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. 2021 Apr 1:13:654509.
doi: 10.3389/fnagi.2021.654509. eCollection 2021.

Western Diet Induces Impairment of Liver-Brain Axis Accelerating Neuroinflammation and Amyloid Pathology in Alzheimer's Disease

Angelika Wiȩckowska-Gacek  1 , Anna Mietelska-Porowska  1 , Dominik Chutorański  1 , Małgorzata Wydrych  1 , Jan Długosz  1 , Urszula Wojda  1
Affiliations

Western Diet Induces Impairment of Liver-Brain Axis Accelerating Neuroinflammation and Amyloid Pathology in Alzheimer's Disease

Angelika Wiȩckowska-Gacek et al. Front Aging Neurosci. .

Abstract

Alzheimer's disease (AD) is an aging-dependent, irreversible neurodegenerative disorder and the most common cause of dementia. The prevailing AD hypothesis points to the central role of altered cleavage of amyloid precursor protein (APP) and formation of toxic amyloid-β (Aβ) deposits in the brain. The lack of efficient AD treatments stems from incomplete knowledge on AD causes and environmental risk factors. The role of lifestyle factors, including diet, in neurological diseases is now beginning to attract considerable attention. One of them is western diet (WD), which can lead to many serious diseases that develop with age. The aim of the study was to investigate whether WD-derived systemic disturbances may accelerate the brain neuroinflammation and amyloidogenesis at the early stages of AD development. To verify this hypothesis, transgenic mice expressing human APP with AD-causing mutations (APPswe) were fed with WD from the 3rd month of age. These mice were compared to APPswe mice, in which short-term high-grade inflammation was induced by injection of lipopolysaccharide (LPS) and to untreated APPswe mice. All experimental subgroups of animals were subsequently analyzed at 4-, 8-, and 12-months of age. APPswe mice at 4- and 8-months-old represent earlier pre-plaque stages of AD, while 12-month-old animals represent later stages of AD, with visible amyloid pathology. Already short time of WD feeding induced in 4-month-old animals such brain neuroinflammation events as enhanced astrogliosis, to a level comparable to that induced by the administration of pro-inflammatory LPS, and microglia activation in 8-month-old mice. Also, WD feeding accelerated increased Aβ production, observed already in 8-month-old animals. These brain changes corresponded to diet-induced metabolic disorders, including increased cholesterol level in 4-months of age, and advanced hypercholesterolemia and fatty liver disease in 8-month-old mice. These results indicate that the westernized pattern of nourishment is an important modifiable risk factor of AD development, and that a healthy, balanced, diet may be one of the most efficient AD prevention methods.

Keywords: Alzheimer's disease; amyloid; astroglia; liver-brain axis; metabolic syndrome; microglia; neuroinflammation; western diet.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Western diet induces hypercholesterolemia (HChol) and obesity. Graphs show Western diet-induced increases of (A) plasma cholesterol level, and (B) body weight, in 4-, 8-, and 12-month-old APPswe mice in WD and WD + LPS groups compared to CTR and LPS. All the data are presented as arithmetic mean ± SD (n = from 3 to 8 animals per experimental group). The statistics were calculated using unpaired ANOVA with Bonferroni's post-hoc test. A p-value of <0.05 was considered a predetermined threshold for statistical significance.
Figure 2
Figure 2
Western diet induces non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Microscopic images of hematoxylin-eosin histological staining of liver tissue from (A) 4-month-old APPswe mice fed with WD for 3 weeks, (B) 8-month-old APPswe mice fed with WD for 5 months, (C) 12-month-old APPswe mice fed with WD for 9 months; dotted arrows—microvesicular steatosis (discrete round lipid vacuoles within hepatocytes), arrows—hypertrophic hepatocytes, #—hepatocellular ballooning and macrovesicular steatosis, *–lobular inflammation; scale bars correspond to 50 μm; magnification: ×20. (D,E) Graphs show comparison of (D) liver weight in WD and CTR mice at 4-, 8-, and 12-months of age and (E) the liver vs. body weight ratio in WD and CTR mice 4-, 8-, and 12-months of age. Data are presented as arithmetic mean ± SD (n = from 3 to 8 animals per experimental group). The statistics were calculated using unpaired Student t-test. A p-value of <0.05 was considered a predetermined threshold for statistical significance.
Figure 3
Figure 3
Western diet induces low-grade systemic inflammation. Graphs show WD-induced increases of (A) blood granulocytes (GRA) concentration in 4- and 8-month-old APPswe mice, (B) monocytes (MON) concentration in 4- and 8-month-old APPswe mice, (C) lymphocytes (LYM) concentration in 8-month-old APPswe mice and (D) total white blood cells (WBC) concentration in 8-month-old APPswe mice. All the data are presented as arithmetic mean ± SD (n = from 3 to 8 animals per experimental group with exception in 8M LPS groups where means were calculated based on n = 2, and these values were not included in statistical analysis). The statistics were calculated using one-way analysis of variance (ANOVA) followed by Bonferroni's post-test or non-parametric Kruskal-Wallis test followed by Dunn's post-test. A p-value of <0.05 was considered a predetermined threshold for statistical significance.
Figure 4
Figure 4
Western diet accelerates astrogliosis in APPswe mouse hippocampus. (A) Graphs show mean GFAP levels detected by immunoblotting in the hippocampal tissue lysates from CTR, LPS, WD, and WD + LPS groups of mice at 4-, 8-, and 12-months of age. Below are representative immunoblots. WD caused increased GFAP levels in the hippocampus of 4-month-old APPswe mice. Data are presented as arithmetic mean of GFAP/actin ± SD (n = from 4 to 8 animals per experimental group). The statistics were calculated using one-way analysis of variance (ANOVA) followed by Bonferroni's post-test or non-parametric Kruskal-Wallis test followed by Dunn's post-test. A p-value of <0.05 was considered a predetermined threshold for statistical significance. (B) Representing immunofluorescence images show the increases in GFAP staining in the hippocampus in WD and WD + LPS mice compared to CTR mice. All mice were 4-month-old; the scale bar corresponds to 50 μm; magnification ×20; green fluorescence—GFAP, blue fluorescence—Hoechst (nuclei).
Figure 5
Figure 5
Western diet accelerates hippocampal microglia activation and impairs their phagocytic function. (A) Graphs show mean areas of the microglia activation marker Iba1 positive staining detected in hippocampal tissue by immunofluorescence microscopy in CTR, LPS, WD, and WD + LPS groups of APPswe mice at 4-, 8-, and 12- months of age. All the data are presented as the arithmetic mean ± SD of Iba1 positive staining area from many different parts and cross-section of the hippocampi from 1 or 2 representative animals per group. The statistics were calculated using non-parametric Kruskal-Wallis test followed by Dunn's post-test. A p-value of <0.05 was considered a predetermined threshold for statistical significance. Graphs demonstrate that Iba1 immunostaining increased in WD and WD + LPS groups compared to both control groups and that the most significant increase was detected in 8-month-old mice. (A') Representative immunofluorescence images of the data shown in A present the increases in Iba1 positive staining in hippocampal tissue from 8-month-old APPswe mice in WD and WD + LPS groups compared to CTR and LPS groups; the scale bar corresponds to 50 μm; magnification: ×20; red fluorescence—Iba1, blue fluorescence—Hoechst (nuclei). Graphs show mean protein levels detected by immunoblotting of microglial anti-inflammatory homeostatic marker P2RY12 (B) and microglial phagocytic marker CD68 (C) in the mouse hippocampal tissue lysates from CTR, LPS, WD, and WD + LPS groups at the age of 4, 8, and 12 months. Below are representative immunoblots. Immunoblotting results show no differences in P2RY12 hippocampal levels among all experimental groups in 4-, 8-, and 12-month-old APPswe mice (B), and decrease of hippocampal CD68 level in 12-month-old WD mice (C). All the data from P2RY12 and CD68 markers are presented as arithmetic mean ± SD (n = from 4 to 8 animals per experiment). Statistical data in (B,C) were calculated using one-way analysis of variance (ANOVA) followed by Bonferroni's post-test or non-parametric Kruskal-Wallis test followed by Dunn's post-test. A p-value of <0.05 was considered a predetermined threshold for statistical significance.
Figure 6
Figure 6
Western diet accelerates deposition of amyloid-β plaques and APP accumulation in the hippocampus. (A) Comparative immunofluorescence images of brain hippocampal tissue from 4-, 8-, and 12-month-old APPswe mice probed with anti-Aβ antibody 6E10 show accelerated formation of Aβ plaques (*) in the hippocampus of 8-month-old APPswe mice fed with WD (WD and WD + LPS groups compared to CTR and LPS control groups). In control APPswe mice fed with standard diet (CTR) or treated with LPS, Aβ plaques were not observed even at the age of 12 months, and as shown in Figure 3 were detected as late as in 16-month-old mice group; the scale bar corresponds to 50 μm; lens magnification ×20; red fluorescence—6E10 (amyloid-β staining), blue fluorescence—Hoechst (nuclei). (B) Graphs show increased levels of APP full-length and its C-terminal end fragments in WD and WD + LPS experimental groups in 4-month-old APPswe mouse hippocampus obtained in Western blot analysis. All the data are presented as arithmetic mean of APP full-length/actin and CTFs/actin ± SD (n = from 4 to 8 animals per experimental group). The statistics were calculated using one-way analysis of variance (ANOVA) followed by Bonferroni's post-test. A p-value of <0.05 was considered a predetermined threshold for statistical significance.
Figure 7
Figure 7
In 16-month-old APPswe mice deposition of amyloid-β plaques in the hippocampus occurs in all experimental groups including animals fed standard diet. The microphotographs show immunofluorescence labeling of Aβ (6E10) in the brain hippocampal tissue of 16-month-old APPswe mice from CTR, LPS, and WD + LPS experimental groups. Aβ plaques (*) are present in all experimental groups at this age; the scale bar corresponds to 50 μm; lens magnification ×20; red fluorescence—6E10 (amyloid-β), blue fluorescence—Hoechst (nuclei).
Figure 8
Figure 8
Timelines presenting the neuropathology cascades of events in APPswe mice fed with standard diet compared to mice fed western diet. (A) Timeline showing the neuropathological cascades of events in the brain of control APPswe mice fed with standard chow diet. (B) Timeline of the WD-induced neuropathological cascades of events in APPswe mouse brain shows the accelerated pathology induced by Western diet feeding.
Figure 9
Figure 9
Scheme summarizing the role of liver function in the relationship between WD-induced metabolic and systemic disturbances, and neuropathological changes in the brain leading to accelerated AD development. APP, amyloid precursor protein; HChol, hypercholesterolemia; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; MetS, metabolic syndrome.
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