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. 2020 Jan:29:101408.
doi: 10.1016/j.redox.2019.101408. Epub 2019 Dec 16.

Vitamin C controls neuronal necroptosis under oxidative stress

Luciano Ferrada  1 , María Jose Barahona  2 , Katterine Salazar  1 , Peter Vandenabeele  3 , Francisco Nualart  4
Affiliations

Vitamin C controls neuronal necroptosis under oxidative stress

Luciano Ferrada et al. Redox Biol. 2020 Jan.

Abstract

Under physiological conditions, vitamin C is the main antioxidant found in the central nervous system and is found in two states: reduced as ascorbic acid (AA) and oxidized as dehydroascorbic acid (DHA). However, under pathophysiological conditions, AA is oxidized to DHA. The oxidation of AA and subsequent production of DHA in neurons are associated with a decrease in GSH concentrations, alterations in glucose metabolism and neuronal death. To date, the endogenous molecules that act as intrinsic regulators of neuronal necroptosis under conditions of oxidative stress are unknown. Here, we show that treatment with AA regulates the expression of pro- and antiapoptotic genes. Vitamin C also regulates the expression of RIPK1/MLKL, whereas the oxidation of AA in neurons induces morphological alterations consistent with necroptosis and MLKL activation. The activation of necroptosis by AA oxidation in neurons results in bubble formation, loss of membrane integrity, and ultimately, cellular explosion. These data suggest that necroptosis is a target for cell death induced by vitamin C.

Keywords: Ascorbic acid; Dehydroascorbic acid; Live cell microscopy; Necroptosis; Neuronal death; Vitamin C.

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Conflict of interest statement

None.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Oxidation of vitamin C induced neuronal death and morphological changes. (A) Schematic of the treatment protocol with vitamin C and oxidative stress induction. (B and I) Intracellular measurement of AA. The control condition represents the intracellular concentration of AA prior to treatment with H2O2. The time corresponds to the time in minutes posttreatment with H2O2. n = 3 biologically independent samples. (C and J) Cell viability at 3 h posttreatment. (D and K) GLUT1 and SVCT2 distribution at 3 h posttreatment. (E) 2D and 3D superresolution SIM of N2a cells at 3 h posttreatment. (F and L) Imaris 3D reconstruction at 3 h posttreatment. (G and N) Morphological analysis in N2a and HN33.11 cells. (H and O) Cell size analysis with the Imaris bounding box tool. (M) 3D morphological alterations of the plasma membrane in HN33.11 cells; the arrows show bubble-like structures. Scale bar 10 μm. Data are shown as the mean ± SEM (two-tail Student's t-tests); n = 3 biologically independent samples. All data are representative of three separate experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Fig. 2
Fig. 2
ROS and apoptosis inhibition do not prevent cell death induced by vitamin C oxidation.(A and C) Intracellular ROS production in N2a and HN33.11 cells. (B and D) Quantification of ROS production with respect to control (n = 2 biologically independent samples). (E and F) Cell viability analysis of ROS inhibition with N-acetyl-cysteine (NAC, 24 h before treatment) in N2a and HN33.11 cells (n = 3 biologically independent samples). (G and H) Cell viability analysis of apoptosis inhibition in N2a cells (n = 3 biologically independent samples). Data are shown as the mean ± SEM (two-tail Student's t-tests); all data are representative of three separate experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n.s., not significant.
Fig. 3
Fig. 3
Vitamin C regulates the expression of RIPK1, RIPK3 and MLKL, stimulating necroptosis. (A and I) mRNA levels of apoptotic and necroptotic genes. (B and J) Expression of RIPK1 and MLKL in N2a and HN33.11 cells. (C and K) RIPK1 expression determined by flow cytometry (50,000 counts). (D and L) Relative quantification of RIPK1 by flow cytometry (n = 2 biologically independent samples). (E and M) MLKL levels determined by flow cytometry (50,000 counts). (F and N) Relative quantification of MLKL by flow cytometry (n = 2 biologically independent samples). (G and O) Analysis of the expression and localization of RIPK1, RIPK3 and MLKL. (H). SIM superresolution of RIPK1 and MLKL in N2a cells treated with AA. Scale bar 10 μm. Data are shown as the mean ± SEM (two-tail Student's t-tests). n = 3 biologically independent samples. The experiment was independently reproduced three times. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n.s. not significant.
Fig. 4
Fig. 4
Vitamin C oxidation induces neuronal necroptosis. (A and G) Necroptosis inhibition with Nec-1 or Nec-1s. (B and H) Microphotographs analyses of MLKL phosphorylation. (C and I) Colocation map of MLKL/P-MLKL determined with Imaris. (D) Nuclear translocation of MLKL by phosphorylation. (E and J) Intensity profile of MLKL/P-MLKL. (F and K) Quantification of MLKL phosphorylation. Scale bar 10 μm. Data are shown as the mean ± SEM (two-tail Student's t-tests). n = 3 biologically independent samples. The experiment was independently reproduced three times. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n.s. not significant.
Fig. 5
Fig. 5
Neuronal vitamin C oxidation induces necroptotic disintegration features. (A and E) Schematic of the live/dead discrimination process. (B, C and D) Bubble formation and features of disintegration during neuronal necroptosis. (F) Shedding of the cytoplasm and features of disintegration during neuronal necroptosis. TOPRO-3 and phalloidin Alexa-488 were used as indicators of cell death by measuring plasma membrane integrity. Scale bar, 10 μm, for cortical neurons, 5 μm.
Fig. 6
Fig. 6
Overexpression of hSVCT2 increases neuronal death induced by vitamin C oxidation. (A and D) SIM superresolution of overexpression of hSVCT2. (B and E) Quantification of cell death in stable N2a and HN33.11 cells transduced with control EGFP (30,000 count). (C and F) Quantification of cell death in stable N2a and HN33.11 cells transduced with hSVCT2wt-EYFP (30,000 count). Scale bar, 10 μm. Data are shown as the mean ± SEM (two-tailed Student's t-tests). n = at least two independent biological replicates. The experiment was independently reproduced three times. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n.s. not significant.
Fig. 7
Fig. 7
MLKL or SVCT2 KO prevents cell death induced by vitamin C oxidation. (A and B) CRISPR/cas9 validation for MLKL and SVCT2. (C and D) Cell viability at 3 h posttreatment in SVCT2−/− N2a cells. (E and F) Quantification of cell death in WT, MLKL−/- and SVCT2−/− N2a cells. (G) Schematic of the protocol used in Fig. H. (H) Cell viability at 3 h posttreatment in SVCT2−/− N2a cells. Data are shown as the mean ± SEM (two-tail Student's t-tests). n = 3 biologically independent samples. Each experiment was independently reproduced three times. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n.s. not significant.
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