Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner

Abstract

It has recently become well-established that there is a connection between Alzheimer's disease pathology and gut microbiome dysbiosis. We have previously demonstrated that antibiotic-mediated gut microbiota perturbations lead to attenuation of Aβ deposition, phosphorylated tau accumulation, and disease-associated glial cell phenotypes in a sex-dependent manner. In this regard, we were intrigued by the finding that a marine-derived oligosaccharide, GV-971, was reported to alter gut microbiota and reduce Aβ amyloidosis in the 5XFAD mouse model that were treated at a point when Aβ burden was near plateau levels. Utilizing comparable methodologies, but with distinct technical and temporal features, we now report on the impact of GV-971 on gut microbiota, Aβ amyloidosis and microglial phenotypes in the APPPS1-21 model, studies performed at the University of Chicago, and independently in the 5X FAD model, studies performed at Washington University, St. Louis.Methods To comprehensively characterize the effects of GV-971 on the microbiota-microglia-amyloid axis, we conducted two separate investigations at independent institutions. There was no coordination of the experimental design or execution between the two laboratories. Indeed, the two laboratories were not aware of each other's experiments until the studies were completed. Male and female APPPS1-21 mice were treated daily with 40, 80, or 160 mg/kg of GV-971 from 8, when Aβ burden was detectable upto 12 weeks of age when Aβ burden was near maximal levels. In parallel, and to corroborate existing published studies and further investigate sex-related differences, male and female 5XFAD mice were treated daily with 100 mg/kg of GV-971 from 7 to 9 months of age when Aβ burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-β deposition, metagenomic, and neuroinflammatory profiles. Finally, studies were initiated at the University of Chicago to evaluate the metabolites in cecal tissue from vehicle and GV-971-treated 5XFAD mice.Results These studies showed that independent of the procedural differences (dosage, timing and duration of treatment) between the two laboratories, cerebral amyloidosis was reduced primarily in male mice, independent of strain. We also observed sex-specific microbiota differences following GV-971 treatment. Interestingly, GV-971 significantly altered multiple overlapping bacterial species at both institutions. Moreover, we discovered that GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway. The metagenomics and metabolomics changes correspond with notable reductions in peripheral pro-inflammatory cytokine and chemokine profiles. Furthermore, GV-971 treatment dampened astrocyte and microglia activation, significantly decreasing plaque-associated reactive microglia while concurrently increasing homeostatic microglia only in male mice. Bulk RNAseq analysis unveiled sex-specific changes in cerebral cortex transcriptome profiles, but most importantly, the transcriptome changes in the GV-971-treated male group revealed the involvement of microglia and inflammatory responses.Conclusions In conclusion, these studies demonstrate the connection between the gut microbiome, neuroinflammation, and Alzheimer's disease pathology while highlighting the potential therapeutic effect of GV-971. GV-971 targets the microbiota-microglia-amyloid axis, leading to the lowering of plaque pathology and neuroinflammatory signatures in a sex-dependent manner when given at the onset of Aβ deposition or when given after Aβ deposition is already at higher levels.

Keywords: Alzheimer’s disease; Microbiome; Microglia; Neuroinflammation; Sodium oligomannate.

Fig. 1

GV-971 reduces amyloidosis in a dose and sex-dependent manner. a University of Chicago experimental design. b Representative images of brains stained with anti-Aβ antibody 3D6 depicting the effect of GV-971 on Aβ plaque burden in APPPS1-21 mice. c Quantification of % area covered by 3D6⁺ Aβ plaques in cortices of APPPS1-21 male mice (n = 10). d Quantification of % area covered by 3D6⁺ Aβ plaques in cortices of APPPS1-21 female mice (n = 9–11). e Washington University in St. Louis experimental design. f Representative images of brains stained with anti-Aβ antibody HJ3.4 showing the effect of GV-971 on Aβ plaque burden in 5XFAD mice. g Quantification of % area covered by HJ3.4⁺ Aβ plaques in cortices of 5XFAD male mice (n = 13). h Quantification of % area covered by HJ3.4⁺ Aβ plaques in cortices of 5XFAD female male (n = 9–12). i Quantification of insoluble and soluble Aβ 40 and 42 isoforms extracted from cortical tissue of male APPPS1-21 mice (n = 10). j Quantification of insoluble and soluble Aβ 40 and 42 isoforms extracted from cortical tissue of female APPPS1-21 mice (n = 9–11). Data presented as SEM. Significance determined using a One-way ANOVA test followed by Tukey’s multiple comparison post hoc test (c, d, i, j), and unpaired t-test (g, h). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig. 2

GV-971 alters microbiome β-diversity in APPPS1-21 male mice. Analysis of bacterial α-diversity from cecal content of University of Chicago APPPS1-21 mice. a Faith phylogenetic diversity, b Shannon index, c Pielou species evenness. d PCoA plot generated by using unweighted unifrac distance metric. Analysis of bacterial α-diversity from cecal content of Washington University in St. Louis 5XFAD mice. e Faith phylogenetic diversity, f Shannon index, g Pielou species evenness. h PCoA plot generated by using unweighted unifrac distance metric. Diversity analyses, including alpha and beta diversity, alpha rarefaction, and group significance were analyzed by QIIME and QIIME2

Fig. 3

GV-971 alters microbiome metabolism in 5XFAD mice. a Representative heat map of significant amino acids abundance in cecal content of 5XFAD mice treated with 100 mg/kg GV-971 or vehicle from Washington University in St. Louis. b Quantification of amino acid abundance in cecal content of 5XFAD male mice (n = 13). c Quantification of amino acid abundance in cecal content of 5XFAD female mice (n = 9–12). d Representative heat map of significant metabolites in the tryptophan, indole pyruvate, and kynurenine pathway. e Quantification of tryptophan, indole pyruvate, and kynurenine pathway metabolite concentrations in cecal content of 5XFAD male mice (n = 13). f Quantification of tryptophan, indole pyruvate, and kynurenine pathway metabolite concentrations in cecal content of 5XFAD female mice (n = 9–12). g Representative heat map of significant primary and secondary bile acids. h Quantification of primary and secondary bile acids concentrations in cecal content of 5XFAD male mice (n = 13). i Quantification of primary and secondary bile acids concentrations in cecal content of 5XFAD female mice (n = 9–12). Data presented as SEM. Significance determined using unpaired t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig. 4

GV-971 modifies cytokine and chemokine levels in peripheral blood and cortical tissues. a Quantification of cytokine and chemokine concentrations in the serum of APPPS1-21 male mice treated with 160 mg/kg GV-971 or vehicle from the University of Chicago (n = 10–11). b Quantification of cytokine and chemokine concentrations in the serum of APPPS1-21 female mice treated with 160 mg/kg GV-971 or vehicle (n = 8–10). c, d Quantification of cytokine and chemokine concentrations in the serum of 5XFAD male mice treated with 100 mg/kg GV-971 or vehicle from Washington University in St. Louis (n = 12–13). e, f Quantification of cytokine and chemokine concentrations in the serum of 5XFAD female mice treated with 100 mg/kg GV-971 or vehicle (n = 9–12). g Quantification of cytokine and chemokine concentrations in the cortical tissue of 5XFAD male mice treated with 100 mg/kg GV-971 or vehicle (n = 12–13). Data presented as SEM. Significance determined using unpaired t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig. 5

GV-971 significantly altered microglia inflammatory activation status surrounding Aβ plaques. a Representative immunofluorescent images of P2ry12⁺ microglia (green) and Clec7a⁺ microglia (red) clustering around 3D6⁺ Aβ plaque (blue) in mice from University of Chicago. b Quantification of the average number of cortical Clec7a⁺ cells within 0.02 mm2 area of 3D6⁺ Aβ plaque in cortices of APPPS1-21 mice treated with 40 mg/kg, 80 mg/kg, or 160 mg/kg GV-971 or vehicle (male n = 12–9, female n = 9–11). c Quantification of the average number of cortical P2ry12⁺ cells within 0.02 mm2 area of 3D6⁺ Aβ plaque in cortices of APPPS1-21 mice treated with 40 mg/kg, 80 mg/kg, or 160 mg/kg GV-971 or vehicle (male n = 12–9, female n = 9–11). Data are presented as mean SEM. Significance was determined using One-way ANOVA test followed by Tukey’s multiple comparison post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig. 6

GV-971 significantly reduces glial inflammation in 9 month old 5XFAD male mice. a Representative images of brains stained for GFAP⁺ astrocytes in 5XFAD mice from Washington University in St. Louis. b, c Quantification of %area covered by GFAP⁺ astrocytes in whole brain, cortices and hippocampus of 5XFAD mice treated with 100 mg/kg GV-971 or vehicle (male = 13, female = 9–12). d Representative images of brains stained for Iba-1⁺ microglia. e, f Quantification of % area covered by Iba-1⁺ microglia in whole brain, cortices and hippocampus of 5XFAD mice treated with 100 mg/kg GV-971 or vehicle (male = 13, female = 9–12). g Representative images of Iba-1⁺ microglia (green) clustering around Aβ plaque (blue). h, i Quantification of the average number of Iba-1⁺ surface within 3 μM radius of Aβ plaque in cortices of 5XFAD mice treated with 100 mg/kg GV-971 or vehicle (male = 13, female = 9–12). j Representative images of Clec7a⁺ microglia (red) clustering around Aβ plaque (blue). k, l Quantification of the average number of Clec7a⁺ surface within 15 μM radius of Aβ plaque in cortices of 5XFAD mice treated with 100 mg/kg GV-971 or vehicle (male = 13, female = 9–12). m Representative images of P2ry12⁺ microglia (red) clustering around Aβ plaque (blue). n, o Quantification of the average number of P2ry12⁺ surface within 15 μM radius of Aβ plaque in cortices of 5XFAD mice treated with 100 mg/kg GV-971 or vehicle (male = 13, female = 9–12). Data presented as SEM. Significance determined using unpaired t-test (d). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig. 7

GV-971 treatment alters cerebral cortex transcriptome profiles in a sex-specific manner. a Gene overlap analysis demonstrating significant (P < 0.01) DEGs n = 728 in Male_Vehicle vs Male_GV, n = 426 DEGs in Female_Vehicle vs Female_GV and n = 730 DEGs in Male_Vehicle vs Female_Vehicle groups. Note only n = 23 DEGs that were common between GV-971-treated male and female groups compared with their respective vehicle-treated controls. b PCA plot. c Heatmap associated with lower DEGs (n = 436, P < 0.01) and higher DEGs (n = 294, P < 0.01) in Vehicle-treated female group compared with Vehicle-treated male group, note the sex-specific differences in DEGs between control male and female groups. Each column in an individual animal, n = 6 mice per group. d Heatmap associated with lower DEGs (n = 437, P < 0.01) and higher DEGs (n = 291, P < 0.01) in GV-971 treated male group compared with Vehicle-treated male group. Notice the female group columns exhibited no apparent differences for these genes. Each column is an individual animal, n = 6 mice per group. e GO biological processes, molecular functions, and KEGG pathways analysis based on the DEGs (P < 0.01) between vehicle-treated male and GV-971-treated male groups. Panel shows heatmap of top 20 pathways associated with lower DEGs in GV-971-treated male group compared with control

Fig. 8

Cytoscape and GSEA analyses using significantly altered genes (P < 0.01) in GV-971 treated male group compared with control. a Cytoscape analysis identified immunity pathway as a major impacted pathway associated with lower DEGs (P < 0.01) in GV-971-treated male compared with control. Most enriched genes are highlighted in darker shade. b Cytoscape analysis identified neuronal development pathway as a major impacted pathway associated with higher DEGs (P < 0.01) in GV-971 treated group compared with control. Most enriched genes are highlighted in darker shade. c GSEA enrichment results showed 9 significantly enriched gene sets out of 44 upregulated gene sets in Vehicle- treated male group compared with GV-971-treated male group