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Impairment of one-carbon metabolism during pregnancy, either due to nutritional deficiencies in B9 or B12 vitamins or caused by specific genetic defects, is often associated with neurological defects, including cognitive dysfunction that persists even after vitamin supplementation. Animal nutritional models do not allow for conclusions regarding the specific brain mechanisms that may be modulated by systemic compensations. Using the Cre-lox system associated to the neuronal promoter Thy1.2, a knock-out model for the methionine synthase specifically in the brain was generated. Our results on the neurobehavioral development of offspring show that the absence of methionine synthase did not lead to growth retardation, despite an effective reduction of both its expression and the methylation status in brain tissues. Behaviors were differently affected according to their functional outcome. Only temporary retardations were recorded in the acquisition of vegetative functions during the suckling period, compared to a dramatic reduction in cognitive performance after weaning. Investigation of the glutamatergic synapses in cognitive areas showed a reduction of AMPA receptors phosphorylation and clustering, indicating an epigenomic effect of the neuronal deficiency of methionine synthase on the reduction of glutamatergic synapses excitability. Altogether, our data indicate that cognitive impairment associated with methionine synthase deficiency may not only result from neurodevelopmental abnormalities, but may also be the consequence of alterations in functional plasticity of the brain. [ABSTRACT FROM AUTHOR]

Methyl-Cobalamin (Cbl) derives from dietary vitamin B12 and acts as a cofactor of methionine synthase (MS) in mammals. MS encoded by MTR catalyzes the remethylation of homocysteine to generate methionine and tetrahydrofolate, which fuel methionine and cytoplasmic folate cycles, respectively. Methionine is the precursor of S-adenosyl methionine (SAM), the universal methyl donor of transmethylation reactions. Impaired MS activity results from inadequate dietary intake or malabsorption of B12 and inborn errors of Cbl metabolism (IECM). The mechanisms at the origin of the high variability of clinical presentation of impaired MS activity are classically considered as the consequence of the disruption of the folate cycle and related synthesis of purines and pyrimidines and the decreased synthesis of endogenous methionine and SAM. For one decade, data on cellular and animal models of B12 deficiency and IECM have highlighted other key pathomechanisms, including altered interactome of MS with methionine synthase reductase, MMACHC, and MMADHC, endoplasmic reticulum stress, altered cell signaling, and genomic/epigenomic dysregulations. Decreased MS activity increases catalytic protein phosphatase 2A (PP2A) and produces imbalanced phosphorylation/methylation of nucleocytoplasmic RNA binding proteins, including ELAVL1/HuR protein, with subsequent nuclear sequestration of mRNAs and dramatic alteration of gene expression, including SIRT1. Decreased SAM and SIRT1 activity induce ER stress through impaired SIRT1-deacetylation of HSF1 and hypomethylation/hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), which deactivate nuclear receptors and lead to impaired energy metabolism and neuroplasticity. The reversibility of these pathomechanisms by SIRT1 agonists opens promising perspectives in the treatment of IECM outcomes resistant to conventional supplementation therapies. [ABSTRACT FROM AUTHOR]

Methionine-dependency is a common feature of cancer cells, which cannot proliferate without constant inputs of exogenous methionine even in the presence of its precursor, homocysteine. The endogenous synthesis of methionine is catalyzed by methionine synthase, which transfers the methyl group of 5-methyltetrahydrofolate (5-methylTHF) to homocysteine in the presence of vitamin B12 (cobalamin, cbl). Diverse mechanisms can produce it, including somatic mutations, aberrant DNA methylation (epimutations) and altered expression of genes. Around twenty somatic mutations have been reported as a cause of methionine dependency. Some of them are contributors but not sufficient on their own to cause methionine dependency. Epigenetic invalidation of MMACHC gene expression triggers methionine dependency of the MeWo-LC1 melanoma cancer cell line. This epimutation is generated by aberrant antisense transcription of the adjacent gene PRDX1. Methionine dependency involves the abnormal expression of 1-CM genes in cancer stem cells. It is related to an increased demand for methionine and SAM, which is not compensated by the increased production of formate by glycine decarboxylase pathway in lung cancer tumor spheres. Tumor spheres of glioblastoma U251 are methionine-dependent through disruption of folate metabolism. The rescue of the growth of glioblastoma stem cells by folate shows the considerable importance to evaluate the influence of supplements and dietary intake of folate on the risk of tumor development, in particular in countries subjected to mandatory food fortification in folic acid. Dietary methionine restriction or the use of methioninase represent promising anticancer therapeutic strategies that deserve to be explored in combination with chemotherapy. • Somatic mutations, epigenetic silencing and altered gene expression can produce methionine dependency. • Cancer stem cells from lung cancer and glioblastoma are methionine dependent through mirrored mechanisms. • The methionine dependency of glioblastoma stem cells can be rescued by folate. [ABSTRACT FROM AUTHOR]

Background The risk of neural tube defects (NTDs) is influenced by nutritional factors and genetic determinants of one-carbon metabolism. A key pathway of this metabolism is the vitamin B-12– and folate-dependent remethylation of homocysteine, which depends on methionine synthase (MS, encoded by MTR), methionine synthase reductase, and methylenetetrahydrofolate reductase. Methionine, the product of this pathway, is the direct precursor of S-adenosylmethionine (SAM), the universal methyl donor needed for epigenetic mechanisms. Objectives This study aimed to evaluate whether the availability of vitamin B-12 and folate and the expression or activity of the target enzymes of the remethylation pathway are involved in NTD risk. Methods We studied folate and vitamin B-12 concentrations and activity, expression, and gene variants of the 3 enzymes in liver from 14 NTD and 16 non-NTD fetuses. We replicated the main findings in cord blood from pregnancies of 41 NTD fetuses compared with 21 fetuses with polymalformations (metabolic and genetic findings) and 375 control pregnancies (genetic findings). Results The tissue concentration of vitamin B-12 (P  = 0.003), but not folate, and the activity (P  = 0.001), transcriptional level (P  = 0.016), and protein expression (P  = 0.003) of MS were decreased and the truncated inactive isoforms of MS were increased in NTD livers. SAM was significantly correlated with MS activity and vitamin B-12. A gene variant in exon 1 of GIF (Gastric Intrinsic Factor gene) was associated with a dramatic decrease of liver vitamin B-12 in 2 cases. We confirmed the decreased vitamin B-12 in cord blood from NTD pregnancies. A gene variant of GIF exon 3 was associated with NTD risk. Conclusions The decreased vitamin B-12 in liver and cord blood and decreased expression and activity of MS in liver point out the impaired remethylation pathway as hallmarks associated with NTD risk. We suggest evaluating vitamin B-12 in the nutritional recommendations for prevention of NTD risk beside folate fortification or supplementation. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin B12 (cobalamin, cbl) is a cofactor of methionine synthase (MTR) in the synthesis of methionine, the precursor of the universal methyl donor S-Adenosylmethionine (SAM), which is involved in epigenomic regulatory mechanisms. We have established a neuronal cell model with stable expression of a transcobalamin–oleosin chimer and subsequent decreased cellular availability of vitamin B12, which produces reduced proliferation, increased apoptosis and accelerated differentiation through PP2A, NGF and TACE pathways. Anti-transcobalamin antibody or impaired transcobalamin receptor expression produce also impaired proliferation in other cells. Consistently, the transcription, protein expression and activity of MTR are increased in proliferating cells of skin and intestinal epitheliums, in rat intestine crypts and in proliferating CaCo2 cells, while MTR activity correlates with DNA methylation in rat intestine villi. Exposure to nitrous oxide in animal models identified impairment of MTR reaction as the most important metabolic cause of neurological manifestations of B12 deficiency. Early vitamin B12 and folate deprivation during gestation and lactation of a ‘dam-progeny’ rat model developed in our laboratory is associated with long-lasting disabilities of behavior and memory capacities, with persisting hallmarks related to increased apoptosis, impaired neurogenesis and altered plasticity. We found also an epigenomic deregulation of energy metabolism and fatty acids beta-oxidation in myocardium and liver, through imbalanced methylation/acetylation of PGC-1alpha and decreased expression of SIRT1. These nutrigenomic effects display similarities with the molecular mechanisms of fetal programming. Beside deficiency, B12 loading increases the expression of MTR through internal ribosome entry sites (IRES) and down-regulates MDR-1 gene expression. In conclusion, vitamin B12 influences cell proliferation, differentiation and apoptosis in brain. Vitamin B12 and folate combined deficiency impairs fatty acid oxidation and energy metabolism in liver and heart through epigenomic mechanisms related to imbalanced acetylation/methylation. Some but not all of these effects reflect the upstream role of vitamin B12 in SAM synthesis. [Copyright &y& Elsevier]

Background: Methionine synthase (MTR) synthesizes methionine from homocysteine, using cobalamin as a cofactor and 5-methyltetrahydrofolate as a cosubstrate. Aim: To determine the influence of homocystine (Hcy, dimer of homocysteine) in the presence of either cobalamin or methionine on the transcription and the activity of methionine synthase in Caco-2, a human adenocarcinoma cell line. Methods: Methionine synthase activity and quantification of its mRNA by real-time RT-PCR were determined in cells cultivated under four differents conditions: Hcy with cobalamin (Hcy+ Cbl+), Hcy with methionine (Hcy+Met+), methionine with Cbl (Met+ Cbl+) and methionine only (Met+). Results: Activity (nmol/h/mg protein) was maximal in cells cultivated in Hcy+Cbl+ (2.45 ± 0.35), compared to cells cultivated in Hcy+Met+ (0.18 ± 0.01, p<0.001), in Met+ Cbl+ (1.60 ± 0.06, p<0.05), and in Met+ (0.40 ± 0.05, p<0.001), suggesting an adaptation of the cells to requirement in synthesized methionine. The mRNA level of MTR in Hcy+ Cbl+ and Hcy+Met+ (2.82 ±0.49 and 3.33 ± 0.48 AU, respectively ) was about 2.5 / 3.0-fold higher than that in Met+ Cbl+ and in Met+ (1.00 ±0.13 and 1.20 ±0.20 AU, respectively, p<0.001). Conclusion: Methionine synthase expression of Caco-2 cell is under a transcriptional control influenced by Hcy. [ABSTRACT FROM AUTHOR]

Background/Aims: The respective influence of folate and vitamin B[sub 12] deficiency on MTR activity and transcription, and on DNA methylation is not clearly established. The aim of this study was to assess the respective influence of folate and vitamin B[sub 12] deficiency on MTR transcription and activity, and on DNA methylation. Methods: Sixty-one rats were administered normal diet or diet deficient in choline, methionine, folic acid and vitamin B[sub 12] . Forty-seven of them underwent total gastrectomy or ileal resection. Results: Low vitamin B[sub 12] was observed only in gastrectomized rats. Low folate was observed in rats under deficient diet. Total MTR activity (holo- + apoenzyme) was lowered only with vitamin B[sub 12] level <200 pmol/l (p = 0.0002), while the ratios of total vs. holo-MTR activity and of transcripts MTR vs. GAPDH (RT-PCR) were unchanged. Vitamin B[sub 12] was the single determinant of low MTR (lower quartile, odds ratio = 15.75, p = 0.0017). Low MTR and low vitamin B[sub 12] were the two determinants of DNA hypomethylation (lower quartile) (odds ratio = 17.07, p = 0.0006, and odds ratio = 7.31, p = 0.006, respectively). Conclusion: Vitamin B[sub 12] affects MTR expression by a non-transcriptional mechanism different from a protective effect on MTR proteolysis. It is also a strong determinant of DNA hypomethylation. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

5,10-Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) are two of the key enzymes in the folate/vitamin B[sub12]-dependent remethylation of homocysteine to methionine. The frequencies of MTHFR single nucleotide polymorphisms (SNPs), 677C→T, 1298A→C, 1317T→ and of MTR, 2756A→G, have been widely studied in Caucasians, but they have never been reported simultaneously in a large population from Sub-Saharan Africa. Presently, we report the prevalence of these SNPs and their relationship to homocysteine in 240 subjects recruited in West Africa. The frequencies of the mutant genotypes 677TT (0.8%) and 1298CC (2%) were lower than that usually observed in Caucasians, while the frequency of the mutant 1317CC was higher (16%). We formed a systematic association of the mutated MTHFR 677C→ with a 1298A/1317T common haplotype. The MTHR mutant genotype 677TT was associated with an intermediate hyperhomocysteinemia (92.4 ± 6.0 µmol/l) higher than that described in Caucasians. The 2756A→G SNP in the MTR was similarly distributed in Africans compared to Caucasians. In conclusion, the MTHFR 677TT or 1298CC genotypes are much rarer in Africans than in Caucasians. The 677TT low frequency may be related to the high effect of this mutation on homocysteine metabolism in the environmental conditions of this African region. [ABSTRACT FROM AUTHOR]

An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG , cblE , and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs. We observed the known interactions of MS with MSR and with MMACHC as well as MMADHC with MMACHC, but we also observed novel interactions for MSR with MMACHC and with MMADHC and MS with MMADHC. Furthermore, we show that the absence of MS or MMACHC expression disrupts the interactions between the other interactome members, in cblC and cblG fibroblasts and in HepG2 cells transfected with siRNAs. Our data show that the processing of Cbl in cytoplasm occurs in a multiprotein complex composed of at least MS, MSR, MMACHC and MMADHC, which could contribute to shuttle safely and efficiently Cbl towards MS. Our data suggest that defective protein-protein interactions among key players of this pathway could contribute to the molecular mechanisms of the cblC, cblG and cblE genetic defects and provide novel insights into our understanding of the pathophysiology of inherited disorders of Cbl metabolism. [ABSTRACT FROM AUTHOR]

Background/Aims: Homocysteine metabolism is linked to DNA methylation, a mechanism potentially involved in the course of hepatitis B virus (HBV) infection. We evaluated the association of determinants of homocysteine metabolism with the outcome of HBV infection. Methods: Four hundred and fifty-five healthy adults from Togo and Benin were tested for HBV serologic markers, HLA DR alleles, folate, vitamin B12, methylenetetrahydrofolate reductase (MTHFR) 677 C→T, 1298 A→C and methionine synthase 2756 A→G polymorphisms. Results: Seventy-eight percent of the study population was anti-HBc positive. Among them, 202 (56.9%) were anti-HBs positive and 58 (16.3%) were HBsAg positive. After stepwise logistic regression, the MTHFR 677 T allele was independently associated with persistence of detectable anti-HBs antibodies (OR: 2.47; 95% CI: 1.29–4.71; p =0.006). The mean HBV DNA level was significantly lower in HBsAg positive subjects carrying the 677 T allele than in those with the 677 CC genotype (1000±1406 vs. 2,400,000±214,000 copies/ml, p =0.005). Beninese origin and HLA-DRB1*09 allele were the other determinants independently associated with favorable outcome of HBV infection. Conclusions: The methylenetetrahydrofolate reductase 677 T allele seems to protect against chronic HBV infection in young African adults. [Copyright &y& Elsevier]

Abstract: Cystathionine beta synthase (CBS) deficiency is a metabolic disorder that is biochemically characterized by severe hyperhomocysteinemia. In order to show the effects of CBS deficiency onto the activity of the enzymes involved in the remethylation pathway, we used the well characterized genetic model of severe hyperhomocysteinemia in mice. We showed that CBS deficiency in mice reduced hepatic methionine synthase and betaine-homocysteine methyltransferase activities, whereas 5,10-methylene tetrahydrofolate reductase activity was increased. [Copyright &y& Elsevier]