Sex-specific hippocampal metabolic signatures at the onset of systemic inflammation with lipopolysaccharide in the APPswe/PS1dE9 mouse model of Alzheimer's disease

Abstract

Systemic inflammation enhances the risk and progression of Alzheimer's disease (AD). Lipopolysaccharide (LPS), a potent pro-inflammatory endotoxin produced by the gut, is found in excess levels in AD where it associates with neurological hallmarks of pathology. Sex differences in susceptibility to inflammation and AD progression have been reported, but how this impacts on LPS responses remains under investigated. We previously reported in an APP/PS1 model of AD that systemic LPS administration rapidly altered hippocampal metabolism in males. Here, we used untargeted metabolomics to comprehensively identify hippocampal metabolic processes occurring at onset of systemic inflammation with LPS (100 µg/kg, i.v.) in APP/PS1 mice, at an early pathological stage, and investigated the sexual dimorphism in this response. Four hours after LPS administration, pathways regulating energy metabolism, immune and oxidative stress responses were simultaneously recruited in the hippocampi of 4.5-month-old mice with a more protective response in females despite their pro-inflammatory and pro-oxidant metabolic signature in the absence of immune stimulation. LPS induced comparable behavioural sickness responses in male and female wild-type and APP/PS1 mice and comparable activation of both the serotonin and nicotinamide pathways of tryptophan metabolism in their hippocampi. Elevations in N-methyl-2-pyridone-5-carboxamide, a major toxic metabolite of nicotinamide, correlated with behavioural sickness regardless of sex, as well as with the LPS-induced hypothermia seen in males. Males also exhibited a pro-inflammatory-like downregulation of pyruvate metabolism, exacerbated in APP/PS1 males, and methionine metabolism whereas females showed a greater cytokine response and anti-inflammatory-like downregulation of hippocampal methylglyoxal and methionine metabolism. Metabolic changes were not associated with morphological markers of immune cell activation suggesting that they constitute an early event in the development of LPS-induced neuroinflammation and AD exacerbation. These data suggest that the female hippocampus is more tolerant to acute systemic inflammation.

Keywords: APP/PS1 mouse model; Alzheimer’s disease; Hippocampus; Inflammation; Lipopolysaccharide; Metabolomics; Methionine; Microglia; Serotonin; Sex differences.

Fig. 1

LPS-induced behavioural suppression at 4 h post-injection is independent of sex or genotype. A) Timeline of the experiment. 4.5-month-old male and female APP/PS1 mice and their wild-type (WT) littermates (n = 5–6) were subjected to baseline assessment of spatial working memory performance and exploratory drive in the spontaneous test as well as food burrowing behaviour prior to receiving a tail vein injection of lipopolysaccharide (LPS, 100 μg/kg) or its vehicle (phosphate buffer saline, PBS). Induced sickness effects were tested at 4 h post-injection in the same tests, prior to blood and tissue collection. At this time point, a significant decrease in core body temperature was observed in males, regardless of their genotype (B). LPS also suppressed food burrowing activity (C) and exploratory drive in the spontaneous alternation test, assessed through the number of arms visits (E), regardless of sex and genotype, but baseline performance for these behavioural measures did not differ between groups (C, D). Female mice overall exhibited lower spontaneous alternation performance than their male counterparts at baseline (F), but LPS had no significant impact on this measure (G). Parametric data are expressed as Means ± SEM. Dots represent individual animals. Post-hoc tests: *p < 0.05; **p < 0.01, ***p < 0.0001 vs PBS or baseline. Food burrowing data were rank-transformed for statistical analysis but represented as non-normalised responses and expressed as Median ± interquartile range. Sickness scores are represented as the difference between pre- and post-injection performance. Within-subjects pairwise comparisons following 3-way ANOVAs: ^#p < 0.05; ^##p < 0.01, ^###p < 0.0001 compared to baseline performance (E).

Fig. 2

LPS-induced plasma cytokines at 4 h post-injection. 4.5-month-old male and female APP/PS1 mice and their wild-type (WT) littermates were challenged with LPS (100µ/kg i.v.) or its vehicle PBS. Their plasma was collected 4 h later, immediately after behavioural assessment, for measurement of induced levels of pro- and anti-inflammatory cytokines. At this time point, a significant increase in circulating Interleukin 6 (IL-6, A), which has both pro- and anti-inflammatory effects, was observed regardless of sex and genotype (A). Levels of the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α) were increased by LPS in females, particularly WT females (B), but the levels of the other pro-inflammatory mediator, interferon gamma (IFN-γ), were unaltered (C). A significant increase in circulating levels of the anti-inflammatory cytokine IL-10, was also observed in females, regardless of genotype (E). Data were rank-transformed for statistical analyses but are expressed as Median ± interquartile range of non-normalised responses. Dots represent individual animals. Pairwise comparisons: *p < 0.05; **p < 0.01, ***p < 0.0001 vs PBS.

Fig. 3

Score plots of Principal Component Analysis (PCA) and two-class Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) models for hippocampal metabolism at 4 h post-injection with LPS or PBS. R²: variance explained, Q²: variance predicted. Dots represent individual animals. A). PCA analysis reveals global metabolic differences between LPS-treated males and females regardless of genotype [PC1 (X axis): R²X[1] = 0.232, Q² = 0.115; PC2 (Y axis): R²X[2] = 0.123, Q² = 0.082). B) Global OPLS-DA model (R² = 0.994, Q2 = 0.886) revealing clear separations between PBS- and LPS-treated mice (X axis: predictive component), but also some separation between LPS-treated male and female mice (Y axis: first orthogonal component), regardless of genotype. C) Accordingly, the class OPLS-DA model comparing genotypes in PBS-treated mice confirmed the lack of clear separation between WT and APP/PS1 mice (R² = 0.697, Q² = 0.244). Predictive component 1 (X axis) vs 2 (Y axis). 2-class OPLS-DA models confirmed the strong differences in hippocampal metabolism due to sex in the absence of immune stimulation, (R² = 0.985, Q² = 0.809; D) as well as the excellent separation between PBS- and LPS-treated mice males (R² = 1.00, Q² = 0.857; E) and females (R² = 1.00, Q2 = 0.863, F). D-F: Predictive (X axis) vs 1st orthogonal (Y axis) component.

Fig. 4

Increased hippocampal tryptophan metabolism 4 h after systemic LPS administration. 4.5-month-old male and female APP/PS1 mice and their wild-type (WT) littermates were challenged with LPS (100µ/kg i.v.) or its vehicle PBS. Schematic representation of the anti-inflammatory serotonin, and pro-inflammatory kynurenine, pathways of tryptophan metabolism. At 4 h post-injection, LPS-treated mice showed significant upregulation of l-tryptophan (A) as well as of 5-Hydroxylindoleacetic acid (B) and N1-Methyl-2-pyridone-5-carboxamide (2PY, C), the end metabolites of the serotonin and kynurenine pathways, respectively. Changes in 2PY levels were negatively correlated to D) rectal temperature in males (r = −0.718, p = 0.0004) which exhibited an hypothermic response to LPS but not females 9r = -0.21, p = 0.37); E) the number of arms visited in the spontaneous alternation test 4 h after the injection in both males (r = −0.837, p < 0.0001) and females (r = -0.791, p < 0.0001); and F) sickness scores for arm visits in both males (r = −0.741, p < 0.0001) and females (r = −0.824, p < 0.0001), suggesting that increased 2PY levels is associated with the severity of LPS-induced sickness. Data are expressed as Means ± SEM. Dots represent individual animals. Discriminant metabolites are highlighted by grey text boxes. Pairwise comparisons following 3-way ANOVAs: *, p < 0.05; **, p < 0.01; **, p < 0.0001 compared to PBS-treated mice of same sex and genotype.

Fig. 5

Reduced hippocampal methionine metabolism 4 h after systemic LPS administration. 4.5-month-old male and female APP/PS1 mice and their wild-type (WT) littermates were challenged with LPS (100 µ/kg i.v.) or its vehicle PBS. Schematic representation of methionine metabolism showing downregulation of 4 key metabolites from this pathway in LPS-treated mice, at 4 h post-injection, regardless of sex or genotype (A–D). Two of these metabolites, l-methionine-S-Oxide (B) and 5′-Methylthioadenosine (D), as well as 2 methionine derivatives involved in taurine metabolism, l-Cystathionine (E) and hypotaurine (F), were also found in significantly reduced levels in females compared to males. Data are expressed as Means ± SEM. Dots represent individual animals. Discriminant metabolites are highlighted by grey text boxes. Pairwise comparisons following 3-way ANOVAs: *, p < 0.05; **, p < 0.01; **, p < 0.0001 compared to PBS-treated mice of same sex and genotype; ⁺⁺, p < 0.01, ⁺⁺⁺, p < 0.0001 compared to males. ^#, p < 0.05; ^##, p < 0.01; ^###, p < 0.0001; compared to PBS-treated males of same genotype.

Fig. 6

Reduced pyruvate metabolism in the hippocampus of APP/PS1 male 4 h after systemic LPS administration. 4.5-month-old male and female APP/PS1 mice and their wild-type (WT) littermates were challenged with LPS (100 µ/kg i.v.) or its vehicle PBS. Schematic representation of the pyruvate metabolic pathway and its links with the sorbitol and glycolate pathways. At 4 h post-injection, LPS-treated APP/PS1 male mice failed to show a reduction in d-sorbitol levels (A), but in contrast, exhibited downregulation of 4 key metabolites of the pyruvate metabolic pathway: 3-Phospho-d-glycerate (B), 2-phosphoglycolate (C), phosphoenolpyruvate (D) and pyruvate (E). Data are expressed as Means ± SEM. Dots represent individual animals. Discriminant metabolites are highlighted by grey text boxes. Pairwise comparisons following 3-way ANOVAs: *, p < 0.05; **, p < 0.01; compared to PBS-treated mice of same sex and genotype. ^#, p < 0.05; ^##, p < 0.01; ^###, p < 0.0001; compared to PBS-treated males of same genotype.

Fig. 7

Reduced methylglyoxal metabolism in the hippocampus of WT and APP/PS1 female 4 h after systemic LPS administration. 4.5-month-old male and female APP/PS1 mice and their wild-type (WT) littermates were challenged with LPS (100 µ/kg i.v.) or its vehicle PBS. Schematic representation of the main pathways regulating methylglyoxal metabolism. At 4 h post-injection, LPS-treated APP/PS1 female mice showed a reduction in lipid metabolism, with downregulation of 5 key metabolites involved in fatty acid and glycerolipid metabolism: hexadecanoic acid (A), octadecanoic acid (B), icosatrienoic acid (C), [FA (17:0)] heptadecanoic acid (D) and sn-Glycerol 3-phosphate (E). This was associated with reduced levels of (d)-S-Lactoylglutathione (F) and (d)-Lactate (G), the reduction products of methylglyoxal. Data are expressed as Means ± SEM. Dots represent individual animals. Discriminant metabolites are highlighted by grey text boxes. Pairwise comparisons following 3-way ANOVAs: *, p < 0.05; **, p < 0.01; ***, p < 0.0001 compared to PBS-treated mice of same sex and genotype. ^#, p < 0.05; compared to PBS-treated males of same genotype.

Fig. 8

Lack of microglial response to LPS in the hippocampus at 4 h post-injection. 4.5-month-old male and female APP/PS1 mice and their wild-type (WT) littermates were challenged with LPS (100 µ/kg i.v.) or its vehicle PBS. Their brain were collected 4 h later, immediately after behavioural assessment, and one hemisphere was processed for immunostaining of Iba1 positive microglia. Representative images of Iba1 immunostaining in the whole hippocampus (A), CA1 (B), CA2 (C), CA3 (D), and dentate gyrus (DG, E) subfields extracted and analysed using a Matlab tool. LPS had no significant effects on microglial density in any hippocampal areas, estimated through the quantification of the percentage area covered by Iba1 positive microglia (F–J) and number of microglial cells per mm² (K–O). The area covered by microglia, was, however, significantly lower in the hippocampus of WT females (F), particularly in the CA2 (H) and CA3 (G) subfields, but lower microglial numbers were only observed in the dentate gyrus (O). Data are expressed as Means ± SEM. Dots represent individual animals. Pairwise comparisons: *p < 0.05; **p < 0.01.