The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet

Abstract

The ketogenic diet (KD) is used to treat refractory epilepsy, but the mechanisms underlying its neuroprotective effects remain unclear. Here, we show that the gut microbiota is altered by the KD and required for protection against acute electrically induced seizures and spontaneous tonic-clonic seizures in two mouse models. Mice treated with antibiotics or reared germ free are resistant to KD-mediated seizure protection. Enrichment of, and gnotobiotic co-colonization with, KD-associated Akkermansia and Parabacteroides restores seizure protection. Moreover, transplantation of the KD gut microbiota and treatment with Akkermansia and Parabacteroides each confer seizure protection to mice fed a control diet. Alterations in colonic lumenal, serum, and hippocampal metabolomic profiles correlate with seizure protection, including reductions in systemic gamma-glutamylated amino acids and elevated hippocampal GABA/glutamate levels. Bacterial cross-feeding decreases gamma-glutamyltranspeptidase activity, and inhibiting gamma-glutamylation promotes seizure protection in vivo. Overall, this study reveals that the gut microbiota modulates host metabolism and seizure susceptibility in mice.

Keywords: 6-Hz seizures; Akkermansia; GGsTop; Kcna1; Parabacteroides; epilepsy; gamma-glutamyl transpeptidase; ketogenic diet; microbiome; microbiota.

Figure 1. The Ketogenic Diet Alters the Gut Microbiota and Protects Against 6-Hz Psychomotor Seizures

(A) Seizure thresholds in response to 6-Hz stimulation in independent cohorts of mice fed the CD or KD for 2, 4, 8, 10 or 14 days (left). n = 8, 6, 9, 20, 6 (CD); 8, 7, 12, 21, 5 (KD). Behavior in representative cohort of seizure-tested mice at 14 days post dietary treatment (right). Yellow line at y=10 seconds represents threshold for scoring seizures, and yellow triangle at 24 mA denotes starting current per experimental cohort. n = 16. (B) Levels of serum glucose in mice fed CD or KD for 2, 4, 8, 10 or 14 days. Data are normalized to serum glucose levels seen in SPF CD mice for each time point. n = 8, 5, 8, 8, 19 (CD); 8, 8, 8, 7, 19 (KD). (C) Levels of serum BHB mice fed CD or KD for 2, 4, 8, 10 or 14 days. n = 8, 13, 8, 8, 37 (CD); 8, 16, 8, 7, 38 (KD). (D) Principal coordinates analysis of weighted (left) and unweighted (right) UniFrac distance based on 16S rDNA profiling of feces from mice fed CD or KD for 0, 4, 8 or 14 days. n = 3 cages/group. (E) Alpha diversity of fecal 16S rDNA sequencing data from mice fed CD or KD for 14 days. n = 3 cages/group. (F) Taxonomic distributions of bacteria from fecal 16S rDNA sequencing data (left). n = 3 cages/group. Relative abundances of Akkermansia muciniphila and Parabacteroides (right). n = 3 cages/group. Data are presented as mean ± SEM. Two-way ANOVA with Bonferroni (A–C, E), Kruskal-Wallis with Bonferroni (F): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s.=not statistically significant. SPF=specific pathogen-free, CD=control diet, KD=ketogenic diet, CC50=current intensity producing seizures in 50% of mice tested, BHB=beta-hydroxybutyrate, OTUs=operational taxonomic units. See also Figure S1 and Tables S1 and S2.

Figure 2. The Microbiota is Required for the Anti-Seizure Effects of the Ketogenic Diet

(A) Seizure thresholds in response to 6-Hz stimulation in SPF, GF or conventionalized GF mice fed CD or KD. n = 13, 18, 12, 6. (B) Serum BHB (left) and glucose (right) levels in SPF, GF or conventionalized GF mice fed CD or KD. n = 37, 38, 19, 8. (C) Seizure thresholds in response to 6-Hz stimulation in SPF mice treated with vehicle or Abx pre-dietary treatment. n = 13, 18, 13. (D) Serum BHB (left) and glucose (right) levels in SPF mice treated with vehicle or Abx predietary treatment. n = 18, 18, 19 (BHB); n = 12, 11, 11 (glucose). Data are presented as mean ± SEM. One-way ANOVA with Bonferroni: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s.=not statistically significant. SPF=specific pathogen-free, GF=germ-free, GF-conv=germ-free conventionalized with SPF microbiota, CD=control diet, KD=ketogenic diet, CC50=current intensity producing seizures in 50% of mice tested. BHB=beta-hydroxybutyrate, veh=vehicle, Abx=antibiotics (ampicillin, vancomycin, neomycin, metronidazole). See also Figure S2.

Figure 3. KD-Associated Bacteria Sufficiently Mediate the Anti-Seizure Effects of the Ketogenic Diet

(A) Fluorescence in situ hybridization for A. muciniphila (MUC1437) and select Bacteroides and Parabacteroides, including P. merdae and P. merdae (BAC303) in colonic lumen from SPF mice fed CD, SPF mice fed KD or A. muciniphila and Parabacteroides-enriched mice fed KD. Scale bar = 25 um. Dotted lines indicate the borders of the intestinal epithelium. n = 3. (B) Seizure thresholds in response to 6-Hz stimulation in SPF mice pre-treated with vehicle or Abx, and colonized with Parabacteroides, Akkermansia muciniphila, both, or Bifidobacterium longum (left). n = 13, 18, 15, 6, 8, 5, 5. Behavior in representative cohort of seizure-tested mice (right). Yellow line at y=10 seconds represents threshold for scoring seizures, and yellow triangle at 24 mA denotes starting current per experimental cohort. n = 12, 16, 8, 25. (C) Seizure thresholds in response to 6-Hz stimulation in GF mice colonized with Parabacteroides and/or A. muciniphila (top). n = 15, 4, 9, 9. Behavior in seizure-tested mice (bottom). n = 17, 19. Data are presented as mean ± SEM. One-way ANOVA with Bonferroni: **p < 0.01, ***p < 0.001, ****p < 0.0001. SPF=specific pathogen-free, GF=germ-free, CD=control diet, KD=ketogenic diet, CC50=current intensity producing seizures in 50% of mice tested, veh=vehicle, Abx=pre-treated with antibiotics (ampicillin, vancomycin, neomycin, metronidazole), Pb=Parabacteroides (P. merdae and P. distasonis), Akk=Akkermansia muciniphila, AkkPb=A. muciniphila, P. merdae and P. distasonis, Bf=Bifidobacterium longum. See also Figure S3.

Figure 4. KD-Associated Bacteria Sufficiently Confer Seizure Protection in Mice Fed the Control Diet

(A) Seizure thresholds in response to 6-Hz stimulation in Abx-treated SPF transplanted with the CD microbiota (CD-FMT) or KD microbiota (KD-FMT) and fed the CD or KD (left). n = 6, 5, 5. Behavior in representative cohort of seizure-tested mice (right). Yellow line at y=10 seconds represents threshold for scoring seizures, and yellow triangle at 24 mA denotes starting current per experimental cohort. n = 12. (B) Seizure thresholds in response to 6-Hz stimulation in SPF mice pre-treated with vehicle or Abx, and colonized with Parabacteroides, Akkermansia muciniphila, both, or Bifidobacterium longum (left). n = 13, 18, 8, 9, 8, 6, 6. Behavior in representative cohort of seizure-tested mice (right). n = 16, 16, 12, 12. (C) Seizure thresholds in response to 6-Hz stimulation in SPF mice orally gavaged with A. muciniphila, P. merdae and P. distasonis, A. muciniphila alone, or heat-killed A. muciniphila and Parabacteroides. (left). n = 6, 6, 4, 3. Behavior in seizure-tested mice (right). n = 15, 20, 8, 8. Data are presented as mean ± SEM. One-way ANOVA with Bonferroni: *p < 0.05, ***p < 0.001, ****p < 0.0001. SPF=specific pathogen-free, CD=control diet, KD=ketogenic diet, CC50=current intensity producing seizures in 50% of mice tested, CD-FMT=transplanted with CD microbiota, KD-FMT=transplanted with KD microbiota, veh=vehicle, Abx=pre-treated with antibiotics (ampicillin, vancomycin, neomycin, metronidazole), Pb=Parabacteroides (P. merdae and P. distasonis), Akk=Akkermansia muciniphila, AkkPb=A. muciniphila, P. merdae and P. distasonis, Bf=Bifidobacterium longum, hk-AkkPb=heat-killed A. muciniphila, P. merdae and P. distasonis. See also Figure S4.

Figure 5. KD-Associated Bacteria Mediate Protection Against Tonic-Clonic Seizures in Response to the Ketogenic Diet

(A) Principal coordinates analysis of weighted UniFrac distances based on 16S rDNA profiling of feces Kcna1−/− mice fed CD or KD for 14 days. n = 7 cages/group. (B) Average taxonomic distributions of bacteria from fecal 16S rDNA sequencing data (left). Relative abundances of Akkermansia muciniphila and Parabacteroides (right). n = 7 cages/group. (C) Representative EEG trace showing stages used to define seizures quantified in (D). (D) Average number of seizures per day (left) and total duration of seizures per day (right) in SPF Kcna1−/− mice treated with vehicle or Abx, colonized with A. muciniphila and Parabacteroides spp. or nothing, and fed CD or KD. n = 2, 8, 6, 12, 9, 3. Data are presented as mean ± SEM. Kruskal-Wallis with Bonferroni (A,B), Mann-Whitney (D): *p < 0.05. SPF=specific pathogen-free, CD=control diet, KD=ketogenic diet, veh=vehicle, Abx=pre-treated with antibiotics (ampicillin, vancomycin, neomycin, metronidazole), AkkPb=A. muciniphila, P. merdae and P. distasonis. See also Figure S5.

Figure 6. Reductions in Peripheral Gamma-Glutamyl Amino Acids and Increases in Hippocampal GABA/Glutamate Ratios are Associated with Diet- and Microbiota-Dependent Seizure Protection

(A) Principal components analysis of colonic lumenal metabolites (top) and serum metabolites (bottom) from SPF mice fed CD, SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonized mice fed KD. n = 8 cages/group. (B) Biochemicals, identified by Random Forests classification of colonic lumenal (left) and serum (right) metabolomes, that contribute most highly to the discrimination of seizure-protected (SPF CD, Abx KD) from seizure-susceptible (SPF KD, AkkPb KD) groups. n = 8 cages/group. (C) Levels of gamma-glutamylated amino acids and cysteine in colonic lumenal contents from SPF mice fed CD, SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonized mice fed KD. n = 8 cages/group. (D) Levels of gamma-glutamyl amino acids and glutamine in sera from SPF mice fed CD, SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonized mice fed KD. n = 8 cages/group. (E) Heatmap of metabolites identified in hippocampi of SPF mice fed CD, SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonized mice fed KD. n = 5. (F) Levels of GABA/glutamate (left) and glutamine (right) in hippocampi of SPF mice fed CD, SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonized mice fed KD. n = 5. Data are presented as mean ± SEM. Two-way ANOVA contrasts (A-D), One-way ANOVA with Bonferroni (E-F): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s.=not statistically significant. CD=control diet, KD=ketogenic diet, SPF=specific pathogen-free, veh=vehicle, Abx=pre-treated with antibiotics (ampicillin, vancomycin, neomycin, metronidazole), AkkPb=A muciniphila, P. merdae and P. distasonis, a.u.=arbitrary units. See also Figure S6 and Tables S3–S6.

Figure 7. The Ketogenic Diet and Bacterial Cross-Feeding Reduces Gamma Glutamyltranspeptidase (GGT) Activity, and GGT Inhibition Sufficiently Confers Seizure Protection

(A) 6-Hz seizure thresholds in response to oral gavage with the GGT inhibitor, GGsTop, in SPF mice fed CD (left). n = 6, 9. Behavior in seizure-tested mice (right). Yellow line at y=10 seconds represents threshold for scoring seizures, and yellow triangle at 24 mA denotes starting current per experimental cohort. n = 16. (B) 6-Hz seizure thresholds in response to supplementation with ketogenic amino acids in Abx-treated SPF mice enriched for A. muciniphila and Parabacteroides (left). n = 5, 6. Behavior in seizure-tested mice (right). n = 12. (C) Total GGT activity per 100 mg feces from SPF CD, SPF KD, AkkPb KD, or AkkPb CD mice (left), and inhibition by GGsTop (right). n = 5. (D) Total GGT activity per 100 mg feces from SPF CD animals treated with vehicle, A. muciniphila and Parabacteroides probiotic, or heat-killed bacteria for bi-daily for 28 days (left), and inhibition by GGsTop (right). n = 5. (E) Levels of live A. muciniphila (Akk) after incubation in CD vs KD culture media or in CD or KD agar overlaid with M9 minimal media containing live P. merdae (PbM) or no bacteria (0). n = 3. (F) Levels of live PbM after incubation in M9 minimal media overlaid on CD or KD agar containing Akk or no bacteria (0). n = 5. (G) GGT activity in P. merdae grown in M9 media overlaid on CD agar containing A. muciniphila or no bacteria at t=24 hrs, and inhibition of GGT activity by GGsTop. n = 5. (H) GGT activity in P. merdae grown in M9 media overlaid on KD agar containing A. muciniphila or no bacteria at t=24 hrs, and inhibition of GGT activity by GGsTop. n = 5. Data are presented as mean ± SEM. Students t-test (A,B), Two-way ANOVA with Bonferroni (C, D), One-way ANOVA with Bonferroni (E-H): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SPF=specific pathogen-free, CD=control diet, KD=ketogenic diet, CC50=current intensity producing seizures in 50% of mice tested, AA=amino acids, veh=vehicle, Abx=pre-treated with antibiotics (ampicillin, vancomycin, neomycin, metronidazole), AkkPb=A. muciniphila, P. merdae and P. distasonis, GGsTop=GGT inhibitor, PbM=Parabacteroides merdae, Akk=Akkermansia muciniphila, M9=minimal media, GGT=gamma-glutamyltranspeptidase, AU=absorbance units. See also Figure S7.