Vitamin C regulates Schwann cell myelination by promoting DNA demethylation of pro-myelinating genes

Abstract

Ascorbic acid (vitamin C) is critical for Schwann cells to myelinate peripheral nerve axons during development and remyelination after injury. However, its exact mechanism remains elusive. Vitamin C is a dietary nutrient that was recently discovered to promote active DNA demethylation. Schwann cell myelination is characterized by global DNA demethylation in vivo and may therefore be regulated by vitamin C. We found that vitamin C induces a massive transcriptomic shift (n = 3,848 genes) in primary cultured Schwann cells while simultaneously producing a global increase in genomic 5-hydroxymethylcytosine (5hmC), a DNA demethylation intermediate which regulates transcription. Vitamin C up-regulates 10 pro-myelinating genes which exhibit elevated 5hmC content in both the promoter and gene body regions of these loci following treatment. Using a mouse model of human vitamin C metabolism, we found that maternal dietary vitamin C deficiency causes peripheral nerve hypomyelination throughout early development in resulting offspring. Additionally, dietary vitamin C intake regulates the expression of myelin-related proteins such as periaxin (PRX) and myelin basic protein (MBP) during development and remyelination after 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.

Keywords: 5-hydroxymethylcytosine; DNA demethylation; Schwann cell; myelin; vitamin C.

Conflict of interest statement

CONFLIC T OF INTEREST

The authors declare no competing interests.

© 2020 International Society for Neurochemistry.

Figures

FIGURE 1

Experimental timeline and design for mouse experiments. In Experiment 1, adult Gulo−/− mice breeding pairs were provided either sufficient (330 mg/L) or deficient (99 mg/L) vitamin C in the drinking water. Sciatic nerves of the resulting pups from the breeding pairs were collected at postnatal day 4 (P4), P9, and P17. For Experiment 2, adult Gulo−/− mice were provided either sufficient (330 mg/L) or deficient (16.5 mg/L) vitamin C for 2.5 months. Mice were then subject to sciatic nerve crush surgery on the lateral nerve while the contralateral nerve was subject to sham surgery. Sham and crush nerves were then collected 14 days post-injury and subjected to histological analysis. N = 3 animals were used in each condition and time point assessed in both experiments

FIGURE 2

Vitamin C deficiency induces persistent hypomyelination in Gulo−/− mice throughout development. (a–c) Semithin sections of sciatic nerves from Gulo−/− mouse pups reared on a vitamin C sufficient (330 mg/L) or deficient (99 mg/L) diet and collected at postnatal day 4 (P4), P9, and P17. G-ratio averages (axon diameter/fiber diameter) are represented by bar graphs with plotted averages for each animal (grey dots, N = 3 animals per time point). Pooled g-ratio calculations from each condition (N > 500) are represented by scatterplot including line of best fit. *p < .05. All data are means ± SEM. Scale bar = 10 μm

FIGURE 3

Vitamin C augments global levels of 5hmC. (a) Immunofluorescence of 5hmC in primary cultured Schwann cells after vitamin C treatment. (b) Quantification of immunofluorescence shows that 10 μM vitamin C treatment modestly increases 5hmC, while 50 μM treatment greatly elevates 5hmC 70% above control levels (N > 250 data points per condition). (c) Heatmap of global 5hmC peaks following vitamin C treatment (7 days, 50 μM) as determined by ChIP-seq. Units are in read counts per million mapped reads. (d) Read counts per million mapped reads across promoter and gene body regions following vitamin C treatment. (e) Heatmap of the relative abundance of reads across promoter and gene body regions following treatment. **p < .01. N = 3 independent cell culture preparations for ChIP-seq experiment. All data are means ± SEM. Scale bar = 20 μm. TSS = transcription start site; TES = transcription end site

FIGURE 4

Vitamin C alters the Schwann cell transcriptome and up-regulates pro-myelinating gene expression and 5hmC content. (a) Heatmap of the relative abundance of reads for all transcripts after vitamin C treatment (7 days). The Venn Diagram represents of the number of differential transcripts called by statistical algorithms edgeR (red) and DESeq2 (yellow). A total of 3,848 transcripts were considered differential by both algorithms. (b) Gene Ontology (GO) pathway analysis of RNA-seq data showing top up-regulated and down-regulated pathways in Schwann cells following vitamin C treatment. Annotations are ranked by combined p value and z scores (Combined Enrichment Score). (c) Relative expression of pro-myelinating genes following vitamin C treatment. Mag expression was below detectable levels. (d) Relative abundance of 5hmC in the promoter and gene body regions of pro-myelinating genes following vitamin C treatment. Data from bar graphs (c and d) represent average fragments per kilobase per million mapped reads (FPKM) expressed as fold change from control. *p < .05, **p < .01. N = 3 independent cell culture preparations for RNA-seq experiment. All data are means ± SEM

FIGURE 5

Vitamin C up-regulates the transcription and 5hmC content of ECM genes. (a) Relative expression of laminin and collagen genes following vitamin C treatment. (b) Relative abundance of 5hmC in the promoter regions of laminin and collagen genes following vitamin C treatment. Lama2 promoter 5hmC was below detectable levels. (c) Relative abundance of 5hmC in the gene body regions of laminin and collagen genes following vitamin C treatment. (d) Phase contrast images of Schwann cells in culture treated with vitamin C (50 μM, 5 days treatment). Lower panels are high-magnification (20X) images showing morphological changes following treatment (Scale bar = 100 μm for top panels. Bottom panels = 50 μm). (e) Live imaging and high-throughput quantification of Schwann cell process length following vitamin C treatment. Images show Schwann cells 48 hr following treatment (Scale bar = 200 μm). Data from bar graphs represent average fragments per kilobase per million mapped reads (FPKM) expressed as fold change from control. *p < .05. N = 3 independent cell culture preparations used for RNA/ChIP seq and Incucyte experiments. All data are means ± SEM

FIGURE 6

Vitamin C regulates the expression of PRX and MBP throughout developmental myelination. (a) Immunofluorescence of PRX in sciatic nerves collected from Gulo−/− mouse pups reared on a vitamin C sufficient (330 mg/L) or deficient (99mg/L) diet and collected at postnatal day 4 (P4), P9, and P17. Scale bar = 10 μm. (b) Immunofluorescence of MBP in sciatic nerves collected from Gulo−/− mouse pups reared on a vitamin C sufficient (330 mg/L) or deficient (99mg/L) diet and collected at postnatal day 4 (P4), P9, and P17. Scale bar = 20 μm. (c) Quantification of PRX expression in the sciatic nerve throughout developmental myelination at three time points assessed. (d) Quantification of MBP expression in the sciatic nerve throughout developmental myelination at three time points assessed. N = 3 animals per time point and condition. *p < .05; **p < .01. All data are means ± SEM

FIGURE 7

Vitamin C regulates the expression of PRX but not MBP during remyelination following injury. (a) Immunofluorescence of PRX in crushed sciatic nerves collected from Gulo−/− mice provided a vitamin C sufficient (330 mg/L) or deficient (16.5 mg/L) diet for 2.5 months and collected at 14 days post-injury (dpi). (b) Immunofluorescence of MBP in crushed sciatic nerves collected from Gulo−/− mice provided a vitamin C sufficient (330 mg/L) or deficient (16.5 mg/L) diet for 2.5 months and collected at 14 days post-injury (dpi). (c) Quantification of PRX expression following sciatic nerve crush in three regions assessed. (d) Quantification of MBP expression following sciatic nerve crush in three regions assessed. N = 3 animals per time point and condition. *p < .05. All data are means ± SEM. Scale bar = 10 μm

FIGURE 8

Working model of the role of vitamin C in Schwann cell myelination. Vitamin C is classically known to promote myelination by serving as a cofactor for P4 hydroxylase which stabilizes collagen during basal lamina formation and is necessary for myelination. Therefore, vitamin C is thought to indirectly promote myelination. Here, we present a novel epigenetic pathway whereby vitamin C promotes myelination via TET-mediated demethylation. Vitamin C induces demethylation in promoter and gene body regulatory regions of genes involved in Schwann cell myelination (Krox20, PRX, and MBP) and ECM organization (Laminin-2 subunits, Collagens I and IV). Demethylation correlates with increased transcription of these genes and promotes their relevant functions. Therefore, vitamin C may promote myelination 1) indirectly through P4 hydroxylase activity, and 2) directly via active demethylation and transcriptional regulation of genes relevant to myelination