Additional file 1 of Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner

Additional file 1: Supplemental Figure 1. Gut microbiome composition differs significantly between University of Chicago and Washington University in St. Louis. Analysis of bacterial α-diversity and β-diversity in fecal content from 9-week-old APPPS1-21 male mice collected at the University of Chicago and Washington University in St. Louis. (a) Shannon index, (b) Pielou species evenness. (d) 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. Data are presented as mean SEM. Significance was determined using Two-way ANOVA . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Supplemental Figure 2. GV-971 targets Aβ plaque halo in a sex-dependent manner. (a) Representative immunofluorescent images of HJ3.4+ Aβ (red) surrounding X34+ Aβ (blue). White * indicates regions of reduced plaque halo. (b,c) Quantification of an average number of HJ3.4 + Aβ surfaces within 5μM X34+ Aβ surface plaque in cortices of 5XFAD mice treated with 100mg/kg GV-971 or vehicle (male = 13, female = 9-12). Data are presented as mean SEM. Significance was determined using unpaired t-test (d). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Supplemental Figure 3. GV-971 alters amino acid metabolism. GC-nCI-MS and PFBBR derivtization heatmap analysis of metabolite abundance in cecal content from 5XFAD mice treated with 100mg/kg GV-971 or vehicle (male = 13, female 9-12). Supplemental Figure 4. GV-971 modifies tryptophan metabolism. LCMS/MS heatmap analysis of tryptophan pathway, indole pathway, and kynurenine pathway metabolite concentrations in cecal content from 5XFAD mice treated with 100mg/kg GV-971 or vehicle (male = 13, female 9-12). Supplemental Figure 5. GV-971 influences primary and secondary bile acid metabolism. LCMS/MS heatmap analysis of primary and secondary bile acid concentrations in cecal content from 5XFAD mice treated with 100mg/kg GV-971 or vehicle (male = 13, female 9-12). Supplemental Figure 6. GV-971 Significantly change peripheral and neuro-cytokine and chemokine production. Pie chart denoting the distribution of cytokine/chemokine production following GV-971 treatment. Chart is characterized into four groups based on expression levels compared to the control groups: Increased, Decreased, Not significant, and Non Detected. (a). Serum analyzed from the University of Chicago APPPS1-21 male and female mice treated with 160mg/kg GV-971. (b). Serum analyzed from Washington University 5XFAD male and female mice treated with 100mg/kg GV-971. (c). Cortical tissue analyzed from Washington University 5XFAD male mice treated with 100mg/kg GV-97. Supplemental Figure 7. GV-971 significantly alters microglia activation and neurodevelopment gene expression. (a). Quantitative PCR analysis of inflammatory, microglial, and neurodevelopment gene expression from bulk cortical tissue of male APPPS1-21 mice treated with GV-971 or vehicle. (b) Quantitative PCR analysis of inflammatory, microglial, and neurodevelopment gene expression from bulk cortical tissue of female APPPS1-21 mice treated with GV-971 or vehicle. Data are presented as mean SEM. Significance was determined using 2 way ANOVA followed by post hoc Tukey’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Supplemental Figure 8. GV-971 alters inflammatory markers in 9 month old 5XFAD male mice. Heat map analysis of bulk RNA in cortices of 5XFAD mice following 100mg/kg GV-971 or vehicle treatment (A) male mice n=13, (B) female mice n= 9-12. Graph generated by hierarchical gene clustering based on groups. Statistical analyses were performed using an unpaired t- test. *, P < 0.05