Acute high folic acid treatment in SH-SY5Y cells with and without MTHFR function leads to gene expression changes in epigenetic modifying enzymes, changes in epigenetic marks, and changes in dendritic spine densities.

Epigenetics are known to be involved in various disorders, including neurobiological disorders like autism. Dietary factors such as folic acid can affect epigenetic marks using methylenetetrahydrofolate reductase (MTHFR) to metabolize folic acid to a one-carbon methyl group. As MTHFR mutations are frequent, it is curious as to whether excess folic acid, with or without functioning MTHFR, could affect gene expression, epigenetics, and neuromorphology. Here, we investigated gene expression and activity of epigenetic modifying enzymes, genome-wide DNA methylation, histone 3 modifications, and dendritic spine densities in SH-SY5Y cells with or without a knockdown of MTHFR and with or without an excess of folic acid. We found alterations to gene expression of epigenetic modifying enzymes, including those associated with disorders like autism. Grouping the epigenetic modifying enzymes by function indicated that gene expression was widely affected for genes that code for enzymes affecting DNA methylation, histone acetylation, histone methylation, histone phosphorylation, and histone ubiquitination when excess folic acid treatment occurred with or without the knockdown of MTHFR. MTHFR was significantly reduced upon excess folic acid treatment whether MTHFR was knocked-down or not. Further, methyl-CpG binding protein 2 expression was significantly decreased with excess folic acid treatment with and without proper MTHFR expression. Global DNA methylation decreased due to the knockdown alone while global hydroxymethylated DNA increased due to the knockdown alone. TET2 expression significantly increased with the MTHFR knockdown alone. Excess folic acid alone induced a decrease in TET3 expression. Excess folic acid induced an increase in dendritic spines without the MTHFR knockdown, but folic acid induced a decrease in dendritic spines when MTHFR was knocked-down. The knockdown alone also increased the dendritic spines significantly. Histone 3 acetylation at lysine 18 was significantly increased when excess folic acid was applied to cells with the MTHFR knockdown, as was histone 3 phosphorylation at serine 10. Broadly, our results indicate that excess folic acid, even with functioning MTHFR, could have detrimental effects on cells.

A 2x folic acid treatment affects epigenetics and dendritic spine densities in SHSY5Y cells.

Many diseases are now associated with aberrant epigenetics and gene expression changes. Epigenetics can be modified by factors like diet. One dietary factor, folic acid, is consumed in various forms including supplements, energy drinks, and fortified grains. It was hypothesized high levels of folic acid would affect gene expression and enzyme activity of chromatin modifying enzymes as well as dendritic spine densities in a commonly utilized neuron model, the SHSY5Y cell. Decreased MBD2 and MECP2 were discovered upon treatment of SHSY5Y cells with a 2x folic acid dose. Corresponding decreases in dendritic spines were apparent in the 2x folic acid treated cells as well. Activity of DNMTs and H3K4 HMTs was altered. Further, H3K4me1, H3K4me3, H3K9Ac, and global DNA methylation were decreased in the 2x folic acid treated cells. Further studies are warranted to determine if the effects of excess folic acid are detrimental to organismal physiology.

A high methyl donor diet affects physiology and behavior in Peromyscus polionotus.

Folic acid and other dietary methyl donors are widely supplemented due to their ability to prevent neural tube defects. Dietary methyl donors are also added to other consumables such as energy drinks due to energy-promoting attributes and other perceived benefits. However, there is mounting evidence that indicates developmental exposure to high levels of dietary methyl donors may have deleterious effects. We assessed whether behavior was affected in the social North American rodent species Peromyscus polionotus exposed to a diet enriched with folic acid, Vitamin B12, choline, and betaine/trimethylglycine(TMG). P. polionotus (PO) animals are very social and exhibit little repetitive behavior, particularly compared to their sister species, P. maniculatus. We assayed the effects of dietary methyl-donor supplementation on anxiety-like repetitive and social behaviors by testing young adult animals for novel cage behavior and in social interaction tests. Animals of both sexes exposed to the diet had increased repetitive behaviors and reduced social interactions. Males exposed to the diet became more aggressive compared to their control counterparts. Since methyl-diet animals were larger than control animals, DEXA scans and hormone analyses were performed. Animals exposed to the diet had increased body fat percentages and experienced hormonal changes typically associated with excess fat storage and anxiety-like behavior changes. Therefore, these data suggest the wide use of these dietary supplements makes further investigation imperative.

Epigenetic mechanisms in schizophrenia.

Epigenetic modifications, including DNA methylation, histone modifications, and non-coding RNAs, have been implicated in a number of complex diseases. Schizophrenia and other major psychiatric and neurodevelopmental disorders are associated with abnormalities in multiple epigenetic mechanisms, resulting in altered gene expression during development and adulthood. Polymorphisms and copy number variants in schizophrenia risk genes contribute to the high heritability of the disease, but environmental factors that lead to epigenetic modifications may either reduce or exacerbate the expression of molecular and behavioral phenotypes associated with schizophrenia and related disorders. In the present paper, we will review the current understanding of molecular dysregulation in schizophrenia, including disruption of the dopamine, NMDA, and GABA signaling pathways, and discuss the role of epigenetic factors underlying disease pathology.

Consequences of dietary methyl donor supplements: Is more always better?

Epigenetic mechanisms are now recognized to play roles in disease etiology. Several diseases increasing in frequency are associated with altered DNA methylation. DNA methylation is accomplished through metabolism of methyl donors such as folate, vitamin B12, methionine, betaine (trimethylglycine), and choline. Increased intake of these compounds correlates with decreased neural tube defects, although this mechanism is not well understood. Consumption of these methyl donor pathway components has increased in recent years due to fortification of grains and high supplemental levels of these compounds (e.g. vitamins, energy drinks). Additionally, people with mutations in one of the enzymes that assists in the methyl donor pathway (5-MTHFR) are directed to consume higher amounts of methyl donors to compensate. Recent evidence suggests that high levels of methyl donor intake may also have detrimental effects. Individualized medicine may be necessary to determine the appropriate amounts of methyl donors to be consumed, particularly in women of child bearing age.

Pleiotropic effects of a methyl donor diet in a novel animal model.

Folate and other methyl-donor pathway components are widely supplemented due to their ability to prevent prenatal neural tube defects. Several lines of evidence suggest that these supplements act through epigenetic mechanisms (e.g. altering DNA methylation). Primary among these are the experiments on the mouse viable yellow allele of the agouti locus (A(vy)). In the Avy allele, an Intracisternal A-particle retroelement has inserted into the genome adjacent to the agouti gene and is preferentially methylated. To further test these effects, we tested the same diet used in the Avy studies on wild-derived Peromyscus maniculatus, a native North American rodent. We collected tissues from neonatal offspring whose parents were fed the high-methyl donor diet as well as controls. In addition, we assayed coat-color of a natural variant (wide-band agouti = A(Nb)) that overexpresses agouti as a phenotypic biomarker. Our data indicate that these dietary components affected agouti protein production, despite the lack of a retroelement at this locus. Surprisingly, the methyl-donor diet was associated with defects (e.g. ovarian cysts, cataracts) and increased mortality. We also assessed the effects of the diet on behavior: We scored animals in open field and social interaction tests. We observed significant increases in female repetitive behaviors. Thus these data add to a growing number of studies that suggest that these ubiquitously added nutrients may be a human health concern.

Peromyscus (deer mice) as developmental models.

Deer mice (Peromyscus) are the most common native North American mammals, and exhibit great natural genetic variation. Wild-derived stocks from a number of populations are available from the Peromyscus Genetic Stock Center (PGSC). The PGSC also houses a number of natural variants and mutants (many of which appear to differ from Mus). These include metabolic, coat-color/pattern, neurological, and other morphological variants/mutants. Nearly all these mutants are on a common genetic background, the Peromyscus maniculatus BW stock. Peromyscus are also superior behavior models in areas such as repetitive behavior and pair-bonding effects, as multiple species are monogamous. While Peromyscus development generally resembles that of Mus and Rattus, prenatal stages have not been as thoroughly studied, and there appear to be intriguing differences (e.g., longer time spent at the two-cell stage). Development is greatly perturbed in crosses between P. maniculatus (BW) and Peromyscus polionotus (PO). BW females crossed to PO males produce growth-restricted, but otherwise healthy, fertile offspring which allows for genetic analyses of the many traits that differ between these two species. PO females crossed to BW males produce overgrown but severely dysmorphic conceptuses that rarely survive to late gestation. There are likely many more uses for these animals as developmental models than we have described here. Peromyscus models can now be more fully exploited due to the emerging genetic (full linkage map), genomic (genomes of four stocks have been sequenced) and reproductive resources.

A genetic map of Peromyscus with chromosomal assignment of linkage groups (a Peromyscus genetic map).

The rodent genus Peromyscus is the most numerous and species-rich mammalian group in North America. The naturally occurring diversity within this genus allows opportunities to investigate the genetic basis of adaptation, monogamy, behavioral and physiological phenotypes, growth control, genomic imprinting, and disease processes. Increased genomic resources including a high quality genetic map are needed to capitalize on these opportunities. We produced interspecific hybrids between the prairie deer mouse (P. maniculatus bairdii) and the oldfield mouse (P. polionotus) and scored meiotic recombination events in backcross progeny. A genetic map was constructed by genotyping of backcross progeny at 185 gene-based and 155 microsatellite markers representing all autosomes and the X-chromosome. Comparison of the constructed genetic map with the molecular maps of Mus and Rattus and consideration of previous results from interspecific reciprocal whole chromosome painting allowed most linkage groups to be unambiguously assigned to specific Peromyscus chromosomes. Based on genomic comparisons, this Peromyscus genetic map covers ~83% of the Rattus genome and 79% of the Mus genome. This map supports previous results that the Peromyscus genome is more similar to Rattus than Mus. For example, coverage of the 20 Rattus autosomes and the X-chromosome is accomplished with only 28 segments of the Peromyscus map, but coverage of the 19 Mus autosomes and the X-chromosome requires 40 chromosomal segments of the Peromyscus map. Furthermore, a single Peromyscus linkage group corresponds to about 91% of the rat and only 76% of the mouse X-chromosomes.

Natural genetic variation underlying differences in Peromyscus repetitive and social/aggressive behaviors.

Peromyscus maniculatus (BW) and P. polionotus (PO) are interfertile North American species that differ in many characteristics. For example, PO exhibit monogamy and BW animals are susceptible to repetitive behaviors and thus a model for neurobehavioral disorders such as Autism. We analyzed these two stocks as well as their hybrids, a BW Y(PO) consomic line (previously shown to alter glucose homeostasis) and a natural P. maniculatus agouti variant (A(Nb) = wide band agouti). We show that PO animals engage in far less repetitive behavior than BW animals, that this trait is dominant, and that trait distribution in both species is bi-modal. The A(Nb) allele also reduces such behaviors, particularly in females. PO, F1, and A(Nb) animals all dig significantly more than BW. Increased self-grooming is also a PO dominant trait, and there is a bimodal trait distribution in all groups except BW. The inter-stock differences in self-grooming are greater between males, and the consomic data suggest the Y chromosome plays a role. The monogamous PO animals engage in more social behavior than BW; hybrid animals exhibit intermediate levels. Surprisingly, A(Nb) animals are also more social than BW animals, although A(Nb) interactions led to aggressive interactions at higher levels than any other group. PO animals exhibited the lowest incidence of aggressive behaviors, while the hybrids exhibited BW levels. Thus this group exhibits natural, genetically tractable variation in several biomedically relevant traits.

Peromyscus as a Mammalian epigenetic model.

Deer mice (Peromyscus) offer an opportunity for studying the effects of natural genetic/epigenetic variation with several advantages over other mammalian models. These advantages include the ability to study natural genetic variation and behaviors not present in other models. Moreover, their life histories in diverse habitats are well studied. Peromyscus resources include genome sequencing in progress, a nascent genetic map, and >90,000 ESTs. Here we review epigenetic studies and relevant areas of research involving Peromyscus models. These include differences in epigenetic control between species and substance effects on behavior. We also present new data on the epigenetic effects of diet on coat-color using a Peromyscus model of agouti overexpression. We suggest that in terms of tying natural genetic variants with environmental effects in producing specific epigenetic effects, Peromyscus models have a great potential.