Obesity Accelerates Alzheimer-Related Pathology in APOE4 but not APOE3 Mice

V. Alexandra Moser; Christian J. Pike

doi:10.1523/ENEURO.0077-17.2017

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Figure 1.
Metabolic outcomes associated with DIO in E3FAD and E4FAD mice. A, Body weights in male E3FAD and E4FAD mice maintained on control (CTL) and Western (WD) diets taken at baseline (week 0) and four-week intervals across the 12-week experimental period. Plasma levels of cholesterol (B) and triglyceride levels (C) in E3FAD and E4FAD mice on control and Western diets at the end of the experimental period. D, Weight of the gonadal fat pads across groups. Relative mRNA expression of macrophage markers (E) CD68 and (F) F4/80 in gonadal fat, as determined by real time PCR. Data show fold differences relative to the E3FAD + control diet group. G, GTT showing blood glucose levels over time after a glucose bolus. H, AUC for the GTT. I, Percentage change in fasting blood glucose levels relative to baseline after 12-weeks of control or Western diet. Data are presented as mean (±SEM) values; n = 7–11/group. E3FAD mice are shown as circles, E4FAD mice are shown as squares; control diet groups are indicated as open symbols or bars, whereas Western diet groups are filled symbols or bars. *, p < 0.05 relative to genotype-matched mice in control diet condition. #, p < 0.05 relative to E3FAD mice in same diet condition.
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Figure 2.
Accumulation of amyloidogenic deposits assessed by Thio-S staining in E3FAD and E4FAD mice across dietary treatments. A, Representative images of Thio-S staining in the subiculum of E3FAD and E4FAD males fed control and Western diets. Scale bar, 50 µm. Numbers of Thio-S positive plaque numbers in E3FAD and E4FAD mice maintained on control and Western diets were quantified in (B) entorhinal cortex, and hippocampal subregions (C) subiculum, (D) CA1, and (E) CA2/3. Data are presented as mean (±SEM) values; n = 7–11/group. E3FAD mice are shown as circles, E4FAD mice are shown as squares; control diet groups are indicated as open symbols, and Western diet groups as filled symbols. *, p < 0.05 relative to genotype-matched mice in control diet condition. #, p < 0.05 relative to E3FAD mice in same diet condition.
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Figure 3.
Accumulation of β-amyloid deposits assessed by immunohistochemistry in E3FAD and E4FAD mice across dietary treatments. A, Representative images of β-amyloid immunoreactivity in entorhinal cortex and hippocampus in E3FAD and E4FAD males maintained on control and Western diets. Scale bar, 100 µm. β-Amyloid burden was quantified as immunoreactivity load in E3FAD and E4FAD mice in control and Western diets groups in (B) entorhinal cortex, and hippocampal subregions (C) subiculum, (D) CA1, and (E) CA2/3. Data are presented as mean (±SEM) values; n = 7–11/group. E3FAD mice are shown as circles, E4FAD mice are shown as squares; control diet groups are indicated as open symbols, and Western diet groups as filled symbols. *, p < 0.05 relative to genotype-matched mice in control diet condition. #, p < 0.05 relative to E3FAD mice in same diet condition.
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Figure 4.
Microglia number and morphologic status assessed by IBA-1 immunohistochemistry in E3FAD and E4FAD mice across dietary treatments. A, Representative images of microglial morphology associated with resting (type 1) and reactive (types 2 and 3) phenotypes. Scale bar, 40 µm. B–E, Densities (cells/mm²) of IBA-1-immunoreactive cells in E3FAD and E4FAD mice on control and Western diets were quantified in (B) entorhinal cortex, and hippocampal subregions (C) subiculum, (D) CA1, and E) CA2/3. F–I) Percentages of all IBA-1-immunoreactive cells scored as having reactive phenotype (types 2 and 3) were quantified in (F) entorhinal cortex, and hippocampal subregions (G) subiculum, (H) CA1, and (I) CA2/3. Data are presented as mean (±SEM) values; n = 7–11/group. E3FAD mice are shown as circles, E4FAD mice are shown as squares; control diet groups are indicated as open symbols, and Western diet groups as filled symbols. *, p < 0.05 relative to genotype-matched mice in control diet condition. #, p < 0.05 relative to E3FAD mice in same diet condition.
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Figure 5.
Astrocyte number and morphologic status assessed by GFAP immunohistochemistry in E3FAD and E4FAD mice across dietary treatments. A, Representative images of astrocyte morphology associated with resting and reactive phenotypes. Scale bar, 50 µm. B–E, Densities (cells/mm²) of GFAP-immunoreactive cells in E3FAD and E4FAD mice on control and Western diets were quantified in (B) entorhinal cortex, and hippocampal subregions (C) subiculum, (D) CA1, and (E) CA2/3. F–I, Percentages of all GFAP-immunoreactive cells scored as having reactive phenotype (type 2) were quantified in (F) entorhinal cortex, and hippocampal subregions (G) subiculum, (H) CA1, and (I) CA2/3. Data are presented as mean (±SEM) values; n = 7–11/group. E3FAD mice are shown as circles, E4FAD mice are shown as squares; control diet groups are indicated as open symbols, and Western diet groups as filled symbols. *, p < 0.05 relative to genotype-matched mice in control diet condition. #, p < 0.05 relative to E3FAD mice in same diet condition.

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Table 1.

Gene targets for the PCR analyses are listed with their corresponding oligonucleotide sequences for the forward and reverse primers

Target gene	Sequence
CD68	Forward: 5’-TTCTGCTGTGGAAATGCAAG-3’ Reverse: 5’-AGAGGGGCTGGTAGGTTGAT-3’
F4/80	Forward: 5’-TGCATCTAGCAATGGACAGC-3’ Reverse: 5’-GCCTTCTGGATCCATTTGAA-3’
HPRT	Forward: 5’-AAGCTTGCTGGTGAAAAGGA-3’ Reverse: 5’-TTGCGCTCATCTTAGGCTTT-3’
SDHA	Forward: 5’-ACACAGACCTGGTGGAGACC-3’ Reverse: 5’-GGATGGGCTTGGAGTAATCA-3’
Neprilysin	Forward: 5’-GAGAAAAGCCCACTTGCTTG-3’ Reverse: 5’-GAAAGACAAAATGGGGCAGA-3’
BACE1	Forward: 5’-TCGCTGTCTCACAGTCATCC-3’ Reverse: 5’-AACAAACGGACCTTCCACTG-3’
IDE	Forward: 5’-TGTTTCCACACACAGGCAAT-3’ Reverse: 5’-ACCTGTGAAAAGCCGAGAGA-3’
CD74	Forward: 5’-CAAGTACGGCAACATGACCC-3’ Reverse: 5’-GCACTTGGTCAGTACTTTAGGTG-3’
GFAP	Forward: 5’-AACGACTATCGCCGCCAACTG-3’ Reverse: 5’-CTCTTCCTGTTCGCGCATTTG-3’
β-Actin	Forward: 5’-AGCCATGTACGTAGCCATCC-3’ Reverse: 5’-CTCTCAGCTGTGGTGGTGAA-3’

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Table 2.

Statistical table

Figure	Kolmogorov-Smirnov test for normality (p value)	Statistical significance
Figure 1A body weight	All groups at all time points are normally distributed (p > 0.05).	Genotype: F_(1,29) = 0.10, p = 0.759 diet: F_(1,29) = 10.51, p = 0.003 interaction: F_(1,29) = 2.68, p = 0.112
Figure 1B plasma cholesterol	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,29) = 2.86, p = 0.103 diet: F_(1,29) = 1.58, p = 0.221 interaction: F_(1,29) = 2.60, p = 0.119
Figure 1C plasma triglycerides	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,29) = 0.56, p = 0.46 diet: F_(1,29) = 2.87, p = 0.102 interaction: F_(1,29) = 1.91, p = 0.179
Figure 1D gonadal fat weight	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,29) = 0.18, p = 0.673 diet: F_(1,29) = 37.04, p < 0.001 interaction: F_(1,29) = 5.01, p = 0.033
Figure 1E CD68	E3FAD CTL N/A E3FAD WD > 0.10 E4FAD CTL = 0.004 E4FAD WD > 0.10	Genotype: F_(1,21) = 0.90, p = 0.353 diet: F_(1,21) = 11.54, p = 0.003 interaction: F_(1,21) = 0.85, p = 0.366
Figure 1F F4/80	E3FAD CTL N/A E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,21) = .09, p = .768 diet: F_(1,21) = 7.02, p = 0.015 interaction: F_(1,21) = 1.19, p = 0.288
Figure 1G glucose (GTT)	All groups at all time points are normally distributed (p > 0.05), except: E4FAD CTL 0 min = 0.002 E4FADWD 15 min = 0.025 E3FAD WD 30 min = 0.011 E4FAD WD 30 min = 0.008	Genotype: F_(1,29) = 0.02, p = 0.886 diet: F_(1,29) = 5.03, p = 0.033 interaction: F_(1,29) = 0.10, p = 0.750
Figure 1H GTT AUC	E3FAD CTL = 0.07 E3FAD WD = 0.097 E4FAD CTL > 0.10 E4FAD WD = 0.033	Genotype: F_(1,29) = .06, p = 0.817 diet: F_(1,29) = 5.73, p = 0.023 interaction: F_(1,29) = 0.12, p = 0.737
Figure 1I percent glucose change	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,29) = .83, p = 0.371 diet: F_(1,29) = 3.84, p = 0.059 interaction: F_(1,29) = 0.90, p = 0.352
Figure 2B Thio-S: entorhinal cortex	E3FAD CTL > 0.10 E3FAD WD = 0.049 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,29) = 50.30, p < 0.001 diet: F_(1,29) = 6.62, p = 0.016 interaction: F_(1,29) = 4.09, p = 0.053
Figure 2C Thio-S: subiculum	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,29) = 59.40, p < 0.001 diet: F_(1,29) = 2.98, p = 0.095 interaction: F_(1,29) = 9.75, p = 0.004
Figure 2D Thio-S: CA1	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,29) = 80.58, p < 0.001 diet: F_(1,29) = 4.95, p = 0.034 interaction: F_(1,29) = 8.41, p = 0.007
Figure 2E Thio-S: CA2/3	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 46.39, p < 0.001 diet: F_(1,29) = 7.41, p = 0.011 interaction: F_(1,29) = 7.32, p = 0.011
Figure 3B Aβ load: entorhinal cortex	E3FAD CTL > 0.10 E3FAD WD = 0.002 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 21.38, p < 0.001 diet: F_(1,29) = 7.83, p = 0.009 interaction: F_(1,29) = 4.91, p = 0.035
Figure 3C Aβ load: subiculum	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 25.40, p < 0.001 diet: F_(1,29) = 11.19, p = 0.002 interaction: F_(1,29) = 0.11, p = 0.742
Figure 3D Aβ load: CA1	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD = 0.036	Genotype F_(1,29) = 37.66, p < 0.001 diet: F_(1,29) = 2.91, p = 0.099 interaction: F_(1,29) = 2.71, p = 0.110
Figure 3E Aβ load: CA2/3	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 47.27, p < 0.001 diet: F_(1,29) = 10.36, p = 0.003 interaction: F_(1,29) = 4.48, p = 0.043
Figure 4B microglia number: entorhinal cortex	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,27) = 9.78, p = 0.004 diet: F_(1,27) = 2.31, p = 0.141 interaction: F_(1,27) = 1.05, p = 0.316
Figure 4C microglia number: subiculum	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,27) = 42.77, p < 0.001 diet: F_(1,27) = 4.20, p = 0.050 interaction: F_(1,27) = 4.75, p = 0.038
Figure 4D microglia number: CA1	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,27) = 51.42, p < 0.001 diet: F_(1,27) = 10.78, p = 0.003 interaction: F_(1,27) = 7.97, p = 0.009
Figure 4E microglia number: CA2/3	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,27) = 21.64, p < 0.001 diet: F_(1,27) = 1.97, p = 0.172 interaction: F_(1,27) = 1.90, p = 0.180
Figure 4F microglia reactivity: entorhinal cortex	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,27) = 109.10, p < 0.001 diet: F_(1,27) = 1.64, p = 0.212 interaction: F_(1,27) = 5.52, p = 0.027
Figure 4G microglial reactivity: subiculum	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL = 0.07 E4FAD WD < 0.001	Genotype F_(1,27) = 19.70, p < 0.001 diet: F_(1,27) = 0.00, p = 0.995 interaction: F_(1,27) = 0.51, p = 0.480
Figure 4H microglial reactivity: CA1	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD = 0.04	Genotype F_(1,27) = 78.70, p < 0.001 diet: F_(1,27) = 5.00, p = 0.034 interaction: F_(1,27) = 11.58, p = 0.002
Figure 4I microglial reactivity: CA2/3	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,27) = 165.70, p < 0.001 diet: F_(1,27) = 21.04, p < 0.001 interaction: F_(1,27) = 32.66, p < 0.001
Figure 5B astrocyte number: entorhinal cortex	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 3.82, p = 0.060 diet: F_(1,29) = 0.29, p = 0.593 interaction: F_(1,29) = 0.41, p = 0.528
Figure 5C astrocyte number: subiculum	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 9.95, p = 0.004 diet: F_(1,29) = 4.79, p = 0.037 interaction: F_(1,29) = 1.04, p = 0.316
Figure 5D astrocyte number: CA1	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 5.88, p = 0.022 diet: F_(1,29) = 3.55, p = 0.069 interaction: F_(1,29) = 0.49, p = 0.489
Figure 5E astrocyte number: CA2/3	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 1.82, p = 0.188 diet: F_(1,29) = 4.26, p = 0.048 interaction: F_(1,29) = 0.02, p = 0.894
Figure 5F astrocyte reactivity: entorhinal cortex	E3FAD CTL > 0.10 E3FAD WD = 0.004 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 46.97, p < 0.001 diet: F_(1,29) = 5.75, p = 0.023 interaction: F_(1,29) = 4.82, p = 0.036
Figure 5G astrocyte reactivity: subiculum	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL = 0.045 E4FAD WD > 0.10	Genotype F_(1,29) = 27.72, p < 0.001 diet: F_(1,29) = 3.13, p = 0.088 interaction: F_(1,29) = 0.00, p = 0.989
Figure 5H astrocyte reactivity: CA1	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 87.49, p < 0.001 diet: F_(1,29) = 23.82, p < 0.001 interaction: F_(1,29) = 2.08, p = 0.160
Figure 5I astrocyte reactivity: CA2/3	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype F_(1,29) = 11.68, p = 0.002 diet: F_(1,29) = 7.83, p = 0.009 interaction: F_(1,29) = 2.405, p = 0.132

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Table 3.

Relative gene expression in hippocampus

Gene	Mean ± SEM	Kolmogorov-Smirnov test for normality (p value)	Statistical significance
BACE1	E3FAD CTL = 1 ± N/A E3FAD WD = 1.53 ± 0.31 E4FAD CTL = 1.32 ± 0.19 E4FAD WD = 1.76 ± 0.41	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,28) = 1.10, p = 0.304 diet: F_(1,28) = 3.44, p = 0.074 interaction: F_(1,28) = 0.03, p = 0.874
Neprilysin	E3FAD CTL = 1 ± N/A E3FAD WD = 1.61 ± 0.79 E4FAD CTL = 0.94 ± 0.30 E4FAD WD = 1.79 ± 0.63	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,28) = 0.02, p = 0.902 diet: F_(1,28) = 2.49, p = 0.126 interaction: F_(1,28) = 0.06, p = 0.802
IDE	E3FAD CTL = 1 ± N/A E3FAD WD = 1.27 ± 0.39 E4FAD CTL = 1.30 ± 0.39 E4FAD WD = 1.12 ± 0.35	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL = 0.01 E4FAD WD > 0.10	Genotype: F_(1,28) = 0.08, p = 0.785 diet: F_(1,28) = 0.00, p = 0.955 interaction: F_(1,28) = 0.49, p = 0.489
CD68	E3FAD CTL = 1 ± N/A E3FAD WD = 1.21 ± 0.29 E4FAD CTL = 1.74 ± 0.30 E4FAD WD = 2.30 ± 0.29	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,28) = 10.75, p = 0.003 diet: F_(1,28) = 1.91, p = 0.178 interaction: F_(1,28) = 0.40, p = 0.532
GFAP	E3FAD CTL = 1 ± N/A E3FAD WD = 1.02 ± 0.11 E4FAD CTL = 1.56 ± 0.21 E4FAD WD = 2.70 ± 0.04	E3FAD CTL > 0.10 E3FAD WD > 0.10 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,28) = 14.26, p < 0.001 diet: F_(1,28) = 0.23, p = 0.634 interaction: F_(1,28) = 0.14, p = 0.712
CD74	E3FAD CTL = 1 ± N/A E3FAD WD = 1.28 ± 0.28 E4FAD CTL = 3.32 ± 0.62 E4FAD WD = 5.04 ± 1.30	E3FAD CTL > 0.10 E3FAD WD = 0.01 E4FAD CTL > 0.10 E4FAD WD > 0.10	Genotype: F_(1,28) = 16.98, p < 0.001 diet: F_(1,28) = 1.86, p = 0.184 interaction: F_(1,28) = 0.96, p = 0.335

Data are presented as mean fold differences (±SEM) relative to E3FAD mice on a control diet. The Kolmogorov-Smirnov test for normality was performed, with p > 0.05 indicating a normal distribution. Genes related to β-amyloid production (BACE-1) and clearance (neprilysin, IDE) showed no significant changes with either diet or genotype, while genes related to glial activation (CD68, GFAP, and CD74) were increased in E4FAD mice on both control and Western diets.