Sex differences in β-amyloid accumulation in 3xTg-AD mice: role of neonatal sex steroid hormone exposure

Abstract

The risk of Alzheimer's disease (AD) is higher in women than in men, a sex difference that likely results from the effects of sex steroid hormones. To investigate this relationship, we first compared progression of β-amyloid (Aβ) pathology in male and female triple transgenic (3xTg-AD) mice. We found that female 3xTg-AD mice exhibit significantly greater Aβ burden and larger behavioral deficits than age-matched males. Next, we evaluated how the organizational effects of sex steroid hormones during postnatal development may affect adult vulnerability to Aβ pathology. We observed that male 3xTg-AD mice demasculinized during early development exhibit significantly increased Aβ accumulation in adulthood. In contrast, female mice defeminized during early development exhibit a more male-like pattern of Aβ pathology in adulthood. Taken together, these results demonstrate significant sex differences in pathology in 3xTg-AD mice and suggest that these differences may be mediated by organizational actions of sex steroid hormones during development.

Figure 1

Female 3xTg-AD mice exhibit higher levels of Aβ than males. Representative images show Aβ immunoreactivity from 12–14 mo female (A, D, G) and male (B, E, H) 3xTg-AD mice in the subiculum (A,B), hippocampus CA1 (D,E), and frontal cortex (G,H). Scale bar = 100μm. Quantification of Aβ immunoreactive load shows significant differences between females (solid bars) and males (open bars) in the subiculum (C) [F (7,46) = 23.72, p < 0.0001], hippocampus CA1 (F) [F (7,46) = 5.88, p = 0.0005], and frontal cortex (I) [F (7,46) = 9.76, p <0.0001]. Representative low magnification images of Aβ immunoreactivity in hippocampus and subiculum of 12–14 mo female (J) and male (K) 3xTg-AD mice show significantly higher numbers of Aβ plaques in females (L) [F (8,43) = 9.25, p < 0.0001]. Scale bar = 250μm. Data show mean values ± SEM. * p < 0.01 relative to males of same age group.

Figure 2

No difference in levels of amyloid precursor protein (APP) C-terminal fragments (CTFs) were observed between male and female 3xTg-AD mice. Representative images show CTF immunoreactivity (CT20 antibody) in the subiculum of male and female non-transgenic C57Bl6/129S and 3xTg-AD mice. Non-transgenic mice (A–E) show low levels of CTF-immunoreactivity while both male (F–H) and female (B–D) mice show similar, high levels of CTF- immunoreactivity. Scale bar = 100μm.

Figure 3

Female 3xTg-AD mice perform more poorly than males on spontaneous alternation behavior (SAB), a hippocampal-dependent working memory task. (A) SAB performance did not decline with age or significantly differ by sex in non-transgenic C57Bl6/129S (WT) mice. (B) We observed an age-related decline in SAB performance in both females (solid bars) [F (3,20) = 0.87, p = 0.002] and males (open bars) [F (3, 7) = 0.67, p = 0.031]. This effect is significantly different between females and males in the 12–14 mo age group [F (3,19) = 6.58, p = 0.0001]. * p < 0.05 from males of same age group. (C) As a control for activity level, the number of arm entries was measured; it was not significantly different across groups [F (3,19) = 0.79, p = 0.56]. Data show mean values ± SEM.

Figure 4

Neonatal flutamide treatment demasculinizes male 3xTg-AD adult brains and TP treatment defeminizes female brains in terms of Aβ pathology in a region-specific manner. Representative photomicrographs display Aβ immunoreactivity visualized in the subiculum (AD), CA1 of hippocampus (F–I), and frontal cortex (K-N). Scale bar = 100μm. Significant treatment differences were observed in quantification of Aβ immunohistochemistry load in the subiculum (E) [F (4,32) = 2.82, p = 0.05], the CA1 (J) [F (4,32) = 17.11, p < 0.0001] and the frontal cortex (O) [F (4,32) = 13.01, p < 0.0001]. Data are represented as mean values ± SEM. * p < 0.05 from opposite sex of same age group.

Figure 5

Neonatal flutamide treatment, but not TP treatment alters hippocampal-dependent behavior on the Y-maze. Neonatally-hormone treated mice exhibited a significant overall treatment effect on Y-maze performance (A) [F (3,30) = 6.37, p = 0.003]. The number of arm entries was not significantly different across groups (B). Data are represented as mean values ± SEM. * p < 0.05 from opposite sex of same age group.