Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

Abstract

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H(2)O(2) in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.

Figure 1. Localized ER photodamage promotes ROS production and stimulation of autophagy in L929 cells. (A) Background-subtracted intracellular DCF fluorescence measured as a function of time for a population after photodynamic treatment with Hyp (25 nM) and (●) 7.8 J•cm⁻² or (○) 0 J•cm⁻² (control) of 572 ± 12-nm light. In the absence of photosensitizer or initial energy dose, negligible changes in DFC fluorescence were observed. The values plotted correspond to the intracellular DCF fluorescence values corrected by the background, since the oxidized probe gradually leaks out the cell with time. (B) Protein carbonylation quantification (nmol of carbonylated protein/mg protein) as a function of time after Hyp-PD stress with an energy dose of 1.1 J•cm⁻² (white light) compared with control. (C) LC3-I-to-LC3-II conversion determined by western blot analysis of whole cell lysates as a function of the initial energy dose (white light), detected 6 h after initial PD insult and quantification of the western blot analysis. Quantifications are performed as the ratio of LC3-II over actin. A representative western blot (n = 3) is shown. (Inset) Levels of ROS detected in the empty-vector transfected cells (i.e., Neo cells), measured by DCF-fluorescence using FACS, at different energy doses (white light). (D) Kinetics of GFP-LC3 puncta formation in a L929 Neo population challenged with 4.5 J•cm⁻² (●) of 572 ± 12-nm light delivered by microscopic PDT, monitored by CTLM, compared with the control (○), i.e., cells with Hyp, but with no initial excitation. (E) Confocal microscopy images corresponding to the formation of GFP-LC3 labeled autophagosomes in L929 Neo(NaSe) cells taken at 6 and 16 h after PD stress (1.1 J•cm⁻², white light) ([CQ] = 5 μM). Scale bar: 10 μm.

Figure 2. Spatio-temporal evolution of autophagy (A) Confocal microscopy images corresponding to the analysis of the autophagic flux for Neo(Nase) cells, taken at 6 and 16 h after PD stress (1.1 J•cm⁻², white light), and for the colocalization of the autophagosomes with ER- and mitochondria-fragments. Calreticulin (CALR) was labeled with the Texas Red fluorophore, whereas TOMM20 with AlexaFluor 647; for clarity of the merged images, images corresponding to TOMM20 are shown in false color red. Arrows indicate ring-shaped autophagosomes. Scale bar: 10 μm. (B) Fluorescence microscopy analysis of the autophagic flux for Neo(NaSe) cells. The graph plots the average of GFP-labeled autophagosomes per cell. Each bar includes the number of autophagosomes that contain/colocalize with ER and mitochondria fragments. Data represent mean ± SD of 4 independent experiments; in each experiment 30 cells were scored per condition.

Figure 3. Protecting mitochondria from oxidative damage blocks Hyp-PD apoptosis. (A) Hyp-induced PD cell death (trypan-blue-positive cells) in L929 Neo or L929 cells overexpressing different antioxidants as a function of the energy dose (white light), 24 after the initial PD insult. (*p < 0.05, **p < 0.01 and ***p < 0.001). (B) Effect of overexpression of SOD2 on the kinetics of cell death, measured by PI uptake using CTLM, as a function of energy dose (572 ± 12-nm light), compared with Neo cells. (C) Effect of antioxidants on cardiolipin peroxidation detected at 6 h after an energy dose of 1.1 J•cm⁻² (white light), measured as percentage of cell population showing loss of NAO fluorescence. (D) Measurement of caspase-3 activity detected at 6 h after an energy dose of 1.1 J•cm⁻² (white light), measured as fluorescence AMC released after caspase-3-fluorescent substrate cleavage. (*p < 0.05, **p < 0.01 and ***p < 0.001). L929 cells overexpressing the selenium dependent enzyme GPX4 were grown in media supplemented with sodium selenite (NaSe) required for the enzyme activity.

Figure 4A–C. Secondary ROS modulate organelle-specific autophagy. (A) Effect of overexpression of antioxidant enzymes on LC3-I-to-LC3-II conversion determined by western blot analysis of whole cell lysates detected 6 h after initial PD insult (1.1 J•cm⁻², white light). A representative western blot (n = 3) is shown. The upper panel is a composite of 3 different lanes (same exposure) of the same gel. (B) Effect of cytosolic membranes-associated and mitochondrial GPX4 (left) and SOD2 (right) on the kinetics of GFP-LC3 puncta formation, compared with the Neo(NaSe) population, in populations challenged with 4.5 J•cm⁻² of 572 ± 12-nm light delivered by microscopic PDT, monitored by CTLM. (C) (Left) Quantification of the number of GFP-labeled autophagosomes detected in the images (D), represented as the average of GFP-LC3 puncta per cell (*p < 0.05, **p < 0.01 and ***p < 0.001) and (right) quantification of the number of autophagosomes that contain/colocalize with ER and mitochondria fragments. Data represent mean ± SD of 4 independent experiments; in each experiment 30 cells were scored per condition.

Figure 4D and E. Secondary ROS modulate organelle-specific autophagy. (D) Confocal microscopy images corresponding to the effect of antioxidants on the number of GFP-labeled autophagosomes detected at 6 and 16 h after Hyp-PD stress as well as the colocalization of the autophagosomes with ER- and mitochondria fragments. Calreticulin (CALR) antibody was labeled with the Texas Red fluorophore, whereas TOMM20 antibody with AlexaFluor 647; for clarity of the merged images, images corresponding to TOMM20 are shown in false color red. (E) Confocal microscopy images corresponding to the changes in mitochondrial morphology that accompany transition from reticulophagy to mitophagy in Neo(NaSe) cells and protection displayed by GPX4. Scale bar: 10 μm.