Poly(ADP-ribose) polymerase-1 protects neurons against apoptosis induced by oxidative stress

In neurons, DNA is prone to free radical damage, although repair mechanisms preserve the genomic integrity. However, activation of the DNA repair system, poly(ADP-ribose) polymerase (PARP-1), is thought to cause neuronal death through NAD+ depletion and mitochondrial membrane potential (delta psi(m)) depolarization. Here, we show that abolishing PARP-1 activity in primary cortical neurons can either enhance or prevent apoptotic death, depending on the intensity of an oxidative stress. Only in severe oxidative stress does PARP-1 activation result in NAD+ and ATP depletion and neuronal death. To investigate the role of PARP-1 in an endogenous model of oxidative stress, we used an RNA interference (RNAi) strategy to specifically knock down glutamate-cysteine ligase (GCL), the rate-limiting enzyme of glutathione biosynthesis. GCL RNAi spontaneously elicited a mild type of oxidative stress that was enough to stimulate PARP-1 in a Ca2+-calmodulin kinase II-dependent manner. GCL RNAi-mediated PARP-1 activation facilitated DNA repair, although neurons underwent delta psi(m) loss followed by some apoptotic death. PARP-1 inhibition did not prevent delta psi(m) loss, but enhanced the vulnerability of neurons to apoptosis upon GCL silencing. Conversely, mild expression of PARP-1 partially prevented to GCL RNAi-dependent apoptosis. Thus, in the mild progressive damage likely occur in neurodegenerative diseases, PARP-1 activation plays a neuroprotective role that should be taken into account when considering therapeutic strategies.     

Poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, protects against experimental immune ovarian failure in mice

The activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) may play an important role in numerous pathological conditions. The aim of the present study was to clarify the role of PARP in the pathogenesis of immune ovarian failure in mice and to examine the possible protective action of PARP inhibitor, 3-aminobenzamide (3-ABA). An experimental ovarian injury induced in mice by immunization with allogenic ovarian extract impaired the meiotic maturation of oocytes and increased apoptosis and necrosis of follicular cells and cells isolated from the spleen, lymph nodes and thymus. The immunization affected blood leukogram indicating the presence of an inflammatory response. Treatment with 3-ABA (1 h before antigen administrations, 0.02 mg/g intraperitoneally, twice a week during the three-week experiment) was effective to prevent meiotic maturation impairment, cell death and inflammation. The decrease in the necrosis of follicular and immune cells after 3-ABA treatment was more pronounced than that in apoptosis. It is concluded that PARP may contribute to immune-mediated ovary injury and PARP inhibitor, 3-ABA, protects against experimental immune ovarian failure, partially via a decrease in necrotic cell death.     

Poly(ADP-ribose) protects vascular smooth muscle cells from oxidative DNA damage

Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis. [BMB Reports 2015; 48(6): 354-359]     

Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

Background

Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress.

Methods

PARP-1(−/−) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function.

Results

PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(−/−), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(−/−) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, l-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(−/−) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(−/−) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(−/−) mice (p < 0.05 vs. PQ treated PARP-1(+/+).

Conclusion

PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.     

Poly(ADP-ribose) polymerase-1 inhibition protects the brain against systemic inflammation

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, but its overactivation can induce cell death. Our aim was to investigate the role of PARP-1 in activation of programmed cell death processes in the brain during systemic inflammation.Our data indicated that lipopolysaccharide (1 mg/kg b.w., i.p.)-evoked systemic inflammation enhanced PARP-1 activity in the mouse brain, leading to the lowering of β-NAD⁺ concentration, to translocation of apoptosis inducing factor from mitochondria to the nucleus, and to enhanced lipid peroxidation. Inhibitor of PARP-1, 3-aminobenzamide (30 mg/kg b.w., i.p.), protected the brain against prooxidative and cell death processes, suggesting involvement of PARP-1 in systemic inflammation-related processes in the brain.     

Poly(ADP-ribose) polymerase-1 inhibition protects the brain against systemic inflammation

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, but its overactivation can induce cell death. Our aim was to investigate the role of PARP-1 in activation of programmed cell death processes in the brain during systemic inflammation.

Our data indicated that lipopolysaccharide (1 mg/kg b.w., i.p.)-evoked systemic inflammation enhanced PARP-1 activity in the mouse brain, leading to the lowering of β-NAD⁺ concentration, to translocation of apoptosis inducing factor from mitochondria to the nucleus, and to enhanced lipid peroxidation. Inhibitor of PARP-1, 3-aminobenzamide (30 mg/kg b.w., i.p.), protected the brain against prooxidative and cell death processes, suggesting involvement of PARP-1 in systemic inflammation-related processes in the brain.     

Disruption of poly (ADP-ribose) polymerase (PARP) protects against stress-evoked immunocompromise.

BACKGROUND: Chronic stress, mediated by adrenal hormones, is a major risk factor in the progression and outcome of human disease. While the secretion of adrenal hormones is known to be the primary endocrine mediator of stress-induced immunocompromise, the molecular mechanisms underlying the immunocompromise remain unspecified. Overproduction of the nuclear enzyme, poly (ADP-ribose) polymerase (PARP) has been implicated in the molecular pathway that leads to cell death by energy depletion following stress. MATERIALS AND METHODS: Wild-type (WT) mice and mice with targeted disruption of the gene encoding PARP-1 (PARP-1 -/-) were subjected to 2 wk daily cold-water swim; splenocyte proliferation, anti-KLH IgG, and serum corticosterone concentrations were assessed. Additional mice of each genotype received daily i.p. injections of dexamethasone (DEX) (0.75 mg/kg) for 2 wk, and splenocyte proliferation and anti-KLH IgG were assessed. RESULTS: Splenocyte proliferation and specific antibody concentrations of stressed WT mice were reduced by ~20% of their pre-stress levels. In contrast, PARP-1 -/- mice maintained normal cell-mediated and humoral immune function following enforced cold-water swim stress. PARP-1 -/- mice also failed to compromise immune function following DEX treatment, whereas WT mice displayed significant reductions of immune function following this treatment. CONCLUSIONS: These results provide support for the involvement of PARP activation in immunological damage following physical stress. These results suggest that glucocorticoid-induced immunosuppression may require the activation of PARP in order for apoptosis of immune cells to take place. Taken together, these results suggest that therapies designed to inhibit PARP may prove valuable in the treatment of stress-related diseases.     

Poly(ADP-ribose) polymerase forms loops with DNA

The interaction between highly purified poly(ADP-ribose) polymerase from calf thymus and different topological forms of pBR322 DNA has been studied by gel retardation electrophoresis and electron microscopy. We show that: (i) in the absence of nicks on DNA the enzyme has a marked affinity for supercoiled (form I) DNA, (ii) in the presence of single stranded breaks poly(ADP-ribose) polymerase preferentially binds to form II, (iii) in all cases enzyme molecules are frequently located at DNA intersections, (iv) a cooperative binding of the enzyme on DNA occurs.     

A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression.     

Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death

PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.     

Poly(ADP-ribose) polymerase: molecular biological aspects.

A number of roles have been ascribed to poly(ADP-ribose) polymerase* including involvement in DNA repair, cell proliferation, differentiation and transformation. Cloning of the gene has allowed the development of molecular biological approaches to elucidate the structure and the function(s) of this highly conserved enzyme. This article will review the recent results obtained in this field.     

Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase

Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.     

The Poly(ADP-ribose) polymerase PARP-1 is required for oxidative stress-induced TRPM2 activation in lymphocytes

TRPM2 cation channels are widely expressed in the immune system and are thought to play a role in immune cell responses to oxidative stress. Patch clamp analyses suggest that TRPM2 channel activation can occur through a direct action of oxidants on TRPM2 channels or indirectly through the actions of a related group of adenine nucleotide 2nd messengers. However, the contribution of each gating mechanism to oxidative stress-induced TRPM2 activation in lymphocytes remains undefined. To better understand the molecular events leading to TRPM2 activation in lymphocytes, we analyzed oxidative stress-induced turnover of intracellular NAD, the metabolic precursor of adenine nucleotide 2nd messengers implicated in TRPM2 gating, and oxidative stress-induced TRPM2-mediated currents and Ca2+ transients in DT40 B cells. TRPM2-dependent Ca2+ entry did not influence the extent or time course of oxidative stress-induced turnover of NAD. Furthermore, expression of oxidative stress-activated poly(ADP-ribose) polymerases (PARPs) was required for oxidative stress-induced NAD turnover, TRPM2 currents, and TRPM2-dependent Ca2+ transients; no oxidant-induced activation of TRPM2 channels could be detected in PARP-deficient cells. Together, our results suggest that during conditions of oxidative stress in lymphocytes, TRPM2 acts as a downstream effector of the PARP/poly(ADP-ribose) glycohydrolase pathway through PARP-dependent formation of ADP-ribose.     

Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.