Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

Gene Environment Interactions in the Etiology of Neural Tube Defects.

Human structural congenital malformations are the leading cause of infant mortality in the United States. Estimates from the United States Center for Disease Control and Prevention (CDC) determine that close to 3% of all United States newborns present with birth defects; the worldwide estimate approaches 6% of infants presenting with congenital anomalies. The scientific community has recognized for decades that the majority of birth defects have undetermined etiologies, although we propose that environmental agents interacting with inherited susceptibility genes are the major contributing factors. Neural tube defects (NTDs) are among the most prevalent human birth defects and as such, these malformations will be the primary focus of this review. NTDs result from failures in embryonic central nervous system development and are classified by their anatomical locations. Defects in the posterior portion of the neural tube are referred to as meningomyeloceles (spina bifida), while the more anterior defects are differentiated as anencephaly, encephalocele, or iniencephaly. Craniorachischisis involves a failure of the neural folds to elevate and thus disrupt the entire length of the neural tube. Worldwide NTDs have a prevalence of approximately 18.6 per 10,000 live births. It is widely believed that genetic factors are responsible for some 70% of NTDs, while the intrauterine environment tips the balance toward neurulation failure in at risk individuals. Despite aggressive educational campaigns to inform the public about folic acid supplementation and the benefits of providing mandatory folic acid food fortification in the United States, NTDs still affect up to 2,300 United States births annually and some 166,000 spina bifida patients currently live in the United States, more than half of whom are now adults. Within the context of this review, we will consider the role of maternal nutritional status (deficiency states involving B vitamins and one carbon analytes) and the potential modifiers of NTD risk beyond folic acid. There are several well-established human teratogens that contribute to the population burden of NTDs, including: industrial waste and pollutants [e.g., arsenic, pesticides, and polycyclic aromatic hydrocarbons (PAHs)], pharmaceuticals (e.g., anti-epileptic medications), and maternal hyperthermia during the first trimester. Animal models for these teratogens are described with attention focused on valproic acid (VPA; Depakote). Genetic interrogation of model systems involving VPA will be used as a model approach to discerning susceptibility factors that define the gene-environment interactions contributing to the etiology of NTDs.

Teratogenicity of valproic acid and its constitutional isomer, amide derivative valnoctamide in mice.

OBJECTIVES: The anticonvulsant valproic acid (VPA) has a known teratogenic effect capable of inducing major congenital malformations and developmental disorders. A comparative teratogenicity study of VPA and its analog valnoctamide (VCD), which is a new generation candidate antiepileptic drug, was carried out using Swiss Vancouver (SWV) mice. METHODS: Pregnant SWV dams were treated with either a single intraperitoneal injection of VPA (1.8 and 2.7 mmol/kg), VCD (1.8 and 2.7 mmol/kg), or vehicle on E8:12 (gestational day:hour). The numbers of implantation and resorption, viable and dead fetuses, and the presence of gross fetal visceral and skeletal abnormalities were determined (E18). Real-time Polymerase chain reaction (RT-PCR) arrays were used to analyze the expression of 84 genes related to the processes of neurogenesis and neural stem cell differentiation. RESULTS: Significant decreases in pregnancy weight gain and the number of live fetuses were observed when VPA was administered at the high dose, whereas the percentage of exencephalic fetuses was significantly increased in VPA treated compared with an equivalent VCD dosage group. There was a dose-related increase in visceral defects in the VPA-exposed fetuses. Missing skull bones and fused vertebrae in fetuses occurred at the high dose of VPA. Three genes (Mtap2, Bmp8b, and Stat3) were significantly upregulated and one (Heyl) was downregulated in samples from VPA-treated dams. CONCLUSIONS: The study demonstrates that the teratogenicity of VPA was significantly greater than that of an equimolar dose of VCD. Four genes (Mtap2, Bmp8b, Stat3, and Heyl) represent candidate target genes for the underlying teratogenic mechanism responsible for VPA-induced malformations.

Formate rescues neural tube defects caused by mutations in Slc25a32.

Periconceptional folic acid (FA) supplementation significantly reduces the prevalence of neural tube defects (NTDs). Unfortunately, some NTDs are FA resistant, and as such, NTDs remain a global public health concern. Previous studies have identified SLC25A32 as a mitochondrial folate transporter (MFT), which is capable of transferring tetrahydrofolate (THF) from cellular cytoplasm to the mitochondria in vitro. Herein, we show that gene trap inactivation of Slc25a32 (Mft) in mice induces NTDs that are folate (5-methyltetrahydrofolate, 5-mTHF) resistant yet are preventable by formate supplementation. Slc25a32gt/gt embryos die in utero with 100% penetrant cranial NTDs. 5-mTHF supplementation failed to promote normal neural tube closure (NTC) in mutant embryos, while formate supplementation enabled the majority (78%) of knockout embryos to complete NTC. A parallel genetic study in human subjects with NTDs identified biallelic loss of function SLC25A32 variants in a cranial NTD case. These data demonstrate that the loss of functional Slc25a32 results in cranial NTDs in mice and has also been observed in a human NTD patient.