Exogenous lipase administration alters gut microbiota composition and ameliorates Alzheimer's disease-like pathology in APP/PS1 mice

Abstract

Alzheimer's disease (AD) represents the most common form of dementia in the elderly with no available disease modifying treatments. Altered gut microbial composition has been widely acknowledged as a common feature of AD, which potentially contributes to progression or onset of AD. To assess the hypothesis that Candida rugosa lipase (CRL), which has been shown to enhance gut microbiome and metabolite composition, can rebalance the gut microbiome composition and reduce AD pathology, the treatment effects in APPswe/PS1de9 (APP/PS1) mice were investigated. The analysis revealed an increased abundance of Acetatifactor and Clostridiales vadin BB60 genera in the gut; increased lipid hydrolysis in the gut lumen, normalization of peripheral unsaturated fatty acids, and reduction of neuroinflammation and memory deficits post treatment. Finally, we demonstrated that the evoked benefits on memory could be transferred via fecal matter transplant (FMT) into antibiotic-induced microbiome-depleted (AIMD) wildtype mice, ameliorating their memory deficits. The findings herein contributed to improve our understanding of the role of the gut microbiome in AD's complex networks and suggested that targeted modification of the gut could contribute to amelioration of AD neuropathology.

Figure 1

Gut-related changes through CRL treatment in APP/PS1 and Wt mice. A Study design: Wt (n = 13–15, 8 months of age) and APP/PS1 (n = 12, 8 months of age) received 5000 FIP/kg CRL for two consecutive months or regular water before sacrifice. B α-diversity analysis in fecal matter analyzed by Shannon and Simpson index. C. β-diversity analysis by Bray–Curtis dissimilarity and distances of fecal matter. D Taxonomy analysis of phyla abundance in fecal matter by alignment with Greengenes database. E Biomarker identification via LEfSe on genus level identified significant genera driving microbial changes in APP/PS1 treated versus untreated animals. F Metabolomics analysis of cecal matter of APP/PS1 groups showed increased abundance of metabolites that are associated with lipid digestion. G Pathway analysis of identified metabolites to determine key pathways activated through lipase administration revealing pathways associated with lipid degradation. H. Gut integrity analysis by transition measurement of oral administered FITC-dextran dye into plasma did not show a significant difference between treatment groups. Significance for α-diversity was assessed by Kruskal–Wallis H test and pairwise comparisons, for β-diversity with PERMANOVA. Cecal metabolomic data and LEfSe results were analyzed using 2-way ANOVA and FDR correction. Gut integrity was evaluated using 2-way ANOVA. Significance: *p < 0.05, **p < 0.01.

Figure 2

Peripheral impact of CRL treatment. A Quantification of immune cell population by flow cytometry analysis. No difference was observed in CD4+CD3+, CD8+CD3+ and their ratio, respectively. B Peripheral inflammation measured by plasma cytokine and chemokine levels. No biologically-relevant significant changes could be determined. C Pathway analysis of untargeted plasma metabolomics data indicating significant changes in UFA and glycerophospholipid metabolism. D Investigation of subclasses of UFA, SFA and UFA/SFA revealed an UFA specific elevation in APP/PS1 mice and reduction post treatment. E ω-3 and ω-6 fatty acid levels measured in plasma. All subtypes showed significant or trending normalization of UFA level post CRL treatment in APP/PS1 mice. F HDL and VLDL/LDL cholesterol and cholesterol esters to analyze CRL’s effect on lipid transport. VLDL/LDL cholesterol and cholesterol ester fraction were both significantly increased. Significance was assessed by 2-way ANOVA and post-hoc Tukey correction. Significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Brain pathological impact of CRL treatment. A Probe and acquisition trial parameters of Barnes maze to assess memory and learning. The probe trial showed a significant improvement for latency to find target hole as well as frequency of target hole investigations for treated mice and a trend for treated WT mice. The acquisition data suggested trends for improved learning in treated APP/PS1 mice. B Immunohistochemical analysis of treatment-dependent effects on microglia activation. Iba1/Congo Red stained cortex showed reduction in astrocytosis and microgliosis (× 20 magnification: four animals per group, four slides per animal, three images per tissue area). C Assessment of amyloid plaque size to associated microglia counts to determine changes in inflammatory response and plaque burden (× 40 magnification: four animals per group, four slides per animal, six images per tissue area). D Enrichment analysis of significant uncorrected cortical transcriptomic data of treated and untreated APP/PS1 mice suggesting microglia and astrocyte activity being altered as well as glycerophospholipid metabolism. E Transcriptomics analysis of brain cell markers of whole cortical tissue revealing treatment-dependent changes in microglia-specific genes in APP/PS1 mice. Significance of probe trial was determined by 1- or 2-way ANOVA and post-hoc Tukey analysis, dependent on the respective parameter, while acquisition data was analyzed using 3-way ANOVA and post-hoc Tukey analysis. 2-way ANOVA and Tukey correction were further applied for immunohistochemical results and 2-way ANOVA and FDR correction for transcriptomics analysis. Significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Multi-Omics integration via rdCV-RF and DIABLO mixOmics analysis. A rdCV-RF analysis displayed as swim lane plot. Each swim lane displays one sample. Circled points indicate misclassified components. Each component was assessed by 200 double cross-validations to analyze class probabilities. B Component correlation of each of the three datasets determined by DIABLO analysis, which maximized the correlated information. C Variable correlation displayed in Circos plot with correlation cut-off of 0.95. Two separate pathways were identified. Positive correlation (brown), negative correlation (black).

Figure 5

Transferability of results via FMTs in AIMD WT mice. A Study design. B α-diversity measured as observed ASVs indicated depletion post antibiotics and bowel cleanse and reshaping post FMT. C β-diversity measured by Bray–Curtis distance. While Sham groups co-located and ABX and PEG groups co-located, animals receiving FMTs were distinct but located separately. The ABX group recovered partially. For analysis, the group receiving Wt + CRL FMT (abnormal α- and β-diversity) was fainted throughout microbial analysis, although no impact on behavioral data was observed. D Taxonomic analysis of phyla levels in fecal samples post FMT. Besides in animals receiving Wt + CRL, no major differences were found between groups. E Barnes maze experiment for examination of differences in memory and learning. Results were normalized to sham group to reduce complexity of analysis. Analysis revealed that AIMD WT mice receiving none, or FMT from untreated APP/PS1 mice, performed similar or worse than all other groups. Significance was determined as in prior experiments of microbiome and Barnes maze analysis. The probe trial results were compared to ABX group only. Significance: *p < 0.05, **p < 0.01, ***p < 0.001.