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.

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Regulated necrosis occurs in various pathophysiological conditions under oxidative stress. Here, we report that receptor-interacting protein kinase 1 (RIPK1), a key player in one type of regulated necrosis (necroptosis), also participates in another type of poly (ADP-ribose) polymerase 1 (PARP1)-dependent regulated necrosis (parthanatos). Various biological signatures of parthanatos were significantly attenuated in Ripk1 mouse embryonic fibroblasts, including PARylation, nuclear translocation of apoptosis-inducing factor, and PARP1-dependent cell death under HO exposure. Hence, we investigated whether RIPK1 regulates the activity of PARP1. RIPK1 activated PARP1 via an interaction with the catalytic domain of PARP1 in the nucleus. Of note, both wild type and kinase-dead mutant RIPK1 induced PARP1 activation and led to PARP1-mediated cell death upon HO insult, demonstrating the kinase-independent regulation of RIPK1 in PARP1 activation. Collectively, our results demonstrate the existence of a kinase-independent role of nuclear RIPK1 in the regulation of PARP1.

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