Characterization of AD-like phenotype in aged APPSwe/PS1dE9 mice

Abstract

Transgenic APPSwe/PS1dE9 (APP/PS1) mice that overproduce amyloid beta (Aβ) are extensively used in the studies of pathogenesis and experimental therapeutics and new drug screening for Alzheimer's disease (AD). However, most of the current literature uses young or adult APP/PS1 mice. In order to provide a broader view of AD-like phenotype of this animal model, in this study, we systematically analyzed behavioral and pathological profiles of 24-month-old male APP/PS1 mice. Aged APP/PS1 mice had reference memory deficits as well as anxiety, hyperactivity, and social interaction impairment. Consistently, there was obvious deposition of amyloid plaques in the dorsal hippocampus with decreased expression of insulin-degrading enzyme, a proteolytic enzyme responsible for degradation of intracellular Aβ. Furthermore, decreases in hippocampal volume, neuronal number and synaptophysin expression, and astrocyte atrophy were also observed in aged APP/PS1 mice. This finding suggests that aged APP/PS1 mice can well replicate cognitive and noncognitive behavioral abnormalities, hippocampal atrophy, and neuronal and astrocyte degeneration in AD patients, to enable more objective and refined preclinical evaluation of therapeutic drugs and strategies for AD treatment.

Keywords: APP/PS1 mice; Aged; Alzheimer’s disease; Hippocampal atrophy; Noncognitive abnormalities; β-amyloid.

Fig. 1

Y-maze test. a Time spent in the novel arm (NA). b The number of entries into the NA. c The total traveling distance during the test. d The mean traveling speed. Data represent means ± SEM from nine mice per group. *P < 0.05 vs. WT mice

Fig. 2

The open field test. a Time spent in the center area. b The number of entries into the center area. c The total traveling distance during the test. d The mean traveling speed. Data represent means ± SEM from nine mice per group. *P < 0.05 vs. WT mice

Fig. 3

The elevated plus maze test. a Time spent in the open arm. b The number of entries into open arm. c The total traveling distance during the test. d The mean traveling speed. Data represent means ± SEM from nine mice per group. *P < 0.05 vs. WT mice

Fig. 4

Social interaction test and social memory test. a Time spent in each chamber in social interaction test. b Time spent exploring novel mice or empty cups. c Time spent in each chamber during the social memory test. d Time spent in exploring novel mouse 1 (stranger 1) or novel mouse 2 (stranger 2). Data represent means ± SEM from nine mice per group. *P < 0.05 vs. WT mice; ^# P < 0.05 vs. empty chamber. ^& P < 0.05 vs. novel mouse 1

Fig. 5

Analyses of hippocampal volume, neuronal number and synaptic protein expression. a Series of Nissl-stained sagittal brain sections showing that the cross area of the dorsal hippocampus was smaller in 24-M aged APP/PS1 mice than WT controls. Note from no. 275 section (about 1.66 mm lateral to the mid-sagittal fissure), the intermediate region of the hippocampus, a transitional zone of the dorsal hippocampal region, and ventral hippocampal region, was observed in APP/PS1 mice but was not present in WT mice until at no. 305 section. Scale bar = 500 μm. b Counting the total number of sections that contain the dorsal hippocampus. c Quantification of the volume of the dorsal hippocampus. d Relative volume of the pyramidal (Py) and granular (Gr) layers (gray matter) and other layers (white matter) between APP/PS1 mice and WT controls. e Representative microimages of neuron-specific nuclear protein (NeuN) immunopositive neurons in the hippocampus CA1 of APP/PS1 mice and WT controls. f Unbiased stereological counting of NeuN-positive neurons in the hippocampus of the two genotypes. g Representative microimages of synaptophysin (SYP) immunoreactivity in the hippocampus of APP/PS1 mice and WT controls. h The quantification analysis of the mean integrated optical density (MIOD) of SYP. i, j Western blotting and densitometry analysis of SYP expression in the hippocampus of the two genotypes. Data represent means ± SEM. Five mice per group for pathological analyses and three mice per group in three independent Western blotting analysis experiments. *P < 0.05 vs. WT controls; ^# P < 0.05 vs. other hippocampal layers

Fig. 6

Analyses of amyloid plaque load and expression of Aβ metabolism enzymes in the hippocampus. a, b Brain sections from 24-M aged APP/PS1 mice and WT controls were stained with thioflavin-S (a) or immunostained with anti-6E10 antibody (b) to label plaques. Few plaques were present in the pyramidal (Py) layer and granular (Gr) layer. c Percentage of brain area occupied by thioflavin-S and 6E10-labeled Aβ deposition in the hippocampus. d Representative microimages of immunostaining for α-secretase (ADAM10), β-secretase (BACE1), γ-secretase (PS1), neprilysin (NEP), and insulin-degrading enzyme (IDE). High immunoreactivity of ADAM10, NEP, and IDE was observed in the Py layer, while expression of BACE1 and PS1 was relatively low. In contrast, in the oriens (Or), radiatum (Rad), and lacunosum moleculare (Lmol) layers, high expression of BACE1 and PS1, but low expression of ADAM10, NEP, and IDE. e The quantification analysis of the mean integrated optical density (MIOD) of ADAM10, BACE1, PS1, NEP, and IDE in the Py plus Gr layers and other hippocampal layers. f, g Western blotting and densitometry analysis of ADAM10, BACE1, PS1, NEP, and IDE expression in the hippocampus of the two genotypes. Data represent means ± SEM from five mice per group for pathological analyses and from three mice per group in three independent Western blotting analysis experiments. *P < 0.05 vs. Py+Gr (Fig. 6c) or WT mice (Fig. 6e and 6g)

Fig. 7

Analyses of reactive gliosis in the hippocampus. a A considerable proportion of GFAP-positive astrocytes exhibited an activated shape (arrow), characterized by larger cell bodies in the hippocampus of 24-month-old WT mice. In contrast, dense GFAP immunoreactive products, lacking the normal morphology of astrocytes, were around Aβ plaques in age-matched APP/PS1 mice. Some astrocytes distant to the Aβ plaques only had few, short processes (arrowhead), indicating occurrence of atrophy. Activated Iba-1-postive microglia with long processes (arrow) were present in both APP/PS1 mice, although those around Aβ plaques became an amoeba-like shape and process disappearance (arrowhead). Activated microglia were also frequently observed in the hippocampus of aged WT mice. b Cell counts of GFAP-positive astrocytes and Iba-1-positive microglia. c Cellular surface of GFAP-positive astrocytes and Iba-1-positive microglia. Data represent mean ± SEM from five mice per group. *P < 0.05 vs. WT controls

Fig. 8

Analysis of BDNF expression in the hippocampus. a Representative microimages of BDNF immunoreactivity in the hippocampus of APP/PS1 mice and WT controls. b The quantification analysis of the mean integrated optical density (MIOD) of BDNF. c, d Western blotting and densitometry analysis of BDNF expression in the hippocampus of the two genotypes. Data represent means ± SEM from five mice per group for pathological analyses and from three mice per group in three independent Western blotting analysis experiments. *P < 0.05 vs. WT controls