TNF-induced necroptosis and PARP-1-mediated necrosis represent distinct routes to programmed necrotic cell death

Programmed necrosis is important in many (patho)physiological settings. For specific therapeutic intervention, however, a better knowledge is required whether necrosis occurs through one single “core program” or through several independent pathways. Previously, the poly(ADP-ribose) polymerase (PARP) pathway has been suggested as a crucial element of tumor necrosis factor (TNF)-mediated necroptosis. Here, we show that TNF-induced necroptosis and the PARP pathway represent distinct and independent routes to programmed necrosis. First, DNA-alkylating agents such as 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or methyl methanesulfonate rapidly activate the PARP pathway, whereas this is a late and secondary event in TNF-induced necroptosis. Second, inhibition of the PARP pathway does not protect against TNF-induced necroptosis, e.g., the PARP-1 inhibitor 3-AB prevented MNNG- but not TNF-induced adenosine-5′-triposphate depletion, translocation of apoptosis-inducing factor, and necrosis. Likewise, olaparib, a more potent and selective PARP-1 inhibitor failed to block TNF-induced necroptosis, identical to knockdown/knockout of PARP-1, pharmacologic and genetic interference with c-Jun N-terminal kinases and calpain/cathepsin proteases as further components of the PARP pathway. Third, interruption of TNF-induced necroptosis by interference with ceramide generation, RIP1 or RIP3 function or by the radical scavenger butylated hydroxyanisole did not prevent programmed necrosis through the PARP pathway. In summary, our results suggest that the currently established role of the PARP pathway in TNF-induced necroptosis needs to be revised, with consequences for the design of future therapeutic strategies.     

Protective effects of the PARP-1 inhibitor PJ34 in hypoxic-reoxygenated cardiomyoblasts

To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD⁺ and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD⁺ and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion. Received 27 July 2006; accepted 26 October 2006     

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5^KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5^KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5^KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5^KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5^KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels.     

Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca<Superscript>2+</Superscript> fluxes in oxidative cell death

Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca²⁺ fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca²⁺ influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca²⁺ fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and proteins.     

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNA^Met. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNA^Met methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.     

Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons

Neuronal loss and neuritic/cytoskeletal lesions (synaptic disconnection and proliferation of dystrophic neurites) represent major dementia-associated abnormalities in Alzheimer’s disease (AD). This study examined the role of oxidative stress as a factor contributing to both the cell death and neuritic degeneration cascades in AD. Primary neuron cultures were treated with H₂O₂ (9–90 μM) or desferrioxamine (2–25 μM) for 24 h and then analyzed for viability, mitochondrial mass, mitochondrial function, and pro-apoptosis and sprouting gene expression. H₂O₂ treatment causes free-radical injury and desferrioxamine causes hypoxia-type injury without free radical generation. The H₂O₂-treated cells exhibited sustained viability but neurite retraction, impaired mitochondrial function, increased levels of the pro-apoptosis gene product CD95/Fas, reduced expression of N2J1-immunoreactive neuronal thread protein and synaptophysin, and reduced distribution of mitochondria in neuritic processes. Desferrioxamine treatment resulted in dose-dependent neuronal loss associated with impaired mitochondrial function, proliferation of neurites, and reduced expression of GAP-43, which has a role in path-finding during neurite outgrowth. The results suggest that oxidative stress can cause neurodegeneration associated with enhanced susceptibility to apoptosis due to activation of pro-apoptosis genes, neurite retraction (synaptic disconnection), and impaired transport of mitochondria to cell processes where they are likely required for synaptic function. In contrast, hypoxia-type injury causes neuronal loss with proliferation of neurites (sprouting), impaired mitochondrial function, and reduced expression of molecules required to form and maintain synaptic connections. Since similar abnormalities occur in AD, both oxidative stress and hypoxic injury can contribute to AD neurodegeneration. Received 24 May 2000; received after revision 7 July 2000; accepted 27 July 2000     

The roles of poly(ADP-ribose)-metabolizing enzymes in alkylation-induced cell death

Poly(ADP-ribose) (PAR) has been identified as a DNA damage-inducible cell death signal upstream of apoptosis-inducing factor (AIF). PAR causes the translocation of AIF from mitochondria to the nucleus and triggers cell death. In living cells, PAR molecules are subject to dynamic changes pending on internal and external stress factors. Using RNA interference (RNAi), we determined the roles of poly(ADP-ribose) polymerases-1 and -2 (PARP-1, PARP-2) and poly(ADP-ribose) glycohydrolase (PARG), the key enzymes configuring PAR molecules, in cell death induced by an alkylating agent. We found that PARP-1, but not PARP-2 and PARG, contributed to alkylation-induced cell death. Likewise, AIF translocation was only affected by PARP-1. PARP-1 seems to play a major role configuring PAR as a death signal involving AIF translocation regardless of the death pathway involved. Received 7 November 2007; received after revision 19 December 2007; accepted 21 December 2007 O. Cohausz, C. Blenn: These two authors contributed equally to this work.     

Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation

Poly-ADP-ribose polymerases (PARPs) use NAD⁺ as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD⁺ detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD⁺ pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD⁺ and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.     

NDRG2 is highly expressed in pancreatic β cells and involved in protection against lipotoxicity

The N-myc downstream-regulated gene 2 (NDRG2) is involved in cell differentiation and apoptosis, but its function in the pancreas remains to be established. Herein we examine the expression and function of NDRG2 in the endocrine pancreas. NDRG2 immunoreactivity was localized mainly in the cytoplasm of pancreatic β cells. When β-TC3 cells were exposed chronically to high levels of free fatty acid (FFA), cell viability was impaired, and Akt and NDRG2 phosphorylation were reduced. NDRG2 is a potential substrate of protein kinase Akt. Overexpression of constitutively active Akt enhanced NDRG2 phosphorylation and abolished the apoptosis induced by FFA in β-TC3 cells, whereas NDRG2 knock-down attenuated Akt-mediated protection of β cells against fatty acid-triggered apoptosis. Collectively, these data indicate that NDRG2 acts as a key molecule in pancreatic β cells and is involved in the Akt-mediated protection of β cells against lipotoxicity.     

Significance and molecular targets of protein kinase A during cAMP-mediated protection of cold stored liver grafts

The use of marginal donor livers is followed by a higher frequency of primary dys- or nonfunction after transplantation. The present study was designed to test the hypothesis that stimulation of the cAMP second-messenger signal pathway might protect the liver from ischemic injury, laying emphasis on the role of protein kinase A-mediated signal transduction.?Rat livers were harvested after 45 min of cardiac arrest and preserved in HTK solution for 24 h. Hepatic integrity was assessed thereafter using a blood-free reperfusion model.?Supplementation of the preservation solution with dibutyryl-cAMP (db-cAMP) promoted phosphorylation of BAD at Ser 112 and concomitantly mitigated mitochondrial release of cytochrome c into the cytosol. Apoptotic cell transformation was evident in reperfused livers by positive TUNEL-staining of sinusoidal lining cells and the detection of cleaved poly(ADP-ribose) polymerase (PARP) in tissue homogenates by western analysis. Treatment with db-cAMP was effective in minimizing both TUNEL staining and PARP cleavage and significantly reduced postischemic enzyme leakage of alanine aminotransferase to one half, while hepatic bile production was enhanced by approximately 60% when compared to untreated livers. This functional improvement was accompanied by a net amelioration of portal vascular conductivity. Inhibition of A kinase-anchoring protein with HT31 completely reversed any of the observed effects obtained by db-cAMP.?We conclude that enhancement of cellular cAMP signal maintains hepatic integrity during and after ischemic preservation which may be attributed to protein kinase A dependent phosphorylation of BAD in line with subsequent inhibition of mitochondria-initiated apoptosis of sinusoidal lining cells. Received 12 July 2001; received after revision 14 August 2001; accepted 14 August 2001     

Geranylgeranylacetone protects human monocytes from mitochondrial membrane depolarization independently of Hsp70 expression

The anti-ulcer drug geranylgeranylacetone (GGA) has been shown to induce the expression of heat shock proteins (HSPs), in particular of Hsp70, in gastric and small intestine cells. In this study, we investigated whether GGA was able to induce Hsp70 in another cell type, human monocytes, which represent a well-established model of Hsp70 expression under oxidative stress. In these cells, GGA had no significant effect either on basal or tobacco smoke-induced Hsp70 expression. We further investigated the effects of GGA on mitochondria, a key organelle of oxidant-mediated cell injury and a putative target for GGA-mediated protection. GGA significantly increased basal mitochondrial membrane polarization and inhibited the decrease in mitochondrial membrane potential of human monocytes exposed to distinct sources of clinically relevant oxidants such as tobacco smoke and y-irradiation. Our results indicate that mitochondria are targets for GGA-mediated protection against oxidative stress in human monocytes, independently of Hsp70.     

Pathophysiology of mitochondrial cell death control

Mitochondria have been recently recognized to play a major role in the control of apoptosis or programmed cell death. Permeabilization of mitochondrial membranes, a decisive feature of early cell death, is regulated by members of the Bcl-2 family which interact with the permeability transition pore complex (PTPC). Thus, the cytoprotective oncoprotein Bcl-2 stabilizes the mitochondrial membrane barrier function, whereas the tumor suppressor protein Bax permeabilizes mitochondrial membranes. The regulation of membrane permeabilization is intertwined with that of the bioenergetic and redox functions of mitochondria. The implications of alterations in the composition of the PTPC and in mitochondrial function for the pathophysiology of cancer (reduced apoptosis) and neurodegeneration (enhanced apoptosis) are discussed.