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Possible Mechanism of Protective Effect of Folic Acid in Neural Tube Defects
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Possible Mechanism of Protective Effect of Folic Acid in Neural Tube Defects

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Highlights
  • Neural tube defects are amongst the most common birth defects
  • H3K27me3 has been recognized as an epigenetic marker during neuronal differentiation.
  • ACat2 is a gene related to folate metabolism
  • An investigation was done to see the effect of folic acid fortification on H3K27me3 expression as well as the association between H3K27me3 and ACat2 in an ATRA-induced NTD rat model

Neural tube defects (NTDs) are serious birth defects of the brain, spine, or spinal cord, which generally occur in the first month of pregnancy and could cause mental retardation, permanent disability or even infant mortality in some cases.

The two most common neural tube defects are spina bifida (split spine) and anencephaly (absence of a major portion of the brain).

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Folate is believed to have protective effects against NTDs as it plays an important role in cell proliferation, differentiation and transcriptional regulation of genes. Past studies have demonstrated that prenatal folic acid supplementation could reduce risk of NTDs by nearly 70%, though the exact mechanism is not yet clear.

Research in rats was undertaken to evaluate the association between the H3K27me3 gene which is believed to be crucial in the regulation of neuronal differentiation and ACat2 (Acyl-coenzyme a-cholesterol acyltransferase 2), to study the protective mechanism of folic acid (FA) in neural tube defects. The ACat2 gene codes for a protein that is involved in folate metabolism.
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Study
  • The study involved 18 female rats, who were randomly divided into 3 groups: healthy control, NTD (Neural Tube Defect) group and FA (Folic Acid) group.  They were mated with male rats, and the day the rats became pregnant was considered as 'embryonic day 0'.
  • The FA group was given supplementation of folic acid two weeks prior to pregnancy. The other two groups were fed with regular food. Both the NTD and FA groups were administered all-trans retinoic acid (ATRA) on embryonic day 10 to induce neural tube defects.
  • On embryonic day 15, neural stem cells (NSCs), the cells that will form the nervous system, were isolated from the rat fetuses and cultured, and cell proliferation was measured using CCK-8 assay method. FA level in the embryonic neural tube was analyzed by ELISA (Enzyme-Linked Immuno Sorbent Assay).
  • Immunocytochemical staining method was used to identify primary and differentiated NSCs.
  • Immunofluorescence and confocal microscopy were used to measure the relative expression of H3K27me3. The total protein of NSCs was quantified by the Western blot analysis method.
  • The relative expression of ACat2 mRNA was calculated using quantitative reverse transcription PCR (qRT-PCR).
  • The relationship between H3K27me3 and FA and ACat2 levels was found using Spearman rank correlation test.
Results
  • Once the NSCs were cultured, they formed numerous Nestin-positive neurospheres, which differentiated into NSE-positive neurons and GFAP-positive astrocytes after 5 days.
  • It was seen that the FA level in FA group was significantly higher as compared to NTD group (P < 0.05), but when compared with controls, it was found to be significantly lower (P < 0.05).
  • Results also demonstrated that FA improved the impaired proliferation and differentiation ability of NSCs.
  • The H3K27me3 level was found to be highest in the NTD group, followed by the FA group and was the least in the control group.
  • The ACat2 protein level in NTD group was significantly lower as compared to the control group (P < 0.05), and was found to be significantly higher after FA fortification (P < 0.05). The ACat2 protein level in FA group was found to be significantly lower when compared to control group (P < 0.05). 
  • H3K27me3 expression was found to be negatively correlated with the FA level  and ACat2 level in the embryonic neural tube.
Conclusion
  • These results reiterate the importance of folic acid in preventing neural tube defects in the fetus. FA supplementation might protect developing nerve cells by reducing H3K27me3 expression which in turn increases ACat2 level.
Reference :
  1. Zhai S. The association and significance of H3K27me3 and a folate metabolic gene ACat2 in neural tube defects. Nutrition Journal 2016; 15:95 DOI: 10.1186/s12937-016-0212-7
Source: Medindia

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