Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder, in which the clinical manifestations may be influenced by genetic and unknown environmental factors. Here we show that ALS-prone Sod1 transgenic (Sod1-Tg) mice have a pre-symptomatic, vivarium-dependent dysbiosis and altered metabolite configuration, coupled with an exacerbated disease under germ-free conditions or after treatment with broad-spectrum antibiotics. We correlate eleven distinct commensal bacteria at our vivarium with the severity of ALS in mice, and by their individual supplementation into antibiotic-treated Sod1-Tg mice we demonstrate that Akkermansia muciniphila (AM) ameliorates whereas Ruminococcus torques and Parabacteroides distasonis exacerbate the symptoms of ALS. Furthermore, Sod1-Tg mice that are administered AM are found to accumulate AM-associated nicotinamide in the central nervous system, and systemic supplementation of nicotinamide improves motor symptoms and gene expression patterns in the spinal cord of Sod1-Tg mice. In humans, we identify distinct microbiome and metabolite configurations—including reduced levels of nicotinamide systemically and in the cerebrospinal fluid—in a small preliminary study that compares patients with ALS with household controls. We suggest that environmentally driven microbiome–brain interactions may modulate ALS in mice, and we call for similar investigations in the human form of the disease.
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The sequencing data has been deposited at the European Nucleotide Archive database with the accession number PRJEB32767.
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We thank the members of the Elinav and Segal laboratories, and members of the DKFZ cancer-microbiome division for discussions, and apologize to authors whose work was not included owing to space constraints. We thank R. Straussman, D. Nejman and E. Hornstein for suggestions and help with experimental methodologies; C. Bar-Nathan for assistance with animal work; S. Belkin for providing reagents and A. Godneva for computational assistance. We thank J. Suez and N. Zmora for extensive editing during production. E.B. is supported by the Weizmann Institute Dean of Faculty fellowship. D.R. received a Levi Eshkol PhD Scholarship for Personalized Medicine by the Israeli Ministry of Science. N.Z. is supported by the Gilead Sciences International Research Scholars Program in Liver Disease. C.K. is supported by an MD fellowship of the Boehringer Ingelheim Fonds. Y.K. is the incumbent of the Sarah and Rolando Uziel Research Associate Chair. H.S. is the Incumbent of the Vera Rosenberg Schwartz Research Fellow Chair. E.S. is supported by the Crown Human Genome Center; the Else Kroener Fresenius Foundation; Donald L. Schwarz, Sherman Oaks, CA; Jack N. Halpern, NeNY; Leesa Steinberg, Canada; and grants funded by the European Research Council and the Israel Science Foundation. E.E. is supported by Y. and R. Ungar; the Abisch Frenkel Foundation for the Promotion of Life Sciences; the Gurwin Family Fund for Scientific Research; the Leona M. and Harry B. Helmsley Charitable Trust; the Crown Endowment Fund for Immunological Research; the Else Kroener Fresenius Foundation; the estate of J. Gitlitz; the estate of L. Hershkovich; the Benoziyo Endowment Fund for the Advancement of Science; the Adelis Foundation; J. L. and V. Schwartz; A. and G. Markovitz; A. and C. Adelson; the French National Center for Scientific Research (CNRS); D. L. Schwarz; The V. R. Schwartz Research Fellow Chair; L. Steinberg; J. N. Halpern; A. Edelheit; and by grants funded by the European Research Council, the Israel Science Foundation and the Helmholtz Foundation. E.E. is the Sir Marc and Lady Tania Feldmann Professorial Chair in Immunology, Weizmann Institute of Science, Israel; is the Director of the Microbiome Research Division, DKFZ, Heidelberg, Germany; is a senior fellow of the Canadian Institute of Advanced Research (CIFAR) and is an international scholar of The Bill and Melinda Gates Foundation and Howard Hughes Medical Institute (HHMI).
E.S. and E.E. are paid consultants at DayTwo and BiomX. None of this work is related to, shared with or licensed to these or any other commercial entity. None of the other authors have competing interests related to this work.
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Peer review information Nature thanks Mani Arumugam, Premysl Bercik, Jeffrey Rothstein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Experimental design. Sod1-Tg and wild-type littermate control mice were untreated or treated with broad-spectrum antibiotics in their drinking water from the age of 40 days until the experimental end point. Testing was performed at age 60, 80, 100, 120 and 140 days. b–j, Motor performance of the mice was assessed at age 60, 80, 100, 120 and 140 days by the rotarod locomotor test (b, e, h), the hanging-wire grip test (c, f, i) and neurological scoring (d, g, j). For b–d: n = 8 Sod1-Tg water-treated mice; n = 6 Sod1-Tg antibiotic-treated mice; n = 5 wild-type water-treated and n = 5 wild-type antibiotic-treated mice. For e–g: n = 5 mice in each group; for h–j: n = 7 Sod1-Tg water-treated mice, n = 7 Sod1-Tg antibiotic-treated mice, n = 4 wild-type water-treated mice and n = 5 wild-type antibiotic-treated mice. Data are mean ± s.e.m. k–m, Linear regression of motor functions over time in Sod1-Tg and wild-type treated mice indicated by the rotarod locomotor test (k), the hanging-wire grip test (l) and neurological scoring (m). n = 20 Sod1-Tg water-treated n = 18 antibiotic-treated mice, n = 14 wild-type water-treated mice and n = 15 wild-type antibiotic-treated mice. n, o, Histological images (n) and cell counts (suggestive of neurons) (o) in the spinal cord of 140-day-old water-treated and antibiotic-treated Sod1-Tg mice. n = 10 Sod1-Tg water-treated mice, n = 5 Sod1-Tg antibiotic-treated mice. The experiment was repeated twice. p, q, MRI of areas of the mouse brain affected by ALS (p) and an image of T2 maps (q). The experiment was repeated twice. r–x, Quantification of T2 relaxation time in water-treated and antibiotic-treated Sod1-Tg mice throughout the course of ALS. n = 9 mice in each group. y, Home-cage locomotion analysis over a period of 46 h, for mice aged 100–101 days (n = 5 mice). z–ae, Distributions of immune cell sub-populations in the small-intestine (z, aa), colon (ab, ac), spinal cord at 50 days old (ad) and 140 days old (ae) in water-treated and antibiotic-treated Sod1-Tg mice. n = 5 mice. af, ag, Survival of specific-pathogen-free and germ-free Sod1-Tg mice and wild-type mice (af) or Sod1-Tg mice (ag) that were spontaneously colonized at age 115 days. The experiment was performed twice, with the results of the different experiments shown in af and ag. For af: n = 13 specific-pathogen-free and 7 germ-free Sod1-Tg mice, 16 wild-type specific-pathogen-free and 6 wild-type germ-free mice; for ag: n = 5 specific-pathogen-free and 8 germ-free Sod1-Tg mice. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. Significance according to a two-tailed Mann-Whitney U-test (b–j), two-tailed t-test (r–x), or Gehan-Breslow (af–ag). Unless stated otherwise, data are mean ± s.e.m.
Extended Data Fig. 2 Microbial compositional dynamics in the Sod1-Tg mouse model throughout the progression of ALS.
a, Taxa summary of bacterial phyla (a) and genera (b) from wild-type and Sod1-Tg mice, obtained by 16S rDNA sequencing of stool samples. c, Relative abundances of genera with significantly different representations in Sod1-Tg mice and wild-type mice. n = 6 mice in each group. d–m, FDR-corrected linear regression analysis of the change in relative bacterial abundance in stool samples of wild-type and Sod1-Tg mice during the progression of ALS. n = 6 Sod1-Tg mice and n = 4 wild-type mice. n, Alpha diversity of the microbiomes of Sod1-Tg mice and wild-type mice. n = 7 Sod1-Tg mice and n = 5 wild-type mice. The experiment was repeated 3 times. o, qPCR-based quantification of total 16S copy-number in 1 ng of DNA extracted from the stool samples of Sod1-Tg mice and wild-type mice. n = 6 Sod1-Tg and n = 5 wild-type mice. p, Taxa summary of bacterial phyla in individual antibiotic-treated wild-type and antibiotic-treated Sod1-Tg mice during the course of ALS. q–v, Weighted UniFrac PCoA on 47-day-old (pre-antibiotic; q) and 60–140-day-old (r–v) mice under a chronic antibiotic regime. n = 7 Sod1-Tg mice and n = 5 wild-type mice. w–ab, Volcano plots of significantly enriched bacterial genera in antibiotic-treated wild-type and antibiotic-treated Sod1-Tg mice during the course of murine ALS-like disease. n = 7 Sod1-Tg mice and n = 5 wild-type mice. ***P < 0.001; **P < 0.01; *P < 0.05. Significance according to an FDR-corrected two-sided FDR-corrected two-tailed Mann–Whitney U-test, or or t-test for coefficient genotype × time (d–m). Unless stated otherwise, data are mean ± s.e.m.
Extended Data Fig. 3 Microbial spontaneous colonization in an ex-germ-free Sod1-Tg mouse model and between two vivaria throughout the progression of ALS.
a, Taxa summary of bacterial genera in individual ex-germ-free wild-type and Sod1-Tg mice undergoing spontaneous bacterial colonization during the course of murine ALS-like disease. b–e, Weighted UniFrac PCoA of ex-germ-free wild-type and Sod1-Tg mice on days 4, 5, 53 and 63 after spontaneous colonization. n = 6 mice in each group. f–i, Volcano plots of significantly enriched bacterial genera in ex-germ-free wild-type mice and ex-germ-free Sod1-Tg mice during the course of the disease on days 4, 5, 53 and 63 after spontaneous colonization. n = 6 mice in each group. j, k, Weighted UniFrac PCoA (j) and alpha diversity (k) of wild-type and Sod1-Tg mice housed in a different non-barrier vivarium (vivarium B, at Ben-Gurion University) at weeks 4, 6, 8 and 12 of age. n = 7–9 Sod1-Tg mice and n = 7–9 wild-type mice. l, m, Individual (l) and averaged (m) taxa summary of bacterial genera in 80-day-old wild-type mice at vivarium A (Weizmann Institute of Science) and vivarium B (Ben-Gurion University). The comparison was performed once, n = 5–8 mice in each group. Significance according to FDR-corrected two-tailed Mann-Whitney U-test. Unless stated otherwise, data are mean ± s.e.m.
Extended Data Fig. 4 Differences in metagenomic faecal microbiomes and a metabolic comparison between wild-type and Sod1-Tg mice.
a, b, PCoA plot of bacterial composition (a), and taxa summary representation at the species level (b) of the gut microbiome of wild-type and Sod1-Tg mice, obtained by metagenomic shotgun sequencing. The experiment was repeated twice. c–n, FDR-corrected linear regression analysis of the change in relative bacterial abundance in stool samples of wild-type and Sod1-Tg mice during the progression of murine ALS-like disease. o, p, Schematic representation of tryptophan metabolism. p, Heat map of the abundances of bacterial genes involved in tryptophan metabolism. q, Heat map of the abundances of bacterial genes involved in the nicotinamide and nicotinate biosynthetic pathway. r–ac, Representative recording (r, t, v, x, z, aa, ab) and quantification (s, u, w, y, ac) of food intake (r, s), water consumption (t, u), respiratory exchange ratio (v, w), O2 consumption (x, y), heat production (z), locomotion (aa) and speed (ab, ac) of 60-day-old wild-type and Sod1-Tg mice. Data are mean ± s.e.m. The experiment was repeated twice. ***P < 0.001; **P < 0.01; *P < 0.05. Significance according to an FDR-corrected two-tailed Mann–Whitney U-test or t-test for coefficient genotype × time (c–n).
Extended Data Fig. 5 Mono-colonization of antibiotic pre-treated Sod1-Tg mice with selected microbiome strains that show a correlation with ALS severity.
a–c, Motor functions of antibiotic pre-treated Sod1-Tg mice treated with PBS (n = 8), E. lenta (EL, n = 7), C. cateniformis (CC, n = 6), P. goldsteinii (PG, n = 6), L. murinus (LM, n = 7), P. distasonis (PD, n = 5), L. gasseri (LG, n = 8), P. melaninogenica (PM, n = 4), or A. muciniphila (AM, ATCC 835, n = 6) as indicated by the rotarod locomotor test (a), the hanging-wire grip test (b) and neurological scoring (c). d–i, Motor functions of antibiotic-pre-treated Sod1-Tg mice treated with PBS or E. tayi (ET, n = 7) (d–f) or S. variabile (SV, n = 7) (g–i). j–k, Motor functions of antibiotic-pre-treated wild-type littermate controls treated with PBS, LM, PD, LG, PM or AM. m, Linear regression of the relative abundance of R. torques (16S rDNA sequencing) in stool samples of Sod1-Tg mice and wild-type mice. n–p, Motor functions of antibiotic-pre-treated wild-type and Sod1-Tg mice treated with PBS or RT, as assessed by the rotarod locomotor test (n), the hanging-wire grip test (o) and neurological scoring (p). n = 18 PBS-treated Sod1-Tg mice; n = 20 RT-treated Sod1-Tg mice; n = 23 PBS-treated and n = 23 PBS-treated or RT-treated wild-type mice. q–y, Assessment of the motor functions of antibiotic-pretreated Sod1-Tg mice and wild-type littermates after treatment with RT in three biological repeats, by the rotarod locomotor test (q, t, w), the hanging wire grip test (r, u, x) and neurological scoring (s, v, y). n = 5–10 mice. z, aa, Histological images (z) and cell counts (suggestive of neurons) (aa) in 140-day-old PBS-treated (n = 5) and RT-treated (n = 3) Sod1-Tg mice. The experiment was repeated 3 times. ab–ah, Brain areas (ab) and the quantification of their T2 relaxation times (ac–ah) of PBS-treated and RT-treated Sod1-Tg mice throughout the course of ALS. The experiment was repeated twice, n = 5 mice in each group. ai, aj, Averaged taxa summary of bacterial genera (ai) and unweighted UniFrac PCoA (aj) of antibiotic-pretreated Sod1-Tg mice treated with PBS (n = 6) or mono-colonized with the above-stated bacteria at 120 days old. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. Significance according to a two-tailed Mann-Whitney U-test (a–l, n–y), two-tailed t-test (ac–ah), or t-test for coefficient genotype × time (m). Unless stated otherwise, data are mean ± s.e.m.
Antibiotic-pretreated Sod1-Tg and wild-type littermate control mice were treated orally with AM (ATCC 835) or PBS as vehicle from 60 days of age until the experimental end point. a–o, At 60, 80, 100, 120 and 140 days old, motor performance of the mice was assessed by the rotarod locomotor test (a, d, g, j, m), the hanging-wire grip test (b, e, h, k, n) and neurological scoring (c, f, i, l, o). In g–i, Sod1-Tg mice were treated with PBS (n = 8), AM (n = 9) P. melaninogenica (n = 4) or L. gasseri (LG, n = 5). n = 4–26 mice. ***P < 0.001; **P < 0.01; *P < 0.05. Significance according to a two-tailed Mann–Whitney U-test. Data are mean ± s.e.m.
Extended Data Fig. 7 The effects of AM treatment on the manifestation of ALS and on microbiome composition in Sod1-Tg mice.
a, b, Histological images (a) and motor-neuron quantification (b) of the spinal cords of 140-day-old PBS-treated and AM-treated Sod1-Tg mice. n = 7 mice in each group. The experiment was repeated twice. c–f, T2 relaxation times in PBS-treated and AM (ATCC 835)-treated and antibiotic-pretreated Sod1-Tg mice at 100 and 140 days old. n = 5 mice in each group. g, Systemic FITC–dextran measurement in 120-day-old wild-type and Sod1-Tg mice treated with PBS (n = 3 wild-type mice and n = 7 Sod1-Tg mice), AM (n = 3 wild-type mice and n = 9 Sod1-Tg mice), P. melaninogenica (PM, n = 5 wild-type mice and n = 4 Sod1-Tg mice) or L. gasseri (LG, n = 5 for both wild-type and Sod1-Tg mice). h, PCoA of bacterial species compositions in Sod1-Tg mice treated with PBS (n = 33) or AM (n = 31). i, j, Relative abundance of AM in Sod1-Tg mice (i) or wild-type mice (j) treated with PBS or AM. n = 8 mice. k, l, Individual (k) and averaged (l) fold-change of AM 16S copy number (analysed by qPCR) in mucosal and luminal samples across the gastrointestinal tract of 140-day-old AM-treated or PBS-treated wild-type and Sod1-Tg mice. n = 5 mice in each group. m, n, Summary of bacterial genera in Sod1-Tg mice (m) or wild-type mice (n) treated with PBS or AM. Antibiotic-pretreated Sod1-Tg and wild-type littermate control mice were treated orally with AM (ATCC 2869) or PBS as a vehicle from 60 days of age until the experimental end point. o–q, At 60, 80, 100, 120 and 140 days old, the motor performance of the mice was assessed by the rotarod locomotor test (o), the hanging-wire grip test (p) and neurological scoring (q). n = 6 PBS-treated Sod1-Tg mice, AM-2869-treated Sod1-Tg mice and AM-2869-treated wild-type mice, and n = 7 wild-type PBS-treated mice. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. Significance according to a two-tailed t-test (c–f), or a two-tailed Mann-Whitney U-test (all others). Unless stated otherwise, data are mean ± s.e.m.
a, b, Immunohistochemical assessment of distal colon mucosa of 140-day-old PBS-treated (a) and AM-treated (BAA-835) (b) antibiotic-pretreated wild-type and Sod1-Tg mice. DNA is stained with SYTOX Green (green) and the mucus is stained with an anti-MUC2C3 antiserum and goat anti-Ig (red). The non-stained area between the epithelium and the outer mucus or luminal bacteria is the inner mucus layer, enabling identification of the bacteria. The experiment was performed once. White arrows represent penetrating crypt bacteria. c, d, Heat-map representation of the total mucus proteomic landscape (c) and AM-related peptides (d). e–j, Quantification of key representative components of mucus. For c–j, n = 4 Sod1-Tg PBS-treated mice and n = 8 Sod1-Tg AM-treated mice, two-tailed Mann–Whitney U-test. p = NS. Data are mean ± s.e.m.
Extended Data Fig. 9 The serum metabolomic profile is affected by antibiotics or by AM treatment in ALS Sod1-Tg mice.
a–c, Heat-map representation of serum metabolites of 100-day-old naive Sod1-Tg and their wild-type littermates (a), water-treated or antibiotic-treated Sod1-Tg mice (b) and PBS-treated or AM-treated Sod1-Tg mice (c). d, Scoring of the top six serum metabolites that were significantly altered by antibiotic treatment in Sod1-Tg mice by their potential to originate from the gut microbiome. n = 8 mice in each group. e–g, Motor performances of Sod1-Tg mice that were treated with phenol sulfate (n = 8) or water (n = 7) administered using subcutaneous osmotic pumps, as indicated by the rotarod locomotor test (e), the hanging-wire grip test (f) and neurological scoring (g). h, i, Non-targeted metabolomics assessment of tryptophan metabolism of water- and antibiotic- treated (h) or PBS- and AM-treated (i) 100-day-old Sod1-Tg mice. n=5 for water- and antibiotic -treated Sod1-Tg mice and n = 7 for PBS- and AM-treated Sod1-Tg mice in each group. j, Nicotinamide levels in the CSF of Sod1-Tg and wild-type mice treated with AM or PBS at the age of 140 days. k, Schematic representation of the microbiome-derived nicotinamide-encoding genes in faecal samples from AM-treated Sod1-Tg mice.*P < 0.05, two-tailed Mann-Whitney U-test. Unless stated otherwise, data are mean ± s.e.m.
a, Serum NAM levels in NAM-treated and vehicle-treated Sod1-Tg mice (n = 10). b–j, Motor performances of Sod1-Tg mice that were treated with NAM or vehicle administered using subcutaneous osmotic pumps, as indicated by the rotarod locomotor test (b, e, h), the hanging-wire grip test (c, f, i) and neurological scoring (d, g, j). n = 9–10 mice in each group. k, Survival analysis of NAM-treated and vehicle-treated Sod1-Tg mice (n = 10 mice in each group). l, Nicotinamide levels in wild-type or ΔnadA E. coli cultures (n = 5). m–o, Motor performances of wild-type E. coli-inoculated or ΔnadA E. coli-inoculated antibiotic-pretreated Sod1-Tg mice as indicated by the rotarod locomotor test (m), the hanging-wire grip test (n) and neurological scoring (o). n = 18 wild-type E.coli-treated Sod1-Tg mice and n = 16 ΔnadA E. coli-treated Sod1-Tg mice. p–x, Motor performances of wild-type E. coli-inoculated or ΔnadA E. coli-inoculated antibiotic-pretreated Sod1-Tg mice indicated by the rotarod locomotor test (p, s, v), the hanging-wire grip test (q, t, w) and neurological scoring (r, u, x). n = 4–8 mice. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. Significance according to a two-tailed Mann–Whitney U-test. Unless stated otherwise, data are mean ± s.e.m.
Extended Data Fig. 11 Uncovering potential downstream motor-neuron modulatory mechanisms of AM and NAM treatments.
a, Heat map of significantly (FDR-corrected two-tailed Mann-Whitney U-test) differentially expressed genes in the spinal cords of NAM-treated Sod1-Tg mice (n = 10 mice). b, Spearman correlation of the log2 fold change of spinal-cord transcripts comparing AM-treated and NAM-treated Sod1-Tg mice, n = 10 mice. c, Comparison of the significantly differentially expressed genes following NAM treatment with the COG database classified into 4 neuropathological groups. The blue shaded area represents the 95% confidence interval of the linear regression curve. d, e, Gene set enrichment distribution of spinal cord transcripts of NAM-treated (d) and AM-treated (e) Sod1-Tg mice sorted by biological process, molecular functions and cellular components. n = 10 mice.
a, Representation of spinal-cord transcripts from Sod1-Tg mice that showed a similar change after treatment with AM and with NAM, and share the GCGCMTGCGCN binding site for the transcription factor NRF-1 (in green). The analysis was performed using the g:Profiler platform49. b, Spinal-cord global transcriptomic analysis in NAM-treated or water-treated Sod1-Tg mice. c, Individual representation of the expression of Slc1a2 (n = 10). d, Spinal-cord global transcriptomic analysis in AM-treated or PBS-treated Sod1-Tg mice. e, Individual representation of the expression of Slc1a2 (n = 7). FDR-corrected two-tailed Mann–Whitney U-test. Data are mean ± s.e.m.
Extended Data Fig. 13 Differences in gut microbiome composition in patients with ALS compared with healthy controls.
a, Relative absence of gut microbiome bacterial species in stool samples of patients with ALS (n = 37) and healthy family members (n = 29). b, Spearman correlation between the abundance of Bifidobacterium pseudocatenulatum and levels of NAM in the serum of patients with ALS (n = 37) and healthy controls (n = 29). c, Relative frequencies of KEGG orthology terms of microbiome-associated genes of the nicotinamide pathway in stool samples of patients with ALS (n = 36) and healthy controls (n = 28). d, Top 97 serum metabolites that are differentially represented between patients with ALS (n = 24) and healthy controls (n = 13). e, Levels of metabolites from the tryptophan and nicotinamide biosynthetic pathways in the serum of patients with ALS (n = 24) and healthy controls (n = 13). f, g, Correlation of NAM levels in serum with clinical scores on the functional rating scale (FRS, f) and bacterial l-aspartate oxidase copies (g) in patients with ALS (n = 60) and healthy controls (n = 33). ***P < 0.0001, **P < 0.01; *P < 0.05. Significance according to a two-tailed Mann–Whitney U-test.
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Blacher, E., Bashiardes, S., Shapiro, H. et al. Potential roles of gut microbiome and metabolites in modulating ALS in mice. Nature 572, 474–480 (2019). https://doi.org/10.1038/s41586-019-1443-5
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