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Cited by 1,182Gut microbiota and healthAutism Spectrum Disorder ResearchGenetics and Neurodevelopmental Disorders

Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice

Open access
Sharon, Gil, Cruz, Nikki Jamie, Kang, Dae-Wook, Gandal, Michael J., Wang, Bo, Kim, Young-Mo, Zink, Erika M., Casey, Cameron P. +16 more·2019·Cell
DOI 10.1016/j.cell.2019.05.004PMC PMC6993574
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Abstract

SUMMARY Autism spectrum disorder (ASD) manifests as alterations in complex human behaviors including social communication and stereotypies. In addition to genetic risks, the gut microbiome differs between typically-developing (TD) and ASD individuals, though it remains unclear whether the microbiome contributes to symptoms. We transplanted gut microbiota from human donors with ASD or TD controls into germ-free mice, and reveal that colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors. The brains of mice colonized with ASD microbiota display alternative splicing of ASD-relevant genes. Microbiome and metabolome profiles of mice harboring human microbiota predict that specific bacterial taxa and their metabolites modulate ASD behaviors. Indeed, treatment of an ASD mouse model with candidate microbial metabolites improves behavioral abnormalities and modulates neuronal excitability in the brain. We propose that the gut microbiota regulates behaviors in mice via production of neuroactive metabolites, suggesting that gut-brain connections contribute to the pathophysiology of ASD.

AI snapshot

Written from the paper’s full text.

TL;DR

Transplanting gut microbiota from children with ASD into germ-free mice was sufficient to induce ASD-like behavioral symptoms in offspring, relative to TD microbiota. The authors link these behaviors to altered brain gene splicing and microbial metabolites, especially lower taurine and 5-aminovaleric acid (5AV), and show that administering these metabolites improves behaviors in BTBR mice.

Key findings

  • Germ-free C57BL/6J mice colonized with ASD donor microbiota produced offspring (oASD) with increased repetitive behavior, decreased locomotion, and decreased communication versus oTD controls; direct social interaction also showed decreased sociability, and effects were more pronounced in male mice.
  • Microbiome analysis identified 31 differentially abundant amplicon sequence variants between oTD and oASD mice, including *Bacteroides ovatus* and *Parabacteroides merdae* enriched in oTD and *Eisenbergiella tayi* enriched in oASD; *E. tayi* correlated with increased repetitive behavior and social deficits.
  • RNA-seq/rMATS found 560 genes with differential alternative splicing events between oASD and oTD brains (PFC: 265, STR: 257, full dataset: 243), with 52 of 560 events in autism-associated genes and 11 present in both SPARK and SFARI datasets.
  • Metabolomics found 27 of 313 detected colon metabolites significantly different in oASD versus oTD mice, including ~50% less taurine and lower 5AV; oral taurine or 5AV from conception reduced repetitive behavior and increased social duration in BTBR mice, and 5AV decreased L5 pyramidal neuron excitability in medial prefrontal cortex.

Methods

  • Human fecal samples from TD and ASD donors, selected using ADOS and GI severity index scores, were transplanted into germ-free C57BL/6J mice; recipient mice were bred, and offspring were behaviorally tested (open field, marble burying, three-chamber sociability, direct social interaction, ultrasonic vocalization) and sampled at P45.
  • The study used 16S rRNA gene sequencing, shotgun metagenomics, RNA-seq with rMATS splicing analysis, untargeted NMR/GC-MS metabolomics, and MIMOSA metabolic modeling; candidate metabolites were administered to BTBR T+ tf/J mice, followed by slice electrophysiology and calcium imaging.
  • Behavioral outcomes were analyzed with longitudinal linear mixed-effects models; microbiome differential abundance used DESeq2 and Random Forest; correlations used Spearman’s rank; splicing events used FDR < 0.1.

Limitations

  • The paper states it is a limited study with 16 donor samples from a pediatric cohort, and that it cannot conclude gut bacteria are entirely causal for ASD symptoms; host genetics and perinatal events may compound risk.
  • The paper states that only human male donor samples were used, so the observed male bias in mouse behavioral effects may be a true effect or an artifact of experimental design; future studies with male and female donors are needed.
  • The paper notes that GI co-morbidities in ASD donors and their correlation with some behaviors may impact mouse outcomes after microbiota transplant; evident from the text, the mechanistic work relies heavily on mouse models, including BTBR mice and germ-free humanized mice, and on correlational predictions from microbiome-metabolome integration.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.cell.2019.05.004

Cites

193 of 193 references verified0 citation errors · 0 not checkable

Statistics

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Claims the paper makes

0 of 58 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Brains of mice colonized with ASD microbiota display alternative splicing of ASD-relevant genes.

    “The brains of mice colonized with ASD microbiota display alternative splicing of ASD-relevant genes.”
    • Also stated in: § ASD Microbiota Promote Extensive Alternative Splicing of ASD-relevant Genes in the Brain · plain
      “These data reveal a major shift in alternative splicing patterns of genes in the brains of oASD mice relative to oTD mice, with an enrichment for ASD-related genes in the differentially spliced subset that mirror findings from human brains.”
    Quote in the paperSubstantial claim
  • Findingown data

    Colonization of germ-free mice with gut microbiota from ASD donors is sufficient to induce hallmark autistic behaviors relative to TD microbiota.

    “We transplanted gut microbiota from human donors with ASD or TD controls into germ-free mice, and reveal that colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors.”
    • Also stated in: § Gut Microbiota from Individuals with ASD is Sufficient to Promote Altered Behaviors in Mice · plain
      “Thus, microbiota transplantation from human ASD donors into mice can transfer ASD relevant behavioral deficits.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The behavioral differences observed in mice colonized with ASD microbiota are due to the microbiota itself and not to other donor-derived factors such as diet, genetics, or GI comorbidities.
    • Rests on an assumption: The human donor fecal samples are representative of the donor's gut microbiota and of ASD/TD status, without other confounding differences between donor groups.
    • Rests on an assumption: The mouse behavioral tests used (marble burying, open-field, ultrasonic vocalization, direct social interaction) measure ASD-relevant behaviors that translate to human ASD symptoms.
  • Findingown data

    Female oASD mice traveled shorter distances than oTD females in the open-field test.

    “Conversely, female oASD mice traveled shorter distances than oTD females in OFT (Figures S2C’, S2H).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The behavioral differences observed in mice colonized with ASD microbiota are due to the microbiota itself and not to other donor-derived factors such as diet, genetics, or GI comorbidities.
    • Rests on an assumption: The mouse behavioral tests used (marble burying, open-field, ultrasonic vocalization, direct social interaction) measure ASD-relevant behaviors that translate to human ASD symptoms.
  • Findingown data

    oASD mice displayed decreased communication compared to oTD controls.

    “we observed increased repetitive behavior, decreased locomotion, and decreased communication in oASD mice compared to mice colonized with samples from TD controls (oTD), as tested by marble burying (MB), open-field testing (OFT), and ultrasonic vocalization (USV), respectively.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The behavioral differences observed in mice colonized with ASD microbiota are due to the microbiota itself and not to other donor-derived factors such as diet, genetics, or GI comorbidities.
    • Rests on an assumption: The mouse behavioral tests used (marble burying, open-field, ultrasonic vocalization, direct social interaction) measure ASD-relevant behaviors that translate to human ASD symptoms.
  • Findingown data

    oASD mice displayed decreased sociability compared to oTD controls in the direct social interaction test.

    “oASD mice “humanized” with fecal microbiota from ASD donors showed increased repetitive behavior, decreased sociability, and decreased locomotion compared to mice colonized with samples from TD controls (Figures 1E–G).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The behavioral differences observed in mice colonized with ASD microbiota are due to the microbiota itself and not to other donor-derived factors such as diet, genetics, or GI comorbidities.
    • Rests on an assumption: The mouse behavioral tests used (marble burying, open-field, ultrasonic vocalization, direct social interaction) measure ASD-relevant behaviors that translate to human ASD symptoms.
  • May combine two claims. 34
  • Classified as probably not a substantial claim. 33

  • May combine two claims.

    Findingown data

    Treatment of an ASD mouse model with candidate microbial metabolites improves behavioral abnormalities and modulates neuronal excitability in the brain.

    “Indeed, treatment of an ASD mouse model with candidate microbial metabolites improves behavioral abnormalities and modulates neuronal excitability in the brain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Eisenbergiela tayi was prevalent among all oASD mice, and absent from oTD groups.

    “Conversely, Eisenbergiela tayi (02b40_Lachnospiraceae and 29857_Lachnospiraceae) was prevalent among all oASD mice, and absent from oTD groups (Figure 2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There were no differences in mouse weight and intestinal barrier function between oTD and oASD mice.

    “However, we found no differences in mouse weight and intestinal barrier function between oTD and oASD mice (Figure S3A–B, Figure S3C–H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bacteroides ovatus and Parabacteroides merdae were prevalent in all oTD samples, and absent from most or all oASD samples.

    “Specifically, Bacteroides ovatus (970ed_Bacteroides ovatus and 054dc_Bacteroides ovatus) and Parabacteroides merdae (4ae7e_Parabacteroides) were prevalent in all oTD samples, and absent from most or all oASD samples.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There were no differences in engraftment or bacterial loads between oTD and oASD mice.

    “Sixty percent of the taxa in donor samples engrafted on average in recipient mice with no differences in engraftment or bacterial loads between oTD and oASD mice (Figure S1H–I, S1J–L).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Specific bacterial taxa differ in engraftment in mice: Actinobacteria and some Firmicutes failed to engraft, while some Bacteroidetes thrived.

    “Moreover, specific taxa, such as Actinobacteria and some Firmicutes, failed to engraft in mice, while others such as some Bacteriodetes thrived (Figure S1F–G).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The core behavioral features in oASD mice were not the result of anxiety or other locomotion deficits.

    “These core features of ASD were not the result of anxiety or other locomotion deficits, as colonized mice spent the same amount of time in the center of an open-field arena and male mice traveled similar distances (Figures S2C–D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mouse behavioral tests used (marble burying, open-field, ultrasonic vocalization, direct social interaction) measure ASD-relevant behaviors that translate to human ASD symptoms.

  • May combine two claims.

    Findingown data

    The Bacteroides (b20cd_Bacteroides) species and P. merdae both correlated with reduced repetitive behavior and increased social behavior.

    “The Bacteroides (b20cd_Bacteroides) species and P. merdae (4ae7e_Parabacteroides) both correlated with reduced repetitive behavior and increased social behavior.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The germ-free mouse model and humanization procedure do not introduce artifacts that confound the behavioral or molecular results.

  • May combine two claims.

    Findingown data

    The dataset of differentially spliced genes was highly enriched for various ribosome binding protein (RBP) targets, but under-enriched in activity dependent transcripts.

    “Interestingly, we found that this dataset of differentially spliced genes was highly enriched for various ribosome binding protein (RBP) targets, but under-enriched in activity dependent transcripts (Figure 3E), suggesting that the gut microbiome can alter splicing programs at the RBP level.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fifty two of the 560 AS events occurred within genes associated with autism, with 11 present in both the SPARK gene list and SFARI Gene dataset, and 52 present in the SFARI gene list.

    “Fifty two of the 560 AS events occurred within genes associated with autism, with 11 present in both the SPARK gene list and SFARI Gene dataset, and 52 present in the SFARI gene list (Figure 3C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Differential splicing events were enriched for genes highly expressed in neurons, and under-enriched for in microglia in oASD mice compared to oTD animals.

    “Differential splicing events were enriched for genes highly expressed in neurons, and under-enriched for in microglia in oASD mice compared to oTD animals (Figure 3D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    16S rRNA gene ASVs that map to E. tayi, but not those that map to P. merdae, were more abundant in ASD individuals from large human cohort studies.

    “Accordingly, we validated that 16S rRNA gene ASVs that map to E. tayi, but not those that map to P. merdae, were more abundant in ASD individuals from large human cohort studies (Figure 2F–G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Three long-noncoding RNAs of unknown function from the PFC, and two additional genes from the combined PFC and STR dataset, displayed differential expression between oTD and oASD mice.

    “Three long-noncoding RNAs of unknown function from the PFC, and two additional genes from the combined PFC and STR dataset, displayed differential expression between groups of mice (Figures S4A–C, Figure S4D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was a significant increase in K13821 and a significant decrease in P5C reductase (K00286) abundance in oASD mice.

    “However, we found a significant increase in K13821 (Figure 5H) and a significant decrease in P5C reductase (K00286) abundance (Figure 5I) in oASD mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    5AV reached the fetal compartment and the fetal brain after oral administration to pregnant mice.

    “While 5AV reached the fetal compartment and the fetal brain (Figure 6B), taurine concentrations did not change with administration suggesting it may act locally in the gut.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hydroxyproline concentrations varied consistently with K00286 abundance, and hypD levels were not significantly decreased in oASD samples.

    “Hydroxyproline concentrations varied consistently with K00286 abundance (Figure S5B), and hypD levels were not significantly decreased in oASD samples (Figure 5G).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    MIMOSA analysis predicted that taurine concentrations may result from differential synthesis potential by three specific species: Alistipes sp. HGB5, Alistipes finegoldii, and Bacteroides xylanisolvens.

    “Interestingly, MIMOSA analysis predicted that taurine concentrations may result from differential synthesis potential by three specific species: Alistipes sp. HGB5, Alistipes finegoldii, and Bacteroides xylanisolvens (Figure S5A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Mice administered 5AV showed increased locomotion, decreased center duration, and increased sociability, with no significant difference in repetitive behavior.

    “Mice administered 5AV showed increased locomotion, decreased center duration (Figure S6F), and increased sociability (Figure S6H), with no significant difference in repetitive behavior (Figure S6G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    L5 pyramidal neurons from mice treated with 5AV, but not taurine, showed significantly decreased excitability.

    “However, L5 pyramidal neurons from mice treated with 5AV, but not taurine, showed significantly decreased excitability as measured by the number of spikes observed as a result of injection of stepwise increasing currents”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither 5AV nor taurine affected the intrinsic membrane properties of neurons or the frequency or amplitude of mEPSCs and mIPSCs, compared to controls.

    “Neither 5AV nor taurine affected the intrinsic membrane properties of neurons or the frequency or amplitude of mEPSCs and mIPSCs, compared to controls (Figure S6I, Figure 6F–G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Analysis of gene splicing revealed extensive differences between oTD and oASD mice, with 11 alternative splicing events in validated high-risk ASD genes.

    “Analysis of gene splicing revealed extensive differences between oTD and oASD mice, with 11 alternative splicing events in validated high-risk ASD genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Taurine and 5AV are GABAA receptor agonists that are increased in the colons of oTD mice.

    “Two examples are taurine, the metabolic product of cysteate or taurocholic acid, and 5AV, the fermentation product of proline—both GABAA receptor agonists that are increased in the colons of oTD mice.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In serum, 130 metabolites were identified by GC-MS out of 255 detected.

    “In serum, a total of 130 metabolites were identified by GC-MS (out of a total of 255 detected).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In colon contents, 67 metabolites were detected and identified by NMR.

    “and 67 metabolites were detected and identified by NMR”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In colon contents, 122 metabolites were identified by GC-MS out of 246 detected.

    “In colon contents, a total of 122 metabolites were identified by GC-MS (out of a total of 246 detected)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Specific gut microbes have demonstrated therapeutic potential in animal models of ASD.

    “Specific gut microbes have demonstrated therapeutic potential in animal models of ASD (Buffington et al., 2016; Hsiao et al., 2013; Sgritta et al., 2018; Tabouy et al., 2018).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Buffington et al., 2016 source

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    • cites Hsiao et al., 2013 source

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    • cites Sgritta et al., 2018

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Changes in the microbiome often result in altered metabolic profiles, impacting the availability and diversity of nutrients and microbial metabolites.

    “changes in the microbiome often result in altered metabolic profiles, impacting the availability and diversity of nutrients and microbial metabolites (Dodd et al., 2017; Melnik et al., 2017; Sharon et al., 2014; Wikoff et al., 2009).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Dodd et al., 2017 source

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    • cites Melnik et al., 2017 source

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    • cites Sharon et al., 2014 source

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    Premisecitation

    Gut bacterial communities differ in mouse models of ASD.

    “as well as in mouse models of ASD (Buffington et al., 2016; Coretti et al., 2017; Hsiao et al., 2013; Kim et al., 2017; Liu et al., 2018; de Theije et al., 2014).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Buffington et al., 2016 source

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    • cites Coretti et al., 2017 source

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    • cites Hsiao et al., 2013 source

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    Premisecitation

    Microbial-based interventions, including fecal transplantation, antibiotics, and probiotics, have shown promise in a limited number of human trials.

    “microbial-based interventions, including fecal transplantation, antibiotics, and probiotics, have shown promise in a limited number of human trials (Grimaldi et al., 2018; Kang et al., 2017b; Sandler et al., 2000).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Grimaldi et al., 2018 source

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    • cites Kang et al., 2017b

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    • cites Sandler et al., 2000 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Fecal microbiome profiles are most divergent in ASD subjects with GI dysfunction.

    “Fecal microbiome profiles are most divergent in ASD subjects with GI dysfunction, a common comorbidity of autism (Chaidez et al., 2014; Gorrindo et al., 2012).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Chaidez et al., 2014 source

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    • cites Gorrindo et al., 2012 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Amino-acid transport and degradation differ between TD and ASD individuals.

    “amino-acid transport and degradation differ between TD and ASD individuals (Aldred et al., 2003; Evans et al., 2008)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Aldred et al., 2003 source

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    • cites Evans et al., 2008 source

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  • May combine two claims.

    Interpretationown data

    Fecal transplantation from human donors into GF recipient mice maintained differences between TD and ASD microbiome profiles, which are vertically transmitted to offspring with fidelity.

    “Collectively, fecal transplantation from human donors into GF recipient mice maintained differences between TD and ASD microbiome profiles, which are vertically transmitted to offspring with fidelity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The decrease in α-diversity upon colonization indicates loss of bacterial species due to sample processing and/or host-incompatibility.

    “We observe a decrease in α-diversity upon colonization of mice (donor to recipients), indicating loss of bacterial species due to sample processing and/or host-incompatibility (Figure S1D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    These data reveal a major shift in alternative splicing patterns of genes in the brains of oASD mice relative to oTD mice, with an enrichment for ASD-related genes in the differentially spliced subset that mirror findings from human brains.

    “These data reveal a major shift in alternative splicing patterns of genes in the brains of oASD mice relative to oTD mice, with an enrichment for ASD-related genes in the differentially spliced subset that mirror findings from human brains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Taurine concentrations did not change with administration, suggesting it may act locally in the gut.

    “While 5AV reached the fetal compartment and the fetal brain (Figure 6B), taurine concentrations did not change with administration suggesting it may act locally in the gut.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    In oTD microbiota, L-proline is produced from Hyp via P5C and subsequently serves as an electron acceptor to produce 5AV; in oASD microbiota, the balance shifts towards production of P5C and glutamate, and away from 5AV.

    “These data suggest that, in oTD microbiota, L-proline is produced from Hyp via P5C and subsequently serves as an electron acceptor to produce 5AV (Figure 5F–I). Conversely, in oASD microbiota, the balance shifts towards production of P5C and glutamate, and away from 5AV (Figure 5F–I).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationown data

    Genistein and daidzein (or the absence of their degradation products) are predicted to contribute to repetitive behavior effects, while lysine, 3AIBA, and genistein are predicted to influence locomotion.

    “In addition, we predict that genistein and daidzein (or the absence of their degradation products) contribute to repetitive behavior effects while lysine, 3AIBA, and genistein are predicted to influence locomotion (Figure 6A).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The germ-free mouse model and humanization procedure do not introduce artifacts that confound the behavioral or molecular results.

  • May combine two claims.

    Interpretationcitation

    The high prevalence of GI dysfunction and evidence of immune imbalance suggest microbiota involvement in ASD.

    “The high prevalence of GI dysfunction (Adams et al., 2011; Chaidez et al., 2014; McElhanon et al., 2014), along with evidence of immune imbalance (Onore et al., 2012), further suggest microbiota involvement in ASD.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Adams et al., 2011 source

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    • cites Chaidez et al., 2014 source

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    • cites McElhanon et al., 2014 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The transcriptional program in ASD brains is distinct from that of TD brains.

    “The transcriptional program in ASD brains is distinct from that of TD brains (Gandal et al., 2018; Parikshak et al., 2016; Voineagu et al., 2011).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Gandal et al., 2018 source

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    • cites Parikshak et al., 2016 source

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    • cites Voineagu et al., 2011 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Altered host genetics and perinatal events, coupled with an altered microbiota, may together influence the etiology of ASD by compounding risks that enhance symptom severity.

    “Factors such as altered host genetics and perinatal events, coupled with an altered microbiota, may together influence the etiology of ASD by compounding risks that enhance symptom severity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The human microbiota can modulate complex behaviors in mice via the production of neuroactive microbial metabolites, with mechanisms that include regulation of E-I balances in the brain.

    “We propose that the human microbiota can modulate complex behaviors in mice via the production of neuroactive microbial metabolites, with mechanisms that include regulation of E-I balances in the brain.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Taurine is essential for brain development and can be produced by neurons and astrocytes or transported through the blood-brain-barrier via TauT.

    “Taurine is essential for brain development (Kilb and Fukuda, 2017; Tochitani, 2017) and can be produced by neurons and astrocytes (Vitvitsky et al., 2011) or transported through the blood-brain-barrier via the Taurine transporter TauT (Benrabh et al., 1995).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Kilb and Fukuda, 2017

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    • cites Tochitani, 2017 source

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    • cites Vitvitsky et al., 2011 source

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  • Classified as probably not a substantial claim.

    Premisecitation

    Gut bacteria can affect gene expression and host behavior through various pathways, including the production of neuroactive small molecules.

    “Gut bacteria can affect gene expression and host behavior through various pathways, including the production of neuroactive small molecules (Hsiao et al., 2013).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Hsiao et al., 2013 source

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  • Classified as probably not a substantial claim.

    Premisecitation

    Recent studies have highlighted the importance of aberrant alternative splicing of mRNA in the brains of subjects with an ASD diagnosis.

    “Recent studies have highlighted the importance of aberrant alternative splicing of mRNA in the brains of subjects with an ASD diagnosis (Corominas et al., 2014; Gandal et al., 2018; Irimia et al., 2014; Parikshak et al., 2016; Voineagu et al., 2011).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Corominas et al., 2014 source

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    • cites Gandal et al., 2018 source

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    • cites Irimia et al., 2014 source

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  • May combine two claims.

    Interpretationown data

    Gut microbes not only regulate gene expression in the mouse brain, but also affect alternative splicing, and can regulate the production of specific isoforms of genes that have been implicated in ASD.

    “These findings suggest that gut microbes not only regulate gene expression in the mouse brain (Hoban et al., 2016; Neufeld et al., 2011; Stilling et al., 2015), but also affect alternative splicing (Stilling et al., 2018), and can regulate the production of specific isoforms of genes that have been implicated in ASD.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Hoban et al., 2016 source

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    • cites Neufeld et al., 2011 source

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    • cites Stilling et al., 2015 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All samples were assigned an identification number that prevented direct identification of the groups to facilitate blinded analysis of samples downstream.

    “All samples were then assigned an identification number that prevented from direct identification of the groups to facilitate blinded analysis of samples downstream.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Social deficits in the CNTNP2-deficient mouse model of autism could be rescued by modulating the excitability of pyramidal neurons.

    “Consistent with these results, social deficits in the CNTNP2-deficient mouse model of autism could be rescued by modulating the excitability of pyramidal neurons (Selimbeyoglu et al., 2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Selimbeyoglu et al., 2017 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    To control for time of day effects, cages of different groups were alternated during behavior testing.

    “Mice were tested during the light phase of the light cycle; to control for time of day effects, cages of different groups were alternated.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    During the initial discovery phase, the experimenter was blinded to the donor but not to the group; in the subsequent validation phase, the experimenter was blinded.

    “During the initial discovery phase, the experimenter was blinded to the donor but not to the group. In the subsequent validation phase, the experimenter was blinded.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Extensive evidence highlights the importance of the E-I balance in neurodevelopmental disorders.

    “Extensive evidence highlight the importance of the E-I balance in neurodevelopmental disorders (Lee et al., 2017; Nelson and Valakh, 2015; Rubenstein and Merzenich, 2003).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Lee et al., 2017 source

      Not checked in the free run.

    • cites Nelson and Valakh, 2015

      Not checked in the free run.

    • cites Rubenstein and Merzenich, 2003 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    To control for effects by the time of collection, mice from different groups were sacrificed in an alternated fashion.

    “To control for effects by the time of collection, mice from different groups were sacrificed in an alternated fashion.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Sample outliers (n=3) were identified and removed based on the RMD-PAV algorithm with a p-value threshold of 0.0001.

    “Sample outliers were identified and removed based on the RMD-PAV algorithm (Matzke et al., 2011) and a p-value threshold of 0.0001; based on this criterion, three samples were removed.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Matzke et al., 2011 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    These GABAA receptor agonists may act to perturb the E-I balance in the brain, specifically during development.

    “These GABAA receptor agonists may act to perturb the E-I balance in the brain, specifically during development.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    When the experimenter was not blinded to the treatment or group, the number of buried marbles was scored by a second experimenter blinded to the treatment/group from overhead images.

    “When the experimenter was not blinded to the treatment or group, the number of buried marbles was scored by a second experimenter blinded to the treatment/group from overhead images taken after the second 10 min session.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    The etiology of ASD may lie in the human genome, the human microbiota, or both via gene-environment interactions.

    “Accordingly, we propose that the etiology of ASD may lie in the human genome, the human microbiota, or both via gene-environment interactions.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Both human and microbial genes are vertically heritable across generations.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A set of reference videos was used to ensure consistency over time.

    “A set of reference videos was used to ensure consistency over time.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The high concordance rates observed between monozygotic twins implicate a strong genetic basis for ASD.

    “The high concordance rates observed between monozygotic twins implicate a strong genetic basis for ASD (Tick et al., 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tick et al., 2016 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Throughout the intestinal permeability procedure, the experimenter was blinded to mouse groups.

    “Throughout the procedure, the experimenter was blinded to mouse groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    We tested and analyzed a subset of representative samples along with additional previously untested samples to ensure there is no bias in sample selection.

    “Subsequently, we tested and analyzed a subset of representative samples (two TD and three ASD) along with additional previously untested samples (one TD and two ASD), to ensure there is no bias in sample selection.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Enrichment of known autism risk genes among differentially spliced genes was assessed using logistic regression, controlling for gene length.

    “Enrichment of known autism risk genes (SFARI: https://gene.sfari.org/, SPARK for Autism: http://spark-sf.s3.amazonaws.com/SPARK_gene_list.pdf) among those exhibiting DS was assessed using logistic regression, controlling for gene length.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All metabolite identification and quantification ions were validated and confirmed to reduce deconvolution errors during automated data processing and to eliminate false identifications.

    “All metabolite identification and quantification ions were validated and confirmed to reduce deconvolution errors during automated dataprocessing and to eliminate false identifications.”
    Quote in the paperNot a substantial claim
61 of 61

  • The paper relies on this without support.

    The behavioral differences observed in mice colonized with ASD microbiota are due to the microbiota itself and not to other donor-derived factors such as diet, genetics, or GI comorbidities.

    “It is possible that GI co-morbidities in the ASD donors and their correlation with some behaviors (Figure 1B, Table S1) may impact outcomes in experimental mice after microbiota transplant.”

  • The paper relies on this without support.

    The human donor fecal samples are representative of the donor's gut microbiota and of ASD/TD status, without other confounding differences between donor groups.

    “Fecal samples from human TD and ASD donors were selected based on Autism Diagnostic Observation Schedule (ADOS) (Gotham et al., 2007) and GI severity index (GSI) scores (Schneider et al., 2006) (Figure 1A–B, Table S1), and transplanted into GF wild-type mice, i.e., “humanized microbiome” mice.”

  • The paper relies on this without support.

    The mouse behavioral tests used (marble burying, open-field, ultrasonic vocalization, direct social interaction) measure ASD-relevant behaviors that translate to human ASD symptoms.

    “oASD mice, colonized with samples from ASD donors, displayed behavioral deficits relevant to ASD, while oMild-ASD mice behaved similar to oTD controls (Figure S2A–E).”

  • The paper relies on this without support.

    Correlations between bacterial taxa or metabolites and behavioral outcomes indicate a potential causal or modulatory relationship rather than mere association.

    “The association of specific bacterial species with oTD or oASD samples, which are also highly correlated with ASD-relevant behaviors, supports the hypothesis that specific bacteria may contribute to various symptoms of ASD.”

  • The paper relies on this without support.

    The germ-free mouse model and humanization procedure do not introduce artifacts that confound the behavioral or molecular results.

    “We transplanted gut microbiota from human donors with ASD or TD controls into germ-free mice, and reveal that colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors.”

  • The paper relies on this without support.

    A direct contribution by the microbiota to the pathophysiology and behavioral outcomes of ASD has not been described previously.

    “While these compelling association studies in humans suggest gut bacteria may impact behavioral symptoms, a direct contribution by the microbiota to the pathophysiology and behavioral outcomes of ASD has not been described previously.”

  • The paper relies on this without support.

    Both human and microbial genes are vertically heritable across generations.

    “However, it is important to consider that both human and microbial genes are vertically heritable across generations.”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Gut microbiota dysbiosis has been implicated in behavioral and neurologic pathologies, such as depression, Alzheimer's disease, Parkinson's disease, and autism spectrum disorder.

    “gut microbiota dysbiosis has been implicated in behavioral and neurologic pathologies, such as depression, Alzheimer's (AD) and Parkinson's (PD) diseases and autism spectrum disorder (ASD) (9, 21, 25–27).”

    From The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication (2020) · cites this paper as [27]

    The Introduction links gut microbiota to ASD and reports that microbiota from patients with depression promoted depressive-like symptoms in mice. It also cites a Parkinson’s disease microbiota study, but does not discuss Alzheimer’s disease. This source therefore supports only part of the list of conditions attributed to the group of citations. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Microbiota transplantation from human ASD donors into mice can transfer ASD-relevant behavioral deficits.

    “Recently, Sharon and collaborators showed that microbiota transplantation from human ASD donors into mice could transfer ASD-relevant behavioral deficits (27).”

    From The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication (2020) · cites this paper as [27]

  • Transplanting fecal microflora from ASD donors to germ-free mice induced autistic behavior-increased repetitive behavior, decreased locomotion and communication-compared to mice with neurotypical donor microbiota.

    “Research on them [114] shows that transplanting fecal microflora from ASD donors to GF mice induced autistic behavior, specifically increased repetitive behavior, decreased locomotion, and communication, compared to mice with NT donors microbiota.”

    From Unraveling the Connections: Eating Issues, Microbiome, and Gastrointestinal Symptoms in Autism Spectrum Disorder (2025) · cites this paper as [114]

  • The altered microbiota of patients could transfer the disease from a human host to a mouse host.

    “Of extreme relevance, the altered microbiota of patients could transfer the disease from a human host to a mouse host (113, 116–120).”

    From The gut-brain axis: how microbiota and host inflammasome influence brain physiology and pathology (2020) · cites this paper as [113]

  • The microbiota composition differs significantly between healthy controls and patients affected by neurodegenerative disorders such as multiple sclerosis, Alzheimer's and Parkinson's diseases, and neuropsychiatric disorders.

    “The microbiota composition differs significantly between healthy controls and patients affected by neurodegenerative disorders (such as multiple sclerosis (MS), Alzheimer’s (AD) and Parkinson’s (PD) diseases) (20, 22, 23, 32, 111–115), and neuropsychiatric disorders (NPS) (30), like major depressive and mood disorders.”

    From The gut-brain axis: how microbiota and host inflammasome influence brain physiology and pathology (2020) · cites this paper as [113]

Cites

Flagged first, then by citations

3 of the 196 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Glenn, 2016, Adapterama I: Universal stubs and primers for thousands of dual-indexed Illumina libraries (iTru & iNext), bioRxiv

Cited by

Sorted by citations

Related

Authors
Sharon, G., Cruz, N. J., Kang, D.-W., Gandal, M. J., Wang, B., Kim, Y.-M., Zink, E. M., Casey, C. P., Taylor, B. C., Lane, C. J., Bramer, L. M., Isern, N. G., Hoyt, D. W., Noecker, C., Sweredoski, M. J., Moradian, A., Borenstein, E., Jansson, J. K., Knight, R., Metz, T. O., Lois, C., Geschwind, D. H., Krajmalnik-Brown, R., & Mazmanian, S. K.
Affiliations
  • California Institute of Technology (US)
  • Arizona State University (US)
  • University of California, Los Angeles (US)
  • Pacific Northwest National Laboratory (US)
  • University of California San Diego (US)
  • University of Southern California (US)
  • University of Washington (US)
  • Santa Fe Institute (US)
Corresponding author
  • Sharon, Gil
  • Mazmanian, Sarkis K.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2019-05
Volume
177
Issue
6
Pages
1600-1618.e17
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2019.05.004
PMC
PMC6993574
OpenAlex
W2948003108
Semantic Scholar
afd469685e7754bfd52bfa454d808bbbec849f79
Cited by
1,182
References
196
Citations by year
2026: 128 · 2025: 187 · 2024: 163 · 2023: 166 · 2022: 189 · 2021: 155 · 2020: 153 · 2019: 37 · 2018: 2
FWCI
44.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • CaltechAUTHORS (California Institute of Technology) (submittedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
  • Simons Foundation Autism Research Initiative
  • Heritage Medical Research Institute
  • Simons Foundation
  • Autism Speaks
  • University of California, San Diego
  • National Institutes of Health
  • Emch Foundation
  • Autism Research Institute
  • Pacific Northwest National Laboratory
  • Brenen Hornstein Autism Research & Education Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
Keywords
autism spectrum disorder, gut microbiota, microbiota transplantation, germ-free mice, autistic behavior, alternative splicing, microbial metabolites, gut-brain axis, neuronal excitability, social communication, stereotypy
MeSH terms
Autism Spectrum Disorder, Gastrointestinal Microbiome, Animals, Bacteria, Behavior, Animal, Behavioral Symptoms, Brain, Disease Models, Animal, Humans, Risk Factors, Mice, Microbiota

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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