Research ArticleGUT MICROBIOTA

Neurogenesis and prolongevity signaling in young germ-free mice transplanted with the gut microbiota of old mice

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Science Translational Medicine  13 Nov 2019:
Vol. 11, Issue 518, eaau4760
DOI: 10.1126/scitranslmed.aau4760
  • Fig. 1 Gut microbiota transplants from old donor mice promote hippocampal neurogenesis in germ-free recipient mice.

    (A) Gut microbiota transplants (MTs) from ~24-month-old or 5- to 6-week-old mice were transplanted into 5- to 6-week-old germ-free young recipient mice. The transplanted mice were subjected to short-term cohousing with donors and then were housed in a controlled environment for 8 or 16 weeks before being euthanized. (B) Shown are representative images of doublecortin (DCX)–stained neurons in the dentate gyrus of young recipient mice receiving a gut microbiota transplant from old or young donors, referred to as old MT recipients or young MT recipients, respectively. DCX staining is red, and DAPI counterstain is blue. The white arrows indicate DCX+ neurons, and the red boxes indicate the area magnified in the two left panels. Scale bars, 100 μm. (C) Shown is quantification of the number of DCX+ neurons in the dentate gyrus of old MT and young MT recipient mice (n = 5 per group). (D and E) Shown is the expression of Sox9 (D) and CD133 (E) mRNA in the hippocampus of old MT and young MT recipient mice (n ≥ 5 per group). (F) Western blot analysis and quantification of BDNF protein expression in the hippocampus of old MT and young MT recipient mice (n = 4 per group). Actin was used as the loading control. (G) Differences in hippocampal metabolites between old MT and young MT recipient mice (n = 8 per group). The model is composed of one predictive (tcv [1]) and one orthogonal (tocv [1]) principal components. Data are reported as means ± SEM. *P calculated using the Student’s t test.

  • Fig. 2 Gut microbiota transplants from old donor mice promote intestinal growth and surface area expansion.

    (A) Representative images of hematoxylin and eosin staining of the small intestine of old MT and young MT recipient mice. Scale bar, 100 μm. (B and C) Quantification of villus length (B) and villus width (C) in the jejunum of old MT and young MT recipient mice (n = 11 per group). (D) Quantification of the length of the intestine from old MT and young MT recipient mice (n = 20 per group). (E) Representative images showing the intestinal length of old MT and young MT recipient mice. (F) Quantification of jejunal crypt depth in old MT and young MT recipient mice (n = 10 per group). (G) Representative images of jejunum tissue sections from old MT or young MT recipient mice, stained with 5-ethynyl-2′-deoxyuridine (EdU) (green) and DAPI counterstain (blue). Scale bars, 200 μm. (H) Quantification of EdU-positive cells in jejunum tissue sections from old MT and young MT recipient mice (n = 10 per group). (I) Shown is the intestinal permeability of old MT and young MT recipient mice measured by the amount of FITC-dextran in blood after oral gavage (n = 5 per group). (J to Q) Shown is the expression of mRNAs encoding claudin 1 (J), occludin (K), zonula occludens-1 [ZO-1; (L)], TNFα (M), IL-6 (N), IL-17 (O), IL-22 (P), and IL-10 (Q) in the ileum of old MT and young MT recipient mice (n ≥ 10 per group in each case). (R) Heatmap showing enrichment scores for the PPAR signaling pathway (KEGG pathway) in the ileum of old MT and young MT recipient mice. Genes with absolute fold changes of >1.5-fold and false discovery rates (FDR) of <0.05 were considered to be differentially expressed. Data are reported as means ± SEM. *P calculated using the Student’s t test; *P < 0.05 is considered significant.

  • Fig. 3 Recipient mice transplanted with old donor gut microbiota show distinct metabolic signatures.

    (A) Heatmap shows enrichment scores for the unfolded protein response (UPR) and triglyceride biosynthesis pathways in the liver of old MT and young MT recipient mice. Genes with absolute fold changes of >1.5-fold and FDR of <0.05 were considered to be differentially expressed. (B) Western blotting and quantification of AMPK activation in the liver of old MT and young MT recipient mice (n = 8 per group). Phosphorylated AMPK (pAMPK), a measure of AMPK activation, was normalized to total AMPK. (C) Western blotting and quantification of SIRT1 protein in the liver of old MT and young MT recipient mice (n = 4 per group). Actin was used as the loading control. (D) Western blotting and quantification of mTOR activation in the liver of old MT and young MT recipient mice (n = 4 per group). Phosphorylated mTOR (p-mTOR), a measure of mTOR activation, was normalized to total mTOR. (E to H) Shown is the expression of mRNAs encoding ACAD1 (E), CPT1 (F), HMGCS1 (G), and HMGCS2 (H) in the liver of old MT and young MT recipient mice (n = 8 per group in each case). Data are reported as means ± SEM. *P calculated using the Student’s t test.

  • Fig. 4 Recipient mice transplanted with old donor gut microbiota show an increase in butyrate-producing bacteria and FGF21.

    (A) Shown is a metagenomic analysis of the gut microbiota of recipient mice receiving either an old donor gut microbiota transplant (old MT) or a young donor gut microbiota transplant (young MT). The labeled data points show microbial species with a twofold or more difference in abundance and P < 0.05. Blue boxes represent enrichment of bacterial species in the gut microbiota of mice receiving old donor transplants, and the green box represents enrichment of bacterial species in the gut microbiota of mice receiving transplants from young donors. (B) Shown is a heatmap of the metagenomic analysis showing enrichment of genes involved in the butyrate production pathway according to the SEED subsystem database (n = 5 per group). (C) Shown is the quantification of butyrate in the feces of young MT and old MT recipients measured using NMR spectroscopy (n ≥ 10 per group). (D) Shown is expression of PPAR-α mRNA in the liver of young and old MT recipient mice (n = 8 per group). (E) Shown is Western blot analysis and quantification of the FGF21 protein in the liver of young and old MT recipient mice (n = 4 per group). Actin was used as the loading control. (F) Enzyme-linked immunosorbent assay (ELISA) assay of serum samples showing the amount of circulating FGF21 in young and old MT recipient mice (n ≥ 14 per group). (G) Shown is the expression of mRNA encoding the FGF21 co-receptor β-klotho in the hippocampus of young and old MT recipient mice (n = 15 per group). Data are reported as means ± SEM. *P calculated using the Student’s t test.

  • Fig. 5 Treating germ-free mice with sodium butyrate resulted in an old MT recipient phenotype.

    (A) Shown is Western blot analysis and quantification of the FGF21 protein in the liver of young germ-free (GF) mice and conventionally housed [pathogen-free (PF) mice; n = 4 per group]. Actin was used as the loading control. (B) ELISA assay of serum samples showing abundance of circulating FGF21 in germ-free and pathogen-free mice (n ≥ 15 per group). (C) Germ-free C57BL/6 mice aged 5 to 6 weeks were orally administered either sodium butyrate (SB) dissolved in PBS or PBS vehicle alone as control daily for 8 weeks. Shown is an ELISA of serum samples measuring circulating FGF21 in sodium butyrate–treated and vehicle-treated mice (n = 7 per group). (D to F) Shown is Western blot analysis (D) and quantification of hepatic FGF21 (E) protein and AMPK activation (F) in sodium butyrate–treated and vehicle-treated (PBS) germ-free mice (n = 4 per group). (G) Shown is Western blot analysis and quantification of BDNF protein expression in the hippocampus of sodium butyrate–treated and vehicle-treated (PBS) germ-free mice (n = 4 per group). Actin was used as the loading control. (H) Representative images showing DCX staining of neurons (red) in the dentate gyrus of sodium butyrate–treated and vehicle-treated germ-free mice (DAPI counterstain, blue). Scale bars, 100 μm. (I) Shown is quantification of DCX+ neurons in the dentate gyrus of sodium butyrate–treated and vehicle-treated germ-free mice (n = 9 per group). (J) Representative images showing the length of the intestine of sodium butyrate–treated and vehicle-treated germ-free mice. (K) Shown is quantification of the lengths of the small intestine and colon of sodium butyrate–treated and vehicle-treated germ-free mice (n = 9 per group). (L) Representative images of periodic acid–Schiff staining of the jejunum of sodium butyrate–treated and vehicle-treated germ-free mice. Scale bars, 100 μm. (M and N) Shown is quantification of villus length (M) and villus width (N) in the jejunum of sodium butyrate–treated and vehicle-treated (PBS) germ-free mice (n = 11 per group). (O) Shown is quantification of jejunal crypt depth of sodium butyrate–treated and vehicle-treated (PBS) germ-free mice (n = 5 per group). (P) Shown is quantification of cells staining positive for EdU in the jejunum of sodium butyrate–treated and vehicle-treated (PBS) germ-free mice (n = 9 per group). (Q) Representative images showing EdU staining (green) with DAPI counterstain (blue) of small intestine tissue sections from sodium butyrate–treated and vehicle-treated germ-free mice (n = 9 per group). Scale bars, 100 μm. Data are reported as means ± SEM. *P calculated using the Student’s t test.

  • Table 1 Altered metabolites in the hippocampus of old MT recipient mice measured using 1H NMR.

    The table lists metabolites, significant chemical shift values and multiplicity, and direction of associated changes. ADP, adenosine diphosphate. Multiplicity key: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet.

    MetaboliteMultiplicityAssociation
    Taurine3.43 (t), 3.26 (t)
    GABA2.29 (t), 3.02 (t), 1.91 (q)
    2.34 (d)
    Lactate1.33 (d), 4.11(q)
    Alanine1.48 (d)
    Glutamate2.05 (m), 2.36 (dd),
    3.77 (t)
    N-acetyl aspartate2.02 (s), 2.70 (dd),
    2.51 (dd)
    Glycerophosphocholine3.78 (m), 3.67 (dd),
    4.36 (m), 3.23 (s)
    Phosphocholine3.22 (s)
    Myoinositol4.06 (t), 3.63 (t),
    3.53 (dd)
    Creatinine3.04 (s), 3.93 (s)
    ADP8.58 (s), 8.27 (s)
    Choline3.20 (s)
    Unknown 17.68 (s)

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/518/eaau4760/DC1

    Materials and Methods

    Fig. S1. Influence of old donor gut microbiota transplants on young germ-free recipients.

    Fig. S2. Systemic effects of old donor gut microbiota transplants on young germ-free recipients.

    Fig. S3. Analysis of neuronal markers in gut microbiota–transplanted recipients and their donors.

    Fig. S4. Assessment of intestinal morphology in gut microbiota–transplanted recipients and their donors.

    Fig. S5. Metabolic effects of old and young donor gut microbiota transplants on germ-free recipients.

    Fig. S6. Comparative analysis of young gut microbiota–transplanted recipient mice and conventionally housed mice with a normal gut microbiota.

    Fig. S7. Impact of young or old donor gut microbiota transplant on old germ-free recipients.

    Fig. S8. Impact of sodium butyrate treatment on germ-free mice.

    Fig. S9. Schematic representation of possible pathways involved in the increased hippocampal neurogenesis and prolongevity signatures observed in old MT recipient mice.

    Table S1. Altered metabolites in the liver of old MT recipient mice measured using 1H NMR.

    Table S2. Details of primers used for RT-PCR analysis of mouse tissues.

    Data file S1. Individual-level data for Fig. 1.

    Data file S2. Individual-level data for Fig. 2.

    Data file S3. Individual-level data for Fig. 3.

    Data file S4. Individual-level data for Fig. 4.

    Data file S5. Individual-level data for Fig. 5.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Influence of old donor gut microbiota transplants on young germ-free recipients.
    • Fig. S2. Systemic effects of old donor gut microbiota transplants on young germ-free recipients.
    • Fig. S3. Analysis of neuronal markers in gut microbiota–transplanted recipients and their donors.
    • Fig. S4. Assessment of intestinal morphology in gut microbiota–transplanted recipients and their donors.
    • Fig. S5. Metabolic effects of old and young donor gut microbiota transplants on germ-free recipients.
    • Fig. S6. Comparative analysis of young gut microbiota–transplanted recipient mice and conventionally housed mice with a normal gut microbiota.
    • Fig. S7. Impact of young or old donor gut microbiota transplant on old germ-free recipients.
    • Fig. S8. Impact of sodium butyrate treatment on germ-free mice.
    • Fig. S9. Schematic representation of possible pathways involved in the increased hippocampal neurogenesis and prolongevity signatures observed in old MT recipient mice.
    • Table S1. Altered metabolites in the liver of old MT recipient mice measured using 1H NMR.
    • Table S2. Details of primers used for RT-PCR analysis of mouse tissues.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual-level data for Fig. 1.
    • Data file S2 (Microsoft Excel format). Individual-level data for Fig. 2.
    • Data file S3 (Microsoft Excel format). Individual-level data for Fig. 3.
    • Data file S4 (Microsoft Excel format). Individual-level data for Fig. 4.
    • Data file S5 (Microsoft Excel format). Individual-level data for Fig. 5.

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