Limits...
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

Loss of mitochondrial membrane potential following exposure to ethidium bromide. 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, mitochondrial membrane potential was assessed 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 two independent replicates with similar results and significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109433&req=5

Figure 3: Loss of mitochondrial membrane potential following exposure to ethidium bromide. 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, mitochondrial membrane potential was assessed 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 two independent replicates with similar results and significance.

Mentions: We examined several aspects of mitochondria following ethidium bromide exposure. Both mitochondrial-specific and total cellular ROS were increased in cells that were exposed to ethidium bromide (Figure 2). Pretreatment of HDF with rapamycin decreased both the baseline levels of cellular ROS and significantly reduced the effect of ethidium bromide on the production of ROS (Figure 2). Similarly, the baseline levels of mitochondrial ROS levels and the mitochondrial ROS induced by ethidium bromide were significantly reduced by rapamycin pretreatment (Figure 2). The percent of cells with depolarized mitochondria was increased following ethidium bromide treatment and this percentage was reduced by pretreatment with rapamycin (Figure 3). Despite the reduction in mitochondrial DNA, mitochondrial mass was increased following ethidium bromide treatment in a dose-dependent manner, while HDF grown in the presence of rapamycin had a greater mitochondrial mass that was not altered by ethidium bromide (Figure 4).


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

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

Loss of mitochondrial membrane potential following exposure to ethidium bromide. 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, mitochondrial membrane potential was assessed 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 two 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 3: Loss of mitochondrial membrane potential following exposure to ethidium bromide. 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, mitochondrial membrane potential was assessed 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 two independent replicates with similar results and significance.
Mentions: We examined several aspects of mitochondria following ethidium bromide exposure. Both mitochondrial-specific and total cellular ROS were increased in cells that were exposed to ethidium bromide (Figure 2). Pretreatment of HDF with rapamycin decreased both the baseline levels of cellular ROS and significantly reduced the effect of ethidium bromide on the production of ROS (Figure 2). Similarly, the baseline levels of mitochondrial ROS levels and the mitochondrial ROS induced by ethidium bromide were significantly reduced by rapamycin pretreatment (Figure 2). The percent of cells with depolarized mitochondria was increased following ethidium bromide treatment and this percentage was reduced by pretreatment with rapamycin (Figure 3). Despite the reduction in mitochondrial DNA, mitochondrial mass was increased following ethidium bromide treatment in a dose-dependent manner, while HDF grown in the presence of rapamycin had a greater mitochondrial mass that was not altered by ethidium bromide (Figure 4).

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