Abstract
Cell death of retinal pigment epithelium (RPE) is characterized as an essential late-stage phenomenon of dry age-related macular degeneration (AMD). The aim of this study was to elucidate the molecular mechanism underlying RPE cell death after exposure to oxidative stress, which occurs often because of the anatomical location of RPE cells. ARPE-19, an established RPE cell line, exhibited necrotic features involving poly (ADP-ribose) polymerase-1 (PARP-1) activation in response to hydrogen peroxide (H2O2). ARPE-19 cells were resistant to H2O2 when PARP-1 was depleted using siRNA or inhibited by a pharmacological inhibitor of PARP-1, olaparib. Our data suggest a causal relationship between PARP-1 activation and ARPE-19 cell death in response to H2O2. Next, we investigated downstream molecular events in PARP-1 activation. Increased mitochondrial depolarization, mitochondrial fission and alterations of the cellular energy dynamics with reduced NAD+ and ATP were observed in H2O2-treated ARPE-19 cells. H2O2-triggered mitochondrial dysfunction was inhibited by olaparib. Nevertheless, translocation of apoptosis-inducing factor (AIF), a biochemical signature for PARP-1-dependent cell death (parthanatos), was not observed in our study. Moreover, the depletion of AIF did not affect the amplitude of cell death, demonstrating the lack of a role for AIF in the death of ARPE-19 cells in response to H2O2. This feature distinguishes the type of death observed in this study from canonical parthanatos. Next, we examined the in vivo role of PARP-1 in a dry AMD animal model system. Histological analysis of the outer nuclear layer in the mouse retina revealed protection against sodium iodate (SI) following treatment with olaparib. Moreover, retina fundus and electroretinograms also confirmed such a protective effect in the SI-treated rabbit. Collectively, we report that AIF-independent PARP-1-dependent necrosis constitutes a major mechanism of RPE cell death leading to retinal degeneration in dry AMD.
Highlights
Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly.[1,2] AMD is classified into wet and dry forms; the dry form is more common than the wet form
Because ARPE-19 cells are proximal to the choroid, we examined cell death following exposure to H2O2 in other cells distal to the choroid, retinal ganglion cells (RGC-5)
RGC-5 cells were more sensitive to H2O2 compared with ARPE-19 cells (Supplementary Figure 2), suggesting that distance from the choroid might correlate with retinal cellular sensitivities to oxygen
Summary
Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly.[1,2] AMD is classified into wet and dry forms; the dry form is more common than the wet form. Retinal pigment epithelium (RPE), a monolayer of pigmented cells, is located between photoreceptor cells and Bruch’s membrane and maintains retinal homeostasis via the transport of nutrients and waste, thereby protecting photoreceptor cells.[8] The pathogenesis of dry AMD involves oxidative stress, mitochondrial dysfunction and inflammation.[9,10,11,12,13] RPE cells are prone to exposure to high-energy light and rich polyunsaturated fatty acids, which are readily oxidized through photonic activation Due to their anatomical localization and metabolic function, RPE cells are continuously exposed to chronic and cumulative oxidative stress and are most severely damaged in progressive dry AMD.[14] RPE degeneration impairs retinal protective measures for the photoreceptor cells and results in their progressive death. Necrotic death in the retinal cells has been studied extensively, and a combination therapy of apoptotic and necrotic inhibitors seems to be promising for the protection of retinal cells
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