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. 2020 Jul 29;6(31):eaba0466.
doi: 10.1126/sciadv.aba0466. eCollection 2020 Jul.

Gut dysbiosis contributes to amyloid pathology, associated with C/EBPβ/AEP signaling activation in Alzheimer's disease mouse model

Chun Chen  1 , Eun Hee Ahn  1 , Seong Su Kang  1 , Xia Liu  1 , Ashfaqul Alam  2 , Keqiang Ye  1
Affiliations

Gut dysbiosis contributes to amyloid pathology, associated with C/EBPβ/AEP signaling activation in Alzheimer's disease mouse model

Chun Chen et al. Sci Adv. .

Abstract

The gut-brain axis is bidirectional, and gut microbiota influence brain disorders including Alzheimer's disease (AD). CCAAT/enhancer binding protein β/asparagine endopeptidase (C/EBPβ/AEP) signaling spatiotemporally mediates AD pathologies in the brain via cleaving both β-amyloid precursor protein and Tau. We show that gut dysbiosis occurs in 5xFAD mice, and is associated with escalation of the C/EBPβ/AEP pathway in the gut with age. Unlike that of aged wild-type mice, the microbiota of aged 3xTg mice accelerate AD pathology in young 3xTg mice, accompanied by active C/EBPβ/AEP signaling in the brain. Antibiotic treatment diminishes this signaling and attenuates amyloidogenic processes in 5xFAD, improving cognitive functions. The prebiotic R13 inhibits this pathway and suppresses amyloid aggregates in the gut. R13-induced Lactobacillus salivarius antagonizes the C/EBPβ/AEP axis, mitigating gut leakage and oxidative stress. Our findings support the hypothesis that C/EBPβ/AEP signaling is activated by gut dysbiosis, implicated in AD pathologies in the gut.

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Figures

Fig. 1
Fig. 1. Microbiome analysis in 5xFAD mouse stool reveals an alteration in the microbial community.
(A) Relative abundance of bacterial phyla determined by HTS analysis (n = 5 per group). (B) Principal coordinate plot (PCoA) (Bray-Curtis index) of microbial community structure in an age-dependent manner in 5xFAD and WT mouse stool. m, months. (C) Boxplot of α-diversity pattern (Chao1) of the microbiota across different age groups in the 5xFAD and WT mouse stool. (D and E) Hierarchical clustering of the core microbial taxa shows alterations of abundances at the genus level across different age groups of 5xFAD and WT mouse. (D) A dendrogram showing clustering of samples across age and genetic backgrounds. (E) Heatmap analysis using taxonomic abundances. Hierarchical clustering was performed using a Spearman’s correlation–based dissimilarity matrices and agglomeration method.
Fig. 2
Fig. 2. Gut microbiomes from aged 3xTg mice activate C/EBPβ/AEP in young 3xTg mice brain, facilitating cognitive dysfunction.
(A) Relative abundance of bacterial phyla determined by HTS analysis (n = 5 per group). (B) PCoA of microbiota community structure in young 3xTg mice cohoused with either aged 3xTg mice or aged WT mice, aged 3xTg mice, and aged WT mice. (C) GI permeability barrier defect, as determined by FITC-dextran translocation in 3xTg mice. 3xTg mice cohoused with aged 3xTg mice developed much severe leaky gut than control 3xTg mice (n = 3 per group). Data represent the means ± SEM; *P < 0.05 compared with control, one-way analysis of variance (ANOVA). (D) Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and processing in the mouse brains. (E) AEP activity assay in the brain lysates (n = 3 per group). Data represent the means ± SEM; **P < 0.01 and ***P < 0.001 compared with control, one-way ANOVA. RFU, relative fluorescence units. (F) Representative EM of the synaptic structures. Red circles indicate the synapses. Scale bars, 1 μm. (G) Quantitative analysis of the synaptic densities in 3xTg mice brains. 3xTg mice cohoused with aged 3xTg mice showed decreased synaptic densities. (n = 5 per group). Data are shown as means ± SEM; **P < 0.01. (H to K) MWM cognitive assays. 3xTg control mice, 3xTg mice cohoused with aged 3xTg mice, or aged WT mice were trained in the water maze more than 5 days. Shown are the means ± SEM latency to platform and the area under curve of latency (AUC latency) (H), means ± SEM swim path distance and the area under curve of distance (AUC distance) (I), the percentage of time spent in the target quadrant in the probe trail (J), and swim speed (K); *P < 0.05, **P < 0.01 compared to control 3xTg mice.
Fig. 3
Fig. 3. C/EBPβ/AEP pathway is escalated in an age-dependent way in 5xFAD mice colon.
(A) IF staining of p-C/EBPβ and MAP2 in colon of 5xFAD mice. Quantitative analysis of p-C/EBPβ–positive cells and MAP2-positive cells. Data represent the means ± SEM; representative data of five samples; ****P < 0.0001. (B) IF staining of C/EBPβ and AEP in the colon of 5xFAD mice. Quantitative analysis of C/EBPβ-positive cells and AEP-positive cells. Data represent the means ± SEM; representative data of five samples; *P < 0.05, ***P < 0.001, and ****P < 0.0001. (C) IF staining of Aβ and cleaved Tau N368 in colon of 5xFAD mice. Quantitative analysis of Aβ-positive cells and cleaved Tau N368–positive cells. Data represent the means ± SEM; representative data of five samples; *P < 0.05 and ****P < 0.0001. (D) IF staining of AT8 and cleaved APP C586 in the colon of 5xFAD mice. Quantitative analysis of AT8-positive cells and cleaved APP C586–positive cells. Data represent the means ± SEM; representative data of five samples. ****P < 0.0001. Scale bars, 20 μm (A to D).
Fig. 4
Fig. 4. Antibiotic treatment represses C/EBPβ/AEP signaling in 5xFAD mice and rescues the cognitive dysfunctions.
(A) Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and processing in the mouse brains. MW, molecular weight. (B) AEP activity assay in the brain lysates from 5xFAD treated with antibiotics or vehicle. Data represent the means ± SEM; representative data of three samples; *P < 0.05 compared with control, one-way ANOVA. (C) Aβ1-42 concentrations in the cortex of 5xFAD treated with antibiotics or vehicle. Data represent the means ± SEM; representative data of four samples; *P < 0.05 compared with control, one-way ANOVA. (D) Anti-Aβ staining of amyloid plaques in the hippocampus of 5xFAD mice brain sections. Scale bar, 200 μm. (E) Quantitative analysis of Aβ-positive plaques. The density of plaques in male 5xFAD mice brain was significantly decreased by antibiotic treatment. Data represent the means ± SEM; representative data of three samples; ***P < 0.001 compared with vehicle, one-way ANOVA. (F) The percentage of time spent in the target quadrant in the probe trail. Mice treated with antibiotics spent more time in the target quadrant than the control mice. Data represent the means ± SEM; representative data of 10 samples; *P < 0.05 compared with control, one-way ANOVA. (G) Area under curve of latency. Data represent the means ± SEM; representative data of 10 samples; **P < 0.01 compared with control, one-way ANOVA. (H) Fear-conditioning tests. Data represent the means ± SEM of n = 10 mice per group; *P < 0.05, one-way ANOVA. CS, conditional stimulus.
Fig. 5
Fig. 5. R13 administration blunts C/EBPβ/AEP signaling in the gut of 5xFAD mice.
(A) Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and processing in the mouse colon. (B) IF staining of C/EBPβ. Scale bar, 20 μm. (C to G) IF staining of AEP and Aβ in colon of 5xFAD mice and the quantification. Scale bars, 200 μm. (H) AEP activity assay of the colon lysates from 5xFAD treated with antibiotics or vehicle. Data represent the means ± SEM; representative data of six samples; *P < 0.05 compared with control, one-way ANOVA. (I) GI permeability barrier defect as determined by FITC-dextran translocation in R13-treated 5xFAD mice and vehicle mice. Data represent the means ± SEM; representative data of four samples; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control, one-way ANOVA. (J) R13 treatment decreased IL-6 but not TNF-α or IL-1β expression. Data represent the means ± SEM; representative data of four samples; ***P < 0.001 compared with control, one-way ANOVA.
Fig. 6
Fig. 6. Gut microbiota analysis by 16S rRNA from the fecal samples of 5xFAD mice, chronically treated with R13.
(A) Relative abundance of bacteria phyla determined by HTS (n ≥ 5 mice per group). (B to E) Mean bacteria abundance of bacterial species. Data represent the means ± SEM; representative data five samples; *P < 0.05 compared with control, one-way ANOVA. (F) PCoA of microbiota community structure in vehicle- and R13-treated 5xFAD mice. (G) In vitro analysis of T1 and 7,8-DHF on bacteria growth. Mean bacteria abundance of bacteria species and phyla was determined. Data represent the means ± SEM; representative data of five samples; *P < 0.05 compared with control; one-way ANOVA.
Fig. 7
Fig. 7. R13-induced probiotic L. salivarius suppresses C/EBPβ/AEP signaling in the brain of 5xFAD mice, decreasing gut leakage.
(A) Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and processing in the mouse brains. LS, L. salivarius. (B) AEP activity assay in the brain lysates from 5xFAD treated with live L. salivarius, boiled L. salivarius, or control. Data represent the means ± SEM; representative data of three samples; *P < 0.05 compared with control, one-way ANOVA. (C) Live L. salivarius treatment decreased IL-6 concentrations in the mouse brains. Data represent the means ± SEM; representative data of three samples; *P < 0.05 compared with control, one-way ANOVA. (D) BDNF concentrations in the brains from 5xFAD treated with live L. salivarius, boiled L. salivarius, or control. Data represent the means ± SEM; representative data of three samples. (E) Aβ1-40 and Aβ1-42 concentrations in 5xFAD mice treated with live L. salivarius, boiled L. salivarius, or control, respectively. Data represent the means ± SEM; representative data of three samples. (F) GI permeability barrier defect, as determined by FITC-dextran translocation in 5xFAD mice treated with live L. salivarius, boiled L. salivarius, or control. Data represent the means ± SEM; representative data of six samples; **P < 0.01 and ***P < 0.001, compared with control, one-way ANOVA. (G to I) IF staining of C/EBPβ and AEP; Aβ/APP C586 and Aβ/ThS on the brain sections from 5xFAD mice treated with live L. salivarius, boiled L. salivarius, or control. Scale bars, 20 μm.
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