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Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion.

Nacarelli T, Azar A, Sell C - Front Endocrinol (Lausanne) (2014)

Bottom Line: Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA.These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate.Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Drexel University College of Medicine , Philadelphia, PA , USA.

ABSTRACT
The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

No MeSH data available.


Related in: MedlinePlus

Ethidium bromide exposure leads to a loss of mitochondrial DNA. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, the relative level of mitochondrial DNA was assessed using real-time PCR analysis as described in Section “Materials and Methods.” Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign. The graph contains a representative experiment of a minimum of three independent replicates with similar results and significance.
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Figure 1: Ethidium bromide exposure leads to a loss of mitochondrial DNA. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, the relative level of mitochondrial DNA was assessed using real-time PCR analysis as described in Section “Materials and Methods.” Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign. The graph contains a representative experiment of a minimum of three independent replicates with similar results and significance.

Mentions: Human diploid fibroblasts were exposed to ethidium bromide at concentrations ranging from 10 to 100 ng/ml for 7 days. Under these conditions, we found a dose-dependent decline in the mitochondrial DNA content (Figure 1). We have previously found that long-term exposure to nanomolar concentrations of rapamycin increases replicative lifespan of HDF, reduces the percent of cells with depolarized mitochondria, and increases the expression of genes associated with mitochondrial biogenesis (25, 29). We utilized this paradigm to examine the response of rapamycin treated cells to a direct mitochondrial insult such as ethidium bromide exposure. When HDF were grown in the presence of 1 nM rapamycin and exposed to ethidium bromide, we found a similar reduction in mitochondrial DNA content, although the initial copy number was lower than control cultures (Figure 1).


Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion.

Nacarelli T, Azar A, Sell C - Front Endocrinol (Lausanne) (2014)

Ethidium bromide exposure leads to a loss of mitochondrial DNA. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, the relative level of mitochondrial DNA was assessed using real-time PCR analysis as described in Section “Materials and Methods.” Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign. The graph contains a representative experiment of a minimum of three independent replicates with similar results and significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109433&req=5

Figure 1: Ethidium bromide exposure leads to a loss of mitochondrial DNA. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, the relative level of mitochondrial DNA was assessed using real-time PCR analysis as described in Section “Materials and Methods.” Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign. The graph contains a representative experiment of a minimum of three independent replicates with similar results and significance.
Mentions: Human diploid fibroblasts were exposed to ethidium bromide at concentrations ranging from 10 to 100 ng/ml for 7 days. Under these conditions, we found a dose-dependent decline in the mitochondrial DNA content (Figure 1). We have previously found that long-term exposure to nanomolar concentrations of rapamycin increases replicative lifespan of HDF, reduces the percent of cells with depolarized mitochondria, and increases the expression of genes associated with mitochondrial biogenesis (25, 29). We utilized this paradigm to examine the response of rapamycin treated cells to a direct mitochondrial insult such as ethidium bromide exposure. When HDF were grown in the presence of 1 nM rapamycin and exposed to ethidium bromide, we found a similar reduction in mitochondrial DNA content, although the initial copy number was lower than control cultures (Figure 1).

Bottom Line: Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA.These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate.Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Drexel University College of Medicine , Philadelphia, PA , USA.

ABSTRACT
The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

No MeSH data available.


Related in: MedlinePlus