Vitamin C Myelin Sheath

. 2018 Jul;66(7):1302-1316.

doi: 10.1002/glia.23306. Epub 2018 Feb 9.

Vitamin C promotes oligodendrocytes generation and remyelination

Affiliations

PMID: 29423921
PMCID: PMC6001564
DOI: 10.1002/glia.23306

Free PMC article

Vitamin C promotes oligodendrocytes generation and remyelination

Yu-E Guo et al. Glia. 2018 Jul .

Free PMC article

Abstract

Oligodendrocyte-formed myelin sheaths play important roles in the neuronal functions in the central nervous system. In demyelinating diseases, such as Multiple Sclerosis, the myelin sheaths are damaged and the remyelinating process is somehow hindered. Restoration of the myelin sheaths requires the differentiation of the oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs). To discover small molecule compounds that might promote the OPC to OL differentiation, a high-throughput screening system is established and L-ascorbyl-2-phosphate (As-2P), a stable form of Vitamin C (Vc), is found to greatly enhance the OPC to OL differentiation. As-2P promotes gradual expression of OL lineage markers, including O4, CNPase and MBP, in a dose- and time-dependent manner. It also facilitates the formation of myelin sheaths in OPC-neuron co-culture. As-2P also promotes the repair of the myelin sheaths in vivo and provides significant therapeutic effect in a cuprizone-mediated demyelination animal model. Interestingly, As-2P's function in promoting OPC differentiation is not related to its antioxidant activity. And an intracellular rather than an extracellular mechanism might be involved. Considering the safe use of Vc as a dietary supplement for many years, it might also be used as an alternative medicine for CNS demyelinating diseases.

Keywords: differentiation; myelin; oligodendrocyte; oligodendrocyte progenitor cell; remyelination; vitamin C.

Figures

**Figure 1**

A NPC‐based phenotypic screening platform to discover inducers of OL differentiation and maturation. (a) Steps of OL differentiation from neurospheres generated from cortical NPCs of mouse E14.5 embryos: Neurosphere formation (NPC medium), OPC differentiation (OPC medium), and OL differentiation and maturation (OL medium). (b) Phase contrast (Top) and immunofluorescent staining of specific markers (Bottom) of NPCs (Nestin), OPCs (NG2), and OLs (MBP). Nuclei were stained with Hoechst. Scale bars, 40 μm. (c) The negative (DMSO) and positive (T3, 100 nM) controls of the high‐throughput screening system (Left). Scatter plot of the primary screen results of 7347 compounds (Right). The dotted line indicates two‐fold increase in the percentage of MBP⁺ cells comparing to DMSO. (d) Representative images of MBP⁺ cells in DMSO, T3, and As‐2P‐treated wells. Nuclei were stained with Hoechst. Scale bars, 100 μm [Color figure can be viewed at
http://wileyonlinelibrary.com
]

**Figure 2**

As‐2P promotes OL differentiation and maturation. (a) Dose‐dependent effect of As‐2P in inducing OPC differentiation into mature OLs. OPCs were treated with As‐2P for 4 days. (b) Time‐dependent effect of As‐2P (150 μM) in inducing OPC differentiation into mature OLs. (c) Mouse NPC‐derived OPCs were differentiated in the presence of As‐2P (150 μM), T3 (100 nM) or the combination of both for 4 days. OLs were stained with antibody against MBP (green). Nuclei were stained with Hoechst (blue). Scale bars, 100 μm; inset 25 μm. (d) Statistical analysis of the MBP⁺ cells in (c). Data are means ± SEM of three independent experiments. *p < .05, **p < .01 (one‐way ANOVA followed by Tukey's multiple comparison test). (e, f) Mouse NPC‐derived OPCs were differentiated in the presence of As‐2P (150 μM) or not for 4 days, cells were subjected to TUNEL (e) and Ki67 (f) staining. (g) Quantification of the TUNEL⁺ cells in (e). (h) Quantification of the Ki67⁺ cells in (f). Data are representative of three independent experiments, means ± SEM (n = 3). *p < .05 (Student's t test). (i, j) Typical morphology of large (>1,000 μm², white arrow and circle), medium (500–1,000 μm², red arrow and circle) and small (250–500 μm², yellow arrow and circle) size OLs. The cells were stained with antibody against MBP (green). Nuclei were stained with Hoechst (blue). Scale bars in (i), 100 μm. Scale bars in J, 20 μm. (k) Statistical analysis of the MBP⁺ cells in (i) [Color figure can be viewed at
http://wileyonlinelibrary.com
]

**Figure 3**

As‐2P promotes gradual expression of OL lineage markers. (a–c) NPC‐derived OPCs were differentiated in the presence of As‐2P (150 μM) or vehicle for 1–5 days. Cells were stained with antibodies against O4 (a), CNPase (b), and MBP (c). Typical morphologies of the cells were presented. Scale bars, 100 μm; inset 25 μm. (d, e) Statistical analysis of total (d) and large (>1000 μm², e) O4⁺ cells presented in (a). (f, g) Statistical analysis of CNPase⁺ (f) and MBP⁺ (g) cells presented in (b and c) [Color figure can be viewed at
http://wileyonlinelibrary.com
]

**Figure 4**

As‐2P enhances myelination in vitro. (a) Representative images of mouse primary NG2⁺‐OPCs treated with vehicle or As‐2P for 4 days and stained for MBP (green) and nuclei (Hoechst, blue). Scale bars, 100 μm. (b) Statistical analysis of the MBP⁺ cells after a 4‐day differentiation of the primary NG2⁺‐OPCs in the presence of various concentrations of As‐2P. Data are representative of three independent experiments, means ± SEM (n = 4). (c, d) Primary OPCs were co‐cultured with DRG‐neurons and treated with vehicle or As‐2P for 6 (c) or 14 (d) days. The cells were then immunostained for NF‐200 (neurofilament, green) and MBP (oligodendrocytes, red). Arrows indicate myelinated axons (double positive for NF‐200 and MBP). Scale bars, 40 μm. (e, f) Statistical analysis of myelinated axons in OPC‐neuron co‐cultures in the presence of various concentrations of As‐2P for 6 (c) or 14 (d) days. Data are representative of three independent experiments, means ± SEM (n=3). (g) Primary OPCs were co‐cultured with DRG‐neurons and treated with vehicle or As‐2P for 14 days and immunostained for Caspr (green) and NF‐200 (blue). Scale bars, 20 μm. (h) Statistical analysis of Caspr⁺ segments in OPC‐neuron cocultures in the presence of various concentrations of As‐2P for 14 days. Data are means ± SEM (n = 3). *p < .05, **p < .01, ***p < .001 versus vehicle control (one‐way ANOVA followed by Dunnett's multiple comparison test) [Color figure can be viewed at
http://wileyonlinelibrary.com
]

**Figure 5**

As‐2P promotes remyelination in vivo in cuprizone‐induced demyelination model. (a) A schematic drawing of the demyelination/remyelination mice model. C57BL/6 mice were given a diet containing 0.2% cuprizone 5 weeks. Following cuprizone withdrawal, the mice were treated with vehicle or As‐2P (200 mg kg⁻¹) for 1, 2, or 3 weeks (wks). (b) Representative images of the corpus callosum region stained with Luxol fast blue after cuprizone and As‐2P treatment. Scale bars, 100 μm. (c) Statistical analysis of the demyelinating areas in (b). Data are means ± SEM (four mice in cuprizone group, three mice in 5 + 1 groups, seven mice in 5 + 2 groups, and ten mice in 5 + 3 groups; four sections from each mouse were analyzed). ***p < .001 versus cuprizone group, ^# p < .05, ^## p < .01, ^### p < .001 versus vehicle group (one‐way ANOVA followed by Tukey's multiple comparison test). (d) Immunofluorescent images of MBP, MOG, and GST‐pi staining in the corpus callosum region isolated from cuprizone‐fed mice treated with As‐2P or vehicle for 3 weeks. Scale bars, 100 μm. (e–g) Quantification of the fluorescent intensity of MBP, MOG and the number of GST‐pi⁺ cells in corpus callosum as presented in (d). Data are means ± SEM (three mice in each group; six sections from each mouse were analyzed). *p < .05, **p < .01, ***p < .001 versus vehicle group (Student's t test). (h) Immunofluorescent images of PDGFRα and OLIG2 staining in the corpus callosum region isolated from cuprizone‐fed mice treated with As‐2P or vehicle for 3 weeks. Scale bars, 100 μm. (i, j) Quantification of the number of PDGFRα⁺ and OLIG2⁺ cells in corpus callosum as presented in (h). Data are means ± SEM (three mice in each group; six sections from each mouse were analyzed). *p < .05, **p < .01 versus vehicle group (Student's t test) [Color figure can be viewed at
http://wileyonlinelibrary.com
]

**Figure 6**

Evaluation of remyelination in cuprizone model with electron microscopy. (a) Representative electron microscopy images of the corpus callosum region isolated from cuprizone‐fed mice treated with As‐2P (200 mg kg⁻¹) or vehicle for 3 weeks. Red asterisk indicates the demyelinating axon and the red arrows indicate the remyelinated axons. (b) Quantification of the myelinated axons from (a). Data are means ± SEM (three mice from each group, six sections from each mouse were analyzed). ***p < .001 versus cuprizone group, ^### p < .001 versus vehicle group (one‐way ANOVA followed by Tukey's multiple comparison test). (c) Quantification of the G ratios of the remyelinated axons in (a). Data are means ± SEM (n = 200, ∼70 axons counted per mouse, three mice per group), ^### p < .001 versus vehicle group (Student's t test). (d) The scatter plot displaying the individual G‐ratio values and axonal size distribution [Color figure can be viewed at
http://wileyonlinelibrary.com
]

**Figure 7**

As‐2P's effect in inducing OL differentiation is independent of its antioxidant property. (a) Effects of two different forms of Vc,
l
‐ascorbic acid (L‐AA, 150 μM) and As‐2P (150 μM), in promoting the differentiation of OLs. Cells were stained with antibody against MBP (green). Nuclei were stained with Hoechst (blue). Scale bars, 100 μm; inset 25 μm. (b) Statistical analysis of MBP⁺ cells in (a); Data are representative of three independent experiments, means ± SEM (n = 4) ***p < .001 versus vehicle control, ^## p < .01, versus L‐AA (one‐way ANOVA followed by Tukey's multiple comparison test). (c) PCR analysis of the mRNA level of Svct1 and Svct2 in NPC‐derived OPCs and differentiated OLs. (d) Western blot analysis showing the protein expression of Svct2 in NPC‐derived OPCs and differentiated OLs. (e) The effect of SVCT2 inhibitor phloretin (PT, 50 μM) on As‐2P‐stimulated generation of MBP⁺ OLs. Cells were stained with antibody against MBP (green). Nuclei were stained with Hoechst (blue). Scale bars, 100 μm; inset 25 μm. (f) Quantification of MBP⁺ cells in (e). Data are representative of three independent experiments, means ± SEM (n = 4) ***p < .001 versus control, ^### p < .001, versus As‐2P (Student's t test). (g) OPCs were cultured in the presence of As‐2P (150 μM), PT (50 μM) or the combination of both for 2 days and the intracellular concentrations of L‐AA were measured by UPLC/Q‐TOF MS. Data are representative of three independent experiments, means ± SEM (n = 4), ^## p < .01, versus As‐2P (Student's t test). (h) NPC‐derived OPCs were differentiated in the presence of various anti‐oxidants for 4 days. Cells were stained with antibodies against MBP (green). (i) Quantification of MBP⁺ cells in (h). (j) ROS measurement at day 2 post differentiation of OPCs treated as indicated [Color figure can be viewed at
http://wileyonlinelibrary.com
]

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Vitamin C Myelin Sheath

Source: https://pubmed.ncbi.nlm.nih.gov/29423921/

Vitamin C Myelin Sheath