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Cited by 304Genetics, Aging, and Longevity in Model OrganismsAntioxidants, Aging, Portulaca oleraceaDementia and Cognitive Impairment Research

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Trimethylamine‐N‐oxide promotes brain aging and cognitive impairment in mice

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Li, Dang, Ke, Yilang, Zhan, Rui, Liu, Changjie, Zhao, Mingming, Zeng, Aiping, Shi, Xiaoyun, Ji, Liang, Cheng, Si, Pan, Bing, Zheng, Lemin, Hong, Huashan·2018·Aging Cell
DOI 10.1111/acel.12768PMC PMC6052480
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Abstract

Summary: Gut microbiota can influence the aging process and may modulate aging‐related changes in cognitive function. Trimethylamine‐N‐oxide (TMAO), a metabolite of intestinal flora, has been shown to be closely associated with cardiovascular disease and other diseases. However, the relationship between TMAO and aging, especially brain aging, has not been fully elucidated. To explore the relationship between TMAO and brain aging, we analysed the plasma levels of TMAO in both humans and mice and administered exogenous TMAO to 24‐week‐old senescence‐accelerated prone mouse strain 8 (SAMP 8) and age‐matched senescence‐accelerated mouse resistant 1 (SAMR 1) mice for 16 weeks. We found that the plasma levels of TMAO increased in both the elderly and the aged mice. Compared with SAMR 1‐control mice, SAMP 8‐control mice exhibited a brain aging phenotype characterized by more senescent cells in the hippocampal CA 3 region and cognitive dysfunction. Surprisingly, TMAO treatment increased the number of senescent cells, which were primarily neurons, and enhanced the mitochondrial impairments and superoxide production. Moreover, we observed that TMAO treatment increased synaptic damage and reduced the expression levels of synaptic plasticity‐related proteins by inhibiting the mTOR signalling pathway, which induces and aggravates aging‐related cognitive dysfunction in SAMR 1 and SAMP 8 mice, respectively. Our findings suggested that TMAO could induce brain aging and age‐related cognitive dysfunction in SAMR 1 mice and aggravate the cerebral aging process of SAMP 8 mice, which might provide new insight into the effects of intestinal microbiota on the brain aging process and help to delay senescence by regulating intestinal flora metabolites.

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Figure 1 The plasma levels of trimethylamine‐N‐oxide (TMAO) increased in the aged group in humans. The plasma was collected from healthy individuals. The levels of plasma TMAO, carnitine, choline, betaine and butyrobetaine were quantified using stable isotope dilution liquid chromatography–tandem mass spectrometry (LC/MS/MS). The levels of plasma choline (a), carnitine (b), betaine (c) and butyrobetaine (d) were unchanged. (e) However, the level of plasma TMAO was significantly higher in the aged group than the young‐ and middle‐aged groups, but there was no significant difference between the young‐ and middle‐aged groups in circulating level of TMAO. (f) Linear regression analysis revealed a tendency that TMAO was positively related to age in the overall studied population. (a–e) Data are shown as the mean ± SD (one‐way ANOVA followed by Tukey's multiple comparison test). Y = young adults (n = 168), M = middle‐aged adults (n = 118), E = elderly adults (n = 141) ***p < .001
Figure 1 The plasma levels of trimethylamine‐N‐oxide (TMAO) increased in the aged group in humans. The plasma was collected from healthy individuals. The levels of plasma TMAO, carnitine, choline, betaine and butyrobetaine were quantified using stable isotope dilution liquid chromatography–tandem mass spectrometry (LC/MS/MS). The levels of plasma choline (a), carnitine (b), betaine (c) and butyrobetaine (d) were unchanged. (e) However, the level of plasma TMAO was significantly higher in the aged group than the young‐ and middle‐aged groups, but there was no significant difference between the young‐ and middle‐aged groups in circulating level of TMAO. (f) Linear regression analysis revealed a tendency that TMAO was positively related to age in the overall studied population. (a–e) Data are shown as the mean ± SD (one‐way ANOVA followed by Tukey's multiple comparison test). Y = young adults (n = 168), M = middle‐aged adults (n = 118), E = elderly adults (n = 141) ***p < .001CC BY · PubMed Central
Figure 2 Trimethylamine‐N‐oxide (TMAO) impaired cognitive performance of the mice. (a) In the Y‐maze, no significant difference in the total number of arms entered, reflecting the total activity of the animals, was observed based on spontaneous alternation among the four groups. (b) The level of spontaneous alternation was significantly reduced in the TMAO groups compared with the control groups (p < .01 in R1 groups, p < .01 in P8 groups). (c and d) In novel object recognition, the percentage of number of visits and the time spent in the novel arm decreased significantly in the TMAO groups compared with the control groups (R1 groups: p < .01 in entries, p < .05 in time; P8 groups: p < .01 in entries, p < .01 in time), (Figure S2) whereas the data obtained from the start arm and other arm were not significantly different. The P8 groups showed a significant decrease in these indexes compared to the R1 groups. (e) On training days, the escape latency in the TMAO groups was significantly longer than that in the control groups in the Morris water maze test. The P8 groups had a longer escape latency compared with the R1 groups. (f) The swimming speed showed no significant difference among the four groups. (g) The TMAO groups crossed significantly more platforms compared with the control groups (p < .01 in R1 groups, p < .01 in P8 groups). (h) The time spent in the platform quadrant was significantly shorter in the TMAO groups than the control groups (p < .01 in R1 groups, p < .01 in P8 groups). (g and h) The P8 groups showed a significant decrease in these indexes compared to the R1 groups. Data are shown as the mean ± SEM. Data from the escape latency and speed in the Morris water maze test were analysed with repeated‐measures two‐way analysis of variance. All other data acquired from behavioural tests were analysed using two‐way ANOVA, and comparisons between two groups were performed using Tukey's multiple comparison test (n = 12 each group, *p < .05, **p < .01)
Figure 2 Trimethylamine‐N‐oxide (TMAO) impaired cognitive performance of the mice. (a) In the Y‐maze, no significant difference in the total number of arms entered, reflecting the total activity of the animals, was observed based on spontaneous alternation among the four groups. (b) The level of spontaneous alternation was significantly reduced in the TMAO groups compared with the control groups (p < .01 in R1 groups, p < .01 in P8 groups). (c and d) In novel object recognition, the percentage of number of visits and the time spent in the novel arm decreased significantly in the TMAO groups compared with the control groups (R1 groups: p < .01 in entries, p < .05 in time; P8 groups: p < .01 in entries, p < .01 in time), (Figure S2) whereas the data obtained from the start arm and other arm were not significantly different. The P8 groups showed a significant decrease in these indexes compared to the R1 groups. (e) On training days, the escape latency in the TMAO groups was significantly longer than that in the control groups in the Morris water maze test. The P8 groups had a longer escape latency compared with the R1 groups. (f) The swimming speed showed no significant difference among the four groups. (g) The TMAO groups crossed significantly more platforms compared with the control groups (p < .01 in R1 groups, p < .01 in P8 groups). (h) The time spent in the platform quadrant was significantly shorter in the TMAO groups than the control groups (p < .01 in R1 groups, p < .01 in P8 groups). (g and h) The P8 groups showed a significant decrease in these indexes compared to the R1 groups. Data are shown as the mean ± SEM. Data from the escape latency and speed in the Morris water maze test were analysed with repeated‐measures two‐way analysis of variance. All other data acquired from behavioural tests were analysed using two‐way ANOVA, and comparisons between two groups were performed using Tukey's multiple comparison test (n = 12 each group, *p < .05, **p < .01)CC BY · PubMed Central
Figure 3 Trimethylamine‐N‐oxide (TMAO) induced the aging process in the hippocampal CA3 region. (a and b) SA‐beta‐GAL was detected in the hippocampal CA3 region. The blue cells indicated SA‐beta‐GAL‐positive cells. (c–f) Double staining with SA‐beta‐GAL and immunohistochemical staining for senescent cells. (c and d) Cells positive for SA‐beta‐GAL and NeuN (a marker of neurons, the red arrow indicates senescent neurons) were found in the CA3 region of the hippocampus. (e) Cells negative for SA‐beta‐GAL but positive for GFAP (a marker of astrocytes, the red arrow indicates astrocytes) were found in the CA3 region of the hippocampus. (f) Cells negative for SA‐beta‐GAL but positive for Iba1/AIF1 (a marker of microglia, the red arrow indicates microglia) were found in the CA3 region of the hippocampus. These results suggested that TMAO could promote cell senescence in the hippocampal CA3 region, and the senescent cells were primarily neurons (n = 6 each group, scale bar: b, d, e, f = 100 μm, a, c = 50 μm)
Figure 3 Trimethylamine‐N‐oxide (TMAO) induced the aging process in the hippocampal CA3 region. (a and b) SA‐beta‐GAL was detected in the hippocampal CA3 region. The blue cells indicated SA‐beta‐GAL‐positive cells. (c–f) Double staining with SA‐beta‐GAL and immunohistochemical staining for senescent cells. (c and d) Cells positive for SA‐beta‐GAL and NeuN (a marker of neurons, the red arrow indicates senescent neurons) were found in the CA3 region of the hippocampus. (e) Cells negative for SA‐beta‐GAL but positive for GFAP (a marker of astrocytes, the red arrow indicates astrocytes) were found in the CA3 region of the hippocampus. (f) Cells negative for SA‐beta‐GAL but positive for Iba1/AIF1 (a marker of microglia, the red arrow indicates microglia) were found in the CA3 region of the hippocampus. These results suggested that TMAO could promote cell senescence in the hippocampal CA3 region, and the senescent cells were primarily neurons (n = 6 each group, scale bar: b, d, e, f = 100 μm, a, c = 50 μm)CC BY · PubMed Central
Figure 4 Trimethylamine‐N‐oxide (TMAO) damaged the ultrastructure of the hippocampal CA1 region. (a and b, the red arrow indicates chemical synapses) Chemical synapses in R1‐C appeared normal: abundant synaptic vesicles were inside the presynaptic membrane. Postsynaptic density was concentrated on the intracellular surface of the opposing postsynaptic membrane. The synaptic cleft appeared narrow and rigidly paralleled with membranes. The structure of synapses in the TMAO groups was damaged compared with that in the control groups, with fewer synaptic vesicles, reduced postsynaptic area, a lower concentration of electron‐dense substances and a wider synaptic cleft. P8‐T showed more damage with a sharply decreased concentration of electron‐dense substances, severely decreased synaptic vesicles and postsynaptic area, obvious widening of the synaptic cleft and swelling of the postsynaptic membrane. (c and d, e and f, the red arrow indicates mitochondria and lipofuscin, respectively) The ultrastructure of cells in the R1‐C group appeared normal; the cells contained abundant normal mitochondria, and little lipofuscin was observed. The structure of cells in the TMAO groups was damaged compared with that of the control groups, with deformed mitochondria, reduced mitochondrial cristae and abundant lipofuscin, especially in the P8‐T group (n = 6 each group, scale bar: a = 1 μm, b = 200 nm, c, e = 2 μm, d, f = 400 nm)
Figure 4 Trimethylamine‐N‐oxide (TMAO) damaged the ultrastructure of the hippocampal CA1 region. (a and b, the red arrow indicates chemical synapses) Chemical synapses in R1‐C appeared normal: abundant synaptic vesicles were inside the presynaptic membrane. Postsynaptic density was concentrated on the intracellular surface of the opposing postsynaptic membrane. The synaptic cleft appeared narrow and rigidly paralleled with membranes. The structure of synapses in the TMAO groups was damaged compared with that in the control groups, with fewer synaptic vesicles, reduced postsynaptic area, a lower concentration of electron‐dense substances and a wider synaptic cleft. P8‐T showed more damage with a sharply decreased concentration of electron‐dense substances, severely decreased synaptic vesicles and postsynaptic area, obvious widening of the synaptic cleft and swelling of the postsynaptic membrane. (c and d, e and f, the red arrow indicates mitochondria and lipofuscin, respectively) The ultrastructure of cells in the R1‐C group appeared normal; the cells contained abundant normal mitochondria, and little lipofuscin was observed. The structure of cells in the TMAO groups was damaged compared with that of the control groups, with deformed mitochondria, reduced mitochondrial cristae and abundant lipofuscin, especially in the P8‐T group (n = 6 each group, scale bar: a = 1 μm, b = 200 nm, c, e = 2 μm, d, f = 400 nm)CC BY · PubMed Central
Figure 5 Trimethylamine‐N‐oxide (TMAO) increased the level of oxidative stress in the hippocampus of the mice. (a) TMAO significantly increased the concentration of H2O2. (b) TMAO significantly inhibited the total SOD activity. Data are shown as the mean ± SEM (n = 6 each group, two‐way ANOVA, comparisons between two groups were performed using Tukey's multiple comparison test. *p < .05, **p < .01)
Figure 5 Trimethylamine‐N‐oxide (TMAO) increased the level of oxidative stress in the hippocampus of the mice. (a) TMAO significantly increased the concentration of H2O2. (b) TMAO significantly inhibited the total SOD activity. Data are shown as the mean ± SEM (n = 6 each group, two‐way ANOVA, comparisons between two groups were performed using Tukey's multiple comparison test. *p < .05, **p < .01)CC BY · PubMed Central
Figure 6 Trimethylamine‐N‐oxide (TMAO) reduced the expression of synaptic plasticity‐related proteins by down‐regulating the activity of the mTOR signalling pathway. (a) Synaptophysin (SYN), postsynaptic density 95 (PSD‐95) and N‐methyl‐d‐aspartate 1 (NMDAR1) were shown by immunohistochemical staining in the hippocampus. The expression levels of SYN, NMDAR1 and PSD‐95 were significantly decreased in the P8‐C group compared with the R1‐C group. There was a significant decrease in the expression of these synaptic‐associated proteins in the TMAO groups compared with their control groups (scale bar = 50 μm). (b) The expression levels of SYN, NMDAR1 and PSD‐95 were assayed by Western blotting. To examine the possible mechanism involved in the decline of synaptic‐associated proteins, three proteins (mTOR, p70s6k and 4EBP2) of the mTOR signalling pathway were assessed by RT–PCR and Western blotting. (Figure S3A) The mRNA levels of mTOR, p70s6k and 4EBP2 were not significantly different in the groups assessed by RT–PCR. (c) Phosphor‐mTOR/mTOR, phosphor‐p70s6k/p70s6k and phosphor‐4EBP1/4EBP2 were monitored by Western blotting. β‐Actin was used as a loading control. The bands in the Western blotting were scanned, and the ratios of optical density of specific bands and β‐actin are illustrated. TMAO inhibited the active form of mTOR, p70s6k and 4EBP2 (phosphorylated). Data are shown as the mean ± SEM (n = 6 each group, two‐way ANOVA, comparisons between two groups were made using Tukey's multiple comparison test. **p < .01). The samples derived from the same experiment and the blots were processed in parallel
Figure 6 Trimethylamine‐N‐oxide (TMAO) reduced the expression of synaptic plasticity‐related proteins by down‐regulating the activity of the mTOR signalling pathway. (a) Synaptophysin (SYN), postsynaptic density 95 (PSD‐95) and N‐methyl‐d‐aspartate 1 (NMDAR1) were shown by immunohistochemical staining in the hippocampus. The expression levels of SYN, NMDAR1 and PSD‐95 were significantly decreased in the P8‐C group compared with the R1‐C group. There was a significant decrease in the expression of these synaptic‐associated proteins in the TMAO groups compared with their control groups (scale bar = 50 μm). (b) The expression levels of SYN, NMDAR1 and PSD‐95 were assayed by Western blotting. To examine the possible mechanism involved in the decline of synaptic‐associated proteins, three proteins (mTOR, p70s6k and 4EBP2) of the mTOR signalling pathway were assessed by RT–PCR and Western blotting. (Figure S3A) The mRNA levels of mTOR, p70s6k and 4EBP2 were not significantly different in the groups assessed by RT–PCR. (c) Phosphor‐mTOR/mTOR, phosphor‐p70s6k/p70s6k and phosphor‐4EBP1/4EBP2 were monitored by Western blotting. β‐Actin was used as a loading control. The bands in the Western blotting were scanned, and the ratios of optical density of specific bands and β‐actin are illustrated. TMAO inhibited the active form of mTOR, p70s6k and 4EBP2 (phosphorylated). Data are shown as the mean ± SEM (n = 6 each group, two‐way ANOVA, comparisons between two groups were made using Tukey's multiple comparison test. **p < .01). The samples derived from the same experiment and the blots were processed in parallelCC BY · PubMed Central

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Metadata

Authors
Li, D., Ke, Y., Zhan, R., Liu, C., Zhao, M., Zeng, A., Shi, X., Ji, L., Cheng, S., Pan, B., Zheng, L., & Hong, H.
Affiliations
  • Union Hospital (CN)
  • Peking University (CN)
  • The Institute of Cardiovascular Sciences and Institute of Systems Biomedicine School of Basic Medical Sciences Peking University Health Science Center Key Laboratory of Molecular Cardiovascular Science Ministry of Education; Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides Ministry of Health; Beijing Key Laboratory of Cardiovascular Receptors Research Beijing China
  • Capital Medical University (CN)
Corresponding author
  • Zheng, Lemin
  • Hong, Huashan
ORCID
Venue
Aging Cell
Publisher
Wiley
Published
2018-05-10
Volume
17
Issue
4
Pages
e12768-e12768
ISSN
  • 1474-9718 (ISSN-L)
  • 1474-9726
DOI
10.1111/acel.12768
PMC
PMC6052480
OpenAlex
W2800864806
Cited by
304
References
49
Citations by year
2026: 32 · 2025: 46 · 2024: 52 · 2023: 50 · 2022: 60 · 2021: 33 · 2020: 19 · 2019: 11 · 2018: 1
FWCI
6.42
Type
journal article
Language
en
Open access
Licence
Locations
  • Aging Cell (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Natural Science Foundation of China (81570448, 91639108, 81370235, 81770272)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Antioxidants, Aging, Portulaca oleracea
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
Keywords
trimethylamine N-oxide, brain aging, cognitive impairment, gut microbiota, cellular senescence, neuronal senescence, hippocampal CA3, mitochondrial dysfunction, superoxide, synaptic damage, synaptic plasticity, mTOR signaling, SAMP8 mice, SAMR1 mice
MeSH terms
Adolescent, Adult, Aged, Animals, Brain, Humans, Male, Methylamines, Middle Aged, Cellular Senescence, Mice, Young Adult, Cognitive Dysfunction

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