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. 2022 Jul 4;23(13):7430.
doi: 10.3390/ijms23137430.

Effects of Dietary n-3 LCPUFA Supplementation on the Hippocampus of Aging Female Mice: Impact on Memory, Lipid Raft-Associated Glutamatergic Receptors and Neuroinflammation

Lucas Taoro-González  1   2 , Daniel Pereda  3   4 , Catalina Valdés-Baizabal  3   4 , Miriam González-Gómez  2   5   6 , José A Pérez  7 , Fátima Mesa-Herrera  8 , Ana Canerina-Amaro  3   4 , Herminia Pérez-González  5 , Covadonga Rodríguez  2   7 , Mario Díaz  6   9   10 , Raquel Marin  3   4
Affiliations

Effects of Dietary n-3 LCPUFA Supplementation on the Hippocampus of Aging Female Mice: Impact on Memory, Lipid Raft-Associated Glutamatergic Receptors and Neuroinflammation

Lucas Taoro-González et al. Int J Mol Sci. .

Abstract

Long-chain polyunsaturated fatty acids (LCPUFA), essential molecules whose precursors must be dietary supplied, are highly represented in the brain contributing to numerous neuronal processes. Recent findings have demonstrated that LCPUFA are represented in lipid raft microstructures, where they favor molecular interactions of signaling complexes underlying neuronal functionality. During aging, the brain lipid composition changes affecting the lipid rafts' integrity and protein signaling, which may induce memory detriment. We investigated the effect of a n-3 LCPUFA-enriched diet on the cognitive function of 6- and 15-months-old female mice. Likewise, we explored the impact of dietary n-3 LCPUFAs on hippocampal lipid rafts, and their potential correlation with aging-induced neuroinflammation. Our results demonstrate that n-3 LCPUFA supplementation improves spatial and recognition memory and restores the expression of glutamate and estrogen receptors in the hippocampal lipid rafts of aged mice to similar profiles than young ones. Additionally, the n-3 LCPUFA-enriched diet stabilized the lipid composition of the old mice's hippocampal lipid rafts to the levels of young ones and reduced the aged-induced neuroinflammatory markers. Hence, we propose that n-3 LCPUFA supplementation leads to beneficial cognitive performance by "rejuvenating" the lipid raft microenvironment that stabilizes the integrity and interactions of memory protein players embedded in these microdomains.

Keywords: aging; diet; hippocampus; lipid rafts; long-chain polyunsaturated fatty acids supplementation; memory; neuroinflammation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Behavioral tests for the four experimental groups. A n-3 LCPUFA-enriched diet improves spatial and recognition memory in aged female mice. (A) Recognition memory was assessed by a novel object recognition task, in which the discrimination ratio was calculated as the difference in exploration time for the novel object minus that for the familiar object divided by the total exploration time. Bars represent the mean ratio ± SEM of 13–18 animals per group. Circles represent each individual measurement. Results were analyzed by a 2-way ANOVA (diet, age) followed by a Tukey’s multiple comparisons test (* p < 0.05; ** p < 0.01). (B) Spatial learning was measured by quantifying the escape time during the 5 days-training. Data were analyzed by a 3-way ANOVA (diet, age, day of training) and individual points in each curve represent the mean ± SEM of 13–16 animals per group. (C) Spatial memory achieved in the 5th training day was analyzed separately by representing the mean escape time ± SEM of the four experimental groups, 12–16 animals per group. Results were analyzed by a 2-way ANOVA (diet, age) followed by a Tukey’s multiple comparisons test (*** p < 0.001).
Figure 2
Figure 2
Differential expression of receptors in lipid raft fractions of the hippocampus. (A) Representative bands obtained by slot blot technique are shown for each condition. Tubulin expression was used as the charge control and flotillin-1 as the control from lipid raft fractions. In all cases, lipid raft fraction (LR) and non-raft fraction (NR) are shown. (B) Effect of age and diet on glutamatergic receptors expression. (C) Effect of age and diet on ERα expression. Bars represent the means of band density ± SEM of 4–12 mice per group (bands: mGluR5 6 m n = 7 and 15 m n = 9; GluN2B 6 m n = 8 and 15 m n = 12; GluA1 6 m n = 5 and 15 m n = 8; Erα 6 m n = 6 and 15 m n = 7). Circles represent each individual measurement. Data were analyzed by a 2-way ANOVA (diet, age) and Tukey’s post hoc test (* p < 0.05, ** p < 0.01). Data analysis was performed after normalization of the normalization of band intensity relative to tubulin.
Figure 3
Figure 3
Microglia changes from young and aged female mice. (A) Representative images of Iba-1 immunostaining. A n-3 LCPUFA-enriched diet normalizes microglia activation induced by aging in the hippocampus of female mice. Immunohistochemistry was performed in fixed horizontal brain slices from 6- and 15-months-old female mice fed CONTROL or n-3 LCPUFA-enriched diets as indicated in the Methods. Scale bar = 40 μm. (B) Microglia perimeter is reduced in the hippocampus of 15-months-old female mice. The n-3 LCPUFA-enriched diet decreases the microglia perimeter in 6-months-old female mice but normalizes it in 15-months-old female mice. Bars represent the mean perimeter length (micrometers) ± SEM from 188–314 microglial cells per group, measured as described in the Methods. Circles represent each individual measurement. Data were analyzed by a 2-way ANOVA (diet, age) and Sidak’s post hoc test (*** p < 0.001). n = 4 animals per group.
Figure 4
Figure 4
Astrocytic changes from young and aged female mice. (A) Representative images of GFAP immunostaining. A n-3 LCPUFA-enriched diet normalizes astrocytes hypertrophy induced by aging in the hippocampus of female mice. Immunohistochemistry was performed in fixed horizontal brain slices from 6- and 15-months-old female mice fed CONTROL or n-3 LCPUFA-enriched diets as indicated in the Methods. Scale bar = 40 μm. (B) Astrocytes covering area is increased in the hippocampus of 15-months-old female mice. A n-3 LCPUFA-enriched diet normalizes the astrocyte’s covering area. Bars represent the mean astrocytes covering area (%) ± SEM from 33–41 40× fields per group, measured as described in the Methods. Circles represent each individual measurement. Data were analyzed by a 2-way ANOVA (diet, age) and Tukey’s post hoc test (*** p < 0.001). n = 4 animals per group.
Figure 5
Figure 5
Hippocampal levels of IL-1β and TNF-α are differentially modulated by age and an n-3 LCPUFA-enriched diet. Hippocampal levels of IL-1β and TNF-α were analyzed by ELISA in young (3–6 months-old) and old (12–15 months-old) female mice. (A) IL-1β levels are increased in the hippocampus of aged mice. The n-3 LCPUFA-enriched diet increased the IL-1β levels in the hippocampus of young mice. (B) Hippocampal TNF-α levels are not modulated by age but the n-3 LCPUFA-enriched diet induced a significant increasing global effect on them. Values are expressed as pg of IL-1β or TNF-α by mg of total protein in the samples, and bars represent the mean ± SEM of 5–7 mice per group. Circles represent each individual measurement. Data were analyzed by a 2-way ANOVA (diet, age) and Tukey’s post hoc test (* p < 0.05).
Scheme 1
Scheme 1
Experimental design. The chronogram shows a summary of the experimental procedures undertaken by the different age groups. Briefly, the 6-month-old LCPUFA group was changed to the LCPUFA-enriched diet 6 weeks before culling and tissue collection, whereas the 15-month-old LCPUFA group were changed 12 weeks before. The behavioral test battery was performed the two weeks prior to culling in all groups targeted. n = 40 animals per group.
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