AAV-mediated expression of mouse or human GLDC normalises metabolic biomarkers in a GLDC-deficient mouse model of Non-Ketotic Hyperglycinemia.

Non-Ketotic Hyperglycinemia (NKH) is a rare inborn error of metabolism caused by impaired function of the glycine cleavage system (GCS) and characterised by accumulation of glycine in body fluids and tissues. NKH is an autosomal recessive condition and the majority of affected individuals carry mutations in GLDC (glycine decarboxylase). Current treatments for NKH have limited effect and are not curative. As a monogenic condition with known genetic causation, NKH is potentially amenable to gene therapy. An AAV9-based expression vector was designed to target sites of GCS activity. Using a ubiquitous promoter to drive expression of a GFP reporter, transduction of liver and brain was confirmed following intra-venous and/or intra-cerebroventricular administration to neonatal mice. Using the same capsid and promoter with transgenes to express mouse or human GLDC, vectors were then tested in GLDC-deficient mice that provide a model of NKH. GLDC-deficient mice exhibited elevated plasma glycine concentration and accumulation of glycine in liver and brain tissues as previously observed. Moreover, the folate profile indicated suppression of folate one­carbon metabolism (FOCM) in brain tissue, as found at embryonic stages, and reduced abundance of FOCM metabolites including betaine and choline. Neonatal administration of vector achieved reinstatement of GLDC mRNA and protein expression in GLDC-deficient mice. Treated GLDC-deficient mice showed significant lowering of plasma glycine, confirming functionality of vector expressed protein. AAV9-GLDC treatment also led to lowering of brain tissue glycine, and normalisation of the folate profile indicating restoration of glycine-derived one­carbon supply. These findings support the hypothesis that AAV-mediated gene therapy may offer potential in treatment of NKH.

The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.

Folate deficiency increases the incidence of dolutegravir-associated foetal defects in a mouse pregnancy model.

BACKGROUND: Dolutegravir (DTG) is a recommended first-line regimen for all people with Human Immunodeficiency Virus (HIV) infection. Initial findings from Botswana, a country with no folate fortification program, showed an elevated prevalence of neural tube defects (NTDs) with peri-conceptional exposure to DTG. Here we explore whether a low folate diet influences the risk of DTG-associated foetal anomalies in a mouse model. METHODS: C57BL/6 mice fed a folate-deficient diet for 2 weeks, were mated and then randomly allocated to control (water), or 1xDTG (2.5 mg/kg), or 5xDTG (12.5 mg/kg) both administered orally with 50 mg/kg tenofovir disoproxil fumarate 33.3 mg/kg emtricitabine. Treatment was administered once daily from gestational day (GD) 0.5 to sacrifice (GD15.5). Foetuses were assessed for gross anomalies. Maternal and foetal folate levels were quantified. FINDINGS: 313 litters (103 control, 106 1xDTG, 104 5xDTG) were assessed. Viability, placental weight, and foetal weight did not differ between groups. NTDs were only observed in the DTG groups (litter rate: 0% control; 1.0% 1xDTG; 1.3% 5xDTG). Tail, abdominal wall, limb, craniofacial, and bleeding defects all occurred at higher rates in the DTG groups versus control. Compared with our previous findings on DTG usage in folate-replete mouse pregnancies, folate deficiency was associated with higher rates of several defects, including NTDs, but in the DTG groups only. We observed a severe left-right asymmetry phenotype that was more frequent in DTG groups than controls. INTERPRETATION: Maternal folate deficiency may increase the risk for DTG-associated foetal defects. Periconceptional folic acid supplementation could be considered for women with HIV taking DTG during pregnancy, particularly in countries lacking folate fortification programs. FUNDING: This project has been funded by Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN275201800001I and award #R01HD104553. LS is supported by a Tier 1 Canada Research Chair in Maternal-Child Health and HIV. HM is supported by a Junior Investigator award from the Ontario HIV Treatment Network.

The actions of methotrexate on endothelial cells are dependent on the shear stress-induced regulation of one carbon metabolism.

Objectives: The disease-modifying anti-rheumatic drug methotrexate (MTX) is recognized to reduce cardiovascular risk in patients with systemic inflammatory diseases. However, the molecular basis for these cardioprotective effects remains incompletely understood. This study evaluated the actions of low-dose MTX on the vascular endothelium. Methods: Human endothelial cells (EC) were studied under in vitro conditions relevant to inflammatory arthritis. These included culture in a pro-inflammatory microenvironment and exposure to fluid shear stress (FSS) using a parallel plate model. Respectively treated cells were analyzed by RNA sequencing and quantitative real-time PCR for gene expression, by immunoblotting for protein expression, by phosphokinase activity arrays, by flow cytometry for cell cycle analyses and by mass spectrometry to assess folate metabolite levels. Results: In static conditions, MTX was efficiently taken up by EC and caused cell cycle arrest concurrent with modulation of cell signaling pathways. These responses were reversed by folinic acid (FA), suggesting that OCM is a predominant target of MTX. Under FSS, MTX did not affect cell proliferation or pro-inflammatory gene expression. Exposure to FSS downregulated endothelial one carbon metabolism (OCM) as evidenced by decreased expression of key OCM genes and metabolites. Conclusion: We found that FSS significantly downregulated OCM and thereby rendered EC less susceptible to the effects of MTX treatment. The impact of shear stress on OCM suggested that MTX does not directly modulate endothelial function. The cardioprotective actions of MTX likely reflect direct actions on inflammatory cells and indirect benefit on the vascular endothelium.

Investigating Genetic Determinants of Plasma Inositol Status in Adult Humans.

BACKGROUND: Myo-inositol (MI) is incorporated into numerous biomolecules, including phosphoinositides and inositol phosphates. Disturbance of inositol availability or metabolism is associated with various disorders, including neurological conditions and cancers, whereas supplemental MI has therapeutic potential in conditions such as depression, polycystic ovary syndrome, and congenital anomalies. Inositol status can be influenced by diet, synthesis, transport, utilization, and catabolism. OBJECTIVES: We aimed to investigate potential genetic regulation of circulating MI status and to evaluate correlation of MI concentration with other metabolites. METHODS: GC-MS was used to determine plasma MI concentration of >2000 healthy, young adults (aged 18-28 y) from the Trinity Student Study. Genotyping data were used to test association of plasma MI with single nucleotide polymorphisms (SNPs) in candidate genes, encoding inositol transporters and synthesizing enzymes, and test for genome-wide association. We evaluated potential correlation of plasma MI with d-chiro-inositol (DCI), glucose, and other metabolites by Spearman rank correlation. RESULTS: Mean plasma MI showed a small but significant difference between males and females (28.5 and 26.9 µM, respectively). Candidate gene analysis revealed several nominally significant associations with plasma MI, most notably for SLC5A11 (solute carrier family 5 member 11), encoding a sodium-coupled inositol transporter, also known as SMIT2 (sodium-dependent myo-inositol transporter 2). However, these did not survive correction for multiple testing. Subsequent testing for genome-wide association with plasma MI did not identify associations of genome-wide significance (P < 5 × 10-8). However, 8 SNPs exceeded the threshold for suggestive significant association with plasma MI concentration (P < 1 × 10-5), 3 of which were located within or close to genes: MTDH (metadherin), LAPTM4B (lysosomal protein transmembrane 4 ß), and ZP2 (zona pellucida 2). We found significant positive correlation of plasma MI concentration with concentration of dci and several other biochemicals including glucose, methionine, betaine, sarcosine, and tryptophan. CONCLUSIONS: Our findings suggest potential for modulation of plasma MI in young adults by variation in SLC5A11, which is worthy of further investigation.

Glycine Cleavage System H Protein Is Essential for Embryonic Viability, Implying Additional Function Beyond the Glycine Cleavage System.

Glycine cleavage system H protein (GCSH) is a component of the glycine cleavage system (GCS), a conserved protein complex that acts to decarboxylate glycine. Mutation of AMT or GLDC, encoding the GCS components aminomethyltransferase and glycine decarboxylase, can cause malformations of the developing CNS (neural tube defects (NTDs) and ventriculomegaly) as well as a post-natal life-limiting neurometabolic disorder, Non-Ketotic Hyperglycinemia. In contrast, it is unclear whether mutation of GCSH contributes to these conditions and we therefore investigated GCSH loss of function in mice. Mice that were heterozygous for a Gcsh null allele were viable and did not exhibit elevated plasma glycine. Moreover, heterozygous mutation of Gcsh did not increase the frequency of NTDs in Gldc mutant embryos. Homozygous Gcsh null mice were not recovered at post-natal stages. Analysis of litters at E8.5-10.5, revealed the presence of homozygous null embryos which were much smaller than littermates and had failed to develop beyond early post-implantation stages with no visible somites or head-folds. Hence, unlike null mutations of Gldc or Amt, which are compatible with embryonic survival despite the presence of NTDs, loss of Gcsh causes embryonic death prior to mid-gestation. Maternal supplementation with formate did not restore embryonic development beyond E7.5, suggesting that the primary cause of lethality was not loss of glycine cleavage activity or suppression of folate one-carbon metabolism. These findings suggest that GCSH has additional roles beyond function in the glycine cleavage system. We hypothesize that GCSH potentially acts in lipoylation of 2-oxoacid dehydrogenase proteins, as reported in bacteria.

Deficient or Excess Folic Acid Supply During Pregnancy Alter Cortical Neurodevelopment in Mouse Offspring.

Folate is an essential micronutrient required for both cellular proliferation through de novo nucleotide synthesis and epigenetic regulation of gene expression through methylation. This dual requirement places a particular demand on folate availability during pregnancy when both rapid cell generation and programmed differentiation of maternal, extraembryonic, and embryonic/fetal tissues are required. Accordingly, prenatal neurodevelopment is particularly susceptible to folate deficiency, which can predispose to neural tube defects, or when effective transport into the brain is impaired, cerebral folate deficiency. Consequently, adequate folate consumption, in the form of folic acid (FA) fortification and supplement use, is widely recommended and has led to a substantial increase in the amount of FA intake during pregnancy in some populations. Here, we show that either maternal folate deficiency or FA excess in mice results in disruptions in folate metabolism of the offspring, suggesting diversion of the folate cycle from methylation to DNA synthesis. Paradoxically, either intervention causes comparable neurodevelopmental changes by delaying prenatal cerebral cortical neurogenesis in favor of late-born neurons. These cytoarchitectural and biochemical alterations are accompanied by behavioral abnormalities in FA test groups compared with controls. Our findings point to overlooked potential neurodevelopmental risks associated with excessively high levels of prenatal FA intake.

Dolutegravir in pregnant mice is associated with increased rates of fetal defects at therapeutic but not at supratherapeutic levels.

BACKGROUND: Dolutegravir (DTG) is a preferred regimen for all people with HIV including pregnant women, but its effects on the fetus are not fully understood. Periconceptional exposure to DTG has been associated with increased rates of neural tube defects (NTDs), although it is unknown whether this is a causal relationship. This has led to uncertainty around the use of DTG in women of reproductive potential. METHODS: Pregnant C57BL/6J mice were randomly allocated to control (water), 1x-DTG (2.5 mg/kg-peak plasma concentration ~3000 ng/ml - therapeutic level), or 5x-DTG (12.5 mg/kg-peak plasma concentration ~12,000 ng/ml - supratherapeutic level), once daily from gestational day 0.5 until sacrifice. DTG was administered with 50 mg/kg tenofovir+33.3 mg/kg emtricitabine. Fetal phenotypes were determined, and maternal and fetal folate levels were quantified by mass-spectrometry. FINDINGS: 352 litters (91 control, 150 1x-DTG, 111 5x-DTG) yielding 2776 fetuses (747 control, 1174 1x-DTG, 855 5x-DTG) were assessed. Litter size and viability rates were similar between groups. Fetal and placenta weights were lower in the 1x-DTG vs. control. Placental weight was higher in the 5x-DTG vs. control. Five NTDs were observed, all in the 1x-DTG group. Fetal defects, including microphthalmia, severe edema, and vascular/bleeding defects were more frequent in the 1x-DTG group. In contrast, defect rates in the 5x-DTG were similar to control. Fetal folate levels were similar between control and 1x-DTG, but were significantly higher in the 5x-DTG group. INTERPRETATION: Our findings support a causal relationship of DTG at therapeutic doses with increased risk for fetal defects, including NTDs at a rate that is similar that reported in the Tsepamo study for women exposed to DTG-based ART from conception. The non-monotonic dose-response relationship between DTG and fetal anomalies could explain the previous lack of fetal toxicity findings from pre-clinical DTG studies. The fetal folate levels suggest that DTG is unlikely to be an inhibitor of folate uptake. FUNDING: This project has been funded with Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN275201800001I.

Regulation of glycine metabolism by the glycine cleavage system and conjugation pathway in mouse models of non-ketotic hyperglycinemia.

Glycine abundance is modulated in a tissue-specific manner by use in biosynthetic reactions, catabolism by the glycine cleavage system (GCS), and excretion via glycine conjugation. Dysregulation of glycine metabolism is associated with multiple disorders including epilepsy, developmental delay, and birth defects. Mutation of the GCS component glycine decarboxylase (GLDC) in non-ketotic hyperglycinemia (NKH) causes accumulation of glycine in body fluids, but there is a gap in our knowledge regarding the effects on glycine metabolism in tissues. Here, we analysed mice carrying mutations in Gldc that result in severe or mild elevations of plasma glycine and model NKH. Liver of Gldc-deficient mice accumulated glycine and numerous glycine derivatives, including multiple acylglycines, indicating increased flux through reactions mediated by enzymes including glycine-N-acyltransferase and arginine: glycine amidinotransferase. Levels of dysregulated metabolites increased with age and were normalised by liver-specific rescue of Gldc expression. Brain tissue exhibited increased abundance of glycine, as well as derivatives including guanidinoacetate, which may itself be epileptogenic. Elevation of brain tissue glycine occurred even in the presence of only mildly elevated plasma glycine in mice carrying a missense allele of Gldc. Treatment with benzoate enhanced hepatic glycine conjugation thereby lowering plasma and tissue glycine. Moreover, administration of a glycine conjugation pathway intermediate, cinnamate, similarly achieved normalisation of liver glycine derivatives and circulating glycine. Although exogenous benzoate and cinnamate impact glycine levels via activity of glycine-N-acyltransferase, that is not expressed in brain, they are sufficient to lower levels of glycine and derivatives in brain tissue of treated Gldc-deficient mice.

Mechanical properties measured by atomic force microscopy define health biomarkers in ageing C. elegans.

Genetic and environmental factors are key drivers regulating organismal lifespan but how these impact healthspan is less well understood. Techniques capturing biomechanical properties of tissues on a nano-scale level are providing new insights into disease mechanisms. Here, we apply Atomic Force Microscopy (AFM) to quantitatively measure the change in biomechanical properties associated with ageing Caenorhabditis elegans in addition to capturing high-resolution topographical images of cuticle senescence. We show that distinct dietary restriction regimes and genetic pathways that increase lifespan lead to radically different healthspan outcomes. Hence, our data support the view that prolonged lifespan does not always coincide with extended healthspan. Importantly, we identify the insulin signalling pathway in C. elegans and interventions altering bacterial physiology as increasing both lifespan and healthspan. Overall, AFM provides a highly sensitive technique to measure organismal biomechanical fitness and delivers an approach to screen for health-improving conditions, an essential step towards healthy ageing.

A new mechanism for cannabidiol in regulating the one-carbon cycle and methionine levels in Dictyostelium and in mammalian epilepsy models.

BACKGROUND AND PURPOSE: Epidiolex™, a form of highly purified cannabidiol (CBD) derived from Cannabis plants, has demonstrated seizure control activity in patients with Dravet syndrome, without a fully elucidated mechanism of action. We have employed an unbiased approach to investigate this mechanism at a cellular level. EXPERIMENTAL APPROACH: We use a tractable biomedical model organism, Dictyostelium, to identify a protein controlling the effect of CBD and characterize this mechanism. We then translate these results to a Dravet syndrome mouse model and an acute in vitro seizure model. KEY RESULTS: CBD activity is partially dependent upon the mitochondrial glycine cleavage system component, GcvH1 in Dictyostelium, orthologous to the human glycine cleavage system component H protein, which is functionally linked to folate one-carbon metabolism (FOCM). Analysis of FOCM components identified a mechanism for CBD in directly inhibiting methionine synthesis. Analysis of brain tissue from a Dravet syndrome mouse model also showed drastically altered levels of one-carbon components including methionine, and an in vitro rat seizure model showed an elevated level of methionine that is attenuated following CBD treatment. CONCLUSIONS AND IMPLICATIONS: Our results suggest a novel mechanism for CBD in the regulating methionine levels and identify altered one-carbon metabolism in Dravet syndrome and seizure activity.

Impaired folate 1-carbon metabolism causes formate-preventable hydrocephalus in glycine decarboxylase-deficient mice.

Ventriculomegaly and hydrocephalus are associated with loss of function of glycine decarboxylase (Gldc) in mice and in humans suffering from non-ketotic hyperglycinemia (NKH), a neurometabolic disorder characterized by accumulation of excess glycine. Here, we showed that ventriculomegaly in Gldc-deficient mice is preceded by stenosis of the Sylvian aqueduct and malformation or absence of the subcommissural organ and pineal gland. Gldc functions in the glycine cleavage system, a mitochondrial component of folate metabolism, whose malfunction results in accumulation of glycine and diminished supply of glycine-derived 1-carbon units to the folate cycle. We showed that inadequate 1-carbon supply, as opposed to excess glycine, is the cause of hydrocephalus associated with loss of function of the glycine cleavage system. Maternal supplementation with formate prevented both ventriculomegaly, as assessed at prenatal stages, and postnatal development of hydrocephalus in Gldc-deficient mice. Furthermore, ventriculomegaly was rescued by genetic ablation of 5,10-methylene tetrahydrofolate reductase (Mthfr), which results in retention of 1-carbon groups in the folate cycle at the expense of transfer to the methylation cycle. In conclusion, a defect in folate metabolism can lead to prenatal aqueduct stenosis and resultant hydrocephalus. These defects are preventable by maternal supplementation with formate, which acts as a 1-carbon donor.

Cellular mechanisms underlying Pax3-related neural tube defects and their prevention by folic acid.

Neural tube defects (NTDs), including spina bifida and anencephaly, are among the most common birth defects worldwide, but their underlying genetic and cellular causes are not well understood. Some NTDs are preventable by supplemental folic acid. However, despite widespread use of folic acid supplements and implementation of food fortification in many countries, the protective mechanism is unclear. Pax3 mutant (splotch; Sp2H ) mice provide a model in which NTDs are preventable by folic acid and exacerbated by maternal folate deficiency. Here, we found that cell proliferation was diminished in the dorsal neuroepithelium of mutant embryos, corresponding to the region of abolished Pax3 function. This was accompanied by premature neuronal differentiation in the prospective midbrain. Contrary to previous reports, we did not find evidence that increased apoptosis could underlie failed neural tube closure in Pax3 mutant embryos, nor that inhibition of apoptosis could prevent NTDs. These findings suggest that Pax3 functions to maintain the neuroepithelium in a proliferative, undifferentiated state, allowing neurulation to proceed. NTDs in Pax3 mutants were not associated with abnormal abundance of specific folates and were not prevented by formate, a one-carbon donor to folate metabolism. Supplemental folic acid restored proliferation in the cranial neuroepithelium. This effect was mediated by enhanced progression of the cell cycle from S to G2 phase, specifically in the Pax3 mutant dorsal neuroepithelium. We propose that the cell-cycle-promoting effect of folic acid compensates for the loss of Pax3 and thereby prevents cranial NTDs.

Glycine decarboxylase deficiency-induced motor dysfunction in zebrafish is rescued by counterbalancing glycine synaptic level.

Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc-/- fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc-/- mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc-/- zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc-/- hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc-/- larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc-/- larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.

Partitioning of One-Carbon Units in Folate and Methionine Metabolism Is Essential for Neural Tube Closure.

Abnormal folate one-carbon metabolism (FOCM) is implicated in neural tube defects (NTDs), severe malformations of the nervous system. MTHFR mediates unidirectional transfer of methyl groups from the folate cycle to the methionine cycle and, therefore, represents a key nexus in partitioning one-carbon units between FOCM functional outputs. Methionine cycle inhibitors prevent neural tube closure in mouse embryos. Similarly, the inability to use glycine as a one-carbon donor to the folate cycle causes NTDs in glycine decarboxylase (Gldc)-deficient embryos. However, analysis of Mthfr-null mouse embryos shows that neither S-adenosylmethionine abundance nor neural tube closure depend on one-carbon units derived from embryonic or maternal folate cycles. Mthfr deletion or methionine treatment prevents NTDs in Gldc-null embryos by retention of one-carbon units within the folate cycle. Overall, neural tube closure depends on the activity of both the methionine and folate cycles, but transfer of one-carbon units between the cycles is not necessary.

Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.

High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring.

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid.

Inositol, neural tube closure and the prevention of neural tube defects.

Susceptibility to neural tube defects (NTDs), such as anencephaly and spina bifida is influenced by genetic and environmental factors including maternal nutrition. Maternal periconceptional supplementation with folic acid significantly reduces the risk of an NTD-affected pregnancy, but does not prevent all NTDs, and "folic acid non-responsive" NTDs continue to occur. Similarly, among mouse models of NTDs, some are responsive to folic acid but others are not. Among nutritional factors, inositol deficiency causes cranial NTDs in mice while supplemental inositol prevents spinal and cranial NTDs in the curly tail (Grhl3 hypomorph) mouse, rodent models of hyperglycemia or induced diabetes, and in a folate-deficiency induced NTD model. NTDs also occur in mice lacking expression of certain inositol kinases. Inositol-containing phospholipids (phosphoinositides) and soluble inositol phosphates mediate a range of functions, including intracellular signaling, interaction with cytoskeletal proteins, and regulation of membrane identity in trafficking and cell division. Myo-inositol has been trialed in humans for a range of conditions and appears safe for use in human pregnancy. In pilot studies in Italy and the United Kingdom, women took inositol together with folic acid preconceptionally, after one or more previous NTD-affected pregnancies. In nonrandomized cohorts and a randomized double-blind study in the United Kingdom, no recurrent NTDs were observed among 52 pregnancies reported to date. Larger-scale fully powered trials are needed to determine whether supplementation with inositol and folic acid would more effectively prevent NTDs than folic acid alone. Birth Defects Research 109:68-80, 2017. © 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc.

Formate supplementation enhances folate-dependent nucleotide biosynthesis and prevents spina bifida in a mouse model of folic acid-resistant neural tube defects.

The curly tail mouse provides a model for neural tube defects (spina bifida and exencephaly) that are resistant to prevention by folic acid. The major ct gene, responsible for spina bifida, corresponds to a hypomorphic allele of grainyhead-like 3 (Grhl3) but the frequency of NTDs is strongly influenced by modifiers in the genetic background. Moreover, exencephaly in the curly tail strain is not prevented by reinstatement of Grhl3 expression. In the current study we found that expression of Mthfd1L, encoding a key component of mitochondrial folate one-carbon metabolism (FOCM), is significantly reduced in ct/ct embryos compared to a partially congenic wild-type strain. This expression change is not attributable to regulation by Grhl3 or the genetic background at the Mthfd1L locus. Mitochondrial FOCM provides one-carbon units as formate for FOCM reactions in the cytosol. We found that maternal supplementation with formate prevented NTDs in curly tail embryos and also resulted in increased litter size. Analysis of the folate profile of neurulation-stage embryos showed that formate supplementation resulted in an increased proportion of formyl-THF and THF but a reduction in proportion of 5-methyl THF. In contrast, THF decreased and 5-methyl THF was relatively more abundant in the liver of supplemented dams than in controls. In embryos cultured through the period of spinal neurulation, incorporation of labelled thymidine and adenine into genomic DNA was suppressed by supplemental formate, suggesting that de novo folate-dependent biosynthesis of nucleotides (thymidylate and purines) was enhanced. We hypothesise that reduced Mthfd1L expression may contribute to susceptibility to NTDs in the curly tail strain and that formate acts as a one-carbon donor to prevent NTDs.