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, 8 (10), e3097

Superoxide Drives Progression of Parkin/PINK1-dependent Mitophagy Following Translocation of Parkin to Mitochondria

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Superoxide Drives Progression of Parkin/PINK1-dependent Mitophagy Following Translocation of Parkin to Mitochondria

Bin Xiao et al. Cell Death Dis.

Erratum in

Abstract

Reactive oxygen species (ROS) and mitophagy are profoundly implicated in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease (PD). Several studies have suggested that ROS are not involved in mitochondrial translocation of Parkin which primes mitochondria for autophagic elimination. However, whether ROS play a role in the execution of mitophagy is unknown. In the present study, we show that carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment induced both mitochondrial depolarization and generation of ROS that were needed for the mitophagy process. Cells failed to proceed to complete mitophagy if CCCP treatment was discontinued even after recruitment of Parkin and autophagy machinery to mitochondria. Notably, treatment of pro-oxidant was able to replace CCCP treatment to take mitophagy forward, while it alone was insufficient to induce translocation of Parkin to mitochondria or autophagic clearance of mitochondria. In addition, an SOD mimetic that attenuated the superoxide level suppressed mitophagy, while an SOD inhibitor accumulated cellular superoxide and promoted mitophagy. Furthermore, blockage of the p38 signaling pathway inhibited mitophagy induced by ROS, suggesting that it may contribute to the activation of ROS-mediated mitophagy. Together, our study sheds light on the link between ROS and mitophagy at a molecular level, and suggests the therapeutic potential of regulating mitophagy through the superoxide-p38-mitophagy axis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CCCP treatment induces recruitment of Parkin and autophagy component to mitochondria and facilitates execution of mitophagy. (a) Representative images of GFP-Parkin expressed HeLa cells treated with CCCP for the indicated times. Cells were stained for p62 (red) and Tom20 (purple). Solid arrowheads indicate mitochondria with both Parkin and p62. Empty arrowheads indicate mitochondria with Parkin but without p62. (b) HeLa cells stably expressing HA-Parkin transfected with GFP-LC3 were treated with DMSO or 10 μM CCCP for 2 h. Cells were stained for Tom20 (red) and HA (purple). Line scans next to the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the lines drawn in the images. (c and f) HeLa cells stably expressing HA-Parkin were treated with CCCP as depicted in (d and g). Cells were stained for Tom20 (green), hsp60 (red) and HA (purple). The arrows in (c) indicate representative cells with reduced mitochondrial mass. The arrows in (f) indicate representative cells with cleared mitochondrial mass. The average percentages of cells with normal, reduced or cleared mitochondrial mass from (c) and (f) were presented in (e) and (h). The error bars represent S.E.M. from three independent experiments; at least 100 cells were analyzed per experiment. Scale bars, 10 μm
Figure 1
Figure 1
CCCP treatment induces recruitment of Parkin and autophagy component to mitochondria and facilitates execution of mitophagy. (a) Representative images of GFP-Parkin expressed HeLa cells treated with CCCP for the indicated times. Cells were stained for p62 (red) and Tom20 (purple). Solid arrowheads indicate mitochondria with both Parkin and p62. Empty arrowheads indicate mitochondria with Parkin but without p62. (b) HeLa cells stably expressing HA-Parkin transfected with GFP-LC3 were treated with DMSO or 10 μM CCCP for 2 h. Cells were stained for Tom20 (red) and HA (purple). Line scans next to the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the lines drawn in the images. (c and f) HeLa cells stably expressing HA-Parkin were treated with CCCP as depicted in (d and g). Cells were stained for Tom20 (green), hsp60 (red) and HA (purple). The arrows in (c) indicate representative cells with reduced mitochondrial mass. The arrows in (f) indicate representative cells with cleared mitochondrial mass. The average percentages of cells with normal, reduced or cleared mitochondrial mass from (c) and (f) were presented in (e) and (h). The error bars represent S.E.M. from three independent experiments; at least 100 cells were analyzed per experiment. Scale bars, 10 μm
Figure 2
Figure 2
Pro-oxidant is able to push forward autophagic clearance of mitochondria. (a) HeLa cells stably expressing HA-Parkin were treated with DMSO, CCCP, antimycin A or H2O2 as depicted in (b). Cells were stained for hsp60 (red) and HA (purple). (c) Cells from (a) were counted as in Figures 1e and h. The error bars represent S.E.M. from three independent experiments. (d) HeLa cells expressing HA-Parkin were treated as indicated and immunoblotted for COX IV and GAPDH. Bottom: Average protein levels of COX IV relative to GAPDH from three independent experiments. (e) HeLa cells expressing HA-Parkin were transfected with scramble or FIP200 siRNA. Seventy-two hours post-transfection, cells were treated with DMSO or CCCP/antimycin A as in (a) for 24 h. Cells were stained for HA (green) and hsp60 (red). NS, not significant; *P<0.05; **P<0.01 (t-test)
Figure 3
Figure 3
Pro-oxidant alone or its priming has little effect on mitophagy. (a) HeLa cells stably expressing HA-Parkin treated with DMSO, H2O2 (100 μM), antimycin A (2 μM) or CCCP (10 μM) for 2 h. Cells were stained for hsp60 (red) and HA (purple). (b) HeLa cells stably expressing HA-Parkin treated with DMSO, H2O2 (100 μM), antimycin A (2 μM) or CCCP (10 μM) for 24 h. Cells were stained for HA (green) and hsp60 (red). GFP-Parkin-overexpressing cells were treated with DMSO or antimycin A for 2 h prior to CCCP treatment for 4 h (c) or 24 h (d). Cells were stained with hsp60 (white). Distribution of Parkin and reduction in mitochondrial mass were quantified on the right. White arrowhead indicates cytosolic Parkin; yellow arrows indicate punctate-shaped Parkin; yellow arrowheads indicate Parkin aggregates. NS, not significant
Figure 4
Figure 4
Superoxide promotes the progression of mitophagy. HeLa cells stably expressing HA-Parkin treated with DMSO, H2O2 (100 μM), antimycin A (2 μM) or CCCP (10 μM) for 2 h. Cells were stained with JC-1 (a) or DHE (b) and subject to flow cytometric analysis. (c) Cells were stained with DCF and washed out prior to treatment of DMSO, H2O2 (100 μM), antimycin A (2 μM) or CCCP (10 μM) for 1 h. Cells were collected and subjected to flow cytometric analysis. HeLa cells stably expressing HA-Parkin were treated with DMSO (d), CCCP for 24 h (e) or CCCP for 2 h followed by the treatment of antimycin A for 22 h (f) in the presence of vehicle, catalase (1000 U/ml), mnTBAP (200 μM) or NAC (2 mM) for the last 22 h. Cells were stained for HA (green) and hsp60 (red). (g) Removal of mitochondrial mass was counted in the cells treated as in (df). (h) Cells treated with DMSO, antimycin A or CCCP in the presence of vehicle, catalase (1000 U/ml), mnTBAP (200 μM) or NAC (2 mM) for 2 h were stained with DHE and subject to flow cytometric analysis. (i) HeLa cells were treated with CCCP (10 μM) for 2 h prior to the treatment of vehicle or DETC (40 μM) for the indicated times. Cells were stained with DHE and subject to flow cytometric analysis. Comparisons of DHE intensity between the cells treated with DETC and the cells treated with vehicle were performed using t-test. (j) Cells were treated with CCCP (10 μM) for 2 h prior to the treatment of DMSO or DETC (40 μM) for 22 h. Cells were immunoblotted for Tim23 and GAPDH. Bottom: Average protein levels of Tim23 relative to GAPDH from three independent experiments. NS, not significant; *P<0.05; **P<0.01 (t-test)
Figure 5
Figure 5
Neither AMPK nor ERK1/2 signaling pathway is responsible for the progression of mitophagy induced by ROS outburst. (a) HeLa cells stably expressing HA-Parkin were treated with CCCP (10 μM), H2O2 (100 μM) or antimycin A (2 μM) for the indicated times. Cells were immunoblotted for phosphorylated AMPKα. (b) HeLa cells stably expressing HA-Parkin were transfected with scramble or PRKAA1 siRNA. Seventy-two hours post-transfection, cells were treated with DMSO or CCCP/antimycin A as in Figure 2b for 24 h. Cells were stained for HA (green) and hsp60 (red). (c) Cells transfected as in (b) were immunoblotted for AMPKα. (d) Cells treated as in (a) were immunoblotted for phosphorylated ERK1/2 and p38. Bottom: Average protein levels of phosphorylated ERK1/2 and phosphorylated p38 relative to GAPDH from three independent experiments. (e) HeLa cells stably expressing HA-Parkin were treated with DMSO, CCCP (10 μM) in the absence or presence of U0126 for 2 h and subject to western blot analysis for phosphorylated ERK1/2. (f) HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 μM) in the presence or absence of U0126 for 24 h. Cells were stained for HA (green) and hsp60 (red). NS, not significant; *P<0.05; **P<0.01 (t-test)
Figure 6
Figure 6
Execution of mitophagy is dependent on p38 signaling. (a) HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP in the absence or presence of 10 μM (fourth row) or 20 μM (fifth row) SB203580 for 24 h and stained for HA (green) and hsp60 (red). (b) Cells with cleared mitochondria in (a) were quantified. (c) HeLa cells stably expressing HA-Parkin were treated with CCCP and SB203580 as indicated, followed by immunoblotting with the indicated antibodies. (d) Cells treated with CCCP and SB203580 as indicated were immunoblotted for COX IV and GAPDH. Bottom: Average protein levels of COX IV relative to GAPDH from three independent experiments. (e) HeLa cells stably expressing HA-Parkin were treated with DMSO, CCCP (10 μM) for 4 h or CCCP (10 μM) for 2 h followed by the treatment of DMSO, H2O2 or antimycin A for 2 h. Cells were collected and immunoblotted for endogenous p38 and phosphorylated p38. Bottom: Average protein levels of phosphorylated p38 relative to p38 from three independent experiments. (f) A schematic diagram of the role of superoxide-induced p38 activation in the execution of mitophagy. Subsequent to the recruitment of Parkin and LC3 to damaged mitochondria, production of superoxide activates the p38 signaling pathway that is required for the execution of mitophagy, leading to degradation of mitochondria in autolysosome (left path). In the absence of activation of the p38 signaling pathway by superoxide, mitophagy is arrested and Parkin dissociates back to the cytosol (right path). *P<0.05; **P<0.01 (t-test).

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