Autophagy and mitochondrial alterations in human retinal pigment epithelial cells induced by ethanol: implications of 4-hydroxy-nonenal

Abstract

Retinal pigment epithelium has a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Autophagy has been shown to exert a protective effect in different cellular and animal models. Thus, in our model, EtOH treatment increases autophagy flux, in a concentration-dependent manner. Mitochondrial morphology seems to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. An increase in 2',7'-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others were confirmed. The characterization of these structures confirmed their nature as aggresomes. Hence, autophagy seems to have a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human retinal pigment epithelial cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases.

Figure 1

Proliferation and cell death of EtOH-treated and non-treated ARPE-19 cells. MTT assay after 24 h of EtOH exposure at different concentrations (a). Ki-67 immunocytochemistry analysis (b). MTT assay along the first 8 h after 600 mM EtOH exposure (c). Flow cytometry measures of V-FITC and PI (PE-A) of control (d), 600 mM (e) and 1200 mM (f) EtOH in ARPE-19 cells. TUNEL analysis of control (g) and 600 mM EtOH (h), cell nuclei stained with DAPI (blue) and TUNEL-positive cells (red). Graphic representation of the percentage of TUNEL-positive cells after EtOH treatment (i). Values are expressed as mean±S.E.M. (N=4). Statistical significance was determined by one-way ANOVA test and Bonferroni analysis (*P<0.05, **P<0.01 and ***P<0.001)

Figure 2

Pro-survival and pro-apoptotic mitochondrial protein expression. Protein expression analyzed by western blot of Bcl-2, Bax, uncleaved caspase-3 and α-actin in control and EtOH-treated ARPE-19 cells (a). Graphic representations of semiquantitative analysis of Bcl-2 (b), Bax (c) and uncleaved caspase-3 (d). Values are expressed as mean±S.E.M. (N=3). Statistical significance was determined by two-tailed Student's t-test (*P<0.05 and **P<0.01)

Figure 3

EtOH treatment increases autophagosome number in ARPE-19 cells. Protein expression analyzed by western blot of LC3-II, p62 and GAPDH (a). Graphic representation of semiquantitative analysis of LC3-II (b) and p62 (c). Cells were transfected with GFP-LC3 plasmid. Expression of GFP-LC3 dots were observed in control cells (d) and treated at 80 mM (e), 200 mM (f), 400 mM (g) and 600 mM EtOH (h). Quantification of GFP-LC3 dots per cell (i) and autophagic cells at different EtOH concentrations (j). Representative analysis of cell death (percentage of TUNEL-positive cells) challenged or not with 80, 200, 400 and 600 mM of EtOH and 3-MA (k). Values are expressed as mean±S.E.M. (N=3). Statistical significance was determined by a two-tailed Student's t-test (*P<0.05, **P<0.01 and ***P<0.001). Scale bars: 12 μm

Figure 4

EtOH exposure increases autophagic flux. Cells were transfected with GFP-LC3 plasmid and treated or not with CQ and EtOH at different concentrations (80, 200, 400 and 600 mM). Histogram shows the number of LC3-GFP dots per cell (a). A summary of the autophagic flux values for synthesis and degradation is shown in table b. Confocal microscopy images of ARPE-19 cells transfected with mRFP-GFP-LC3 showing autophagosomes (yellow dots) and autolysosomes (red dots) (c). Quantification of autolysosomes (d). Values are expressed as mean±S.E.M. (N=3). Statistical significance was determined by a two-tailed Student's t-test (*P<0.05 and **P<0.01). Scale bars: 12 μm

Figure 5

Mitochondrial fragmentation and distance to autophagosomes in ARPE-19 cells. ARPE-19 cells were transfected with pDsRed2-Mito plasmid. Mitochondrial morphology was observed in control (a) and 600 mM (b) EtOH in ARPE-19 cells. Histogram shows the percentage of cells with fragmented mitochondrial patterns (c). ARPE-19 cells were transfected with GFP-LC3 and pDsRed2-Mito plasmids. Expression of pDsRed2-Mito and GFP-LC3 plasmids was observed in control (d) and EtOH-treated cells at 80 mM (e), 200 mM (f), 400 mM (g) and 600 mM (h). Histogram shows the distance between mitochondria and autophagosome (i). Incubation with vinblastine of ARPE-19 cells. Colocalizations of mitochondria and LC3 in the presence of 600 mM EtOH in ARPE-19 cells (white arrows in j and k). Histogram shows the colocalizations per cell in the presence or absence of 10 μM vinblastine after 600 mM EtOH exposure (l). Values are expressed as mean±S.E.M. (N=3). Statistical significance was determined by a two-tailed Student's t-test (*P<0.05, **P<0.01 and ***P<0.001). Scale bars: 12 μm

Figure 6

Determination of DCFH fluorescence and lipid peroxidation: HAE and MDA. Quantification of DCFH fluorescence (a) and lipid peroxidation (b) in control and EtOH-treated ARPE-19 cells. Positive correlation between DCFH fluorescence and lipid peroxidation (c). Values are expressed as mean±S.E.M. (N=3). Statistical significance was determined by one-way ANOVA test and Bonferroni analysis (*P<0.05 and **P<0.01)

Figure 7

4-HNE aggresomes induced by EtOH and its degradation by autophagy in ARPE-19 cells. 4-HNE-positive cells (green) are indicated by white arrows in both untreated (a) and EtOH-treated ARPE-19 cells (b) after 24 h. Significant differences between control and treated cells are observed at different concentrations (c). 4-HNE-positive labeling was densely accumulated forming large ball-shaped aggregates in the vicinity of the nucleus (d, g and j). Ubiquitin (e), gamma tubulin (h) and HDAC6 (k) positive labeling (red). Colocalization of 4-HNE with ubiquitin (f), gamma tubulin (i) and HDAC6 (l). 4-HNE-positive labeling in the presence of the solvent DMSO (m) and Nocodazole (1 μg/ml) (n). 4-HNE aggregate colocalize with LC3 dots after EtOH exposure (o–q). Cells were challenged with or without EtOH and 3-MA, after fixation 4-HNE immunostaining was performed. Histogram shows the percentage of cells with 4-HNE inclusion with or without EtOH and 3-MA exposure (r). Values are expressed as mean±S.E.M. (N=3). Statistical significance was determined by a two-tailed Student's t-test (*P<0.05; **P<0.01; ***P<0.001). Scale bars on (a, b): 50 μm; (d–q): 12 μm

Figure 8

Optical and transmission electron microscopy in ARPE-19 cells. Nuclei of control cells with lax chromatin and multiple nucleoli (a). EtOH-treated cells at 600 mM show reduced cytoplasm and numerous vacuoles (arrows in b). Vacuoles can aggregate as clusters (arrows in c). Asterisk corresponds to a spherical area with less affinity for toluidine blue (d). Eventually, some EtOH-treated cells show absence of cytoplasm organelles (arrow in e). Under the electron microscope, control cells present long mitochondria with dense matrices and tubular crests (arrows in f). EtOH-treated cells present dilated mitochondria and spherical shape (arrows in g). Mitochondria inside vesicles next to ER were also observed (h). Vacuoles with heterogeneous content (arrows in i) and filamentous structures (arrows in j). Mitochondria show heterogeneous dense bodies and irregular crests (k) close to a double membrane vesicle. Scale bars: a–e: 25 μm; f–j: 0,5 μm; k: 1 μm