The Effect of a High-Fat Diet on Brain Plasticity, Inflammation and Cognition in Female ApoE4-Knockin and ApoE-Knockout Mice

Abstract

Apolipoprotein E4 (ApoE4), one of three common isoforms of ApoE, is a major risk factor for late-onset Alzheimer disease (AD). ApoE-deficient mice, as well as mice expressing human ApoE4, display impaired learning and memory functions and signs of neurodegeneration. Moreover, ApoE protects against high-fat (HF) diet induced neurodegeneration by its role in the maintenance of the integrity of the blood-brain barrier. The influence of a HF diet on the progression of AD-like cognitive and neuropathological changes was assessed in wild-type (WT), human ApoE4 and ApoE-knockout (ApoE-/-) mice to evaluate the modulatory role of ApoE in this process. From 12 months of age, female WT, ApoE4, and ApoE-/- mice were fed either a standard or a HF diet (19% butter, 0.5% cholate, 1.25% cholesterol) throughout life. At 15 months of age mice performed the Morris water maze, evaluating spatial learning and memory. ApoE-/- showed increased spatial learning compared to WT mice (p = 0.009). HF diet improved spatial learning in WT mice (p = 0.045), but did not affect ApoE4 and ApoE-/- mice. Immunohistochemical analyses of the hippocampus demonstrated increased neuroinflammation (CD68) in the cornu ammonis 1 (CA1) region in ApoE4 (p = 0.001) and in ApoE-/- (p = 0.032) mice on standard diet. HF diet tended to increase CD68 in the CA1 in WT mice (p = 0.052), while it decreased in ApoE4 (p = 0.009), but ApoE-/- remained unaffected. A trend towards increased neurogenesis (DCX) was found in both ApoE4 (p = 0.052) and ApoE-/- mice (p = 0.068). In conclusion, these data suggest that HF intake induces different effects in WT mice compared to ApoE4 and ApoE-/- with respect to markers for cognition and neurodegeneration. We propose that HF intake inhibits the compensatory mechanisms of neuroinflammation and neurogenesis in aged female ApoE4 and ApoE-/- mice.

Fig 1. Immunohistochemistry.

Representative images of (A) DCX staining (2.5x objective), (B) PSD95 staining (2.5x objective). Black = visual cortex, white = sensory cortex, yellow = stratum radiatum, blue = inner molecular layer, red = outer molecular layer, green = stratum lucidum, (C) GLUT-1 staining (5x objective), and (D) CD68 staining (5x objective). Scale bars represent 500μm.

Fig 2. Body weight during the experimental phase.

HF diet increased body weight pre- and post-testing in both ApoE4 (p<0.001) and WT (p<0.001) mice compared to CTRL diet, but not in ApoE-/-. Furthermore, the body weight increase due to HF diet was significantly stronger in ApoE4 compared to WT (p = 0.002). Values represent mean±SEM; WT CTRL n = 8, WT HF n = 8, ApoE4 CTRL n = 8, ApoE4 HF n = 8, ApoE-/- CTRL n = 5, ApoE-/- HF n = 6. *p≤0.05. ApoE = apolipoprotein E; CTRL = control; HF = high-fat; WT = wild-type.

Fig 3. MWM acquisition and probe (0-30s).

(A) Both groups displayed a learning effect (p = 0.000). HF fed mice showed improved spatial learning compared to CTRL diet (p = 0.045). (B) Both groups displayed a learning effect (p = 0.000). ApoE-/- demonstrated improved spatial learning compared to WT mice (p = 0.009). There was no difference in spatial learning between diets. (C) No significant differences were found in the probe phase of the MWM caused by either genotype or diet (p>0.05). (D) No significant differences were found in the probe phase of the MWM caused by either genotype or diet (p>0.05). ApoE-/- mice displayed a trend for increased time spent in platform area (p = 0.059). Values represent mean±SEM; WT CTRL n = 6, WT HF n = 7, ApoE4 CTRL n = 8, ApoE4 HF n = 8, ApoE-/- CTRL n = 6, ApoE-/- HF n = 6. *p≤0.05. ApoE = apolipoprotein E; CTRL = control; HF = high-fat; MWM = Morris water maze; WT = wild-type.

Fig 4. CD68 immunoreactivity.

In the CA1, we observed a genotype × diet interaction (p = 0.001), there was a trend for increased inflammation in WT after HF supplementation (p = 0.052). Inflammation in ApoE4 mice significantly decreased after HF supplementation (p = 0.009). ApoE4 mice on CTRL demonstrated increased inflammation compared to WT mice on CTRL (p = 0.001). ApoE-/- compared to WT mice showed a genotype × diet interaction (p = 0.010). ApoE-/- on CTRL demonstrated increased inflammation when compared to WT mice on CTRL (p = 0.032). In the CA3, a genotype × diet interaction was observed (p = 0.024) but no significant effect on inflammation caused by genotype or diet when comparing ApoE4 to WT mice. Furthermore, ApoE-/- compared to WT mice showed a genotype × diet interaction (p = 0.023). No significant effects on inflammation caused by genotype or diet were found though. In the DG, we observed a genotype × diet interaction (p = 0.047) when comparing ApoE4 to WT mice. There was a trend for increased inflammation in ApoE4 mice on CTRL diet compared to WT mice on CTRL (p = 0.063). In addition, ApoE-/- displayed increased inflammation when compared to WT mice (p = 0.029). Values represent mean±SEM; WT CTRL n = 5, WT HF n = 7, ApoE4 CTRL n = 7, ApoE4 HF n = 5, ApoE-/- CTRL n = 4, ApoE-/- HF n = 6. *p≤0.05, #0.05

Fig 5. DCX-positive cells in the DG.

ApoE4 and ApoE-/- mice display a trend for increased adult neurogenesis compared to WT mice (p = 0.052 and p = 0.068 respectively). Values represent mean±SEM; WT CTRL n = 4, WT HF n = 6, ApoE4 CTRL n = 4, ApoE4 HF n = 7, ApoE-/- CTRL n = 4, ApoE-/- HF n = 7. #0.05

Fig 6. PSD95 immunoreactivity.

PSD95 was analyzed as a synaptic marker in the VIS, SEN, IML, OML, SL and SR of the hippocampus. No significant genotype nor diet effects were found (p>0.05). In the SEN, ApoE^-/- mice showed a slight trend (p = 0.064) for decreased PSD95 expression. Values represent mean±SEM; WT CTRL n = 8, WT HF n = 8, ApoE4 CTRL n = 8, ApoE4 HF n = 7, ApoE^-/- CTRL n = 5, ApoE^-/- HF n = 7. #0.05<p<0.07. ApoE = apolipoprotein E; CTRL = control; HF = high-fat; IML = inner molecular layer; OML = outer molecular layer; PSD = post-synaptic density; SEN = sensory cortex; SL = stratum lucidum; SR = stratum radiatum; VIS = visual cortex; WT = wild-type.

Fig 7. GLUT-1 immunoreactivity.

GLUT-1 density was analyzed as a measure for capillary density. No differences in capillary density, caused by either genotype nor diet, were observed (p>0.05). Values represent mean±SEM; WT CTRL n = 6, WT HF n = 6, ApoE4 CTRL n = 6, ApoE4 HF n = 6, ApoE^-/- CTRL n = 4, ApoE^-/- HF n = 4. ApoE = apolipoprotein E; CA1 = cornu ammonis 1; CA3 = cornu ammonis 3; CTRL = control; DG = dentate gyrus; GLUT = glucose transporter; HF = high-fat; WT = wild-type.