Retinal pigment epithelial cells undergoing mitotic catastrophe are vulnerable to autophagy inhibition

The increased mitochondrial DNA damage leads to altered functional capacities of retinal pigment epithelial (RPE) cells. A previous study showed the increased autophagy in RPE cells caused by low concentrations of rotenone, a selective inhibitor of mitochondrial complex I. However, the mechanism by which autophagy regulates RPE cell death is still unclear. In the present study, we examined the mechanism underlying the regulation of RPE cell death through the inhibition of mitochondrial complex I. We report herein that rotenone induced mitotic catastrophe (MC) in RPE cells. We further observed an increased level of autophagy in the RPE cells undergoing MC (RPE-MC cells). Importantly, autophagy inhibition induced nonapoptotic cell death in RPE-MC cells. These findings indicate that autophagy has a pivotal role in the survival of RPE-MC cells. We next observed PINK1 accumulation in the mitochondrial membrane and parkin translocation into the mitochondria from the cytosol in the rotenone-treated RPE-MC cells, which indicates that increased mitophagy accompanies MC in ARPE-19 cells. Noticeably, the mitophagy also contributed to the cytoprotection of RPE-MC cells. Although there might be a significant gap in the roles of autophagy and mitophagy in the RPE cells in vivo, our in vitro study suggests that autophagy and mitophagy presumably prevent the RPE-MC cells from plunging into cell death, resulting in the prevention of RPE cell loss.Cell death is a process that is both complementary and antagonistic to cell division in order to maintain tissue homeostasis, and cell death has a pivotal role in several physiological processes and diseases.¹ The most extensively studied category, apoptosis, is characterized by the massive activation of caspases, chromatin condensation, and a reduction in cell volume. Necrosis is characterized by an increase in cell volume, the swelling of organelles, and the rupture of the plasma membrane and is largely considered an accidental, uncontrolled type of cell death.² Necroptosis is a regulated necrotic cell death that is triggered by broad caspase inhibition in the presence of death receptor ligands and is characterized by necrotic cell death morphology. Autophagy is a degradative lysosomal pathway that is characterized by the accumulation of cytoplasmic material in the vacuoles for bulk degradation by lysosomal enzymes. Although autophagy has a pivotal role in cell survival, increased autophagic activity is often associated with cell death.² Mitotic catastrophe (MC) is a type of cell death that results from a failure to undergo mitosis after DNA damage, leading to tetraploidy or endopolyploidy. Cells undergoing MC usually form large cells with multiple micronuclei.³Retinal pigment epithelial (RPE) cells form a single layer of cells adjacent to the photoreceptor outer segment (POS) of the retina, and these cells have pivotal roles in the maintenance of the POS cells. RPE cell death is a significant factor in several ocular pathological conditions, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). AMD is a progressive degeneration of the macula and is broadly classified as either dry or wet. The dry form of AMD is more common and is characterized by the presence of drusen in the macula. Mitochondrial DNA variants of respiratory complex I are associated with an increased risk of AMD.⁴ Because damage to and the death of RPEs are crucial and perhaps even triggering events in AMD,⁵ protection against RPE cell death could delay the onset of AMD. Conversely, RPE cells significantly contribute to the formation of the epiretinal membrane in PVR. Thus, the induction of RPE cell death in the epiretinal membranes could be a new approach to inhibit cellular proliferation in PVR.⁶ Most studies concerning RPE cell death in the context of these ocular pathological conditions have focused on two types of cell death, apoptosis and necrosis.Although advances have been made in the understanding of RPE cell death, there is little information concerning the role of autophagy in the RPE cell death associated with these ocular pathological conditions. Each day, RPE cells phagocytose and digest the distal parts of the POS, which are ultimately degraded in the lysosomes.^{7, 8, 9} The interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.⁹ In the RPE cells of old eyes, this physiological lysosomal load may be further increased to remove damaged material, and insufficient digestion of the damaged macromolecules and organelles by old RPE cells will lead to progressive accumulation of biological ‘garbage'', such as lipofuscin.¹⁰ Thus, abnormalities in the lysosome-dependent degradation of shed POS debris can contribute to the degeneration of RPE cells. A previous study suggested that age-related changes in autophagy may underlie the genetic susceptibility found in AMD patients and may be associated with the pathogenesis of AMD.¹⁰ However, the mechanism by which autophagy regulates RPE cell demise in AMD is still unclear. The role of autophagy in the proliferation of the RPE cells in PVR and its regulation as a therapeutic strategy for PVR have not been documented yet.Rotenone, a natural isoflavonoid produced by plants, is a selective and stoichiometric inhibitor of mitochondrial complex I.¹¹ More specifically, rotenone blocks NADH oxidation by the NADH-ubiquinone oxide reductase enzymatic complex, which results in the inhibition of mitochondrial respiration and a reduction in ATP synthesis.^{12, 13, 14} Rotenone treatment also results in the production of reactive oxygen species (ROS), eventually leading to cell death.^{15, 16} Several studies have shown that rotenone causes an accumulation of autophagic vacuoles, perhaps in response to the inhibition of mitochondrial function and the generation of oxidative stress.^{17, 18, 19} Irrespective of that activity of rotenone has been lively studied in various cells, the effect of rotenone on RPE cells has rarely been studied. A previous study using an in vitro system revealed that low concentrations of rotenone resulted in mtDNA damage in RPE cells and suggested that the increased autophagy caused by rotenone treatment in aged RPE cells could affect the formation of drusen and AMD.¹⁰ However, the mechanism by which rotenone regulates RPE cell demise remains unclear.We undertook this study to elucidate the mechanism regulating the demise of RPE cells that are damaged by mitochondrial complex I inhibition. We report herein that rotenone induces MC in RPE cells. Additionally, we show that RPE cells undergoing mitotic catastrophe (RPE-MC cells) induced by mitochondrial complex I inhibition are vulnerable to autophagy inhibition.