ERβ mediates sex-specific protection in the App-NL-G-F mouse model of Alzheimer's disease

Abstract

Background: Menopausal loss of neuroprotective estrogen is thought to contribute to the sex differences in Alzheimer's disease (AD). Activation of estrogen receptor beta (ERβ) can be clinically relevant since it avoids the adverse systemic effects of ERα activation. However, very few studies have explored ERβ-mediated neuroprotection in AD, and no information on its contribution to the sex differences in AD exists. In the present study, we specifically explored the role of ERβ in mediating sex-specific protection against AD pathology in the App^NL-G-F knock-in mouse model of amyloidosis, and if surgical menopause (ovariectomy) modulates pathology in this model.

Methods: We treated male and female App^NL-G-F knock-in mice with the clinically relevant and selective ERβ agonist LY500307. A subset of the females was ovariectomized prior to treatment. Y-maze and contextual fear conditioning tests were used to assess memory performance, and biochemical assays such as qPCR, immunohistochemistry, Western blot, and multiplex immunoassays, were used to evaluate amyloid pathology.

Results: We found that Female App^NL-G-F mice had higher soluble Aβ levels in cortex and hippocampus than males and more activated microglia. ERβ activation protected against amyloid pathology and cognitive decline in both male and female App^NL-G-F mice. Although ovariectomy increased soluble amyloid beta (Aβ) in cortex and insoluble Aβ in hippocampus, as well as sustained neuroinflammation after ERβ activation, it had otherwise limited effects on pathology. We further identified that ERβ did not alter APP processing, but rather exerted its protection at least partly via microglia activation in a sex-specific manner.

Conclusion: Combined, we provide new understanding to the sex differences in AD by demonstrating that ERβ protects against AD pathology differently in males and females, warranting reassessment of ERβ in combating AD.

Keywords: APP knock-in; Alzheimer’s disease; Amyloidosis; Estrogen receptor beta; Microglia; Sex differences; Sex hormone.

Fig. 1

ERβ activation improves cognitive behavior in App^NL−G−F male and female mice. A Treatment regime of App^NL−G−F mice. B Representative image of Y-maze arena. C Percent Y-maze arm alterations and D total number of arm entries of male (left) and female (right) App^NL−G−F mice treated with vehicle or ERβ agonist LY500307 (LY) (n = 7–10). E Diagram showing the fear conditioning paradigm. F Percent context-associated freezing time of male (left) and female (right) App^NL−G−F mice (n = 6–9) in the contextual fear conditioning test. Cued-associated freezing time in the contextual fear conditioning test of G male and H female App^NL−G−F mice before cue (baseline) and upon cue (tone) in a different cage context (n = 6–9). Female mice were either ovariectomized (OVX) or sham operated (Sham). * P < 0.05, ** P < 0.01, *** P < 0.001. Unpaired t-test was used for males and 2-way ANOVA for females followed by uncorrected Fisher’s LSD test for multiple comparisons. Overall significant main effects of treatment or OVX are indicated

Fig. 2

Less Aβ pathology in App^NL−G−F male and female mice after ERβ activation. A Immunohistochemical representation of amyloid plaques in frontal and motor cortex (FT/M), somatosensory and visual cortex (Ss/Vis) and hippocampus (Hippoc) of male App^NL−G−F mice after vehicle or LY treatment. B Quantification of number of plaques per 100 µm² (n = 4–6) and C percent plaque area (n = 4–6) in male App^NL−G−F mice. D Similar as in A, immunohistochemical representation of amyloid plaques in different brain regions of female App^NL−G−F mice after vehicle or LY treatment. E Quantification of number of plaques per 100 µm² (n = 4–5) and F percent plaque area (n = 4–9) in female App^NL−G−F mice. G Linear regression analysis comparing effect size from LY treatment (vehicle vs. LY) on number of Aβ plaques in relation to average number of ERβ positive cells per 100 µm² in different brain regions of male and female mice (n = 4–6). H Soluble and (I) insoluble Aβ42 levels in male cortex (Ctx, left) and hippocampus (Hippoc, right) (n = 3–4). (J) Soluble and (K) insoluble Aβ₄₂ levels in female cortex (left) and hippocampus (right) (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired t-test was used for males and 2-way ANOVA for females followed by uncorrected Fisher’s LSD test for multiple comparisons. Overall significant main effects of treatment or OVX are indicated. Scale bars = 100 µm

Fig. 3

ERβ activation does not alter APP processing. Western blot analysis of full-length APP (FL-APP), β-CTF and α-CTF, Aβ peptide, and β-actin in A cortex and B hippocampus of female and male App^NL−G−F mice after vehicle (V) or LY treatment, as well as after sham surgery or ovariectomy (OVX in females). Quantification of C FL-APP relative to β-actin, D β-CTF relative to FL-APP, E β-CTF relative to actin, and F Aβ relative to FL-APP in male (left), and female (right), cortex (Ctx) (top), and hippocampus (bottom) (n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance was determined using unpaired t-test for males and 2-way ANOVA for females followed by uncorrected Fisher’s LSD test for multiple comparisons. Overall significant main effects of treatment or OVX are indicated

Fig. 4

ERβ activation modulates microglia activation in a sex-specific manner in App^NL−G−F mice. A Representative immunofluorescence images of male App^NL−G−F hippocampus stained with the amyloid stain AmyloGlo (magenta), Iba1 (green), and CD68 (white) after vehicle or LY treatment. Yellow dotted area (left) indicates magnified region of interest (right). Arrowheads indicate microglia with lower CD68 levels. Scale bar 100 µm (left) and 50 µm (right). Quantification in male App^NL−G−F mice of B number of Iba1 cells per 100 µm² (n = 5–6), C percent CD68 +, Iba1 + double positive cells (n = 4–5), and D percent microglia within 20 µm radius of plaque edge (n = 5–6). E Representative immunofluorescence images of female App^NL−G−F hippocampus stained with AmyloGlo (magenta), Iba1 (green), and CD68 (white) after vehicle or LY treatment, as well as after sham surgery or ovariectomy (OVX). Yellow dotted area (left) indicates magnified region of interest (right). Arrowheads indicate microglia with lower CD68 levels. Scale bar 100 µm (left) and 50 µm (right). Quantification in female App^NL−G−F mice F number of Iba1 cells per 100 µm² (n = 4), (G percent CD68 +, Iba1 + double positive cells (n = 4), and H percent plaque-associated microglia (n = 4). * P < 0.05, *** P < 0.001. Unpaired t-test was used for males and 2-way ANOVA for females followed by uncorrected Fisher’s LSD test for multiple comparisons. Overall significant main effects of treatment are indicated

Fig. 5

Microglial and proinflammatory markers are altered upon ERβ activation in a sex-specific manner. A Representative immunofluorescence image of ERβ and Iba1 co-staining (arrowheads) in WT (left) and Esr2-KO (right) male cortex (dotted rectangle: magnified area), and B in male App^NL−G−F cortex upon vehicle or LY treatment (scale bars = 50 µm). C Quantification and D comparison of percent ERβ positive microglia in male and female App^NL−G−F brains (cortex and hippocampus) upon OVX and/or LY treatment (n = 4). Expression of the proresolving microglial markers E Trem2, and F Cx3cr1 relative to housekeeping gene Rplp0 in male (left) and female (right) App^NL−G−F hippocampus after vehicle or LY treatment, as well as after sham surgery or OVX in females (n = 3–7). Multiplex ELISA analysis of the inflammatory markers G CXCL1 (KC/GRO), H IL-12p70, and I IL-10, in male (left) and female (right) APP^NL−G−F hippocampus after vehicle or LY treatment, as well as after sham surgery or ovariectomy (OVX in females) (n = 4–6). * P < 0.05, ** P < 0.01, *** P < 0.001. Unpaired t-test was used for males and 2-way ANOVA for females followed by uncorrected Fisher’s LSD test for multiple comparisons. Overall significant main effects of treatment or OVX are indicated