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. 2024 May 25;25(11):5754.
doi: 10.3390/ijms25115754.

Temporal Characterization of the Amyloidogenic APPswe/PS1dE9;hAPOE4 Mouse Model of Alzheimer's Disease

Martine B Grenon  1   2 , Maria-Tzousi Papavergi  1   3 , Praveen Bathini  1 , Martin Sadowski  4 , Cynthia A Lemere  1
Affiliations

Temporal Characterization of the Amyloidogenic APPswe/PS1dE9;hAPOE4 Mouse Model of Alzheimer's Disease

Martine B Grenon et al. Int J Mol Sci. .

Abstract

Alzheimer's disease (AD) is a devastating disorder with a global prevalence estimated at 55 million people. In clinical studies administering certain anti-beta-amyloid (Aβ) antibodies, amyloid-related imaging abnormalities (ARIAs) have emerged as major adverse events. The frequency of these events is higher among apolipoprotein ε4 allele carriers (APOE4) compared to non-carriers. To reflect patients most at risk for vascular complications of anti-Aβ immunotherapy, we selected an APPswe/PS1dE9 transgenic mouse model bearing the human APOE4 gene (APPPS1:E4) and compared it with the same APP/PS1 mouse model bearing the human APOE3 gene (APOE ε3 allele; APPPS1:E3). Using histological and biochemical analyses, we characterized mice at three ages: 8, 12, and 16 months. Female and male mice were assayed for general cerebral fibrillar and pyroglutamate (pGlu-3) Aβ deposition, cerebral amyloid angiopathy (CAA), microhemorrhages, apoE and cholesterol composition, astrocytes, microglia, inflammation, lysosomal dysfunction, and neuritic dystrophy. Amyloidosis, lipid deposition, and astrogliosis increased with age in APPPS1:E4 mice, while inflammation did not reveal significant changes with age. In general, APOE4 carriers showed elevated Aβ, apoE, reactive astrocytes, pro-inflammatory cytokines, microglial response, and neuritic dystrophy compared to APOE3 carriers at different ages. These results highlight the potential of the APPPS1:E4 mouse model as a valuable tool in investigating the vascular side effects associated with anti-amyloid immunotherapy.

Keywords: Alzheimer’s disease; amyloid-related imaging abnormalities (ARIAs); apolipoprotein E; cerebral amyloid angiopathy; cholesterol; human APOE-targeted replacement mice.

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Conflict of interest statement

C.A.L. received, as a gift-in-kind, the anti-pGlu-3 Aβ antibody from Probiodrug AG (now Vivoryon Therapeutics NV). The other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Aβ deposition in hippocampus and prefrontal cortex of APPPS1:E4 and APPPS1:E3 mice. (A–C) Hemibrain sections from APPPS1:E4 and APPPS1:E3 mice stained with Thioflavin-S for fibrillar Aβ. (A) Representative microphotographs of hippocampus. Scale bar: 500 μM. (B,C) Quantification of fibrillar protein aggregates in hippocampus. (B) Analysis of main effect of age in APPPS1:E4 mice (n = 3–7 mice/group). (C) Analysis of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–11 mice/group). (D–I) Hemibrain sections from APPPS1:E4 and APPPS1:E3 mice stained with S97 pAb for general Aβ. (D) Representative microphotographs of hippocampus. Scale bar: 500 μM. (E,F) Quantification of general Aβ deposition in hippocampus. (E) Analysis of main effect of age in APPPS1:E4 mice (n = 3–5 mice/group). (F) Analysis of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 2–9 mice/group). (G) Representative microphotographs of prefrontal cortex. Scale bar: 50 μM. (H,I) Quantification of general Aβ deposition in prefrontal cortex. (H) Analysis of main effect of age in APPPS1:E4 mice (n = 3–6 mice/group). (I) Analysis of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–10 mice/group). (J–M) Biochemical quantification of common Aβ species with MSD 4G8 Aβ Triplex ELISA on guanidine hydrochloride-extracted whole-hemibrain homogenates from APPPS1:E4 and APPPS1:E3 mice. Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice on levels of insoluble (J) Aβx-40 and (K) Aβx-42 (n = 3–7 mice/group). (L) Analysis of main effect of age in APPPS1:E4 mice on Aβx-42/40 ratio (n = 7 mice/group). (M) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice on Aβx-42/40 ratio (n = 7 mice/group). Overall, Aβ deposition significantly increased with age in both hippocampal and cortical areas of brain in APPPS1:E4 mice. Data are presented as mean ± SEM. APPPS1:E4 age effect was analyzed by two-way ANOVA followed by Tukey’s HSD post-test (simple effects within sex). With sexes pooled, genotype effect was analyzed by two-way ANOVA followed by post hoc Sidak’s correction (simple effects within age). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. HC: hippocampus; PFC: prefrontal cortex.
Figure 2
Figure 2
PGlu-3 Aβ deposition and biochemical detection in hippocampus and prefrontal cortex of APPPS1:E4 and APPPS1:E3 mice. (A–F) Hemibrain sections from APPPS1:E4 and APPPS1:E3 mice stained with K17 IgG2b mAb for pGlu-3-Aβ. (A) Representative microphotographs of hippocampus. Scale bar: 500 μM. (B,C) Quantification in hippocampus. (B) Analysis of main effect of age in APPPS1:E4 mice (n = 7–11 mice/group). (C) Analysis of genotype in 16-month APPPS1:E4 (n = 7) and APPPS1:E3 (n = 3) mice. (D) Representative microphotographs of prefrontal cortex. Scale bar: 50 μM. (E,F) Quantification in prefrontal cortex. (E) Analysis of main effect of age in APPPS1:E4 mice (n = 7–11 mice/group). (F) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–10 mice/group). (G,H) Biochemical quantification of pGlu-3 Aβ levels with IBL N3pE-42 ELISA on guanidine hydrochloride-extracted whole-hemibrain homogenates from APPPS1:E4 and APPPS1:E3 mice. (G) Analysis of main effect of age in APPPS1:E4 mice (n = 7–11 mice/group). (H) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–10 mice/group). Overall, pGlu-3 Aβ deposition increased significantly at 16 months of age in both hippocampal and cortical areas of brain in female and male APPPS1:E4 mice. Data are presented as mean ± SEM. With sexes pooled, APPPS1:E4 age effect was analyzed with one-way ANOVA followed by post hoc Tukey’s correction. When sex effect was significant within age groups, analysis was conducted by two-way ANOVA followed by post hoc Tukey’s correction. With sexes pooled, genotype effect was analyzed by two-way ANOVA followed by post hoc Sidak’s correction (simple effects within age) or pair-wise comparisons with Alternative Welch’s t-test. * p < 0.05; *** p < 0.001; **** p < 0.0001. HC: hippocampus; PFC: prefrontal cortex.
Figure 3
Figure 3
Cerebral amyloid angiopathy (CAA) and microhemorrhage. (A–C) Hemibrain sections from APPPS1:E4 and APPPS1:E3 mice stained with Thioflavin-S for fibrillar Aβ. (A) Representative microphotographs from whole sagittal sections from 16-month-old APPPS1:E4 and APPPS1:E3 mice. Arrowheads indicate long segments of CAA-laden vessels. Scale bar: 100 μM. (B,C) Semi-quantitative analysis of vascular Aβ deposition. (B) Analysis of main effect of age in APPPS1:E4 mice (n = 7–10 mice/group). (C) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–10 mice/group). (D,E) Hemibrain sections from APPPS1:E4 and APPPS1:E3 mice stained with Perls’ Prussian blue for microhemorrhage. (D) Representative microphotographs of whole sagittal sections. Arrowheads show hemosiderin-positive staining. Scale bar: 100 μM. (E) Semi-quantitative analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice with hemosiderin-positive staining (n = 3–10 mice/group). Data are presented as mean ± SEM. With sexes pooled, APPPS1:E4 age effect was analyzed with non-parametric Kruskal–Wallis test followed by Dunn’s post-test. Genotype effect was analyzed by two-way ANOVA followed by post hoc Sidak’s correction (simple effects within age). * p < 0.05. HC: hippocampus.
Figure 4
Figure 4
ApoE and astrogliosis. (A–R). Hemibrain sections from APPPS1:E4 and APPPS1:E3 mice were stained with Amylo-Glo for fibrillar Aβ, anti-apoE mAb for apoE, and anti-GFAP mAb for astrocytes. Representative microphotographs of (A) hippocampus (HC; left panel) and (B) frontal cortex (FC; right panel). Scale bar: 100 μM. (C,D) Quantification of apoE immunoreactivity in hippocampus. (C) With sexes pooled, analysis of main effect of age in APPPS1:E4 mice (n = 6–11 mice/group). (D) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–9 mice/group). (E,F) Quantification of apoE immunoreactivity in frontal cortex. (E) Analysis of main effect of age in APPPS1:E4 mice (n = 1–3 mice/group). (F) Analysis of genotype in 16-month APPPS1:E4 and APPPS1:E3 mice (n = 3–6 mice/group). (G,H) Quantification of Amylo-Glo and apoE colocalization in hippocampus, normalized by positive staining of Amylo-Glo. (G) With sexes pooled, analysis of main effect of age in APPPS1:E4 mice (n = 6–11 mice/group). (H) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–9 mice/group). (I,J) Quantification of Amylo-Glo and apoE colocalization in frontal cortex, normalized by immunoreactivity of Amylo-Glo. (I) With sexes pooled, analysis of main effect of age in APPPS1:E4 mice (n = 6–7 mice/group). (J) Analysis of main effect of genotype in 8- and 16-month-old APPPS1:E4 and APPPS1:E3 mice (n = 3–7 mice/group). (K,L) Quantification of GFAP immunoreactivity in hippocampus. (K) With sexes pooled, analysis of main effect of age in APPPS1:E4 mice (n = 5–9 mice/group). (L) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–9 mice/group). (M,N) Quantification of GFAP immunoreactivity in frontal cortex. (M) Analysis of main effect of age in APPPS1:E4 mice (n = 2–5 mice/group). (N) Analysis of main effect of genotype in 8- and 16-month APPPS1:E4 and APPPS1:E3 mice (n = 3–7 mice/group). (O) Quantification of Amylo-Glo and GFAP colocalization in hippocampus, normalized by positive staining of Amylo-Glo. Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–9 mice/group). (P) Quantification of Amylo-Glo and GFAP colocalization in frontal cortex, normalized by immunoreactivity of Amylo-Glo. Analysis of main effect of genotype in 8- and 16-month APPPS1:E4 and APPPS1:E3 mice (n = 3–7 mice/group). (Q) Quantification of apoE and GFAP colocalization in hippocampus, normalized by immunoreactivity of GFAP. Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–9 mice/group). (R) Quantification of apoE and GFAP colocalization in frontal cortex, normalized by immunoreactivity of GFAP. Analysis of main effect of genotype in 8- and 16-month APPPS1:E4 and APPPS1:E3 mice (n = 3–7 mice/group). (S,T) Biochemical quantification of apoE levels following Liao et al.’s 2015 protocol on guanidine hydrochloride-extracted whole-hemibrain homogenates from APPPS1:E4 and APPPS1:E3 mice. (S) Analysis of main effect of age in APPPS1:E4 mice (n = 7–11 mice/group). (T) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 3–11 mice/group). Data are presented as mean ± SEM. Colocalization data are presented in arbitrary units. APPPS1:E4 age effect was analyzed by one-way ANOVA followed by post hoc Tukey’s analysis or two-way ANOVA followed by Tukey’s HSD post-test (simple effects within sex). With sexes pooled, genotype effect was analyzed by two-way ANOVA followed by post hoc Sidak’s correction (simple effects within age) or pair-wise comparisons with Alternative Welch’s t-test. Due to low n-value, frontal cortex data were not evaluated for 12-month-old APPPS1:E3 mice. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. HC: hippocampus; FC: frontal cortex.
Figure 5
Figure 5
Inflammatory biomarkers in mouse plasma. (A–D) Quantification of IL-1β levels. (A) Analysis of main effect of age in APPPS1:E4 mice (n = 6–11 mice/group). Analysis of genotype in APPPS1:E4 and APPPS1:E3 mice at (B) 8 months (n = 4–5 mice/group), (C) 12 months (n = 2–6 mice/group), and (D) 16 months (n = 3–11 mice/group). (E–H) Quantification of IL-6 levels. (E) Analysis of main effect of age in APPPS1:E4 mice (n = 6–11 mice/group). Analysis of genotype in APPPS1:E4 and APPPS1:E3 mice at (F) 8 months (n = 4–5 mice/group), (G) 12 months (n = 2–6 mice/group), and (H) 16 months (n = 3–11 mice/group). (I,J) Quantification of KC/GRO (IL-8-related protein in rodents) levels. (I) Analysis of main effect of age in APPPS1:E4 mice (n = 6–11 mice/group). (J) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 2–11 mice/group). (K,L). Quantification of TNF-α levels. (K) Analysis of main effect of age in APPPS1:E4 mice (n = 6–11 mice/group). (L) Analysis of main effect of genotype in APPPS1:E4 and APPPS1:E3 mice (n = 2–11 mice/group). Data are presented as mean ± SEM. With sexes pooled, APPPS1:E4 age effect was analyzed with one-way ANOVA followed by post hoc Tukey’s correction or non-parametric ANOVA (Kruskal–Wallis) followed by Dunn’s post-test. With sexes pooled, genotype effect was analyzed by two-way ANOVA followed by post hoc Sidak’s correction (simple effects within age) or pair-wise comparisons with Alternative Welch’s t-test. * p < 0.05; ** p < 0.01.
Figure 6
Figure 6
The microglial response in the hippocampus and prefrontal cortex of APPPS1:E4 and APPPS1:E3 mice throughout aging. (A) IBA1, CD68, and Amylo-Glo staining in the hippocampal regions of APPPS1:E4 and APPPS1:E3 mice at 8 months of age. Scale bar: 100 μM. (B–E) Phagocytic microglia in the hippocampus (B) and frontal cortex (D) of APPPS1:E4 and APPPS1:E3 mice at 8 months of age were quantified by the % colocalization area of CD68 and IBA1 staining. Plaque-associated lysosomal activity in the hippocampus (C) and frontal cortex (E) of APPPS1:E4 and APPPS1:E3 mice at 8 months of age was quantified by the % colocalization area of CD68 and Amylo-Glo staining (n = 4–9 mice/genotype group). (F) IBA1, CD68, and Amylo-Glo staining in the frontal cortex of APPPS1:E4 and APPPS1:E3 mice at 8 months of age. Scale bar: 100 μM. (G) IBA1, CD68, and Amylo-Glo staining in the hippocampus and frontal cortex of APPPS1:E4 mice at 12 and 16 months of age. Scale bar: 100 μM. (H–K) The quantification of phagocytic microgliosis (H,I) and plaque-associated lysosomal activity (J,K) in the hippocampus (H,J) and frontal cortex (I,K) of APPPS1:E4 mice, indicating an aging effect. (n = 7–11 mice/age group). Overall, a significant genotype effect is only observed at 8 months of age, while the microglia of APPPS1:E4 mice had an impaired response to plaque deposition during aging. Data are presented as mean ± SEM. Colocalization data are presented in arbitrary units. The genotype effect was analyzed by Alternative Welch’s test. The age effect was analyzed by one-way ANOVA followed by post hoc Tukey’s correction. * p < 0.05; ** p < 0.01. HC: hippocampus; FC: frontal cortex; AMG: Amylo-Glo.
Figure 7
Figure 7
Lysosomal dysfunction and neuritic dystrophy in the hippocampus and frontal cortex of APPPS1:E4 and APPPS1:E3 mice. (A) SMI-31, LAMP1, and Amylo-Glo staining in the hippocampus and frontal cortex of APPPS1:E4 mice and the frontal cortex of APPPS1:E3 mice at 8 months of age (left panel), at 12 months of age (middle panel), and at 16 months of age (right panel). Scale bar: 100 μM. (B–D) Lysosomal accumulation in the hippocampus (B) and frontal cortex (C,D) of APPPS1:E4 and APPPS1:E3 mice was quantified by the % area of LAMP1+ immunoreactivity and indicated an effect of aging in APPPS1:E4 mice (B,C) (n = 5–8 mice/age group) and a genotype effect in the frontal cortex of APPPS1:E4 and APPPS1:E3 mice at the three different age points (D) (n = 2–8 mice/genotype group). (E,F) Plaque-associated LAMP1+ immunoreactivity in the hippocampus (E) and frontal cortex (F) of APPPS1:E4 mice was quantified by the % colocalization area of LAMP1 and Amylo-Glo staining and indicated an effect of aging in APPPS1:E4 mice (E,F) (n = 5–8 mice/age group). (G,H) Proteasomal dysfunction in neurites in the hippocampus (G) and frontal cortex (H) of APPPS1:E4 mice was quantified by the % colocalization area of LAMP1 and SMI31 staining and indicated an aging effect in both brain areas (G,H) (n = 5–8 mice/age group). (I–K) The accumulation of dystrophic neurites in the hippocampus (I) and frontal cortex (J,K) of APPPS1:E4 and APPPS1:E3 mice was quantified by the % area of SMI31+ immunoreactivity and indicated an effect of aging in APPPS1:E4 mice (I,J) (n =5–8 mice/age group) and a genotype effect in the frontal cortex of APPPS1:E4 and APPPS1:E3 mice at the three different age points (K) (n = 2–8 mice/genotype group). (L,M) Plaque-associated neuritic dystrophy was quantified by the % colocalization area of SMI-31 and Amylo-Glo staining and showed an effect of age in the hippocampus (I) and frontal cortex (J) in APPPS1:E4 mice (n = 5–8 mice/age group). Data are presented as mean ± SEM. Colocalization data are presented in arbitrary units. The age effect was analyzed by one-way ANOVA followed by Tukey’s post hoc correction or by Kruskal–Wallis followed by Dunn’s post hoc correction. The genotype effect was analyzed by two-way ANOVA followed by post hoc Sidak’s correction or by Welch’s test. * p < 0.05; ** p < 0. 01. HC: hippocampus; FC: frontal cortex; AMG: Amylo-Glo.
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