Limits...
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 affected the accumulation of PINK1 and translocation of Parkin.The lysates of treated GM00637 cells were immunoblotted with anti-PINK1 or anti-Hsp60 (a loading control of mitochondrial proteins) (a). Alternatively, the expression of PINK1 in GM00637 cells was detected by immunofluorescence analysis and visualized with an Axio Observer Z1 fluorescence microscope (b). The translocation of Parkin was determined by the expression of Parkin after cell fractionation. GAPDH, a loading control of cytoplasmic proteins; Hsp60 and COX IV, loading controls of mitochondrial proteins. C: cytoplasmic fraction; M: mitochondrial fraction (c). The colocalizations of Parkin with either mitochondria (labeled by COX IV or Hsp60) (d), the autophagosomal marker LC3B (e), or PINK1 (f) were imaged using a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Bar graphs show the ratios of Parkin/mitochondria, Parkin/LC3B and Parkin/PINK1 colocalizations from 20–45 cells/condition compiled from three experiments. Values are mean ± SD, *p < 0.05 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835770&req=5

f3: Spermidine affected the accumulation of PINK1 and translocation of Parkin.The lysates of treated GM00637 cells were immunoblotted with anti-PINK1 or anti-Hsp60 (a loading control of mitochondrial proteins) (a). Alternatively, the expression of PINK1 in GM00637 cells was detected by immunofluorescence analysis and visualized with an Axio Observer Z1 fluorescence microscope (b). The translocation of Parkin was determined by the expression of Parkin after cell fractionation. GAPDH, a loading control of cytoplasmic proteins; Hsp60 and COX IV, loading controls of mitochondrial proteins. C: cytoplasmic fraction; M: mitochondrial fraction (c). The colocalizations of Parkin with either mitochondria (labeled by COX IV or Hsp60) (d), the autophagosomal marker LC3B (e), or PINK1 (f) were imaged using a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Bar graphs show the ratios of Parkin/mitochondria, Parkin/LC3B and Parkin/PINK1 colocalizations from 20–45 cells/condition compiled from three experiments. Values are mean ± SD, *p < 0.05 vs. control.

Mentions: PINK1 accumulates on the outer membranes of dysfunctional mitochondria when mitophagy is initiated. Under normal conditions, it is rapidly degraded. As shown in Fig. 3a, full-length PINK1 was induced after GM00637 cells were treated with 50 μM spermidine for 4, 8 and 12 h or CCCP for 4 and 8 h. This observation is confirmed by immunofluorescence analysis of PINK1 after treating the cells with 50 μM spermidine for 8 h or CCCP for 4 h (Fig. 3b). Subsequently, PINK1 induced the translocation of Parkin from the cytoplasm to the mitochondria as shown by the increased Parkin expression in mitochondria after cell fractionation (Fig. 3c) and colocalization of Parkin with aggregated mitochondria (labeled by COX IV) (Fig. 3d). Moreover, immunofluorescence analysis revealed that Parkin and endogenous LC3 also colocalized after spermidine (50 μM, 8 h) or CCCP treatments (50 μM, 4 h) (Fig. 3e). Notably, Parkin also colocalized with PINK1 (Fig. 3f). Quantitative analysis of Parkin/mitochondria, Parkin/LC3B and Parkin/PINK1 colocalizations confirmed the above observations.


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 affected the accumulation of PINK1 and translocation of Parkin.The lysates of treated GM00637 cells were immunoblotted with anti-PINK1 or anti-Hsp60 (a loading control of mitochondrial proteins) (a). Alternatively, the expression of PINK1 in GM00637 cells was detected by immunofluorescence analysis and visualized with an Axio Observer Z1 fluorescence microscope (b). The translocation of Parkin was determined by the expression of Parkin after cell fractionation. GAPDH, a loading control of cytoplasmic proteins; Hsp60 and COX IV, loading controls of mitochondrial proteins. C: cytoplasmic fraction; M: mitochondrial fraction (c). The colocalizations of Parkin with either mitochondria (labeled by COX IV or Hsp60) (d), the autophagosomal marker LC3B (e), or PINK1 (f) were imaged using a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Bar graphs show the ratios of Parkin/mitochondria, Parkin/LC3B and Parkin/PINK1 colocalizations from 20–45 cells/condition compiled from three experiments. Values are mean ± SD, *p < 0.05 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Spermidine affected the accumulation of PINK1 and translocation of Parkin.The lysates of treated GM00637 cells were immunoblotted with anti-PINK1 or anti-Hsp60 (a loading control of mitochondrial proteins) (a). Alternatively, the expression of PINK1 in GM00637 cells was detected by immunofluorescence analysis and visualized with an Axio Observer Z1 fluorescence microscope (b). The translocation of Parkin was determined by the expression of Parkin after cell fractionation. GAPDH, a loading control of cytoplasmic proteins; Hsp60 and COX IV, loading controls of mitochondrial proteins. C: cytoplasmic fraction; M: mitochondrial fraction (c). The colocalizations of Parkin with either mitochondria (labeled by COX IV or Hsp60) (d), the autophagosomal marker LC3B (e), or PINK1 (f) were imaged using a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Bar graphs show the ratios of Parkin/mitochondria, Parkin/LC3B and Parkin/PINK1 colocalizations from 20–45 cells/condition compiled from three experiments. Values are mean ± SD, *p < 0.05 vs. control.
Mentions: PINK1 accumulates on the outer membranes of dysfunctional mitochondria when mitophagy is initiated. Under normal conditions, it is rapidly degraded. As shown in Fig. 3a, full-length PINK1 was induced after GM00637 cells were treated with 50 μM spermidine for 4, 8 and 12 h or CCCP for 4 and 8 h. This observation is confirmed by immunofluorescence analysis of PINK1 after treating the cells with 50 μM spermidine for 8 h or CCCP for 4 h (Fig. 3b). Subsequently, PINK1 induced the translocation of Parkin from the cytoplasm to the mitochondria as shown by the increased Parkin expression in mitochondria after cell fractionation (Fig. 3c) and colocalization of Parkin with aggregated mitochondria (labeled by COX IV) (Fig. 3d). Moreover, immunofluorescence analysis revealed that Parkin and endogenous LC3 also colocalized after spermidine (50 μM, 8 h) or CCCP treatments (50 μM, 4 h) (Fig. 3e). Notably, Parkin also colocalized with PINK1 (Fig. 3f). Quantitative analysis of Parkin/mitochondria, Parkin/LC3B and Parkin/PINK1 colocalizations confirmed the above observations.

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