Abnormal gut microbiota composition contributes to cognitive dysfunction in SAMP8 mice

Gaofeng Zhan, Ning Yang, Shan Li, Niannian Huang, Xi Fang, Jie Zhang, Bin Zhu, Ling Yang, Chun Yang, Ailin Luo, Gaofeng Zhan, Ning Yang, Shan Li, Niannian Huang, Xi Fang, Jie Zhang, Bin Zhu, Ling Yang, Chun Yang, Ailin Luo

Abstract

Alzheimer's disease is characterized by cognitive dysfunction and aging is an important predisposing factor; however, the pathological and therapeutic mechanisms are not fully understood. Recently, the role of gut microbiota in Alzheimer's disease has received increasing attention. The cognitive function in senescence-accelerated mouse prone 8 (SAMP8) mice was significantly decreased and the Chao 1 and Shannon indices, principal coordinates analysis, and principal component analysis results were notably abnormal compared with that of those in senescence-accelerated mouse resistant 1 (SAMR1) mice. Moreover, 27 gut bacteria at six phylogenetic levels differed between SAMP8 and SAMR1 mice. In a separate study, we transplanted fecal bacteria from SAMP8 or SAMR1 mice into pseudo germ-free mice. Interestingly, the pseudo germ-free mice had significantly lower cognitive function prior to transplant. Pseudo germ-free mice that received fecal bacteria transplants from SAMR1 mice but not from SAMP8 mice showed improvements in behavior and in α-diversity and β-diversity indices. In total, 14 bacteria at six phylogenetic levels were significantly altered by the gut microbiota transplant. These results suggest that cognitive dysfunction in SAMP8 mice is associated with abnormal composition of the gut microbiota. Thus, improving abnormal gut microbiota may provide an alternative treatment for cognitive dysfunction and Alzheimer's disease.

Keywords: Alzheimer’s disease; SAMP8; cognitive dysfunction; gut microbiota; pseudo germ-free mice.

Conflict of interest statement

CONFLICTS OF INTEREST: CY received research support from B. Braun. Other authors declared no conflicts of interest.

Figures

Figure 1
Figure 1
MWMT for SAMR1 and SAMP8 mice. (A) The schedule for MWMT and fecal sample collection. Seven days after accommodation, SAMR1 and SAMP8 mice were scheduled for MWMT and probe trial on days 1–6. On day 7, fecal samples were collected for 16S rRNA gene sequencing and fecal bacteria transplant. (B) Body weight (T-test, P > 0.05). (C) The trace graph of SAMR1 and SAMP8 mice in MWMT. (D) Escape latency (two-way ANOVA; Time: F4,20 = 6.062, P < 0.01; Group: F1,5 = 8.802, P < 0.05; Interaction: F4,20 = 3.476, P < 0.05). (E) Escape path length (two-way ANOVA. Time: F4,20 = 11.32, P < 0.001; Group: F1,5 = 4.834, P = 0.0792; Interaction: F4,20 = 3.271, P < 0.05). (F) Platform crossing (t-test, P < 0.05). (G) Time spent in each quadrant (t-test, P < 0.05). ANOVA: analysis of variance; MWMT: Morris water maze test. Data are shown as mean ± SEM (n = 6). *P < 0.05, **P < 0.01.
Figure 2
Figure 2
Differential profiles of the gut microbiota between SAMR1 and SAMP8 mice. (A) Heat map of differential levels of bacteria between the groups. (B) Chao 1 index (t-test, P < 0.01). (C) Shannon index (t-test, P < 0.05). (D) Simpson index (t-test, P > 0.05). (E) PCoA analysis of gut bacteria data (Bray–Curtis dissimilarity). (F) PCA analysis of gut bacteria data. α-diversity data are shown as mean ± SEM (n = 10). PCA: principal component analysis; PCoA: principal coordinates analysis. *P < 0.05, **P < 0.01.
Figure 3
Figure 3
Differential levels of the gut bacteria between SAMR1 and SAMP8 mice. (A) Phylum Deferribacteres (t-test, P < 0.05). (B) Class Deferribacteres (t-test, P < 0.05). (C) Class Deltaproteobacteria (t-test, P < 0.001). (D) Order Deferribacterales (t-test, P < 0.05). (E) Order Desulfovibrionales (t-test, P < 0.001). (F) Order Mollicutes RF9 (t-test, P < 0.01). (G) Family Clostridiales vadinBB60 group (t-test, P < 0.05). (H) Family Desulfovibrionaceae (t-test, P < 0.001). (I) Family Christensenellaceae (t-test, P < 0.001). (J) Family Family XIII (t-test, P < 0.001). (K) Family Deferribacteraceae (t-test, P < 0.001). (L) Family Ruminococcaceae (t-test, P < 0.001). (M) Genus Mucispirillum (t-test, P < 0.05). (N) Genus Serratia (t-test, P < 0.05). (O) Genus Family XIII AD3011 group (t-test, P < 0.001). (P) Genus Christensenellaceae R-7 group (t-test, P < 0.01). (Q) Genus Subdoligranulum (t-test, P < 0.05). (R) Genus Desulfovibrio (t-test, P < 0.001). (S) Genus Ruminiclostridium 9 (t-test, P < 0.01). (T) Genus Coprococcus 1 (t-test, P < 0.05). (U) Genus Ruminococcaceae UCG 004 (t-test, P < 0.05). (V) Genus Lachnospiraceae NK4A136 group (t-test, P < 0.05). (W) Genus Lachnospiraceae oscillibacter (t-test, P < 0.001). (X) Species Desulfovibrio sp. UNSW3caefatS (t-test, P < 0.001). (Y) Species uncultured Deferribacteraceae bacterium (t-test, P < 0.05). (Z) Species uncultured Bacteroidales bacterium (t-test, P < 0.001). (AA) Species Unidentified (t-test, P < 0.05).
Figure 4
Figure 4
Effects of SAMR1 and SAMP8 mice fecal microbiota transplant on behavior in pseudo germ-free mice. (A) Schedule of fecal microbiota transplant on behavioral testing in pseudo germ-free mice. Mice were first treated by administering large doses of antibiotic solution for 14 consecutive days. Thereafter, mice were orally treated with fetal microbiota of SAMR1 and SAMP8 mice. MWMT was performed on days 29–33, and the probe trial was performed on day 34. On day 35, fecal samples were collected for 16S rRNA gene sequencing testing. (B) Body weight (Two-way ANOVA; Time: F2,14 = 25.59, P < 0.001; Group: F3,21 = 0.1132, P = 0.9514; Interaction: F6,42 = 0.6019, P = 0.7272.). (C) The trace graph of mice in MWMT. (D) Escape latency (two-way ANOVA; Time: F4,28 = 17.23, P < 0.001; Group: F3,21 = 3.27, P < 0.05; Interaction: F12,84 = 0.9568, P = 0.4961). (E) Escape path length (two-way ANOVA; Time: F4,28 = 32.04, P < 0.001; Group: F3,21 = 8.668, P < 0.001; Interaction: F12,84 = 0.7349, P = 0.7137.). (F) Platform crossing (one-way ANOVA; F3,28 = 8.745, P < 0.001.). (G) Time spent in each quadrant (one-way ANOVA; F3,28 = 9.133, P < 0.001). ANOVA: analysis of variance; MWMT: Morris water maze test. Data are shown as mean ± SEM (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
α-diversity and β-diversity of fecal microbiota transplant in pseudo germ-free mice. (A) Chao 1 index (One-way ANOVA; F3,24 = 21.28, P < 0.001). (B) Observed species index (One-way ANOVA; F3,24 = 17.29, P < 0.001). (C) PD whole tree (One-way ANOVA; F3,24 = 16.76, P < 0.001). (D) Shannon index (One-way ANOVA; F3,24 = 50.19, P < 0.001). Data are shown as mean ± SEM (n = 7). *P < 0.05, **P < 0.01, ***P < 0.001. (E) PCoA analysis of gut bacteria data (Bray–Curtis dissimilarity). (F) NMDS analysis of gut bacteria data. NMDS: non-metric multi-dimensional scaling; PCoA: principal coordinates analysis.
Figure 6
Figure 6
Effects of fecal microbiota transplant on levels of gut microbiota in pseudo germ-free mice. (A) Phylum Firmicutes (One-way ANOVA; F3,24 = 34.61, P < 0.001). (B) Class Betaproteobacteria (One-way ANOVA; F3,24 = 37.98, P < 0.001). (C) Order Bacteroidales (One-way ANOVA; F3,24 = 17.77, P < 0.001). (D) Order Burkholderiales (One-way ANOVA; F3,24 = 41.43, P < 0.001). (E) Order Clostridiales (One-way ANOVA; F3,24 = 36.92, P < 0.001). (F) Family Alcaligenaceae (One-way ANOVA; F3,24 = 34.95, P < 0.001). (G) Family Lachnospiraceae (One-way ANOVA; F3,24 = 40.75, P < 0.001). (H) Family Prevotellaceae (One-way ANOVA; F3,24 = 26.69, P < 0.001). (I) Genus Lachnospiraceae NK4A136 group (One-way ANOVA; F3,24 = 30.02, P < 0.001). (J) Genus Moryella (Fisher’s exact test; P < 0.001). (K) Genus Parasutterella (One-way ANOVA; F3,24 = 37.64, P < 0.001). (L) Genus Peptococcus (Fisher’s exact test; P < 0.001). (M) Genus Prevotellaceae NK3B31 group (Fisher’s exact test; P < 0.05). (N) Species Lachnospiraceae bacterium 615 (Fisher’s exact test; P < 0.05). ANOVA: analysis of variance

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