Myelin as a regulator of development of the microbiota-gut-brain axis

Abstract

Myelination in the peripheral and central nervous systems is critical in regulating motor, sensory, and cognitive functions. As myelination occurs rapidly during early life, neonatal gut dysbiosis during early colonization can potentially alter proper myelination by dysregulating immune responses and neuronal differentiation. Despite common usage of antibiotics (Abx) in children, the impact of neonatal Abx-induced dysbiosis on the development of microbiota, gut, brain (MGB) axis, including myelination and behavior, is unknown. We hypothesized that neonatal Abx-induced dysbiosis dysregulates host-microbe interactions, impairing myelination in the brain, and altering the MGB axis. Neonatal C57BL/6 mice were orally gavaged daily with an Abx cocktail (neomycin, vancomycin, ampicillin) or water (vehicle) from postnatal day 7 (P7) until weaning (P23) to induce gut dysbiosis. Behavior (cognition; anxiety-like behavior), microbiota sequencing, and qPCR (ileum, colon, hippocampus and pre-frontal cortex [PFC]) were performed in adult mice (6-8 weeks). Neonatal Abx administration led to intestinal dysbiosis in adulthood, impaired intestinal physiology, coupled with perturbations of bacterial metabolites and behavioral alterations (cognitive deficits and anxiolytic behavior). Expression of myelin-related genes (Mag, Mog, Mbp, Mobp, Plp) and transcription factors (Sox10, Myrf) important for oligodendrocytes were significantly increased in the PFC region of Abx-treated mice. Increased myelination was confirmed by immunofluorescence imaging and western blot analysis, demonstrating increased expression of MBP, SOX10 and MYRF in neonatally Abx-treated mice compared to sham controls in adulthood. Finally, administration of the short chain fatty acid butyrate following completion of the Abx treatment restored intestinal physiology, behavior, and myelination impairments, suggesting a critical role for the gut microbiota in mediating these effects. Taken together, we identified a long-lasting impact of neonatal Abx administration on the MGB axis, specifically on myelin regulation in the PFC region, potentially contributing to impaired cognitive function and bacterial metabolites are effective in reversing this altered phenotype.

Fig. 1.

Study Design. C57BL/6J mice were orally gavaged with antibiotic (Abx) cocktail or water (vehicle) from postnatal (P) day 7 to P23. From P24 to P50, mice were given normal water or tributyrin. Upon euthanasia, fecal pellets were used to analyze gut bacterial community analysis via 16s Illumina sequencing. Tissue samples such as prefrontal cortex (PFC), hippocampus, and ileum were used for qPCR or western blot. Immunofluorescence was performed in the medial-PFC. Ileal physiology was analyzed via Ussing chambers. Behavioral tests were performed to assess cognition and anxiety-like behavior. Neurogenesis was analyzed via immunofluorescence of dorsal hippocampus.

Fig. 2.

Neonatal Abx administration causes persistent gut dysbiosis in adulthood. (A) Shannon and Simpson indexes for α-diversity, (B) Bray-Curtis, Jaccard, and UniFrac (weighted and unweighted) indexes for β-diversity, (C) Phylum abundance and (D) Family abundance. Holm-Sidak; **p < 0.01, ***p < 0.001; N = 12.

Fig. 3.

Neonatal Abx administration causes ileal pathophysiology without causing intestinal inflammation in adulthood. Ussing chamber studies assessed (A) short circuit current [Isc], (B) conductance (G) and (C) FITC flux in the ileum (N = 11–16). (D) Expression of immune-related genes (cytokines, pattern recognition receptors) in the ileum by qPCR (N = 8). Females denoted as light grey circles, and males as dark grey squares. Students T-test, *p < 0.05, **p < 0.01.

Fig. 4.

Neonatal Abx administration leads to behavioral deficits and myelin dysregulation in adulthood. (A) Novel object recognition task. (B) Light/dark box; (i) % time spent in the light, (ii) Frequency of transitions. (C) Open field test; (i) Distance travelled, (ii) Frequency of transitions, (iii) Time in inner zone. Expression of myelin-associated genes in the prefrontal cortex (PFC; D) and hippocampus (E). N = 14–16 for behavioral tests; N = 7–8 for qPCR; N = 5 for IF. (F) Gene expression (N = 8) and IF (N = 5) for MBP in the PFC region. DAPI in blue and MBP in red. Scale bar 20 µm. Unpaired T-test *p < 0.05. Females denoted as light grey circles, and males as dark grey squares. Students T-test, *p < 0.05, **p < 0.01.

Fig. 5.

Neonatal Abx administration leads to maturation and differentiation of oligodendrocytes. (A) mRNA expression of oligodendrocyte lineage-specific markers in the pre-frontal cortex (PFC) (N = 7–8). (B) Immunofluorescence for SOX10 (63x) expression in the PFC (N = 4). DAPI in blue and SOX10 in red. Scale bar 20 µm. Unpaired T-test *p < 0.05; co-localisation staining of OLIG2 and SOX10 (40x) in the PFC region (N = 4). DAPI in blue, SOX10 in red, OLIG2 in green, merge in yellow). Scale bar 50 µm. (C) Western blot and densitometric analysis for MYRF, SOX10 and β-actin (N = 5). Females denoted as light grey circles, and males as dark grey squares. Student’s T-test *p ≤ 0.05.

Fig. 6.

Neonatal Abx administration causes dysregulation of IGF signaling and neurogenesis. (A) BDNF gene expression in the pre-frontal cortex (PFC) (N = 6–8) (B) Expression of DCX (green) and Ki67 (red) in the hippocampus (20x) (N = 6). Females denoted as light grey circles, and males as dark grey squares. Students T-test, *p ≤ 0.05, **p < 0.01.

Fig. 7.

Tributyrin supplementation rescues behavior, intestinal physiology, and myelin dysregulation in adulthood. (A) Short chain fatty acid concentrations in fecal pellets as measured by mass spectrometry. (B) Ussing chamber studies for short-circuit current (Isc), conductance (G) and flux of FITC dextran. (C) Novel object recognition (NOR) task. (D) Light/dark box; (i) % time spent in the light, (ii) Frequency of transitions. (E) Open field task (OFT); (i) Distance travelled, (ii) Frequency of transitions, (iii) Time in inner zone. N = 11–14 (F) Expression of myelin-related genes in the pre-frontal cortex (PFC) (N = 8–9). (G) Western blot and densitometric analysis for MYRF, SOX10 and GAPDH in the PFC (N = 5). (H) Immunofluorescence of SOX10 (63x) in the PFC region (N = 4). DAPI in blue and SOX10 in red. Scale bar 20 µm. (I) Hippocampal Bdnf mRNA expression (N = 8–9). (J) Immunofluorescence of MBP (63x) in the PFC region (N = 3) DAPI in blue and MBP in red. Scale bar 20 μm. Females denoted as light grey circles, and males as dark grey squares. Student’s T-test. *p < 0.05.