PAE reduces γ-secretase activity in B6129 and 3xTg-AD mice at birth.

(A) Western analysis of cortical lysates from B6129 controls (lanes 1-6) and PAE-B6129 (lanes 7-19) mice at postnatal day 0 (P0) shows no change in PS1 CTF levels between groups, and increased levels of APP CTFs in PAE-B6129 mice compared to B6129 controls. (B) Western blot analysis of cortical lysates from B6129 controls (lanes 1-6) and PAE-B6129 (lanes 7-19) mice at P0 shows increased levels of NICD in PAE-B6129 mice compared to controls. (C) Quantification of APP CTFs, PS1 CTF, and NICD shows that levels of APP CTFs (p = 0.0026) and NICD (p = 0.0315) are significantly increased compared to non-alcohol-exposed B6129. Quantification of PS1 CTF levels shows no significant differences between PAE-B6129 and B6129 controls (p = 0.2269). Unpaired Student’s t-test. (D) Western analysis of cortical lysates from B6129 controls (lanes 1-5), 3xTg-AD controls (lanes 6-11), and PAE-3xTg-AD (lanes 12-16) mice at P0 shows no change in PS1 CTF levels between groups, and increased levels of APP CTFs in 3xTg-AD controls and PAE-3xTg-AD mice compared to B6129 controls. (E) Western analysis of cortical lysates from B6129 controls (lanes 1-5), 3xTg-AD controls (lanes 6-11), and PAE-3xTg-AD (lanes 12-16) mice at P0 shows increased levels of NICD in 3xTg-AD controls and PAE-3xTg-AD PAE-B6129 mice compared to B6129 controls. (F) Quantification of APP CTFs, PS1 CTF, and NICD shows that levels of APP CTFs in 3xTg-AD controls (p = 0.0395) and in PAE-3xTg-AD (p = 0.0011) were significantly higher compared to B6129 controls. Quantification of PS1 CTF levels shows no significant differences between 3xTg-AD controls (p = 0.9923), PAE-3xTg-AD (p = 0.8980), and B6129 controls. Levels of NICD in PAE-3xTg-AD (p = 0.0087) are significantly higher compared to B6129 controls. One-way ANOVA with Tukey’s post hoc multiple comparisons. All data represent mean ± SEM. *p < 0.05, **p < 0.01. Sample size indicated in parentheses next to each experimental group label.

PAE-B6129 and PAE-3xTg-AD mice show high expression of APP CTFs in cortical regions at birth.

(Top Panel) Representative images of cortical sagittal sections immunoassayed using antibodies specific for the C-terminal fragment of APP. Accumulation of APP CTFs is evident in the somatosensory cortex (SSCx), in layers II to VI, of PAE-B6129, 3xTg-AD, and PAE-3xTg-AD mice compared to B6129 controls. Quantification of fluorescence signal shows a significant increase in PAE-B6129 (p = 0.0216), and PAE-3xTg-AD (p = 0.0261) compared to B6129 controls. (Bottom Panel) Hippocampal sagittal representative images showing accumulation of APP CTFs mostly in CA1, CA2/3, and modest increased immunoreactivity in dentate gyrus (DG). Quantification of fluorescence signal shows a significant increase in PAE-B6129 (p = 0.0030), 3xTg-AD (p = 0.0088), and PAE-3xTg-AD (p = 0.0105) compared to B6129 controls. One-way ANOVA with Tukey’s post hoc multiple comparisons. All data represent mean ± SEM. *p < 0.05, **p < 0.01. Sample size indicated in parentheses next to each experimental group label. Scale bars = 50 μm.

PAE impairs learning and memory in B6129 mice at 3 months of age.

(A) Morris water maze experimental timeline: Mice are individually acclimated to handling during days 1 to 3. The hidden platform training and acquisition phase is conducted from days 4 to 10-11, for a total of 7-8 days. Probe and visible platform tests are performed one day after completion of the acquisition phase, on days 12-13. Brains are harvested immediately following the behavioral study, on days 14-15. (B) During the hidden platform version of the Morris water maze test, PAE-B6129 mice at 3 months of age display significantly longer latencies compared to B6129 controls (p = 0.0036). At the pre-symptomatic stage (3 months of age), 3xTg-AD controls did not show significant differences in escape latencies compared to B6129 controls (p = 0.2153). Two-way ANOVA with Tukey’s post hoc multiple comparisons of the main columns means. All data represent mean ± SEM **p < 0.01. Sample size indicated in parentheses next to each experimental group label.

PAE reduces γ-secretase activity in B6129 mice at 3 months of age.

A) Western analysis of cortical lysates from B6129 controls (lanes 1-3) and PAE-B6129 (lanes 4-19) mice at 3 months of age shows no change in PS1 CTF levels between groups and increased levels of APP CTFs in PAE-B6129 mice compared to B6129 controls. (B) Western blot analysis of cortical lysates from B6129 controls (lanes 1-4) and 3xTg-AD controls (lanes 5-17) mice at 3 months of age shows no change in levels of PS1 CTF and increased levels of APP CTFs in 3xTg-AD mice compared to B6129 controls. (C-D) Quantification of cortical levels of APP CTFs and PS1 CTF shows that (C) levels of APP CTFs are significantly increased in the cortex of PAE-B6129 (p = 0.065) compared to B6129 controls, and levels of PS1 CTF show no significant differences between PAE-B6129 (p = 0.0725) and B6129 controls. (D) Levels of APP CTFs (p = 0.0097) and PS1 CTF (p = 0.0165) in 3xTg-AD cortex are significantly increased compared to B6129 controls. Unpaired Student’s t-test. (E) Western analysis of hippocampal lysates from B6129 controls (lanes 1-3) and PAE-B6129 (lanes 4-19) show no changes in PS1 CTF level, and increased levels of APP CTFs in PAE-B6129 compared to B6129 controls. (F) Western analysis of hippocampal lysates from B6129 controls (lanes 1-4) and 3xTg-AD controls (lanes 5-17) shows increased levels of PS1 CTF and APP CTFs in 3xTg-AD mice compared to B6129 controls. (G-H) Quantification of hippocampal levels of APP CTFs and PS1 CTF shows that (G) levels of APP CTFs in PAE-B6129 (p = 0.0170) are significantly increased compared to B6129 controls. Levels of PS1 CTF were not significantly different between groups (p = 0.4578). (H) Levels of APP CTF in 3xTg-AD controls (p = 0.0096) were significantly higher compared to B6129 controls. Levels of PS1 CTF in 3xTg-AD (p = 0.0165) are significantly higher compared to B6129 controls. Unpaired Student’s t-test. All data represent mean ± SEM. *p < 0.05, **p < 0.01. Sample size indicated in parentheses next to each experimental group label.

PAE impairs learning and memory and γ-secretase activity in B6129 mice at 6 months of age.

During the hidden platform version of the Morris water maze test, PAE-B6129 mice at 6 months of age display significantly longer escape latencies compared to B6129 controls (p < 0.0001). Similarly, 3xTg-AD controls (p < 0.0001) show significant differences in escape latencies compared to B6129 controls. The difference in escape latency between PAE-B6129 and 3xTg-AD mice reached modest statistical significance (p = 0.0220). Two-way ANOVA with Tukey’s post hoc multiple comparisons of the main columns means. All data represent mean ± SEM *p < 0.05 ****p < 0.0001. Sample size indicated in parentheses next to each experimental group label.

PAE reduces γ-secretase activity in the hippocampus of B6129 mice at 6 months of age.

(A) Western analysis of cortical lysates from B6129 controls (lanes 1-5) and PAE-B6129 (lanes 6-19) mice at 6 months of age shows no change in PS1 CTF and APP CTFs levels between groups (B) Western blot analysis of cortical lysates from B6129 controls (lanes 1-4) and 3xTg-AD controls (lanes 5-17) mice at 6 months of age shows no change in levels of PS1 CTF and increased levels of APP CTFs in 3xTg-AD mice compared to B6129. (C-D) Quantification of cortical levels of APP CTFs and PS1 CTF shows that (C) levels of APP CTFs (p = 0.1219) and PS1 CTF (p = 0.4461) are not significantly different in the cortex of PAE-B6129 compared to non-alcohol-exposed B6129 (D) Levels of APP CTFs (p = 0.0067) and PS1 CTF (p = 0.0165) in 3xTg-AD cortex are significantly increased compared to B6129. Unpaired Student’s t-test. (E) Western analysis of hippocampal lysates from B6129 controls (lanes 1-3) and PAE-B6129 (lanes 4-19) shows no changes in PS1 CTF level, and increased levels of APP CTFs in PAE-B6129 compared to B6129 controls. (F) Western analysis of hippocampal lysates from B6129 controls (lanes 1-4) and 3xTg-AD controls (lanes 5-17) shows increased levels of PS1 CTF and APP CTFs in 3xTg-AD mice compared to B6129 controls. (G-H) Quantification of hippocampal levels of APP CTFs and PS1 CTF shows that (G) levels of APP CTFs in PAE-B6129 (p = 0.0094) are significantly increased compared to B6129 controls. Levels of PS1 CTF were not significantly different between groups (p = 0.7407). (H) Levels of APP CTF in 3xTg-AD controls (p = 0.0004) are significantly higher compared to B6129 controls. Levels of PS1 CTF in 3xTg-AD (p = 0.0393) are significantly higher compared to B6129 controls. Unpaired Student’s t-test. All data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Sample size indicated in parentheses next to each experimental group label.

PAE alters learning and memory in 3xTg-AD mice in the pre-symptomatic stage.

(A) During the hidden platform version of the Morris water maze test, PAE-3xTg AD mice at 4 months of age (pre-symptomatic stage) display significantly longer escape latencies compared to B6129 controls (p = 0.0005). 3xTg-AD controls (p = 0.0299) show less significant differences in escape latencies compared to B6129 controls. Two-way ANOVA with Tukey’s post hoc multiple comparisons of the main columns means. All data represent mean ± SEM *p < 0.05 ***p < 0.001. Sample size indicated in parentheses next to each experimental group label.

Isocaloric liquid diet formulas for 1 liter preparation (w/w)

List of antibodies used in this study