Changes to gut amino acid transporters and microbiome associated with increased E/I ratio in Chd8+/- mouse model of ASD-like behavior

Abstract

Autism spectrum disorder (ASD), a group of neurodevelopmental disorders characterized by social communication deficits and stereotyped behaviors, may be associated with changes to the gut microbiota. However, how gut commensal bacteria modulate brain function in ASD remains unclear. Here, we used chromodomain helicase DNA-binding protein 8 (CHD8) haploinsufficient mice as a model of ASD to elucidate the pathways through which the host and gut microbiota interact with each other. We found that increased levels of amino acid transporters in the intestines of the mouse model of ASD contribute to the high level of serum glutamine and the increased excitation/inhibition (E/I) ratio in the brain. In addition, elevated α-defensin levels in the haploinsufficient mice resulted in dysregulation of the gut microbiota characterized by a reduced abundance of Bacteroides. Furthermore, supplementation with Bacteroides uniformis improved the ASD-like behaviors and restored the E/I ratio in the brain by decreasing intestinal amino acid transport and the serum glutamine levels. Our study demonstrates associations between changes in the gut microbiota and amino acid transporters, and ASD-like behavioral and electrophysiology phenotypes, in a mouse model.

Fig. 1. Chd8^+/− mice show abnormalities in the brain and intestine and ASD-like behaviors.

a Chd8 mutant construction and the sequences of wild-type and mutant alleles. b Full-length CHD8 protein expression in the whole brain and the gut of the mice at embryonic day 14.5 (E14.5) as well as in the hypothalamus of the mice at week 12. n = 8, 4, 7,4, 6 and 6 mice, respectively. c Weights of the whole brain and cerebrum of 12-week-old mice. n = 26, 24, 12, and 8 mice, respectively. d Lengths of the small intestine (SI) and colon of 12-week-old mice. n = 7 and 8 mice, respectively. e Body weights of the mice at weeks 4, 12, and 18. n = 20 (Week 4/Chd8^+/+), 23 (Week 4/Chd8^+/−), 12 (Week 12/Chd8^+/+), 11 (Week 12/Chd8^+/−), 14 (Week 18/Chd8^+/+), and 13 (Week 18/Chd8^+/−) mice, respectively. f Percentages of interaction time (left) and the preference index (right) in the social novelty preference session of the three-chamber social interaction test. n = 24 and 20 mice, respectively. g Representative traces (left) and the percentages of time in the center in the open-field test (right). n = 26 and 24 mice, respectively. h Percentages of time spent in the light box in the light/dark box test. n = 26 and 22 mice, respectively. i Percentages of interaction time (left) and the preference index (right) in the novel object recognition test. Nov novel object, Old old object. n = 25 and 24 mice, respectively. Source data are provided as a Source Data file. Quantitative data are shown as the mean ± SEM. Statistical analysis was determined by the two-tailed Mann–Whitney test (b, c, d, f (right panel), g, h and i (right panel)) and two-way ANOVA with two-tailed Turkey’s test for multiple comparisons (e, f (left panel) and i (left panel)). Significance was indicated by P value. n.s. means no significant difference.

Fig. 2. The E/I imbalance in the Chd8^+/− mice is associated with abnormalities in both the brain and intestine.

a, b Representative traces (top), cumulative distribution plots (bottom) and bar graphs (bottom) showing the amplitude and frequency of mEPSCs (a) and mIPSCs (b) of pyramidal cells in neocortical layer V of adult mice (weeks 8–9). n = 3 mice per group. Four to six data points per mouse. n = 13 (mEPSCs/Chd8^+/+), 12 (mEPSCs/Chd8^+/−), 14 (mIPSCs/Chd8^+/+), and 15 (mIPSCs/Chd8^+/−) data points, respectively. c Levels of L-glutamate (μg/g), glutamate/GABA, and L-glutamine (μg/g) in the cerebrum of 12-week-old mice detected by the targeted metabolomics assay. n = 13 and 12 mice, respectively. d Level of L-glutamine (μg/ml) in the serum of 12-week-old mice detected by the targeted metabolomics assay. n = 12 and 10 mice, respectively. e GSEA plot showing the enrichment of the amino acid transmembrane transporter activity pathway in small intestinal cells. f UMAP plot of single-cell transcriptome profiles of intestines from 12-week-old mice. g, h Highlights of enterocyte cells expressing Slc6a19 (g) and Slc7a8 (h) in the UMAP plot (left), and comparison of the expression levels of Slc6a19 and Slc7a8 between the Chd8^+/+ and Chd8^+/− mice (right) (n = 4 mice). The bar plots represent the mean of abundance of Slc6a19 (top) and Slc7a8 (bottom) in enterocyte cells (n = 1527 and 3445 cells in Chd8^+/+ and Chd8^+/− mice, respectively). Error bars represent 95% confidence interval (CI) of mean value. i Immunofluorescence staining of SLC6A19 and SLC7A8 in the ileum of the mice at week 12. Scale bar, 20 μm. Each symbol represents one image; six to seven images per mouse. n = 17 and 22 symbols, respectively. j Western blotting analysis of SLC6A19 and SLC7A8 protein levels in the small intestine of mice at week 12 (n = 6 mice). Source data are provided as a Source Data file. Quantitative data are shown as the mean ± SEM. Statistical analysis was determined by the two-tailed Mann–Whitney test (a–d, g–j). Significance was indicated by P value (a–d, g–j). n.s. means no significant difference.

Fig. 3. Increased levels of alpha defensins contribute to gut microbiota dysbiosis in the Chd8^+/− mice.

a–d Metagenomic sequencing of the gut microbiota from 8-week-old mice. n = 12 and 11 mice, respectively. a PCA plots (species, Total Sum Scaling-Transformed). b Microbiota composition (genus). c Volcano plots. d LDA plots. e qPCR analysis in the small intestine of 12-week-old mice. n = 12 and 10 mice, respectively. f ELISA of proteins in the small intestinal, cecal, and colonic contents of 12-week-old Chd8^+/+ mice (n = 5). g ELISA of defensin α1 in the small intestinal (SI) and colonic contents (Colon) of 12-week-old mice. n = 12, 11, 14, and 13 mice, respectively. h Schematic diagram for the defensin α1 gavage. i–k A 16S rRNA sequencing of gut microbiota from 63-day-old mice (n = 10 mice). i Microbiota composition (phylum). j PCA plots (genus, Total Sum Scaling-Transformed) (ANOSIM test). k BC distance (Total Sum Scaling-Transformed) between D_W (defensin α1-treated mice and Chd8^+/+ mice), H_W (water-treated mice and Chd8^+/+ mice), and D_K (defensin α1-treated mice and Chd8^+/− mice). Box plots were based on 350 data points and showed center line as median, box limits as upper and lower quartiles, whiskers as 1.5 × interquartile range and dots as outliers. l Schematic diagram for the defensin α1 and α2 gavage, and qPCR analysis of B. uniformis in the feces. n = 9 and 10 mice, respectively. m Schematic diagram for the GM6001 administration. n ELISA of defensin α1 in the cecal contents (left), and qPCR analysis of B. uniformis in the feces (right). n = 9, 6, 5, 12, 7, and 7 mice, respectively. o Multiplexed determination of cytokine levels from colon of 12-week-old mice (pg/mg protein). n = 11 and 10 mice, respectively. Source data are provided as a Source Data file. Ellipses in (a and j) were drawn around each group’s centroid (95%). Quantitative data are shown as the mean ± SEM. Statistical analysis was determined by the two-tailed Mann–Whitney test (e, g, l, and o), one-way ANOVA with two-tailed Tukey’s multiple comparison test (n), and two-way ANOVA with Turkey’s test for multiple comparisons (f). Significance was indicated by P value.

Fig. 4. B. uniformis improves the ASD-like behaviors and restores E/I imbalance in the Chd8^+/− mice.

a Schematic diagram for co-housing experiments. b qPCR analysis of B. uniformis in the feces from 8-week-old mice. n = 26, 16, and 8 mice, respectively. c Percentages of time spent in the light box in the light/dark box test. n = 27, 15, and 8 mice, respectively. d Percentages of the interaction time (left) and the preference index (right) in the social novelty preference session of the three-chamber social interaction test. n = 26, 18, and 8 mice, respectively. e Schematic diagram for B. uniformis gavage. f Percentages of the interaction time (left) and the preference index (right) in the social novelty preference session of the three-chamber social interaction test. n = 12, 11, and 11 mice, respectively. g Representative traces (left) and percentages of time in the center (right) in the open-field test. n = 10, 14, and 11 mice, respectively. h Percentages of time spent in the light box in the light/dark box test. n = 15, 8, and 9 mice, respectively. i, j Representative traces (top), cumulative distribution plots (bottom) and bar graphs (bottom) showing the amplitude and frequency of the mEPSCs (e) and the mIPSCs (f) of pyramidal cells in neocortical layer V. n = 4, 4, and 5 mice, respectively. Four to six data points per mouse. For mEPSCs, n = 17, 16, and 20 data points, respectively; for mIPSCs, n = 18, 22, and 25 data points, respectively. k Levels of L-glutamate (μg/g), glutamate/GABA (Glu/GABA), and L-glutamine (μg/g) in the cerebrum detected by targeted metabolomics assays. n = 16, 9, and 9 mice, respectively. Quantitative data are shown as the mean ± SEM. Statistical analysis was determined by one-way ANOVA with two-tailed Tukey’s multiple comparison test (b, c, d (right panel), f (right panel), g, h, i, j, k) and two-way ANOVA with two-tailed Turkey’s test for multiple comparisons (d (left panel) and f (left panel)). Significance was indicated by P value.

Fig. 5. B. uniformis reduces the expression of intestinal amino acid transporters in the Chd8^+/− mice.

a Serum levels of L-glutamine in of 12-week-old mice detected by targeted metabolomics assays. n = 18, 18, and 11 mice, respectively. b–d Bulk RNA-seq analysis of the small intestine of 12-week-old mice. There were n = 3, n = 3, and n = 5 mice in the KN, KB, and WN groups, respectively (WN: Chd8^+/+ mice gavaged with PBS; KN: Chd8^+/− mice gavaged with PBS; KB: Chd8^+/− mice gavaged with B. uniformis). b Venn diagram illustrating the count of DE genes between KN_WN and DE genes between KB_WN. The DE genes were determined under a strict threshold of adjusted P < 0.01 and |log2(cf)| > 0.585. c Euclidean distance of KB_KN (Chd8^+/− mice gavaged with PBS and Chd8^+/− mice gavaged with B. uniformis), KB_WN, and KN_WN. The two-tailed Mann–Whitney test was used to calculate the significance. Box plots were based on 350 data points and showed center line as median, box limits as upper and lower quartiles, whiskers as 1.5 × interquartile range and dots as outliers. d GSEA plot shows the enrichment of the amino acid transmembrane transporter activity pathway in small intestinal cells of the Chd8^+/− mice gavaged with PBS compared to the Chd8^+/− mice gavaged with B. uniformis. e Immunofluorescence staining of SLC6A19 and SLC7A8 in the ileum of the mice at week 12. Scale bar, 20 μm. n = 3 mice per group. Each symbol represents one image; seven to nine images per mouse. n = 28, 26, and 21 symbols, respectively. f Western blotting analysis of the SLC6A19 and SLC7A8 protein levels in the small intestine of the mice at week 12. n = 9, 7, and 11 mice, respectively. Quantitative data are shown as the mean ± SEM. Statistical analysis was determined by one-way ANOVA with two-tailed Tukey’s multiple comparison test (a, e, f). Significance was indicated by P value.

Fig. 6. S-Benzyl-L-cysteine improves the ASD-like behaviors and restores E/I imbalance in the Chd8^+/− mice.

a Schematic diagram showing S-benzyl-L-cysteine and nimesulide as inhibitors of the intestinal amino acid transporter SLC6A19. b Schematic diagram for supplementation of S-benzyl-L-cysteine and nimesulide in the drinking water of 8-week-old Chd8^+/+ mice for 14 days. n = 10, 11, and 10 mice, respectively. c Serum levels of L-glutamine detected by the targeted metabolomics assays (n = 10 mice). d Levels of L-glutamine and L-glutamate in the whole brain detected by the targeted metabolomics assays (n = 10 mice). e Schematic diagram for supplementation of S-benzyl-L-cysteine in the drinking water of 21-day-old Chd8^+/+ and Chd8^+/− mice. f Percentages of the interaction time (left) and the preference index (right) in the social novelty preference session of the three-chamber social interaction test. n = 19, 10, and 10 mice, respectively. g Percentages of time spent in the light box in the light/dark box test. n = 21, 11, and 11 mice, respectively. h Representative traces (left) and percentages of time in the center (right) in the open-field test. n = 21, 11, and 11 mice, respectively. i Levels of L-glutamate, glutamate/GABA (Glu/GABA) and L-glutamine in the cerebrum and the level of L-glutamine in the serum detected by targeted metabolomics assays. n = 20, 10, and 10 mice, respectively. Source data are provided as a Source Data file. Quantitative data are shown as the mean ± SEM. Statistical analysis was determined by the two-tailed Mann–Whitney test (d), one-way ANOVA with two-tailed Tukey’s multiple comparison test (c, f (right panel), g, h, i), and two-way ANOVA with two-tailed Turkey’s test for multiple comparisons (f (left panel)). Significance was indicated by P value.

Fig. 7. Summary of the microbiota–gut–brain axis in the CHD8 mouse model of ASD.

Chd8^+/− mice show increased expression of the intestinal transporters SLC6A19 and SLC7A8, resulting in more glutamine transported to the serum. Elevated serum glutamine increases the levels of glutamine and glutamate in the brain, contributing to the E/I imbalance and ASD-like behaviors in the Chd8^+/− mice. Gut microbiota dysbiosis characterized by a low abundance of Bacteroides in the Chd8^+/− mice is induced by high levels of intestinal α-defensins. In addition, supplementation with B. uniformis improves the E/I imbalance and restores ASD-like behaviors in the Chd8^+/− mice by decreasing the intestinal amino acid transport of glutamine and thus reducing the glutamine and glutamate levels in the brain.