Age-Related Changes in Lipidome Of Rat Frontal Cortex And Cerebellum Applied in Old Age

2.2. Effect of Aging and Methionine Restriction in the Frontal Cortex Lipidome

In the frontal cortex, our lipidomics analysis detected a total of 11,029 molecular features from both ionization modes (positive and negative), once baseline correction, peak picking, and peak alignment were applied on acquired data. After quality control assessment, fifiltering, and correcting the signal, 763 features remained, which were used for statistical analysis. Using the whole lipidome, principal component analysis (PCA) showed that the three first components (PC1, PC2, and PC3) explained 36.8% of the variability of the samples. This analysis showed that the adult group lipidome differed with respect to the aged groups, and that the MetR group was also clearly differentiated, suggesting that aging and diet may be determinants in the defifinition of the lipodome in the frontal cortex (Figure 2A). Multivariate statistics applied only to adult and aged groups revealed that the changes in the central cortex lipidome were minor during aging (Supplementary Figure S1B). 

Each colored cell on the map corresponds to a relative concentration value, with samples in columns and compounds in rows. N (Adult) = 7, n (Aged) = 7, n (Aged+MetR) = 7.

Partial least-squares discriminant analysis (PLS-DA) was able to clearly separate the three groups (data not shown), but permutation tests (1000 repeats) yielded a not signifificant p value (p = 0.17), indicating that it was not an optimal model. Hierarchical clustering using all lipid species detected showed no specifific trend when the whole lipidome was analyzed (Supplementary Figure S2B). However, a heatmap of relative intensity changes for the 25 lipid species with the lower p value was then composed to visualize possible clustering (Figure 2B), and a pattern emerged suggesting that although all three groups grouped perfectly, diet was the most important factor determining the frontal cortex lipidome. The heatmap of metabolite abundances showed that the Adult+MetR group had a specifific profifile compared with the other two groups. Lipidomics analysis demonstrated the existence of specifific changes in the lipidome of the frontal cortex during aging and MetR applied in old age.

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Thus, when we searched for specifific aging and diet biomarkers, we found 81 lipid species to be statistically different between groups (Table 5, Supplementary Materials, , accessed on 16 November 2021). Among all the signifificant molecules, 34 were annotated, and 41 were not identifified. Among identifified lipid species, we described 1 FA, 11 GL, 14 GP, 5 SP, 1 prenol lipid (PL), and 2 SL. According to the differences between groups, we assigned a biological meaning for the differential lipid species consisting of three concepts: healthy aging, aging, and diet. Healthy aging was considered the lipid species that followed the post hoc: Aged 6 = Aged+MetR and Adult. To consider a lipid species as biomarker of aging, the post hocs were: Adult 6 = Aged and Aged+MetR. Finally, biomarkers of the effect of MetR were considered: Aged+MetR 6 = Aged and Adult. Following this rule, we found 25 biomarkers of aging (1 identifified as FA, 4 as GL, 3 as GP, 1 as PL and 1 as SL), 33 biomarkers of MetR diet (1 identifified as GL, 6 as GP, 4 as SP and 1 as SL) (Supplementary Figure S3A), and 7 biomarkers of healthy aging (2 identifified as GL and 1 as PL). Changes in the fatty acid profifile of total lipids in the aged frontal cortex were even more limited than in the cerebellum. Thus, only a decreased content of 24:6n-3 (19%), decreased delta-6 desaturase activity (24%), and increased peroxisomal beta-oxidation (23%) in the aged frontal cortex were observed (Tables 6 and 7). No additional changes were detected, including the oxidation-derived protein damage markers, in the aged frontal cortex compared to the young group (Table 6). MetR diet in old age neither modifified nor introduced additional modififications to the minor changes verifified in the aged group (Tables 6–8). 

All compounds are putatively annotated compounds based upon physicochemical properties and/or spectral similarity with public/commercial spectral libraries [29]. a Identity (ID) based on exact mass, retention time (RT), and MS/MS spectrum; b ID based on exact mass and RT; c ID based on MS/MS spectrum; (d) ID based on exact mass. FA: fatty acyls, GL: glycerolipids, GP: glycerophospholipids, SP: sphingolipids, SL: sterol lipids. Ag: Aged, R: Aged+MetR, A: Adult, n.i: no information. n (Adult) = 7, n (Aged) = 7, n (Aged+MetR) = 7. 

For details, see Table 3. Data are expressed as mean ± SEM. a comparison between aged and aged+MetR with the adult group. * p < 0.05, ** p < 0.01. n (Adult) = 8, n (Aged) = 8, n (Aged+MetR) = 8. 

For details, see Table 4. Data are expressed as mean ± SEM. b comparison between aged and aged+MetR groups. * p < 0.05. Units: µmol/mol lysine. n (Adult) = 7, n (Aged)= 8, n (Aged+MetR) = 8. 

3. Discussion

Lipids are key components of brain structure that play a crucial role in main brain functions. However, there is a lack of studies analyzing the effects of aging and anti-aging interventions applied in old age on lipid composition in distinct regions of the rat brain, and using a lipidomics approach. The rat frontal cortex and cerebellum are brain regions that participate in motor and cognitive functions. Interestingly, these region-dependent functions can also be extended to humans, suggesting a functional unit across mammalian phylogeny. Consequently, it may be hypothesized that the molecular bases of the aging process and potential changes in the lipidome may be common between species, thus converting rodents into a good experimental model to extrapolate mechanisms of brain aging in humans [19,31–34]. In this context, in our study, a comprehensive lipidomic analysis of the rat frontal cortex and cerebellum was performed to learn the age-related changes of lipids in these regions, as well as to assess the effect of an anti-aging intervention, methionine restriction, applied in old age in order to evaluate whether the effect verifified at a young age in brains by this dietary intervention [20–25] can also be reproduced at old age.

In the healthy adult rat brain, it seems that some fatty acid traits are shared in a cross-regional way. Thus, the fatty acid average chain length (ACL) is maintained at about 18 carbon atoms, in both regions, and the main SFAs are 16:0 and 18:0, whereas for PUFA 18:1n-9, 20:4n-6, and 22:6n-3 are predominant. Interestingly, these fifindings are analogous to what is observed in human brain [35], suggesting the maintenance of basic rules in the fatty acid profifile of the nervous system in, at least, mammalian species. This idea is not, however, contradictory to the existence of interregional differences. Thus, the rat cerebellum is more enriched in UFAs—with a predominance of MUFAs, followed by PUFAn-3 and, fifinally, PUFAn-6—compared to the frontal cortex. This profifile determines that the cerebellum presents a higher PI and, consequently, greater vulnerability to lipid peroxidation than the frontal cortex does. In addition, the rat cerebellum shows a greater steady-state level of protein oxidative damage with respect to the frontal cortex, indicating higher oxidative conditions in the cerebellum. However, and surprisingly, the lipoxidationderived protein damage (expressed by the MDAL marker) was lower in the cerebellum than in the frontal cortex, suggesting the existence of effificient protective mechanisms that may be mediated, paradoxically, by the PUFA 22:6n-3, which, despite a high oxidative potential, is also an indirect antioxidant mediator inducing the expression of antioxidant systems and related pathways [36]. This might explain the better protection of the effects of aging and neurodegenerative diseases classically attributed to the cerebellum [26,27]. Minor but signifificant changes were detected for the fatty acid profifiles with aging, with more substantial changes observed in the cerebellum (increased MUFA and decreased PUFA contents) in contrast to the frontal cortex, which showed a more sustained composition throughout the adult lifespan of rats. These observations are in line with previous studies in rats [15], as well as humans [37–40], suggesting that the maintenance of a fatty acid profifile throughout the adult lifespan is a key prerequisite to ensuring optimal neuronal integrity and, surely, brain structure and function. In this line, the steady-state levels of different protein damage markers are also sustained during adult life. Reinforcing this idea, alterations in lipid profifiles and protein damage are associated with the onset and development of diverse neurodegenerative diseases such as Alzheimer’s [3,12,41]. Signifificant changes were also observed with a lipidomics approach. However, these changes are minor again since they represent around 10% (60 out of 665 lipid species for cerebellum, and 81 out of 763 for frontal cortex) of the detected lipidome for both rat brain regions during aging, but with opposing changes in some cases between regions, and with a partial and reversible effect derived from MetR. Globally, the cerebellum seems to be more affected by the aging process, whereas the frontal cortex shows more changes according to diet applied. Remarkably, the most affected lipid classes were, for both regions, ether-triacylglycerols, diacylglycerols, phosphatidylethanolamine N-methylated, alkenylPE (plasmalogens), ceramides, and cholesterol esters. The observed changes in these lipid classes require special attention because the metabolic pathways and cell mechanisms behind them can be crucial in brain aging.

There are four functional categories associated with the different lipid classes identifified: biosynthesis of membrane structural components, bioenergetics, antioxidant protection, and bioactive lipids. Thus, diacylglycerols and ceramides are components of cell membranes (specififically located in lipid rafts) and lipid mediators that participate in the regulation of a broad diversity of cell mechanisms and pathways, including protein kinase activities, cytoskeletal organization, cell survival, autophagy, and control of neuronal communication, among several others [42–45]. Plasmalogens are structural components of cell membranes that also play a role in the formation and stability of lipid raft microdomains, as well as in diverse cell functions including cholesterol transport, membrane fusion events, and vesicular function [46,47], but they also have antioxidant properties [46] that help to maintain membrane integrity. This antioxidant property is probably also shared by ethertriacylglycerols (TG-O), but at the lipid droplet level; the presence of this lipid class in rat brain was identifified for the fifirst time in this work. Triacylglycerides and cholesteryl esters are bioenergetic compounds that compose the lipid droplets, and they are also present in neural cells [48]. It is in this context we propose that the presence of ether-triacylglycerides in lipid droplets has an antioxidant property that preserves the integrity of this fat storage organelle. Finally, phosphatidylethanolamine N-methylated is a precursor for biosynthesis of phosphatidylcholine, a structural component in cell membranes, in a reaction mediated by phosphatidylethanolamine-N-methyltransferases, which uses metabolites generated in the methionine cycle as a substrate [49]. The observed changes during aging in different lipid species suggest the involvement, to a greater or lesser extent, of specifific cell functions related to membrane structure, bioenergetics, antioxidant defense, and cell signaling. Both regions, the cerebellum and frontal cortex, share loss of antioxidant potential, deterioration in bioenergetic systems, increased ceramide content, and defects in the diacylglycerol-phosphatidic acid signaling pathway. In contrast, aging also differentially affects the cerebellum and frontal cortex, with the biosynthesis of phosphatidylcholine from the methylation of phosphatidylethanolamine (increased in cerebellum, but decreased in frontal cortex) and the cholesteryl ester content (decreased in cerebellum, and increased in frontal cortex) being the main interregional dissociation found. Methionine restriction, as an anti-aging intervention applied in old age, partially reverses changes induced by aging. Thus, in the cerebellum MetR seems to reverse changes in glycerolipids (especially ether-triacylglycerols), plasmalogens, and phosphatidylethanolamine N-methylated; whilst in the frontal cortex, MetR preferentially also affects phophatidylethanolamine N-methylated, ceramides, and cholesteryl esters. Metabolite identifification is still the bottleneck of LC-MS-based metabolomics/lipidomic studies and has some limitations [50]. Public databases are still incomplete, and several compounds do not have an associated MS/MS spectrum. Furthermore, most of the compound MS/MS spectra available are predicted. In our study, 90% of annotated compounds matched two or more criteria according to the Metabolomics Standards Initiative [29], but most of them (60%) are annotated based on exact mass and retention time because there is no available MS/MS spectrum in public databases. We may conclude that the rat cerebellum and frontal cortex have effificient mechanisms to preserve most lipid profifiles of their cell membranes throughout their adult lifespan in order to maintain brain structure and function, and that part of the small changes that take place during aging may be partially reversed with methionine restriction applied in old age. 

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