The gut metabolite indole-3-propionic acid activates ERK1 to restore social function and hippocampal inhibitory synaptic transmission in a 16p11.2 microdeletion mouse model

doi:10.1186/s40168-024-01755-7

. 2024 Mar 28;12(1):66.

doi: 10.1186/s40168-024-01755-7.

The gut metabolite indole-3-propionic acid activates ERK1 to restore social function and hippocampal inhibitory synaptic transmission in a 16p11.2 microdeletion mouse model

Jian Jiang^#¹, Dilong Wang^#^{1

2}, Youheng Jiang^#¹, Xiuyan Yang¹, Runfeng Sun³, Jinlong Chang¹, Wenhui Zhu¹, Peijia Yao¹, Kun Song⁴, Shuwen Chang⁵, Hong Wang⁵, Lei Zhou³, Xue-Song Zhang⁶, Huiliang Li⁷, Ningning Li^{8

9

10}

Affiliations

¹ Tomas Lindahl Nobel Laureate Laboratory, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, China.
² Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
³ Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China.
⁴ Brain Research Centre, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.
⁵ The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen-Hong Kong Institute of Brain Science Shenzhen Fundamental Research Institutions, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
⁶ Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, USA. xuesong.zhang@rutgers.edu.
⁷ Wolfson Institute for Biomedical Research, Division of Medicine, Faculty of Medical Sciences, University College London, London, UK. huiliang.li@ucl.ac.uk.
⁸ Tomas Lindahl Nobel Laureate Laboratory, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, China. linn29@mail.sysu.edu.cn.
⁹ China-UK Institute for Frontier Science, Shenzhen, China. linn29@mail.sysu.edu.cn.
¹⁰ Department of Anesthesiology, The Afliated Hospital of Youjiang Medical University for Nationalities, Baise, China. linn29@mail.sysu.edu.cn.

^# Contributed equally.

PMID: 38549163
PMCID: PMC10976717
DOI: 10.1186/s40168-024-01755-7

The gut metabolite indole-3-propionic acid activates ERK1 to restore social function and hippocampal inhibitory synaptic transmission in a 16p11.2 microdeletion mouse model

Jian Jiang et al. Microbiome. 2024.

. 2024 Mar 28;12(1):66.

doi: 10.1186/s40168-024-01755-7.

Authors

Affiliations

¹ Tomas Lindahl Nobel Laureate Laboratory, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, China.
² Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
³ Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China.
⁴ Brain Research Centre, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.
⁵ The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen-Hong Kong Institute of Brain Science Shenzhen Fundamental Research Institutions, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
⁶ Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, USA. xuesong.zhang@rutgers.edu.
⁷ Wolfson Institute for Biomedical Research, Division of Medicine, Faculty of Medical Sciences, University College London, London, UK. huiliang.li@ucl.ac.uk.
⁸ Tomas Lindahl Nobel Laureate Laboratory, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, China. linn29@mail.sysu.edu.cn.
⁹ China-UK Institute for Frontier Science, Shenzhen, China. linn29@mail.sysu.edu.cn.
¹⁰ Department of Anesthesiology, The Afliated Hospital of Youjiang Medical University for Nationalities, Baise, China. linn29@mail.sysu.edu.cn.

^# Contributed equally.

PMID: 38549163
PMCID: PMC10976717
DOI: 10.1186/s40168-024-01755-7

Abstract

Background: Microdeletion of the human chromosomal region 16p11.2 (16p11.2 $^{+ / -}$ ) is a prevalent genetic factor associated with autism spectrum disorder (ASD) and other neurodevelopmental disorders. However its pathogenic mechanism remains unclear, and effective treatments for 16p11.2 $^{+ / -}$ syndrome are lacking. Emerging evidence suggests that the gut microbiota and its metabolites are inextricably linked to host behavior through the gut-brain axis and are therefore implicated in ASD development. Despite this, the functional roles of microbial metabolites in the context of 16p11.2 $^{+ / -}$ are yet to be elucidated. This study aims to investigate the therapeutic potential of indole-3-propionic acid (IPA), a gut microbiota metabolite, in addressing behavioral and neural deficits associated with 16p11.2 $^{+ / -}$ , as well as the underlying molecular mechanisms.

Results: Mice with the 16p11.2 $^{+ / -}$ showed dysbiosis of the gut microbiota and a significant decrease in IPA levels in feces and blood circulation. Further, these mice exhibited significant social and cognitive memory impairments, along with hyperactivation of hippocampal dentate gyrus neurons and reduced inhibitory synaptic transmission in this region. However, oral administration of IPA effectively mitigated the histological and electrophysiological alterations, thereby ameliorating the social and cognitive deficits of the mice. Remarkably, IPA treatment significantly increased the phosphorylation level of ERK1, a protein encoded by the Mapk3 gene in the 16p11.2 region, without affecting the transcription and translation of the Mapk3 gene.

Conclusions: Our study reveals that 16p11.2 $^{+ / -}$ leads to a decline in gut metabolite IPA levels; however, IPA supplementation notably reverses the behavioral and neural phenotypes of 16p11.2 $^{+ / -}$ mice. These findings provide new insights into the critical role of gut microbial metabolites in ASD pathogenesis and present a promising treatment strategy for social and cognitive memory deficit disorders, such as 16p11.2 microdeletion syndrome. Video Abstract.

Keywords: Autism; GABA; Gut microbiota metabolite; Indole-3-propionic acid; Mapk3; Social deficits.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
16p11.2^+/- mice exhibited altered microbial composition and declined synthesis and circulation of IPA. A The mice breeding strategies. B Schematic diagram for experimental design. C–E 16S rRNA gene sequencing of gut microbiota of 8-week-old WT and 16p11.2^+/- mice. (C) Principal coordinate analysis (PCoA) plot from feces of two groups (WT: n = 7 mice; 16p11.2^+/-: n = 9 mice). α-diversity was measured by Chao (D) and Shannon (E) indexes. F–H Untargeted metabolomics was performed on feces of 8-week-old WT and 16p11.2^+/- mice. (F) Orthogonal partial least squares discriminant analysis (OPLS-DA) were used to reflect the differences between metabolites in the WT and 16p11.2^+/- groups. (G) The metabolites with significant differences were screened. (H) Multiple correlation coefficients suggested that IPA was most associated with structural dysbiosis (WT: n = 7 mice; 16p11.2^+/-: n = 9 mice). I, J The expression levels of several genes associated with IPA production were decreased in 16p11.2^+/- mice. (I) Schematic representation of tryptophan metabolism leading to IPA. (J) The expression levels of *fldB*, *fldH*, and *acdA* were reduced in 16p11.2^+/- mice (WT: n = 8 mice; 16p11.2^+/-: n = 8 mice). K qPCR analysis showed the levels of *C. sporogenes*, the main producer of IPA, were decreased in 16p11.2^+/- mice (WT: n = 15 mice; 16p11.2^+/-: n = 11 mice). L The level of IPA in feces of mice was detected (WT: n = 6 mice; 16p11.2^+/-: n = 6 mice). M The level of IPA in serum of mice was detected (WT: n = 9 mice; 16p11.2^+/-: n = 9 mice). Data are presented as mean ± SEM, and Student’s t test was applied. *p < 0.05, ***p < 0.001, ****p < 0.0001. Detailed statistical information is presented in Additional file 2: Table S1

**Fig. 2**
16p11.2^+/- mice exhibited deficits in social novelty and recognition memory comparad to WT controls. A-D Three-chamber test (TCT). (A) Schematic of the sociability phase of TCT (E: empty cage; S1: stranger mouse) and representative trajectories of mice. (B) 16p11.2^+/- mice exhibited comparable sociability compared to WT mice (WT: n = 10 mice; 16p11.2^+/-: n = 9 mice. Two-way ANOVA). (C) Schematic of the social novelty phase of TCT (S1: stranger mouse; S2: novel stranger mouse) and representative trajectories of mice. (D) 16p11.2^+/- mice exhibited social novelty deficit (WT: n = 10 mice; 16p11.2^+/-: n = 9 mice. Two-way ANOVA). E Direct social interaction (DSI) test. 16p11.2^+/- mice showed a significant reduction in social time compared to WT mice (WT: n = 11 mice; 16p11.2^+/-: n = 10 mice. Student’s t test). F-I Novel object recognition (NOR) test. (F) Schematic of the habituation phase of NOR test (Obj1: object1; Obj2: object2) and representative trajectories of mice. (G) 16p11.2^+/- mice and WT mice showed no obvious preference over two identical objects (WT: n = 10 mice; 16p11.2^+/-: n = 9 mice. Two-way ANOVA). (H) Schematic of the recognition phase of NOR test (Obj1: object1; Nov: novel object) and representative trajectories of mice. (I) 16p11.2^+/- mice exhibited no obvious preference over novel objects (WT: n = 10 mice; 16p11.2^+/-: n = 9 mice. Two-way ANOVA). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s.: not significant. Detailed statistical information is presented in Additional file 2: Table S1

**Fig. 3**
Inhibitory synaptic transmission dysfunction in the hippocampus dentate gyrus (DG) of 16p11.2^+/- mice. A Hippocampal neurons in 16p11.2^+/- mice were hyperactivated after the TCT. The red puncta indicate c-Fos⁺ neurons in the DG of hippocampus. Scale bar: 30 μm. B Quantification of the numbers of c-Fos⁺ neurons (WT: n = 6 mice; 16p11.2^+/-: n = 5 mice. Student’s t test). C Representative sIPSCs traces from granule cells in hippocampus. Scale bars: 5 s, 10 pA. D Cumulative distribution of sIPSCs amplitude (WT: n = 914 events from 9 cells of 3 mice; 16p11.2^+/-: n = 556 events from 6 cells of 5 mice. Kolmogorov-Smirnov test). E Cumulative distribution of sIPSCs frequency (WT: n = 914 events from 9 cells of 3 mice; 16p11.2^+/-: n = 556 events from 6 cells of 5 mice. Kolmogorov-Smirnov test). F High-magnification confocal planes of VGLUT1-expressing perisomatic puncta on neurons of WT and 16p11.2^+/- mice. Scale bar: 5 μm. G Graph showing the density of VGLUT1 puncta on the perisomatic region of hippocampal neurons (WT: n = 33 cells from 3 mice; 16p11.2^+/-: n = 31 cells from 3 mice. Student’s t test). H GAD65/67 immunoreactivity in the hippocampus of WT and 16p11.2^+/- mice. I Graph showing the density of GAD65/67 puncta on the perisomatic region of hippocampal neurons (WT: n = 20 cells from 3 mice; 16p11.2^+/-: n = 22 cells from 3 mice. Student’s t test). J The level of glutamate (Glu) in hippocampus of mice was detected (WT: n = 7 mice; 16p11.2^+/-: n = 6 mice. Student’s t test). K The level of GABA in hippocampus of mice was detected (WT: n = 8 mice; 16p11.2^+/-: n = 7 mice. Student’s t test). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and n.s.: not significant. Detailed statistical information is presented in Additional file 2: Table S1

**Fig. 4**
IPA rescued the social novelty deficits and cognitive impairment in 16p11.2^+/- mice. A Schematic diagram for experimental design. B Representative trajectories of mice in the social novelty phase of the TCT. C IPA rescued social novelty deficits of 16p11.2^+/- mice in the TCT (WT + Vehicle: n = 20 mice; WT + IPA: n = 20 mice; 16p11.2^+/- + Vehicle: n = 18 mice; 16p11.2^+/- + IPA: n = 18 mice. Two-way ANOVA). D IPA improved social interaction time of 16p11.2^+/- mice in the DSI test (WT + Vehicle: n = 14 mice; WT + IPA: n = 15 mice; 16p11.2^+/- + Vehicle: n = 14 mice; 16p11.2^+/- + IPA: n = 14 mice. Two-way ANOVA). E Representative trajectories of mice in the recognition phase of the NOR test. F IPA restored cognitive impairment of 16p11.2^+/- mice but had no significant effect on WT mice in NOR test (WT + Vehicle: n = 20 mice; WT + IPA: n = 20 mice; 16p11.2^+/- + Vehicle: n = 18 mice; 16p11.2^+/- + IPA: n = 18 mice, Two-way ANOVA). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, and n.s.: not significant. Detailed statistical information is presented in Additional file 2: Table S1

**Fig. 5**
IPA mitigated the imbalance of hippocampal inhibitory transmission in 16p11.2^+/- mice. A The red puncta indicate c-Fos⁺ neurons in the DG of the hippocampus. Scale bar: 50 μm. B IPA can improve the overactivation of hippocampal neurons in 16p11.2^+/- mice (WT + Vehicle: n = 7 mice; WT + IPA: n = 4 mice; 16p11.2^+/- + Vehicle: n = 6 mice; 16p11.2^+/- + IPA: n = 5 mice. Two-way ANOVA). C Representative sIPSCs traces from granule cells in hippocampus of mice, scale bars: 5 s, 10pA. D Cumulative distribution of sIPSCs amplitudes. E Cumulative distribution of sIPSC frequencies (16p11.2^+/- + Vehicle: n = 1814 events from 9 cells of 5 mice; 16p11.2^+/- + IPA: n = 3169 events from 11 cells of 4 mice. Kolmogorov-Smirnov test). F Single confocal planes of punctate GAD65/67 surrounding neurons in the DG of the hippocampus, scale bar: 5 μm. G Graph showing the density of GAD65/67 puncta on the perisomatic region of hippocampal neurons (WT + Vehicle: n = 68 cells from 5 mice; WT + IPA: n = 35 cells from 4 mice; 16p11.2^+/- + Vehicle: n = 49 cells from 5 mice; 16p11.2^+/- + IPA: n = 52 cells from 5 mice. Two-way ANOVA). H IPA restored the level of GABA in hippocampus of 16p11.2^+/- mice (n = 5 per group. Two-way ANOVA). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001. Detailed statistical information is presented in Additional file 2: Table S1

**Fig. 6**
IPA enhanced ERK1/2 phosphorylation in hippocampus of 16p11.2^+/- mice. A Schematic of the 16p11.2 deletion region and the synonymous region in mouse chromosome 7. B Heat map of 23 expressed genes within 16p11.2 fragment in mouse hippocampus (n = 3 per group). C Protein-protein interaction (PPI) network of differential genes (screening criteria, p < 0.05) in the hippocampus of WT and 16p11.2^+/- mice. Nodes were size-scaled by degree. D *Mapk3* expression was decreased in the hippocampus of 16p11.2^+/- mice as assessed by RT-qPCR, and IPA could not increase its expression (n = 4 per group. Two-way ANOVA). E The representative Western blots showed IPA promoted the phosphorylation of ERK1/2 in hippocampus of 16p11.2^+/- mice. F-I Quantification of Western blot analysis showed that IPA did not change the expression levels of ERK1 (F) and ERK2 (G) in the hippocampus of 16p11.2^+/- mice, but significantly increased the phosphorylation level of ERK1/2 (H, I) (WT + Vehicle: n = 8 mice; WT + IPA: n = 8 mice; 16p11.2^+/- + Vehicle: n = 8 mice; 16p11.2^+/- + IPA: n = 7 mice. Two-way ANOVA). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and n.s.: not significant. Detailed statistical information is presented in Additional file 2: Table S1

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References

1. Lord C, Brugha TS, Charman T, Cusack J, Dumas G, Frazier T, Jones EJH, Jones RM, Pickles A, State MW, et al. Autism spectrum disorder. Nat Rev Dis Primers. 2020;6(1):5. doi: 10.1038/s41572-019-0138-4. - DOI - PMC - PubMed
1. Lord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J. Autism spectrum disorder. The Lancet. 2018;392(10146):508–520. doi: 10.1016/S0140-6736(18)31129-2. - DOI - PMC - PubMed
1. Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, Yamrom B, Yoon S, Krasnitz A, Kendall J, et al. Strong association of de novo copy number mutations with autism. Science. 2007;316(5823):445–449. doi: 10.1126/science.1138659. - DOI - PMC - PubMed
1. Chung WK, Roberts TP, Sherr EH, Snyder LG, Spiro JE. 16p11.2 deletion syndrome. Curr Opinion Genet Dev. 2021;68:49–56. doi: 10.1016/j.gde.2021.01.011. - DOI - PMC - PubMed
1. Weiss LA, Shen Y, Korn JM. Association between microdeletion and microduplication at 16p11.2 and autism. 2008. - PubMed

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[1] Lord C, Brugha TS, Charman T, Cusack J, Dumas G, Frazier T, Jones EJH, Jones RM, Pickles A, State MW, et al. Autism spectrum disorder. Nat Rev Dis Primers. 2020;6(1):5. doi: 10.1038/s41572-019-0138-4. - DOI - PMC - PubMed

[2] Lord C, Brugha TS, Charman T, Cusack J, Dumas G, Frazier T, Jones EJH, Jones RM, Pickles A, State MW, et al. Autism spectrum disorder. Nat Rev Dis Primers. 2020;6(1):5. doi: 10.1038/s41572-019-0138-4. - DOI - PMC - PubMed

[3] Lord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J. Autism spectrum disorder. The Lancet. 2018;392(10146):508–520. doi: 10.1016/S0140-6736(18)31129-2. - DOI - PMC - PubMed

[4] Lord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J. Autism spectrum disorder. The Lancet. 2018;392(10146):508–520. doi: 10.1016/S0140-6736(18)31129-2. - DOI - PMC - PubMed

[5] Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, Yamrom B, Yoon S, Krasnitz A, Kendall J, et al. Strong association of de novo copy number mutations with autism. Science. 2007;316(5823):445–449. doi: 10.1126/science.1138659. - DOI - PMC - PubMed

[6] Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, Yamrom B, Yoon S, Krasnitz A, Kendall J, et al. Strong association of de novo copy number mutations with autism. Science. 2007;316(5823):445–449. doi: 10.1126/science.1138659. - DOI - PMC - PubMed

[7] Chung WK, Roberts TP, Sherr EH, Snyder LG, Spiro JE. 16p11.2 deletion syndrome. Curr Opinion Genet Dev. 2021;68:49–56. doi: 10.1016/j.gde.2021.01.011. - DOI - PMC - PubMed

[8] Chung WK, Roberts TP, Sherr EH, Snyder LG, Spiro JE. 16p11.2 deletion syndrome. Curr Opinion Genet Dev. 2021;68:49–56. doi: 10.1016/j.gde.2021.01.011. - DOI - PMC - PubMed

[9] Weiss LA, Shen Y, Korn JM. Association between microdeletion and microduplication at 16p11.2 and autism. 2008. - PubMed

[10] Weiss LA, Shen Y, Korn JM. Association between microdeletion and microduplication at 16p11.2 and autism. 2008. - PubMed

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The gut metabolite indole-3-propionic acid activates ERK1 to restore social function and hippocampal inhibitory synaptic transmission in a 16p11.2 microdeletion mouse model

Affiliations

The gut metabolite indole-3-propionic acid activates ERK1 to restore social function and hippocampal inhibitory synaptic transmission in a 16p11.2 microdeletion mouse model

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