Acetate supplementation rescues social deficits and alters transcriptional regulation in prefrontal cortex of Shank3 deficient mice

Abstract

Background: The pathophysiology of autism spectrum disorder (ASD) involves genetic and environmental factors. Mounting evidence demonstrates a role for the gut microbiome in ASD, with signaling via short-chain fatty acids (SCFA) as one mechanism. Here, we utilize mice carrying deletion to exons 4-22 of Shank3 (Shank3^KO) to model gene by microbiome interactions in ASD. We identify SCFA acetate as a mediator of gut-brain interactions and show acetate supplementation reverses social deficits concomitant with alterations to medial prefrontal cortex (mPFC) transcriptional regulation independent of microbiome status.

Methods: Shank3^KO and wild-type (Wt) littermates were divided into control, Antibiotic (Abx), Acetate and Abx + Acetate groups upon weaning. After six weeks, animals underwent behavioral testing. Molecular analysis including 16S and metagenomic sequencing, metabolomic and transcriptional profiling were conducted. Additionally, targeted serum metabolomic data from Phelan McDermid Syndrome (PMS) patients (who are heterozygous for the Shank3 gene) were leveraged to assess levels of SCFA's relative to ASD clinical measures.

Results: Shank3^KO mice were found to display social deficits, dysregulated gut microbiome and decreased cecal levels of acetate - effects exacerbated by Abx treatment. RNA-sequencing of mPFC showed unique gene expression signature induced by microbiome depletion in the Shank3^KO mice. Oral treatment with acetate reverses social deficits and results in marked changes in gene expression enriched for synaptic signaling, pathways among others, even in Abx treated mice. Clinical data showed sex specific correlations between levels of acetate and hyperactivity scores.

Conclusion: These results suggest a key role for the gut microbiome and the neuroactive metabolite acetate in regulating ASD-like behaviors.

Figure 1:. Deletion of the Shank3 Gene Results in a Dysregulated Gut Microbiome and Metabolome

(A) Study design for experiment 1 and cecal content collection. Shank3 ^KO mice show decreased microbiome diversity by Simpson. (B) and Shannon. (C) diversity metrics. (D) Donut charts showing the relative Phylum composition of Wt and Shank3^KO mice. (E) Relative composition of bacteria from the Firmicutes phylum was decreased in Shank3^KO. (F) but no significant difference in Bacteroidetes. (G) The ratio of Firmicutes to Bacteroidetes is significantly decreased in Shank3^KO mice. (H) Relative abundance of phylogenies containing Lactobacillus showed decreases across all taxonomic levels, n = 6–7 mice per genotype for 16S analysis. (I) Functional KEGG pathway counts for Fatty Acid degradation and (J) Phenylalanine metabolism were both significantly increased in Shank3^KO mice, n = 7–8 mice per genotype for Shotgun metagenomic analysis. (K) Targeted metabolomics shows levels of Short Chain Fatty Acid (SCFA) Acetic Acid are significantly decreased in Shank3^KO mice. (L) Levels of amino acid Phenylalanine were significantly increased in Shank3^KO mice. n = 6–8 mice per group for cecal targeted metabolomic analysis. All data presented as mean ± SEM *p<0.05 **p<0.01 ***p<0.001. Male and female mice were used in all analysis.

Figure 2:. Antibiotic Treatment Throughout Development Exacerbates Metabolic and Social Deficits Caused by Shank3 Deletion

(A) Study design for Experiment 2 and mPFC collection. (B) Wild-type and Shank3^KO mice show normal body weight growth over time regardless of genotype or treatment n = 4–7 mice per genotype for weight gain analysis. (C) mice of both genotypes drink the same amount of control or Abx treated water; n = 12 mice per treatment group. (D) Antibiotics result in marked depletion of microbial diversity in both Wt and Shank3^KO mice by Shannon diversity index. (E) Unweighted PCOA plot of beta diversity shows a marked shift induced by antibiotic treatment in both genotypes. (F) Levels of Acetic Acid were decreased in control Shank3^KO mice and further decreased by Abx treatment n = 3– 8 mice for both 16S and targeted metabolomic analysis. (G) In the three-chambered social interaction task, Wt-H₂O mice spent increased time in the chamber containing the novel social interactor while Shank3^KO-H₂O did not show a significant preference for the social interactor. Treatment with Abx caused Wt mice to lose social preference and exacerbated social deficits in KO mice, n = 9–19 mice per genotype and treatmen.t (H) Shank3^KO mice show decreased locomotion in 60-minute open field not affected by antibiotics. (I) No genotype or treatment differences are seen on time in center of an open field, n = 12–19 mice per genotype and treatment for open field. (J) In the elevated O-maze no significant differences in total time spent in the open arms is found. (K) Shank3^KO-H₂O mice display increased frequency of entries into the open arm of the elevated zero maze, n = 11–19 mice per genotype and treatment. All data presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Male and female mice were used for all analysis.

Figure 3:. Effects of Shank3 Deletion and Antibiotic Treatment on mPFC Gene Expression.

(A) Volcano plot of gene expression changes comparing Wt-H₂O and Wt-Abx. (B) Select gene pathways regulated in Abx treated Wt mice. Dotted line at 1.3 indicates significance (FDR corrected p<0.05). (C) Volcano plot of gene expression changes comparing Wt-H₂O and Shank3^KO-H₂O mice. (D) Select pathways that are predicted to be regulated in Shank3^KO mice. (E) Volcano plot of gene expression changes comparing KO-H₂O and KO-Abx. (F) Select pathways that are predicted to be regulated in KO-Abx mice. (G) Venn diagram of all significant genes in Wt-Abx and KO-Abx relative to Wt-H₂O controls shows the two genotypes have highly disparate responses to prolonged microbiome depletion. (H) Gene ontology analysis of regulated genes in Wt and KO mice following Abx. (I) Top Transcription Factors (TF) predicted to be upstream from differentially expressed genes in Wt-Abx and KO-Abx groups relative to Wt-H₂O controls. n = 3–6 mice per genotype and treatment group. Male and female mice were used for all analysis

Figure 4:. Acetate Replenishment Rescues Social Deficits Caused by Shank3 Deletion Independent of an Intact Microbiome

(A) Study design for Experiment 3 and mPFC collection. (B) Acetate treatment interacts with genotype to slightly alter microbiome diversity by Shannon diversity index. n = 6–7 mice per group for 16S analysis. (C) In the three-chambered social interaction task, Wt-H₂O and Wt-Acetate mice spent increased time in the chamber containing the novel social interactor (blue and green bars). Shank3^KO-H₂O did not show a significant preference for the social interactor at baseline as previous (pink bars). Acetate treatment rescues this social deficit in Shank3^KO mice (purple bars). n = 6–15 mice per genotype and treatment. (D) Acetate treatment also reverses locomotor activity deficit in Shank3^KO in a 60 minute open field task. (E) No genotype or treatment effects on time spent in center of open field in a 60 minute test session. n = 6–16 mice per genotype and treatment for open field. (F) Shank3^KO control and acetate treated spend more time spent in open arms of elevated zero maze. (G) Shank3 ^KO-H₂O mice display increased frequency of entries into the open arm, but not those treated with acetate n = 12–20 mice per genotype and treatment for zero maze. (H) Treatment with acetate does not reverse Abx effects on microbiome diversity by Shannon diversity index. n = 6–7 mice per group for 16S analysis. (I) In the three-chambered social interaction task, Wt-Abx mice do not show a significant preference for the novel social interactor (blue bars). Treatment with acetate in microbiome depleted (Ab/Ac) mice reverse this phenotype (green bars). Shank3 ^KO-Abx mice again demonstrate an aversion for the social stimulus (red bars), but this is reversed when acetate is supplemented to the antibiotics (Ab/Ac – purple bars). n = 4–10 mice per genotype and treatment for 3 chamber. (J) Abx or Ab/Ac do not alter Shank3 genotype effects on locomotion in a 1-hour open field test session. (K) No genotype or treatment effects on time spent in center of open field in a 60-minute test session. n = 12–19 mice per genotype and treatment for open field. (L) Treatment with Abx or Ab/Ac does not alter Shank3 genotype effects on time spent in open arms of the o-maze. (M) Wt Ab/Ac mice display decreased frequency of entries into the open arm of the elevated zero maze compared to Wt-Abx mice. n = 10–14 mice per genotype and treatment for zero maze. All data presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Male and female mice were used in all analysis.

Figure 5:. Treatment with acetate induces unique transcriptional patterns in the mPFC

(A-D) Volcano plots of all Acetate and Ab/Ac treatment groups relative to Wt-H₂O controls. (E) Venn diagram of all genes significantly regulated in Wt-Acetate and KO-Acetate relative to Wt-H₂O. (F) Heatmap of fold change expression of all genes from the previous panel (G) Venn diagram of all genes significantly regulated in Wt-Ab/Ac and KO-Ab/Ac relative to Wt-H₂O. (H) Heatmap of fold change expression of all genes from the previous panel (I) Select pathways predicted to be enriched in uniquely regulated genes in Wt and KO mice from the four treatment groups. (J) Transcription factors predicted to be upstream of significantly regulated genes relative to Wt-H₂O controls. n = 4–6 mice per genotype and treatment group. Male and female mice were used for all analysis.

Figure 6:. Weighted Gene Network Analysis Reveals Acetate Treatment Alters Transcription of Genes involved in metabolism, Immune system and Synpatic Function

(A) Dendrogram of WGCNA analysis. (B) Module-trait relationship table showing modules of interest. Each cell reports the Pearson correlation value and if significant the p-value in parentheses. Columns describe the variable, and the rows show the module name (C) Heatmap showing unsupervised hierarchical clustering of all genes in Purple module. (D) Gene ontology enrichment of the differentially expressed genes in Purple module. (E) Protein-protein interaction (PPI) network enrichment for the Purple module from the STRING database. (F) Select pathways from full PPI network enrichment for the purple module from STRING database. (G-J) Heatmap, gene ontology enrichment and PPI network enrichment for genes in Brown module. (K-N) Heatmap, gene ontology enrichment and PPI network enrichment for genes in Green Yellow module. n = 4–6 mice per group. Male and female mice were used for all analysis.

Figure 7:. Serum Levels of Acetate Correlate with ABC Hyperactivity in Male PMS Patients

(A) Study Design: Clinical scores as well as serum was collected from 32 PMS probands and 28 unaffected family controls. Serum was then analyzed for levels of 8 SCFAs using liquid chromatography-mass spectrometry (LC/MS) (B) Heatmap showing a significant correlation between Vineland Communication scores and levels of Isobutyric acid and propanoic acid n =30) (C) Heatmap showing a significant negative correlation between Vineland Communication scores and levels of Isobutyric acid and propanoic acid in females only (n =17) and significant negative correlation between ABC hyperactivity scores and Acetic Acid levels in males ( n= 13) (D) Spearman’s correlation confirming significant negative correlation between levels of acetic acid and ABC hyperactivity in males (R= −0.5824). *p < 0.05; **p < 0.01