Activated SIRT1 contributes to DPT-induced glioma cell parthanatos by upregulation of NOX2 and NAT10.

Parthanatos is a type of programmed cell death dependent on hyper-activation of poly (ADP-ribose) polymerase 1 (PARP-1). SIRT1 is a highly conserved nuclear deacetylase and often acts as an inhibitor of parthanatos by deacetylation of PARP1. Our previous study showed that deoxypodophyllotoxin (DPT), a natural compound isolated from the traditional herb Anthriscus sylvestris, triggered glioma cell death via parthanatos. In this study, we investigated the role of SIRT1 in DPT-induced human glioma cell parthanatos. We showed that DPT (450 nmol/L) activated both PARP1 and SIRT1, and induced parthanatos in U87 and U251 glioma cells. Activation of SIRT1 with SRT2183 (10 µmol/L) enhanced, while inhibition of SIRT1 with EX527 (200 µmol/L) or knockdown of SIRT1 attenuated DPT-induced PARP1 activation and glioma cell death. We demonstrated that DPT (450 nmol/L) significantly decreased intracellular NAD+ levels in U87 and U251 cells. Further decrease of NAD+ levels with FK866 (100 µmol/L) aggravated, but supplement of NAD+ (0.5, 2 mmol/L) attenuated DPT-induced PARP1 activation. We found that NAD+ depletion enhanced PARP1 activation via two ways: one was aggravating ROS-dependent DNA DSBs by upregulation of NADPH oxidase 2 (NOX2); the other was reinforcing PARP1 acetylation via increase of N-acetyltransferase 10 (NAT10) expression. We found that SIRT1 activity was improved when being phosphorylated by JNK at Ser27, the activated SIRT1 in reverse aggravated JNK activation via upregulating ROS-related ASK1 signaling, thus forming a positive feedback between JNK and SIRT1. Taken together, SIRT1 activated by JNK contributed to DPT-induced human glioma cell parthanatos via initiation of NAD+ depletion-dependent upregulation of NOX2 and NAT10.

The molecular mosaic of regulated cell death in the cardiovascular system.

Cell death is now understood to be a highly regulated process that contributes to normal development and tissue homeostasis, alongside its role in the etiology of various pathological conditions. Through detailed molecular analysis, we have come to know that all cells do not always die in the same way, and that there are at least 7 processes involved, including: apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, and autophagy-mediated cell death. These processes act as pieces in the mosaic of cardiomyocyte cell death, which come together depending on context and stimulus. This review details each individual process, as well as highlights how they come together to produce various cardiac pathologies. By knowing how the pieces go together we can aim towards the development of efficacious therapeutics, which will enable us to prevent cardiomyocyte loss in the face of stress, both reducing mortality and improving quality of life.

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

Programmed Non-Apoptotic Cell Death in Hereditary Retinal Degeneration: Crosstalk between cGMP-Dependent Pathways and PARthanatos?

Programmed cell death (PCD) is a highly regulated process that results in the orderly destruction of a cell. Many different forms of PCD may be distinguished, including apoptosis, PARthanatos, and cGMP-dependent cell death. Misregulation of PCD mechanisms may be the underlying cause of neurodegenerative diseases of the retina, including hereditary retinal degeneration (RD). RD relates to a group of diseases that affect photoreceptors and that are triggered by gene mutations that are often well known nowadays. Nevertheless, the cellular mechanisms of PCD triggered by disease-causing mutations are still poorly understood, and RD is mostly still untreatable. While investigations into the neurodegenerative mechanisms of RD have focused on apoptosis in the past two decades, recent evidence suggests a predominance of non-apoptotic processes as causative mechanisms. Research into these mechanisms carries the hope that the knowledge created can eventually be used to design targeted treatments to prevent photoreceptor loss. Hence, in this review, we summarize studies on PCD in RD, including on apoptosis, PARthanatos, and cGMP-dependent cell death. Then, we focus on a possible interplay between these mechanisms, covering cGMP-signaling targets, overactivation of poly(ADP-ribose)polymerase (PARP), energy depletion, Ca2+-permeable channels, and Ca2+-dependent proteases. Finally, an outlook is given into how specific features of cGMP-signaling and PARthanatos may be targeted by therapeutic interventions.

Flow cytometric detection of hyper-polarized mitochondria in regulated and accidental cell death processes.

Shikonin induced necroptosis in Jurkat cells were identified flow cytometrically by the up-regulation of RIP3 in live cells and that a proportion of these cells underwent other forms of regulated cell death (RCD) which included parthanatos (< 10%), or cleaved PARP (< 10%) and DNA Damage (> 30%). Live necroptotic cells also possessed functioning mitochondria with hyper-polarized mitochondria membrane potential and generated a fivefold increase in cellular reactive oxygen species (ROS) which was resistant to inhibition by zVAD and necrostatin-1 (Nec-1). After loss of plasma membrane integrity these dead necroptotic cells then showed a higher incidence of parthanatos (> 40%), or cleaved PARP (> 15%) but less DNA Damage (< 15%). Inhibition of shikonin induced apoptosis and necroptosis by zVAD and Nec-1 respectively resulted in live necroptotic cells with an increased incidence of cleaved PARP and reduced levels of DNA Damage respectively. Dead necroptotic cells then showed a reduced incidence of parthanatos and DNA Damage after inhibition by zVAD and Nec-1 respectively. A high proportion of these dead necroptotic cells (30%) which lacked plasma membrane integrity also displayed functioning hyper-polarized mitochondria with high levels of cellular ROS and thus had the capacity to influence the outcome of RCD processes rather than just been the end product of cell death, the necrotic cell. Flow cytometry can thus measure multiple forms of RCD and the level of cellular ROS and MMP which highlights the inter-connection between cell death processes and that a single cell may simultaneously display multiple forms of RCD.

Drug antagonism and single-agent dominance result from differences in death kinetics.

Cancer treatment generally involves drugs used in combinations. Most previous work has focused on identifying and understanding synergistic drug-drug interactions; however, understanding antagonistic interactions remains an important and understudied issue. To enrich for antagonism and reveal common features of these combinations, we screened all pairwise combinations of drugs characterized as activators of regulated cell death. This network is strongly enriched for antagonism, particularly a form of antagonism that we call 'single-agent dominance'. Single-agent dominance refers to antagonisms in which a two-drug combination phenocopies one of the two agents. Dominance results from differences in cell death onset time, with dominant drugs acting earlier than their suppressed counterparts. We explored mechanisms by which parthanatotic agents dominate apoptotic agents, finding that dominance in this scenario is caused by mutually exclusive and conflicting use of Poly(ADP-ribose) polymerase 1 (PARP1). Taken together, our study reveals death kinetics as a predictive feature of antagonism, due to inhibitory crosstalk between cell death pathways.

PARP1 inhibition alleviates injury in ARH3-deficient mice and human cells.

Poly(ADP-ribosyl)ation refers to the covalent attachment of ADP-ribose to protein, generating branched, long chains of ADP-ribose moieties, known as poly(ADP-ribose) (PAR). Poly(ADP-ribose) polymerase 1 (PARP1) is the main polymerase and acceptor of PAR in response to DNA damage. Excessive intracellular PAR accumulation due to PARP1 activation leads cell death in a pathway known as parthanatos. PAR degradation is mainly controlled by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor hydrolase 3 (ARH3). Our previous results demonstrated that ARH3 confers protection against hydrogen peroxide (H2O2) exposure, by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) nuclear translocation. We identified a family with an ARH3 gene mutation that resulted in a truncated, inactive protein. The 8-year-old proband exhibited a progressive neurodegeneration phenotype. In addition, parthanatos was observed in neurons of the patient's deceased sibling, and an older sibling exhibited a mild behavioral phenotype. Consistent with the previous findings, the patient's fibroblasts and ARH3-deficient mice were more sensitive, respectively, to H2O2 stress and cerebral ischemia/reperfusion-induced PAR accumulation and cell death. Further, PARP1 inhibition alleviated cell death and injury resulting from oxidative stress and ischemia/reperfusion. PARP1 inhibitors may attenuate the progression of neurodegeneration in affected patients with ARH3 deficiency.

Astragaloside IV reduces neuronal apoptosis and parthanatos in ischemic injury by preserving mitochondrial hexokinase-II.

Cerebral ischemia induces neuronal cell death in different ways and mitochondrial dysfunction is an important cause. Astragaloside IV (AIV) is a natural saponin abandent in Astragalus membranaceus and this study aims to find if AIV protects neuronal survival via preserving mitochondrial hexokinase-II (HK-II). Glutamate stimulation induced HK-II dissociation from mitochondria and impaired mitochondrial function, indicated by the opening of the mitochondrial permeability transition pore, the collapse of mitochondrial membrane potential and reduced mitochondrial oxygen consumption ratio in neurons. Accompanied with apoptosis, oxidative DNA damage, PAR formation and nuclear translocation of apoptosis inducing factor (AIF) indicated the presence of parthanatos. AIV activated Akt and protected mitochondrial HK-II via promoting the binding of Akt to HK-II and protected hexokinase activity with improved glycolysis. As a consequence of preserved mitochondrial HK-II, AIV reduced the release of pro-apoptotic proteins and AIF, resultantly protected neurons from apoptosis and parthanatos. Moreover, the neuroprotective effects of AIV were also reproduced in mice subjected to middle cerebral artery occlusion to support the findings in vitro. Together, these results showed that glutamate excitotoxicity impaired mitochondrial HK-II and simultaneously induced apoptosis and parthanatos owing to mitochondrial dysfunction. AIV activated Akt to promote HK-II binding to mitochondria, and the structural and functional integrity of mitochondria contributed to protecting neurons from apoptosis and DNA damage. These findings address the important role of mitochondrial HK-II in neuronal protection.

LPS protects macrophages from AIF-independent parthanatos by downregulation of PARP1 expression, induction of SOD2 expression, and a metabolic shift to aerobic glycolysis.

In inflamed tissues or during ischemia-reperfusion episodes, activated macrophages produce large amounts of reactive species and are, thus, exposed to the damaging effects of reactive species. Here, our goal was to investigate the mechanism whereby activated macrophages protect themselves from oxidant stress-induced cell death. Hydrogen peroxide-treated mouse bone marrow-derived macrophages (BMDM) and THP-1 human monocyte-derived cells were chosen as models. We found a gradual development of resistance: first in monocyte-to-macrophage differentiation, and subsequently after lipopolysaccharide (LPS) exposure. Investigating the mechanism of the latter, we found that exposure to intense hydrogen peroxide stress causes poly(ADP-ribose) polymerase-1 (PARP-1) dependent programmed necrotic cell death, also known as parthanatos, as indicated by the protected status of PARP-1 knockout BMDMs and the protective effect of the PARP inhibitor PJ-34. In hydrogen peroxide-treated macrophages, however, apoptosis inducing factor (AIF) proved dispensable for parthanatos; nuclear translocation of AIF was not observed. A key event in LPS-mediated protection against the hydrogen peroxide-induced AIF independent parthanatos was downregulation of PARP1 mRNA and protein. The importance of this event was confirmed by overexpression of PARP1 in THP1 cells using a viral promoter, which lead to stable PARP1 levels even after LPS treatment and unresponsiveness to LPS-induced cytoprotection. In BMDMs, LPS-induced PARP1 suppression lead to prevention of NAD+ depletion. Moreover, LPS also induced expression of antioxidant proteins (superoxide dismutase-2, thioredoxin reductase 1 and peroxiredoxin) and triggered a metabolic shift to aerobic glycolysis, also known as the Warburg effect. In summary, we provide evidence that in macrophages intense hydrogen peroxide stress causes AIF-independent parthanatos from which LPS provides protection. The mechanism of LPS-mediated cytoprotection involves downregulation of PARP1, spared NAD+ and ATP pools, upregulation of antioxidant proteins, and a metabolic shift from mitochondrial respiration to aerobic glycolysis.