Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Dec 14;9(12):1276.
doi: 10.3390/antiox9121276.

Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro

Francisca Espinoza  1 , Rocío Magdalena  1 , Natalia Saldivia  1 , Nery Jara  1 , Fernando Martínez  1 , Luciano Ferrada  1 , Katterine Salazar  1 , Felipe Ávila  2 , Francisco Nualart  1   3
Affiliations

Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro

Francisca Espinoza et al. Antioxidants (Basel). .

Abstract

The reduced form of vitamin C, ascorbic acid (AA), has been related with gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron-astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown. To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells. AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells that efficiently capture DHA. In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. We demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.

Keywords: GLUT1; SVCT2; astrocytes; bystander effect; neurites; neuron; neuronal differentiation; retinoic acid; vitamin C; vitamin C recycling.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Adhered neurospheres showed SVCT2 expression mainly in differentiated neuronal cells. (A) Confocal microscopy with tile-scan 3D and rendering analysis of NEs stained with anti-SVCT2 (red) and anti-βIII tubulin (green). Inset. A detailed analysis of neurosphere processes. Scale bar, 100 µm. (B) NE confocal microscopy analysis using antibodies against GFAP, nestin, and SOX2 and nuclear staining with Hoechst. Scale bar, 50 µm. (C) PCR analysis of SVCT2 and GLUT1 mRNA. Line 1: cDNA obtained from control NEs adhered for 24 h. Line 2: Control reaction without reverse transcriptase. Line 3: Postnatal day 8 rat brain cDNA (positive control). Line 4: Negative control (water). (D) Western blot analysis with anti-GLUT1 and anti-SVCT2 antibodies in extracts of control NEs 24 h postadhesion. (E) Confocal microscopy and merge analysis using anti-SVCT2 (red) and anti-βIII-tubulin (green) antibodies and nuclear staining with Hoechst. Right side. Digital zoom images of the depicted zone in E. Scale bar, 50 µm. (F) SVCT2 and GLUT1 transporter detection by 3D-SIM superresolution (SIM-SR) microscopy analysis in control NEs adhered for 24 h. Left side. βIII-tubulin (green) and SVCT2 (red) detection. Right side. βIII-tubulin (green) and GLUT1 (white) detection. Scale bar, 10 µm. (G) SVCT2 and GLUT1 transporter detection by SIM superresolution (SIM-SR) microscopy analysis in control NEs adhered for 24 h. Left side. Digital magnification of βIII tubulin/SVCT2 positive processes. Right side. Digital magnification of βIII tubulin/GLUT1 positive processes. Scale bar, 2 µm. (H) AA uptake in NEs adhered (24 h, control) in the presence or absence of sodium. All data are representative of three separate experiments. Parametric Student’s t-test, n.s., not significant. (I) DHA uptake analysis in NEs adhered (24 h, control) in the absence or presence of 20 µM cytochalasin B (GLUT1 inhibitor). All data are representative of three separate experiments. Parametric Student’s t-test, ** p < 0.01.
Figure 2
Figure 2
AA stimulated neuronal differentiation in adhered NEs. (A) Protocol for neuronal differentiation stimulated by AA or RA in adhered NEs. (B,E,H,K) Immunocytochemistry analysis of βIII tubulin (green) in NEs treated with 10 µM RA. Scale bar, 30 µm. (C,F,I,L) Immunocytochemistry analysis of βIII tubulin (green) in NEs treated with 100 µM AA for 24 to 72 h. Scale bar, 30 µm. (D,G,J,M) Neurite volume quantification. All data are representative of three separate experiments. Parametric ANOVA statistics analysis, Tukey posttest, ** p < 0.01; *** p < 0.001; n.s., not significant.
Figure 3
Figure 3
Treatment of NEs differentiated in vitro with DHA impacted neuritic growth. (A) Viability analysis of NEs treated with AA for 24, 48 and 72 h. All data are representative of three separate experiments. Nonparametric ANOVA statistical analysis, Tukey posttest, * p < 0.05; n.s., not significant. (B) Intracellular AA concentration during AA treatment. All data are representative of three separate experiments. Parametric ANOVA statistical analysis, Tukey posttest, * p < 0.05; ** p < 0.01. (C) DHA treatment protocol used in NE adhesion. (D–F) βIII tubulin (green) analysis in NEs treated with 100 µM AA for 24 h (B) or 100 µM AA for 24 h followed by 100 µM DHA until 48 h (E) or until 72 h (F). Scale bar, 60 µm. (G) NE neurite quantification at each treatment time point. A “neurite” was considered a cellular extension positive for βIII tubulin and at least 10 µm in length. All data are representative of three separate experiments. Parametric ANOVA statistical analysis, Tukey posttest, *** p < 0.001.
Figure 4
Figure 4
Coculture of HL60 cells with NEs maintained neurites in vitro after AA treatment. (A) PCR analysis of GLUT1 mRNA. RNA extracts obtained from HL60 cells (line 1), negative RT-PCR control (line 2), and positive control using human U87 cell line samples (line 3). (B) Western blot analysis for GLUT1 expression in HL60 cells. (C) GLUT1 immunocytochemical detection in HL60 cells. Scale bar, 30 µm. (D) Uptake of 100 µM DHA for 5 min in the presence of Na+, 20 µM cytochalasin B or 5 µM WZB117 (GLUT1 specific inhibitor). All data are representative of three separate experiments. Statistical analysis, parametric ANOVA test, followed by Tukey’s test, *** p < 0.001. (E) Protocol used to recycle vitamin C with HL60 cells. (F,G) Immunocytochemistry analysis of βIII tubulin in neurospheres treated with AA for 72 h without (F) or cocultured with HL60 cells after 24 h of treatment (G). Scale bar, 100 µm. (H,I) High-power image of NEs depicted in (F,G), respectively. Scale bar, 50 µm. (J) Quantification of the neurites of NEs until 72 h of treatment with 100 µM AA in the presence of HL60 cells or the WZB117 inhibitor (WZB). All data are representative of three separate experiments. Statistical analysis, parametric ANOVA test, followed by Tukey’s test, * p < 0.05; ** p < 0.01.
Figure 5
Figure 5
Vitamin C recycling using astrocytes, maintained neurite outgrowth under long-term AA treatment. (A,E) PCR analysis of GLUT1 mRNA. RNA extracts obtained from U87 cells (A) and cortical astrocytes (E)(line 1), negative RT-PCR control (line 2), and positive control using complete brain extract (line 3). (B,F) Western blot analysis of GLUT1 in U87 cell (B) and cortical astrocyte extracts (F). (C,G) Immunocytochemistry analysis of GLUT1 (green) in U87 cells (C) and astrocytes (G). Scale bar, 15 µm. (D,H) Uptake of 100 µM DHA for 5 min in the presence of 20 µM Na+, cytochalasin B or WZB117 in U87 cells (D) and astrocytes (H). All data are representative of three separate experiments. Parametric ANOVA, followed by Tukey’s test, * p <0.05; ** p <0.01. (I) Experimental model of mixed cultures of NEs with U87 cells or cortical astrocytes at 7 DIV, which were treated with AA for 72 h. (J,L) Immunocytochemical and confocal tile-scan microscopy analyses of βIII tubulin (green), GFAP (red) and nuclear staining with Hoechst, in mixed NEs cultured with cortical astrocytes. Scale bar, 100 µm. High-power image of NEs (L). Scale bar, 30 µm. (K) Immunocytochemical and confocal microscopy analyses of βIII tubulin (green) and nuclear staining with Hoechst, in control NEs treated with AA for 72 h in the absence of astrocytes. Scale bar, 30 µm. (M,N) Total NEs with neurites generated at 72 h in mixed cultures of NEs with U87 cells (M) or cortical astrocytes (N). All data are representative of three separate experiments. Parametric Student’s t-test was used for statistical analysis, ** p < 0.01; *** p < 0.001.
Figure 6
Figure 6
DHA accumulation impaired neurite outgrowth through redox imbalance. (A–C) Representative histogram of CellROX® dye detection (APC channel) in control NEs (PBS, pink) and in NEs treated with 100 µM AA (light blue) for 36 (A), 48 (B) and 72 h (C), respectively. Fluorescence intensity quantification of CellROX® in the control (PBS) and treated (AA) conditions is shown at the bottom. All data are representative of three separate experiments. Student’s t-test was used, * p < 0.05. (D–F) Flow cytometry analysis was performed using ThioBrightTM Green probe for intracellular GSH detection in control NEs (pink) and in NEs treated with AA (light blue) for 36 h (D), 48 h (E) and 72 h (F). The representative histogram of the detection of the probe through the FITC channel (GSH) is shown at the top. The quantification of fluorescence intensity in control samples (PBS) compared to treated samples (AA) at different times analyzed is shown in the lower part. Analysis was performed on three independent experiments; 20,000 events were recorded per condition. Student’s t-test was used, * p < 0.05; n.s., not significant. (G) Carboxymethyl-lysine (CML) immunoblot detection in control NE samples (lane 1) and NEs treated with AA (lane 2). All data are representative of three separate experiments. (H) Oxy-Blot analysis of protein carbonylation in extracts from control NEs (lane 1) and from those treated for 72 h with 100 µM AA (lane 2). All data are representative of three separate experiments. (I) Densitometry analysis of the CML and Oxy-Blot analyses is shown as a percentage of increase with respect to the control (PBS). All data are representative of three separate experiments.
See this image and copyright information in PMC

References

    1. Barnes A.P., Polleux F. Establishment of axon-dendrite polarity in developing neurons. Annu. Rev. Neurosci. 2009;32:347–381. doi: 10.1146/annurev.neuro.31.060407.125536. - DOI - PMC - PubMed
    1. Takano T., Xu C., Funahashi Y., Namba T., Kaibuchi K. Neuronal polarization. Development. 2015;142:2088–2093. doi: 10.1242/dev.114454. - DOI - PubMed
    1. Oga T., Elston G.N., Fujita I. Postnatal Dendritic Growth and Spinogenesis of Layer-V Pyramidal Cells Differ between Visual, Inferotemporal, and Prefrontal Cortex of the Macaque Monkey. Front. Neurosci. 2017;11:118. doi: 10.3389/fnins.2017.00118. - DOI - PMC - PubMed
    1. Ge W.P., Miyawaki A., Gage F.H., Jan Y.N., Jan L.Y. Local generation of glia is a major astrocyte source in postnatal cortex. Nature. 2012;484:376–380. doi: 10.1038/nature10959. - DOI - PMC - PubMed
    1. Tabata H. Diverse subtypes of astrocytes and their development during corticogenesis. Front. Neurosci. 2015;9:114. doi: 10.3389/fnins.2015.00114. - DOI - PMC - PubMed

LinkOut - more resources