Derangement of hepatic polyamine, folate, and methionine cycle metabolism in cystathionine beta-synthase-deficient homocystinuria in the presence and absence of treatment: Possible implications for pathogenesis.

Cystathionine beta-synthase deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. Our knowledge of the metabolic changes induced in HCU are based almost exclusively on data derived from plasma. In the present study, we present a comprehensive analysis on the effects of HCU upon the hepatic metabolites and enzyme expression levels of the methionine-folate cycles in a mouse model of HCU. HCU induced a 10-fold increase in hepatic total homocysteine and in contrast to plasma, this metabolite was only lowered by approximately 20% by betaine treatment indicating that this toxic metabolite remains unacceptably elevated. Hepatic methionine, S-adenosylmethionine, S-adenosylhomocysteine, N-acetlymethionine, N-formylmethionine, methionine sulfoxide, S-methylcysteine, serine, N-acetylserine, taurocyamine and N-acetyltaurine levels were also significantly increased by HCU while cysteine, N-acetylcysteine and hypotaurine were all significantly decreased. In terms of polyamine metabolism, HCU significantly decreased spermine and spermidine levels while increasing 5'-methylthioadenosine. Betaine treatment restored normal spermine and spermidine levels but further increased 5'-methylthioadenosine. HCU induced a 2-fold induction in expression of both S-adenosylhomocysteine hydrolase and methylenetetrahydrofolate reductase. Induction of this latter enzyme was accompanied by a 10-fold accumulation of its product, 5-methyl-tetrahydrofolate, with the potential to significantly perturb one­carbon metabolism. Expression of the cytoplasmic isoform of serine hydroxymethyltransferase was unaffected by HCU but the mitochondrial isoform was repressed indicating differential regulation of one­carbon metabolism in different sub-cellular compartments. All HCU-induced changes in enzyme expression were completely reversed by either betaine or taurine treatment. Collectively, our data show significant alterations of polyamine, folate and methionine cycle metabolism in HCU hepatic tissues that in some cases, differ significantly from those observed in plasma, and have the potential to contribute to multiple aspects of pathogenesis.

Reduced Shmt2 Expression Impairs Mitochondrial Folate Accumulation and Respiration, and Leads to Uracil Accumulation in Mouse Mitochondrial DNA.

BACKGROUND: Adequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in nuclear dTMP synthesis leads to increased uracil misincorporation into DNA, affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in nontransformed tissues have been understudied. OBJECTIVES: This study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA (mtDNA) integrity and mitochondrial function in mouse tissues. METHODS: Liver mtDNA content, and uracil content in liver mtDNA, were measured in Shmt2+/- and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid; C) or a modified diet lacking folic acid (0 mg/kg folic acid) for 7 wk. Shmt2+/- and Shmt2+/+ mouse embryonic fibroblast (MEF) cells were cultured in defined culture medium containing either 0 or 25 nM folate (6S-5-formyl-tetrahydrofolate, folinate) to assess proliferative capacity and mitochondrial function. Chi-square tests, linear mixed models, and 2-factor ANOVA with Tukey post hoc analyses were used to analyze data. RESULTS: Shmt2 +/- mice exhibited a 48%-67% reduction in SHMT2 protein concentrations in tissues. Interestingly, Shmt2+/- mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/- mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/- mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. CONCLUSIONS: This study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis in mice, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.

Taurine alleviates repression of betaine-homocysteine S-methyltransferase and significantly improves the efficacy of long-term betaine treatment in a mouse model of cystathionine ß-synthase-deficient homocystinuria.

Classical cystathionine ß-synthase-deficient homocystinuria (HCU) is a life-threatening inborn error of sulfur metabolism. Treatment for pyridoxine-nonresponsive HCU involves lowering homocysteine (Hcy) with a methionine (Met)-restricted diet and betaine supplementation. Betaine treatment efficacy diminishes significantly over time due to impairment of betaine-Hcy S-methyltransferase (BHMT) function. Little is known regarding the regulation of BHMT in HCU. Using a betaine-responsive preclinical mouse model of HCU, we observed that this condition induces a 75% repression of BHMT mRNA, protein and enzyme activity, and significant depletion of hepatic betaine levels. BHMT repression was proportional to plasma Hcy levels but was not observed in mouse models of homocystinuria due to either methylenetetrahydrofolate reductase or Met synthase deficiency. Both Met supplementation and chemically induced glutathione depletion exacerbated hepatic BHMT repression in HCU mice but not wild-type (WT) controls. Conversely, cysteine treatment normalized hepatic BHMT expression in HCU mice but had no effect in WT control animals. Taurine treatment induced BHMT expression in HCU mice by 5-fold and restored maximal lowering of Hcy levels during long-term betaine treatment with a concomitant normalization of inflammatory cytokine expression and a significantly improved coagulative phenotype. Collectively, our findings indicate that adjuvantial taurine treatment has the potential to significantly improve clinical outcomes in HCU.-Maclean, K. N., Jiang, H, Phinney, W. N., Keating, A. K., Hurt, K. J., Stabler, S. P. Taurine alleviates repression of betaine-homocysteine S-methyltransferase and significantly improves the efficacy of long-term betaine treatment in a mouse model of cystathionine ß-synthase-deficient homocystinuria.

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.

Cross-Reactive Antibodies to SARS-CoV-2 and MERS-CoV in Pre-COVID-19 Blood Samples from Sierra Leoneans.

Many countries in sub-Saharan Africa have experienced lower COVID-19 caseloads and fewer deaths than countries in other regions worldwide. Under-reporting of cases and a younger population could partly account for these differences, but pre-existing immunity to coronaviruses is another potential factor. Blood samples from Sierra Leonean Lassa fever and Ebola survivors and their contacts collected before the first reported COVID-19 cases were assessed using enzyme-linked immunosorbent assays for the presence of antibodies binding to proteins of coronaviruses that infect humans. Results were compared to COVID-19 subjects and healthy blood donors from the United States. Prior to the pandemic, Sierra Leoneans had more frequent exposures than Americans to coronaviruses with epitopes that cross-react with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). The percentage of Sierra Leoneans with antibodies reacting to seasonal coronaviruses was also higher than for American blood donors. Serological responses to coronaviruses by Sierra Leoneans did not differ by age or sex. Approximately a quarter of Sierra Leonian pre-pandemic blood samples had neutralizing antibodies against SARS-CoV-2 pseudovirus, while about a third neutralized MERS-CoV pseudovirus. Prior exposures to coronaviruses that induce cross-protective immunity may contribute to reduced COVID-19 cases and deaths in Sierra Leone.