Transplantation of fecal microbiota from APP/PS1 mice and Alzheimer's disease patients enhanced endoplasmic reticulum stress in the cerebral cortex of wild-type mice

Abstract

Background and purpose: The gut-brain axis is bidirectional and the imbalance of the gut microbiota usually coexists with brain diseases, including Alzheimer's disease (AD). Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a core lesion in AD and persistent ER stress promotes AD pathology and impairs cognition. However, whether the imbalance of the gut microbiota is involved in triggering the ER stress in the brain remains unknown.

Materials and methods: In the present study, fecal microbiota transplantation (FMT) was performed with gut microbiota from AD patients and APP/PS1 mice, respectively, resulting in two mouse models with dysregulated gut microbiota. The ER stress marker protein levels in the cerebral cortex were assessed using western blotting. The composition of the gut microbiota was assessed using 16S rRNA sequencing.

Results: Excessive ER stress was induced in the cerebral cortex of mice after FMT. Elevated ER stress marker proteins (p-perk/perk, p-eIF2α/eIF2α) were observed, which were rescued by 3,3-dimethyl-1-butanol (DMB). Notably, DMB is a compound that significantly attenuates serum trimethylamine-N-oxide (TMAO), a metabolite of the gut microbiota widely reported to affect cognition.

Conclusion: The findings indicate that imbalance of the gut microbiota induces ER stress in the cerebral cortex, which may be mediated by TMAO.

Keywords: Alzheimer’s disease; ER stress; FMT; TMAO; gut microbiota.

FIGURE 1

Antibiotic cocktail administration did not affect endoplasmic reticulum (ER) stress in the cerebral cortex of wild-type (WT) mice. (A) Experimental design of antibiotic cocktail-treated WT mice (mice were administered an antibiotic cocktail by gavage). (B) Representative immunoblots showing p-perk and p-eif2a levels in the cerebral cortex on day 3 of the experiment; n = 3 for each group. (C) Representative immunoblots on day 17 of the experiment showed p-perk and p-eif2a levels in the cerebral cortex. Data were compared between the two groups using a two-tailed Student’s t-test.

FIGURE 2

Cortical endoplasmic reticulum (ER) stress marker protein and serum hs-CRP, LPS, and TMAO levels in wild-type (WT) mice after fecal microbiota transplantation (FMT). (A) Experimental design of FMT-treated mice. (B) Representative immunoblots showing p-perk and p-eif2a levels in the cerebral cortex on day 17 of the experiment. (C) Serum hs-CRP levels after FMT. (D) Serum LPS levels after FMT. (E) Serum TMAO levels after FMT. P < 0.05 was set as the threshold for significance by one-way ANOVA followed by post-hoc comparisons using LSD’s test for multiple groups’ comparisons, *p < 0.05 and **p < 0.01.

FIGURE 3

Alterations in the gut microbiota of wild-type (WT) recipient mice after fecal microbiota transplantation (FMT). (A) Principal coordinates analysis (PCoA) plots based on unweighted UniFrac distances. (B) Alterations in relative abundance (RA) of gut microbiota based on the heatmap. (C) RA bar plots of gut microbiota between groups at the phylum and family levels. P < 0.05 was set as the threshold for significance by one-way ANOVA followed by post-hoc comparisons using LSD’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, *** p < 0.001, and ****p < 0.0001.

FIGURE 4

Trimethylamine-N-oxide (TMAO) inhibition with DMB reversed endoplasmic reticulum (ER) stress in the cerebral cortex. (A) Experimental design of DMB-treated mice. (B) Serum TMAO levels after DMB treatment. (C–E). Representative immunoblots on day 17 of the experiment showed p-perk and p-eif2a levels in the cerebral cortex. P < 0.05 was set as the threshold for significance by one-way ANOVA followed by post-hoc comparisons using LSD’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, and ****p < 0.0001.