The gut microbiome-derived metabolite trimethylamine N-oxide modulates neuroinflammation and cognitive function with aging

Abstract

Aging is associated with declines in cognitive performance, which are mediated in part by neuroinflammation, characterized by astrocyte activation and higher levels of pro-inflammatory cytokines; however, the upstream drivers are unknown. We investigated the potential role of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) in modulating neuroinflammation and cognitive function with aging. Study 1: In middle-aged and older humans (65 ± 7 years), plasma TMAO levels were inversely related to performance on NIH Toolbox Cognition Battery tests of memory and fluid cognition (both r² = 0.07, p < 0.05). Study 2: In mice, TMAO concentrations in plasma and the brain increased in parallel with aging (r² = 0.60), suggesting TMAO crosses the blood-brain barrier. The greater TMAO concentrations in old mice (27 months) were associated with higher brain pro-inflammatory cytokines and markers of astrocyte activation vs. young adult mice (6 months). Study 3: To determine if TMAO independently induces an "aging-like" decline in cognitive function, young mice (6 months) were supplemented with TMAO in chow for 6 months. Compared with controls, TMAO-supplemented mice performed worse on the novel object recognition test, indicating impaired memory and learning, and had increased neuroinflammation and markers of astrocyte activation. Study 4: Human astrocytes cultured with TMAO vs. control media exhibited changes in cellular morphology and protein markers consistent with astrocyte activation, indicating TMAO directly acts on these cells. Our results provide translational insight into a novel pathway that modulates neuroinflammation and cognitive function with aging, and suggest that TMAO might be a promising target for prevention of neuroinflammation and cognitive decline with aging.

Keywords: Astrocyte activation; Brain; Cognitive impairment; Inflammation; Memory.

Fig. 1

Plasma TMAO is higher in middle-aged to older (MA/O) adults and associated with lower memory and fluid cognition. a Plasma concentrations of TMAO in young (20–27 years; N = 22) and MA/O (50–79 years; N = 103) adults. Data are mean ± standard deviation (SD). *p < 0.05 vs. young adults. b–d Within MA/O adults (N = 77), plasma concentrations of TMAO (naturally log-transformed due skewness) were inversely and independently related to performance on National Institutes of Health Toolbox Cognition Battery assessments of working memory (b), episodic memory (c), and fluid cognition (d; composite of working and episodic memory, attention, processing speed, and executive function). Results of unadjusted linear regression models are shown with 95% confidence intervals

Fig. 2

Aging proportionally increases plasma and brain TMAO levels. Concentrations of TMAO in plasma (a) and whole-brain lysates (b) from young (6 months; N = 9) and old (27 months; N = 14) mice. Data are mean ± standard error of the mean (SEM). *p < 0.05 vs. young. c Plasma and brain concentrations of TMAO are highly correlated

Fig. 3

Aging is accompanied by neuroinflammation and astrocyte activation. In whole-brain lysates from young (6 months; N = 9) and old (27 months; N = 14) mice, a concentrations of pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)α) and anti-inflammatory cytokine (IL-10); b protein abundance of phosphorylated and total nuclear factor kappa B (NF-κB) normalized to GAPDH, with representative Western blot images generated from WES electropherograms shown on the right; and c protein abundance of lipocalin 2 (LCN2), an established marker of astrocyte activation, with representative Western blot images shown below, were determined. Data are mean ± SEM. *p < 0.05 vs. young; ^†p < 0.10 vs. young

Fig. 4

Chronic supplementation with TMAO in young mice impairs cognitive function. In young mice fed control chow (CON; N = 8–9) or chow supplemented with 0.12% TMAO (N = 10–11) for 6 months, a concentrations of TMAO in plasma (left) and whole-brain lysates (right), b exploration index (percent of time out of the 5-min test spent exploring the novel vs. both objects), c the ratio of interactions with the novel vs. familiar object, and d the latency time until the first interaction with the familiar object on the novel object recognition test of spatial memory and learning were determined. Data are mean ± SEM. *p < 0.05 vs. CON

Fig. 5

Chronic supplementation with TMAO in young mice induces neuroinflammation and astrocyte activation. In whole-brain lysates from young mice fed control chow (CON) or chow supplemented with 0.12% TMAO for 6 months, a concentrations of pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α) and anti-inflammatory cytokine (IL-10); b protein abundance of phosphorylated and total nuclear factor kappa B (NF-κB) normalized to GAPDH, with representative Western blot images generated from WES electropherograms shown on the right; and c protein abundance of lipocalin 2 (LCN2), an established marker of astrocyte activation, with representative Western blot images shown below, were determined. Data are mean ± SEM. *p < 0.05 vs. CON. ^†p < 0.10 vs. CON

Fig. 6

TMAO induces astrocyte activation. Protein abundance of the reactive astrocyte markers (a lipocalin 2, LCN2, and b CD44) in human astrocytes treated with 100 μM TMAO vs. control (CON) media for 24 h. Representative immunofluorescence images are shown below. N = 3/condition. *p < 0.05 vs. CON. c Representative brightfield images of human astrocytes showing differences in morphology (especially stellation) in cells cultured for 24 h with 100 μM TMAO vs. control media