritic plaques, hippocampal Aβ, and thioflavine-S-positive deposits in APP^{V717F +/−} TG mice expressing mouse, human, or no apoE. (A) Hippocampal neuritic plaques were prominent in APP^{V717F +/−}, apoE^+/+ mice at 12 months and increased by more than 3-fold by 15 months of age (n = 4, both time points). In APP^{V717F +/−}, apoE^−/− mice (n = 6, both time points), neuritic plaques were virtually absent and did not increase in number between 12 and 15 months of age (*, P < 0.0001 compared with apoE^−/−). Neuritic plaques were identified with the de Olmos method. (B) The total volume of Aβ-IR deposits in the right hippocampus was determined in APP^{V717F +/−} TG mice that were apoE^+/+ (n = 4) or apoE^−/− (n = 6) at 12 months of age and apoE^+/+ (n = 4) or apoE^−/− (n = 6) at 15 months of age. Volume of Aβ deposits was determined by using unbiased stereological methods. There was no statistical difference in the size of the right hippocampus between any of the groups of mice (data not shown). (C) Aβ ELISA for total Aβ and Aβ₄₂ was assessed in the left hippocampus from the same mice used in A. *, P < 0.05, comparing apoE^+/+ with apoE^−/−. The total volume of Aβ-IR deposits in the right hippocampus (D) and in the molecular layer of dentate gyrus (E) was determined in APP^{V717F +/−} TG mice that were either apoE3^+/− (line 37, n = 6) or apoE4^+/− (line 22, n = 9) at 15 months of age. Volume of Aβ deposits was determined by using unbiased stereological methods. There was no statistical difference in the size of the right hippocampus or the molecular layer of the dentate gyrus between the groups (data not shown). (F) The mean density of thioflavine-S-positive plaques was assessed in the molecular layer of the dentate gyrus of the same mice. *, P < 0.05 (D and E); *, P < 0.001, comparing apoE3^+/− with apoE4^+/− (F). Data in B, D, E, and F were log-transformed and tested for normality before statistical analysis.

Figure 4

Human apoE restores the pattern of Aβ-IR deposits in the hippocampus of APP^{V717F +/−} TG mice to that seen in animals expressing mouse apoE. Photomicrographs are shown of Aβ-IR deposits representative of the median values of their respective groups from APP^{V717F +/−} TG mice either apoE^+/+ (A and B), apoE^−/− (C and D), apoE4^+/− (E and F), or apoE3^+/− (G and H) at 15 months of age. Although all mice expressing mouse apoE and some expressing human apoE at this age had Aβ-IR deposits in the molecular layer of the dentate gyrus (between arrowheads in B, D, F, and H), in the absence of apoE, Aβ-IR deposits were never observed in this layer. Significantly more E4-expressing (eight of nine, 89%) as compared with E3-expressing (two of six, 33%) mice developed Aβ deposition in the molecular layer. [Bar in H = 150 μm (A, C, E, and G) and 60 μm (B, D, F, and H).]

Figure 5

Fibrillar Aβ deposits and neuritic plaques develop in the presence of human apoE. Coronal sections through the hippocampus of an APP^{V717F +/−}, apoE4^+/− (A, B, D, and E) and an APP^{V717F +/−}, apoE^−/− mouse at 15 months of age were stained with thioflavine-S (A, B, and C) or the de Olmos silver degeneration method. Thioflavine-S-positive plaques were observed in the molecular layer of the dentate gyrus (A) and corpus callosum (B) of this apoE4-expressing mouse whereas no thioflavine-S-positive deposits were found in apoE^−/− mice (C). In addition to fibrillar Aβ, neuritic plaques also were found in the hippocampus of apoE4-expressing mice (D and E). [Bar in C = 180 μm (A–D); bar in E = 30 μm (E).] (F) Representative Western blot analysis of apoE, APP, and LRP in hippocampal lysates of APP^{V717F +/−} TG mice expressing apoE3 (line 37) or apoE4 (line 22) at 15 months of age. There were no significant differences in the level of apoE, APP, or LRP between the apoE3- and apoE4-expressing mice, as shown here qualitatively or quantitatively assessed by densitometric analysis (data not shown).