Sex- and brain region-specific acceleration of β-amyloidogenesis following behavioral stress in a mouse model of Alzheimer's disease

Abstract

Background: It is hypothesized that complex interactions between multiple environmental factors and genetic factors are implicated in sporadic Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. Importantly, recent evidence reveals that expression and activity levels of the β-site APP cleaving enzyme 1 (BACE1), which initiates amyloid-β (Aβ) production, are elevated in AD brains. In this study, we investigated a molecular mechanism by which sex and stress interactions may accelerate β-amyloidogenesis and contribute to sporadic AD.

Results: We applied 5-day restraint stress (6 h/day) to the male and female 5XFAD transgenic mouse model of AD at the pre-pathological stage of disease, which showed little amyloid deposition under non-stressed control conditions. Exposure to the relatively brief behavioral stress increased levels of neurotoxic Aβ42 peptides, the β-secretase-cleaved C-terminal fragment (C99) and plaque burden in the hippocampus of female 5XFAD mice but not in that of male 5XFAD mice. In contrast, significant changes in the parameters of β-amyloidosis were not observed in the cerebral cortex of stressed male or female 5XFAD mice. We found that this sex- and brain region-specific acceleration of β-amyloidosis was accounted for by elevations in BACE1 and APP levels in response to adverse stress. Furthermore, not only BACE1 mRNA but also phosphorylation of the translation initiation factor eIF2α (a proposed mediator of the post-transcriptional upregulation of BACE1) was elevated in the hippocampus of stressed female 5XFAD mice.

Conclusions: Our results suggest that the higher prevalence of sporadic AD in women may be attributable to the vulnerability of female brains (especially, the hippocampus) to stressful events, which alter APP processing to favor the β-amyloidogenesis through the transcriptional and translational upregulation of BACE1 combined with elevations in its substrate APP.

Figure 1

Effects of behavioral stress on Aβ42 levels in the hippocampus and cerebral cortex of 5XFAD mice. Male and female mice were exposed to restraint stress for 6 h per day during 5 consecutive days and were sacrificed for analysis 24 h after the last stress treatment. Levels of total Aβ42 were quantified by sandwich ELISA of guanidine extracts of hippocampal and cortical samples and expressed in nanograms per milligram of total protein (n = 5-8 mice per group). Note that Aβ42 levels are increased specifically in the hippocampus of stressed female 5XFAD mice as compared with non-stressed controls (*p < 0.05). All data are presented as mean ± SEM.

Figure 2

Effects of behavioral stress on Aβ load in the hippocampus of 5XFAD mice. Mice were exposed to restraint stress for 6 h per day during 5 consecutive days and were sacrificed for analysis 24 h after the last stress treatment. Brain sections from male (A, B) and female (D, E) 5XFAD mice were immunostained with the 6E10 anti-Aβ antibody. Shown are representative photomicrographs of the hippocampus of 5XFAD mice with (B, E) or without (A, D) stress treatments. Scale bar = 500 μm. Percentage area occupied by amyloid deposits in the hippocampus of male (C) and female (F) 5XFAD mice was measured for quantification (n = 6-7 mice per group). Note that hippocampal plaque burden is significantly increased in stressed female but not male 5XFAD mice as compared with non-stressed controls (* p < 0.05). All data are presented as mean ± SEM.

Figure 3

Effects of behavioral stress on APP processing in the hippocampus of 5XFAD mice. Mice were exposed to restraint stress for 6 h per day during 5 consecutive days and were sacrificed for analysis 24 h after the last stress treatment. Representative immunoblots of protein extracts from hippocampal homogenates of male (A) and female (B) 5XFAD mice with and without exposure to stress are shown along with those of non-stressed wild-type controls. Intensities of immunoreactive bands were quantified by phosphorimaging and were expressed as percentage of wild-type controls for BACE1 (C, D) or as percentage of non-stressed 5XFAD levels for full-length APP (E, F) and C99 (G, H). Data are presented as mean ± SEM for male (C, E, G) and female (D, F, H) subjects (n = 4-9 mice per group). ^#p < 0.05 vs. non-stressed wild-type controls, *p < 0.05 vs. non-stressed 5XFAD mice.

Figure 4

Effects of behavioral stress on phospho-eIF2α levels in the hippocampus and cerebral cortex of 5XFAD mice. Male and female mice were exposed to restraint stress for 6 h per day during 5 consecutive days and were sacrificed for analysis 24 h after the last stress treatment. Representative immunoblots of phosphorylated eIF2α (p-eIF2α) and total eIF2α for hippocampal homogenates of female 5XFAD mice with and without exposure to stress are shown along with those of non-stressed wild-type controls. Intensities of p-eIF2α-immunoreactive bands were quantified by phosphorimaging and were expressed as percentage of wild-type controls (n = 3-9 mice per group). Note that p-eIF2α levels, but not total eIF2α levels, are increased specifically in the hippocampus of stressed female 5XFAD mice as compared with non-stressed wild-type controls (^#p < 0.05) and non-stressed 5XFAD mice (*p < 0.05). All data are presented as mean ± SEM.

Figure 5

Effects of behavioral stress on BACE1 mRNA levels in the hippocampus of female 5XFAD mice. Mice were exposed to restraint stress for 6 h per day during 5 consecutive days and were sacrificed for real-time qPCR analysis 24 h after the last stress treatment. BACE1 mRNA levels were significantly elevated in the hippocampus of stressed female 5XFAD mice as compared with that of non-stressed 5XFAD controls (*p < 0.05) (n = 4 mice per group). All data are presented as mean ± SEM.