Reduction of Abeta amyloid pathology in APPPS1 transgenic mice in the absence of gut microbiota

Abstract

Alzheimer's disease is the most common form of dementia in the western world, however there is no cure available for this devastating neurodegenerative disorder. Despite clinical and experimental evidence implicating the intestinal microbiota in a number of brain disorders, its impact on Alzheimer's disease is not known. To this end we sequenced bacterial 16S rRNA from fecal samples of Aβ precursor protein (APP) transgenic mouse model and found a remarkable shift in the gut microbiota as compared to non-transgenic wild-type mice. Subsequently we generated germ-free APP transgenic mice and found a drastic reduction of cerebral Aβ amyloid pathology when compared to control mice with intestinal microbiota. Importantly, colonization of germ-free APP transgenic mice with microbiota from conventionally-raised APP transgenic mice increased cerebral Aβ pathology, while colonization with microbiota from wild-type mice was less effective in increasing cerebral Aβ levels. Our results indicate a microbial involvement in the development of Abeta amyloid pathology, and suggest that microbiota may contribute to the development of neurodegenerative diseases.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Comparison of the gut microbiota between conventionally raised (CONVR-) APPPS1 and wild type (WT) mice analyzed with multiple t-tests together with the Holm-Sidak method to correct for multiple comparisons.

(a) Mean sequence relative abundance of gut microbial taxa at phylum level in CONVR-APPPS1 and WT mice, aged 1 month (n = 6 and n = 8 respectively), 3.5 months (n = 7 and n = 8) and 8 months (n = 6 and n = 7). Bacteroides, Firmicutes, Verrucomicrobia and Tenericutes were significantly different between 8 month-old APPPS1 and WT mice (p < 0.001), as well as Proteobacteria (p < 0.01) and Actinobacteria (p < 0.01). No significant differences were found in the younger mice. (b) At genus level, 4 microbial taxa with relative abundance >5% were significantly different between 8 month-old CONVR-APPPS1 and WT mice (***p < 0.001), while younger mice did not show any significant differences. (c) Rarefaction curves (α-diversity vs. sequencing effort) and weighted Unifrac PCoA plot to compare phylogenetic distance matrices of CONVR-APPPS1 and WT mice. Species richness was significantly increased in the 8 month-old APPPS1 mice (p < 0.05, observed species test in QIIME with FDR correction for multiple comparisons). Younger mice did not show significant differences in α-diversity. Clustering of CONVR-APPPS1 mice in the PCoA was significantly separated from the WT mice at 8 months of age (p < 0.001 for both the ANOSIM and the Adonis non-parametric test in QIIME). Experiments were performed three times, and data represent mean values of a representative experiment.

Figure 2. Reduction of Aβ levels in brain and blood of GF-APPPS1 transgenic mice.

(a–d) Levels of Aβ38, Aβ40, Aβ42 and Aβ42/Aβ40 ratio measured by ELISA in 3.5 and 8 month-old conventionally-raised (CONVR-APPPS1) and germ-free APPPS1 mice (GF-APPPS1). Levels of Aβ assessed by western blot in 3.5 month-old (n = 5) (e) and in 8 month-old mice (n = 6). Full blots are shown in Supplementary Fig. 6(f). Plasmatic levels of soluble Aβ40 (g), Aβ42 (h) and ratio Aβ42/Aβ40 (i) by ELISA. Experiments were performed three times, and data represent mean values of a representative experiment. Data represent mean ± SEM. Statistical differences between germ free and conventionally-raised mice: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Reduced amyloid load in GF-APPPS1 transgenic mice.

(a) Thioflavin T-stained brain sections encompassing the cortex and hippocampus of a 3.5 and 8 month-old CONVR-APPPS1 and age-matched GF-APPPS1 mice (scale bar: 150 um, all panels have the same magnification). (b) Quantification of amyloid load on Thioflavin-stained sections demonstrates a significant reduction of cerebral amyloid plaques in young and aged GF-APPPS1 mice (n = 5 and n = 6 respectively) in both cortex and hippocampus, as compared to CONVR-APPPS1 mice (n = 5 and n = 6 respectively). Statistical differences between GF- and CONVR-APPPS1 mice: *p < 0.05, **p < 0.01, ***p < 0.001. Shown are mean ± SEM.

Figure 4. Colonization of 4 month-old GF-APPPS1 mice with the microbiota from aged CONVR-APPPS1 (COLOAD-APPPS1) and CONVR-wild type mice (COLOWT-APPPS1).

After 8 weeks of colonization, levels of cerebral soluble Aβ38 (a), Aβ40 (b), Aβ42 (c) and ratio Aβ42/Aβ40 (d) were evaluated by ELISA in (6 month-old) GF-APPPS1, COLOWT-APPPS1, COLOAD-APPPS1 and CONVR-APPPS1 mice. Levels of Aβ assessed by western blot in 6 month-old GF-APPPS1 and COLOWT-APPPS1 mice (n = 6) (e) and in 6 month-old COLOAD-APPPS1, COLOWT-APPPS1 animals and CONVR-APPPS1 (n = 4) (f) Full blots are shown in Supplementary Fig. 7. Plasmatic levels of soluble Aβ40 (g), Aβ42 (h) and ratio Aβ42/Aβ40 (i) by ELISA. Description of used genotypes (j). Experiments were performed three times, and data represent mean values of a representative experiment. Data represent mean ± SEM. Statistical differences between germ free and conventionally-raised mice: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Comparison of the gut microbiota between COLOWT-APPPS1 and COLOAD-APPPS1 mice at 6 weeks after colonization.

(a) Mean sequence relative abundance of gut microbial taxa at phylum level in COLOWT-APPPS1 and COLOAD-APPPS1 mice (n = 6 and n = 6 respectively). No significant differences were observed at this time point. (b) At genus level, the 4 bacterial taxa that differed in the aged CONVR-APPPS1 and CONVR-WT mice showed similar trends regarding Akkermansia abundance in the colonized mice, but did not reach statistical significance. Experiments were performed three times, and data represent mean values of a representative experiment. Data represent mean ± SEM. Rarefaction curves (α-diversity vs. sequencing effort) and weighted Unifrac PCoA plot to compare phylogenetic distance matrices of (c) COLOWT-APPPS1 and (d) COLOAD APPPS1 mice at day 1, day 4 and week 2, 4 and 6 of colonization. Significant differences in α-diversity were found between COLOAD-APPPS1 and COLOWT-APPPS1 mice at Day 1 (p < 0.05) as well as between COLOAD-APPPS1 at Day 1 and Day 4, Week 2, 4 and 6 (all p < 0.05).