Limits...
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.

Show MeSH
Analysis of mitochondrial morphology by live cell imaging and expression levels of proteins involved in mitochondrial fusion and fission prior to and after serum removal(A) Live cell confocal fluorescence imaging of serum-grown and serum-deprived PC12-ND6 cells. PC12-ND6 cells were first transfected with the mito-GFP vector to label the mitochondrial mass and then switched to a serum-free medium for the indicated periods of time. Mitochondrial morphology was assessed by confocal fluorescence microscopy using a ×100 oil objective and an environmental chamber to keep the CO2 level and temperature constant. The left panels show GFP-labelled mitochondria, while the middle panels show the merge with the corresponding DIC pictures. The right panels show high magnification of the labelled mitochondria (large arrows for elongated mitochondria and small for rod-like mitochondria). Scale bar, 5 μm. (B) High magnification of serum-deprived PC12-ND6 cells transfected with the mito-GFP vector. This image illustrates the range of mitochondrial length, suggestive of a dynamic fusion–fission activity even after 15 days of serum deprivation. Scale bar, 5 μm. (C) Expression profile of protein involved in the fusion–fission process of mitochondria. Immunoblot analyses were performed using whole-cell extracts from serum-grown control PC12 and PC12-ND6 cells and equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of each Western blot panel. The immunoblots are representative of at least three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates. (D) Full-length PINK1 protein remains expressed in serum-deprived PC12-ND6 cells, even after 15 days of serum deprivation. Immunoblot analysis was performed using whole-cell extracts from PC12-ND6 cells grown in a serum-free medium for the indicated periods of time. Equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of the Western blot panels. Results shown are representative of three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874871&req=5

Figure 3: Analysis of mitochondrial morphology by live cell imaging and expression levels of proteins involved in mitochondrial fusion and fission prior to and after serum removal(A) Live cell confocal fluorescence imaging of serum-grown and serum-deprived PC12-ND6 cells. PC12-ND6 cells were first transfected with the mito-GFP vector to label the mitochondrial mass and then switched to a serum-free medium for the indicated periods of time. Mitochondrial morphology was assessed by confocal fluorescence microscopy using a ×100 oil objective and an environmental chamber to keep the CO2 level and temperature constant. The left panels show GFP-labelled mitochondria, while the middle panels show the merge with the corresponding DIC pictures. The right panels show high magnification of the labelled mitochondria (large arrows for elongated mitochondria and small for rod-like mitochondria). Scale bar, 5 μm. (B) High magnification of serum-deprived PC12-ND6 cells transfected with the mito-GFP vector. This image illustrates the range of mitochondrial length, suggestive of a dynamic fusion–fission activity even after 15 days of serum deprivation. Scale bar, 5 μm. (C) Expression profile of protein involved in the fusion–fission process of mitochondria. Immunoblot analyses were performed using whole-cell extracts from serum-grown control PC12 and PC12-ND6 cells and equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of each Western blot panel. The immunoblots are representative of at least three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates. (D) Full-length PINK1 protein remains expressed in serum-deprived PC12-ND6 cells, even after 15 days of serum deprivation. Immunoblot analysis was performed using whole-cell extracts from PC12-ND6 cells grown in a serum-free medium for the indicated periods of time. Equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of the Western blot panels. Results shown are representative of three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates.

Mentions: Since a strong link exists between mitochondrial morphology and bioenergetic functions, with an elongated morphology linked to the normal metabolic state and cell survival and a fragmented morphology (spheroid-like) associated with compromised respiratory activities and programmed cell death (reviewed by Karbowski and Youle, 2003; Okamoto and Shaw, 2005; Chan 2006), we evaluated by confocal live cell imaging whether PC12-ND6 cells maintained a healthy mitochondrial population at distinct time points of serum deprivation. PC12-ND6 cells were first transfected by electroporation using the mito-GFP vector, which encodes the GFP with the mitochondrial targeting sequence from subunit VIII of human COX, and were then serum-deprived after 24 h of transfection for defined periods of time. Figure 3(A) shows that serum-grown PC12-ND6 cells displayed a preponderance of elongated mitochondria, as previously reported by immunocytochemistry using fixed PC12-ND6 cells (Baxter et al., 2009). After 48 h of serum deprivation, most mitochondria showed a short rod-like morphology and a few showed an elongated morphology, while spheroid-like fragmented mitochondria were rare (Figure 3A). A similar overall mitochondrial morphology was sustained after 6–9 days of serum deprivation (Figure 3A). PC12-ND6 cells, which were serum-deprived for 15 days, displayed elongated mitochondria (Figure 3B), suggestive of an ongoing dynamic mitochondrial fusion–fission process, associated with proper mitochondrial functions. Collectively, our flow cytometry analysis and confocal live cell imaging have revealed that NeuroD6 not only maintains the mitochondrial biomass, but also a properly regulated mitochondrial fusion–fission dynamics throughout the different phases of long-term serum deprivation.


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)

Analysis of mitochondrial morphology by live cell imaging and expression levels of proteins involved in mitochondrial fusion and fission prior to and after serum removal(A) Live cell confocal fluorescence imaging of serum-grown and serum-deprived PC12-ND6 cells. PC12-ND6 cells were first transfected with the mito-GFP vector to label the mitochondrial mass and then switched to a serum-free medium for the indicated periods of time. Mitochondrial morphology was assessed by confocal fluorescence microscopy using a ×100 oil objective and an environmental chamber to keep the CO2 level and temperature constant. The left panels show GFP-labelled mitochondria, while the middle panels show the merge with the corresponding DIC pictures. The right panels show high magnification of the labelled mitochondria (large arrows for elongated mitochondria and small for rod-like mitochondria). Scale bar, 5 μm. (B) High magnification of serum-deprived PC12-ND6 cells transfected with the mito-GFP vector. This image illustrates the range of mitochondrial length, suggestive of a dynamic fusion–fission activity even after 15 days of serum deprivation. Scale bar, 5 μm. (C) Expression profile of protein involved in the fusion–fission process of mitochondria. Immunoblot analyses were performed using whole-cell extracts from serum-grown control PC12 and PC12-ND6 cells and equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of each Western blot panel. The immunoblots are representative of at least three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates. (D) Full-length PINK1 protein remains expressed in serum-deprived PC12-ND6 cells, even after 15 days of serum deprivation. Immunoblot analysis was performed using whole-cell extracts from PC12-ND6 cells grown in a serum-free medium for the indicated periods of time. Equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of the Western blot panels. Results shown are representative of three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874871&req=5

Figure 3: Analysis of mitochondrial morphology by live cell imaging and expression levels of proteins involved in mitochondrial fusion and fission prior to and after serum removal(A) Live cell confocal fluorescence imaging of serum-grown and serum-deprived PC12-ND6 cells. PC12-ND6 cells were first transfected with the mito-GFP vector to label the mitochondrial mass and then switched to a serum-free medium for the indicated periods of time. Mitochondrial morphology was assessed by confocal fluorescence microscopy using a ×100 oil objective and an environmental chamber to keep the CO2 level and temperature constant. The left panels show GFP-labelled mitochondria, while the middle panels show the merge with the corresponding DIC pictures. The right panels show high magnification of the labelled mitochondria (large arrows for elongated mitochondria and small for rod-like mitochondria). Scale bar, 5 μm. (B) High magnification of serum-deprived PC12-ND6 cells transfected with the mito-GFP vector. This image illustrates the range of mitochondrial length, suggestive of a dynamic fusion–fission activity even after 15 days of serum deprivation. Scale bar, 5 μm. (C) Expression profile of protein involved in the fusion–fission process of mitochondria. Immunoblot analyses were performed using whole-cell extracts from serum-grown control PC12 and PC12-ND6 cells and equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of each Western blot panel. The immunoblots are representative of at least three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates. (D) Full-length PINK1 protein remains expressed in serum-deprived PC12-ND6 cells, even after 15 days of serum deprivation. Immunoblot analysis was performed using whole-cell extracts from PC12-ND6 cells grown in a serum-free medium for the indicated periods of time. Equal loading was verified using an anti-GAPDH antibody. Relevant molecular masses (in kDa) are indicated on the left side of the Western blot panels. Results shown are representative of three independent experiments. Indicated quantification values are specific to the blot shown, with an S.D. less than 10% compared with the corresponding replicates.
Mentions: Since a strong link exists between mitochondrial morphology and bioenergetic functions, with an elongated morphology linked to the normal metabolic state and cell survival and a fragmented morphology (spheroid-like) associated with compromised respiratory activities and programmed cell death (reviewed by Karbowski and Youle, 2003; Okamoto and Shaw, 2005; Chan 2006), we evaluated by confocal live cell imaging whether PC12-ND6 cells maintained a healthy mitochondrial population at distinct time points of serum deprivation. PC12-ND6 cells were first transfected by electroporation using the mito-GFP vector, which encodes the GFP with the mitochondrial targeting sequence from subunit VIII of human COX, and were then serum-deprived after 24 h of transfection for defined periods of time. Figure 3(A) shows that serum-grown PC12-ND6 cells displayed a preponderance of elongated mitochondria, as previously reported by immunocytochemistry using fixed PC12-ND6 cells (Baxter et al., 2009). After 48 h of serum deprivation, most mitochondria showed a short rod-like morphology and a few showed an elongated morphology, while spheroid-like fragmented mitochondria were rare (Figure 3A). A similar overall mitochondrial morphology was sustained after 6–9 days of serum deprivation (Figure 3A). PC12-ND6 cells, which were serum-deprived for 15 days, displayed elongated mitochondria (Figure 3B), suggestive of an ongoing dynamic mitochondrial fusion–fission process, associated with proper mitochondrial functions. Collectively, our flow cytometry analysis and confocal live cell imaging have revealed that NeuroD6 not only maintains the mitochondrial biomass, but also a properly regulated mitochondrial fusion–fission dynamics throughout the different phases of long-term serum deprivation.

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