Fig 1.
Global 16S metagenomics profiling.
A) Schematic representation of study design (upper panel); Abundance profiles of microbes (percentage) in mice stool at the phylum taxonomic rank (bottom panel); samples were named as reported in experimental design. B) The Firmicutes/Bacteroidetes ratio was calculated in each animal at the indicated time point T0, T1 and T2, and plotted individually (upper panels) or grouped (bottom panel). An ANOVA analysis was performed using all groups. *p<0,05; *** p<0,001. It is important to note that a statistical difference was only observed compared WT T1 vs WT T2 groups, and WT T2 vs AD T2 groups. C) Relative abundance profiles of microbes in mice stool at family taxonomic rank. D) Boxplots showing alpha diversity (Shannon index) values across experimental conditions at family taxonomic rank. E) PCA plot based on Jensen-Shannon divergence values across experimental conditions at family taxonomic rank. * p<0.05.
Fig 2.
AD progression drives global perturbations in the microbiota.
A) Differences at the genus taxonomic rank in mice stool microbiota between T2 and T0 time points, for AD samples. Plots show alpha diversity (Shannon index) for the samples considered and the corresponding heatmap of the differentially modulated genera. B) Differences at the genus taxonomic rank in mice stool microbiota between T1 and T2 time points, for AD samples. Plots show alpha diversity (Shannon index) for the samples considered and the corresponding heatmap of the differentially modulated genera. C) Differences at the genus taxonomic rank in mice stool microbiota between WT samples and AD samples at T2 time points. Plots show alpha diversity (Shannon index) for the samples considered and the corresponding heatmap of the differentially modulated genera.
Fig 3.
Metabolic profile at six months in AD mouse.
A) Score plot of principal component analysis (PCA) showing two distinct groups, the WT (light blue) and the AD mouse model (red). B) Hierarchical clustering heat-map of the most modulated metabolites, C) box plot of modulated molecules at six months (T2), expressed as ‘normalized intensity’, corresponding to normalized abundance of the molecules reported in the heatmap (panel B). * p<0.05; ** p<0.01; *** p<0.001.
Fig 4.
Short-chain fatty acids modulations over time.
Box plots with p-values of acetic acid, butanoic acid, 3-methyl butanoic acid, valeric acid, propanoic acid, and 2-methyl propanoic acid at weaning (T0), at two months (T1), and at six months (T2). * p<0.05; ** p<0.01; *** p<0.0005.
Fig 5.
Metabolic profile of small molecules from aqueous phase extraction of AD mouse fecal samples, at six months.
A) Score plot of principal component analysis (PCA) showing two distinct groups, the WT (light blue) and the AD mouse model (red). B) Hierarchical clustering heat-map of the most modulated metabolites. C) Box plot of modulated amino acids and phloretic acid at six months (T2), expressed as ‘normalized intensity’, corresponding to normalized abundance of the molecules reported in the heatmap (panel B). * p<0.05; ** p<0.01; *** p<0.0005.
Fig 6.
A) Workflow of the study. Fecal samples from WT and 3xTG mice at weaning, two and six months were collected and analyzed using metabolomic and metagenomic approaches. Metabolome and metagenome profiles were then analyzed and integrated to explore microbiome variations potentially associated with the progression of the disease. B) Circos plot of Spearman correlation between metabolite-phyla (left panel) and class-metabolites-phyla (right panel). A positive correlation is distinguished by red lines, while a negative correlation by green lines. C) Hierarchical heat maps with Spearman correlation between bacterial genera and metabolites (left panel), and with bacterial genera and metabolites-classes (right panel). The red color indicates a positive correlation, while the blue color indicates a negative correlation. The darker color indicates more statistical significance. Symbol * and ** indicates correlation coefficients smaller than 0.05 or 0.01, respectively.
Fig 7.
Functional network analysis of microbiota-metabolome correlations.
Metabolites and microbes are represented as red circles and blue ovals. Their positive and negative correlations are indicated using red and green lines, respectively. In the upper panel is reported the network correlation between metabolites and genera, while in the bottom panel is reported the functional network between metabolite-classes and phyla.