Molecular Differences and Similarities Between Alzheimer's Disease and the 5XFAD Transgenic Mouse Model of Amyloidosis

Abstract

Transgenic (Tg) mouse models of Alzheimer's disease (AD) have been extensively used to study the pathophysiology of this dementia and to test the efficacy of drugs to treat AD. The 5XFAD Tg mouse, which contains two presenilin-1 and three amyloid precursor protein (APP) mutations, was designed to rapidly recapitulate a portion of the pathologic alterations present in human AD. APP and its proteolytic peptides, as well as apolipoprotein E and endogenous mouse tau, were investigated in the 5XFAD mice at 3 months, 6 months, and 9 months. AD and nondemented subjects were used as a frame of reference. APP, amyloid-beta (Aβ) peptides, APP C-terminal fragments (CT99, CT83, AICD), β-site APP-cleaving enzyme, and APLP1 substantially increased with age in the brains of 5XFAD mice. Endogenous mouse tau did not show age-related differences. The rapid synthesis of Aβ and its impact on neuronal loss and neuroinflammation make the 5XFAD mice a desirable paradigm to model AD.

Keywords: AICD; Alzheimer’s disease; BACE1; amyloid precursor protein; presenilin; tau; transgenic mice.

Figure 1

Levels of soluble and insoluble human Aβ as quantified by ELISA in 3-month-old, 6-month-old, and 9-month-old 5XFAD Tg mice. Notes: (A) Tris-soluble Aβ40 in pg/mg total protein. (B) Tris-soluble Aβ42 in pg/mg total protein. (C) GHCl-soluble Aβ40 in ng/mg total protein. (D) GHCl-soluble Aβ42 in ng/mg total protein. (E) Total Aβ (Tris-soluble and GHCl-soluble Aβ40 + Aβ42) in ng/mg total protein. Statistical analysis was performed using a Kruskal–Wallis test followed by Dunn’s multiple comparison test (*P = 0.05–0.01; **P = 0.01–0.001). The horizontal bars represent the mean of the four cases from each age group. For further details see Table 2. Formulations: Tris = 20 mM Tris-HCl, 5 mM EDTA, pH 7.8 buffer; GHCl = 5 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.0 buffer. Abbreviations: f, female; m, male; Aβ, amyloid-beta; ELISA, enzyme-linked immunosorbent assay; FAD, familial Alzheimer’s disease; GHCI, guanidine hydrochloride; EDTA, ethylenediaminetraacetic acid.

Figure 2

Western blot analysis of male 5XFAD Tg mice at 3 months, 6 months, and 9 months of age, as well as human SAD and NDC gray matter from the frontal cortex. Notes: (A) The mature form of BACE1 shown at ~70 kDa. (B) Full-length presenilin-1 (~55 kDa). (C) Full-length APP as detected by 22C11. (D) Full-length APLP1. (E) The adaptor protein Fe65. (F) CT99 and CT83 of APP as detected by mCT20APP. SE and LE are both shown. The putative AICD (denoted by an arrow) can be detected in older mice by antigen retrieval and by greatly increasing the antibody concentration and total protein per lane as detected with mCT20APP (G) and pCT20APP (H) antibodies. A purified CT–APP peptide of 57 amino acids (rPeptide, Athens, GA, USA) is shown on the right side of the blots in (G) and (H). (I) Full-length ApoE is observed at ~34 kDa. All blots were stripped and reprobed with actin (shown below each primary antibody) as quantitative protein loading control. A total of 40 μg of total protein was loaded into each lane for (A–F) and (I), while 65 μg of total protein was loaded into each lane for (G) and (H). For antibody details, see Table 1. The molecular weight markers are shown to the left of each blot and are reported in kDa. For further details, see Table 3. Abbreviations: BACE, β-site APP-cleaving enzyme; APLP, amyloid-precursor-like protein; CT, C-terminal; APP, amyloid precursor protein; ApoE, apolipoprotein E; SAD, sporadic Alzheimer’s disease; NDC, nondemented controls; m, months; SE, short exposure; LE, long exposure.

Figure 3

ELISA quantification of endogenous mouse tau at 3 months, 6 months, and 9 months of age. Notes: (A) Tris-soluble tau in ng/mg total protein. (B) GHCl-soluble tau in ng/mg total protein. (C) Total tau (Tris + GHCl soluble fractions) in ng/mg total protein. Statistical analysis was performed using a Kruskal–Wallis test followed by Dunn’s multiple comparison test. The horizontal bars represent the mean of the 4 cases from each age group. For further details see Table 2. Formulations: Tris = 20 mM Tris-HCl, 5 mM EDTA, pH 7.8 buffer; GHCl = 5 M guanidine hydrochloride 50 mM Tris-HCl, pH 8.0 buffer. Abbreviations: NS, not significant; f, female; m, male; ELISA, enzyme-linked immunosorbent assay; GHCI, guadinine hydrochloride; EDTA, ethylenediaminetraacetic acid.