Folate-mediated One-carbon Metabolism Research Articles

Folate-Mediated One-Carbon Metabolism in the Crustacean Copepod Calanus finmarchicus: Identification of Transcripts and Relative Expression across Development

Folate, also known as vitamin B9, plays a crucial role in the one-carbon (1C) metabolism, a conserved pathway from microbes to humans. The 1C metabolism, consisting of the folate and methionine cycles, is essential in many biological processes such as nucleotide and protein biosynthesis, cell proliferation, and embryonic development. Despite its functional role, little is known about the 1C metabolism in crustaceans. As part of an ongoing effort to characterize important pathways in Calanus finmarchicus, the biomass-dominant zooplankton in much of the North Atlantic Ocean, we identified transcripts encoding the 1C metabolism enzymes. Using an in silico workflow consisting of a transcriptome mining, reciprocal blasts, and structural analyses of the deduced proteins, we identified the entire set of enzymes in both cycles. The majority encoded for full-length proteins and clustered with homologs from other species. Stage-specific expression was reported, with several transcripts showing high expression in the naupliar stage (e.g., 10-FTHFD, SHMT2) while some methyltransferases (e.g., BHMT, SHMT, DNMT) were more expressed in adults. Overall, this study provides a set of genes which can be used as potential biomarkers of development and reproduction and can be tested in other zooplankters to assess ocean health status monitoring.

Interactions between folate metabolism-related nutrients and polymorphisms on colorectal cancer risk: a case-control study in the Basque country.

Folate-mediated one-carbon metabolism (FOCM) plays an important role in colorectal carcinogenesis. Previous studies have assessed the role of folate-mediated one-carbon metabolism (FOCM)-related gene-diet interaction in the aetiology of colorectal cancer (CRC), however, the results remained inconclusive. Thus, this study aimed to investigate dietary factors and genetic variants related to FOCM, as well as potential nutrient-gene and nutrient-lifestyle interactions, on CRC risk. This observational study included 229 patients diagnosed with CRC and 229 age- and sex-matched subjects as controls from a population-based bowel cancer screening program. Conditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (95%CI) for CRC risk. A Bonferroni-corrected threshold of α = 0.005 was considered significant, and P values less than 0.05 were considered to be suggestive of an association. After Bonferroni correction, a high dietary intake of betaine was associated with a decreased risk of CRC in the adjusted model (OR, 95% CI: 0.21, 0.10-0.40, P < 0.001). Two SNPs, rs1476413 and rs17824591, exhibited significant gene-diet interactions with total choline ad vitamin B12 intakes, respectively, in adjusted models (total choline, tertile 3 vs. 1, OR, 95% CI: 0.25, 0.11-0.66, Pinteraction = 0.012; vitamin B12, tertile 2 vs. tertile 1, OR, 95% CI: 2.48, 1.04-5.00, Pinteraction = 0.003). These findings suggest that betaine intake and interactions between some dietary factors and variants in MTHFR and MTHFD1 genes have an influence on CRC risk in the population studied. If these results are confirmed, specific nutritional intervention strategies could be designed.

The Heterogeneous Impact of Prediagnostic Folate Intake for Fluorouracil-Containing Induction Chemotherapy for Head and Neck Cancer.

Fluorouracil (FU) exerts its antitumor activity by inhibiting folate-mediated one-carbon metabolism. Evidence that folate may play a role in the carcinogenic process via folate-mediated one-carbon metabolism has given rise to the hypothesis that pre-diagnostic folate intake may induce heterogeneous chemosensitivity to FU-containing induction chemotherapy (IC) in head and neck cancer. To assess this hypothesis, we conducted a cohort study to investigate whether the association between prediagnostic dietary folate intake and cancer survival differed between treatment regimens with and without FU-containing IC in 504 cases of locally advanced (stage III/IV) HNSCC, using an epidemiologic database combined with clinical data. In total, 240 patients were treated with FU-containing IC followed by definitive treatment, and 264 patients were treated with definitive treatment alone. Definitive treatment is defined as (1) the surgical excision of a tumor with clear margins, with or without neck lymph node dissection; or (2) radiotherapy with or without chemotherapy. In the overall cohort of the FU-containing IC group, a higher folate intake was significantly associated with better overall survival (adjusted hazard ratios (HRs) for the highest compared to the lowest folate tertiles (HRT3-T1) = 0.42, 95%CI, 0.25-0.76, Ptrend = 0.003). Conversely, no apparent association between prediagnostic folate intake and survival was observed with definitive treatment alone (HRT3-T1: 0.83, 95%CI, 0.49-1.42, Ptrend = 0.491)). A consideration of the cumulative dose of FU-containing IC showed that the survival impact of prediagnostic folate intake differed statistically significantly by treatment regimen (Pinteraction = 0.012). In conclusion, an association between prediagnostic folate intake and HNSCC survival significantly differed by FU-containing IC. This finding indicates that in the carcinogenic process, folate status causes HNSCC to be heterogenous in terms of one-carbon metabolism.

Acetylation of aldehyde dehydrogenase ALDH1L2 regulates cellular redox balance and the chemosensitivity of colorectal cancer to 5-fluorouracil

Folate-mediated one-carbon metabolism (FOCM) is crucial in sustaining rapid proliferation and survival of cancer cells. The folate cycle depends on a series of key cellular enzymes, including aldehyde dehydrogenase 1 family member L2 (ALDH1L2) that is usually overexpressed in cancer cells, but the regulatory mechanism of ALDH1L2 remains undefined. In this study, we observed the significant overexpression of ALDH1L2 in colorectal cancer (CRC) tissues, which is associated with poor prognosis. Mechanistically, we identified that the acetylation of ALDH1L2 at the K70 site is an important regulatory mechanism inhibiting the enzymatic activity of ALDH1L2 and disturbing cellular redox balance. Moreover, we revealed that sirtuins 3 (SIRT3) directly binds and deacetylates ALDH1L2 to increase its activity. Interestingly, the chemotherapeutic agent 5-fluorouracil (5-Fu) inhibits the expression of SIRT3 and increases the acetylation levels of ALDH1L2 in colorectal cancer cells. 5-Fu-induced ALDH1L2 acetylation sufficiently inhibits its enzymatic activity and the production of NADPH and GSH, thereby leading to oxidative stress-induced apoptosis and suppressing tumor growth in mice. Furthermore, the K70Q mutant of ALDH1L2 sensitizes cancer cells to 5-Fu both invitro and invivo through perturbing cellular redox and serine metabolism. Our findings reveal an unknown 5-Fu-SIRT3-ALDH1L2 axis regulating redox homeostasis, and suggest that targeting ALDH1L2 is a promising therapeutic strategy to sensitize tumor cells to chemotherapeutic agents.

Folate-mediated one-carbon metabolism as a potential antifungal target for the sustainable cultivation of microalga Haematococcus pluvialis

BackgroundMicroalgae are widely considered as multifunctional cell factories that are able to transform the photo-synthetically fixed CO2 to numerous high-value compounds, including lipids, carbohydrates, proteins and pigments. However, contamination of the algal mass culture with fungal parasites continues to threaten the production of algal biomass, which dramatically highlights the importance of developing effective measures to control the fungal infection. One viable solution is to identify potential metabolic pathways that are essential for fungal pathogenicity but are not obligate for algal growth, and to use inhibitors targeting such pathways to restrain the infection. However, such targets remain largely unknown, making it challenging to develop effective measures to mitigate the infection in algal mass culture.ResultsIn the present study, we conducted RNA-Seq analysis for the fungus Paraphysoderma sedebokerense, which can infect the astaxanthin-producing microalga Haematococcus pluvialis. It was found that many differentially expressed genes (DEGs) related to folate-mediated one-carbon metabolism (FOCM) were enriched in P. sedebokerense, which was assumed to produce metabolites required for the fungal parasitism. To verify this hypothesis, antifolate that hampered FOCM was applied to the culture systems. Results showed that when 20 ppm of the antifolate co-trimoxazole were added, the infection ratio decreased to ~ 10% after 9 days inoculation (for the control, the infection ratio was 100% after 5 days inoculation). Moreover, application of co-trimoxazole to H. pluvialis mono-culture showed no obvious differences in the biomass and pigment accumulation compared with the control, suggesting that this is a potentially algae-safe, fungi-targeted treatment.ConclusionsThis study demonstrated that applying antifolate to H. pluvialis culturing systems can abolish the infection of the fungus P. sedebokerense and the treatment shows no obvious disturbance to the algal culture, suggesting FOCM is a potential target for antifungal drug design in the microalgal mass culture industry.

Sources and Sinks of Serine in Nutrition, Health, and Disease.

Amino acid dysregulation has emerged as an important driver of disease progression in various contexts. l-Serine lies at a central node of metabolism, linking carbohydrate metabolism, transamination, glycine, and folate-mediated one-carbon metabolism to protein synthesis and various downstream bioenergetic and biosynthetic pathways. l-Serine is produced locally in the brain but is sourced predominantly from glycine and one-carbon metabolism in peripheral tissues via liver and kidney metabolism. Compromised regulation or activity of l-serine synthesis and disposal occurs in the context of genetic diseases as well as chronic disease states, leading to low circulating l-serine levels and pathogenesis in the nervous system, retina, heart, and aging muscle. Dietary interventions in preclinical models modulate sensory neuropathy, retinopathy, tumor growth, and muscle regeneration. A serine tolerance test may provide a quantitative readout of l-serine homeostasis that identifies patients who may be susceptible to neuropathy or responsive to therapy.

Nitric oxide modulates folate-mediated one-carbon metabolism and mitochondrial energy levels of peaches during cold storage.

Folate-mediated one-carbon metabolism (FOCM) is closely associated with postharvest preservation. This study investigated the effects of exogenous nitric oxide (NO) on FOCM, storage quality, energy metabolism, and mitochondrial membrane integrity in cold-storage peach fruit. In this experiment, peaches were soaked with 1.5 mmol L-1S-nitrosoglutathione (GSNO) as NO donor, and the negative treatment (NT) solution containing 5 μmol L-1 carboxy-PTIO (c-PTIO, NO scavenger), 200 μmol L-1 NG-Nitro-L-arginine methyl ester (L-NAME, NO synthase-like enzyme inhibitor), and 200 μmol L-1 sodium tungstate dihydrate (nitrate reductase inhibitor) and stored at 0°C. The results showed that NO decreased the activity of S-adenosylmethionine synthase and S-adenosylhomocysteine hydrolase and increased the activity of methionine sulfoxide reductase A, as well as the content of N5-methyl-THF, the ratio of tetrahydrofolate (THF), homocysteine, methionine, S-adenosylmethionine (SAM), and SAM to S-adenosylhomocysteine compared with the control, indicating that NO effectively increased FOCM flux by affecting the activity of FOCM enzymes. Meanwhile, NO increased the activities of H+-ATPase, Ca2+-ATPase, cytochrome c oxidase, succinate dehydrogenase, and the contents of adenosine triphosphate and adenosine diphosphate, and maintained high energy charge in peaches during storage. NO retarded the increase in mitochondrial permeability transition, reactive oxygen species content, and the decrease in mitochondrial membrane fluidity, membrane potential, and swelling. NT treatment exhibited the opposite results. In conclusion, these results suggested that NO could induce the accumulation of folate and FOCM flux and maintain mitochondrial energy levels, which might be responsible for maintaining the quality of peaches during cold storage.

Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity.

Adequate thymidylate [deoxythymidine monophosphate (dTMP) or the "T" base in DNA] levels are essential for stability of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Folate and vitamin B12 (B12) are essential cofactors in folate-mediated one-carbon metabolism (FOCM), a metabolic network which supports synthesis of nucleotides (including dTMP) and methionine. Perturbations in FOCM impair dTMP synthesis, causing misincorporation of uracil (or a "U" base) into DNA. During B12 deficiency, cellular folate accumulates as 5-methyltetrahdryfolate (5-methyl-THF), limiting nucleotide synthesis. The purpose of this study was to determine how reduced levels of the B12-dpendent enzyme methionine synthase (MTR) and dietary folate interact to affect mtDNA integrity and mitochondrial function in mouse liver. Folate accumulation, uracil levels, mtDNA content, and oxidative phosphorylation capacity were measured in male Mtr+/+ and Mtr+/- mice weaned onto either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a folate-deficient (FD) diet (lacking folic acid) for 7 weeks. Mtr heterozygosity led to increased liver 5-methyl-THF levels. Mtr+/- mice consuming the C diet also exhibited a 40-fold increase in uracil in liver mtDNA. Mtr+/- mice consuming the FD diet exhibited less uracil accumulation in liver mtDNA as compared to Mtr+/+ mice consuming the FD diet. Furthermore, Mtr+/- mice exhibited 25% lower liver mtDNA content and a 20% lower maximal oxygen consumption rates. Impairments in mitochondrial FOCM are known to lead to increased uracil in mtDNA. This study demonstrates that impaired cytosolic dTMP synthesis, induced by decreased Mtr expression, also leads to increased uracil in mtDNA.

Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/− male to generate Shmt2+/+ (wild-type) or Shmt2+/− (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.ResultsHomozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/− MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.ConclusionsThe results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.

Overview and New Insights into the Metabolic Syndrome: Risk Factors and Emerging Variables in the Development of Type 2 Diabetes and Cerebrocardiovascular Disease.

Metabolic syndrome (MetS) is considered a metabolic disorder that has been steadily increasing globally and seems to parallel the increasing prevalence of obesity. It consists of a cluster of risk factors which traditionally includes obesity and hyperlipidemia, hyperinsulinemia, hypertension, and hyperglycemia. These four core risk factors are associated with insulin resistance (IR) and, importantly, the MetS is known to increase the risk for developing cerebrocardiovascular disease and type 2 diabetes mellitus. The MetS had its early origins in IR and syndrome X. It has undergone numerous name changes, with additional risk factors and variables being added over the years; however, it has remained as the MetS worldwide for the past three decades. This overview continues to add novel insights to the MetS and suggests that leptin resistance with hyperleptinemia, aberrant mitochondrial stress and reactive oxygen species (ROS), impaired folate-mediated one-carbon metabolism with hyperhomocysteinemia, vascular stiffening, microalbuminuria, and visceral adipose tissues extracellular vesicle exosomes be added to the list of associated variables. Notably, the role of a dysfunctional and activated endothelium and deficient nitric oxide bioavailability along with a dysfunctional and attenuated endothelial glycocalyx, vascular inflammation, systemic metainflammation, and the important role of ROS and reactive species interactome are discussed. With new insights and knowledge regarding the MetS comes the possibility of new findings through further research.

Cohort profile: Biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer recurrence and survival – the FOCUS Consortium

PurposeThe overarching goal of the FOCUS (biomarkers related to folate-dependent one-carbon metabolism in colorectal cancer (CRC) recurrence and survival) Consortium is to unravel the effect of folate and folate-mediated one-carbon...

Profiling the Influence of Gene Variants Related to Folate-Mediated One-Carbon Metabolism on the Outcome of In Vitro Fertilization (IVF) with Donor Oocytes in Recipients Receiving Folic Acid Fortification.

Nutritional status and gene polymorphisms of one-carbon metabolism confer a well-known interaction that in pregnant women may affect embryo viability and the health of the newborn. Folate metabolism directly impacts nucleotide synthesis and methylation, which is of increasing interest in the reproductive medicine field. Studies assessing the genetic influence of folate metabolism on IVF treatments have currently been performed in women using their own oocytes. Most of these patients seeking to have a child or undergoing IVF treatments are advised to preventively intake folate supplies that restore known metabolic imbalances, but the treatments could lead to the promotion of specific enzymes in specific women, depending on their genetic variance. In the present study, we assess the influence of candidate gene variants related to folate metabolism, such as Serine Hydroxymethyltransferase 1 SHMT1 (rs1979276 and rs1979277), Betaine-Homocysteine S-Methyltransferase BHMT (rs3733890), Methionine synthase reductase MTRR (rs1801394), Methylenetetrahydrofolate reductase MTHFR (rs1801131 and rs1801133), methionine synthase MTR (rs12749581), ATP Binding Cassette Subfamily B Member 1 ABCB1 (rs1045642) and folate receptor alpha FOLR1 (rs2071010) on the success of IVF treatment performed in women being recipients of donated oocytes. The implication of such gene variants seems to have no direct impact on pregnancy consecution after IVF; however, several gene variants could influence pregnancy loss events or pregnancy maintenance, as consequence of folic acid fortification.

Comparative profiling of single-cell transcriptome reveals heterogeneity of tumor microenvironment between solid and acinar lung adenocarcinoma

BackgroundThe diversity of histologic composition reflects the inter- and intra-tumor heterogeneity of lung adenocarcinomas (LUADs) macroscopically. Insights into the oncological characteristics and tumor microenvironment (TME) of different histologic subtypes of LUAD at the single-cell level can help identify potential therapeutic vulnerabilities and combinational approaches to improve the survival of LUAD patients.MethodsThrough comparative profiling of cell communities defined by scRNA-seq data, we characterized the TME of LUAD samples of distinct histologic subtypes, with relevant results further confirmed in multiple bulk transcriptomic, proteomic datasets and an independent immunohistochemical validation cohort.ResultsWe find that the hypoxic and acidic situation is the worst in the TME of solid LUADs compared to other histologic subtypes. Besides, the tumor metabolic preferences vary across histologic subtypes and may correspondingly impinge on the metabolism and function of immune cells. Remarkably, tumor cells from solid LUADs upregulate energy and substance metabolic activities, particularly the folate-mediated one-carbon metabolism and the key gene MTHFD2, which could serve as a potential therapeutic target. Additionally, ubiquitination modifications may also be involved in the progression of histologic patterns. Immunologically, solid LUADs are characterized by a predominance of exhausted T cells and immunosuppressive myeloid cells, where the hypoxic, acidified and nutrient-deprived TME has a non-negligible impact. Discrepancies in stromal cell function, evidenced by varying degrees of stromal remodeling and fibrosis, may also contribute to the specific immune phenotype of solid LUADs.ConclusionsOverall, our research proposes several potential entry points to improve the immunosuppressive TME of solid LUADs, thereby synergistically potentiating their immunotherapeutic efficacy, and may provide precise therapeutic strategies for LUAD patients of distinct histologic subtype constitution.

Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid β-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.

Presence of Blastocystis in gut microbiota is associated with cognitive traits and decreased executive function

Growing evidence implicates the gut microbiome in cognition. Blastocystis is a common gut single-cell eukaryote parasite frequently detected in humans but its potential involvement in human pathophysiology has been poorly characterized. Here we describe how the presence of Blastocystis in the gut microbiome was associated with deficits in executive function and altered gut bacterial composition in a discovery (n = 114) and replication cohorts (n = 942). We also found that Blastocystis was linked to bacterial functions related to aromatic amino acids metabolism and folate-mediated pyrimidine and one-carbon metabolism. Blastocystis-associated shifts in bacterial functionality translated into the circulating metabolome. Finally, we evaluated the effects of microbiota transplantation. Donor’s Blastocystis subtypes led to altered recipient’s mice cognitive function and prefrontal cortex gene expression. In summary, Blastocystis warrant further consideration as a novel actor in the gut microbiome-brain axis.

Impaired Mitochondrial Folate Metabolism Reduces Mitochondrial Respiration in Mouse Muscle Progenitor Cells

ObjectivesMitochondrial dysfunction is a central contributor to aging. Mitochondrial DNA mutations increase with aging and are associated with low serum folate. Furthermore, both increased age and low serum folate are suggested to increase skeletal muscle fiber loss and impair muscle function. However, no research has focused on the metabolic pathway linking the causal relationship. Data from our lab indicate that folate-mediated one-carbon metabolism may play a role, as loss of the mitochondrial specific isozyme (serine hydroxymethyltransferase 2, SHMT2) and folate depletion both result in mitochondrial DNA instability and impaired mitochondrial function. Furthermore, serum folate and SHMT2 gene expression levels decrease with aging, potentially providing a causal mechanism for age-associated mitochondrial DNA mutations and skeletal muscle impairments. To date, little is known about the relationship between loss of SHMT2 enzyme expression and folate status in skeletal muscles.MethodsTibialis anterior muscles were harvested from 10-week-old wild-type (Shmt2+/+) and heterozygous (Shmt2+/−) mice that were weaned onto either a folate-sufficient diet or a modified diet lacking folic acid. Skeletal muscles used to isolate primary muscle progenitor cells (MPCs) were isolated from 20-week-old Shmt2+/+ and Shmt2+/− mouse hindlimbs. MPCs were cultured to 80–90% confluency and then differentiated in low-serum medium for 7–10 days. Skeletal muscles were examined for mitochondrial DNA content and MPCs were examined for mitochondrial function.ResultsHeterozygous loss of Shmt2 resulted in an 80% reduction in SHMT2 protein levels in mouse tibialis anterior muscle. Mice fed a folate-depleted diet for 7 weeks exhibited a 25% reduction in mitochondrial DNA content compared to the folate-sufficient group. In MPCs, reduced Shmt2 expression impaired basal and maximal respiration. Furthermore, protein levels of mitochondrial NADH dehydrogenase 3 were reduced and protein levels of PGC1α and mitofusin 2 were elevated with Shmt2 heterozygosity in MPCs.ConclusionsOur results demonstrate that folate depletion reduced mitochondrial DNA content in tibialis anterior skeletal muscle and loss of SHMT2 impairs MPCs mitochondrial respiration and may influence mitochondrial biogenesis and dynamics.Funding SourcesPresident's Council of Cornell Women Award.

Impaired Folate-Mediated One-Carbon Metabolism in Type 2 Diabetes, Late-Onset Alzheimer's Disease and Long COVID.

Impaired folate-mediated one-carbon metabolism (FOCM) is associated with many pathologies and developmental abnormalities. FOCM is a metabolic network of interdependent biosynthetic pathways that is known to be compartmentalized in the cytoplasm, mitochondria and nucleus. Currently, the biochemical mechanisms and causal metabolic pathways responsible for the initiation and/or progression of folate-associated pathologies have yet to be fully established. This review specifically examines the role of impaired FOCM in type 2 diabetes mellitus, Alzheimer’s disease and the emerging Long COVID/post-acute sequelae of SARS-CoV-2 (PASC). Importantly, elevated homocysteine may be considered a biomarker for impaired FOCM, which is known to result in increased oxidative–redox stress. Therefore, the incorporation of hyperhomocysteinemia will be discussed in relation to impaired FOCM in each of the previously listed clinical diseases. This review is intended to fill gaps in knowledge associated with these clinical diseases and impaired FOCM. Additionally, some of the therapeutics will be discussed at this early time point in studying impaired FOCM in each of the above clinical disease states. It is hoped that this review will allow the reader to better understand the role of FOCM in the development and treatment of clinical disease states that may be associated with impaired FOCM and how to restore a more normal functional role for FOCM through improved nutrition and/or restoring the essential water-soluble B vitamins through oral supplementation

Evidence of the folate-mediated one-carbon metabolism pathway genes in controlling the non-syndromic oral clefts risks.

The folate-mediated one-carbon metabolism pathway is thought to play an important role in the etiology of non-syndromic oral clefts (NSOFC), although none of the genes in this pathway has shown significant signals in genome-wide association studies (GWAS). Recent evidence indicated that enhanced understanding could be gained by aggregating multiple SNPs effect simultaneously into polygenic risk score (PRS) to assess its association with disease risks. This study is aimed to assess the association between the genetic effect of folate-mediated one-carbon metabolism pathway and NSOFC risks using PRS based on a case-parent trio design. A total of 297 SNPs mapped from 18 genes in the folate-mediated one-carbon metabolism pathway were aggregated from a GWAS of 2458 case-parent trios recruited from an international consortium. We found a PRS based on the folate-mediated one-carbon metabolism pathway was significant among all NSOFC trios (OR=1.95, 95% CI: 1.66-2.28, p=2.39×10-16 ), as well as two major subtypes, non-syndromic cleft lip with or without cleft palate (NSCL/P) trios (OR=1.71, 95% CI: 1.50-1.96, p=7.66×10-15 ) and non-syndromic cleft palate only (NSCPO) trios (OR=1.51, 95% CI: 1.36-1.68, p=2.1×10-14 ). Similar results were also observed in further subgroup analyses stratified into Asian and European trios. The averaged PRS of the folate-mediated one-carbon metabolism pathway varied between the NSOFC case group and its comparison group (p<0.05) with higher average PRS in the cases. Moreover, the top 5% pathway PRS group had 2.25 (95% CI: 1.85-2.73) times increased NSOFC risk, also 3.09 (95% CI: 2.50-3.81) and 2.06 (95% CI: 1.39-3.02) times increased risk of NSCL/P and NSCPO compared to the remainder of the distribution. The results of our study confirmed the folate-mediated one-carbon metabolism pathway was important in controlling risk to NSOFC and this study enhanced evidence towards understanding the genetic risks of NSOFC.

Inorganic Arsenic Exposure and Gut Microbiota in a Birth Cohort from Central Taiwan

BACKGROUND AND AIM: Increasing evidence supports the association between composition and abundance of gut bacteria and human diseases (such as obesity and type 2 diabetes). Certain environmental toxicants, such as inorganic arsenic (iAs), may alter the equilibrium and diversity of human gut microbiota. Reversely, gut microbiota might affect the toxicokinetics of toxicants such as absorption and distribution. As folate-mediated one-carbon metabolism is crucial in fetus and children’s health and development, this study aimed to investigate the associations between iAs exposure, gut microbiota, and folate-related functional pathways in children. METHODS: Eighty children previously recruited in central Taiwan (2012–2014) were followed in 2015–2017. The structured questionnaire was interviewed with urinary sample collected at baseline and follow-up for pregnant women and children, and feces specimens were collected in children aged 3‒4 years. Urinary concentration of iAs species was quantified, and DNA was extracted from feces specimen. The composition and abundance of gut microbiota, and microbial functional profiles were sequenced by whole metagenome shotgun sequencing. We utilize the sum of urinary concentrations of arsenate, arsenate, monomethyl- and dimethyl arsenic acids (ΣiAs) as the index of total iAs exposure. RESULTS:High level of total iAs exposure (upper tertile, ΣiAs ≥ 21.06 μg/L) had a significantly lower abundance of Parabacteroides distasonis but the higher abundance of Roseburia intestinalis and Eubacterium rectale as compared to mid and lower tertile. Total iAs exposure was significantly associated with a decreased abundance of Parabacteroides distasonis (β = -0.021) after adjustment for sex and environmental tobacco smoke. Importantly we also found a significantly decreased function of N10-formyl-tetrahydrofolate biosynthesis (β = -0.040) and folate transformations II (β = -0.040) in relation to total iAs exposure. CONCLUSIONS:Exposure to iAs may alter the abundance of gut microbiota associated with human health, and it also had a negative association with folate-related functions. KEYWORDS: Heavy metals, Microbiome, Epidemiology

Reduced Methionine Synthase (Mtr) Expression, a Model of Vitamin B12 Deficiency, Leads to Uracil Accumulation in Mitochondrial and Nuclear DNA

ObjectivesIntegrity of both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) is affected by nutritional and environmental factors. Folate and B12 are water-soluble B-vitamins that act as cofactors in folate-mediated one-carbon metabolism (FOCM), a series of one-carbon transfer reactions that support several essential cell processes including nucleotide biosynthesis. Impairments in FOCM, such as folate or vitamin B12 deficiency, can disturb thymidylate (dTMP, the “T” base in DNA) synthesis and lead to uracil misincorporation to DNA. Both folate and B12 deficiency as well as genetic polymorphisms affecting FOCM function are associated with increased uracil accumulation and DNA damage in nDNA. However, dTMP synthesis occurs in multiple cellular compartments (cytosol, nucleus, and mitochondria), and it is not well defined how FOCM impairments affects mtDNA integrity and nucleotide pool balance between compartments. MethodsCurrently, we are using in vivo and in vitro experiments to assess nucleotide synthesis and uracil accumulation in a functional model of vitamin B-12 deficiency with decreased expression of methionine synthase (Mtr), one of two B12-requiring enzymes. Tissues from Mtr+/+,+/– mice and Mtr+/+,+/- mouse-derived cells were exposed to either folate-replete or folate-deplete conditions to explore the combined effects of folate and B12 deficiency on nucleotide synthesis capacity and integrity of both nDNA and mtDNA. ResultsWe have developed and validated a novel real-time PCR-based assay to quantify uracil misincorporation into mtDNA. In mouse liver, we observed a significant increase of uracil in mtDNA with decreased Mtr expression as well as an interaction between Mtr genotype and folate status. Mouse embryonic fibroblasts (MEFs) demonstrated perturbed dTMP synthesis and increased uracil accumulation in nDNA with decreased Mtr expression. ConclusionsThese data suggest that functional B12 deficiency, as modeled by decreased Mtr expression, disrupts nucleotide synthesis in more than one cellular compartment and increases uracil accumulation not only in nDNA, but also in mtDNA. Reduced Mtr expression may cause a redistribution of folates in the cytoplasm which impacts mitochondrial folate levels, and subsequently mitochondrial dTMP synthesis. Funding SourcesN/A.