Increasing Maternal or Post-Weaning Folic Acid Alters Gene Expression and Moderately Changes Behavior in the Offspring

Background Studies have indicated that altered maternal micronutrients and vitamins influence the development of newborns and altered nutrient exposure throughout the lifetime may have potential health effects and increased susceptibility to chronic diseases. In recent years, folic acid (FA) exposure has significantly increased as a result of mandatory FA fortification and supplementation during pregnancy. Since FA modulates DNA methylation and affects gene expression, we investigated whether the amount of FA ingested during gestation alters gene expression in the newborn cerebral hemisphere, and if the increased exposure to FA during gestation and throughout the lifetime alters behavior in C57BL/6J mice. Methods Dams were fed FA either at 0.4 mg or 4 mg/kg diet throughout the pregnancy and the resulting pups were maintained on the diet throughout experimentation. Newborn pups brain cerebral hemispheres were used for microarray analysis. To confirm alteration of several genes, quantitative RT-PCR (qRT-PCR) and Western blot analyses were performed. In addition, various behavior assessments were conducted on neonatal and adult offspring. Results Results from microarray analysis suggest that the higher dose of FA supplementation during gestation alters the expression of a number of genes in the newborns’ cerebral hemispheres, including many involved in development. QRT-PCR confirmed alterations of nine genes including down-regulation of Cpn2, Htr4, Zfp353, Vgll2 and up-regulation of Xist, Nkx6-3, Leprel1, Nfix, Slc17a7. The alterations in the expression of Slc17a7 and Vgll2 were confirmed at the protein level. Pups exposed to the higher dose of FA exhibited increased ultrasonic vocalizations, greater anxiety-like behavior and hyperactivity. These findings suggest that although FA plays a significant role in mammalian cellular machinery, there may be a loss of benefit from higher amounts of FA. Unregulated high FA supplementation during pregnancy and throughout the life course may have lasting effects, with alterations in brain development resulting in changes in behavior

A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI, the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca(2+).Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme.NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI.We propose that W542 and Ca(2+) are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca(2+) is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis

New Inhibitors of Scrapie-Associated Prion Protein Formation in a Library of 2,000 Drugs and Natural Products

Transmissible spongiform encephalopathies (TSEs) are fatal, untreatable neurodegenerative diseases associated with the accumulation of a disease-specific form of prion protein (PrP) in the brain. One approach to TSE therapeutics is the inhibition of PrP accumulation. Indeed, many inhibitors of the accumulation of PrP associated with scrapie (PrP(Sc)) in scrapie-infected mouse neuroblastoma cells (ScN(2)a) also have antiscrapie activity in rodents. To expedite the search for potential TSE therapeutic agents, we have developed a high-throughput screening assay for PrP(Sc) inhibitors using ScN(2)a cells in a 96-well format. A library of 2,000 drugs and natural products was screened in ScN(2)a cells infected with scrapie strain RML (Chandler) or 22L. Forty compounds were found to have concentrations causing 50% inhibition (IC(50)s) of PrP(Sc) accumulation of ≤10 μM against both strains. Seventeen had IC(50)s of ≤1 μM against both strains. Several classes of compounds were represented in the 17 most potent inhibitors, including naturally occurring polyphenols (e.g., tannic acid and tea extracts), phenothiazines, antihistamines, statins, and antimalarial compounds. These 17 compounds were also evaluated in a solid-phase cell-free hamster PrP conversion assay. Only the polyphenols inhibited the cell-free reaction, and their IC(50)s were near 100 nM. Several of the new PrP(Sc) inhibitors cross the blood-brain barrier and thus have potential to be effective after TSE infection reaches the brain. The fact that many are either approved human drugs or edible natural products should facilitate their use in animal testing and clinical trials

Microarray Analysis Reveals Higher Gestational Folic Acid Alters Expression of Genes in the Cerebellum of Mice Offspring—A Pilot Study

Folate is a water-soluble vitamin that is critical for nucleotide synthesis and can modulate methylation of DNA by altering one-carbon metabolism. Previous studies have shown that folate status during pregnancy is associated with various congenital defects including the risk of aberrant neural tube closure. Maternal exposure to a methyl supplemented diet also can alter DNA methylation and gene expression, which may influence the phenotype of offspring. We investigated if higher gestational folic acid (FA) in the diet dysregulates the expression of genes in the cerebellum of offspring in C57BL/6 J mice. One week before gestation and throughout the pregnancy, groups of dams were supplemented with FA either at 2 mg/kg or 20 mg/kg of diet. Microarray analysis was used to investigate the genome wide gene expression profile in the cerebellum from day old pups. Our results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups. Several transcription factors, imprinted genes, neuro-developmental genes and genes associated with autism spectrum disorder exhibited altered expression levels. These findings suggest that higher gestational FA potentially dysregulates gene expression in the offspring brain and such changes may adversely alter fetal programming and overall brain development

Single-base resolution of mouse offspring brain methylome reveals epigenome modifications caused by gestational folic acid

BACKGROUND: Epigenetic modifications, such as cytosine methylation in CpG-rich regions, regulate multiple functions in mammalian development. Maternal nutrients affecting one-carbon metabolism during gestation can exert long-term effects on the health of the progeny. Using C57BL/6 J mice, we investigated whether the amount of ingested maternal folic acid (FA) during gestation impacted DNA methylation in the offspring’s cerebral hemispheres. Reduced representation bisulfite sequencing at single-base resolution was performed to analyze genome-wide DNA methylation profiles. RESULTS: We identified widespread differences in the methylation patterns of CpG and non-CpG sites of key developmental genes, including imprinted and candidate autism susceptibility genes (P <0.05). Such differential methylation of the CpG and non-CpG sites may use different mechanisms to alter gene expressions. Quantitative real time reverse transcription-polymerase chain reaction confirmed altered expression of several genes. CONCLUSIONS: These finding demonstrate that high maternal FA during gestation induces substantial alteration in methylation pattern and gene expression of several genes in the cerebral hemispheres of the offspring, and such changes may influence the overall development. Our findings provide a foundation for future studies to explore the influence of gestational FA on genetic/epigenetic susceptibility to altered development and disease in offspring

The schematic diagram of the experimental design for this study.

<p>The schematic diagram of the experimental design for this study.</p

A: Quantitative RT-PCR confirmation of microarray data.

<p>Relative expression of the <i>Cpn2</i>, <i>Zfp353</i>, <i>Htr4</i>, <i>Nkx6-3</i> and <i>Xist</i> transcripts in the cerebral hemispheres of male newborn pups, from mothers supplemented with FA at 4 mg/kg of diet. The results were normalized to <i>Hprt</i> transcript expression, and expressed as relative values in comparison to corresponding transcripts from newborn pups with FA at 0.4 mg/kg diet. Results represent means ± S.D; asterisks denote statistically significant change (***<i>P</i><0.001, **<i>P</i><0.01 and *<i>P</i><0.05). <b>B</b>): <b>Quantitative RT PCR confirmation of microarray data</b>. Relative expression of the <i>Vgll2</i>, <i>Leprel1</i>, <i>Nfix</i> and <i>Slc17a7</i> transcripts from the cerebral hemispheres of female newborn pups, from mothers supplemented with FA at 4 mg/kg of diet. The results were normalized to <i>Gapdh</i> transcript expression, and were expressed as relative values in comparison to corresponding transcripts from newborn pups with FA at 0.4 mg/kg diet. Results represent means ± S.D; asterisks denote statistically significant change (***<i>P</i><0.001, **<i>P</i><0.01 and *<i>P</i><0.05).</p

High maternal FA alters the expression of Slc17a7p and Vgll2p in the cerebral hemispheres of female newborn pups.

<p>Total cell lysates from the cerebral hemispheres of female (F) newborn pups were prepared from both 0.4 mg/kg and 4 mg/kg FA diet groups and proteins were resolved on 10% Tris/HEPES/SDS-polyacrylamide gels. The blot was probed with Slc17a7p antibody (<b>A</b>, <b>B</b>) or with Vgll2p antibody (<b>C</b>, <b>D</b>). To confirm equal protein loading, the membranes were re-probed with GAPDH/β-tubulin antibodies. The left panel represents one representative blot and the right panel shows densitometric evaluation of two independent experiments. Densitometric evaluation of the bands were calculated by using the Image J software and normalized to the densities of GAPDH//β-tubulin. The values corresponding to 0.4 mg/kg FA diet were arbitrarily set to 100%, and the 4 mg/kg values are presented relative to this. The densitometric values represent the mean, and the error bar represents the inter-variability among independent experiments.</p