Biochemical and morphological characterization of the AβPP/PS/tau triple transgenic mouse model and its relevance to sporadic Alzheimer's disease

Abstract

Transgenic (Tg) mouse models of Alzheimer's disease (AD) have been genetically altered with human familial AD genes driven by powerful promoters. However, a Tg model must accurately mirror the pathogenesis of the human disease, not merely the signature amyloid and/or tau pathology, as such hallmarks can arise via multiple convergent or even by pathogenic mechanisms unrelated to human sporadic AD. The 3 × Tg-AD mouse simultaneously expresses 3 rare familial mutant genes that in humans independently produce devastating amyloid-β protein precursor (AβPP), presenilin-1, and frontotemporal dementias; hence, technically speaking, these mice are not a model of sporadic AD, but are informative in assessing co-evolving amyloid and tau pathologies. While end-stage amyloid and tau pathologies in 3 × Tg-AD mice are similar to those observed in sporadic AD, the pathophysiological mechanisms leading to these lesions are quite different. Comprehensive biochemical and morphological characterizations are important to gauge the predictive value of Tg mice. Investigation of AβPP, amyloid-β (Aβ), and tau in the 3 × Tg-AD model demonstrates AD-like pathology with some key differences compared to human sporadic AD. The biochemical dissection of AβPP reveals different cleavage patterns of the C-terminus of AβPP when compared to human AD, suggesting divergent pathogenic mechanisms. Human tau is concomitantly expressed with AβPP/Aβ from an early age while abundant extracellular amyloid plaques and paired helical filaments are manifested from 18 months on. Understanding the strengths and limitations of Tg mouse AD models through rigorous biochemical, pathological, and functional analyses will facilitate the derivation of models that better approximate human sporadic AD.

Fig. 1

Histological assessment of 6 month-old 3×Tg-AD mice. Coronal mouse brain sections (30 μm) were prepared from 6 month-old male 3×Tg-AD mice (n = 4). Histochemistry/IHC was performed to detect the following: human AβPP A4 using the Y188 monoclonal antibody (A, D), extracellular Aβ₁-₄₂ accumulation using the 12F4 monoclonal antibody (B, E), intracellular Aβ₁-₄₂ accumulation by microwave and buffer retrieval using polyclonal anti Aβ₁-₄₂ antibody (C, F), amyloid fibrils using Congo red staining (G, K) and polarized light for birefringence (J), human tau^{P301 L} transgene using the HT7 monoclonal antibody (H, L), and paired helical filaments using the PHF-1 monoclonal antibody (I, M). Photomicrographs in panels A, B, C, G, H, and I were obtained at 10×, where scale bar in I represents 200 μm. Photomicrographs in panels D, E, F, J, K, L, and M were obtained at 100×, where scale bar in M represents 10 μm.

Fig. 2

Histological assessment of 12 month-old 3×Tg-AD mice. Coronal mouse brain sections (30 μm) were prepared from 12 month-old male 3×Tg-AD mice (n = 4). Histochemistry/IHC was performed to detect the following: human AβPP A4 using the Y188 monoclonal antibody (A, D), extracellular Aβ₁-₄₂ accumulation using the 12F4 monoclonal antibody (B, E), intracellular Aβ₁-₄₂ accumulation by microwave and buffer retrieval using polyclonal anti Aβ₁-₄₂ antibody (C, F), amyloid fibrils using Congo red staining (G, K) and polarized light for birefringence (J), human tau^{P301 L} transgene using the HT7 monoclonal antibody (H, L), and paired helical filaments using the PHF-1 monoclonal antibody (I, M). Photomicrographs in panels A, B, C, G, H, and I were obtained at 10×, where scale bar in I represents 200 μm. Photomicrographs in panels D, E, F, J, K, L, and M were obtained at 100×, where scale bar in M represents 10 μm.

Fig. 3

Histological assessment of 18 month-old 3×Tg-AD mice. Coronal mouse brain sections (30 μm) were prepared from 18 month-old male 3×Tg-AD mice (n = 4). Histochemistry/IHC was performed to detect the following: human AβPP A4 using the Y188 monoclonal antibody (A, D), extracellular Aβ₁-₄₂ accumulation using the 12F4 monoclonal antibody (B, E), intracellular Aβ₁-₄₂ accumulation by microwave and buffer retrieval using polyclonal anti Aβ₁-₄₂ antibody (C, F), amyloid fibrils using Congo red staining (G, K) and polarized light for birefringence (J), human tau^{P301 L} transgene using the HT7 monoclonal antibody (H, L), and paired helical filaments using the PHF-1 monoclonal antibody (I, M). Photomicrographs in panels A, B, C, G, H, and I were obtained at 10 ×, where scale bar in I represents 200 μm. Photomicrographs in panels D, E, F, J, K, L, and M were obtained at 100 ×, where scale bar in M represents 10 μm.

Fig. 4

Scatter plots of Aβ, tau, and p-tau ELISAs in 3×Tg-AD mice. Levels of Aβ₄₀ (A) and Aβ₄₂ (B) extracted with Tris-buffer. Quantification of Aβ₄₀ (C) and Aβ₄₂ (D) after extraction with GHCl. Total human tau (E) and p-tau (F) were determined by ELISA as well. Each data point corresponds to one mouse. mo = months; GHCl = guanidine hydrochloride; M = male; F = female p-tau (S396) = phosphorylated tau at serine 396.

Fig. 5

Western blot analysis of 3×Tg-AD mice at 7, 12–16, and 18 months, 3 human AD and 3 human NDC cases using RIPA homogenates of gray matter. All blots were re-probed for β-actin as a loading control. A) 22C11 is an antibody against amino acids 66–81 of AβPP. 2) CT9AβPP detects the last 9 amino acids of AβPP. C) Tau (HT7 clone) recognized amino acids 159–163 of human tau. mo = months; Tg = transgenic; wt = wild-type; AD = Alzheimer’s disease; NDC = non-demented control.

Fig. 6

HPLC and Western blots of 7 month-old 3×Tg-AD mice. Fractions 1, 2, and 3 from the FPLC were each run on a C8 column developed with 0–60% water/acetonitrile containing 0.1% TFA. A total of 8 fractions were collected and submitted to WB analysis using antibodies against Aβ₄₀ (A), Aβ₄₂ (B), CT9AβPP (C) and tau (D). Lanes 9 in the Aβ₄₀ (A) and Aβ₄₂ (B) WB contain a purified peptide as a standard. HPLC = high performance liquid chromatography; FPLC = fast protein liquid chromatography; min = minutes.

Fig. 7

HPLC and Western blots of 12 to 16 month-old 3×Tg-AD mice. Fractions 1, 2, and 3 from the FPLC were each run on a C8 column developed with 0–60% water/acetonitrile containing 0.1% TFA. A total of 8 fractions were collected and submitted to WB analysis using antibodies against Aβ₄₀ (A), Aβ₄₂ (B), CT9AβPP (C), and tau (D). Lanes 9 in the Aβ₄₀ (A) and Aβ₄₂ (B) WB contain a purified peptide as a standard. HPLC = high performance liquid chromatography; FPLC = fast protein liquid chromatography; min = minutes.

Fig. 8

HPLC and Western blots of 18 month-old 3×Tg-AD mice. Fractions 1, 2, and 3 from the FPLC were each run on a C8 column developed with 0–60% water/acetonitrile containing 0.1% TFA. A total of 8 fractions were collected and submitted to WB analysis using antibodies against Aβ₄₀ (A), Aβ₄₂ (B), CT9AβPP (C), and tau (D). Lanes 9 in the Aβ₄₀ (A) and Aβ₄₂ (B) WB contain a purified peptide as a standard. HPLC = high performance liquid chromatography; FPLC = fast protein liquid chromatography; min = minutes.