Perinatal alpha linolenic acid availability alters the expression of genes related to memory and to epigenetic machinery, and the Mecp2 DNA methylation in the whole brain of mouse offspring

Many animal and human studies indicated that dietary n3 fatty acids could have beneficial roles on brain development, memory, and learning. However, the exact mechanisms involved are far from being clearly understood, especially for alpha linolenic acid (ALA), which is the precursor for the  n 3 elongation and desaturation pathways. This study investigated the alterations induced by different intakes of flaxseed oil (containing 50 per cent  ALA), during gestation and lactation, upon the expression of genes involved in neuro genesis, memory related molecular processes, and DNA methylation, in the brains of mouse off spring at the end of lactation. In addition, DNA methylation status for the same genes was investigated. Maternal flaxseed oil supplementation during lactation increased the expression ofMecp2, Ppp1cc, and Reelin, while decreasing the expression of Ppp1cb and Dnmt3a. Dnmt1 expression was decreased by postnatal flaxseed oil supplementation but this effect was offset by ALA deficiency during gestation. Mecp2 DNA methylation was decreased by maternal ALA deficiency during gestation, with amore robust effect in the lactation deficient group. In addition, linear regression analysis revealed positive correlations between Mecp2, Reelin, and Ppp1cc, between Gadd45b, Bdnf, and Creb1, and between Egr1and Dnmt1, respectively. However, there were no correlations, in any gene, between DNA methylation and gene expression. In summary, the interplay between ALA availability during gestation and lactation differentially altered the expression of genes involved in neuro genesis and memory, in the whole brain of the offspring at the end of lactation. The Mecp2 epigenetic status was correlated with ALA availability during gestation. However, the epigenetic status of the genes investigated was not associated with transcript levels, suggesting that either the regulation of these genes is not necessarily under epigenetic control, or that the whole brain model is not adequate for the exploration of epigenetic regulation in the context of this study. (Authors abstract)

Memory is the process by which new information is being encoded, stored, and then retrieved. Specificity in gene expression, protein synthesis and structural properties of neurons and synapses, is required for all three stages. n3 polyunsaturated fatty acids could improve learning and memory, and also alter gene expression and proteome profiles in the whole brain or certain brain regions, such as cerebrum and hippocampus, therefore altering neurogenesis and synaptic plasticity. ALA availability has been shown to be important for brain development and its function during gestation and early postnatal life.   During the perinatal period, the neonate’s brain is experiencing a substantial acceleration in growth, cellular proliferation, and neuronal and glial differentiation. Also, perinatal ALA deficiency induced gene expression changes in the brain of adult rats.  In mice, ALA availability during gestation and lactation altered cell proliferation, early neuronal differentiation, and apoptosis, in the hippocampus of the offspring. These outcomes were associated with postnatal ALA supplementation, but were offset by gestational ALA deficiency. Perinatal ALA availability was associated with epigenetic alterations in Fads2 DNA methylation in maternal and offspring livers.  Recent studies indicated the importance of epigenetic mechanisms in the alteration of neuronal gene expression patterns, which are required for synaptic plasticity or memory formation.  Epigenetic mechanisms include inter related processes such as DNA methylation, histone modifications and RNA interference.  In the current study, we explored the impact of maternal perinatal ALA availability on the expression of memory associated genes in offspring brains, and the associated modifications in DNA meth-ylation. Because memory and learning are complex processes that are not confined to only a brain area this study sought to first determine, in a whole brain model, whether such alterations could be detected, and whether this approach could warrant additional and more specific explorations.

The findings of this study support the hypothesis that maternal ALA availability during pregnancy and lactation alters the expression of memory associated genes and of those involved in the DNA methylation machinery, in the offspring brain at P19. In addition, ALA deficiency decreased the DNA methylation within the Mecp2 promoter, but not within intron. Interestingly, among the six genes that had substantial alterations in their transcript levels, five (Reelin, Ppp1cb, Ppp1cc, Dnmt3a, and Mecp2 )were dependent exclusively on postnatal maternal ALA availability (regardless gestational ALA intakes), while Dnmt1 was decreased only in the CS group, indicating that postnatal ALA supplementation triggered its decreased expression, which was nevertheless offset by maternal ALA deficiency during gestation. The alterations induced by ALA availability to DNA methyltransferases (Dnmt1 and Dnmt3a), and to Mecp2 expression were more specific. Dnmt1 expression was decreased only in the CS group but not in the DS group, indicating that the influence of postnatal ALA supplementation upon Dnmt1 is offset by gestational ALA deficiency. Dnmt1 is a maintenance DNA methyl transferase required for the propagation of DNA methylation patterns to replicated DNA.

In the present study Mecp2 was over expressed by post natal ALA supplementation, and the increase was more robust statistically in the DS group. Because the increase in the CS group was not statistically significant due to one outlier, it is difficult to ascertain whether gestational ALA exposure was an independent factor. While Mecp2 promoter DNA methylation was decreased in the DD group as compared with either CC or CS groups, but not when compared with the DS group, one might speculate that its methylation status would be dependent primarily on the gestational ALA availability, rather than its postnatal intake levels. The mechanisms by which ALA could induce alterations in gene expression are poorly understood. While ALA is a precursor for the synthesis of other n3 PUFAs, it can also have a distinct role in cell signaling.   ALA’s role in the regulation of gene expression is mediated either through peroxisome proliferator activated receptor (PPAR-dependent) mechanisms, PPAR independent mechanisms, or by altering the phosphorylation of mitogen activated protein kinases (MAPKs) involved in cell proliferation, differentiation and apoptosis. The activation of the ERK dependent MAPK pathway component plays a central role in cell proliferation and differentiation via the nuclear c fos activation mechanisms on gene expression. The expression of DNA methyl transferases is, in part, regulated by the activation of one or more MAPK pathways. ERK MAPK inhibitors down regulate Dnmt1 and Dnmt3a. However, another MAPK pathway (JNK dependent phosphorylation) may also be required for Dnmt1 promoter activation via c jun phosphorylation (within the AP 1 transcription complex). Since ALA availability alters the content of EPA and DPA in the brains of the offspring, it is not clear whether ALA’s role in altering the expression of genes is direct (via signaling), or due to alterations in other minus 3 species.  The study suggested that ALA availability during gestation and lactation differentially altered the expression of genes involved in memory formation and neurogenesis, and in the epigenetic regulation of gene expression. Some of these alterations could be the result of the interplay between ALA availability in the two developmental periods, while others depended exclusively on postnatal ALA supplementation. With the notable exception of Mecp2 promoter methylation, ALA availability did not alter the methylation of the aforementioned genes, as measured in P19 whole brain extracts. Also, Mecp2 methylation was not correlated with its expression. Whether potential correlations between gene expression and DNA methylation could be altered by ALA availability in specific brain areas, it remains to be investigated further. (Editors comments)