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The neurogenic basic helix-loop-helix transcription factor NeuroD6 confers tolerance to oxidative stress by triggering an antioxidant response and sustaining the mitochondrial biomass.

Uittenbogaard M, Baxter KK, Chiaramello A - ASN Neuro (2010)

Bottom Line: In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress.The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1alpha, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1.Thus our collective results support the concept that the NeuroD6-PGC-1alpha-SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Regenerative Biology, George Washington University Medical Center, 2300 I Street N.W., Washington, DC 20037, U.S.A. anaaec@gwumc.edu

ABSTRACT
Preserving mitochondrial mass, bioenergetic functions and ROS (reactive oxygen species) homoeostasis is key to neuronal differentiation and survival, as mitochondria produce most of the energy in the form of ATP to execute and maintain these cellular processes. In view of our previous studies showing that NeuroD6 promotes neuronal differentiation and survival on trophic factor withdrawal, combined with its ability to stimulate the mitochondrial biomass and to trigger comprehensive antiapoptotic and molecular chaperone responses, we investigated whether NeuroD6 could concomitantly modulate the mitochondrial biomass and ROS homoeostasis on oxidative stress mediated by serum deprivation. In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress. Using a combination of flow cytometry, confocal fluorescence microscopy and mitochondrial fractionation, we found that NeuroD6 sustains mitochondrial mass, intracellular ATP levels and expression of specific subunits of respiratory complexes upon oxidative stress triggered by withdrawal of trophic factors. NeuroD6 also maintains the expression of nuclear-encoded transcription factors, known to regulate mitochondrial biogenesis, such as PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha), Tfam (transcription factor A, mitochondrial) and NRF-1 (nuclear respiratory factor-1). Finally, NeuroD6 triggers a comprehensive antioxidant response to endow PC12-ND6 cells with intracellular ROS scavenging capacity. The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1alpha, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1. Thus our collective results support the concept that the NeuroD6-PGC-1alpha-SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress.

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Constitutive expression of NeuroD6 prevents ROS production upon serum deprivation(A, B) Live cell confocal images of control PC12 and PC12-ND6 cells grown in the presence or absence of serum for 24 h before being labelled with the MitoSOX™ dye (red) and the nuclear counterstain Hoechst 33342 (blue). Images are representative of three independent experiments. The left panels illustrate MitoSOX™-labelled cells before (t = 0) and after serum deprivation (t = 24 h), while the right panels show the merge with the corresponding DIC pictures (scale bar, 10 μm). (C) Quantification of ROS production at different time points of serum deprivation for control PC12 and PC12-ND6 cells. The mean fluorescence intensity of MitoSOX™ labelling was measured in the presence or absence of serum for 6 and 24 h in control PC12 and PC12-ND6 cells and for 48 h in PC12-ND6 cells. At the outset of serum deprivation, we did not observe any statistical difference of ROS production between control PC12 and PC12-ND6 cells (P = 0.6074). The graph represents the averages from 150 cells from three independent experiments. ‡P = 0.0077 as compared with serum-grown control PC12 cells; *P = 0.0098 as compared with serum-deprived control PC12 cells for 6 h; **P = 0.0004 as compared with serum-deprived control PC12 cells for 24 h; ***P = 0.0207 as compared with untreated PC12-ND6 cells.
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Figure 5: Constitutive expression of NeuroD6 prevents ROS production upon serum deprivation(A, B) Live cell confocal images of control PC12 and PC12-ND6 cells grown in the presence or absence of serum for 24 h before being labelled with the MitoSOX™ dye (red) and the nuclear counterstain Hoechst 33342 (blue). Images are representative of three independent experiments. The left panels illustrate MitoSOX™-labelled cells before (t = 0) and after serum deprivation (t = 24 h), while the right panels show the merge with the corresponding DIC pictures (scale bar, 10 μm). (C) Quantification of ROS production at different time points of serum deprivation for control PC12 and PC12-ND6 cells. The mean fluorescence intensity of MitoSOX™ labelling was measured in the presence or absence of serum for 6 and 24 h in control PC12 and PC12-ND6 cells and for 48 h in PC12-ND6 cells. At the outset of serum deprivation, we did not observe any statistical difference of ROS production between control PC12 and PC12-ND6 cells (P = 0.6074). The graph represents the averages from 150 cells from three independent experiments. ‡P = 0.0077 as compared with serum-grown control PC12 cells; *P = 0.0098 as compared with serum-deprived control PC12 cells for 6 h; **P = 0.0004 as compared with serum-deprived control PC12 cells for 24 h; ***P = 0.0207 as compared with untreated PC12-ND6 cells.

Mentions: Based on our recent finding that serum-grown PC12-ND6 cells produce more ATP than the serum-grown control PC12 cells (Baxter et al., 2009), combined with the fact that the main source of electron leakage originates from the mitochondrial ETC to reduce O2 to the predominant form of ROS superoxide anions (Turrens, 1997; Demin et al., 1998), we investigated whether such an increased energy production would result in higher levels of superoxide anions (O2−) than that in control PC12 cells. To address this question, we measured relative levels of O2− using the redox-sensitive dye, MitoSOX™, which is a fluorogenic dye for highly selective detection of superoxide anions. Since the MitoSOX™ dye is live-cell-permeant and selectively targeted to the mitochondria, we labelled live control PC12 and PC12-ND6 cells with the MitoSOX™ dye and nuclear counterstain Hoechst 33342 and we measured by confocal microscopy the amount of red fluorescence from oxidized MitoSOX™ dye, as an indicator of mitochondrial O2− levels (Johnson-Cadwell et al., 2007). Figure 5 shows that serum-grown PC12-ND6 cells did not display higher levels of O2− than serum-grown control PC12 cells.


The neurogenic basic helix-loop-helix transcription factor NeuroD6 confers tolerance to oxidative stress by triggering an antioxidant response and sustaining the mitochondrial biomass.

Uittenbogaard M, Baxter KK, Chiaramello A - ASN Neuro (2010)

Constitutive expression of NeuroD6 prevents ROS production upon serum deprivation(A, B) Live cell confocal images of control PC12 and PC12-ND6 cells grown in the presence or absence of serum for 24 h before being labelled with the MitoSOX™ dye (red) and the nuclear counterstain Hoechst 33342 (blue). Images are representative of three independent experiments. The left panels illustrate MitoSOX™-labelled cells before (t = 0) and after serum deprivation (t = 24 h), while the right panels show the merge with the corresponding DIC pictures (scale bar, 10 μm). (C) Quantification of ROS production at different time points of serum deprivation for control PC12 and PC12-ND6 cells. The mean fluorescence intensity of MitoSOX™ labelling was measured in the presence or absence of serum for 6 and 24 h in control PC12 and PC12-ND6 cells and for 48 h in PC12-ND6 cells. At the outset of serum deprivation, we did not observe any statistical difference of ROS production between control PC12 and PC12-ND6 cells (P = 0.6074). The graph represents the averages from 150 cells from three independent experiments. ‡P = 0.0077 as compared with serum-grown control PC12 cells; *P = 0.0098 as compared with serum-deprived control PC12 cells for 6 h; **P = 0.0004 as compared with serum-deprived control PC12 cells for 24 h; ***P = 0.0207 as compared with untreated PC12-ND6 cells.
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Figure 5: Constitutive expression of NeuroD6 prevents ROS production upon serum deprivation(A, B) Live cell confocal images of control PC12 and PC12-ND6 cells grown in the presence or absence of serum for 24 h before being labelled with the MitoSOX™ dye (red) and the nuclear counterstain Hoechst 33342 (blue). Images are representative of three independent experiments. The left panels illustrate MitoSOX™-labelled cells before (t = 0) and after serum deprivation (t = 24 h), while the right panels show the merge with the corresponding DIC pictures (scale bar, 10 μm). (C) Quantification of ROS production at different time points of serum deprivation for control PC12 and PC12-ND6 cells. The mean fluorescence intensity of MitoSOX™ labelling was measured in the presence or absence of serum for 6 and 24 h in control PC12 and PC12-ND6 cells and for 48 h in PC12-ND6 cells. At the outset of serum deprivation, we did not observe any statistical difference of ROS production between control PC12 and PC12-ND6 cells (P = 0.6074). The graph represents the averages from 150 cells from three independent experiments. ‡P = 0.0077 as compared with serum-grown control PC12 cells; *P = 0.0098 as compared with serum-deprived control PC12 cells for 6 h; **P = 0.0004 as compared with serum-deprived control PC12 cells for 24 h; ***P = 0.0207 as compared with untreated PC12-ND6 cells.
Mentions: Based on our recent finding that serum-grown PC12-ND6 cells produce more ATP than the serum-grown control PC12 cells (Baxter et al., 2009), combined with the fact that the main source of electron leakage originates from the mitochondrial ETC to reduce O2 to the predominant form of ROS superoxide anions (Turrens, 1997; Demin et al., 1998), we investigated whether such an increased energy production would result in higher levels of superoxide anions (O2−) than that in control PC12 cells. To address this question, we measured relative levels of O2− using the redox-sensitive dye, MitoSOX™, which is a fluorogenic dye for highly selective detection of superoxide anions. Since the MitoSOX™ dye is live-cell-permeant and selectively targeted to the mitochondria, we labelled live control PC12 and PC12-ND6 cells with the MitoSOX™ dye and nuclear counterstain Hoechst 33342 and we measured by confocal microscopy the amount of red fluorescence from oxidized MitoSOX™ dye, as an indicator of mitochondrial O2− levels (Johnson-Cadwell et al., 2007). Figure 5 shows that serum-grown PC12-ND6 cells did not display higher levels of O2− than serum-grown control PC12 cells.

Bottom Line: In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress.The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1alpha, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1.Thus our collective results support the concept that the NeuroD6-PGC-1alpha-SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Regenerative Biology, George Washington University Medical Center, 2300 I Street N.W., Washington, DC 20037, U.S.A. anaaec@gwumc.edu

ABSTRACT
Preserving mitochondrial mass, bioenergetic functions and ROS (reactive oxygen species) homoeostasis is key to neuronal differentiation and survival, as mitochondria produce most of the energy in the form of ATP to execute and maintain these cellular processes. In view of our previous studies showing that NeuroD6 promotes neuronal differentiation and survival on trophic factor withdrawal, combined with its ability to stimulate the mitochondrial biomass and to trigger comprehensive antiapoptotic and molecular chaperone responses, we investigated whether NeuroD6 could concomitantly modulate the mitochondrial biomass and ROS homoeostasis on oxidative stress mediated by serum deprivation. In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress. Using a combination of flow cytometry, confocal fluorescence microscopy and mitochondrial fractionation, we found that NeuroD6 sustains mitochondrial mass, intracellular ATP levels and expression of specific subunits of respiratory complexes upon oxidative stress triggered by withdrawal of trophic factors. NeuroD6 also maintains the expression of nuclear-encoded transcription factors, known to regulate mitochondrial biogenesis, such as PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha), Tfam (transcription factor A, mitochondrial) and NRF-1 (nuclear respiratory factor-1). Finally, NeuroD6 triggers a comprehensive antioxidant response to endow PC12-ND6 cells with intracellular ROS scavenging capacity. The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1alpha, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1. Thus our collective results support the concept that the NeuroD6-PGC-1alpha-SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress.

Show MeSH
Related in: MedlinePlus