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ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts.

Qi Y, Qiu Q, Gu X, Tian Y, Zhang Y - Sci Rep (2016)

Bottom Line: Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells.These results suggest that ATM drives the initiation of the mitophagic cascade.These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy.

View Article: PubMed Central - PubMed

Affiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.

ABSTRACT
The ATM (ataxia telangiectasia mutated) protein has recently been proposed to play critical roles in the response to mitochondrial dysfunction by initiating mitophagy. Here, we have used ATM-proficient GM00637 cells and ATM-deficient GM05849 cells to investigate the mitophagic effect of spermidine and to elucidate the role of ATM in spermdine-induced mitophagy. Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells. However, in GM05849 cells or GM00637 cells pretreated with the ATM kinase inhibitor KU55933, the expression of full-length PINK1 and the translocation of Parkin are blocked, and the colocalization of Parkin with either LC3 or PINK1 is disrupted. These results suggest that ATM drives the initiation of the mitophagic cascade. Our study demonstrates that spermidine induces mitophagy through ATM-dependent activation of the PINK1/Parkin pathway. These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy.

No MeSH data available.


Related in: MedlinePlus

Spermidine induced an ATM-dependent mitochondrial depolarization.The treated GM00637 (a) and GM05849 cells (b) were stained with TMRM and MTG for 20 min simultaneously and images were taken with a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Fluorescence intensity was quantified from stacks of images through the entire thickness of cells which were from three independent experiments. MTG/TMRM is indicative of the level of depolarization (c). Values are mean ± SD (n = 3), *p < 0.05, compared with control.
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f1: Spermidine induced an ATM-dependent mitochondrial depolarization.The treated GM00637 (a) and GM05849 cells (b) were stained with TMRM and MTG for 20 min simultaneously and images were taken with a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Fluorescence intensity was quantified from stacks of images through the entire thickness of cells which were from three independent experiments. MTG/TMRM is indicative of the level of depolarization (c). Values are mean ± SD (n = 3), *p < 0.05, compared with control.

Mentions: To examine the effect of spermidine on MMP, GM00637 and GM05849 cells were treated with 50 μM spermidine for 2 h, followed by incubation with TMRM and MTG (200 nM) simultaneously for 20 min in complete growth medium. Confocal microscopy images revealed an accumulation of MTG and TMRM in the mitochondria, and fluorescence intensity was quantified. In such MTG and TMRM co-loaded cells, depolarizing mitochondria turn from red to green, and the ratio of MTG/TMRM is indicative of the level of depolarization. The control GM00637 cells were in a highly polarized status, as indicated by the stronger fluorescence of TMRM and decreased fluorescence intensity ratio. In contrast, 50 μM spermidine significantly decreased the fluorescence of TMRM (Fig. 1a) and robustly increased fluorescence intensity ratio of MTG/TMRM (Fig. 1c), implying a depolarized status of mitochondria in GM00637 cells. The mitochondria uncoupler CCCP (carbonyl cyanide 3-chlorophenylhydrazone) as a positive control showed a more potent effect. In contrast, GM05849 cells displayed slight depolarization, even in the control group, and spermidine has no obvious effect on MMP as shown by the similar TMRM fluorescence to that of control cells and approximately equal fluorescence intensity ratio of MTG/TMRM (Fig. 1b,c).


ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts.

Qi Y, Qiu Q, Gu X, Tian Y, Zhang Y - Sci Rep (2016)

Spermidine induced an ATM-dependent mitochondrial depolarization.The treated GM00637 (a) and GM05849 cells (b) were stained with TMRM and MTG for 20 min simultaneously and images were taken with a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Fluorescence intensity was quantified from stacks of images through the entire thickness of cells which were from three independent experiments. MTG/TMRM is indicative of the level of depolarization (c). Values are mean ± SD (n = 3), *p < 0.05, compared with control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835770&req=5

f1: Spermidine induced an ATM-dependent mitochondrial depolarization.The treated GM00637 (a) and GM05849 cells (b) were stained with TMRM and MTG for 20 min simultaneously and images were taken with a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Fluorescence intensity was quantified from stacks of images through the entire thickness of cells which were from three independent experiments. MTG/TMRM is indicative of the level of depolarization (c). Values are mean ± SD (n = 3), *p < 0.05, compared with control.
Mentions: To examine the effect of spermidine on MMP, GM00637 and GM05849 cells were treated with 50 μM spermidine for 2 h, followed by incubation with TMRM and MTG (200 nM) simultaneously for 20 min in complete growth medium. Confocal microscopy images revealed an accumulation of MTG and TMRM in the mitochondria, and fluorescence intensity was quantified. In such MTG and TMRM co-loaded cells, depolarizing mitochondria turn from red to green, and the ratio of MTG/TMRM is indicative of the level of depolarization. The control GM00637 cells were in a highly polarized status, as indicated by the stronger fluorescence of TMRM and decreased fluorescence intensity ratio. In contrast, 50 μM spermidine significantly decreased the fluorescence of TMRM (Fig. 1a) and robustly increased fluorescence intensity ratio of MTG/TMRM (Fig. 1c), implying a depolarized status of mitochondria in GM00637 cells. The mitochondria uncoupler CCCP (carbonyl cyanide 3-chlorophenylhydrazone) as a positive control showed a more potent effect. In contrast, GM05849 cells displayed slight depolarization, even in the control group, and spermidine has no obvious effect on MMP as shown by the similar TMRM fluorescence to that of control cells and approximately equal fluorescence intensity ratio of MTG/TMRM (Fig. 1b,c).

Bottom Line: Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells.These results suggest that ATM drives the initiation of the mitophagic cascade.These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy.

View Article: PubMed Central - PubMed

Affiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.

ABSTRACT
The ATM (ataxia telangiectasia mutated) protein has recently been proposed to play critical roles in the response to mitochondrial dysfunction by initiating mitophagy. Here, we have used ATM-proficient GM00637 cells and ATM-deficient GM05849 cells to investigate the mitophagic effect of spermidine and to elucidate the role of ATM in spermdine-induced mitophagy. Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells. However, in GM05849 cells or GM00637 cells pretreated with the ATM kinase inhibitor KU55933, the expression of full-length PINK1 and the translocation of Parkin are blocked, and the colocalization of Parkin with either LC3 or PINK1 is disrupted. These results suggest that ATM drives the initiation of the mitophagic cascade. Our study demonstrates that spermidine induces mitophagy through ATM-dependent activation of the PINK1/Parkin pathway. These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy.

No MeSH data available.


Related in: MedlinePlus