Iduna protects HT22 cells from hydrogen peroxide-induced oxidative stress through interfering poly(ADP-ribose) polymerase-1-induced cell death (parthanatos)

Abstract

Oxidative stress-induced cell death is common in many neurological diseases. However, the role of poly(ADP-ribose) polymerase-1-induced cell death (parthanatos) has not been fully elucidated. Here, we found that hydrogen peroxide (H₂O₂) could lead to PARP-1 activation and apoptosis-inducing factor nuclear translocation in a concentration dependent manner. Iduna, as a novel regulator of parthanatos, was also induced by H₂O₂. Down-regulation of Iduna by genetic ablation promoted H₂O₂-induced cell damage. Up-regulation of Iduna reduced the loss of mitochondrial potential and ATP and NAD + production, but did not affect the mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio, and Caspase-9/Caspase-3 activity. In contrast, overexpression of Iduna inhibited activation of PARP-1 and nuclear translocation of AIF. Further study showed that PARP-1 specific inhibitor, DPQ, blocked the protective effect of Iduna against H₂O₂-induced oxidative stress. Moreover, in the presence of proteasome inhibitor (MG-132) or ubiquitin E1 inhibitor (PYR-41), protective effect of Iduna was significantly weaken. These results indicate that Iduna acts as a potential antioxidant by improving mitochondrial function and inhibiting oxidative stress-induced parthanatos, and these protective effects are dependent on the involvement of ubiquitin–proteasome system.

Introduction

Reactive oxygen species (ROS), including superoxide, hydroxyl radicals and peroxides such as hydrogen peroxide (H₂O₂), are formed by incomplete one-electron reduction of oxygen in normal or aberrant metabolic processes, which can disrupt cellular function and membrane integrity by attacking proteins, deoxynucleic acids, and lipid membranes [1], [2]. Overproduction of ROS is an essential feature of cellular damage following oxidative stress. Accumulating studies have shown that oxidative stress-induced cell damage is common in the etiology of several neurobiological disorders such as stroke [3], [4], traumatic brain injury [5], [6], Alzheimer's disease [7], [8], and Parkinson's disease [9], [10]. In these pathological processes, oxidative stress leads to many biological consequences including cell death [11]. Compared to ROS-induced apoptosis and necrosis, the mechanism of ROS induced non-apoptotic/necrotic cell death has not been well investigated.

Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), as a nuclear enzyme, plays an important role in DNA repair and perception of DNA damage [12], [13]. In some cases, however, PARP-1 is also involved in death program. PARP-1-dependent cell death is different from classical subtypes of cell death, such as apoptosis, necrosis, and autophagy [12], [13], which is defined as parthanatos [14]. Although parthanatos is similar to apoptosis, it does not cause apoptotic body formation or small scale DNA fragmentation and cannot be rescued by pan-caspase inhibitor, such as z-VAD-fmk and boc-aspartyl-fmk (BAF) [15]. In addition to PARP-1, apoptosis inducing factor (AIF) is also a key factor that mediates parthanatos. As a mitochondrial flavoprotein, AIF releases into cytoplasm following dissipation of mitochondrial membrane potential caused by PARP-1 activation, and enters the nucleus to induce parthanatos [12]. Inhibition of PARP-1 and AIF by pharmacological inhibitor or gene deletion has a significantly protective effect in many oxidative stress-associated cell injury, suggesting that parthanatos may play a pivotal role in oxidative stress [15], [16].

PAR modification (PARsylation) of proteins by PARP-1 is an important cellular signaling during parthanatos [17], [18], [19]. Iduna (encoded by Rnf146) is a novel gene (RING) finger protein that contains a WWE domain. Iduna acts as a PAR-dependent E3 ligase that regulates binding and ubiquitination of PARsylated and PAR binding proteins via its PAR binding motif (PBM) [20]. Iduna was recently shown to protect brain from glutamate excitotoxicity by inhibition of parthanatos [21]. However, it is unknown whether Iduna could interfere oxidative stress-induced parthanatos. Therefore, in this study, we investigated the effect of Iduna on the H₂O₂-induced oxidative stress and parthanatos.