Fto Deficiency Reduces Anxiety- and Depression-Like Behaviors in Mice via Alterations in Gut Microbiota

Abstract

Depression and obesity have high concurrence within individuals, which may be explained by sharing the same risk factors, including disruption of the intestinal microbiota. However, evidence that delineated the causal connections is extremely scarce. Methods: Mice lacking fat mass- and obesity-associated gene (Fto) were generated. Fto-deficient and wild-type control mice were subjected to novel conditions with or without chronic unpredictable mild stress (CUMS) for 6 weeks. Some mice were treated with antibiotics via their drinking water for 6 weeks in order to deplete their microbiota. Behavioral tests were performed to evaluate anxiety- and depression-like behaviors. 16S rRNA amplicon and metagenomic sequencing were employed to analyse fecal microbiota. Plasma levels of inflammatory cytokines and lipopolysaccharides (LPS) were also compared. Results: Deletion of Fto led to lower body weight and decreased anxiety- and depression-like behaviors, Fto+/- mice were also less susceptible to stress stimulation, highlighting the essential role of Fto in pathogenesis of depression. With regard to gut microbiota, Fto deficiency mice harbored specific bacterial signature of suppressing inflammation, characterized with higher abundance of Lactobacillus, lower Porphyromonadaceae and Helicobacter. Critically, behavioral alterations of Fto^+/- mice are mediated by shift in gut microbiota, as such changes can be partially attenuated using antibiotics. Exposure to CUMS increased serum IL-6 level while Fto deficiency reduced its level, which may be explained by a lower LPS concentration. Conclusion: Together, our findings uncover the roles of Fto on depression and provide insights into microbiota-related biological mechanisms underlying the association between obesity and depression.

Keywords: Fto; Depression; Lactobacillus; Microbiota; Obesity.

Figure 1

Experimental schedule of the present study (11 weeks). Briefly, all animals were brought to the experimental room and adapted for 1 week prior to stress stimulation. Approximately 48 adult male mice were subjected to various tests and conditions during the following 6 weeks: Wild-type (WT) and heterozygous (HZ) mice received no treatment, while mice in the WT+CUMS and HZ+CUMS groups were subjected to chronic unpredictable mild stress (CUMS) for 6 weeks. In the WT+CUMS+antibiotics and HZ+CUMS+antibiotics groups, mice were subjected to the CUMS protocol for 6 weeks, during which they were offered drinking water mixed with multiple antibiotics. All animals underwent a series of behavioral tests during week 8. In the following 2 weeks, the mice were again subjected to different protocols. EPM: elevated plus maze; FST: forced swimming test; OFT: open-field test; TST: tail-suspension test.

Figure 2

Fto-knockout mice exhibited alterations in anxiety- and depression-like behaviors. (A) Fto-knockout mice spent more time on open arms in EPM; (B) more time in center in OFT; (C) less time in immobility in FST; (D) There was no significant difference of immobility time in TST; (E) Fto HZ mice spent more time on open arms after restraint stress in EPM; (F) Fto HZ mice spent less time in immobility after restraint stress in FST. EPM: elevated plus maze; FST: forced swim test; HZ: heterozygous; OFT: open-field test; TST: tail-suspension test. These data are presented as the mean±s.e.m. *P < 0.05 **P < 0.01 ***P < 0.001, between two groups, as measured by independent samples t-test.

Figure 3

Fto-knockout mice harbored a specific gut microbial signature to suppress inflammation. (A) PCA revealed a trend of separation by genotype (WT, n=8; KO, n=8). (B) Microbial distribution at the genus level. (Lactobacillus is marked in red). (C) Relationship of OTUs with different abundances. Lactobacillus (OTU3) was negatively correlated with almost all other OTUs with different relative abundances observed between the two groups. (D) Correlation of Lactobacillus abundance with behavioral indices. EPM: elevated plus maze; FST: forced swim test; KO: knockout; OFT: open-field test; OTU: operational taxonomic unit; PCA: principal component analysis; TST: tail suspension test; WT: wild-type.

Figure 4

Lactobacillus plays a key role in altering behavior. (A) Changes in microbial species composition were analyzed via metagenomics sequencing (WT, n=7; KO, n=7). (B) Heatmap of the Spearman's rank correlation coefficient between behavioral indices and Lactobacillus species. Lactobacillus species showed were significantly higher in KO group by using Wilcoxon rank sum test. n=14, *P< 0.05 **P<0.01 ***P<0.001. (C) Analysis for KEGG pathway using Lefse. Functional divergence between gut microbiota of WT and KO mice. FST: forced swim test; FT: floating time; IT: immobility time; KO: knockout; L: Lactobacillus; OA: open arms; TST: tail suspension test; WT: wild-type.

Figure 5

Fto deficiency-induced hyposensitivity to anxiety and depression is largely dependent on gut microbiota. (A) Fto heterozygous (HZ) mice spent more time on the open arms during the EPM test after CUMS. Our analyses confirmed that these changes were not due to alterations in basal locomotor activity. However, the difference between the two groups was reduced following antibiotic treatment. There was no significant difference in open arm preference. (B) Fto HZ mice spent more time in the central area during the OFT. Our analyses again confirmed that these changes were not due to alterations in basal locomotor activity, and that the difference between the two groups was reduced following antibiotic treatment. There was no significant difference in fecal pellets. (C) Fto HZ mice spent less time being immobile during the FST, although the difference between the two groups was reduced following antibiotic treatment. (D) There was no difference in immobility time during the TST between the groups. Data are presented as mean±s.e.m. The multi-factor analysis of variance(ANOVA; genotype×treatment) revealed no significant interaction between factors for Time on open arms, Time in center and immobility time in FST, TST. Arm entries and Distance travelled are covariates for time on open arms and time in center respectively. It revealed a significant effect of genotype for time on open arms (P<0.0001), time in center (P=0.03), immobility time in FST (P=0.01) and a significant effect of treatment for time on open arms(P=0.001), open arms preference(P<0.0001), fecal pellets(P<0.0001), immobility time in FST(P=0.006). *P< 0.05 **P<0.01 ***P<0.001 Difference between the two groups was measured according to independent samples t-test. Abs: Antibiotics; CUMS: chronic unpredictable mild stress; EPM: elevated plus maze; FST: forced swim test; IT: Immobility Time; OFT: open-field test; TST: tail-suspension test.

Figure 6

Inflammatory responses closely mirrored the observed behavioral changes. (A) Expression of IL-1β, IL-6, IL-17, IL-4, IL-10, and IL-22. The expression of IL-6 increased significantly after CUMS, and was lower in the Fto HZ group. Data are presented as mean±s.e.m. The multi-factor analysis of variance(ANOVA; genotype×treatment) revealed a significant interaction between factors for IL-6 (P=0.001). It revealed a significant effect of genotype for LPS (P<0.0001), IL-6 (P=0.002) and IL-10 (P=0.035). It also revealed a significant effect of treatment for IL-6(P=0.018). (B) Levels of serum LPS were much lower in the HZ group. Data with '*' indicate a significant difference (P<0.05) between the two groups according to the independent samples t-test. Abs: Antibiotics; CUMS: chronic unpredictable mild stress; HZ: heterozygous; IL: interleukin; LPS: lipopolysaccharide; WT: wild-type.

Figure 7

Schematic overview of the phenotype observed following global deletion of Fto. Fto deletion reduces anxiety- and depression-like behaviors under novel conditions and increases resistance to stress—changes that are accompanied by a specific gut bacterial signature to suppress inflammation, which characterized with higher abundance of Lactobacillus, lower Porphyromonadaceae and Helicobacter. Moreover, this higher abundance of Lactobacillus attenuates stress-induced dysbiosis and decreases the inflammatory response, which in turn participates in generating the behavioral phenotype observed in the present study.