UC Berkeley

Vitamin B12 and folate are substrates and cofactors for various enzymes involved in one carbon metabolism, which is important for DNA/RNA synthesis, amino acid interconversions and the methylation cycle. Two enzymes, methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) play pivotal roles determining the direction of one carbon flow. MTHFR catalyzes the intracellular synthesis of 5-methyl-THF from 5, 10-methylene-THF. MS is one of two B12-dependent mammalian enzymes and catalyzes the remethylation of homocysteine to methionine and the concurrent demethylation of 5-methyl-THF to THF.

MTHFR knockout mice show a variable phenotype with reduced survival. The MS knockout is early embryonic lethal. MTHFR/MS double knockout mice survive and have similar phenotypes as MTHFR null mice, which proved that the MS null mice die from extreme folate deficiency due to a methyl-folate trap.

The common phenotypes shared by the MTHFR null and MS/MTHFR double null mice include retarded growth, hyperhomocysteinemia and a significantly low SAM/SAH ratio. Histological studies revealed cerebellum neuropathology and impaired male reproductive system in knockout mice. Microarray analysis showed that the expression of many genes was altered in MTHFR null and MS/MTHFR double null mice. Several subsets of induced or repressed genes encoded proteins involved in iron metabolism, lipid metabolism, SUMOylation, growth hormone/IGF pathway, stress responses, etc.

We also studied some of the consequences of vitamin B12 deficiency in wild type, MTHFR null and MS heterozygous mice. B12 deficiency resulted in increased plasma homocysteine, elevated plasma total cholesterol and triglyceride, and significantly up-regulated hepatic ApoC1 and ApoC3 expression in wild type and MS heterozygous mice. MTHFR null mice maintained lower plasma cholesterol and triglycerides, repressed hepatic ApoC1/C3 expression on both B12-deficient and sufficient diets, despite their very high plasma homocysteine, which indicated that the elevated homocysteine was not responsible for the changes in blood lipid profiles found in B12-deficient wild type or MS heterozygous mice.

Both folate deficiency and vitamin B12 deficiency can result in megaloblastic anemia, but the latter also causes subacute combined disease (SCD), a chronic demyelination disease, but its underlying mechanism is not understood. Although SCD occurs in B12 deficient humans and other primates, it has never been observed in rodents placed on B12-deficient diets. In animals with defective MS or MTHFR, the effects of B12 deficiency on myelin turnover were quantitatively studied by measuring the kinetics of synthesis and turnover of galactocerebroside (GalC), a myelin lipid, using a GC/MS stable isotope method. Vitamin B12 deficiency significantly decreased initial myelination rates in MS heterozygote and young wild type mice, and significantly inhibited remyelination after cuprizone-induced demyelination in adult MS heterozygotes and wild type mice. The MTHFR null mouse had a lower apparent remyelination rate compared with MS heterozygote and wild type mice, but remyelination in the MTHFR null was not affected by vitamin B12 deprivation.

We also investigated the effect of replacing folic acid with 5-methyl-THF in mice null for both MTHFR and MS. In this artificial methyl folate trap situation, MS/MTHFR double null mice developed megaloblastic anemia, and some of them showed a mild myelin defect with increased expression of TNF-alpha and NF-kappaB. The existence of megaloblastic macrocytic anemia and an early-phase neuropathy in MS/MTHFR double null mice fed on 5-methyl-THF implies that the accumulation of methyl-folate that occurs in the methyl folate trap plays a direct mechanistic role and is at least partly responsible for the development of both symptoms of vitamin B12 deficiency.

The final part of my study focused on differences in folate-related biochemical metabolites in inbred mouse strains and the identification of genes variants or modifier genes associated with these differences. Mouse brain and liver SAM and SAH, plasma homocysteine and cysteine, liver and plasma total folate were measured. Some showed significant differences among inbred mouse strains. Genomic loci associated with metabolite differences were identified by quantitative trait loci analysis and are now available for further scanning for modifier gene polymorphisms.