Sex-specific effects of microbiome perturbations on cerebral Aβ amyloidosis and microglia phenotypes.

We demonstrated that an antibiotic cocktail (ABX)-perturbed gut microbiome is associated with reduced amyloid-β (Aβ) plaque pathology and astrogliosis in the male amyloid precursor protein (APP)SWE /presenilin 1 (PS1)ΔE9 transgenic model of Aβ amyloidosis. We now show that in an independent, aggressive APPSWE/PS1L166P (APPPS1-21) mouse model of Aβ amyloidosis, an ABX-perturbed gut microbiome is associated with a reduction in Aβ pathology and alterations in microglial morphology, thus establishing the generality of the phenomenon. Most importantly, these latter alterations occur only in brains of male mice, not in the brains of female mice. Furthermore, ABX treatment lead to alterations in levels of selected microglial expressed transcripts indicative of the "M0" homeostatic state in male but not in female mice. Finally, we found that transplants of fecal microbiota from age-matched APPPS1-21 male mice into ABX-treated APPPS1-21 male restores the gut microbiome and partially restores Aβ pathology and microglial morphology, thus demonstrating a causal role of the microbiome in the modulation of Aβ amyloidosis and microglial physiology in mouse models of Aβ amyloidosis

Sex‐specific changes in amyloidosis and neuro‐immune modulation in response to alterations in the gut microbiome

BackgroundMicroglia, resident macrophages of CNS constantly screen the brain and engage in pathological processes by changing their morphology, expressing various antigens and become phagocytic. This activation causes the release of a wave of chemical mediators that promote the neuroinflammatory milieu. Thus, microglial homeostasis represents a highly plastic multifaceted response, finely tuned by the nature of the stimulus and the molecular repertoire involved. Studies from Sisodia lab have shown that antibiotic (ABX) mediated alterations of the gut microbiome in Thy1‐APPSwe.PS1L166Pmice (APPPS1‐21), resulted in a significant decrease in amyloidosis and altered microglial phenotypes in male mice. To investigate the influence of gut microbiome in modulating microglial function in a sex‐specific manner, we generated transgenic mice that would allow us to assess microglial RNA‐Protein networks and address the molecular mechanisms of CNS immunomodulation.MethodWe generated ‘Thy1‐APP.PS1‐RiboTag’ by crossing APPPS1‐21 mice to RiboTag mice wherein a CD11b promoter drives expression of ribosomal protein L10a (RpL10) that is fused to FLAG/EGFP at the amino‐terminus. Using our established protocol, we altered the microbiome of male and female mice (n=12) by orally gavaging them with ABX from day 14‐21, followed by low dose ABX in drinking water till they are sacrificed (7 wks). Vehicle treated mice were used as controls along with WT‐RiboTag mice. Immunoprecipitation of cortical homogenates with anti‐FLAG was performed to pull‐down actively translated mRNA and proteome in microglia following histopathology.ResultWe evaluated Aβ pathology and found that ABX‐treatment led to reduced amyloidosis in male mice with no significant changes in the female. Analysis of the mRNAs and the newly synthesized peptides resulted in a snapshot of the dynamic translational state of microglial ribosomes. We observed 21 upregulated and 22 downregulated proteins between the WT‐RiboTag and Th1‐APP.PS1‐RiboTag in male mice and 16 upregulated and 70 downregulated in female. Abx treatment resulted in upregulation of 39 and downregulation of 37 proteins in males while the females showed 51 and 9 respectively.ConclusionWe find marked differences in the mRNA and proteins associated with microglial phenotypes expressed in a sex‐specific manner as a function of changes in the gut microbiome.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163849/1/alz045243.pd

Translational profiling identifies sex-specific metabolic and epigenetic reprogramming of cortical microglia/macrophages in APPPS1-21 mice with an antibiotic-perturbed-microbiome

Background: Microglia, the brain-resident macrophages perform immune surveillance and engage with pathological processes resulting in phenotype changes necessary for maintaining homeostasis. In preceding studies, we showed that antibiotic-induced perturbations of the gut microbiome of APPPS1-21 mice resulted in significant attenuation in Aβ amyloidosis and altered microglial phenotypes that are specific to male mice. The molecular events underlying microglial phenotypic transitions remain unclear. Here, by generating ‘APPPS1-21-CD11br’ reporter mice, we investigated the translational state of microglial/macrophage ribosomes during their phenotypic transition and in a sex-specific manner. Methods: Six groups of mice that included WT-CD11br, antibiotic (ABX) or vehicle-treated APPPS1-21-CD11br males and females were sacrificed at 7-weeks of age (n = 15/group) and used for immunoprecipitation of microglial/macrophage polysomes from cortical homogenates using anti-FLAG antibody. Liquid chromatography coupled to tandem mass spectrometry and label-free quantification was used to identify newly synthesized peptides isolated from polysomes. Results: We show that ABX-treatment leads to decreased Aβ levels in male APPPS1-21-CD11br mice with no significant changes in females. We identified microglial/macrophage polypeptides involved in mitochondrial dysfunction and altered calcium signaling that are associated with Aβ-induced oxidative stress. Notably, female mice also showed downregulation of newly-synthesized ribosomal proteins. Furthermore, male mice showed an increase in newly-synthesized polypeptides involved in FcγR-mediated phagocytosis, while females showed an increase in newly-synthesized polypeptides responsible for actin organization associated with microglial activation. Next, we show that ABX-treatment resulted in substantial remodeling of the epigenetic landscape, leading to a metabolic shift that accommodates the increased bioenergetic and biosynthetic demands associated with microglial polarization in a sex-specific manner. While microglia in ABX-treated male mice exhibited a metabolic shift towards a neuroprotective phenotype that promotes Aβ clearance, microglia in ABX-treated female mice exhibited loss of energy homeostasis due to persistent mitochondrial dysfunction and impaired lysosomal clearance that was associated with inflammatory phenotypes. Conclusions: Our studies provide the first snapshot of the translational state of microglial/macrophage cells in a mouse model of Aβ amyloidosis that was subject to ABX treatment. ABX-mediated changes resulted in metabolic reprogramming of microglial phenotypes to modulate immune responses and amyloid clearance in a sex-specific manner. This microglial plasticity to support neuro-energetic homeostasis for its function based on sex paves the path for therapeutic modulation of immunometabolism for neurodegeneration.</p

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

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.</p

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