Fig 1.
In the methionine cycle, homocysteine is remethylated to methionine through the methionine synthase enzyme (MTR) with vitamin B12 as a cofactor, or necessary catalyst for MTR activity. Methionine is then converted to SAM, which donates a methyl group and is converted to S-adenosylhomocysteine (SAH), which is then converted back to homocysteine. Histone and DNA methyltransferases transfer the methyl group from SAM to amino acids and bases on histone amino-terminal tails and to the 5 position of cytosine residues in DNA [11]. When MTR enzymatic activity is inhibited by reactive oxygen and nitrogen species, BHMT uses betaine to remethylate homocysteine to methionine. Betaine can be taken in through the diet or synthesized through the oxidation of choline in mitochondria. Abbreviations: Hcy, homocysteine; Met, methionine; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; BHMT, betaine homocysteine methyltransferase; ROS, reactive oxygen species; RNS, reactive nitrogen species; HMTs, histone methyltransferases; DNMTs, DNA methyltransferases.
Fig 2.
BHMT is present in oligodendrocytes.
(A) Representative confocal images acquired at 60x showing immunofluorescent staining for BHMT (green) and OLIG2 (red) in immature MO3.13 oligodendrocytes. Nuclei are stained with DAPI (blue). Area outlined in white was enlarged and split channels are shown (right). BHMT immunoreactivity is co-localized in oligodendrocytes also stained with OLIG2. (B) Representative confocal images acquired at 60x showing immunofluorescent staining for BHMT (green) and OLIG2 (red) in mature MO3.13 oligodendrocytes. Nuclei are stained with DAPI (blue). Area outlined in white was enlarged and is shown (right). (C) Representative confocal images acquired at 40X showing immunofluorescent staining for BHMT (green) and MBP (red) in mature primary rat oligodendrocytes. Nuclei are stained with DAPI (blue). Area outlined in white was enlarged and shown to the right.
Fig 3.
BHMT is present in a human MS lesion.
(A) Postmortem MS tissue stained for PLP. A white matter lesion is evident. Area shown in (B) is from boxed white matter lesion area on adjacent section. Brightfield images taken at 40x show colocalization of OLIG2 (blue alkaline phosphatase staining) and BHMT immunoreactivity (brown DAB precipitate) denoted by arrows on cortical tissue sections. Area outlined in black was enlarged.
Fig 4.
The BHMT-betaine pathway is involved in regulating differentiation in oligodendrocytes.
(A) Representative Western blot performed with control and SNP treated MO3.13 oligodendrocytes. Lysates were probed for BHMT and the cytoplasmic marker GAPDH. Error bars are expressed as SEM. **, p = 0.007. (B) Changes in gene expression were confirmed by qRT-PCR for select transcripts that play a role in regulation of OPC differentiation. qRT-PCR was performed with RNA isolated from control MO3.13 oligodendrocytes and oligodendrocytes treated with 400 μM SNP, 1 mM betaine, or SNP/betaine. Relative expression levels with controls set at 1 are shown. Error bars indicate SEM, n = 9. P values shown were determined by a two-tailed Students t-test. Additionally, a Mann-Whitney U nonparametric test shows that SOX10 expression treated with SNP and betaine is increased, p = 0.016.
Fig 5.
The BHMT–betaine pathway regulates HMT and DNMT activity.
(A) Representative Western blot images for BHMT, DNMT3a, and H3K4me3 from nuclear chromatin fractions of MO3.13 oligodendrocytes treated with 400 μM SNP. Lanes are denoted “C” for control and “S” for SNP treated. n = 3. (B) Co-IP analysis of protein-protein interactions between BHMT and DNMT3a in MO3.13 oligodendrocytes. Co-IPs were performed using an antibody to BHMT. Flow-through (FT) and IP samples were run on a Western blot to identify DNMT3a interactions with BHMT. Normal rabbit serum (IgG) was used as a negative control. n = 2. (C) Quantitation of HMT activity on H3K4 in control MO3.13 oligodendrocytes and oligodendrocytes transfected with siRNA to BHMT, with and without 1 mM betaine. Error bars indicate SEM, n = 6. (D) Quantitation of total DNMT activity in control MO3.13 oligodendrocytes and oligodendrocytes transfected with siRNA to BHMT, with and without 1 mM betaine. Error bars indicate SEM, n = 6.