Early-onset formation of parenchymal plaque amyloid abrogates cerebral microvascular amyloid accumulation in transgenic mice

Abstract

The fibrillar assembly and deposition of amyloid β (Aβ) protein, a key pathology of Alzheimer disease, can occur in the form of parenchymal amyloid plaques and cerebral amyloid angiopathy (CAA). Familial forms of CAA exist in the absence of appreciable parenchymal amyloid pathology. The molecular interplay between parenchymal amyloid plaques and CAA is unclear. Here we investigated how early-onset parenchymal amyloid plaques impact the development of microvascular amyloid in transgenic mice. Tg-5xFAD mice, which produce non-mutated human Aβ and develop early-onset parenchymal amyloid plaques, were bred to Tg-SwDI mice, which produce familial CAA mutant human Aβ and develop cerebral microvascular amyloid. The bigenic mice presented with an elevated accumulation of Aβ and fibrillar amyloid in the brain compared with either single transgenic line. Tg-SwDI/Tg-5xFAD mice were devoid of microvascular amyloid, the prominent pathology of Tg-SwDI mice, but exhibited larger parenchymal amyloid plaques compared with Tg-5xFAD mice. The larger parenchymal amyloid deposits were associated with a higher loss of cortical neurons and elevated activated microglia in the bigenic Tg-SwDI/Tg-5xFAD mice. The periphery of parenchymal amyloid plaques was largely composed of CAA mutant Aβ. Non-mutated Aβ fibril seeds promoted CAA mutant Aβ fibril formation in vitro. Further, intrahippocampal administration of biotin-labeled CAA mutant Aβ peptide accumulated on and adjacent to pre-existing parenchymal amyloid plaques in Tg-5xFAD mice. These findings indicate that early-onset parenchymal amyloid plaques can serve as a scaffold to capture CAA mutant Aβ peptides and prevent their accumulation in cerebral microvessels.

Keywords: Alzheimer Disease; Amyloid; Cerebral Vascular; Pathology; Plaque; Protein Misfolding; Transgenic Mice.

FIGURE 1.

Measurement of Aβ peptide levels in brains of Tg-SwDI, Tg-5xFAD, and bigenic Tg-SwDI/Tg-5xFAD mice. The levels of soluble and insoluble Aβ40 and Aβ42 peptides were measured in mouse forebrain extracts of 3- (A), 6- (B), and 9-month-old (C) Tg-SwDI mice (black bars), Tg-5xFAD mice (blue bars), and bigenic Tg-SwDI/Tg-5xFAD mice (red bars) by ELISA analysis, as described under “Experimental Procedures.” The levels of soluble Aβ oligomers were measured by ELISA (D) and quantitative dot blot analysis using OC antibody (E and F) in the different mice at 3, 6, and 9 months of age, as described under “Experimental Procedures.” Data are mean ± S.D. of eight to 10 mice of each genotype at each age. a, p < 0.05; b, p < 0.01; c, p < 0.001; d, p < 0.0001; ns, not significant.

FIGURE 2.

Progressive accumulation of Aβ deposition in Tg-SwDI, Tg-5xFAD, and bigenic Tg-SwDI/Tg-5xFAD mouse brains. Shown are representative images of 3- to 9-month-old transgenic mouse brain sections immunostained for Aβ as described under “Experimental Procedures.” A–C, Tg-SwDI mice. D–F, Tg-5xFAD mice. G–I, bigenic Tg-SwDI/Tg-5xFAD mice. Scale bars = 1 mm. Regional cerebral Aβ deposition in the cortex (J), thalamus (K), hippocampus (L), and subiculum (M) of 3-, 6-, and 9-month-old Tg-SwDI mice (black bars), Tg-5xFAD mice (blue bars), and bigenic Tg-SwDI/Tg-5xFAD mice (red bars) was determined by image analysis of Aβ immunostaining. Data are mean ± S.D. of eight mice of each genotype at each age. a, p < 0.05; b, p < 0.01; c, p < 0.001; d, p < 0.0001; ns, not significant.

FIGURE 3.

Progressive accumulation of fibrillar amyloid deposition in Tg-SwDI, Tg-5xFAD, and bigenic Tg-SwDI/Tg-5xFAD mouse brains. Shown are representative images of 3- to 9-month-old transgenic mouse brain sections stained for fibrillar amyloid using thioflavin S (green) as described under “Experimental Procedures.” A–C, Tg-SwDI mice. D–F, Tg-5xFAD mice. G–I, bigenic Tg-SwDI/Tg-5xFAD mice. Scale bars = 1 mm. Regional fibrillar amyloid deposition in the cortex (J), thalamus (K), hippocampus (L), and subiculum (M) of 3-, 6-, and 9-month-old Tg-SwDI mice (black bars), Tg-5xFAD mice (blue bars), and bigenic Tg-SwDI/Tg-5xFAD mice (red bars) was determined by image analysis of thioflavin S staining. Data are mean ± S.D. of eight mice of each genotype at each age. a, p < 0.05; b, p < 0.01; c, p < 0.001; d, p < 0.0001; ns, not significant.

FIGURE 4.

Altered fibrillar amyloid deposition in bigenic Tg-SwDI/Tg-5xFAD mouse brains. Shown are representative images of 3- to 9-month-old transgenic mouse brain sections stained for fibrillar amyloid using thioflavin S (green) and immunolabeled for cerebral blood vessels using an antibody to collagen IV (red), as described under “Experimental Procedures.” A–C, Tg-SwDI mice. D–F, Tg-5xFAD mice. G–I, bigenic Tg-SwDI/Tg-5xFAD mice. Scale bars = 50 μm. The amount of cerebral microvascular amyloid (J) and the size distribution of all fibrillar amyloid deposits in 3- (K), 6- (L), and 9-month-old (M) Tg-SwDI mice (black bars), Tg-5xFAD mice (blue bars), and bigenic Tg-SwDI/Tg-5xFAD mice (red bars) was determined in the subiculum region using stereological principles, as described under “Experimental Procedures.” Data are the mean ± S.D. of eight mice per group. a, p < 0.05; b, p < 0.01; c, p < 0.001; d, p < 0.0001; ns, not significant.

FIGURE 5.

Increased neuronal loss and microglial activation in bigenic Tg-SwDI/Tg-5xFAD mouse brains. Shown are representative images from 6-month-old transgenic mouse brain sections immunolabeled for neurons using an antibody to NeuN, for activated microglia using mAb5D4, and stained for fibrillar amyloid using thioflavin S (ThS). A, D, G, and J, images of neuronal staining in cortical layers II–VI. Scale bars = 100 μm. B, E, H, and K, images of neuronal staining in cortical layer V. Scale bars = 50 μm. C, F, I, and L, images of thioflavin S staining for fibrillar amyloid (green) and immunolabeling for activated microglia (red). Scale bars = 50 μm. The numbers of activated microglia (M) and neurons (N) in cortical layer V in wild-type mice (gray bars), Tg-SwDI mice (black bars), Tg-5xFAD mice (blue bars), and bigenic Tg-SwDI/Tg-5xFAD mice (red bars) were determined using stereological principles as described under “Experimental Procedures.” Data are mean ± S.D. of five to eight mice per group. a, p < 0.05; b, p < 0.01; c, p < 0.001; d, p < 0.0001; ns, not significant.

FIGURE 6.

Analysis of non-mutated Aβ and Dutch/Iowa CAA mutant Aβ accumulation in Tg-SwDI, Tg-5xFAD, and bigenic Tg-SwDI/Tg-5xFAD mouse brains. A, a dot blot analysis was performed to demonstrate that rabbit polyclonal antibody to human Aβ (pAb-Aβ) recognizes both non-mutated Aβ and Dutch/Iowa CAA mutant Aβ, whereas mAb4G8 only recognizes non-mutated Aβ. Brain sections obtained from the different transgenic mouse lines at 9 months of age were immunolabeled with pAb-Aβ (red, B, E, and H), mAb4G8 (green, C, F, and I), and merged (D, G, and J). Dutch/Iowa CAA mutant Aβ deposits in Tg-SwDI mice are immunolabeled with pAb-Aβ (B) but not mAb4G8 (C). Non-mutated Aβ deposits were immunolabeled with both pAb-Aβ (E) and mAb4G8 (F). Aβ plaque core deposits in bigenic Tg-SwDI/Tg-5xFAD mice were immunolabeled with both pAb-Aβ (H) and mAb4G8 (I). The merged image shows a halo of Aβ deposits around the periphery of plaques that were not immunolabeled with mAb4G8, suggesting that they are composed of Dutch/Iowa CAA mutant Aβ (J). Scale bars = 50 μm.

FIGURE 7.

Non-mutated Aβ42 amyloid fibril seeds promote the assembly of Dutch/Iowa CAA mutant Aβ40 fibrils. Freshly prepared solutions of Dutch/Iowa CAA mutant Aβ40 (20 μm) were incubated in the absence (red trace) or presence of 10 μm (purple trace) or 20 μm (green trace) of non-mutated Aβ42 fibrillar seeds. The rate and extent of fibril formation was assessed by thioflavin T fluorescence measurements. The fluorescence curves of the non-mutated Aβ42 fibril seeds with added Dutch/Iowa CAA mutant Aβ40 exhibit two components. The rapid component is attributed to the growth of CAA mutant Aβ40 fibrils on the non-mutated Aβ42 seeds. The slower component is attributed to fibril formation of CAA mutant Aβ40 following nucleation of the monomeric peptide. Non-mutated Aβ42 fibril seeds in the absence of added monomeric CAA mutant Aβ peptide do not exhibit a change in fluorescence (blue trace). Data are mean ± S.D. of triplicate determinations.

FIGURE 8.

Non-mutated parenchymal amyloid deposits in Tg-5xFAD mice promote the accumulation of injected biotin-labeled Dutch/Iowa CAA mutant Aβ. Biotin-labeled Dutch/Iowa CAA mutant Aβ40 (A–D) and biotin (E–H) were injected into the hippocampal region of 6-month-old Tg-5xFAD mice. Brain sections were prepared, fibrillar amyloid was visualized by staining with thioflavin S (green), and biotin-labeled Dutch/Iowa CAA mutant Aβ40 or biotin alone was detected using Texas Red-labeled streptavidin (red). A–C and E–G, scale bars = 50 μm. D and H, scale bars = 12.5 μm.