[PubMed] [Google Scholar]Gorres KL, & Raines RT (2010). injury in mice. Taken together, these results suggest that vitamin C cooperatively promotes myelination through 1) increased DNA demethylation and transcription of pro-myelinating genes, and 2) its known role in stabilizing collagen helices to form the basal lamina that is necessary for myelination. gene which encodes the enzyme responsible for metabolizing S-Adenosylmethione, the methyl donor responsible for DNA methylation exhibits rampant global methylation and hypomyelination of peripheral nerves (Varela-Rey et al., 2014). This suggests a negative correlation between methylation and myelination whereby hypermethylation results in hypomyelination. Conversely, molecular agents which promote DNA demethylation, like vitamin C, may subsequently thwart methylation levels to promote myelination. In this study, we investigated the epigenetic role of vitamin C in Schwann cell myelination. Vitamin C treatment of primary cultured Schwann cells induced massive changes in transcription and a global increase in genomic 5hmC. Vitamin C up-regulated the transcription of 10 myelin-related genes which exhibited concurrent changes in both promoter and gene body 5hmC content. Using a mouse model relevant to L-Valyl-L-phenylalanine human vitamin C metabolism, we found that dietary vitamin C deficiency impaired peripheral myelination throughout early development. Finally, we found that dietary vitamin C intake was necessary for the expression of periaxin (PRX) and myelin basic protein (MBP), critical components of the myelin sheath, throughout development, and during remyelination after injury. Overall, these results suggest that vitamin C promotes Schwann cell myelination not only through collagen stability but also through direct epigenetic regulation of myelin-related genes. 2 |.?METHODS This study was not pre-registered with OSF Registries. 2.1 |. Materials The following primary antibodies were used in this study: PRX (Sigma cat#HPA001868, RRID:AB_2172440, 1:1,000), MBP (Novus Biologicals cat#MAB42282, 1:500), 5hmC (Active L-Valyl-L-phenylalanine Motif cat#39769, RRID:AB_10013602, 1:1,000). Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, RRID:AB_2768317, 1:1,000), and Alexa Fluor 488-conjugated goat anti-mouse IgG (Invtirogen, RRID:AB_2768316, 1:1,000) secondary antibodies were used for all immunofluorescence-based experiments. 2.2 |. Animals C57BL/6 background mice containing a functionally inactive Gulonolactone oxidase (= 3 animals were L-Valyl-L-phenylalanine used in each condition and time point assessed in both experiments 2.3 |. Cell culture Primary cultured Schwann cells were isolated from 3-month-old Fisher rat (RRID:RGD_734478) sciatic nerves as previously described (Bacallao & Monje, 2015). Rats were of both sexes, of normal weight (200g), had access to food and water at all times, and were house in a cage containing five rats. Rats were sacrificed by CO2 asphyxiation followed by cervical dislocation GNG7 and sciatic nerve collection. Nerves were cut into small segments, incubated in vitro for 10 days in DMEM medium containing 10% heat-inactivated FBS, and allowed to degenerate. Degenerated nerve explants were dissociated using a 0.25% dispase/0.05% collagenase solution and the resulting cell suspension was plated on poly-l-lysine (PLL)-coated dishes (Sigma). Purified primary Schwann cells were expanded up to passage one in DMEM media containing 10 nM neuregulin (recombinant heregulin-1) and 2 L-Valyl-L-phenylalanine M forskolin. Experiments were performed on Schwann cells between 3 and 6 passages and plated on glass coated in PLL and laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane. Schwann cells were isolated from at least five different rats and used for all downstream experiments. Schwann cells were grown for experiments in DMEM supplemented with 10% FBS and without neuregulin or forskolin. In vitro experiments using vitamin C were performed using 50 M sodium ascorbate unless otherwise stated in the text. 2.4 |. Semithin sectioning Sciatic nerves were dissected from Vitamin C sufficient and deficient Gulo?/? mice and resin embedded for semithin sectioning as described previously (Rebelo et al., 2018). Nerves were fixed in 3% glutaraldehyde 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4C and subsequently placed in 30% sucrose until processed. Nerves were post-fixed with 2% osmium tetroxide and dehydrated using a graded ethanol series (50%, 75%, 95%, and 100%). After dehydration, nerves were embedded in.